International Conference on Microbial Biofilms 11 - 13 May...

Biofilms 6 International Conference

on Microbial Biofilms

11 - 13 May 2014 Vienna, Austria

Biofilms 6

We are grateful for the support by our sponsors and co-operation partners.They greatly contribute to the success of Biofilms 6.

Program

Biofilms 6 International Conference

on Microbial Biofilms

11 - 13 May 2014 Vienna, Austria

Welcome and General Information

Information for Presenters Social Events

Program overview

Biofilms 6

Welcome

to the Biofilms 6 Conference

Microbial research is on the rise and more than ever biofilm research cuts across the boundaries of disciplines. Biofilms 6 will advance our understanding of microbial biofilms with a certain focus on environmental and technical systems. More than 300 scientists from more than 30 different nations will attend the conference to share their science with you. Biofilms 6 builds upon previous meetings in Paris (2014), Winchester (2010), Munich (2008), Leipzig (2006) and in Osnabrück (2004).

May I wish you a most successful and stimulating meeting and a pleasant stay in Vienna.

Tom Battin

General Information

National Organising CommitteeRegina SommerAndreas FarnleitnerHolger DaimsTom J. Battin International Scientific Committe Marvin Whiteley (USA)Paul Wilmes (LUX)Yehuda Cohen (SGP)Kevin Foster (GBR)Cristian Picioreanu (NDL)Harald Horn (GER)Thomas Neu (GER)Paul Stoodley (USA)Romain Briandet (FRA)Hans-Curt Flemming (GER)

Conference Office Gerry Schneider Margarethe Jurenitsch Benedikt Burkhardt Ulla Schröttner-BerningNikolaus Ortner

EventmanagementUniversity of ViennaUniversitätsring 11010 ViennaAustria

Biofilms 6 Artwork Emanuella Delignon Lino Battin Layout of the Conference Program Julia Kubetschka Petra Kohlmayr

Program

The University of Vienna

Situated on the Ringstrasse, the main building of the University of Vienna was designed in the style of historicism by architect Heinrich von Ferstel and inaugurated in 1884. Alongside the central administration, the main building also houses the University Lib-rary, as well as several institutes and administrative bodies (the research and teaching facilities of the University of Vienna are located at 60 different sites throughout the city).

Due to its central location in the historical center of Vienna and to its excellent mass transit connections, the main building of the University of Vienna is a very sought-af-ter venue for national and international congresses and conventions. Over 25 audito-ria equipped with state-of-the-art technology are available for any number of different events, while the Small Festival Room, with its magnificent ambience, offers a splendid backdrop for banquets. In the main Assembly Hall, in the two lateral Assembly halls, as well as in the Arcade Courtyard, frequent exhibitions are organized.

The “collected knowledge” of the University of Vienna is available at the University Li-brary. More than six million volumes and over 10,000 electronic media on about 72 ki-lometers of bookshelves await their readers. The Great Festival Room offers a dignified backdrop for graduation ceremonies and other festive functions. The ceiling is decorated with reproductions of faculty paintings by the painters Gustav Klimt and Franz Matsch.

Conference VenueUniversity of Vienna, Main Building

Venue addressUniversity of ViennaUniversitätsring 11010 Vienna

Registration deskThe eventmanagement-team of the University of Vienna will be pleased to help you with all inquires regarding registration, congress material and the conference program. Please do not hesitate to contact the team members if there is anything they can do to make your stay more enjoyable.

Registration feeThe fee includes the Icebreaker, workshop material, coffee breaks and the Conference Dinner.

Paticpant identification – badgesAll participants of the conference are kindly requested to wear their personal badge at all conference events. The access to the scientific sessions, lunch and coffee breaks, and social events is restricted to those who have registered.

Certificate of attendanceThe certificate of attendance will be provided when registering for the conference.

ExhibitorsBiofilms 6 is pleased to welcome a number of prominent com-panies that all exhibt instruments relevant to biofilm research. You ample opportunity to get involved with the exhibitors du-ring coffee breaks in the area of the Small Ceremonial Chamber (Kleiner Festsaal).

Biofilms 6

WiFi internet accessThe University of Vienna provides free wireless internet access for Biofilms 6 partici-pants. You can connect via u:connect (unencrypted), WiFi-Vouchers will be provided when registering for the conference.

Coffee breaks and lunchesDuring breaks, coffee, tea, water and snacks will be served in the exhibitor area around the Small Ceremonial Chamber (Kleiner Festsaal). Lunches will be served in the Arcade Courtyard (Arkadenhof) with the Posters. Coffee, tea, snacks and lunches are included in the registration fee. Beverages and snacks outside the regular breaks are not inclu-ded in the registration fee and can be purchased.

Prices and tipsMenu prices usually include service and taxes. In restaurants, a tip of approximately 5-10% is appreciated.

LoungeThe Senatsaal will also serve as a lounge where participants can relax or meet their colleagues and friends. For brief usage, a few computers will be provided adjacent to the conference office.

Important phone numbersEmergency number 112Fire brigade 122Police 133Ambulance 144Medical service 141

PharmacyThe nearest pharmacy is located on Universitätsstraße 10 (open: Mon-Fri 8.00 am – 6.00 pm, Sat 8.00 am – noon). The same opening times apply to most pharmacies in Vienna. For telephone information about the 24-hour pharmacy standby service call +43 1 1455.

SmokingDue to the non-smoking policy in public buildings, smoking is prohibited in all congress venues. There are some smoking areas in the Arcade Courtyard of the main building.

Program

Public transportationThe best way to discover Vienna is by public transport. The transport system comprises a dense network of trams, buses, underground trains and trains.

The following tickets are available:

Single-ride ticket EUR 2.10 at vending machines EUR 2.20 on trams only24-hour ticket EUR 7.1048-hour ticket EUR 12.4072-hour ticket EUR 15.40Week card EUR 15.80 valid from Monday to MondayVienna card EUR 21.90 72-hour ticket, reduced rates for guided tours, at restaurants

TaxiThe main taxi companies in Vienna can be reached on: +43 1 31300 or +43 1 40100 or +43 1 60160. There is a taxi stand across the street from the main building of the University in Schottengasse.

Tourist infoParticpants will receive a city map with their conference materials when registration. The nearest tourist info point is located in the city centre at Albertinaplatz (at the corner of Maysedergasse), and is open daily from 9.00 am to 7.00 pm. For more information about sightseeing in Vienna, please refer to the Vienna tourism website: www.wien.info/en.

Biofilms 6

Information for Presenters

Language of presentationsBiofilms 6 will bring together scientists from more than 30 nations. English is the official conference language.

Oral presentationsEach talk including discussion is allocated a time slot of 15 minutes. We recommend that presentations last 12 minutes followed by one or two questions. Computers for PowerPoint or PDF presentations are available in all lecture rooms. If you prepare a PowerPoint presentation, please make sure that it is compatible with Microsoft Power-Point 2007 for Windows. It is recommended to use standard fonts for preparing the pre-sentations to minimize the risk of distorted layout. If you need any particular additional equipment, please let us know in advance by contacting [email protected].

Please make sure to transfer your presentation files to the respective computer in the lecture room at least 1 hour before the start of your session, using a USB memory stick. Preferably, you should do this in the morning or in the break before the session. Ask the technical assistants in the lecture rooms for help. They will be present 30 minutes before each session and during the session. Note that a presentation with your own laptop computer is not possible.

Poster presentationsPoster presenters are asked to prepare their posters in DIN A0 portrait Format (841 mm x 1189 mm maximum).

There will be three poster sessions: Sunday, May 11th 2014, 5:30 pm – 8:00 pmMonday, May 12th 2014, 11:55 am – 2:10 pmTuesday, May 13th 2014, 11:55 am – 2:00 pm

Poster sessions take place in the Arcade Court (Arkadenhof). The number of each poster within each session can be found in the program. Please put your poster up latest by 18:00 on Sunday 11 May. Authors are kindly ask to be present at their poster during the sessions. We recommend preparation of handouts (miniature versions of the poster) for interested colleagues.

ExhibitonThe exhibition is located on the first floor of the University Building around the Small Ceremonial Chamber (Kleiner Festsaal) just beside the Plenary Room.

Exhibition days11.– 13. May 2014

Exhibition hoursSunday, May 11th: 1:00 pm – 6:00 pmMonday, May 12th: 8:00 am – 6:00 pmTuesday, May 13th: 8:00 am – 4:00 pm

Program

Social Events

Welcome PartyThe Biofilms 6 Welcome Party takes place on Sunday, 11 May, starting at 17:30. The Arcade Courtyard (Arkadenhof) of the University of Vienna provides the best setting for the Welcome Party during which posters can be discussed as well. Drinks and small snacks will be served.

Conference Dinner GARTENPALAIS LiechtensteinFürstengasse 1 1090 Wien

The Conference Dinner takes place in the Gartenpalais Liechtenstein — one of the fi-nest Baroque venues in Vienna and surrounded by glorious gardens. Life Baroque music will frame a welcome drink in the Sala Terrena, after which we will proceed to the Her-cules Hall to launch the new “NPJ Biofilms and Microbiomes” Journal with prominent contributions from Nature Publishing Group and Nanyang Technological University, Singapore. Next we will enjoy delicious Austrian cuisine. Fine electronic music in the Sala Terrena, drinks and a lounge will close the event.

Conference Dinner GARTENPALAIS LiechtensteinFürstengasse 11090 Wien

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Sunday, May 11th 2014

10:00-12:50 Registration and coffee

12:50 WelcomeTom Battin (University of Vienna)

13:00-13:40 Bird‘s eye view

13:00 Hans-Curt Flemming (University of Duisburg-Essen)

Biofilms – how microbes organize their social life

13:20 Farooq Azam (Scripps Institution of Oceanography)

Ocean as microbial biofilm

13:40-14:45 Evolution and ecology of biofilms – Alan Decho (Chair)

13:40 Kevin Foster (Oxford University)

Cooperation and competition in microbial communities

14:00 Knut Drescher (Princeton University)

Solutions to the Public Goods Dilemma in Bacterial Biofilms

14:15 Stacy Apollo (The University of Texas at Austin)

Bacterial fight and flight responses enhance virulence in a polymicrobial infection

14:30 Byarmuglo Bihter (Ben Gurion University)

Colicins role in Escherichia coli biofilms

14:45-15:30 Coffee break

15:30-17:05 New models for biofilm structure and function – Hans-Curt Flemming (Chair)

15:30 Jan-Ulrich Kreft (University of Birmingham)

Why develop generic individual-based models?

15:50 Phil Stewart (Bozeman, MT)

Reaction-Diffusion Theory Applied to Microbial Biofilms Associated with Chronic Infections

16:05 Eberl Hermann (University of Guelph)

Cross-diffusion in biofilms

16:20 Li Chunyan (Kalrsruhe Institute of Technology)

Tradeoff between mass transfer and actual biofilm surface area on carriers in moving bed biofilm reactors - Combination of optical coherence tomography imaging with two-dimensional modeling

16:35 Liu Li (Technical University of Denmark)

Biofilm model to provide in silico predictions of micropollutant biodegradation

16:50 Pechaud Yoan (Université de Toulouse)

Understanding the role of EPS distribution in biofilm and its consequence on cohesiveness and detachment in thick and mature biofilms: an experimental and modelling study

17:05-17:25 Bird‘s eye view

17:05 Paul Stoodley (The Ohio State University)

The mechanical properties of bacterial biofilms as a strategy for survival in ancient and modern environments

17:30-20:00 Welcome Party and Posters

Program

Monday, May 12th 2014

08:30-09:50 Omics for the study of biofilm structure and function – Jean-Marc Ghigo (Chair)

08:30 Paul Wilmes (University of Luxem-bourg)

Resolving microbial niche breadths in situ through integrated omics

08:50 Marvin Whitely (University of Texas at Austin)

Can‘t we all just get along: How cooperation and conflict shape polymicrobial infections

09:05 Keith Tuner (University of Texas at Austin)

Genetic requirements in spatially organized polymicrobial wound infections

09:20 Pascal Cosette (CNRS) Molecular Evaluation of biofilm influence on neighboring planktonic bacteria

09:35 Mia Bengtsson (Univeristy of Vienna) Impact of shading on the food web structure of phototrophic stream biofilms elucidated by metatranscriptomics


10:20-12:10 From genes to biofilm dynamics and back – Paul Wilmes (Chair)

10:20 Jean-Marc Ghigo (Institut Pasteur) Sniffing the crowds: airborne volatile molecules produced by bacterial communities

10:40 Karin Sauer (Binghamton University) It‘s all about taste and sense: Outside-in signaling mechanism of the NicD taste-like receptor required for nutrient-induced dispersion by Pseudomonas aeruginosa

10:55 Scott Rice (SCELSE NTU Singapore) The molecular mechanism and effects of superinfection of Pseudomonas aeruginosa by the filamentous phage Pf4

11:10 Hasmik Hayrapetyan (TI Food and Nutrition)

RpoN plays a pleiotropic role in Bacillus cereus affecting its lifestyle and biofilm formation

11:25 Banin Ehud (Bar-Ilan University) NanoRNase impacts biofilm formation and c-di-gmp signal transduction through its activity on pGpG

11:40 Pilar Sanchez-Vizuette (INRA) Coordinated interplay between submerged biofilm and liquid-air pellicles in Bacillus subtilis

11:55-14:10 Lunch and Posters

14:10-15:15 The extracellular space of biofilms – Roman Stocker (Chair)

14:10 Thomas Neu (Helmholtz Centre for Environmental Research)

The biofilm matrix — magic or mystic?

14:30 Rikke Meyer (Aarhus University) Differential role of eDNA, proteins, and polysaccharides in cell-cell and cell-substrate adhesion by three Staphylococcus species

14:45 David Stopar (University of Ljubljana) Nanostructure of extracellular space in biofilms

15:00 Thomas Brauge (ANSES) Exolopysaccharide characterization of extracellular matrix of Listeria monocytogenes biofilm.

15:15 Kim Se Yeon (INRA) Vascularisation of the biofilm matrix by hyper-motile bacteria


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16:00-17:35 Unveiling the unseen: new and emerging technologies in biofilm research – Paul Stoodley (Chair)

16:00 Roman Stocker (Massachusetts Institu-te of Technology)

Biofilms in flow: A reductionist, microfluidic approach

16:20 Julia Bruchmann (Karlsruhe Institute of Technology (KIT))

Novel microfluidic biosensor for online monitoring of biofilm formation

16:35 Alexander Rickard (University of Michi-gan)

A high-throughput microfluidic dental plaque biofilm system

16:50 Matthew Marshall (Pacific Northwest National Laboratory)

Chemical Imaging of Biofilms: The Integration of Synchrotron Imaging, Electron Microscopy and Nuclear Magnetic Resonance (NMR) Technologies

17:05 Jodi Connell (University of Texas at Austin)

Micro-3D Printing of Bacterial Communities

17:20 Holger Daims (University of Vienna) Picking them one by one: Raman-based sorting and single-microcolony genomics of uncultured microdiverse nitrifiers in activated sludge flocs

19:00 Conference Dinner

Tuesday, May 13th 2014

08:30-10:05 Biophysics and environmental consequences of microbial biofilms – Jan-Ulrich Kreft (Chair)

08:30 William Sloan (University of Glasgow)

Modelling interactions between hydrodynamics and dispersal that help shape biofilm community composition across a range of scales

08:50 Regina Viduthalai (Aarhus University)

Surface physicochemistry and ionic strength affects eDNA‘s role in bacterial adhesion to abiotic surfaces

09:05 Neda Davoudi (University of Kaiserslautern)

Adsorption of Sugars, Proteins, and Bacteria: A Multimethod Study for a Better Understanding of Biofilm Formation

09:20 Florian Blauert (Karlsruhe Institute of Technology)

A new attempt to measure biofilm rheology by optical coherence tomography

09:35 Patrick de Martino (Université de Cergy-Pontoise)

Amyloids modify the viscoelastic properties of biofilm model matrices

09:50 Thibaut Saur (Laboratoire de Biotechnologie de l‘Environnement)

Impact of the shear stress on initial bacterial adhesion


10:40-11:55 Applied water systems – Thomas Neu (Chair)

10:40 Edo Bar-Zeev (Yale University) Life under Hydraulic Pressure: Biofouling Development and Architecture

10:55 Arda Gülay (Denmark Technical university)

Mineral coating creates internal porosity and supports microbial activity in rapid sand filters treating groundwaters

11:10 Silvia Cervero (University of Barcelona)

The association with Acanthamoeba spp. affects the UV-sensitivity of Legionella pneumophila

11:25 Wim Hijnen (KWR Watercycle Research Institute)

Hardness of water and Calcium affects biofilm formation on PVC, glass and a thin-composite membrane

11:40 Olivier Habimana (University College Dublin)

Trapped in the matrix: the impact of Natural Organic Matter fouling on bacterial adhesion under full-scale Nanofiltration processes

Program

11:55-14:00 Posters and Lunch

14:00-15:05 Biofilms remixed: Microbial mats, stromatolites and soils – William Sloan (Chair)

14:00 Alan Decho (University of South Carolina)

Biofilms Remixed: Microbial mats and stromatolites

14:20 Dagmar Woebken (University of Vienna)

Investigating N2 fixation activity in photosynthetic microbial mats at the single-cell level

14:35 Clemens Karwautz (Helmholtz Zentrum München)

Massive microbial biofilms in a mineral spring cavern dominated by methane and iodine

14:50 Pedro Rodrigues Frade (University of Vienna)

Linking the composition of coral surface mucus to the structure and function of its associated microbial biofilm community

15:05-15:30 Young Investigator Awards – Regina Sommer (Medical University of Vienna)

15:30-15:40 Biofilms7 – Nuno Azevedo (University of Porto)

15:40 Closing

Biofilms 6

Biofilms 6

Abstracts of Invited Speakers 11- 13 May 2014, Vienna, Austria

Invited Talks

Session: Bird's eye view

Biofilms - how microbes organize their social life Flemming, Hans-Curt (Biofilm Centre, Faculty of Chemistry, Essen, GER) The world is full of microorganisms, they are everywhere. But only a tiny fraction of them lives as singles while the vast majority prefers to live in aggregates which are collectively termed as “biofilms”. They occur on solid surfaces exposed to water or humidity, but also at the air-water interface and also on living tissue. They are found even in extreme environments, from frozen to boiling water, from pH-value 1-14, in nuclear power plants, in space stations, and even in disinfection pipelines. Biofilms are the oldest forms of life on Earth with fossils dating back over 3 billion years. In biofilms, photosynthetic microorganisms evolved, producing oxygen as a “waste gas” and turning the originally reducing atmosphere into an oxidizing one. This caused a first environmental disaster for the ancient anaerobic bacteria who were dominant before, because oxygen was toxic to them. Photosynthesis changed the planet and is ultimately the basis of our life. In biofilms, the microbes are embedded in a slimy matrix of extracellular polymeric substances (EPS), consisting of hydrated polysaccharides, proteins, DNA and lipids1. Why is life in that matrix so popular? The reason is that the formation of this matrix results in so-called “emergent properties”. This means that the matrix is a collective effect (more than the sum of its parts) and living in it differs profoundly from solitary life of the cells – biofilms bear aspects of a tissue, but also profound differences. Embedded in that matrix, biofilm cells are much more resistant to disinfectants and other stress factors such as antibiotics, toxic metals, heat or irradiation. In the matrix, steep gradients exist, e.g., of oxygen concentration, pH-value, redox potential, nutrients and products, resulting in “microbial landscapes”2. That generates a wide variety of habitats and, thus, a large biodiversity, which is another ecological advantage. Particularly interesting is the fact that the matrix is full of extracellular enzymes which are produced by the biofilm organisms and retained in their close proximity. The result is an external digestion system which allows the breakdown of insoluble materials much larger than the microbes. Biofilms represent the “global cleaning company” for organic carbon and are, thus, involved not only in the biogeochemical cycles of carbon but also of oxygen, nitrogen, phosphorus, sulfur and most metals. Their biodegradation properties are exploited technically in biofiltration and waste water treatment. The matrix allows for the formation of stable microconsortia in which strong synergistic cooperation of different species can develop; nitrification is one example. The organisms communicate with each other and organize their interactions in a way which can be described as “sociomicrobiology”3. In infectious processes, this is of strong relevance, because the expression of virulence factors is regulated by “quorum sensing”, indicating when a critical number of cells capable to attack host tissue is reached. This is performed by small molecules which are retained in the matrix; their concentration is sensed as a signal. On the other hand, biofilms are the place of strong competition, and it can be assumed, that the concept of infection has evolved along competition when one cells attack another one. ...

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Ocean as Microbial Biofilm Azam, Farooq (Scripps Institution of Oceanography University of California San Diego, La Jolla, USA) Microbes are a major component of ocean biomass, abundance, biodiversity and metabolic activity, therefore, it is important to understand how their in situ activity structures the ocean ecosystems, and response to climate change. Despite major strides in describing microbial diversity and genomic predictions of activity we lack knowledge of microbes’ interactions with other microbes and the architecture of the habitat in which these interactions play out. Considerable data now show that seawater is replete with nanometer to millimeter scale gels, fiber networks and refractory organic matter creating an organic matter continuum within which seemingly free bacteria and Archaea as well as the traditional organic matter hotspots are dynamically embedded. This architecture is created and maintained by microbes’ interactions and EPS production—constituting the ocean’s “connectome” and “interactome”. We suggest cumulatively conceptualizing the ocean’s microbiome and organic matter continuum as a having basic characteristics of a biofilm. Indeed, the global ocean’s biofilm may have been an evolutionary force throughout much of history of life in the sea. This context may help understand and predict the structure and biogeochemical dynamics of the future ocean in response to climate change. The Mechanical Properties of Bacterial Biofilms as a Strategy for Survival in Ancient and Modern Environments. Stoodley, Paul (nCATS, Faculty of Engineering and the Environment (FEE) / Center for Microbial Interface Biology, Departments of Microbial Infection and Immunity, and, Southhampton, GBR); Rmaile, Amir (nCATS, Faculty of Engineering and the Environment (FEE) / Philips Research, Oral Healthcare Research, Eindhoven, GBR); Fabbri, Stefania (nCATS, Faculty of Engineering and the Environment (FEE), University of Southampton, GBR); Carugo, Dario (Bioengineering Science Research Group, (FEE), University of Southampton, GBR); Aspiras, Marcelo (Philips Oral Healthcare Inc. (POH), Bothell, USA); De Jager, Marko (Bioengineering Science Research Group, (FEE), University of Southampton, GBR); Ward, Marilyn (Philips Oral Healthcare Inc. (POH), Bothell, USA); Thurner, Philipp J. (Bioengineering Science Research Group, FEE / Institute of Lightweight Design and Structural Biomechanics, AUT); Decho, Alan W. (University of South Carolina, Environmental Health Sciences, Columbia, South Carolina , USA); Noffke, Nora (Old Dominion University, Ocean, Earth and Atmospheric Sciences, Norfolk, Virginia, USA) Viscoelasticity has been shown to be a conserved mechanical feature of bacterial biofilms formed by a wide diversity of species as well as for polymicrobial biofilms. More specifically biofilms behave as viscoelastic liquids meaning that they can deform elastically or through flow. The viscoelastic properties are dominated by the extracellular polymeric slime (EPS) matrix, which has multiple functions, including provision of a physical structure for the communities of cells as well as a protective role against antimicrobials and environmental stresses. Previously, it was shown in laboratory experiments that biofilms can flow in ripple formations across surfaces, driven by momentum transfer from the overlying aqueous flow. Similar ripple-like structures have been observed in modern environments and ancient fossils suggesting that viscoelasticity is an ancient

Invited Talks

adaptation to surface associated life in the presence of flowing water. To date, the response of biofilms to fluid flow has been recorded over relatively long time scales. By using high-speed imaging we have revealed that model dental biofilms also behave as viscoelastic liquids when impacted by high-velocity water and air droplets. These images show that biofilms can flow over surfaces at a high velocity, and after being “shredded” by the impacting fluid, can quickly coalesce into a single mass if they are not removed, supporting the hypothesis that the viscoelastic properties are important for survival on surfaces. To effectively remove biofilm from surfaces through fluid flow the generated flow and shear rates need to be beyond the physiological limits of biofilm adaptation to survival in normal flows and/or sustained for long enough to push biofilms over the edge.

Session 1: The extracellular space of biofilms

The biofilm matrix - magic or mystic? Neu, Thomas (Helmholtz Centre for Environmental Research / UFZ, Magdeburg, Magdeburg, GER) The presence of an extracellular matrix in microbial communities associated with interfaces forming bio-films and bio-aggregates is well accepted. Over time more and more matrix constituents were identified. Based on their properties various functions were suggested. Reasonable details are known from pure culture studies of few strains investigated precisely such as Escherichia, Bacillus, Pseudomonas, Streptococcus, etc.. Nevertheless, in environmental microbial communities the tremendous diversity of microbial species and as a consequence of matrix characteristics represents a demanding task. In most cases the biofilm matrix was examined by means of extraction with subsequent bulk chemical analysis or by in situ imaging usually after lectin-staining in combination with laser microscopy. This presentation will address major aspects of the biofilm matrix and identifies several issues regarding matrix properties and functions. Finally suggestions are made to further progress studies with focus on the extracellular space in microbial communities. In any case, despite 40 years of biofilm research, the characterisation of the matrix in biofilm systems remains a grand challenge and the adjectives “magic” (Christensen & Characklis 1990) and “mystic” (Cooksey 1992) are still valid.

Session 2: New models for biofilm structure and function

Why develop generic individual-based models? Kreft, Jan-Ulrich (University of Birmingham, Birmingham, GBR) In order to understand the effect of different environments on microbes and their interactions, it is useful to be able to simulate microbes in different environments, everything else being equal. For example, comparing biofilms with chemostats helps to understand the effect of spatial structure, which is often very important. Another reason for developing generic models for many different

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studies rather than specific models for each specific case study is ‘structural robustness’. If it is convenient for researchers to test the effect of including various processes in their model on the question of interest, they are more likely to do so than when they have to invest half a year or more on implementing the process in their own specific model without knowing whether this process is important. Often, the consequence is that structural robustness of models is not tested, but this may well be more important than investigating parameter sensitivity (equations/processes remain the same, only parameters are varied).

An example illustrating the utility of developing a model for both chemostats and biofilms is a study of plasmid dynamics. Plasmids vary in host range. Some bacterial plasmids are ‘specialists’ with a narrow host range (NHR) while others are generalists with a broad host-range (BHR). We assume that the BHR strategy should have a greater fitness burden for the host as frequent swapping between hosts should not allow enough time for co-evolution between any plasmid and host pair to minimise fitness costs. So what are the advantages of having a broad host range in a biofilm or chemostat? We obtained some surprising results for chemostats. A costly NHR plasmid that can survive in a single species community cannot survive in a multispecies community. Further, adding a competing BHR plasmid helps the NHR plasmid to survive under a range of conditions but the BHR plasmid can out-compete the NHR plasmid if it has a sufficiently high transfer rate. In the individual-based chemostat model, where dilution is stochastic, we observe bottlenecks leading to the quick extinction of one or both types of plasmids. In biofilms or structured communities we find that both plasmids can co-exist under conditions where they could not in the well-mixed chemostats, but the BHR strategy is always the better competitor. This is due to the ability of the BHR plasmid to cross boundaries between patches occupied by different hosts. Overall, spread of plasmid encoded antibiotic resistance genes is facilitated when they are present on both broad and

narrow host range plasmids, and on broad host range plasmids in spatially structured systems.

An example illustrating the utility of using a model with more processes than absolutely required for a specific case study is the evaluation of fitness benefits and costs of damage segregation versus repair in unicellular organisms. Damage segregation can occur during cell division, and leads to aging of the cell that retains the damaged material. Repair is the alternative. A range of models have been developed specifically to investigate this question, demonstrating that damage segregation is the better strategy. However, these models either did not consider repair properly, or did not consider that cells are growing between divisions. The latter is a critical oversight because repair of damage increases the pool of active proteins catalysing growth, and therefore provides an advantage to cells only if they are assumed to grow in the model. A model including growth and

repair processes makes the opposite prediction that repair is better than aging.

Generic models make it convenient to check the effect of different environments or additional processes that one might otherwise decide to ignore when time is running out on a project,

potentially affecting conclusions.

Invited Talks

Session 3: Unveiling the unseen: new and emerging technologies in biofilm research

Biofilms in flow: A reductionist, microfluidic approach Stocker, Roman (Massachusetts Institute of Technology Civil and Environmental Engineering , USA) One philosophy to cope with the immense complexity of biofilms is to adopt the most controlled approach available and focus on selected processes. Microfluidic technology is perhaps the best enabler of this reductionist approach, as it allows one to accurately control the geometry of the environment, the rates of flow and nutrient delivery, the shear stress and also, as I will show, the biofilm's footprint. Here I will illustrate the power of microfluidics by reporting new findings on the effects of fluid flow on (i) the early stages of biofilm formation and (ii) the disruption of middle-aged biofilms. First, I will demonstrate that fluid flow can have a counterintuitive effect on cell attachment to surfaces. Using microfluidic experiments with accurately controlled shear rates combined with single-cell tracking, we found that the coupling of shear and motility induces trapping of bacteria in the high-shear regions of the flow, and thus a strong preferential accumulation of cells in the immediate vicinity of the surface. Consequently, we observe faster surface coverage as shear rate increases. Second, I will show that the application of strong impulsive shear associated with the passage of a controlled air plug over precisely patterned biofilm patches in a microchannel, results in a new mode of biofilm disruption, whereby a characteristic, semi-regular (Swiss cheese) pattern of 'holes and levees' forms. EPS staining together with a microscale spatial correlation analysis demonstrate that holes open where attachment is weak, and that 'levees' are composed of 'mechanical persisters', cells residing in areas with high EPS concentration (presumably, the first settlers) that act as strongholds against mechanical insult. These examples aim to show that the precise control over environmental conditions inherent in the microfluidic approach can afford new mechanistic insights into otherwise complex biofilm processes.

Session 4: Evolution and ecology of biofilms

Cooperation and competition in microbial communities Foster, Kevin (Oxford University, Oxford, GBR) Since Darwin, evolutionary biologists have been fascinated by cooperative behavior. Honeybee workers labor their whole life without reproducing, birds make alarm calls, and humans often help one another. One major group that remains relatively unexplored, however, is the microbes whose full spectrum of sociality only recently came to light. Microbes often live in large dense groups where one cell can strongly affect the survival and reproduction of others. But do microbes typically help or harm those around them and can we identify the factors that promote cooperation over competition? We study these questions using a diversity of systems, including computer simulations, pseudomonad bacteria and budding yeast. We find that single-genotype patches

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naturally emerge in microbial groups, which creates favorable conditions for cooperation within a particular genotype. Experimental evolution in bacteria shows that this process drives extremely strong natural selection for cooperative adaptations that can be understood at the molecular scale. Moreover, some microbes actively adjust both genotypic assortment and investment into social traits in a way that promotes cooperation within a genotype. However, our work on interactions between different microbial genotypes suggests that, here, the evolution of competitive phenotypes is more likely than cooperation. This leads us to a simple model - the genotypic view - that predicts microbes will evolve to help their own genotype but harm most other strains and species that they meet.

Session 5: Omics for the study of biofilm structure and function

Resolving microbial niche breadths in situ through integrated omics Wilmes, Paul (ATTRACT Research Fellow , Esch-sur-Alzette) Microbial communities (biofilms) are complex and dynamical systems that are primarily structured according to their members’ ecological niches. Resource availability and usage govern population sizes and structures. High-resolution “meta-omics” offer exciting prospects to investigate microbial populations in their native environment. In particular, micorbial community integrated omics, by allowing simultaneous resolution of fundamental niches (genomics) and realized niches (transcriptomics, proteomics and metabolomics), can resolve microbial lifestyles (generalist versus specialist lifestyle strategies) in situ. We have recently developed the necessary wet- and dry-lab methodologies to carry out systematic molecular measurements of microbial consortia over space and time, and to integrate and analyze the resulting data at the population-level. We have applied these methods to oleaginous mixed microbial assemblages, which are found on the surface of anoxic biological wastewater treatment tanks. Coupled metabolomics and 16S rRNA gene-based deep sequencing demonstrate that the community-wide lipid accumulation phenotype is associated with the dominance of Candidatus Microthrix spp. By integrating population-level genomic reconstructions with transcriptomic and proteomic data, we found that the dominance of this microbial generalist population results from finely tuned resource usage and optimal foraging behavior. Moreover, the fluctuating environmental conditions constrain the accumulation of variations, leading to a genetically homogeneous population likely due to fitness trade-offs. In summary, our integrated omic study of oleaginous mixed microbial communities demonstrates that natural microbial population sizes and structures are intricately linked to resource usage and that differing microbial lifestyle strategies explain the varying degrees of within-population genetic heterogeneity observed in metagenomic datasets. Therefore, the niche breadths of different populations have to be considered as important factors for understanding the ecological and evolutionary processes governing microbial population sizes and structures in situ.

Invited Talks

Session 6: From genes to biofilm dynamics and back

Sniffing the crowds: airborne volatile molecules produced by bacterial communities Ghigo, Jean-Marc (Institut Pasteur, Paris, FRA) Bacteria produce and sense a high diversity of signals to adapt their behaviors to changing environments. Among them, volatile compounds produced by fungi and plants were shown to modify bacterial activities. However, besides being perceived as odors, attractive scents or pollutants, by animal’s olfactory receptors or specialized detection devices, the influence of bacterial volatile molecules on bacteria themselves remained poorly explored. I will present our recent investigation of how volatile compounds produced by high-cell density cultures or biofilms can contribute or result from the development of bacterial communities.

Session 7: Biophysics and environmental consequences of microbial biofilms

Modelling interactions between hydrodynamics and dispersal that help shape biofilm community composition across a range of scales. Sloan, William (University of Glasgow, Glasgow, GBR) In any open biological community there are a few basic biological processes that shape the community composition: births, deaths, immigration and emigration. Of course there are myriad factors that influence the rate at which each of these occur in different species within a community. For bacterial biofilms, our understanding and growing ability to quantify these factors has been furnished by laboratory experimentation often at very-small scale in comparison to the system that is ultimately of interest.

Here we present circumstantial evidence that in drinking water distribution networks, where biofilm control is of paramount importance, and in river networks, where biofilms mediate biogeochemical cycling, limitations in the hydrodynamic dispersal of bacterial between locations has a strong influence on the community composition. Thus, where biofilms reside in a hydraulically connected network, immigration and emigration at the local scale can have a strong effect on the biodiversity patterns across the network. In most biofilm models, conceived on the basis of laboratory biofilms, these mechanisms assume less importance than the environmental and biological factors affecting births and deaths and are often modelled as flow-dependent biomass loss/gain terms. We present evidence and models for more complex dynamic interactions between dispersal and flow regimes. Developing a better understanding of bacterial dispersal mechanisms in networks should allow for a more robust quantification of risk in, for example, pathogen occurrence in drinking water or

disturbance of river ecosystems, which should inform network management strategies.

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Session 8: Biofilms remixed: Microbial mats, stromatolites and soils

Biofilms Remixed: Microbial mats and stromatolites Decho, Alan W. (University of South Carolina, USA) Microbial mats once dominant life on Earth. Stromatolites and other forms of microbial mats comprise the earliest fossil vestiges of life. They are also interesting, present-day systems to observe biofilms. The very high diversities of microbial species, and highly-structured spatial patterns, arguably exemplify the apex of microbial cooperation (or partitioning). The combined interactions of not just microbial species, but different functional groups (clades) result in development of large macrostructures having structurally-organized precipitation of CaCO3 laminae. The microspatial management of different forms of EPS forges spatially-controlled precipitation of CaCO3through either inhibition or enhancement of precipitation, depending on microbial orogeny. The formation of lithfied laminae, themselves, has been suggested by others to represent a primitive, multicellular effort by microbes to develop support structures, analogous to eukaryote shells and bones. Lithified laminae in present-day open-water Bahamian stromatolites offer physical stability against intense current and wave action. Laminae may have contributed different roles to microbial communities eons ago by blocking intense UV-irradiation, as shown by the strong absorbance of UV wavelengths by different carbonate forms (i.e., calcite, aragonite, vaterite). How do the thousands of species/strains coexist in microspatial proximity? Chemical sensing and communication occurs and may be linked to the geochemistry of diel (day/night) cycles. Finally, the resiliency of mat systems during desiccation begins to illustrate EPS-linked survival tactics by both bacteria and archaea, and the possibility of chemical communication messages lasting months at a time. Mats in anhydrophilic hypersaline systems experience alternating periods (e.g., months) of low-salinities, and periods of high-salinities often followed by desiccation. The EPS contain abundant lipid vesicles in their EPS and the formation of ‘microbial glass’ occurs during the desiccation process. These may play roles in the preservation of microbial cells, extracellular signals and enzymes. Intense microbial warfare is also exemplified by production of diverse antagonistic chemicals, which is providing a source of novel and potentially powerful antibiotics.

This overview talk will focus on previous and ongoing research using extant marine stromatolites and other mats from hypersaline ponds tropical carbonate environments in the Bahamas to

illustrate different properties used by biofilms to manage their respective fluctuating environments.

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Abstracts of oral and poster presentations 11- 13 May 2014, Vienna, Austria

Abstracts of oral presentations Session 1: The extracellular space of biofilms ................................................................................................................................ 1 Session 2: New models for biofilm structure and function .............................................................................................................. 5 Session 3: Unveiling the unseen: new and emerging technologies in biofilm research .................................................................... 10 Session 4: Evolution and ecology of biofilms................................................................................................................................ 15 Session 5: Omics for the study of biofilm structure and function ................................................................................................... 17 Session 6: From genes to biofilm dynamics and back .................................................................................................................... 20 Session 7: Biophysics and environmental consequences of microbial biofilms ............................................................................... 24 Session 8: Biofilms remixed: Microbial mats, stromatolites and soils ............................................................................................ 30 Session 9: Applied water systems................................................................................................................................................ 32

Abstracts of poster presentations Session 1: The extracellular space of biofilms .............................................................................................................................. 37 Session 2: New models for biofilm structure and function ............................................................................................................ 48 Session 3: Unveiling the unseen: new and emerging technologies in biofilm research .................................................................... 52 Session 4: Evolution and ecology of biofilms................................................................................................................................ 72 Session 5: Omics for the study of biofilm structure and function ................................................................................................... 94 Session 6: From genes to biofilm dynamics and back .................................................................................................................. 103 Session 7: Biophysics and environmental consequences of microbial biofilms ............................................................................. 113 Session 8: Biofilms remixed: Microbial mats, stromatolites and soils .......................................................................................... 131 Session 9: Applied water systems.............................................................................................................................................. 139 Session 10: Medical systems and anti-biofouling ....................................................................................................................... 153

Abstracts of oral presentations

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Session 1: The extracellular space of biofilms

Exolopysaccharide characterization of extracellular matrix of Listeria monocytogenes biofilm. BOURDIN, GRAZIELLA (ANSES, BOULOGNE SUR MER, FRA); BRAUGE, THOMAS (ANSES, BOULOGNE SUR MER, FRA); SADOVSKAYA, IRINA (ULCO, BOULOGNE SUR MER, FRA); FAILLE, CHRISTINE (INRA, VILLENEUVE D'ASCQ, FRA); MAES, EMMANUEL (USTL UGSF, VILLENEUVE D'ASCQ, FRA); GUERARDEL, YANN (USTL UGSF, VILLENEUVE D'ASCQ, FRA) Listeria monocytogenes (L. monocytogenes ) is a pathogenic bacterium implicated in foodborne infections cause listeriosis, an uncommon disease but with a mortality rate of 12.7% in humans (EFSA, 2011) . Seventeen serotypes are distinguished in the species L. monocytogenes , the most widespread in the foodborne infection are serotypes 1/2a (serogroup 1/2) and 4b (serogroup 4). This bacterium is capable of binding to biotic or abiotic surfaces and secretes an extracellular matrix and protective adhesive enabling it to form biofilms. This extracellular matrix is composed of exopolysaccharides , proteins, DNA, and water (Combrouse et al., 2013). Some work was done on the characterization of exopolysacharide but only parietal (Uchikawa et al.,1986). In order to better characterize biofilms L. monocytogenes, we characterized the exopolysaccharides secreted in the extracellular matrix of biofilm of serogroup 1/2 and 4 strains of L. monocytogenes versus the exopolysaccharide parietal and exopolysaccharide excreted into the culture medium of the biofilm.

Biofilms of four serogroup 1/2 strains and two serogroup 4 strains of L. monocytogenes were grown on polystyrene in a poor carbon source medium MCDB 202 (Hébraud and Guzzo, 2000) at 37 ° C.

After 48 hours of incubation, the exopolysaccharides were extracted from bacterial cell walls, of the extracellular matrix and the culture medium (Sadovskaya et al, . 2005). These were analyzed by gas chromatography - mass spectrometry (GC MS) and nuclear magnetic resonance 900 MHz

2549 and lmo 2550 genes were sequenced for the serogroup 1/2 strains of L. monocytogenes for confirmation of the observations in RMN.

For five strains, we observed that the major exopolysaccharides secreted in the extracellular matrix and in the culture medium of L. monocytogenes biofilm were structurally identical to the cell wall serogroup teichoic acid described in 1986 by Uchikawa et al. (1986). For one strain, DSS 1130 BFA2 (serogroup1/2 by PCR), we identified a teichoic acid with rhamnose but without the N -acetylglucosamine substitution or other polysaccharide. We sequenced the lmo 2549 and lmo 2550 genes of the serogroup 1/2 strains versus the sequences of the EGD-e 1/2 serogroup strain. We identified a mutation on the lmo2550 gene sequence for the DSS 1130 BFA2 strain and a premature codon stop leading to a truncated exopolysaccharides without N acetylglucosamine ramification. These results obtained with this strain have allowed us to demonstrate that the teichoic acid and excreted in the matrix of the biofilm culture medium of L. monocytogenes were from potentially the same biosynthetic pathway as teichoic acids parietal which has never been demonstrated for other bacterial genus. These results have allowed us to better characterize the extracellular matrix of biofilms of L. monocytogenes which helps to better understand and manage the health risk associated with this bacterium in industries.


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Differential role of eDNA, proteins, and polysaccharides in cell-cell and cell-substrate adhesion by three Staphylococcus species Meyer, Rikke (Aarhus University, Aarhus C, DNK); Okshevsky, Mira (Aarhus University, Aarhus C, DNK); Zeng, Guanghong (Aarhus University, Aarhus C, DNK) The diversity in mechanisms for bacterial attachment and biofilm formation is the overarching challenge for development of strategies to combat biofilms. Understanding the quantitative contribution of different cell surface adhesins biofilm formation is therefore valuable for designing new approaches to biofilm prevention. In this study, we combine microfluidic flow-cell studies with single-cell analyses to understand how polysaccharides, extracellular DNA (eDNA), and proteins contribute to bacterial adhesion and aggregation on abiotic surfaces. We quantified initial adhesion, cell aggregation, and single-cell adhesion forces of Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus xylosus in the presence and absence of DNase, dispersin, or subtilisin, which cleave extracellular DNA, polysaccharides and proteins, respectively. Initial adhesion of S. aureus to glass was strongly affected by DNase and by subtilisin, but not by dispersin. This was shown by quantifying adhered cells in a microfluidic flow system and by challenging the adhesion strength of these cells by passing the surface through an air/liquid interface. This treatment lead to detachment of almost all cells treated with DNase or subtilisin, indicating weak adhesion. Single-cell force spectroscopy measures the force needed to detach a cell from the surface after a few seconds of contact, and we used this method to quantify adhesion forces by the same cell before and after enzyme treatment. Our analyses confirmed a substantial (>90% ) weakening of the adhesion force after DNase or subtilisin treatment. Hence eDNA and proteins are the most important adhesins for initiating S. aureus biofilms. S. epidermidis was strongly affected by all enzyme treatments, which in addition to impairing adhesion to glass, also prevented the formation of aggregates and streamers observed abundantly in control samples. One could speculate that making use of several types of adhesins would enable an organism to form biofilms even if one type is missing, but in this case it appears that removal of any one of these adhesins prevents biofilm formation.

S. xylosus. Adhesion to glass was almost unaffected by any of the enzyme treatments, and even passing the surface through an air/liquid interface could not remove the adhered cells despite enzyme treatments. Despite the successful colonization of glass surface, the ability of S. xylosus to form aggregates was strongly affected by DNase and dispersin treatments, hence eDNA and polysaccharides were essential for cell-cell interactions. We showed that proteins, polysaccharides and eDNA contribute differently to the adhesion of three Staphylcococcus species, underlining the need to either tailor biofilm prevention strategies to a specific species, or to combine several strategies to target a broader spectrum of microorganisms. Among the three enzymatic treatments used in this study, we found that removal of eDNA had the most general impact, as it weakened adhesion forces and lowered the adhesion rate of S. epidermidis and S. aureus to glass surfaces, while it also weakened cell-cell interactions and hampered aggregation in all species. Dispersin was most effective against S. epidermidis and S. xylosus, whereas subtilisin was most effective against S. aureus. eDNA and polysaccharides appeared to work in concert to form streamers and aggregates, as removal of either prevented their formation. Hence this work has spurred further investigations into the interdependence of eDNA and polysaccharides in bacterial attachment and biofilm formation.


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Vascularisation of the biofilm matrix by hyper-motile bacteria KIM, Se Yeon (INRA , MASSY, FRA); DESCHAMPS, Julien (INRA, Massy, FRA); Li, Yingbo (INRA, MIAJ, , Jouy en Josas, FRA); Trubuil, Alain ( INRA, , Jouy en Josas, FRA); Lapadatescu, Carmen (Naturatech, LEVALLOIS-PERRET , FRA); Gruss, Alexandra (INRA, Massy, FRA); Piard, Jean-Christophe (INRA, Massy, FRA); Briandet, Romain (INRA, Massy, FRA) Se Yeon Kim1; Julien Deschamps1; Yingbo Li2; Alain Trubuil2; Carmen Lapadatescu3; Alexandra Gruss1; Jean-Christophe Piard1; Romain Briandet1. 1 INRA, UMR1319 Micalis, 25 avenue République, 91300, France 2 INRA, MIAJ, Domaine de Vilvert, 78352, France 3 Naturatech, 7 rue Aristide Briand, 92300, France Biofilms are everywhere in our environment, in our gardens, to the hospital in our bathrooms and refrigerators, with benefits or dangers. But microorganisms forming biofilms are they all completely immobilized in the organic matrix? We recently highlight the existence of flagellated bacteria able to infiltrate and "vascularize" these microbial biostructures by digging a network of transitory tunnels in the matrix [1]. We discovered that a subpopulation of planktonic bacilli is propelled by flagella to tunnel deep within a biofilm structure. Swimmers create transient pores that increase macromolecular transfer within the biofilm. Irrigation of the biofilm by swimmer bacteria may improve biofilm bacterial fitness by increasing nutrient flow in the matrix. However, we showed that the opposite may also occur (i.e., swimmers can exacerbate killing of biofilm bacteria by facilitating penetration of toxic substances from the environment). We combined these observations with the fact that numerous bacteria produce antimicrobial substances. We demonstrated with a set of mutants that motile bacilli expressing a bactericide can also kill a heterologous biofilm population, Staphylococcus aureus in this case, and then occupy the newly created space. From the basis of this proof of concept, we screened 350 natural bacilli isolates for swimming ability and production of antibiofilm agents. In order to quantify the isolates performance, and propose synergetic cocktails of strains, a dedicated confocal time-lapse image-tracking pipeline was developed by computer scientists. All together, these findings identify microbial motility as a determinant of the biofilm landscape and add motility to the complement of traits contributing to rapid alterations in biofilm populations. Acknowledgments: ANR-12- ALID-0006 GreenSwimmers References: [1] Houry, A. et al. Proc Natl Acad Sci U S A. 2012 Aug 7;109(32):13088-93. Nanostructure of extracellular space in biofilms Stopar, David (University of Ljubljana, Ljubljana, SVN); Dogsa, Iztok (University of Ljubljana, Ljubljana, , SVN); Tom¿ic, Matija (University of Ljubljana, Ljubljana, , SVN); Benigar, Elizabeta (University of Ljubljana, Ljubljana, SVN); Stojkovic, Biljana (University of Ljubljana, Ljubljana, , SVN); Sretenovic, Simon (University of Ljubljana, Ljubljana, , SVN); Jamnik, Andrej (University of Ljubljana, Ljubljana, SVN); Poberaj, Igor (niversity of Ljubljana, lj, SVN) On a molecular level, structure and dynamics of biofilm extracellular space is not well understood. Recently, the role of levan in stabilization of biofilm structure in B. subtilis has been reported. Since levan is an extremely soluble (up to 60 % w/v), polydisperse, low viscosity, nonionic fructan polymer, it is surprising that levan can stabilize biofilm structure. In this study structure and dynamics of levan isolated from B. subtilis biofilms have been studied by SAXS, SLS, and DLS that give detail dynamic and structural information on nm to mm scale. Levan structure was further studied by sound velocity measurements, rheology, determination of


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polymer branching, and microscopy. To induce levan transition from low viscosity Newtonian to high viscosity pseudoplastic flow, observed in biofilms, levan was mixed with DNA. Mixing of levan with DNA caused instantaneous stable aggregation of levan on a micro scale. Phase behavior of semi-dilute mixtures of DNA and levan were studied with fluorescence and DIC microscopy. The marked inhomogeneous behavior of levan and DNA mixtures was further studied by microrheology using optical tweezers both inside and outside the phase separated levan aggregates. We were able to determine viscosity as well as viscoelastic behavior of levan within a microsized domain. The results indicate that microviscosity of levan significantly increased within the microdomain, whereas viscosity of DNA decreased relative to pure levan and DNA solutions at the same concentration. This suggests that microrheology of extracellular space is complex and is not a simple sum of its components. When levan and DNA mixtures were treated with DNAse, the microdomain aggregates of levan dissolved and the mixture behaved as a low viscous Newtonian fluid. On a molecular level this demonstrates the essential role of eDNA in stability of levan based biofilms. The SAXS, DLS, SLS, and optical tweezers provide powerful research tools to study biofilm nanostructures and could be applied to other biofilm extracellular matrices as well.


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Reaction-Diffusion Theory Applied to Microbial Biofilms Associated with Chronic Infections Stewart, Phil (Center for Biofilm Engineering, Bozeman, MT, USA) Reaction-diffusion theory has been successfully applied to understand microscale chemical gradients, ecological niches, and substrate fluxes in wastewater treatment and environmental biofilms. There are many fewer examples of the adaptation of this theory to systems of medical relevance. Here reaction-diffusion models are applied to gain insight into aspects of biofilm infection and persistence. Four case studies involving limitation for either oxygen, glucose, or iron, or accumulation of an acid waste product, are illustrated with comparisons of experimental measurements and theoretical simulations. The penetration of oxygen into a Pseudomonas aeruginosa biofilm was simulated and measured experimentally using microelectrodes under conditions of ambient air and also pure oxygen. Measured oxygen profiles were well-described by theory and concord with the theoretical prediction that oxygen penetration depth increases as the square root of the bulk oxygen concentration. This result could have direct relevance to understanding biofilm dynamics in response to hyperbaric oxygen therapy or to reduced oxygen tension such as could occur in an ischemic limb. Specific growth rate within a Klebsiella pneumoniae biofilm was simulated in a spatially complex two-dimensional domain of variable cell density based on a published experiment. Predicted growth rates manifested as a clear spatial gradient within the biofilm ranging from 0.71 h-1 (84% of the maximum growth rate) to just 0.025 h-1 (3% of maximum). Simulated and experimental growth rate patterns showed good agreement. This result may aid in understanding antibiotic tolerance arising from significant proportions of non-growing bacteria in biofilms. A Pseudomonas aeruginosa strain containing an inducible green fluorescent protein was used to visualize and quantify microscale spatial patterns of protein synthetic activity within hemispherical biofilm cell clusters formed in a flow cell. Anabolic activity was localized around the periphery of cell clusters at the biofilm-nutrient medium interface. Quantified patterns of protein synthetic activity were described well by a first order reaction-diffusion model in spherical coordinates. The mimimal medium used was not supplemented with trace elements, and additional calculations implicate iron as the limiting substrate. The induction of an acid-induced gene in a sugar-fermenting staphylococcal biofilm is considered. It is shown that the gene is predicted to be maximally expressed in an interior stratum of the biofilm. Together, these examples show that reaction-diffusion theory can be applied to shed light on the chemical and physiological heterogeneity that likely contributes to the pathogenesis and persistence of biofilm infections. Cross-diffusion in biofilms Eberl, Hermann (University of Guelph, Guelph ON, CAN) The past fifteen years or more have seen a huge amount of activity in biofilm modelling, on the mesoscale, i.e. the range of hundreds of microns to several millimetres. In particular much of this effort was directed toward biofilm growth at the colony level and how biofilm colonies respond to their physical environment, i.e. on structure-function-dynamics relationships. The mathematical models that have been developed utilise a wide range of mathematical and computational machinery and concepts, ranging from stochastic individual based models, to


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lattice based cellular automata, and fully deterministic continuum models, and include several hybrids between these concepts. These models take either (i) the viewpoint of a biofilm as a spatial structured population, i.e. the view of a biofilm as an ecological system, or (ii) the viewpoint of a biofilm as a mechanical object, e.g. a complex fluid. Since biofilms have been characterised as both, either approach seems viable. By and large, the predictions of biofilm structure and their response to environmental conditions such a local substrate or antimicrobial concentrations given by these very different model agree very well. Despite this, relatively little is known about the relationship between these different models classes.

In this presentation we will show how both viewpoints can be brought together under one roof. We will show, how starting from either viewpoint mentioned above, (i) the biofilm as a structured population, or (ii) the biofilm as a mechanical object, one can arrive at the same mathematical model of biofilm growth on the colony level. In the first approach (i), starting point is a lattice based modeling framework that is commonly used in Theoretical Ecology to derive macroscopic models of individual movement on the microscopic scale (cellular automata biofilm models are related to this approach). In the second variant (ii) the starting point is the principle of conservation of mass and momentum and the assumption that a biofilm is a complex fluid. In the multi-species case the resulting model in both cases is a fully deterministic degenerate diffusion-reaction system with cross-diffusion. We will also show that in the single species case we can derive the corresponding continuum model also from an individual based model (this result is due to Philip Murray, Dundee).

We explore this multi-species model in extensive computer simulations with a particular emphasis on the role of cross-diffusion for spatio-temporal mixing of species. For this we consider three systems: (a) competition between species for a nutrient, (b) allelopathic control under substrate limitations, and (c) biofilm exposure to antimicrobials. In all three cases, our results indicate that the cross-diffusion effect plays an important role for local mixing and the distribution of cells within the biofilm. However, our results also show that overall, on the colony level, these effects are of minor importance and stratifying models that over estimate mixing give similar global results.

Tradeoff between mass transfer and actual biofilm surface area on carriers in moving bed biofilm reactors --Combination of optical coherence tomography imaging with two-dimensional modeling Wagner, Michael (Karlsruhe Institute of Technology, Kalrsruhe, GER); Lackner, Susanne (Karlsruhe Institute of Technology, Karlsruhe, GER); Horn, Harald (Karlsruhe Institute of Technology, Karlsruhe, GER); Li, Chunyan (Kalrsruhe Institute of Technology, Karlsruhe, GER) Imaging and modeling are two major approaches to investigate biofilm structure and to understand the underlying physical and biochemical processes. These approaches are often

geometry as the basis to model biofilm detachment (Böl et al., 2009). In a previous study conducted by the authors, biofilm imaging data acquired by means of Optical Coherence Tomography (OCT) were transferred into COMSOL Multiphysics (COMSOL 4.2a, Comsol Inc, Burlington, MA, www.comsol.com) to model the flow around the biofilm located inside a biofilm carrier (Li et al., 2013). It proved the combination of imaging with OCT and modeling provides a


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good opportunity for a better understanding of processes on the meso-scale. As a continuation of the aforementioned theoretical study, the current study improved the existing methodology to simulate substrate removal of real moving bed biofilm carriers out of a lab-scale MBBR. To validate the model, the simulation results were compared with measured results of that lab-scale MBBR which was operated aerobically with glucose as the sole substrate for biofilm growth.

Originally invented for medical diagnostics, optical coherence tomography has been introduced into biofilm research to reliably monitor biofilm development and structure at the mm-scale (meso-scale). It enables fast, in situ and non-invasive three-dimensional imaging of biofilms. In this study, a GANYMEDE spectral domain OCT (Thorlabs, Dachau, Germany) was used to image the biofilm structure that developed on the carriers in the lab-scale heterotrophic MBBR over 40 days. Volumetric OCT datasets were taken throughout the cultivation to follow the biofilm evolvement. Images at different stages of biofilm development (day 8, day 17 and day 28) showing different amounts of biomass served as structural templates within the model. OCT images were binarized and imported into COMSOL Multiphysics for simulation. Laminar flow parallel to the carrier surface and substrate removal by heterotrophic bacteria were considered in the model. At steady state, the flow field in the bulk liquid and around the visualized biofilm structure as well as the substrate consumption (ASM1, Henze et al. 2000) was modeled. The results showed that thin biofilm in the carrier led to higher biofilm surface but thick mass transfer boundary layer and thick biofilm led to less biofilm surface but thinner mass transfer boundary layer. The main results are:

The OCT images deliver well defined surface structures of biofilms growing within the carriers.

Experimental results show that after exceeding a certain biofilm thickness the total turnover in a MBBR is constant and independent of the actual biofilm surface area.

The simulation results proved that the turnover is dependent on the trade-off between mass transfer and actual biofilm surface.

Reference Böl, M., Möhle, R.B., Haesner, M., Neu, T.R., Horn, H., and Krull, R. (2009). 3D finite element model of biofilm detachment using real biofilm structures from CLSM data. Biotechnol Bioeng, 103(1), 177-186. Li, C., Wagner, M., Lackner, S., Horn, H. (2013). Combination of optical coherence tomography imaging with two-dimensional modeling of flow and substrate transport in and around biofilms grown on fluidized carriers. Oral presentation. 9th International Conference on Biofilm Reactors, Paris, France. Henze, M., Gujer, W., Mino, T., van Loosdrecht, M. (2000). Activated sludge models ASM1, ASM2, ASM2d and ASM3. IWA Scientific and Technical Report No. 9. IWA Publishing, London, UK.


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Biofilm model to provide in silico predictions of micropollutant biodegradation Liu, Li (Technical University of Denmark, Department of Environmental Engineering, Lyngby, DNK); Smets, Bart F. (Department of Environmental Engineering, Kgs.Lyngby, DNK); Helbling, Damian E. (Department of Environmental Engineering / Eawag Swiss Federal Institute of Aquatic Science and Technology, DNK); E. Kohler, Hans-Peter (Eawag, Swiss Federal Institute of Aquatic Science and Technology, Dübendorf, CHE) Pesticides and their derived degradation products are frequently found in groundwater at trace

of expensive physico-chemical remediation techniques in drinking water production plants. Specific pure strains had been applied to remove pesticides from contaminated water, either through natural attenuation involving indigenous microbial community or in dedicated treatment systems involving indigenous community or foreign strains applied by bioaugmentation. The existence of indigenous community will cause competition of assimilable organic carbon (AOC) in oligotrophic environment. The concentration of AOC largely determines the microbiological stability of drinking water, and can also affect the subsequent pesticide-degrading activity, especially at micropollutant concentrations. So far, it has not been clear how the growth and survival of pesticide degraders in biofilm systems is affected by the presence of AOC and an indigenous community because of the limitation of experimental methods. Mathematical modeling can be a helpful tool to explore mechanisms governing such processes, which are very difficult to investigate experimentally. At the same time, mathematical simulation can provide a wide-range scenarios where real data are sparse. In silico predictions, based on measured parameters, can then be verified with some experimental results. Computationally, we introduced a general mathematical modelling framework to capture the essential phenomena associated with the biodegradation of compounds present at trace concentrations in oligotrophic water systems considering the effects of other assimilable carbon in the system, and the effects of an indigenous community in the environment. Then we constructed a multispecies biofilm model in a rapid sand filter reactor based on this modelling framework. The one-dimensional model includes dual-substrate Monod kinetics for a steady-state biofilm with multiple dissolved components. The model framework considers growth, lysis and decay mechanism, which describes the relationships among six model components: STP, SAOC,Z, SAOC,other, XB,Z, XB,BC, XI. Here we separate the SAOC into two parts, the concentration of the AOC that can be utilized by XB,Z is simulated as a state variable SAOC,Z, while SAOC,other is that part of the SAOC not accessible by the specific degrader. Death is typically simulated in a first-order fashion. In total, seven microbial processes were considered. Using the modelling framework we investigate each of the isolated processes layer by layer through different scenarios, representing increasingly complex situations: scenario 1 describes bacterial growth and specific substrate utilization in isolation; scenario 2 captures the effect of indigenous bacterial community on the survival of specialized metabolic degraders; scenario 3

influence of AOC; scenario 4 captures all processes and all variables. To gain insight into micropollutent removal, we also simulated the effluent concentrations of the biofilm reactor while varying the influent concentration. Furthermore, the obtained results and developed model concepts incorporate the known mechanisms controlling pesticide degradation with specific strain in biofilm system in general.


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Understanding the role of EPS distribution in biofilm and its consequence on cohesiveness and detachment in thick and mature biofilms: an experimental and modelling study Pechaud, Yoan (Université de Toulouse; INSA, UPS, INP; LISBP, Toulouse, FRA); Derlon, Nicolas (EAWAG, Dübendorf, CHE); Queinnec, Isabelle (CNRS ; LAAS, Toulouse, FRA); Bessiere, Yolaine (Université de Toulouse; INSA, UPS, INP; LISBP, Toulouse, FRA); Paul, Etienne (Université de Toulouse; INSA, UPS, INP; LISBP, Toulouse, FRA) The control of biofilm physical and mechanical properties is of chief importance for predicting wastewater treatment biofilm reactors (WWT) or biofilm treatment strategy efficiencies in industrial water network1.

In the last decade, investigation of the biofilm structure at the microscale using Laser Scanning Microscopy or microsensors have shown that biofilms are stratified in terms of activities, porosity, extracellular polymeric substances (EPS) and thus on mechanical properties. However, these studies are scarce and no experimental data reports the influence of growth conditions on EPS production and the consequence of the local biochemical composition on physical and mechanical properties of thick and mature biofilms. Only few works theoretically explore these relations 2,3. Nevertheless, the correct representation of the stratification of porosity and EPS depending on operating conditions is a crucial issue to elaborate the optimal strategies for fighting against biofilms in industry or for increasing activities of biofilm in WWT.

The main objective of the present work is, based on experimental observations, to use a conceptual biofilm model for studying the chief role of the spatial distribution of EPS (proteins and polysaccharides) on the structure and cohesiveness of a biofilm. The model structure evaluation and a rough calibration is achieved by confrontation to experimental results of biofilm growth in a Couette-Taylor reactor type under contrasted growth conditions (various SOLR, carbon substrate, degrees of nitrogen limitation, shear stress). This conceptual model describes the influence of several growth conditions on the stoichiometry of proteins and polysaccharides production and consequently on the stratification of these components, on the resulting porosity and hence on detachment.

The model correctly describes the key experimental trends of the biofilm structuration, i.e, the stratification and dynamics of proteins and polysaccharides, the stratification of porosity and the dynamics of biofilm thickness. For example, the model is able to represent the effect of a growth limitation by nitrogen on the local and global ratio of proteins and polysaccharides. By considering the biofilm as a composite material, the model is able to describe the effect of the shear stress on the local porosity and on the concentration of the various components and hence the local apparent cohesiveness governing detachment. When the shear stress is high the biofilm becomes denser and thinner and less stratified in terms of components and of the resulting activity and cohesiveness.

References: 1 Pechaud et al. (2012). Chem. Eng. Sci. 80, 109 118. 2 Laspidou and Rittmann. (2004). Water Research 38 (14-15):3362-3372. 3 Xavier et al. (2005). Microbiology. 3817-3832.


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Session 3: Unveiling the unseen: new and emerging technologies in biofilm research A high-throughput microfluidic dental plaque biofilm system Rickard, Alexander H. (Department of Epidemiology, School of Public Health, University of Michigan, Michigan, USA); Dowd, Scot E. (Molecular Research LP (MR DNA), Shallowater, USA); Samarian, Derek (Department of Epidemiology, School of Public Health, University of Michigan, Michigan, USA); Chludzinski, Jeffrey (Department of Epidemiology, School of Public Health, University of Michigan, Michigan, USA); Delli, Joseph (W. Nuhsbaum Inc., McHenry, USA); Battista, John (Fluxion, San Francisco, USA); Nance, William C. (Department of Epidemiology, School of Public Health, University of Michigan, Michigan, USA) Objectives: Few model systems are amenable to developing multi-species biofilms in parallel under environmentally germane conditions. This is a problem when evaluating the potential real-world effectiveness of antimicrobials in the laboratory. One such antimicrobial is cetylpyridinium chloride (CPC), which is used in numerous over-the-counter oral healthcare products. The aim of this work was to develop a high-throughput microfluidic system that is combined with a confocal laser scanning microscope (CLSM) to quantitatively evaluate the effectiveness of CPC against oral multi-species biofilms grown in human saliva.

Methods: Twenty-four-channel BioFlux microfluidic plates were inoculated with pooled human saliva and fed filter-sterilized saliva for 20 h at 378C. The bacterial diversity of the biofilms was evaluated by bacterial tagencoded FLX amplicon pyrosequencing (bTEFAP). The antimicrobial/anti-biofilm effect of CPC (0.5% 0.001% w/v) was examined using Live/Dead stain, CLSM and 3D imaging software.

Results:Theanalysis of biofilmsbybTEFAPdemonstratedthat theycontainedgeneratypically foundinhumandental plaque. These included Aggregatibacter, Fusobacterium, Neisseria, Porphyromonas, Streptococcus and Veillonella. Using Live/Dead stain, clear gradations in killing were observed when the biofilms were treated with CPC between 0.5%and 0.001%w/v. At 0.5%(w/v) CPC, 90%of the total signalwas fromdead/damaged cells. Belowthis concentration range, less killing was observed. In the 0.5% 0.05% (w/v) range CPC penetration/killing was greatest and biofilmthickness was significantly reduced.

Conclusions: This work demonstrates the utility of a high-throughput microfluidic CLSM system to grow multispecies oral biofilms, which are compositionally similar to naturally occurring biofilms, to assess the effectiveness of antimicrobials.

Keywords: multi-species biofilm, confocal scanning laser microscopy, Live/Dead staining, microfluidics, pyrosequencing


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Novel microfluidic biosensor for online monitoring of biofilm formation Bruchmann, Julia (Karlsruhe Institute of Technology (KIT), Eggenstein-Leopoldshafen, GER); Sachsenheimer, Kai (Karlsruher Institute of Technology (KIT), Eggenstein-Leopoldshafen, GER); Kleintschek, Tanja (Karlsruher Institute of Technology (KIT), Eggenstein-Leopoldshafen, GER); Rapp, Bastian E. (Karlsruher Institute of Technology (KIT), Eggenstein-Leopoldshafen, GER); Schwartz, Thomas (Karlsruher Institute of Technology (KIT), Eggenstein-Leopoldshafen, GER) Controlling and monitoring of biofilm formation are still demanding tasks. Established biofilm research methods mainly provide destructive end-point analysis. Therefore, we developed a new sensor system for characterizing biofilm formation online in a microfluidic flow system by two parameters: attached biomass and biofilm activity.

Our newly developed microfluidic biosensor is based on electrical impedance spectroscopy and parallel measurement of amperometric current which allows the real-time monitoring of biofilm formation processes. Biofilm biomass and activity are recorded in a non-destructive manner. Thereby increasing impedance correlates with an increase in biomass attached to the electrode and increase in amperometric measured current corresponds to a higher respiratory activity of the biofilm. These features were proven by microscopic time-lapse experiments and exo-enzymatic activity measurements. Integration of a reference channel allows minimizing environmental oscillations. The microfluidic properties of the senor enable parallel screening of different bacteria as well as biofilm affecting substances by providing 48 parallel flow channels with each containing two electrodes. A direct RNA extraction out of the channels allows transcriptome analysis on desired time-points.

Using this setup we were able to monitor biofilm development of different Gram-positive and Gram-negative bacterial species including Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Staphylococcus aureus, Bacillus subtilis, and even complex waste water biofilms. Screening of strain collections identified different biofilm formation potentials of the strains. Furthermore, the device enables monitoring of agents with biocidal or biofilm destabilizing effects. Loosening of the EPS matrix leads to a destabilized biofilm which can be monitored online by a decrease in impedance signal. Toxicity effects of biocides cause inactivation of the biofilm and therefore a decrease in the amperometric signal is observed. Regrowth after treatment reveals the presence of persister cells and is indicated by a recovery of both signals.

Overall, this sensor provides a tool for real-time monitoring biofilm formation and allows a rapid screening of biofilm influencing substances in a microfluidic system.

Ongoing development of the sensor targets the implementation into technical systems, where biofilm formation is a demanding problem and early on monitoring is necessary.


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Picking them one by one: Raman-based sorting and single-microcolony genomics of uncultured microdiverse nitrifiers in activated sludge flocs Daims, Holger (Department für Mikrobiologie und Ökosystemforschung, Wien, AUT); Lee, Tae Kwon (Department für Mikrobiologie und Ökosystemforschung, Wien, AUT); Kitzinger, Katharina (Department für Mikrobiologie und Ökosystemforschung, Wien, AUT); Dorninger, Christiane (Department für Mikrobiologie und Ökosystemforschung, Wien, AUT); Schmid, Markus (Department für Mikrobiologie und Ökosystemforschung, Wien, AUT); Wagner, Michael (Department für Mikrobiologie und Ökosystemforschung, Wien, AUT) Recent advances in DNA sequencing have revealed an unexpected diversity of closely related, but genomically divergent, coexisting bacteria in planktonic and sessile communities. This enormous microdiversity has revolutionized our view of microbial communities and has fueled discussions and research on microbial speciation and genetic population structure, ecological niche differentiation, and the importance of diversity for the stability of microbial communities and processes. However, actual phenotypic differences among microdiverse populations have mainly been demonstrated for cultured isolates under lab conditions, which do not reflect the situation in complex communities, and the microdiversity of uncultured or very slow-growing organisms remains an elusive phenomenon. Indeed, the cultivation of numerous single strains of such recalcitrant organisms for phenotypic tests would be an almost hopeless endeavor. The problem is also not easily solved by molecular techniques because community-wide metagenomics allows only the assembly of composite genomes from microdiverse populations, but can usually not assign sequences to single strains for more specific functional predictions. Here we address this challenge by using uncultured nitrifiers (ammonia- and nitrite-oxidizing bacteria) as model organisms and activated sludge from a wastewater treatment plant (WWTP) as a spatially complex model ecosystem. In a recent study we discovered a huge microdiversity of nitrifier populations in WWTPs and showed by cultivation-independent in situ techniques that this microdiversity is functionally relevant. To characterize these populations in much more detail, we developed a novel approach to identify the microcolonies of nitrifiers in situ by Raman microspectroscopy, which provides a chemical fingerprint of single cells or cell aggregates. Single nitrifier microcolonies are separated from activated sludge by laser tweezing, and the microcolony metagenomes are sequenced by techniques used for single-cell genomics. As each microcolony contains hundreds to thousands of target cells, typical problems of single-cell genomics are significantly reduced. After contig assembly and binning, the (partial) nitrifier genomes from single microcolonies are ready for downstream analyses such as functional annotation and population genetics. We show that this "single-microcolony" genomics pipeline is highly efficient and report on novel nitrifier genomic sequences obtained by this approach. Intriguingly, the genomes of microbial symbionts or predators of nitrifiers, which are often attached to the sorted microcolonies, are co-sequenced and can shed new light on interactions between nitrifiers and other microbes. Genome-based hypotheses on functional traits of the microdiverse populations are subsequently tested by in situ methods to study microbial physiology such as FISH-microautoradiography, FISH-Raman microspectroscopy, and FISH-NanoSIMS. In summary, single-microcolony genomics combined with functional analyses will substantially increase our knowledge on the genomic plasticity and biology of nitrifiers and has a high potential to further illuminate the evolutionary and functional basis of bacterial microdiversity. As Raman-based sorting is a flexible tool and can be applied to other microbial groups, our approach opens new general perspectives for future studies of uncultured populations in flocs and environmental or medical biofilms.


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Micro-3D Printing of Bacterial Communities Connell, Jodi (The University of Texas at Austin, Austin, TX, USA); Ritschdorff, Eric (The University of Texas at Austin, Austin, TX, USA); Kim, Jiyeon (The University of Texas at Austin, Austin, TX, USA); Bard, Allen (The University of Texas at Austin, Austin, TX, USA); Shear, Jason (The University of Texas at Austin, Austin, TX, USA); Whiteley, Marvin (The University of Texas at Austin, Austin, TX, USA) Bacteria communicate via short-range physical and chemical signals, interactions known to mediate quorum sensing, sporulation, and other adaptive phenotypes. Although most in vitro studies examine bacterial properties averaged over large populations, the levels of key molecular determinants of bacterial fitness and pathogenicity (e.g., oxygen, quorum-sensing signals) may vary over micrometer scales within small, dense cellular aggregates believed to play key roles in disease transmission. A detailed understanding of how cell cell interactions contribute to pathogenicity in natural, complex environments will require a new level of control in constructing more relevant cellular models for assessing bacterial phenotypes. Here, we describe a microscopic three-dimensional (3D) printing strategy that enables multiple populations of bacteria to be organized within essentially any 3D geometry, including adjacent, nested, and free-floating colonies. In this laser-based lithographic technique, microscopic containers are formed around selected bacteria suspended in gelatin via focal cross-linking of polypeptide molecules. After excess reagent is removed, trapped bacteria are localized within sealed cavities formed by the cross-linked gelatin, a highly porous material that supports rapid growth of fully enclosed cellular populations and readily transmits numerous biologically active species, including polypeptides, antibiotics, and quorum-sensing signals. Using this approach, we show that a picoliter-volume aggregate of Staphylococcus aureus can display substantial resistance to β-lactam antibiotics by enclosure within a shell composed of Pseudomonas aeruginosa. This novel 3D printing technology provides the unprecedented ability to organize multiple populations at defined positions and distances to investigate key questions regarding the spatial requirements for bacterial interactions and intercellular communication. This highly adaptable method and can be interfaced with other emerging analytical techniques to establish new experimental platforms for characterizing how spatial organization impacts interactions within microbial populations at the molecular level. Here, we introduce one example of the many conceivable integrated approaches, where this innovative 3D printing strategy is used in combination with scanning electrochemical microscopy (SECM) to create a quantitative, spatiotemporal map of the production of a quorum-sensing-regulated redox-active secondary metabolite, pyocyanin, by P. aeruginosa in confined aggregates containing ≤104 cells in real-time with microscale 3D resolution. The initial studies presented here, probing the onset of pyocyanin production as small populations of P. aeruginosa grow over time as well as the distances over which neighboring communities of mutant strains can sense and respond to one another, establish the utility of this integrated approach for characterizing how spatial parameters influence microbial behaviors.


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Chemical Imaging of Biofilms: The Integration of Synchrotron Imaging, Electron Microscopy and Nuclear Magnetic Resonance (NMR) Technologies Marshall, Matthew (Pacific Northwest National Laboratory, Richland, WA, USA) In most environments the majority of microorganisms live in structured biofilm communities comprised of microbial cells and extracellular polymeric substance (EPS). Using synchrotron-based x-ray imaging and high-resolution electron microscopy (EM), we have shown copious amounts of highly hydrated EPS was produced during microbial metal reduction and likely played a key role in metal capture and precipitation. A multi-faceted, multi-scale approach using a combination of synchrotron-based imaging, EM, and non-invasive nuclear magnetic resonance (NMR) imaging will yield a high spatial resolution, chemical image of a biofilm community in its nearest-to-native state as it influences biogeochemical reactions such as the redox-transformation of multivalent metals. For pore- and/or community-scale biofilm investigations in complex environments, x-ray microtomography produced three-dimensional renderings of hydrated Shewanella biofilms and visualized a delicate internal structure and network of pores within biofilms. These pores (e.g., fluid perfusion channels) indicate regions where water, nutrients, or electrolytes can flow through biofilm biomass and may influence pore-scale biogeochemical reactions. We are now computing the distribution of biomass to demonstrate the relative importance of nutrient advection and diffusion for sustenance across the biomass. Non-invasive nuclear magnetic resonance (NMR) imaging will test our predictions of nutrient flow and diffusive properties. We also correlated EM capabilities with scanning transmission x-ray microscopy (STXM) and synchrotron-based Fourier transform-infrared (FTIR) microimaging to produce high-sensitivity, chemical images that correspond to EM images of the biofilm at the nanometer scale. We have developed techniques that facilitate correlative imaging and spectroscopy studies in the absence of chemicals that interfere with native biofilm chemistry. These techniques have been used to collect STXM and FTIR chemical images of biofilms to show spatially resolved chemical gradients within biofilms. The integration of these techniques will provide detailed, high-resolution visualizations of chemical/elemental information that will strengthen our understanding of how biofilms interact with redox transformable metals to influence local biogeochemical reactions at many scales.


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Solutions to the Public Goods Dilemma in Bacterial Biofilms Drescher, Knut (Princeton University, Princeton, USA); Nadell, Carey (Princeton University, Princeton, USA); Stone, Howard (Princeton University, Princeton, USA); Wingreen, Ned (Princeton University, Princeton, USA); Bassler, Bonnie (Princeton University, Princeton, USA) Bacteria often live in densely populated surface-bound communities, termed biofilms. Biofilm-dwelling cells rely on secretion of extracellular substances to construct their communities and to capture nutrients from the environment. Many of these secreted factors behave as cooperative public goods: they can be exploited by nonproducing cells. The means by which public-good-producing bacteria avert exploitation to evolve these cooperative behaviors are largely unknown. Using experiments with Vibrio cholerae, which secretes extracellular enzymes to digest its primary food source, the solid polymer chitin, we show that the public goods dilemma may be solved by two very different, biophysical mechanisms: cells can produce thick biofilms that confine the goods to producers, or fluid flow can remove soluble products of chitin digestion, denying access to nonproducers. Both processes are unified by limiting the distance over which enzyme-secreting cells provide benefits to neighbors, resulting in preferential benefit to nearby clonemates and allowing kin selection to favor public good production. Our results demonstrate new mechanisms that promote the evolution of cooperative behaviors in biofilms. Colicins role in Escherichia coli biofilms Byarmuglo, Bihter (Ben Gurion University, Midreshet Ben Gurion, ISR) In the never ending battles for resources and space bacteria use every means at their disposal, but by far the most abundant weapons are target specific antibiotics, the bacteriocins. These are proteinaceous antibiotics produced against close relatives to the producing cell, competing with it for the same resources, thus bacteriocins play a major role in bacterial interactions. In Escherichia coli populations the interactions are often mediated by bacteriocins named colicins, so termed after the producing species. Colicins have been studied for almost a century and their ecological role, evolution, structure and function are well documented. However, the vast majority of studies examined E. coli residing in planktonic cultures though in their natural environment, i.e., the gastrointestinal tract, this species form biofilms. Hence, we wished to examine how well the traits and interactions described in planktonic E. coli cultures reflect on the colicins produced in biofilms? To answer this question we examined colicin expression in planktonic and biofilm environments at the single cell level using fluorescent protein tagging and expression analyses. We also imposed aerobic and anaerobic conditions, as cells residing in the gastrointestinal tract experience both, and attempted to monitor colicin expression under these conditions. We predicted that colicin expression would increase in biofilm environments as was found in other species, such as Streptococcus mutans strains that express bacteriocins solely in biofilm cultures. We further speculated that anaerobic growth conditions would augment colicin expression as demonstrated in sessile Pseudomonas aeruginosa. Five-folds increase in colicin expression was monitored in biofilm compared to planktonic E. coli cells. Upon examining the colicinogenic biofilms at a single cell level we observed two distinct


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subpopulations: small groups of cells formed hot-spots expressing colicins that are randomly distributed in the matrix, while the majority of the population silenced colicin expression. Further analyses suggest that phenotypic noise may play a role in bacterial interactions and that colicins expression is differentially expressed in a structured population to avert invaders while avoiding the high cost associated with expression. We further tested colicin expression in planktonic and sessile E. coli cultures under anaerobic conditions and noted that colicin expression was augmented in planktonic cultures by an order of magnitude, but biofilms cultivated with or without oxygen did not differ in their colicins expression. This may suggest that as portions of any biofilm is anaerobic or oxygen limited, colicins did not evolved to respond to shifts in oxygen concentrations. We conclude that bacterial defense systems are mediated by phenotypic noise evolved to balance security and cost to the population responding to structure and culture conditions. These results could facilitate the development of means that could be used to control pathogenic E. coli biofilms in the gastrointestinal tract or the urinary tract. Bacterial fight and flight responses enhance virulence in a polymicrobial infection Stacy, Apollo (The University of Texas at Austin, Austin, TX, USA); Everett, Jake (Texas Tech University Health Sciences Center, Lubbock, TX, USA); Jorth, Peter (The University of Texas at Austin, Austin, TX, USA); Trivedi, Urvish (Texas Tech University Health Sciences Center, Lubbock, TX, USA); Rumbaugh, Kendra P. (Texas Tech University Health Sciences Center, Lubbock, TX, USA); Whiteley, Marvin (The University of Texas at Austin, Austin, TX, USA) Bacteria rarely live in isolation and instead engage in interactions with neighboring microbes that alter the fitness of both the individual and the community. These interactions are of clinical relevance because they can result in enhanced colonization and persistence of bacteria at the infection site, a phenomenon known as polymicrobial synergy. An environment where synergy has been especially noted is the oral cavity, where a diversity of microbes coexist in surface-attached biofilm communities. We have been investigating interactions between the Gram-negative periodontal pathogen Aggregatibacter actinomycetemcomitans (Aa) and the Gram-positive oral commensal Streptococcus gordonii (Sg). These organisms display enhanced survival in an in vivo infection model due to defined metabolic interactions involving Aa cross-feeding on the Sg metabolite L-lactate. However, Sg and other streptococci also produce copious amounts of hydrogen peroxide (H2O2), an antimicrobial. Although Aa benefits in co-culture due to the presence of Sg-produced lactate, it is not fully understood how this interaction can proceed in the presence of Sg-produced H2O2. Here we demonstrate that Aa possesses two complementary responses to H2O2

enzyme that dissolves Aa biofilms. Using a murine abscess infection model, we show that both of these responses are required for Sg to promote Aa virulence. While the role of KatA is to detoxify H2O2 during co-infection, 3D spatial analysis of mixed infections revealed that DspB is required for Aa to spatially organize itself at an optimal distance (>4 µm) from Sg, which we propose allows cross-feeding yet reduces exposure to inhibitory levels of H2O2. These results reveal that an antimicrobial produced by a human commensal bacterium enhances the virulence of a pathogenic bacterium by modulating its spatial location in the infection site.


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Session 5: Omics for the study of biofilm structure and function Molecular Evaluation of biofilm influence on neighboring planktonic bacteria Nigaud, Yohan (UMR6270 CNRS, Mont Saint Aignan, AUT); Ben Mlouka, Amine (UMR6270 CNRS, Mont Saint Aignan, AUT); Chan Tchi Tsong, Philippe (IRIB - PISSARO Proteomics Facility, Mont Saint Aignan, FRA); Corbin, Agnes (Nonlinear Dynamics, Courtaboeuf, FRA); Vaudry, David (U982 INSERM - PISSARO Proteomics Facility, Mont Saint Aignan, FRA); Cosette, Pascal (UMR6270 CNRS - PISSARO Proteomics Facility, Mont Saint Aignan, FRA); Jouenne, Thierry (UMR6270 CNRS - PISSARO Proteomics Facility, Mont Saint Aignan, FRA) If the phenomena of bacterial communication have been widely studied in particular in sessile microbial communities, few studies have so far been dedicated to the intercellular communication between planktonic and biofilm bacteria. We showed here, by the use of an original bioreactor combined to proteomic analysis, that planktonic Pseudomonas aeruginosa cells regulate their gene expression, and therefore potentially their behavior, in response to the presence of a biofilm in their close environment. Our results indicate that the presence of a P. aeruginosa biofilm modifies a few percent of the total proteome of planktonic cells present near the biofilm. This alteration mainly concerns proteins involved in the response to oxidative stress, the repair to DNA damage or the synthesis of virulence factors. Such observations suggest that that the free cells react to the presence of a biofilm as if they face a "dangerous" environment.We propose that the biofilm, to protect itself, secretes oxidizing agents and other virulence factors that may affect both the physiology of foreign cells but also cells of its own species.

We showed that this communication " planktonic - biofilm cells" was not based on the perception by planktonic cells of quorum sensing signaling molecules. To seek for the molecules responsible for this communication signal, we initiated a differential study to characterize low molecular weight compounds that can diffuse from the compartment in which the biofilm stands (Bm) to the compartment of free floating bacteria (Pc). To this aim, we placed a 72 hours biofilm in the Bm compartment and only glucose phosphate buffer (TPG) in the Pc compartment. After 24 hours of incubation, the TPG was collected and its composition chemically assessed by nano-chromatography coupled to mass spectrometry. Alongside this experiment, we also attempted to analyze the composition of the supernatant of planktonic P. aeruginosa cells. We detected a set of peaks common to the two experimental conditions corresponding to metabolites produced by planktonic cells. However, the study also gives evidences of compounds whose abundance was altered in the presence of a biofilm. Examination of the fragmentation patterns of these compounds is in progress for identification.

The highlighted molecules will serve for further investigations to evaluate the incidence of their exposure to planktonic bacteria.


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Genetic requirements in spatially organized polymicrobial wound infections Turner, Keith (The University of Texas at Austin, Austin, TX, USA); Everett, Jake (Texas Tech University Health Sciences Center, Lubbock, TX, USA); Gabrilska, Rebecca (Texas Tech University Health Sciences Center, Lubbock, TX, USA); Rumbaugh, Kendra (Texas Tech University Health Sciences Center, Lubbock, TX, USA); Whiteley, Marvin (The University of Texas at Austin, Austin, TX, USA)

Biofilm-like structures are common in bacterial infections, suggesting that a structured group lifestyle underlies much of the physiology of bacteria in vivo. One particularly significant example of this is infections in chronic wounds, which contribute significantly to high healthcare costs in both the developed and developing world. These chronic infections are often polymicrobial, and the opportunistic pathogens Pseudomonas aeruginosa and Staphylococcus aureus are among the most commonly isolated bacteria from infected chronic wounds. Yet many features of the spatial organization and physiology of these bacteria during chronic wound infection remain unclear. Here, we use confocal microscopy to show that the spatial structure of P. aeruginosa and S. aureus in biofilm-like chronic wound infections is organized, with different species occupying different sites within the wound, and dynamic, changing during wound healing. To investigate the genetic requirements for chronic pathogenesis of P. aeruginosa in wound infections, we combined high-throughput sequencing-mediated transcriptome profiling (RNA-seq) and genome-wide insertion mutant fitness profiling (Tn-seq) to characterize gene expression and fitness determinants in a murine model of chronic wound infection. Generally we discovered that expression of a gene in vivo is not correlated with its importance for fitness, with the exception of metabolic genes. By combining metabolic models generated from in vivo gene expression data with mutant fitness profiles, we determined the nutritional requirements for P. aeruginosa colonization and persistence in chronic wounds. Specifically, we found that long-chain fatty acids represent the primary carbon source for P. aeruginosa in chronic wounds, and that wounds are nutrient-rich requiring P. aeruginosa to biosynthesize only purines, folate, and riboflavin during infection. Interestingly, we also found that flagellar motility, thought to be a requirement for P. aeruginosa biofilm formation in vitro, was dispensable in chronic wound infections, suggesting that genetic requirements for biofilm development can be conditional. Finally, we will detail advances made using genomic approaches to dissect the genetic bases for P. aeruginosa and S. aureus coinfection in chronic wounds. Our results provide novel insight into the genetic requirements for, and spatial organization in, P. aeruginosa and S. aureus polymicrobial chronic wound infections and demonstrate the power of using both gene expression and fitness profiling for probing bacterial virulence and persistence in biofilm-like infections. Can't we all just get along: How cooperation and conflict shape polymicrobial infections Whiteley, Marvin (Univ. of Texas at Austin, Cedar Park, USA)

Most bacterial infections are biofilm-associated and polymicrobial, and the presence of multiple microbes in an infection results in delayed healing, increased antibiotic resistance, and poor clinical outcomes. This is thought to be due in part to mutualistic and antagonistic interactions between bacteria; thus to fully understand these infections it is essential to view them through an ecological lens. Here I will discuss the use of high-throughput sequencing-based genomic methods such as RNA sequencing and transposon sequencing to study polymicrobial biofilm communities both in animal models and in human infections. These studies have yielded novel


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insights into bacterial behavior and community structure during infection. Our work suggests that metabolic interactions underpin the stability of disease-associated microbial communities, and point to "keystone" metabolic pathways as potential therapeutic targets for a variety of infections. Impact of shading on the food web structure of phototrophic stream biofilms elucidated by metatranscriptomics Bengtsson, Mia (Department für Limnologie und Ozeanographie, AUT); Wagner, Karoline (University of Vienna, Vienna, AUT); Urich, Tim (University of Vienna, Vienna, AUT); Burns, Nancy (University of Vienna, Vienna, AUT); Schwab, Clarissa (University of Vienna, Vienna, AUT); Battin, Tom (University of Vienna, Vienna, AUT)

Phototrophic stream biofilms are highly diverse communities featuring multiple trophic levels with organisms representing all three domains of life. It has previously been difficult to study natural biofilms in their entirety due to their inherent diversity of microscopic organisms. Whereas molecular techniques such as PCR amplification of ribosomal RNA genes and subsequent sequencing has shed light on mainly bacterial community members, eukaryotic microorganisms such as protists and fungi have been overlooked due to the lack of suitable PCR primers. In addition, microscopic identification of eukaryotic microorganisms, including algae, is often difficult and laborious. Therefore, we designed an experiment to study community dynamics in natural stream biofilms using metatranscriptomics, targeting ribosomal RNA transcripts of all three domains of life. We hypothesized that manipulation of light, the primary resource of these biofilms, would alter the structure of the foodweb, shifting the community from an algal-dominated state under high light conditions, featuring organisms adapted to grazing on and degrading fresh algal material, to a more detritus-based community under low light conditions, with organisms adapted to utilization of dead algal material and terrestrially derived dissolved organic matter. We grew phototrophic stream biofilms under different light conditions ranging from full light to 73% light exclusion in the stream Oberer Seebach in Lunz am See, Austria. Transferring the biofilms to microcosms allowed us to measure several parameters such as gross primary production, nutrient uptake and DOC uptake before we harvested the biofilms for RNA extraction. Extracted RNA was subjected to random-hexamer primed reverse transcription into cDNA, which was sequenced using Ion Torrent technology. We generated in total 10.4 million RNA reads and used the small subunit (SSU) rRNAs for deep 3-domain community profiling with the CREST toolbox. 68% of SSU rRNA transcripts originated from Eukaryota, 31.8% from Bacteria and <0.1% from Archaea. Diatoms of the genera Achnanthes, Nitzschia and Navicula were the most abundant phototrophic organisms closely followed by Cyanobacteria related to the genus Leptolyngbya. Among the protists, the Cercozoa dominated. The shading treatment had clear impacts on the composition of the phototrophic portion of the community and also had cascading effects which were evident on certain bacterial groups, for example the Planctomycetes. Overall however, the biofilm foodweb structure was relatively stable across the different light treatments, indicating a robust assemblage that is to some extent independent of light availability. This study shows that metatranscriptomic techniques are extremely powerful when analyzing complex biofilm communities. Further, the compositional robustness of our studied biofilms raises interesting ecological questions concerning a homeostatic versus dynamic nature of microbial communities.


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RpoN plays a pleiotropic role in Bacillus cereus affecting its lifestyle and biofilm formation Hayrapetyan, Hasmik (TI Food and Nutrition, Wageningen); Nierop Groot, Masja (TI Food and Nutrition, Wageningen); Tempelaars, Marcel (Wageningen University, Wageningen); Abee, Tjakko (TI Food and Nutrition, Wageningen) Alternative transcriptional regulators mediate adaptation of bacterial cells to changing environmental conditions by controlling expression of specific repertoires of genes under tightly controlled conditions. Sigma factor 54 (RpoN ) is a unique transcriptional regulator in terms of its molecular mechanism and the multitude of reported processes it regulates in different microorganisms. Functionality of sigma 54 requires specific activator proteins, called enhancer binding proteins (EBPs), and hydrolysis of ATP for initiation of transcription. These specialised EBPs finetune regulation by directing expression of sub regulons, depending on the trigger. A role of sigma 54 has been experimentally validated for different functionalities in various, mainly Gram negative, species. A Bio-IT driven search suggested a broader role of sigma54 as central regulator in the physical interaction of bacteria with their environment, including virulence and biofilm formation. In order to study the role of sigma 54 in Bacillus cereus we constructed a markerless deletion mutant in the strain ATCC 14579. The mutant was dramatically affected in many different cellular functions including anaerobic growth, motility, biofilm formation, sporulation and toxin production compared to its WT counterpart. Interestingly, the tendency of the WT to form air-liquid interface biofilms on stainless steel was altered in the mutant which preferentially formed a submerged biofilm albeit at reduced levels. Most of the observed phenotypes could be restored by complementation with a plasmid-encoded copy of the RpoN gene, confirming that these processes are under control of RpoN. Gene expression profiles obtained for the WT, deletion mutant, and the complemented mutant grown in BHI in aerated and static conditions largely supported the phenotypes observed and provided more insight in the function of sigma 54 in B. cereus. The affected transcriptomes, and phenotypes in the mutant confirm the predicted pleiotropic role of sigma 54, governing directly or indirectly the expression of a number of genes involved in a plethora of cellular processes required under different environmental conditions. Coordinated interplay between submerged biofilm and liquid-air pellicles in Bacillus subtilis SANCHEZ-VIZUETE, Pilar (INRA, Massy, FRA); BRIDIER, Arnaud (Irstea, Antony, FRA); HERRY, Jean Marie (INRA, Massy, FRA); AYMERICH, Stéphane (INRA, Jouy-en-Josas, FRA); LE COQ, Dominique (CNRS, Jouy-en-Josas, FRA); BRIANDET, Romain (INRA, Massy, FRA) Bacillus subtilis is a non-pathogenic bacterium, largely used as model organism for the study of the genetic regulation of sporulation, metabolism and biofilm formation processes. Most of the genetic mechanisms involved in B. subtilis community behavior were analyzed thought the analysis on the biofilm formed at the liquid-air interface, the so-called pellicle. The aim of this work is to focus on B. subtilis submerged surface colonization, and the interplay between communities associated to both solid and air interfaces. The behavior of a B. subtilis strain recently isolated from a medical environment, GFP-labeled, was analyzed non invasively and at single cell resolution in polystyrene 96-well microtiter plates using 3 complementary


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techniques: time-lapse confocal imaging was used to analyze the colonization of the solid interface, flow-cytometry for planktonic cells, and epifluorescence for pellicle formation. This set of techniques allowed identifying coordinated sequences of colonization of each these interfaces. In addition, spatio-temporal patterns of gene expression involved in biofilm lifecycles such as motility (e.g. hag which encodes flagellin and genes involved in cell separation), matrix production (e.g. epsA-O operon, tapA-sipW-tasA operon and bslA) and low oxygen conditions genes have been investigated using transcriptional GFP fusions mutants. Results suggested that oxygen content was a major factor triggering the population interplay between the two interfaces. Those results also demonstrated the coexistance of subpopulations with dramatically different phenotypes in a model as simple as a well of a microtiter plate, stressing the importance of single cell and populational analysis to understand biofilm properties and their fantastic plasticity in face to harsh conditions.

Acknowledgment: P. Sanchez-Vizuete is the recipient of a PhD grant from the Ile-de-France Council (DIM Astrea).

NanoRNase impacts biofilm formation and c-di-gmp signal transduction through its activity on pGpG Cohen, Dorit (Bar-Ilan University, Ramat-Gan, ISR); Banin, Ehud (Bar-Ilan University, Ramat-Gan, ISR) The second messenger bis- -cyclic-dimeric-guanosine monophosphate (c-di-GMP) controls diverse cellular processes among the Bacteria. In this domain of life, c-di-GMP is synthesized by diguanylate cyclases (DGCs) and degraded by c-di-GMP-specific phosphodiesterases (PDEs). Nearly 80% of these PDEs are predicted to depend on the catalytic function of EAL domains, which hydrolyse a single phosphodiester group in c-di-GMP to produce 5'-phosphoguanylyl-(3',5')-guanosine (pGpG). However, to degrade pGpG and prevent its accumulation, bacterial cells require another enzyme, the identity of which remains unknown. Here we identify that the highly conserved enzyme oligoribonuclease (Orn) is responsible for pGpG degradation. A Pseudomonas aeruginosa Δorn mutant had high intracellular c-di-GMP levels, and consequently, this strain overexpressed extracellular polysaccharides, aggregated and overproduced biofilm. Although recombinant Orn degraded small RNAs in vitro, this enzyme had a proclivity for degraincluding pGpG. Corresponding with this activity, P. aeruginosa Δorn cells possessed highly elevated pGpG levels. We found that pGpG inhibited the activity of EAL-dependent PDEs in vitro. This inhibition occurred at pGpG concentrations below those detected in Δorn cells. These data suggest that elevated levels of pGpG may exert feedback inhibition on PDEs in vivo, thereby increasing intracellular c-di-GMP. We propose that Orn is an evolutionarily conserved nanoRNAse that degrades pGpG and that this activity is fundamental to bacterial c-di-GMP signal transduction.


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The molecular mechanism and effects of superinfection of Pseudomonas aeruginosa by the filamentous phage Pf4 Rice, Scott (SCELSE, Singapore, SGP); Hui, Janice (The Centre for Bio-Innovation, Sydney, AUS); Mukherjee, Manisha (SCELSE, Singapore, SGP); Huron, Vanessa (CMB, Sydney, AUS); McElroy, Kerensa (CMB, Sydney, AUS); Thomas, Torsten (CMB, sydney, AUS); McDougald, Diane (SCELSE, Singapore, SGP); Kjelleberg, Staffan (SCELSE, Singapore, SGP) The filamentous prophage, Pf4, of Pseudomonas aeruginosa plays a significant role in biofilm development and virulence. The phage contributes to the establishment of small colony variants during biofilm formation. Many of these phenotypes have been linked to the appearance of a superinfective form of the Pf4 phage that kills the host.

Superinfective as a consequence of a limited number of mutations in a gene and its promoter that has homology to other phage repressor genes. During biofilm dispersal, roughly 20-40% of the population are morphotypic variants. Meta-genome sequencing of the dispersal population revealed that most of the observed mutations occurred in the phage genome, and that these mutations correspond to those associated with the superinfective phenotype. Establishment of the superinfective form of the phage was linked to a functional mismatch repair system and the primary oxidative stress response protein, OxyR and was induced by oxidative stress. Superinfection results in relatively few changes in host protein expression, limited primarily to changes in efflux pump and siderophore production. This was further supported by qRT-PCR and phenotypic data.

It's all about taste and sense: Outside-in signaling mechanism of the NicD taste-like receptor required for nutrient-induced dispersion by Pseudomonas aeruginosa Basu Roy, Ankita (Binghamton University, Binghamton, USA); Petrova, Olga (Binghamton University, Binghamton, USA); Sauer, Karin (Binghamton University, Binghamton, USA) The formation of bacterial biofilms, or surface-associated communities is a developmental process that is initiated with surface attachment by planktonic cells. Biofilms return to the planktonic state upon dispersion, a process in which sessile, surface-attached organisms liberate themselves from the biofilm. This is made apparent by biofilm microcolonies having central voids, and their transition to the free-living state, enabling bacteria to spawn new communities in multiple locales. Dispersion occurs in response to various environmental cues including amino acids, sugars, and nitric oxide. Moreover, in P. aeruginosa the dispersion response has been shown to require BdlA and two phosphodiesterases (PDE; RbdA, DipA), and to coincide with increased PDE activity and a reduction of the cellular c-di-GMP levels. However, little is known about how these proteins interact to translate sensing of dispersion cues into the modulation of the intracellular c-di-GMP pool to enable dispersion. Moreover, no protein that either directly senses or responds to carbon sources, thereby controlling c-di-GMP turnover, has been identified. Using glutamate-induced dispersion as a model, we report that induction of dispersion in response to nutrients including glutamate and glucose is independent of the cue being utilized as carbon and energy sources. Instead, dispersion-inducing nutrient cues are sensed via membrane bound NicD composed of a periplasmic 7TMR-DISMED2 domain, a cytoplasmic GGDEF domain and a membrane spanning 7TMR domain. 7TMR-DISMED2 domains are sensory domains predicted to bind carbohydrates and their derivatives. Nutrient induced


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dispersion required the periplasmic sensory domain of NicD, with glutamate-sensing by NicD resulting in NicD dephosphorylation and increased NicD cyclase activity. Moreover, NicD was found to form a complex with DipA and BdlA. Upon glutamate sensing, active NicD contributes to the non-processive proteolysis and activation of BdlA via phosphorylation of BdlA and by generating temporarily elevated c-di-GMP levels. Active BdlA not only recruits a second PDE, namely RbdA, to the complex but furthermore significantly enhances the PDE activity of DipA. This ultimately results in the overall reduction of c-di-GMP levels and thus, dispersion. Further evidence of the NicD, BdlA, and DipA working in concert and participating in a signaling cascade required for dispersion was obtained using dispersion assays. While overexpression of nicD in ?nicD, dipA in ?dipA and bdlA in ?bdlA restored biofilm dispersion by these mutant strains to wild-type levels, overexpression of nicD in ?bdlA or ?dipA, and overexpression of dipA in ?bdlA or ?nicD mutant strains did not restore biofilm dispersion to wild-type levels. Overall, our findings point at an outside-in signal transduction mechanism to relay environmental dispersion cues into the modulation of intracellular c-di-GMP levels, with signal transduction requiring the sensory 7TMR-DISMED2 domain, DGC activity of NicD, BdlA, DipA PDE activity, and posttranslational modification. While such an outside-in signaling mechanism has not been previously reported in P. aeruginosa, the signal transduction cascade is reminiscent of eukaryotic signal transduction pathways that incorporate signal processing across compartments via multiprotein complexes and temporal posttranslational modifications. In fact, NicD/BdlA/DipA complex mimics both in structure and function signal transduction mechanism by seven-transmembrane-region containing receptors (7TMRs), also referred to as G protein-coupled receptors, that play central roles in eukaryotic signal transduction, in particular metabotropic glutamate receptor that function as excitatory neurotransmitters and/or taste receptors.


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Surface physicochemistry and ionic strength affects eDNA's role in bacterial adhesion to abiotic surfaces R. Regina, Viduthalai (Aarhus University, Aarhus, DNK); R. Lokanathan, Arcot (Aalto University, Aalto); J. Modrzynski, Jakub (Copenhagen University, Frederiksberg, DNK); S. Sutherlan, Duncan (Aarhus University, Aarhus, DNK); L. Meyer, Rikke (Aarhus University, Aarhus, DNK) Extracellular DNA (eDNA) is an important structural component of biofilms formed by many bacteria, but few reports have focused on its role in initial bacterial adhesion. The initial adhesion of bacteria is dictated by long and short range forces, such as Liefshitz van der Waals and electric double layer forces, and Lewis acid-base interactions, acting between the cell and the surface. The physico-chemistry of the two approaching surfaces defines the extent of these forces, but may be altered by the property of the surrounding medium. Similarly, the conformation of biopolymers, such as DNA, can be influenced by ionic strength and composition of the medium. The aim of this study was to investigate the role of eDNA in bacterial adhesion to abiotic surfaces, and determine to which extent eDNA-mediated adhesion depends on the physicochemical properties of the surface and surrounding liquid.

subsequently quantified the effect of eDNA on the adhesion of Staphylococcus xylosus to glass surfaces functionalised with different chemistries resulting in variable hydrophobicity and charge. Bacterial adhesion experiments were carried out at three different ionic strengths (I = 0.7 M, 0.18 M, and 0.015 M). Removal of eDNA from S. xylosus cells by DNase treatment did not alter the zeta potential, but rendered the cells more hydrophilic. DNase treatment impaired adhesion of cells to glass surfaces, but the adhesive properties of S. xylosus were regained within 30 minutes if DNase was not continuously present, implying a continuous release of eDNA in the culture. eDNA promoted adhesion of S. xylosus to hydrophilic surfaces with hydroxyl and carboxyl groups, but only at low and intermediate I. This indicates that eDNA is more adhesive at low I, possibly due to its extended conformation at low salt concentraions. In contrast, eDNA lowered adhesion to positively charged surfaces with amine groups at low I, but not at high I. Adhesion to hydrophobic surfaces with neutrally charged fluorine functional groups was promoted by eDNA irrespective of I. Our results indicate that eDNA-mediated bacterial adhesion to abiotic surfaces is affected by

n, the surface charge, and the thickness of the electric double layer. These parameters are strongly affected by pH and/or ionic strength. Hence eDNA is a versatile adhesin that can promote bacterial adhesion to a variety of surfaces, but as most surfaces in nature are

environments with low ionic strength.


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IMPACT OF THE SHEAR STRESS ON INITIAL BACTERIAL ADHESION Saur, Thibaut (INRA - LBE, NARBONNE, FRA); Morin, Emilie (Laboratoire de Biotechnologie de l'Environnement, Narbonne, FRA); Habouzit, Frédéric (Laboratoire de Biotechnologie de l'Environnement, Narbonne, FRA); Bernet, Nicolas (Laboratoire de Biotechnologie de l'Environnement, Narbonne, FRA); Escudié, Renaud (Laboratoire de Biotechnologie de l'Environnement, Narbonne, FRA) Hydrodynamic strength plays a key role in microbial adhesion. This first step of biofilm formation constitutes a critical stage in biofilm development and management (Liu & Tay 2002). A better understanding of the impact of shear on microbial adhesion is crucial to prevent detrimental biofilm formation for sanitary or economic reasons - or in contrary develop a beneficial biofilm system such as in environmental engineering. Adhesion involves transport of bacteria to the substratum surface and their attachment. Hydrodynamic conditions can have paradoxical and complicated effects on adhesion (Busscher & Van der Mei 2006). On the one hand, improving mixing and fluid velocity in the liquid phase enhances access of bacteria to the substratum. On the other hand, shear also increases promoting detachment forces. In the literature, many studies devoted to the impact of shear on adhesion were realized in pure culture and under a laminar flow. The novelty of this work lies in that it was done for mixed culture in a turbulent flow which is more relevant for bioprocess engineering. Both qualitative and quantitative aspects of adhesion were characterized to better understand the role of shear on bacterial adhesion. Microbial adhesion was carried out in rotating annular reactors (RAR) inoculated with diluted activated sludge. Hydrodynamic conditions of such reactors are well-known and the shear stress can be estimated (Racina & Kind 2006). Shear stresses ranging from 0.09 to 7.4 Pa, were tested. High Reynolds numbers ensured a turbulent flow. In the reactors, plastic coupons located on the inner rotating cylinder, were used. After a two-hour contact time, coupons were withdrawn and analyzed. One part of the coupons was used to assess the structure of microbial communities using a fingerprinting technique (PCR-SSCP). The other part of the coupons was soaked in a DAPI staining solution and observed in epifluorescence microscopy. Images were recorded and processed to obtain the surface coverage, the number of attached particles per surface area and the average size of attached particles. The analysis of SSCP profiles showed that (i) whatever the hydrodynamic conditions, all the adhered communities were significantly different from the inoculum and (ii) a progressive modification of the attached community and a decreasing diversity index from low to high shear. Besides, the coverage, which basically corresponds to the number of bacteria attached on the substratum surface, revealed a greater adhesion at high shear stress. But the variation with shear was not linear and a plateau appeared from medium to high values. These data suggest that, at low to medium shears, mass transport is preponderant on detachment. This results in an increase in the coverage. Then a plateau is reached and both antagonist effects could compensate each other. However, despite these quantitative similarities according to the coverage, different qualitative features regarding the spatial organizations were observed. As indicated by the number and the size of detected objects on the pictures, at higher shear stresses bacteria gathered to form


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clusters and this phenomenon intensified with increasing shear. Hydrodynamic conditions influence not only the number of attached bacteria, but also their distribution on the substratum surface and the microbiological composition of the early biofilm. Busscher, H.J. & Van der Mei, H.C., 2006. Clinical Microbiology Reviews, 19(1), pp.127-141. Liu, Y. & Tay, J.-H., 2002. Water Research, 36(7), pp.1653 1665. Racina, A. & Kind, M., 2006. Experiments in Fluids, 41(3), pp.513 522. A new attempt to measure biofilm rheology by optical coherence tomography Blauert, Florian (Karlsruhe Institute of Technology - Chair of Water Chemistry and Water Technology, Karlsruhe, GER); Wagner, Michael (Karlsruhe Institute of Technology - Chair of Water Chemistry and Water Technology, Karlsruhe, GER); Horn, Harald (Karlsruhe Institute of Technology - Chair of Water Chemistry and Water Technology, Karlsruhe, GER) Whereas the activity or function of biofilms is mainly driven by the microbial community hosted in a polymeric matrix on the one side the physical properties of microorganisms and polymeric matrix mainly influence shape and mechanical stability of biofilms. The latter thereby has a huge influence on mass transfer as well as detachment processes at the bulk biofilm interface. Biofilm rheology has been studied widely, often on-line by microscopy. In the study presented simple stress-strain and creep experiments were conducted on multi-species biofilms grown in a flow cell at laminar flow. Structural deformation of the biofilm caused by hydrodynamic shear stress (τw) was visualized by means of optical coherence tomography (OCT). OCT is an emerging non-invasive imaging technique, which measures a depth profile (A-scan) from translucent tissues. Consecutive A-scans provide a cross section of biofilms (B-scan), which can be combined to a volumetric representation (C-scan). Moreover, C-scans can be analyzed to determine biofilm characteristics such as porosity, roughness and distribution of cavities. OCT has been introduced into biofilm research due to its ease of use and the capability to monitor on-line in situ and non-invasively the biofilm structure at the mm-scale (mesoscale). The biofilm structure can be visuali -range, overcoming the disadvantage of measuring only local biofilm properties at the microscale. In a new attempt on-line monitoring by means of acquiring OCT B-scans along the flow direction was used to measure biofilm strain and creep. Biofilms were grown in 12 cm long flow cells made of PMMA with a cross-section area of 2 × 1 mm2 at defined hydrodynamic and nutritional conditions. By changing the flow velocity, hydrodynamic conditions were altered to expose biofilms to shear stresses over a wider range. Biofilms were grown at low shear stress τw= 0.01 N/m2 (Re = 3) and shear stress experiments were conducted ranging from τw = 0.01 N/m2 to τw = 2.9 N/m2 (Re = 860). At τw = 1.6 N/m2 ε =

f deformation was α = 3°. After each shear test the biofilm returned into its original shape showing an elastic response. Measuring displacement within biofilms by confocal laser scanning microscopy (CLSM) is only possible by addition of fluorescent particles (Stoodley et al., 1999). With the help of OCT it is now possible to visualize structural changes within the whole biofilm, closing the gap between microscopic techniques and mechanical measurements such as spinning disc rheometry (Towler et al., 2003) or fluid dynamic gauging (Möhle et al., 2007). OCT offers new possibilities for monitoring biofilm growth and will help to gain a deeper understanding of


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biofilm rheology at hydrodynamic stress without addition of interfering substances such as fluorescent substances. Reference: Möhle, R. B., Langemann, T., Haesner, M., Augustin, W., Scholl, S., Neu, T. R. & Horn, H. (2007). Structure and shear strength of microbial biofilms as determined with confocal laser scanning microscopy and fluid dynamic gauging using a novel rotating disc biofilm reactor. Biotechnology and Bioengineering, 98(4), 747 755. Stoodley, P., Lewandowski, Z., Boyle, J. D., & Lappin-Scott, H. M. (1999). Structural deformation of bacterial biofilms caused by short-term fluctuations in fluid shear: an in situ investigation of biofilm rheology. Biotechnology and Bioengineering, 65(1), 83 92. Towler, B. W., Rupp, C. J., Cunningham, A. B., & Stoodley, P. (2003). Viscoelastic properties of a mixed culture biofilm from rheometer creep analysis. Biofouling, 19(5), 279 85. Adsorption of Sugars, Proteins, and Bacteria: A Multimethod Study for a Better Understanding of Biofilm Formation Müller-Renno, Christine (University of Kaiserslautern, Kaiserslautern, GER); Kratz, Fabian (University of Kaiserslautern, Department of Physics, Kaiserslautern, GER); Rösch, Christina (University of Kaiserslautern, Department of Physics, Kaiserslautern, GER); Davoudi, Neda (University of Kaiserslautern, Department of Physics, Kaiserslautern, GER); Köhler, Oliver (University of Kaiserslautern, Department of Physics, Kaiserslautern, GER); Umanskaya, Natalia (University of the Saarland, Department of Operative Dentistry and Periodontology, Homburg, GER); Schlegel, Christin (Institute of Bioprocess Engineering, University of Kaiserslautern, Kaiserslautern, GER); Muffler, Kai (Institute of Bioprocess Engineering, University of Kaiserslautern,, Kaiserslautern, GER); Ulber, Roland (Institute of Bioprocess Engineering, University of Kaiserslautern, Kaiserslautern, GER); Hannig, Mathias (University of the Saarland, Department of Operative Dentistry and Periodontology, Homburg, GER); Ziegler, Christiane (University of Kaiserslautern, Department of Physics, Kaiserslautern, GER) In biofilm formation biological systems from the nano- to the microscale are involved: sugars, proteins, lipids, nucleic acids, and bacterial cells. Whereas protein adsorption is a widely studied field and some studies on bacterial adsorption exist, there is little known about the other components and their interplay. No standardized method exists to date to investigate the biofilm formation and the interaction of its molecular components with the substrate or to each other. Here we used different analytical methods, Scanning Force Microscopy/Spectroscopy (SFM/SFS), Dynamic Contact Angle (DCA) Measurements, BCA (bicinchionic acid assay), Quartz Crystal Microbalance (QCM), to study the adsorption of the sugars dextran and glucose, the proteins bovine serum albumin (BSA) and lysozyme, as well as the bacterium Paracoccus seriniphilus. The investigated substrate was mainly titanium, in addition to gold, ceramic, composite, PMMA, PTFE and natural enamel. An increased sugar adsorption with increasing sugar concentration at pH 7 could be detected by QCM. In a subsequent experiment the quartz substrates are immersed in phosphate buffer. Glucose shows a complete desorption while a rest of dextran stays on the surface. Over the


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whole investigated pH range (pH 4-13) dextran does not show a charge (with the exception of pH 13), determined by zetapotential measurements. Thus no electrostatic forces govern the sugar adhesion and adsorption at pH 7. Van-der-Waals and other forces seem to be the main factors. This is in contrast to many proteins. In the case of BSA and lysozyme Coulomb forces play an important role, as well as the softness of the proteins and the wettability of the surface. In scanning force spectroscopy measurements BSA shows a decreased adhesion force with increasing pH with the exception of pH 9 at which BSA has a very soft conformation. With increasing hydrophobic properties of the solid surface the measured adhesion force of BSA increases but it is still lower than at very low pH values with attractive Coulomb interaction forces. Furthermore BCA allows the distinction between loosely and firmly adsorbed proteins.

Since sugars and proteins are parts of the extracellular polymeric substances (EPS, matrix formed by bacteria) and of the cell surface also their interaction to each other is important in biofilm formation studies. A preadsorbed dextran layer fosters a pronounced BSA adsorption with a nearly completely irreversible character as determined by QCM. Also a preadsorbed BSA layer shows an increased dextran adsorption compared to the interaction with the bare surface. In the following desorption experiment in phosphate buffer dextran shows only a partly reversible desorption behavior. In addition the protein protein interaction is a key factor in the biofilm formation. While sugars increase the amount of adsorbed protein and vice versa, a preadsorbed BSA layer reduces the protein adhesion force if compared to the bare surface (by SFS).

Additionally first adhesion force measurements between bacterial cells and the substrate were performed (by SFS). In general the adsorption and desorption behavior of the bacteria differs as a function of the used medium during the cell culture as determined by DCA. In addition shear forces easily detach the loosely bound part of the biofilm while the initial adsorbing layers (mostly composed of sugars and proteins) stick extremely firm to the surface. In summary a complex image of the biofim formation, the involved forces and the influencing parameters is obtained by the use of different analytical methods. All these results serve as basis for medical and technological applications of materials in a biological surrounding for which a biofilm formation is desired (biofilm reactor) or should be avoided (dental implants).

Amyloids modify the viscoelastic properties of biofilm model matrices Di Martino, Patrick (Université de Cergy-Pontoise, Cergy-Pontoise, FRA); LEMBRE, Pierre (Université de Cergy-Pontoise, Cergy-Pontoise, FRA); Vendrely, Charlotte (Université de Cergy-Pontoise, Cergy-Pontoise, FRA) We designed artificial biofilm matrices made of polysaccharides and self-assembled peptides to study the influence of bacterial amyloids onto biofilm matrix mechanical properties. We used model polysaccharides methylcellulose and alginate and peptides derived from the amyloid proteins CsgA and FapC found in Enterobacteriaceae and Pseudomonas, respectively. The peptides were selected through the TANGO software prediction of protein aggregation and formation of amyloid fibers. Self-assembly properties of the peptides were demonstrated by infrared spectroscopy and atomic force microscopy analysis. The influence of amyloids on the gelation process of model matrices was monitored using dynamic oscillatory rheological characterization with a plate-plate rheometer. The presence of amyloids was compatible with the sol/gel transition of alginate but modified the properties of the network. The loss modulus


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remained low and relatively unchanged but the storage modulus was decreased upon addition of the peptide to alginate indicating a decrease of the elasticity. The Methylcellulose matrices were characterized by a temperature-dependant gelation. The gelation temperature was higher and the curves obtained for the matrix made of methylcellulose with the amyloid peptide were different from those of the pure methylcellulose gel. At the end of the experiment, the loss modulus was around twice lower in the presence of the peptide and the viscous modulus was unchanged. The creep response to shear of the matrices was investigated using spinning disk rheometry. The character of the strain response for alginate gels without peptide under a shear stress showed a typical viscoelastic nature. Adding the amyloid peptide with alginate modified drastically the properties of the gel: the complex modulus G* and the viscosity were increased. Moreover, the viscous loss was totally recovered for the gel in presence of the peptide, in contrast to alginate without amyloids. The methylcellulose matrices also showed a viscoelastic nature. The stress was not reversible showing that some bonds maintaining the gel structure have been broken by the stress. The incorporation of the peptide inside the model matrix lead a very strong effect with a diminution of about 50% of the complex modulus G* and a relatively unmodified viscosity. Moreover, the stress of the methylcellulose gel with the peptide was about three times higher than the value reached for the gel made of pure methylcellulose. In conclusion the presence of amyloids in a polysaccharide matrix influenced the matrix deformation under shear stress and modified the matrix elastic response. Our results suggest that the secretion of amyloids could be for the biofilm a way of adaptation to environmental changes requiring modulation of elasticity and deformability.


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Session 8: Biofilms remixed: Microbial mats, stromatolites and soils Linking the composition of coral surface mucus to the structure and function of its associated microbial biofilm community Rodrigues Frade, Pedro (Department für Limnologie und Ozeanographie, AUT); Herndl, Gerhard (Dep. Limnology and Oceanography, Vienna, AUT) At the interface between the coral host epithelium and the seawater environment, coral surface mucus supports a microbial biofilm that acts as barrier against a wide range of environmental stresses. The main goal of this study was to link the dynamics in the composition of the coral mucus layer to related changes in the structure and function of its associated prokaryotic community. The mucus composition of several Caribbean coral species has been analyzed. Glycosyl composition analysis by GC-MS resolved two phylogenetically broad groups of host species where the main mucus carbohydrate was either galactose or fucose and mannose. GC-MS analyses of the total lipid extracts of mucus revealed a species-specific composition in terms of relative amounts of esters, sterols and fatty acids. Interestingly, no bacterial-derived lipid biomarkers were found. Species-specific differences in the thickness of the mucus layer are hypothesized to offer differing niches for microbial propagation. Massive parallel amplicon-based metagenomic 16S-rRNA (gene) analysis (Roche454 GS-FLX+ platform) was carried out on 132 mucus samples collected from the coral species along a depth gradient. Results suggest that mucus-associated prokaryotic communities vary substantially among host species with substantial differences in the community structure between Bacteria and Archaea. While Bacteria seem to be more dependent on host-related factors, Archaea are more related to environmental factors. Furthermore, there is evidence for a role of mucus carbohydrate fractionation in the response of the associated prokaryotic community. Mucus carbohydrate composition relates to mucus stability and thickness, and particularly to the diversity and community structure of the associated biofilm. This constitutes a mechanism to be further explored, potentially explaining the host-specificity of the associated microbial biofilms. Investigating N2 fixation activity in photosynthetic microbial mats at the single-cell level Woebken, Dagmar (University of Vienna, Division of Microbial Ecology, Vienna, AUT) Photosynthetic microbial mats are laminated, self-sustaining ecosystems with vast phylogenetic and functional diversity that exhibit steep physico-chemical gradients on the millimeter scale. N2 fixation is a key process in these mats that supports the nitrogen demands associated with high primary production. This process has been intensively studied for decades by biogeochemical assays and molecular approaches, such as the sequencing of the functional gene for N2 fixation (dinitrogenase reductase, nifH). However, N2 fixation can be regulated from the transcriptional to the post-translational level, thus the gene distribution or even expression might not accurately reflect the groups actively contributing to N2 fixation. As such the identity of the active diazotroph(s) in these ecosystems remains unknown. Previous investigations of microbial mats inhabiting the intertidal zone at Laguna Ojo de Liebre, Mexico, suggested that cyanobacteria (but not the visually dominant Lyngbya spp.-related cyanobacteria) and non-photosynthetic bacteria such as delta-proteobacterial sulfate reducers contributed to N2 fixation. In our study of the same mats, we used a combined approach of


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manipulation experiments with inhibitors, nifH sequencing and single-cell isotope analysis to identify members of the diazotroph community that actively contributed to N2 fixation along with their associated degree of activity. Acetylene reduction assays with specific metabolic inhibitors suggested that both sulfate reducers and cyanobacteria contributed to N2 fixation, while 15N2 tracer experiments at the bulk level only supported a contribution of cyanobacteria. Potential diazotrophic bacteria were identified by targeted nifH gene and transcript sequencing. Cyanobacterial and nifH cluster III sequences (including delta-proteobacterial sulfate reducers) dominated the nifH gene pool, while the nifH transcript pool was dominated by sequences related to Lyngbya spp.. The in situ N2 fixation activity of cyanobacteria and delta-proteobacteria was tested by 15N2 incubation experiments and subsequent single-cell isotope analysis through high-resolution secondary ion mass spectrometry (NanoSIMS). These measurements revealed that cyanobacteria were enriched in 15N with the highest enrichment being detected in Lyngbya spp. filaments (on average 4.4 at% 15N), whereas the delta-proteobacteria were close to natural abundance. Our combined data provide evidence that cyanobacteria, especially Lyngbya spp., actively contributed to N2 fixation in the investigated intertidal mats, while we could not find support for N2 fixation activity of the targeted delta-proteobacterial sulfate reducers. This work demonstrates that the combination of biogeochemical, molecular and single-cell techniques are powerful tools to define key functional populations in complex microbial communities. Massive microbial biofilms in a mineral spring cavern dominated by methane and iodine Karwautz, Clemens (Helmholtz Zentrum München, Neuherberg, GER); lueders, tillmann (Helmholtz Zentrum München, Neuherberg, GER) Most subsurface ecosystems are limited by their supply of energy and carbon from the surface. Yet, cave ecosystems can harbor unique microbial life, isolated from photoautotrophic primary production, supported purely by in situ chemoautotrophic processes. Here, we investigated massive microbial biofilms formed in a cavern fed by an iodine rich mineral spring with high geogenic methane loading. Methane, by far the most abundant organic gas in the atmosphere, plays a dynamic part of the global carbon cycle. Its role as very potent climate active compound has increased interest in the control of methane oxidation. Microbes oxidizing methane (e.g. bacteria, fungi, archaea) under aerobic and anaerobic conditions are central to the biological removal. High gas emissions within the spring water itself and elevated concentrations within the cave atmosphere were measured. The massive biomass produced by the microbes and exopolysaccharide slime completely covered the walls and ceilings of the cave, the latter bearing bacterial snottites of up to 10 cm length. We aim to unravel the role of methane and potentially also methyl halides (iodomethane) as electron donors in this unique habitat. A surprisingly diverse microbial community within these biofilms was found, applying molecular fingerprints and in-depth 454 pyrotag sequencing. The dominating role of methane as biofilm substrate was confirmed. Several taxa known to be involved in the utilization of C1-compounds such as methylotrophic Methylotenera spp. and methanotrophic Methylobacter spp. were identified. Furthermore, functional gene analysis of the methyltransferase (CmuA) suggested the presence of methyl halide oxidizing bacteria. Longitudinal and vertical gradients in the cavern were reflected in biofilm community composition and stoichiometry giving insights on potential redox processes and biogeochemical fluxes. The role of methanogenic Archaea (Methanomicrobia) detected in cavern sediments and a potential for parallel aerobic and anaerobic methane oxidation remains to be unraveled. In summary, we provide first insights into a unique subsurface biofilm system, and discuss a possible coupling of microbial methane and iodine cycling.


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Session 9: Applied water systems

Hardness of water and Calcium affects biofilm formation on PVC, glass and a thin-composite membrane Hijnen, Wim (KWR Watercycle Research Institute, Nieuwegein); Schulz, Frederik (KWR Watercycle Research Institute, Nieuwegein); Brouwer-Hanzens, A.H. (KWR Watercycle Research Institute, Nieuwegein); Harmsen, D.J.H. (KWR Watercycle Research Institute, Nieuwegein); Cornelissen, E.R. (KWR Watercycle Research Institute, Nieuwegein); Bereschenko, L.A. (KWR Watercycle Research Institute, Nieuwegein) Biofilm formation has been observed in water systems causing technical problems and microbiological water quality problems. Biofouling is mainly ruled by the availability of nutrients in the water for growth and maintenance. Removal of nutrients is the most suitable approach to reduce biofouling but also causes high costs due to extensive pretreatment. Biofilm stability is partially determined by EPS composed of a sticky negatively-charged network of polysaccharides,proteins and ribonucleic acids.The resistant of this network to mechanical and chemical stress is influenced by the presence Ca2+ which forms permanent junction points in the EPS matrix. Reducing hardness of water is a common process in water treatment to optimize water quality to minimize Ca2+ fouling,but the process may also be of benefits to reduce biofouling problems in water transport systems and spiral-wound membranes. In this study we evaluated the effect of water softening on biofilm formation and the specific role of calcium in this process. The biofilm formation and biofouling process was studied in (i) a small scale biofilm production unit (BPU) and (ii) a three element spiral-wound membrane pilot (350 L/h; SWP). The BPU was loaded with PVC-P and glass coupons and the membrane pilot with RO membranes.Both systems were supplied with pre-filtered drinking water with and without an ion-exchange treatment. The Ca2+ conc. was reduced from 36 to 0.3 mg/l.The biofilm formation was induced by the internal organic compounds in the PVC-P and by dosing 10 µg acetate-C/l. Biofilm formation was monitored with ATP (active biomass pg ATP/cm²) and with carbohydrates as part of the extracellular polymeric substances (EPS) using the sulfuric/phenol method (µg glucose-eq./cm²).With DNA analyses using a terminal restriction fragment length polymorphism (T-RFLP) the influence of water softening on microbial populations in the biofilms was assessed. Over a period of 112 days the biofilm formation was monitored on the PVC-P and glass coupons installed in the BPU. Since ion-exchange reduces also other positively charged ions in the water the specific role of Calcium was verified in a second run with PVC-P coupons by dosing Ca2+ to the softened water.In the SWP the biofouling was monitored on base of the increase of pressure drop, the reduction of permeate flux and the autopsy of the fouled membranes after reaching of a certain pressure drop. The biofilm formation on PVC-P coupons showed no effect of softening on the ATP production, but a significant retardation in the CH production rate was observed. The CH production rate on the PVC-P coupons supplied with (i) drinking water (DW) and (ii) softened water (SW) + Ca2+ ranged from 3.9 6.1 µg/cm2.d, whereas the CH production rate on the coupons supplied with SW was 1.2 µg/cm2.d. The biofilm formation on the glass coupons showed from the start reduced biomass concentrations (ATP and CH) in the coupons supplied with softened water. In


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the SWP supplied with drinking water the pressure drop increased after a lag-phase of 10 days and the exponential fouling rate was 0.15 LN Pi.d-1. Softening of the drinking water retarded the biofouling process in the membrane. Pressure drop increase started after 20 days and the exponential fouling rate was 0.11 LN P.d-1.Autopsy of the membranes revealed lower CH and ATP content in this element compared to the drinking water element. With cleaning tests it was demonstrated that the biofilm on the SW membrane was removed with higher efficacy than the biofilm on the DW membrane. The T-RFLP profiles of the isolated DNA samples revealed a clear difference in bacterial populations(41% difference in DNA content) between biofilms grown in DW and SW. Conclusion of the study was that softening reduces the rate of biofouling in water systems. The results of the BPU showed the prominent role of the divalent ion Ca2+ in this process which caused a decline in carbohydrate production in the biofilm. The association with Acanthamoeba spp. affects the UV-sensitivity of Legionella pneumophila Cervero, Silvia (Departament de Microbiologia, Facultat de Biologia, Universitat de Barcelona, Barcelona, AUT); Sommer, Regina (Medical University Vienna, Institute for Hygiene and Applied Immunology, Vienna, AUT); Araujo, Rosa M. (Departament de Microbiologia, Facultat de Biologia, Universitat de Barcelona, Barcelona, ESP) Drinking water distribution systems are complex environments that host microbial biofilms and may impair the water quality. This applies in particular to domestic hot water systems representing a source for of human infections by legionellae. Thus in many countries national standards were established on how to operate technical water systems. These standards also include disinfection techniques which are applied in case that elevated concentrations of legionellae occur. However, when insufficiently applied, the survival of the bacteria can promote a rapid re-colonization of the system. The two methods primarily used for disinfection of water systems are thermal treatment and chlorination. Another disinfection method is the application of UV radiation.

In the present work we investigated the UV-253.7 nm sensitivity of L. pneumophila and one of its natural hosts, Acanthamoeba spp., representing prominent members of biofilms in water systems. Special focus was laid on the question if the association with amoebae influences the UV sensitivity of legionellae.

By means of a strictly controlled laboratory UV irradiation apparatus five strains of Legionella spp. and two strains of free-living amoeba of the genera Acanthamoeba, including both the trophozoites and the cysts, as well as two co-cultures of L. pneumophila with the Acanthamoeba strains.

No significant differences in the UV inactivation behaviour were observed among Legionella strains tested. A 3 log reduction was reached with a UV fluence of around 45 J/m². UV irradiation was less effective against free-living amoebae; a 3 log reduction required up to 990 J/m². As expected, cysts were much more UV resistant than trophozoites. Remarkably, the results showed that the association of L. pneumophila with free-living amoebae increased their UV resistance resulting in a reduced efficacy of UV disinfection process. This finding


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demonstrates that relationships between different microorganisms as occurring in biofilms of water systems have to be taken into account by defining the operational conditions of disinfection processes.

Life under Hydraulic Pressure: Biofouling Development and Architecture Bar-Zeev, Edo (Department of Chemical and Environmental Engineering, Yale University, New Haven, USA); Zodrow, Katherine R (Department of Chemical and Environmental Engineering, New Haven, USA); Elimelech, Menachem (Department of Chemical and Environmental Engineering, New Haven, USA) Biofilm development, architecture and physiochemical state drastically vary, depending on the nutrients oxygen and shear stresses present. Here we studied the role hydraulic pressure plays in shaping biofilms on reverse osmosis (RO) membranes. Dynamic bench-scale experiments were carried out for 48 hours by pressurizing (up to 55 bar) artificial seawater containing a model bacteria, Pseudoalteromonas atlantica. Total organic carbon, protein, polysaccharide and bacterial samples were routinely collected from the feed reservoir to test the effect of feedwater characteristics on biofilm initiation and development. At the end of each experiment, membranes were imaged by confocal laser scanning microscopy to determine biofilm three-dimensional (3-D) complexity and analyzed by atomic force microscopy to quantify biofilm elastic properties. Concurrently, membrane subsections were also tested for biofilm density and biochemical composition. Our results suggest that polysaccharides (up to 175 µg GX ml-1) secreted from the planktonic culture were the primary cause of biofilm initiation, irreversible attachment of bacteria and expedited biofilm growth. Biofouling that developed under these high hydraulic pressures (55 bar), appeared as pliable sheets, 30-90 um thick (depending on carbon source availability) and composed mainly by a packed layer of live cells. Our results lead us to posit that severe hydraulic stress results in the development of a dense and reinforced biofilm layer that was characterized by low 3-D complexity. The study insights shed new light on the nature of biofilm life in pressurized cross-flow systems. Trapped in the matrix: the impact of Natural Organic Matter fouling on bacterial adhesion under full-scale Nanofiltration processes Habimana, Olivier (School of Chemical and Bioprocess Engineering, University College Dublin, Dublin); Heffernan, Rory (School of Chemical and Bioprocess Engineering, University College Dublin, Dublin); Semião, Andrea J.C. (School of Engineering, The University of Edinburgh, Edinburgh, GBR); Safari, Ashkan (School of Chemical and Bioprocess Engineering, University College Dublin, Dublin); Casey, Eoin (School of Chemical and Bioprocess Engineering, University College Dublin, Dublin) Nanofiltration (NF) is becoming an increasingly important process for the production of clean water. The performance of this process is however routinely affected by biofouling, which involves the initial adhesion of microorganisms and their subsequent biofilm development on the membranes. While Natural Organic Matter (NOM) is known to form a conditioning layer on the membrane surface during Nanofiltration, the role of these organic layers during biofouling has gained much attention in the past few years. Although studies have shown that the presence of Transparent Exopolymer Particles (TEPs) in the feed solution was a key factor in membrane


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biofouling during filtration, little is known on the role of NOM/TEP conditioning films during the initial stages of biofouling under full scale Nanofiltration processes. Hence, the purpose of this study was to assess the influence of Humic acid, and sodium alginate conditioning layers on the initial adhesion of bacterial cells under full-scale nanofiltration processes, by altering permeate flux conditions.

Model fouling layers of Humic acid, with and without Sodium Alginate, were first formed on the surface of a NF 90 Nanofiltration membrane. Deposition experiments using a mCherry-tagged Pseudomonas fluorescens model strain were conducted for 30 minutes inside Membrane Fouling Simulators (MFS) devices at different permeate flux conditions under a crossflow-filtration system. Ex-situ analysis of membranes was systematically performed using epi-fluorescence microscopy to establish the level of surface coverage on membranes. The morphological and physical attributes of NOM/TEP fouling layers were characterized prior to P. fluorescens adhesion, using confocal microscopy and atomic force spectroscopy.

Results indicate that NOM and TEP layers lead to a 2.5-fold reduction in surface coverage compared to deposition onto clean membranes under identical permeate flux conditions. Interestingly, bacterial surface coverage was significantly heterogeneous from samples taken from the inlet/mid and outlet regions of the same membrane. This shows that under permeate flux conditions the (NOM/TEP) fouling layer actively recruits and entrap cells, thereby reducing the amount of non-deposited cells as liquid flows from the inlet to outlet of the MFS device. Moreover entrapped cells were found to be randomly dispersed within the NOM/TEP fouling matrix as opposed to a homogenous deposited layer on clean membranes. Work is currently underway to investigate the NOM/TEP morphological and physical attributes. The implication of these findings may help better understand initial instances of biofouling under nanofiltration processes, in which embedded cells may develop as microcolony niches within the NOM/TEP layers.

Mineral coating creates internal porosity and supports microbial activity in rapid sand filters treating groundwaters Gülay, Arda (Denmark Technical university, Copenhagen, DNK); Tatari, Karolina (Denmark Technical university, Copenhagen, DNK); Musovic, Sanin (DTU, Copenhagen, DNK); Mateiu, Ramona (DTU, Copenhagen, DNK); Albrechtsen, Hans-Jørgen (DTU, Copenahgen, DNK); Smets, Barth F. (DTU, Copenhagen, DNK) Biological rapid sand filters are often used to produce drinking water from high quality groundwaters. Here, microbial communities grow attached on the surfaces of the filter media. Granular materials such as silica sand and anthracite are the most widely used filter materials. Although rapid sand filters are periodically subject to back washing, a coating layer may develop on the surface of the original filter material due to the aggregation and transformation of various oxy/hydroxide and carbonate precipitates. Such mineral coating has been observed on filter materials in many waterworks, especially in plants treating groundwater with elevated iron and manganese content. However, the role of these mineral coatings has only been investigated in abiotic context (adsorption capacity, surface area, chemical content). Since microbial communities are attached in these systems, mineral coatings are likely to affect their activity and distribution. In this study, mineral coatings were observed in a rapid sand filter


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together with a strong positive correlation between amounts of coating mass and DNA density. We examined whether the coating layer affected microbial activity indirectly due to physicochemical alterations or directly, by affecting (i) microbial abundance, (ii) spatial microbial distribution, (iii) microbial diversity or (iv) microbial community composition. Hence, we applied several experimental techniques including 454 pyrosequencing, quantitative PCR, confocal scanning laser microscopy, low and high vacuum electron microscopy, bio-kinetic assays and concurrent physical and chemical analyses on filter material with different mineral coating densities taken from different depth in a long term operated rapid sand filter). Focusing on nitrification as a model microbial process and nitrifiers as a model microbial guild, we showed that mineral coatings on filter media directly and positively link with nitrification activity, microbial abundance and spatial distribution of the microbial communities. Microbes and exocellular materials were readily materialized in the internal pore structure of the mineral phases. Our results provide the first insight into the interactions between the biotic and abiotic phases in drinking water rapid sand filter systems.

Abstracts of poster presentations

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Session 1: The extracellular space of biofilms P001 - The role of ecotin in the biofilm matrix of P. aeruginosa Tseng, Boo Shan (University of Washington School of Medicine, Seattle, USA); Parsek, Matthew R. (University of Washington School of Medicine, Seattle, USA) Biofilm bacteria are encased in a protective, self-produced extracellular matrix that is poorly characterized. Although proteins have long been known to be an important part of the matrix, very few biofilm matrix proteins of P. aeruginosa have been identified and little is known about their functions. We hypothesize that the biofilm retains extracellular enzymatic proteins to functionalize the activated matrix, which would play various roles in biofilm formation and antimicrobial tolerance. To identify potential proteins in the matrix of P. aeruginosa, we performed a screen for proteins enriched in the biofilm matrix relative to the total biofilm proteome. Here we describe one hit, ecotin (PA2755), a serine protease inhibitor that was approximately 4-fold over-represented in the biofilm extracellular protein sample in two different runs of the screen. Confirming this hit, we show that ecotin co-precipitates with the biofilm exopolysaccharide Psl. Since our work shows that ecotin protects planktonic P. aeruginosa from neutrophil elastase invitro, we are currently investigating whether ecotin can protect biofilms from protease attack and the role of ecotin in biofilm formation.

P002 - Composition of matrix extracellular of Listeria monocytogenes biofilm GRAZIELLA, BOURDIN (ANSES, BOULOGNE SUR MER, FRA); SADOVSKAYA, IRINA (ULCO, BOULOGNE SUR MER, FRA); FAILLE, CHRISTINE (INRA, VILLENEUVE D'ASCQ, FRA); GUERARDEL, YANN (USTL UGSF, VILLENEUVE D'ASCQ, FRA) Listeria monocytogenes is an opportunist pathogen that causes listeriosis. Few data on the matrix composition of L. monocytogenes biofilms are available. However it is well-known that the extracellular matrix plays an important role in biofilm development, resistance to chemicals and to detachment The aim of the study was to characterize the ability of L. monocytogenes strains to form biofilm (strain lineage, growth medium and incubation temperature), using complementary methods. For the first time, a quantitative analysis of total carbohydrate, protein and eDNA content in the biofilm matrix was carried out . Total biomass of 27 strains of L. monocytogenes belonging to lineages I or II was evaluated in different conditions (temperature and medium) by using crystal violet assay. Carbohydrate concentrations were determined by phenol-sulfuric acid assay. The protein content was measured using the Bio-Rad Protein Assay. The eDNA concentration was determined by OD260nm. Lineage II strains produced significantly more biofilm than lineage I strains. Biofilm quantities were greater in MCDB 202 vs. TSBYE medium (confirmed by Scanning Electron Microscopy (SEM) analysis) and at 37°C vs. 22°C. Conversely, cultivable bacteria were enumerated in greater quantities in TSBYE than in MCDB 202 medium. These opposite results would suggest the presence of uncultivable and/or dead bacteria in the biofilm. The SEM investigation established that L. monocytogenes biofilms produce extracellular matrix in both media at 37°C. The amount of exopolymers in the extracellular matrix and the pH values were significantly higher in TSBYE than in MCDB 202 medium. The exception was the ScottA strain which presented similar pH


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values and exopolymers content in both media . Proteins were the most abundant exopolymer component, followed by DNA and polysaccharides the least. The characterization of L. monocytogenes biofilm composition may help to develop new strategies to prevent the formation and promote the detachment of biofilms. P003 - Haloarchaeal Biofilms - Widespread and Complex as their Bacterial Counterparts Fröls, Sabrina (Technische Universität Darmstadt , Darmstadt, GER); Dyall-Smith, Mike (Charles Sturt University, Wagga Wagga, AUS); Pfeifer, Felicitas (Technische Universität Darmstadt, Darmstadt, GER) Complex microbial communities i.e. biofilms are recognized to be the predominant microbial mode of life in nature and found in a remarkable spectrum of habitats. In the past, most investigations concerning biofilms were limited to the members of the domainBacteria, but the use of archaea specific probes demonstrated that biofilms in nature are often mixed microbial communities formed by species of Bacteria and Archaea (reviewed in Fröls, 2013). For example halophilic archaea were identified within mixed communities of biofilms from sediments around an underwater fresh spring in the Dead Sea (Ionescu et al., 2012),howevernothing was known about the nature of haloarchaeal biofilms. We developed a fluorescence-based live-cell adhesion assay to examine biofilm formation by twenty different haloarchaea, including species of Halobacterium, Haloferax and Halorubrum, as well as novel natural isolates from an Antarctic salt lake. Thirteen of the twenty tested strains significantly adhered (P-value < 0.05) to a plastic surface. Examination of haloarchaeal biofilms grown on glass surfaces by differential interference contrast, fluorescence and confocal microscopy showed two types of biofilm structures. Carpet-like, multi-layered biofilms containing micro- and macrocolonies were formed by strains of Halobacterium salinarum and the Antarctic isolatet-ADL strain DL24. In contrast a second type of biofilm, characterised by large aggregates of cells adhering to surfaces, was formed by Hfx. volcanii DSM 3757T and Hrr. lacusprofundi DL28. Analyses of the biofilm composition formed by the strongly adhesive haloarchaeal strains revealed the presence of extracellular polymeric substances, such as eDNA and glycoconjugates. Our analysis demonstrated that the ability of surface adhesion is widely distributed in haloarchaea, whereas the structures and compositions of the biofilms formed is diverse between the haloarchaeal species tested. Fröls, S. (2013) Archaeal biofilms: widespread and complex. Biochem Soc Trans 41: 393-398. Fröls, S., Dyall-Smith, M., and Pfeifer, F. (2012) Biofilm formation by haloarchaea. Environ Microbiol 14: 3159-3174. Ionescu, D., Siebert, C., Polerecky, L., Munwes, Y.Y., Lott, C., Hausler, S. et al. (2012) Microbial and chemical characterization of underwater fresh water springs in the Dead Sea. PLoS One 7: e38319.


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P004 - In-vitro micro-structural characterization and designer solvents used to describe role of polysaccharides in Pseudomonas aeruginosa biofilm Seviour, Thomas (Singapore Centre for Environmental Life Sciences Engineering, Singapore, AUS); Chuen, Su (Singapore Centre for Environmental Life Sciences Engineering, Singapore, SGP); Weerachanchai, Piyarat (Nanyang Technological University, Singapore, SGP); Lee, Jong-Min (Nanyang Technological University, Singapore, SGP); Kjelleberg, Staffan (Singapore Centre for Environmental Life Sciences Engineering, Singapore, SGP) The polysaccharides of P. aeruginosa biofilms, PEL, PSL and alginate, have been implicated in many biofilm functions including structure, signaling and biofilm differentiation. Functional studies into the P. aeruginosa polysaccharides, however, have suffered from being observational rather than quantitative, and from not having been demonstrated on isolated polysaccharide fractions. We developed complementary approaches to overcome both challenges, comprising microrheological assay and solubilisation by designer solvents. By observing the fine movement of latex microspheres infused into biofilms, biofilm mechanics were described at a microscale. Differential contributions of PEL and PSL to stages of the biofilm life cycle were elucidated in-vitro. A significant role was thus described quantitatively, specifically for PEL, in biofilm spreading and expansion.

While consistent with previous, albeit non-quantiative studies, the second part of the challenge has not been addressed owing to lack of methodology. Hence, basic knowledge of PEL chemistry is lacking. In fact the same is largely the case for PSL. While a structure for PSL has been assigned, the constituent used for structural assignment was chosen on the basis of high solubility and analytical convenience rather than functional importance. Studying extracellular polymeric substances in biofilms is paradoxical. While high solubility is required for isolation, the poorly soluble constituents are those that should be targeted when seeking functionally importance. Much work is still required to understand the polysaccharides even of the well documented P. aeruginosa . The situation with mixed species biofilms is even more challenging. Identifying solvents to maximize polysaccharide solubility is critical. By measuring transfer of biofilm matrix constituents into the solvent phase as solvent viscosity changes, several ionic liquids (ILs) were identified, based on their Hildebrand solubility constants, that can achieve higher PEL solubility than other solvents. ILs are green solvents that can be designed for optimum substrate solubility and toxicity.

Using these ILs, key compositional features of PEL were elucidated to explain its role in biofilm spreading and expansion. High solubilities achieved by ILs on other biofilms suggest that ILs can be used to isolate a range of recalcitrant biofilm polysaccharides. Additionally, identifying innocuous solvents that can achieve high biofilm solubilities could lead to novel biofilm mitigation strategies for a range of industrial, environmental and medical situations that avoid potential secondary problems associated with other control strategies like dispersal (i.e. re-colonisation and release of more virulent form of bacteria).


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P005 - DNA is an essential component of the Campylobacter jejuni biofilm extracellular matrix Brown, Helen (Institute of Food Research, Norwich, GBR); Reuter, Mark (Institute of Food Research, Norwich, GBR); Betts, Roy (Campden BRI, Chipping Campden, GBR); van Vliet, Arnoud (Institute of Food Research, Norwich, GBR) Introduction: The majority of bacteria exist in either single or multi-species biofilms. Biofilm growth allows increased tolerance to starvation, antimicrobials and survival in food chain relevant conditions. Campylobacter jejuni is one of the leading causes of infectious intestinal disease in the developed world and is known to form biofilms. We have previously shown that C. jejuni biofilm formation is increased in aerobic conditions, suggesting biofilm formation has a role in tolerance to stress responses. Although interest in biofilm formation by Campylobacter species, particularly C. jejuni, has increased in recent years, there is still relatively little known about C. jejuni biofilm formation. This is particularly true for the C. jejuni biofilm extracellular matrix (ECM). Extracellular DNA (eDNA) has shown to be an important component in the ECM of many bacterial species. In this study we investigated the importance of eDNA within the C. jejuni biofilm, and if degradation of eDNA was able to lead to biofilm removal from abiotic surfaces.

Results: We have shown that eDNA is present in the supernatant of C. jejuni cultures throughout the growth cycle. DAPI-staining of a biofilm with GFP-positive C. jejuni NCTC 11168 showed that bacteria in C. jejuni biofilms were surrounded by eDNA, suggesting that eDNA has the potential to be a substantial component of the ECM. Degradation of eDNA by DNase I treatment in mature C. jejuni biofilms led to the rapid and complete removal of the biofilm, without compromising C. jejuni viability. The extracellular DNase-expressing C. jejuni strain RM1221, and its cell-free supernatant, were capable of degrading mature C. jejuni NCTC 11168 biofilms in co-culture assays. Molecular investigation of this mechanism is currently under investigation.

Conclusion: These results suggest that the eDNA of the C. jejuni biofilm can be targeted to disrupt C. jejuni biofilms. This is a significant finding which may lead to more efficient sanitisation of food processing equipment, and ultimately lower levels of C. jejuni contamination within the food-chain.

P006 - GFP-labeled carbohydrate-binding module as a novel tool for probing exopolysaccharides in Escherichia coli biofilm Ojima, Yoshihiro (Osaka university, Toyonaka, JPN) Keywords: Escherichia coli, exopolysaccharide, carbohydrate-binding module Summary of key findings: A novel fluorescent probe for exopolysaccharides (EPShs) was constructed by conjugating a carbohydrate-binding module (CBM) 3, from Paenibacillus curdlanolysticus, with the green fluorescence protein (GFP-CBM3). The GFP-CBM3 fused protein exhibited strong affinity to microcrystalline cellulose. Moreover, GFP-CBM3 specifically bound to cell-dense microcolonies in the developing process of Escherichia coli biofilms on a solid surface. The fused protein can be an alternative tool for specifying EPShs produced by E. coli cells. Background and relevance: As the main constituents of E. coli matrices, polysaccharides are largely responsible for the architecture of the biofilm. E. coli cells involved in cell-to-cell and cell-to-surface interactions produce a protective matrix of poly-β-1,6-N-acetyl-D-glucosamine (PGA). Some articles demonstrated that the co-expression of cellulose and thin aggregative fimbriae leads to


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forming the networks which tightly packed cells in inert matrices (Mol Microbiol, 39:1452, 2001). For analyzing the biofilm structure, confocal laser scanning microscopy (CLSM) combined with fluorescent dyes is a powerful tool. Using this technique, researchers can identify specific EPShs in the extracellular matrices of biofilms (Infect Immun, 75:4728, 2007). On the other hand, the CBMs exhibit strong affinity to carbohydrates. Based on their amino acid sequences, the CBMs have been classified into 68 similarly-structured families (http://www.cazy.org). Because of their binding specificities to various polysaccharide matrices, the CBMs have been widely applied in biotechnology. For example, polysaccharides in plant cells have been targeted and identified by the CBM (Anal Biochem, 326:49, 2004). Given their specific binding properties, we considered that the CBM could be exploited as a marker of EPShs produced by bacteria. In this study, we fused GFP with CBM3 from P. curdlanolyticus B-6 (Appl Environ Microbiol, 77:4260, 2011) and assessed the effectiveness as a probe of EPSh in E. coli biofilms. Results and discussion: SDS-PAGE showed that the bands GFP, CBM3 and GFP-CBM3 proteins appeared at approximately 27, 16 and 43 kDa, respectively, indicating that the fused protein was properly expressed in transformed E. coli Rosetta pLysS cells. Subsequently, the carbohydrate-binding capability of GFP-CBM3 was confirmed with Avicel, a typical microcrystalline cellulose, suggesting that the GFP-CBM3 was a suitable fluorescent marker protein for cellulosic materials. Next, biofilms of E. coli BW25113 and MG1655 strains were incubated with the GFP-CBM3. As a result, GFP-CBM3 fluorescence appeared to be localized to cell-dense areas in the microcolonies of both strains. To better assess the performance of GFP-CBM3 fused protein, we also stained the EPShs with wheat germ agglutinin-Oregon 488 dye, a conventional chemical for staining biofilm matrices. In CLSM images, the staining of the core areas of microcolonies was consistent with that of GFP-CBM3 fused protein. This finding highlights the efficacy of GFP-CBM3 as a new marker protein for probing EPShs in biofilm. Though the specific carbohydrates targeted by the GFP-CBM3 in E. coli microcolonies were not elucidated, we confirmed that, besides cellulose, this fused protein bound with strong affinity to chitosan. The binding of CBM3a to chitin was also reported (Appl Environ Microbiol, 61:1980, 1995). The chitin and chitosan (poly-D-glucosamine derivative) are chemically similar to PGA, a main constituent of EPShs in E. coli biofilms (J Mol Sci, 14:4560, 2013). In this context, our constructed GFP-CBM3 protein is expected to target cellulose and PGA in the EPShs formed by E. coli K-12 derived strains. P007 - Understanding and harnessing the power of biofilm matrix: What we have learned from Shewanella biofilms Ding, Yuanzhao (Singapore Centre on Environmental Life Sciences Engineering, Singapore, SGP); Ng, Chun Kiat (Singapore Centre on Environmental Life Sciences Engineering, Singapore, SGP); Cao, Bin (Singapore Centre on Environmental Life Sciences Engineering, and School of Civil and Environmental Engineering, Singapore, SGP) An improved understanding of biofilm structure and function is crucial for developing new strategies to eradicate detrimental biofilms or to engineer next generation beneficial biofilms. To this end, key genetic and molecular determinants of biofilm formation have been elucidated in several model organisms. In contrast, molecular components and interactions defining biofilm matrix structure and function remain less explored. Further, little has been done to harness the power of the biofilm matrix to engineer beneficial biofilms for environmental and energy applications. We have used biofilms of Shewanella sp. as a model system and applied


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biochemical analysis, molecular biology, proteomics, and RNA-sequencing to unravel molecular components and their interactions in stabilizing the biofilm matrix. We have also applied a multidisciplinary approach interfacing materials science, biofilm biology, and nanotechnology to engineer the biofilm matrix for an improved biofilm performance.

Matrix-associated proteins Matrix-associated proteins extracted from biofilms grown in a hollow-fiber membrane biofilm reactor were identified by LC-MS/MS proteomics. A total of 58 extracellular and outer membrane proteins were identified in the biofilm matrix. These included proteins that contribute to key physiological biofilm processes, such as the biofilm-promoting protein BpfA. In addition, 20 redox proteins were found in the matrix. Among the detected redox proteins were c-type cytochromes, MtrC and OmcA, which have been implicated in extracellular electron transfer. Among the identified matrix-associated proteins, BpfA is a gigantic protein (2768 amino acids, ~285 kDa) with glycine-rich domains for Ca2+ binding and which features ~100 amino acid tandem repeats characterizing Bap-family proteins. These proteins are predominantly loosely associated with the outer membrane. We constructed an in-frame deletion mutant and further explored the role of BpfA in biofilm formation and matrix stabilization.

Engineering a highly cohesive biofilm matrix for improved performance Although biofilm-based bioprocesses have been increasingly used in various applications, long-term robust and efficient biofilm performance remains one of the main bottlenecks. Recently, we showed that the cohesiveness and performance of Shewanella oneidensis biofilms in decontaminating chromium (Cr)-contaminated water, i.e., Cr(VI) immobilization, are enhanced by disrupting putrescine biosynthesis. This finding suggests a role for putrescine in S. oneidensis through mediating biofilm matrix disassembly, by altering the structural configuration of the extracellular polymeric substances, and offers a novel strategy in biofilm engineering for enhanced bioprocesses.

Matrix-enabled nanocatalytic biofilms for contaminant removal Microorganisms have the potential to change the oxidation state of metals and these microbial processes have opened up a new window for novel applications including biosynthesis of metal nanomaterials. In a recent study, we found that nanoparticles that are generated extracellularly can have a high catalytic activity. Through exploiting the activity of the biofilm matrix, we further fabricated hybrid biofilm-nanocrystal systems or nanocatalytic biofilms via in situ formation of nanocrystals in the biofilm matrix. This enables the generation of a biofilm-based nanocatalytic activity, complementing the inherent biofilm biocatalytic traits, for the development of novel environmentally friendly multifunctional catalytic systems exhibiting both bio- and nano-catalytic activities. To prevent the loss of catalytic activity through biofilm detachment, we further engineered the biofilms through increasing cyclic-di-GMP level to minimize the active dispersal of biofilm cells so that the hybrid catalytic systems display long-term activity.


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P008 - Extracellular components that define Rhizobium leguminosarum biofilm matrix architecture Abdian, Patricia (Laboratorio de Microbiología Molecular y Celular, Fundación Instituto Leloir, Buenos Aires, ARG); Vozza, Nicolás (Laboratorio de Microbiología Molecular y Celular, Fundación Instituto Leloir, Buenos Aires, ARG); Caramelo, Julio (Laboratorio de Biología Estructural y Celular, Fundación Instituto Leloir, Buenos Aires, ARG); von Bilderling, Catalina (Centro de Microscopías Avanzadas, Universidad de Buenos Aires, Buenos Aires, ARG); Russo, Daniela (Laboratorio de Microbiología Molecular y Celular, Fundación Instituto Leloir, Buenos Aires, ARG); Pietrasanta, Lía (Centro de Microscopías Avanzadas, Universidad de Buenos Aires, Buenos Aires, ARG); Zorreguieta, Angeles (Laboratorio de Microbiología Molecular y Celular, Fundación Instituto Leloir, Buenos Aires, ARG) Rhizobia are soil bacteria that interact with leguminous plants inducing nitrogen-fixing root nodules. They grow in varied environmental conditions producing different types of biofilms. We study the biofilms formed by R. leguminosarum in vitro , a process probably relevant to the attachment of cells to soil particles. The typical architecture of a mature biofilm consists of layers of cells attached to each other by lateral interactions, forming clusters interlaced by water channels. The main component of the matrix is an acidic exopolysaccharide (EPS) that provides support to the biofilm structure. Moreover R. leguminosarum produces a capsular polysaccharide (CPS) that seems involved in surface attachment at the onset of biofilm formation. Proteins secreted by a type I secretion system are also involved. Interestingly, some of them possess one or more cadherin-like domains that confer carbohydrate binding ability. Among them, we have characterized RapA2, a unipolar calcium binding lectin that specifically recognizes the EPS; and the Ply glycanases that regulate the length of EPS molecules. A characteristic distribution of EPS molecules length is important to build up an ordered polysaccharide network. A model depicting the interaction of RapA, Ply glycanases and acidic polysaccharides during the progress of biofilm matrix development is presented.

P009 - Biofilm extracellular polysaccharides of Pseudomonas aeruginosa have dual roles as public and private goods Irie, Yasuhiko (University of Bath, Bath, GBR) The extracellular polysaccharides (EPS) are crucial components of microbial biofilms. They have been hypothesised to play pivotal roles in the social evolution of biofilm development, but direct experimental evidence to demonstrate these are limited. EPS have been suggested to be public goods, where their production is metabolically costly, but are secreted as shared resources by mediating adhesion of the entire mixed population to surfaces. In this study, we inspect whether Pseudomonas aeruginosa EPS have biological roles as public goods or private goods in biofilm cultures. Our results suggest that PSL polysaccharides have features of both public and private goods, as PEL polysaccharides appear to function completely as private goods. These data highlight that biofilm EPS cannot be generalised to fit models, and that it is important to customise the parameters based on specific experimental evidence.


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P010 - Antifungal agents induces alteration in the matrix composition of candida glabrata biofilms Rodrigues, Célia (Minho University, Departement of Biological Engineering Department, Braga ); Fonseca, Elza (Minho University, Departement of Biological Engineering Department, Braga , AUT); Gonçalves, Bruna (Minho University, Departement of Biological Engineering Department, Braga ); Bogas, Diana (Minho University, Departement of Biological Engineering Department, Braga ); Silva, Sónia (Minho University, Departement of Biological Engineering Department, Braga ); Azeredo, Joana (Minho University, Departement of Biological Engineering Department, Braga ); Henriques, Mariana (Minho University, Departement of Biological Engineering Department, Braga ) Candida glabrata has emerged as the second most prevalent pathogen, after Candida albicans, in mucosal and invasive fungal infection. Its ability to form biofilms has been considered one of the most important virulence factors, since they present a high resistance to antifungal agents used in fungal infections treatment. Moreover, there is a lack of information about the physiological response of C. glabrata biofilms to antifungal agents. Thus, the aim of this study was to evaluate the effect of different antifungal agents on C. glabrata biofilm composition and the influence in related resistance genes expression. For that C. glabrata biofilms were formed in the presence of fluconazole (Flu), voriconazole (Vrz) and amphotericin B (AmB). Biofilm matrix composition was evaluated in terms of polysaccharides, proteins and ergosterol and ERG genes expression was also assessed.

As expected C. glabrata biofilms are more resistant to Flu, Vrz and AmB than planktonic cells. . Although in a strain dependent manner, polysaccharides were increased in the presence of the antifungals, in opposition to proteins, which decreased in the presence of AmB and Vrz. Due to the interaction of these agents with ergosterol, even in different ways, we evaluated, for the first time, the presence of this compound in the extracelluar matrix. It was noticed that ergosterol was, in fact, present in all the matrices and in general it increased with the presence of the drugs. Therefore, there is an obvious answer of biofilm cells to the stress induced by the different agents, that caused and alteration of matrix composition.

In order to determine if the increased concentration of ergosterol in the biofilm matrix was caused by an up-regulation of proteins responsible for ergosterol synthesis, ERG expression was evaluated.. Although ERG gene expression was very strain dependent is was possible to verify that some genes, as ERG11 and ERG6, were upregulated in biofilm cells. Interestingly one strain was unable to express ERG genes when grown in the presence of Vrz.

It was then possible to conclude that biofilm cells upon exposure to antifungal agents overexpress ERG genes, which seems to contribute to an increase in ergosterol concentration in the biofilm matrix.


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P011 - The effect of low molecular weight cationic peptides on Staphylococcus epidermidis adhesion. Eroshenko, Daria (Institute of Ecology and Genetics of Microorganisms UB RAS, Perm, RUS); Lemkina, Larisa (Institute of Ecology and Genetics of Microorganisms UB RAS, Perm, RUS); Morozov, Ilia (Institute of Continuous Media Mechanics UB RAS, Perm, RUS); Korobov, Vladimir (Institute of Ecology and Genetics of Microorganisms UB RAS, Perm, RUS) Introduction: The formation of bacterial film is one of the major causes of the device-associated infections. The key step in the development of such infections is bacterial adhesion to the implant surface. In this regard, the methods to suppress the earliest stages in the biofilm formation, especially the of bacteria-target surface interaction, are becoming very relevant. Aim: Study the effect of the low molecular weight cationic peptides on an S.epidermidis 33 adhesion to hydrophobic and hydrophilic surfaces. Methods: Strain S. epidermidis 33 was cultivated in LB medium on a shaker at 37°C to the mid-log phase and twice washed with 0.14 M NaCl (pH 7.2). Live or heat-killed (20 min) bacterial cells (107 CFU/ml) were suspended in solutions of antibacterial peptides warnerin or hominin (0.5 µg/ml), the bacterial suspensions were incubated into polystyrene or glass Petri dishes statically at 37°C for 30 min. The peptide-free bacterial suspension was used as the control. Peptides were obtained from the culture media of collection strains S. warneri KL-1 and S. hominis KLP-1 according to the method described previously [1]. Another experiment involved the pretreatment of polystyrene surface with the peptide solutions. For this purpose the Petri dishes were supplemented with peptide solutions (64 µg/ml) and stored under the static conditions at 37°C for 60 min. Then liquid was carefully aspirated and the suspension of living log-phase cells of S. epidermidis 33 (107 CFU/ml, 0.14 M NaCl) were added into pretreated polystyrene Petri dishes and incubated statically at 37°C for 30 min. In the control the Petri dishes were pretreated by 0.14 M NaCl. The number of adherent cells was evaluated after their

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Results: The presence of both cationic peptides in the medium caused the lowering in the number of adhered cells to both hydrophobic and hydrophilic surfaces. Meanwhile, in the warnerin presence the cells adhesion was reduced by 25% and 40% on polystyrene and glass, respectively. More pronounced results were found under the hominin action, the cells adhesion was suppressed by 56% and 70%, respectively. Importantly, the used peptide concentrations did not reduce the number of viable cell. Both antibacterial peptides did not affect on the heat-killed bacterial cell sorption. The pretreatment of the polystyrene surface by peptides resulted in a significant decline in the number of adhered cells namely by 49% and 65% after the warnerin or hominin pretreatment, respectively. The number of the viable cell remained constant under the same conditions. Conclusion: The results obtained evidence for that low molecular weight cationic peptides warnerin and hominin reduced the bacterial adhesion to abiotic hydrophobic and hydrophilic surfaces. This work was supported by grants from RFBR (12-04-01431-а and 14-04-00687) andthe UB RAS

(12-P-4-1002, 12-I-4-1033 and 12-M-14-2035).


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1. Korobov V.P., Lemkina L.M., Polyudova T.V., Akimenko V.K. Isolation and Characterization of New Low-Molecular Antibacterial Peptide of the Lantibiotics Family. Microbiology, 2010, 79 (2), 206-215.

P012 - Pseudomonas fluorescens SBW25 reveals AHL-dependent quorum sensing behaviour in culture and biofilms Moshynets, Olena (Institute of Molecular Biology and Genetics of National Academy of Sciences of Ukraine, Kiev, UKR); Rogalsky, Sergey (Institute of Bioorganic Chemistry and Petrochemistry of National Academy of Sciences of Ukraine, Kiev, UKR); Pokholenko, Ianina (Institute of Molecular Biology and Genetics of National Academy of Sciences of Ukraine, Kiev, UKR); Irodov, Dmitry (Institute of Molecular Biology and Genetics of National Academy of Sciences of Ukraine, Kiev, UKR); Karakhim, Sergey (Palladin Institute of Biochemistry of National Academy of Sciences of Ukraine , Kiev, UKR); Spiers, Andrew (The SIMBIOS Centre & School of Science, Engineering and Technology, Abertay University, Dundee, GBR) Pseudomonas fluorescens SBW25 forms a cellulose-based biofilm at the air-liquid interface of liquid microcosms without the apparent involvement of AHL-based quorum signalling typically associated with biofilm-formation in pseudomonads. However, we have recently identified in the SBW25 genome a potential AHL-synthase ( PFLU0008 ), and a AHL-dependent tyrosine phosphatase (PFLU0050), suggesting that AHL synthesis and response are possible in SBW25. In this work, we have investigated the response of SBW25 to N-dodecanoyl-L-homoserine lactone (DHL) by looking at siderophore production and biofilm-formation. Our results demonstrate that siderophore expression, biofilm thickness, eDNA and eRNA levels within biofilms are increased by exogenous DHL. Furthermore, although biofilm-formation appeared to be enhanced by DHL, cellulose expression within biofilms seems to be repressed. In P. aeruginosa, TpbA negatively regulates the diguanylate cyclase (DGC) TpbB to lower c-di-GMP levels; although SBW25 does not have a TpbB homologue, we hypothesize that PFLU0050 negatively regulates an unidentified DGC to lower c-di-GMP levels and reduce cellulose expression. One possible target is WspR, responsible for the over-production of cellulose in the Wrinkly Spreader biofilm. This work is the first to provide experimental proof that SBW25 is capable of responding to quorum signals and to suggest that cellulose expression during biofilm-formation may also be AHL-regulated.

P013 - Exopolymer diversity and the role of levan in Bacillus subtilis biofilms Dogsa, Iztok (University of Ljubljana, Biotechnical Faculty, Dept. Food Science and Technology,Chair of Microbiology, Ljubljana, SVN); Brloznik, Mojca (University of Ljubljana, Biotechnical Faculty, Dept. Food Science and Technology, Chair of Microbiology, Ljubljana, SVN); Mandic-Mulec, Ines (University of Ljubljana, Biotechnical Faculty, Dept. Food Science and Technology, Chair of Microbiology, Ljubljana, SVN); Stopar, David (University of Ljubljana, Biotechnical Faculty, Dept. Food Science and Technology, Chair of Microbiology, Ljubljana, SVN) Exopolymeric substances (EPS) are important for biofilm formation and their chemical composition may influence biofilm properties. To explore these relationships the chemical composition of EPS isolated from Bacillus subtilis NCIB 3610 biofilms grown either in sucrose-rich (SYM) or sucrose-poor (MSgg and Czapek) media was studied. Marked differences in


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composition of EPS polymers isolated from biofilms or from spent media below the biofilms were observed. The polysaccharide levan dominated the EPS of SYM grown biofilms, while EPS from biofilms grown in sucrose-poor media contained significant amounts of proteins and DNA in addition to polysaccharides. Very large polymers (Mw>2000 kDa) were found only in biofilms, while smaller polymers (Mw<200 kD) dominated in spent media. Biofilms of the eps knockout mutants were significantly thinner than those of the tasA knockout. The thickness of biofilms of defective tasA and eps mutants was partially compensated in the sucrose-rich SYM medium. Consistently, sucrose supplementation of Czapek and MSgg media induced the production of levan and increased thickness and stability of biofilms compared to non-supplemented controls. This study shows for the first time that levan, although not essential for biofilm formation, can be a structural and stabilizing component of B. subtilis floating biofilms.


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P014 - Cell wall deformation of different Staphylococcus aureus strains Van der Mei, Henny (University Medical Center Groningen, Groningen) Adhesion to a surface is a survival mechanism for bacteria, since nutrients have the tendency to accumulate at surfaces. Bacterial adhesion is generally recognized as the first step in biofilm formation. For the human host, the ability of a bacterium to adhere is a definite virulence factor, because it stimulates the organism to produce extracellular polymeric substances (EPS) through which they embed themselves in a protective matrix. Bacterial adhesion to surfaces is accompanied by cell-wall deformation that may extend to the lipid membrane with an impact on the antimicrobial susceptibility of the organisms. Cell-wall deformation upon adhesion is difficult to measure, except for ?pbp4 mutants, deficient in peptidoglycan cross-linking. Here we explore surface-enhanced-fluorescence to measure cell-wall deformation of staphylococci adhering on gold surfaces. Adhesion-related-fluorescence-enhancement depended on the distance of the bacteria to the surface and the residence-time of the adhering bacteria. A model is forwarded based on the adhesion-related-fluorescence-enhancement of green-fluorescent microspheres, through which the distance to the surface and cell-wall deformation of adhering bacteria can be calculated from their residence-time dependent adhesion-related-fluorescence-enhancement. The distances between adhering bacteria and a surface, including condensation of their EPS layer, decreased up to 60 min after adhesion, followed by cell-wall deformation. Cell-wall deformation was independent on the integrity of the EPS layer and proceeded fastest for a ?pbp4 strain. Cell wall deformations arising from the measurement of adhesion-related fluorescence enhancement could be validated with atomic force microscopic (AFM) measurements of cell wall deformation, provided care was taken to carefully match the conditions under which the AFM experiments are carried out with the naturally occurring adhesion forces. As an important advantage of using surface-enhanced-fluorescence, the number of bacteria involved in a single analysis is much larger than can be obtained using more microscopic methods, like AFM.

P015 - A modeling and simulation study of the interaction between biofilms and suspended bacteria in a nitrifying biofilm reactor Masic, Alma (Eawag, Swiss Federal Institute of Aquatic Science and Technology, Duebendorf, CHE); Eberl, Hermann (Biophysics Interdepartmental Program and Dept. Mathematics and Statistics, University of Guelph , Guelph, ON, CAN) Biofilm models already have a long history in the fields of wastewater engineering and mathematical biology, where they have mostly been used to increase process understanding, but also for design and optimization of wastewater processes. In their seminal paper in 1986, Wanner and Gujer introduced the one-dimensional continuous dynamic multiple substrate multiple species biofilm model, which has since become a standard model, particularly in wastewater engineering. On the other hand, the Activated Sludge Models (ASMs) have dominated the modeling of suspended bacteria in the form of activated sludge. Traditional biofilm models typically do not take into account the existence and potential contribution of any suspended biomass, which is always present due to detachment and (re-)attachment of biomass, thereby assuming that such biomass is negligible.


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Early attempts were made by Freter et al. to model a chemostat with both sessile and suspended growth of bacteria. Lately, models that combine biofilms with activated sludge have arisen motivated by real-life processes in wastewater treatment, which are designed with the contribution of both biomass types in mind. However, the presence of suspended biomass in a biofilm reactor can instead be considered a mere side-effect of the process design. In this work, we study the interactions between biofilms and suspended bacteria in a continuous-flow stirred tank biofilm reactor with two-step nitrification as the selected biological process by presenting a mathematical model that couples the reactor mass balance for the suspended populations and the substrates with a Wanner-Gujer type biofilm model. The exchange of biomass through detachment and attachment is not yet fully understood, wherefore these processes are often approximated by relatively simple expressions. The aims of the study are to investigate the effects on the model performance by the interactions between biofilm and suspended biomass through detachment and (re-)attachment. In consequence, we investigate whether suspended biomass should be taken into account in a biofilm model, and if so under which circumstances, or if the contribution of the suspended biomass indeed can be neglected. The structural and parametrical complexity of the model only allows extensive numerical simulations instead of rigorous analysis of its dynamics. Our results show that the intermediate compounds and processes in the nitrification model, such as nitrite concentration, are notably affected by changes in the coefficient in the detachment rate, while changes in the attachment rate show no pronounced effects. Further results allow us to conclude that suspended biomass should be taken into account explicitly in a biofilm model if the objectives of the study include any of the intermediate steps and compounds in the nitrification process. On the other hand, overall reactor performance criteria do not require a more complex model. In general, variables that are primarily involved in the second step of nitrification are more susceptible to changes in attachment and detachment rates.

P016 - Microbiology's N-body problem: How should we estimate the rate of metabolite exchange in spatially structured populations? Clegg, Robert (University of Birmingham, Birmingham, GBR); Dyson, Rosemary (University of Birmingham, Birmingham, AUT); Kreft, Jan-Ulrich (University of Birmingham, Birmingham, GBR) No microbe is an island: exchange of solutes between microbes is crucial to nutrient cycles in both natural and man-made environments. However, directly measuring the rate of exchange within observed populations is difficult experimentally, and detailed modelling requires knowledge of all positions and kinetics.

As solutes are often transported by diffusion, reducing the physical distance between partners can greatly increase the rate of exchange and so also increase the productivity of the communities. E.g., microbial degradation of organic matter to methane often requires rapid transfer of hydrogen or formate from producers to consumers.

Ecologists and engineers interested in this problem typically estimate the rate of solute exchange between groups using the average distance between a cell of one type and its nearest neighbour of the other. This statistic is a valid estimator in the detection and classification of


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spatial patterns, but its reliability in estimation of exchange rate is untested. The uncertainty in estimating the rate of exchange affects many topics in microbial ecology and biochemical engineering: our computational approach seeks to correct this by determining the most reliable statistical estimator in a scientifically rigorous manner.

P017 - Validation of Standard Methods for Growing and Efficacy Testing Biofilms Goeres, Darla (Abo Akademi University/Center for Biofilm Engineering, Turku) Biofilm research has matured into an established academic field of study. Through the diligent combined efforts of individuals from academia and industry, regulatory agencies are beginning

-biofilm formulations and treatment strategies. Validated biofilm standard test methods are required before regulatory agencies will consider registering anti-biofilm products. The Standardized Biofilm Methods Laboratory (SBML) at the Center for Biofilm Engineering has been working on the development and validation of standard biofilm test methods for over 10 years. This presentation will describe the strategy the SBML has adopted for the development of biofilm test methods, provide an overview of the five methods which have been approved by ASTM and show the results from two multi-laboratory collaborative studies.

est Method for Testing Disinfectant Efficacy against Pseudomonas aeruginosa standardized biofilm disinfectant efficacy test method. Initial ruggedness testing of the MBEC method suggested the assay was rugged (i.e., insensitive) to small changes to the protocol with respect to four parameters: incubation time of the bacteria, treatment temperature, sonication duration, and sonication power. Based upon the favorable ruggedness test results, the method underwent an 8-laboratory collaborative study. For this study 8 concentrations of 3 disinfectants (a non-chlorine oxidizer, a phenolic, and a quaternary ammonium compound) were applied to biofilms following ASTM Method E2799. The analysis of the data showed the repeatability and reproducibility of the untreated control biofilms were acceptably small at 0.33 and 0.67 Log10(CFU/cm2), respectively. The repeatability standard deviations (SD) of the biofilm log reductions after application of the 24 concentration and disinfectant combinations ranged from 0.22 to 1.61, and the reproducibility SDs ranged from 0.27 to 1.70. Finally, the test method was statistically significantly responsive to the increasing treatment concentrations (p-value < 0.00005) for the 3 disinfectants tested in the study.

Pseudomonas aeruginosa was the second approved standardized biofilm disinfectant efficacy test method. Method E2871

determining the efficacy of liquid disinfectants against biofilm bacteria. A 6- laboratory collaborative study was completed using three classes of disinfectants (sodium hypochlorite oxidizer, a phenolic, and quaternary-alcohol compound) at two concentrations (presumed high and low efficacy) against a Pseudomonas aeruginosa ATCC 15442 biofilm grown on borosilicate glass coupons. The repeatability and reproducibility SDs for the untreated control coupons were acceptably small at 0.22 and 0.24 Log10(CFU/cm2), respectively. The repeatability SDs for the log reductions ranged from 0.58 to 1.45, and the reproducibility SDs ranged from 0.89 to 1.67. The method was statistically significantly responsive to the increasing efficacy level for the


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oxidizer (p-value = 0.002) and quaternary-alcohol compound (p-value = 0.013), but not the phenol (p-value = 0.170).


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Session 3: Unveiling the unseen: new and emerging technologies in biofilm research

P018 - Probing bacterial adhesion at the single-cell level Zeng, Guanghong (iNANO, Aarhus University, Aarhus, DNK); Müller, Torsten (JPK Instruments AG, Berlin, GER); Meyer, Rikke L. (iNANO, Aarhus University, Aarhus, DNK) Bacteria initiate attachment to surfaces with the aid of different extracellular proteins and polymeric adhesins. To quantitatively analyse the cell-cell and cell-surface interactions provided by bacterial adhesins, it is essential to go down to single cell level where cell-to-cell variation can be considered. We have developed a simple and versatile method to make single-cell bacterial probes for measuring single cell adhesion by force spectroscopy using atomic force microscopy (AFM). A single-cell probe was readily made by picking up a bacterial cell from a glass surface by approaching a tipless AFM cantilever coated with the commercial cell adhesive CellTakTM. We applied the method to study adhesion of living cells to abiotic surfaces at the single-cell level.

Immobilisation of single bacterial cells to the cantilever was stable for several hours, and viability was confirmed by Live/Dead staining and fluorescence microscopy. This method for preparing a single-cell AFM probe offers control of the cell immobilization, and therefore holds advantages over the commonly used approach where multiple cells are immobilized at random positions by submerging the cantilever in a bacterial suspension. The method provides a general platform for investigating single cell interactions of bacteria with different surfaces and other cells by AFM force spectroscopy, thus improving our understanding of the mechanisms of bacterial attachment.

To explore the influence of biological and physicochemical parameters on the adhesion force, we explored the bond formation and adhesive strength of four different bacterial strains towards three abiotic substrates with variable hydrophobicity and surface roughness. The adhesion force and final rupture length were dependent on bacterial strains, surfaces properties, and time of contact. Staphylococcus xylosus DSM 20266 and Staphylococcus epidermidis DSM 20044 showed much higher adhesion forces than Pseudomonas fluorescens AH1, but bond strengthening by P. fluorescens (2 s) was faster than for the staphylococci (10 s) . Escherichia coli DSM 429, which was the only strain unable to form biofilm,showed almost no adhesion to any surface. The differences between staphylococci and P. fluorescens in adhesion pattern reflect their differences in the composition of extracellular adhesins. Both adhesion force and rupture length were significantly smaller on mica compared to glass. Staphylococci adhere stronger on fresh glass than on hydrophilic glass, while the weaker adhesion by P. fluorescens was similar on both types of glass. These results confirmed the importance of surface hydrophobicity in bacterial adhesion.

This study has demonstrated that single-cell force spectroscopy allows quantitative measurement of the adhesion of different bacteria toward surfaces of different physicochemical properties. We expect that this method can be combined with bacterial mutation or enzymatic treatment to knock off specific surface components, thus providing more insights into the contribution of various surfaces components in bacterial initial attachment.


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P019 - Formation and quantification of biofilm for industrial anti-biofilm product evaluation/development Stiefel, Philipp (Empa, St. Gallen, CHE); Altenried, Stefanie (Empa, St. Gallen, CHE); Ren, Qun (Empa, St. Gallen, CHE); Thöny-Meyer, Linda (Empa, St. Gallen, CHE) Up to now, there are no standardized evaluation methods for biofilm, probably due to the complexity of various biofilm structures and compositions. Different methods have been reported for biofilm formation and assessment. Generally, the kind of microorganism and the conditions of biofilm formation are selected based on the requirement of the developed "anti-biofilm" products. Furthermore, appropriate biofilm quantification methods have to be chosen, including detection of viable cells, total cells, total biomass or specific compounds of the extracellular polymeric substances (EPS) such as polysaccharides and proteins. In this study, we systematically assessed and compared several reported methods for the quantification of biofilms formed in 96-well microtiter plates. Pseudomonas aeruginosa and Staphylococcus aureus were used as the model test organisms. To study which method is suitable for biofilm cleaning and/or for bacterial killing tests, the formed biofilm was treated with cleaning detergents or biocides. The cleaning and killing effects of the reagents against biofilm were evaluated by the mentioned assays. The advantages and disadvantages of the different methods will be discussed in detail.

P020 - The choice of growth medium affects Deinococcus geothermalis biofilm morphology and EPS composition Frösler, Jan (University of Duisburg-Essen, Essen, GER); Wingender, Jost (University of Duisburg-Essen, Essen, GER); Flemming, Hans-Curt (University of Duisburg-Essen, Essen, GER) The bacterium Deinococcus geothermalis is considered a primary biofilm former. It is able to adhere to and multiply on various submerged abiotic surfaces such as polystyrene, stainless steel, glass, or polyethylene, where it forms tenacious biofilms [1,2]. As reported for D. geothermalis strain E50051, adhesion does not seem to be mediated by either capsules or slimes, but rather by extracellular glycosylated proteins resembling adhesion threads [3]. Little is known about the EPS and other extracellular features of the type strain DSM 11300.

In this study, biofilms of D. geothermalis DSM 113000 were grown on cellulose mixed esters membranes (pore size 0.45 µm) placed on solid R2A medium or tryptic soy agar (TSA), respectively. After incubation at 45 °C for 2 days, water-unsaturated biofilms were obtained, which were pink (R2A) or orange (TSA) pigmented.

The EPS of both biofilm types were isolated using a cation-exchange resin (Dowex) and analysed for their biochemical composition. The presence of polysaccharides, proteins and extracellular DNA in the EPS of D. geothermalis biofilms was confirmed. EPS yield per cm2 was significantly higher for TSA biofilms compared with R2A biofilms (proteins: 9.7-fold, polysaccharides: 2.1-fold, eDNA: 5.3-fold). On R2A, polysaccharides were found to be the dominant fraction of EPS, with a protein/polysaccharide/eDNA ratio of 10:16:1, whereas proteins dominated in the EPS of biofilms grown on TSA (21:7:1).


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Scanning electron microscopy revealed the presence of an amorphous EPS matrix covering the TSA-grown biofilms. Such a matrix was not evident on the surface of biofilms grown on R2A. The adhesion threads reported for strain E50051 [3] were found in neither of the two biofilm types investigated here, but may be present in deeper regions within the biofilm. Preliminary evidence for this was obtained by using fluorescently-labelled lectins in combination with confocal laser scanning microscopy. Lectin ACA of Amaranthus caudatus, previously shown to bind to the adhesion threads of D. geothermalis E50051 [4], was reactive to the cell surface of some cells below the biofilm surface. In accord with observations made by Peltola and colleagues [4], lectin DBA of Dolichos biflorus interacted with extracellular, patchy structures present in the biofilm core.

Both biofilm types showed strong adhesive properties: Cell-to-substrate adhesion was pronounced in R2A biofilms. Detachment of these biofilms from the cellulose membrane by mechanical force (e.g., scraping, shaking, sonication) was only partially successful. Detachment was easier for TSA biofilms; however, this type exhibited strong cell-to-cell interactions, thus forming aggregates which withstood dispersion by a variety of treatments (e.g. mixing, sonication, heat, surfactants), but could be dispersed by the addition of the proteolytic enzyme proteinase K. This indicates the involvement of proteins as an important structural component of the EPS.

References: [1] Kolari M., Nuutinen J., Salkinoja-Salonen M.S. (2001): Mechanisms of biofilm formation in paper machine by Bacillus species: the role of Deinococcus geothermalis. J Ind Microbiol Biotechnol 27, 343-351. [2] Kolari M., Schmidt U., Kuismanen E., Salkinoja-Salonen M.S. (2002): Firm but slippery attachment of Deinococcus geothermalis. J Bacteriol 184, 2473-2480. [3] Saarimaa C., Peltola M., Raulio M., Neu T.R., Salkinoja-Salonen M.S., Neubauer P. (2006): Characterization of adhesion threads of Deinococcus geothermalis as type IV pili. J Bacteriol 188, 7016-7021. [4] Peltola M., Neu T.R., Raulio M., Kolari M., Salkinoja-Salonen M.S. (2008): Architecture of Deinococcus geothermalis biofilms on glass and steel: a lectin study. Environ Microbiol 10, 1752-1759.

P021 - High Content Screening (HCS) Confocal imaging: new perspectives in biofilm research Deschamps, Julien (INRA, Massy, FRA); Herry, Jean-Marie (INRA, Massy, FRA); Canette, Alexis (inra, Massy, FRA); Trotier, Elsa (INRA, Massy, FRA); Piard, Jean-Christophe (inra, Massy, FRA); Kim, Se yeon (INRA, Massy, FRA); sanchez-vizuette, pilar (inra, Massy, FRA); bridier, arnaud (irstea, antony, FRA); Briandet, Romain (INRA, Massy, FRA) The functional properties of biofilms are intimately related to their spatial architecture. Their resistance to antimicrobial agents due to diffusion and/or reaction delays is a telling example of the importance of the matrix shape and the three-dimensional organization of cells. Moreover, microorganisms within biofilms respond to heterogeneous local environmental conditions because of the chemical gradients that are generated by the three-dimensional structure of these communities. Thus, the structural heterogeneity of biofilms leads to different gene


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expression patterns and specific physiological activities for the cells within the structure, resulting in the emergence of novel and global community functions. Structural data are therefore of prime importance to better understand the complex behavioral and survival strategies of biofilms and ultimately to improve the control of these biological structures.

Confocal laser scanning microscopy (CLSM) is one of the tools most widely used at present to study biofilm structure because it enables the direct in situ and non-destructive investigation of native multicellular structures using specific fluorescent markers.

The emergence of fully automated High Screening Content (HCS) systems, associated with large scale image quantification, radically amplifies the flow of available biofilm structural data. We will present in this poster examples of new types of information that are accessible with these new pipelines, such as large scale biofilms structural dynamics, 4D genes expression patterns, spatial interaction patterns between species and cohesion measurements. Meta-analysis of the direct and indirect variates generates by HCS-CLSM will participate to a better biological understanding of biofilms traits.

Acknowledgement: Part of the presented data were generated in the framework of the FP7 SUSCLEAN project.

P022 - Light sheet fluorescence correlation spectroscopy for developmental biology of model biofilms Kandaswamy, Kumaravel (Singapore Centre on Environmental Life Sciences Engineering (SCELSE), Singapore, SGP); Karampatzakis, Andreas (NUS Centre for Bioimaging Sciences (CBIS), Singapore, SGP); Wohland, Thorsten (NUS Centre for Bioimaging Sciences (CBIS), Singapore, SGP); Rice, Scott A. (Singapore Centre on Environmental Life Sciences Engineering (SCELSE), Singapore, SGP); Cohen, Yehuda (Singapore Centre on Environmental Life Sciences Engineering (SCELSE), Singapore, SGP) Assessment of spatiotemporal dynamics of cells and physiochemical parameters during the course of biofilm developmental cycle requires highly sensitive imaging tools, which will enable us to rapidly visualize and quantify the cell dynamics at various spatial scales in a three-dimension (3D). In this study we present a single molecule detection technique using Single Plane Illumination Microscopy (SPIM) with camera based fluorescence correlation spectroscopy (FCS). SPIM-FCS technique rapidly address the spatiotemporal dynamics of single cells during the course of biofilm development cycle ranging from initial attachment to dispersal. We also present our proof of concept results in implementing SPIM-FCS to measure 3D diffusion in a Pseudomonas aeruginosa biofilm. This technique allowed to measure diffusion in biofilms

SPIM-FCS serves as a promising tool to uncover dynamic cellular events as correlated to the diffusion processes inside the biofilm and thereby providing new levels of understanding biofilm development.

P023 - Innovative imaging techniques for analysis of natural biofilms on ceramic filters Matthies, Kerstin (Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen, GER); Brenner-Weiß, Gerald (Institute of Functional Interfaces, Department of Interface Microbiology,


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Eggenstein-Leopoldshafen, GER); Guthausen, Gisela (Institute for Mechanical Process Engineering and Mechanics, Department for Process Machines, Karlsruhe, GER); Diedel, Ralf (Forschungsinstitut für Anorganische Werkstoffe Glas / Keramik GmbH, Höhr-Grenzhausen, GER); Obst, Ursula (Institute of Functional Interfaces, Department of Interface Microbiology, Eggenstein-Leopoldshafen, GER)

Especially in developing countries people suffer from waterborne diseases. Point-of-use (POU) water treatment often is the only probable way to get safe drinking water. One of the most effective and appropriate technologies for drinking water treatment in poor regions is the use of ceramic water filters like thWHO. The filters are mostly coated with silver (colloidal or in form or silver nitrate) for an additional bactericidal effect. These filters are already manufactured and used in many developing countries (e.g. Cambodia, Indonesia, Ghana and Nicaragua). They show good disinfection capacities. However, the flow rate tends to decrease after time. One reason is clogging due to inorganic material from the raw water. Another reason might be a biofilm formation in spite of the silver layer. Experiments proved that silver is leaching from these filters in relatively high dosages. It is known that silver does not only kill bacteria but also has toxic effects for humans. Therefore, it might be useful to develop a filter without adding toxic substances. In a BMBF funded project in Southern Java, Indonesia, we are developing a recipe to produce such filters from locally available material in local potteries. To analyse the effect of silver on a potential biofilm formation, we conducted a study comparing biofilm formation on untreated ceramic material and ceramic material coated with silver nitrate.

Werkstoffe Glas / Keramik -Grenzhausen, Germany). In a set of laboratory experiments, we let a natural biofilm population that was previously isolated from a small river, grow on the ceramic material. Afterwards, the biofilm was analysed using innovative imaging techniques (e.g. MRI, Micro CT). In the poster, we will present techniques used for analysis and findings of the study.

P024 - Novel impedimetric senzor for detection of Staphylococcus epidermidis biofilms Jakubec, Martin (Dairy research institute Ltd., Praha 6, CZE); Kadlec, Robert (Dairy research institute Ltd., Praha 6, CZE) The ability of Staphylococcus epidermidis to form biofilm and its persistence presents serious clinical problems e.g. colonization of catheters and cannulas. Early detection of adherence and biofilm formation could be crucial step in prevention of deterioration of patient's health. We have tested a novel approach based on measurement of impedance spectra using e-plate (microtiter plate with gold interdigitated electrodes) for detection of biofilm formation. Forty strains of Staphylococcus epidermidis clinical isolates were used. These strains were characterized as biofilm negative or as biofilm positive by presence of ica locus and microtiter plate test (using crystal violet as detection agent). Based on principal component analysis we have shown that biofilm positive and biofilm negative strains have different impedance spectra and we have been able to classify biofilm positivity or negativity with 90% success rate. This


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study was supported by COST project LD14123 and by Ministry of Agriculture of the Czech Republic QJ1310258.

P025 - Continuous investigation of biofilm formation on micro structured surfaces Mulansky, Susan (Dresden University of Technology, Dresden, GER); Bley, Thomas (Dresden University of Technology , Dresden, GER); Boschke, Elke (Dresden University of Technology, Dresden, GER) Anti-adhesive surfaces are of importance in areas, such as in food industry, marine science, engineering and public health. Nature provides many examples of mechanisms to control fouling. They can be copied (biomimetic) or tailored (bio-inspired) to solve problems with fouling on artificial structures [1]. In order to minimize the potential for biofouling it is important to understand the mechanisms of the first stages of biofilm formation. To study these mechanisms and to characterize anti-adhesive materials and surfaces a modular flow cell system with a parallel plate flow chamber has been established [3]. It is designed and used for online observation of biofilm formation by a fluorescence microscope in continuous flow on opaque surfaces. The laminar flow ensures the maintenance of highly controlled conditions for biofilm growth. The flow cell can be easily and rapidly adapted to meet different requirements. An approach to reduce initial surface contamination, which may lead to subsequent reduction in biofilm formation, is to generate structures that reduce the number of attachment points of the microorganisms to the surface. The anti-adhesive properties are related to the dimensions of the tested organism. According to Scardino et al. biofilm forming organisms that are larger than the dimension of the generated structure will have reduced adhesion strength due to fewer attachment points, micro-refuges and protection from hydrodynamic forces [2]. We present results of experiments aimed at studying primary adhesion the first step of biofilm formation on circular structures with a scale smaller than 500 nm produced by electrochemical precision etching. As a test organism a gfp labeled E. coli strain was used.

Literature [1] A. J. Scardino, and R. de Nys, Mini review: Biomimetic models and bioinspired surfaces for fouling control, Biofouling, vol. 27, no. 1, 2011/01/01, 2010, pp. 73-86. [2] A. J. Scardino, E. Harvey, and R. De Nys, Testing attachment point theory: diatom attachment on microtextured polyimide biomimics, Biofouling, vol. 22, no. 1-2, 2006, pp. 55-60. [3] K. Wagner, S. Friedrich, C. Stang et al., Initial phases of microbial biofilm formation on opaque, innovative anti-adhesive surfaces using a modular microfluidic system, Engineering in Life Sciences, 2013, pp. 1-9.

P026 - Label-free monitoring of the efficacy and the impact of multiple agents on bacterial biofilm formation through Differential Digital Holographic Microscopy. Barbau, Jérémie (OVIZIO Imaging Systems NV/SA, Brussels, BEL); Henneghien, Joël (OVIZIO Imaging Systems NV/SA, Brussels, BEL); Haumont, Denis (Ovizio Imaging Systems, Brussels, BEL); Jooris, Serge (OVIZIO Imaging Systems, Brussels, BEL); Godissart, Patricia (GlaxoSmithKline, Rixensart, BEL); Ysebaert, Carine (GlaxoSmithKline, Rixensart, BEL); Neou,


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Sereiwaddhana (GlaxoSmithKline, Rixensart, BEL); Hermand, Philippe (GlaxoSmithKline, Rixensart, BEL) Biofilm formation is an alternative way for organisms to grow and interact together in order to improve their survival in specific environments. These matrix-enclosed accretions are antibiotic and antibody-resistant, which makes them difficult to break up without a mechanical action. Additionally, aggregates of microorganisms are involved in a wide variety of infections, such as urinary track infection, middle-ear or lung infections.

The evaluation of new treatment is performed by measuring the biofilm coverage on a multi-well containing various concentrations of agents directed against the biofilm. The device used is an automated inverted fluorescence microscope: the AxioVert (Zeiss). This system has a 3 axis motorized stage that allows the acquisition of a complete well area, where the biofilm-forming bacteria are labeled with a fluorescent probe in order to detect them. The system is currently semi-automated and an operator is required for the definition of the scanning area.

oLine inverted DDHM microscope and able to automatically scan a multi-well plate containing biofilms. The DDHM technology (1) provides outstanding contrast and high spatial resolution (compared to classic Digital Holographic Microscopy), (2) allows optical refocusing post-acquisition (improving acquisition speed and reliability) and (3) provides quantitative phase information.

In DDHM, the well surface is scanned and the confluence of the biofilm is computed on threshold-

-invasive, easier and also faster assay development. The acquisition is automated and takes less than 3 minutes per 8 wells, the data analysis requires 10 minutes in total, which is comparable to the AxioVert times.

A side-by-side study of both technologies has demonstrated that the DDHM technology is able to properly measure the width and the height of the biofilm compared to the measurement of the coverage of the biofilm with AxioVert. In the light of these observations, it seems that the oLine from OVIZIO is a cost-effective and reliable alternative for an automated assessment of biofilm disruption.

Recent developments at OVIZIO enabled the possibility to combine both methods: biofilm (structure) monitoring through acquisition of the quantitative phase information and detection of the (living) bacteria through fluorescence detection. Furthermore, given the large depth of view and the non-invasive approach, the DDHM methodology offers also opportunities for time-lapse experiments.

P027 - GSDIM nanoscopy - a new approach for assessment of adhered microorganisms Neu, Thomas (Helmholtz Center for Environmental Research - UFZ, Magdeburg, GER); Kuhlicke, Ute (Helmholtz Center for Environmental Research - UFZ, Magdeburg, GER) Diffraction limited widefield microscopy and confocal laser scanning microscopy (CLSM) have a resolution limit of ≈ 250 nm. In the meantime several techniques are commercially available


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achieving an improved lateral resolution. The three techniques comprise structured illumination microscopy (SIM) ≈ 120 nm, stimulated emission depletion microscopy (STED) ≈ 70 nm and blink microscopy (many acronyms) ≈ 30 nm. We used one version of blink microscopy, ground state depletion with individual molecule return (GSDIM), for imaging adhered bacteria. GSDIM is based on the detection of individual fluorochromes whose blinking behavior is fine-tuned by two lasers and a chemical cocktail. As imaging can be done in the TRIF or Epi mode only, 3d resolution is limited even with new systems having a certain 3d capability. Recording of blinking signals as large diffraction-limited spots over time together with calculation of the spot center reveals a point-like pattern making up the final image. In this study the application of the technique for assessment of adhered microorganisms is demonstrated. Finally the pro and cons of GSDIM against the other nanoscopy techniques (SIM, STED) and conventional CLSM are compared.

P028 - Indoor stream mesocosms: Versatile tools in biofilm ecology Norf, Helge (Helmholtz-Centre for Environmental Research - UFZ, Magdeburg, GER); Arndt, Hartmut (University of Cologne, Cologne, GER); Weitere, Markus (Helmholtz-Centre for Environmental Research - UFZ, Magdeburg, GER) Microbial biofilms are important components of running water ecosystems. Hence, empirical analysis of the factors that regulate both the assemblage structure and ecological functioning of microbial biofilms is an important pillar of current limnological research. Stream mesocosms represent versatile tools in biofilm ecology; they allow building a bridge between the controllability and manipulability of laboratory experiments and the naturalness and complexity of field studies. Indoor stream mesocosms are operated as bypasses to surface waters via pumping devices, which is the used to continually supply ecological experiments. This facilitates experimental manipulation of selected environmental factors (e.g. light regime, nutrient load, flow velocity) while the ambient hydrochemical background of the field water remains unchanged. Different types of indoor stream mesocosms as well as their applications will be presented. Particular attention will be paid to first results obtained in experiments conducted in MOBICOS (mobile aquatic mesocosm), a new research infrastructure of six container-based indoor stream mesocosms exposed along a land use gradient within the Harz mountains (Saxony-Anhalt, Germany).

P029 - Bacterial emboli: novel biofilm-like structures formed during phytopathogenesis Gogolev, Yuriy (Kazan Institute of Biochemistry and Biophysics RAS, Kazan, RUS); Daminova, Amina (Kazan Institute of Biochemistry and Biophysics RAS, Kazan, RUS); Gorshkov, Vlsdimir (Kazan Institute of Biochemistry and Biophysics RAS, Kazan, RUS); Ageeva, Marina (Kazan Institute of Biochemistry and Biophysics RAS, Kazan, RUS); Petrova, Olga (Kazan Institute of Biochemistry and Biophysics RAS, Kazan, RUS); Mikshina, Polina (Kazan Institute of Biochemistry and Biophysics RAS, Kazan, RUS); Tarasova, Nadezhda (Kazan Institute of Biochemistry and Biophysics RAS, Kazan, RUS); Salnikov, Vadim (Kazan Institute of Biochemistry and Biophysics RAS, Kazan, RUS); Gubaev, Rim (Kazan Institute of Biochemistry and Biophysics RAS, Kazan, RUS); Gogoleva, Natalia (Kazan Institute of Biochemistry and Biophysics RAS, Kazan, RUS); Burygin, Gennadiy (IBPPM RAS, Saratov, RUS)


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Microbes in natural ecosystems exist as complex communities forming microcolonies, biofilms and special phenotypes adapted to particular ecological niches. For plant pathogenic bacteria their host-plants, which are composed of different cells and tissues varying in terms of physical, chemical and phytoimmune properties, form heterogenous and dynamic systems. In our study on the example of Pectobacterium atrosepticum SCRI1043 (Pba) it was shown that such dynamism serves as a driving force, leading to the dissociation of microbial population. Among various microbial phenotypes formed in planta, we have characterized the one that forms special biofilm-

spatial orientation, and have a peculiar way of formation. It is not related to the attachment of microbial cells to the plant cell wall, as typical for biofilms of xylem-existing bacteria, but is followed by the formation of gel in the vessels and pilus-like structures on the bacterial surface. The gel is represented by partially degraded and modified plant cell wall polysaccharides, namely 100 kDa fragments of rhamnogalacturonan I, containing hydrophobic compounds (presumably, phenolics) that may provide cross-linking between carbohydrates. Formation of the gel enables bacteria to anchor in highly diffusive environment (lumen of the xylem vessel) and to form a "dam".

Additionally, Pba exopolysaccharides that were described in our study were visualised within the bacterial emboli using immunocytochemistry, suggesting their role in formation of these structures. The resultant bacterial emboli represent very tight structures that do not have any channels or cavities and totally occlude xylem vessels. Such organization is likely to be necessary for providing bacterial downward vascular translocation that was monitored in our experiments and is suggested to be crucial for vertical and horizontal bacterial transmission.

Considerable problem in the study of bacterial emboli - the difficulty to isolate them from the plant tissues and differentiate from the rest of the population - was overcome by using laser microdissection. This permitted to unravel the differences in expression level of several genes in bacterial emboli and in bacteria that colonize parenchyma. Our investigation shows that bacterial emboli are a new type of organized microbial community and their formation represents a crucial step in colonization of the plant by Pba.

References: Gorshkov V, Daminova A, Ageeva M, Petrova O, Gogoleva N, Tarasova N, Gogolev Y. Protoplasma. 2013. Article in Press DOI: 10.1007/s00709-013-0546-3.

Acknowledgments: This study was supported by the Russian Foundation for Basic Research, research project nos. 14-04-01750 and 14-04-01828.

P030 - Determination of ATP as an indicator of the physiological state of biofilm cells of nitrile utilizing bacteria Maksimova, Yuliya (Institute of Ecology and Genetics of Microorganisms UB RAS, Perm, RUS); Maksimov, Aleksandr (Institute of Ecology and Genetics of Microorganisms UB RAS, Perm, RUS); Oleneva, Mariya (Perm State National Research University, Perm, RUS); Demakov, Vitalii (Institute of Ecology and Genetics of Microorganisms UB RAS, Perm, RUS)


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Changing of the ATP concentration in a cell indicates a stressful state of microorganisms. The assessment of the impact of various environmental factors, including the toxic substances, as well as the stages of biofilm formation on the cell energetic state can be achieved by determining the intracellular ATP by bioluminescent method. The results have shown that adhesion leads to a decrease of intracellular ATP by an order of magnitude, probably due to the energy costs for the initiation of biofilm formation, which is accompanied by metabolic rearrangements.

To determine the viability of adherent cells , samples were transferred to fresh culture medium and the ATP content was determined after 2, 24 and 48 hours. It was demonstrated that the ATP content per cell after 2 hours in fresh culture medium was reduced even compared with the intracellular ATP after adhesion. However, in fresh medium intracellular ATP of 24 and 48-hour biofilm began to increase, reflecting the possible accumulation of biomass, confirming the viability of adherent cells.

The effect of toxic substrates and products of nitrile hydrolysis (acrylonitrile, acrylamide, acrylic acid) on the intracellular ATP content from biofilms was studied. Under the 20-min exposure of 1.7M acrylamide on Rhodococcus planktonic cells the intracellular ATP content decreased in more than half of the control, whereas the ATP concentration in biofilm cells significantly increased. Acrylonitrile in the amount of 1.3M caused 50% reduction in ATP content in Pseudomonas planktonic cells, whereas in biofilm cells this was not changed as compared to the control. Thus, biofilms of nitrile utilizing bacteria are more resistant to toxic substances and have higher adaptive capacity than planktonic cells. The work was supported by the RFBR grant 13-04-96050 and a grant of the program in Molecular and Cell Biology of the Russian Academy of Sciences.

P031 - Application of nucleic acid mimics in fluorescence in situ hybridization for the visualization of naturally-occurring biofilms with minimal disruption Fontenete, Silvia (LEPABE, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto); Azevedo, Andreia S. (LEPABE, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto); Rocha, Rui (LEPABE, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto); Santos, Rita S. (LEPABE, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto); Almeida, Carina (1LEPABE, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto); Cerqueira, Laura (LEPABE, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto); Guimarães, Nuno (LEPABE, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto); Azevedo, Nuno F. (LEPABE, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto) Fluorescent in situ hybridization (FISH) is now a widely established technique to discriminate microbial populations within biofilms. However, the standard FISH procedure that employs DNA probes typically involves steps that will likely alter the biofilm structure, such as the use of enzymes for permeabilization and high temperatures for the hybridization. The first type of nucleic acid mimics applied to FISH were the peptide nucleic acids (PNA). Due to the smaller


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size and uncharged nature of these molecules, the diffusion through the biofilm matrix and cell envelope was facilitated and permeabilization steps were less aggressive. More recently, we

-O-methyl-RNA molecules. While maintaining many of the advantages of PNA, these molecules offer a greater flexibility in design as individual residues can be intercalated with DNA bases at precise locations to, for instance, increase mismatch recognition and control the temperature at which the probes hybridize. Employing these mimics, we were able to successfully carry out a full FISH procedure at 37ºC and low pH to specifically visualize H. pylori in a tissue matrix in conditions resembling the human stomach. In addition, many of the compounds that are normally part of the hybridization solution were found not to be essential to obtain a high signal-to-noise ratio. Current work is focused on trying to avoid a fixation/permeabilization step and in further testing the limitations of the method in terms of temperatures and other parameters. The proof-of-concept demonstrated here paves the way for the emergence of other FISH-based methods that will be minimally disruptive and directly applicable to other types of naturally-occurring biofilms. References: Almeida, C., Azevedo, N.F., Santos, S., Keevil, C.W. and Vieira, M.J. (2011) Discriminating multi-species populations in biofilms with peptide nucleic acid fluorescence in situ hybridization (PNA FISH). PloS One 6(3), e14786. Fontenete, S., Guimaraes, N., Leite, M., Figueiredo, C., Wengel, J. and Azevedo, N.F. (2013) Hybridization-based detection of Helicobacter pylori at human body temperature using advanced locked nucleic acid (LNA) probes. PloS One 8(11), e81230.

P032 - Low oxygen environment favours Pseudomonas aeruginosa biofilm formation and metabolic activity Dumitrache, Romeo (Ryerson University, Toronto, ON., Toronto, CAN); Kroukamp, Otini (Ryerson University, Toronto, ON., Toronto, CAN); Wolfaardt, Gideon (Ryerson University, Toronto, ON., Toronto, CAN) Under limited or no oxygen conditions bacterial biofilms are commonly viewed to develop at a slower rate than under aerobicity. The aim of this study was to compare aerobic and anaerobic biofilms with respect to their overall metabolic output, accumulation on surfaces, sessile-to-planktonic cell yield and carbon utilization preferences. The present study involved the use of a carbon dioxide evolution measurement system (CEMS) to monitor, in real-time, the metabolic response of Pseudomonas aeruginosa biofilms. The CEMS technique, a non-invasive approach for the measurement of whole biofilm activity under continuous medium flow, proved very sensitive and responsive to changes in biofilm respiratory rates as conditions were altered during aerobic or anaerobic growth. Using a defined minimal media with citrate as the sole carbon source, microaerophilic and anaerobic biofilms had a similar or up to two times higher metabolic output (µmoles CO2/hour) than their aerobic counterparts; and their activity showed a dependency on the use of chemical reducing agents in the culture medium. The lag phase, from inoculation to exponential growth, as shown by CEMS profiles, was comparable between all growth conditions when inoculum preparation was matched with the biofilm culturing. Viable counts of effluent collected from the P. aeruginosa biofilms at steady state metabolic output showed similar numbers (10^7 to 10^8 CFU/ml) for both the aerobic and anaerobic environments. The real-time tracking of whole biofilm activity is a systemic approach that

Confocal laser microscopy performed on 72 hours old biofilms revealed higher average


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thickness, biovolume and maximum thickness for anaerobic biofilms; suggesting that P. aeruginosa favored microaerophilic/anaerobic growth where it reached higher metabolic rates with elevated cell yields.

The structural and functional differences revealed between anaerobic and aerobic biofilms studied in situ and real-time could help expand the understanding of the biofilms interactions and adaptation mechanisms. One such example is antibiotic treatment where the effectiveness of the killing is growth dependent.

P033 - Micro-3D printing of bacterial communities Connell, Jodie (ZZZ); Ritschdorff, Eric T. (ZZZ); Kim, Jiyeon (ZZZ); Bard, Allen J (ZZZ); Shear, Jason B. (ZZZ); Whiteley, Marvin (ZZZ) Micro-3D printing of bacterial communitiesBacteria communicate via short-range physical and chemical signals, interactions known to mediate quorum sensing (QS) and other adaptive phenotypes. Although most in vitro studies examine bacterial properties averaged over large populations, levels of key molecular determinants of fitness and pathogenicity may vary over micrometer scales within small, dense aggregates believed to play key roles in disease transmission. Understanding how these interactions contribute to pathogenicity in nature requires a new level of control in constructing more relevant models for assessing phenotypes. We describe a three-dimensional (3D) printing strategy for organizing populations within any geometry, including adjacent, nested, and free-floating colonies. This laser-based lithographic technique builds microtraps around bacteria suspended in gelatin. After printing, cells are localized within sealed traps formed by cross-linked gelatin, a porous material that supports growth of enclosed populations and readily transmits numerous bioactive species. We show that a pL-sized Staphylococcus aureus aggregate displays substantial resistance to β-lactam antibiotics when enclosed within a shell containing Pseudomonas aeruginosa. Micro-3D printing can organize populations at defined positions and distances to investigate fundamental questions regarding the spatial requirements for microbial interactions. This method can be interfaced with other analytical techniques to develop platforms to characterize how spatial organization impacts interactions at the molecular level. We introduce one combined approach, where micro-3D printing and scanning electrochemical microscopy (SECM) are coupled to create a quantitative, spatiotemporal map of pyocyanin (PYO), a QS-regulated redox- active secondary metabolite produced by P. aeruginosa, within aggregates of ≤104 cells. These initial studies probing PYO production as small populations of P. aeruginosa grow and the requirements for neighboring communities of mutant strains can sense and respond to one another, establish the value of this approach for characterizing how spatial parameters influence bacteria.

P034 - Environmental microbiology at single cell level, coupling identity and metabolic functions of single cells with NanoSIMS Ehrke, Ulrich (Cameca GmbH, Unterschleissheim, GER)


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1H.-U. Ehrke, 2F. Hillion, 2F. Horréard 1CAMECA GmbH, Carl-von-Linde-Str. 42, 85716 Unterschleissheim/Munich, Germany, 2CAMECA, 29 Quai des Grésillons, 92622 Gennevilliers Cedex - France,

We describe an instrumental and methodology development aiming at coupling single microorganism's phylogenetic identity with measurements of metabolic activities, and of fluxes of elements in environmental samples, including microbial mats, soil, water, sediment or other ecosystems. The technique helps linking specific metabolic pathways with individual microbial populations.

The coupling of in situ hybridization (ISH) techniques with nanometer-scale Secondary Ion Mass Spectrometry (NanoSIMS) after incubation in an isotopically enriched substrate allows single-cell metabolic analysis of uncultured microbial phylogenic groups. The additional information accessible with this method includes patterns of substrate sharing between cells, variations in metabolic rates between members of the same community, and temporal and physical separation of particular intracellular activities. Symbioses and elemental transfers can be studied at individual cell level. SIMS is based on the sputtering and ionization of surface atoms by a focused beam of primary ions scanned across a sample. Ejected secondary ions are mass filtered in a magnetic sector and detected in a multicollection. The lateral resolution goes down to 50nm and permits single cell isotopic and elemental analysis. It can be used in order to obtain in one single measurement: 1) the phylogenetic identity of single cells through the visualization of oligonucleotide probe hybridization signal (EL-FISH, similar to FISH but replacing fluorescent oligonucleotide by an elementally labeled (with F, I, Br, Au…) genetic probe, 2) the quantitative measurement of the metabolic activity of these individual cells by using stable isotope (i.e. 13C, 15N, 18O, 33S, …) or radioactive (i.e. 14C, 33P,…) labeling during the incubation. Alternately or in complement, any other epifluorescence microscopy methods, including FISH, can be used before the NanoSIMS isotope ratio measurement, in order to correlate phylogenic identities and metabolisms. After a description of the method and instrumentation we will show application of the FISH-SIMS and EL-FISH-SIMS in different fields including: - Microbiological study of micron-scale sulfur cycling within hypersaline microbial mats. Dissolved sulfide within the mats was analyzed for its abundance and d34S isotopic composition using SIMS and correlated with CARD-FISH imaging, - Validation by FISH and NanoSIMS of metabolic reconstruction of anoxic carbon flux in photosynthetic microbial mats as revealed by metatranscriptomics, - Identification of a novel cyanobacterial group as active diazotrophs in a coastal microbial mat using NanoSIMS analysis. References: [1] Micron-scale mapping of sulfur cycling across the oxycline of a cyanobacterial mat: a paired nanoSIMS and CARD-FISH approach. David Fikes et al. The ISME Journal (2008) 2, 749 759 [2] Identification of a novel cyanobacterial group as active diazotrophs in a coastal microbial


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mat using NanoSIMS analysis. Dagmar Woebken et al. The ISME Journal (2012), 1 13 [3] Anoxic carbon flux in photosynthetic microbial mats as revealed by metatranscriptomics. Luke C Burow et al. The ISME Journal (2012), 1 13

P035 - Qualitative and quantitative high-throughput screening of biofilms by automated fluorescence microscopy Schiebel, Juliane (BTU Cottbus , Senftenberg, GER); Böhm, A. (BTU Cottbus , Senftenberg, GER); Nitschke, J. (BTU Cottbus , Senftenberg , GER); Weinreich, J. (BTU Cottbus , Senftenberg, GER); Rödiger, S. (BTU Cottbus , Senftenberg, GER); Schierack, P. (BTU Cottbus , Senftenberg, GER) Biofilms are communities of microorganisms which live in a self-produced matrix of extracellular polymeric substances. This alternative lifestyle enables survival in different environmental niches which is constituted by their resistance to immune defense and antibiotics. Once bacteria form a biofilm in the host, infections are hard to treat and can change into chronic infections. Therefore it is important to develop tools to investigate biofilms for therapy improvement.

The aim of this work is to establish a method for the fully automated large-scale screening of biofilms which allows the comparison of different strains, the impact of different conditions during biofilm formation and most importantly the determination of the effects from different antibiotics.

We enhanced our previous published VideoScan technology to perform high-throughput screening of biofilms. This technology is based on fully automated fluorescence microscopy. The camera focuses automatically and captures images which are processed via sophisticated digital image processing methods. VideoScan enables the analysis of multiplex assays such as microbead or cell-based assays [Rödiger et al. 2013; Frömmel et al. 2013].

We use a 96 well plate format for the formation of biofilms. In a study we screened 20 strains for biofilm formation where one strain was found as a strong biofilm former. Our reference strain

-M. Ghigo, Pasteur Institute Paris). After 16h of incubation biofilms were stained with SYTO-9 and propidium iodide for the Live/Dead staining followed by a VideoScan analysis. This analysis represents a two-step evaluation realized by our software and image processing. In the first step which indicates the global fluorescence Live (coumarin)/Dead (cyanine 3) ratio, single images are taken and assembled to an overview picture of Live/Dead stained bacteria. The second step is a fine-grained analysis of the biofilm. This contains the counting of bacteria in the live and dead channel in a specific layer and area of the biofilm. Furthermore we developed a tool to count bacteria in different z-stacks of the biofilm. In addition we tested different antibiotics (Amp, Cam, Km, Gent, Tc) in different concentrations to look for the changes in the Live/Dead ratio.

With our VideoScan technology it is possible to study biofilms in a fully automated large-scale screening. The use of the 96 well format constitutes a cheap, reproducible and simple method. With this high-throughput platform we can check bacteria for biofilm formation under various conditions. This time-saving technology could facilitate the evaluation of biofilms regarding to


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applied antibiotics.

P036 - New force tools and high speed atomic force microscopy for microbiology research Müller, Torsten (JPK Instruments AG, Berlin, GER); Barner, Jörg (JPK Instruments AG, Berlin, GER); Haschke, Heiko (JPK Instruments AG, Berlin, GER); Henze, Thomas (JPK Instruments AG, Berlin, GER); Stamov, Dimita (JPK Instruments AG, Berlin, GER); Pettersson, Carmen (JPK Instruments AG, Berlin, GER) The heterogeneity of the microbial universe is one reason for the huge number of different applications and investigations in microbiology research. There are important modern research issues in the area of medicine, due to the potential of microbial pathogens to cause diseases, and increasing antibiotic resistance due to their widespread misuse. This increasing multi-resistance and the search for alternative substances is one of the main driving forces to investigate bacterial/microbial metabolism and its response to drugs [1].

Atomic force microscopy (AFM) is a technique that enables a variety of interesting measurements for microbiology research. The microbes can be measured in their native environment and the microbial surface structure can be measured down to the nanometer scale. AFM imaging can be used to investigate the surface architecture as well as the surface changes due to interactions with drugs and the environment [2]. In addition to information about the microorganism itself, the interactions between different microorganisms or between cell and surface can also be investigated [3]. However, for the new AFM instrumentation there are two important requests to consider: I imaging of structural dynamics of protein/molecule systems under near physiological conditions; II reducing the well-known drawbacks for challenging samples that have steep edges, or are soft, sticky, or loosely attached to the surface [4]. Therefore, we developed two new AFM tools: I- NanoWizard® ULTRA Speed AFM, and II- Quantitative Imaging (QI™). With N anoWizard® ULTRA Speed AFM fast AFM imaging of approximately 1 frame per second can be seamlessly combined with advanced optical methods such as confocal, TIRF, or STED microscopy. Individual molecule dynamics can now be studied

More than half a century after the first high-resolution electron microscopy images of collagen type I banding of 67 nm have been reported, now with the NanoWizard® ULTRA Speed AFM we could gain a high-resolution temporal insight into the dynamics of collagen I fibril formation and its characteristic 67 nm banding hallmark. The literature still abounds with conflicting data regarding the models of its fibril formation, structural intermediates, and kinetics. AFM is the only currently available high-resolution imaging technique amongst many to offer insight into the collagen I fibrillogenesis by operating in situ.

Quantitative Imaging (QI™) is based on fast force curves and combines nano-topography with the opportunity of obtaining mechanical properties simultaneously. The QI™ tip movement algorithm prevents lateral forces and controls the vertical forces for nondestructive imaging at each pixel [4]. Next to the classical information, such as, topography and adhesion, contact point images, Young´s moduli images, or even recognition events can be analyzed as published recently for living cells [5], bacteria [6] and single biomolecules. To demonstrate the performance and flexibility QI™ mode we investigated topography, adhesion properties, and


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s local distribution of diverse microbial samples (virus particles, yeast cells, E. coli bacteria) with different sizes, shapes and nanomechanical properties.

[1] G. Longo et al. Nature Nanotechnol. 8/7, 469-538, 2013 [2] Liu S., Wang Y., Scanning 32, 61-73, 2010 [3] Dufrêne Y. F., J. Bacteriol. 184, 5205-5213, 2002 [4] H. Haschke, T. Jähnke, Laser Focus World, 48, 2012 [5] L . Chopinet, C. Formosaa, M.P. Rols, R.E. Duval, E. Dague, Micron 48, 26-33, 2013 [6] S. Dhahri, M. Ramonda, C. Marliére, Plos-One 8/4 :e61663, 2013

P037 - Quantification of adherent bacteria using different genome integrated fluorescence markers Gutt, Beatrice (Empa, St. Gallen, CHE); Neff, Laura (Empa, St. Gallen, CHE); Thöny-Meyer, Linda (Empa, St. Gallen, CHE); Ren, Qun (Empa, St. Gallen, CHE) To assess bacterial adhesion and biofilm formation on a surface it is important to quantify biofilms with reliable in situ methods. Many different stains, dyes or markers for the detection of biofilm have been developed. Most of the quantification methods are based on counting cells. This is done either by direct staining on the surface, or by scratching the cells off prior to staining. These methods often require multistep procedures involving washing steps to remove surplus dye. Hence, this is likely to cause detachment of biofilm. A more straightforward approach for quantification of bacterial adhesion should be the ability to make use of engineered strains to emit light directly on the surface. Because of this the need of any washing step becomes redundant.

We therefore use genome integrated gene cassettes encoding fluorescence proteins for in situ quantification of bacterial adhesion. It is well known that genome integration leads to a stable, long-term expression of a gene of interest in comparison to plasmid based gene expression. As biofilm model organisms we selected the standard hospital pathogens Staphylococcus aureus (ATCC 6538) and Pseudomonas aeruginosa (ATCC 15442). To differentiate these bacterial cells, various gene encoding fluorescence proteins, namely GFP (green fluorescent protein), Ds Red (Discosoma red fluorescent protein), YFP (yellow fluorescent protein) and CFP (cyan fluorescent protein), were introduced. The work reported here describes the preparation of expression constructs, and their use for chromosomal integration for development of fluorescent S. aureus and P. aeruginosa model strains. The adhesion of the obtained strains on surfaces is evaluated by in situ fluorescence measurement of GFP, DsRed, YFP and CFP.

P038 - Tracing spontaneous mutation in biofilms by new confocal microscopy technology Yang, Rebekah Jiayue (University of Tsukuba, Tsukuba, JPN); Sakai, Ryosuke (University of Tsukuba, Tsukuba, JPN); Shiojima, Yutaro (University of Tsukuba, Tsukuba, JPN); Hamada, Masakaze (University of Tsukuba, Tsukuba, JPN); Toyofuku, Masanori (University of Tsukuba, Tsukuba, JPN); Yawata, Yutaka (University of Tsukuba, Tsukuba, JPN); Tateda, Kazuhiro (Toho University School of Medicine, Tokyo, JPN); Nomura, Nobuhiko (University of Tsukuba, Tsukuba, JPN)


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Microbial biofilms cause many problems in industrial and medical fields. It is known that biofilms exhibit increased resistance to stress compared with free-living microbes, and the physical removal of biofilms is difficult. On the other hand, biofilms can be beneficial in wastewater treatment (e.g., activated sludge). Therefore, the regulation of biofilms is necessary. Biofilms are heterogeneous and various spontaneous mutants are generated in biofilms. Some of these mutants have extremely high resistance to antibiotics and stress. Interestingly, the frequency of spontaneous mutation in biofilms is very high. It is thought that this diversity may help to protect biofilms when they are exposed to various environmental changes. Thus, an investigation of spontaneous mutations that occur in biofilms would contribute to the control of biofilms. Despite this fact, the behaviours of spontaneous mutants in biofilms are still not fully understood. The questions of when and where spontaneous mutants emerge in biofilms remain unanswered because the technology used to detect these mutants is limited. In this study, we attempted to visualize the spontaneous mutants that emerge in biofilms and investigate their behaviours. Pseudomonas aeruginosa biofilms contain many types of spontaneous mutants, and mucoid mutants are some of the most well known of these. It is known that mucoid mutants overproduce extracellular polysaccharide alginate and dominate within P. aeruginosa biofilms that cause infections in the lungs of patients with cystic fibrosis (CF). However, the questions of when and where mucoid mutants emerge within biofilms and how these mutants dominate within biofilms are unknown. Therefore, we chose to focus our research on mucoid mutants. We have explored a new confocal laser microscopy technology: continuous-optimizing confocal reflection microscopy (COCRM), which enables us to visualize the bacterial cell without fluorescence stain. In this study, we combined COCRM with mutation detection vector to successfully visualize the emergence and the localization of mucoid mutants in biofilms. We next examined the mechanism by which the mucoid strain dominates within biofilms, and we found that this dominance occurs under certain conditions. Our study suggest that mucoid mutants emerge in biofilms and dominate under certain environments.

P039 - A novel bench-top flume to grow stream biofilms in reproducible hydraulic environments Niederdorfer, Robert (Department for Limnolgy and Bio-Oceanography, University of Vienna, Vienna, AUT); Gernand, Anna (Department for Limnolgy and Bio-Oceanography, University of Vienna, Vienna, AUT); Adlassnig, Wolfram (Core Facility Cell Imaging and Ultrastructure Research, University of Vienna, Vienna, AUT); Battin, Tom (Department for Limnolgy and Bio-Oceanography, University of Vienna, Vienna, AUT) It is now established that hydraulics imposes a major control on biofilm architecture in stream ecosystems. Hydraulic conditions are highly dynamic and heterogeneous in space and time in streams. Here we present and describe a novel flume design that allows us to reproducibly study the architecture of biofilms under different hydraulic conditions relevant for streams. Bench-top flumes (length: 1.5 m) were designed to accommodate turbulent, transitional and laminar conditions along a single flow continuum thereby ensuring the same inoculum and chemical environment. In these flumes, we grew phototrophic biofilms mimicking the benthic zone of a small stream. We imaged the architecture of the biofilms in each of these hydraulic environments and using microelectrodes we established vertical profiles and derived gross primary production and respiration. Our results suggest that hydraulics is indeed a significant


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control on biofilm architecture with major repercussions on biofilm functioning. Our findings underscore the usefulness of flumes to study biofilm structure-function coupling and highlight the physical basis of biofilm ecology and related ecosystem processes.

P040 - Time-lapse confocal microscopy of gel-entrapped bacteria as models of infection Pitts, Betsey (Center for Biofilm Engineering, Bozeman Montana, USA) Some biofilm infections, such as those in the cystic fibrosis lung and chronic dermal wounds, do not involve a foreign metal or polymer surface to which the biofilm attaches. Instead, microorganisms are distributed as small aggregates in a layer of mucus or necrotic tissue. To simulate these structures in vitro, green fluorescent protein-tagged Staphylococcus aureus was seeded into low-melting temperature agarose gels which were then cast into films or hemispherical shapes with a characteristic dimension on the order of one millimeter. Growth, antimicrobial treatment, and regrowth were observed by time-lapse confocal microscopy using an environmental chamber to expose the gel biofilm to medium and maintain a constant relative humidity and temperature of 37oC. Bacteria grew within the gel during 24 h creating small aggregates approximately 10-30 microns in diameter. Quantitative image analysis was used to measure the integrated biomass during the growth phase. The specific growth rate during the first 12 hours of incubation was 0.4 h-1. In some experiments, bacterial growth ruptured the gel causing the dramatic release of a cloud of planktonic cells. Young gel biofilms were more susceptible to antimicrobial treatments than were older biofilms. For example, a 5h old gel biofilm exposed to nisin for 1 h subsequently lost all green fluorescence indicating a complete loss of membrane integrity. In contrast, 24 h old gel biofilms lost some green fluorescence but then sometimes exhibited robust regrowth from the interior of the gel structure. The gel biofilm system was also used to ascertain the penetration of nisin into the structure by first loading bacterial cells with a fluorescent dye (via staining with calcein-AM. The loss of red calcein fluorescence at the center of the gel biofilm confirmed the penetration and action of the peptide. Agarose gel biofilms are transparent and ideally suited to investigation by confocal microscopy. The experiments described above are each illustrated by compelling time-lapse video.

P041 - A high efficiency sensor device to collect Listeria monocytogenes biofilms from food industry surfaces Gião, Maria Salomé (University of Southampton, Southampton, GBR); Blanc, Séverine (40-30, Grenoble, FRA); Porta, Sonia (ainia, Valencia, ESP); Belenguer, José (ainia, Valencia, ESP); Keevil, Charles William (University of Southampton, Southampton, GBR) Listeria monocytogenes is a foodborne pathogen that is commonly associated with contaminated ready-to-eat products. Therefore, cross-contamination of these food products is one of the biggest concerns in the food industry as it has been shown that L. monocytogenes is resilient to surface cleaning procedures and can develop into biofilms. Indeed, this pathogen has been isolated from several food industry surfaces, such as walls, work benches and cutting utensils. Listeria affects mainly susceptible people, such as pregnant women, elderly, newborns and immunocompromised people. Although the incidence of this pathogen is low, it has the highest mortality rate among the foodborne pathogens, with approximately one third of infected


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cases ending in death. The current methods for isolation and detection of Listeria from food industry surfaces are still very limited, with recovery rates as low as 10% and results, which rely on culture methods, taking several days to be obtained. In the FP7 project Biolisme a sensor was developed to collect and detect L. monocytogenes by integrating a sample collector and detector in the same device. The collection of cells is achieved by injecting compressed air and water that detach cells when hitting the surface. The flow mixed with cells is then collected in a chamber. The presence of Listeria is tested using antibody technology. The aim of this work was to test the collecting part of the device to recover L. monocytogenes biofilms from stainless steel (SS) and polytetrafluoroethylene (PTFE) surfaces and study the membrane integrity of collected cells. For that L. monocytogenes biofilms were grown in brain heart infusion (BHI) broth for 4, 24 and 48 hours and 7 days on SS and PTFE coupons. The effect of injecting compressed air mixed with water at 2, 3 and 4 bar was tested for 30 and 60 seconds on SS and for 60 seconds on PTFE. Control coupons were also used where cells were recovered using a cell scraper. The recovered cells were analysed for cultivability by plating onto BHI agar and for membrane integrity by staining with a Live/Dead® BacLightTM bacterial viability kit. Coupon surfaces were visualized in situ after staining with DAPI and cells that remained on the surface directly quantified. Results showed that by using this device cells are easier to recover from PTFE surfaces, with a recovery rate of over 99%. Recovery rates from SS surfaces are slightly lower but more than 95% of sessile cells can still be removed. In general, recovery of cells from SS ranged, on average, from 1.3 x 106 cells cm-2 (4 hour-old biofilms) to 6.3 x 107 cells cm-2 (7 day-old biofilms). Cells collected from biofilms grown on PTFE varied between 8.3 x 105 cells cm-

2 (4 hour-old biofilms) and 5.1 x 108 cells cm-2 (7 day-old biofilms). Compared to the controls, it is possible using this device to recover more cells from biofilms than when using the cell scraper, showing that this method is highly efficient. Moreover, recovered cells maintained membrane integrity and cultivability which confirms the validity of using specific antibodies against surface exposed epitopes to rapidly detect L. monocytogenes. This will ensure that tedious culture techniques can be replaced by this method, which has the advantage to detect viable but non-cultivable cells which we have previously shown to be present in some Listeria biofilms. This work presents a more efficient alternative to the current sampling methods and can help promote a safer food industry environment.

P042 - Visualizing synthetic dental biofilm on teeth using micro computed tomography Vyas, Nina (University of Birmingham, Birmingham, GBR); Walmsley, Damien (University of Birmingham, Birmingham, GBR); Pecheva, Emilia (University of Birmingham, Birmingham, GBR); Dehghani, Hamid (University of Birmingham, Birmingham, GBR); Grover, Liam (University of Birmingham, Birmingham, GBR); Sammons, Rachel (University of Birmingham, Birmingham, GBR) Background: Dental plaque biofilms and calculus form naturally over time on tooth surfaces and dental implants. These need to be regularly removed in order to prevent periodontal diseases. Deep cleaning methods such as ultrasonic scaling are used to remove biofilm which cannot be removed by everyday dental hygiene. It is therefore important to investigate the effects of ultrasonic scaling instruments in order to understand the different processes involved in removing dental biofilm and calculus, so more efficient instrumentation can be developed in the future. Bacterial biofilm is difficult to grow in a homogenous layer, and the unpredictable nature of its growth means that consistent results cannot be obtained each time. It could therefore be


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advantageous to use a material which has similar properties to biofilms, which can be used instead to image biofilm disruption from ultrasonic scalers. The aim of this project is to investigate the possibility of using microCT to image biofilms, initially by using an artificial biofilm as a model system.

Methods: Robinson et al. (2014) [1] have formulated a hydrogel for mimicking biofilm in the root canal, created by dissolving 3 g of gelatine and 0.06 g of hyaluronan (sodium hyaluronate 95%,) in 45 ml of filtered water at 50?C. In this work, two variations of hydrogel were produced; one as

2% potassium iodide), to investigate whether this could enhance the contrast in microCT. The hydrogels were then centrifuged at 3000rpm for 10 minutes to remove air bubbles, and left to solidify at room temperature. In order to see whether the hydrogel can be imaged on teeth using microCT, a plastic model tooth (Frasaco, Germany) and a natural tooth crown were imaged in microCT before and after application of the hydrogel (stained and unstained). The pre- and post- scans were carried out with a SkyScan 1172 micro-CT scanner (SkyScan, Kontich, Belgium). Due to the low x-ray absorption coefficient of hydrogel (and biofilm), it is difficult to visualise it on radio-opaque materials such as bone. Therefore dual energy scans were performed for the tooth

Results: After superimposing the pre- and post scans, the unstained hydrogel could be clearly visualised in 3D on the tooth model. Dual energy analysis also enabled segmentation of the stained hydrogel on the tooth crown. Other hydrogels will be developed in future work which mimic oral plaque or calculus more accurately. Disruption of the hydrogel by ultrasonic scalers will then be investigated by imaging before and after scaling via microCT. The method will then be used to image and evaluate disruption of real plaque.

Conclusion: This work shows the potential of micro-CT to image a biofilm-mimicking hydrogel for the purpose of investigating biofilm disruption. The use of dual energy imaging in microCT to visualise low density biofilm-like materials on teeth is also presented. This novel application of microCT will provide new insights into exactly how ultrasonic scalers remove plaque, which will eventually lead to improved ultrasonic scaler tip design.

[1] R.G. Macedo, J. P. Robinson, B. Verhaagen, A. D. Walmsley, M. Versluis, P. R. Cooper & L. W. M. van der Sluis, International Endodontic Journal, 2014


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P043 - Density of founder cells affects spatial pattern formation and cooperation in B. subtilis biofilms van Gestel, Jordi (University of Groningen, Theoretical Biology Group, Groningen); Weissing, Franz J (University of Groningen, Theoretical Biology Group, Groningen); Kuipers, Oscar P (University of Groningen, Molecular Genetics Group, Groningen); Kovacs, Akos T (Friedrich Schiller University of Jena, Terrestrial Biofilms Group, Jena, GER) Many cells inhabiting a biofilm express cooperative traits, like the secretion of extracellular polysaccharides (EPS). These traits enhance biofilm properties, such as stress resilience or colony expansion, while being costly to the cells that express them. In well-mixed populations cooperation is difficult to achieve, because non-cooperative individuals can reap the benefits of cooperation without having to pay the costs. The physical process of biofilm growth can however result in spatial patterns that segregate cooperative from non-cooperative individuals. This segregation makes it impossible for non-cooperative cells to exploit the benefits of cooperation.

Here we examine the interaction between spatial pattern formation and cooperation in Bacillus subtilis biofilms. We show, by mathematical modeling and experimentally, that the density of cells at the onset of biofilm growth affects pattern formation during biofilm growth. At low initial cell densities co-cultured strains strongly segregate in space, while at high initial cell densities they do not. As a consequence, EPS-producing cells have a competitive advantage over non-cooperative mutants when biofilms are initiated with a low density of founder cells, while EPS-deficient cells have an advantage at high assortment. This study thereby disentangles the important interaction between spatial pattern formation and the evolution of microbial cooperation.

P044 - Legionella biofilm colonization and formation is enhanced after intra-amoeba multiplication. Bigot, Renaud (Universite de Poitiers, Poitiers, FRA); Bertaux, Joanne (Universite de Poitiers, Poitiers, FRA); Berjeaud, jean-marc (Universite de Poitiers, Poitiers, FRA) Legionella pneumophila , a facultative intracellular bacterium, is the causative agent of legionellosis. In the environment this pathogenic bacterium colonizes the biofilms as well as amoebae which provide a rich environment for the replication of Legionella. When seeded on pre-formed biofilms, L. pneumophila was able to establish and survive and was only found at the surface of the biofilms. Different phenotypes were observed when the L. pneumophila used to implement pre-formed biofilms or to form mono-species biofilms, were cultivated in a laboratory liquid culture medium or had grown intracellulary within the amoeba. Indeed, the bacteria, which developed within the amoeba, formed clusters when deposited on a solid surface. Moreover, our results demonstrate that multiplication inside the amoeba increased the capacity of L. pneumophila to produce polysaccharides and therefore enhanced its capacity to establish biofilms. Finally, it was shown the clusters formed by L. pneumophila were probably related to the secretion of a chemotaxis molecular agent which induced the Biofilm phenotype.


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P045 - Evolutionary cooperation in Salmonella biofilms C S, Srinandan (SASTRA University, Thanjavur, IND); Elango, Monalisha (Indian Institute of Science, Bangalore, IND); Gnanadhas, Divya Prakash (Indian Institute of Science, Bangalore, IND); Chakravortty, Dipshika (Indian Institute of Science, Bangalore, IND) Salmonella is known to form biofilm pellicle in the air-liquid interface, submerged biofilm on the substratum surfaces and also persistent biofilms in the gall stones. Salmonella cells produce cellulose, curli and BapA protein as extracellular polymeric substances (EPS) to form a biofilm where the secreted matrix materials could act as a public good. Cheater cells (STM∆csgD) do not invest to produce EPS, but exploit the matrix produced by the wild type cells (STM 14028) to stay in the biofilm. No significant difference was found during planktonic growth between the wild type Salmonella Typhimurium (STM 14028) and STM∆csgD cells under shaking incubation conditions at 37oC. However, estimation of pathogen burden and competitive index measurements indicated that the CsgD-dependent growth was important for the bacteria in different steps during pathogenesis. Productivity of the biofilm as determined by dry weight, decreased in the co-culture of STM14028 and STM∆csgD cells, but a more pronounced effect of the cheater cells was on the pellicle than submerged biofilm. A low value of pair cross-corre ∆csgD cells in the biofilm. The weakened biofilm harboring cheater cells were significantly susceptible to chlorine and ciprofloxacin treatment. This work demonstrates that the csgD-dependent matrix produced by Salmonella is a public good and a proof-of-principle strategy that evolutionary cooperation could first be targeted to weaken the biofilm before administering antimicrobials.

P046 - The influence of prior modes of growth, temperature, nutrients and substrate surface on biofilm formation by Campylobacter jejuni Teh, Amy Huei Teen (Monash University, Selangor, MYS); Lee, Sui Mae (Monash University, Selangor, MYS); Dykes, Gary (Monash University, Selangor, MYS) Campylobacter jejuni is one of the most frequent causes of bacterial gastrointestinal food-borne infection worldwide. It has fastidious growth requirements for atmosphere, nutrients and temperature. Biofilm formation by C. jejuni may play a significant role in its survival through the food chain. The influence of prior mode of growth (planktonic or sessile), temperature (37°C and 42°C) and nutrient conditions (nutrient broth and Muller-Hinton broth) on biofilm formation by eight C. jejuni strains on different abiotic surfaces (stainless steel, glass and polystyrene) was examined. Hydrophobicity, auto-aggregation and the presence of virulence genes in the strains were also determined. The results showed that cells grown in sessile culture generally showed more biofilm formation (p<0.05) as compared to their planktonic counterparts. Growth at different temperatures did not affect biofilm formation (p>0.05) but it was higher in lower nutrient media (p<0.05). The C. jejuni strains were able to form biofilms on abiotic surfaces with the highest amount formed on glass, followed by polystyrene and then stainless steel. None of the strains demonstrated strong, complex or structured biofilm formation. No clear trend was observed between the presence of virulence genes, hydrophobicity and auto-aggregation, and biofilm formation by the strains.


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P047 - Organo-mineral complexation alters carbon and nitrogen cycling within streambed biofilms Hunter, William (Department für Limnologie und Ozeanographie, Wien, AUT); Veuger, Bart (Royal Netherlands Institute for Sea Research, Yerseke); Wanek, Wolfgang (University of Vienna, Vienna, AUT); Prommer, Judith (University of Vienna, Wien, AUT); Mooshammer, Maria (University of Vienna, Vienna, AUT); Battin, Tom (University of Vienna, Vienna, AUT) Inland waters are of global biogeochemical importance receiving carbon inputs of ~ 4.8 Pg C y-1. Of this 12 % is buried, 18 % transported to the oceans, and 70 % supports aquatic secondary production. An important aspect of OM burial and transport is the formation of organo-mineral complexes within aquatic systems and their potential use as a carbon (C) and nitrogen (N) sources within the streambed microbial biofilms. Organo-mineral particles form by sorption of dissolved OM to freshly eroded mineral surfaces and may contribute to ecosystem-scale particulate OM fluxes. We tested the availability of mineral-sorbed OM as a C & N source for streambed biofilms vs. streamwater microbial assemblages. Organo-mineral particles were constructed in vitro by sorption of 13C:15N-labelled amino acids to hydrated kaolin particles, and microbial degradation of these particles compared with equivalent doses of 13C:15N-labelled free amino acids. Organo-mineral sorption had a significant effect upon microbial activity, restricting C and N mineralization by both biofilm and streamwater assemblages. Distinct community-specific response were observed, with both dissolved and mineral-stabilized amino acids playing an enhanced role in the metabolism of the streamwater microbial community. Mineral-sorption of amino acids differentially affected C & N metabolism, restricting the mineralization of amino acid-derived N to ammonia and altering C and N incorporation into microbial biomass. The present study demonstrates that organo-mineral complexes restrict microbial degradation of OM, limiting the availability of organic N sources. Consequently organo-mineral interactions alter carbon and nitrogen cycling dynamics both within streambed biofilms and streamwater microbial assemblages.

P048 - The evolution of biofilm-forming Wrinkly Spreaders in glass bead columns and static microcosms leads to subtle differences in wrinkleality and fitness Udall, Yvette (SIMBIOS Centre, Dundee, GBR); Hapca, Simona (SIMBIOS Centre, Dundee, GBR); Spiers, Andrew (SIMBIOS Centre, Dundee, GBR) Adaptive radiation of bacteria has been investigated using Pseudomonas fluorescens SBW25 in static liquid microcosms where the biofilm-forming mutant known as the Wrinkly Spreader arises having significant fitness advantage over the ancestral strain . Wrinkly Spreaders also appear in evolving populations developing on partially-saturated glass bead columns (GBCs) constantly irrigated with nutrients, with competitive fitness (W) advantages in GBCs of 1.28 1.78. An environment effect was discernible in the quantitative wrinkleality (colony expansion, reversion rate, growth in static microcosms, biofilm attachment levels and strength) and W in static microcosms of mutants isolated from GBCs and static microcosms (P ≤ 0.0496), suggesting that the two environments had selected for subtly different classes of Wrinkly Spreader. An investigation of W using a GLM approach revealed a significant interaction between environment and growth, attachment and reversion (P ≤ 0.044). Furthermore, when the interaction effects were investigated in more detail, differences in how each environment interacted with the covariates were found. This analyses suggest that these environments


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provide divergent pressures that result in the selection of subtly different classes of Wrinkly Spreader, illustrating how environmental differences may influence the evolutionary trajectories of adaptive lineages originating from the same ancestral population.

P049 - Warming modulates the phototrophic river biofilm ability to denitrify Boulêtreau, Stéphanie (University Paul Sabatier, Toulouse, FRA); Lyautey, Emilie (University of Savoie, Le Bourget du Lac, FRA); Touron - Bodilis, Aurélie (EDF R&D, Chatou, AUT); Delattre, Cécile (EDF R&D, Chatou, FRA); Mastrorillo, Sylvain (University Paul Sabatier, Toulouse, FRA); Garabetian, Frederic (University of Bordeaux, Arcachon, FRA) Phototrophic river biofilms (PRB) develop in shallow running waters where they are largely exposed to warming. They are recognized as in-stream processors of natural or anthropogenic compounds and efficient actors of the nitrate elimination service of denitrification.

The aims of this study were: (i) to show whether and how moderate water warming (+2.5°C) drives bacterial structuring and diversity and how bacterial diversity affects denitrification enzymatic activity in PRB and (ii) to quantify the short-term sensitivity of denitrification enzyme activity in PRB to a large temperature range (1-31°C).

We used water warming associated to nuclear power plant thermal plume to produce 6- and 21-day-old natural PRB assemblages in two similar environmental conditions but temperature. Bacterial community composition was assessed using ARISA. Denitrifier community abundance and denitrification gene mRNA levels were estimated by q-PCR and qRT-PCR, respectively, of 5 genes encoding catalytic subunits of the denitrification key enzymes. Denitrification enzyme activity (DEA) was measured by the acetylene-block assay. A mean water warming of 2.5 °C was sufficient to produce contrasted total bacterial and denitrifier communities and to also affect DEA. The warming-induced changes differed, increasing or decreasing DEA in 6- or 21-day-old biofilms, showing that PRB maturation controls the bacterial and denitrifying community response to temperature.

The short-term sensitivity of DEA was explored on natural PRB submitted to 24-hour laboratory incubations at four temperatures ranging from 1 to 31°C. On this large range of temperature, DEA could be fitted to an exponential function of incubation temperature, yielding mean (±standard error) activation energy of 1.0 (±0.2) eV and Q10 of 4 (±1.6). This demonstrated the high and variable temperature dependence of denitrification as compared to other community-level metabolisms such as respiration or photosynthesis.

Together these results suggest that indirect effects of warming through changes in bacterial community composition, coupled to the strong direct effect of temperature on DEA, could modulate dissolved nitrogen removal by denitrification in rivers and streams.


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P050 - Interspecific diversity is more important than intraspecific diversity for performance of a mixed community biofilm Lee, Kai Wei Kelvin (SCELSE/NTU, Singapore, SGP) Bacterial biofilms can be found in most settings, where their eradication represents a major challenge. In particular, the formation of genetic variants within biofilms has been shown to increase their resistance to various stresses. However, biofilms in nature are multi-species communities and the relationship between interspecies interactions and variant formation has not been studied.

Here, Pseudomonas aeruginosa, Pseudomonas protegens and Klebsiella pneumoniae mono-species biofilms generated stable variants with unique colony morphology. Whole genome sequencing of each variant (> 40 fold coverage) showed that they have one to four genetic mutations on genes such as pilT. In addition, these variants also differed from their respective parental strains in phenotypes such as motility, biofilm formation capability and siderophore production. When co-cultured with their respective parental strains, the variants outcompeted their parental strains, making up 65-99% of the total biovolume. Despite their enhanced fitness, their frequencies decreased in mixed-species biofilms. Cell-free effluent from P. aeruginosa and K. pneumoniae mono-species biofilms could reduced the frequency of P. protegens variants, suggesting that variant formation is regulated by extracellular cues. Furthermore, substituting the parental strain with its variant reduced the resistance of the mixed biofilm community to sodium dodecyl sulfate by 45%.

This study demonstrates that interspecies variation is favored in a community at the expense of intraspecies (self-generated) variation, reflecting altruistic behavior in a biofilm community and may in part explain why the parental strains are the dominant forms isolated from the environment.

P051 - TXRF and 2D µ-XRF analyses analyses - Methods to determine the element behavior of biofilms grown in mining drainage water Mages, Margarete (Helmholtz Centre for Environmental Research UFZ, Magdeburg, GER) Mages, M.1, von Tümpling, W1., Waldschläger2, U., Baborowski, M. 1

1 UFZ Centre for Environmental Research Leipzig-Halle, Department of River Ecology Magdeburg, Brückstrasse 3a, D-39114, Magdeburg, Germany 2 Bruker Nano GmbH, 12489 Berlin, Am Studio 2D, Germany

In natural surface waters classified to be in a good ecological status elements are ubiquitous distributed on nearly natural levels. Under this condition the element accumulation rate on Biofilms can be seen also as natural process.

Human impacts like mining activities are a reason for significant higher element concentrations in waters and or contents sediments. Biofilms as boundary layer between sediments and the water phase existing in a very reactive phase of such water bodies. Often limited quantity of


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biofilms as sample material are the reason that time dependent accumulation and desorption processes of heavy metals and arsenic on biofilms are very difficult to describe.

Because of the small amount of sample material in the µg range needed for the simultaneous determination of elements ? atomic number 16 with the Total Reflection X-ray Fluorescence (TXRF), biofilm samples grown on natural and on artificial materials have been used to describe the time dependent element accumulation behavior. Investigation presented had been done on biofilms exposed on drainage water from a key gallery of former copper mining activities near the Schlenze (small tributary of the Saale River in central Germany). The drainage water was taken as point source for the element and salt pollution of the River Saale with constant environment conditions (conductivity 47 mS/cm, oxygen content 10 mg/L, pH 7.5, water temperature 12°C and extreme low nutrient content). For an assessment of these results, samples from stream Schlenze upstream the confluence with the drainage water, a small tributary of the Saale River in central Germany, were analyzed, too.

In general the results of element investigation on biofilms from the adit Schlüsselstollen have shown high accumulation rates on lead, copper and zinc and nickel. Based on the calculated accumulation factors for the element contents of the Schlenze biofilms the following enrichment can be stated: Pb > Cu > Zn > Ni > Fe > Ca. In more detail: For the carrier grown biofilm could be observed that after a fast first accumulation of Pb, Ni, Zn and Cu a relatively rapid desorption of Pb and Ni and a significant increase in the element content of Zn and Cu took place after two weeks. 8 Weeks after laying out the carriers a plateau state has been reached for the investigated elements.

Beyond these analyses the 2D Micro X-ray Fluorescence (2D µ-XRF), M4 Tornado, Bruker Nano Ltd, Berlin, Germany, had been used to describe the element distribution and accumulation pattern in the biofilm samples itself. As an outcome of the scan it can be stated, that the elements like Al, K, Ca, Si and Mn are adsorbed on or embedded in inorganic colloids. Pb, Fe, Cu S und Zn are embedded more in organic materials.

P052 - Species sorting during biofilm assembly by artificial substrates deployed in a cold seep system Qian, Pei-Yuan (Hong Kong University of Science and Technology, Hong Kong, HKG); Zhang, Weipeng (Hong Kong University of Science and Technology, Hong Kong, HKG); Wang, Yong (Hong Kong University of Science and Technology, Hong Kong, HKG) In the marine environment, bioflms are often referred to as microfouling, and the effect of substrate type on biofilm formation is an important and controversial issue. On the other hand, studies focusing on biofilm assembly in deep-sea environments are rarely conducted. In this study, following our earlier work, biofilms were developed on different substrates in two locations, a brine pool and nearby bottom water (NBW) adjacent to the Thuwal cold s eep II in the Red Sea for different durations , to examine the effects of substrate type o n microbial community assembly. The composition s of the microbial communities of the 51 biofilms and waters were revealed by classification of pyrosequenced 16S rRNA gene amplicons . T ogether with the microscopic characteristics of the biofilms, t he results are indicative of a stronger selection effect on microbial assembly by substrates in the brine pool, in which there was a clear


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difference between the community structure of the water and the biofilms of different ages, compared with the NBW . Moreover, the selection effect by substrate type was stronger in earlier stage than later stage of biofilm development. Further analysis taking species content and effect of substrate type into account suggests that all these results are well consistent with hypotheses proposed in the framework of species sorting theory, which states that the substrate coupled species sorting during biofilm assembly is dictated by habitat condition, duration time and source community structure. Therefore, this study has shed light on control strategy of biofilm associated marine fouling and provided evidence for the ecological theories under development toward understanding the formation of deep-sea biofilms.

P053 - Light control of biofilm food webs: Results from stream mesocosm experiments along an eutrophication gradient. Geisthardt, Steffen (Helmholtz Zentrum für Umweltforschung UFZ, Magdeburg, GER); Norf, Helge (Helmholtz Zentrum für Umweltforschung UFZ, Magdeburg, GER); Weitere, Markus (Helmholtz Zentrum für Umweltforschung UFZ, Magdeburg, GER) Biofilms mediate a large part of microbial nutrient turnover and, furthermore, form the trophic basis for many aquatic consumers. While primary producers (e.g. bacteria, microalgae) within biofilms are the primary determinants for biofilm biomass accrual, protozoa such as ciliates and amoeba act as consumers of primary production, that may also link organic carbon from basal to higher trophic levels. In order to test the role of the light regime on biofilm food webs in near-natural (i.e. non-impacted) and agricultural streams, biofilms were cultivated under standardized conditions in mobile aquatic mesocosms (MOBICOS) exposed at two contrasting stream sites. We tested the hypothesis that both biofilm-dwelling primary producers and protozoan consumers are nutrient-limited in natural (oligotrophic) streams, whereas light-limitation as well as efficient grazing by macroinvertebrates is crucial for limiting microbial biofilm growth in agricultural (eutrophic) streams. The hypotheses were tested by selectively manipulating light intensity before the background of site-specific differences in nutrient loads. Biofilms were harvested over two to four weeks and analyzed using chemical analyses (biofilm ash free dry mass, chlorophyll a content, utrient stoichiometry) as well as microscopic analyses of microbial abundances and assemblage compositions. Confirming our hypothesis regarding agricultural streams, both primary producers and microbial consumers were enhanced by light. In addition, macroinvertebrates could be shown to graze upon, but not to control, biofilm standing stocks. Surprising results were obtained at the near-natural test site, in that both producer and consumer abundances responded positively to increased illumination, indicating absence of nutrient limitation even at this presumably oligotrophic stream site. Particulary ciliate community composition and dominance of single taxa showed distinct patterns reflecting both the nutrient load and the (light-regulated) autotrophic index of the particular stream site.


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P054 - Microbial communities in stream biofilms under flow wax and wane Timoner, Xisca (Catalan Institute for Water Research and University of Girona, Girona, ESP); Borrego, Carles M (Catalan Institute for Water Research and University of Girona, Girona, ESP); Acuña, Vicenç (Catalan Institute for Water Research, Girona, ESP); Sabater, Sergi (Catalan Institute for Water Research and University of Girona, Girona, ESP) Biofilms dwelling in Mediterranean streams face strong variations in stream flow. Water flow is interrupted during long summer droughts causing a complete desiccation of the streambed. Biofilms are therefore exposed to long periods of dehydration and intense solar radiation that may affect their structure and functioning. To characterize the effects of flow intermittency on biofilm microbial communities we analyzed bacterial and archaeal communities inhabiting epilithic, epipsammic and hyporheic streambed compartments through pyrotag sequencing of the SSU rRNA gene in a small, forested Mediterranean stream along a hydrological cycle that included drying, non-flow (112 days) and rewetting phases. The biofilm harbored different bacterial communities on each streambed compartment. Epipsammic and hyporheic hosted similar bacterial communities (~60% of shared OTUs) and were more diverse than epilithic ones. Major changes occurred in the epilithic probably due to its higher exposure to humidity loss in comparison to sandy compartments (epipsammic and hyporheic). Approximately 36% of bacterial taxa in epilithic biofilms were considered sensitive to desiccation, whereas these taxa only constituted a 15% in the epipsammic biofilms. Hyporheic biofilms maintained a stable bacterial community through the whole hydrological cycle, ~ 86% of the OTUs were considered tolerant to desiccation. During the non-flow phase the occurrence of opportunistic taxa (~ 40%)

period mainly in the epilithic. These changes were consistent with the increased contribution of sequences affiliated to Firmicutes during the non-flow phase and a concomitant decrease of cyanobacterial sequences (prevalent during the wetted phases). Sequences affiliated to Actinobacteria and Alphaproteobacteria were consistently found in the epipsammic and hyporheic compartments for the whole period and accordingly considered resistant to desiccation. Diversity decreased in all biofilm compartments due to flow intermittency, but flow resumption restored the structure of bacterial biofilm communities observed before the non-flow phase. The effects of desiccation on biofilm communities were higher than those caused by rewetting since the number of sensitive OTUs ranged from 20% to 15%. In all biofilms high tolerance to rewetting was observed (~50%). Concerning archaeal biofilm communities, preliminary results indicated a predominance of Thaumarchaeota (i.e. Nitrososphaerales) in all compartments suggesting an important but hitherto unknown contribution to autotrophic ammonia oxidation in streambed biofilms. Methanogenic archaea and uncultured members of both the Euryarchaeota (i.e. Thermoplasmatales) and the Crenarchaeota (i.e. Miscellaneous Crenarchaeotic Group) were also important components of epipsammic and hyporheic biofilms raising questions about their potential implication to organic carbon cycling. Overall, the physical configuration of the stream compartments greatly influences the response of their indigenous microbial communities to flow intermittency. Drought drives the selection of desiccation-resistant bacteria and archaea that might be crucial in maintaining the ecosystem functions during the non-flow phase. Analogously, flow resumption might restore microbial species that were prevalent before desiccation, therefore biofilms subjected to flow intermittency show a high resilience. Understanding the dynamics of microbial communities subjected to flow intermittency might be crucial to unravel the potential effects of Climate


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Change on the biogeochemical cycles mediated by biofilms, and therefore the associated ecological implications for the stream ecosystem.

P055 - Development of a mixed species biofilm with Campylobacter jejuni and Clostridium perfringens under microaerobic conditions Hilbert, Friederike (University of Veterinary Medicine Vienna, Vienna, AUT); Sofka, Dmitri (University of Veterinary Medicine Vienna, Vienna, AUT); Szostak, Michael (University of Veterinary Medicine Vienna, Vienna, AUT) In addition to its clinical relevance, biofilm formation is frequently necessary for the survival of foodborne pathogens in stressful environments inside and outside the host. Most foodborne pathogens have been found to form biofilms. Under specific conditions, Campylobacter, a fastidious microaerobic bacterium, forms biofilm structures. However, not much is known about

Campylobacter regularly encounters many different microorganisms commonly involved in biofilm formation, e.g. Firmicutes like Lactobacillales, Bacillales, and Clostridiales; Enterobacteriales like Escherichia coli; and Pseudomonadales. Recent studies on so-called mixed biofilms, which are considered more important in nature than single species biofilms, have addressed whether individual species in mixed biofilms act in neutralism, amensalism, competition, antagonism, commensalism or mutualism. It seems likely that all these effects play a part in mixed biofilms and may even co-occur in a single multi-species biofilm. We have reported that Campylobacter can benefit from a specific bacterial-bacterial interaction with Pseudomonas under aerobic conditions. We now show that Campylobacter supports the growth of strictly anaerobic Clostridia spp. under microaerobic conditions within a naturally formed mixed biofilm. The study was undertaken with Campylobacter jejuni NCTC 11168 and Clostridium perfringens DSM 756 as well as a Clostridium food isolate. Incubation was performed in micro-well plates under microaerobic conditions, which are advantageous for the growth of thermophilic Campylobacter spp. but do not allow the strictly anaerobic Clostridium spp. to grow. Incubation of mixed cultures (Campylobacter jejuni and Clostridium perfringens) allowed growth of both species within a biofilm that was not assembled in single-species wells. Biofilm formation was studied using scanning electron microscopy. The quantity of each species within the biofilm was found to be strictly time dependent. Interestingly, biofilm formation induced coccoid forms of Campylobacter jejuni while reducing culturability, suggesting that bacteria entered a viable but non-culturable state. We hypothesize that the interaction between Campylobacter and Clostridium spp. within the intestinal tract, a good portion of which features microaerobic conditions, is highly important as Campylobacter spp. can pave the way for the growth of Clostridium perfringens, which may cause intestinal infections in humans and animals likewise such as enteritis necroticans.

P056 - Inter-annual trends in stream biofilm bacterial community composition. Lewis, Gillian (University of Auckland, Auckland, NZL); Lau, Kelvin E. (Centre for Microbial Innovation, School of Biological Sciences, NZL) Biofilm from subsets of a panel of stream sites sampled annually in late summer for 4 years (2010 -2013) across New Zealand (between 35 47oS and 165-180oE) have been evaluated for


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the influence of location and catchment land use (Rural, Urban, Undisturbed (native forest), Exotic forest) on bacterial community composition.

In all 380 sites were sampled on 1 or more occasion with 53 sites sampled in 3 or more years. DNA extracts were analysed for 16sRDNA intergenic spacer region diversity (ARISA) as a surrogate to compare bacterial community composition, diversity and richness. For each site 4 -5 replicate samples were collected and analysed separately returning 150 350 peaks per ARISA profile. Data was compared within and between years and within and between regions and broad land uses to evaluate similarity of populations.

There is considerable variation apparent within stream biofilm communities sampled over the 4 years with a strong and significant grouping by year. Principal component analysis plots suggest a shift in community composition over the period with 2012 and 2013 being most similar and 2011 the most different year. Within each year there is strong evidence of regional difference in biofilm bacterial communities with an overall latitudinal trend. There is also a slight influence of landuse type most evident within region. The greatest biofilm bacterial community differences existed between urban and native land use influenced streams.

Analysis for specific community components related to a year, region or landuse was inconclusive although urban stream biofilms gave some indications of a distinctive population profile.

P057 - Cadmium stimulates biofilm formation by Staphylococcus epidermidis Wu, Xueqing (Utrecht University, Utrecht, AUT) Soil contamination with cadmium results in an uptake of this heavy metal by plants and subsequent in an undesirable exposure of humans and animals. While in general adverse health effects of the cadmium are associated with its ability to accumulate in renal tissue, increasing attention is paid to the interaction of heavy metals like cadmium with commensal opportunistic pathogens. A typical example is the metal inducible TCS (Two component system) allowing an adaptive response of bacteria to changing environmental conditions, but also conveying by alterations in gene expression to resistance of bacteria to various classes of antibiotics. TCS regulates also the expression of various quorum sensing genes determining the expression of virulence factors and biofilm formation. Here we describe the effects of cadmium on the biofilm formation by Staphylococcus epidermidis (ATCC 35984), an opportunistic pathogens and well-known type strain for biofilm investigations. Bacteria were in vitro cultured for 24 hours in the absence or presence of cadmium at different concentrations: 0.39, 0.78, 1.56 or 3.13 µM. Biofilm formation was measured by routine safranin staining followed by OD measurement using a spectrophotometer at 540nm wave length. Bacterial viability in the biofilm was determined by using a live/dead assay kit (LIVE/DEAD® Biofilm Viability Kit). Images were recorded by a camera coupled to a confocal microscope (Leica TCS SPE-II, Mannheim, Germany) and analysed with Image J program. Confocal microscopy images were also used to measure biofilm thickness. qRT PCR was used to determine the mRNA expression of molecular markers for S. epidermidis biofilm formation (autolysin E atlE; extracellular matrix binding protein embp), maturation ( intercellular adhesion A and B icaA and icaB) and dispersion (accessory gene regulator B agrB, a regulator of quorum sensing system) in planktonic and biofilm bacteria.


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Results show that S. epidermidis biofilm formation was stimulated (P < 0.001) after exposure to 1.56 and 3.13 µM cadmium, which was confirmed by increased (P < 0.001) biofilm thickness (23 and 22 µm, respectively) when compared to non-treated controls (17.8 µm). Concomitantly, bacterial viability in the biofilm was decreased (P < 0.001) after exposure to cadmium at concentrations of 1.56 (72%) and 3.13 (52%) µM when compared to controls (89%). Up-regulation of atlE (10.5 folds), embp (5.4 folds), icaA (2.6 folds), icaB (2.9 folds) and agrB (3.9 folds) after exposure to 3.13 µM cadmium was observed in planktonic bacteria. No gene dysregulation was observed in biofilm bacteria. In conclusion, at concentrations of 1.56 or 3.13 µM, cadmium stimulates S. epidermidis biofilm formation and adversely affects cell viability. Modification in gene expression was observed in planktonic bacteria but not in those enclosed in the biofilm.

Key words: Cadmium;planktonic bacteria; biofilm formation; gene expression; viability.

P058 - Polymicrobial interactions between Pseudomonas aeruginosa and Staphylococcus aureus influence biofilm formation Armbruster, Catherine (University of Washington, School of Medicine, Seattle, USA); Wolter, Daniel (University of Washington, School of Medicine, Seattle, USA); Parsek, Matthew (University of Washington, School of Medicine, Seattle, USA); Hoffman, Lucas (University of Washington, School of Medicine, Seattle, USA) Introduction: S. aureus (Sa) and P. aeruginosa (Pa) are two of the most prevalent pathogens in the airways of people with cystic fibrosis (CF). Co-infection with both organisms occurs commonly and has been associated with significantly increased morbidity and mortality rates in this patient population. Previous studies have demonstrated interactions between these two species that impact growth and metabolism. However, little is known regarding the impact of polymicrobial interactions on biofilm formation, a bacterial community lifestyle suggested to predominate within CF lungs and to result in persistent infections. We sought to determine whether Sa could modulate Pa biofilm formation in co-cultures.

Methods and Results: Biofilm formation was examined in 96 well microtiter plates using the crystal violet assay with mono and co-cultures of Pa (laboratory strains PAO1 and PA14 and CF clinical isolates) and various strains of Sa. Co-cultures of Pa with Sa resulted in two opposing phenotypes depending on Pa strain: a significant and rapid increase in biofilm biomass (induction) with PAO1, but a significant reduction in biomass (inhibition) with PA14. Both biofilm induction and inhibition were also observed during co-culture of Sa with Pa CF clinical isolates. These results were replicated using Sa cell-free culture supernatants, suggesting that Sa produces an extracellular signal(s) that differentially regulates biofilm formation. Further analysis demonstrated that Pa biofilm induction was attributable to a heat-stable, small molecule, while Pa biofilm inhibition was due to a large molecular weight protein. Using a bioassay-guided fractionation approach followed by LC-MS/MS on Sa supernatant, we identified Staphylococcal Protein A (SpA) as the secreted protein that inhibits strain-specific Pa biofilm formation on abiotic surfaces. Deletion of the gene encoding SpA, spa, in Sa prevented strain-specific Pa biofilm inhibition, and exogenous addition of purified Spa to the growth media led to strain-specific biofilm inhibition of Pa. To define the mechanism of the strain-specific differences in Pa biofilm induction and inhibition, we sequenced the genomes of two clonal CF Pa isolates that respond divergently to the presence of Sa. CF Pa isolates that are inhibited for


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biofilm formation in Sa culture supernatants contained a large genomic deletion encompassing the entire Psl polysaccharide biosynthetic operon. Deletion of a gene required for Psl production (pslD) in isolates with the biofilm induction phenotype resulted in biofilm inhibition upon exposure to Sa supernatant. Complementation of the psl operon in PA14, a Psl and biofilm inhibited strain, protected the strain from biofilm inhibition by Sa supernatant. Finally, purified SpA added exogenously inhibited biofilm formation by a Psl Pa isolate, but not a clonally-related Psl+ isolate.

Conclusion: Sa is able to differentially modulate Pa biofilm formation in a Pa strain-specific manner using at least two secreted products. Pa producing the Psl polysaccharide (Psl+) are induced to form increased biofilm in response to an as-yet unidentified Sa product, whereas Psl Pa are inhibited for biofilm formation by the Sa secreted protein, SpA. SpA also promotes cell-to-cell adhesion and biofilm formation in Sa, and has previously been shown to be abundantly expressed in the CF lung. The effect of SpA on Psl Pa may represent an additional, interspecies social role for SpA that is beneficial for one or both species when co-infecting the CF lung. Understanding how interspecies interactions can influence behaviors that confer persistence and virulence, such as biofilm formation, in these species may lead to a better understanding of chronic lung infection and disease progression in CF.

P059 - Chitin grown biofilms of Vibrio cholerae are grazing resistant due to quorum-sensing regulated chitin metabolism Sun, Shuyang (Singapore Centre on Environmental Life Sciences Engineering, Singapore, SGP); Tay, Martin (Singapore Centre on Environmental Life Sciences Engineering, Singapore, SGP); McDougald, Diane (Singapore Centre on Environmental Life Sciences Engineering, Singapore, SGP) Vibrio cholerae persists in the environment by forming biofilms on chitinous surfaces of zooplankton. Chitin-grown biofilms have access to nutrients and are resistant to many stresses encountered in the marine environment. Predation by heterotrophic protists has a major impact on the survival of V. cholerae and biofilm formation is the main defensive strategy that is expressed under predation stress. Here, we investigated the role of chitin in V. cholerae grazing resistance and demonstrate the quorum sensing (QS) regulation of chitin metabolism provides resistance to predation.

V. cholerae wild type and QS mutant biofilms grown on chitin flakes were exposed to the bacteriotrophic surface-feeding flagellate Rhynchomonas nasuta, and grazing resistance was reflected by biofilm biomass and enumeration of R. nasuta. In addition, the concentration of ammonium produced during chitin degradation was determined. Toxicity of ammonium to R. nasuta was measured as numbers of surviving flagellates in biofilm supernatants and in control medium supplemented with ammonium. Finally, RNA-Seq was used to elucidate the QS regulation of genes involved in chitin metabolism and anti-protozoal activity.

Data show that both WT and QS mutant strains formed more biofilm biomass in the presence of chitin. The growth of R. nasuta was inhibited by WT biofilms grown on chitin flakes compared with non-chitin controls, while the inhibition was attenuated in QS mutant biofilms. Ammonium accumulated in supernatants of V. cholerae biofilms grown on chitin, and the supernatants of


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WT biofilms and medium with ammonium supplementation were inhibitory against R. nasuta. The supernatants of the QS mutant biofilms were lower in ammonium concentration and less toxic to R. nasuta. RNA-Seq revealed that the majority of genes involved in chitin metabolism and chemotaxis were down-regulated in QS mutant biofilms.

Therefore, chitin association not only provides V. cholerae nutrient for growth, but is also a protective niche for long-term persistence of V. cholerae, where QS regulation is critical for chitin metabolism and anti-protozoal activity.

P060 - Bacterial interactions in an intertidal temperate mudflat biofilm of the French Atlantic coast Doghri, Ibtissem (LIENSs Université de La Rochelle, La Rochelle cedex 01, FRA); Lanneluc, Isabelle (LIENSs Université de La Rochelle, La Rochelle cedex 01, FRA); Sablé, Sophie (LIENSs Université de La Rochelle, La Rochelle cedex 01, FRA) Intertidal mudflats are extremely productive areas and may provide up to 50% of the primary and secondary production of estuaries (Underwood and Kromkamp, 1999), especially because of the formation of diatom-dominated biofilms at their surface (Falciatore and Bowler, 2002). Benthic biofilms are mostly composed of water, diatoms, bacteria, eukaryotic microbes and inorganic particles entangled in an extracellular polymeric substances (EPS) matrix (Yallop et al., 2000). In intertidal benthic ecosystems, previous research has shown that diatoms can stimulate the growth of bacteria by secretion of EPS that can be used as a carbon source by the bacterial community (Hanlon et al., 2006). However, no studies have been performed on the bacterial interactions in this kind of biofilms. The aim of our study was to determine and understand the interactions between the bacteria of a microphytobenthic biofilm. The bacteria used in this study were isolated from mudflat biofilms collected from Marennes-Oléron Bay (Atlantic Coast of France) at low tide. One of these bacteria, of the Flavobacterium genus, was able to form a stable biofilm. We used this strain as a model, and we tested the effect of the culture supernatants of the other mudflat strains on biofilm formation and the growth of the Flavobacterium strain. Several supernatants were shown to have activities against this strain. No surfactant or pH effects were detected. The time and heat stabilities and the molecular nature of the active molecules were evaluated.

References:

Falciatore, A., Bowler, C., 2002. Revealing thermolecular secrets of marine diatoms. Annual Review of Plant Biology 53, 109 130.

Hanlon ARM, Bellinger B, Haynes K, Xiao G, Hofmann TA, Gretz MR, Ball AS, Osborn M, Underwood GJC (2006) Dynamics of extracellular polymeric substances (EPS) production and loss in an estuarine, diatom-dominated, microbial biofilm over a tidal emersion-immersion period. Limnol Oceanogr 51:79 93

Underwood, G.J.C., Kromkamp, J., 1999. Primary production by phytoplankton and microphytobenthos in estuaries. Advances in Ecological Research 29, 93 153.


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Yallop, M.L., Paterson, D.M., Wellsbury, P., 2000. Inter-relationships between rates of microbial production, exopolymer production, microbial biomass and sediment stability in biofilms of intertidal sediments. Microbial Ecology 39, 116 127.

P061 - Biofilm Growth Mode Optimizes Carrying Capacity in Cooperative Resource Sharing via Product Inhibition Syntrophy Fields, Matthew (Montana State University, Bozeman, USA); Brileya, Kristen A. (Department of Microbiology and Immunology/Center for Biofilm Engineering, Bozeman, USA); Camilleri, Laura B. (Department of Microbiology and Immunology/Center for Biofilm Engineering, Bozeman, USA) Sulfate-reducing bacteria (SRB) can be syntrophic in the absence of sulfate, particularly with methanogens that can consume the inhibitory H2 and this interaction is important to numerous anaerobic environments. A binary continuous culture approach was used to characterize population structuring within a syntrophic biofilm formed by the SRB Desulfovibrio vulgaris Hildenborough and the methanogenic archaeum Methanococcus maripaludis. Under the tested conditions, monocultures of D. vulgaris formed thin, stable biofilms but monoculture M. maripaludis did not, and these results showed that biofilm initiation was dependent upon the bacterium. Under syntrophic conditions, the biofilm was seeded from a planktonic inoculum that had a ratio of 4 to 1 (DvH:Mm). Confocal laser scanning microscopy of intact biofilm confirmed the role of D. vulgaris in forming a scaffold for the methanogenic biofilm, while intermediate and steady-state images revealed that M. maripaludis was recruited to the biofilm with both single cells and large micro-colonies interspersed throughout the framework of D. vulgaris. M. maripaludis produced more carbohydrate (uronic acid, hexose, and pentose) as a monoculture compared to coculture biofilm, and this result suggested an altered carbon flux. The lactate-

had structure that most likely facilitated mass transfer of H2 and lactate yet maximized biomass with a more even population distribution. The entire biofilm biomass was metabolically active, as shown by staining with a redox dye. Close interactions in structured biofilm also allowed efficient transfer of H2 to M. maripaludis, and H2 was only detected in cocultures where the SRB was a mutant deficient in biofilm formation (∆pilA). Total syntrophic biomass was equivalent in lactate-limited and lactate-excess conditions when a biofilm was present, but in the absence of biofilm, total biomass was significantly reduced. The results suggest that multispecies biofilms create an environment conducive to resource sharing that can result in an increased carrying capacity for cooperative populations.

ENIGMA (www.enigma.lbl.gov) National Center for Genome Resources, Santa Fe, NM

P062 - Shifts in microbial community structure and function in light and dark grown biofilms: when the interactions are major drivers? Romaní, Anna M. (University of Girona, Insitute of Aquatic Ecology, Girona, ESP); Borrego, Carles M. (Group of Molecular Microbial Ecology, University of Girona and ICRA, Girona, ESP); Díaz-Villanueva, Verónica (Lab. Limnología, INIBIOMA-CONICET, Bariloche, ARG); Freixa, Anna


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(Institute of Aquatic Ecology, University of Girona, Girona, ESP); Gich, Frederic (Group of Molecular Microbial Ecology, University of Girona, Girona, ESP); Ylla, Irene (Institute of Aquatic Ecology, University of Girona, Girona, ESP) Microorganisms coexist and interact within biofilms making use of their respective capabilities. The biofilm provides both physical refuge and nutrients from within the polymeric matrix. At the same time, microorganisms compete for available resources from the flowing water and/or the main biofilm. In light-grown biofilms, microbial heterotrophs preferentially use and uptake metabolites released by primary producers, such as algal exudates and lysis by-products. This autotrophic-heterotrophic link reduces the uptake of dissolved organic compounds from the flowing water pool in biofilms growing under illumination, in comparison to those growing in the dark, which appear to be more depending on external organic resources. In this regard, biofilm autotrophic-heterotrophic interactions have mainly been defined in one direction, describing the positive effect of autotrophs on heterotrophic prokaryotes. However, autotrophs can also benefit from heterotrophs. Algae require special vitamins for their growth which are basically synthesized by heterotrophic bacteria. Bacteria may also release extracellular enzymes that degrade complex organic molecules not transportable across cell membranes, making different organic and inorganic compounds available. This autotrophic-heterotrophic relationship has been generally defined as mutualism or co-operation within the biofilm. However, at initial phases of biofilm formation, the great difficulties faced by algae to initiate the biofilm development without accompanying heterotrophic bacteria suggest that a facilitation interaction occurs at this first stage. During biofilm formation, autotrophic-heterotrophic ecological interactions may change from facilitation to mutualism, while competition may also occur specially in mature biofilms. Further insights into interactions in biofilms are highlighted in a recent laboratory experiment where light and dark biofilm were grown and changes in prokaryotic community composition (analyzed by massively parallel sequencing) and functioning (assessed by analyzing extracellular enzymatic activities and carbon substrates utilization) have been intensively analyzed. In this study, changes in the biomass development of the different groups (primary producers, prokaryotes, ciliates and rotifers) were analyzed to investigate the biofilm response to a water temperature increase of 2ºC. Interestingly, at the initial phases of biofilm development (7-day old), there was a higher proportion of archaea than bacteria in the colonizing biofilm than in the mature one (28-day old) suggesting a colonizing role of archaea. However, at the initial phases (7-day old biofilms) neither light nor temperature treatments were relevant for selecting a specific bacterial community but both variables affected composition and function of mature biofilms. In dark-grown biofilms, changes in the prokaryotic community composition due to warming were mainly related to rotifer grazing but no significant changes were observed in functional fingerprints. In light-grown biofilms, warming also affected protozoan densities but its effect on prokaryotic density and composition was less evident. In contrast, heterotrophic metabolic activities in light-grown biofilms under warming showed a decrease in the functional richness and diversity towards a specialized use of several carbohydrates. Results suggest that prokaryotes are functionally redundant in dark-grown biofilms but functionally plastic in biofilms incubated under illumination. The more complex and self-serving light-grown biofilm determines a more buffered response to environmental changes (such as temperature) than dark-grown biofilms. Results also evidenced the key role of protozoans and small metazoans in shaping the prokaryotic biofilm community composition


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raising the necessity to carefully evaluate grazing activities when modeling biofilm interactions at the microbial level.

P063 - Biofilm Growth and Particle Size Relationships Altenburg, Sara (Montana State University, Bozeman, USA); Zelaya, Anna (Montana State University, Bozeman,, USA); Arkin, Adam P. (Lawrence Berkeley National Laboratory, Berkeley,, CAN); Fields, Matthew (Montana State University, Bozeman, USA)

Sulfate-reducing-bacteria (SRB) occur naturally in a variety of anaerobic environments where sediments are present. In order to investigate the impact of physical surface scale on microbial interactions occurring in anaerobic habitats, attempts were made to standardize the growth of Desulfovibrio biofilm on various particle sizes using modified biofilm reactors. The standard coupon holders were modified to contain a mass of particles with continuous access to nutrients and Desulfovibrio culture, thus providing a surface for biofilm formation that could be easily removed at the end of the study period. The reactor systems have used environmental isolates, Desulfovibrio RCH1 (Hanford) and Desulfovibrio FW1012B (Oak Ridge) to characterize growth on glass beads (30

and ranged from 1500 to 58 to 20 cm-1

was 25-fold and 50-fold greater for the intermediate and largest sized particles, respectively, compared to the smallest. A similar trend was observed for biofilm carbohydrate (17- and 40-fold increased) compared to the smallest bead size. However, the overall biofilm carbohydrate to protein ratio was similar for the tested particle sizes (0.11, 0.07, 0.09, respectively). Because the tested particle sizes are significantly larger than the dimension of cells, we propose that initial colonization of beads is not mass transfer limited. For the particle sizes tested, the amount of biofilm per unit area decreased with the particle size even as surface area/volume increased.

ENIGMA-http://enigma.lbl.gov

P064- Biofilms and the adaptation of Thiomonas spp. to arsenite Farasin, Julien (GMGM - UMR 7156, Strasbourg, FRA); Freel, Kelle (GMGM - UMR 7156, Strasbourg, FRA); Peres, Martina (GMGM - UMR 7156, Strasbourg, FRA); Bertin, Philippe N. (GMGM - UMR 7156, Strasbourg, FRA); Arsène-Ploetze, Florence (GMGM - UMR 7156, Strasbourg, FRA) Biofilms are known to be involved in the resistance and tolerance to toxic levels of metal ions1, and can increase the genetic diversity via HGT2 or by inducing higher mutations rates3. Sites of acid mine drainage (AMD) contain high concentrations of toxic metals and are an ideal location to study microbial ecology and evolution4 . Several strains of Thiomonas sp. have been isolated from the arsenic-rich AMD of Carnoulès (Gard, France), and a recent comparative genomic study suggested that they evolved through gain or loss of genomic islands5. Another set of experiments demonstrated that Thiomonas sp. biofilm architecture varies in the absence or presence of arsenite (As(III)) and that variants more resistant than the initial population could emerge6. Thus, biofilms may have played a role in the adaptation of Thiomonas sp. In our current work, we have continued to elucidate the mechanisms whereby life in a biofilm could promote adaptation to As(III) in Thiomonas spp. First, we determined that the strains studied vary concerning mobility and the ability to form biofilms. Then, we assessed the viability and resistance to As(III) of the isolates, and found clear differences between biofilms vs.


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planktonic growth conditions, as well as various concentrations of As(III). Finally, the rate of variants with a higher resistance to As(III) appear to be more important in biofilm than in planktonic form. Further studies, including comparative genomics between variants, will allow us to better understand the genetic background which might confer higher tolerance to As(III).

Bibliography 1. Harrison, J. J., Ceri, H. & Turner, R. J. Multimetal resistance and tolerance in microbial biofilms. Nat. Rev. Microbiol. 5, 928 938 (2007). 2. Madsen, J. S., Burmolle, M., Hansen, L. H. & Sorensen, S. J. The interconnection between biofilm formation and horizontal gene transfer. FEMS Immunol. Med. Microbiol. 65, 183 195 (2012). 3. Conibear, T. C. R., Collins, S. L. & Webb, J. S. Role of Mutation in Pseudomonas aeruginosa Biofilm Development. PLOS ONE 4, (2009). 4. Baker, B. J. & Banfield, J. F. Microbial communities in acid mine drainage. Fems Microbiol. Ecol. 44, 139 152 (2003). 5. Arsene-Ploetze, F. et al. Structure, Function, and Evolution of the Thiomonas spp. Genome. Plos Genet. 6, e1000859 (2010). 6. Marchal, M. et al. Subinhibitory Arsenite Concentrations Lead to Population Dispersal in Thiomonas sp. Plos One 6, e23181 (2011).

P065 - Coaggregation among Rhodococcus and Acinetobacter strains isolated from the food industry Møretrø, Trond (The Norwegian Institute of Food, Fishery and Aquaculture Research , Aas, NOR); Sharifzadeh, Shahab (Nofima, the Norwegian Institute of Food, Fisheries and Aquaculture Research, Aas, NOR); Rickard, Alexander H. (The University of Michigan, School of Public Health, Department of Epidemiology, Ann Arbor, USA); Heir, Even (Nofima, the Norwegian Institute of Food, Fisheries and Aquaculture Research, Aas, NOR); Langsrud, Solveig (Nofima, the Norwegian Institute of Food, Fisheries and Aquaculture Research, Aas, NOR) Different types of bacteria may bind specifically to each other and form coaggregates, which may have implications for the spatial organisation of biofilms and recalcitrance to antimicrobials.

In the present study coaggregation between strains of Acinetobacter and Rhodococcus that were isolated from the food processing surfaces was investigated. Three strains of Rhodococcus and eight Acinetobacter were grown in tryptic soy broth at 30 °C, washed, the resulting suspensions mixed, and formation of coaggregates was observed visually and spectrophotometrically. The mechanisms of coaggregation were studied by treatment of suspensions with heat and enzymes and in competitive binding assays with different sugars.

Strain specific coaggregation was observed. One strain of Rhodococcus formed coaggregates with two strains of Acinetobacter and another strain of Rhodococcus. Stronger coaggregation was observed for cells cultivated at 30 vs 20 °C, in TSB vs R2A growth medium and for cells from exponential or early stationary phase vs late stationary phase. The formation of coaggregates was promoted in the presence of mineral salts. For three of the strains the coaggregation factor seems to be proteinaceous.


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Coaggregation was observed between bacteria from food industry, and has previously been shown to be important for structure of oral biofilms. Further studies are needed to determine the impact of coaggregation on survival of bacteria in the food industry.

P066 - Toxic biofilms - building a better biofilm while keeping the neighbours out King, Andrew (Centre for Immunology and Infection and the Department of Biology, University of York, York, GBR); Ruhe, Zachary (Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, USA); Low, David (Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, USA); Hayes, Christopher (Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, USA); van der Woude, Marjan (Centre for Immunology and Infection, Hull York Medical School and the Department of Biology, University of York, York, GBR) Biofilms are a highly competitive environment in which bacteria have to grow in enforced extended contact with other bacterial strains, both in the environment and during infections. Such a situation presents an opportunity in which expression of contact-dependent toxins could play a significant role in increasing competitive fitness. A novel toxin family of this type comprises the contact-dependent inhibition of growth (CDI) toxin systems which, to date, have been studied almost exclusively in planktonic systems. Here we describe the role of CDI toxin systems in biofilm formation. Understanding this toxin family will increase our knowledge of inter-species competition in naturally occurring biofilms.

Bacteria forming a biofilm are faced with two potentially conflicting challenges. Firstly, maximising biofilm formation rate to obtain the benefits of the biofilm mode of growth in the shortest possible time. Adhesion to a surface and to each other help meet this challenge. Secondly, bacteria have to out-compete other strains that are present in the natural environment. Inhibiting growth of competitors will facilitate success in this regard. We will present data showing that a CDI system present in E. coli enhances not only inter-strain competitiveness, but also biofilm formation.

The CDI system was initially identified in E. coli strain EC93 by Low and colleagues. We undertook studies to examine the role of this CDI system in competition during biofilm formation, using a flow cell system. These experiments showed that CDI is an effective toxin system under these growh conditions. However, during these studies it became apparent that EC93 strains with a CDI toxin system deletion had a significant reduction in their rate of biofilm formation. Further studies showed only the core of the CdiA protein was required to confer adhesin-like properties through enhanced cell-cell interactions. Increased adhesion between cells presumably aids biofilm formation both by minimising losses to shedding and by allowing capture of shed cells from nearby clonal microcolonies. However, capture of cells from closely related strains may be detrimental due to increased competition for shared resources. This may be counterbalanced by toxin activity.

CDI toxins include a wide variety of toxin types and, in order to increase understanding of their different potential roles in biofilm competition, analyses of two toxin types were undertaken. Using time-lapse microscopy and computational analysis, the time course of target cell intoxication by EC93 (proton motive force disruption) and Ec869o11 (nuclease) CDI toxins was


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quantified at the single cell level. Our analysis shows that the dynamics of target cell intoxication are remarkably different depending on the class of CDI toxin. Altering early stage biofilm formation by CDI cell intoxication is predicted to significantly impact mature biofilm formation between closely related strains. The effect of different dynamics of intoxication is discussed with simulation data.

In summary, our data show that the EC93 CDI system has two distinct properties that together confer advantages on the strain in biofilm formation and inter-strain competition. Work is ongoing to correlate the single cell level toxin data to competition outcomes in bacterial flow cell biofilms.

P067 - The biofilm granulation mechanisms depend on the predominant bacterial populations involved Weissbrodt, David G. (ETH Zürich, Zürich, CHE); Neu, Thomas (Helmholtz Centre for Environmental Research - UFZ, Magdeburg, GER); Holliger, Christof (Ecole Polytechnique Fédérale de Lausanne, Lausanne, CHE) Introduction Aerobic granular sludge used for intensified biological wastewater treatment is based on fast-settling mobile biofilms with a gel-like consistence,called "granules",formed from activated sludge flocs (Seviour et al. 2012). Fundamental aspects of the mechanisms of microbial selection and self-immobilisation as spherical biofilms have recently been studied in detail (Weissbrodt et al. 2013).In an approach combining biofilm engineering, molecular microbial ecology, and laser scanning microscopy, we provide insights in the relationships between bacterial selection mechanisms, predominant populations involved,and resulting biofilm architectures in the granular sludge microbiome. Methods Biofilm granulation mechanisms were followed in anaerobic-aerobic sequencing batch reactors inoculated with activated sludge and operated under conditions that selected for populations displaying distinct physiologies.Bacterial population dynamics were analyzed using the PyroTRF-ID molecular workflow combining community fingerprinting by terminal-restriction fragment length polymorphism and amplicon-pyrosequencing targeting the v1-v3 region of the 16S rRNA gene pool (Weissbrodt et al. 2012).Microbial structures were followed over time by laser scanning microscopy (Neu et al 2010)combined with overall biomass staining using Rhodamine 6G,fluorescence lectin-binding analysis of glycoconjugate matrices, and 16S rRNA based fluorescence in situ hybridization targeting predominant phylotypes. Results Under wash-out conditions selecting for a fast-settling biomass and non-limiting amount of biodegradable organic substrate in the aeration phase, fast-growing heterotrophs affiliating with Zoogloea spp. proliferated and formed smooth homogeneous granular biofilms by swelling and outgrowth of microbial colonies around flocs.Under mid-mesophilic temperature and low-aeration shear stress, filamentous structures of Burkholderiales relatives penetrated outside aggregates and resulted in unfavorable slow-settling fluffy architectures. As soon as granular sludge accumulated in the system and organic loads were fully removed anaerobically prior to


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aeration, slower-growing populations of nitrifiers and of polyphosphate-("Ca. Accumulibacter")and glycogen-accumulating organisms("Ca.Competibacter") proliferated from granule cores outwards as heterogeneous compact colonies inside the zoogloeal continuous matrices.Enrichment cultures of the two latter guilds validated the mechanism of development of granular conglomerates by proliferation as compact clusters in the floc structure.Mature granular biofilms that removed all nutrients biologically exhibited a relatively high bacterial diversity (H'>3) comprising side populations with tentative metabolisms of exopolysaccharide production and consumption related to Xanthomonadaceae,Sphingomonadales,Rhizobiales,and Sphingobacteriales,respectively.The structural gelling agent of these multi-species biofilms was composed of a complex mixture of glycoconjugate matrices as revealed by lectine-binding analysis on different levels of resolution going from the biofilm continuum,to large microbial clusters,microcolonies,and cells. Conclusion and Outlook The combination of high-resolution datasets of microbial ecology and microscopy highlighted that the biofilm granulation mechanisms depend on the predominant organisms selected by the operation conditions and on their physiological properties. Granular sludge biofilms might in addition be considered as complex ecosystems of glycoconjugate producers and consumers. References Neu TR, Manz B,Volke F,Dynes JJ,Hitchcock AP & Lawrence JR (2010) FEMS Microbiol Ecol 72(1):1-21. Seviour T, Yuan Z,van Loosdrecht MCM & Lin Y (2012) Water Res 46(15): 4803-4813. Weissbrodt DG,Neu TR,Kuhlicke U,Rappaz Y & Holliger C (2013) Front Microbiol 4:175. Weissbrodt DG,Shani N, Sinclair L,Lefebvre G,Rossi P,Maillard J, Rougemont J & Holliger C (2012) BMC Microbiol 12:306. P068 - Development of three-dimensional structure of biofilms in the River Rhine and colonization with microbes Scherwass, Anja (University of Cologne, Biocenter, General Ecology, Cologne, GER); Budde, Heidrun (University of Cologne, Biocenter, Cologne, GER); Arndt, Hartmut (University of Cologne, Biocenter, Cologne, GER) The structure and development of microbial biofilms in the River Rhine were investigated over a period of six months. The 3D-Structure was analysed by staining the exopolymeric matrix with fluorochromes (DTAF for staining of polysaccarides and proteins) and by further analysis at the Laser-Scanning-Microscope. Additonally, the occurrence of the bacterial community within the microbial biofilms was observed. We investigated short-term (3 weeks) as well as longterm (up to 6 months) exposed substrates regarding the developed biofilm. A variety of three-dimensional structures could be registered and several charastics parameters were analysed quantitatively (like e.g. coverage of substrate, height of the biofilm etc.) and compared between the two different durations of exposure.


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P069 - Spatial patterns of biodiversity and community composition in stream biofilms Besemer, Katharina (Institut für Philosophie, AUT); Singer, Gabriel (Abteilung für Ökohydrologie, Leibniz-Institut für Gewässerökologie und Binnenfischerei, Berlin, GER); Quince, Christopher (School of Engineering, University of Glasgow, Glasgow, GBR); Bertuzzo, Enrico (Laboratory of Ecohydrology, École polytechnique fédérale de Lausanne, Lausanne, CHE); Battin, Tom J. (University of Vienna, Department of Limnology and Oceanography, Vienna, AUT) Biofilms dominate microbial life in streams and fulfill critical ecosystem functions therein. The dendritic landscapes formed by streams and rivers create the spatial template for biodiversity patterns. It remains yet unknown whether the geometry of fluvial networks constrains the patterns of microbial diversity similar to that of larger organisms. We investigated microbial biodiversity and community composition in benthic biofilms from 114 streams using 454 pyrosequencing to elucidate the role of network topology and environmental variation for microbial diversity patterns at the network level. We found that alpha diversity and evenness were reduced downstream of confluences compared to the tributary pairs upstream. At network scale, alpha diversity decreased from headwaters downstream, obviously contrasting biodiversity patterns reported for higher organisms. Further, alpha diversity was related to dissolved organic carbon properties and the predominance of benthic cyanobacteria. Beta diversity was higher in headwaters than in larger streams, which is consistent with patterns reported from invertebrates and evokes dispersal limitation as a potential driver of community composition. Our findings extend our understanding of the relevance of fluvial network geometry for biodiversity patterns to the microbial world.

P070 - Influence of low temperature and presence on morphology of Pseudomonas fluorescens biofilms H. Puga, Carmen (Univ. Complutense of Madrid, Madrid, ESP); SanJosé, Carmen (Univ. Complutense of Madrid, Madrid, ESP); Orgaz, Belén (Univ. Complutense of Madrid, Madrid, ESP) In the food industry, refrigeration is a selective advantage for psychrotrophs´ proliferation and persistence. Pseudomonas fluorescens and Listeria monocytogenes are frequently found in food industry facilities due to their capacity of growing at low temperatures and ability to form biofilms. Single species L. monocytogenes biofilms are generally modest in terms of matrix production. The association with P. fluorescens, that is clearly superior in this respect, may counter its limitations. For further insight into this, mixed biofilms with P. fluorescens and several strains of L. monocytogenes (a persistent meat industry strain and Scott A) were obtained at 20 ºC and 4ºC.

structural parameters together with the matrix/cells ratio were calculated. The effect of the association Pseudomonas-Listeria seems to reduce the volume of the biofilm compared with the monospecies Pseudomonas biofilms, although total counts were higher in dual-species biofilms, suggesting that the presence of Listeria not only did not contribute to increase matrix levels but also implied structural changes. The matrix/cells ratio was slightly higher in old biofilms, especially at 20º C, probably due to the dispersal of cells from the top layers. These results suggest that both the ecological relationships and certain external factors, such as


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temperature, might influence not only community behavior but also structural patterns and thus biocide effectiveness in biofilms.

P071 - Isolation and Biochemical Characterization of Underwater Adhesives from Diatoms Poulsen, Nicole (TU Dresden, Dresden, GER); Buhmann, Matthias (TU Dresden, Dresden, GER); Kröger, Nils (TU Dresden, Dresden, GER) Diatoms are unicellular algae that have the impressive capability to colonize any natural and man-made submersed surfaces. There is great technological interest in both mimicking and preventing diatom adhesion, yet the responsible biomolecules have so far remained unidentified. Surface adhesion of diatoms belonging to the raphid pennate type requires the secretion of adhesive mucilage strands through a dedicated slit in the silica cell wall, which is termed the raphe. Adhering cells can then move across the surface, which is accomplished through an actin and myosin dependent cytoskeletal motor that translocates the adhesive mucilage strands in a rearward direction through the raphe thereby moving the cell forward. When the adhesive mucilage strands reach the end of the raphe they are released and left as a trail behind the moving cell. Previous attempts to characterize the adhesive trails from diatoms have been hampered by the difficulty of removing the cells from the adhesive material. We have developed a method for isolation of diatom adhesive material, that allowed us to determine its amino acid and carbohydrate composition free of cellular contaminations. The adhesive material isolated from two model fouling diatoms (Amphora coffeaeformis and Craspedostauros australis) show differences in their amino acid and carbohydrate compositions, but also share characteristic features including a high content of uronic acids, the predominance of hydrophilic amino acid residues, and the presence of 3,4-dihydroxyproline, an extremely rare amino acid. Notably, proteins bearing dihydroxyphenylalanine (Dopa), which mediate underwater adhesion of mussels, are absent from the adhesive material of diatoms. The data on the composition of diatom adhesives are consistent with an adhesion mechanism based on complex coacervation of polyelectrolyte like biomolecules.


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Session 5: Omics for the study of biofilm structure and function

P072 - Bacterial mats from a closed uranium mine in Kowary. Tomczyk-Zak, Karolina (Institute of Biochemistry and Biophysics PAS, Warsaw, POL); Obszanska, Katarzyna (Institute of Biochemistry and Biophysics PAS, Warsaw, POL); Skrobot, Joanna (Institute of Biochemistry and Biophysics PAS, Warsaw, POL); Zielenkiewicz, Urszula (Institute of Biochemistry and Biophysics PAS, Warsaw, POL) In a closed uranium mine in Kowary located in Silesia ( south-west Poland ) two types of microbial mats were discovered: one developing on the water surface (UM) and the other underwater (MU). In this area, the content of uranium is varied and generally low in both heaps and waters. In our study, we examined the ion content of mats surface, their bacterial diversity and autotrophic potential. SEM-EDS analysis showed intrusions of rare earth elements inside the mats and the presence of numerous bacteria with manganese ions on their surface. Culture-dependent and independent methods were performed to analyze the biodiversity of the mats. The culture independent strategy deployed pyrosequencing of 16S rRNA amplified from total DNA isolated from the mat samples. Autochthonous microorganisms from uranium mine can be used in uranium bioleaching and bioremediation. Therefore, the autotrophic potential of microorganisms present in the mats was studied. Fragments of the cbbL gene of the large subunit of ribulose-1,5-bisphosphate carboxylase were amplified from total DNA of the mats and pyrosequenced.

In the culture dependent strategy, we isolated and identified 51 different strains from UM and 26 different strains from MU. Our observations showed that the mat developing underwater (MU) was much less diverse than the one developing on the water surface (UM), which was probably related to limited concentration of oxygen in water. Both mats were dominated by different Pseudomonas strains. The rest of identified bacteria were classified to the following genera: Leifsonia (10%), Arthrobacter (10%), Janthinobacterium (4%), Microbacterium (2%), Cellulomonas (2%), Brachybacterium (2%) from UM mat and Flavobacterium (4%), Stenotrophomonas (4%), Aeromonas (4%) from MU mat. In both cases about 10% of isolated microorganisms remained unclassified. Actinobacteria isolates were further characterized in terms of growth conditions, the ability to utilize different carbon sources, sensitivity to antibiotics and secondary antibacterial metabolites secretion. All tested strains showed antibacterial activity against Bacillus subtilis and Staphylococcus aureus . Our research also demonstrated resistance to five popular antibiotics in the majority of the Actinobacteria isolates.

P073 - Ser/Thr/Tyr phosphorylation map of non-extracellular and extracellular proteins of Pseudomonas aeruginosa PA14 strain OUIDIR, Tassadit (UMR 6270, Polymères, Biopolymères, Surfaces Laboratory, Mont Saint Aignan, FRA); Jarnier, Frédérique (UMR 6270, Polymères, Biopolymères, Surfaces Laboratory, Mont Sait Aingan, FRA); Cosette, Pascal (UMR 6270, Polymères, Biopolymères, Surfaces Laboratory, Mont Saint Aignan, FRA); Jouenne, Thierry (UMR 6270, Polymères, Biopolymères, Surfaces Laboratory, Mont Saint Aingnan, FRA); Hardouin, Julie (UMR 6270, Polymères, Biopolymères, Surfaces Laboratory, Mont Saint Aignan, FRA)


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Pseudomonas aeruginosa is a well-known opportunistic, nosocomial pathogen that causes chronic infections in immunocompromised individual. Its virulence is related to its immense battery of virulence factors, which are regulated by a complex hierarchy of regulators1These virulence determinants are involved in colonization , survival and tissues invasion. Since protein phosphorylation is known to be involved in signal transduction and bacterial virulence2, the characterization of the Ser/Thr/Tyr phosphorylated proteins of P. aeruginosa is essential for gaining insights into the phosphoproteins involved in this pathogenesis.

In this work, we undertook a proteomic approach to establish the repertory of both non-extracellular and extracellular phosphorylated proteins on Ser/Thr/Tyr of PA14 strain. We used phosphopeptides enrichment through titanium dioxide and high resolution/high accuracy liquid chromatography-mass spectrometry to characterize phosphoproteins. In the case of the non-extracellular proteins, we identified 73 phosphopeptides (83 phosphosites) belonging to 63 proteins. Among them, all the proteins involved in a cell-surface signaling (CSS) system were, for the first time, characterized as phosphorylated. This CSS system seems to be involved in the response to the inducing signal and in the regulation of virulence. In the second part, we focused, for the first time, on the characterization of the phosphorylated extracellular proteins of the PA14 strain. Twenty-eight phosphoproteins were identified on Ser/Thr/Tyr residues. Major enzymes (i.e., elastase LasB, LasA protease, chitin binding protein,…) known as potent virulence factors in P. aeruginosa were identified and interestingly, they presented multiple phosphorylation sites.

This proteomic investigation provided a total of 73 phosphoproteins of P. aeruginosa (55 new bacterial phosphorylated proteins) which constituted the foundation for further investigations, and noticeably for mutagenesis studies to confirm or not the involvement of these phosphorylations in the virulence of the P. aeruginosa PA14 strain. In fact, the demonstration of a direct relationship between Ser/Thr/Tyr phosphoproteins and bacterial virulence presents a great importance in deciphering the molecular and cellular mechanisms that underlie pathogenesis.

1 : Luckett et al., (2012) PLoS Pathog 8(8): e1002854. doi:10.1371/journal.ppat.1002854. 2 : Ge et al., (2011) Proteomics 11 : 1449 1461.

P074 - Bacterial surface sensing: Phenotype of adherent bacteria depends on abiotic surface properties Bruzaud, Jérôme (INRA, AgroParisTech, UMR1319 Micalis B2HM, Massy, FRA); Guilbaud, Morgan (AgroParisTech, INRA, UMR Micalis Equipe Bioadhésion Biofilm et Hygiène des Matériaux, Massy, FRA); Chevalier, Sylvie (Normandie Université, Université de Rouen, Laboratoire de Microbiologie Signaux et Micro-environnement EA 4312, Evreux, FRA); Maillot, Emeline (Normandie Université, Université de Rouen, Laboratoire de Microbiologie Signaux et Micro-environnement EA 4312, Evreux, FRA); Guillot, Alain (INRA, PAPPSO, Jouy-en-Josas, FRA); Monnet, Véronique (INRA, PAPPSO, Unité de Biochimie Bactérienne, UR477, Jouy-en-Josas, FRA); Herry, Jean-Marie (INRA, AgroParisTech, UMR Micalis Equipe Bioadhésion Biofilm et Hygiène des Matériaux, Massy, FRA); Bellon-Fontaine, Marie-Noëlle (AgroParisTech, INRA, UMR Micalis Equipe Bioadhésion Biofilm et Hygiène des Matériaux, Massy, FRA)


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Biofilms are organized communities of aggregated bacteria that are found on most surfaces in natural and industrial environments, featuring a high resistance to external stress and could potentially become a threat to public health when involving pathogenic strains. Among the strategies developed to control biofilm formation, the prevention of surface colonization by bacteria requires the study of bacterial adhesion. In this field, a recent study showed that surface softness induces change in protein expression of colonizing adherent cells, prior to the

the surface and modify its phenotype was investigated. We studied the phenotype of adherent Pseudomonas aeruginosa, a pathogenic model for biofilm formation, to different substratum in the early phase of surface colonization to determine if the bacterial physiology could be modified by abiotic surface properties. Adhesion of bacteria in physiological water onto three materials of different wettability (stainless steel, polyethylene terephthalate and glass) were performed and followed by virulence evaluation on a vegetal model, growth rate quantification and total proteome analysis by liquid chromatography coupled to tandem mass spectrometry. Data revealed reduction in production of virulence factors and outer-membrane proteins on stainless steel compared to the other substrates associated with a reduced phytopathogenic virulence on endive leaves. Total proteome analysis showed a substantial bacterial response to surface with 89 proteins differentially produced between the three conditions. In conclusion, we showed that the phenotype of adherent bacteria depends on the substratum properties. Protein expression and virulence are both influenced by the properties of the surface.

properties that are involved in such response should now be investigated in order to better understand what triggers observed phenotype changes.

P075 - Molecular diversity of microbial biofilms in a small stream, Czech Rebublic Buriánková, Iva (Faculty of Science, Palacky University, Olomouc-Holice, CZE); Brablcova, Lenka (Faculty of Science, Palacky University, Olomouc, CZE); Rulík, Martin (Faculty of Science, Palacky University, Olomouc, CZE) Molecular diversity of bacteria in different biofilm substrata (stream sediment, underwater tree

Czech Republic. The biofilm communities were analyzed by PCR amplification, cloning, and sequencing, fluorescence in situ hybridization and denaturant gradient gel electrophoresis. Sequencing analysis revealed phylotypes affiliated with nine bacterial orders mainly with Burkholderiales and Acidobacteriales members. All applied techniques detected considerable differences among biofilm substrata studied. The stream stone was the substratum where bacterial community was the most diverse. The authors are thankful to the European Social Fund and state budget of the Czech Republic. This work was partly supported by project CZ.1.07/2.2.00/28.0032, which is financed by the previously stated funding agencies.


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P076 - Comparative proteomic study of development of Mycobacterium smegmatis biofilm on the solid organic surfaces Petrackova, Denisa (Institute of Microbiology AS CR, v.v.i., Prague 4, CZE); Ramaniuk, Olga (Institute of Microbiology AS CR, v.v.i., Prague 4, CZE); Bezouskova, Silvia (Institute of Microbiology AS CR, v.v.i., Prague 4, CZE); Banada, Oldrich (Institute of Microbiology AS CR, v.v.i., Prague 4, CZE); Kofronova, Olga (Institute of Microbiology AS CR, v.v.i., Prague 4, CZE); Halada, Petr (Institute of Microbiology AS CR, v.v.i., Prague 4, CZE); Weiser, Jaroslav (Institute of Microbiology AS CR, v.v.i., Prague 4, CZE) Biofilm formation is one of the most common bacterial survival strategies . Majority of bacterial species are able to form these three-dimensional structures, including pathogens like Mycobacterium tuberculosis. Representatives of Mycobacterium genus widely occur in the nature and they can also cause serious problems when they accommodate in medical devices and artificial organs in the human body.

Non-pathogenic and fast growing Mycobacterium smegmatis mc2 155 was used as a model organism. First part of work was focused on the biofilm morphology. The morphology of 3 or 6-day-old colonies of M. smegmatis were investigated by using optical stereo microscope and scanning electron microscope (SEM). The bacterial colonies were grown on agar or on agar covered with cellophane sheet. Cellophane was chosen as an alternative surface due to its properties and successful use in cultivation of streptomycetes, close relatives of Mycobacteria.

Second part of work was focused on the analysis of cytoplasmatic proteomes of M. smegmatis. Protein samples from the agar and cellophane cultures and from planktonic culture of bacteria grown in liquid medium of the same composition were isolated. A proteomic analysis approach, based on 2D electrophoresis, was used to identify and characterize expression levels of proteins associated with development of M. smegmatis biofilm. Using subtractive analyses (software PDQuest 8.0.1, BioRad) we predicted proteins which might play crucial role during planktonic growth and biofilm formation. The sets of proteins detected by qualitative and quantitative analyses were compared using Venn diagrams. As a result, we recognized 2 proteins that might be specific for recognition and adhesion of bacteria to the cellophane sheet. Other 2 proteins are specific for on the solid surfaces grown biofilm. Quantitative analysis revealed 9 proteins with different expression, 6 proteins of these were up-regulated during biofilm formation.

Our results show that, although morphological differences of colonies grown on agar and agar covered with cellophane are remarkable the changes in protein profiles of these cells are much less significant. Understanding of gene expression background of observed morphological differences would require further investigation of cell wall proteomes and on them exposed proteins.


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P077 - COMPARATIVE MEMBRANE PROTEOMIC ANALYSIS BETWEEN Staphylococcus epidermidis CECT 231 GROWN IN BIOFILM AND PLANKTONIC CONDITIONS Alkorta, Itziar (University of the Basque Country, Leioa, ESP); Águila-Arcos, Sandra (University of the Basque Country, Leioa, ESP); Ding, Shujing (The Medical Research Council Mitochondrial Biology Unit, Cambridge , GBR); Carroll, Joe (The Medical Research Council Mitochondrial Biology Unit, Cambridge , AUT); Aloria, Kerman (Universidad del País Vasco UPV/EHU, Leioa, ESP); Arizmendi, Jesus Mari (University of the Basque Country, Leioa, ESP); Fearnley, Ian (The Medical Research Council Mitochondrial Biology Unit, Cambridge , GBR); Walker, John E. (The Medical Research Council Mitochondrial Biology Unit, Cambridge , GBR); Goñi, Félix M. (Universidad del País Vasco UPV/EHU, Leioa, ESP) Staphylococcus epidermidis has emerged as one of the major nosocomial pathogens associated with infections of implanted medical devices. The most important factor in the pathogenesis of these infections is the formation of bacterial biofilms. Since bacteria grown in biofilms are more resistant to antibiotics and to the immune defense system than planktonic bacteria, antimicrobial therapy fails without removal of the implanted device. In this study three proteomic approaches were performed to investigate membrane proteins associated to biofilm formation: i) sample fractionation by gel electrophoresis, followed by isotopic labelling and LC-MS/MS analysis, ii) in-solution sample preparation, followed by isotopic labelling and LC-MS/MS analysis, and iii) in-solution sample preparation and label-free LC-MS/MS analysis. This is the first comparative membrane proteomic study between S. epidermidis CECT 231 cultivated under biofilm and planktonic conditions. We found that cells in biofilms expressed higher levels of five proteins associated with pathogenesis (accumulation associated protein, staphylococcal secretory antigen, signal transduction protein TRAP, ribonuclease Y and phenol soluble molubdin beta 1). A further analysis of these proteins is required to understand their role in the biofilm formation as targets to develop new strategies for prevention and treatment of biofilm infections.

P078 - PROTEOMICS STUDY ON THE EXTRACELLULAR PROTEOME OF BURKHOLDERIA CENOCEPACIA GROWN UNDER BIOFILM AND PLANKTONIC CONDITIONS Goza, Agata (Ghent University, Gent, BEL); Brackman, Gilles (Laboratory of Pharmaceutical Microbiology, Gent, BEL); Coenye, Tom (Laboratory of Pharmaceutical Microbiology, Gent, BEL); Valvano, Miguel (Queen's University Belfast, Centre for Infection and Immunity, Belfast, GBR); Devreese, Bart (Laboratory for Biochemistry and Biomolecular Engineering L-ProBE, Gent, BEL) Burkholderia cenocepacia has emerged as problematic cystic fibrosis pathogen due to antibiotic resistance and high transmissibility. It develops in biofilms on lung epithelial cells and can survive intracellularly in macrophages, influencing antibiotic susceptibility.

Recent evidence suggests cooperation between the type II and type VI bacterial secretory pathways in intracellular bacterial survival (Rosales-Reyes, 2012). However, little is known about the actual extracellular proteins secreted by these two systems.

The main objective of this study was to characterize the secreted protein repertoire of T2SS and T6SS of B. cenocepacia K56-2 under the biofilm and planktonic mode of growth.


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The extracellular proteins present in the culture supernatant of B. cenocepacia WT, ∆T2SS, ∆T6SS strain and grown under biofilm conditions employing a CDC bioreactor model with a synthetic CF sputum medium, were analyzed by Gel-LC-MS/MS using an LTQ-FTICR mass spectrometry system.

Comparing secreted proteins from various strains revealed their dependency from T2SS or T6SS in a quorum sensing dependent manner. Apart of known T2SS-dependent virulence factors like ZmpA, ZmpB and phospholipase C, we identified a range of hydrolytic enzymes and solute transporters. For instance, we discovered a putative chitinase that might interact with host cell glycans contributing to a pathogenic potential of bacteria. Moreover, some proteins do exhibit clear moonlighting properties.

Rosales-Reyes R, Skeldon AM, Aubert DF, Valvano MA. Cell Microbiol.14(2):255-73

P079 - DNase and proteinase K impair Listeria monocytogenes biofilm formation and induce dispersal of pre-existing biofilms Nguyen, Uyen (McMaster University, Hamilton, CAN); Burrows, L.L. (Dep. of Biochemistry and Biomedical Sciences / Michael G. DeGroote Institute for Infectious Diseases Research, Hamilton, CAN) Introduction: Current sanitation methods in the food industry are not always sufficient for prevention or dispersal of Listeria monocytogenes biofilms, which can result in food product contamination. Here, we determined if prevention of adherence or dispersal of existing biofilms could occur if biofilm matrix components were disrupted enzymatically.

Methods: L. monocytogenes 568 biofilms were grown on 96-peg lids for 72h in the presence or absence of DNase or proteinase K. In the biofilm dispersal assay, biofilms were grown for 72 h and then treated with enzymes for various incubation times (5 min 24 h). Biofilms were quantified using crystal violet staining. Scanning electron microscopy was used to visualize the amount of biofilm remaining following treatment with 50-food-grade type 304H stainless steel.

Results: Addition of DNase during biofilm formation reduced attachment (<50% of control) to

biofilm dispersal, with <25% biofilm remaining compared to control. In contrast, addition of proteinase K completely inhibited biofilm formation and 72 h biofilms - including those grown under stimulatory conditions - Generally-regarded-as-safe proteases bromelain and papain were less effective dispersants than proteinase K. In a time course assay, complete dispersal of L. monocytogenes biofilms from both polystyrene and type 304H food-grade stainless steel occurred within 5 min at concentrations ≥

Conclusions: These data suggest that both DNA and proteins are required for L. monocytogenes biofilm development and maintenance, and that these components of the biofilm matrix can be targeted for effective prevention and removal of biofilms.


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P080 - Morphological and proteomic analysis of early stage air-liquid interface biofilm formation in Mycobacterium smegmatis Weiser, Jaroslav (Institute of Microbiology v.v.i., Prague 4, CZE); Sochorova, Zuzana (Institute of Microbiology v.v.i., Prague 4, CZE); Petrácková, Denisa (Institute of Microbiology v.v.i., Prague 4, CZE); Sitarová, Barbora (Institute of Microbiology v.v.i., Prague 4, CZE); Buriánková, Karolína (Institute of Microbiology v.v.i., Prague 4, CZE); Bezousková, Silivia (Institute of Microbiology v.v.i., Prague 4, CZE); Benada, Oldrich (Institute of Microbiology v.v.i., Prague 4, CZE); Kofronová, Olga (Institute of Microbiology v.v.i., Prague 4, CZE); Janecek, Jirí (Institute of Microbiology v.v.i., Prague 4, CZE); Halada, Petr (Institute of Microbiology v.v.i., Prague 4, CZE) The formation of biofilms by bacteria is a key strategy in the colonisation of natural environments, including plant and animal hosts. During the last decade, a number of studies dealing with aspects of morphology and regulation of biofilm development in mycobacteria have been published. In almost all of these studies, only biofilms that had grown for more than 48 hours attached to solid surface or floating on the air-liquid interface were considered.

We studied the early stages of pellicle formation by Mycobacterium smegmatis on the surface of a liquid medium (air-liquid, A-L interface). Using optical and scanning electron microscopy, we showed the formation of a compact biofilm pellicle from micro-colonies during the period of 8 to 30 hours. The cells in the pellicle changed size and their cell division pattern during this period. Based on our findings, we created a model of M. smegmatis A-L early pellicle formation showing the coordinate growth of cells in the micro-colonies and in the homogenous film between them, where the accessibility to oxygen and nutrients is different. A proteomic approach utilising high-resolution two-dimensional gel electrophoresis (2-D GE), in combination with mass spectrometry (MS)-based protein identification, was used to analyse the protein expression profiles of the different morphological stages of the pellicle. The identified proteins formed four expression groups; the most interesting of these groups contained the proteins with highest expression in the beginning stage of biofilm formation, when the floating micro-colonies start to form the compact pellicle and their size, as well as thickness, significantly decreases. The majority of these proteins, including GroEL1, are involved in cell wall synthesis or modification, mostly through the involvement of mycolic acid biosynthesis, and their expression correlated with the changes in the rigidity of the bacterial cell wall observed by SEM.

Because biofilms are the source of many chronic infections, regulation of their development represents an excellent drug target. Although M. smegmatis is the non-pathogenic species, it is suggested that the way of biofilm formation is similar also for the pathogenic ones, like M. tuberculosis or M. avium. Therefore, understanding the principles of the early stages of biofilm formation, where the cells are more susceptible to the drugs, might be very useful.

P081 - Functional and structural responses of stream biofilms to varying resources Wagner, Karoline (University of Vienna, Department of Limnology and Oceanography , Vienna, AUT); Bengtsson, Mia M (University of Vienna, Department of Limnology and Oceanography , Vienna, AUT); Besemer, Katharina (University of Vienna, Department of Limnology and Oceanography, Vienna, AUT); Sieczko, Anna (University of Vienna, Department of Limnology and Oceanography , Vienna, AUT); Burns, Nancy R (University of Vienna, Department of Limnology


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and Oceanography, Vienna, AUT); Battin, Tom J (University of Vienna, Department of Limnology and Oceanography, Vienna, AUT) It is generally recognized that most of the organic matter processing in streams occurs in the hyporheic zone, the saturated streambed where groundwater and stream surface water mix. The hyporheic zone offers a large surface area for colonization by microbial biofilms, the dominant mode of microbial life and the major contributors to organic matter retention and transformation in streams. An important characteristic of stream sediments is that they are highly interconnected, receiving organic matter inputs from terrestrial and aquatic ecosystems. Autochthonous, labile organic matter inputs are supplied by the primary producers in the stream surface waters, whereas groundwater inputs or surface run off can contribute an allochthonous, recalcitrant organic matter input. The composition and functioning of microbial communities in the hyporheic zone may therefore be largely influenced by these allochthonous and autochthonous organic matter sources and might have a profound influence on organic matter processing in streams with impacts on large- scale carbon fluxes. In our experiment, hyporheic microorganisms, contained in bioreactors, were exposed to a putatively recalcitrant organic matter plant extract, simulating an allochthonous input to the stream ecosystem (e.g. leaf litter fall, terrestrial organic matter input). Downwelling of exudates from benthic algae into the hyporheic zone was mimicked by the addition of different labile organic matter sources (glucose, glucose+NO3+PO4 and an algal extract). We used 454-pyrosequencing of the 16S

bioavailability of the different organic matter sources. The activity of 9 extracellular enzymes was measured as a proxy for microbial function and to detect functional shifts in response to varying resources. Our bioreactor experiment demonstrated that allochthonous organic matter inputs strongly influence community composition, whereas autochthonous additions have less profound effects on community composition. Allochthonous and autochthonous organic matter additions did not change community functioning, indicating functional redundancy of the hyporheic microbial communities. This has notable consequences for the functioning of stream ecosystems.

P082 - Application of LC-MS global metabolomics approach for the comparison of the PAO1 biofilm and planktonic cells Ng, Wei Ling (SCELSE, Singapore, SGP); Woo, Yissue (SCELSE, Singapore, SGP); Periasamy, Saravanan (SCELSE, Singapore, SGP); Nesatyy, Victor (Singapore Centre on Environmental Life Sciences Engineering (SCELSE), Singapore, SGP); Swarup, Sanjay (SCELSE, Singapore, SGP); Chow, Chung Ping (Waters Singapore, Singapore, SGP); Rice, Scott (SCELSE, Singapore, SGP); Kjelleberg, Staffan (SCELSE, Singapore, SGP) It is known that microorganisms, forming a natural part of the Earth, ecology exist in free fBiofilms composed from the Pseudomonas aeruginosa have been implicated in a variety of diseases including cystic fibrosis and nosocomial infections. Thus it is vital to understand the

of treatment. Previous comparisons of the planktonic cells to their biofilm counterparts on the genomic level showed significant changes in expression of the genes involved in quorum


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sensing, motility, and production of polysaccharides. Proteomics investigation of the P.putida and P.aeruginosa revealed significant differences between biofilm and free cell mode. Use of modern metabolomics approaches would provide another dimension to the ongoing efforts to pin-point differences between planktonic and biofilm modes. Previous studies attempting to

P. aeruginosa were using NMR. Comparisons between planktonic and biofilm modes of growth and consequent principal component analysis showed similarity of the metabolite profiles in the spend media. At the same time significant differences in chemical shifts of intracellular metabolic profiles were observed between planktonic and biofilm cells themselves. However, limited NMR sensitivity and dynamic range results in quite narrow coverage of experimental metabolome space. This restricts application of NMR to the detection of most abundant metabolites present at concentrations greater than 3-

In this study, we have used High Resolution Accurate Mass Spectrometry (HRAMS) global metabolomics approach to pin-point differences on metabolic level between these two modes of growth in P. aeruginosa. Intracellular metabolites from the respective biofilm and planktonic cells samples grown in stationary environment were separated by Ultra Performance Liquid Chromatography (UPLC) and detected by Waters Xevo G2-S qTOF and Orbitrap Velos Pro mass spectrometers. The resulting LC-MS profiles were analized by the XCMSonline, Sieve, Genedata and TransOmics softwares. Our data showed distinct differences in the LC-MS profiles for biofilm and planktonic metabolite cell extracts. In particular, Orbitrap data analyzed by Sieve software revealed 161 features with the fold change more than 5 and p-value less than 0.05 assigned to 129 putative metabolites by METLIN database search and mapped to 61 metabolites in KEGG database. Similar analysis of the data produced by Xevo qTOF using XCMSonline data processing software revealed 671 features with the fold change more than 5 and p-value less than 0.005, with 622 of those features showing changes in the intensity in the range from 5-100 fold change. The majority of the most drastic differences associated with changes in the biofilm endo-metabolome were observed in the retention time region from 10 to 16 minute indicating on the hydrophobic nature of the eluting compounds, which is consistent with the biofilms adherence to the variety of surfaces. Search of 671 masses against METLIN database resulted in the assignment to 225 putative metabolites responsible for the observed differences. Further analysis and identification of masses with most prominent differences is underway. Similar analysis will be repeated for the PAO1 cells grown in the flow-through devices.


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P083 - Manganese influences cellular processes in Bacillus subtilis biofilms Mhatre, Eisha (Terrestrial Biofilms Group, Jena, GER); Troszo, Agnieszka (Molecular Genetics, Groningen Bio-molecular Sciences and Biotechnology Institute, Groningen); Kuipers, Oscar P (University of Groningen, Molecular Genetics Group, Groningen); Kovács, Ákos T. (Terrestrial Biofilms Group, Institute of Microbiology, Jena, GER) Bacterial biofilms are dynamic and structurally complex communities, involving cell-to-cell interactions. In recent years, there have been reports of many environmental signals inducing such complex structures in Bacillus subtilis biofilms through the membrane bound kinases. These signaling molecules are often media components, molecules produced by cells themselves as well as other interacting species or due to depletion of some molecules in the vicinity of cell. In our studies we see the effect of extracellular manganese (Mn2+) on biofilm formation and other differentiation processes in B. subtilis. KinD kinase was previously described to mediate Mn2+ derived signals in presence of glycerol during biofilm induction [1]. Mn2+ is also one of the essential components of biofilm media used for B. subtilis. Comparison of colony biofilms in the presence or absence of Mn2+ using microarray reveals that genes involved in biofilm formation are down regulated in absence of Mn2+. Mn2+ also affects the genes involved in distinct pathways of various cellular processes. Biofilm formation was examined when mutants were created in selected genes identified in the microarray, putatively involved in processes like fatty acid-S-CoA biosynthesis, sporulation, and Fe-transport. Pellicle morphology on air-liquid interphase and colony biofilm formation are altered in absence of Mn2+, while certain mutants showed granular pellicle. Further, yngI mutant, involved in surfactin biosynthesis showed smooth colony morphology that lacks complex architectures. Our aim is to also understand the role of Mn2+ in other biofilm related traits like motility, antimicrobial production, stress and sporulation. Thus, the microarray study not only highlights the importance of Mn2+ during biofilm development but also opens a window to identify genes with Mn2+-dependent expression that could be related to biofilm formation.

[1] Shemesh M, Chai Y. 2013. A combination of glycerol and manganese promotes biofilm formation in Bacillus subtilis via histidine kinase KinD signaling. J. Bacteriol. 195:2747 2754.

P084 - Analysis of anaerobic simulated gene expression in Escherichia coli biofilm Bayramoglu, Bihter (Ben Gurion University of the Negev, Midreshet Ben Gurion, ISR); Herzberg, Moshe (Ben Gurion University of the Negev, Midreshet Ben Gurion, ISR); Gillor, Osnat (Ben Gurion University of the Negev, Midreshet Ben Gurion, ISR) Escherichia coli is a facultative anaerobe inhabiting the intestines of warm-blooded animals and reptiles. In the intestine it metabolizes traces of oxygen thus providing an oxygen-free environment to the obligate anaerobic bacterial communities that constitute the majority of the gut microbiom. Although E. coli is one of the best characterized model organisms, attempts to grow it as biofilm in anaerobic conditions were not successful. We demonstrate the cultivation of E. coli biofilm under strict anaerobic conditions using a flow cell developed in our lab. We characterized the E. coli anaerobic biofilm comparing and contrasting its properties and


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expression profile to biofilm grown under aerobic conditions. In addition, we compared the response to anaerobiosis in planktonic cultures exploring patterns and links to the biofilms. We performed whole genome sequencing and analysis to compare transcriptomes of aerobically and anaerobically E. coli cells growing in either planktonic or biofilm settings. We further characterized the physico-chemical properties of the extra polymeric substances of the biofilms in an attempt to link them to the expression profile. Using the information derived from this sequencing effort, we considered the implications of biofilm grown anaerobically to colon enteric bacteria. The differences observed between biofilm grown under aerobic and anaerobic conditions were more pronounced then the differences in planktonic cell cultures cultivated under the same conditions. Moreover, the expression of most genes was down-regulated when cells were grown in anaerobic biofilm compared to aerobic conditions. In contrast, gene expression of planktonic cells growing in aerobic and anaerobic conditions was either enhanced or reduced. We have noted a sharp decrease mostly in genes encoding for protein synthesis related enzymes suggesting that anaerobic conditions impose severe stress on E. coli cells limiting their activity to survival. However, the analysis of the physico-chemical properties of the extra polymeric substances revealed no apparent differences between biofilms grown in aerobic and anaerobic conditions. Sessile E. coli cultures residing in the intestine are subjected to many forms of environmental stress, including oxygen-limitation. Here we suggest that unlike planktonic cells, E. coli biofilm grown under strict anaerobic conditions impose severe stress upon the cells, which may explain the difficulties encountered when attempting to examine these conditions. We suggest that anaerobic E. coli ctivity in order to survive. The results derived from this study may help elucidate factors and mechanisms enabling E. coli cells to populate the intestine that have otherwise been overlooked.

P085 - Biofilm formation of Lactobacillus plantarum WCFS1 dynamic flow vs. static conditions Fernández Ramírez, Mónica (Top Institute Food and Nutrition, Wageningen); Nierop Groot, Masja (Topp Institute Food and Nutrition, Wageningen); Smid, Eddy (Top Institute Food and Nutrition, Wageningen); Abee, Tjakko (Tops Institute Food and Nutrition, Wageningen) Background : Lactobacillus ssp. are generally associated with their beneficial roles in food fermentation processes. However, representatives of this genus are also known in food industry as spoilage bacteria. Spoilage of food products is often linked to the presence of biofilms on processing equipment. In the food industry, production lines, including pipes for pumping products like juices, dressings and milk, can be contaminated with lactobacilli causing spoilage. To mimic the dynamics of industrial conditions, we applied dynamic flow-cells to study biofilm formation by Lactobacillus plantarum.

Cell envelope characteristics of the biofilm forming microbes are thought to be crucial for initial attachment and further biofilm development. Therefore we analysed biofilm formation in static and in dynamic flow conditions by L. plantarum WCFS1 (wild type) and mutants each lacking one of the four capsular polysaccharide clusters (cps) and a mutant lacking the gene encoding sortase (srtA).


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Aim: The objective of this study is to evaluate the biofilm forming capacity under static and dynamic flowing conditions of L. plantarum WCFS1 and mutants lacking one or more gene clusters for capsular polysaccharide synthesis (Δcps1, Δcps2, Δcps3, and Δcps4) and a mutant lacking the sortase encoding gene (ΔsrtA) which is predicted to direct and anchor twenty seven surface proteins covalently to the peptidoglycan (Kleerebezem, et al. 2003).

Results: L. plantarum WCFS1 and selected spoilage related isolates form biofilms of high cell density under dynamic conditions even when the dilution factor exceeds the growth rate by a factor ten. The capacity to form dense biofilms was highly dependent on the presence of the cps1 gene cluster. Deletion of this particular cluster resulted in a reduction of biofilm cell numbers by 2 orders of magnitude. This effect was specific for dynamic conditions, as in static conditions the number of cells in the biofilm was comparable to the wild type. While crystal violet staining suggested increased matrix synthesis in static conditions, this was not observed under dynamic conditions. The relevance of sortase dependent proteins on the cell surface was evaluated using a srtA mutant. Surprisingly, under static conditions the ability to form biofilms was impaired, whereas under dynamic flow conditions biofilm formation was not significantly changed in the sortase mutant compared to the wild type.

Conclusions: L. plantarum WCFS1 is able to form biofilms under high flow rate conditions, yielding high cell densities. The modulation of surface properties using cps and srtA mutants showed that sortase-dependent proteins influence biofilm formation under static conditions but not dynamic flow conditions, whereas the presence of capsular polysaccharide cluster 1 seems to be important for biofilm formation in dynamic flow conditions but not in static conditions.

P086 - Characterization of an alpha-L-fucosidase in Tannerella forsythia and its role in biofilm formation Megson, Zoe Anne (Universität für Bodenkultur Wien, Department of NanoBiotechnology / NanoGlycobiology unit, Vienna, AUT); Koerdt, Andrea (Universität für Bodenkultur Wien, Department of NanoBiotechnology / NanoGlycobiology unit, Vienna, AUT); Schuster, Heinrich (Universität für Bodenkultur Wien, Department of NanoBiotechnology / NanoGlycobiology unit, Vienna, AUT); Messner, Paul (Universität für Bodenkultur Wien, Department of NanoBiotechnology / NanoGlycobiology unit, Vienna, AUT); Schäffer, Christina (Universität für Bodenkultur Wien, Department of NanoBiotechnology / NanoGlycobiology unit, Vienna, AUT) Tannerella forsythia (Tf) is a Gram-negative, filamented, anaerobic oral pathogen, member of the so called red complex of bacteria which cause a set of inflammatory diseases named periodontitis, affecting millions of people worldwide. The effects on the periodontium include loss of the alveolar bone around the teeth, swelling and bleeding of the gum and in more severe cases, loss of teeth. Periodontitis has also been linked to systemic inflammation and to an increased risk of stroke, heart attacks and atherosclerosis, amongst others. Tf possesses a unique cell surface (S)-layer composed of two different co-assembling glycoproteins [1]. Mutant strains of the S-layer proteins and the glycans they carry give insight into the importance of this S-layer in attachment, biofilm formation and disease progression. In addition, the structure of the glycan somewhat imitates that of host glycoproteins, having terminal sialic acid derivatives and fucose [2]. The importance of this finding resonates also in the robust repertoire of glycosidases which Tf and other oral bacteria express. Sialic acid


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residues present on both bacterial outer leaflets and host surfaces are cleaved off by host and bacterial neuraminidases to uncover sialic acid-masked epitopes for adhesion, triggering a multispecies biofilm formation [3]. In Tf in particular, this behaviour was observed in NanH sialidase mutants which had hindered attachment and growth on surfaces coated with salivary glycoproteins, giving this enzyme a crucial role in biofilm formation [4]. As the other key terminal sugar on host glycoproteins is fucose, we propose that the annotated alpha-L-fucosidase in Tf has a similar purpose. Here we present the characterization of the recombinant protein expressed in E. coli, by elucidating its specificity using various standard substrates and blood group oligosaccharides. We also discuss initial biofilm and attachment assays using the knock-out mutant, deltafuc. Complex salivary glycoproteins are considered the major source of nutrients for the oral supragingival microbiota and so the in vitro model glycoproteins used here are mucin from bovine submaxillary glands and fetuin from fetal bovine serum, as a non-fucosylated control in our biofilm assays. This work is supported by the Austrian Science Fund FWF project P24317-B22 (to C. S.) and the

- 1. Sekot, G., et al. Arch. Microbiol. (2012) 194: 525-539. 2. Posch, G., et al. J. Biol. Chem. (2011) 286: 38714-38724. 3. Stafford, G., et al. Mol. Oral Microbiol. (2012) 27: 11-22. 4. Roy, S., et al. Microbiology (2011) 157: 3195-3202.

P087 - The Possible Pathogenic Roles of Moraxella catarrhalis Biofilm-induced Nitrite Reduction Johnson, Candice (US Food and Drug Administration, Bethesda, USA); Mocca, Brian (US Food and Drug Administration, Bethesda, USA); Yin, Dandan (US Food and Drug Administration, Bethesda, USA); Wang, Wei (US Food and Drug Administration, Bethesda, USA) The classification of Moraxella catarrhalis has been changed from a commensal microorganism to a disease causing pathogen in humans. M. catarrhalis is one of the three most common bacterial causes for acute otitis media (AOM) and infection often relapses in infants and young children (Murphy, 2009). The WHO estimates that the global AOM incidence rate is about 709 million cases each year with 51% of those cases occurring in children under 5. In addition, M. catarrhalis forms biofilms on the mucosal surfaces of the middle ears of children experiencing otitis media with effusions (OME, Hall-Stoodley et al. 2006). However, there is no clear understanding of pathogenic roles of M. catarrhalis biofilms. Bacteria resident in biofilm have been demonstrated with different characteristics from those in planktonic growth form, including altered growth rates and gene expression profiles, increased resistance to antibiotics and innate immunity. In the case of M. catarrhalis, this bacterium significantly increases the expression of its truncated denitrification pathway during biofilm growth in vitro in comparison to planktonic growth conditions (Wang et al. 2007). This study investigates the effects of nitrite reduction on M. catarrhalis biofilm formation as well as on host cells in vitro. Using a live-cell monitoring system we monitored the biofilm formation and dispersion of a M. catarrhalis wild type strain and its denitrification mutant strains in media with or without nitrite over a 24hr period of culturing. It is evident that M. catarrhalis nitrite consumption/nitric oxide production has an effect on biofilm formation. Our data showed that bacterial nitrite reduction was


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activated during biofilm formation, and the reduction of nitrite seemed to cause biofilm dispersion in later stages of culturing. More importantly, the biofilm-induced nitrite reduction also induced adjacent host cell death, suggesting a possible pathogenic role of M. catarrhalis biofilms.

P088 - The ornithine lipid biosynthetic operon regulates the biofilm formation and virulence-related phenotypes in Pseudomonas aeruginosa Kim, Soo-Kyoung (Pusan National University, Busan, KOR) Ornithine lipids (OLs) are bacterial membrane lipids that are widely found in outer membrane of many gram-negative bacteria, whereas it is not detected in Eukarya and Archaea. Pseudomonas aeruginosa, an opportunistic pathogen has an operon annotated for the function of ornithine lipid biosynthesis, olsBA. olsBA encodes acyltransferases and works in two steps for the OL biosynthesis, in which OlsB transfers an acyl group to ornithine to make lyso-ornithine lipid and OlsA converts the lyso-ornithine lipid into ornithine lipid by another acyl-group transfer. OLs are reported to increase in phosphorus-free culture condition and olsBA operon of P. aerugnosa is induced in phosphate-limiting condition. While OLs were suggested to reduce the toxic effect of endotoxin probably functioning as an antagonist, a recently study showed that the mutation of this operon had no effect on the virulence of P. aeruginosa. In this study we found that the overexpression of olsBA operon modulated some virulence related-phenotypes of P. aeruginosa, including quorum sensing regulation, biofilm formation, and motility. Interestingly, the olsBAoverexpressing P. aeruginosa cells induced calcium release of animal cells, implying that it may modulate the physiology of host cells.

P089 - Structure-activity relationships in thiophenone Lund Witsø, Ingun (Department of Oral Biology, University of Oslo, Oslo, NOR); Valen Rukke, Håkon (Nordic Institute of Dental Materials, Oslo, NOR); Assev, Synnøve (Department of Oral Biology, University of Oslo, Oslo, NOR); Benneche, Tore (Department of Chemistry, University of Oslo, Oslo, NOR); Lönn-Stensrud, Jessica (Department of Oral Biology, UNiversity of Oslo, Oslo, NOR); Aamdal Scheie, Anne (Department of Oral Biology, University of Oslo, Oslo, NOR) Bacterial biofilms represent a burden to the society due to enhanced resistance to antibiotics, biocides and the immune systems. There are to day few available means to effectively prevent or treat biofilm-related problems, such as infections, biofouling or biofilm-induced corrosion. Bacteria in a biofilm communicate with neighbouring cells via various signalling molecules, auto-inducers. The auto-inducer interacts with cognate receptors initiating a signalling cascade leading to regulation of several genes, many of which are involved in biofilm formation and virulence. In this study we used the marine bacterium Vibrio harveyi as a model organism. V. harveyi produces the auto-inducer AI-2, which interacts with the LuxP receptor, leading to regulation of genes involved in biofilm and bioluminescence. Thiophenones are synthetic sulphur analogues of furanones. Several studies indicate that thiophenones inhibit biofilm formation by interfering with bacterial communication. Thus the AI-2 receptor LuxP could be a potential target for intervention. The present aim was to study structure-activity relationships of three different groups of thiophenones with focus on potential to inhibit biofilm formation, planktonic growth, and bioluminescence, and to assess their binding affinity to LuxP. METHODS:


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V. harveyi BB120 were grown in Sea-water medium in microtiter plates for 5 h. The biofilm mass was assessed by the safranine method and growth was measured by optical density. BIC50 and PIC50 (the concentration inhibiting biofilm formation and growth, respectively by 50 %) were determined. V. harveyi MM32, a non-AI-2 producing LuxP reporter strain was used to study the effect of thiophenone on DPD (synthetic AI-2) induced bioluminescence. The various thiophenones were added to the incubation mixture at 2.5 µM and the relative reduction was calculated. The docking software PyrX was used to predict the binding affinity of the thiophenones to LuxP. The Pymol software was used to visualize the docking and to determine amino-acid interactions between the thiophenones and LuxP. RESULTS: All thiophenones tested reduced biofilm formation, but with varying BIC50 values (< 5 µM to 37 µM). The PIC50 values varied from 9 µM to >100 µM. All thiophenones reduced bioluminescence, and the reduction varied from 73 % to 97 %, depending on the chemical structure of the thiophenone. The most effective thiophenones in inhibiting bioluminescence were also the ones most effective in reducing biofilm formation. The activity of the thiophenone seemed to be dependent on the chemical structure. There was a clear binding affinity for the thiophenones to LuxP, and several of the thiophenones appeared to bind to several of the same amino acids as AI-2, in the LuxP binding pocket. There was, however, no clear correlation between the biofilm inhibition and the LuxP binding affinity. CONCLUSION: This study show that thiophenones interfere with biofilm formation and bioluminescence in V. harveyi, and might act as an antagonist, probably by binding to the LuxP receptor. Thiophenone represent a promising agent for biofilm related problems as bacterial infections, biofouling and biofilm-induced corrosion. However, the chemical composition of the thiophenone is important for its activity, and further work is needed to find the molecules with the most effective chemical structure.

P090 - UV-inducible Aggregate Formation in Hyperthermophilic Archaea Mediates Exchange and Repair of Chromosomal DNA Schleper, Christa (Archaea Biology and Ecogenomics Division, Department of Ecogenomics and Systems Biology, Vienna, AUT); Albers, Sonja-Verena (Max-Planck-Institute for Terrestrial Microbiology, Marburg, GER); Ajon, Malgorazeta (Max-Planck-Institute for Terrestrial Microbiology, Marburg, GER); Fröls, Sabrina (Archaea Biology and Ecogenomics Division, Department of Ecogenomics and Systems Biology, Vienna, AUT) Species of the archaeal genus Sulfolobus reside in terrestrial acidic hot springs in virtually any volcanic environment on Earth. Although adapted to extreme growth conditions (80°C, pH 3), these organisms experience also stressful conditions and contain proteins involved in various mechanisms of DNA repair, highlighting the importance of these processes for all forms of life (Fröls et al. Biochem Soc Trans. 2009, 37:36-41 ). When exposed to sublethal doses of UV-light, Sulfolobus quickly forms species-specific cellular aggregates in a period of 6 to 8 hours while DNA replication is arrested (Fröls et al., Mol Microbiol. 2008;70:938-52) . These aggregates dissolve after about 8 hours, when the cells resume growth and chromosomal DNA breaks are no more visible in the cultures. Aggregate formation is mediated by a strongly UV-inducible type IV pilus system. Using chromosomal markers and gene-knockouts we have demonstrated that the UV-induced cellular aggregation mediates chromosomal marker exchange with high frequency (Ajon and Fröls et al., Mol Microbiol. 2011, 82:807-17) . Recombination rates exceeded those of uninduced cultures by up to three orders of magnitude. Since pilus knockout strains showed


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decreased survival upon UV-treatment, we conclude that the UV-inducible DNA transfer process and subsequent homologous recombination represents an important mechanism to maintain chromosomal integrity in Sulfolobus. It will be interesting to see, if this UV-inducible aggregate formation and DNA exchange is found more widespread in Bacteria and Archaea and might also increase recombination events and horizontal gene transfer rates in natural populations.

P091 - Quorum Sensing Inhibition of Pseudomonas aeruginosa virulence and biofilm formation by the Marine Bacterium Cellulophaga sp. E6 Lafleur, John (Alpert School of Medicine at Brown University, Providence, USA); Costa, Stephen (UMASS Dartmouth, North Dartmouth, USA); MacLean, Jason (UMASS Dartmouth, North Dartmouth, USA); Bitzer, Adam (UMASS Dartmouth, North Dartmouth, USA); Neto, Catherine (UMASS Dartmouth, North Dartmouth, AUT); Silby, Mark (UMASS Dartmouth, North Dartmouth, USA) Quorum sensing (QS) is a means of cell-cell communication, found among many species of bacteria, which allows coordinated control of group behaviors such as virulence and biofilm formation. QS involves secretion of a diffusible signal (inducer) which results in coordinated, population-level gene regulation once threshold concentrations of the inducer are reached. Inhibition of QS has been proposed as a strategy to interfere with virulence. Here we report the isolation of a marine bacterium capable of inhibiting quorum sensing by the opportunistic pathogen P. aeruginosa, which quorum-senses with acylated homoserine lactones (AHLs). Bacteria were collected from the surface of seaweeds from the coastal waters of Buzzards Bay, Massachusetts, and cultivated on marine agar. Using a luciferase reporter which detects long chain AHLs we screened 300 isolates for the ability to interfere with AHL detection. Among several candidates, one isolate was chosen for further analysis. Sequencing of the 16S rDNA indicated the inhibitory bacterium was a Cellulophaga sp., which we have designated as strain E6. Using luciferase-based reporter constructs specific for AHLs of different chain length, we have shown that Cellulophaga sp. E6 is inhibits QS based on AHLs with chains of four, eight, and 12 carbons. P. aeruginosa uses short- and long-chain based QS systems (Rhl and Las, respectively) to control virulence gene expression and biofilm formation. Supernatant from Cellulophaga sp. E6 culture reduced expression of the Las-dependent virulence factor elastase (lasB) and reduced biofilm formation in a dose-dependent manner. Activity-guided purification of the QS inhibitory activity has shown that the target molecule is smaller than 1000 Da, water soluble, and stable to temperatures of 50?C. This study and others suggest that microbial communities may be a rich source of QS inhibitors. The anti-QS molecule produced by Cellulophaga sp. E6 is a potent inhibitor of QS-controlled virulence factors in P. aeruginosa, supporting the growing contention that QS inhibitors represent a promising therapeutic addition for treatment of bacterial infections.


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P092 - Biofilm triggering properties of chemically synthesized gramicidin variants Gallegos Monterrosa, Ramses (Friedrich Schiller University of Jena, Terrestrial Biofilms Group, Jena, GER); Jadhav, Kirtikumar B. (Friedrich Schiller University of Jena, Institute of Organic and Macromolecular Chemistry, Jena, GER); Arndt, Hans-Dieter (Friedrich Schiller University of Jena, Institute of Organic and Macromolecular Chemistry, Jena, GER); Kovacs, Akos T. (Friedrich Schiller University of Jena, Terrestrial Biofilms Group, Jena, GER) The process of biofilm formation in diverse bacteria has been shown to specifically involve the response to self-generated secreted small molecules, i.e. quorum-sensing. Bacillus subtilis is a gram-positive model bacterium for studying biofilm formation. One of the interesting properties of this bacterium is that it differentiates into several subpopulations of specialized cell types in response to different environmental cues while forming provides the whole population with several benefits, including a heterogeneous gene expression that provides B. subtilis with a high survivability if the colony is suddenly faced against strong adverse conditions. This differentiation is controlled by a complex gene regulatory network that is centered on the activation of the global regulator Spo0A, which can be phosphorylated through a phosphorelay dependent on the activity of various histidine kinases (KinA, B, C, and D). The phosphorylated/non-phosphorylated ratio of Spo0A determines the developmental pathways that a given cell will follow, thus determining its social role in the biofilm.

Biofilm formation is initiated in response to various environmental and cellular signals, few of which have been studied. We have found that several structurally diverse secondary metabolites that were originally isolated for their antimicrobial properties, can promote biofilm formation in B. subtilis. These small-molecule natural products cause pore formation, altered membrane permeability, and potassium leakage. Loss of potassium in the cells is sensed by the membrane integrated histidine kinase KinC, which triggers the production of the extracellular matrix via Spo0A. One of the identified small-molecules that induce matrix production through this mechanism is the peptide antibiotic gramicidin A, which is derived from soil-dwelling bacteria, e.g. Brevibacillus brevis. Here, we tested numerous synthetic variants of this compound that were specifically designed to understand the effect of residue content and conformational properties of gramicidin A on antimicrobial activity, for their ability to trigger the expression of biofilm related genes. Using fluorescence reporters and air-liquid interphase biofilm model, we followed the effect of these different molecules on the biofilm development of B. subtilis.

P093 - Role of the accessory gene regulator system during biofilm formation of Listeria monocytogenes EGDe Zetzmann, Marion (University of Ulm, Ulm, GER); Endres, Jasmin (University of Ulm, Ulm, GER); Sedlag, Anne (University of Ulm, Ulm, GER); Riedel, Christian (University of Ulm, Ulm, GER) Listeria monocytogenes is able to form biofilms on polystyrene, glass and stainless steel under various conditions. The process of biofilm formation is partly regulated by the accessory gene regulator (agr) system. This system consists of the two component system AgrAC, the pre-peptide (AgrD) and AgrB, which processes AgrD into the autoinducing peptide (AIP). An agrD deletion mutant of L. monocytogenes EGDe (ΔagrD) displayed a defect in biofilm formation under limiting conditions (10-fold diluted BHI broth at 25 °C) whereas under nutrient rich conditions (BHI broth at 37 °C) the biofilm formation was not different from the wild type (WT)


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strain. To gain further insights into the agr-dependent and -independent phenotypes, biofilm formation of L. monocytogenes was analyzed under various conditions. Biofilm formation in 10-fold diluted BHI was inhibited by addition of glucose for both the WT and the ΔagrD mutant. No differences between the WT and the ΔagrD mutant was observed for cell surface hydrophobicity. However, the reduced biofilm formed by the ΔagrD strain was at least partially due to a defect in initial attachment to hydrophobic surfaces. Moreover under all conditions biofilm formation was reduced by addition of pronase or periodate indicating the importance of proteins and sugars in the biofilm formation process.

P094 - Genetic Variation and Phenotypic Plasticity of Pseudoalteromonas lipolytica Biofilms Wang, Xiaoxue (South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, CHN) Pseudoalteromonas is a genus of gamma-proteobacteria and it is widespread in the ocean. Many marine bacteria produce exopolysaccharides as a strategy for forming biofilms to survive in adverse environments. It had been proposed that the phenotypic diversity generated in biofilm can benefit the biofilm communities survive in natural extreme habits. Here we isolated a marine Pseudoalteromonas lipolytica strain (named SCSIO 04301), which can form both a solid-liquid interface biofilm and a liquid-air interface pellicles. Colonies in the biofilm communities were screened for changes of morphology, and mutants with a wrinkled phenotype and mutants with dark-brown color were isolated in two-day old SCSIO 04301 pellicles. Motility was abolished in the color mutant while more pellicles were formed in the wrinkled and color mutants. 16S rRNA sequencing results confirmed that these mutants are variants of SCSIO 04301. Thus, a genetic change has occurred in the wrinkled mutant and in the color mutant. A whole genome re-sequencing method was used to locate the mutation at the DNA level. A total of twelve single-base mutations were identified in the color mutant. Among them, one gene hmgA, involved in pyomelanin synthesis, had a change at amino acid position 197 (from Gly to Asp). HmgA is an enzyme (homogentisate 1,2-dioxygenase) involved in the pathway of phenylalanine and tyrosine catabolism, and it converts homogentisic acid into 4-maleylacetoacetate instead of pyomelanin. Earlier studies have shown that inactivation of the hmgA gene leads to pyomelanin hyperproduction in other bacteria. To test whether this non-synonymous mutation in hmgA is the cause of the hyperproduction of dark-brown pigment, we complemented the wild-type hmgA to the color mutant, and as expected, the color mutant can restore to the wild-type phenotype which no longer generates pyomelanin. Therefore, our results demonstrate that genetic and phenotypic variation have occurred in the pellicles of Pseudoalteromonas lipolytica, and inactivating hmgA leads to the arising of hyperproducer of pyomelanin variants in biofilms. Furthermore, we observed a high mutation rate of single nucleotide changes in biofilm community, probably due to a reduction of mismatch-repair activity. Further studies on the mechanism of point mutation and the benefits/cost of such variant in biofilms are needed.


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P095 - Multiple nitric oxide sensors are involved in the regulation of seeding dispersal in Pseudomonas aeruginosa biofilms Barraud, Nicolas (University of New South Wales, Sydney, AUS); Hui, Janice (The University of New South Wales, Sydney, AUS); Rice, Scott (The University of New South Wales, Sydney, AUS); Kjelleberg, Staffan (The University of New South Wales, Sydney, AUS) In most environments bacteria adopt a biphasic life cycle, with a sessile surface-attached biofilm phase and a motile, planktonic dispersal phase. Dispersal is essential to allow for colonization of new surfaces, and is critical for species stability. Recently, the simple gas and ubiquitous biological signal nitric oxide (NO) was identified as a key regulator of biofilm dispersal, broadly conserved across species. NO was shown to trigger a signaling pathway involving the intracellular secondary messenger cyclic di-GMP (c-di-GMP), which in turn activates a range of effectors that result in dispersal. Further NO was also linked with the activation of the superinfective form of the Pf4 prophage in P. aeruginosa, leading to cell death and phenotypic variation in mature biofilms, however this regulation does not seem to be linked to the c-di-GMP pathway.

The aim of this study was to elucidate the mechanisms used for sensing NO signals in biofilms of the model organism P. aeruginosa. Because NO stimulates phosphodiesterase (PDE) activity in cell free extracts and NO signals lead to decreased intracellular c-di-GMP, it is likely that a sensor of NO is associated with a c-di-GMP related gene. Twenty six single knockout mutants in c-di-GMP PDE genes were screened in batch assays for NO-mediated dispersal. The potential role of protein cysteine thiols in sensing NO, via S-nitrosylation, which has emerged in recent years as a principal mechanism by which endogenous NO influences many cellular processes in eukaryotes, was also assessed by using a biotin switch technique adapted for biofilm studies.

The results revealed that mutants in two PDE, dipA and rbdA, were impaired in the dispersal response mediated by NO, suggesting that these are involved in sensing NO signals. The analysis of S-nitrosylated proteins by avidin pulldown and mass spectrometry analysis revealed almost 200 proteins that were S-nitrosylated in biofilms after dispersal induced by NO compared to control biofilms. In particular, the oxidative stress response regulator OxyR, for which the dimeric form has been reported to bind a promoter region of the prophage in P. aeruginosa, was shown here to be S-nitrosylated during NO-mediated dispersal. Five-day-old biofilms formed by an oxyR mutant showed increased levels, up to 4-log difference, of the superinfective phage compared to the wild type. This suggests a role for OxyR in mediating the interaction between NO signaling and the establishment of superinfective phage in P. aeruginosa. In contrast the oxyR mutant was not impaired in NO-mediated stimulation of PDE activity. Overall the data suggest that multiple parallel regulatory pathways are mediated by NO for the control of seeding dispersal in mature biofilms, activating c-di-GMP signaling and the transition to a free swimming phenotype, and in parallel inducing a superinfective prophage linked to phenotypic variation.


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P096 - Activity of Atmospheric Pressure Plasma against Pseudomonas aeruginosa Biofilms: Efficacy, Mechanism and Tolerance Gilmore, Brendan (Queen's University Belfast, Belfast, GBR) The aim of this study is to investigate the efficacy of atmospheric pressure non-thermal plasma (APNTP) for bacterial inactivation in planktonic and biofilm modes of growth. Furthermore, identification of cold plasma cellular and extracellular targets and characterization of activity responsible plasma-generated species were performed to elucidate the mechanism of APNTP-mediated bacterial destruction. In this study we have investigated the phenotypic and genotypic factors mediating elevated tolerance to atmospheric pressure, non-thermal plasma observed in P. aeruginosa biofilms.

Pseudomonas aeruginosa biofilms were treated with an in-house designed APNTP jet. Cell viability was evaluated by colony counting, XTT assay and live/dead staining followed by confocal microscopy. Interactions of plasma with intracellular and extracellular components were also investigated including that with DNA, catalytic enzymes, lipids, cell wall and constituents of extracellular matrix. Nine clinical P. aeruginosa strains in the biofilm mode of growth and three mutant strains (fleQ, pelA and mucA) were evaluated.

Treatment with APNTP jet was sufficient to completely eradicate Pseudomonas aeruginosa in both planktonic and biofilm modes of growth within 4-10-minutes, confirmed by XTT assay, viable count and confocal microscopy. A multiple cellular target destruction mechanism is proposed. Although APNTP produced mixture of electrons, ions, electromagnetic radiation in addition to several reactive oxygen and nitrogen species (RONS), it was found that certain RONS mediated antimicrobial APNTP activity against P. aeruginosa. Mucoid strains of P. aeruginosa exhibited increased phenotypic resistance compared with non-mucoid strains. This may be related to the protective role of overproduced alginate rich matrix in mucoid strains. Overproduction of the polysaccharide matrix, led to higher tolerance to plasma treatment than pelA and fleQ mutants. This elevated tolerance confirms the role of the polysaccharide matrix in sequestration of antibacterial species produced by the plasma. The effects of exogenous DNA and alginate, both alone and in combination, were studies and found to significantly reduce the plasma0mediated antibacterial activity of this non-thermal plasma source.

Conclusions: Growing evidence has been progressively indicating potential of using APNTP in various antimicrobial applications, including chronic biofilm-associated infections. The role of biofilm matrix components in biofilm-mediated tolerance to this treatment requires careful attention for optimization of this technology in overcoming biofilms.


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P097 - Surface conditioning allows efficient attachment of Campylobacter jejuni biofilms to abiotic surfaces Brown, Helen (Institute of Food Research, Norwich, GBR); Reuter, Mark (Institute of Food Research, Norwich, GBR); Betts, Roy (Campden BRI, Chipping Campden, GBR); van Vliet, Arnoud (Institute of Food Research, Norwich, GBR) Introduction: Campylobacter jejuni is one of the leading causes of infectious intestinal disease in the developed world, and its public health and economic burden makes reducing its prevalence a priority. Biofilms are a well-known method for environmental survival and transmission, and we have previously shown that in laboratory conditions, C. jejuni biofilm formation is increased in aerobic conditions. This suggests that biofilm formation in C. jejuni may be a response to adverse environmental conditions and is a potential mechanism of survival in the food chain. The initial attachment of biofilms to abiotic surfaces is complex process which

within food-chain settings. Here we have investigated biofilm formation of C. jejuni on abiotic surfaces in the presence of meat juices, representing a food-chain relevant model. Results: A metabolic dye (TTC)-based staining method, and microscopy were used to show that surface conditioning both supports C. jejuni growth and significantly enhances biofilm formation in C. jejuni and C. coli. This effect was observed with several C. jejuni and C. coli isolates, and surprisingly also in a C. jejuni flaAB mutant deficient in biofilm formation. SEM confirmed that chicken juice was able to attach to abiotic surfaces and form a conditioning layer. Images also showed that C. jejuni attached preferentially to the chicken juice particulates, rather than directly the surface. In high concentrations of chicken juice, biofilm formation was not only enhanced due to increased ability of the C. jejuni to attach to the abiotic surfaces, but also by a significant increase in growth further enhancing biofilm formation. The conditioning layer formed by the chicken juice showed a high degree of persistence, and even following washing of the abiotic surface was still able to increase C. jejuni attachment and subsequent biofilm formation. Discussion: Meat juices are present throughout the food-chain and are not only a source of bacterial contamination, but may also offer a protective environment for microbes, allowing increased attachment and subsequent biofilm formation and nutritional support. This study shows that chicken juice is able to enhance and support both C. jejuni and C. coli biofilm formation. A greater understanding of the mechanisms underlying biofilm formation in food matrices will undoubtedly aid in reducing the burden of bacterial contamination of the food chain, and ultimately safer food.

P098 - Development of an agar based soil replication system for the fundamental study of corrosion in soil due to biofilms Spark, Amy (CSIRO, Caulfield North, AUS); Ward, Liam (RMIT University, Melbourne, AUS); Cole, Ivan (CSIRO, Clayton, AUS); Marney, Donavan (CSIRO, Highett, AUS); Law, David (RMIT University, Melbourne, AUS) Biological corrosion is a process whereby deterioration of a metal occurs directly or indirectly due to the activity of, and interaction with, living organisms such as bacteria or algae. Primarily corrosion occurs in the presence of a biofilm on the surface of the metal in question. Micro-organisms both individually and in biofilms have been shown to influence corrosion to occur at


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much higher rates than normally anticipated, leading to premature and sometimes catastrophic failure. This phenomenon is known as microbial influenced corrosion (MIC) and is commonly seen as pitting on a surface. While it is well established that MIC is predominately due to the influence of biofilms composed of both single and multiple species, much research work is focused on the effects of platonic bacteria on metals in solutions.

Seventy percent of Australian drinking water distribution pipes are made of iron based materials which are highly susceptible to both general corrosion and localised corrosion due to the action of biofilms. General corrosion is well understood and easily negated through increasing the thickness of pipe walls and coating the external surface of pipes. Localised corrosion due to MIC is not well understood and difficult to predict. It is the main cause of pipe failure through pitting on either the internal or external surface which leads to leaks and/or bursts which are both difficult and expensive to locate and repair. The internal surface of the pipe is exposed to drinking water which is a well researched electrochemical system both with and without bacteria present. Drinking water pipes are primarily buried pipes and thus the external surface is exposed to soil. Very little is known of how bacteria influence corrosion of the external surface of pipes, in part because it is difficult to replicate the soil environment in the laboratory.

Fundamental studies of MIC in soil are most commonly undertaken in solutions containing nutrients to simulate a soil environment. This does not take into account the physical structure of soil and resultant electrochemical interactions between the soil, steel and microbes. Using varying concentrations of microbiological agar, an alternative system for the study of MIC in soils is being developed. To date 1010 carbon steel has been exposed to two different concentrations of agar with no nutrients other than sodium chloride and the electrochemical response measured. The concentrations of agar are that used for plating microbes (15g/L) and to test for motility (4g/L). Future work will incorporate Pseudomonas as a model biofilm forming organism to determine the effectiveness of the system for MIC studies.

P099 - Adsorption of indicator microorganisms by riverbed biofilms and the effect of light Masuike, Ayano (Osaka Kyoiku University, Kashiwara, JPN); Hirotani, Hiroshi (Osaka Kyoiku University, Kashiwara, JPN) During the field study on indicator microorganisms in riverbed biofilms, we observed a decline of F specific phages in the biofilms developed on the stones sampled from the shaded location compared to those from locations exposed to the sunlight. Furthermore, the ratios of enterococci in biofilm/water were greater than E. coli and phages.

Two sampling points were set along the Ishikawa River, Osaka Japan, both at the bridge. Stones covered with natural biofilms were sampled just beneath the bridge, where it was always in the shades, and from close-by uncovered points, where it was always exposed to the sunlight whenever it is shining. Water samples were sampled at the same time. Sampling was done twice in October and December, 2013.

Biofilms were aseptically brushed off from the stone and this biofilms samples were subject to microbial tests, as well as water samples. F specific phages were enumerated by single layer


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plaque counts using Salmonella Typhimurium WG49 as a host. Escherichia coli and enterococci were enumerated by Colilert18 and Enterolert, respectively.

F specific phages, which are known to behave similar to some of the pathogenic viruses, were detected in all the biofilm samples measured. Greater amount of the phages were detected in biofilms developed in the uncovered location compare to those in the shades by 0.7 to 1.2 logs. Visually, algal abundances were similar among biofilms developed in the shades or light. The effect of UV light, if any, should decrease phages counts, although there was no significant decrease in phage counts in the biofilms by UV light as examined by the laboratory experiment. Pretreatment by UV light did not result in significant difference in the absorption of phage particles by biofilm matrix. Temperature should be much the same since there was a continuous flow of river water through the sampling location area. Therefore we set up a hypothesis that light (other than UV) affect the natural riverbed biofilms to enhance adsorption of phage particles.

Indicator microorganisms in biofilm and water were examined, and biofilm/water ratios were calculated. E. coli and enterococci indicated a similar ratio values, i.e. 102 104, whereas enterococci indicated 105 107. Enterococci may be concentrated in the biofilms, survive longer in the biofilms, or grow in the biofilms. Much care must be taken in the usage of enterococci to detect fecal contamination in river water, when there is abundant biofilm development in the riverbed environment.

P100 - Compressive force in pellicles Raspaud, Eric (CNRS, Orsay, FRA) Biofilm formation is not a simple accumulation of organic matter on a surface. The self-organization creates complex architectures where the progressive accumulation of matter could compress the biomass. In turn the inner compressive force could act on the edifice organization and on the morphology. We have worked with a strain commonly found in many environments and more precisely with wild strains of Bacillus subtilis. These strains form floating pellicles that we have analyzed experimentally using a specific force setup in order to demonstrate the existence of inner compressive forces and to quantify them. I will present here our forces data and show how they are responsible for a change in the pellicles morphology.

P101 - Paradigm shift in biofouling control in MBR for wastewater treatment: bacterial quorum quenching Lee, Chung-Hak (School of chemical & Biological Engineering, Seoul National University, Seoul, KOR) Membrane bioreactor (MBR) for sewage treatment ha s made rapid progress over twenty -five years . The compound annual growth rate in MBR world market is forecasted 20.2 % ($1,400 Millions in 2014), while that in MBR world treatment volume is 28.2% (4.2 millions m3 in 2014) over ten years (2008-2018), respectively. One of the major reasons is its unique features solving water shortage problems that are in close association with global climate change.


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However, the propagation of MBR has been hampered, particularly by membrane biofouling, which is closely associated with the deposition of microbial flocs from mixed liquor by convectional flow as well as with the naturally attached microbial growth on the membrane surface. M uch current R&D on MBR is related to analysis and control of membrane bio fouling, which is also the chronic challenge for operation of all membrane types. It is because biofouling is closely associated with high energy demand and thus a main drawback of MBR over conventional sewage treatment processes.

We are now standing at a turning point on this issue of biofouling from partial, visible, macro-physicochemical approaches to holistic, invisible, molecular biological ones. In other words, we have to listen carefully to interactive voices of microorganisms that take an active part on the stage of MBR to solve its intrinsic problem, biofouling.

Recently, novel molecular biological approaches have been attempted to control the biofouling with quorum quenching ( QQ ) concept - disruption of quorum sensing (QS), i.e., cell to cell communications between microorganisms. Various ways of QQ developed so far for MBR for sewage treatment will be presented from both mechanistic and practical view point.

References 1) Yeon, K.M. et al. (2009), Quorum s ensing: A n ew b iofouling c ontrol p aradigm in a m embrane b ioreactor for a dvanced w astewater t reatment. Environ . Sci . Technol . 43, 380 385. 2) Oh, H . S . et al. (2013) , Biofouling Inhibition in MBR by Rhodococcus sp. BH4 Isolated From Real MBR Plant . Applied Microbiology and Biotechnology, 97(23), 10223-10231 . 3) Kim, S .R.et al. (2013) , Biofouling Control with Bead-Entrapped Quorum Quenching Bacteria in MBR: Physical and Biological Effects . Environ . Sci . Technol . 47, 836-842 . 4) Cheong , W.S. et al. (2014) , Design of Quorum Quenching Microbial Vessel to Enhance Cell Viability for Biofouling Control in MBR . J. of Microbiology and Biotechnology, 24 (1), 97-105 .

P102 - Biofilm and ESBLs production in environmental Escherichia coli. Cornejova, Tatiana (University of Veterinary Medicine and Pharmacy in Ko¿ice, Kosice, SVK); Kmetova, Marta (Faculty of Medicine, Pavol Jozef Safarik University in Kosice, Kosice, SVK); Kmet, Vladimir (Institute of Animal Physiology Slovak Academy of Sciences, Kosice, SVK) Objective: We studied the formation of a biofilm in 15 strains of ESBLs in E.coli isolated from arteficial solid surfaces, polystyrene sherics and from water in urban wastewater station.

Methods: Fifteen phenotype ESBL positive E. coli strains (CLSI 2008 ) were isolated from arteficial solid surfaces, polystyrene sherics and from water in wastewater station. To detect the biofilm formation we used crystal violet and resasurin methods with Maxisorb plates. The presence of CTX-M groups, CMY-2, qnrS , integron 1 and Tn3 were determined by PCR.

Results: The majority of ESBL producing E.coli had weak biofilm production and belonged to commensal phylogenetic group. However one E.coli strain 17 B (isolated from polystyrene sperics) with moderate biofilm activity contained four resistance genes CTX M-1, CTX M-2, CMY-2 and qnrS together with integron 1 and transposon 3.


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Conclusions: Strains with biofilm production connected with presence of CTX-M group, CMY-2, qnrS genes associated with integron 1 and Tn3 represent a potential environmental health risk. This study was supported by Slovak grants APVV-0009-10 and VEGA 2/0014/13

P103 - Coupling of electrochemistry and molecular biology for the analysis of biofilms involved in Amazonian carbon steel biocorrosion Vastra, Margaux (Laboratoire des matériaux et molécules en milieux amazonien, Université des Antilles et de la Guyane, UAG-UMR ECOFOG, Cayenne, GUF); SALVIN, Paule (UMR ECOFOG, laboratoire L3MA, Cayenne, GUF); ROOS, Christophe (UMR ECOFOG, laboratoire L3MA, Cayenne, GUF) The biocorrosion is a process that causes damages on metallic structures worldwide [ 1 ] . In French Guiana, very expensive reparations have been done on the Larivot Bridge in 2009, which is the only link between the East and the West economic zones: the East including the Cayenne port (the main one) and airport; the West, the Guiana Space Centre, which is the European spaceport in Kourou. The biocorrosion is supposed to be responsible for the bridge failure. This last has been built over the estuary of the Cayenne River, an Amazonian River: this brackish river is rich in nutrients and the air and water temperatures are constant year-round, respectively around 26°C. The mechanisms variety, coming from the organisms diversity present into the biofilm, make this corrosion phenomenon both efficient and difficult to assess. The species stratification is key in this phenomenon [ 2 ] . In temperate environment, the prokaryotes considered as responsible for biocorrosion are the sulfate reducing prokaryotes (SRP), but the EPS-producing prokaryotes and the iron oxidizing bacteria (IOB) can also be involved [ 3 ] . These metabolic groups belong to a great variety of genus. The aim of the study is to understand how biofilm present on the bridge conducts to its failure and what are the biocorrosion mechanisms which occur. The biocorrosion will be studied in this Amazonian environment. The tropical biocorrosion characterized by identified phenomena and parameters can then be compared to biocorrosion of temperate medium. In this study, carbon steel (S355) plates have been immerged in the estuary of the Cayenne River. During two months, four plates were collected every 10 days. The biofilm development was electrochemically followed (impedance and polarization curve). The formed biofilms were studied by PCR-DGGE concerning its diversity and by FISH concerning the spatial organization.The first results showed a great diversity of bacteria and phytoplankton but a small diversity of Archaea. This diversity is supposed to become more and more organized over time which will be shown by FISH. This project was supported by the European Union, the Centre

-10-LABX-25-01).

[1] M. C. Moura, E. V. Pontual, P. M. Paiva, and L. C. B. B. Coelho, Microbial pathogens and strategies for combating them: science, technology and education. Vol. 1. Formatex Research Center, 2013, ch. An outline to Corrosive Bacteria., pp. 11 22.

International Microbiology, vol. 8, pp. 169 180, 2005.


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-reducing bacteria - new views of an Applied and Environmental Microbiology , 2013. [Online]. Available: http://-

aem.asm.org/content/early/2013/12/03/AEM.02848-13.abstract

P104 - "Extremofilms" - Biofilm formation of the thermoacidophilic archaeon Sulfolobus acidocaldarius and response towards 1-butanol exposure Benninghoff, Jens (University of Duisburg-Essen, Essen, GER); Wingender, Jost (University of Duisburg-Essen, Essen, GER); Siebers, Bettina (University of Duisburg-Essen, Essen, GER) In contrast to Bacteria, knowledge of archaeal biofilms is still limited. The ability of biofilmformation has been reported for haloarchaea (Fröls et al., 2012) and crenarchaea (Koerdt et al., 2010). In this study the effect of organic solvents, i. e. 1-butanol, on biofilms of the thermoacidophilic, obligate aerobic crenarchaeon Sulfolobus acidocaldarius with optimal growth at 76 °C and pH 3 is investigated. S. acidocaldarius is adapted to extreme environments, genetically tractable and able to form biofilms. As biofilms often have a higher resistance

biotechnological applications like whole cell biocatalysis with biofilms. The aim of this study is to analyze the response of S. acidocaldarius extremofilms to organic solvents, here 1-butanol exposure. Therefore S. acidocaldarius biofilm formation was analyzed in different static incubation systems (e. g. microtiter plates, and µ-dishes) as well as on different substrata like glass and polystyrene. A 96-well polystyrene microtiter plate assay was developed to determine the effect of i) 1-butanol on biofilm formation as well as ii) established biofilms of S. acidocaldarius. The biofilm and planktonic cells were quantified using crystal violet as well as OD measurements. Cell viability was determined using a newly developed resazurin assay. Inhibition of biofilm formation was observed at a concentration of 2 % [v/v] 1-butanol. For established biofilms (three days old) addition of 3 % [v/v] 1-butanol results in effects such as detachment of cells from the surface and loss of metabolic activity of remaining biofilm cells. However, already at subinhibitory concentration obvious changes of the biofilm and cell phenotype were observed for established biofilms: i) enhanced carbohydrate content in biofilms (visualization by fluorescently labeled lectins and confocal laser scanning microscopy), ii) aggregation of cells grown on glass slides (crystal violet staining, light microscopy), iii) loss

domain of life - the Archaea - , as well as the response towards organic solvents will be presented and the potential for future biotechnological applications will be discussed. - Fröls, S, Dyall-Smith, M and Pfeifer, F, Environmental Microbiology (2012) Biofilm formation by haloarchaea. 14, 3159 3174 - Koerdt, A, Gödeke, J, Berger, J, Thormann, KM and Albers, S-V (2010) Crenarchaeal biofilm formation under extreme conditions. PLoS ONE 5(11)


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P105 - Characterization of a sulfo-oxidant biofilm involved in concrete corrosion Etienne, PAUL (INSA, Toulouse, FRA); Peyre Lavigne, M. (Université de Toulouse; INSA, UPS, INP; LISBP, Toulouse, FRA); Bertron, A. (Université de Toulouse; INSA, UPS ; LMDC, Toulouse , FRA); Etienne, PAUL (INSA, Toulouse, FRA); Cockx, Arnaud (Université de Toulouse; INSA, UPS, INP; LISBP, Toulouse , FRA); Foussard, J.N. (Université de Toulouse; INSA, UPS, INP; LISBP, Toulouse , FRA) The biofilm deterioration of cementitious materials is observed in various industrial domains (sewage network, storage tank). In the sewage network, the concrete deterioration can lead to dysfunctions in the wastewater treatment by clear water intrusion (rainy days, groundwater), to sanitary problems due to wastewater leaking into the groundwater, and to a need for considerable financial investment in restoration (Zhang et al., 2008). In a sewage network, hydrogen sulfur (H2S) produced in the wastewater volatilizes and then condenses in the upper part of the pipe where a biofilm is growing. H2S in the condensed water is chemically transformed into reduced sulfur compounds (S0, S2O3

2- etc…) which are then oxidized by sulfur-oxidizing bacteria (SOB) into sulfate (SO4

2-). It thus leads to a biological acidification of the local medium in contact with the cementitious material (Hvitved-Jacobsen et al., 2013). In order to elaborate more resistant materials or methods for minimizing corrosion, it is necessary to get more knowledge about the link between the biological activities and the microbial selection in the biofilm and the degradation mechanisms occurring in the cement for various cementitious materials. In this work, a pilot with two pipes in parallel fed with a same feeding solution is used. The inner surface pipes is inoculated by activated sludge to study the sulfur-oxidizing selection depending on the composition of two types of cementitious lining. Biofilm activities and microbial composition are characterized through mass balances for the former and 16S rDNA extraction and pyrosequencing for the later. The experimental plan includes the comparison of: - An abiotic pipe system and a biotic pipe system in order to confirm the biofilm role in deteriorations, - The effect of different reduced sulfur compounds in the feed (as sulfur ion S2-, thiosulfate S2O3

2- and tetrathionate S4O62-) on the SOB activities and on the lixiviation of

cementitious linings, - The effect of the type of two cementitious materials (Blast-Furnace-Slag Cement and Calcium-Aluminate Cement) on biofilm activities, biofilm structure and lixiviation products. This work emphasizes the difference in the dissolution rate depends on (i) the presence of a biofilm or not (ii) the type of substrate (S2-, S2O3

2- or S4O62-) used (iii) the materials composition

and structure. It also clearly demonstrates that the dissolution of the cementitious linings, linked to the inlet load, is SOB activity dependent. For S2- feeding, SOB growth is limited due to the loose of the substrate by chemical oxidation and due to competition for nitrogen with nitrifiers. Using a S2O3

2- feeding, the production of S4O62- intermediate leads to a spatial

colonization of SOB in the biofilm along the pipe. The type of the cementitious lining induced different development of the biofilm, in terms of dry matter, water affinity, covering ratio and microbial diversity.

Keywords: Biofilm, Sulfur-oxidizing bacteria, microbial selection, Cementitious deterioration

References: Hvitved-Jacobsen, T., Vollertsen, J., Nielsen, A.H., 2013. Sewer Processes: Microbial and


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Chemical Process Engineering of Sewer Networks, Second Edition. CRC Press. Zhang, L., De Schryver, P., De Gusseme, B., De Muynck, W., Boon, N., Verstraete, W., 2008. Chemical and biological technologies for hydrogen sulfide emission control in sewer systems: A review. Water Res. 42, 1 12.

P106 - MEMORY EFFECT ON BIOFILM DEVELOPMENT: BIOFILM HISTORY STRONGER THAN ENVIRONMENTAL CONDITIONS Saur, Thibaut (INRA - LBE, NARBONNE, FRA); Milferstedt, Kim (Laboratoire de Biotechnologie de l'Environnement, Narbonne, FRA); Santa-Catalina, Gaëlle (Laboratoire de Biotechnologie de l'Environnement, Narbonne, FRA); Bernet, Nicolas (Laboratoire de Biotechnologie de l'Environnement, Narbonne, FRA); Escudié, Renaud (Laboratoire de Biotechnologie de l'Environnement, Narbonne, FRA) Hydrodynamic conditions are one key parameter acting on biofilms. It is commonly assumed that hydrodynamic conditions shape biofilm morphology by inducing sloughing events, erosion or compression (Van Loosdrecht et al. 2002). Biofilm community structure can also directly or indirectly be influenced by hydrodynamics conditions (Rochex et al. 2008). But it remains unclear if a change in hydrodynamic conditions affects mature biofilm morphology and/or community structure. In the literature, biofilms are mainly studied either under constant shear, e.g. comparing two reactors where biofilms developed at two different shear rates, or as erosion tests as in Paul et al (2012) . In our study, we exposed biofilms to variable hydrodynamic conditions where the applied shear changed over time. Once matured, we exchanged biofilms with different shear histories and exposed them to a change in the hydrodynamic conditions. By this way, it is possible to study the adaptation of physical and microbiological structures of biofilms after a switch in the hydrodynamic conditions. Biofilms developed on coupons of a size of 5 cm2 in three bubble column reactors with a volume of 5 L. The coupons can be withdrawn for sampling or could be exchanged from one reactor to another. Reactors were inoculated with aerobic activated sludge and continuously fed with a synthetic solution at a hydraulic retention time of 65 minutes. A first reactor was operated under stable hydrodynamic conditions. In the other two reactors, hydrodynamic perturbations were generated by an 8-fold increase of the air flow rate for 12 minutes either hourly or weekly. After 30 days of reactor operation, coupons were exchanged between reactors in order to see the impact of a hydrodynamic change on mature biofilms. Dynamics of biofilm morphology were analysed using automated image analysis (Milferstedt et al. 2013). At the same time, biofilm community structure was assessed using molecular fingerprinting (PCR-SSCP). We found that biofilms grown under three different hydrodynamic conditions developed a unique morphology and biofilm community. When transplanting biofilms into a new environment, biofilm morphology and community development continued on trajectories imposed by biofilm history and was not influenced by a change in hydrodynamic conditions. Whatever the nature of the hydrodynamic switch - from highly perturbed to unperturbed or from unperturbed to highly perturbed biofilms structure did not adapt to their new environments and kept the features acquired during the growth phase. We call this property memory effect. Further research is on-going to study the required time to acquire this memory effect and how


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long a biofilm memorizes its history. Van Loosdrecht, M.C.M. et al., 2002. Antonie van Leeuwenhoek, 81(1-4), pp.245 56. Milferstedt, K. et al., 2013. PloS one, 8(11), p.e80692. Paul, E. et al., 2012. Water research, 46(17), pp.5499 508. Rochex, A. et al., 2008. Water Research, 42(20), pp.4915 4922. P107 - Can Benthic Biofilm/Sediment in an Oligotrophic River Act as a Reservoir of Pathogens? Comparing Behavior in Large and Lab-Scale Flumes

Walters, Evelyn (Temple University, Philadelphia, PA, USA); Müller, Elisabeth (Technische Universität München, Garching, GER); Kätzl, Korbinian (Ruhr Universität Bochum, Bochum, GER); Schwarzwälder, Kordula (Technische Universität München, München, GER); Rutschmann, Peter (Technische Universität München, München, GER); Horn, Harald (Karlsruhe Institute of Technology, Karlsruhe, GER) Although microbial contamination of surface waters has been an issue for decades, there are still several aspects of bacterial survival and transport that remain unclear. Under certain tropical and even temperate conditions,stream sediments and biofilms have been shown to harbor fecal indicator bacteria (FIB) at concentrations much higher than the overlying water (Rehmann and Soupir, 2009; Schultz-Fademrecht et al., 2008). For a variety of reasons including a higher availability of soluble organic matter and nutrients as well as protection from predators and UV light rays, bed sediments can represent a hospitable environment in which autochthonous microorganisms can survive longer than in the water column (Davies et al.,1995; Jamieson et al.,2005). In this study, both indoor lab-scale flumes and large-scale, outdoor flow-through flume systems were used to investigate the fate of E.coli and enterococci in the benthic sediment/biofilm of an oligotrophic river.

The lab-scale flume was fabricated with PVC and had a working length of 1.2m, width of 10cm,

temperature was maintained at 12°C. The water had to be filtered before use to remove any larvae or larger particles that could not be supported by the system. Sediment having a grain size of 4 - 15 mm were collected from the Isar River and used as substrata in the lab-scale flume. To facilitate sampling, 13 substratum cages (l = 4.5 cm, w = 10 cm, d = 3 cm) were built from stainless steel (mesh size 0.8 cm).The cages were divided into three equal compartments with solid stainless steel walls for triplicate testing. The cages were filled with the collected pebbles and placed in the middle of the flume. The remaining sections before and after the substratum cages were filled-in with loose pebbles so as to maintain a constant bed depth over the length of the flume.

The large-scale flume located outdoors was built with concrete (l=12m, w=0.5m, d=0.5m) and had a total system volume of approximately 13m3.The water used in the flume was taken from the Obernach River, a branch of the Isar River, and had an average temperature of 8 °C. Original sediment from the river (15 30mm) was used and again, to assist with sampling,was placed in 13 substratum cages (w=30cm, l = 15 cm, d = 3 cm).The remainder of the flume bed was filled in with original sediment. Month-long experiments in both systems were conducted by initially spiking the flume beds overnight with municipal wastewater. After, the flumes were operated


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with fresh river water and sediment samples were periodically removed.The sediment samples were tested for FIB with both MPN and qPCR techniques.

Upon addition of wastewater to the lab-scale flume, concentrations in the substratum cages peaked at 105 MPN gDM−1. Thereafter, a first-order removal rate was seen characterized by removal rate coefficients for E. coli and enterococci of 0.6 and 0.3 d−1, respectively.In contrast to the lab experiments, similar first-order decay coefficients were observed for both FIB (kec = 0.8 d−1; kent = 0.7 d−1) in the large flume.To better understand why enterococci persisted twice as long as E. coli in the lab flume, but disappeared at the same rate in the large-scale flume, qPCR was performed.Here it was seen that while the copy numbers of enterococci in the lab flume remained fairly constant,there was a rapid decrease in copy numbers in the large-scale flume.This suggests that bed sediment and biofilm erosion as well as the influence of grazers were responsible for FIB removal from the sediments in the large-scale flume, whereas in the lab flume FIB inactivation caused removal.These results indicate that hydraulic parameters such as bed shear stress as well as the presence of macroinvertebrates in a system are both important factors to consider when designing flumes depicting fast-flowing, alpine streams.

P108 - Epilithic river biofilms - structure and composition examined by laser microscopy and digital image analysis Neu, Thomas (Helmholtz Centre for Environmental Research - UFZ, Magdeburg, GER); Kuhlicke, Ute (Helmholtz Centre for Environmental Research - UFZ, Magdeburg, GER); Kamjunke, Norbert (Helmholtz Centre for Environmental Research - UFZ, Magdeburg, GER) River biofilms are hot spots of microbial activity and substrate turnover. They are dominated by phototrophic microorganisms such as algae and cyanobacteria as well as a polymer matrix. In frame of an investigation on carbon turnover in small rivers along a land-use gradient, the biofilm structure on rocks (top and bottom) was examined by multichannel laser microscopy. Parameters recorded included reflection (minerals), autofluorescence (Chl A, phycobiline), bacteria (nucleic acid stained) and glycoconjugates (fluorescence lectin-binding analysis). Digital image data sets were analyzed by differential visualization, quantification in axial direction, co-localization of autofluorescence and biofilm structural parameters. For this purpose several software tools were employed. The results showed an extreme heterogeneity in terms of the colonization patterns. With respect to the river continuum, flow velocity and hydrodynamics, rock biofilms revealed biofilm structures very likely determined by exposure history. The complex community comprised a variety of algal species, cyanobacteria and heterotrophic bacteria growing directly on the substratum forming a base biofilm. In addition, filamentous bacteria growing out of the base biofilm and diverging EPS glycoconjugate patterns were found. Based on biofilm structural features, the study suggests a spatial and differential separation of the polymer matrix and microbial community across the biofilm landscape.


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P109 - Screening of non-biocidal coatings: establishment of a high-throughput method for testing bacterial adhesion on textiles Schmidt-Emrich, Sabrina (Empa, St. Gallen, CHE); Amberg, Caroline (Swissatest Testmaterialien AG, St. Gallen, CHE); Katzenmeier, Heinz (Sanitized AG, Burgdorf, CHE); Thöny-Meyer, Linda (Empa, St. Gallen, CHE); Ren, Qun (Empa, St. Gallen, CHE) Hygienic and non-smelling textiles are more and more requested by consumers. Conventional solutions are usually based on biocidal products. Even though the use of biocides is considered as safe owning to strict regulations, due to environmental or health concerns there is a huge demand on the market for new biocide-free technologies to improve the hygienic status and reduce the formation of unpleasant odors on apparel textiles. The goal of this study was to establish a high-throughput screening method to assess the non-biocidal and non-bacterial adhesive properties of different textile coatings. A microplate assay based on Syto9 staining has been developed to quantify the reduction of bacterial attachment. 40 samples of coated polyester and cotton were subsequently evaluated for their anti-bacterial and anti-adhesive activities. The most-promising textile coatings were further investigated in a second screening series with a focus on different concentrations of coating materials. The impact of washing on non-bacterial adhesive properties of the selected coatings was also studied (after 5, 10, and 20 washing cycles). Finally, a coating was found which led to an 80% reduction of bacterial adhesion on the coated textile compared to the non-coated one. This coating was also demonstrated to be washing resistant after 20 washing cycles.

P110 - The Carbon Monoxide-Releasing Molecule CORM-3 is a potent inhibitor of planktonic, but not biofilm growth of Pseudomonas aeruginosa Chen, Chenfei (Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, GBR); Mann, Brian (University of Sheffield, Sheffield, GBR); Poole, Robert (Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, GBR) The overuse of antibiotics to treat cystic fibrosis (CF) lung infections has led to high levels of antibiotic resistance. Novel antimicrobial agents that could replace or complement current therapies are needed to fight chronic infections in CF patients. The advent of carbon monoxide-releasing molecules (CO-RMs) has been useful in investigating the physiological effects of CO by allowing the controlled release of CO to specific targets in mammalian systems. Recent evidence shows that the bactericidal activity of CO-RMs against a number of species is more effective compared to CO gas. Here, we applied CORM-3 [ Ru(CO)3Cl(glycinate )], a ruthenium-based, water-soluble carbon monoxide-releasing molecule, to biofilms using a novel nebulizer technique in conjunction with a Modified Robbins Device, and compared these effects with laboratory planktonic cultures of the Gram-negative pathogen Pseudomonas aeruginosa. We demonstrate that CORM-3 kills planktonic cells in laboratory culture. Although CORM-3 inhibits surface-associated growth of P. aeruginosa by both preventing biofilm maturation and killing bacteria within the established biofilm, cells growing in a biofilm are more resistant to CORM-3 than planktonic cells. CORM-3 treatment has a similar bactericidal effect to colistin, a commonly used antibiotic for P. aeruginosa lung infections.


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P111 - Analysis of the influence of growth conditions on the competition between biological aggregate morphotypes: biofilms, flocs and streamers Pechaud, Yoan (Université de Toulouse; INSA, UPS, INP; LISBP, Toulouse, FRA); Peyre Lavigne, Matthieu (Université de Toulouse; INSA, UPS, INP; LISBP, Toulouse, FRA); Bessiere, Yolaine (Université de Toulouse; INSA, UPS, INP; LISBP, Toulouse, FRA); Queinnec, Isabelle (CNRS ; LAAS , Toulouse, FRA); Paul, Etienne (Université de Toulouse; INSA, UPS, INP; LISBP, Toulouse, FRA) The morphological and physical properties of biofilms, and the fractions of each morphotypes are very important for the stability and performance of a biofilm reactor1 and strongly impact on the efficiency of biofilm removal in industrial water pipes2.

In this study, we focus in analyzing the influence of operating conditions on the competition between 3 aggregate morphotypes in biofilm reactors: biofilms, flocs and streamers.

The main objectives are: (i) to improve understanding of the mechanisms controlling the competition between the three microbial morphotypes and (ii) to analyze the dynamics of streamers formation. The influence of the substrate residual concentration, the surface organic loading rate (SOLR) and the carbon source on the fractions of the various morphotypes and their impact on the substrate removal efficiency is assessed. In order to modify the residual concentration of substrate in the bulk without changing the SOLR, the hydraulic retention time (HRT, i.e. the average time spent by the liquid in the reactor and is equal in our condition to the suspended solid retention time) was changed. The HRT governs the dilution rate of substrates and thus the substrate concentration in the liquid but also the detached particles retention time. Moreover, for streamers, the morphological properties (ratio length/diameter of the streamers, amplitude of oscillation) and the kinematic parameter (frequency of oscillation) were quantified at two times of their development.

At low HRT (3h) which induced lower carbon substrate concentrations in the bulk, development of streamers is favored and suspended particles in the liquid and biofilm are disfavored. On contrary for high HRT (20h), detached particles and biofilms were the dominant aggregate morphotypes. This difference of predominance in morphotypes has been attributed to three main factors: the SRT of SS, the residual concentration in the bulk liquid and the hydrodynamic conditions of growth (mass transfer and shear stress). Turbulent conditions and a low substrate concentration considerably favored the development of streamers which benefit from the higher external mass transfer. Indeed compared to biofilms the flapping movement and the higher surface to volume ratio of streamers increase the global external mass transfer. In some

the necessity to consider such morphotypes in further studies since they can have a significant impact on treatment efficiency. Concerning the dynamics of streamers formation, a densification of the streamers was observed over time (30 days of growth). From these results and theoretical considerations, assumptions on the mechanisms responsible for the development of streamers are proposed.

Key-words: Biofilm, streamers, transport, detachment, competition.


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References: 1 Filali et al. (2012). Water Sci. Technol. 65, 289 295. 2 Stoodley et al. (1998). Biotechnol. Bioeng. 57, 536 544.

P112 - Impact of tortuous flow on bacteria streamer developement in microfuidic system during filtration : implication of EPS Marty, Aurelie (University of Toulouse - LGC - UMR 5503, Toulouse cedex 9, FRA); Causserand, Christel (University of Toulouse - LGC - UMR 5503, Toulouse cedex 9, FRA); roques, christine (Faculty of Pharmacy - University Paul Sabatier - LGC - UMR 5503, Toulouse, FRA); Bacchin, Patrice (University of Toulouse - LGC - UMR 5503, Toulouse cedex 9, FRA) Because of their ability to attach to surfaces, bacteria often form biofilms i.e. complex assemblies of packed bacteria bound by biopolymers and linked to surfaces. In membrane retention applications, porous media with high surface to volume ratio provide an appropriate environment for the attachment of bacteria and biofilm formation. The way in which bacterial communities colonize flow in porous media is of importance, but basic knowledge on the dynamic of this phenomena is still missing. The aim of this work is to develop microfluidic experiments in order to progress in the understanding of bacteria capture in filters and membranes. PDMS microfluidic devices mimicking filtration processes have been developed to allow a direct dynamic observation of bacteria across 10 or 20 µm width microchannels. When filtered in such devices, bacteria behave surprisingly: Escherichia coli, Pseudomonas aeruginosa or Staphylococcus aureus accumulate in the downstream zone of the filter and form large streamers which oscillate in the flow. In this study, streamer formation (200µm long) is put in evidence for bacteria suspension in non nutritive conditions in less than 1 h. This result is totally

captured in the bottleneck zone and are accumulated in the upstream zone. Observations within different flow geometries (straight channels, connected channels, and staggered row pillars) show that the bacteria streamer development is influenced by the flow configuration and particularly by the presence of tortuosity within the microchannels zone. These results are discussed at the light of 3D flow simulations. In confined systems and in laminar flow, there is secondary flow (z-velocities) superimposed to the streamerwise motion (in xy plane). The presence of the secondary flow in the microsystems has an effect on the bacterial adhesion. Thin EPS filaments were formed and transported by the flow within the microchannels and then stretched to the middle of the channel by the secondary flow at the microchannels outlet. These filaments act as a fishing line and at least as a fishing net. A scenario in three steps is established to describe the formation of the streamers and to explain the positive effect of tortuous flow on the development kinetics.

P113 - Bacterial friction as part of adhesion to surfaces Swartjes, Jan (University medical center groningen, Groningen) Bacterial friction as part of adhesion to surfaces. Jan J.T.M. Swartjes, Deepak H. Veeregowda, Yun Chen, Prashant K. Sharma, Henny C. van der Mei and Henk J. Busscher


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Department of Biomedical Engineering, University Medical Center Groningen, The Netherlands Bacterial adhesion is problematic in many diverse applications. The mechanism of bacterial adhesion has been described theoretically considering non-specific Lifshitz-Van der Waals, electrostatic, acid-base and hydrophobic interactions and specific interactions initiated by ligand-receptor interactions. However, the actual situation is far more complicated, leaving the exact mechanisms unknown. The forces that actually arise between bacteria and surfaces have been studied using Atomic Force Microscopy and have been related to bacterial adhesion. Bacterial adhesion mostly occurs in systems under the influence of fluid flow and the combination of normally-oriented adhesion forces and convective fluid flow parallel to a surface will lead to friction forces. The influence of the friction forces, however, has never been considered in bacterial adhesion in general or in the reduction of bacterial adhesion by surface modification. Here we studied the friction between Streptococcus mutans and salivary condition films (SCFs) to obtain the lateral adhesion forces. Comparison between S. mutans with and without antigen I/II showed that the specific ligand-receptor interactions between S. mutans and SCFs are direction-dependent, causing lateral adhesion forces to be much larger than the adhesion forces perpendicular to the surface. In the oral cavity, where there is constant flow of saliva, the increased lateral adhesion force gives S. mutans a great advantage to adhere. Additional to the friction forces between S. mutans and SCFs, we determined both the normally-oriented adhesion and friction forces between a Staphylococcus epidermidis strain and two distinct, self-assembling-monolayers of polyethylene-glycol applied at different concentrations. We observed friction forces measured in this study which were orders of magnitude larger than the calculated shear forces acting on immobilized staphylococci. Considering that shear forces arising from lateral convection are much smaller than the measured friction forces, we conclude that the friction forces are large enough to oppose the fluid shear forces and contribute to immobilization of a bacterium on the surface, needed for subsequent bond-maturation. Summarizing, inclusion of friction forces provides a more complete mechanism of bacterial adhesion to surfaces than can be obtained considering only normally-oriented adhesion forces and will help to unveil additional options in the search for materials properties and coatings that resist bacterial adhesion.

P114 - Microbial engineers in the aquatic sedimentary habitat Gerbersdorf, Sabine (University of Stuttgart, Institute for Modelling Hydraulic and Environmental Systems , Stuttgart, GER); Schmidt, Holger (Institute of Modelling Water and Environmental Systems, Stuttgarg, GER); Thom, Moritz (Institute of Modelling Water and Environmental Systems, Stuttgarg, GER); Wieprecht, Sikle (Institute of Modelling Water and Environmental Systems, stu, GER) The ETDC (Erosion, Transport, Deposition, Consolidation) cycle of sediments is crucial for the ecological and commercial health of aquatic habitats. It is now commonly accepted that the organisms inhabiting natural sediments mediate their erosive response. This presentation addresses microbial colonization and secretion of EPS (extracellular polymeric substances) to influence the stability of the sediment bed and the characteristics of the eroded sediment flocs;

fascinating function has been recognized since long in the marine habitat (with a focus on microalgae and microphytobenthic mats), little is known from freshwaters. Generally, the


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interactions between biological growth and environmental abiotic parameters have been rarely addressed in a comprehensive manner. Here, we will firstly show that the bacterial stabilization capacity is much higher than previously thought but will concentrate then on results from naturally diverse freshwater biofilm; obtained

Sediment entrainment and flocculation mediated by microbial produced extracellular polymeric

freshwater biofilm (increases up to 11 times as compared to controls) and its influence on the floc sizes, forms and settling velocity after entrainment to further impact sediment transport. Varying abiotic conditions proofed to have effects on the microbial stabilization potential: delayed development and stabilization under high flow velocities, while deploying highest adhesiveness and stability under good light conditions. Despite the important influence of light, the steepest increase in sediment stability happened in most of the experiments during the first 2 weeks. This was closely related to an increase in bacterial cell numbers, while the photoautotrophic microalgae developed mainly between week 3 and 5, giving evidence on the different successional stages of biofilm growth and their importance to functionality (here biostabilization). Thereby, changes in bacterial diversity, community dynamic and functional organization seem to be closely linked to the appearance (and types) of microalgae, unravelling simultaneously effects of competition and mutual dependence to impact EPS quantity and quality. The two main EPS components sugars and proteins were both significantly related to the increase in adhesiveness of the biofilm, sediment stability and floc formation; however we have evidence that the proteins might be the even better marker for biostabilization. We will briefly present the methods that are decisive to address biostabilization properly: the MagPI (Magnetic Particle Induction), straight erosion flumes (SETEG channel) and Gust Microcosm for floc erosion. KEYWORDS: biostabilization, adhesion, sediment stability, flocculation, microbial ecology, diatoms, bacteria P115 - Investigation of the biofilm-flow interactions with three-dimensional Particle Tracking Velocimetry, Optical Coherence Tomography and Confocal Laser Scanning Microscopy Carrel, Maxence (ETH Zürich IfU Chair for Environmental Fluid Mechanics, Zurich, CHE); Hartmann, Manuel (ETH Zurich IfU Chair of Environmental Fluid Mechanics, Zurich, CHE); Derlon, Nicolas (Eawag Process Engineering, Duebendorf, CHE); Morgenroth, Eberhard (ETH Zurich IfU Chair of Process Engineering in Urban Water Management / Eawag Process Engineering, Zurich, CHE); Holzner, Markus (ETH Zurich IfU Chair of Environmental Fluid Mechanics, Zurich, CHE) The hydrodynamic conditions play a key role in the definition of the microbial landscape formed by biofilms. Depending on the environmental conditions, biofilms develop a variety of different physical structures which have been studied and presented in the last decades (clusters, mushroom-like, ripples, streamers etc.). It is known that biofilms develop these morphologies in order to better cope with the environment, e.g. to reduce the drag force they are exposed to (streamers) or to increase the nutrient mass transfer. But further investigation of the coupling between the hydrodynamics, the biofilm structure and the nutrient transport is required in order to provide a better understanding of the physical mechanisms influencing the growth of biofilms.


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This work aims at investigating the biofilm-flow interactions by means of subsequent flow (three dimensional particle tracking (3DPTV)) and biofilm (optical coherence tomography (OCT) and confocal laser scanning microscopy (CLSM)) imaging. This innovative combination of analytical tools allows a three dimensional investigation of the coupling between hydrodynamic conditions, nutrient concentration, biofilm growth and morphology. With this aim, experiments were performed with natural biofilms growing under aerobic conditions on profiled plates in flow cells submitted to different conditions, with Reynolds Numbers and nutrient concentrations varying over three orders of magnitude (Re = 5 500 and CCOD = 20 2000 mg/L). Roughness elements with (semi-) spherical shape were mounted on one side-wall of the flow cell and the resulting flow pattern is highly three-dimensional.

The overlaid results from the 3DPTV, the CLSM and the OCT provide a basis for the assessment of biofilm morphological heterogeneities and their relation to the hydrodynamic conditions and nutrient supply. Heterogeneities were observed inside of the same flow cells due to differences in the local flow field caused by the roughness elements. These results also allow to identify recirculation regions exhibiting higher mixing and kinetic energy. Important differences were also observed over the different order of magnitudes of Reynolds Numbers, where streamers were observed to grow for the highest Reynolds Numbers.

The results obtained as well as further systematic investigations should contribute to a better understanding of the biofilm-flow interactions. This could for instance allow the verification and optimization of existing flow-biofilm models and allow a better characterization of the coupling between the hydrodynamics and the biofilm structure.

P116 - Properties physic-chemical of Listeria monocytogenes and transfer bacteria GRAZIELLA, BOURDIN (ANSES, BOULOGNE SUR MER, FRA); LELEU, GUYLAINE (ANSES, BOULOGNE SUR MER, FRA); INGLEBERT, GAELLE (ANSES, BOULOGNE SUR MER, FRA); FAILLE, CHRISTINE (INRA, VILLENEUVE D'ASCQ, FRA) Listeria monocytogenes is commonly found to be associated with equipment surfaces in food industry environments. The transfer of bacteria depends on environmental and intrinsic factors. But no works study the residual bacteria on the surface (conveyor belts) after the contact between food and adherent bacteria, and the physicochemical properties which are involved in these phenomena. This study was designed to evaluate the ability of adherent Listeria monocytogenes to cross-contaminate foods in contact, and to identify which bacterial properties would affect this transfer to food. Stainless steel surfaces were contaminated in static condition and successive blotting of the contaminated surfaces were performed with slices of agar. The initial removal rate and the percentage of residual bacteria were calculated. Both parameters were then confronted to data on bacterial surface properties. The strains of L. monocytogenes were electronegative and hydrophilic with the variation in the hydrophilic character in function of the strains and the incubation temperature. Significant differences were observed between the strains and between the temperature of bacterial adhesion for the initial slope (P=0.0000). We had analysed the correlation between different variables (physico-chemical parameters, adhesion, initial slope) and the similarity between the strains which would make it possible to classify them in distinct groups. We had data processing by PCA. Two components were extracted and counted for 46.1% of the variability in the original


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data. The first component represented 24.23% of the total variation of the data. The risk of food-borne infection associated with cross-contamination depends on two factors: the level of contamination on the surfaces and the probability of its transfer of the foods being consumed .

P117 - Assessing the Optimum Geometry of Pre-formed Aggregates in a Biofilm Melaugh, Gavin (Univeristy of Edinburgh, Edinburgh , GBR) Until recently, the initial stage of biofilm development was thought to predominantly involve the attachment of planktonic cells to the surface of interest. However, recent studies involving Pseudomonas aeruginosa and Staphylococcus aureus, have found that planktonic cells can form suspended aggregates and exhibit much of the phenotypical behaviour of attached biofilms, e.g. packing density, extracellular DNA, comparable growth rates, matrix structure, ability to survive lethal treatments of antibiotics, and mutation frequency. It seems probable that initial colonisation of the surface by single planktonic cells may also be accompanied by attachment of pre-existing aggregates that are already in a biofilm-like mode of growth. Since it is likely that those cells that begin biofilm-life in a preformed aggregate may develop structure differently, it is of fundamental interest to investigate the initial configuration of cell aggregates on the surface, and the influence that these aggregates have on the overall structure on the biofilm. For example: cells within a pre-formed aggregate may have either better or worse access to nutrients; the presence of aggregates might affect clonal structure of the biofilm; and considering that an aggregate is likely to have a higher initial cell density, one might expect aggregate cells to initiate cooperation through quorum sensing much earlier than cells that are randomly distributed on the surface.

Using agent based computer simulations, we investigate whether there is an optimum configuration for a clump of cells of size N to arrange on the surface, considering competitive effects from a surrounding mat of individual cells. The fitness of these clumps is assessed using a surface contact angle as the order parameter. Results reveal that there is an interplay between initial surface coverage and aggregate height. For a given density of surrounding cells, there is a critical angle at which clump height becomes more important than surface coverage. Our results show a direct correlation between this angle and the density (competition) of the surrounding cells.


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Session 8: Biofilms remixed: Microbial mats, stromatolites and soils

P118 - Spatial structuring of diatom communities of biofilms fouling a marine fast ferry Longyear, Jennifer (AkzoNobel, International Paint Ltd, Gateshead, GBR) Ship hull fouling by biofilms results in a significant fuel penalty to the global marine shipping industry. However, little is known about the nature of these marine biofilms in situ. Data in the literature primarily relate to biofilms collected from panels or coupons on static immersion, rather than biofilms growing in the dynamic environment created by a ship underway. In this study, biofilm samples were collected from a newly dry-docked fast ferry with an operating speed of 25 knots. Samples were scraped from spatially referenced locations along a horizontal (180m, near the waterline) and a vertical (5.5m, waterline to turn of the bilge) transect of the hull. The diatomaceous biofilms were digested, leaving clean frustules for species identification by light microscopy. The diatom communities were characterised by high diversity, particularly at the nose of the vessel. The vertical transect revealed three community types, two of which were also observed in the horizontal transect. The spatial extent of these community types suggests a sampling strategy for future ship biofilm research in face of the practical limitations of access to ships and the scale of the surface area of interest.

P119 - The effects of a 100-year flood on bacterial activity and community composition associated with particles in a river-floodplain system. Sieczko, Anna (Department für Limnologie und Ozeanographie, AUT); Mayr, Magdalena (Department of Limnology and Bio-Oceanography, Wien, AUT); Demeter, Katalin (Department of Limnology and Bio-Oceanography, Wien, AUT); Teubner, Irene (Department of Limnology and Bio-Oceanography, Wien, AUT); Peduzzi, Peter (Department of Limnology and Bio-Oceanography, Wien, AUT) Floods are considered as important events, which maintain various functions of river-floodplain systems. During such disturbances, floodplain systems affect downstream particle transfer, intercepting large amounts of transported terrestrial material. Such particles can be hot spots of increased bacterial activity and diversity, especially after the flood when favorable conditions are established. However, the significance of episodic flood pulses for bacterial activity and

remains largely unknown. The effects of a 100-year flood on bacterial community composition (BCC) and enzymatic utilization of organic matter were studied in the Danube Floodplain National Park (Austria). The consequences of disconnection of floodplain side-arms after the flood were emphasized. Extracellular enzyme activity (EEA) was assessed using spectroscopic techniques, and bacterial community composition was analyzed by TRFLP (terminal restriction fragment length polymorphism). The flood introduced significant amounts of particles into the floodplain side-arms. During the flood organic constituents of particles comprised only a small fraction (12%) of total suspended solids. However, after the flood, when side-arms were disconnected from the Danube, particles contained on average 40% of organic material. The activity of particle-associated enzymes was


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significantly reduced during the flood. The BCCs within the floodplain were found to be more similar during flood conditions. However, after the flood there was opportunity for pronounced synthesis of enzymes associated with particles. Also, the similarity of the BCCs decreased what indicates shifts in community composition after the disconnection of floodplain side-arms. Changing hydrological conditions prevailingly affected the utilization of terrestrial, lignin-derived, refractory organic matter.

P120 - EPS and cell labeling in multispecies biofilms collected in contrasted environments MICHEL, Caroline (BRGM, Orléans, FRA); Garrido, Francis (BRGM, Orléans, FRA); Devau, Nicolas (BRGM, Orléans, FRA); Guichard, Alan (BRGM, Orléans, FRA); Tambosco, Jennifer (BRGM, Orléans, FRA); Hellal, Jennifer (BRGM, Orléans, FRA) The composition and organization of environmental multispecies biofilms in terms of EPS and cells were investigated using fluorescent microscopy. For this, two multispecies biofilms were collected, one from an anaerobic culture favoring chlorate reducing bacteria from a contaminated site and one from a small stream in French Guyana (Crique Combat). The first biofilm is thus believed to be less complex (in terms of microbial diversity) than the second one. Biofims were fixed in OCT, freezed at -80°C, and then cut (cryotom, 10 µm, -18°C). The biofilms were then treated with several cell targeting fluorescent labels (DAPI, SYBRgreen), extracellular DNA (DDAO) and exopolysaccharides (lectins, Fluorescent Brightener). Double and triple stainings were applied, and the influence of staining order was tested. The objectives of this work were thus to establish a staining protocol and study the influence of biofilm origin and composition on labeling. First results showed that 3 washing steps with PBS 1X buffer for OCT elimination and between each staining step were needed to label EPS and cells with several different markers. Washing steps with PBS gave better observation in fluorescent microscopy than those with acetone (usually used to eliminate OCT). Cells from the biofilm collected in the reactor were better stained with DAPI than with SYBERgreen as a wider range of cell morphologies could be observed using DAPI. On the opposite, the labeling of the microorganisms composing the biofilm from the Guyanese stream was better with SYBERgreen staining. The choice of the fluorescent staining for cells thus depends on the biofilm. No labeling was obtained with DDAO, suggesting either the absence of eDNA in both biofilms or the need to enhance the protocol for eDNA staining. Results also showed that exopolymeric staining of the Guyanese biofilm leads to the obtention of a « cloud », whereas exopolysaccharides from the bioreactor biofilm rather form a capsule around cells. Results also suggested that when several fluorescent markers were used, results can vary according to the order in which the markers were tested. The labeling of cells and EPS of multispecies biofilms has thus to be adapted to the biofilm as the choice of the various markers vary according to the biofilm.

P121 - Contribution to the study of the Biofilm plants in endemic zone of buruli ulcer in C�te d'Ivoire Boni cisse, catherine (institut pasteur de Côte d'Ivoire, ABIDJAN, CIV) BACKGROUND Bacterial biofilms pose actually a major problem because of their responsibility in human pathology. Mycobacterium ulcerans, the pathogen agent of Buruli ulcer, is a bacterium found in


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the environment as biofilms. In order to contribute to the improvement of the understanding of conditions for survival in the environment of M. ulcerans, The overall objective of this study was to determine the bacterial composition of the biofilm of M. ulcerans in plants, in an endemic

ng the biofilm, to determine the bacterial load, to analyze the terms of coexistence between mycobacteria and bacteria found on plants composing the biofilm.

METHODS In terms of methodology, the collection of plant samples was conducted at the edge of surface water from the river of Banco, located in the village Adiopodoumé. The research of mycobacteria was performed at the Adiopodoumé site of Institut Pasteur in Côte genome of M. ulcerans, and the culture on Lowenstein-Jensen. The search for non fastidious bacteria composing the biofilm was performed in the laboratory of Bacteriology-Virology of the University Hospital of Yopougon.

RESULTS The genome of M. ulcerans was found in 30.76% of cases. The primary culture yielded a bacterial biofilm that consisted of four non-fastidious bacteria: E. colisp, Klebsiella, Vibrionaceae, Pseudomonas aeruginosa, and the subculture, mainly, yielded the genus Pseudomonas (59.49%), then the Vibrionaceae (40.51%). Mean bacterial loads of bacteria forming the biofilm tended to decrease from subculture to primary culture with the disappearance of the bacteria of genus Escherichia and Klebsiella, and the bacterial reverse predominance between Vibrionaceae and Pseudomonas aeruginosa for Pseudomonas. The study of the correlation between positive cultures on Lowenstein-Jensen and the presence of M. ulcerans genome showed that in 80% (8/10) of cases of positive cultures on Lowenstein-Jensen, the genome of M. ulcerans had been identified. In only 25% of the samples where M. ulcerans has been identified, no other bacterial presence was observed.

CONCLUSION This characterization study of bacterial biofilm of M. ulcerans shows that this is a heterogeneous biofilm, consisting of minority mycobacteria that are different from M. ulcerans, and not fastidious bacteria with which it coexists in symbiosis, in particular Pseudomonas aeruginosa.

Keywords: Mixed biofilm, M. ulcerans

P122 - Spatial patterns of carbonate biomineralization in biofilms Li, Xiaobao (Northwestern University, Evanston, USA); Chopp, David (Northwestern University, Evanston, USA); Russin, William (Northwestern University, Evanston, USA); Brannon, Paul (Northwestern University, Evanston, USA); Packman, Aaron (Northwestern University, Evanston, USA) Microbial carbonate biomineralization is an important process in a number of natural, clinical and engineered environments. Biomineralization is closely associated with biofilms, which are surface-attached microbial communities that have heterogeneous microenvironments. Biofilm heterogeneity should regulate spatial patterns of carbonate biomineralization, and conversely biomineralization strongly influences biofilm heterogeneity. However, the interactions between


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biomineralization and biofilm heterogeneity are largely unexplored because it is difficult to directly observe biomineralization in biofilms. Here we present a method for in situ, real-time imaging of biomineralization in biofilms and use it to show that Pseudomonas aeruginosa biofilms produce morphologically distinct carbonate deposits that substantially modify biofilm properties. Patterns of carbonate biomineralization produced in situ were substantially different from those caused by accumulation of particles produced by abiotic precipitation. Contrary to the common expectation that mineral precipitation in biofilms is primarily a surface process, we found that biomineralization started at the base of the biofilm. The carbonate deposits grew over time, deforming biofilm morphology and increasing biofilm permeability. These findings provide detailed information on spatial patterns of biomineralization in biofilms relevant to a diverse array of poorly understood problems, including microbial carbon sequestration, identification of biological signatures in the rock record, and formation of harmful renal and urinary calculi and calcified pulmonary nodules. The imaging method presented here can be broadly applied in conjunction with existing fluorescent imaging methods to assess feedbacks between biomineralization and other basic biofilm processes including nutrient and substrate delivery, metabolic heterogeneity, antimicrobial susceptibility, and mechanical stability.

P123 - About the life in an acidic snottite Krause, Susanne (Karlsruhe Institute of Technology, Karlsruhe, GER); Ziegler, Sibylle (Karlsruhe Institute of Technology, Karlsruhe, GER); Geiger, Katharina (Karlsruhe Institute of Technology, Karlsruhe, GER); Gescher, Johannes (Karlsruhe Institute of Technology, Karlsruhe, GER) Acid mine drainage is an environmental thread characterized mainly by acidification of lakes, rivers and streams, which is driven by the oxidation of pyrite. This process is accelerated by chemolithoautotrophic microorganisms that are often the only primary producers in these ecosystems. In this work snottite biofilms depending on pyrite oxidation as energy source were analysed. They are growing in stalactite-like structures on the ceiling of an abandoned pyrite mine and water with a pH of 2.3 drops constantly trough these biofilms. The water shows sulfate and iron concentrations of up to 200 mM and 60 mM, respectively. The matrix of the biofilms is composed of carbohydrates and bio-/geochemical products of pyrite oxidation. Using XANES measurements, the ferric iron and sulfate containing mineral jarosite was identified as one of the major anorganic constituents of the snottite. Microautoradiography (MAR)-FISH with 14CO2 indicated carbon dioxide fixation mainly in the outer parts of the biofilms. 16S rDNA analyses displayed a diverse microbial community consisting of bacteria (predominantly Acidithiobacillus and Leptospirillum) and archaea (mostly uncultured Thermoplasmatales and ARMAN). In situ oxygen-measurements revealed rapidly decreasing oxygen concentrations from the outer rim towards the center of the biofilm. In fact only the outer 700 µm of the biofilm contained detectable oxygen concentration, whereas the remaining inner parts were anoxic. XANES spectra as well as CARD-FISH-pictures in comparison to the oxygen measurements indicated that the most important factor for the distribution of bacteria and archaea is oxygen. Surprisingly, so far uncultured archaea of the genus Thermoplasmatales and ARMAN could only be detected in the anoxic inner parts. They do not seem to be involved in primary production, while known chemolithoautotrophic bacteria of the genus Leptospirillum could be detected only in the outer oxic parts.


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Based on the described analyses of the biofilm constituents and the community, an anaerobic medium was developed for the enrichment of the so far uncultured archaea. It contained ferric iron, an organic carbon source, an H2/CO2 atmosphere as well as autoclaved biofilm as undefined supplement. CARD-FISH pictures and PCR of 16S rDNA analyses show the enrichment of so far uncultured Thermoplasmatales. After a prolonged incubation time, organisms belonging to the ARMAN genus could be detected in high numbers. Several successful transfers and medium adjustments showed that the cultures do not depend on the autoclaved biofilm and on H2 in the headspace. In contrast, it became apparent that organic carbon as well as Fe(III)SO4 are necessary compounds of the medium. Still the most surprising results were the good enrichments of the enigmatic nanoorganisms of the ARMAN genus in the enrichment cultures. In contrary to so far existing results from other mining areas that suggest an aerobic lifestyle, it appeared that the detected ARMAN grow under anoxic conditions. Nevertheless, an isolation of the organisms was so far not possible, which indicates interspecies dependences which we were so far not able to resolve.

P124 - Effects of D-amino acids on Campylobacter jejuni biofilm formation Elgamoudi, Bassam (University of Leicester, Leicester, GBR); Ketley, Julian (University of Leicester, Leicester, GBR) Biofilm formation may play an important role in Campylobacter pathogenesis and transmission. D-Amino Acids (D-AAs) have been found to interfere with biofilm formation and promote dispersion and thus may be a possible strategy for biofilm control. The aim of this study was to investigate the effect of various D-AAs on C. jejuni biofilm formation and maintenance. Crystal violet based (1) biofilm inhibition and (2) biofilm dispersion assays were used along with fluorescence microscopy and confocal laser scanning microscopy (CLSM) to investigate the effect of D-AAs. We found that the C. jejuni biofilm significantly decreased after treatment with all tested D-AAs. D-Alanine, D-methionine and D-Tyrosine were the most effective in decreasing the biofilm (reductions compared to untreated controls of 75%, 66% and 74% respectively) while D-serine and D-tryptophan were the least effective (47% and 41%, respectively). Interestingly, D-Tyrosine and D-Tryptophan were observed to both inhibit and disperse the C. jejuni biofilm whereas D-Methionine, D-Alanine and D-Serine were only found to have an effect in the biofilm dispersion assay. Our findings indicate that D-AAs effect biofilm formation in Campylobacter and may provide an additional tactic in the control of Campylobacter transmission in poultry.

P125 - Long-range electrical interaction turns surface layer of marine sediments into a super biofilm


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van Bijsterveldt, Celine (Department of Ecosystem Studies, Royal Netherlands Institute for Sea Research (NIOZ), Yerseke); Zetsche, Eva-Maria (Laboratory for Analytical, Environmental and Geo-Chemistry, Vrije Universiteit Brussel (VUB), Brussels, BEL); Meysman, Filip (Department of Ecosystem Studies, Royal Netherlands Institute for Sea Research (NIOZ), Yerseke) Interaction and communication between micro-organisms is crucial for biofilm functioning and is traditionally thought to occur by molecular diffusion of signal molecules (e.g. quorum sensing). Diffusion however limits the length scale over which micro-organisms can rapidly interact with each other to a few micrometres. Here, we present a newly discovered bacterium that is capable of interacting over centimetre distances by means of electrical currents. These filamentous sulphur oxidizing bacteria (Desulfobulbaceae) were first discovered in laboratory experiments, and have been found under natural conditions in coastal sediments along the North Sea coast. Biogeochemical profiling shows that oxygen reduction at the sediment-seawater interface is linked to sulphide oxidation in the deeper sediment. Experiments have shown that the electron transport across this centimetre gap takes place along the longitudinal axis of filamentous bacteria. This long-range electron transport system entails a whole new way in which bacteria interact, thus turning the first few centimetres of marine sediments into a sort of extended biofilm with highly efficient interaction properties. To gain a better understanding

techniques, including digital holographic microscopy. Here we present our first results of how these long filamentous bacteria might interact with their environment by excreting wire-like extracellular polymeric substances.

P126 - Community of nitrile utilizing bacteria of active sludge flocs in biological treatment facilities Maksimov, Aleksandr (INSTITUTE OF ECOLOGY AND GENETICS OF MICROORGANISMS UB RAS, Perm, RUS); Vasiliev, Dmitriy (INSTITUTE OF ECOLOGY AND GENETICS OF MICROORGANISMS UB RAS, Perm, RUS); Pavlova, Yulia (Perm State National Research University, Perm, RUS); Maksimova, Yulia (INSTITUTE OF ECOLOGY AND GENETICS OF MICROORGANISMS UB RAS, Perm, RUS); Demakov, Vitalyi (INSTITUTE OF ECOLOGY AND GENETICS OF MICROORGANISMS UB RAS, Perm, RUS) Flocs of active sludge represent communities of predominantly heterotrophic aerobic microorganisms that are similar to biofilms. It is established that these are dominated by the number and variety of fast-growing aerobic proteobacteria. From the activated sludge flocs of the treatment facilities 40 gram-negative isolates of heterotrophic bacteria, utilizing the cyanopyridines as the sole source of carbon and nitrogen were isolated. By the results of sequenced fragments of the 16S rRNA gene assay the isolates with more than 99% of homology were identified as belonging to genera Acinetobacter, Alcaligenes, Delftia, Ochrobactrum, Pseudomonas, Stenotrophomonas, Xanthobacter. PCR analysis revealed that 13 out of 40 isolates have a sequence nearly 1070 bp being homologous to nitrilase genes, previously detected in A. faecalis JM3 GenBank, D13419.1. All of isolates possessed amidase activity, which ranged from 0.5 to 46.3 mmol/g/h. Among those strains capable of transforming aromatic and aliphatic nitriles there were detected: Acinetobacter sp. 11h and Alcaligenes spp. OSV, which nitrile hydrase activity during the transformation of 3-cyanopyridine amounted 4.4% and 0.4 mmol/g/h, respectively. The presence of bacteria capable of utilizing the aromatic nitriles, in


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active sludge flocs is probably fixed property of their metabolism and is not associated with the income of nitrile compounds in wastewaters. It is known that nitrile transforming bacteria are widely distributed in nature and their frequent occurrence is associated with the presence of metabolic pathways of amino acids, in which aldoxime dehydratase and nitrile transforming enzymes are functioning, as well as with the plants releasing nitriles into environment. Disposal of 3-cyanopyridine, apparently, follows nitrilase and nitrile hydratase-amidase pathway with subsequent oxidation of nicotinic acid via 2.5-dihydroxipyridine to fumaric acid.

This work was supported by RFBR grant 13-04-96050 and the RAS program of Molecular and Cell Biology.

P127 - The phototrophic community of submarine ikaite columns in Greenland: Dynamics of O2, pH, Light and Photosynthesis in an Extreme Environment Trampe, Erik (Marine Biological Section, University of Copenhagen, Helsingor, DNK); Larsen, Jens E. N. (Marine Biological Section, University of Copenhagen, Helsingør, DNK); Kühl, Michael (Marine Biological Section, University of Copenhagen, Helsingør, DNK) We present a detailed characterization of the physicochemical conditions and photosynthetic activity of phototropic biofilm thriving in submarine ikaite tufa columns in Ikkafjord (SW Greenland). The columns are formed under very specific conditions from different carbonate minerals, in particular ikaite, a hexahydrate form of calcium carbonate. In situ underwater microsensor profiling of O2, pH and light dynamics revealed an extreme environment comprised by temperatures <6°C, low light, and gradients in pH changing from pH 8 on the surface, rising to pH>10 only two cm inside a column. Dense green bands of phototrophs inhabiting the ~2 cm outer layer of the freshly deposited ikaite matrix were revealed from cross sections of the columns. Underwater O2measurements as well as variable chlorophyll fluorescence imaging of photosynthesis gives first proofs of oxygenic photosynthesis in this extreme environment.

P128 - Comparison of microbial biofilm properties among three different river sediment particle sizes Rulik, Martin (Faculty of Science, Palacky University in Olomouc, Olomouc, CZE); Badurová, Pavlína (Faculty of Science, Palacky University in Olomouc, Olomouc, CZE); Brablcová, Lenka (Faculty of Science, Palacky University in Olomouc, Olomouc, CZE); Buriánková, Iva (Faculty of Science, Palacky University in Olomouc, Olomouc, CZE); Bednarík, Adam (Faculty of Science, Palacky University in Olomouc, Olomouc, CZE) Sediment bacterial biofilms play an important role in river benthic systems because they comprise a large fraction of the total benthic biomass that contributes significantly to the turnover of organic matter within the sediment. However, compared to the relative homogenous sediments of lakes, there is a heterogenous mixture of particles of different sizes, origins, and surface features within river sediments. This heterogeneity represents very often a problem coupled with sampling and sediments analysis, because measurements of bacterial numbers or metabolic activities in marine and estuarine sediments have exhibited a strong inverse correlation with sediment grain size, implying a surface area dependence. Thus, abundance of various prokaryotic taxa, their activities, biovolumes and biomasses on three differentially sized


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sediment fractions were studied in order to determine whether an inverse relationship among the particle sizes (< 1, 1-3 and 3-5,6 mm in diameter) and various parameters exists. Results of this study were in concordance with previous studies which emphasized the importance of smallest size of sediment fraction. Although relative amount of sediment grains smaller than 1 mm ranged from 3.96 to 49.80 % across all the sites, all biofilm characteristics were associated much more with this sediment fraction. This finding also implies that for the next routine microbiological analyses it is necessary to ensure that sufficient amount of fine sediment particles is there otherwise we may underestimate the measured microbiological parameters. The authors are thankful to the European Social Fund and state budget of the Czech Republic. This work was partly supported by project CZ.1.07/2.2.00/28.0032, which is financed by the previously stated funding agencies.


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Session 9: Applied water systems P129 - The fate of Pseudomonas aeruginosa and Legionella pneumophila in copper-containing water Dwidjosiswojo, Zenyta (Biofilm Centre, University of Duisburg-Essen, Essen, GER); Richard, Jessica (IWW, Water Centre, Mülheim a. d. Ruhr, GER); Flemming, Hans-Curt (Biofilm Centre, University of Duisburg-Essen, Essen, GER); Dopp, Elke (IWW, Water Centre, Mülheim a. d. Ruhr, GER); Wingender, Jost (Biofilm Centre, University of Duisburg-Essen, Essen, GER) Copper plumbing materials can be the source of copper ions in drinking water supplies [1]. For the pathogens P. aeruginosa and Legionella pneumophila, inactivation of planktonic cells as well as of biofilms by copper ions has been a topic of research [2, 3]. Copper-mediated inactivation of bacteria was usually studied based on culture-based methods, but these do not necessarily verify cell death. The current study reveals that copper-stressed planktonic cells of P. aeruginosa [4] and L. pneumophila, although not detectable with the cultural methods designed for their detection, maintain essential viability markers (e. g. membrane integrity, 16S rRNA). Thus, they cannot be considered to be dead. Integrated in a biofilm, P. aeruginosa could even tolerate higher copper concentrations up to 100 mM before losing culturability. The minimal inhibitory concentration was found to be about 20 fold higher than that found for planktonic cells. Addition of the copper chelator sodium diethyldithiocarbamate (DDTC) to non-culturable copper-stressed P. aeruginosa resulted in the return of the bacteria to a culturable state (resuscitation) within 14 d. Preliminary results indicate that non-culturable cells of L. pneumophila could be resuscitated upon co-culture with Acanthamoeba castellanii. Therefore, copper might be considered as an inducer of the viable but non-culturable (VBNC) state in bacteria. The hygienic importance of pathogens in the VBNC state is still unclear and presents a key issue of this study. To investigate the cytotoxicity and genotoxicity of P. aeruginosa, human bronchial epithelium cells (BEAS-2B) were exposed to P. aeruginosa in the VBNC state and after resuscitation. Determining the membrane integrity (trypan blue staining), mitochondrial activity (MTT assay), cell proliferation (exCELLigence) and chromosomal damage (micronucleus test), the vitality of BEAS-2B was analyzed. It could be shown that P. aeruginosa in the VBNC state were not harmful towards human bronchial epithelium cells (BEAS-2B), whereas resuscitated P. aeruginosa showed the same cytotoxic and genotoxic effect as the original strain. From a health perspective, the relevance of pathogens in the VBNC state may be underestimated, because it has to be taken into account that they can regain their virulence and are able to initiate infection when they revert to the culturable state. Thus, pathogens in the VBNC state represent an infectious potential when present in drinking water systems.

Acknowledment This work was financially supported by the German Federal Ministry of Education and Research (grant number 02WT1157). The authors thank Conny Rosengarten and Melanie Gerhards for their excellent technical assistance.

References [1] WHO (World Health Organization): Guidelines for drinking-water quality. Fourth edition, Geneva 2011, World Health Organization.


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[2] Huang, H.-I., Shih, H.-Y., Lee, C.-M., Yang, T.C., Lay, J.-J., Lin, Y.E., 2008. In vitro efficacy of copper and silver ions in eradicating Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Acinetobacter baumannii: implications for onsite disinfection for hospital infection control. Water Res. 42, 73 80. [3] Lin, Y. E., Vidic, R. D., Stout, J. E., Yu, V. L., 2002. Negative effect of high pH on biocidal efficacy of copper and silver ions in controlling Legionella pneumophila. Appl. Environ. Microbiol. 68, 2711-2715. [4] Dwidjosiswojo, Z., Richard, J., Moritz, M. M., Dopp, E., Flemming, H.-C., Wingender, J., 2011. Influence of copper ions on the viability and cytotoxicity of Pseudomonas aeruginosa under conditions relevant to drinking water environments. Int. J. Hyg. Environ. Health 214, 485-492.

P130 - A matter of life and death: the significance of permeate flux on bacterial adhesion and subsequent cell-wall integrity under Nanofiltration Habimana, Olivier (School of Chemical and Bioprocess Engineering, University College Dublin, Dublin); Semião, Andrea J.C. (School of Engineering, The University of Edinburgh, Edinburgh, GBR); Casey, Eoin (School of Chemical and Bioprocess Engineering, University College Dublin, Dublin) Nanofiltration (NF) is a high pressure membrane filtration process increasingly applied in drinking water treatment and water reuse processes. NF typically rejects divalent salts, organic matter and micropollutants. However, the efficiency of NF is adversely affected by membrane biofouling, during which microorganisms adhere to the membrane and proliferate to create a biofilm. Although much has been done in studying membrane performance caused by biofouling, few studies have gone as far as examining the mechanisms of biofouling at its earliest stage.

To understand the initial stages of biofouling during NF processes, deposition experiments using a mCherry-tagged Pseudomonas fluorescens model strain were conducted in cross-flow and dead-end filtration conditions under varying permeate fluxes ranging from 0.5 up to 120 L.h-1.m-2, using six commercial NF membranes each having different surface properties. Ex-situ analysis of membranes were systematically performed using epi-fluorescence microscopy to establish the level of surface coverage on membranes as well as, the degree of cell damage following post staining procedures with SYTOX® green.

Results from this study show that initial bacterial adhesion is strongly dependent on permeate flux conditions, where increased adhesion was obtained with increased permeate flux, regardless of membrane tested, until a maximum of 40% coverage was reached. Moreover, adhered P. fluorescens cells were shown to have incurred structural damage and collapse as a result of high fluid shear and convective fluxes at the membrane-liquid interface following high permeate flux conditions during NF processes. Cells adhering to membranes over the course of NF undergo substantial levels of stress affecting their structural integrity, ultimately leading to the release of cytoplasmic material onto the membrane, an important precursor to biofilm formation.

These results emphasise not only the importance of conducting adhesion and biofouling experiments under realistic permeate flux conditions for the evaluation of claimed antifouling


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membranes, but conjointly identify cell collapse as an integral part of membrane initial fouling.

P131 - Dicloroacetic acid metabolism by anaerobic bacteria from natural drinking water biofilm, Deh II gen identification. Viancha, Valerie (Universidad de los Andes, Bogotá, COL); Perez, Alma (Universidad de los Andes, Bogotá, COL); Lemus, Mildred (Universidad de los Andes, Bogotá, COL); Martinez, Juliana (Universidad de los Andes, Bogotá, COL); Rodriguez, Manuel (Universidad de los Andes, Bogotá, COL) In low carbon concentration from natural source in treated water, microorganisms in biofilms could take advantage on other organic substances as dicloroacetic acid, a xenobiotic compound predominant in haloacetics acids (HAA) group. HAA are organic compounds generated by natural organic matter and disinfectant reaction, normally found in drinking water systems, which are controlled in some countries by water rule for causing probable effects on health. From tank biofilm enrichment with 1mM dicloroacetic acid, some cultures were isolated in R2A to assess degradation kinetic and dehalogenase genes presence. To achieve that, Deh genes were amplified by PCR with a pair of degenerate primers. Deh genes presence was determined from DNA extraction in 10 isolated cultures but only Deh II gen was detected in Xantohobacter flavus. After research in the Swiss Prot (NCBI) proteins database, the similarity with Xanthobacter autotrophicus´s acid haloacid delahogenase was found. Therefore, the DCA degradation mechanisms that could be used by Xantohobacter flavus depends on enzyme-substrate direct action leading a halogen release, according to Deh II enzyme activity. Furthermore, sequence amino-acid comparison showed some conserved residues including those of dehalogenases catalysis (R41, S118, K151 y Y157) and motive II, a secondary structure that characterize delahogenase haloacid protein family. Finally, dichloroacetic degradation capacity by drinking water network biofilm was determined, and an approachment to enzymatic action was made with Deh II genes identification. Key words: Haloacetic acids, biodegradation, Deh genes, biofilms

P132 - Multispecies biofilm: a strategy to survive in industrial water system? Gillmann, Antoine (Merck Millipore, Molsheim, FRA); Cleiss-Arnold, J. (UMR 7156, Université de Strasbourg-CNRS, Laboratoire Génétique Moléculaire, Strasbourg, FRA); Lièvremont, D. (UMR 7156, Université de Strasbourg-CNRS, Laboratoire Génétique Moléculaire, Strasbourg, FRA); Chollet, R. (Merck Millipore, Application group, Molsheim, FRA); Lett, M.C. (UMR 7156, Université de Strasbourg-CNRS, Laboratoire Génétique Moléculaire, Strasbourg, FRA) The water flowing through the distribution systems in pharmaceutical and food industries is subject to numerous decontamination processes such as reverse osmosis, filtration and UV sterilization. Despite the use of these drastic methods, some bacterial genera have developed coping mechanisms and remain viable in the treated water. These bacterial infections could make finished products unfit for human consumption and represent a significant economic loss to the industry.

A survival strategy allowing bacteria to colonize an environment is the attachment to a surface and its development as biofilm. These natural biofilms generally consist of several species. Thus,


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from strains isolated from treated water, we made several co-cultures and the resulting biofilms were quantified by crystal violet. We showed that the amount of biofilm varied considerably between different combinations of bacterial strains. Interestingly, the amount of biofilm has doubled in the co-culture of Methylobacterium sp. and Paenibacillus sp. In addition, attraction and dispersion in solid medium were performed for different combinations of bacteria. These results confirm our previous observations that there are interactions between some micro-organisms isolated from treated water.

In order to determine the nature of the interactions within biofilms (mutualism, antagonism, competitiveness or commensalism) a FISH technique was performed on multi-species biofilms. Given that the spatial organization of microorganisms may be dependent on their interactions. Preliminary results showed that strains of Methylobacterium sp. and Paenibacillus sp. are retrieved and structured. Further, to understand the role of each bacterium, the carbohydrate composition of the different biofilms was characterized using different FITC-labelled lectins. This study provides evidence that different mechanisms are involved in the formation of multi-species biofilms potentially implicated in survival in hostile environments such as water distribution systems.

P133 - Investigation of induced viable but nonculturable Legionella pneumophila (VBNC) by reactive chlorine disinfectant stress and there danger for sanitation Fischer, Sebastian Wolfgang (Institute for Hygiene and Public Health, University of Bonn, Germany , Bonn, GER); Kuriakose, Sapuna (Institute for Hygiene and Public Health, University of Bonn, Germany , Bonn, GER); Gemein, Stefanie (Institute for Hygiene and Public Health, University of Bonn, Germany , Bonn, GER); Gebel, Jürgen (Institute for Hygiene and Public Health, University of Bonn, Germany , Bonn, GER); Exner, Martin (Institute for Hygiene and Public Health, University of Bonn, Germany , Bonn, GER) Background: Legionella pneumophila a drinking water pathogen, responsible for legionellosis, are capable, like several bacteria strains, of entering the VBNC (viable but non culturable) state under certain environmental stress conditions. The detection of bacteria in such states is difficult as they fail to grow on culture media. This detection problem is often associated with a supposed safety that can in fact be dangerous because cells in this physiological state are alive and often still infectious. Furthermore these states are characterized by low levels of metabolic activity and their ability to be culturable again. If subletal dosages induce the formation of such states in Legionella it is an underrated danger in public health and for sanitation treatment that should be taken into account in future.

Methods: Two different chlorine releasing agents were used for 60 min in a concentration range of 0.1 1.2 mg/l free chlorine to determine the sublethal concentration which might induce Legionella pneumophila entering the VBNC state. To identify the induction of VBNC states in Legionella pneumophila after treatment, the quantitative suspension test according to DIN EN 13623 was used. The results were compared with multiple culture independent methods assessing the membrane integrity, protein synthesis (16s rRNA degradation), respiratory and esterase activity. To confirm the induced VBNC by resuscitation we use Amoeba Acanthamoeba castellanii as model organism.


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Results: Exposure to a chlorine concentration higher than 0.2 mg/l yielded no colony forming units (cfu) on culture media and no respiratory activity of Legionella pneumophila. However a complete loss of the cell membrane integrity and esterase activity was observed by only at three times higher chlorine concentration. The entering of VBNC states by L. pneumophila could be found at a concentration range between 0.25-0.3 mg/l free chlorine, were no cfu exist but viability markers still indicates cell viability under this experimental conditions. By the way non of the tested concentrations leads to a decrease of 16s rRNA signal. The resuscitation tests are still in progress.

Discussion: The presence of 16s rRNA in the cells after treatment with chlorine show that it is important to use different kinds of viability markers to detect VBNC. Using only one sole marker could lead to false positive results. Therefore the used unique combination of different viability markers made it possible to characterize VBNC states at a drinking water relevant concentration level between 0.25-0.3 mg/l free chlorine under reserve of the resuscitation results.

Outlook: Actual we perform the resuscitation tests and investigate the possible protection survival mechanisms against the disinfectant agents by gene expression analysis. Further it is planned to study the infectivity of VBNC Legionella pneumophila.

P134 - Biofilms in drinking water installations - Analysis and quantification of biofilms in drinking water pipes Nguyen, Cam Phuong (Institute of Chemistry and Biological Chemistry, ZHAW, Wädenswil, CHE); Gattlen, Jasmin (Institute of Chemistry and Biological Chemistry, ZHAW, Wädenswil, CHE); Lüscher, Marcel (GF IRG AG, Sissach, CHE); Füchslin, Hans Peter (Bachema AG, Schlieren, CHE); Krebs, Walter (Institute of Chemistry and Biological Chemistry, ZHAW, Wädenswil, CHE) In Switzerland, purified drinking water is generally of good microbial quality; therefore, treatment of the water network with chlorine is usually not necessary. Nevertheless, it is of great interest to minimize microbial contamination and regrowth during water distribution. Nowadays, plastic water pipes are widely installed in sanitary facilities owing to simplified installation and cheaper production costs. However, when plastic pipes get in direct contact with water, low-molecular substances (primarly organic carbon) migrate into the water body and possibly influencing its taste or microbial state. The Swiss Federal Institute of Aquatic Science and Technology (Eawag) has therefore developed an analytical method package (BioMig) consisting of a) an established substance migration technique for different plastics, determination of b) the total and assimilable carbon (TOC and AOC) content and c) the biofilm formation potential. Microbial availability of AOC directly correlates with the biofilm formation potential. Planktonic as well as biofilm forming cells, which were detached from the test surface with a sonication tip, were then quantified with a flow cytometer (Kötzsch et al. 2010). However, the BioMig test only focusses on the analysis of several thin test coupons (test surface: 1 cm2). Goal of this study was to further develop the BioMig package to evaluate substance migration, biofilm formation potential and to quantify biofilm forming microbes directly in installed domestic water pipes.


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To simulate real-life domestic installations, a model tubing system has been constructed consisting of a 50 meters long PE-Xc (irradiation cross-linked polyethylene) tube, in which drinking water with a constant flow rate of 47 ml/min was passing through. Cell concentrations in the aqueous phase at the in- and outlet as well as in the biofilms at the beginning and the end of the model tubing system were measured during 39 days. Because a sonication tip was not appropriate to remove cells from the inner surface of the pipe samples, an ultrasound toothbrush was applied. This methodical adaptation was gentle and effective to detach all the cells. We could show that the adapted BioMig method can be applied for quantifying biofilms on the inner surfaces of drinking water pipes. These methods will allow the prediction of the biofilm formation potential of each pipe material before its introduction on the market minimizing the contamination of the drinking water. Kötzsch S, Egli T, Bucheli-Witschel M. 2010. Beurteilung von Kunststoffen in Kontakt mit Trinkwasser BioMig: Ein verbessertes Methodenpaket zur Bestimmung des Verkeimungspotentials. GWA 9:797-810.

P135 - Retention, survival and growth of Escherichia coli and Campylobacter jejuni in dual-species biofilms with P. aeruginosa under nutrient-limited conditions Culotti, Alessandro (Northwestern University, Evanston, IL, USA) Biofilms are a concern in engineered water distribution systems, as they have been found to harbour microorganisms as diverse as enteric viruses, cysts of protozoa, pathogenic bacteria and fecal coliforms. Residence within biofilms can provide microorganisms with access to higher concentrations of nutrients and protection from environmental stresses and chemical disinfectants. Biological interactions with indigenous biofilms have been shown to influence the survival of fastidious bacteria in harsh environments. However, the role of biofilms in the persistence of pathogens in natural and engineered aquatic systems is not yet well understood. Here, we present our findings on the growth of Escherichia coli, a common fecal indicator organism, and Campylobacter jejuni, a fastidious microaerophilic bacterium, in dual-species biofilms with Pseudomonas aeruginosa in microfluidic flow cells. In mono-culture, E. coli formed sparse biofilms with very small, discontinuous microcolonies in mono-culture. However, the introduction of P. aeruginosa triggered a dramatic growth response that enabled extensive lawn-like E. coli biofilm formation. E. coli consistently overgrew pre-established P. aeruginosa biofilms and also colonized the interior of P. aeruginosa clusters. We also observed this behavior in co-inoculated experiments, and in experiments where P. aeruginosa was introduced to pre-established E. coli biofilms. The results were surprising, as P. aeruginosa is known to be a robust biofilm-forming microorganism that produces a variety of antimicrobial agents. In mono-culture, C. jejuni was incapable of forming biofilms after 3 days. In experiments where C. jejuni was introduced to pre-established P. aeruginosa biofilms however, small C. jejuni clusters were observed on the surface of P. aeruginosa biofilms after one day of growth. Three days after inoculation, C. jejuni continued to grow in small clusters on the surface of P. aeruginosa biofilms. These results demonstrate that pre-existing P. aeruginosa biofilms can facilitate C. jejuni growth under bulk oxic conditions for a period of at least three days. The results presented


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here should contribute to the understanding of pathogen retention in aquatic biofilms and help identify potential sources of pathogen contamination in water distribution systems.

P136 - Comparison of iron depositing bacterial communities in technical water systems Schroeder, Josephin (Technische Universität Berlin, Berlin, GER) Groundwater is the preferred source for drinking water in Germany. Wells extracting groundwater are often vulnerable to clogging. Oxidized iron compounds are being deposited in the biofilm due to the growth and activity of bacteria inside the well, which in turn can lead to a loss of production capacity, reduced water quality and may result in expensive maintenance. Bacterial communities of various water wells were determined for a better understanding of the mechanism behind. Traditional cultivation methods, microscopy and molecular techniques, including PCR-DGGE, 16S rDNA genomic clone library and 454-pyrosequencing were used.

Consequently, ochreous samples from two different sites were analyzed, (i) water wells at opencast mining sites, and (ii) drinking water wells of Berliner Wasserbetriebe. Phylogenetic analysis of the isolates revealed that most of the strains were unaffiliated to already identified iron bacteria. Most of the isolates occurred in the phylum of Actinobacteria. Based on 16S rDNA, molecular analysis showed a high bacterial diversity in different water wells but also within one well. It appears that each well has its own bacterial community. Members of Proteobacteria dominated microbial populations, as demonstrated by 16S rDNA clone library and 454-pyrosequencing. Most of them were related to Beta- and Deltaproteobacteria.

Iron precipitating bacteria are native to the soil and incrustation might be the result of ever changing conditions due to the well pumping. Nutritional resources released into the well increased the build-up of biofilm formation on the inner well surface. However, our results have shown that the diversity of iron precipitating bacteria is higher than previously thought and that some of the isolated strains belong to groups, which have not yet been associated with iron deposition.

P137 - Influence of water temperature on hygienically relevant bacteria in drinking water biofilms Wagner, Janine (Biofilm Centre, University of Duisburg-Essen, Essen, GER); Grobe, Susanne (IWW, Water Centre, Mülheim a. d. Ruhr, GER); Schaule, Gabriela (IWW, Water Centre, Mülheim a. d. Ruhr, GER); Wingender, Jost (Biofilm Centre, University of Duisburg-Essen, Essen, GER) Drinking water biofilms predominantly consist of environmental bacteria without any relevance for human health. However, hygienically relevant bacteria may occasionally incorporate and persist in these biofilms [1]. Due to global warming, increasing raw water and soil temperatures may lead to elevated temperatures in drinking water distribution systems [2]. In this study, the influence of water temperature on the incorporation and persistence of the hygienically relevant bacterial species Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Legionella pneumophila in drinking water biofilms was investigated. Biofilms were grown on coupons of elastomeric (EPDM) and plastic (polyethylene PE80) material in modified RotoTorque annular reactors. Continuous flow-through of the reactors was


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performed with UV-disinfected drinking water from a surface water treatment plant with low assimilable organic carbon (AOC, 6 µg Cacetate/L), and, for nutrient rich conditions, water supplemented with tryptic soy broth (AOC: 150 µg Cacetate/L). The reactors were operated at water temperatures in the range of 8 °C to 29 °C. After 14 d, they were inoculated with E. coli and K. pneumoniae, and separately with P. aeruginosa and L. pneumophila and operated for another 28 d. Quantification of general bacteria was conducted by determination of total cell counts, while the target organisms were quantified by standard cultural methods and by culture-independent fluorescence in situ hybridization (FISH). Levels of total cell counts were hardly affected by temperature. However, under oligotrophic conditions total cell counts slightly decreased on PE at 21 °C. The incorporation (number of culturable cells in biofilms 1 d after inoculation) of E. coli was the same at all temperatures for biofilms on EPDM under oligotrophic conditions. On PE incorporation decreased at temperatures > 21 °C. For K. pneumoniae the same effect was observed on PE and EPDM. Incorporation of P. aeruginosa increased at temperatures > 21 °C in biofilms on EPDM and PE. On both materials, incorporation of L. pneumophila was largely similar at temperatures > 8 °C. Nutrient rich conditions did not affect the incorporation of E. coli and K. pneumonia. Incorporation of P. aeruginosa increased on EPDM at temperatures > 21 °C whereas the incorporation of L. pneumophila decreased at temperatures > 21 °C. Persistence (period in which the organisms could be detected by cultural methods) in biofilms was not influenced by water temperature, except for L. pneumophila, whose persistence on PE was enhanced at temperatures > 21 °C under oligotrophic conditions. Nutrient addition shortened the period of culturability of L. pneumophila, whereas E. coli and K. pneumoniae were significantly longer detectable under nutrient rich conditions. P. aeruginosa was always culturally detectable, regardless of water temperature and nutrient situation. With FISH, E. coli and K. pneumoniae were detectable over 28 d under all conditions, and concentrations were significantly higher than those detected by cultural methods, indicating that these bacteria may exist in a viable but nonculturable state in drinking water biofilms. In this study it could be shown, that water temperature influenced incorporation and persistence of hygienically relevant bacteria in drinking water biofilms. However, further research is required to elucidate the influence of water temperature on the transition of bacteria from the culturable to the VBNC state or vice versa.

[1] Wingender, J., Flemming, H.-C., Int. J. Hyg. Environ. Health (2011), 214, 417-423 [2] Meuleman, A. F. M., Cirkel, G., Zwolsman, G.J.J., Water Sci.Technol. (2007), 56(4), 137-144

P138 - Lack of an autochthonous biofilm causes a rapid regrowth of P. aeruginosa in drinking water systems Meier, Thomas (DVGW-Forschungsstelle TUHH, Hamburg, GER); Bendinger, Bernd (DVGW-Forschungsstelle TUHH, Hamburg, GER) Household installations may play a substantial role in waterborne diseases since they can provide favorable conditions for persistence and growth of pathogens. In cases of contaminated drinking water systems an extreme regrowth of P. aeruginosa is often observed shortly after disinfection measures were performed. This unexpected phenomenon deteriorates the hygienic state in the drinking water system and may lead to a risk to the consumer. Following important


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questions arise: What is the cause of this extreme regrowth, which impact factors play a key role and by which measures can this be prevented?

Materials and methods: Semi-industrial test rigs using a close to practice consumption profile were used in long term experiments simulating household drinking water systems at 12 and 37°C and with different nutrient concentrations. Commonly used pipe materials were selected and applied in the test rigs e.g. EPDM1, HD PE-Xc2, and stainless steel. After establishing an autochthonous drinking water biofilm in the pipes, experimental contaminations with P. aeruginosa were conducted. After an operation time of 7 weeks a cleaning by impuls flushing and subsequent disinfection with oxidative disinfectants (up to 50 mg/L NaOCl or ClO2) was performed. Parameters monitored in biofilm and water phase were total cell counts (TCC) and concentrations of the pathogen according to standard cultivation methods and qPCR. Additional laboratory experiments were performed in order to simulate the situation in a pipe shortly after disinfection. Therefore, pipes containing an intact drinking water biofilm and pipes with biofilm treated by disinfection were used. A defined number of starved P. aeruginosa was introduced into the pipes, simulating an upstream contamination source. After 16 h stagnation at 37°C, biofilm and water phase were sampled and analyzed by above mentioned methods.

Results: The experiments in the test rigs verified the regrowth phenomenon shortly after disinfection: compared to the initial state a strong regrowth of P. aeruginosa was observed under specific conditions (37°C, elevated nutrient concentration) shortly after disinfection procedures were completed. The lab experiments with EPDM, stainless steel and HD PE-Xc showed that in presence of an autochthonous biofilm P. aeruginosa er phase while about 10 % of the added pathogens remained in the biofilm. In presence of a damaged biofilm or in absence of a biofilm P. aeruginosa multiplied fast in the water phase and reached 10 to 100fold concentrations compared to results from pipes with intact biofilms. Also, the numbers of P. aeruginosa in the biofilms on the pipe surfaces increased by a factor of 10 to 1000 compared to the numbers in intact biofilms.

Conclusions: Based on these observations, the presence of an autochthonous biofilm can limit the growth of P. aeruginosa in drinking water systems while the pathogen is able to multiply rapidly to high numbers in its absence. The biofilm seems to be a competitor for nutrients and space on the pipe surface. If the biofilm is removed or damaged by disinfection, P. aeruginosa is the r-selected species and exhibits strong regrowth in the whole drinking water system. These findings emphasize the importance of removal of the source for contamination with pathogens before disinfection measures are performed. If this disinfection is insufficient, the hygienic situation in a drinking water system may get worse than before.

This work was financially supported by BMBF grant number 02WT1154.

1 Peroxide cross-linked ethylene propylene diene monomer in compliance with DVGW W 270 and KTW category C (as per 2007) 2 High density electron ray cross-linked polyethylene


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P139 - The effect of silver nanoparticles on the viability of Pseudomonas aeruginosa biofilms Pillen, Alexa Margareta (University Duisburg-Essen, Essen, GER); Flemming, Hans-Curt (University Duisburg-Essen, Essen, GER); Wingender, Jost (University Duisburg-Essen, Essen, GER) Silver nanoparticles (AgNPs) displaying antimicrobial properties due to the release of silver ions are of high interest for protection of medical devices and materials as well as consumer products. The effect of silver on bacteria is usually determined by culture methods, monitoring the decrease of colony counts of planktonic cells, without considering the efficacy of silver towards biofilms. However, culture-dependent methods do not account for bacteria which have entered the viable-but-non-culturable (VBNC) state. A bacterial cell in the VBNC state fails to grow under routine bacteriological cultivation conditions, but is in fact alive and still has metabolic activity [1]. Recently, copper ions were found to induce this state in the opportunistic pathogen Pseudomonas aeruginosa [2], but it is unknown if silver ions can also trigger the VBNC state in bacteria.

In this study, the influence of AgNPs and silver nitrate on established biofilms and planktonic cells of P. aeruginosa was investigated, using 24 h-old attached biofilms and planktonic bacteria that were separately exposed to AgNPs or silver nitrate in deionized water for 24 h. Parameters for the characterization of the silver-exposed bacteria were culturability (plate counts), total cell counts (DNA staining), cell membrane integrity (LIVE/DEAD staining), ribosomal RNA (fluorescence-in-situ-hybridization) and ATP content (bioluminescence assay).

As determined by the cultivation method, AgNPs turned out to be less effective than silver ions. A concentration of 25 µg/mL Ag from silver nitrate led to a decrease of colony counts of planktonic bacteria by more than > 7 log units, whereas 500 µg/mL Ag from AgNPs were necessary to achieve the same effect. The biofilm mode of growth protected the bacteria and rendered them significantly less susceptible to silver ions and AgNPs than planktonic bacteria.

Non-culturable P. aeruginosa exposed to silver showed clear signs of viability when investigated by culture-independent methods. The cells still displayed intact DNA and ribosomal RNA, maintained their membrane integrity and contained significant amounts of ATP. Therefore, it was concluded that silver ions and AgNPs in concentrations, at which culturability was inhibited, induced the VBNC statein both planktonic and biofilm cells of P. aeruginosa.

[1] J.D. Oliver, FEMS Microbiol. Rev. 34, 415 (2010) [2] Z. Dwidjosiswojo, J. Richard, M.M. Moritz, E. Dopp, H.-C. Flemming, J. Wingender, Int. J. Hyg. Environ. Health 6, 485 (2011)

Acknowledgement: This research project was funded by the Federal Ministry of Education and Research.


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P140 - Introducing optical coherence tomography as a new tool to visualize the (bio)fouling development in a modified membrane fouling simulator West, Stephanie (Chair of Water Chemistry and Water Technolog, Engler-Bunte-Institut, Karlsruhe Institute of Technology, Karlsruhe, GER); Horn, Harald (Chair of Water Chemistry and Water Technolog, Engler-Bunte-Institut, Karlsruhe Institute of Technology, Karlsruhe, GER); Wagner, Michael (Chair of Water Chemistry and Water Technolog, Engler-Bunte-Institut, Karlsruhe Institute of Technology, Karlsruhe, GER) Biofouling is a serious problem in the operation of membrane systems such as reverse osmosis membrane modules often used in water treatment. Especially, the growth of biofilm inside the feed spacer channel has been identified as the dominating fouling process. The biofouling development is typically followed on the macroscale measuring the inlet and outlet pressure as well as the permeate flux. However, a detailed understanding of the biofilm formation, its structure and characteristics can mainly be achieved ex situ through the examination of fouled membrane (modules). In situ investigations are possible at modified membrane testing systems (Lerch et al., 2012) and with sophisticated imaging techniques as shown by Vrouwenvelder et al. (2009) who applied magnetic resonance imaging to revealed the flow characteristics and biomass distribution in a fouled feed spacer channel. Recently, optical coherence tomography (OCT) has been introduced in biofilm research, because of several advantages compared to other imaging modalities. OCT is non-invasive, fast, in situ applicable and visualizes representative volumes of several mm³ with a resolution at the µm-range (Wagner et al., 2010). In the current study, OCT was used to monitor the biofilm developing in the feed spacer channel of a lab-scale membrane fouling simulator (MFS; L = 280 mm, B = 40 mm) mimicking a reverse osmosis membrane module. Three optical windows were installed to allow for the in situ visualization of the (bio)fouling structure during full operation (maximal operating pressure = 10 bar). Two MFS were operated simultaneously at identical substrate (glucose) and nutritional conditions as well as hydrodynamic flow conditions growing a heterotrophic multi-species wastewater biofilm. MFS 1 was operated with a 44 mil diamond spacer whereas MFS 2 was operated with a 44 mil parallel spacer. Within six days the pressure drop along the feed channel increased to 0.24 bar (MFS 1) and 0.17 bar (MFS 2), respectively. This macroscopic behavior was confirmed acquiring 3D OCT datasets (C-scan) of 8×8×1.5 mm³ at a resolution of 20×20×2.1 µm. Biofilm growth started at the intersection of spacer fibers. From these seeding points filamentous biofilm structures developed which later on attached to the membrane surface. Additionally, biomass accumulation was observed downstream these intersections. A digital image analysis of OCT C-scans revealed an almost continuous increase in the amount of the biomass within the visualized volume. At the last day of operation MFS 1 (diamond spacer) showed an increase in the amount of biomass volume of 11% whereas MFS 2 (parallel spacer) was characterized by an increase of 7%. Based on the results it can be assumed that the diamond spacer is more prone to biofouling than the parallel spacer. Compared to 2D imaging techniques such as stereo and digital microscopy, OCT allows a highly resolved 3D visualization, evaluation and quantification of the fouling characteristics in MFS. It has to be highlighted that OCT was applied in situ without disturbing the cultivation. Furthermore, the MFS were slightly modified assuming that the gained results can extend previous studies. Because of its ease of use and low administrative demand, OCT could in future be the favorite imaging technique to investigate membrane fouling in more detail. References


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Lerch, A., Siebdrath, N., Berg, P., Heijnen, M., Gitis, V., Uhl, W., 2012. Desalin. Water Treat. 42, 181 188. Vrouwenvelder, J.S., Graf von der Schulenburg, D. a, Kruithof, J.C., Johns, M.L., van Loosdrecht, M.C.M., 2009. Water Res. 43, 583 594. Wagner, M., Taherzadeh, D., Haisch, C., Horn, H., 2010. Biotechnol. Bioeng. 107, 844 853.

P141 - Spatial and temporal dynamics of bacterial biofilm communities along the processing pathway in a large-scale seawater reverse osmosis desalination plant Levi, Adi (Bar-Ilan University, Ramat Gan, ISR); Bar-Zeev, Edo (Bar-Ilan University, Ramat Gan, ISR); Elifantz, Hila (Bar-Ilan University, Ramat Gan, ISR); Berman, Tom (Kinneret Limnological Laboratory, Israel Oceanographic and Limnological Research, Migdal , ISR); Berman Frank, Ilana (Bar-Ilan University, Ramat Gan, ISR) Biofouling impacts seawater reverse osmosis (SWRO) desalination plants by directly reducing filtration efficiency, increasing energetic demands and incurring further costs. Here we examined the spatial and temporal composition and dynamics of the bacterial communities along the treatment stages of a large-scale SWRO facility as a first step to developing sustainable biofilm reduction solutions. The bacterial community structure of both water samples and surface-attached biofilm was followed annually by seasonal sampling at the ADOM desalination facility (Ashkelon, Israel) in February, May, September and November 2011. Bacterial community composition throughout the desalination facility was determined by 16S rRNA 454 pyrosequencing. Our results show that while seasonal variations were reflected in the ambient water-sample communities, surface biofilm samples were not impacted by season. The biofilm community within the rapid sand filtration (RSF), micronic filters (MF) and reverse osmosis (RO) membranes were significantly different from the ambient water-sample populations. Moreover, surface biofilm samples significantly differed from one another while the bacterial populations within the water samples were similar along the treatment pathway. Proteobacteria- (water 80-90% and surface 40-60%) dominated all sampling stations. RO biofilm community structure was closely related to the water samples with high abundance of Alphaproteobacteria SAR11 cluster. The microbial communities on the RO shifted from Alphaproteobacteria (winter and fall) to Gamaproteobacteria dominance (spring-summer), primarily due to increased abundance of the moderate halophilic genus Kangiella combined with a reduction in SAR11 cluster. RSF and MF samples had the highest Shannon diversity values (5.06-5.95), while microbial diversity of the RO biofilm was much lower (2.27-3.91) resulting from the hypersaline conditions, high pressure, and intense shear forces on the RO membrane. The unique niche of the RO and its distinctive bacterial population reduce the chance for successful proliferation of RSF or MF bacteria on to the RO membranes. Our results clarify the dynamic interactions between the ambient source planktonic bacteria, biofilm development along the pretreatment stages and the subsequent biofouling of RO membranes; thus, facilitating future regulation of biofouling for desalination industries.


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P142 - Oxygen profiles in aerobic granules during SBR cycles Brunner, Fabian (Karlsruhe Institute of Technology - Chair of Water Chemistry and Water Technology, Karlsruhe, GER); Lackner, Susanne (Karlsruhe Institute of Technology - Chair of Water Chemistry and Water Technology, Karlsruhe, GER); Horn, Harald (Karlsruhe Institute of Technology - Chair of Water Chemistry and Water Technology, Karlsruhe, GER) Aerobic granules, spherical aggregated biomass without artificial carrier, will play an important part in the future of wastewater treatment. A huge advantage of aerobic granules compared to activated sludge is, that aerobic and anaerobic processes can occur in one reactor at the same time. This is because, oxygen will only penetrate the outer layer of large granules. Up to now there are many studies regarding different aspects of aerobic granulation such as influence of operations strategies (Rocktäschel et al., 2013) and characteristics of single granules (Tay et al., 2002). Mainly such studies look either at the average activity of granules within a reactor system or at single inactive granules outside the reactor. The latter is the case when morphology or microbial distribution is observed. et al., 2007; Li et al., 2008). For further understanding of the processes within granules it is important to observe the activity of a granule that is running through a sequencing batch reactor (SBR) cycle. This opens up the possibility to study the behavior of parameters inside granules, while the bulk phase is constantly changing. Moreover, processes can be studied over a long period of time. Therefore a microfluidic device was developed to conduct microelectrode measurements of oxygen, pH and ORP in time and space (means the z-axis of a granule). This device will be connected to an SBR. Recirculation of the bulk phase of the reactor through the microfluidic device ensures identical operation conditions in the reactor and the microfluidic device. Through this experimental set-up it is possible to analyze microbial processes directly in granules under reactor conditions.

Chiu, Z.C., Chen, M.Y., Lee, D.J., Wang, C.H., Lai, J.Y., 2007. Oxygen diffusion in active layer of aerobic granule with step change in surrounding oxygen levels. Water Res. 41, 884 92. Li, Y., Liu, Y., Shen, L., Chen, F., 2008. DO diffusion profile in aerobic granule and its microbiological implications. Enzyme Microb. Technol. 43, 349 354. Rocktäschel, T., Klarmann, C., Helmreich, B., Ochoa, J., Boisson, P., Sørensen, K.H., Horn, H., 2013. Comparison of two different anaerobic feeding strategies to establish a stable aerobic granulated sludge bed. Water Res. Tay, J.H., Liu, Q.S., Liu, Y., 2002. Characteristics of aerobic granules grown on glucose and acetate in sequential aerobic sludge blanket reactors. Environ. Technol. 23, 931 6.

P143 - Insights into colmation-causing biofilms of a groundwater catchment Braun, Burga (TU Berlin, Berlin, GER); Schröder, Josephin (TU Berlin, Berlin, GER); Szewzyk, Ulrich (TU Berlin, Berlin, GER) Groundwater is of great importance as a source of drinking water. The extraction of groundwater can suffer from colmation as a result of clogging of interstitial pore spaces. Colmation, as indicated by the reduction of the hydraulic conductivity of porous media, is a physical, chemical and, mainly, a microbiological process. It can result in complete blockage of a well and lead to costly well cleanup and rehabilitation, which have important economic impacts.


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Microbiological colmation is caused by biofilms, i.e., matrix-enclosed microbial populations that are ubiquitous at any interface. The aim of this project was to determine the phylogenetic diversity of colmation-causing bacteria in different water wells in a groundwater catchment to help prevent and remediate the colmation. Samples from infiltration wells, extraction wells and different control points were examined to provide an overview of the composition of the biofilms and to identify the responsible bacterial groups. For this purpose, we isolated different bacteria and grouped them into distinct OTU´s as determined by ARDRA. Phylogenetic analysis based on 16S rRNA gene sequencing revealed that most of the strains were affiliated to Pseudomonas veronii or to Sphingomonas sp. 16S rDNA genomic clone libraries of samples derived from different sample points. In addition, 454 pyrosequencing data indicated Rhodoferrax and Pseudomonas as some of the most abundant genera. Based on the correlation of these data with physicochemical water parameters, an indicator system to prevent colmation should be established. The new established q-PCR allows the rapid detection and quantification of the responsible bacteria and facilitates fast screening of samples.


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Session 10: Medical systems and anti-biofouling

P144 - Antimicrobial activity against Streptococcus mutans of the synthetic peptide Aurein 1.2 extracted from Litoria aurea Carneiro, Victor (Federal University of Ceara, Sobral, BRA); Lorenzón, Esteban (Univ. Estadual Paulista - UNESP, Araraquara, BRA); Cilli, Eduardo (Univ. Estadual Paulista - UNESP, Araraquara, BRA); van der Mei, Henny (University Medical Center Groningen , Groningen); Busscher, Henk (University Medical Center Groningen , Groningen) Dental caries is considered one of the biggest health problem in the world due to its high incidence. It is caused by oral biofilms comprising mutans streptococci, most notably Streptococcus mutans. However, in saliva and other natural environments there are molecules that are known as antimicrobial agents with a high biocidal activity against various pathogenic microorganisms, called antimicrobial peptides. Moreover, few or no reports are available on resistance amongst bacterial strains against antimicrobial peptides. This study evaluates the antimicrobial activity of the synthetic peptide Aurein 1.2 on the planktonic growth of a collection of oral bacterial strains, such as S. salivarius, S. mitis, S. sanguis, S. oralis and S. mutans. This peptide is a member of the Aurein´s family, which was isolated firstly from the skin secretions of Litoria aurea. Aurein 1.2 was obtained through solid-phase peptide synthesis using Fmoc/tBu. The peptide was purified in RP-HPLC and characterized by HPLC and electrospray mass spectrometry.The antimicrobial activity of Aurein 1.2 was determined through micro-dilution method in microtiter plates yielding a time-kill curve. The peptide showed activity against several streptococcal strains, in particular on S. mutans. Also bacterial zeta potentials and surface morphology were determined of peptide-treated and untreated bacteria. Particulate microelectrophoresis yielded small changes in bacterial zeta potentials when treated with 0.5, 1 and 2-fold MIC concentrations of the peptide. AFM imaging exhibited morphological differences on the bacterial surfaces when treated with different concentrations of Aurein 1.2. These results indicate that the antimicrobial effect of Aurein 1.2 causes damage to the bacterial membrane. Positive charges from Aurein 1.2 peptide can interact with negative membrane charges in streptococcal cell walls to eventually induce cell lysis. Considering the strong activity of Aurein 1.2 against S. mutans, the peptide might be particularly suitable to restore a healthy oral microflora rather than by a-selective killing of bacteria, as done by many current generations of oral antimicrobials.

Keywords: Streptococcus mutans , antimicrobial peptides and Aurein 1.2

P145 - Highly reactive, superacidic surfaces as novel antifouling materials Gehring, Julia (Universität Konstanz; FB Chemie; AG Polarz, Konstanz, GER) Highly reactive, superacidic surfaces as novel antifouling materials: the perspective of mesoporous organosilica nanoparticles Julia Gehring1, David Schleheck2, Sebastian Polarz3 1Julia Gehring, University of Konstanz, Department of Chemistry, D-78457 Konstanz, Germany, tel.: 07531-882006, email: [email protected] 2David Schleheck, University of Konstanz, Department of Biology, D-78457 Konstanz, Germany 3Sebastian Polarz, University of Konstanz, Department of Chemistry, D-78457 Konstanz, Germany Abstract Combining high


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internal surface area with tailor-made surface properties is pivotal for granting advanced functional properties in many areas like heterogeneous catalysis, electrode materials, membranes, or biomimetics [1] and potentially for novel antifouling materials. In this respect, organic-inorganic hybrid nanostructures and in particular mesoporous organosilica materials are ideal systems. We prepared mesoporous solids via a new sol gel building block comprising sulfonic acid (R-SO3H). The degree of organic modification is not only maximal (100%), it was also proven that the novel material exhibits superacid properties. Furthermore, an aerosol assisted method [2] is applied for generating this material in the form of mesoporous, spherical nanoparticles with substantial colloidal stability. Here we show results of the first experiments addressing the antibacterial and antifouling effect of the sulfonic-acid, mesoporous organosilica nanoparticles [4] . It is demonstrated that the superacid character is required for exhibiting sufficient antifouling activity. Introducing a further functional group such as thiol (SH) into the superacidic highly porous silica material will extend the area of application tremendously. In particular it is known that thiol functionalities bind silver (Ag) nanoparticles as well as silver ions (Ag+) and we want to create such a new more complex system in the field of antifouling surfaces. [1] a) B. Bhushan , Phil. Trans. Roy. Soc. A 2009 , 367 , 1445 1486 b) P. Fratzl , J. R. Soc. Interfaces 2007 , 4 , 637 642 [2] a) Y. F. Lu , H. Y. Fan , A. Stump , T. L. Ward , T. Rieker , C. J. Brinker, Nature 1999 , 398 , 223 226 b) Y. F. Lu , H. Y. Fan , N. Doke , D. A. LoyR. A. Assink , D. A. LaVan , C. J. Brinker, J. Am. Chem. Soc. 2000 , 122 ,5258 5261 [3] a) K.-i. Shimizu , E. Hayashi , T. Hatamachi , T. Kodama ,Y. Kitayama, Tetrahedron Lett. 2004 , 45 , 5135 5138 b) A. Corma , H. Garcia , Adv. Synth. Catal. 2006 , 348 , 1391 1412 [4] J.Gehring, D. Schleheck, S. Polarz; Adv. Funct. Mater. 2013, 24, 8, 1140-1150 P146 - THE ROLE OF BACTERIAL INFECTIONS BIOFILMS ON MALE INFERTILITY IN AL-ANBAR PROVINCE OF IRAQ Farhan, Abbas (University of Al-Anbar, College of Medicine, Department of Microbiology, Ramadi, IRQ); Al-Janabi, Abbas (University of Al-Anbar, Department of Microbiology, IRQ); Jubair, Abdul Sattar (AL-Ramadi Teaching Hospital, Ramadi, IRQ) Background: Bacterial infections Biofilms on male infertility has always been in the field of debate due to scarce analysis tools to examine seminal fluid specimens as a result of which these infectious processes leads to deterioration of spermatogenesis, impairment of sperm function and/or obstruction of the seminal tract. Aims & Objective: In the current study we investigated the role of bacterial infection Biofilms in male factor infertility in Al-Anbar Province, West of Iraq through detection of abnormal sperms and other factor pertains to male infertility. Material and Methods: Seminal fluid from six hundred volunteer males was investigated for infertility by the detection of abnormal sperms using the WLJY-9000 TYPE WEILI Color Sperm Analysis System and the Neubauer counting chamber. Results: From the six hundreds patients investigated for infertility, it was found that 408 (68%) patients had a positive culture for pathogenic bacteria, of different species. The results indicate that 32.0% had sperm density less than twenty million per millilitre. The oligospermic were 23.0%, severe oligospermic 0.17% and Azoospermia 8.83%. Asthenospermia was reported to be


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76.33% and Teratospermia 86.16% respectively. Conclusion: Seminal fluid infection Biofilms increases with decreasing sperm density, motility and morphology. The prevalence of abnormal sperm indices and bacterial infection is high with Klebsiella spp. infection. Hence, treatment measures should be taken properly in the management of male factor infertility. Key-Words: ; Bacterial Infection Biofilms.WLJY-9000 TYPE WEILI Color Sperm Analysis System; Seminal Fluid; Male Infertility

P147 - Effects of fibronectin coating on bacterial adherence in a co-culture model of pathogenic bacteria and osteoblast progenitor Wu, Dongni (Université de Cergy-Pontoise, Cergy-Pontoise, FRA); Hindié, Mathilde (Université de Cergy-Pontoise, Cergy-Pontoise, FRA); Gallet, Olivier (Université de Cergy-Pontoise, Cergy-Pontoise, FRA); Di Martino, Patrick (Université de Cergy-Pontoise, Cergy-Pontoise, FRA) Biochemical functionalization provides a suitable environment for the surrounding living tissue and improves biocompatibilty. Bacterial infection on functionalized biomaterials is the major limitation to long-term use of implants and prostheses. Bacterial adherence to the surface of implants is a critical first step of biofilm formation and infection development. Nevertheless, it is essential to determine how the functionalization process can impact bacterial infection by increasing target number for bacterial adherence and biofilm formation on the material. We developed an in vitro model of co-culture of pathogenic bacteria and osteoblast progenitor cells to study the cells and bacteria behavior in contact with a functionalized template. Calibrated suspensions of bacterial and human cells were mixed and incubated at 37°C in wells of cell-culture plates from 3 to 6 hours before analysis. P. aeruginosa PAOI, S. aureus CIP4.83, STRO-1+

A cells, and glass coverslips coated with purified plasmatic human fibronectin (Fn) placed in multiwell plates were used for adherence tests. We analyzed both the STRO-1+A cells adhesion and bacterial adherence to glass. Finally, bacterial adherence to human cells with or without Fn coating was monitored. Fn coating improved STRO-1+ A cells adherence to glass. After 3 hours of co-culture, the presence of bacteria did not modify the STRO-1+ A cells adherence to glass. After 6 hours of co-culture, a decrease of STRO-1+ A cells adherence to glass and a cytotoxic effect were observed. The two bacterial strains tested adhered efficiently to glass and to human cells. Bacterial adherence to both surfaces was higher for the S. aureus CIP4.83 strain than for the P. aeruginosa PAOI strain. Fn coating significantly enhanced bacterial adherence to glass and to STRO-1+ A cells. The adherence increase was higher for CIP4.83 than for PAOI. Confocal laser scanning microscopy observations after plasmatic Fn coating revealed cellular Fn overexpression by STRO-1+ A cells. Since cellular Fn is a receptor for P. aeruginosa and S. aureus adherence to human cells, such Fn overexpression could explain the increase of bacterial adherence to STRO-1+ A cells. In conclusion, the co-culture model developed in this study was efficient to determine the impact of the material surface functionalization on colonization by human osteoprogenitor cells and pathogenic bacteria during the first hours post-implantation. Plasmatic Fn coating increased both STRO-1+ A cells and bacterial adherence to glass but also bacterial adherence to human cells. Thus, use of Fn coating may increase the risk of bacterial adherence and biofilm formation on a material in the presence of bacterial contamination.


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P148 - IMPACT OF LOW-DOSAGE IRRADIATION ON BIOFILM-FORMATION BY CORROSION-RELEVANT BACTERIA Boretska, Mariia (Zabolotny Institute of Microbiology and virology of NASU, Kyiv, UKR); Pareniuk, Olena (Ukrainean Institute of Agricaltural Radiobiology, Chabany, UKR); Guga, Yurij (SE"RADMA" L.V. Pisarzhevky Institute of NAS of Ukraine, Kyiv, UKR); Spiers, Andrew (SIMBIOS Centre and School of Science, Engineering and Technology, Dundee, GBR) At nuclear hazard sites, such as the Chernobyl reactor sarcophagus, radiation is one of the main factors influencing microbial communities, including those involved in microbially influenced corrosion (MIC) of metal structures. By studying the impact of radiation on biofilm formation by model corrosion-relevant bacteria, it may be possible in the future to predict changes in corrosion-associated bacterial communities and the extent of MIC at nuclear hazard sites. It is likely that the composition and function of multi-species biofilms may change when exposed to the stress of ionizing radiation, enhancing or decreasing corrosion activity on different structures. To address these issues, biofilm formation by the corrosion-relevant heterotrophic bacteria Pseudomonas pseudoalcaligenes0 and Stenotrophomonas maltophilia were studied after exposure to a range of radiation dosages. Altered planktonic cell morphologies and biofilm architectures on submerged glass surfaces were noted after low irradiation (5Gy) at the microcolony (1 hour), macrocolony (1day) and mature biofilm (3days) developmental stages. Irr adiated cellsformed atypical biofilms with conglomerates and filamentous exopolymeric slime strands that were not observed in control experiments. These observations suggest that the exposure to what is normally considered as insignificant levels of irradiation is sufficient to alter biofilm formation of corrosion-relevant bacteria. Such low dosage radiation may have significant impact on soil microbial communities in nuclear hazard sites, potentially altering the MIC of exposed metal structures, their stability and service life.

P149 - The interaction of Pseudomonas aeruginosa biofilms with inflammatory cytokines and immune cells Rahman, Tamanna (University of Nottingham, Nottingham, NFK) Colonization and persistent lung infection by pathogens, mainly Pseudomonas aeruginosa (PA), is an important cause of morbidity and mortality in Cystic Fibrosis patients. PA persists inside the lungs by forming drug-resistant biofilms. Quorum sensing (QS), a cell to cell communicating system has an important role in the PA biofilm development and resistance to antibiotics and host defence. Investigation into the behaviour of bacterial cells within these structures and their response to components of the innate immune response will give us an insight into novel approaches to combat infection.

We aim to investigate the effect of inflammatory mediators such as cytokines and immune cells on the development of PA biofilms. For this the BioFlux system has been used to generate biofilms of PA expressing green fluorescent protein. After incubation with cytokines such as GM-CSF and IFN- ɣ or labelled purified human monocytes and macrophages changes in the characteristic of the biofilms were analysed using confocal microscope and COMSTAT software. No obvious reduction of PA biofilm was observed on response to GM-CSF and IFN- ɣ. No change was observed in the levels of QS molecules extracted from supernatants of PA biofilm after incubation with GM-CSF and IFN- ɣ. These results indicate that both of these cytokines do not have direct affect PA biofilm formation. Preliminary results indicate that macrophages, unlike


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monocytes, attached to PA biofilms. Further experiments will be conducted to confirm these observations and to determine how these cytokines, alone or in combination with macrophages and monocytes, could be used to control/modulate biofilm formation.

P150 - fungal biofilms and infectious risks by venous access BEKKAL BRIKCI-BENHABIB, Ouassila (University of Tlemcen /LAPSAB, TLEMCEN , DZA); boucherir-otmani, zahia (University of Tlemcen /LAPSAB, TLEMCEN , DZA); BOUCHERIT, kebir (University of Tlemcen /LAPSAB, TLEMCEN , DZA) Background and Objectives: Several studies have shown that Candida species have a capacity to generate biofilms adhering medical devices such as intravenous catheters. In this context, it appears important to study the biofilm formation capability of Candida isolates on peripheral venous catheter and their implication on antifungal resistance

Methods: All samples were taken from implanted peripheral venous catheters for 72 hours or more days. They are removed directly from patients hospitalized at hospital of west Algeria. The biofilm formation of Candida strains were formed in vitro by using the microtiter plate method following the protocol described by Ramage et al.,(2001) and were compared to their planktonic countreparts by using NCCLS M27-A

Results: Biofilm formation by Candida species may play an important role in pathogenesis. they are 4 to 32 times more resistant to amphotericin B compared to their planktonic equivalents.

Conclusion: Our study shows the problems caused by resistance of strains isolates on intravenous catheters . In their biofilm state, Candida spp.( Candida albicansand Candida krusei, C.parapsilosis and C.famata) isolates are much less susceptible to amphotericin B compared to their antifungal susceptibility in the planktonic forms.

P151 - Synergistic inhibition of the Pseudomonas aeruginosa biofilm formation by the combination of antibiotics with a new Quorum Sensing inhibitor Furiga, Aurélie (Laboratoire de Génie Chimique (UMR 5503), Université Paul Sabatier, Université de Toulouse, Toulouse cedex 9, FRA); Lajoie, Barbora (Laboratoire de génie Chimique (UMR 5503), Université Paul Sabatier, Université de Toulouse, Toulouse cedex 9, FRA); El Hage, Saloma (Laboratoire de Génie Chimique (UMR 5503), Université Paul Sabatier, Université de Toulouse, Toulouse cedex 9, FRA); Baziard, Genevieve (Laboratoire de Génie Chimique (UMR 5503), Université Paul Sabatier, Université de Toulouse, Toulouse cedex 9, FRA); Roques, Christine (Laboratoire de Génie Chimique (UMR 5503), Université Paul Sabatier, Université de Toulouse, Toulouse cedex 9, FRA) Pseudomonas aeruginosa can cause severe lung infections in patients with cystic fibrosis, particularly in establishing a resistant structured form called biofilm. Biofilm formation is mainly regulated by the communication system of the Quorum Sensing (QS), controlled by the


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natural N-acyl homoserine lactones (HSL) molecules. Thus, the aim of our study is to design potent HSL analogs to inhibit this biofilm development. Among new analogs based on the C4-HSL structure, compound C11 (N-pymimidyl butanamide) showed a significant inhibition of biofilm formation in a dose-dependent manner, coupled with an absence of cytotoxicity on lung cells. Its inhibitory activity was preserved on a biofilm developed under anaerobic conditions, set up to approximate the in vivo colonization conditions by the bacterium. Then, C11 was tested in association with antipyocyanic antibiotics and a significant synergistic effect was obtained with ciprofloxacin, tobramycin and colistin, both under aerobic and anaerobic conditions. Actually, the effect of different combination of C11 with antibiotics on P. aeruginosa virulence gene expression is analyzed by qRT-PCR. Thus, this study should allow to determine the best combination of effective molecules against the P. aeruginosa biofilm and to define optimal conditions for further in vivo investigations. This work is supported by Vaincre la Mucoviscidose.

P152 - Actinobacillus pleuropneumoniae can acquire pyridines compounds from other swine pathogens and form or incorporate into biofilms with other swine pathogens. Loera Muro, Abraham (Universidad Autónoma de Aguascalientes, Aguascalientes, MEX); Jacques, Mario (Université de Montréal, Saint Hyacinthe, CAN); Avelar Gonzalez, Francisco Javier (Universidad Autónoma de Aguascalientes, Aguascalientes, MEX); Labrie, Josée (Université de Montréal, Saint Hyacinthe, CAN); Tremblay, Yannick D.N. (Université de Montréal, Saint Hyacinthe, CAN); Oropeza Navarro, Ricardo (Instituto de Biotecnología, UNAM, Cuernavaca, MEX); Guerrero Barrera, Alma Lilián (Universidad Autónoma de Aguascalientes, Aguascalientes, MEX) Actinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumonia that causes great economic losses in the pig industry. Several respiratory infections are associated with biofilm formation, and A. pleuropneumoniae has the ability to form biofilms in vitro. The goal of this study was to determine the capacity of A. pleuropneumoniae to form multi-species biofilms with other swine pathogens (Streptococcus suis, Bordetella bronchiseptica, Pasteurella multocida, Staphylococcus aureus and Escherichia coli). A. pleuropneumoniae was able to form strong two-species biofilms in the presence of S. suis, B. bronchiseptica and S. aureus under growth conditions that are not favorable for A. pleuropneumoniae (BHI media without NAD supplementation). With P. multocida or E. coli, A. pleuropneumoniae formed a weak biofilm. In all cases, viable bacteria were recovered from the two-species biofilms. A. pleuropneumoniae was also able to incorporate into pre-formed biofilms of S. aureus, S. suis or E. coli, under conditions that favour biofilm formation by S. aureus, S. suis or E. coli but not for A. pleuropneumoniae. In conclusion, our data suggest that A. pleuropneumoniae is able to acquire pyridines compounds from other swine pathogens and form or incorporate into biofilms with other swine pathogens.

P153 - Effects of anthranilate and indole on the Pseudomonas aeruginosa biofilm formation Kim, Soo-Kyoung (Pusan National University, Busan, KOR) Indole has been reported to enhance the biofilm formation of P. aeruginosa. Although quorum sensing (QS) is important in the biofilm formation of P. aeruginosa, the enhancement of biofilm formation by indole was QS-independent and none of QS regulators such as LasR, RhlR, QscR,


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and PqsR were activated by indole. Instead, a QS-related regulator, AntR was significantly activated by indole. AntR controls the metabolism of anthranilate, a precursor of tryptophan and PQS. Since both anthranilate and indole are aromatic compounds that are produced from tryptophan degradation, we investigated the anthranilate effect on the biofilm formation of P. aeruginosa. Interestingly, anthranilate enhanced the biofilm formation at early stage of biofilm development by augmenting the initial attachment of cells, but it destabilized the biofilm structure at later stage, making flat biofilm. Distinctively, indole structured robust biofilm and accelerated the biofilm development, advancing the dispersion. Co-treatment of anthranilate and indole activated AntR additively and also enhanced the biofilm formation additively at early stage. But at later stage, the biofilm enhancement by indole was dampened by anthranilate effect. The anthranilate effect on the biofilm formation was QS-independent, since the QS mutant still showed the enhanced attachment at early stage and flattening the biofilm structure at later stage in the presence of anthranilate.

P154 - Efficacy of chlorhexidine digluconate & eucalyptus oil based mouthwashes against peri-implantitis associated biofilms Narendrakumar, Krunal (University of Birmingham, Birmingham, GBR); Sammons, Rachel (University of Birmingham, Birmingham, GBR); Tony, Worthington (University of Aston, Birmingham, GBR); Martin, Richard (University of Aston, Birmingham, GBR); Addison, Owen (University of Birmingham, Birmingham, GBR) INTRODUCTION Eucalyptus oil (EO) enhances permeation of chlorhexidine digluconate (CHG) through bacterial biofilms. The major component of the oil is 1,8-cineole1 and in combination with CHG results in synergistic antimicrobial activity2 and could therefore potentially be used for disinfection of Ti dental implants. Previous work has shown that the synergistic antimicrobial activity of CHG/EO has improved the penetrative properties and rapid antimicrobial efficacy on biofilms associated with skin colonisation3. The aim of the study was to determine if a mouthwash formulation containing EO enhances the bactericidal effect on Streptococcus sanguinis grown on grade IV commercially pure titanium (CpTi).

Sample preparation CpTi grade IV titanium discs (n=24) were polished to a smooth p4000 finish and placed in a bacterial biofilm reactor. The biofilms of S. sanguinis were grown anaerobically for 48 hours at 37C and conditioned with artificial saliva for a further 24 hours at 37C

Bacterial viability after exposure The CpTi grade IV discs (n=24) were placed into separate universal tubes: 8 containing 2ml of distilled water (control), 8 containing 2 ml of the formulation without EO and 8 containing 2ml of the test formulation with EO, for 30 seconds. The solutions were removed and replaced with PBS and then the adherent bacteria were detached by sonication for 10 min and vortex mixing for 15 secs. Viable bacterial cell counts were determined by serial dilution and subsequent culture on blood agar. Data were analysed by a T-test.

RESULTS AND DISCUSSION A significant reduction in the viability of S sanguinis biofilm was observed after exposure to the


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test formulation after 30 seconds compared to the control formulation (P ≤0.05). This suggests that the addition of EO in CHG- based mouthwashes could potentially be used as a non-invasive therapy in the treatment of peri-implantitis.

CONCLUSION Bacterial viability of S sanguinis biofilms is significantly reduced with EO used in combination with CHG due synergistic antimicrobial activity and enhanced penetration into bacterial biofilms

REFERENCES 1. Cimanga K. et al., Ethnopharm. (2002) 79; 213-220. 2. Karpenen T.J. et al., JAC. (2008). 62; 1031-1036. 3. Hendry E.R. et al., BMCID. (2009). 13: 1219-1225.

P155 - Genetic requirements in spatially organized polymicrobial wound infection Turner, Keith H. (Department of Molecular Biosciences, The Univ. of Texas at Austin, Austin, USA); Everett, Jake (Department of Surgery, Texas Tech University Health Sciences Center,, USA); Gabrilska, Rebecca (Department of Surgery, Texas Tech University Health Sciences Center, USA); Rumbaugh, Kendra (Department of Surgery, Texas Tech University Health Sciences Center, USA); Whiteley, Marvin (Department of Molecular Biosciences, The University of Texas at Austin, Austin, USA) Biofilm-like structures are common in bacterial infections, suggesting that a structured group lifestyle underlies much of the physiology of bacteria in vivo. One particularly significant example of this is infections in chronic wounds, which contribute significantly to high healthcare costs in both the developed and developing world. These chronic infections are often polymicrobial, and the opportunistic pathogens Pseudomonas aeruginosa and Staphylococcus aureus are among the most commonly isolated bacteria from infected chronic wounds. Yet many features of the spatial organization and physiology of these bacteria during chronic wound infection remain unclear. Here, we use confocal microscopy to show that the spatial structure of P. aeruginosa and S. aureus in biofilm-like chronic wound infections is organized, with different species occupying different sites within the wound, and dynamic, changing during wound healing. To investigate the genetic requirements for chronic pathogenesis of P. aeruginosa in wound infections, we combined high-throughput sequencing-mediated transcriptome profiling (RNA-seq) and genome-wide insertion mutant fitness profiling (Tn-seq) to characterize gene expression and fitness determinants in a murine model of chronic wound infection. Generally we discovered that expression of a gene in vivo is not correlated with its importance for fitness, with the exception of metabolic genes. By combining metabolic models generated from in vivo gene expression data with mutant fitness profiles, we determined the nutritional requirements for P. aeruginosa colonization and persistence in chronic wounds. Specifically, we found that long-chain fatty acids represent the primary carbon source for P. aeruginosa in chronic wounds, and that wounds are nutrient-rich requiring P. aeruginosa to biosynthesize only purines, folate, and riboflavin during infection. Interestingly, we also found that flagellar motility, thought to be a requirement for P. aeruginosa biofilm formation invitro, was dispensable in chronic wound infections, suggesting that genetic requirements for biofilm development can be conditional. Finally, we will detail advances made using genomic approaches to dissecting the genetic bases for P. aeruginosa and S. aureus coinfection in chronic wounds. Our results provide novel insight into the genetic requirements for, and spatial organization in, P. aeruginosa and S. aureus


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polymicrobial chronic wound infections and demonstrate the power of using both gene expression and fitness profiling for probing bacterial virulence and persistence in biofilm-like infections. P156 - Biofilm-induced biodeterioration of polyurethane coatings: Impact of nutrients and polyurethane chemistry Zingarelli, Sandra (Air Force Research Laboratory, Wright-Patterson AFB, OH, USA); Barlow, Daniel (US Naval Research Laboratory, Washington, DC, USA); Biffinger, Justin (US Naval Research Laboratory, Washington, DC, USA); Lloyd, Nadeau (Air Force Research Laboratory, Wright-Patterson AFB, OH, USA); Babson, David (US Naval Research Laboratory, Washington, DC, USA); Pirlo, Russell (US Naval Research Laboratory, Washington, DC, USA); Drake, Carrie (Air Force Research Laboratory, Wright-Patterson AFB, OH, USA); Russell, John (US Naval Research Laboratory, Washington, DC, USA); Goodson, Wendy (Air Force Research Laboratory, Wright-Patterson AFB, OH, USA) Microbial biofilms frequently contaminate surfaces and cause degradation of polymeric coatings that are intended to protect against environmental degradation. Historically, investigations of biodeterioration of polyurethane coatings have focused on identification and characterization of the organisms and esterases involved in degradation. The role of biofilms and the impact of biofilm physiology in the degradation process have largely been ignored. Microbes capable of polymer degradation are ubiquitous in the environment, yet only affect polymers under some circumstances. Is degradation driven by biofilm physiology, polyurethane chemistry, or both? The goal of our research is to define parameters that result in polyurethane biodeterioration by Pseudomonad biofilms, with a focus on nutrient sources and polyurethane chemistry. Eleven different Pseudomonad strains were screened for polyurethane-degrading activity as a function of carbon source, using a zone-of-clearing assay in which a colloidal polyester polyurethane (Impranil®-DLN) was dispersed into agar plates in a defined medium background. Generally, non-preferred carbon sources (citrate, pyruvate) resulted in maximal esterase activity, while preferred carbon sources (glucose) inhibited activity, suggesting that carbon catabolite repression may be involved in regulating esterase regulation. Esterase activity was maximal in Pseudomonas protegens (formerly fluorescens) Pf-5 compared to all other organisms tested, so Pf-5 was used for further study. Due to the limited sensitivity and qualitative nature of zone-of-clearing assays, we next developed a microATR-FTIR (attenuated total reflectance-Fourier transform infrared) spectroscopy method to better quantitate and define the relationship between Pf-5 biofilm physiology and polyurethane coatings. ZnSe IR windows coated with thin films of polyurethane were placed in contact with biofilms grown on traditional agar plates with specific medium backgrounds. Surface chemical analysis of the coatings after biofilm removal showed that degradation proceeds through preferential loss of the ester component, and the C=O peak could be used to quantitate differences in degradation among conditions. As with the zone-of-clearing assays, growth of the biofilm on glucose inhibited degradation, while growth on citrate promoted degradation. The microATR-FTIR assay was also used to compare degradation of Impranil, Irogran (non-colloidal polyester polyurethane), and AS-P108 (polyether polyurethane) after exposure to Pf-5 biofilms grown on Luria-Bertani and M9 media. Under these conditions, Pf-5 only degraded the colloidal polyester polyurethane. Thus, both biofilm physiology and polyurethane chemistry contribute to the ability of Pf-5 to degrade polyurethanes.


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P157 - Cranberry products can decrease E.coli adherence in Urinary Catheters. Cevallos, Manuel (Grand Canyon University, Phoenix, USA); Trautner, Barbara (Baylor College of Medicine, Houston, USA) Background: An estimated 4 million patients are subjected yearly to urinary catheterization. 600,000 patients/year develop UTI intra-nosocomial in USA, 66% to 86% of them are associated to urinary catheterization; Catheter Associated Urinary Tract Infection (CAUTI) is a recognized public health problem. Cranberry products (proanthocyanidins, PAC) have an inhibitory effect on the adherence of E. coli to uroepithelial cells as been showed before. We hypothesized that cranberry products could likewise decrease adherence of uropathogenic E. coli to urinary catheters, and we could develop methods to image the inhibitory effect on biofilms on urinary catheters. Poster presented in 5th ASM Biofilm Conference, November 2009, Cancun, Mexico.

Methods: Silicone urinary catheters were incubated overnight with E. coli CFT073 with different

(TheraCran™), which were dissolved in either filter-sterilized human urine (n=6) or Luria-Bertani (LB) broth (n=12).Part A: Catheter fluorescent microscopy imaging (CFT073 expressing GFP) were performed using Nikon A1sR advanced Laser Scanning Confocal Microscope, live cell imaging, Objective 20X. Part B: Catheter after incubation was sonicated, then sonicate fluid was dilution plated. Part Cwas tested as active compound in catheter bacterial adherence (n=3). Part D: Growth curve assays were performed. Part E: Hemagglutination assays were performed with by E. coli CFT073 Grown in 0, 100, and 200 ug/ml PAC and human RBC.

Results: Fluorescent microscopy imaging from catheters showed a decreased number of colonies after inoculation with cranberry The presence of cranberry capsule extracts in the LB media decreased the adherence of E. coli to urinary catheters in a dose-dependent manner (measured in cfu/cm of catheter): From 1.9 X105 for 0, to 4.4 X104 PAC (P<0.05, at 50, 100, and 200 ug/ml Wilcoxon Rank Sum Test). Results were similar in urine: 6.6 X105 cfu/mL for 0, 5.0 X104 for 100, 6.3 X104 vs100 and for 0 vs. 200, Wilcoxon Rank Sum Test). Growth curves in LB showed identical doubling times in different PACs concentrations; showing an effect of decrease adherence not

adherence of E. coli CFT073 to catheters. Hemagglutination was inhibited at higher concentrations of cranberry products in both LB and Urine.

Conclusions: The presence of cranberry compounds in either LB or urine significantly decreased the adherence of a urinary pathogenic strain of E. coli to urinary catheters. This effect cannot be attributed totally to PAC but may involve P-fimbriae.

Future horizonswe have in urine? Is there any relationship in bacterial adherence with Cranberry product origin? If we increase urine flow, could we change adherence rate? Since effect is P-fimbriae related, which are the genes affected? Are those similar to other bacteria? Will we able to coat urinary catheters with PACs derivates? Discussion based in evidence will be provided.


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P158 - Profiling the resistome of an opportunistic pathogen Murray, Justine (University of Texas at Austin, AUSTIN, USA) The Gram-negative opportunistic pathogen Pseudomonas aeruginosa causes various acute and chronic infections in immunocompromised individuals and accounts for 15% of all nosocomial infections. P. aeruginosa is both intrinsically resistant and can acquire resistance to multiple antimicrobial classes, however, the numerous mechanisms of resistance remain poorly understood. Transcriptional profiling and genetic screening have been employed in an effort to identify genes involved in antimicrobial resistance, colstudies analyze wild-type gene expression and mutant fitness of P. aeruginosa grown in the presence of single antimicrobials. While these studies have provided insight into the mechanisms of antimicrobial resistance, individually these approaches do not allow for the exhaustive comparison of resistomes from a genome-wide perspective. By clustering RNA sequencing and transposon sequencing data, respectively, we aim to construct functional gene networks and phenotypic networks that provide a global view of gene expression and mutant fitness. This proposal seeks to couple next-generation sequencing methods and network analyses to evaluate the resistomes of P. aeruginosa for a suite of antimicrobials and thereby determine the extent to which these resistomes are similar. We hypothesize that networks can be used to provide a better mechanistic understanding of the highly resistant nature of P. aeruginosa, which could lead to improved infection treatment. We predict that antimicrobials with significantly dissimilar resistomes will represent synergistic antimicrobial combinations. These studies will potentially identify new drug targets and combination therapies. P159 - Effect of cationic peptide warnerin on staphylococcal biofilms forming on titanium surfaces with diamond-like coating Korobov, Vladimir (Institute of Ecology and Genetics of Microorganisms UB RAS, Perm, RUS); Trakhtenberg, Ilia (Institute of Metal Physics UB RAS, Ekaterinburg, RUS); Rubstein, Anna (Institute of Metal Physics UB RAS, Ekaterinburg, RUS); Lemkina, Larisa (Institute of Ecology and Genetics of Microorganisms UB RAS, Perm, RUS) Wide use of titanium in manufacturing of various medicinal replacement devices is conditioned by marked chemical inertness and biocompatibility of this metal. However, long-term use of titanium implants is frequently accompanied by the development of bacterial biofilms thereon. This identifies the need to protect titanium surfaces from bacterial cell sorption and to find ways of slowing down such type of bacterial colonization.

Subject. Investigation of antibacterial effects of low-molecular weight cationic peptide warnerin [1] on coagulase-negative staphylococcus biofilms formed on titanium plates prior to and after deposition of diamond-like coating.

Methods. Surface preparation of titanium plates (10x10x1 mm) involved polishing, purification in ethanol in ultrasonic bath (80 W), and final cleaning via bombardment by argon ions. Diamond-like carbon (DLC) coatings up to 50 nm were deposited by vacuum pulsed arc graphite sputtering. For the purpose of microbiological investigation autoclaved titanium plates (1.5 atm, 121°C, 60 min) were introduced in LB medium containing S. epidermidis 33 (107 CFU/ml), incubated for 1, 3 and 5 days at 37°C, after that those were thrice washed with 10 mM phosphate buffer (pH 7.2).


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One part of washed biofilm-coated plates was placed in warnerin solution (128 mkg/ml) for 24 hours, the control was left without any treatment. Viability of cell elements in biofilms was determined using Cell Proliferation Assay system (Promega, USA), and biofilm biomass - by gentian violet binding to cell walls and biofilm matrix. Absorbance of extracts was measured by spectrophotometer at wavelengths of 570 and 490 nm, respectively.

Results. Deposition of DLC coating on titanium plates did not result in significant increase in plate surface roughness. Most rapid increase in bacterial biomass occurred in first hours of incubation, and bacterial biomass augmentation possessed linear character within 2-5 days of incubation. Modification of titanium surface by overlaying of diamond-like coating reduces the biomass of developing staphylococcal films and viable cell by 1.5-2-fold. The treatment of formed biofilms on titanium plates by low-molecular weight cationic peptide warnerin reduced the staphylococcal biostructure amount by 4-5-fold.

Conclusions. Results of investigation allow recommending surface implant modification using titanium with carbon diamond-like nano-films to reduce the possible complications from staphylococcal biofilm formation. Low-molecular weight cationic peptide warnerin can be used to prevent formation and degradation of already established biofilms of these bacteria.

This work was supported by grants from RFBR (12-04-01431-а and 14-04-00687), the UB RAS (12-P-4-1002, 12-I-4-1033 and 12-M-14-2035) and Perm government grant «Development of fundamental basis for creation of new generation of functional endoprosthetics of soft tissues with improved biocompatibility».


P160 - Motility of Pseudomonas aeruginosa contributes to the SOS-inducible biofilm formation Wei, Tao (College of Science and Mathematics. Southwest Baptist University., Missouri , USA); Chellappa, Shakinah T (College of Science and Mathematics. Southwest Baptist University., Missouri , USA); Marediaa, Reshma (College of Science and Mathematics. Southwest Baptist University., Missouri , USA); Phipps, Kara (College of Science and Mathematics. Southwest Baptist University., Missouri , USA); Haskins, William E (College of Science and Mathematics. Southwest Baptist University., Missouri , USA) DNA damaging antibiotics, such as ciprofloxacin, induce biofilm formation and the SOS response through auto-cleavage of SOS-repressor LexA in Pseudomonas aeruginosa . However, the biofilm-SOS connection remains poorly understood. It was investigated with the 96-well and lipid biofilm assays. The effects of ciprofloxacin were examined on biofilm stimulation of the SOS mutant and the wildtype strains. The stimulation observed in the wildtype in which SOS was induced was reduced in the mutant in which LexA was made non-cleavable (LexAN) and thus SOS un-inducible. Therefore, the stimulation appeared to involve SOS. The possible mechanisms of the inducible biofilm formation were explored by subproteomic analysis of outer membrane fractions extracted from biofilms. The data predicted an inhibitory role of LexA on flagellum


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function. This premise was tested first by functional and morphological analyses of the flagellum-based motility. The flagellum-swimming motility decreased in the LexAN strain treated with ciprofloxacin. Second, t he motility-biofilm assay was performed, which tested cell migration and biofilm formation. The results showed that the wild-type biofilm increased significantly over the LexAN. These results suggest that LexA repression of motility, which is the initial event of biofilm development, contributes to repression of the SOS-inducible biofilm formation

P161 - Influence of atmosphere on oral biofilm regrowth after interdental cleaning Gottenbos, Bart (Philips Research, Eindhoven); Scheffers, Lucas (Philips Research, Eindhoven); van de Ven, Cindy (Philips Research, Eindhoven); Hötzl, Sandra (Philips Research, Eindhoven) Traditionally oral hygiene devices are tested for plaque reduction. Dental professionals are realizing however that not only the amount of plaque determines whether the patient develops oral disease, but that the microbial compositionof that plaque is of equal or even greater importance. High levels of anaerobic Gram-negative bacteria are often associated with gum disease, as many of these produce endotoxins and proteases. Therefore the presence of O2 may be an important factor to maintain a healthy balance by suppressing Gram-negative anaerobes and favoring the more benign Gram-positive (facultative) aerobes. Interdental cleaning using string floss and interdental brushes is seen by many users as an inconvenient chore, and therefore only a minority of the people performs it regularly. Recently a new powered hand-held device was introduced aimed at making interdental cleaning easier by removing plaque using a burst of high-velocity air and micro-droplets. Studying plaque composition changes in vivo is a challenge due to the large variation between subjects. Therefore we developed an in vitro model of oral biofilm starting from plaque collected from volunteers, creating a uniform biofilm model for testing treatment effects. It was verified with in situ grown "interproximal" plaque samples that the in vitro biofilm was comparable in mechanical behavior and composition. The aim of the current in vitro study was to follow how oral biofilms grow back in three different atmospheres after cleaning with high-velocity air and micro-droplets, looking at changes in both biofilm amount and species composition.

M&M. Biofilms were grown anaerobically for 48h from plaque samples pooled from 4 persons in an artificial saliva medium containing mucin. The biofilms were treated in an anatomical model of the interproximal space with the Philips Sonicare AirFloss or received no treatment and were re-incubated for an additional 24h in anaerobic (5% H2, 5% CO2, 90% N2), microaerophilic (5% O2, 10% CO2, 85% N2) or aerobic (5% CO2, 20% O2, 75% N2) atmosphere. Biofilms were analyzed using optical coherence tomography (OCT) and confocal laser scanning microscopy (CLSM). Proteins and specific Gram-negative anaerobes were quantified using BCA and qPCR assays, respectively.

Results. Initially almost all biofilm was removed, leaving only thin colony patches behind. OCT showed that the treated biofilms were thinner after re-growth in oxygenated atmospheres and led to a more open biofilm structure compared to purely anaerobic regrowth. Fluorescent Gram stain CLSM indicated that the levels of Gram-negative bacteria were lower in the treated biofilms regrown in oxygenated atmospheres. It was confirmed with qPCR that specific Gram-negative anaerobes were significantly reduced in treated biofilms regrown in presence of


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oxygen, while there was no reduction effect of the treatment in anaerobic atmosphere. Interestingly, non-treated control biofilms did show no difference in species composition or biomass in different atmospheres. This is likely caused by the consumption of oxygen in the top of these thicker biofilms, rendering the bulk of the biofilm essentially anaerobic.

Conclusion. It can be concluded that the atmosphere plays an important role in biofilm morphology and species composition in in vitro interproximal biofilms growing back after thinning down with a microburst of high-velocity air and micro-droplets. As oxygen is present in most locations in the oral cavity, and the new air assisted cleaning device makes the interproximal plaque biofilm accessible to that oxygen, interdental cleaning using this device may cause suppression of anaerobes, leading to a healthierplaque ecology with respect to gum health. More in vitro and clinical research is needed to confirm these beneficial plaque composition changes.

P162 - Identification of Novel Small Molecule Antagonists of Diguyanylate Cyclases (DGC) Inhibiting Biofilms in a Broad-Spectrum Manner Sambanthamoorthy, Karthik (Walter reed army institute of research, Silver Spring, USA) Bacterial biofilm formation causes significant industrial economic loss and high morbidity and mortality in medical settings. Because biofilms have a high tolerance for treatment with antimicrobials, it is critical to develop new approaches to prevent biofilm formation. In our study to identify inhibitors of diguyanylate cyclases (DGC), we identified 2 molecules, LP 3134 and rottlerin, with broad spectrum antibiofilm activities. Here, we present the results of a small molecule analog screen to identify analogs of LP 3134 and rottlerin with antibacterial and antibiofilm efficacies. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) experiments on Staphylococcus aureus revealed that rottlerin has MIC and MBC values of 0.5 and 1 ug/ml, respectively. Biofilm experiments performed on an osteomyelitis isolate of S. aureus, UAMS-1, generated an IC50 of 4.6 uM which was further confirmed by confocal microscopy. In addition, we also found that rottlerin possessed antibacterial and antibiofilm activities against Pseudomonas aeruginosa and Acinetobacter baumannii at higher concentrations. A SMILES query program to identify molecules structurally similar to rottlerin yielded pentamethyl chroman (PC), oxychalcone (OC) and dimethylchromene (DC). PC, OC and DC were also able to inhibit S. aureus biofilm development at concentrations ranging 50-100 uM. The SMILES search on LP 3134 yielded 16 analogs and majority of them inhibited A. baumannii biofilms. With a diminishing pipeline of antibiotics for treating biofilm based infections, our results strongly suggest that these aforementioned novel biofilm inhibitors have potential utility for controlling biofilms in both medical and industrial settings.

P163 - Virulence strategies of Proteus mirabilis during catheter-associated urinary tract infection Maierl, Mario (University of Applied Sciences, Graz, AUT); Jörger, Michael (University of Applied Sciences, Graz, AUT); Siebenhofer, David (University of Applied Sciences, Graz, AUT); Zechner, Ellen (University of Graz, Graz, AUT); Reisner, Andreas (University of Applied Sciences, Graz, AUT)


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Catheter-associated urinary tract infections (CAUTI) are the most frequent infections in hospitals and other health care facilities. Proteus mirabilis is the most important pathogen that colonizes the bladder during long-term catheterization. Colonization of the bladder is associated with formation of a crystalline biofilm leading to catheter blockage, stone formation and severe complications for the patient. The relevance of Proteus mirabilis biofilm formation on urethral catheters for CAUTI pathogenicity as well as the mechanism underlying efficient bladder colonization of Proteus mirabilis are largely unknown. To identify molecular factors of P. mirabilis required for catheter colonization in vitro under conditions mimicking the catheterized urinary tract, random mutagenesis of CAUTI isolate P. mirabilis HI4320 was performed. Of ~5500 mutants screened, eight mutants attenuated in static biofilm formation on a polystyrene surface were identified. The disrupted genes encode proteins involved in assembly of surface structures and secreted proteins (dsbA, mrpB, zapB) as well as metabolic enzymes (icd, sdhB, PMI3232). Among the biofilm deficient mutants initially identified only those affecting genes involved in assembly of MR/P fimbriae or ambient temperature fimbriae (ATF) were outcompeted when challenged with the parental strain in a dynamic catheterized bladder model in vitro irrigated with urine. Subsequent analysis using site-specific knockout mutants revealed that MR/P fimbriae, but not ATF, nor genes potentially involved in c-di-GMP synthesis or signaling, are important for Foley catheter colonization and biofilm formation in vitro. Orientation of the invertible element controlling MR/P expression in bacterial populations harvested from the colonized catheterized bladder in vitro suggest that the vast majority (>80%) of catheter colonizing cells express MR/P fimbriae. Our data supports the conclusion that MR/P fimbriae are a crucial virulence factor for efficient catheter colonization in patients experiencing CAUTI due to P. mirabilis. To identify novel virulence determinants of P. mirabilis crucial for colonization of urine and urethral catheters during CAUTI in a dynamic catheterized bladder model in vitro signature-tagged mutagenesis (STM) was applied. Of ~1800 STM mutants screened, 19 mutants were found to be significantly outcompeted by the parent strain in the catheterized bladder model. Only three of these mutants were also attenuated for biofilm formation in a static biofilm model. Swarming was not found to be necessary for catheter colonization. Using single gene knockouts and expression analysis we identified genes affecting urease activity, aerobic respiration control, response to osmolarity changes, extracytoplasmic stress, and key metabolic pathways as determinants for efficient bladder and catheter colonization by P. mirabilis in vitro. Our results suggest that biofilm formation is an important virulence strategy of P. mirabilis during CAUTI and indicate molecular pathways that support bladder colonization by Proteus mirabilis, providing therapeutic targets for combating CAUTIs.

P164 - Characterization of biofilms from Escherichia coli isolated from urinary tract infections in Mexico. Ramirez, Flor (Universidad Autónoma de Aguascalientes, Aguascalientes, MEX); Marquez-Diaz, Francisco (Centenario Hospital Miguel Hidalgo, Aguascalientes, MEX); Garneau, Philippe (Universite de Montreal, Quebec, CAN); Harel, Josee (Universite de Montreal, Quebec, AUT); Guerrero Barrrera, Alma Lilian (Universidad Autónoma de Aguascalientes, Aguascalientes, MEX); Avelar González, Francisco Javier (Universidad Autónoma de Aguascalientes, Aguascalientes, MEX)


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Uropathogenic Escherichia coli (UPEC) is a cause of 80% to 90% of the urinary tract infections (UTIs) in community and hospital acquired infections. UTIs are the most common bacterial infections worldwide and cause of high morbidity and elevate health cost treatment. Biofilm formation plays an important role for E. coli to cause recurrent UTI since allows the strains to survival and persist long time in the urinary tract and endure killing by antibiotics and host immune responses. In this study we characterized a subset of 30 UPEC isolated from urinary tract infections strains by the ability to form biofilm, congo red assay, antimicrobial susceptibility to 20 antibiotics, phylogroup and virulence factors (papC, sfaS, afa/draBC, yfcV, agn43, fyuA, chuA, kpsMII, and vat) and explored possible associations amongst them. The investigated isolates were frequently resistant with 27 (90%) isolates displaying resistance to antimicrobial agents, including cephalosporins and fluoroquinolones. While 18 (60%) isolates displayed multidrug resistance phenotypes. Most of the studied isolates belonged to group D, 14 (47 %) and to group B2, 11 (37 %). Biofilm was produced by 26 (87 %) of the strains. Strong biofilm-producing strains were associated with isolates from community-acquired infections (P =0.045). Congo red assay demonstrated that the production of curli and cellulose varies among isolates and the production was associated with biofilm formation (P =0.038). Non particular phylogroup or virulence genes were associated with strong biofilm formation. The virulence genes: fyuA and chuA were significantly more prevalent among strains, indicating the role of iron acquisition systems causing UTIs. Expression of curli and cellulose can contribute to biofilm formation of UPEC strains and therapeutic approach to eliminate the curli expression and cellulose could be an useful treatment to eliminate biofilms in patients with UPEC biofilm producing isolates from community-acquire infections.

P165 - Effect of carbon source and concentration on streptomycin susceptibility of Pseudomonas aeruginosa biofilms Jackson, Lindsay (Ryerson University, Toronto, CAN); Kroukamp, Otini (Ryerson University, Toronto, CAN); Wolfaardt, Gideon (Ryerson University, Toronto, CAN) Pseudomonas aeruginosa is an opportunistic pathogen with a propensity to form biofilms, and is linked to approximately 15-20% of nosocomial infections. In biofilm, bacteria can withstand antibiotic concentrations 10-1000 times greater than their planktonic counterparts. Whether in biofilm or planktonic state, environmental factors play an important role in antibiotic susceptibility. Studies performed on P. aeruginosa in planktonic systems have demonstrated that the minimum inhibitory concentration of colistin and polymyxin B varied greatly depending on the carbon source in the medium. We have used a CO2 evolution measurement system to delineate real time metabolic response to antibiotic exposure in continuous flow biofilms. Stable biofilms of P. aeruginosa (PA01) were exposed to streptomycin (an aminoglycoside that targets protein synthesis) for 4 hours after which the flow of sterile growth medium was resumed. Three different carbon sources (glucose, citrate, and pyruvate) were used to test the effect of carbon source and carbon concentration on PAO1 biofilm susceptibility to streptomycin. Antibiotic susceptibility was monitored by changes in CO2 production: a susceptible biofilm was unable to

metabolism remained at or above steady state levels during exposure. Recovery time of the biofilm metabolism back to steady state levels following an antibiotic exposure was faster when metabolism was at or above steady state throughout the antibiotic exposure. Our results demonstrate that carbon concentration is inversely proportional to PAO1 biofilm susceptibility


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during the streptomycin exposure. In addition, biofilm metabolism during a streptomycin exposure may serve as an indicator of bacterial streptomycin susceptibility.

P166 - Removal kinetics of bacterial biofilms potentially formed in commonly used equipment in agro-industries yahaya, sylla (institut pasteur, Abidjan, CIV) This work deals with removal kinetics of bacterial biofilms potentially formed in commonly used equipment in agro-industries. Two bacterial species Pseudomonas and Bacillus with a strong ability to form biofilms in which the almost permanent presence is reported in the food industry (meat, milk, vegetables...) were chosen as relevant models for the study. Moreover Bacillus, potentially pathogenic bacteria can form spores which are known to strongly adhere to solid surfaces and for their ability to withstand adverse environments as thermal and chemical detrimental conditions (detergents, pasteurization or sterilization temperatures). Bacillus species are known to pose a major health risk for agro-industry is contamination of food by the technical equipment itself. Two main areas were considered in the field of food processing equipment cleaning. At first a kinetic approach was implemented to study the removal of biofilms from surfaces during cleaning operations. Thus respective roles of the mechanical action (wall shear stress under turbulent flow regime), the chemical action (different NaOH concentrations) and the combination of both chemical and mechanical actions were studied. A biphasique model was proposed to describe cleaning kinetics according to the observation of two very different phases which we interpreted as the elimination of two different fractions of the biofilm. A more or less fast detachment in 1 to 5 min would correspond to the three-dimensional elimination of structure with more or less exopolymeric materials followed by a slow detachment of bacteria cells directly in contact with solid surfaces. In the case of Bacillus, can represent about 90 % of the total biofilm. Spores removal appeared to be a two phases phenomenon as the whole biofilm. However, the removal was essentially observed during the short first phase of the kinetics. Preliminary data on mixed biofilms with both species Bacillus and Pseudomonas had shown an increase in their resistance to cleaning comparing to the corresponded monospecies biofilm counterpart. This is really significant for Pseudomonas. The second developed theme considered as a consequence of the above one deals with possible equipment re-contamination by Bacillus spores removed during cleaning operations. Post contamination of food processing equipment during cleaning was thus observed in pipes located at different places in the cleaning loop, as well as in more complex materials as a mixproof valve commonly encountered in Dairies. This work allowed us to identify all the conditions explaining such phenomenon. Equipment hygienic design issues appeared to be here

Keywords: Biofilms, removal kinetics, Cleaning-in-place, hygiene, Pseudomonas fluorescens, Bacillus sp., spores, hygienic design


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P167 - Streptococcus mutans biofilms microspray detachment using an in-vitro IP space model Fabbri, Stefania (University of Southampton, southampton, GBR); Johnston, David A. (Biomedical Imaging Unit, School of Medicine, University of Southampton,, Southampton , GBR); Carugo, Dario (Bioengineering research group, Faculty of Engineering and the Environment, University of Southampton, Southampton , GBR); de Jager, Marko (Philips Research, Eindhoven); Rmaile, Amir (Philips Research, Eindhoven); Ward, Marilyn (Philips Oral Healthcare, Bothell, WA, USA); Aspiras, Marcelo (Philips Oral Healthcare, Bothell, WA, USA); Stoodley, Paul (Department of Microbial Infection and Immunity, Department of Orthopaedics, The Ohio State College of Medicine , Columbus OH, USA) Objectives: Cariogenic bacteria such as Streptococcus mutans in dental plaque biofilms are responsible for caries development. S. mutans UA159 biofilm was used to assess biofilm microspray induced detachment inside an in vitro interproximal (IP) space model. We also assessed the potential for microspray for delivery of dentifrices into the biofilm using fluorescent µbeads.

Methods: Biofilms were grown on 1cm length or full length (6 cm) glass microscope slides on a mucin containing medium (BHI +2% sucrose, +1% mucin) for 3 days. For the biofilm microspray detachment experiments, to simulate the IP-space two biofilm colonized slides were placed, inside a rectangular plastic holder, in a parallel position with a gap of 1 mm. A Philips Sonicare AirFloss was used to assess biofilm detachment strength by shooting a 130 ± 0.03 µl (n=11) burst into the IP-space. Removal was recorded using a high speed (HS) camera and the movies were analysed with ImageJ. Biofilm removal was performed by shooting water or air. For the

-modified or amine-modified polystyrene latex beads were incorporated inside the AirFloss at a concentration of 108 beads/mL. Full length biofilm colonized slides were shot with the AirFloss positioned perpendicular to the slide, rinsed once with PBS and fixed with 4% PFA. Afterwards, the samples were stained with SYTO 63 and imaged under confocal microscope.

Results: HS movies showed the AirFloss (using water) had two distinct burst phases. The first was a 7.7 ms (±1.5 ms, n=7) jet-like stream with an average initial velocity (ūi) of 35.5 m/s (±7.0 m/s, n=20), the second was a 52,6 ms (±10.5 ms, n=7) spray of water droplets (ūi=11.1 m/s ±2.2 m/s, n=60). Regarding the biofilm microspray detachment experiments, HS videos and image analysis showed a macroscopic zone of clearance (CZ) where the AirFloss removed 34.8 mm2 in 24 ms with water and and 14.4 mm2 in 3.75 ms with air. Using ImageJ a biofilm removal velocity (vbiofilm) in which the front of the zone of clearance as it moved down the slide, was measured as function of the time (n=1): the vbiofilm, using water and air was 0.8 m/s at 4 ms and then increased to 2.8 at 6 ms, remaining constant until the end of the burst. For air only, the velocity increased to a maximum of 2 m/s at 3.5 ms and then decreased to 1.1 at 6 ms. Then, it remained constant until the end of the burst. Additionally, after the burst with only air, there was biofilm recoil which reduced the CZ back to 3.97 mm2 after a further 1s. There was no visible recoil with water suggesting that the biofilm had been forced off the end of the slide. Confocal microscopy showed that the reduction in biofilm biomass in the CZ was 97.8% (±2.7, n=2), 99.8 % (±0.2, n=3) and 99.4% (±0.5, n=3) at 1 mm, 5 mm and 8 mm from the shooting edge respectively. SEM microscopy revealed there was still a thin layer of biofilm left in the CZ. Confocal images also showed that some beads settled on the surface and others inside the first layers of the biofilm.


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Conclusion: As expected, the CZ area and the vbiofilm were lower shooting only air than water, which could be explained by lower shear stresses and lack of the surface tension effects of a travelling solid air-water interface. However, the AirFloss burst was sufficient to remove significant amounts of biofilm for almost the entire length of the 1 cm slide. The presence of recoil showed that the biofilm was flowing like a viscous fluid and underlined the importance, when using physical removal, to maintain the force for long enough to push the biofilm off the surface. While the beads suggest that fluid were being driven into the biofilm, regardless of charge, future work will use higher concentrations and different beads sizes. Finally, NaF will be incorporated inside the AirFloss in order to study the delivery of fluoride into the biofilm.

P168 - Effect of Disinfectants on Clinically Relevant Bacteria Under Planktonic and Biofilm Conditions Swift, Dean (Biolennia, Toronto, CAN); Dhyani, R. (ZZZ); Del Re, D. (ZZZ); Mair, R. (ZZZ); Ikeno, C. (ZZZ); Legner, M. (ZZZ); Cvitkovitch, D.G. (ZZZ) Background/Objective: Microbial biofilms are now recognized as playing a major role in the progression of infection and disease. Current research has shown that biofilms are more difficult to eradicate than their planktonic counterparts; however, the majority of standardized methods used to test the efficacy of disinfectants rely on the use of planktonic bacterial cultures. Recently, a new experimental device has been developed to determine the minimum biofilm-eliminating concentration (MBEC) of antimicrobial agents and disinfectants: the Calgary Biofilm Device (CBD). The MBEC Assay allows for rapid, high-throughput assessment of the antibiofilm activity of antibiotics, disinfectants, biocides and metals at varying concentrations. The main objectives of this study are to compare the effectiveness of various disinfectants on bacteria grown planktonically and in biofilms, and to compare the minimum inhibitory concentration (MIC) and MBEC methods for testing the efficacy of disinfectants. Methods: Overnight cultures of Pseudomonas aeruginosa MPAO1, Bacillus subtilis JH642and clinical isolates of Escherichia coli and Staphylococcus aureus were grown aerobically in brain heart infusion (BHI) medium at 37 ° C. For MIC assays, diluted overnight cultures were added to 96-well plates containing serially diluted disinfectants including ethanol, bleach, glutaraldehyde and several commercial products. The plates were incubated for 24 hours and visually inspected for growth, spot plated and quantitatively measured at OD590nm. For the MBEC assay, biofilms were grown in the CBD for 48 hours. The MBEC lids were then placed in a similar serially diluted 96-well plate containing disinfectants and incubated for 24 hours. The biofilms were subsequently washed twice in phosphate-buffered saline and re-immersed in fresh BHI, sonicated, incubated for 24 hours and quantitatively measured at OD590nm for regrowth. Both assays were performed in triplicate. MIC and MBEC values were determined as the lowest concentration of disinfectant that inhibited growth of the bacteria. Results: Each strain exhibited different susceptibility profiles to the disinfectants tested. B. subtilis was the most resistant, while the clinical isolates were most susceptible. In addition, biofilms were more resistant to the disinfectants compared to planktonic cultures. Conclusions: Since biofilms are the primary mode of growth for most bacteria, it is important to recognize their role in the vast majority of medically relevant infections. The results of this study support the use of the MBEC method to test the efficacy of disinfectants, as it presents the most relevant results of antimicrobial activity. This will allow for further development of standardized


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test methods that more accurately reflect conditions found in the field, thus leading to more effective strategies for controlling the spread of infection.

P169 - Use of a calcifying biofilm model system to investigate efficiency of disruption with piezo electric scalers Pecheva, Emelia (University of Birmingham, Birmingham, GBR); Vyas, Nina (Universty of Birmingham, Birmingham, GBR); Walmsley, Damien (Univeristy of Birmingham, Birmingham, GBR); Brown, Judith (University of Bath, Bath, AUT); Price, Gareth (University of Bath, Bath, GBR); Sammons, Rachel (Univeristy of Birmingham, Birmingham, GBR) Background: Bacterial biofilms on teeth and dental implants may lead to dental caries or periodontal disease with eventual tooth loss or other serious infections of the oral cavity. Oral biofilms may also go on to mineralise, forming calculus deposits above and below the gum line that have to be removed mechanically. Ultrasonic powered instruments are used for biofilm and calculus disruption due to their efficiency, greater comfort for the patient, as well as the presence of cavitation and streaming within the water flow, which increases the cleaning efficiency. Our research aims to investigate the biofilm disruption ability of a piezoelectric dental scaler using tips with different shapes and position in relation to the biofilm on flat surfaces and within cavities.

Methods: A non-pathogenic species of Gram-negative Serratia (NCIMB40259), which has been extensively characterised with respect to its ability to form biofilms on various surfaces and to undergo biomineralisation forming calcium deficient hydroxyapatite deposits[1] that chemically resemble calculus, was used. The Serratia was grown within a transparent silicone rubber model of a dental root canal with side canal (main canal dimensions: 20 x 1.5 to 0.5mm (length x diameter); side canal 10mm x 0.2mm situated 3mm from the apex of the main canal) and on glass and titanium surfaces for subsequent disruption. Scanning electron microscopy was used to investigate bacterial penetration and biofilm formation within the artificial canal and energy dispersive spectroscopy (EDS) to confirm mineralisation. A Satelec (piezoelectric) P5XS (Newtron, Acteon, France; working frequency 29kHz) ultrasonic dental scaler with tips of differing width (1, 2 and 10P) with water irrigation was used to disrupt the biofilm for 30 seconds. The tips were applied with their front side and tip end facing down operating at a fixed power setting giving a tip horizontal displacement of 15µm. Cavitation was evaluated using luminol and biofilm disruption by comparison of photographic images using image analysis.

Results: Serratia penetrated throughout the model root canal and formed calcium phosphate deposits in the main canal. On glass and titanium an area of disruption developed when the biofilm was approached with the front side of the tips. When the tips were oriented side on, only

perpendicularly with only the end in contact with the biofilm. The cavitation fields observed by use of luminol corresponded to this result, with the strongest response at the front curve of each tip, and less response at the end or side edges. The most effective removal occurred with the thinnest tip (10P) and the least effective with the broadest (2).

Conclusion: Serratia biofilm provides a useful model for investigation of the efficiency of ultrasonic dental instruments. Tip width, position and angle all influence biofilm disruption. The


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three tips remove the biofilm on glass and titanium only when their front side is used and the tip sides or tip end do not yield a disruption area under the conditions used here. Further work will investigate disruption of mineralised biofilms within the root canal. This work was funded by EPSRC Grant EP/J014060. [1] Medina Ledo et al, J Mater Sci Mater Med, 2008; 19: 3419-3427

P170 - Bacterial fight and flight responses enhance virulence in a polymicrobial infection Apollo, Stacy (Department of Molecular Biosciences, The University of Texas at Austin, USA); Everett, Jake (Department of Surgery, Texas Tech University University Health Sciences Center, USA); Jorth, Peter (Department of Molecular Biosciences, The University of Texas at Austin, USA); Trivedi, Urvish (Department of Surgery, Texas Tech University University Health Sciences Center, USA); Rumbaugh, Kendra P. (Department of Surgery, Texas Tech University University Health Sciences Center, USA); Whiteley, Marvin (Department of Molecular Biosciences, The University of Texas at Austin, USA) The oral pathogen Aggregatibacter actinomycetemcomitans (Aa) resides in infection sites with many microbes including commensal streptococci such as Streptococcus gordonii (Sg). During infection, Sg promotes the virulence of Aa by producing its preferred carbon source, L-lactate, a phenomenon referred to as cross-feeding. However, as with many streptococci, Sg also produces high levels of the antimicrobial hydrogen peroxide (H2O2), leading to the question of how Aa deals with this potent antimicrobial during co-infection. Here we demonstrate that Aa possesses two complementary responses to H2O2

enzyme that dissolves Aa biofilms. Using a murine abscess infection model, we show that both of these responses are required for Sg to promote Aa virulence. While the role of KatA is to detoxify H2O2 during co-infection, three-dimensional (3D) spatial analysis of mixed infections revealed that DspB is required for Aa to spatially organize itself at an optimal distance (>4 µm) from Sg, which we propose allows cross-feeding yet reduces exposure to inhibitory levels of H2O2. In addition, these behaviors benefit not only Aa but also Sgthe fitness of the community. These results reveal that an antimicrobial produced by a human commensal bacterium enhances the virulence of a pathogenic bacterium by modulating its spatial location in the infection site. P171 - From mouth to model: survival of native oral biofilm in biofilm reactors Klug, Barbara (University of Graz, Medical University of Graz, Graz, AUT); Westendorf, Christian (University of Graz, Graz, AUT); Grube, Martin (University of Graz, Graz, AUT); Wimmer, Gernot (Medical University of Graz, Graz, AUT); Santigli, Elisabeth (Medical University of Graz, Graz, AUT) Background: Processes and interactions in oral biofilm are difficult to interpret when concepts are based on observations of simplified experimental setups. Model systems are mostly limited by the number of bacterial species observed and artificial media composition reflects natural conditions insufficiently. So far, only few studies tried to use native oral biofilm for in vitro studies to avoid these limitations in bacterial species, yet, culturing this complex biofilm remains complicated. Aims:


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Our aim was to develop a workflow to grow native oral biofilm in vivo, transfer it to the laboratory and incubate it in biofilm reactors. Survival of native oral biofilm under laboratory conditions and changes of bacterial composition over time were observed. Methods: Standardized human dentin disks embedded in oral splints were used as biofilm carriers. 6 disks were fixed on the inside of the splint facing the teeth buccally. Appliances were fitted individually to the lower jaw of 20 male non-smoking volunteers. Splints were worn for 48h continuously. Omittance of tooth brushing and alcohol consumption contributed to an undisturbed biofilm formation. The appliances were removed from the mouth directly in the lab and immediately transferred into pre-warmed Brain Heart Infusion (BHI) medium. Dentine disks were taken out and placed into biofilm reactors. Fed with BHI medium biofilm was incubated for another 48h in vitro. Live-dead staining was performed and evaluated with confocal laser scanning microscopy to monitor survival rates of the biofilm directly after removal (t0) and after incubation of 1h (t1), 24h (t2) and 48h (t3), respectively. Changes of the bacterial diversity over time were analyzed by 454 pyrosequencing. Survival curves were calculated from the respective confocal stack data with an in-house-programmed Matlab script. Pyrosequencing data was processed with Acacia and QIIME 1.8.0. Results and Discussion: Survival curves started with an increase of bacteria 1h after incubation (t1) probably due to increased nutrition supply in BHI medium. The proportion of bacteria then settled down and reached the level from t0 after 48 hours. Compositional shifts during in vitro growth were revealed with 454 pyrosequencing comparing t0 and t3. Major phyla found were Firmicutes (86.58%), Bacteroidetes (5.34%), Proteobacteria (3.99%) and Actinobacteria (1.21%). Fusobacteria (0.64%), Cyanobacteria (0.58%) and TM7 (0.1%) represented groups around 0.1%. Bacterial diversity decreased over 48h but was not lost completely. Streptococci dominated the biofilm with 60% at the start going up to over 90% in some samples at t3. Clostridia also multiplied strongly. Conclusion: Survival of oral biofilm for 48h under laboratory conditions can be assured. Environmental variables such as temperature shifts, changes in nurture, interaction with the host and aggressions from external bacteria are likely to have further impact on bacterial survival and oral biofilm diversity. Appropriate simulations need to be addressed in further studies.

P172 - Evaluation of biofilm formation by Corynebacterium diphtheriae strains isolated from classical and atypical infections Leandro Rama Gomes, Débora (IFRJ , Rio de Janeiro, BRA); Vieira Faria, Yuri (Universidade do Estado do Rio de Janeiro - UERJ, Rio de Janeiro, BRA); Lugão Lacerda, Géisica (Universidade do Estado do Rio de Janeiro - UERJ, Rio de Janeiro, BRA); Seabra, Sérgio Henrique (Centro Universitário Estadual da Zona Oeste - UEZO, Rio de Janeiro, BRA); Hirata Júnior, Raphael (Universidade do Estado do Rio de Janeiro - UERJ, Rio de Janeiro, BRA); Mattos-Guaraldi, Ana Luíza (Universidade do Estado do Rio de Janeiro - UERJ, Rio de Janeiro, BRA) Although classical diphtheria is a model of extracellular mucosal pathogenesis, reports of invasive infections due to Corynebacterium diphtheriae have been increasing in the literature. Endocarditis is a biofilm-associated infection where bacteria adhere to heart valves and subsequently form biofilms, leading to heart valve vegetations and valvular destruction. Biofilm


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formation by pathogenic microorganisms is increasingly recognized as an important virulence trait, since it allows them to become persistent colonizers and increase their resistance to antimicrobial agents. We have reported that a biofilm-producing C. diphtheriae strain was isolated from a percutaneous nephrostomy catheter infection . Therefore, t he present work aimed to investigate the biofilm formation capacity of C. diphtheriae strains isolated from both classical and atypical infections (such as endocarditis) . Five C. diphtheriae subsp. mitis and two C. diphtheriae subsp. gravis strains were investigated: one obtained from the Centers for Disease Control and Prevention (CDC, Atlanta, GA, USA) (CDC E-8392 strain - mitis / tox+ /suc-), one isolated from throat of a patient with classical diphtheria (241 strain - mitis / tox+ / suc+) and five isolated from blood of patients with endocarditis (HC01, HC02 and HC03 strains - mitis / toxsup>- / suc+; HC04 strain - gravis / tox- / suc- and HC06 strain - gravis / tox- / suc-) in Rio de Janeiro, Brazil. C. diphtheriae endocarditis strains produced significantly more biofilm on different surfaces than classical diphtheria strains (P < 0.0001) and were classified as strong biofilm producers. The structural architecture of C. diphtheriae biofilms was also investigated by both scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). The results show that C. diphtheriae is able to form biofilm at different intensities: HC04 strain produced some rough cell clusters on polystyrene coverslips, while a patchy coverage and confluent growth areas was observed for HC06 strain. In conclusion, the strong ability of biofilm formation may play a significant role in the pathogenesis of endocarditis by C. diphtheriae.

P173 - Phenotypic drug discovery applied to the exploration of anti-biofilm activity from a flavonoids chemical library: an anti-biofilm data repository now freely available Manner, Suvi (Pharmaceutical Sciences, Department of Biosciences, Åbo Akademi University, Turku); Skogman, Malena ( Pharmaceutical Sciences, Department of Biosciences, Abo Akademi University, Turku); Goeres, Darla (Center for Biofilm Engineering, Bozeman, USA); Vuorela, Pia (Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Helsinki); Fallarero, Adyary (Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Helsinki) Given the widespread presence of bacterial biofilms in Nature, it seems logical to hypothesize that natural organisms have likely developed anti-biofilm defenses. Natural compounds with unequalled chemical diversity provide significant value for drug discovery, especially in the area of anti-infectives. One of the most popular classes of natural compounds is the flavonoids. Flavonoids are plant secondary metabolites with polyphenolic nature that have been extensively claimed to possess a variety of biological activities, including antibacterial effects on suspended cells. However, fewer reports have focused on their anti-biofilm properties. Bacterial biofilms are typically much less susceptible to antimicrobials than single-cells and the efficacy of conventional antibiotics against them is very limited, typically requiring very high concentrations and repeated doses, which in turn, triggers toxicity and resistance. Thus, the need for novel anti-biofilm compounds is immense.

In this contribution, an improved methodological workflow for anti-biofilm screens of chemical libraries was developed and applied to explore a commercial collection of 500 natural and synthetic flavonoids for inhibitory activity against Staphylococcus aureus biofilms. After the primary screening, flavonoids were classified as inactive (443), moderately active (47) or highly active (10). Further, two synthetic flavans were identified as the most potent anti-biofilm


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compounds (leads) when a selection process combining activity and selectivity was utilized. This resulted in a large and freely available bioactivity dataset of flavonoids (http://www.mdpi.com/1422-0067/14/10/19434), which is, to the best of our knowledge, the largest repository of anti-biofilm data collected for a single group of natural compounds so far. The database was divided according to the activity criteria: moderately active and highly active leads. For the moderately active leads, structures, PubChem IDs and general information on bioactivities was compiled in this database, whereas for the highly active leads bioactivity reports (PubChem Bioassays and PubMed) were also included. Further, follow-up inhibition and efficacy studies were performed to determine the selectivity of the lead flavonoids. Anti-biofilm potencies (IC50 and IC90) as well as effects on planktonic bacteria (MIC and MBC) were measured. Altogether, this contribution represents the first systematic exploration of anti-biofilm activity of a large collection of different types of flavonoids identifying several new flavonoids with anti-biofilm activity and providing a great amount of new bioactivity data that expands the knowledge on flavonoids from a drug discovery perspective. Moreover, it offers an improved methodological workflow for anti-biofilm screens taking into account the connections between antibacterial and anti-biofilm effects. Hence, this work could serve as a model for future studies of natural compounds that are known to be antibacterial.

P174 - Cationic peptide warnerin damages Staphylococcus epidermidis-Vanr biofilms Kononova, Lyudmila (Institute of Ecology and Genetics of Microorganisms UB RAS, Perm, RUS); Korobov, Vladimir (Institute of Ecology and Genetics of Microorganisms UB RAS, Perm, RUS) Bacteria of genus Staphylococcus are the most frequent cause of catheter-associated infections due to rapid occupation of medical device surfaces with biofilm structure formation. Being surrounded by intercellular matter bacteria possess increased resistance to various external factors and compounds including new antibiotics. In this respect of particular value and actuality appears to be the search for novel methods suppressing formation and persistence of antibiotic-resistant bacterial biofilms.

Subject. The sensitivity analysis of S. epidermidis 33 (lab-museum) biofilms and of in vitro selected its vancomycin-resistant variant (Vanr) to low-molecular weight cationic peptide warnerin [1].

Methods. Bacterial biofilm formation was implemented in flat-bottom polystyrene plates ⁰ C without stirring for 2 days. For this purpose

bacteria grown on liquid LB medium were diluted with fresh medium up to 107 CFU/ml, and each microplate well was inoculated with 100 µl of this dilution. To evaluate the effect of warnerin on bacterial film formation by S. epidermidis 33, those wells were concurrently filled with warnerin (100 µg/ml), and wells with S. epidermidis 33-Vanr were supplemented with this peptide (100-400 µg/ml). After cultivation films were carefully twice washed with 0.14 M NaCl, dried in air at room temperature and stained with 0.1% gentian violet for biomass measuring. Amount of living cells in biofilms of both strains was evaluated according to tetrazolium

hydrophobicity of bacterial cell surfaces was evaluated by BATH-test with hexadecane application.


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Results. It was found thatunlike initial strain S. epidermidis 33 Vanr formed weaker biofilms. Probably this is conditioned by pronounced decrease in cell surface hydrophobicity of this strain (11±4%) as compared to initial strain (45±10%). It was revealed that low-molecular weight cationic peptide warnerin significantly suppressed the biofilm biomass accumulation of parent strain S. epidermidis 33, whereas its effect on biofilm formation of S. epidermidis 33 Vanr was less pronounced. However, it should be noted that the increase of warnerin in growth medium of Vanr-strain results in decrease in formed biofilm biomass, as well as the number of metabolically active cells as compared to biofilms of parent strain.

Conclusion. Low-molecular weight peptide warnerin declines the intensity of biofilm formation by vancomycin-resistant variant of S. epidermidis 33, and possesses the ability to promote the cleavage of established biofilms of this strain.

This work was supported by grants from RFBR (12-04-01431-а and 14-04-00687) and the UB RAS (12-P-4-1002, 12-I-4-1033 and 12-M-14-2035).


P175 - Influence of different physico-chemical parameters on the biofilm formation in metal working fluids Langbein, Jennifer (Fraunhofer IPK, Berlin, GER); Steiner, Nora (Fraunhofer Institut für Produktionsanlagen und Konstruktionstechnik, Berlin, GER); Stahl, Ulf (Fachgebiet Mikrobiologie und Genetik, Institut für Biotechnologie, Technische Universität Berlin, Berlin, GER); Oberschmidt, Dirk (Fraunhofer Institut für Produktionsanlagen und Konstruktionstechnik / Institut für Werkzeugmaschinen und Fabrikbetrieb, Technische Universität Berlin, Berlin, GER) Water miscible metalworking fluids (MWF) are used in several industries in processing metal pieces. Despite the addition of biocides some microorganisms (MO) are able to survive and form biofilms in the MWF tanks and pipelines, which recontaminate freshly prepared MWF. Microbial contamination should be avoided in order to save money, time, energy and re-sources and additionally to exclude a potential risk of human health of the machine operators. The aim of this work was to establish an in vitro model in analogy to real machine systems, which allows the generation of realistic biofilms to test the impact of a number of physico-chemical parameters on the biofilm formation. The effects of the interacting parameters metal-species, temperature, mixing and nutritional addition and time on the biofilm formation have been analyzed by fluorescence- and atomic force microscopy. Therefore, stripes with a defined size, consisting of glass, elemental copper (Cu), alloyed aluminium (Alu) as well as alloyed stainless steel (Sta) have been put into small chambers, which were filled with mixed population-infected MWF. Apart from the influence of the parameters on the biofilm development, a possible correlation between the percentage of viable MO in the biofilm and the colony forming units (CFU) in the MWF was investigated.

The addition of glucose enhanced the biofilm formation tendentiously, whereas time showed no significant influence. The effects of temperature and turbidity have been essentially influenced


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by the metal species: for the biofilm formation on glass and Sta temperature and turpidity were beneficial, whereas on Cu and Alu not. Glass had in comparison with the metals no toxic effect on the MO in the biofilms. The highest toxic effects on the MO were caused by Alu, followed by Cu, Sta showed only minor effects.

A correlation between planktonic MO in the MWF and biofilm-MO has been detected. The more MO within the biofilms died the more planktonic CFU could be detected in the MWF tendentiously. This indicated quorum sensing, where single MO tried to leave the microbial consortium due to hostile surroundings to colonize a more life supportive place. Up to now no study has investigated the biofilm formation in MWF all too specific. Within this work, for the first time realistic in vitro biofilms in MWF could be generated successfully, in order to test the impacts of physico-chemical parameters on biofilm formation in metal working fluids.

P176 - Comparison of food soils on the adhesion of Listeria monocytogenes on stainless steel and research of culture media models Overney, Anaïs (Laboratory of Food Safety, French Agency for Food, Environmental and occupational Health Safety (Anses), Maisons-Alfort, FRA); Chassaing, Danielle (Laboratory of Food Safety, French Agency for Food, Environmental and occupational Health Safety (Anses), Maisons-Alfort, FRA); Carpentier, Brigitte (Laboratory of Food Safety, French Agency for Food, Environmental and occupational Health Safety (Anses), Maisons-Alfort, FRA); Firmesse, Olivier (Laboratory of Food Safety, French Agency for Food, Environmental and occupational Health Safety (Anses), Maisons-Alfort, FRA) The persistence of Listeria monocytogenes in food processing industries, in other words its presence for long periods despite correct and frequent applications of hygiene operations [1], is a major problem. In fact, persistent cells may be responsible for outbreaks of listeriosis with dramatic consequences [2]. Bacterial persistence is coming from its growth, so it is important to experiment with food soils (nutriments which are present in food plants) or with culture media models. However, the use of food soils has some drawbacks: their quality is not homogeneous from one batch to another, they are not autoclavable, cannot be kept for a long time and generate a high background noise for the microscopic observations and the flow cytometric analysis.

The first aim of this study is to compare food soils (the smoked salmon juice and the meat exudate) on the basis of several criteria : (i) the topography of adherent bacterial populations after 24 hours at 25°C on stainless steel surface observed by epifluorescence microscopy, (ii) the adhesion forces of these adherent populations estimated with detachment curves after swabbing succession and (iii) the physiological state of these attached cells including the quantification of total cells by qPCR, viable cells by PMA-qPCR and culturable cells by CFU enumeration.

Then, based on the same criteria and taking into account also the bacterial growth in liquid media, culture media models as close as possible food soils were sought to overcome the drawbacks regarding their use.


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According to our results, the behaviour of L. monocytogenes on stainless steel surface is highly -cultur

been shown on stainless steel using meat exudate while all viable cells were culturable using smoked salmon juice. Moreover, on the basis of our criteria and after validation with four strains of L. monocytogenes, the smoked salmon juice can be substituted with a modified culture medium, contrary to the meat exudate.

[1] Carpentier, B., Cerf, O. (2011). Review - Persistence of Listeria monocytogenes in food industry equipment and premises. International Journal of Food Microbiology, 145, 1-8.

[2] Todd, E.C.D., Notermans, S. (2011). Surveillance of listeriosis and its causative pathogen, Listeria monocytogenes. Food Control, 22, 1484-1490.

P177 - AROMATIC POLYKETIDES AS INHIBITORS OF STAPHYLOCOCCUS AUREUS BIOFILMS Manner, Suvi (Åbo Akademi University, Turku); Oja, Terhi (Åbo Akademi University, Turku); Goeres, Darla (Center for Biofilm Engineering, Bozeman, USA); Vuorela, Pia (University of Helsinki, Helsinki); Fallarero, Adyary (University of Helsinki, Helsinki) The polyketide class of secondary metabolites produced by bacteria, fungi and plants, includes a large number of medicinally important compounds. Aromatic polyketides, a subclass of polyketides with diverse pharmacological properties, includes compounds with anti-parasite, anticancer and antibacterial activity. These are produced by e.g. soil-dwelling bacteria as chemical warfare against other microbial species competing for the same resources. Considering that the aromatic polyketide compound class includes numerous antibiotics, it is interesting to note that there are no reports about the anti-biofilm activity of these compounds.

The aim of this study was to screen a small library of closely related aromatic polyketides for the anti-biofilm activity against S. aureus, and further, to determine the structural basis for the activity. First, the compounds were screened for the effects on biofilm viability and biomass utilizing a two-phased screening strategy. Thereafter, the anti-biofilm potencies, as well as the effects on planktonic bacteria were measured for the lead compounds. Moreover, the killing efficacy was assessed using the log reduction (log R) assay. Based on the results, some structural features correlating with high anti-biofilm activity were indeed identified.

The 96-well plate assay utilized in the project is a well-established method for screening of compound libraries for the anti-biofilm activity. In order to obtain some mechanistic insights on the effect of the lead compounds, the static method was optimized for S. aureus biofilms, as the method is very well suited for the analysis of the early stages of biofilm formation. Interestingly, several effective anti-biofilms with IC50 values in the low micromolar range were identified. The lead compounds showed high inhibitory activity on biofilm formation and some of them were also able to act on existing biofilms.


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P178 - Characteristics of Staphylococcus aureus biofilm producer isolates capable of nasal colonization. Buzzola, Fernanda (Instituto de Microbiología y Parasitología Médica, UBA-CONICET, Buenos Aires, ARG) The nasal vestibule is the most frequent carriage location by Staphylococcus aureus and this is a risk factor for infection in hospitalized patients. More than 50% of the healthy adult population shows persistent or intermittent nasal colonization by S. aureus. The remaining individuals are rarely or never colonized by this pathogen. S. aureus is the causative agent of a diverse array of acute and chronic infections owing to its ability to attach to a surface and form a biofilm. The polysaccharide intercellular adhesin (PIA) encoded by the icaADBC operon play an important role in staphylococcal biofilms matrix production. The aim of the present study was analyse the biofilm production of S. aureus nasal isolates typed by molecular and phenotypic methods. S. aureus isolates were collected from nasal swabs based on the standard prophylactic procedure at admission to two hospitals in Argentina. Of the 258 individuals screened, 62 (24%) tested positive for S. aureus nasal colonization. Forty-one isolates were registered as slime-positive by Congo red agar plates. In vitro quantification of biofilm production permitted to classify 19 and 15 isolates as high (HP) and moderate (MP) biofilm producers, respectively. The 28 isolates resting showed be weak or non-producer (NP) of biofilm. Treatment with proteinase K virtually disrupted preformed biofilms for the total of isolates. Whereas treatment with DNAse I did not affect biofilm detachment of 38% of the isolates. The quantity of PIA production was determined by ELISA method. Only four nasal isolates classified as HP and MP produced significantly (p<0.001, ANOVA) less PIA (A490=0.20 - 0.62 ± 0.08) than the prototypic strain SA113 (2.64 ± 0.25). The 62 isolates were icaA positives by PCR method. The clonal distribution of nasal isolates was studied by pulsed-field gel electrophoresis (PFGE). A total of 19 pulsotypes were arbitrarily defined by a similarity threshold of 85%. Thirty-nine of the isolates were grouped into 5 major pulsotypes (A, G, J, K and M). The isolates identified as pulsotype A (9/12) were mostly non-biofilm formers and those clustered into pulsotypes G and J were mainly HP and MP. Additionally, FTIR fingerprinting analysis revealed the differences among nasal isolates on the basis of their phenotype. Most of the HP isolates (13/19) were grouped into 3 FTIR types (B1, B2, and B4). More than 50% of the MP isolates were arranged mainly into A1 and A2 FTIR types. The allelic variations in the agr genes were determined in order to investigate its influence on biofilm production. The four agr types were found among nasal S. aureus isolates studied. Twenty-two HP and MP isolates showed to be agr type II (p=0.0004, Chi square). All isolates with agr type IV was non-biofilm producers. The findings of this study show the characteristics of biofilm and the clonal relatedness among S. aureus nasal colonizing strains.

P179 - Candida albicans and Candida glabrata sharing a live in vaginal environment Silva, Sónia (Minho University, Departement of Biological Engineering Department, Braga ); Alves, Carlos Tiago (Minho University, Departement of Biological Engineering Department, Braga ); Pereira, Leonel (Minho University, Departement of Biological Engineering Department, Braga ); Wei, Xiao-Qing (Cardiff University,Tissue Engineering & Reparative Dentistry, Cardiff); Williams, David W (Cardiff University,Tissue Engineering & Reparative Dentistry, Cardiff, GBR); Henriques, Mariana (Minho University, Departement of Biological Engineering Department, Braga ); Azeredo, Joana (Minho University, Departement of Biological Engineering Department, Braga )


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Candidal vulvovaginitis (CV) is an infection of the mucous membranes of the vagina by Candida. Up to 75% of woman will have this infection at same point in their lives and approximately 5% will have recurring episodes. Candida species are found naturally in the vagina, and are usually harmless. However, conditions in the vagina may change resulting in an environment that facilitates Candida infection. However, it remains unclear what specific changes in the vaginal environment, or amongst the Candida, are key to promoting CV.

The current study examined the interaction and expression of virulence factors by two frequent Candida species colonising the vagina, namely Candidaalbicans and C. glabrata. In vitro infection studies were performed using a reconstituted human vaginal epithelium (RHVE), which facilitated examining the effect of altered environmental factors on Candida virulence. Confocal laser scanning microscopy showed that in single species infection, C. albicans was an extensive colonizer and invader of the RHVE, which was in direct contrast to C. glabrata . However, increased colonization and invasion of the RHVE by C. glabrata was evident in dual species infection. Furthermore, in dual species infection, expression of the C. glabrata epithelial adhesin (EPA) family of genes was considered less relevant to the infection process than expression of C. albicans virulence genes (HWP1; ALS and Phospholipases B and D family). Interestingly, up-regulation of ALS3 and HWP1 by C. albicans was evident in dual species infection. When the vaginal environment was modified by changing like pH or increasing progesterone concentration, decreased RHVE colonization by both species occurred together with reduced hyphal production by C. albicans and down regulation of HWP1. In conclusion, this work demonstrated that dual species infection of RHVE results in enhanced pathogenicity, which in turn, is attenuated by environmental changes.

P180 - Effect of lysozyme on Candida biofilm formation Sebaa, Sarra (Université Libre de Bruxelles, Brussels, BEL); Boucherit-Otmani, Zahia (University of Aboubekr Belkaïd-Tlemcen, Tlemcen, DZA); Hamouda, Assia (Haute Ecole Francisco Ferrer, Brussels, BEL); Courtois, Philippe (Université Libre de Bruxelles, Brussels, BEL) Candida albicans is a commensal yeast found in the oral environment, capable of causing candidosis in medically compromised hosts. Candida is also able to develop biofilms on dentures, which is frequently found in elderly; moreover, biofilm presence may be associated to denture stomatitis. In vitro several salivary proteins (as histatin, lactoferrin, lactoperoxidase and lysozyme) have been described to present an inhibitory effect on Candida growth. This presentation aims at evaluating the effect of one of these proteins, namely lysozyme, on the development of Candida biofilms. This investigation was performed on 6 wild strains isolated from dentures and on the ATCC 10231 reference strain, always at the third subculture. Yeast biofilms were allowed to grow during 24 hours in 96-well plates (250 x103 cells per wall) in liquid Sabouraud medium with increasing concentration of lysozyme from 3 to 1000 µg per ml. The yeast growth was evaluated by turbidimetry and the attached biomass was quantified by the cristal- l. Data were compared with control cultures without lysozyme. Negative controls (without yeast) attested the absence of accidental contamination during the handling. Lysozyme presented a biphasic effect on biofilm formation while it does not affect yeast growth tested by turbidimetry. Indeed,


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lysozyme acted as a biofilm promotor at the highest concentration tested (1 mg / ml) and as a biofilm limitating-factor at the lowest concentration (3 µg / ml). At 1 mg/ml, in the ATCC 10231 strain, the attached biomass represented from 141.9 to 638.5 % (N = 7) of that observed for the positive control (mean ± SEM: 316.1 ± 63.9 %, median: 303.2 %, gaussian distribution). In the 6 clinical strains isolated from dentures, the attached biomass ranged from 57.3 to 3825.0 % (N = 6) of that observed for the respective control (mean: 825.7 %, median: 205.2 %, not-gaussian distribution). But at lysozyme 3 µg/ml, the ATCC 10231 attached biomass ranged from 25.6 to 97.4 % (N = 7) of that observed for the positive control (mean ± SEM: 62.7 ± 10.3 %, median: 69.8 %, gaussian distribution); in the 6 wild strains isolated from dentures, it ranged from 36.5 to 114.8 % (N = 6) of that observed in the respective control (mean: 75.3 ± 11.0 %, median: 72.6 %, gaussian distribution). The difference of attached biomass between the two tested lysozyme concentrations is significant as well for the reference as the clinical strains (Mann-Withney test, p < 0.05). The increase of the biofilm amount in the presence of 1 mg / ml lysozyme approximates significance (Mann-Whithney test: p = 0,0651) in clinical strains and is significant in the reference strain (p = 0.0017). In contrast, the decrease of attached yeast in the presence of 3 µg / ml lysozyme is significant for all strains (t-test < 0.05). The inhibitory effect of the lysozyme was observed for concentrations down to 30 µg / ml considered as physiological concentrations in saliva while the biofilm growth effect was found with non physiological doses, above 100 µg / ml. These data emphasize the necessity to develop control strategies and to implement them in clinical trials before elaborating hygiene products enriched with exocrin proteins, especially with lysozyme. Further studies should extend this investigation to other Candida species and to bacteria present in the oral biofilms.

This study was supported by a grant from the Xenophilia Funds (Université Libre de Bruxelles, Brussels, Belgium) promoting a collaboration with the University of Aboubekr Belkaïd-Tlemcen (Algeria). Corresponding author: Dr Ph. Courtois, Université Libre de Bruxelles, Faculty of Medicine (CP 626), route de Lennik 808, B-1070 Brussels, Belgium. [email protected]

P181 - Use of 96-well microtiter plates to reproduce medical biofilms Gomes, Luciana (FEUP - Faculty of Engineering Univ. Porto, Porto); Moreira, Joana (FEUP - Faculty of Engineering Univ. Porto, Porto); Araújo, José (FEUP - Faculty of Engineering Univ. Porto, Porto); Miranda, João (FEUP - Faculty of Engineering Univ. Porto, Porto); Simões, Manuel (FEUP - Faculty of Engineering Univ. Porto, Porto); Melo, Luís (FEUP - Faculty of Engineering Univ. Porto, Porto); Mergulhão, Filipe (FEUP - Faculty of Engineering Univ. Porto, Porto) Biofilm formation is of paramount importance in the development of a large number of human diseases. In order to develop strategies to prevent or eliminate these biofilms there is a need to reproduce them in vitro so that they can be used in various assays like, for instance, antimicrobial susceptibility tests. However, these in vitro biofilms and this can only be achieved if they are formed in the same hydrodynamic conditions that are found inside the human body. The main goal of this work is to assess if agitated 96-well microtiter plates are adequate for this purpose and to define the operational conditions to be used in the simulation of different medical biofilms. Escherichia coli was chosen as model organism for biofilm formation in microtiter plates due to its presence in several medical biofilms. Computational fluid dynamics (CFD) was used to


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determine the shear strain rate distribution inside a 96-well microtiter plate for shaking frequencies ranging from 50 to 200 rpm and orbital diameters from 25 to 100 mm. Scanning electron microscopy (SEM) was used to assess the biofilm distribution in the wells and cell morphology. Both features were correlated with the numerical results from the CFD simulations. The CFD results indicate that the shear strain rates under which the biofilms develop change drastically along the cylindrical wall. The higher shear strain rates below the air-liquid interface are associated with the formation of dispersed cell aggregates, while lower shear strain rates result in a homogeneous distribution of single cells on the wall. The 96-well microtiter plate is a powerful platform to simulate the shear strain rates that are found in several biomedical systems as long as the operating conditions are carefully set. Moreover, the uneven distribution of the shear strain rates in 96-well plates captures the real strain rate variation that occurs in different sites of the human body unlike other biofilm platforms where the shear strain rate is constant.

P182 - Carbonmonoxid releasing nanofibers as a potential anti-biofilm treatment Klinger, Mareike (Universitätsklinikum Jena, Zentrum für Infektionsmedizin und Krankenhaushygiene, CSCC, Jena, GER); Makarewicz, Oliwia (Universitätsklinikum Jena, Zentrum für Infektionsmedizin und Krankenhaushygiene, CSCC, Jena, GER); Pletz, Mathias W. (Universitätsklinikum Jena, Zentrum für Infektionsmedizin und Krankenhaushygiene, CSCC, Jena, GER) In the clinic day routine, biofilms are formed on various surfaces like tubes and artificial devices, but also on epithelia (bladder, lung, mucosa ), or on open wounds. According to the WHO, biofilms coincide with more than 80 % of all nosocomial bacterial infections. Accrued biofilms are almost impossible to eradicate by common drug treatments since microbes embedded in a biofilm benefit from the protective biofilm environment exhibiting elevated tolerance to antibiotics and increased protection against the immune system. Additionally, since most antibiotics target metabolic processes they are inefficient in killing persister cells in deeper biofilm layers. Therefore, alternative therapeutic strategies that interfere with biofilm integrity would offer great benefits for clinical treatment.

Staphylococcus aureus present one the most abundant biofilm forming pathogen, often related with various implant-associated infections and skin wound infections. We aimed to eradicate S. aureus biofilms by controlled photo-stimulated (405 nm and 480 nm) release of toxic CO that have been incorporated in nanofibers. The water insoluble and photoactive CO releasing molecule dimanganese decacarbonyl (CORM-1) has been non-covalently embedded into poly(L-lactide-co-D/L-lactide) nanofibers via electro-spinning to enable bioavailability and water accessibility of CORM-1. Anti-biofilm tests were performed in chamber slides and analyzed by confocal laser scanning microscopy (CLSM) as well as evaluated using the in-house software that phenotypically and statistically determine the living and dead cells.

First results showed that the controlled release of CO effectively eliminate the bacteria of existing biofilms. Since the toxic effect of CO base on the impaired electron transport chain, emergence of resistance mechanisms is unlikely. Moreover, CO might also impact persister cells. Thus CO-nanofibers might be an interesting possibility for the antimicrobial treatment, e.g. of wound infection.


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P183 - Biofilm production and glutaraldehyde 2% tolerance by planktonic and sessile forms of Corynebacterium striatum Souza, Cassius (Universidade do Estado do Rio de Janeiro - UERJ, Rio de Janeiro, BRA); Franceschi Mota, Higor (Universidade do Estado do Rio de Janeiro - UERJ, Rio de Janeiro, BRA); Ramos, Juliana (Universidade do Estado do Rio de Janeiro - UERJ, Rio de Janeiro, BRA); Simpson-Louredo, Liliane (Fundação Oswaldo Cruz - Fiocruz, Rio de Janeiro, BRA); Souza, Monica Cristina (Universidade do Estado do Rio de Janeiro - UERJ, Rio de Janeiro, BRA); Vieira Faria, Yuri (Centro Universitário Estadual da Zona Oeste - UEZO, Rio de Janeiro, BRA); Seabra, Sérgio Henrique (Centro Universitário Estadual da Zona Oeste - UEZO, Rio de Janeiro, BRA); Vianna Vieira, Verônica (Fundação Oswaldo Cruz - Fiocruz, Rio de Janeiro, BRA); Napoleão, Fátima (Universidade do Estado do Rio de Janeiro - UERJ, Rio de Janeiro, BRA); Silva Santos, Cíntia (Universidade do Estado do Rio de Janeiro - UERJ, Rio de Janeiro, BRA); Leandro Rama Gomes, Débora (IFRJ, Rio de Janeiro, BRA); Hirata Júnior, Raphael (Universidade do Estado do Rio de Janeiro - UERJ, Rio de Janeiro, BRA); Oliveira Moreira, Lílian (Universidade Federal do Rio de Janeiro - UFRJ, Rio de Janeiro, BRA); Mattos-Guaraldi, Ana Luíza (Universidade do Estado do Rio de Janeiro - UERJ, Rio de Janeiro, BRA) Corynebacterium striatum is a potentially pathogenic microorganism with the ability to produce nosocomial outbreaks. However, there are few studies focusing on virulence factors that may contribute to corynebacterial healthcare-associated infections. The importance of biofilms in nosocomial infections has increased and many studies have evaluated the effects of antimicrobial agents on these surface-attached communities. Accordingly, we investigated the biofilm production on abiotic surfaces and the susceptibility to biocidal products on planktonic and sessile forms of 14 (12 multiresistant-MDR and 02 susceptible-MDS to antibiotics) C. striatum strains PFGE-types I-MDR, II-MDR, III-MDS and IV-MDS isolated during a nosocomial outbreak, Rio de Janeiro, Brazil. The PFG- MDR I and II types (n=10 and n=2) were predominantly isolated from patients undergoing endotracheal intubation procedures (n=10) on intensive care and medical surgical units. Results showed that C. striatum is able to adhere to hydrophilic (glass) and hydrophobic (plastic) abiotic surfaces at different intensities. The scanning electron microscopy assays showed the production of mature biofilms on polyurethane catheters by representatives of pulsotypes I, II, III and IV. Susceptibility profiles of C. striatum planktonic forms to glutaraldehyde 2% were assessed by the Time Killtest at incubation times of 5, 10, 15 and 30 min.MDR and MDS C. striatum strains showed tolerance to the biocide at different levels (1 to 7 x 103cfu/ml) and periods of time (4 to 30 min). C. striatum (all pulsotypes) remained capable of producing biofilm on different abiotic substrates even in the presence of glutaraldehyde 2% (30 min). This result i ndicates that both planktonic and sessile forms of MDR and MDS C. striatum epidemic and endemic clones could survive the treatment with biocidal products. In conclusion, biofilm formation together with tolerance to biocides frequently used in nosocomial environment may contribute to the establishment and dissemination of healthcare-associated infections caused by C. striatum, independent of antibiotics susceptibility profiles. P184 - NEW APPROACH FOR PROPIONIBACTERIUM ACNES BIOFILM TREATMENT IN ACNE VULGARIS : MYRTACINE ® ANTI-BIOFILM EFFICACY roques, christine (Faculty of Pharmacy - University Paul Sabatier - LGC - UMR 5503, Toulouse, FRA); Feuillolay, Catherine (Fonderephar, Toulouse, FRA); Le Gac, Céline (Fonderephar,


185

Toulouse, FRA); Luc, Joëlle (Pierre Fabre Dermo-Cosmétique - Microbiology Lab, R&D , Castanet tolosan, FRA) Recent works present evidence of P. acnes growing as a biofilm in cutaneous follicles. This formation of clusters is now considered as an explanation for in vivo resistance of P. acnes to the main antimicrobials prescribed in acne vulgaris. Our objective was to explore this hypothesis and propose a new therapeutic approach focusing on anti-biofilm activity. At first, we designed specific in vitro models (dynamic : Campanac et al, 2002; and static) able to promote the growth of adhered bacteria. The loss of sensibility of P. acnes biofilm (48h) towards erythromycin, clindamycin and doxycycline was then checked considering sensible and resistant strains. Even if P. acnes biofilm appears as limited microcolonies a very high level of resistance has been observed especially with erythromycin and clindamycin and whatever strain (S or R) tested. In a second step, the activity of Myrtacine® (Mediterranean Myrtle extract Botanical Expertise P. Fabre) against biofilm formation and mature biofilm (48h) was evaluated using the dynamic model. Myrtacine® expresses an antibacterial activity on planktonic P. acnes with MIC ranging from 0.31 to 3.12 mg/L (strains eryS and eryR). Considering anti-biofilm activity, we noted an inhibition of biofilm formation (addition of Myrtacine® at D0) and a significant effect on mature biofilm (48h) since 1 min of contact. This potent therapeutic effect was checked using the static model for Myrtacine® concentrations ranging from 0.03% to 0.0001%. A significant and dose-dependent anti-biofilm effect was observed even at concentration under MIC, i.e. 0.001%. At least, the interest of the combination of Myrtacine® with antibiotics was explored. A synergistic efficacy was noted when erythromycin (1000mg/L) or clindamycin (500mg/L) were added to 0.001% Myrtacine®. These results are in accordance with preliminary in vivo studies on Myrtacine® indicating a reduction in acne vulgaris symptoms. An in vivo experiment was in progress to demonstrate the impact of Myrtacine® on P. acnes counts and biofilm formation.

This study received financial support from Lab Pierre Fabre, France.

P185 - Pulsed-plasma surface treatment of polyurethane plates reduces intensity of forming staphylococcal biofilms Shardakov, Igor (Institute of Continuous Media Mechanics UB RAS, Perm, RUS); Kondyurin, Aleksey (Perm State National Research University, Perm, RUS); Osorgina, Irina (School of Physics, University of Sidney , Sidney, AUS); Solodko, Vladislav (Institute of Continuous Media Mechanics UB RAS, Perm, RUS); Lemkina, Larisa (Institute of Ecology and Genetics of Microorganisms UB RAS, Perm, RUS); Morozov, Ilia (Institute of Continuous Media Mechanics UB RAS, Perm, RUS); Korobov, Vladimir (Institute of Ecology and Genetics of Microorganisms UB RAS, Perm, RUS) Wide use of polyurethane in manufacturing of various medicinal replacement devices is conditioned by its high biocompatibility. However, this property contributes to the bacterial cells adhesion on polyurethane surface with following the formation of biofilm structures and the development of so-called implant-associated infections. This identifies the need to find ways to reduce the sorption characteristics of the devices surfaces made from polyurethane, which is extremely important for the production of medical products.


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Purpose. Analysis of the biofilm formation of S.epidermidis on the polyurethane surface plates after pulsed plasma treatment.

Methods. The object of the research is a polyurethane coupons. The material is produced from the copolymer comprised of poly(propylene glycol) and poly(tetramethylene oxide) with toluene diisocyanate. Heated and vacuumed prepolymer with melt linker is filled in a plane form coated with surfactant and then cured by heating. The ready samples are immersed to toluene for several days and dried. Modification is produced by nitrogen plasma immersion ion implantation. Two treatment modes are used for the samples. The first mode supposes the value of pulse voltage of 20 kV with frequency of 50 Hz during 800 seconds. The magnitude of ion implantation fluence for this mode is 1016 ions/cm2. Applied pulse voltage for the second mode is 5 kV, frequency 200 Hz, ion fluence 1015 ions/cm2. This mode produces less ion fluence with less energy. The treated surfaces were characterized with atomic-force and optical microscopy. For the purpose of microbiological investigations ultraviolet pretreated samples (15x15 mm) were introduced in LB medium containing S. epidermidis 33 (107 CFU/ml), incubated for 24-72 hours at 37 ° C, after that those were thrice washed with 10 mM phosphate buffer (pH 7.2) and biofilms was determined by using gentian violet binding to cell walls and matrix. The absorbance of the ethanol extracts was measured at 570 nm.

Results. Analysis of the structure and properties of polyurethane samples by atomic force microscopy found significant changes in their physicochemical properties with an increase of the surface structures hardness of the investigated samples after pulse-plasma treatment from 2 MPa to 30 MPa. Nonspecific binding of the gentian violet after weak plasma polyurethane treatment decreased by 70-75%, whereas after strong treatment by 80-85%. The usable polyurethane treatments of plates result in the significant reduce the biomass of the adsorbed bacterial cells and the forming biofilms. After a weak polyurethane treatment their biomass in 72 hours was reduced by 58-60 % and after a strong pulsed plasma processing by 70-73 % compared to untreated (control) plates.

Conclusion. The pulsed-plasma surface treatment of polyurethane products leads to a significant reduction in adhesion of S. epidermidis cell and the oppression of formation biofilm by these bacteria.

This work was supported by grants from RFBR (12-04-01431-а), the UB RAS (12-P-4-1002 and 12-M-14-2035) and Perm Education and Science Ministry grant for International Scientific Group «Development of fundamental basis for creation of new generation of functional endoprosthetics of soft tissues with improved biocompatibility».

P186 - Inhibition of multispecies biofilms by a fluoridated copolymer denture material Al-Sammarraie, Sufian (Newcastle University, Newcastle upon Tyne, GBR); Jakubovics, Nick Jakubovics (Newcastle University, Newcastle upon Tyne, IOT); German, Mattew (Newcastle University, Newcastle upon Tyne, IOT); Rolland, Sarah (Newcastle University, Newcastle upon Tyne, IOT) Objectives: Increasing numbers of patients are wearing removable oral appliances, which contribute to increase of oral diseases such as candidiasis and root caries. Fluoride is one of the


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most effective agents for caries control. In this study , we evaluated the efficacy of a new fluoride-releasing copolymer material for reducing single- and mixed-species biofilm formation on oral appliances.

Methods: Discs of a copolymer of methyl methacrylate and 2-hydroxyethyl methacrylate with polymethyl methacrylate powder were produced by chemically-activated free radical polymerisation. For one group, 30% NaF powder was added at the expense of some of the PMMA. Samples were fitted into two polysulfone Modified Robbins Devices. Single- and mixed-species biofilms of Candida albicans, Streptococcus mutans and Lactobacillus casei were cultured for 48h, harvested, and microorganisms were quantified by total viable counts (TVCs) and quantitative PCR (qPCR). Scanning electron microscopy and confocal laser microscopy were used to visualise the biofilms.

Results: The inclusion of fluoride within the copolymer resulted in significantly reduced colonisation by C. albicans, S. mutans and L. casei in mixed-species biofilms (T Test, p<0.05). However, when microorganisms were grown in single-species biofilms, all the three organisms were not significantly (T Test, p>0.05) reduced by fluoride. In mixed species colonization, thick biofilms were formed by these microorganisms on non-fluoridated samples while small patchy biofilms were found on fluoridated samples.

Conclusions: A model system has been developed for culturing single- and multiple-species biofilms on dental materials. The inclusion of fluoride within the material resulted in a reduction in multispecies biofilm formation, but not in single-species biofilms. These studies highlight the need to study mixed-species systems, and demonstrate the potential for the fluoride-containing material against complex oral biofilms. Further work will aim to elucidate the mechanism by which fluoride specifically targets mixed-species biofilms.

A

Aamdal Scheie, Anne (Department of Oral Biology, University of Oslo, Oslo, NOR) ................................... 107

Abdian, Patricia (Laboratorio de Microbiología Molecular y Celular, Fundación Instituto Leloir, Buenos Aires, ARG) 43

Abee, Tjakko (TI Food and Nutrition, Wageningen) ............ 20

Abee, Tjakko (Tops Institute Food and Nutrition, Wageningen) ........................................................... 104

Acuña, Vicenç (Catalan Institute for Water Research, Girona, ESP) .......................................................................... 79

Addison, Owen (University of Birmingham, Birmingham, GBR) ....................................................................... 159

Adlassnig, Wolfram (Core Facility Cell Imaging and Ultrastructure Research, University of Vienna, Vienna, AUT) .......................................................................... 68

AG Polarz, Konstanz, GER) ............................................ 153

Ageeva, Marina (Kazan Institute of Biochemistry and Biophysics RAS, Kazan, RUS) ...................................... 59

Águila-Arcos, Sandra (University of the Basque Country, Leioa, ESP) ................................................................ 98

Ajon, Malgorazeta (Max-Planck-Institute for Terrestrial Microbiology, Marburg, GER) .................................... 108

Albers, Sonja-Verena (Max-Planck-Institute for Terrestrial Microbiology, Marburg, GER) .................................... 108

Albrechtsen, Hans-Jørgen (DTU, Copenahgen, DNK) ......... 35

Al-Janabi, Abbas (University of Al-Anbar, Department of Microbiology, IRQ) ................................................... 154

Alkorta, Itziar (University of the Basque Country, Leioa, ESP) ................................................................................. 98

Almeida, Carina (1LEPABE, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto) ........................................................................ 61

Aloria, Kerman (Universidad del País Vasco UPV/EHU, Leioa, ESP) .......................................................................... 98

Al-Sammarraie, Sufian (Newcastle University, Newcastle upon Tyne, GBR) ...................................................... 186

Altenburg, Sara (Montana State University, Bozeman, USA) ................................................................................. 87

Altenried, Stefanie (Empa, St. Gallen, CHE) ...................... 53

Alves, Carlos Tiago (Minho University, Departement of Biological Engineering Department, Braga ) .............. 180

Amberg, Caroline (Swissatest Testmaterialien AG, St. Gallen, CHE) ....................................................................... 124

Apollo, Stacy (Department of Molecular Biosciences, The University of Texas at Austin, USA) ........................... 173

Araújo, José (FEUP - Faculty of Engineering Univ. Porto, Porto) ...................................................................... 182

Araujo, Rosa M. (Departament de Microbiologia, Facultat de Biologia, Universitat de Barcelona, Barcelona, ESP) .... 33

Arizmendi, Jesus Mari (University of the Basque Country, Leioa, ESP) ................................................................ 98

Arkin, Adam P. (Lawrence Berkeley National Laboratory, Berkeley,, CAN).......................................................... 87

Armbruster, Catherine (University of Washington, School of Medicine, Seattle, USA) .............................................. 82

Arndt, Hans-Dieter (Friedrich Schiller University of Jena, Institute of Organic and Macromolecular Chemistry, Jena, GER) ............................................................... 110

Arndt, Hartmut (University of Cologne, Biocenter, Cologne, GER) .......................................................................... 91

Arndt, Hartmut (University of Cologne, Cologne, GER) ...... 59

Arsène-Ploetze, Florence (GMGM - UMR 7156, Strasbourg, FRA) .......................................................................... 87

Aspiras, Marcelo (Philips Oral Healthcare, Bothell, WA, USA) ............................................................................... 170

Assev, Synnøve (Department of Oral Biology, University of Oslo, Oslo, NOR) ...................................................... 107

Avelar Gonzalez, Francisco Javier (Universidad Autónoma de Aguascalientes, Aguascalientes, MEX) ...................... 158

Avelar González, Francisco Javier (Universidad Autónoma de Aguascalientes, Aguascalientes, MEX) ...................... 167

AYMERICH, Stéphane (INRA, Jouy-en-Josas, FRA) ............ 20

Azeredo, Joana (Minho University, Departement of Biological Engineering Department, Braga ) ........................ 44, 180

Azevedo, Andreia S. (LEPABE, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto) ........................................................................ 61

Azevedo, Nuno F. (LEPABE, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto) ........................................................................ 61

B

Babson, David (US Naval Research Laboratory, Washington, DC, USA).................................................................. 161

Bacchin, Patrice (University of Toulouse - LGC - UMR 5503, Toulouse cedex 9, FRA) ............................................. 126

Badurová, Pavlína (Faculty of Science, Palacky University in Olomouc, Olomouc, CZE) .......................................... 137

Banada, Oldrich (Institute of Microbiology AS CR, v.v.i., Prague 4, CZE) ........................................................... 97

Banin, Ehud (Bar-Ilan University, Ramat-Gan, ISR) ........... 21

Barbau, Jérémie (OVIZIO Imaging Systems NV/SA, Brussels, BEL) .......................................................................... 57

Bard, Allen (The University of Texas at Austin, Austin, TX, USA) .......................................................................... 13

Bard, Allen J (ZZZ) .......................................................... 63

Barlow, Daniel (US Naval Research Laboratory, Washington, DC, USA).................................................................. 161

Barner, Jörg (JPK Instruments AG, Berlin, GER) ................ 66

Barraud, Nicolas (University of New South Wales, Sydney, AUS) ........................................................................ 112

Bar-Zeev, Edo (Bar-Ilan University, Ramat Gan, ISR) ....... 150

Bar-Zeev, Edo (Department of Chemical and Environmental Engineering, Yale University, New Haven, USA) ........... 34

Bassler, Bonnie (Princeton University, Princeton, USA) ..... 15

Basu Roy, Ankita (Binghamton University, Binghamton, USA) ................................................................................. 22

Battin, Tom (Department for Limnolgy and Bio-Oceanography, University of Vienna, Vienna, AUT) ...... 68,

19, 74, 92, 100

Battista, John (Fluxion, San Francisco, USA) .................... 10

Bayramoglu, Bihter (Ben Gurion University of the Negev, Midreshet Ben Gurion, ISR) ....................................... 103

Baziard, Genevieve (Laboratoire de Génie Chimique (UMR 5503), Université Paul Sabatier, Université de Toulouse, Toulouse cedex 9, FRA) ............................................. 157

Bednarík, Adam (Faculty of Science, Palacky University in Olomouc, Olomouc, CZE) .......................................... 137

BEKKAL BRIKCI-BENHABIB, Ouassila (University of Tlemcen /LAPSAB, TLEMCEN , DZA) ....................................... 157

Belenguer, José (ainia, Valencia, ESP) .............................. 69

Bellon-Fontaine, Marie-Noëlle (AgroParisTech, INRA, UMR Micalis Equipe Bioadhésion Biofilm et Hygiène des Matériaux, Massy, FRA) .............................................. 95

Ben Mlouka, Amine (UMR6270 CNRS, Mont Saint Aignan, AUT) .......................................................................... 17

Benada, Oldrich (Institute of Microbiology v.v.i., Prague 4, CZE) ........................................................................ 100

Bendinger, Bernd (DVGW-Forschungsstelle TUHH, Hamburg, GER) ........................................................................ 146

Bengtsson, Mia (Department für Limnologie und Ozeanographie, AUT) ................................................. 19

Bengtsson, Mia M (University of Vienna, Department of Limnology and Oceanography , Vienna, AUT) ............. 100

Benigar, Elizabeta (University of Ljubljana, Ljubljana, SVN) 3

Benneche, Tore (Department of Chemistry, University of Oslo, Oslo, NOR) ...................................................... 107

Benninghoff, Jens (University of Duisburg-Essen, Essen, GER) ........................................................................ 119

Bereschenko, L.A. (KWR Watercycle Research Institute, Nieuwegein) .............................................................. 32

Berjeaud, jean-marc (Universite de Poitiers, Poitiers, FRA) 72

Berman Frank, Ilana (Bar-Ilan University, Ramat Gan, ISR) ............................................................................... 150

Berman, Tom (Kinneret Limnological Laboratory, Israel Oceanographic and Limnological Research, Migdal , ISR) ............................................................................... 150

Bernet, Nicolas (Laboratoire de Biotechnologie de l'Environnement, Narbonne, FRA) ....................... 25, 121

Bertaux, Joanne (Universite de Poitiers, Poitiers, FRA) ...... 72

Bertin, Philippe N. (GMGM - UMR 7156, Strasbourg, FRA) . 87

Bertron, A. (Université de Toulouse ................................ 120

Bertuzzo, Enrico (Laboratory of Ecohydrology, École polytechnique fédérale de Lausanne, Lausanne, CHE) .. 92

Besemer, Katharina (Institut für Philosophie, AUT) ........... 92

Besemer, Katharina (University of Vienna, Department of Limnology and Oceanography, Vienna, AUT) .............. 100

Bessiere, Yolaine (Université de Toulouse ................... 9, 125

Betts, Roy (Campden BRI, Chipping Campden, GBR) . 40, 114

Bezousková, Silivia (Institute of Microbiology v.v.i., Prague 4, CZE) ........................................................................ 100

Bezouskova, Silvia (Institute of Microbiology AS CR, v.v.i., Prague 4, CZE) ........................................................... 97

Biffinger, Justin (US Naval Research Laboratory, Washington, DC, USA) .............................................. 161

Bigot, Renaud (Universite de Poitiers, Poitiers, FRA) ......... 72

Bitzer, Adam (UMASS Dartmouth, North Dartmouth, USA) ............................................................................... 109

Blanc, Séverine (40-30, Grenoble, FRA) ............................ 69

Blauert, Florian (Karlsruhe Institute of Technology - Chair of Water Chemistry and Water Technology, Karlsruhe, GER) ................................................................................. 26

Bley, Thomas (Dresden University of Technology , Dresden, GER) .......................................................................... 57

Bogas, Diana (Minho University, Departement of Biological Engineering Department, Braga ) ................................ 44

Böhm, A. (BTU Cottbus , Senftenberg, GER) ...................... 65

Boni cisse, catherine (institut pasteur de Côte d'Ivoire, ABIDJAN, CIV) ......................................................... 132

Boretska, Mariia (Zabolotny Institute of Microbiology and virology of NASU, Kyiv, UKR) .................................... 156

Borrego, Carles M (Catalan Institute for Water Research and University of Girona, Girona, ESP) ............................... 79

Borrego, Carles M. (Group of Molecular Microbial Ecology, University of Girona and ICRA, Girona, ESP) ................ 85

Boschke, Elke (Dresden University of Technology, Dresden, GER) .......................................................................... 57

Boucherir-Otmani, Zahia (University of Tlemcen /LAPSAB, TLEMCEN , DZA) ...................................................... 157

BOUCHERIT, kebir (University of Tlemcen /LAPSAB, TLEMCEN , DZA) ...................................................... 157

Boucherit-Otmani, Zahia (University of Aboubekr Belkaïd-Tlemcen, Tlemcen, DZA) ........................................... 181

Boulêtreau, Stéphanie (University Paul Sabatier, Toulouse, FRA) .......................................................................... 75

BOURDIN, GRAZIELLA (ANSES, BOULOGNE SUR MER, FRA)1

Brablcova, Lenka (Faculty of Science, Palacky University, Olomouc, CZE) ................................................... 96, 137

Brackman, Gilles (Laboratory of Pharmaceutical Microbiology, Gent, BEL) ............................................ 98

Brannon, Paul (Northwestern University, Evanston, USA) 133

BRAUGE, THOMAS (ANSES, BOULOGNE SUR MER, FRA) ..... 1

Braun, Burga (TU Berlin, Berlin, GER)............................. 151

Brenner-Weiß, Gerald (Institute of Functional Interfaces, Department of Interface Microbiology, Eggenstein-Leopoldshafen, GER) .................................................. 55

Briandet, Romain (INRA, Massy, FRA) .......................... 3, 54

BRIANDET, Romain (INRA, Massy, FRA) ............................ 20

Bridier, Arnaud (Irstea, Antony, FRA) ......................... 20, 54

Brileya, Kristen A. (Department of Microbiology and Immunology/Center for Biofilm Engineering, Bozeman, USA) .......................................................................... 85

Brloznik, Mojca (University of Ljubljana, Biotechnical Faculty, Dept. Food Science and Technology, Chair of Microbiology, Ljubljana, SVN) ..................................... 46

Brouwer-Hanzens, A.H. (KWR Watercycle Research Institute, Nieuwegein) .............................................................. 32

Brown, Helen (Institute of Food Research, Norwich, GBR) 40,

114

Brown, Judith (University of Bath, Bath, AUT) ................. 172

Bruchmann, Julia (Karlsruhe Institute of Technology (KIT), Eggenstein-Leopoldshafen, GER) ................................ 11

Brunner, Fabian (Karlsruhe Institute of Technology - Chair of Water Chemistry and Water Technology, Karlsruhe, GER) ............................................................................... 151

Bruzaud, Jérôme (INRA, AgroParisTech, UMR1319 Micalis B2HM, Massy, FRA) .................................................... 95

Budde, Heidrun (University of Cologne, Biocenter, Cologne, GER) .......................................................................... 91

Buhmann, Matthias (TU Dresden, Dresden, GER) .............. 93

Buriánková, Iva (Faculty of Science, Palacky University in Olomouc, Olomouc, CZE) .......................................... 137

Buriánková, Iva (Faculty of Science, Palacky University, Olomouc-Holice, CZE) ................................................ 96

Buriánková, Karolína (Institute of Microbiology v.v.i., Prague 4, CZE) .................................................................... 100

Burns, Nancy (University of Vienna, Vienna, AUT) ............ 19,

100

Burrows, L.L. (Dep. of Biochemistry and Biomedical Sciences / Michael G. DeGroote Institute for Infectious Diseases Research, Hamilton, CAN) .......................................... 99

Burygin, Gennadiy (IBPPM RAS, Saratov, RUS) ................. 59

Busscher, Henk (University Medical Center Groningen , Groningen) .............................................................. 153

Buzzola, Fernanda (Instituto de Microbiología y Parasitología Médica, UBA-CONICET, Buenos Aires, ARG) ............................................................................... 180

Byarmuglo, Bihter (Ben Gurion University, Midreshet Ben Gurion, ISR) ............................................................... 15

C

C S, Srinandan (SASTRA University, Thanjavur, IND) ......... 73

Camilleri, Laura B. (Department of Microbiology and Immunology/Center for Biofilm Engineering, Bozeman, USA) .......................................................................... 85

Canette, Alexis (inra, Massy, FRA) .................................... 54

Cao, Bin (Singapore Centre on Environmental Life Sciences Engineering, and School of Civil and Environmental Engineering, Singapore, SGP) ..................................... 41

Caramelo, Julio (Laboratorio de Biología Estructural y Celular, Fundación Instituto Leloir, Buenos Aires, ARG) 43

Carneiro, Victor (Federal University of Ceara, Sobral, BRA) ............................................................................... 153

Carpentier, Brigitte (Laboratory of Food Safety, French Agency for Food, Environmental and occupational Health Safety (Anses), Maisons-Alfort, FRA) ......................... 178

Carrel, Maxence (ETH Zürich IfU Chair for Environmental Fluid Mechanics, Zurich, CHE) .................................. 128

Carroll, Joe (The Medical Research Council Mitochondrial Biology Unit, Cambridge , AUT) ................................... 98

Carugo, Dario (Bioengineering research group, Faculty of Engineering and the Environment, University of Southampton, Southampton , GBR) ........................... 170

Casey, Eoin (School of Chemical and Bioprocess Engineering, University College Dublin, Dublin) .. 34, 140

Causserand, Christel (University of Toulouse - LGC - UMR 5503, Toulouse cedex 9, FRA) ................................... 126

Cerqueira, Laura (LEPABE, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto) ........................................................................ 61

Cervero, Silvia (Departament de Microbiologia, Facultat de Biologia, Universitat de Barcelona, Barcelona, AUT) .... 33

Cevallos, Manuel (Grand Canyon University, Phoenix, USA) ............................................................................... 162

Chakravortty, Dipshika (Indian Institute of Science, Bangalore, IND) ......................................................... 73

Chan Tchi Tsong, Philippe (IRIB - PISSARO Proteomics Facility, Mont Saint Aignan, FRA) ................................ 17

Chassaing, Danielle (Laboratory of Food Safety, French Agency for Food, Environmental and occupational Health Safety (Anses), Maisons-Alfort, FRA) ......................... 178

Chellappa, Shakinah T (College of Science and Mathematics. Southwest Baptist University., Missouri , USA) .......... 164

Chen, Chenfei (Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, GBR) ............................................................................... 124

Chevalier, Sylvie (Normandie Université, Université de Rouen, Laboratoire de Microbiologie Signaux et Micro-environnement EA 4312, Evreux, FRA)......................... 95

Chludzinski, Jeffrey (Department of Epidemiology, School of Public Health, University of Michigan, Michigan, USA) . 10

Chollet, R. (Merck Millipore, Application group, Molsheim, FRA) ........................................................................ 141

Chopp, David (Northwestern University, Evanston, USA) . 133

Chow, Chung Ping (Waters Singapore, Singapore, SGP) .. 101

Chuen, Su (Singapore Centre for Environmental Life Sciences Engineering, Singapore, SGP) ....................... 39

Cilli, Eduardo (Univ. Estadual Paulista - UNESP, Araraquara, BRA) ........................................................................ 153

Clegg, Robert (University of Birmingham, Birmingham, GBR) ................................................................................. 49

Cleiss-Arnold, J. (UMR 7156, Université de Strasbourg-CNRS, Laboratoire Génétique Moléculaire, Strasbourg, FRA) ........................................................................ 141

Cockx, Arnaud (Université de Toulouse .......................... 120

Coenye, Tom (Laboratory of Pharmaceutical Microbiology, Gent, BEL) ................................................................. 98

Cohen, Dorit (Bar-Ilan University, Ramat-Gan, ISR) .......... 21

Cohen, Yehuda (Singapore Centre on Environmental Life Sciences Engineering (SCELSE), Singapore, SGP) ........ 55

Cole, Ivan (CSIRO, Clayton, AUS) ................................... 114

Connell, Jodi (The University of Texas at Austin, Austin, TX, USA) ......................................................................... 13

Connell, Jodie (ZZZ) ........................................................ 63

Corbin, Agnes (Nonlinear Dynamics, Courtaboeuf, FRA) .... 17

Cornejova, Tatiana (University of Veterinary Medicine and Pharmacy in Ko¿ice, Kosice, SVK) .............................. 117

Cornelissen, E.R. (KWR Watercycle Research Institute, Nieuwegein) .............................................................. 32

Cosette, Pascal (UMR 6270, Polymères, Biopolymères, Surfaces Laboratory, Mont Saint Aignan, FRA) ............. 94

Cosette, Pascal (UMR6270 CNRS - PISSARO Proteomics Facility, Mont Saint Aignan, FRA) ................................ 17

Costa, Stephen (UMASS Dartmouth, North Dartmouth, USA) ............................................................................... 109

Courtois, Philippe (Université Libre de Bruxelles, Brussels, BEL) ........................................................................ 181

Culotti, Alessandro (Northwestern University, Evanston, IL, USA) ........................................................................ 144

Cvitkovitch, D.G. (ZZZ) .................................................. 171

D

Daims, Holger (Department für Mikrobiologie und Ökosystemforschung, Wien, AUT) ............................... 12

Daminova, Amina (Kazan Institute of Biochemistry and Biophysics RAS, Kazan, RUS) ...................................... 59

Davoudi, Neda (University of Kaiserslautern, Department of Physics, Kaiserslautern, GER) ..................................... 27

de Jager, Marko (Philips Research, Eindhoven) ............... 170

Dehghani, Hamid (University of Birmingham, Birmingham, GBR) .......................................................................... 70

Del Re, D. (ZZZ) ............................................................ 171

Delattre, Cécile (EDF R&D, Chatou, FRA) .......................... 75

Delli, Joseph (W. Nuhsbaum Inc., McHenry, USA) ............. 10

Demakov, Vitalii (Institute of Ecology and Genetics of Microorganisms UB RAS, Perm, RUS) .................. 60, 136

Demeter, Katalin (Department of Limnology and Bio-Oceanography, Wien, AUT) ....................................... 131

Derlon, Nicolas (Eawag Process Engineering, Duebendorf, CHE) ........................................................................ 128

Derlon, Nicolas (EAWAG, Dübendorf, CHE) ......................... 9

Deschamps, Julien (INRA, Massy, FRA)............................. 54

DESCHAMPS, Julien (INRA, Massy, FRA) ............................ 3

Devau, Nicolas (BRGM, Orléans, FRA) ............................ 132

Devreese, Bart (Laboratory for Biochemistry and Biomolecular Engineering L-ProBE, Gent, BEL) ............ 98

Dhyani, R. (ZZZ) ............................................................ 171

Di Martino, Patrick (Université de Cergy-Pontoise, Cergy-Pontoise, FRA) ......................................................... 155

Di Martino, Patrick (Université de Cergy-Pontoise, Cergy-Pontoise, FRA) ........................................................... 28

Díaz-Villanueva, Verónica (Lab. Limnología, INIBIOMA-CONICET, Bariloche, ARG) .......................................... 85

Diedel, Ralf (Forschungsinstitut für Anorganische Werkstoffe Glas / Keramik GmbH, Höhr-Grenzhausen, GER) .......................................................................... 55

Ding, Shujing (The Medical Research Council Mitochondrial Biology Unit, Cambridge , GBR) ................................... 98

Ding, Yuanzhao (Singapore Centre on Environmental Life Sciences Engineering, Singapore, SGP) ....................... 41

Doghri, Ibtissem (LIENSs Université de La Rochelle, La Rochelle cedex 01, FRA) ............................................. 84

Dogsa, Iztok (University of Ljubljana, Biotechnical Faculty, Dept. Food Science and Technology,Chair of Microbiology, Ljubljana, SVN) ..................................... 46

Dogsa, Iztok (University of Ljubljana, Ljubljana, , SVN) ....... 3

Dopp, Elke (IWW, Water Centre, Mülheim a. d. Ruhr, GER) ............................................................................... 139

Dorninger, Christiane (Department für Mikrobiologie und Ökosystemforschung, Wien, AUT) ............................... 12

Dowd, Scot E. (Molecular Research LP (MR DNA), Shallowater, USA) ...................................................... 10

Drake, Carrie (Air Force Research Laboratory, Wright-Patterson AFB, OH, USA) .......................................... 161

Drescher, Knut (Princeton University, Princeton, USA) ...... 15

Dumitrache, Romeo (Ryerson University, Toronto, ON., Toronto, CAN) ............................................................ 62

Dwidjosiswojo, Zenyta (Biofilm Centre, University of Duisburg-Essen, Essen, GER) .................................... 139

Dyall-Smith, Mike (Charles Sturt University, Wagga Wagga, AUS) ......................................................................... 38

Dykes, Gary (Monash University, Selangor, MYS) .............. 73

Dyson, Rosemary (University of Birmingham, Birmingham, AUT) .......................................................................... 49

E

E. Kohler, Hans-Peter (Eawag, Swiss Federal Institute of Aquatic Science and Technology, Dübendorf, CHE) ........ 8

Eberl, Hermann (Biophysics Interdepartmental Program and Dept. Mathematics and Statistics, University of Guelph , Guelph, ON, CAN) ...................................................... 48

Eberl, Hermann (University of Guelph, Guelph ON, CAN) ..... 5

Ehrke, Ulrich (Cameca GmbH, Unterschleissheim, GER) .... 63

El Hage, Saloma (Laboratoire de Génie Chimique (UMR 5503), Université Paul Sabatier, Université de Toulouse, Toulouse cedex 9, FRA) ............................................. 157

Elango, Monalisha (Indian Institute of Science, Bangalore, IND) .......................................................................... 73

Elgamoudi, Bassam (University of Leicester, Leicester, GBR) ............................................................................... 135

Elifantz, Hila (Bar-Ilan University, Ramat Gan, ISR) ........ 150

Elimelech, Menachem (Department of Chemical and Environmental Engineering, New Haven, USA) ............ 34

Endres, Jasmin (University of Ulm, Ulm, GER) ................ 110

Eroshenko, Daria (Institute of Ecology and Genetics of Microorganisms UB RAS, Perm, RUS) .......................... 45

Escudié, Renaud (Laboratoire de Biotechnologie de l'Environnement, Narbonne, FRA) ....................... 25, 121

Etienne, PAUL (INSA, Toulouse, FRA) ............................. 120

Etienne, PAUL (INSA, Toulouse, FRA) ............................. 120

Everett, Jake (Department of Surgery, Texas Tech University Health Sciences Center,, USA) .................................. 160

Everett, Jake (Texas Tech University Health Sciences Center, Lubbock, TX, USA) ........................................ 16, 18, 173

Exner, Martin (Institute for Hygiene and Public Health, University of Bonn, Germany , Bonn, GER) ................. 142

F

Fabbri, Stefania (University of Southampton, southampton, GBR) ....................................................................... 170

FAILLE, CHRISTINE (INRA, VILLENEUVE D'ASCQ, FRA) 1, 37,

129

Fallarero, Adyary (Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Helsinki)175,

179

Farasin, Julien (GMGM - UMR 7156, Strasbourg, FRA) ...... 87

Farhan, Abbas (University of Al-Anbar, College of Medicine, Department of Microbiology, Ramadi, IRQ) ................ 154

FB Chemie .................................................................... 153

Fearnley, Ian (The Medical Research Council Mitochondrial Biology Unit, Cambridge , GBR)................................... 98

Fernández Ramírez, Mónica (Top Institute Food and Nutrition, Wageningen) ............................................ 104

Feuillolay, Catherine (Fonderephar, Toulouse, FRA)........ 184

Fields, Matthew (Montana State University, Bozeman, USA) ................................................................................. 87

Fields, Matthew (Montana State University, Bozeman, USA) ................................................................................. 85

Firmesse, Olivier (Laboratory of Food Safety, French Agency for Food, Environmental and occupational Health Safety (Anses), Maisons-Alfort, FRA) ................................... 178

Fischer, Sebastian Wolfgang (Institute for Hygiene and Public Health, University of Bonn, Germany , Bonn, GER) ............................................................................... 142

Flemming, Hans-Curt (Biofilm Centre, University of Duisburg-Essen, Essen, GER) ...................... 53, 139, 148

Fonseca, Elza (Minho University, Departement of Biological Engineering Department, Braga , AUT) ........................ 44

Fontenete, Silvia (LEPABE, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto) ........................................................................ 61

Foussard, J.N. (Université de Toulouse ........................... 120

Franceschi Mota, Higor (Universidade do Estado do Rio de Janeiro - UERJ, Rio de Janeiro, BRA) ......................... 184

Freel, Kelle (GMGM - UMR 7156, Strasbourg, FRA) ........... 87

Freixa, Anna (Institute of Aquatic Ecology, University of Girona, Girona, ESP) ................................................... 85

Fröls, Sabrina (Archaea Biology and Ecogenomics Division, Department of Ecogenomics and Systems Biology, Vienna, AUT) ............................................................ 108

Fröls, Sabrina (Technische Universität Darmstadt , Darmstadt, GER) ........................................................ 38

Frösler, Jan (University of Duisburg-Essen, Essen, GER) .... 53

Füchslin, Hans Peter (Bachema AG, Schlieren, CHE) ....... 143

Furiga, Aurélie (Laboratoire de Génie Chimique (UMR 5503), Université Paul Sabatier, Université de Toulouse, Toulouse cedex 9, FRA) ............................................. 157

G

Gabrilska, Rebecca (Department of Surgery, Texas Tech University Health Sciences Center, USA) ............. 18, 160

Gallegos Monterrosa, Ramses (Friedrich Schiller University of Jena, Terrestrial Biofilms Group, Jena, GER) .......... 110

Gallet, Olivier (Université de Cergy-Pontoise, Cergy-Pontoise, FRA) ......................................................... 155

Garabetian, Frederic (University of Bordeaux, Arcachon, FRA) .......................................................................... 75

Garneau, Philippe (Universite de Montreal, Quebec, CAN) ............................................................................... 167

Garrido, Francis (BRGM, Orléans, FRA) ........................... 132

Gattlen, Jasmin (Institute of Chemistry and Biological Chemistry, ZHAW, Wädenswil, CHE) ......................... 143

Gebel, Jürgen (Institute for Hygiene and Public Health, University of Bonn, Germany , Bonn, GER) ................. 142

Gehring, Julia (Universität Konstanz .............................. 153

Geiger, Katharina (Karlsruhe Institute of Technology, Karlsruhe, GER) ....................................................... 134

Geisthardt, Steffen (Helmholtz Zentrum für Umweltforschung UFZ, Magdeburg, GER) .................... 78

Gemein, Stefanie (Institute for Hygiene and Public Health, University of Bonn, Germany , Bonn, GER) ................. 142

Gerbersdorf, Sabine (University of Stuttgart, Institute for Modelling Hydraulic and Environmental Systems , Stuttgart, GER) ........................................................ 127

German, Mattew (Newcastle University, Newcastle upon Tyne, IOT) ................................................................ 186

Gernand, Anna (Department for Limnolgy and Bio-Oceanography, University of Vienna, Vienna, AUT) ....... 68

Gescher, Johannes (Karlsruhe Institute of Technology, Karlsruhe, GER) ....................................................... 134

Gião, Maria Salomé (University of Southampton, Southampton, GBR) .................................................... 69

Gich, Frederic (Group of Molecular Microbial Ecology, University of Girona, Girona, ESP) ............................... 85

Gillmann, Antoine (Merck Millipore, Molsheim, FRA) ...... 141

Gillor, Osnat (Ben Gurion University of the Negev, Midreshet Ben Gurion, ISR) ....................................................... 103

Gilmore, Brendan (Queen's University Belfast, Belfast, GBR) ............................................................................... 113

Gnanadhas, Divya Prakash (Indian Institute of Science, Bangalore, IND) ......................................................... 73

Godissart, Patricia (GlaxoSmithKline, Rixensart, BEL) ....... 57

Goeres, Darla (Abo Akademi University/Center for Biofilm Engineering, Turku) ................................................... 50

Goeres, Darla (Center for Biofilm Engineering, Bozeman, USA) ............................................................... 175, 179

Gogolev, Yuriy (Kazan Institute of Biochemistry and Biophysics RAS, Kazan, RUS) ...................................... 59

Gogoleva, Natalia (Kazan Institute of Biochemistry and Biophysics RAS, Kazan, RUS) ...................................... 59

Gomes, Luciana (FEUP - Faculty of Engineering Univ. Porto, Porto) ...................................................................... 182

Gonçalves, Bruna (Minho University, Departement of Biological Engineering Department, Braga ) ................ 44

Goñi, Félix M. (Universidad del País Vasco UPV/EHU, Leioa, ESP) .......................................................................... 98

Goodson, Wendy (Air Force Research Laboratory, Wright-Patterson AFB, OH, USA) .......................................... 161

Gorshkov, Vlsdimir (Kazan Institute of Biochemistry and Biophysics RAS, Kazan, RUS) ...................................... 59

Gottenbos, Bart (Philips Research, Eindhoven) ............... 165

Goza, Agata (Ghent University, Gent, BEL) ....................... 98

GRAZIELLA, BOURDIN (ANSES, BOULOGNE SUR MER, FRA) ......................................................................... 37, 129

Grobe, Susanne (IWW, Water Centre, Mülheim a. d. Ruhr, GER) ........................................................................ 145

Grover, Liam (University of Birmingham, Birmingham, GBR) ................................................................................. 70

Grube, Martin (University of Graz, Graz, AUT) ................. 173

Gruss, Alexandra (INRA, Massy, FRA) ................................. 3

Gubaev, Rim (Kazan Institute of Biochemistry and Biophysics RAS, Kazan, RUS) ....................................................... 59

GUERARDEL, YANN (USTL UGSF, VILLENEUVE D'ASCQ, FRA) ............................................................................. 1, 37

Guerrero Barrera, Alma Lilián (Universidad Autónoma de Aguascalientes, Aguascalientes, MEX) .............. 158, 167

Guga, Yurij (SE .............................................................. 156

Guichard, Alan (BRGM, Orléans, FRA) ............................ 132

Guilbaud, Morgan (AgroParisTech, INRA, UMR Micalis Equipe Bioadhésion Biofilm et Hygiène des Matériaux, Massy, FRA) ............................................................... 95

Guillot, Alain (INRA, PAPPSO, Jouy-en-Josas, FRA) .......... 95

Guimarães, Nuno (LEPABE, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto) ........................................................................ 61

Gülay, Arda (Denmark Technical university, Copenhagen, DNK) ......................................................................... 35

Guthausen, Gisela (Institute for Mechanical Process Engineering and Mechanics, Department for Process Machines, Karlsruhe, GER) ......................................... 55

Gutt, Beatrice (Empa, St. Gallen, CHE) ............................. 67

H

H. Puga, Carmen (Univ. Complutense of Madrid, Madrid, ESP) .......................................................................... 92

Habimana, Olivier (School of Chemical and Bioprocess Engineering, University College Dublin, Dublin) .. 34, 140

Habouzit, Frédéric (Laboratoire de Biotechnologie de l'Environnement, Narbonne, FRA) ............................... 25

Halada, Petr (Institute of Microbiology AS CR, v.v.i., Prague 4, CZE) .............................................................. 97, 100

Hamada, Masakaze (University of Tsukuba, Tsukuba, JPN) 67

Hamouda, Assia (Haute Ecole Francisco Ferrer, Brussels, BEL) ........................................................................ 181

Hannig, Mathias (University of the Saarland, Department of Operative Dentistry and Periodontology, Homburg, GER) ................................................................................. 27

Hapca, Simona (SIMBIOS Centre, Dundee, GBR) ............... 74

Hardouin, Julie (UMR 6270, Polymères, Biopolymères, Surfaces Laboratory, Mont Saint Aignan, FRA) ............. 94

Harel, Josee (Universite de Montreal, Quebec, AUT) ........ 167

Harmsen, D.J.H. (KWR Watercycle Research Institute, Nieuwegein) .............................................................. 32

Hartmann, Manuel (ETH Zurich IfU Chair of Environmental Fluid Mechanics, Zurich, CHE) .................................. 128

Haschke, Heiko (JPK Instruments AG, Berlin, GER) ........... 66

Haskins, William E (College of Science and Mathematics. Southwest Baptist University., Missouri , USA) .......... 164

Haumont, Denis (Ovizio Imaging Systems, Brussels, BEL) .. 57

Hayes, Christopher (Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, USA) ............................................................ 89

Hayrapetyan, Hasmik (TI Food and Nutrition, Wageningen)20

Heffernan, Rory (School of Chemical and Bioprocess Engineering, University College Dublin, Dublin)........... 34

Heir, Even (Nofima, the Norwegian Institute of Food, Fisheries and Aquaculture Research, Aas, NOR) ........... 88

Helbling, Damian E. (Department of Environmental Engineering / Eawag Swiss Federal Institute of Aquatic Science and Technology, DNK) ...................................... 8

Hellal, Jennifer (BRGM, Orléans, FRA)............................ 132

Henneghien, Joël (OVIZIO Imaging Systems NV/SA, Brussels, BEL) .......................................................................... 57

Henriques, Mariana (Minho University, Departement of Biological Engineering Department, Braga ) ........ 44, 180

Henze, Thomas (JPK Instruments AG, Berlin, GER) ............ 66

Hermand, Philippe (GlaxoSmithKline, Rixensart, BEL) ....... 57

Herndl, Gerhard (Dep. Limnology and Oceanography, Vienna, AUT) .......................................................................... 30

HERRY, Jean Marie (INRA, Massy, FRA) ............................ 20

Herry, Jean-Marie (INRA, AgroParisTech, UMR Micalis Equipe Bioadhésion Biofilm et Hygiène des Matériaux, Massy, FRA) .......................................................... 54,95

Herzberg, Moshe (Ben Gurion University of the Negev, Midreshet Ben Gurion, ISR) ....................................... 103

Hijnen, Wim (KWR Watercycle Research Institute, Nieuwegein) .............................................................. 32

Hilbert, Friederike (University of Veterinary Medicine Vienna, Vienna, AUT) .................................................. 80

Hindié, Mathilde (Université de Cergy-Pontoise, Cergy-Pontoise, FRA) ......................................................... 155

Hirata Júnior, Raphael (Universidade do Estado do Rio de Janeiro - UERJ, Rio de Janeiro, BRA) ................. 174, 184

Hirotani, Hiroshi (Osaka Kyoiku University, Kashiwara, JPN) ............................................................................... 115

Hoffman, Lucas (University of Washington, School of Medicine, Seattle, USA) .............................................. 82

Holliger, Christof (Ecole Polytechnique Fédérale de Lausanne, Lausanne, CHE) ......................................... 90

Holzner, Markus (ETH Zurich IfU Chair of Environmental Fluid Mechanics, Zurich, CHE) .................................. 128

Horn, Harald (Chair of Water Chemistry and Water Technolog, Engler-Bunte-Institut, Karlsruhe Institute of Technology, Karlsruhe, GER) ............ 6, 26, 122, 149, 151

Hötzl, Sandra (Philips Research, Eindhoven)................... 165

Hui, Janice (The Centre for Bio-Innovation, Sydney, AUS) . 22,

112

Hunter, William (Department für Limnologie und Ozeanographie, Wien, AUT) ........................................ 74

Huron, Vanessa (CMB, Sydney, AUS) ................................ 22

I

Ikeno, C. (ZZZ) .............................................................. 171

INGLEBERT, GAELLE (ANSES, BOULOGNE SUR MER, FRA) ............................................................................... 129

INSA, UPS ..................................................................... 120

INSA, UPS, INP ................................................. 9, 120, 125

Irie, Yasuhiko (University of Bath, Bath, GBR) ................... 43

Irodov, Dmitry (Institute of Molecular Biology and Genetics of National Academy of Sciences of Ukraine, Kiev, UKR) ................................................................................. 46

J

J. Modrzynski, Jakub (Copenhagen University, Frederiksberg, DNK) ................................................... 24

Jackson, Lindsay (Ryerson University, Toronto, CAN) ...... 168

Jacques, Mario (Université de Montréal, Saint Hyacinthe, CAN) ....................................................................... 158

Jadhav, Kirtikumar B. (Friedrich Schiller University of Jena, Institute of Organic and Macromolecular Chemistry, Jena, GER) ............................................................... 110

Jakubec, Martin (Dairy research institute Ltd., Praha 6, CZE) ................................................................................. 56

Jakubovics, Nick Jakubovics (Newcastle University, Newcastle upon Tyne, IOT) ....................................... 186

Jamnik, Andrej (University of Ljubljana, Ljubljana, SVN) ..... 3

Janecek, Jirí (Institute of Microbiology v.v.i., Prague 4, CZE) ............................................................................... 100

Jarnier, Frédérique (UMR 6270, Polymères, Biopolymères, Surfaces Laboratory, Mont Sait Aingan, FRA) .............. 94

Johnson, Candice (US Food and Drug Administration, Bethesda, USA) ........................................................ 106

Johnston, David A. (Biomedical Imaging Unit, School of Medicine, University of Southampton,, Southampton , GBR) ....................................................................... 170

Jooris, Serge (OVIZIO Imaging Systems, Brussels, BEL) ..... 57

Jörger, Michael (University of Applied Sciences, Graz, AUT) ............................................................................... 166

Jorth, Peter (Department of Molecular Biosciences, The University of Texas at Austin, USA) ..................... 16, 173

Jouenne, Thierry (UMR 6270, Polymères, Biopolymères, Surfaces Laboratory, Mont Saint Aingnan, FRA) ..... 17, 94

Jubair, Abdul Sattar (AL-Ramadi Teaching Hospital, Ramadi, IRQ)......................................................................... 154

K

Kadlec, Robert (Dairy research institute Ltd., Praha 6, CZE) ................................................................................. 56

Kamjunke, Norbert (Helmholtz Centre for Environmental Research - UFZ, Magdeburg, GER) ............................ 123

Kandaswamy, Kumaravel (Singapore Centre on Environmental Life Sciences Engineering (SCELSE), Singapore, SGP) ......................................................... 55

Karakhim, Sergey (Palladin Institute of Biochemistry of National Academy of Sciences of Ukraine , Kiev, UKR) . 46

Karampatzakis, Andreas (NUS Centre for Bioimaging Sciences (CBIS), Singapore, SGP) ................................ 55

Karwautz, Clemens (Helmholtz Zentrum München, Neuherberg, GER) ...................................................... 31

Katzenmeier, Heinz (Sanitized AG, Burgdorf, CHE) ......... 124

Kätzl, Korbinian (Ruhr Universität Bochum, Bochum, GER) ............................................................................... 122

Keevil, Charles William (University of Southampton, Southampton, GBR).................................................... 69

Ketley, Julian (University of Leicester, Leicester, GBR) ... 135

Kim, Jiyeon (The University of Texas at Austin, Austin, TX, USA) ......................................................................... 13

Kim, Jiyeon (ZZZ) ............................................................ 63

KIM, Se Yeon (INRA , MASSY, FRA) ..................................... 3

Kim, Se yeon (INRA, Massy, FRA) ..................................... 54

Kim, Soo-Kyoung (Pusan National University, Busan, KOR) ....................................................................... 107, 158

King, Andrew (Centre for Immunology and Infection and the Department of Biology, University of York, York, GBR) .. 89

Kitzinger, Katharina (Department für Mikrobiologie und Ökosystemforschung, Wien, AUT) ............................... 12

Kjelleberg, Staffan (Singapore Centre for Environmental Life Sciences Engineering, Singapore, SGP) . 22, 39, 101, 112

Kleintschek, Tanja (Karlsruher Institute of Technology (KIT), Eggenstein-Leopoldshafen, GER) ................................ 11

Klinger, Mareike (Universitätsklinikum Jena, Zentrum für Infektionsmedizin und Krankenhaushygiene, CSCC, Jena, GER) ........................................................................ 183

Klug, Barbara (University of Graz, Medical University of Graz, Graz, AUT) ............................................................... 173

Kmet, Vladimir (Institute of Animal Physiology Slovak Academy of Sciences, Kosice, SVK) ........................... 117

Kmetova, Marta (Faculty of Medicine, Pavol Jozef Safarik University in Kosice, Kosice, SVK) ............................. 117

Koerdt, Andrea (Universität für Bodenkultur Wien, Department of NanoBiotechnology / NanoGlycobiology unit, Vienna, AUT) .................................................... 105

Kofronova, Olga (Institute of Microbiology AS CR, v.v.i., Prague 4, CZE) ................................................... 97, 100

Köhler, Oliver (University of Kaiserslautern, Department of Physics, Kaiserslautern, GER) ..................................... 27

Kondyurin, Aleksey (Perm State National Research University, Perm, RUS) ............................................. 185

Kononova, Lyudmila (Institute of Ecology and Genetics of Microorganisms UB RAS, Perm, RUS) ........................ 176

Korobov, Vladimir (Institute of Ecology and Genetics of Microorganisms UB RAS, Perm, RUS) .......... 45, 176, 185

Korobov, Vladimir (Institute of Ecology and Genetics of Microorganisms UB RAS, Perm, RUS) ........................ 163

Kovacs, Akos T (Friedrich Schiller University of Jena, Terrestrial Biofilms Group, Jena, GER) ......................... 72

Kovacs, Akos T. (Friedrich Schiller University of Jena, Terrestrial Biofilms Group, Jena, GER) ............... 103, 110

Kratz, Fabian (University of Kaiserslautern, Department of Physics, Kaiserslautern, GER) ..................................... 27

Krause, Susanne (Karlsruhe Institute of Technology, Karlsruhe, GER) ....................................................... 134

Krebs, Walter (Institute of Chemistry and Biological Chemistry, ZHAW, Wädenswil, CHE) ......................... 143

Kreft, Jan-Ulrich (University of Birmingham, Birmingham, GBR) .......................................................................... 49

Kröger, Nils (TU Dresden, Dresden, GER) .......................... 93

Kroukamp, Otini (Ryerson University, Toronto, CAN) ....... 168

Kroukamp, Otini (Ryerson University, Toronto, ON., Toronto, CAN) ......................................................................... 62

Kühl, Michael (Marine Biological Section, University of Copenhagen, Helsingør, DNK) ................................... 137

Kuhlicke, Ute (Helmholtz Center for Environmental Research - UFZ, Magdeburg, GER) ..................................... 58, 123

Kuipers, Oscar P (University of Groningen, Molecular Genetics Group, Groningen) ................................ 72, 103

Kuriakose, Sapuna (Institute for Hygiene and Public Health, University of Bonn, Germany , Bonn, GER) ................. 142

L

L. Meyer, Rikke (Aarhus University, Aarhus, DNK) ............. 24

LAAS , Toulouse, FRA) ............................................... 9, 125

Labrie, Josée (Université de Montréal, Saint Hyacinthe, CAN) ............................................................................... 158

Lackner, Susanne (Karlsruhe Institute of Technology - Chair of Water Chemistry and Water Technology, Karlsruhe, GER) .................................................................... 6, 151

Lafleur, John (Alpert School of Medicine at Brown University, Providence, USA) .................................... 109

Lajoie, Barbora (Laboratoire de génie Chimique (UMR 5503), Université Paul Sabatier, Université de Toulouse, Toulouse cedex 9, FRA) ............................................. 157

Langbein, Jennifer (Fraunhofer IPK, Berlin, GER) ............ 177

Langsrud, Solveig (Nofima, the Norwegian Institute of Food, Fisheries and Aquaculture Research, Aas, NOR) ........... 88

Lanneluc, Isabelle (LIENSs Université de La Rochelle, La Rochelle cedex 01, FRA) ............................................. 84

Lapadatescu, Carmen (Naturatech, LEVALLOIS-PERRET , FRA) ............................................................................ 3

Larsen, Jens E. N. (Marine Biological Section, University of Copenhagen, Helsingør, DNK)................................... 137

Lau, Kelvin E. (Centre for Microbial Innovation, School of Biological Sciences, NZL) ........................................... 80

Law, David (RMIT University, Melbourne, AUS) ............... 114

LE COQ, Dominique (CNRS, Jouy-en-Josas, FRA) .............. 20

Le Gac, Céline (Fonderephar, Toulouse, FRA) ................. 184

Leandro Rama Gomes, Débora (IFRJ , Rio de Janeiro, BRA) ............................................................................... 174

Leandro Rama Gomes, Débora (IFRJ, Rio de Janeiro, BRA) ............................................................................... 184

Lee, Chung-Hak (School of chemical & Biological Engineering, Seoul National University, Seoul, KOR).. 116

Lee, Jong-Min (Nanyang Technological University, Singapore, SGP) ......................................................... 39

Lee, Kai Wei Kelvin (SCELSE/NTU, Singapore, SGP) .......... 76

Lee, Sui Mae (Monash University, Selangor, MYS) ............. 73

Lee, Tae Kwon (Department für Mikrobiologie und Ökosystemforschung, Wien, AUT) ............................... 12

Legner, M. (ZZZ) ........................................................... 171

LELEU, GUYLAINE (ANSES, BOULOGNE SUR MER, FRA) . 129

LEMBRE, Pierre (Université de Cergy-Pontoise, Cergy-Pontoise, FRA) ........................................................... 28

Lemkina, Larisa (Institute of Ecology and Genetics of Microorganisms UB RAS, Perm, RUS) .......... 45, 163, 185

Lemus, Mildred (Universidad de los Andes, Bogotá, COL) 141

Lett, M.C. (UMR 7156, Université de Strasbourg-CNRS, Laboratoire Génétique Moléculaire, Strasbourg, FRA) 141

Levi, Adi (Bar-Ilan University, Ramat Gan, ISR) ............... 150

Lewis, Gillian (University of Auckland, Auckland, NZL) ..... 80

Li, Chunyan (Kalrsruhe Institute of Technology, Karlsruhe, GER) ............................................................................ 6

Li, Xiaobao (Northwestern University, Evanston, USA) .... 133

Li, Yingbo (INRA, MIAJ, , Jouy en Josas, FRA) ...................... 3

Lièvremont, D. (UMR 7156, Université de Strasbourg-CNRS, Laboratoire Génétique Moléculaire, Strasbourg, FRA) 141

LISBP, Toulouse , FRA) .................................................. 120

LISBP, Toulouse, FRA) ....................................... 9, 120, 125

Liu, Li (Technical University of Denmark, Department of Environmental Engineering, Lyngby, DNK) .................... 8

Lloyd, Nadeau (Air Force Research Laboratory, Wright-Patterson AFB, OH, USA) .......................................... 161

LMDC, Toulouse , FRA) .................................................. 120

Loera Muro, Abraham (Universidad Autónoma de Aguascalientes, Aguascalientes, MEX) ...................... 158

Longyear, Jennifer (AkzoNobel, International Paint Ltd, Gateshead, GBR) ...................................................... 131

Lönn-Stensrud, Jessica (Department of Oral Biology, UNiversity of Oslo, Oslo, NOR) .................................. 107

Lorenzón, Esteban (Univ. Estadual Paulista - UNESP, Araraquara, BRA) ..................................................... 153

Low, David (Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, USA) ............................................................ 89

Luc, Joëlle (Pierre Fabre Dermo-Cosmétique - Microbiology Lab, R&D , Castanet tolosan, FRA) ............................. 184

lueders, tillmann (Helmholtz Zentrum München, Neuherberg, GER) ...................................................... 31

Lugão Lacerda, Géisica (Universidade do Estado do Rio de Janeiro - UERJ, Rio de Janeiro, BRA) ......................... 174

Lund Witsø, Ingun (Department of Oral Biology, University of Oslo, Oslo, NOR) ...................................................... 107

Lüscher, Marcel (GF IRG AG, Sissach, CHE) .................... 143

Lyautey, Emilie (University of Savoie, Le Bourget du Lac, FRA) .......................................................................... 75

M

MacLean, Jason (UMASS Dartmouth, North Dartmouth, USA) ............................................................................... 109

MAES, EMMANUEL (USTL UGSF, VILLENEUVE D'ASCQ, FRA) ................................................................................... 1

Mages, Margarete (Helmholtz Centre for Environmental Research UFZ, Magdeburg, GER) ................................. 76

Maierl, Mario (University of Applied Sciences, Graz, AUT) 166

Maillot, Emeline (Normandie Université, Université de Rouen, Laboratoire de Microbiologie Signaux et Micro-environnement EA 4312, Evreux, FRA) ......................... 95

Mair, R. (ZZZ) ............................................................... 171

Makarewicz, Oliwia (Universitätsklinikum Jena, Zentrum für Infektionsmedizin und Krankenhaushygiene, CSCC, Jena, GER) ........................................................................ 183

Maksimov, Aleksandr (Institute of Ecology and Genetics of Microorganisms UB RAS, Perm, RUS) .......................... 60

Maksimov, Aleksandr (INSTITUTE OF ECOLOGY AND GENETICS OF MICROORGANISMS UB RAS, Perm, RUS) ............................................................................... 136

Maksimova, Yulia (INSTITUTE OF ECOLOGY AND GENETICS OF MICROORGANISMS UB RAS, Perm, RUS) .............. 136

Maksimova, Yuliya (Institute of Ecology and Genetics of Microorganisms UB RAS, Perm, RUS) .......................... 60

Mandic-Mulec, Ines (University of Ljubljana, Biotechnical Faculty, Dept. Food Science and Technology, Chair of Microbiology, Ljubljana, SVN) ..................................... 46

Mann, Brian (University of Sheffield, Sheffield, GBR) ...... 124

Manner, Suvi (Åbo Akademi University, Turku) ............... 179

Manner, Suvi (Pharmaceutical Sciences, Department of Biosciences, Åbo Akademi University, Turku) ............. 175

Marediaa, Reshma (College of Science and Mathematics. Southwest Baptist University., Missouri , USA) .......... 164

Marney, Donavan (CSIRO, Highett, AUS) ........................ 114

Marquez-Diaz, Francisco (Centenario Hospital Miguel Hidalgo, Aguascalientes, MEX) ................................. 167

Marshall, Matthew (Pacific Northwest National Laboratory, Richland, WA, USA) .................................................... 14

Martin, Richard (University of Aston, Birmingham, GBR) . 159

Martinez, Juliana (Universidad de los Andes, Bogotá, COL) ............................................................................... 141

Marty, Aurelie (University of Toulouse - LGC - UMR 5503, Toulouse cedex 9, FRA) ............................................. 126

Masic, Alma (Eawag, Swiss Federal Institute of Aquatic Science and Technology, Duebendorf, CHE) ................. 48

Mastrorillo, Sylvain (University Paul Sabatier, Toulouse, FRA) .......................................................................... 75

Masuike, Ayano (Osaka Kyoiku University, Kashiwara, JPN) ............................................................................... 115

Mateiu, Ramona (DTU, Copenhagen, DNK) ....................... 35

Matthies, Kerstin (Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen, GER) ................................ 55

Mattos-Guaraldi, Ana Luíza (Universidade do Estado do Rio de Janeiro - UERJ, Rio de Janeiro, BRA) ............. 174, 184

Mayr, Magdalena (Department of Limnology and Bio-Oceanography, Wien, AUT) ....................................... 131

McDougald, Diane (Singapore Centre on Environmental Life Sciences Engineering, Singapore, SGP) ................. 22, 83

McElroy, Kerensa (CMB, Sydney, AUS) ............................. 22

Megson, Zoe Anne (Universität für Bodenkultur Wien, Department of NanoBiotechnology / NanoGlycobiology unit, Vienna, AUT) .................................................... 105

Meier, Thomas (DVGW-Forschungsstelle TUHH, Hamburg, GER) ........................................................................ 146

Melaugh, Gavin (Univeristy of Edinburgh, Edinburgh , GBR) ............................................................................... 130

Melo, Luís (FEUP - Faculty of Engineering Univ. Porto, Porto) ............................................................................... 182

Mergulhão, Filipe (FEUP - Faculty of Engineering Univ. Porto, Porto) ............................................................ 182

Messner, Paul (Universität für Bodenkultur Wien, Department of NanoBiotechnology / NanoGlycobiology unit, Vienna, AUT) .................................................... 105

Meyer, Rikke (Aarhus University, Aarhus C, DNK) ............... 2

Meyer, Rikke L. (iNANO, Aarhus University, Aarhus, DNK) . 52

Meysman, Filip (Department of Ecosystem Studies, Royal Netherlands Institute for Sea Research (NIOZ), Yerseke) ............................................................................... 136

Mhatre, Eisha (Terrestrial Biofilms Group, Jena, GER) ..... 103

MICHEL, Caroline (BRGM, Orléans, FRA) ........................ 132

Mikshina, Polina (Kazan Institute of Biochemistry and Biophysics RAS, Kazan, RUS) ...................................... 59

Milferstedt, Kim (Laboratoire de Biotechnologie de l'Environnement, Narbonne, FRA) ............................. 121

Miranda, João (FEUP - Faculty of Engineering Univ. Porto, Porto) ...................................................................... 182

Mocca, Brian (US Food and Drug Administration, Bethesda, USA) ....................................................................... 106

Monnet, Véronique (INRA, PAPPSO, Unité de Biochimie Bactérienne, UR477, Jouy-en-Josas, FRA) ................... 95

Mooshammer, Maria (University of Vienna, Vienna, AUT) .. 74

Moreira, Joana (FEUP - Faculty of Engineering Univ. Porto, Porto) ...................................................................... 182

Møretrø, Trond (The Norwegian Institute of Food, Fishery and Aquaculture Research , Aas, NOR) ........................ 88

Morgenroth, Eberhard (ETH Zurich IfU Chair of Process Engineering in Urban Water Management / Eawag Process Engineering, Zurich, CHE) ............................ 128

Morin, Emilie (Laboratoire de Biotechnologie de l'Environnement, Narbonne, FRA) ............................... 25

Morozov, Ilia (Institute of Continuous Media Mechanics UB RAS, Perm, RUS) ................................................ 45, 185

Moshynets, Olena (Institute of Molecular Biology and Genetics of National Academy of Sciences of Ukraine, Kiev, UKR) ................................................................. 46

Muffler, Kai (Institute of Bioprocess Engineering, University of Kaiserslautern,, Kaiserslautern, GER) ...................... 27

Mukherjee, Manisha (SCELSE, Singapore, SGP) ................ 22

Mulansky, Susan (Dresden University of Technology, Dresden, GER) ........................................................... 57

Müller, Elisabeth (Technische Universität München, Garching, GER) ........................................................ 122

Müller, Torsten (JPK Instruments AG, Berlin, GER) ........... 52

Müller, Torsten (JPK Instruments AG, Berlin, GER) ........... 66

Müller-Renno, Christine (University of Kaiserslautern, Kaiserslautern, GER) .................................................. 27

Murray, Justine (University of Texas at Austin, AUSTIN, USA) ............................................................................... 163

Musovic, Sanin (DTU, Copenhagen, DNK) ......................... 35

N

Nadell, Carey (Princeton University, Princeton, USA) ........ 15

Nance, William C. (Department of Epidemiology, School of Public Health, University of Michigan, Michigan, USA) . 10

Napoleão, Fátima (Universidade do Estado do Rio de Janeiro - UERJ, Rio de Janeiro, BRA) ..................................... 184

Narendrakumar, Krunal (University of Birmingham, Birmingham, GBR) ................................................... 159

Neff, Laura (Empa, St. Gallen, CHE) ................................. 67

Neou, Sereiwaddhana (GlaxoSmithKline, Rixensart, BEL) .. 57

Nesatyy, Victor (Singapore Centre on Environmental Life Sciences Engineering (SCELSE), Singapore, SGP) ...... 101

Neto, Catherine (UMASS Dartmouth, North Dartmouth, AUT) ............................................................................... 109

Neu, Thomas (Helmholtz Center for Environmental Research - UFZ, Magdeburg, GER) ..................................... 58, 123

Neu, Thomas (Helmholtz Centre for Environmental Research - UFZ, Magdeburg, GER) ............................................. 90

Ng, Chun Kiat (Singapore Centre on Environmental Life Sciences Engineering, Singapore, SGP) ....................... 41

Ng, Wei Ling (SCELSE, Singapore, SGP) ......................... 101

Nguyen, Cam Phuong (Institute of Chemistry and Biological Chemistry, ZHAW, Wädenswil, CHE) ......................... 143

Nguyen, Uyen (McMaster University, Hamilton, CAN) ....... 99

Niederdorfer, Robert (Department for Limnolgy and Bio-Oceanography, University of Vienna, Vienna, AUT) ....... 68

Nierop Groot, Masja (Topp Institute Food and Nutrition, Wageningen) ..................................................... 20, 104

Nigaud, Yohan (UMR6270 CNRS, Mont Saint Aignan, AUT) 17

Nitschke, J. (BTU Cottbus , Senftenberg , GER) ................. 65

Nomura, Nobuhiko (University of Tsukuba, Tsukuba, JPN) . 67

Norf, Helge (Helmholtz Zentrum für Umweltforschung UFZ, Magdeburg, GER) ....................................................... 78

Norf, Helge (Helmholtz-Centre for Environmental Research - UFZ, Magdeburg, GER) ............................................... 59

O

Oberschmidt, Dirk (Fraunhofer Institut für Produktionsanlagen und Konstruktionstechnik / Institut für Werkzeugmaschinen und Fabrikbetrieb, Technische Universität Berlin, Berlin, GER) ................................. 177

Obst, Ursula (Institute of Functional Interfaces, Department of Interface Microbiology, Eggenstein-Leopoldshafen, GER) .......................................................................... 55

Obszanska, Katarzyna (Institute of Biochemistry and Biophysics PAS, Warsaw, POL) .................................... 94

Oja, Terhi (Åbo Akademi University, Turku) ..................... 179

Ojima, Yoshihiro (Osaka university, Toyonaka, JPN) .......... 40

Okshevsky, Mira (Aarhus University, Aarhus C, DNK) .......... 2

Oleneva, Mariya (Perm State National Research University, Perm, RUS) ................................................................ 60

Oliveira Moreira, Lílian (Universidade Federal do Rio de Janeiro - UFRJ, Rio de Janeiro, BRA) ......................... 184

Orgaz, Belén (Univ. Complutense of Madrid, Madrid, ESP) . 92

Oropeza Navarro, Ricardo (Instituto de Biotecnología, UNAM, Cuernavaca, MEX) ........................................ 158

Osorgina, Irina (School of Physics, University of Sidney , Sidney, AUS) ............................................................ 185

OUIDIR, Tassadit (UMR 6270, Polymères, Biopolymères, Surfaces Laboratory, Mont Saint Aignan, FRA) ............. 94

Overney, Anaïs (Laboratory of Food Safety, French Agency for Food, Environmental and occupational Health Safety (Anses), Maisons-Alfort, FRA) ................................... 178

P

Packman, Aaron (Northwestern University, Evanston, USA) ............................................................................... 133

Pareniuk, Olena (Ukrainean Institute of Agricaltural Radiobiology, Chabany, UKR) .................................... 156

Parsek, Matthew (University of Washington, School of Medicine, Seattle, USA) ........................................ 37, 82

Paul, Etienne (Université de Toulouse ........................ 9, 125

Pavlova, Yulia (Perm State National Research University, Perm, RUS) .............................................................. 136

Pechaud, Yoan (Université de Toulouse ...................... 9, 125

Pecheva, Emelia (University of Birmingham, Birmingham, GBR) ....................................................................... 172

Pecheva, Emilia (University of Birmingham, Birmingham, GBR) ......................................................................... 70

Peduzzi, Peter (Department of Limnology and Bio-Oceanography, Wien, AUT) ....................................... 131

Pereira, Leonel (Minho University, Departement of Biological Engineering Department, Braga ) .............................. 180

Peres, Martina (GMGM - UMR 7156, Strasbourg, FRA)....... 87

Perez, Alma (Universidad de los Andes, Bogotá, COL) ..... 141

Periasamy, Saravanan (SCELSE, Singapore, SGP) ........... 101

Petrackova, Denisa (Institute of Microbiology AS CR, v.v.i., Prague 4, CZE) ........................................................... 97

Petrácková, Denisa (Institute of Microbiology v.v.i., Prague 4, CZE) ........................................................................ 100

Petrova, Olga (Binghamton University, Binghamton, USA) 22

Petrova, Olga (Kazan Institute of Biochemistry and Biophysics RAS, Kazan, RUS) ...................................... 59

Pettersson, Carmen (JPK Instruments AG, Berlin, GER) ..... 66

Peyre Lavigne, M. (Université de Toulouse .............. 120, 125

Pfeifer, Felicitas (Technische Universität Darmstadt, Darmstadt, GER) ........................................................ 38

Phipps, Kara (College of Science and Mathematics. Southwest Baptist University., Missouri , USA) .......... 164

Piard, Jean-Christophe (INRA, Massy, FRA) .................. 3, 54

Pietrasanta, Lía (Centro de Microscopías Avanzadas, Universidad de Buenos Aires, Buenos Aires, ARG) ........ 43

Pillen, Alexa Margareta (University Duisburg-Essen, Essen, GER) ........................................................................ 148

Pirlo, Russell (US Naval Research Laboratory, Washington, DC, USA) ................................................................. 161

Pitts, Betsey (Center for Biofilm Engineering, Bozeman Montana, USA)........................................................... 69

Pletz, Mathias W. (Universitätsklinikum Jena, Zentrum für Infektionsmedizin und Krankenhaushygiene, CSCC, Jena, GER) ........................................................................ 183

Poberaj, Igor (niversity of Ljubljana, lj, SVN) ...................... 3

Pokholenko, Ianina (Institute of Molecular Biology and Genetics of National Academy of Sciences of Ukraine, Kiev, UKR) ................................................................. 46

Poole, Robert (Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, GBR) ............................................................................... 124

Porta, Sonia (ainia, Valencia, ESP) ................................... 69

Poulsen, Nicole (TU Dresden, Dresden, GER) .................... 93

Price, Gareth (University of Bath, Bath, GBR).................. 172

Prommer, Judith (University of Vienna, Wien, AUT) ........... 74

Q

Qian, Pei-Yuan (Hong Kong University of Science and Technology, Hong Kong, HKG) .................................... 77

Queinnec, Isabelle (CNRS .......................................... 9, 125

Quince, Christopher (School of Engineering, University of Glasgow, Glasgow, GBR) ............................................ 92

R

R. Lokanathan, Arcot (Aalto University, Aalto) .................. 24

R. Regina, Viduthalai (Aarhus University, Aarhus, DNK) .... 24

Rahman, Tamanna (University of Nottingham, Nottingham, NFK) ........................................................................ 156

Ramaniuk, Olga (Institute of Microbiology AS CR, v.v.i., Prague 4, CZE) ........................................................... 97

Ramirez, Flor (Universidad Autónoma de Aguascalientes, Aguascalientes, MEX)............................................... 167

Ramos, Juliana (Universidade do Estado do Rio de Janeiro - UERJ, Rio de Janeiro, BRA) ....................................... 184

Rapp, Bastian E. (Karlsruher Institute of Technology (KIT), Eggenstein-Leopoldshafen, GER) ................................ 11

Raspaud, Eric (CNRS, Orsay, FRA) .................................. 116

Reisner, Andreas (University of Applied Sciences, Graz, AUT) ............................................................................... 166

Ren, Qun (Empa, St. Gallen, CHE) ...................... 53, 67, 124

Reuter, Mark (Institute of Food Research, Norwich, GBR) . 40,

114

Rice, Scott (SCELSE, Singapore, SGP) ...................... 55, 101

Rice, Scott (SCELSE, Singapore, SGP) .............................. 22

Rice, Scott (The University of New South Wales, Sydney, AUS) ........................................................................ 112

Richard, Jessica (IWW, Water Centre, Mülheim a. d. Ruhr, GER) ........................................................................ 139

Rickard, Alexander H. (Department of Epidemiology, School of Public Health, University of Michigan, Michigan, USA) ................................................................................. 10

Rickard, Alexander H. (The University of Michigan, School of Public Health, Department of Epidemiology, Ann Arbor, USA) .......................................................................... 88

Riedel, Christian (University of Ulm, Ulm, GER) .............. 110

Ritschdorff, Eric (The University of Texas at Austin, Austin, TX, USA) .................................................................... 13

Ritschdorff, Eric T. (ZZZ) ................................................. 63

Rmaile, Amir (Philips Research, Eindhoven) ................... 170

Rocha, Rui (LEPABE, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto) ....... 61

Rödiger, S. (BTU Cottbus , Senftenberg, GER) ................... 65

Rodrigues Frade, Pedro (Department für Limnologie und Ozeanographie, AUT) .................................................. 30

Rodrigues, Célia (Minho University, Departement of Biological Engineering Department, Braga ) ................ 44

Rodriguez, Manuel (Universidad de los Andes, Bogotá, COL) ............................................................................... 141

Rogalsky, Sergey (Institute of Bioorganic Chemistry and Petrochemistry of National Academy of Sciences of Ukraine, Kiev, UKR) .................................................... 46

Rolland, Sarah (Newcastle University, Newcastle upon Tyne, IOT) ......................................................................... 186

Romaní, Anna M. (University of Girona, Insitute of Aquatic Ecology, Girona, ESP) ................................................. 85

ROOS, Christophe (UMR ECOFOG, laboratoire L3MA, Cayenne, GUF) ......................................................... 118

Roques, Christine (Faculty of Pharmacy - University Paul Sabatier - LGC - UMR 5503, Toulouse, FRA) ............... 184

Roques, Christine (Laboratoire de Génie Chimique (UMR 5503), Université Paul Sabatier, Université de Toulouse, Toulouse cedex 9, FRA) ..................................... 126, 157

Rösch, Christina (University of Kaiserslautern, Department of Physics, Kaiserslautern, GER) ................................. 27

Rubstein, Anna (Institute of Metal Physics UB RAS, Ekaterinburg, RUS) .................................................. 163

Ruhe, Zachary (Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, USA) ............................................................ 89

Rulik, Martin (Faculty of Science, Palacky University in Olomouc, Olomouc, CZE) .......................................... 137

Rulík, Martin (Faculty of Science, Palacky University, Olomouc, CZE) ........................................................... 96

Rumbaugh, Kendra (Department of Surgery, Texas Tech University Health Sciences Center, USA) 16, 18, 160, 173

Russell, John (US Naval Research Laboratory, Washington, DC, USA).................................................................. 161

Russin, William (Northwestern University, Evanston, USA) ............................................................................... 133

Russo, Daniela (Laboratorio de Microbiología Molecular y Celular, Fundación Instituto Leloir, Buenos Aires, ARG) 43

Rutschmann, Peter (Technische Universität München, München, GER) ........................................................ 122

S

S. Sutherlan, Duncan (Aarhus University, Aarhus, DNK) .... 24

Sabater, Sergi (Catalan Institute for Water Research and University of Girona, Girona, ESP) ............................... 79

Sablé, Sophie (LIENSs Université de La Rochelle, La Rochelle cedex 01, FRA) ........................................................... 84

Sachsenheimer, Kai (Karlsruher Institute of Technology (KIT), Eggenstein-Leopoldshafen, GER) ....................... 11

SADOVSKAYA, IRINA (ULCO, BOULOGNE SUR MER, FRA) .. 1,

37

Safari, Ashkan (School of Chemical and Bioprocess Engineering, University College Dublin, Dublin) .......... 34

Sakai, Ryosuke (University of Tsukuba, Tsukuba, JPN) ...... 67

Salnikov, Vadim (Kazan Institute of Biochemistry and Biophysics RAS, Kazan, RUS) ...................................... 59

SALVIN, Paule (UMR ECOFOG, laboratoire L3MA, Cayenne, GUF)........................................................................ 118

Samarian, Derek (Department of Epidemiology, School of Public Health, University of Michigan, Michigan, USA) . 10

Sambanthamoorthy, Karthik (Walter reed army institute of research, Silver Spring, USA) .................................... 166

Sammons, Rachel (University of Birmingham, Birmingham, GBR) ......................................................... 70, 159, 172

SANCHEZ-VIZUETE, Pilar (INRA, Massy, FRA) .................. 20

Sanchez-Vizuette, pilar (inra, Massy, FRA) ....................... 54

SanJosé, Carmen (Univ. Complutense of Madrid, Madrid, ESP) .......................................................................... 92

Santa-Catalina, Gaëlle (Laboratoire de Biotechnologie de l'Environnement, Narbonne, FRA) ............................. 121

Santigli, Elisabeth (Medical University of Graz, Graz, AUT) ............................................................................... 173

Santos, Rita S. (LEPABE, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto) ........................................................................ 61

Sauer, Karin (Binghamton University, Binghamton, USA) .. 22

Saur, Thibaut (INRA - LBE, NARBONNE, FRA) ........... 25, 121

Schäffer, Christina (Universität für Bodenkultur Wien, Department of NanoBiotechnology / NanoGlycobiology unit, Vienna, AUT) .................................................... 105

Schaule, Gabriela (IWW, Water Centre, Mülheim a. d. Ruhr, GER) ........................................................................ 145

Scheffers, Lucas (Philips Research, Eindhoven) .............. 165

Scherwass, Anja (University of Cologne, Biocenter, General Ecology, Cologne, GER) .............................................. 91

Schiebel, Juliane (BTU Cottbus , Senftenberg, GER) .......... 65

Schierack, P. (BTU Cottbus , Senftenberg, GER) ................ 65

Schlegel, Christin (Institute of Bioprocess Engineering, University of Kaiserslautern, Kaiserslautern, GER) ....... 27

Schleper, Christa (Archaea Biology and Ecogenomics Division, Department of Ecogenomics and Systems Biology, Vienna, AUT) ............................................... 108

Schmid, Markus (Department für Mikrobiologie und Ökosystemforschung, Wien, AUT) ............................... 12

Schmidt, Holger (Institute of Modelling Water and Environmental Systems, Stuttgarg, GER) ................... 127

Schmidt-Emrich, Sabrina (Empa, St. Gallen, CHE) .......... 124

Schröder, Josephin (TU Berlin, Berlin, GER) ................... 151

Schroeder, Josephin (Technische Universität Berlin, Berlin, GER) ........................................................................ 145

Schulz, Frederik (KWR Watercycle Research Institute, Nieuwegein) .............................................................. 32

Schuster, Heinrich (Universität für Bodenkultur Wien, Department of NanoBiotechnology / NanoGlycobiology unit, Vienna, AUT) .................................................... 105

Schwab, Clarissa (University of Vienna, Vienna, AUT)........ 19

Schwartz, Thomas (Karlsruher Institute of Technology (KIT), Eggenstein-Leopoldshafen, GER) ................................ 11

Schwarzwälder, Kordula (Technische Universität München, München, GER) ........................................................ 122

Seabra, Sérgio Henrique (Centro Universitário Estadual da Zona Oeste - UEZO, Rio de Janeiro, BRA) ........... 174, 184

Sebaa, Sarra (Université Libre de Bruxelles, Brussels, BEL) ............................................................................... 181

Sedlag, Anne (University of Ulm, Ulm, GER) ................... 110

Semião, Andrea J.C. (School of Engineering, The University of Edinburgh, Edinburgh, GBR) ........................... 34, 140

Seviour, Thomas (Singapore Centre for Environmental Life Sciences Engineering, Singapore, AUS) ....................... 39

Shardakov, Igor (Institute of Continuous Media Mechanics UB RAS, Perm, RUS) ................................................. 185

Sharifzadeh, Shahab (Nofima, the Norwegian Institute of Food, Fisheries and Aquaculture Research, Aas, NOR) .. 88

Shear, Jason (The University of Texas at Austin, Austin, TX, USA) .......................................................................... 13

Shear, Jason B. (ZZZ) ...................................................... 63

Shiojima, Yutaro (University of Tsukuba, Tsukuba, JPN) .... 67

Siebenhofer, David (University of Applied Sciences, Graz, AUT) ........................................................................ 166

Siebers, Bettina (University of Duisburg-Essen, Essen, GER) ............................................................................... 119

Sieczko, Anna (Department für Limnologie und Ozeanographie, AUT) ................................................ 131

Sieczko, Anna (University of Vienna, Department of Limnology and Oceanography , Vienna, AUT) ............. 100

Silby, Mark (UMASS Dartmouth, North Dartmouth, USA) 109

Silva Santos, Cíntia (Universidade do Estado do Rio de Janeiro - UERJ, Rio de Janeiro, BRA) ......................... 184

Silva, Sónia (Minho University, Departement of Biological Engineering Department, Braga ) ................................ 44

Silva, Sónia (Minho University, Departement of Biological Engineering Department, Braga ) .............................. 180

Simões, Manuel (FEUP - Faculty of Engineering Univ. Porto, Porto) ...................................................................... 182

Simpson-Louredo, Liliane (Fundação Oswaldo Cruz - Fiocruz, Rio de Janeiro, BRA) ................................................. 184

Singer, Gabriel (Abteilung für Ökohydrologie, Leibniz-Institut für Gewässerökologie und Binnenfischerei, Berlin, GER) ............................................................... 92

Sitarová, Barbora (Institute of Microbiology v.v.i., Prague 4, CZE) ........................................................................ 100

Skogman, Malena ( Pharmaceutical Sciences, Department of Biosciences, Abo Akademi University, Turku) ............. 175

Skrobot, Joanna (Institute of Biochemistry and Biophysics PAS, Warsaw, POL) ..................................................... 94

Smets, Bart F. (Department of Environmental Engineering, Kgs.Lyngby, DNK)................................................... 8, 35

Smid, Eddy (Top Institute Food and Nutrition, Wageningen) ............................................................................... 104

Sochorova, Zuzana (Institute of Microbiology v.v.i., Prague 4, CZE) ........................................................................ 100

Sofka, Dmitri (University of Veterinary Medicine Vienna, Vienna, AUT) .............................................................. 80

Solodko, Vladislav (Institute of Continuous Media Mechanics UB RAS, Perm, RUS) ................................................. 185

Sommer, Regina (Medical University Vienna, Institute for Hygiene and Applied Immunology, Vienna, AUT) .......... 33

Souza, Cassius (Universidade do Estado do Rio de Janeiro - UERJ, Rio de Janeiro, BRA) ....................................... 184

Souza, Monica Cristina (Universidade do Estado do Rio de Janeiro - UERJ, Rio de Janeiro, BRA) ......................... 184

Spark, Amy (CSIRO, Caulfield North, AUS) ...................... 114

Spiers, Andrew (SIMBIOS Centre and School of Science, Engineering and Technology, Dundee, GBR) .............. 156

Spiers, Andrew (SIMBIOS Centre, Dundee, GBR) ............... 74

Spiers, Andrew (The SIMBIOS Centre & School of Science, Engineering and Technology, Abertay University, Dundee, GBR) ......................................................................... 46

Sretenovic, Simon (University of Ljubljana, Ljubljana, , SVN) ................................................................................... 3

Stacy, Apollo (The University of Texas at Austin, Austin, TX, USA) ......................................................................... 16

Stahl, Ulf (Fachgebiet Mikrobiologie und Genetik, Institut für Biotechnologie, Technische Universität Berlin, Berlin, GER) ........................................................................ 177

Stamov, Dimita (JPK Instruments AG, Berlin, GER) ........... 66

Steiner, Nora (Fraunhofer Institut für Produktionsanlagen und Konstruktionstechnik, Berlin, GER) .................... 177

Stewart, Phil (Center for Biofilm Engineering, Bozeman, MT, USA) ........................................................................... 5

Stiefel, Philipp (Empa, St. Gallen, CHE) ............................ 53

Stojkovic, Biljana (University of Ljubljana, Ljubljana, , SVN) 3

Stone, Howard (Princeton University, Princeton, USA) ...... 15

Stoodley, Paul (Department of Microbial Infection and Immunity, Department of Orthopaedics, The Ohio State College of Medicine , Columbus OH, USA) ................. 170

Stopar, David (University of Ljubljana, Biotechnical Faculty, Dept. Food Science and Technology, Chair of Microbiology, Ljubljana, SVN) ..................................... 46

Stopar, David (University of Ljubljana, Ljubljana, SVN) ....... 3

Sun, Shuyang (Singapore Centre on Environmental Life Sciences Engineering, Singapore, SGP) ....................... 83

Swartjes, Jan (University medical center groningen, Groningen) .............................................................. 126

Swarup, Sanjay (SCELSE, Singapore, SGP) ..................... 101

Swift, Dean (Biolennia, Toronto, CAN) ............................ 171

Szewzyk, Ulrich (TU Berlin, Berlin, GER) ........................ 151

Szostak, Michael (University of Veterinary Medicine Vienna, Vienna, AUT) .............................................................. 80

T

Tambosco, Jennifer (BRGM, Orléans, FRA) ..................... 132

Tarasova, Nadezhda (Kazan Institute of Biochemistry and Biophysics RAS, Kazan, RUS) ...................................... 59

Tatari, Karolina (Denmark Technical university, Copenhagen, DNK) ......................................................................... 35

Tateda, Kazuhiro (Toho University School of Medicine, Tokyo, JPN) .......................................................................... 67

Tay, Martin (Singapore Centre on Environmental Life Sciences Engineering, Singapore, SGP) ....................... 83

Teh, Amy Huei Teen (Monash University, Selangor, MYS) .. 73

Tempelaars, Marcel (Wageningen University, Wageningen) ................................................................................. 20

Teubner, Irene (Department of Limnology and Bio-Oceanography, Wien, AUT) ....................................... 131

Thom, Moritz (Institute of Modelling Water and Environmental Systems, Stuttgarg, GER) ................... 127

Thomas, Torsten (CMB, sydney, AUS) ............................... 22

Thöny-Meyer, Linda (Empa, St. Gallen, CHE) ...... 53, 67, 124

Timoner, Xisca (Catalan Institute for Water Research and University of Girona, Girona, ESP) ............................... 79

Tomic, Matija (University of Ljubljana, Ljubljana, , SVN) ..... 3

Tomczyk-Zak, Karolina (Institute of Biochemistry and Biophysics PAS, Warsaw, POL) .................................... 94

Tony, Worthington (University of Aston, Birmingham, GBR) ............................................................................... 159

Touron - Bodilis, Aurélie (EDF R&D, Chatou, AUT) ............. 75

Toyofuku, Masanori (University of Tsukuba, Tsukuba, JPN) 67

Trakhtenberg, Ilia (Institute of Metal Physics UB RAS, Ekaterinburg, RUS) .................................................. 163

Trampe, Erik (Marine Biological Section, University of Copenhagen, Helsingor, DNK) ................................... 137

Trautner, Barbara (Baylor College of Medicine, Houston, USA) ........................................................................ 162

Tremblay, Yannick D.N. (Université de Montréal, Saint Hyacinthe, CAN)....................................................... 158

Trivedi, Urvish (Department of Surgery, Texas Tech University University Health Sciences Center, USA) ... 173

Trivedi, Urvish (Texas Tech University Health Sciences Center, Lubbock, TX, USA) .......................................... 16

Troszo, Agnieszka (Molecular Genetics, Groningen Bio-molecular Sciences and Biotechnology Institute, Groningen) .............................................................. 103

Trotier, Elsa (INRA, Massy, FRA) ...................................... 54

Trubuil, Alain ( INRA, , Jouy en Josas, FRA) ......................... 3

Tseng, Boo Shan (University of Washington School of Medicine, Seattle, USA) .............................................. 37

Turner, Keith (The University of Texas at Austin, Austin, TX, USA) .......................................................................... 18

Turner, Keith H. (Department of Molecular Biosciences, The Univ. of Texas at Austin, Austin, USA) ........................ 160

U

Udall, Yvette (SIMBIOS Centre, Dundee, GBR) .................. 74

Ulber, Roland (Institute of Bioprocess Engineering, University of Kaiserslautern, Kaiserslautern, GER) ....... 27

Umanskaya, Natalia (University of the Saarland, Department of Operative Dentistry and Periodontology, Homburg, GER) .......................................................................... 27

Urich, Tim (University of Vienna, Vienna, AUT) .................. 19

V

Valen Rukke, Håkon (Nordic Institute of Dental Materials, Oslo, NOR) ............................................................... 107

Valvano, Miguel (Queen's University Belfast, Centre for Infection and Immunity, Belfast, GBR) ......................... 98

van Bijsterveldt, Celine (Department of Ecosystem Studies, Royal Netherlands Institute for Sea Research (NIOZ), Yerseke) .................................................................. 136

van de Ven, Cindy (Philips Research, Eindhoven) ............ 165

van der Mei, Henny (University Medical Center Groningen , Groningen) .............................................................. 153

Van der Mei, Henny (University Medical Center Groningen, Groningen) ................................................................ 48

van der Woude, Marjan (Centre for Immunology and Infection, Hull York Medical School and the Department of Biology, University of York, York, GBR) .................... 89

van Gestel, Jordi (University of Groningen, Theoretical Biology Group, Groningen).......................................... 72

van Vliet, Arnoud (Institute of Food Research, Norwich, GBR) ......................................................................... 40, 114

Vasiliev, Dmitriy (INSTITUTE OF ECOLOGY AND GENETICS OF MICROORGANISMS UB RAS, Perm, RUS) .............. 136

Vastra, Margaux (Laboratoire des matériaux et molécules en milieux amazonien, Université des Antilles et de la Guyane, UAG-UMR ECOFOG, Cayenne, GUF).............. 118

Vaudry, David (U982 INSERM - PISSARO Proteomics Facility, Mont Saint Aignan, FRA) ............................................. 17

Vendrely, Charlotte (Université de Cergy-Pontoise, Cergy-Pontoise, FRA) ........................................................... 28

Veuger, Bart (Royal Netherlands Institute for Sea Research, Yerseke) .................................................................... 74

Viancha, Valerie (Universidad de los Andes, Bogotá, COL)141

Vianna Vieira, Verônica (Fundação Oswaldo Cruz - Fiocruz, Rio de Janeiro, BRA) ................................................. 184

Vieira Faria, Yuri (Centro Universitário Estadual da Zona Oeste - UEZO, Rio de Janeiro, BRA) ........................... 184

Vieira Faria, Yuri (Universidade do Estado do Rio de Janeiro - UERJ, Rio de Janeiro, BRA) ....................................... 174

von Bilderling, Catalina (Centro de Microscopías Avanzadas, Universidad de Buenos Aires, Buenos Aires, ARG) ........ 43

Vozza, Nicolás (Laboratorio de Microbiología Molecular y Celular, Fundación Instituto Leloir, Buenos Aires, ARG) 43

Vuorela, Pia (Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Helsinki) 175

Vuorela, Pia (University of Helsinki, Helsinki) ................. 179

Vyas, Nina (University of Birmingham, Birmingham, GBR) . 70

Vyas, Nina (Universty of Birmingham, Birmingham, GBR) 172

W

Wagner, Janine (Biofilm Centre, University of Duisburg-Essen, Essen, GER) ................................................... 145

Wagner, Karoline (University of Vienna, Department of Limnology and Oceanography , Vienna, AUT) ............. 100

Wagner, Karoline (University of Vienna, Vienna, AUT) ....... 19

Wagner, Michael (Chair of Water Chemistry and Water Technolog, Engler-Bunte-Institut, Karlsruhe Institute of Technology, Karlsruhe, GER) .................................... 149

Wagner, Michael (Department für Mikrobiologie und Ökosystemforschung, Wien, AUT) ............................... 12

Wagner, Michael (Karlsruhe Institute of Technology - Chair of Water Chemistry and Water Technology, Karlsruhe, GER) .......................................................................... 26

Wagner, Michael (Karlsruhe Institute of Technology, Kalrsruhe, GER) ........................................................... 6

Walker, John E. (The Medical Research Council Mitochondrial Biology Unit, Cambridge , GBR) ............. 98

Walmsley, Damien (Univeristy of Birmingham, Birmingham, GBR) ....................................................................... 172

Walmsley, Damien (University of Birmingham, Birmingham, GBR) ......................................................................... 70

Walters, Evelyn (Temple University, Philadelphia, PA, USA) ............................................................................... 122

Wanek, Wolfgang (University of Vienna, Vienna, AUT) ...... 74

Wang, Wei (US Food and Drug Administration, Bethesda, USA) ....................................................................... 106

Wang, Xiaoxue (South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, CHN) ....... 111

Wang, Yong (Hong Kong University of Science and Technology, Hong Kong, HKG) .................................... 77

Ward, Liam (RMIT University, Melbourne, AUS) .............. 114

Ward, Marilyn (Philips Oral Healthcare, Bothell, WA, USA) ............................................................................... 170

Weerachanchai, Piyarat (Nanyang Technological University, Singapore, SGP) ......................................................... 39

Wei, Tao (College of Science and Mathematics. Southwest Baptist University., Missouri , USA) ........................... 164

Wei, Xiao-Qing (Cardiff University,Tissue Engineering & Reparative Dentistry, Cardiff) ................................... 180

Weinreich, J. (BTU Cottbus , Senftenberg, GER) ................ 65

Weiser, Jaroslav (Institute of Microbiology AS CR, v.v.i., Prague 4, CZE) ........................................................... 97

Weiser, Jaroslav (Institute of Microbiology v.v.i., Prague 4, CZE) ........................................................................ 100

Weissbrodt, David G. (ETH Zürich, Zürich, CHE) ................ 90

Weissing, Franz J (University of Groningen, Theoretical Biology Group, Groningen) ......................................... 72

Weitere, Markus (Helmholtz Zentrum für Umweltforschung UFZ, Magdeburg, GER) ......................................... 59, 78

West, Stephanie (Chair of Water Chemistry and Water Technolog, Engler-Bunte-Institut, Karlsruhe Institute of Technology, Karlsruhe, GER) .................................... 149

Westendorf, Christian (University of Graz, Graz, AUT) ..... 173

Whiteley, Marvin (The University of Texas at Austin, Austin, TX, USA) ................................... 13, 16, 18, 63, 160, 173

Whiteley, Marvin (Univ. of Texas at Austin, Cedar Park, USA) ................................................................................. 18

Wieprecht, Sikle (Institute of Modelling Water and Environmental Systems, stu, GER) ............................ 127

Williams, David W (Cardiff University,Tissue Engineering & Reparative Dentistry, Cardiff, GBR) ........................... 180

Wimmer, Gernot (Medical University of Graz, Graz, AUT) . 173

Wingender, Jost (Biofilm Centre, University of Duisburg-Essen, Essen, GER) ........................................... 139, 145

Wingender, Jost (University of Duisburg-Essen, Essen, GER) ................................................................. 53, 119, 184

Wingreen, Ned (Princeton University, Princeton, USA) ...... 15

Woebken, Dagmar (University of Vienna, Division of Microbial Ecology, Vienna, AUT) ................................. 30

Wohland, Thorsten (NUS Centre for Bioimaging Sciences (CBIS), Singapore, SGP) .............................................. 55

Wolfaardt, Gideon (Ryerson University, Toronto, CAN) ..... 62,

168

Wolter, Daniel (University of Washington, School of Medicine, Seattle, USA) .............................................. 82

Woo, Yissue (SCELSE, Singapore, SGP) ........................... 101

Wu, Dongni (Université de Cergy-Pontoise, Cergy-Pontoise, FRA) ........................................................................ 155

Wu, Xueqing (Utrecht University, Utrecht, AUT) ................ 81

Y

yahaya, sylla (institut pasteur, Abidjan, CIV) ................... 169

Yang, Rebekah Jiayue (University of Tsukuba, Tsukuba, JPN) ................................................................................. 67

Yawata, Yutaka (University of Tsukuba, Tsukuba, JPN) ...... 67

Yin, Dandan (US Food and Drug Administration, Bethesda, USA) ........................................................................ 106

Ylla, Irene (Institute of Aquatic Ecology, University of Girona, Girona, ESP) .............................................................. 85

Ysebaert, Carine (GlaxoSmithKline, Rixensart, BEL) .......... 57

Z

Zechner, Ellen (University of Graz, Graz, AUT) ................ 166

Zelaya, Anna (Montana State University, Bozeman,, USA) . 87

Zeng, Guanghong (Aarhus University, Aarhus C, DNK) ........ 2

Zeng, Guanghong (iNANO, Aarhus University, Aarhus, DNK) ................................................................................. 52

Zetsche, Eva-Maria (Laboratory for Analytical, Environmental and Geo-Chemistry, Vrije Universiteit Brussel (VUB), Brussels, BEL) ................................... 136

Zetzmann, Marion (University of Ulm, Ulm, GER) ............ 110

Zhang, Weipeng (Hong Kong University of Science and Technology, Hong Kong, HKG) .................................... 77

Ziegler, Christiane (University of Kaiserslautern, Department of Physics, Kaiserslautern, GER) ................................. 27

Ziegler, Sibylle (Karlsruhe Institute of Technology, Karlsruhe, GER) ....................................................... 134

Zielenkiewicz, Urszula (Institute of Biochemistry and Biophysics PAS, Warsaw, POL) .................................... 94

Zingarelli, Sandra (Air Force Research Laboratory, Wright-Patterson AFB, OH, USA) .......................................... 161

Zodrow, Katherine R (Department of Chemical and Environmental Engineering, New Haven, USA) ............ 34

Zorreguieta, Angeles (Laboratorio de Microbiología Molecular y Celular, Fundación Instituto Leloir, Buenos Aires, ARG) ................................................................ 43

Vienna is supplied with natural spring water. In case of repairs of the spring water mains, major pipe leaks or extremely high water consumption during hot spells, groundwater is additionally fed into the pipe network.

Directly into the city without pumping stations Via underground pipelines that partly cut through rock, the spring water reaches Vienna powered solely by gravity, without one single pumping station.

The spring water originates in the Lower Austrian Limestone Alps. The spring zone of the 1st Spring Water Main extends across the Schneeberg, Rax and Schneealpe mountains, while that of the 2nd Spring Water Main comprises the Hochschwab massif. The protection zones designated for water resource conservation encompass an area of approx. 700 square kilometres.

Phot

os: M

A 31

/Sas

cha

Jaka

b, H

oude

k

Natural spring water for Vienna

Beza

hlte

Anz

eige

Facts and figures

• Number of inhabitants of Vienna supplied: 1.8 million• 1st Spring Water Main: 220,000 cubic metres/day• 2nd Spring Water Main: 217,000 cubic metres/day• Available groundwater volume: 142,000 cubic

metres/day• Aqueducts (1st and 2nd SWM): 130• Number of reservoirs: 31• Total storage volume: 1.65 million cubic metres• Pumping stations: 24• Average daily consumption: 370,000 cubic metres• Average daily consumption/person: 130 litres

Find more about Vienna’s water supply: www.wien.gv.at/english/ environment/watersupply/

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