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International Burkholderia cepacia Working Group

17th

Annual Meeting

April 10 – April 13, 2013

Ann Arbor, Michigan

This meeting has been possible by the generous contributions of

IBCWG 2013 Ann Arbor, MI April 2013

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Wednesday, April 10, 2013

6:00 – 8:00 p.m.

Registration / Meet and Greet Reception – Hors d’Oeuvres

Thursday, April 11, 2013

7:30 – 8:15 a.m.

Breakfast & Registration

8:15 – 8:30 a.m. Welcome – Dr. John LiPuma, Dr. Joanna Goldberg and Dr. Amal Amer

8:30 – 9:30 a.m. KEYNOTE SPEAKER Dr. Vaughn Cooper

POPULATION GENOMICS OF EXPERIMENTALLY EVOLVED BURKHOLDERIA BIOFILMS

9:40 – 10:40 a.m. Session 1: The variation of Bcc isolates

Moderators: Joanna Goldberg and Tom Coenye

9:40 – 10:00 a.m. 1. EXPLORING THE CLINICAL IMPACT OF BURKHOLDERIA CEPACIA COMPLEX STRAINS ISOLATED FROM INDUSTRIAL PRODUCTS

José Degrossi, Agustina Lopez de Volder, Verónica Pioli, Fabricio Rugnone, Eduardo Vadell, Laura Galanternik, Sergio Teves and Mirta Franco

10:00 – 10:20 a.m. 2. PSEUDOMONAS AERUGINOSA METABOLITES INDUCE A TRANSIENT COLONY MORPHOTYPE IN SOME SPECIES OF THE BURKHOLDERIA CEPACIA COMPLEX

Steve P. Bernier, Courtney Hum, and Michael G. Surette

10:20 – 10:40 a.m. 3. COMPARATIVE PHYLOGENOMIC ANALYSIS OF THE BURKHOLDERIA MULTIVORANS ISOLATES FROM PEDIATRIC AND ADULT CYSTIC FIBROSIS PATIENTS

Vijaya Brahma, Julio Diaz Caballero, Shawn Clark, Valerie Waters, Yvonne Yau, Pauline Wang, Lijie Zhang, Sylva Donaldson, D. Elizabeth Tullis, David S. Guttman and David M. Hwang.


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10:40 -11:00 a.m. Coffee break

11:00 a.m. – 12:40 p.m. Session 2. The variation of Bcc isolates II

Moderators: Annette Vergunst and Siobhán McClean

11:00 – 11:20 a.m. 4. PHENOTYPIC AND GENOTYPIC CHARACTERIZATION OF SEQUENTIAL BURKHOLDERIA MULTIVORANS ISOLATES FROM AN ADULT WITH CYSTIC FIBROSIS Shawn T. Clark, Julio Diaz Caballero, Vijaya Brahma, Pauline W. Wang, Sylva L. Donaldson, Yvonne C. Yau, Valerie J. Waters, D. Elizabeth Tullis, David S. Guttman and David M. Hwang.

11:20 – 11:40 a.m. 5. TOBRAMYCIN SUSCEPTIBILITIES OF BURKHOLDERIA CEPACIA COMPLEX ISOLATES FROM PEDIATRIC AND ADULT CYSTIC FIBROSIS PATIENTS

Valerie Waters, Yvonne Yau Anina Ratjen, Jill Wettlaufer , Larissa Matukas, Felix Ratjen , Elizabeth Tullis

11:40 – 12:00 p.m. 6. EPIDEMIC B. MULTIVORANS STRAINS IN BELGIAN CF PATIENTS

C. Peeters, A. De Bel, J. Willekens, A. Malfroot, D. Pierard, P. Vandamme

12:00 – 12:20 p.m. 7. GENETIC VARIATION OF BURKHOLDERIA DOLOSA IN SINGLE CLINICAL SAMPLES PROVIDES A SIGNATURE OF SELECTIVE PRESSURES

Tami D. Lieberman, Kelly B. Flett, Thomas R. Martin, Alexander J. McAdam, Gregory P. Priebe, and Roy Kishony

12:20 – 12:40 p.m. 8. A 15 YEAR STUDY OF BURKHOLDERIA INFECTION IN PERSONS WITH CYSTIC FIBROSIS Amy A. Bumford, Theodore Spilker, Bridget Foster, Jennifer Krot, Linda M. Kalikin and John J. LiPuma

12:40 – 2:00 p.m.

Lunch


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2:00 – 3:40 p.m.

Session 3. Gene expression responses

Moderators: Vittorio Venturi and Pam Sokol

2:00 – 2:20 p.m. 9. MANNITOL ELICITS A GENOME-WIDE RESPONSE IN BURKHOLDERIA MULTIVORANS THAT IMPACTS ON NUMEROUS VIRULENCE TRAITS

Carmen C. Denman and Alan R. Brown

2:20 – 2:40 p.m. 10. LIFE AT LOW-OXYGEN TENSION IN B. CENOCEPACIA H111

Gabriella Pessi, Rubina Braunwalder, Alexander Grunau, Ulrich Omasits, Christian H. Ahrens and Leo Eberl

2:40 – 3:00 p.m. 11. GENE EXPRESSION CHANGES OF BURKHOLDERIA CENOCEPACIA ST32 WHEN CULTURED IN CF SPUTUM AND SERUM

Lucie Kalferstova, Michal Kolar, Pavel Drevinek

3:00 – 3:20 p.m. 12. HIGH THROUGHPUT SCREENING FOR GROWTH INHIBITORS OF BURKHOLDERIA CENOCEPACIA

Carrie Selin, Eric Brown and Silvia T. Cardona

3:20 – 3:40 p.m. 13. GENE-SPECIFIC SILENCING OF BURKHOLDERIA CEPACIA COMPLEX WITH PHOSPHORODIAMIDATE MORPHOLINO OLIGOMERS

Kimberly Marshall-Batty, Lacey Harbor, Bruce L. Geller and David E. Greenberg

3:40 -4:00 p.m. Coffee break


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4:00 – 5:40 p.m. Session 4. Virulence factors

Moderators: Eshwar Mahenthiralingam and Alan Brown

4:00 – 4:20 p.m. 14. COMPARATIVE GENOMIC ANALYSIS DEFINES THE BURKHOLDERIA CENOCEPACIA CORE GENOME AND CANDIDATE VIRULENCE GENES

Erick Cardenas, Linda M. Kalikin, Jean-Marie Rouillard, Jiangchao Zhao, Patrick S. Chain, Erdogan Gulari, James M. Tiedje, and John J. LiPuma

4:20 – 4:40 p.m. 15. THE ROLE OF BURKHOLDERIA DOLOSA FLAGELLA IN HOST COLONIZATION

Deborah Yoder-Himes, Damien Roux, David Skurnik, Mihaela Gadjeva, Stephen Lory

4:40 – 5:00 p.m. 16. INVESTIGATION OF GENES AND REGULATORY ELEMENTS INVOLVED IN PRODUCTION OF NOVEL ANTIFUNGAL COMPOUNDS IN BURKHOLDERIA CENOCEPACIA H111

Christian Jenul, Aurelien Carlier, Pamela A. Sokol and Leo Eberl

5:00 – 5:20 p.m.

17. GENETIC ANALYSIS OF A BROAD-HOST RANGE TAILOCIN IN BURKHOLDERIA CENOCEPACIA

Iris Duarte, Guichun W. Yao, John J. LiPuma, Ryland F. Young and Carlos F. Gonzalez

5:20 – 5:40 p.m.

6:00 p.m.

18. IDENTIFICATION OF NOVEL BURKHOLDERIA CEPACIA COMPLEX ANTIGENS BY IMMUNOPROTEOMICS AND INVESTIGATION OF THEIR ROLES IN VIRULENCE Ruth Dennehy, Cristobal Mujica, Miguel A. Valvano, Maire Callaghan and Siobhán McClean

Free time / Dinner on your own


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Friday, April 12, 2013

7:30 – 8:30 a.m.

Breakfast

8:40– 10:20 a.m. Session 5: Biofilms and quorum sensing

Moderators: Elizabeth Tullis and David Hwang

8:40 – 9:00 a.m. 19. INVOLVEMENT OF TOXIN-ANTITOXIN MODULES IN TOLERANCE OF BURKHOLDERIA CENOCEPACIA BIOFILMS

Heleen Van Acker, Hans J. Nelis and Tom Coenye

9:00 – 9:20 a.m. 20. NEW INSIGHTS INTO THE BDSF AND THE AHL-BASED QUORUM SENSING SYSTEMS OF B. CENOCEPACIA H111

Nadine Schmid, Gabriella Pessi, Yinyue Deng, Claudio Aguilar, Ulrich Omasits, Lian-Hui Zhang, Christian H. Ahrens and Leo Eberl

9:20 – 9:40 a.m. 21. ANTI-BIOFILM ROLE OF EXOPOLYSACCARIDES PRODUCED BY BURKHOLDERIA CENOCEPACIA

Elena Pellizzoni, Fabio Ravalico, Denis Scaini, Roberto Rizzo, Paola Cescutti

9:40 – 10:00 a.m. 22. AHL QUORUM SENSING IN THE ENVIRONMENTAL AND POTENTIALLY BENEFICIAL PLANT-ASSOCIATED BURKHOLDERIA CLUSTER

Bruna G. Coutinho, Birgit Mitter, Chouhra Talbi, Angela Sessitsch, Eulogio J. Bedmar, Nigel Halliday, Euan K. James, Miguel Cámara and Vittorio Venturi

10:00 – 10:20 a.m. 23. TEASING APART THE MULTIPLE ROLES OF A CzcR/CzcS-LIKE TWO-COMPONENT SYSTEM IN BURKHOLDERIA CENOCEPACIA: VIRULENCE, QUORUM SENSING AND HEAVY METAL RESISTANCE

Matthew T. Robinson and Alan R. Brown

10:20 – 10:40 a.m. Coffee Break


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10:40 a.m. – 12:20 p.m.

Session 6. Innate immunity and intracellular lifestyle

Moderators: Paola Cescutti and Leo Elberl

10:40 – 11:00 a.m. 24. PROTEINS INVOLVED IN HOST CELL ATTACHMENT ARE PROTECTIVE ANTIGENS AGAINST BCC INFECTION.

Minu Shinoy, Marc Healy, Bernard Mahon, Máire Callaghan and Siobhán McClean

11:00 – 11:20 a.m. 25. DEFECTIVE AUTOPHAGY ACTIVITY CONTRIBUTES TO INCREASED IL-1β PRODUCTION DURING BURKHOLDERIA CENOCEPACIA INFECTION

Basant A. Abdulrahman, Mia Tazi, Anwari Akhter, Kyle Caution, Hoda Hassan, Miguel A. Valvano, Benjamin Kopp, and Amal O. Amer

11:20 – 11:40 a.m. 26. AUTOPHAGY STIMULATION BY IFN-γ IMPROVES BURKHOLDERIA CENOCEPACIA CLEARANCE IN HUMAN CYSTIC FIBROSIS MACROPHAGES

Ben Kopp, Kaivon Assani, and Amal Amer

11:40 -12:00 p.m. 27. A BURKHOLDERIA CENOCEPACIA GENE ENCODING A NON-FUNCTIONAL PHOSPHOTYROSINE PHOSPHATASE IS REQUIRED FOR THE DELAYED MATURATION OF THE BACTERIA-CONTAINING VACUOLES IN MACROPHAGE

Angel Andrade and Miguel Valvano

12:00-12:20 p.m. 28. MODELING OF THE INNATE IMMUNE RESPONSE TO INFECTION WITH BACTERIA BELONGING TO THE BURKHOLDERIA CEPACIA COMPLEX USING ZEBRAFISH EMBRYOS

Jennifer Mesureur, Julien Rougeot, David O’Callaghan, Annemarie Meijer, Annette Vergunst

12:20 – 2:00 p.m. 2:00 p.m.

Lunch

Free time to visit Ann Arbor

Dinner on your own


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Saturday, April 13, 2013

7:30 – 8:30 a.m. Breakfast

8:40 – 10:20 a.m. Session 7. Modeling and biology of Bcc

Moderators: David Greenberg and Silva Cardona

8:40 – 9:00 a.m. 29. MODELLING BURKHOLDERIA CEPACIA COMPLEX ROOT COLONIZATION IN VITRO AS AN IMPORTANT ENVIRONMENTAL TRAIT

J. Cristian Vidal-Quist, Louise A. O’Sullivan, Annaëlle Desert, Amanda S. Fivian-Hughes, Angela M. Marchbank, Coralie Millet, T. Hefin Jones, Andrew J. Weightman, Hilary J. Rogers, Colin Berry and Eshwar Mahenthiralingam

9:00 – 9:20 a.m. 30. REAL TIME ANALYSIS OF BURKHOLDERIA CENOCEPACIA ST32 ISOLATES IN A ZEBRAFISH EMBRYO MODEL

Klara Vilimovska Dedeckova, Pavel Drevinek, Annette Vergunst

9:20 – 9:40 a.m. 31. EXPLORING THE TOPOLOGY OF ArnT FROM BULKHOLDERIA CENOPACIA

Faviola Tavares-Carreón and Miguel A Valvano

9:40 – 10:00 a.m. 32. STRUCTURAL DIFFERENCES BETWEEN THE GENOMES OF BURKHOLDERIA CENOCEPACIA STRAINS K56-2 AND J2315

Andrea M. Sass, Silvia Cardona, Miguel A.Valvano, Tom Coenye, Eshwar Mahenthiralingam

10:00 – 10:20 a.m. 33. COMPARATIVE FUNCTIONAL ANALYSIS OF METABOLIC ABILITIES OF BCC SPECIES USING RECONCILED GENOME-SCALE METABOLIC RECONSTRUCTIONS

Jennifer Bartell, Phillip Yen, John Varga, Joanna Goldberg and Jason Papin

10:20 – 10:40 a.m. Coffee Break


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10:40 a.m. – 12:00 p.m. Session 8. The complexity of Bcc genetics

Moderators: Peter Vandamme and Amal Amer

10:40 – 11:00 a.m. 34. INVESTIGATING FACTORS INVOLVED IN THE MAINTENANCE OF THE ‘CHROMOSOME 3’ MEGAPLASMID

Kirsty Agnoli, Carmen Frauenknecht and Leo Eberl

11:00 – 11:20 a.m. 35. GLOBAL MAPPING OF PRIMARY TRANSCRIPTION START SITES IN THE BURKHOLDERIA CENOCEPACIA J2315 GENOME BY DIFFERENTIAL RNA SEQUENCING

Andrea M Sass, Heleen van Acker, Konrad Förstner, Jörg Vogel, Tom Coenye

11:20 a.m. – 12:00 p.m. Closing remarks – Discussion about next meeting

12:00 p.m. Lunch

Adjourn


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ABSTRACTS

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EXPLORING THE CLINICAL IMPACT OF BURKHOLDERIA CEPACIA COMPLEX

STRAINS ISOLATED FROM INDUSTRIAL PRODUCTS

José Degrossi1, Agustina Lopez de Volder

1, Verónica Pioli

1, Fabricio Rugnone

2, Eduardo

Vadell1, Laura Galanternik

3, Sergio Teves

1, 2 and Mirta Franco

1

1 Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Buenos Aires. Argentina

2 Laboratorio Proanalisis SA. Buenos Aires. Argentina

3 Hospital de Niños “Dr. Ricardo Gutierrez”. Buenos Aires. Argentina

Contact: [email protected]

Members of the Burkholderia cepacia complex (Bcc) are frequent contaminants of industrial

products like pharmaceuticals, cosmetics and house cleaners.

Although the transmission of Bcc from environmental sources to patients has been reported, the

establishment of industrial contaminated products as a reservoir for infections remains unclear.

In order to elucidate the potential clinical impact of Bcc isolated from industrial sources, we

studied strains of B. cepacia, B. vietnamiensis, B. contaminans and B. lata (n=15) recovered

from contaminated products and purified water employed in different industries.

A genetic comparison by RAPD analysis with Bcc isolates recovered from patients, and

pathogenicity of the industrial strains using four infection models (alfalfa, Galleria mellonella,

Drosophila melanogaster and Dictyostelium discoideum) were performed.

60 % of the industrial isolates displayed similar RAPD pattern profiles to clinical isolates.

Regarding pathogenicity, results obtained with different models were not correlative. Most

isolates displayed virulence in at least one model. B. vietnamiensis and B. lata isolates were the

less virulent studied strains. Among B. contaminans isolates, those recovered from contaminated

products in which preservative agents are present, resulted more virulent than isolates recovered

from purified water.

These results reinforce the idea that industrial products should be considered a Bcc reservoir with

potential clinical impact. On the other hand, due to the differences observed among the employed

models, a multi host infection model is suggested for pathogenicity studies of industrial isolates.


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2

PSEUDOMONAS AERUGINOSA METABOLITES INDUCE A TRANSIENT COLONY

MORPHOTYPE IN SOME SPECIES OF THE BURKHOLDERIA CEPACIA COMPLEX

Steve P. Bernier1, Courtney Hum

1, and Michael G. Surette

1,2

1

Farncombe Family Digestive Health Research Institute, Department of Medicine, Faculty of

Health Sciences, McMaster University, Hamilton, Ontario, Canada 2

Department of Biochemistry and Biological Sciences, Faculty of Health Sciences, McMaster

University, Hamilton, Ontario, Canada

Contact: Steve P. Bernier, [email protected]

Background. In multi-species communities, metabolite-mediated interactions have the potential

to trigger polymicrobial-specific behaviours that are typically cryptic in mono-species cultures.

In this study, we use the cystic fibrosis pathogens Pseudomonas aeruginosa and the

Burkholderia cepacia complex (Bcc) as an in vitro model of polymicrobial communities to

demonstrate that metabolites from P. aeruginosa trigger a biological response in some Bcc

species leading to a transient colony morphotype.

Results. Representative strains from nine species of the Bcc (B. cepacia, B. multivorans, B.

cenocepacia, B. stabilis, B. vietnamiensis, B. dolosa, B. ambifaria, B. anthina, and B. pyrrocinia)

were initially plated onto agar medium-containing low concentrations (0.5 - 1.0) of spent

medium derived from a 24-h culture of P. aeruginosa to determine whether different colony

morphotypes would arise. Strains from B. multivorans, B. cenocepacia, and B. dolosa exhibited a

colony morphotype change upon exposure to P aeruginosa extracellular metabolites. The

observed morphotype is specific to P. aeruginosa metabolites since Bcc strains fail to respond in

a similar manner to both their own and Escherichia coli spent medium. Further, the morphotype

change is transient and not due to a mutation since wild-type colony morphotype is restored

when plated back onto regular agar medium (no spent medium). To broadly identify Bcc genes

and pathways essential for responding to P. aeruginosa metabolites, a random transposon

mutagenesis was performed in parallel on two responsive strains from the species B. multivorans

and B. dolosa. Disruption of genes coding for lipoproteins, hypothetical proteins, anti-sigma

factors, transcriptional regulators, histidine kinase, type II secretion system, and type IV pili

completely abolished the colony morphotype change in response to active P. aeruginosa spent

medium. An important overlap was observed between the mutagenesis screens of both B.

multivorans and B. dolosa with approximately 50 of the hits being located within a 16-kb

region coding for the biogenesis of type IV pili. Investigations are ongoing to identify the

bioactive molecule present in P. aeruginosa spent medium as well as the characterization of Bcc

behaviours associated with the described polymicrobial-specific colony morphotype.

Conclusions/relevance. Our study demonstrates that bacterial behaviours can emerge in

polymicrobial communities which are cryptic in mono-species cultures. Understanding

molecular mechanisms leading to polymicrobial-specific traits may reveal how multi-species

interactions collectively contribute to pathogenesis and persistence in the host.

mailto:[email protected]


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3

COMPARATIVE PHYLOGENOMIC ANALYSIS OF THE BURKHOLDERIA

MULTIVORANS ISOLATES FROM PEDIATRIC AND ADULT CYSTIC FIBROSIS

PATIENTS

Vijaya Brahma1 , Julio Diaz Caballero2, Shawn Clark 1,3, Valerie Waters4, Yvonne Yau4 ,

Pauline Wang5, Lijie Zhang5, Sylva Donaldson5, D. Elizabeth Tullis6, David S. Guttman2,5 and

David M. Hwang.1,3

1 Latner Thoracic Surgery Research Laboratories, University Health Network, Toronto ON

2 Department of Cell & Systems Biology, University of Toronto, Toronto ON

3 Department of Laboratory Medicine & Pathobiology, University of Toronto, Toronto ON

4 Department of Infectious Diseases, Hospital for Sick Children, Toronto ON

5 Centre for the Analysis of Genome Evolution & Function, University of Toronto, Toronto ON

6 Division of Respirology, St. Michael‟s Hospital, Toronto, ON

Contact: David Hwang, [email protected]

David Guttman, [email protected]

Burkholderia multivorans is an emerging opportunistic pathogen in individuals with cystic

fibrosis (CF). Current repositories are very limited with respect to B. multivorans whole genome

sequences, particularly from clinical strains. To better understand the genetic diversity of B.

multivorans infections in CF, we have sequenced and assembled the genomes of B. multivorans

isolates from pediatric and adult CF patients. Genomic DNA was sequenced with paired-end

Miseq sequencing. The resulting reads were assembled using CLC de novo assembler and

reference mapped with the Burrows Wheeler Assembler (BWA). Variant calling was performed

with Samtools and the identified variants were characterized with CLC Genomics. Initial

analysis of genome sequences from our isolates found significant differences compared to B.

multivorans ATCC 17616, an environmental isolate. Ongoing analysis will further define core

and accessory genomes in B. multivorans, delineating the diversity between isolates infecting

different patients and address the question whether or not the environmental, pediatric and adult

CF isolates follow distinct evolutionary paths.




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4

PHENOTYPIC AND GENOTYPIC CHARACTERIZATION OF SEQUENTIAL

BURKHOLDERIA MULTIVORANS ISOLATES FROM AN ADULT WITH CYSTIC

FIBROSIS

Shawn T. Clark1,2

, Julio Diaz Caballero3, Vijaya Brahma

2, Pauline W. Wang

4, Sylva L.

Donaldson4, Yvonne C. Yau

1,5, Valerie J. Waters

6, D. Elizabeth Tullis

7, David S. Guttman

3,4 and

David M. Hwang1,2

1 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada

2 Latner Thoracic Surgery Research Laboratories, University Health Network, Toronto, Canada

3 Department of Cell & Systems Biology, University of Toronto, Toronto, Canada

4 Centre for the Analysis of Genome Evolution & Function, University of Toronto, Toronto,

Canada 5 Department of Pediatric Laboratory Medicine, Hospital for Sick Children, Toronto, Canada

6 Department of Pediatrics, Infectious Diseases, Hospital for Sick Children, Toronto, Canada

7 Respirology, University of Toronto, Toronto, Canada

Contact: Shawn Clark, [email protected]

Pulmonary infection by members of the Burkholderia cepacia complex (BCC) remains a

feared condition in the cystic fibrosis (CF) community because of their high intrinsic resistance

and association with poor patient outcome. Recent studies into the diversity and dynamics of

dominant CF pathogen populations have identified clonal-relatedness and phenotypic

heterogeneity among many chronic infection isolates. At present, similar examinations into BCC

populations are limited. The objective of this study was to survey adaptive changes in an

infecting B. multivorans population during periods of pulmonary exacerbation. We present a

preliminary analysis of the phenotypic and genotypic attributes of 20 isolates recovered from

sequential sputum samples produced by an adult CF patient chronically infected by

B. multivorans. Three non-mucoid colony morphotypes, including small colony variants, were

identified and confirmed as B. multivorans by initial 16S rDNA sequencing. Phenotypic

characterization identified that all isolates were non-motile, able to produce rhamnolipids and

later isolates demonstrated an increased ability to form biofilms in vitro. Additionally, isolates

could be grouped into two distinct phenotypic profiles based on differences in both β-lactam

resistance and growth patterns in LB. The complete genome of each isolate was sequenced by

paired-end MiSeq sequencing and comparative genomic analysis is currently being performed.



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5

TOBRAMYCIN SUSCEPTIBILITIES OF BURKHOLDERIA CEPACIA COMPLEX

ISOLATES FROM PEDIATRIC AND ADULT CYSTIC FIBROSIS PATIENTS

Valerie Waters 1

, Yvonne Yau 2

, Anina Ratjen 1

, Jill Wettlaufer 1

, Larissa Matukas 3

, Felix Ratjen 4, Elizabeth Tullis

5

1Division of Infectious Diseases, Department of Pediatrics, The Hospital for Sick Children,

University of Toronto, Toronto, Canada.

2Division of Microbiology, Department of Pediatric Laboratory Medicine, The Hospital for Sick

Children, University of Toronto, Toronto, Canada. 3Division of Microbiology, St. Michael‟s Hospital, University of Toronto, Toronto, Canada.

4Division of Respirology, Department of Pediatric Laboratory Medicine, The Hospital for Sick

Children, University of Toronto, Toronto, Canada. 5Division of Respirology and Keenan Research Centre of Li Ka Shing Knowledge Institute,

Department of Medicine, St. Michael‟s Hospital, University of Toronto, Toronto, Canada.

Contact: Valerie Waters, [email protected]

Background: Pulmonary infection with Burkholderia cepacia complex (BCC) is associated with

increased lung function decline and mortality in patients with cystic fibrosis (CF) but there are

currently no effective chronic maintenance antimicrobial therapies for this condition. Although

BCC is intrinsically resistant to aminoglycosides via efflux pumps, for example, new inhaler

devices such as the podhaler can deliver very high levels of tobramycin inhalation powder (TIP)

to the lung that may be able to overcome these mechanisms of resistance. The goal of this study

was to determine the tobramycin minimum inhibitory concentrations (MICs) of a large collection

of BCC isolates in relation to the tobramycin sputum concentrations known to be achievable by

TIP (up to 1979 µg/g).

Methods: This was a 1 year (Sept 2011-Dec 2012) prospective observational study of pediatric

and adult CF patients infected with BCC attending the Hospital for Sick Children and St

Michael‟s Hospital CF clinic. BCC were isolated from sputum and genomovars were determined

using recA typing. Conventional broth microdilution susceptibility testing was done on cultures

grown planktonically by inoculating 105

CFU/ml into wells with Mueller-Hinton broth with

tobramycin at increasing concentrations: 10, 100,200, 400, 800, 1600 and 3200 µg/ml.

Results: A total of 89 BCC isolates were collected from 46 patients (8 pediatric, 38 adult). Of

the 89 BCC isolates, 64 (72%) were B. cenocepacia, 17 (19%) were B. multivorans and 8 were

unknown. The median tobramycin MIC was 200 µg/ml (range 10-3200 µg/ml) overall (median

tobramycin MIC for B. cenocepacia=200 µg/ml; for B. multivorans=100 µg/ml). When the MIC

of only the first isolate per patient was examined, the median tobramycin MIC was 100 µg/ml.

The change in tobramycin MIC in individual patients over time was examined; 21 patients had

repeated isolates (range 2-4 isolates). In 15 of the 21 patients (71%), the tobramycin MIC of the

first isolate compared to that of the last isolate was within 1 dilution. Fourteen patients had

multiple morphotypes from one sample tested; in the majority of cases (10/14, 71%) the

tobramycin MICs of different morphotypes were within 1 dilution of each other.

Conclusions: This preliminary in vitro study demonstrates that for the majority of BCC isolates

infecting both pediatric and adult CF patients, the minimum concentration required to inhibit

these organisms is 200 µg/ml. This suggests that TIP delivered via podhaler may be able to

achieve a ten-fold increase in tobramycin sputum concentration above the planktonic MIC.

Further susceptibility testing growing BCC as a biofilm is required to confirm these findings. In

addition, clinical trials are currently underway to determine therefore whether TIP administration

via podhaler can decrease the sputum bacterial density and improve lung function in CF patients

infected with BCC.



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6

EPIDEMIC B. MULTIVORANS STRAINS IN BELGIAN CF PATIENTS

C. Peeters1, A. De Bel

2, J. Willekens

3,4, A. Malfroot

3, D. Pierard

2, P. Vandamme

1

1Laboratory of Microbiology Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent,

Belgium 2Department of Microbiology and Infection Control, Universitair Ziekenhuis Brussel, Vrije

Universiteit Brussel (VUB), Laarbeeklaan 101, B-1090 Brussels, Belgium 3Cystic Fibrosis Clinic, Universitair Ziekenhuis Brussel, Belgium

4Department of Pediatrics, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel (VUB),

Laarbeeklaan 101, B-1090 Brussels, Belgium

Contact : Charlotte Peeters, [email protected]

Burkholderia cepacia complex (Bcc) bacteria have emerged as significant respiratory pathogens

in CF patients due to the risk of cepacia syndrome (a fatal necrotizing pneumonia with

bacteraemia), the organism‟s innate multiresistance to antibiotics, and the transmissibility of

bacterial strains among patients by social contact. Of the 17 established species within the Bcc,

B. cenocepacia and B. multivorans dominate in CF. While B. cenocepacia strains have been

responsible for large epidemics among CF patients and are often extremely virulent, only a

limited number of B. multivorans outbreak strains and rare cases of cepacia syndrome caused by

B. multivorans have been reported. However, recent epidemiological surveys reveal that the

proportion of CF patients infected with B. multivorans is rising (e.g. UK and USA), or that B.

multivorans is the most prevalent Bcc CF pathogen (e.g. Belgium, France, New-Zealand).

Since January 2011 a National Reference Center is charged with the surveillance of respiratory

infections caused by non-fermenting Gram-negative bacilli in Belgian CF patients. This project

aims to identify isolates from CF patients and study the epidemiology and virulence of Bcc

bacteria in order to improve infection control guidelines.

During the two-year period 2011-2012, 88 Bcc isolates were identified using recA gene sequence

analysis and 56 isolates (64%) belonged to B. multivorans. Multilocus Sequence Typing (MLST)

was used to assess the epidemiology of the Bcc isolates and revealed the occurrence of two B.

multivorans strains among multiple Belgian CF patients. The two clusters comprised five and

twelve patients, respectively. Additionally, many unique B. multivorans strains were found,

suggesting acquisition from environmental sources.

In conclusion, this study demonstrated the presence of two epidemic B. multivorans clones in the

Belgian CF population.

This research was supported by the Special Research Council of Ghent University and National

Health Insurance (RIZIV/INAMI).



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7

GENETIC VARIATION OF BURKHOLDERIA DOLOSA IN SINGLE CLINICAL

SAMPLES PROVIDES A SIGNATURE OF SELECTIVE PRESSURES

Tami D. Lieberman1, Kelly B. Flett

2, Thomas R. Martin

3, Alexander J. McAdam

4, Gregory P.

Priebe2,5

, and Roy Kishony1,6

1Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.

2Division of Infectious Diseases, Department of Medicine, Boston Children‟s Hospital; and

Harvard Medical School, Boston, MA 02115, USA. 3Division of Respiratory Diseases, Department of Medicine, Boston Children‟s Hospital and

Harvard Medical School, Boston, MA 02115, USA 4Department of Laboratory Medicine, Boston Children‟s Hospital and Harvard Medical School,

Boston, MA 02115, USA. 5Division of Critical Care Medicine, Department of Anesthesiology, Perioperative and Pain

Medicine; Boston Children‟s Hospital; and Harvard Medical School, Boston, MA 02115, USA. 6School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138,

USA.

Contact: Tami Lieberman, [email protected]

Bacterial pathogens acquire mutations during infection as they adapt to challenges posed by the

environment within the human host. Recent genomic studies tracking bacterial evolution within

individual patients have identified mutations responsible for antibiotic resistance, the rate at

which mutations accumulate, and specific bacterial genes and pathways under selective pressure.

Such studies typically use sequencing of a single colony isolate from each clinical sample, giving

only a limited picture of the diversity of the pathogen population at the time of sampling. Here,

using colony re-sequencing and deep population sequencing of sputum isolates of Burkholderia

dolosa from people with cystic fibrosis, we identify extensive intrastrain genomic diversity

within individual clinical samples. We find that mutations rarely fix within a patient's pathogen

population—instead, diversifying lineages coexist for many years. When strong selection is

acting on a gene, multiple adaptive mutations arise yet coexist, generating lasting allele diversity

that provides a signature of past selection. In the pathogen populations we studied, genes

involved in outer-membrane components, iron scavenging and antibiotic resistance all showed

signs of within-patient selection. These results offer a general approach for diagnosing selective

pressures acting on a pathogen within the human host using single clinical samples.


17

8

A 15 YEAR STUDY OF BURKHOLDERIA INFECTION IN PERSONS WITH CYSTIC

FIBROSIS

Amy A. Bumford, Theodore Spilker, Bridget Foster, Jennifer Krot, Linda M. Kalikin and John J.

LiPuma

Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor,

USA

Contact: John LiPuma, [email protected]

Between 1997 and 2011, the Burkholderia cepacia Research Laboratory and Repository

(BcRLR) at the University of Michigan received 14,580 isolates that we identified as species in

the B. cepacia complex (Bcc) or B. gladioli. These isolates had been recovered from 2,670

persons with CF attending 433 CF care centers in the United States. All Bcc species, except for

B. ubonensis, were identified. Analysis also suggested the presence of several novel Bcc species.

B. cenocepacia, B. multivorans, and B. gladioli were the most commonly identified species in

each year, with the annual incidence of B. cenocepacia showing a steady decline during the

study period, and the incidence of B. gladioli increasing in recent years. Transient or persistent

co-infection with more than a single species was noted, as well as super-infection with one

infecting species being supplanted by another. Chronic infection of greater than 14 years

duration with B. cenocepacia, B. multivorans, B. dolosa, or B. arboris was observed in several

patients. Genotyping analyses on a large subset of isolates using repetitive element BOX-PCR

identified previously described „epidemic‟ strains, including B. cenocepacia PHDC, the B.

cenocepacia Midwest clone, and B. dolosa SLC6. Several novel strains shared by multiple

patients were also identified. Active surveillance genotyping is now routinely performed to

monitor for evidence of outbreaks and to support infection control efforts at referring care

centers. Strain genotyping is also a critical element contributing to our understanding of

Burkholderia global epidemiology, as well as the natural history and clinical outcomes of

infection.


18

9

MANNITOL ELICITS A GENOME-WIDE RESPONSE IN BURKHOLDERIA

MULTIVORANS THAT IMPACTS ON NUMEROUS VIRULENCE TRAITS

Carmen C. Denman1 and Alan R. Brown

1

1Biosciences, College of Life & Environmental Sciences, University of Exeter, Exeter. UK.

Contact: Alan Brown, [email protected]

A dried-powder formulation of mannitol is approved as an osmolyte therapy for use in cystic

fibrosis patients. It is widely-documented that mannitol promotes exopolysaccharide (EPS)

production by members of the Burkholderia cepacia complex (BCC). In the present study we

sought to assess the wider impact that mannitol has on Burkholderia multivorans. Parallel studies

of representative wildtype B. multivorans (ATCC 17616 and C1576) and EPS-deficient isogenic

mutants of each strain enabled us to determine which components of the mannitol response were

associated with EPS production, and which were apparently independent of EPS production. In

addition, microarray (ATCC 17616) or RNA-seq (C1576) analysis was performed to characterize

the transcriptional response of the wildtype B. multivorans to mannitol. Our studies revealed a

significant whole-genome response to mannitol that impacts on a multitude of virulence-

associated traits, including biofilm formation, biotic & abiotic adherence, motility, antibiotic

resistance and epithelial cell invasion. In the majority of cases, this mannitol response was

observed equally in the wildtype and the isogenic EPS-deficient mutants, indicating it to be

largely independent of EPS production. Strain-to-strain variation was observed in the extent to

which EPS contributed to particular phenotypes, most notably adherence. Genome sequencing of

C1576 identified fimbrial and afimbrial adhesin loci that underlie the enhanced adherence

observed with this strain following growth in mannitol. In conclusion, these studies highlight the

wide-ranging effect that mannitol has on members of the BCC, and suggest that close monitoring

of BCC-infected patients receiving mannitol therapy would appear prudent.



19

10

LIFE AT LOW-OXYGEN TENSION IN B. CENOCEPACIA H111

Gabriella Pessi1, Rubina Braunwalder

1, Alexander Grunau

1, Ulrich Omasits

2, Christian H.

Ahrens2 and Leo Eberl

1

1Institute of Plant Biology, Department of Microbiology, UZH, Zürich, Switzerland

2Institute of Molecular Life Sciences, UZH, Zürich, Switzerland

Contact : [email protected]

B. cenocepacia H111 is an opportunistic pathogen originally isolated from a cystic fibrosis (CF)

patient. In CF infections, bacteria grow to high densities in a mucopurulent material that is

limited in oxygen. Pseudomonas aeruginosa, the dominant pathogen in CF patients, is known to

grow and survive under oxygen-limited to anaerobic conditions by using microaerobic

respiration, denitrification and fermentative pathways. In contrast, inspection of the B.

cenocepacia H111 genome sequence suggested that it is an obligate aerobic and non-fermenting

bacterium. Accordingly, our study of growth under different oxygen concentrations

demonstrated that H111 is able to grow with as little as 0.1% O2 but not under anaerobic

conditions. A phenotypical analysis showed that H111 produced more biofilms and pellicles

under microaerobic conditions (0.5% O2, i.e. conditions that mimic infection), and was

consequently more resistant to several antibiotics. To obtain a more comprehensive view of the

molecular mechanisms underlying physiological adaptation to an oxygen-limited environment

RNA-Seq and shotgun proteomics analyses were performed on cultures of B. cenocepacia H111

grown under microaerobic (0.5% O2) and aerobic (21% O2) conditions. For further

characterization and analysis of mutant strains, we have chosen two transcriptional units that are

highly expressed and up-regulated in microaerobically growing cells: i) an FNR-like

transcriptional regulator and ii) a rpoS-related sigma factor.


20

11

GENE EXPRESSION CHANGES OF BURKHOLDERIA CENOCEPACIA ST32 WHEN

CULTURED IN CF SPUTUM AND SERUM

Lucie Kalferstova1, Michal Kolar

2, Pavel Drevinek

1

1 Department of Medical Microbiology and Department of Pediatrics, 2

nd Medical School,

Charles University, Prague, Czech Republic. 2 Department of Genomics and Bioinformatics, Institute of Molecular Genetics, Academy of

Science of the Czech Republic, Prague, Czech Republic

Contact: Lucie Kalferstova, [email protected]

The aim of this study was to compare gene expression in two pairs of ST32 clinical isolates that

were subjected to cultivation in two different conditions, characteristic for chronic B.

cenocepacia infection in CF patients. ST32 strain is known to be a problematic epidemic strain,

which caused a serious outbreak at the Prague CF centre.

While one isolate per pair was recovered from blood at the time of cepacia syndrome, the other

was isolated from sputum one month prior to cepacia syndrome. Each isolate was incubated in

triplicate in: (i) CF sputum (minimal medium supplemented with 10% pooled CF sputum), (ii)

CF heat-inactivated serum and (iii) the basal salt medium (BSM) supplemented with glucose and

casein amino acids, generating in total samples for 36 microarrays: 3 growth conditions (sputum;

serum; BSM) x 4 isolates (sputum isolates from patients A and B; blood isolates from patients A

and B) x 3 replicates.

Bacterial cells were harvested after 4 hours of cultivation; their RNA was extracted and enriched

using Trizol, RiboPure and MICROBEnrich kits (Life Technologies). The conversion to cDNA,

fluorescent labeling and hybridization to the microarray was processed according to protocol for

microarrays analysis. The data mining was performed in GeneSpring v 12.0 (Agilent

Technologies).

We used a 4x44k custom array (Agilent Technologies) designed for closely related J2315 strain

since the whole genome sequence of ST32 is still unavailable. However, data from an ongoing

ST32 genome sequencing allowed to identify 5739 chip probes matching the putative CDS in

ST32 (~ 80% of all putative ST32 CDS) and 1049 probes for intergenic regions.

Numbers of genes with significantly altered expression are summarized in Tab.1. We noted that

the variable “growth condition” generated a pattern that was affected by the factor “patient” to

considerably lesser extent than the variable “origin of isolate” [an overlap between gene lists was

37% (comparisons #1 vs. #2), 51% (#3 vs. #4), 21% (#5 vs. #6) and 11% (#7 vs. #8)]. Now we

focus on the analysis of the latter variable with the ultimate goal to reveal gene expression

changes specific to origin of isolate and to septicemia, a last and critical stage of Burkholderia

infection. Tab. 1

condition compared

Comparison #

origin of isolate

growth condition

isolate from patient

origin of isolate

growth condition

isolate from patient

2-way ANOVA, p 0.05, 2-fold change up

down

growth condition

1 blood serum A

vers

us

blood sputum A 330 146 184

2 blood serum B blood sputum B 286 112 174

3 sputum serum A sputum sputum A 429 165 264

4 sputum serum B sputum sputum B 412 210 202

origin of isolate

5 blood serum A sputum serum A 135 104 31

6 blood serum B sputum serum B 127 47 80

7 blood sputum A sputum sputum A 116 79 37

8 blood sputum B sputum sputum B 140 86 54

Supported by IGA MZ NT12405-5, MSMT LD11029


21

12

HIGH THROUGHPUT SCREENING FOR GROWTH INHIBITORS OF

BURKHOLDERIA CENOCEPACIA

Carrie Selin1, Eric Brown

2 and Silvia T. Cardona

1,3

1Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada

2Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON,

Canada 3Department of Medical Microbiology, University of Manitoba, Winnipeg, MB, Canada


There is an urgent need for the discovery and development of new antimicrobial treatments for

Burkholderia cepacia complex infections. One approach to address this need is high-throughput

screening of small molecule libraries against whole cells to discover new, active scaffolds that

could stimulate drug discovery.

We used a whole-cell based assay to screen the Canadian Compound Collection (CCB) small

molecule library for growth inhibitory activity against B.cenocepacia K56-2. The screen used

bacterial growth in 96-well plate format, measured by optical density at 600 nm, to evaluate the

effect of 30,259 compounds. From 206 primary actives with <0.7 percent residual growth and <-

17.5 B-Score, we selected and confirmed the growth inhibitory activity of 72 compounds, named

herein Bce bioactives. The top twenty most active compounds consisted of antibiotics used in

clinics to treat Burkholderia infections, known antibiotics that are not commonly used, and

disinfectants. The remaining compounds were clustered by structure and prioritized for further

research according to novelty, growth inhibitory activity, solubility, and potential as multitarget

compounds. Thiophen, pyrazine and maleimide derivatives were among the selected small

molecules.

Our work illustrates the challenges of finding novel compounds with antibacterial activity

against B. cepacia complex and highlights the utility of whole cell-based high throughput screens

to identify bioactives as a starting point of antibacterial drug discovery.


22

13

GENE-SPECIFIC SILENCING OF BURKHOLDERIA CEPACIA COMPLEX WITH

PHOSPHORODIAMIDATE MORPHOLINO OLIGOMERS

Kimberly Marshall-Batty1, Lacey Harbor

1, Bruce L. Geller

2 and David E. Greenberg

1

1Department of Internal Medicine, University of Texas Southwestern, Dallas, TX, USA

2Department of Microbiology, Oregon State University, Corvallis, OR, USA


Background: Members of the Burkholderia cepacia complex (Bcc) can be difficult to treat in

patients with cystic fibrosis (CF) and chronic granulomatous disease (CGD) given their inherent

antibiotic resistance. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs)

are synthetic DNA/RNA analogues that silence expression of specific genes and prevent

translation. We studied whether PPMOs targeted to essential genes in Bcc are active in vitro and

in vivo.

Methods: A PPMO targeting the gene acpP was tested in in vitro and in vivo assays.

Bacterial strains were grown overnight and for in vitro assays, were diluted to a final

concentration of 5 x 105 CFU/mL. Blood was collected from normal donors and CF patients and

neutrophils were purified. For ex vivo studies, PPMOs were added to infected neutrophils (PMN)

one hour post-infection. For in vivo experiments, X-linked CGD mice (gp91phox

) that were 16-20

weeks old were infected intranasally with Burkholderia multivorans. Mice were treated with

various doses of PPMO intraperitoneally or through aerosol delivery. Both fixed-end point and

survival studies were undertaken and tissue burden of organism as well as pathologic findings

were determined.

Results: A PPMO targeting acpP is growth inhibitory across a variety of members of the Bcc.

This includes a pan-resistant B. cenocepacia outbreak isolate where there was a >3-log reduction

in CFU after 24 hour incubation with PPMO while incubation with peptide alone or a scrambled

sequence PPMO (Scr) had no effect on growth. The addition of the AcpP PPMO augments both

normal and CF PMN killing at both 2 and 4 hours post-infection. To better mimic the clinical

presentation and treatment of Bcc infections in humans, CGD mice were infected with B.

multivorans intranasally. Mice were infected with 6 x 105 CFU of B. multivorans IN and then

received one dose of AcpP PPMO or placebo (Scr) via an Aerogen nebulizer 6-hours post-

infection. All mice were euthanized at 24 hours and CFU/g of lung determined. A single, delayed

300 μg dose of AcpP PPMO reduced CFU by 90% compared to Scr PPMO treated mice

(p<.005). Mice were then infected with B. multivorans as above and treated with aerosolized

AcpP or Scr PPMO at 1,6 and 24 hours post-infection and then daily thereafter through day 7.

Mice received a single IP dose of PPMO at 24, 48 and 72 hours post-infection. While 50% of

AcpP PPMO treated mice survived through day 7, 100% of mice treated with Scr PPMO were

euthanized by day 5.

Conclusions: Gene-specific targeting of Bcc using PPMOs are effective in vitro and in vivo.

Targeted pulmonary delivery of PPMOs could be a viable therapeutic approach in treating

patients with Bcc infections.


23

14

COMPARATIVE GENOMIC ANALYSIS DEFINES THE BURKHOLDERIA

CENOCEPACIA CORE GENOME AND CANDIDATE VIRULENCE GENES

Erick Cardenas1, Linda M. Kalikin

2, Jean-Marie Rouillard

3, Jiangchao Zhao

2, Patrick S. Chain

4,5,

Erdogan Gulari3, James M. Tiedje

1, and John J. LiPuma

2

1Center for Microbial Ecology, Michigan State University, East Lansing, Michigan, USA

2Departments of Pediatrics and , University of Michigan, Ann Arbor, Michigan, USA

3Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan, USA

4Department of Energy Joint Genome Institute, Walnut Creek, California, USA

5Los Alamos National Laboratory, Los Alamos, New Mexico, USA


Burkholderia cenocepacia is a naturally occurring soil bacteria that can live in a wide range of

ecosystems. It is also an opportunistic pathogen in cystic fibrosis (CF) patients where it can lead

to chronic, and sometimes lethal respiratory infections. Our understanding of the pathogenicity

of B. cenocepacia has been shaped by epidemiological data in CF patients, as well as

experimental data using a variety of biological models of infection. However, little attention has

been given to the characterization of the intra-species genetic diversity as a way to understand its

pathogenicity. We used comparative genomic hybridization to study the distribution of over

8,000 genes in a set of 187 B. cenocepacia strains. This collection included clinical isolates,

including representatives from the ET12, PHDC, and Midwest epidemic lineages, and

environmental isolates. Hierarchical cluster analysis of array present/absent calls grouped

isolates broadly by recA lineages and in smaller clusters by epidemic lineage. Almost half

(49.1%) of the array genes belonged to the species core, defined as presence in at least 90% of all

187 strains. Gene functions associated with metabolism (COG categories C, E, F, G, H, I P, and

Q) represented the largest proportion (36.7%) of the core genome. Analysis of 165 genes

previously reported to have a potential role in Burkholderia virulence found that almost half

(48.0%) were part of the species core. While virulence genes were preferentially found in strains

from the epidemic lineages compared to non-epidemic strains, no virulence gene was exclusive

to one epidemic lineage, or to the epidemic strains at all. The lack of a strong association of

genes with the “epidemic” trait may be interpreted as a characteristic of opportunistic pathogens.

Nevertheless, we were able to find novel genes associated with epidemic strains in the collection.

Our results support the hypothesis that B. cenocepacia is a generalist, and that the versatility in

lifestyles it possesses is due to a large and adaptable genome that naturally harvests the potential

for pathogenesis.



24

15

THE ROLE OF BURKHOLDERIA DOLOSA FLAGELLA IN HOST COLONIZATION

Deborah Yoder-Himes1,2

, Damien Roux3, David Skurnik

3, Mihaela Gadjeva

3, Stephen Lory

2

1 Department of Biology, University of Louisville, Louisville, Kentucky, USA

2 Department of Microbiology and Immunobiology, Harvard Medical School, Boston,

Massachusetts, USA 3

Channing Lab, Brigham and Women‟s Hospital, Boston, Massachusetts, USA


Burkholderia dolosa is the causative agent of an epidemic at the Children‟s Hospital Boston

cystic fibrosis clinic that spanned from 1999 to the present day. Over 40 patients have been

infected and over 7 have died from cepacia syndrome. Very little is known about the virulence

of B. dolosa. The genome sequence revealed a number of loci that are not found in most

Burkholderia cepacia complex members but are found in the closely related B. pseudomallei and

B. mallei human and horse pathogens, including genes encoding lateral flagella. By screening a

number of B. dolosa isolates, we found that the genes encoding the lateral flagella are found in

all but one epidemic strains, sporadically in non-epidemic clinical strains, and not in the

environmental isolate. To investigate the role of the polar and lateral flagellin in a host system,

we infected mice intranasally with wild-type B. dolosa, mutant strains in either flagellar system,

wild-type Pseudomonas aeruginosa, or a P. aeruginosa flagellin mutant and tracked the

clearance of these strains, the recruitment of immune cells and the production of cytokines in

response to these strains over time. We found that B. dolosa strains persist longer in the lung

compared to P. aeruginosa strains and recruit fewer neutrophils; however, all B. dolosa strains

recruit cells that may play an active role in suppressing the immune system. We also found that

B. dolosa strains produced far fewer cytokines than P. aeruginosa strains with little difference

seen between the B. dolosa wild-type and mutant strains. We conclude that B. dolosa may be

actively avoiding or suppressing the immune system in order to proliferate and persist in a host.


25

16

INVESTIGATION OF GENES AND REGULATORY ELEMENTS INVOLVED IN

PRODUCTION OF NOVEL ANTIFUNGAL COMPOUNDS IN BURKHOLDERIA

CENOCEPACIA H111

Christian Jenul1, Aurelien Carlier

1, Pamela A. Sokol

2 and Leo Eberl

1

1Department of Microbiology, Institute of Plant Biology, University of Zürich, Zürich,

Switzerland 2Department of Microbiology, Immunology, and Infectious Diseases, University of Calgary,

Calgary, Alberta, Canada

Contact : Leo Eberl, [email protected]

Species of the Burkholderia cepacia complex (Bcc) are known for their ability to suppress fungal

pathogens by production of various antifungal compounds. Previous work has shown that in B.

cenocepacia K56-2 a large gene cluster that is present on the megaplasmid pC3 directs the

biosynthesis of an antifungal lipopeptide. Unexpectedly, we observed that a pC3-cured

derivative of B. cenocepacia H111 still displayed antifungal activity, indicating that H111

synthesizes a previously unrecognized antifungal compound. Our objective was to elucidate what

genes and regulatory elements are responsible for the different antifungal phenotypes of H111

and K56-2. Further we seeked to elucidate the nature of the bioactive compound involved in

antifungal activity of strain H111.

We found that the CepIR quorum sensing system regulates antifungal activity in H111, but not in

K56-2. Comparison of transcriptomics data of H111 and K56-2 quorum sensing mutants led to

the identification of a gene cluster comprising seven genes (H111 antifungal metabolite cluster),

that are predicted to be involved in secondary metabolism and that are differently regulated in

K56-2 and H111. In silico analysis of the gene cluster revealed that hamD encodes a non-

ribosomal peptide synthetase (NRPS). Mutation of hamD in H111 completely abrogated

antifungal activity and this phenotype could be rescued by expression of hamD from a plasmid.

This finding suggests that the ham gene cluster is involved in the biosynthesis of a so far

uncharacterized antifungal compound in H111.


26

17

GENETIC ANALYSIS OF A BROAD-HOST RANGE TAILOCIN IN BURKHOLDERIA

CENOCEPACIA

Iris Duarte1, 4

, Guichun W. Yao1, 4

, John J. LiPuma2

, Ryland F. Young3,4

and Carlos F.

Gonzalez1,4

1 Department of Plant Pathology and Microbiology, Texas A&M University, College Station,

USA 2 Department of Pediatrics and Communicable Diseases, University of Michigan Medical

School, Ann Arbor, USA

3 Department of Biochemistry and Biophysics, Texas A&M University, College Station, USA

4Center for Phage Technology, Texas A&M University, College Station, USA

Contact: Carlos F. Gonzalez, [email protected]

Members of the Burkholderia cepacia complex (Bcc) are opportunistic human pathogens that

exhibit broad-spectrum antibiotic resistance. High-molecular weight (HMW) bacteriocins, like

the well-studied R- and F-type pyocins (tailocins) from Pseudomonas aeruginosa, closely

resemble bacteriophage (phage) tails. Tailocins are attractive candidates for novel antibacterial

strategies because they can be genetically manipulated like phages and can also be deployed with

defined dosage, since they do not propagate in therapeutic use.

An 18 X 18 test matrix of B. cenocepacia clinical isolates was used to identify tailocin

Bcep0425, which exhibited broad host range activity against members of the Bcc. Electron

microscopy studies confirmed the phage tail-like structure of Bcep0425. The major tailocin

proteins were resolved using SDS-PAGE and four major protein species were subjected to N-

terminal sequence analysis. The host genome was sequenced and, using the sequence

information from the major proteins, the tailocin locus was identified and annotated in detail.

The tailocin cassette was found to be integrated into a tRNAArg

gene. The Bcep0425 integrase

gene was identified by significant sequence similarity to the integrase of the well-studied

temperate Salmonella podophage P22. Most Bcep0425 proteins showed homology to the tail

proteins of the paradigm E. coli myophage P2. A lysis cassette encoding an endolysin, i-spanin

and o-spanin was identified by sequence homology. Two genes, 26 and 27, were experimentally

confirmed as encoding the holin and antiholin, respectively, by phenotypic analysis of deletions

and complemented deletions. Bcep0425 holin contained four transmembrane domains (N-in and

C-in), defining a new holin topological class. Targeted mutagenesis of genes involved in the

biosynthesis of the bacterial lipopolysaccharide (LPS) in B. cenocepacia strain K56-2 was

conducted to determine that LPS is the putative receptor for Bcep0425. The molecular

characterization of Bcep0425 indicates that it was derived from a defective prophage.

Considering its broad host range, unprecedented for tailocins, Bcep0425 has significant potential

for antibacterial therapy.


27

18

IDENTIFICATION OF NOVEL BURKHOLDERIA CEPACIA COMPLEX ANTIGENS

BY IMMUNOPROTEOMICS AND INVESTIGATION OF THEIR ROLES IN

VIRULENCE

Ruth Dennehy1, Cristobal Mujica

2, Miguel A. Valvano

2, Maire Callaghan

1 and Siobhán

McClean1

1Centre of Microbial Host Interactions, Institute of Technology Tallaght, Dublin.

2Centre of Infection and immunity, Queens University Belfast.


Virulence factors make direct contact with host cells and so are among the primary antigen

targets of the host immune system. Therefore, proteomic analysis using serum from

Burkholderia cepacia complex (Bcc) colonised patients can lead to identification of novel

virulence factors. The aim of this study was to identify virulence factors from two of the most

clinically relevant Bcc species, B. cenocepacia and B. multivorans, using an immunoproteomic

approach. Two-dimensional blots were probed with serum from CF patients that had been

infected with Bcc and compared to blots probed with serum from patients with no history of Bcc

infection. Twelve B. cenocepacia and 14 B. multivorans immunogenic proteins were identified

using MALDI-Tof mass spectrometry. Of the proteins identified, six were common to both

species. Two proteins were chosen for further examination: an outer membrane lipoprotein

(BCAL3204), previously shown by us to be involved in attachment to CF lung epithelial cells

(CFBEs) and a hypothetical protein (BCAS0292), located in a virulence gene cluster.

Targeted deletion mutants were constructed in the B. cenocepacia strain, K56-2, with the aim of

further examining the role of these proteins in the pathogenesis of Bcc. The mutants were

examined in vivo using the Galleria mellonella virulence model. The virulence of the

BCAL3204 mutant was significantly reduced (p < 0.02) in comparison to the wild type, while a

slight decrease in virulence was observed in the BCAS0292 mutant. Both mutants also displayed

a substantial reduction in adhesion to CF lung epithelial cells compared to the wild-type strain,

indicating that both proteins may play a role in bacterial attachment to host cells.

Further investigation into the role of these immunogenic proteins will provide a better

understanding of the role they play in the pathogenesis of Bcc, which could lead to their

development as potential vaccine antigens or as drug targets for anti-virulence therapies.

Funded by Science Foundation Ireland and EU COST Action BM1003



28

19

INVOLVEMENT OF TOXIN-ANTITOXIN MODULES IN TOLERANCE OF

BURKHOLDERIA CENOCEPACIA BIOFILMS

Heleen Van Acker1, Hans J. Nelis

1& Tom Coenye

1

1

Laboratorium voor Farmaceutische Microbiologie, Universiteit Gent, Gent, België

Contact : Heleen Van Acker, [email protected]

Burkholderia cenocepacia is an opportunistic pathogen that can cause severe lung infections in

cystic fibrosis patients. Infections are often difficult to treat because of its innate resistance and

its capacity to form biofilms. Biofilms play an important role in the recalcitrance of infections

due to their tolerance. Tolerance is an ability to survive antibiotic treatment without expressing a

resistance mechanism. Although the molecular basis of tolerance is still largely unknown, toxin-

antitoxin modules (TA) are thought to play a role. The toxin (T) is a protein that inhibits an

important cellular function such as translation or replication and which can form an inactive

complex with an antitoxin (AT). Bactericidal antibiotics kill cells by corrupting the target

functions. Inhibition of these functions would prevent antibiotics from corrupting their targets

and would give rise to tolerant persister cells. In the present study we wanted to investigate

whether these TA modules play a role in B. cenocepacia biofilm tolerance towards different

antibiotics.

Using bioinformatics tools, 15 pairs of genes were identified as possible TA modules in the

genome of B. cenocepacia J2315 (score ≥ 60). We also found 4 other possible TA modules by

blast analysis. Based on microarray data, which indicated that these genes were upregulated in

biofilms treated with tobramycin, RNA of treated and untreated B. cenocepacia J2315 biofilms

and planktonic cultures was extracted and analyzed by qPCR. The expression of these TA

modules in treated sessile and planktonic cultures was compared to that in untreated cultures.

Mutants in which a toxin or antitoxin-coding gene was overexpressed under control of a

rhamnose inducible promoter were constructed and evaluated for growth, resistance, biofilm

formation and response to antibiotics. We constructed 14 toxin-overexpressing and 3 antitoxin

overexpressing mutants.

Generally the toxins showed a higher basal expression in sessile cells than in planktonic cells,

but the expression patterns were similar for most T-AT pairs. Four toxins were upregulated in all

conditions tested, whereas only one toxin was systematically downregulated. Operons showing

similarity with mazEF, relBE or mqsRA and five other TA modules were significantly

upregulated in biofilms and planktonic cultures after treatment with tobramycin but not after

treatment with ciprofloxacin. None of the operons was upregulated in both conditions after

treatment with ciprofloxacin. Others were only upregulated in sessile or in planktonic cultures,

which indicates that planktonic and sessile cells may have different mechanisms to regulate the

relative amounts of T/AT.

Overexpression of toxins had different effects on growth, biofilm formation and the number of

surviving cells after treatment. For example, overexpression of RelE or MazF resulted in a

prolonged lag phase and more cells surviving antibiotic treatment whereas overexpression of an

undefined antitoxin (BCAM0258) resulted in less biofilm formation. These results indicate that

various TA modules may play a role in tolerance and persistence but that their exact contribution

depends on the mode of growth and the antibiotic used.


29

20

NEW INSIGHTS INTO THE BDSF AND THE AHL-BASED QUORUM SENSING

SYSTEMS OF BURKHOLDERIA CENOCEPACIA H111

Nadine Schmid1*

, Gabriella Pessi1*

, Yinyue Deng2*

, Claudio Aguilar1, Ulrich Omasits

3, Lian-Hui

Zhang2, Christian H. Ahrens

3 and Leo Eberl

1

1Department of Microbiology, Institute of Plant Biology, University of Zurich, Switzerland

2Institute of Molecular and Cell Biology, Singapore

3Institute of Molecular Life Sciences, University of Zurich, Switzerland

* These authors contributed equally to this work


Burkholderia cenocepacia H111, a CF isolate, employs two quorum-sensing (QS) systems to

control the expression of genes associated with virulence and biofilm formation. While the N-

acyl homoserine lactone (AHL)-based QS system has been fairly well analyzed, little is known

about the more recently discovered cis-2-dodecenoic acid (BDSF)-dependent signaling system.

Recent work showed that the regulatory effect of BDSF on gene expression is mediated through

alteration of the intracellular c-di-GMP level. RpfR, the protein responsible for this effect, is a

bifunctional enzyme with both diguanylate-cyclase and phosphodiesterase activity. Upon binding

of BDSF to its PAS-domain, the phosphodiesterase activity is stimulated and as a consequence

the cellular c-di-GMP level is lowered.

Phenotypic characterization of QS mutants and transcriptome analyses revealed that the BDSF

and AHL stimulons partially overlap. In spite of the fact that BDSF positively regulates the AHL

levels, our data suggest that both signal molecules are required for maximal expression of the

large surface protein bapA and the lectin operon bclACB. In accordance with this observation,

the defects in biofilm formation, protease activity and swarming motility of BDSF- and AHL-

deficient double mutant can only be restored to wild type level upon the addition of both signal

molecules. This suggests that the two QS systems are not hierarchically arranged but rather

operate in parallel.


30

21

ANTI-BIOFILM ROLE OF EXOPOLYSACCARIDES PRODUCED BY

BURKHOLDERIA CENOCEPACIA

Elena Pellizzoni, Fabio Ravalico, Denis Scaini, Roberto Rizzo, Paola Cescutti

Dipartimento di Scienze della Vita, Università degli Studi di Trieste, via L. Giorgeri 1, Ed. C11,

34127 Trieste

Contact : Paola Cescutti, [email protected]

The preferential mode of growth of microbes in nature and disease is within a biofilm and there

are strong indications that bacteria of the BCC develop biofilms in the CF lung. The biofilm

matrix surrounds the microbial cells and confers an increased resistance to antibiotics, biocides

and host defenses. It is an active macromolecular scaffold composed mainly by proteins,

extracellular DNA and polysaccharides (PLS), but the exact composition may vary from species

to species and depends on cultivation media. We focussed our attention on B. cenocepacia

BTS2, a CF clinical isolate and an abundant biofilm producer. The aim of our investigation was

to determine the influence of growth media on the physico-chemical properties of the biofilm

matrix, with particular attention to polysaccharides (PLS), often regarded as the main matrix

components. Five different media were used: four common bacteriological media and the

synthetic cystic fibrosis medium (SCFM) which simulates the in vivo situation. Proteins and PLS

content were determined, and NMR established the structure of the latter. Biofilm morphology

was visualized by CLSM while AFM was used to investigate the thickness of the matrix. The

evaluation of PLS content in biofilms showed that media promoting abundant biofilm formation

exhibited the lowest PLS content. In particular, cepacian, the PLS synthesized by the majority of

BCC strains in YEM medium, was associated with scanty biofilm development. Interestingly, it

was recently reported that the extent of mucoidy in BCC is inversely correlated with the rate of

decline in lung function. These observations prompted an investigation of the role of the PLS

produced by B. cenocepacia strain BTS2 in biofilms. Cepacian had two main effects: when

mixed with the bacterial culture it impaired biofilm development, while its addition to mature

biofilms caused their partial detachment. These properties could be exploited for impeding

and/or removing biofilms.


31

22

AHL QUORUM SENSING IN THE ENVIRONMENTAL AND POTENTIALLY

BENEFICIAL PLANT-ASSOCIATED BURKHOLDERIA CLUSTER

Bruna G. Coutinho1,2

, Birgit Mitter3, Chouhra Talbi

4, Angela Sessitsch

3, Eulogio J. Bedmar

4,

Nigel Halliday5, Euan K. James

6, Miguel Cámara

5 and Vittorio Venturi

1

1Bacteriology Group, International Centre for Genetic Engineering & Biotechnology,

Padriciano 99, 34149 Trieste, Italy 2The Capes Foundation, Ministry of Education of Brazil, Cx postal 250, Brasilia, DF 70.040-

020, Brazil 3AIT Austrian Institute of Technology GmbH, Bioresources Unit, A-3430 Tulln, Austria

4Department of Soil Microbiology and Symbiotic Systems Estación Experimental del Zaidín,

CSIC, Apartado Postal 419, 18080-Granada, Spain 5School of Molecular Medical Sciences, Centre for Biomolecular Sciences, University of

Nottingham, Nottingham NG7 2RD, UK 6The James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK


The genus Burkholderia is composed of functionally diverse species and it can be divided into

several clusters. One of these, designated as the plant-beneficial-environmental (PBE)

Burkholderia cluster, is formed by non-pathogenic species, which in most cases have been found

to be associated with plants. It was previously established that all members of the PBE group

share an N-acyl-homoserine lactone (AHL) quorum-sensing (QS) system designated BraI/R that

produces and responds to 3-oxo-C14-HSL. Moreover, some of them also possess a second AHL

QS system, designated XenI2/R2, producing and responding to 3-hydroxy-C8-HSL (OHC8-

HSL). We performed LC-ESI-MS/MS analysis to determine which AHL molecules are produced

by each QS system of this group of bacteria. Results showed that XenI2/R2 is mainly responsible

for the production of OHC8-HSL and that the BraI/R system is involved in the production of

several different AHLs. This analysis also revealed that B. phymatum STM815 produces greater

amounts of AHLs than the other species tested. Further studies showed that the BraR protein of

B. phymatum is more promiscuous than other BraR proteins, responding equally well to several

different AHL molecules, even at low concentrations. Transcriptome studies with B. xenovorans

LB400 and B. phymatum STM815 revealed that the BraI/R regulon is species-specific, with

exopolysaccharide production being the only common phenotype regulated by this system in the

PBE cluster. In addition, BraI/R was shown not to be important for plant nodulation by B.

phymatum spp. nor for endophytic colonization and growth promotion of maize by B.

phytofirmans PsJN.


32

23

TEASING APART THE MULTIPLE ROLES OF A CzcR/CzcS-LIKE TWO-

COMPONENT SYSTEM IN BURKHOLDERIA CENOCEPACIA: VIRULENCE,

QUORUM SENSING AND HEAVY METAL RESISTANCE

Matthew T. Robinson1 and Alan R. Brown

1

1Biosciences, College of Life & Environmental Sciences, University of Exeter, Exeter. UK.

Contact: Alan Brown, [email protected]

Two-component systems (TCSs) are key regulators of bacterial adaptation to extracellular

stimuli and frequently play a pivotal role in the regulation of virulence. We have previously

reported that inactivation of a CzcR/CzcS-like TCS in Burkholderia cenocepacia results in

hypersensitivity to heavy metals and attenuated virulence (Robinson & Brown, IBCWG 2012).

Here we report further characterization of the virulence-related phenotypes associated with this

CzcR/CzcS-like TCS, and how this TCS facilitates an effective response to heavy metal stress

(using zinc as a model). We confirm that both biofilm formation and intracellular survival within

macrophages are dramatically-impaired as a consequence of czcRS deletion, phenotypes that can

be fully restored by complementation. Despite the profound attenuation observed in the ΔczcRS

mutant, transcriptional profiling suggests that only a modest set of genes are actually regulated

by the TCS in response to zinc. However, studies have revealed that in the absence of zinc, the

czcRS genes are required for the full activation of the quorum sensing network, explaining the

profound virulence attenuation observed in the ΔczcRS mutant. Whilst these observations suggest

that the CzcR transcriptional regulator possesses basal activity in the absence of TCS activation

by zinc, transcriptional profiling has also confirmed the pivotal role that this CzcR/CzcS-like

TCS plays in ensuring cellular homeostasis in the presence of zinc. We conclude that the

CzcR/CzcS-like TCS in B. cenocepacia directly and indirectly regulates diverse cellular

processes within the cell that contribute to virulence and heavy metal resistance.



33

24

PROTEINS INVOLVED IN HOST CELL ATTACHMENT ARE PROTECTIVE

ANTIGENS AGAINST BCC INFECTION.

Minu Shinoy1, Marc Healy

2, Bernard Mahon

2, Máire Callaghan

1 and Siobhán McClean*

1

1Centre of Microbial Host Interactions Institute of Technology Tallaght, Dublin 24 Ireland.

2Department of Biology, National University of Ireland, Maynooth, Ireland.


Bcc is rarely eradicated once a patient has been colonised, therefore vaccination against this

pathogen may represent a better therapeutic option. Traditionally good vaccine candidates are

those which are involved in host cell attachment. We have developed a method to identify the

bacterial proteins that are involved in the attachment of Burkholderia multivorans and

Burkholderia cenocepacia to lung epithelial cells. Bacterial proteins were extracted from B.

multivorans strains (LMG13010 or C1962) or B. cenocepacia strains (BC-7 or C1394), separated

on 2-D gels and transferred to a membrane, before probing with lung epithelial cells (CFBE41o-

or 16HBE14o- cells). Proteins involved in attachment were identified by MALDI-ToF MS. A

total of twenty proteins were identified by 2-DE from all the four strains of Bcc. Out of these,

seven proteins were found to be common to both species, but only two were common to all four

strains.

These two proteins were selected for further study and were cloned into E. coli, expressed and

purified by chromatography. In both cases the recombinant E. coli strain showed more binding

than the wild type (12.5- and 8.7- fold), confirming that these proteins play a role in binding to

epithelial cells. Immunoproteomic analysis using serum from Bcc colonised CF patients

confirmed that both proteins are expressed in vivo and each one elicits a potent humoral

response. Groups of mice were immunized individually (i.p.) with the recombinant antigens and

challenged 5 days after the booster with either B. multivorans or B. cenocepacia. Mice

immunised with BcOMP1 were protected from B. cenocepacia infection (15 Ln reductions in

lung CFU in immunized mice) and B. multivorans infection (7 Ln reductions in lung CFU).

Immunization with BcOMP2 protected against B. cenocepacia with a reduction in lung bacterial

counts by 7 Ln (60,000 fold reduction in bacterial counts, p=0) and protected against B.

multivorans by a four-Ln reduction, (p<0.013). Both antigens induced potent antigen-specific

antibody responses (IgG titres ~ 1 x 106). The ratio of IgG2a to IgG1 antibodies in response to

BcOMP1 was indicative of a potent Th1 response, while the IgG2a: IgG1 ratio in response to

BcOMP2 was indicative of a mixed Th1/Th2 response. In conclusion, both antigens are

involved in bacterial adhesion to lung cells and are likely to be good vaccine candidates in a

multi-subunit vaccine.


34

25

DEFECTIVE AUTOPHAGY ACTIVITY CONTRIBUTES TO INCREASED IL-1β

PRODUCTION DURING BURKHOLDERIA CENOCEPACIA INFECTION

Basant A. Abdulrahman1, Mia Tazi

1, Anwari Akhter

1, Kyle Caution

1, Hoda Hassan

1, Miguel A.

Valvano2, Benjamin Kopp

3, and Amal O. Amer

1

1

Division of Pulmonary, Allergy, Critical Care and Sleep Medicine, Center for Microbial Interface Biology and the Department of Internal Medicine, Ohio State University. Columbus, OH, USA. 2 Centre for Human Immunology, Department of Microbiology and Immunology, and The

Department of Medicine, University of Western Ontario, London, Ontario, N6A 5C1, Canada. 3 Division of Pediatric Pulmonology, Nationwide Children‟s Hospital, Columbus, OH 43205.

Contact: Amal Amer, [email protected]

IL-1β is expressed as a precursor inactive molecule in response to a Toll-Like Receptor-mediated

signal, and is later cleaved by caspase-1 to yield active IL-1β. Caspase-1 is activated within the

inflammasome complex in response to a Nod-Like-receptor (NLR) signal. The biological

activities of IL-1β include promoting inflammatory responses and leukocyte infiltration.

Recently, autophagy has emerged as an important innate immune response to infection and is

required to limit IL-1β production. Hence, insufficient autophagy has been implicated in disease

conditions characterized by excessive IL-1β production such as Crohn‟s disease and arthritis.

Autophagy is an evolutionary conserved, catabolic process that involves the entrapment of

cytoplasmic components within characteristic vesicles for their delivery to and degradation

within lysosomes. Autophagy is regulated via a group of genes called AuTophaGy related Genes

(ATGs) and is executed at basal levels in all cells for maintaining cellular integrity. IL-1β level is

elevated in bronchoalveolar lavages (BALs) of cystic fibrosis (CF) patients which suffer from

exacerbated inflammation leading to lung tissue damage. However, the underlying mechanism

leading to IL-1β exacerbated production is not understood. Additionally, the source of IL-1β is

not clear. Here we show that CF macrophages produce significantly high amounts of IL-1β when

infected with B. cenocepacia. IL-1β pro-form is produced downstream of TLR4 and is cleaved to

its active form via the pyrin inflammasome. We also show that IL-1β exacerbated production is

due to defective autophagy in CF macrophages. Moreover, B. cenocepacia down regulates

autophagy molecules upon infection of CF macrophages. Thus, IL-1β production is reduced in

response to autophagy stimulating agents. Interestingly, recent studies demonstrated that

epithelial cells do not release IL-1β in response to B. cenocepacia. Together, these data suggest

that IL-1β is primarily produced by activated macrophages and this production is uncontrolled in

CF macrophages due to impaired autophagy activity.



35

26

AUTOPHAGY STIMULATION BY IFN-γ IMPROVES BURKHOLDERIA

CENOCEPACIA CLEARANCE IN HUMAN CYSTIC FIBROSIS MACROPHAGES

Ben Kopp1,2

, Kaivon Assani2, and Amal Amer

3

1Section of Pediatric Pulmonology, Nationwide Children‟s Hospital, Columbus, OH USA

2Center for Microbial Pathogenesis, The Research Institute at Nationwide Children‟s Hospital,

Columbus, OH USA 3Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH

Contact : Ben Kopp, [email protected]

Introduction: Burkholderia cenocepacia is the most feared bacteria for cystic fibrosis (CF)

patients to acquire due to a severe spectrum of disease with diminished long term survival due to

ineffective antibiotic treatment. Autophagy is a physiologic process that involves engulfing non-

functional organelles and delivering them to the lysosome for degradation, but also plays a role

in eliminating intracellular pathogens. Deficiencies in autophagy have been shown in CF, and a

murine CF model infected with B. cenocepacia demonstrates reduced bacterial load and

inflammation when pre-treated with the autophagy stimulator rapamycin. Therefore, we propose

that induction of autophagy in human CF macrophages infected with B. cenocepacia will

improve bacterial clearance, limiting chronic inflammatory stimulus and host injury.

Methods: Peripheral blood monocyte derived macrophages were obtained from 20 CF and 20

non-CF donors at the Nationwide Children‟s Hospital. Macrophages were infected with B.

cenocepacia clinical isolate strain k56-2 at an MOI of 10 and treated with autophagy inducers

rapamycin or IFN-γ and for 4 and 24 hours. Host-bacterial interactions were determined with

confocal microscopy. Autophagic flux was monitored through the use of autophagy inhibitors

combined with protein turnover detected on western blotting.

Results: CF macrophages have diminished IFN-γ production in supernatants after stimulation

with B. cenocepacia compared to controls. CF macrophages infected with B. cenocepacia

demonstrate reduced bacterial load with a 24 hour IFN-γ treatment, nearing non CF bacterial

load levels. Initiation of autophagy is seen in CF macrophages after 4 hours of IFN γ treatment

by the co-localization of bacteria with the autophagy marker LC3. Autophagy co-localization

was not demonstrated in untreated CF macrophages. Bacterial loads are still similar for CF

macrophages between a 4 hour treatment and no treatment, but diminish greatly by 24 hours.

Cell death is reduced by 48 hours after IFN-γ treatment. IL-1β production is significantly

reduced with rapamycin treatment, with a trend towards reduction with IFN-γ. There is a

significantly higher proportion of bacteria co-localizing with the autophagy docking molecule

P62/SQSTM1 (p62) in CF macrophages after treatment. Non CF macrophages demonstrate

decreased bacterial load compared to untreated CF macrophages, early autophagy initiation with

co-localization of bacteria to autophagosomes, and sustained clearance of bacteria through 4 or

24 hours regardless of IFN-γ treatment. Treatment with rapamycin also diminishes bacterial load

in CF macrophages. The addition of 3-MA to block autophagosome formation negates the

benefits of IFN-γ in CF macrophages.

Conclusions: Autophagy stimulation improves human CF macrophage clearance of B.

cenocepacia with subsequent reductions in host inflammation and cell death. The mechanisms

by which it enhances autophagic flux in CF patients are the focus of ongoing studies, but are

suggestive of enhanced autophagosome formation.


36

27

A BURKHOLDERIA CENOCEPACIA GENE ENCODING A NON-FUNCTIONAL PHOSPHOTYROSINE PHOSPHATASE IS REQUIRED FOR THE DELAYED MATURATION OF THE BACTERIA-CONTAINING VACUOLES IN MACROPHAGES

Angel Andrade1 and Miguel Valvano

1,2

1Department of Microbiology and Immunology. Centre for Human Immunology, University of

Western Ontario, London, Ontario, Canada. N6A 5C1. 2Centre for Infection and Immunity. Queen's University of Belfast, Belfast, UK.


Burkholderia cepacia complex (Bcc) are a group of highly problematic opportunistic pathogens

causing chronic infections in patients with cystic fibrosis (CF) (1). The Bcc members display

high levels of intrinsic antibiotic resistance, persistence in the presence of antimicrobials, and

intracellular survival capabilities. The intracellular persistence of Burkholderia cenocepacia (a

member of the Bcc) has been associated with subverting the host cell endocytic pathway to

prevent the acidification and fusion of the phagolysosome. Previous reports showed that

intracellular B. cenocepacia arrests the assembly of the NADPH oxidase complex on the

phagocytic vacuole (BcCV) membrane, and impairs the activation of Rab7. Although delay of

NADPH oxidase assembly depends on a defect in Rac1 and Cdc42 activation, the mechanism by

which B. cenocepacia precludes fusion of the phagosome with lysosomes and persists within the

macrophage remains poorly defined. Because bacterial phosphatases have been associated with

virulence, contributing to the intracellular survival of pathogens in this work we focused on

investigate a possible role of tyrosine phosphatases in B. cenocepacia pathogenesis. Four genes

encoding predicted low molecular weight protein tyrosine phosphatases (PTP´s) were identified

in B. cenocepacia genome (BCAM0208, BCAM0628, BCAM0857 and BCAL2200). First, the four

4 ptp genes were cloned into a plasmid and expressed in B. cenocepacia to evaluate their

secretion as putative virulence factors. Secretion assays indicated that under the evaluated

conditions, BCAM0628 is the only PTP secreted by B. cenocepacia, pointing toward a potential

role of BCAM0628 interacting with a tyrosine phosphorylated host substrate. Using pulsed

macrophages with the Fluorescein Isothiocyanate-Dextran or LAMP-1 as probes for lysosomal

fusion markers showed differences between parental B. cenocepacia BCAM0628 strains.

Suggesting a contribution of BCAM0628 delaying the maturation of the BcCV´s. However,

gentamicin protection assays showed no difference in survival between wild type strain and any

ptp mutant strain. Recombinant histidine-taged versions of BCAM0628 and BCAL2200

were purified to homogeneity and phosphatase activity was evaluated using the general

chromogenic substrate pNPP. Phosphatase activity assays indicate that BCAM0628 is an inactive

enzyme most likely due to amino acid substitution in the catalytic core of the protein. However,

this is the only PTP that becomes secreted to the supernatant and is likely to have an effect on the

intracellular infection. Together, our data support a role of BCAM0628 affecting the host

machinery involved in late endosome maturation.


37

28

MODELING OF THE INNATE IMMUNE RESPONSE TO INFECTION WITH

BACTERIA BELONGING TO THE BURKHOLDERIA CEPACIA COMPLEX USING

ZEBRAFISH EMBRYOS

Jennifer Mesureur1, Julien Rougeot

2, David O‟Callaghan

1, Annemarie Meijer

2, Annette

Vergunst1

1INSERM U1047. Université de Montpellier 1. UFR Médecine, Nimes, France

2Institute of Biology, Leiden University, Leiden, The Netherlands


Chronic respiratory infection in cystic fibrosis patients is characterized by a high level of pro-

inflammatory cytokines, leukocyte infiltration, and inflammation in the lungs due to colonization

by pathogenic bacteria. Particularly infections caused by Burkholderia cepacia complex (Bcc)

bacteria can progress into fatal respiratory inflammation and septicaemia, and infections with

these bacteria are associated with poor prognosis for patients. Several lines of evidence, using

murine and cell culture models, have shown that B. cenocepacia induces a particularly high pro-

inflammatory response (including secretion of the neutrophil chemo attractant IL-8, and IL-1β),

and that bacterial virulence factors, including LPS and flagellin, play a role in this. Importantly,

after phagocytosis, Bcc have been shown to manipulate macrophage host cell biology in vitro,

leading to bacterial survival and intracellular multiplication, and this intracellular survival

strategy has been proposed to contribute significantly to pathogenesis.

We are using zebrafish embryos to study in detail the role of host phagocytes in bacterial

virulence and the mechanism of induction of an excessive pro-inflammatory response in vivo.

We have reported that B. cenocepacia K56-2 belonging to the epidemic ET12 lineage survives

and multiplies in macrophages of zebrafish embryos prior to dissemination, and systemic fatal

infection. Our recent data show that K56-2 induces a very rapid bacterial dose-dependent strong

increase in pro-inflammatory gene expression that is much higher than the response elicited by

heat-killed K56-2 or a less pathogenic strain such as B. stabilis LMG14294, which causes

persistent infection. Using a combination of transgenic reporter fish lines, morpholino gene

knock down, deep sequencing of RNA and bacterial mutants, we are now further dissecting the

role of host phagocytes, and bacterial factors and host signaling pathways that lead to the

excessive pro-inflammatory response. We will further report on the importance of an

intramacrophage stage for progress of infection in this in vivo model, and our current analysis of

the host transcriptome in virulent versus persistent infection.


38

29

MODELLING BURKHOLDERIA CEPACIA COMPLEX ROOT COLONIZATION IN

VITRO AS AN IMPORTANT ENVIRONMENTAL TRAIT

J. Cristian Vidal-Quist1, Louise A. O‟Sullivan

1, Annaëlle Desert

1, Amanda S. Fivian-Hughes

2,

Angela M. Marchbank1, Coralie Millet

1, T. Hefin Jones

1, Andrew J. Weightman

1, Hilary J.

Rogers1, Colin Berry

1 and Eshwar Mahenthiralingam

1*

1 Organisms and Environment Division, Cardiff School of Biosciences, Cardiff University,

Cardiff, UK. 2

Imperial College, London, UK.

*Contact: [email protected]

Burkholderia cepacia complex (Bcc) bacteria occur widely in the natural environment and are

particularly dominant around the plant rhizosphere. This region around the roots is the site of

many biotechnologically useful interactions including the rhizoremediation of toxic pollutants

and the secretion of bioactive metabolites that promote the control of pathogenic fungi or

nematodes. We assessed the fitness of different Bcc species in the rhizosphere by developing an

in vitro model of root colonization using the pea (Pisum sativum) and the model plant,

Arabidopsis thaliana. A panel of 26 genetically diverse Bcc strains representing 9 different

species and different sources (clinical versus environmental) was assembled. Their rhizosphere

interactions were then systematically compared using the two species of plant. Initial

development of the A. thaliana microcosm was greatly facilitated by the use of a lux-tagged

Burkholderia vietnamiensis G4 reporter strain. Bacteria colonising the roots was determined

using viable counts and by direct observation through bioluminescence or electron microscopy.

All Bcc species colonized the roots of both plant models in vitro at levels significantly greater

than that seen for two non-rhizosphere control species, Staphylococcus aureus and Escherichia

coli K12. In general, the source of the Bcc strain, either clinical or environmental, did not

influence their root colonization phenotype. However, for species such as Burkholderia

ambifaria, strains of an environmental origin possessed greater rhizosphere colonization rates

than clinical isolates. In previous studies, the genetic basis for pea root colonization was assessed

using signature-tagged-mutagenesis of B. vietnamiensis G4. Further exploration of the genetics

of Burkholderia rhizosphere colonisation was performed with the A. thaliana model using five

mutated Bcc strains (representing 5 species) which lacked their third chromosomal replicon.

Deletion of this replicon representing greater than 1 Mb of DNA (1000 genes), did not

significantly alter the ability of these strains to colonize A. thaliana roots. Overall our data

indicate that the ability to colonize the rhizosphere is an intrinsic trait of Burkholderia cepacia

complex bacteria.



39

30

REAL TIME ANALYSIS OF BURKHOLDERIA CENOCEPACIA ST32 ISOLATES IN A

ZEBRAFISH EMBRYO MODEL

Klara Vilimovska Dedeckova1, Pavel Drevinek

1, Annette Vergunst

2

1Charles University, 2

nd Faculty of Medicine,

Department of Medical Microbiology, Prague,

Czech Republic 2

INSERM U1047, Universite´ de Montpellier 1, UFR Médecine, Nimes, France

Contact: Klara Vilimovska Dedeckova, [email protected]

Burkholderia cenocepacia sequence type 32 (ST32) represents one of the globally distributed

strains from the Burkholderia cepacia complex (Bcc), which infected 30% of the Czech cystic

fibrosis (CF) patients. The aim of our study was to analyse virulence potential of several of these

ST32 isolates in an animal model, and to identify specific changes in virulence that might occur

over time.

Using a zebrafish embryo infection model, earlier we found large differences between 31

different ST32 isolates isolated from 14 different patients in their ability to cause embryo death.

Fourteen isolates were virulent (most embryos were killed between 3 and 6 dpi). Thirteen

isolates showed intermediate virulence, with between 50-80% mortality at 6 dpi. Four isolates

were not very virulent, and most embryos were still alive at 6 dpi, the end point of the infection

experiments. Interestingly, isolates originating from cepacia syndrome seemed less virulent in

the model than isolates that originated from earlier time points of chronic and/or exacerbated

stages of infection in the same patient.

The zebrafish embryo model is highly amenable to real time in vivo imaging of infection with

fluorescently labeled bacteria. Intravenous microinjection of virulent strains, such as K56-2, is

followed by rapid phagocytosis of bacteria by macrophages (Vergunst et al. 2010), bacterial

intracellular survival and multiplication from 7 hpi onwards, followed by bacterial systemic

dissemination and rapid (2 dpi) embryo death. Less virulent strains however are unable to

multiply efficiently, or unable to disseminate and do not cause fatal infection.

Based on our previous mortality assays in zebrafish embryos we analyzed some of the ST32

isolates in more detail in real time using fluorescence microscopy. We will discuss our findings

showing that a less-virulent isolate (as defined by the previous zebrafish experiment results),

originating from a patient with cepacia syndrome is unable to survive and replicate in

macrophages, whereas an isolate from preceding stages of infection of the same patient was

highly virulent in zebrafish. Further, our preliminary data suggest differences in phagocytic

behaviour by macrophages between an intermediately virulent cepacia syndrome isolate and its

virulent counterpart from the same patient. We will discuss how the zebrafish model may help us

in defining potential differences between isolates during the subsequent stages of infection, and

how this may be combined with other genomic tools to better understand the virulence potential

of ST32.

Supported by: GAUK 307311; IGA MZ NT12405-5; MS MT LD11029



40

31

EXPLORING THE TOPOLOGY OF ArnT FROM Bulkholderia cenopacia

Faviola Tavares-Carreón1 and Miguel A Valvano

1,2.

1Department of Microbiology and Immunology, Centre for Human Immunology, University of

Western Ontario, London, Ontario, Canada. N6A 5C1 2Centre for Infection and Immunity. Queen's University of Belfast, Belfast, UK.


Burkholderia cenocepacia is an opportunistic pathogen displaying high resistance to

antimicrobial peptides and polymyxins. This pathogen cause severe lung infections in patients

with cystic fibrosis, which are difficult to eradicate. Lipopolysaccharide (LPS) is embedded in

the outer leaflet of the outer membrane of Gram-negative bacteria and contributes to the

structural integrity of the bacterial cell. LPS is a complex molecule consisting of lipid A, core

oligosaccharide (OS), and in some strains, O antigen and acts as endotoxin to elicit strong

inflammatory responses in mammals.

LPS may also be modified by the addition of sugars or phosphate groups, which allows

adaptation to stress condition or resistance to several antibiotics including antimicrobial

peptides. In many Gram-negative bacteria, L-Ara4N (4-amino-4-deoxy-L-arabinose) is added to

lipid A conferring increased resistance to antimicrobial peptides. In B. cenocepacia ArnT protein

modify the LPS by adding L-Ara4N at two different positions: the phosphates in the lipid A and

Ko (D-glycero-D-talo-oct-2-ulosonic acid) residue in the core OS. These modifications in B.

cenocepacia are also critical for bacterial viability. Therefore, we are interested in investigating

the structure-function of ArnT as this protein could be a possible target to develop novel

antibiotics. Understanding the structure function of ArnT, an integral membrane protein that

exerts its enzymatic function on the periplasmic side of the membrane, requires elucidation of its

topology. So far, experimentally based topological model had just been proposed for ArnT from

Salmonella enterica serovar Typhimurium. The topology of ArnT from B. cenocepacia has not

been characterized and also functional regions or residues in the protein are unknown. Therefore,

the elucidation of an accurate topological map of ArnT is essential to understand its function. In

this study, we began to characterize the topology of the ArnT protein from B. cenocepacia. The

C-terminal of ArnT is the less conserved region of this protein and we observed that is essential

for their function and to confer polymyxin B resistance. Interestingly, through LacZ and PhoA

fusions we identified that C-terminal of ArnT is located in the periplasm, which is opposite from

reported for ArnT of Salmonella. Now, we are focus on get a refined topological model of B.

cenocepacia ArnT protein by PEGylation assay. PEGylation is a method for assessing

topological accessibilities through protein modification by the covalent attachment with

monomethoxy-polyethylene glycol-maleimide (mPEG-Mal), and detection using a gel shift

assay. With this method, will be able to provide an ArnT topological map that can be extended to

other integral membrane proteins involved in Lipid A modification.


41

32

STRUCTURAL DIFFERENCES BETWEEN THE GENOMES OF

BURKHOLDERIA CENOCEPACIA STRAINS K56-2 AND J2315

Andrea M. Sass1, Silvia Cardona

3, Miguel A.Valvano

4, Tom Coenye

1, Eshwar

Mahenthiralingam2

1Laboratorium voor Farmaceutische Microbiologie, Universiteit Gent, Gent, Belgium

2Organisms and Environment Division, Cardiff School of Biosciences, Cardiff University,

Cardiff, Wales, UK 3Department of Microbiology, University of Manitoba, Winnipeg, Canada

4Centre for Infection and Immunity, Queen's University of Belfast, Belfast, UK

Contact : Andrea Sass, [email protected]

For molecular biological studies involving Burkholderia cenocepacia, strain K56-2 has often

been used due to its relatively high amenability for genetic manipulation compared to the

genome reference strain J2315. However, a complete genome sequence for K56-2 is not yet

available. Three datasets of high-throughput genome sequencing data are currently available at

NCBI for strain K56-2, all derived from sequencing at the Craig Venter Institute. We examined

these sequences and tested several methods for sequencing assembly. The best results, i.e. the

smallest number of contigs and the largest scaffold, were achieved with multi-span (3 kb and 8

kb) paired end Titanium 454 sequencing data, assembled with the GS assembler which uses the

Newbler algorithm. The assembly produced 798 contigs with an average size of 10580

nucleotides and an N50 contig size of 20000 nucleotides, organised in 7 large scaffolds with

together 7.25 Mb; the largest scaffold being 3.2 Mb in size.

Burkholderia genomes are large and rich in genes occurring in multiple copies, such as rRNA

genes and transposases, but these facts alone cannot account for the relatively high contig

number. To analyse the cause of the high contig number, mapping to the reference genome

sequence of B. cenocepacia J2315 was performed.

The genomes of both strains appear to be largely similar. Certain large genomic regions present

in J2315 are not present in K56-2. For example, the duplicated region on the large replicon of

J2315 (BCAL0969-1928 are identical to BCAL2845-2901) is not duplicated in K56-2. The

duplication within J2315 had let to an inversion within the large replicon. Genomic islands GI3

(BCAL0409a-0420) and GI8 (BCAL2475-2600) on the large replicon of J2315 are not present in

K56-2. A phage and adjacent genes on small replicon of J2315 are deleted from K56-2

(BCAS0440-BCAS0571). Additionally, numerous transposases and SNPs within the K56-2

genome could be mapped with relatively high probability.

Many contig breaks appear to be caused by genes that are present in K56-2, but contain regions

without mapped reads which are apparently not caused by deletion events. More often than not

these regions are correlated with an above average GC-content of >80%. This points to a locally

high-GC content impairing one or several steps within the sequencing workflow, leaving high-

GC regions un-sequenced and probably also causing locally increased number of sequencing

errors. Increased occurrence of sequencing errors can in turn lead to mis-assemblies.

In conclusion, the assembly of a high quality draft genome does not appear possible from the

currently available K56-2 sequencing data. Extensive further sequencing across contig gaps,

potential misassemblies and all transposases will need to be performed to enable accurate

assembly. Sequencing should be repeated with a method able to give reliable results for high-GC

DNA regions, ideally in combination with a method providing reads with higher average length

than transposase genes. A draft assembly, in multiple contigs, and a list of transposases and

SNPs with their location is available for basic comparisons of J2315 with K56-2.



42

33

COMPARATIVE FUNCTIONAL ANALYSIS OF METABOLIC ABILITIES OF BCC

SPECIES USING RECONCILED GENOME-SCALE METABOLIC

RECONSTRUCTIONS

Jennifer A. Bartell*1, Phillip Yen*

1, John J. Varga

2, Joanna B. Goldberg

2 and Jason A. Papin

1

1Biomedical Engineering, University of Virginia, Charlottesville, VA, USA

2Pediatric, Emory University, Atlanta, GA, USA


Motivation: Cystic fibrosis patients show increased morbidity when infected with species of the

Burkholderia cepacia complex. Studies suggest B. multivorans is the most prevalent Bcc

pathogen in CF, while B. cenocepacia is considered more virulent with higher associated risk of

cepacia syndrome and death. Based on our prior work with Pseudomonas aeruginosa, we

hypothesized that unique metabolic capacities could contribute to B. cenocepacia‟s increased

virulence and sought to quantify functional differences between species using metabolic

modeling.

Methods: To investigate potential mechanisms for this differential virulence and quantitatively

compare metabolic capacity, we built genome scale metabolic reconstructions using genome

annotations of B. cenocepacia J2315 (BC) and B. multivorans ATCC17616 (BM). The

reconstructions connect reactions and metabolites using a stoichiometric matrix and link genes to

their corresponding reactions. Reaction activity is predicted using linear optimization to

maximize the flux through a Burkholderia-specific biomass synthesis reaction (representing

growth). We used a free computational resource to produce initial reconciled draft

reconstructions. By then curating the reconstructions using literature and databases (MetaCyc,

KEGG, etc), we ensured that any differences between the final models were biologically relevant

rather than artifacts of model syntax. We used Biolog and M9 minimal media growth screening

to guide our reconstruction of amino acid catabolism. We also incorporated lipid production

pathways from a previously published smaller model of B. cenocepacia, and expanded their

reconstruction of virulence-related pathways.

Results: BC model iPY1520 accounts for 1520 genes and 1512 reactions, while BM model

iJB1301 accounts for 1301 genes and 1434 reactions. The 122 unique reactions in iPY1520 and

60 unique reactions in iJB1301 are concentrated in fatty acid synthesis, galactose metabolism,

and starch metabolism, but unique reactions were spread through more than 40 canonical KEGG

pathways. iPY1520 and iJB1301 predict 74 and 80 essential genes respectively for growth on

rich media in silico, and match available growth screening data at an accuracy of 84% and

85.7%, respectively. iPY1520 shows enhanced capacity to produce an array of virulence factors,

functional differences in several amino acid catabolic pathways, and a greater number of

metabolic isozymes, suggesting potential increased metabolic adaptability. These models

account for hundreds of additional genes and reactions compared to similar curated

reconstructions. They can be integrated with transcriptomic, proteomic, and metabolomic data to

functionally characterize metabolic differences for specific growth conditions and adapted to

related Bcc strains of interest, enabling genome scale comparative study of metabolism for these

important pathogens.


43

34

INVESTIGATING FACTORS INVOLVED IN THE MAINTENANCE OF THE

‘CHROMOSOME 3’ MEGAPLASMID

Kirsty Agnoli1, Carmen Frauenknecht

1 and Leo Eberl

1

1Department of Microbiology, Institute of Plant Biology, University of Zürich.


We have previously reported that Bcc chromosome 3 is a non-essential megaplasmid in

many complex members, and suggested that this element be renamed pC3. To date,

representatives of all Bcc species except B. cepacia have been cured of pC3. Amongst these

pC3-null derivatives are three B. cenocepacia strains (H111, MCO-3 and HI2424), however the

highly transmissible ET-12 lineage strain K56-2 remained resistant to such curing.

Integration of an origin of transfer into K56-2 pC3 allowed its transfer into an H111 pC3-

null derivative. K56-2 pC3 could not be cured from this strain, suggesting that the resistance of

K56-2 pC3 to curing was most likely due to the presence of efficient toxin-antitoxin systems

(TASs), rather than essential genes. A scan of putative pC3 contigs from K56-2 using the web-

based TAS finder RASTA-bacteria revealed the presence of two candidate TASs and one lone

antitoxin. Heterologous introduction of the antitoxin components allowed K56-2 to be cured of

pC3 using plasmid incompatibility. Investigations into the pathogenicity of pC3-null K56-2 are

on-going.

To investigate the stability of pC3, a derivative of B. cenocepacia H111 was constructed,

bearing a trimethoprim resistance gene controlled by a tightly regulated LacI repressable

promoter on chromosome 1, and on pC3 lacI. This strain was used to evaluate the rate of pC3

loss under nutrient-rich growth conditions. The rate of loss was calculated at 0.000147

/generation, and it was found that although the experimentally measured exponential growth

rates of wt and pC3-null H111 appeared identical, the pC3-null derivative had a slight growth

advantage over its parent strain (0.017 %).

The prevalence of pC3 was assessed by screening the 111 Bcc isolates available within

our laboratory collection for the presence of pC3 using PCR, pulsed-field gel electrophoresis and

Southern hybridisation. Only five strains were found which appeared to lack pC3, suggestive of

high selective pressures for its maintenance within the Bcc. The nature of these pressures is

currently under investigation.


44

35

GLOBAL MAPPING OF PRIMARY TRANSCRIPTION START SITES IN THE

BURKHOLDERIA CENOCEPACIA J2315 GENOME BY DIFFERENTIAL RNA

SEQUENCING

Andrea M Sass1, Heleen van Acker

1, Konrad Förstner

2, Jörg Vogel

2, Tom Coenye

1

1Laboratorium voor Farmaceutische Microbiologie, Universiteit Gent, Gent, Belgium

2Institute for Molecular Infection Biology, University of Würzburg, Würzburg, Germany

Contact: Andrea Sass, [email protected]

Since the event of transcriptomics by high-throughput sequencing, an unexpected complexity

within prokaryotic transcriptomes has been revealed. In particular, large numbers of small non-

coding RNAs have been discovered due to RNA-sequencing, and have since been found to play

an important role in regulation of gene expression in bacteria.

To analyse the transcriptome of B. cenocepacia for presence of non-coding RNAs, RNA

extracted from biofilm-grown cells was sequenced with a recently developed method aiming at

specifically identifying primary transcription start sites. RNA sequencing was performed without

fragmentation and mRNA enrichment. cDNA was generated by priming from only the 3‟-end,

and sequencing was performed from only the 5‟-end. The majority of sequencing reads located

therefore at the 5‟-end of transcripts. Prior to cDNA generation, a portion of the RNA samples

was treated with an processive exonuclease specific for 5‟-ends with a monophosphate.

Most mRNAs and non-coding RNAs carry a 5‟-triphosphate, whereas most rRNAs and tRNAs

are processed and carry a 5‟-monophosphate. Exonuclease treatment leads to an enrichment of

primary transcripts and a depletion of processed RNA. Comparison of the exonuclease-treated

sequencing libraries with untreated libraries (differential RNA sequencing) allows therefore to

precisely map the first nucleotide of a transcript, the transcription start site (TSS), by identifying

sites with peaks in read-coverage that increase in magnitude in the exonuclease-treated sample

portion. This allows more accurate analysis of 5‟-UTRs of annotated genes and of promoter

elements such as Pribnow boxes, and identification of primary transcribed non-coding RNAs.

Applied to B. cenocepacia J2315 biofilms, this method revealed high numbers of TSS in

intergenic regions, associated with adjacent genes or orphan. In most cases of intergenic regions

harbouring transcription initiation, multiple TSS occurred on both strands, regardless of the

orientation of adjacent genes. TSS also occurred in high numbers within annotated genes, in

sense and antisense direction. Antisense transcription generally occurred on a large scale.

Numerous putative non-coding RNAs could be identified, and the criteria for defining non-

coding RNAs by this method could be evaluated.

In addition to mapping TSSs and putative non-coding RNAs, the differential RNA sequencing

dataset allowed identification of genes that are expressed in a B. cenocepacia J2315 biofilm, by

evaluating reads mapping to the first 100 bases upstream of a primary TSS of annotated genes.

Numerous primary TSS were located upstream of the first base of an annotated gene, indicating

putatively misannotated genes. TSS mapping also allowed definition of genomic islands as

sequence regions with a locally increased number of intergenic TSS, and in this way additional

previously unannotated genomic islands could be identified.


1

Int'l Burkholderia cepacia Working Group Conference Roster

April 10 - April 13, 2013 University of Michigan, Ann Arbor, MI 48109

NAME TITLE ORGANIZATION E-MAIL ADDRESS ADDRESS TELEPHONE Agnoli, Kirsty

Post-doctoral researcher

University of Zurich

[email protected]

Zollikerstrasse 107 , Zurich, ZH CH-8008 USA

(+41) 0446348268

Amer, Amal Associate Professor

Ohio State University [email protected] 4647 Wilkin Court , New Albany, OH 43054 USA

614 247 1566

Andrade, Angel Postdoctoral Fellow

University of Western Ontario

[email protected] 1151 Richmond Street DSB, Room 3014, Department of Microbiology and Immunology, London, ON N6A 5C1 CANADA

(519) 661-3433

Bartell, Jennifer Grad Student University of Virginia [email protected] 425 Lane Rd MR5 Rm 2041, Charlottesville, VA 22908 USA

8045147986

Bernier, Steve Postdoctoral Fellow

McMaster University [email protected] 1280,Main St. West Department of Medicine, McMaster University, Rm HSC 3N4, Hamilton, ON L8S4K1 Canada

905-525-9140 ext:21905

Brahma, Vijaya Postdoctoral Researcher

University of Toronto [email protected] 101 College Street Latner Thoracic Research Laboratories,Lab 2-501, Toronto, ON M5G 1L7 CANADA

16477807123

2

Brown, Alan Dr University of Exeter [email protected] Geoffrey Pope Building, Stocker Road Biosciences, Exeter, EXETER EX4 4QD UK

01392725526

Burns, Jane Professor Seattle Children's Research Institute/Univ of Washington

[email protected] 1900 Ninth Avenue C9S8-8th Floor, Seattle, WA 98101 USA

206-987-2073

Cardenas, Erick Post Doc, Fellow Michigan State University

[email protected] Dept. of Microbiology & Immununology 2350 Health Sciences Mall, Vancouver, V6T 1Z3 Canada

809-822-5646

Cardona, Silvia Associate Professor

University of Manitoba [email protected] 45 Chancellors Circle Department of Microbiology, Winnipeg, MB R3T 2N2 Canada

1-204-474-8997

Cescutti, Paola Dr Univ Trieste [email protected] via L. Giorgieri 1, Ed C11 Dept Life Sciences, Trieste, - 34127 Italy

+39 040 5588755

Chaparro, Cecilia Respirologist University of Toronto [email protected] 485 University Av CSB - 11C-1190, Toronto, On M5M 3J6 USA

416-340-4996

Clark, Shawn Graduate Student

University of Toronto [email protected] 101 College Street TMDT-2nd Floor, Toronto, ON M5G 1L7 Canada

905-334-6101

Coenye, Tom Prof. Ghent University [email protected] Harelbekestraat 72 , Gent, - 9000 Belgium

+3292648141

3

Cooper, Vaughn Assoc Professor of Microbiology and Genetics

University of New Hampshire

[email protected] 46 College Rd 212 Rudman Hall, University of New Hampshire, Durham, NH 03824 USA

6039887590

Dedeckova, Klara Msc./PhD. student

Charles University in Prague, 2nd Faculty of Medicine

[email protected] V Úvalu 84 , Prague, CZ 150 06 Czech Republic

2 2443 2026

Degrossi, José Ph.D. Universidad de Buenos Aires

[email protected] Junín 956 , Buenos Aires, 1113 Argentina

54114964-8257

Dennehy, Ruth Postgraduate student

ITT Dublin [email protected] Old Blessington road CASH centre, Dublin, Ireland Ireland

00353857120947

Eberl, Leo Prof. University of Zurich [email protected] Zollikerstrasse 107 Dept. of Microbiology, Zurich, ZH 8008 CH

+41446348220

Goldberg, Joanna Professor Emory University School of Medicine

[email protected] 1510 Clifton Rd NE Suite 3009, Atlanta, GA 30322 USA

404-727-6760

Gonzalez, Carlos Professor Texas A&M University [email protected] 120Peterson; 2132 TAMU Department of Plant Pathology and Microbiology, College Station, TX 77843-2132 USA

979-220-6622

Greenberg, David Assistant Professor

University of Southwestern Medical Center

[email protected] 5323 Harry Hines Boulevard Y9.206C MC 9113, Dallas, TX 75390 USA

214-648-2458

4

Hwang, David Pathologist University of Toronto [email protected] 200 Elizabeth Street Department of Pathology, Toronto, ON M5G 2C4 Canada

416-340-4988

Jenul, Christian PhD student University of Zurich [email protected] Zollikerstrasse 107 , Zürich, ZH 8008 Switzerland

0041 44 63 48227

Juarez-Perez, Victor Scientific Advisor ALAXIA Company [email protected] batiment Adénine 60 avenue Rockefeller, Lyon, 69008 FRANCE

33 437 532 640

Kalferstova, Lucie MSc/PhD student

2nd Faculty of Medicine, Charles University in Prague

[email protected] V Uvalu 84 , Prague, CZ 15006 Czech Republic

+420 2 2443 2026

Kopp, Benjamin Assistant Professor

Nationwide Children's Hospital/The Ohio State University

[email protected] 700 Children's Drive , Columbus, OH 43205 USA

6142262497

Lieberman, Tami PhD Student Harvard Medical School

[email protected] 200 Longwood Avenue, WA 536 , Boston, MA 02115

732-501-6444

Lopez De Volder, Agustina

PhD Student Universidad de Buenos Aires

[email protected] Faultad de Farmacia y Bioquimica Viamonte 444, Buenos Aires CUIT, 30-54666656-1 Argentina

54 011 49648200

Mahenthiralingam, Eshwar

Professor Cardiff University [email protected] Main Building, Museum Avenue Rm. 023, Cardiff, SG CF10 3AT USA

+442920875875

McClean, Siobhan Dr ITT Dublin [email protected] Old Blessington Road Centre of Microbial Host Interactions, Tallaght, D24 Ireland

+35314042794

5

Peeters, Charlotte PhD student Ghent University [email protected] Ledeganckstraat 35 , Gent, Fl 9000 Belgium

+32.9.264.5130

Pessi Ahrens, Gabriella

Dr University of Zürich/ Institut of Plant Biology

[email protected] Winterthurerstrasse 190 Microbiology Department, Zürich, ZH 8057 Switzerland

0041446352904

Priebe, Greg Assoc. Prof. of Anesthesia

Boston Children's Hospital

[email protected] 300 Longwood Ave Bader 634, Boston, MA 02115 USA

617-794-0175

Sass, Andrea Dr. Ghent University [email protected] Harelbekestraat 72 , Gent, - 9000 Belgium

+3292648141

Schmid, Nadine MSc Institute of Plant Biology,University of Zurich

[email protected] Zollikerstrasse 107 , Zürich, ZH 8008 Switzerland

+41795626477

Sokol, Pam Professor Emeritus

University of Calgary [email protected] 3330 Hospital Dr NW Department ofMicrobiology, Immunology and Infectious Disease, Calgary, AB T2N4N1 Canada

480-982-0532

Tavares-Carreon, Faviola

Dr. University of Western Ontario

[email protected] 1151 Richmond Street DSB, Room 3014, Department of Microbiology and Immunology, London, ON N6A5C1 CANADA

(519) 661-3433

Tiedje, James M. University Distinguished Professor and Director

Michigan State University

[email protected] Michigan State University 540 Plant and Soil Science Building, East Lansing, MI 48824-1325 USA

517-353-9021

6

Tullis, Elizabeth CF Clinic Director

University of Toronto [email protected] 83 Humberview Road , Toronto, ON M6S 1W9 Canada

416 864 5406

Van Acker, Heleen PhD student Ghent University [email protected] Harelbekestraat 72 , Gent, - 9000 Belgium

+3292648141

Vandamme, Peter Professor Ghent University [email protected] Ledeganckstraat 35 , Gent, Fl 9000 Belgium

+32.9.264.5113

Venturi, Vittorio Group Leader ICGEB [email protected] Padriciano 99 Bacteriology, Trieste, I 34149 Italy

+390403757319

Vergunst, Annette Dr Universite de Montpellier/ INSERM

[email protected] 186 chemin du Carreau de lanes INSERM U1047, UFR Medecine, Nimes, 30908 France

+33466028157

Waters, Valerie Assistant Professor

Hospital for Sick Children

[email protected] 555 University Avenue , Toronto, ON M5G 1X8 Canada

416-813-7654

Yoder-Himes, Deborah

Assistant Professor

University of Louisville [email protected] 139 Life Sciences Building , Louisville, KY 40292 USA

502-852-0991

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