�������� ����� ��

Initial adhesion of Listeria monocytogenes to fine polished stainless steelunder flow conditions is determined by prior growth conditions

Anne Skovager, Marianne Halberg Larsen, Josue Leonardo Castro-Mejia,Michael Hecker, Dirk Albrecht, Ulf Gerth, Nils Arneborg, Hanne Ingmer

PII: S0168-1605(13)00200-6DOI: doi: 10.1016/j.ijfoodmicro.2013.04.014Reference: FOOD 6177

To appear in: International Journal of Food Microbiology

Received date: 2 February 2013Revised date: 18 April 2013Accepted date: 19 April 2013

Please cite this article as: Skovager, Anne, Larsen, Marianne Halberg, Castro-Mejia,Josue Leonardo, Hecker, Michael, Albrecht, Dirk, Gerth, Ulf, Arneborg, Nils, Ingmer,Hanne, Initial adhesion of Listeria monocytogenes to fine polished stainless steel underflow conditions is determined by prior growth conditions, International Journal of FoodMicrobiology (2013), doi: 10.1016/j.ijfoodmicro.2013.04.014

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

1

Initial adhesion of Listeria monocytogenes to fine polished stainless steel under flow conditions

is determined by prior growth conditions

Anne Skovager a

Marianne Halberg Larsen b

Josue Leonardo Castro-Mejia a

Michael Hecker c

Dirk Albrecht c

Ulf Gerth c

Nils Arneborg a

Hanne Ingmer b

a Department of Food Science, Section for Food Microbiology, Faculty of Sciences, University of

Copenhagen, Denmark

b Department of Veterinary Disease Biology, Section for Microbiology, Faculty of Medical and

Health Sciences, University of Copenhagen, Denmark.

c Institute of Microbiology, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

2

Abstract

Listeria monocytogenes is a food-borne pathogen known to persist in food production

environments, where it is able to attach and form biofilms, potentially contaminating food product

ready for consumption. In this study the first step in the establishment of L. monocytogenes in a

food-processing environment was examined, namely the initial adhesion to stainless steel under

specific dynamic flow conditions. It was found that the intrinsic ability of L. monocytogenes to

adhere to solid surfaces under flow conditions is dependent on nutrient availability. Addition of L-

leucine to growth medium altered the fatty acid composition of the L. monocytogenes cells and

increased adhesion. The growth conditions resulting in the highest adhesion (growth medium with

added glucose) had cells with the highest electron donating and lowest electron accepting

properties, whereas growth conditions resulting in lowest adhesion (growth medium with added

mannose) had cells with the lowest electron donating properties and highest electron accepting

properties. The highest and lowest adhesion conditions correlated with differences in expression of

cell surface protein of L. monocytogenes and among these the autolysin amidase (Ami). This study

implies that food composition influences the adhesion of L. monocytogenes to solid surfaces during

dynamic flow conditions.

Keywords

Listeria monocytogenes, initial adhesion, carbohydrates, L-leucine, surface proteins, fatty acids

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

3

Introduction

One of the pathogens of particular concern in food processing environments is Listeria

monocytogenes, that causes listeriosis in humans (Painter and Slutsker, 2007). Although cases of

listeriosis are relatively rare; the mortality rate is high and at risk are immunocompromised, elderly

and the foetus of pregnant women (Bhunia, 2008; Todd and Notermans, 2011). L. monocytogenes

has an ability to persist in various food producing environments (Ortiz et al., 2010; Carpentier and

Cerf, 2011). Strains have been found to persist from months to several years in pig slaughter houses

and processing facilities (Ortiz et al., 2010), in fermented meat sausage production sites (Ferreira et

al., 2011), in cheese production environments (Fox et al., 2011) and in fish slaughter houses (Wulff

et al., 2006). It has recently been suggested, that it may not be strains of L. monocytogenes with

unique properties that lead to persistence, but harborage sites in food industry premises and

equipment, where cleaning is difficult and nutrients are available, that makes L. monocytogenes able

to grow and persist (Carpentier and Cerf, 2011). The first step in L. monocytogenes establishment at

a harborage site is its ability to attach to solid surfaces. Adhesion is a multifactorial process, and the

degree of adhesion may be dependent on many factors such as solid surface properties and

environmental conditions (Ploux et al., 2010). Some studies show that persistent strains of L.

monocytogenes adhere to surfaces and form biofilms more easily than strains not associated with

persistence, implying that adherence and biofilm formation on surfaces are important for

persistence of L. monocytogenes in the food processing environment (Lundén et al., 2000; Norwood

and Gilmour, 1999;). If a biofilm is formed, it may protect microorganisms against cleaning and

disinfection (Chavant et al., 2004) and allow pathogens like L. monocytogenes to establish in the

food processing environment. Even a limited number of L. monocytogenes cells may eventually

form biofilm potentially contaminating food products (Møretrø and Langsrud, 2004; Shi and Zhu,

2009; Simões et al., 2010).

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

4

Although little is known about the physiological conditions that predispose L.

monocytogenes for persistence, nutrient availability may influence the intrinsic ability of L.

monocytogenes to adhere (Palmer et al., 2007). Cell surface characteristics differ in response to

variations in growth condition and have also been found to influence adhesion and biofilm

formation (Briandet et al., 1999a; Briandet et al., 1999b; Chavant et al., 2002; Di Bonaventura et al.,

2008; Gordesli and Abu-Lail, 2012; Jensen et al., 2007; Tresse et al., 2006; ; Zhou et al., 2012). It

has been shown that different growth temperatures, growth media, and storage temperatures,

influence the physiochemical properties of L. monocytogenes and thereby adhesion and biofilm

formation (Briandet et al., 1999a; Briandet et al., 1999b; Chavant et al., 2002; Di Bonaventura et al.,

2008; Stepanović et al., 2004). While carbon sources (glucose, cellobiose, fructose, mannose and

trehalose) do not influence attachment to stainless steel under static conditions, an increase in

ammonium chloride and decrease in iron concentration in the growth medium result in a decreased

attachment (Kim and Frank, 1994). In contrast, a similar study found biofilm development to be

influenced by the presence of mannose and trehalose and various levels of phosphate and amino

acids (Kim and Frank, 1995). Additionally, Briandet et al. (1999b) showed that addition of glucose

to trypticase soy broth supplemented with 6g yeast extract (TSYE) alters the physicochemical

properties of L. monocytogenes. Neither of these studies evaluated how specific nutrients, such as

carbohydrates and amino acids, influence the very initial adhesion process in combination with how

the specific growth conditions influence cell physiochemical properties; and if high or low adhesion

ability, due to specific nutrient availability, could be due to other factors such as alteration of

surface protein expression and fatty acid composition.

Studies evaluating the influence of growth conditions and nutrient availability on

adhesion have primarily been done under static conditions and have been evaluated after extended

periods of time (Briandet et al., 1999a; Briandet et al., 1999b; Chavant et al., 2002; Di Bonaventura

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

5

et al., 2008; Kim and Frank, 1994; Stepanović et al., 2004). However, in food production systems L.

monocytogenes are often exposed to sheer stress conditions, and adhesion under such conditions is

critical for biofilm establishment (Doijad et al., 2011; Gudbjörnsdóttir et al., 2004; Perni et al.,

2006; Silva et al., 2003; ). L. monocytogenes has been isolated form high shear environments, such

as vats and pipes in milk processing environments (Doijad et al., 2011; Perni et al., 2007; Silva et

al., 2003) and cooking facilities, flow lines, and RTE-food production drains in meat processing

environments (Gudbjörnsdóttir et al., 2004). Application of flow perfusion systems allows

determination of real-time initial adhesion at single cell level, making it possible to monitor the

very initial adhesion step (Skovager et al., 2012). The aim of the present study was to examine the

influence of single nutrient components (mannose, glucose and L-leucine) on the ability of L.

monocytogenes to adhere to fine polished stainless steel under flow; and the contribution of cellular

macromolecules to the process.

Materials and methods

Strain and growth conditions

The GFP labelled, fluorescent Listeria monocytogenes strain EGDe/pNF8 (strain EGDe was

obtained from Werner Goebel (Biozentrum)) (Fortinea et al., 2000; Larsen et al., 2006) was used in

the present study. The strain was maintained on Tryptone Soya Agar (TSA) (Oxoid) supplemented

with 5 µg/ml erythromycin (erm) at 5°C, inoculated into 100 ml Tryptone Soya Broth (TSB)

(Oxoid) with a total of 1% (w/v) glucose and 5 µg/ml erm, and grown at 37°C with agitation (225

rpm) for 24 h. Subsequently, each strain was re-inoculated (25 µL per 10 ml) into fresh TSB (Difco,

without dextrose) but containing 5 µg/ml erm and different nutrients namely either 2.5 g/L glucose

or 2.5 g/L mannose (designated glu-medium and man-medium, respectively) or supplemented

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

6

additionally with 100 mM L-Leucine (designated glu+leu-medium and man+leu-medium,

respectively). Cells were grown for 22 h (stationary phase) at 37°C with agitation (225 rpm). The

cell culture was washed twice in 0.15 M NaCl (Merck) (4000 x g, 5 min).

Preparation for flow perfusion experiments

Surface characteristics and preparation of fine polished stainless steel coupons

Fine polished stainless steel, SS 304 (FPSS) (Outo Kumpu, Sheffield, UK) was prepared as

described in Skovager et al. (2012). In short, FPSS was cut into coupons (7.5 cm x 3.5 cm) using a

guillotine. The surface characteristics of the fine polished stainless steel can be found in Skovager et

al. (2012). The steel coupons were soaked and rinsed in acetone overnight, after which they were

rinsed in 96 % alcohol for 5 min. Finally, the steel coupons were rinsed with distilled water and air

dried, standing on a table overnight. CoverWell Perfusion chambers (622503, PC3L-0.5,

CoverWell, Grace Bio-Labs, Inc) were glued (Super attak, Loctite, Henkel Norden AB) onto the

surfaces. Silicone tubes (Watson Marlow Alitea) were glued on the inlet (ID: 1.6 mm; Wall: 1.6

mm) and the outlet (ID: 4.8 mm; Wall: 1.6 mm) of the perfusion chambers (Skovager et al., 2012).

Flow perfusion experiments

The optical density (600 nm) of the prepared cell suspension was adjusted to 0.100 ±0.005,

corresponding to a cell density of approx. 8·107 CFU/ml, determined by pour plating on TSA

medium (Oxoid) + 5 µg/ml erm.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

7

The same setup and procedure as described by Skovager et al. (2012) was used. However, another

epi-fluorescence microscope (Zeiss Axioplan 2, Carl Zeiss; mercury lamp: HBO100w) and digital

camera (Leica DFC340 FX) controlled by the LAS Software V3.6.0 (Leica) were used. Fluorescent

images were captured after 1 min, 3 min, 5 min, 10 min and 15 min of perfusion. For each image,

cells were excited for 4-6 seconds (excitation filter BP450-490; beam splitter FT 510 and emission

filter BP 515-5650) visualizing GFP labelled cells. For each of three biological replicates and for

each growth condition, triplicate surfaces were used. Wall shear stress was set to 0.10Pa (shear

rate~100s-1

) corresponding to a flow rate of 0.75 ml/min. Image analysis and calculation of initial

adhesion rate (IAR) were made as described by Skovager et al. (2012).

Microbial Adhesion To Solvents (MATS) analysis

The MATS -analysis was carried out as described previously (Bellon-Fontaine et al., 1996), with

minor modifications as described by Skovager et al. (2012). In short, L. monocytogenes was grown

as previously described. Stationary phase cells were harvested (4000 x g, 5 min) and the supernatant

discarded. The cells were washed twice in 0.15 M NaCl (4000 x g, 5 min), and re-suspended in 0.15

M NaCl to OD400 ≈ 0.8 (A0 value) (UV-1800 Shimadzu spectrophotometer). Two ml of cell

suspension was added to a test tube with 0.4 ml of one of the following solvents, chloroform

(Merck), hexadecane (Sigma-Aldricht), ethyl acetate (Merck), or decane (Fluka). The cell

suspensions were vortexed with the solvent for 1 min. The emulsions were left to stand for 15 min

to allow phase separation, and the OD400 (A value) of the aqueous phase was measured. Affinity of

the cells for each solvent was calculated by use of equation 1:

% affinity = 100 (1 - A/A0) (1)

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

8

A0 and A are the OD400 values of the cell suspension in the aqueous phase before and after mixing,

respectively. Analysis were carried out in triplicate.

Determination of cell size and sedimentation velocity

Stationary phase cells were harvested and washed twice in 0.15 M NaCl (4000 x g, 5 min), placed

on an objective glass slide under the microscope with a 100 x magnification (Zeiss), and an image

was acquired. Cell length and width were measured automatically by use of ImageJ and calibrated

to a µm scale using a micrometer (Leica). In each experiment, 70-82 cells were analysed. An

estimation of the sedimentation velocity of the cells was calculated by using Stokes law:

Vs = (D2

g Δρ) / (18µ) (2)

Where D is the cell diameter calculated as an average of cell length and width, g is gravity (9.807

m/s2), Δp, cell density minus water density (89 kg /m

3) and µ, viscosity of water (9.04 · 10

-4 kg / (m

s)). A cell density of 1089 kg/m3 was adopted from experiments with the Gram positive, rod-shaped

Bacillus cereus (Fukushima et al., 2007), since no such value seems to be available for L.

monocytogenes.

Extraction and analysis of fatty acids

Fatty acid analyses were carried out by the Identification Service of the DSMZ, Braunschweig,

Germany. The prepared cell suspensions were centrifuged (4000 x g, 10 min) and the cell pellet was

frozen at -80°C. Fatty acid methyl esters were obtained from 40 mg cell pellet by saponification,

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

9

methylation and extraction using minor modifications of the method of Miller (1982) and

Kuykendall et al. (1988). The fatty acid methyl esters mixtures were separated using Sherlock

Microbial Identification System (MIS) (MIDI, Microbial ID, Newark, DE 19711 U.S.A.) which

consisted of an Agilent model 6890N gas chromatograph fitted with a 5% phenyl-methyl silicone

capillary column (0.2 mm x 25 m), a flame ionization detector, Agilent model 7683A automatic

sampler, and a HP-computer with MIDI data base (Hewlett-Packard Co., Palo Alto, California,

U.S.A.). Peaks were automatically integrated and fatty acid names and percentages calculated by

the MIS Standard Software (Microbial ID). The gas chromatographic parameters were as follows:

carrier gas, ultra-high-purity hydrogen; column head pressure 60 kPa; injection volume 2 µl;

column split ratio, 100:1; septum purge 5 ml/min; column temperature, 170 to 270°C at 5°C/min;

injection port temperature, 240°C; and detector temperature, 300°C (Kuykendall et al., 1988;

Kämpfer and Kroppenstedt, 1996).

Extraction of cell wall proteins

L. monocytogenes cells were prepared as described above and cell wall proteins were extracted as

described by Schaumburg et al. (2004) with minor modifications. Cells (400ml medium) were

centrifuged (4000 x g) for 12 min. Pelleted cells were re-suspended in 1 mL 1 M Tris, pH 7.5 and

incubated for 30 min at 37°C under gentle shaking (225 rpm). Cells were centrifuged at 4000 x g

for 12 min. The supernatant containing the solubilised cell wall associated proteins was removed

and stored at -20°C. The proteins were precipitated in ice cold acetone over night at -20°C. The

proteins were centrifuged (15000 x g, 4°C, 15 min) and the pellet was solubilized in 8M urea.

Protein concentration was determined by use of 2D Quant kit (80-6483-56, GE Healthcare Life

Science).

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

10

Aminopeptidase C assay

To verify the purity of cell wall proteins, the protein extract was screened for the absence of

cytoplasmic contaminants. Therefore, p-nitroanilide derivatives that can be converted by the strictly

cytoplasmic enzyme aminopeptidase C (PepC) in a photometrically measurable reaction at 405 nm

were added to the surface fractions. This was done as described by Schaumburg et al. (2004). Ten

µL of freshly prepared cell wall extract were added to 190 µL 20 mM Tris-HCl, pH 7.4, in a

microtiter-plate. After mixing with 2 µL L-arginine-p-nitroanilide (Sigma) in 20 mM Tris, pH 7.4,

samples were immediately assayed at 405 nm for 10 min (Thermo Scientific Varioskan® Flash,

Cat. no. N06355). As positive control, 5 units of amino-peptidase from Aeromonas proteolytica

(Sigma) in 20 mM Tris, pH 7.4 were used instead of cell wall fractions.

2-D PAGE and mass spectrometry

For iso-electrical focusing (IEF), the Multiphore II system from GE Healthcare was used with 200

μg of protein sample loaded onto Immobiline dry strips (7 cm, pH 4–7; GE Healthcare). For the

second dimension, 12% SDS-PAGE gels were run with Mini-Protean cells (Bio-Rad). After

separation, the protein gels were fixed with 40% v/v ethanol and 10% v/v acetic acid for 1 h and

subsequently stained with Coomassie blue R-250 and de-stained (25% methanol, 7.5% acetic acid).

2-D-protein gel analysis was performed with 3 biological replicates.

For mass spectrometry identification, the protein spots were excised from the stained

2-D gel using a sterile pipette tip. Cut spots were transferred into 96 well microtiter plates. The

tryptic digest with subsequent spotting on a MALDI-target was carried out automatically with the

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

11

Ettan Spot Handling Workstation (Amersham Biosciences, Uppsala, Sweden) using a modified

standard protocol (Eymann et al., 2004).

The MALDI-TOF measurement was carried out on the 4800 MALDI TOF/TOF

Analyzer (Applied Biosystems, Foster City, CA, USA). This instrument is designed for high

throughput measurement, being automatically able to measure the samples, calibrate the spectra,

and analyze the data using the 4000 Explorer™ Software V3.5.3. The spectra were recorded in a

mass range from 900 to 3700 Da with a focus mass of 2000 Da. For one main spectrum 25 sub-

spectra with 100 shots per sub-spectrum were accumulated using a random search pattern. If the

autolytical fragment of trypsin with the mono-isotopic (M+H)+ m/z at 2211.104 reached a signal to

noise ratio (S/N) of at least 10, an internal calibration was automatically performed as one-point-

calibration using this peak. The standard mass deviation was less than 0.15 Da. If the automatic

mode failed the calibration was carried out manually.

From the TOF-spectra the three strongest peaks were measured. For one main

spectrum, 20 sub-spectra with 125 shots per sub-spectrum were accumulated using a random search

pattern. The internal calibration was automatically performed as one-point-calibration with the

mono-isotopic Arginine (M+H)+ m/z at 175,119 or Lysine (M+H)+ m/z at 147,107 reached a signal

to noise ratio (S/N) of at least 5. The peak lists were created by using the script of the GPS

Explorer™ Software Version 3.6 (build 332). Settings for TOF-MS were a mass range from 900 to

3700 Da, a peak density of 20 peaks per 200 Da, maximal 65 peaks per spot and an S/N ratio of 15.

The TOF-TOF-MS settings were a mass range from 60 to Precursor - 20 Da, a peak density of 50

peaks per 200 Da and maximal 65 peaks per precursor. The peak list was created for an S/N ratio of

10. For database search the Mascot search engine Version: 2.1.04 (Matrix Science Ltd, London,

UK) with a specific Listeria monocytogenes NCBI based sequence database was used. Protein

scores greater than 66 were considered significant (p < 0.05).

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

12

Image analysis of 2D gels

2D gel images were imported and analysed using ImageMaster™ 2D Platinum version 5.0 (GE

Healthcare). The parameters used for optimum spot detection were: Smooth, 2; Min. Area, 41; and

Saliency, 6. The spots of interest within each gel were paired with a reference gel and automatically

matched according to their landmarks. Spot quantification was carried out by means of their relative

spot volume (spot volume percentage as normalized data). Subsequently, the relative spot volume

ratios, derived from the different growth conditions, were used to analyse the protein expressions.

Statistical analysis

Statistical analysis of IAR, MATS and cell size were carried out using one-way ANOVA, and

multiple comparisons between groups were made using LSD (Least significant difference) test in

the SAS software (SAS Institute, Inc.). Probabilities below 0.05 were considered significant.

Results and discussion

Initial adhesion of L. monocytogenes is influenced by growth with specific nutrients

Adhesion is the first step in the establishment of L. monocytogenes in food processing environment

and it may be crucial for persistence and development of biofilms (Lundén et al., 2000; Norwood

and Gilmour, 1999). Recently, a flow perfusion system was combined with fluorescence

microscopy to determine real-time initial adhesion to non-transparent stainless steel surfaces under

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

13

flow conditions (Skovager et al. 2012). Bacterial cells were grown in TSB medium with defined

carbohydrate composition (glucose or mannose) and amino acid composition (± L-leucine). The

lowest IAR was observed for cells grown in medium added mannose and it was significantly

(p<0.05) lower than for all other growth conditions (Table 1). The significant (p<0.05) highest IAR

was found for cells grown in glu+leu-medium. Thus, the type of carbohydrate available during L.

monocytogenes growth clearly affects the initial adhesion under flow conditions. Similar differences

were not recognized in a previous study (Kim and Frank, 1994), which could be due to static

conditions employed in that experiment. Further, it was observed that addition of an amino acid (L-

leucine) to the growth medium resulted in an increase in IAR compared to when only a

carbohydrate (glucose or mannose) was added (Table 1), demonstrating that L-leucine has a specific

effect on adhesion. This finding agrees with a previous study (Kim and Frank, 1995) and indicates

that amino acids influence adhesion both under static and flow conditions.

Effect of nutrients on physicochemical properties of L. monocytogenes

To address if the differences in adhesion, due to specific nutrients availability, were related to

changes in physicochemical properties, the hydrophobicity and electron donating and electron

accepting abilities were monitored by use of MATS - analysis. A high affinity to non-polar solvents

(decane and hexadecane) indicates high surface hydrophobicity and low affinity indicates low cell

surface hydrophobicity. Measuring the electron donating/accepting properties is based on

comparison between cell affinities towards two pairs of solvents; each pair comprising a polar and a

nonpolar solvent. The polar solvent is either an electron acceptor (e.g. chloroform) or an electron

donor (e.g. ethyl acetate). By comparing the cell affinity to solvent pair, comprising chloroform,

indicates the ability of a cell surface to donate electrons. By comparing the cell affinity to the

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

14

solvent pair, comprising ethyl acetate, indicates the ability of the cell surface to accept electrons

(Bellon-Fontaine et al., 1996). The affinity towards hydrocarbons is dependent on the pH and the

ionic composition of the cell suspension, as electrostatic interactions may influence the interaction

between the adhesions of the microorganism to the hydrocarbons in solution (Busscher et al., 1995;

Van der Mei et al., 1995). In this study, the same cell suspension, for all MATS analysis as well as

initial adhesion experiments, was used, thus rendering our results comparable. As previously

observed (Briandet et al., 1999a; Briandet et al., 1999b), L. monocytogenes cells, under all growth

conditions in the present study, had higher tendency to donate electrons from their cell surface than

to accept electrons; i.e. the chloroform-hexadecane values were higher than the ethyl acetate-decane

values (Table 2). The growth conditions that resulted in the highest tendency to donate electrons

from the cell surface were the glu-medium (27.9%) and glu+leu (24.3%) medium. The electron

donating capability was low for all growth conditions, however highest for cells grown with

man+leu medium (-9.9%) and man-medium (-15.2%).

Addition of glucose (2.5 g/L) to the growth medium results in better adhesion, less

electron accepting and more electron donating properties of L. monocytogenes compared to addition

of mannose and this effect was independent on the presence of L-leucine. Such effect has not

previously been observed. It has been assumed that more electron donating properties of the cells

may result in less adhesion to FPSS, due to the FPSS having more electron donating than accepting

properties (Briandet et al., 1999a; Skovager et al., 2012; Van Oss, 1993). Glucose added to TSYE

medium have previously been found to result in more electron donating properties (Briandet et al.,

1999b). Additionally, it has been found that there may be a relationship between L. monocytogenes

affinity to ethyl acetate and adhesion to stainless steel (Briandet et al., 1999b). However, such

relationship was not found in the present study. This could be due to the hydrodynamic forces

dominating under flow conditions, neglecting the predictability of adhesion in relation to

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

15

physiochemical interactions (Katsikogianni and Missirlis, 2010). Additionally, this study implies

that specific cellular macromolecules may be involved in the adhesion ability. This could also

explain why no relationship was seen between adhesion and hydrophobicity of L. monocytogenes

related to nutrient availability (Table 1 and Figure 1). In general, the affinity to decane and

hexadecane of the cells grown under different growth conditions, were in the range 9.9-39.5%,

indicating moderate to low hydrophobicity of the cell surface. Cells grown in glu+leu-medium

appeared most hydrophobic, as the affinity towards decane is significantly higher (p<0.05) for

glu+leu-medium (27.4%) compared to the other growth conditions, whereas the significant lowest

affinity towards decane was found for cells grown in man+leu-medium (9.9%) (Figure 1).

Specific nutrients influence on cell size and sedimentation velocity of L. monocytogenes

As the shear rate in the flow system is lower than approximately 300s-1

, and the size of L.

monocytogenes cell is greater than 0.2 µm, gravity becomes important for the deposition of

particles/bacteria in a flow system (Adamczyk and van de Ven, 1981; Adams and Moss, 2004;

Yiantsios and Karabelas, 2003). Therefore, the effect of cell size and sedimentation velocity of L.

monocytogenes as influenced by the growth conditions was examined. Listeria monocytogenes

grown in glu+leu-medium had an average cell length of 2.39 µm and was significantly (p<0.05)

smaller than L. monocytogenes cells grown in the other media (Table 2). The sedimentation

velocity, calculated on the basis of cell length and width, was greatest when grown in glu+leu-

medium (0.16 µm/s). As the cell size of the cells grown in glu-medium, man-medium and man+leu-

medium were similar, the sedimentation velocity was the same for these growth conditions. The

smaller cell size of L. monocytogenes, grown in glu+leu-medium, could account for best adhesion,

compared to the other growth conditions. The dimensions of the smaller cells may fit better into the

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

16

structures of the fine polished stainless steel (Skovager et al., 2012), however this may be

counteracted by the effect of gravity, that may influence the adhesion under flow conditions

(Adamczyk and Van de Ven, 1981; Li et al., 2011a; Li et al., 2011; Yiantsios and Karabelas, 2003).

This could indicate that the geometrical fit of the cells in the structures of the FPSS is more

important for the adhesion than the gravity of the cells.

Influence of specific nutrients on fatty acid composition

Fatty acid analysis was conducted to examine the effect of the different growth media on fatty acid

composition and potential influence on the cell surface characteristics and adhesion (Table 3). The

sum of anteiso (C15:0 and C17:0) fatty acids was decreased when the L. monocytogenes cells were

grown with L-leucine compared to when grown without added L-leucine. Furthermore, the sum of

iso (C17:0 and C15:0) fatty acids and the sum of iso (C14:0 and C16:0) fatty acids were increased

compared to when no L-leucine was added to the growth medium. There was no difference in fatty

acid composition when L. monocytogenes cells was grown with different sugars (glucose and

mannose); however a slightly higher sum of iso (C14:0 and C16:0) fatty acids was seen when cells

was grown in glu+leu-medium. The anteiso/iso ratio was markedly lower when L-leucine was

added to the growth medium (ratio: 0.6) compared to when no L-leucine was added to the medium

(ratio: 6.8) (Table 3).

It has been suggested by Gianotti et al. (2008) that cell fatty acid composition and

lipid metabolism are involved in the adhesion of L. monocytogenes to surfaces. They found that

acid stress changed the fatty acid composition and adhesion to glass surfaces. The present study

confirms that there may be such a relationship, however as a consequence of nutrient availability.

Addition of L-leucine to the growth medium, a precursor for synthesis of odd-numbered iso fatty

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

17

acids of L. monocytogenes (Annous et al., 1997; Julotok et al., 2010), was found both to alter the

fatty acid composition (decreased ante/iso ratio) of the L. monocytogenes and to improve its ability

to adhere to stainless steel. Alteration in fatty acid composition of Listeria has previously been

found to alter the hydrophobic characteristics and the fluidity of the L. monocytogenes cell (Julotok

et al., 2010; Moorman et al., 2008), which potentially could influence the adhesion ability. In the

present study, the change in fatty acid composition and initial adhesion could not be correlated to

the hydrophobic and the electron donating/accepting characteristics of the L. monocytogenes cells,

although these factors are believed to play a role in adhesion (McLandsborough et al., 2006).

However, the change from higher to lower anteiso/iso ratio may result in more rigid cell membrane

(Julotok et al., 2010) allowing it to stick more or better into the surface features of the stainless

steel. Changes in cell rigidity as a consequence of altered fatty acid composition, could also be

interfering with the membrane and membrane associated functions (Tasara and Stephan, 2006),

potentially resulting in altered adhesion ability.

Influence of specific nutrients on surface protein expression

The adhesive properties of bacterial cells may also be influenced by various surface proteins.

Therefore cell surface protein expression was analysed of L. monocytogenes cells grown in glu+leu-

medium and man-medium corresponding in highest and lowest IAR, respectively. The cell surface

protein extracts were examined for the presence of aminopeptidase C to ensure that no

contamination from the cytoplasm had occured during cell wall extraction (Schaumburg et al.,

2004), and no enzymatic activity was observed (data not shown).

The surface proteins expressed differently on the 2D-gels (see Figure S1,

supplementary material) from the two different growth conditions were identified and their relative

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

18

spot volume ratios were determined (Table 4). It was observed that autolysin and oligopeptide ABC

transporter protein are more highly expressed when L. monocytogenes is grown in glu+leu-medium

compared to man-medium. On the contrary, subunit of Dps, Sod, glyceraldehyde-3-phosphate

dehydrogenase, GroEL, EF-Tu and dihydrodipicolinate reductase proteins were present in greater

amounts in cells grown in man-medium compared to glu+leu-medium (Table 4). The majority of

these proteins have previously been isolated from the cell surface of L. monocytogenes (Sod,

autolysin, glyceraldehyde-3-phosphate dehydrogenase, GroEL, elongation factor Tu, oligopeptide

ABC transporter) (Mujahid et al., 2007; Schaumburg et al., 2004;), although some of these proteins

may be considered as cytoplasmic proteins (glyceraldehyde 3-phosphat dehydrogenase,

elongationfactor Tu, GroEL, Sod) (Mujahid et al., 2007). Mujahid et al. (2007) suggested that

cytoplasmic proteins identified on bacterial cell surfaces may have “moonlighting” functions which

may include stimulation of adhesion to surfaces.

For L. monocytogenes, it is generally known that flagella play a role in the initial

establishment on solid surfaces (Tresse et al., 2006; Vatanyoopaisarn et al., 2000), however they are

not likely to contribute to adhesion in the present study as they are not expressed at 37°C

(Vatanyoopaisarn et al., 2000). However, it was observed that the surface protein, autolysin was

expressed at conditions with enhanced adhesion (glu+leu-medium). Autolysin (Ami) is an autolytic

amidase associated with the cell surface of L. monocytogenes (McLaughlan and Foster, 1998;

Milohanic et al., 2004) and deletion of the corresponding gene leads to severe impairment of initial

attachment to glass and the subsequent development of a mature biofilm by L. monocytogenes on

polystyrene microtiter plates (Kumar et al., 2009). In vivo, autolysin contributes to virulence of L.

monocytogenes (Cabanes et al., 2004; Milohanic et al., 2001; Pinto et al., 2012), and stimulates

adhesion of L. monocytogenes to eukaryotic cells via cell anchoring (Milohanic et al., 2001). Thus,

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

19

enhanced expression of the autolysin could explain the increased adhesion to FPSS in the flow

system.

Conclusion

In conclusion, it was demonstrated that specific nutrients (glucose, mannose and L-leucine)

influence the intrinsic ability of L. monocytogenes to adhere to solid surfaces during flow

conditions, and thereby to establish in a food production environment where shear conditions are

common. Several surface properties were affected by conditions that stimulate adhesion including

the electron donating/accepting properties of the cell surface, the fatty acid composition and the

expression of surface associated proteins. Thus, initial adhesion of L. monocytogenes during flow

conditions appears to be a multifactorial process to which several surface properties are

contributing.

Acknowledgments

This work was supported by the Danish Research Council for Technology and Production Science,

grant no. 274-08-0291. We thank Annette Tschirner for excellent technical assistance.

References

Adamczyk, Z-., Van de Ven, T.G.M., 1981. Deposition of particles under external forces in laminar

flow through parallel-plate and cylindrical channels. Journal of Colloid and Interface Science 80,

340-356.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

20

Adams, M.R., Moss, M.O., 2004. Bacterial agents of foodborne illness. In: Food Microbiology. The

Royal Society of Chemistry, Cambridge.

Annous, B.A., Becker, L.A., Bayles, D.O., Labeda, D.P., Wilkinson, B.J., 1997. Critical role of

anteiso-C15:0 fatty acid in the growth of Listeria monocytogenes at low temperatures. Applied and

Environmental Microbiology 63, 3887-3894.

Bellon-Fontaine, M.-N., Rault, J., Van Oss, C.J., 1996. Microbial adhesion to solvents: a novel

method to determine the electron-donor/electron-acceptor or Lewis acid-base properties of

microbial cells. Colloids and Surfaces B: Biointerfaces 7, 47-53.

Bhunia, A.K., 2008. Listeria monocytogenes. In: Foodborne Microbial Pathogens. Springer, New

York, USA.

Briandet, R., Leriche, V., Carpentier, B., Bellon-Fontaine, M.N., 1999a. Effects of the growth

procedure on the surface hydrophobicity of Listeria monocytogenes cells and their adhesion to

stainless steel. Journal of Food Protection 62, 994-998.

Briandet, R., Meylheuc, T., Maher, C., Bellon-Fontaine, M.N., 1999b. Listeria monocytogenes

Scott A: cell surface charge, hydrophobicity, and electron donor and acceptor characteristics under

different environmental growth conditions. Applied and Environmental Microbiology 65, 5328-

5333.

Busscher, H.J., Van de Belt-Gritter, B., Van der Mei, H.C., 1995. Implications of microbial

adhesion to hydrocarbons for evaluating cell surface hydrophpicity 1. Zeta potentials of

hydrbocarons. Colloids and Surfaces B: Biointerfaces 5, 111-116.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

21

Cabanes, D., Dussurget, O., Dehoux, P., Cossart, P., 2004. Auto, a surface associated autolysin of

Listeria monocytogenes required for entry into eukaryotic cells and virulence. Molecular

Microbiology 51, 1601-1614.

Carpentier, B., Cerf, O., 2011. Review - Persistence of Listeria monocytogenes in food industry

equipment and premises. International Journal of Food Microbiology 145, 1-8.

Chavant, P., Gaillard-Martinie, B., Hébraud, M., 2004. Antimicrobial effects of sanitizers against

planktonic and sessile Listeria monocytogenes cells according to the growth phase. FEMS

Microbiology Letters 236, 241-8.

Chavant, P., Martinie, B., Meylheuc, T., Hebraud, M., 2002. Listeria monocytogenes LO28 :

surface physicochemical properties and ability to form biofilms at different temperatures and

growth phases. Applied and Environmental Microbiology 68, 728-737.

Di Bonaventura, G., Piccolomini, R., Paludi, D., D’Orio, V., Vergara, A., Conter, M., Ianieri, A.,

2008. Influence of temperature on biofilm formation by Listeria monocytogenes on various food-

contact surfaces: relationship with motility and cell surface hydrophobicity. Journal of Applied

Microbiology 104, 1552-1561.

Doijad, S., Barbuddhe, S.. B., Garg, S., Kalekar, S., Rodrigues, J., D’Costa, D., Bhosle, S.,

Chakraborty, T., 2011. Incidence and genetic variability of Listeria species from three milk

processing plants. Food Control 22, 1900-1904.

Eymann, C., Dreisbach, A., Albrecht, D., Bernhardt, J., Becher, D., Gentner, S., Tam, L.T., Büttner,

K., Buurman, G., Scharf, C., Venz, S., Völker, U., Hecker, M., 2004. A comprehensive proteome

map of growing Bacillus subtilis cells. Proteomics 4, 2849-76.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

22

Ferreira, V., Barbosa, J., Stasiewicz, M., Vongkamjan, K., Switt, A.M., Hogg, T., Gibbs, P.,

Teixeira, P., Wiedmann, M., 2011. Diverse geno- and phenotypes of persistent Listeria

monocytogenes isolates from fermented meat sausage production facilities in Portugal. Applied and

Environmental Microbiology 77, 2701-2715.

Fortinea, N., Trieu-Cuot, P., Gaillot, O., Pellegrini, E., Berche, P., Gaillard, J.L., 2000.

Optimization of green fluorescent protein expression vectors for in vitro and in vivo detection of

Listeria monocytogenes. Research in Microbiology 151, 353-360.

Fox, E., Hunt, K., O’Brien, M., Jordan, K., 2011. Listeria monocytogenes in Irish Farmhouse

cheese processing environments. International Journal of Food Microbiology 145, S39-S45.

Fukushima, H., Katsube, K., Hata, Y., Kishi, R., Fujiwara, S., 2007. Rapid separation and

concentration of food-borne pathogens in food samples prior to quantification by viable-cell

counting and real-time PCR. Applied and environmental microbiology 73, 92-100.

Gianotti, A., Serrazanetti, D., Sado Kamdem, S., Guerzoni, M.E., 2008. Involvement of cell fatty

acid composition and lipid metabolism in adhesion mechanism of Listeria monocytogenes.

International Journal of Food Microbiology 123, 9-17.

Gordesli, F.P., Abu-Lail, N.I., 2012. The role of growth temperature in the adhesion and mechanics

of pathogenic L. monocytogenes: an AFM study. Langmuir 28, 1360-1373.

Gudbjörnsdóttir, B., Suihko, M.-L., Gustavsson, P., Thorkelsson, G., Salo, S., Sjöberg, a.-M.,

Niclasen, O., Bredholt, S., 2004. The incidence of Listeria monocytogenes in meat, poultry and

seafood plants in the Nordic countries. Food Microbiology 21, 217-225.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

23

Jensen, A., Larsen, M.H., Ingmer, H., Vogel, B.F., Gram, L., 2007. Sodium chloride enhances

adherence and aggregation and strain variation influences invasiveness of Listeria monocytogenes

strains. Journal of Food Protection 70, 592-599.

Julotok, M., Singh, A.K., Gatto, C., Wilkinson, B.J., 2010. Influence of fatty acid precursors,

including food preservatives, on the growth and fatty acid composition of Listeria monocytogenes at

37 and 10 degrees C. Applied and Environmental Microbiology 76, 1423-1432.

Katsikogianni, M.G., Missirlis, Y.F., 2010. Bacterial adhesion onto materials with specific surface

chemistries under flow conditions. Journal of Materials Science: Materials in Medicine 21, 963-

968.

Kim, K.Y., Frank, J.F., 1994. Effect of growth nutrients on attachment of Listeria monocytogenes to

stainless steel. Journal of Food Protection 57, 720-726.

Kim, K.Y., Frank, J.F., 1995. Effect of nutrients on biofilm formation by Listeria monocytogenes

on stainless steel. Journal of Food Protection 58, 24-28.

Kumar, S., Parvathi, A., George, J., Krohne, G., Karunasagar, I., 2009. A study on the effects of

some laboratory-derived genetic mutations on biofilm formation by Listeria monocytogenes. World

Journal of Microbiology and Biotechnology 25, 527-531.

Kuykendall, L.D., Roy, M.A., Neill, J.J.O., Devine, T.E., 1988. Fatty acids, antibiotic resistance,

and deoxyribonucleic acid homology groups of Bradyrhizobiurn japonicum.International Journal of

Systematic Bacteriology 38, 358-361.

Kämpfer, P., Kroppenstedt, R.M., 1996. Numerical analysis of fatty acid patterns of coryneform

bacteria and related taxa. Canadian Journal of Microbiology 42, 989-1005.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

24

Larsen, M.H., Kallipolitis, B.H., Christiansen, J.K., Olsen, J.E., Ingmer, H., 2006. The response

regulator ResD modulates virulence gene expression in response to carbohydrates in Listeria

monocytogenes. Molecular microbiology 61, 1622-35.

Li, J., Busscher, H.J., Norde, W., Sjollema, J., 2011a. Analysis of the contribution of sedimentation

to bacterial mass transport in a parallel plate flow chamber. Colloids and Surfaces B: Biointerfaces

84, 76-81.

Li, J., Busscher, H.J., Van der Mei, H.C., Norde, W., Krom, B.P., Sjollema, J., 2011b. Analysis of

the contribution of sedimentation to bacterial mass transport in a parallel plate flow chamber: part

II: use of fluorescence imaging. Colloids and Surfaces B: Biointerfaces 87, 427-432.

Lundén, J.M., Miettinen, M.K., Autio, T.J., Korkeala, H.J., 2000. Persistent Listeria monocytogenes

strains show enhanced adherence to food contact surface after short contact times. Journal of Food

Protection 63, 1204-1207.

McLandsborough, L., Rodriguez, A., Pérez-Conesa, D., Weiss, J., 2006. Biofilms: at the interface

between biophysics and microbiology. Food Biophysics 1, 94-114.

McLaughlan, A.M., Foster, S.J., 1998. Molecular characterization of an autolytic amidase of

Listeria monocytogenes EGD. Microbiology 144, 1359-1367.

Miller, L.T., 1982. Single derivatization method for routine analysis of bacterial whole-cell fatty

acid methyl esters, including hydroxy acids. Journal of Clinical Microbiology 16, 584-586.

Milohanic, E., Jonquie, R., Glaser, P., Dehoux, P., Jacquet, C., Berche, P., Cossart, P., Gaillard, J. -

l., 2004. Sequence and binding activity of the autolysin-adhesin Ami from epidemic Listeria

monocytogenes 4b. Infection and Immunity 72, 4401-4409.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

25

Milohanic, E., Jonquières, R., Cossart, P., Berche, P., Gaillard, J.L., 2001. The autolysin Ami

contributes to the adhesion of Listeria monocytogenes to eukaryotic cells via its cell wall anchor.

Molecular Microbiology 39, 1212-1224.

Moorman, M.A., Thelemann, C.A., Zhou, S., Pestka, J.J., Linz, J.E., Ryser, E.T., 2008. Altered

hydrophobicity and membrane composition in stress-adapted Listeria innocua. Journal of Food

Protection 71, 182-185.

Mujahid, S., Pechan, T., Wang, C., 2007. Improved solubilization of surface proteins from Listeria

monocytogenes for 2-DE. Electrophoresis 28, 3998-4007.

Møretrø, T., Langsrud, S., 2004. Listeria monocytogenes: biofilm formation and persistence in

food-processing environments. Biofilms 1, 107-121.

Norwood, D.E., Gilmour, A., 1999. Adherence of Listeria monocytogenes strains to stainless steel

coupons. Journal of Applied Microbiology 86, 576-582.

Ortiz, S., López, V., Villatoro, D., López, P., Dávila, J.C., Martínez-Suárez, J.N. V., 2010. A 3-year

surveillance of the genetic diversity and persistence of Listeria monocytogenes in an Iberian pig

slaughterhouse and processing plant. Foodborne Pathogens and Disease 7, 1177-1184.

Painter, J., Slutsker, L., 2007. Listeriosis in humans, in: Ryser, E.T., Marth, E.H. (Eds.), Listeria,

Listeriosis, and Food Safety. Marcel Dekker, New York, pp. 85-109.

Palmer, J., Flint, S., Brooks, J., 2007. Bacterial cell attachment, the beginning of a biofilm. Journal

of Industrial Microbiology & Biotechnology 34, 577-588.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

26

Perni, S., Aldsworth, T.G., Jordan, S.J., Fernandes, I., Barbosa, M., Sol, M., Tenreiro, R.P.,

Chambel, L., Zilhão, I., Barata, B., Adrião, A., Faleiro, M.L., Andrew, P.W., Shama, G., 2007. The

resistance to detachment of dairy strains of Listeria monocytogenes from stainless steel by shear

stress is related to the fluid dynamic characteristics of the location of isolation. International Journal

of Food Microbiology 116, 384-390.

Perni, S., Jordan, S.J., Andrew, P.W., Shama, G., 2006. Biofilm development by Listeria innocua in

turbulent flow regimes. Food Control 17, 875-883.

Pinto, E., Marques, N., Andrew, P.W., Faleiro, M.L., 2012. Over-production of P60 family proteins,

glycolytic and stress response proteins characterizes the autolytic profile of Listeria monocytogenes.

Advances in Microbiology 02, 181-200.

Ploux, L., Ponche, A., Anselme, K., 2010. Bacteria/material interfaces: role of the material and cell

wall properties. Journal of Adhesion Science and Technology 24, 2165-2201.

Schaumburg, J., Diekmann, O., Hagendorff, P., Bergmann, S., Rohde, M., Hammerschmidt, S.,

Jänsch, L., Wehland, J., Kärst, U., 2004. The cell wall subproteome of Listeria monocytogenes.

Proteomics 4, 2991-3006.

Shi, X., Zhu, X., 2009. Biofilm formation and food safety in food industries. Trends in Food

Science & Technology 20, 407-413.

Silva, I.M.M., Almeida, R.C.C., Alves, M.A.O., Almeida, P.F., 2003. Occurrence of Listeria spp. in

critical control points and the environment of Minas Frescal cheese processing. International

Journal of Food Microbiology 81, 241-248.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

27

Simões, M., Simões, L.C., Vieira, M.J., 2010. A review of current and emergent biofilm control

strategies. LWT - Food Science and Technology 43, 573-583.

Skovager, A., Whitehead, K., Siegumfeldt, H., Ingmer, H., Verran, J., Arneborg, N., 2012.

Influence of flow direction and flow rate on the initial adhesion of seven Listeria monocytogenes

strains to fine polished stainless steel. International Journal of Food Microbiology 157, 174-81.

Stepanović, S., Cirković, I., Ranin, L., Svabić-Vlahović, M., 2004. Biofilm formation by

Salmonella spp. and Listeria monocytogenes on plastic surface. Letters in Applied Microbiology 38,

428-432.

Tasara, T., Stephan, R., 2006. Cold stress tolerance of Listeria monocytogenes: A review of

molecular adaptive mechanisms and food safety implications. Journal of Food Protection 69, 1473-

84.

Todd, E.C.D., Notermans, S., 2011. Surveillance of listeriosis and its causative pathogen, Listeria

monocytogenes. Food Control 22, 1484-1490.

Tresse, O., Lebret, V., Benezech, T., Faille, C., 2006. Comparative evaluation of adhesion, surface

properties, and surface protein composition of Listeria monocytogenes strains after cultivation at

constant pH of 5 and 7. Journal of Applied Microbiology 101, 53-62.

Van der Mei, H.C., Van de Belt-Gritter, B., Busscher, H.J., 1995. Implications of microbial

adhesion to hydrocarbons for evaluating cell surface hydrophobicity 2. Adhesion mechanisms.

Colloids and Surfaces B: Biointerfaces 5, 117-126.

Van Oss, C.J., 1993. Acid-base interfacial interactions in aqueous media. Colloids and Surfaces A:

Physicochemical and Engineering Aspects 78, 1-49.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

28

Vatanyoopaisarn, S., Nazli, A., Dodd, C.E., Rees, C.E., Waites, W.M., 2000. Effect of flagella on

initial attachment of Listeria monocytogenes to stainless steel. Applied and Environmental

Microbiology 66, 860-863.

Wulff, G., Gram, L., Ahrens, P., Vogel, B.F., 2006. One group of genetically similar Listeria

monocytogenes strains frequently dominates and persists in several fish slaughter- and

smokehouses. Applied and Environmental Microbiology 72, 4313-4322.

Yiantsios, S.G., Karabelas, A.J., 2003. Deposition of micron-sized particles on flat surfaces: effects

of hydrodynamic and physicochemical conditions on particle attachment efficiency. Chemical

Engineering Science 58, 3105-3113.

Zhou, Q., Feng, X., Zhang, Q., Feng, F., Yin, X., Shang, J., Qu, H., Luo, Q., 2012. Carbon

catabolite control is important for Listeria monocytogenes biofilm formation in response to nutrient

availability. Current Microbiology 65, 35-43.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

29

Table 1. Initial adhesion rates (IAR) of L. monocytogenes EGDe/pNF8 to fine polished stainless

steel (FPSS) after growth in different media at 37°C (22±1h). Values with different capital letters in

superscripts were significantly different (P<0.05). The IAR values should be multiplied by 105.

Values are means (± standard deviation) of three biological replicates on triplicate surfaces.

Initial adhesion rates (IAR) [cells / (min cm

2)]s

Growth media IAR

glu-medium 3.39 (±0.37)B

man-medium 2.00(±0.68)D

glu+leu-medium 4.29(±0.32)A

man+leu-medium 2.58(±0.29)C

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

30

Table 2. Results of Microbial Adhesion to Solvents (MATS) analysis, cell size

a and sedimentation

velocity. The analyses were made after the L. monocytogenes EGDe/pNF8 cells had been grown in

different media.

MATSb Sedimentation

Growth media

Chloroform-

Hexadecane (%)

Ethyl acetate-

Decane (%)

Average

cell lengthc [µm] ESV

d [µm/s]

glu-medium 27.9 -17.1 2.63(±0.07)A

0.16

man-medium 15.7 -15.2 2.71(±0.09) A

0.16

glu+leu-medium 24.3 -22.1 2.39(±0.08) B

0.14

man+leu-medium 16.9 -9.9 2.63(±0.07) A

0.16

a Values with different capital letters in superscripts, within a column, were significantly different

(P<0.05).

b MATS, Microbial Adhesion to Solvents. Values are differences of MATS-pairs (chloroform-

hexadecane and ethyl acetate-decane) from Fig 1.

c Values are means of 70-82 cells ± standard error of the mean.

d ESV, Estimated sedimentation velocity. Sedimentation velocities were estimated using the average

cell diameter (average of cell length and wide) in Stokes law (see materials and methods).

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

31

Table 3. Fatty acid composition of L. monocytogenes EGDe/pNF8 cells grown in different media at 37°C (22h±1h). Values are means for

two independent experiments ± standard deviations. Some minor fatty acid components are not shown. The “sum” colums gives the sum of

the values in the two columns to the left.

Growth

condition

% (wt/wt) of total fatty acids

Anteiso/

Iso -

ratio

%

BCFAa

Anteiso-C15:0 Anteiso-C17:0 Sum Iso-C15:0 Iso-C17:0 Sum Iso-C14:0 Iso-C16:0

Su

m

C14:0 C16:0 Sum

glu-medium 46.16 ± 0.12 38.65 ± 0.17

84.8

1 7.29 ± 0.08 3.42 ± 0.05 10.71 0.40 ± 0.01 1.21 ± 0.08 1.61 0.35 ± 0.05 1.21 ± 0.08 1.56 6.9 99

man-medium 45.44 ± 0.49 38.88 ± 0.64

84.3

2

7.33 ± 0.23 3.44 ± 0.21 10.77 0.40 ± 0.02 1.27 ± 0.14 1.67 0.38 ± 0.06 1.27 ± 0.14 1.65 6.8 98

glu + leu-medium 22.82 ± 1.60 11.96 ± 0.33

34.7

8

42.07 ±

1.31 14.79 ± 1.29 56.86 1.23 ± 0.15 1.99 ± 0.15 3.22 0.90 ± 0.13 1.99 ± 0.15 2.89 0.6 98

man+leu-medium 23.15 ±0.18 12.07 ±0.15 35.2

2

42.54 ±0.62 16.02 ±0.17 58.56 0.79 ±0.06 2.78 ±0.21 3.57 0.41 ±0.00 1.14 ±0.02 1.55 0.6 99

a BCFA, Branched chain fatty acid

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

32

Table 4. Identification of cell surface proteins of L. monocytogenes EGDe/pNF8 expressed in

glu+leu-medium (G) and man-medium (M). The cell surface proteins were separated by 2D- gel

electrophoresis and identified by MALDI TOF/TOF Analyzer/analysis.

Spot

No.

Protein name

MWa

[kDa]

pIb

Protein

Scorec

G / M

Relative spot

volume ratio (%)d

M:G

G1/M1 Chain B, X-ray structure of Dps from L.

monocytogenes.

18.0 4.82 263/255 f

G2/M2 Superoxide dismutase, Sod 22.6 5.28 318/291 2.2

G3/M3 Autolysin, amidase 63.8 9.70 333/434 0.2

G4/M4 Glyceraldehyde-3-phosphate dehydrogenase 36.3 5.20 54/141 -

G5/M5 Chaperone protein GroEL 57.3 4.72 447/310 3.8

G6/G6 Elongation factor Tu, EF-Tu 43.3 4.81 655/613 1.8

G7/G7 Oligopeptide ABC transporter, oligopeptide-binding

protein

58.3 4.87 511/482 0.7

G8/G8 Dihydrodipicolinate reductase 28.9 5.20 264/235 f

a MW, molecular weight of protein.

b pI, isoelectric point.

c Protein scores greater than 66 have been considered significant (p<0.05) identified.

d Relative spot volume ratio represents 1 pair of gels. Similar relative spot volume ratios were

observed for triplicate gels made from biological replicates. Relative spot volume ratios < 1 indicate

that protein expression was increased in glu+leu-medium compared to man-medium.

f The intensity of G spots was too low to determine the relative spot volume; however, the intensity

of M spots was higher.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

33

Figure 1. Affinities of the L. monocytogenes EGDe/pNF8 suspended in 0.15 M NaCl solution to

four different solvents used in MATS (Microbial Adhesion to Solvents) analysis. The analysis was

made after cells were grown in different growth media. Values were means ± standard deviation

(vertical bars) of three solvent affinity measurements of three biological measurements. The

columns for each type of solvent with different capital letters in superscript, were significantly

different (P<0.05). The columns representing a specific solvent are compared separately for each

growth condition.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

34

Highlights Anne Skovager manuscript:

we examined initial adhesion to stainless steel at specific dynamic flow conditions

the intrinsic ability of L. monocytogenes to adhere is dependent on nutrient availability

highest adhesion was provided by cells with the highest electron donating properties

highest adhesion correlated with specific surface proteins expression

We propose food composition influences the adhesion of L. monocytogenes