Biofilm Formation, Attachment, and Cell Hydrophobicity of

J Korean Soc Appl Biol Chem (2013) 56, 207−220

DOI 10.1007/s13765-012-3253-4

Biofilm Formation, Attachment, and Cell Hydrophobicity of Foodborne Pathogensunder Varied Environmental Conditions

Na-Young Choi · Bo-Ram Kim · Young-Min Bae · Sun-Young Lee

Received: 8 November 2012 / Accepted: 20 February 2013 / Published Online: 30 April 2013

© The Korean Society for Applied Biological Chemistry and Springer 2013

Abstract Biofilm formation, attachment and cell hydrophobicity

of foodborne pathogens, including Listeria monocytogenes,

Pseudomonas aeruginosa, and Staphylococcus aureus were

investigated under various environmental conditions such as

sodium chloride (0.5–7.0%, w/v), glucose (0.25–10.0%, w/v), pH

(6.0–6.8), temperature (25 and 37oC), incubation time (24 and 6

h), and nutrients trypic soy broth (TSB) and diluted TSB (1:10).

Biofilm formation for 24 h at 25 and 37oC and attachment for 30

min and 6 h on the surface of polystyrene were measured by the

crystal violet staining method. Cell hydrophobicity of pathogens

for 6 and 24 h at 25 and 37oC was conducted using the modified

bacterial adherence to hydrocarbons method (mBATH). Biofilm

formation and attachment of pathogens were highly influenced by

the addition of glucose and sodium chloride compared to pH. The

biofilm of all pathogens formed in TSB was greater than that in

diluted TSB. Biofilm formations of S. aureus and P. aeruginosa at

37oC were greater than that at 25oC. However, biofilm formation

of L. monocytogenes was not significantly affected by temperature.

Levels of L. monocytogenes hydrophobicity were influenced by

adding glucose and sodium chloride at 37oC, whereas levels of

hydrophobicity for other pathogens were significantly different

depending on the glucose condition (p <0.05). The results demonstrate

that biofilm formation, attachment, and hydrophobicity of pathogens

were affected by environmental conditions such as the addition of

glucose and sodium chloride. However, factors affecting biofilm

formation and cell hydrophobicity differed depending on the

pathogen type.

Keywords attachment · biofilm formation · cell hydrophobicity ·

environment factor · foodborne pathogens

Introduction

Contamination of food with pathogenic bacteria arising from the

processing environment is a significant food hygiene and safety

issue. Numerous studies have shown that Listeria monocyteogenes,

Staphylococcus aureus, and Pseudomonas aeruginosa are capable

of adhering and forming biofilms in food-processing environments

(Sundheim et al., 1992; Blackman and Frank, 1996; Mettler and

Carpentier, 1998; Poulsen, 1999; Holah and Gibson, 2000).

Pseudomonas spp. are spoilage bacteria commonly found in the

food industry. P. aeruginosa, an opportunistic pathogen, has a high

tendency to form biofilms and is resistant to disinfectants (Poulsen,

1999). In our previous study investigating the biofilm formation of

various pathogens (not published data), L. monocyteogenes, S.

aureus, and P. aeruginosa were generally strong biofilm formers

compared with others (Escherichia coli O157:H7, Cronobacter

sakazakii, Salmonella Typhimurium, and Bacillus cereus). The

formation of biofilms creates major problems in the food industry,

because they represent an important source of contamination for

foodstuffs, leading to food spoilage or disease transmission.

Particularly, biofilms allow microorganism to persist in the

environment and resist desiccation, ultraviolet light, and treatment

with antimicrobial and sanitizing agents (Borucki et al., 2003;

Folsom and Frank, 2006). L. monocytogenes strains that persist in

food industry environments form thicker biofilms than isolates

found sporadically (Lunden et al., 2000) indicating that biofilm

formation is important for the survival of pathogens in the food

industry.

Environmental factors, including pH, temperature, nutrient

composition, and bacterial characteristics play important roles in

the phenotype change from planktonic cells to the sessile form

(Herald and Zottola, 1988). Bacteria have a natural tendency to

adhere to surfaces as a survival mechanism, and bacterial

colonization of solid surfaces has been described as a basic and

natural bacterial strategy in a wide variety of environments (Hunt

N.Y. Choi · B.R. Kim · Y.M. Bae · S.Y. Lee (�)Department of Food Science and Technology, Chung-Ang University, 72-1Nae-ri, Daedeok-myeon, Anseong-si 456-756, Republic of KoreaE-mail: [email protected], [email protected]

ORIGINAL ARTICLE

208 J Korean Soc Appl Biol Chem (2013) 56, 207−220

et al., 2004). Møretrø and Langsrud (2004) reported that

environmental factors, including temperature, sugar, salt, pH, and

nutrients, which are common in foods and food-processing

environments, have impacts on L. monocytogenes adhesion and

biofilm formation. Bacterial attachment to surfaces is influenced

by physicochemical properties of the environment (temperature

and pH), surface (hydrophobicity), and the microorganism

(hydrophobicity, flagellation, and motility) (Herald and Zottola,

1988; Hood and Zottala, 1995; Chavant et al., 2002; Gorski et al.,

2003; Moltz and Martin, 2005; Folsom et al., 2006). Cell surface

hydrophobicity responds to a wide variety of environmental

factors and appears to be involved in cell-to-cell interactions;

adherence of bacteria to solid surfaces; partitioning at liquid-

liquid, solid-liquid or liquid-air interfaces; and resistance of cells

to specific treatments by organic solvents or antibiotics (Rosenberg

et al., 1980; Rosenberg and Doyle, 1990; Vanhaeche et al., 1990),

which are essential to various technologies and natural processes.

Jana et al. (2000) demonstrated that hydrophobicity of Pseudomonas

fluorescens differs depending on culture age, pH, and temperature.

Pathogens such as L. monocyteogenes, S. aureus, and P.

aeruginosa are responsible for major foodborne outbreaks due to

their wide distribution in the environment and presence in the food

industry. However, no studies have evaluated cell hydrophobicity,

biofilm formation and attachment of these pathogens under

different environmental conditions. Therefore, the present study

was conducted to investigate biofilm formation, attachment, and

hydrophobicity of L. monocyteogenes, S. aureus, and P. aeruginosa

on the surface of polystyrene under various environmental conditions,

including different sodium chloride and glucose concentrations,

pHs, temperatures, incubation times, and nutrients.

Fig. 1 Biofilm formation of Listeria monocytogenes on the surface of polystyrene incubated at 25oC for 24 h in tryptic soy broth (TSB) (A) anddiluted TSB (1:10) (B), at 37oC for 24 h in TSB (C) and diluted TSB (1:10) (D). * means are significantly different compared to control (p <0.05).

J Korean Soc Appl Biol Chem (2013) 56, 207−220 209

Materials and Methods

Bacterial strains and growth conditions. Three pathogens of 2–

3 strains each, including P. aeruginosa (ATCC 10145 and ATCC

15692), L. monocytogenes (ATCC 7644, ATCC 19114, and ATCC

19115), and S. aureus (ATCC 49444, ATCC 12692, and ATCC

12600) were obtained from the bacterial culture collection of

Chung-Ang University (Anseong, Korea) for use in this study. P.

aeruginosa. L. monocytogenes, and S. aureus, which may be

present in food, beverages, and food processing facilities, were

cultivated in tryptic soy broth (TSB: Difco Laboratories, USA)

without dextrose and diluted TSB (1:10) broth supplemented with

various concentrations of sodium chloride (0.5–7.0%, w/v),

glucose (0.25–10.0%, w/v), and pH values (6.0, 6.4, and 6.8) for

24 h at 25 and 37oC. All cultures were maintained on tryptic soy

agar (Difco Laboratories) slants at 4oC and subcultured monthly.

Biofilm formation assay. Each strain of pathogens was cultured

individually in TSB and 1:10 TSB for 24 h at 25 and 37oC. Sterile

96-well polystyrene plates were filled with 90 µL TSB and diluted

TSB (1:10) and inoculated with 10 µL of overnight-cultured

pathogens (ca. 109 CFU/mL) to form biofilms on the surface of

96-well microtiter plates. Negative control wells containing only

TSB and diluted TSB (1:10) were included in each assay. The

pathogens were incubated at 37oC for 24 h. After discarding the

medium in the microtiter plate by inversion of the plate, the wells

were rinsed three times with distilled water (200 µL/well). After

air-drying, the wells were stained with 50 µL of 0.5% crystal

violet for 5 min. Excess stain was removed by washing (5×) with

distilled water (200 µL/well). Dye bound to adherent cells was

destained with or without 50 µL of 99% ethanol, and the optical

density of each well was determined at 595 nm using a spectro-

photometer (Specronic 20 Genesys, Spectronic Instruments, USA).

Fig. 2 Attachment of Listeria monocytogenes on the surface of polystyrene incubated for 30 min in tryptic soy broth (TSB) (A) and in diluted TSB(1:10) (B) for 6 h in TSB (C) and in diluted TSB (1:10) (D). * means are significantly different compared to control (p <0.05).


Bacterial adherence to hydrocarbons (BATH) assay on

polystyrene microtiter plates. The modified BATH method

(Goulter et al., 2010) was used in this study. Three strains of each

pathogen were harvested at the exponential (6 h) and stationary

phases (24 h) at 25 and 37oC, collected by centrifugation (1000 ×

g for 10 min), washed twice, and resuspended in phosphate

buffered saline (PBS) (pH 7.2). The OD of the suspension was

adjusted with PBS to 1.0 (±0.2) at 595 nm. Fifty microliters of

bacterial cell suspensions (Ac) was measured using a spectro-

photometer, and 90 µL of bacterial cell suspension was overlaid

with 30 µL of n-nonane (Alfa Aesar, UK) as the hydrocarbon and

was added to ammonium sulfate (Kanto Chemical, Japan) at the

final concentration of 2 M. The suspensions were vortexed for 5

min (Ab). A 96-well plate containing 50 µL of the untreated cell

suspension was used as the control (Ac). The 96-well plate was

allowed to stand at room temperature for 30 min. Following the

incubation, 50 µL of the lower aqueous layer was removed, and

the OD600nm was measured. The ratio of the absorbance of the

bacterial assay tubes (Ab) to the control suspension (Ac) was

calculated as a percentage of bound cells to the hydrocarbon using

the following formula; cell hydrophobicity (%) = (Ac – Ab)/Ac ×

100

Attachment of pathogens to polystyrene. The growth of pathogens

was monitored by measuring the absorbance at 600 nm (A600)

using a spectrophotometer, and the cells were incubated until the

late exponential phase (A600 of 0.9). Five milliliters of the cultures

were centrifuged (1800 × g, 10 min), washed twice with PBS (pH

7.0), diluted to attain an A600 of approximately 0.1 with PBS,

followed by addition of 100 µL of the diluted cells to each well of

a 96-well microtiter plate. The microtiter plate was allowed to

stand for 30 min or 6 h at room temperature, after which the

supernatant, including the planktonic cells, was removed from

each well. Each well was gently washed twice using aliquots of

150 µL of deionized water. After washing, the plate was air-dried

for 5 min, followed by addition of 50 µL of 0.5% crystal violet

solution, and the cells were allowed to stain for 5 min. The excess

Fig. 3 Hydrophobicity of Listeria monocytogenes incubated at 25oC for 6 h in tryptic soy broth (TSB) (A) and diluted TSB (1:10) (B), at 25oC for24 h in TSB (C) and in diluted TSB (1:10) (D). * means are significantly different compared to control (p <0.05).


crystal violet solution was removed by gently washing the wells

four times with 150 µL of sterile deionized water. The attached

cells were air-dried for 10 min, and 50 µL of 95% ethanol was

placed in each well. Crystal violet was sufficiently eluted by

pipetting, and the initial adherence to each well was measured at

A595.

Statistical analysis. All experiments were repeated three times

with duplicate samples. Data were analyzed by analysis of

variance with the Generalized Linear Model procedure of SAS

(version 9.1, SAS Institute Inc., USA) for a completely randomized

design. Duncan’s multiple-range test was applied to establish

differences between means for each parameter.

Results

The ability of L. monocytogenes to produce biofilm on the surface

of polystyrene in TSB and diluted TSB (1:10) for 24 h at 25 and

37oC is presented in Fig. 1. Biofilm formation of L. monocytogenes

increased significantly in all media supplemented with glucose

(0.25–5%) (p <0.05). In particular, L. monocytogenes biofilm was

produced at significantly higher levels in all media supplemented

with 1% glucose than those in media without glucose (p <0.05).

At 25oC, the level of L. monocytogenes biofilm formation in

medium supplemented with 2% sodium chloride was higher than

that of medium without sodium chloride. A medium with pH of

6.0 had a significant effect on L. monocytogenes biofilm formation

(p <0.05). L. monocytogenes formed higher levels of biofilm in

TSB than those in diluted TSB (1:10). No significant difference in

L. monocytogenes biofilm levels was observed for the biofilms

formed at 25 and 37oC.

When L. monocytogenes was cultivated in TSB and diluted

TSB (1:10), attachment to the polystyrene surface was significantly

affected by attachment time (p <0.05) (Fig. 2). Attachment of L.

Fig. 4 Hydrophobicity of Listeria monocytogenes incubated at 37oC for 6 h in tryptic soy broth (TSB) (A) and diluted TSB (1:10) (B), at 37oC for24 h in TSB (C) and in diluted TSB (1:10) (D). * means are significantly different compared to control (p <0.05).


monocytogenes for 30 min on the polystyrene surfaces was greater

in TSB supplemented with (0.5 and 5%) sodium chloride and in

TSB (1:10) supplemented with 5% sodium chloride, 1% glucose,

and adjusted to a pH of 6.8 compared to that under other

conditions. Attachment of L. monocytogenes on the polystyrene

surface for 6 h was influenced by the addition of glucose in TSB

and diluted TSB (1:10). In addition, attachment in diluted TSB

(1:10) supplemented with sodium chloride for 6 h was significantly

higher than the other bacteria tested (p <0.05).

Levels of L. monocytogenes hydrophobicity differed depending

on sodium chloride, glucose, pH, temperature, incubation time,

and nutrients (Figs. 3 and 4). Similar to the biofilm formation

results, L. monocytogenes cell hydrophobicity levels were significantly

influenced by the addition of glucose to diluted TSB (1:10) at 25

and 37oC, regardless of incubation time (p <0.05). A high level of

hydrophobicity was observed in both TSB and diluted TSB (1:10)

supplemented with 0.5% sodium chloride after 6 h at 25oC (Fig.

3). In particular, when L. monocytogenes was incubated in TSB

supplemented with sodium chloride at 37oC for 24 h, the levels of

L. monocytogenes hydrophobicity were significantly higher than

those of the other bacteria (p <0.05) (Fig. 4).

The ability of P. aeruginosa to produce biofilm in TSB and

diluted TSB (1:10) for 24 h at 25 and 37oC is shown in Fig. 5. P.

aeruginosa produced higher levels of biofilm in TSB compared to

those in diluted TSB (1:10). On the other hand, biofilm formed by

P. aeruginosa in diluted TSB (1:10) at 37oC for 24 h was not

significantly different compared to that of the control. P. aeruginosa

biofilm formation was enhanced when the glucose concentration

was increased from 5 to 10% in all media except for diluted TSB

at 37oC. Biofilm formation of P. aeruginosa was influenced by the

Fig. 5 Biofilm formation of Pseudomonas aeruginosa on the surface of polystyrene incubated at 25oC for 24 h in tryptic soy broth (TSB) (A) and indiluted TSB (1:10) (B), at 37oC for 24 h in TSB (C) and in diluted TSB (1:10) (D). * means are significantly different compared to control (p <0.05).


addition of sodium chloride in all media except for diluted TSB

(1:10) at 37oC.

P. aeruginosa demonstrated high levels of attachment after 30

min in TSB supplemented with glucose (Fig. 6). Levels of attachment

of P. aeruginosa after 30 min were influenced by the pH level of

the media and showed higher levels of attachment at the pH of 6.4

than that of the other pHs tested. Attachment of P. aeruginosa for

6 h on the polystyrene surfaces was greater in diluted TSB (1:10)

supplemented with 0.5% sodium chloride and a pH of 6.8 than

that in diluted TSB (1:10). No significant difference in the P.

aeruginosa attachment levels were observed when incubated in

TSB for 6 h.

Hydrophobicity of P. aeruginosa incubated at 25oC for 6 and 24

h in TSB and diluted TSB (1:10) is shown in Fig. 7. Levels of P.

aeruginosa hydrophobicity incubated at 25oC for 6 and 24 h in

TSB and diluted TSB (1:10) were not significantly different

except for results after the 6 h incubation in diluted TSB (1:10).

Levels of P. aeruginosa hydrophobicity incubated for 6 h in diluted

TSB (1:10) were influenced by the addition of 1 and 5% glucose.

Levels of P. aeruginosa hydrophobicity in TSB supplemented

with glucose at 37oC were significantly higher than that of the

others except for P. aeruginosa incubated for 24 h in TSB (p

<0.05) (Fig. 8).

No significant differences in S. aureus biofilm formation were

observed at 37oC in any of the media (Fig. 9.). However, S. aureus

formed greater levels of biofilm compared with that of the control

at 25oC when sodium chloride was added at concentrations of 2–

7% into TSB, and glucose was added to diluted TSB (1:10) at

concentrations of 0.25–5%. Biofilm levels of S. aureus formed at

37oC were greater than those formed at 25oC. S. aureus produced

high levels of biofilm in TSB compared to those in diluted TSB

(1:10).

Fig. 6 Attachment of Pseudomonas aeruginosa on the surface of polystyrene incubated for 30 min in tryptic soy broth (TSB) (A) and in diluted TSB(1:10) (B), for 6 h in TSB (C) and in diluted TSB (1:10) (D), * means are significantly different compared to control (p <0.05).


Significant differences in attachment of S. aureus on the

polystyrene surface were observed when S. aureus was adhered in

TSB and diluted TSB (1:10) supplemented with sodium chloride

(p <0.05) (Fig. 10). In particular, S. aureus in diluted TSB (1:10)

adjusted to pH of 6.4 for 30 min and 6 h displayed high levels of

attachment on the polystyrene surface than other pH levels.

Biofilm formation and attachment of S. aureus were highly

influenced by the addition of sodium chloride compared to those

of other pathogens. Additionally, S. aureus incubated at 37oC for

6 and 24 h in TSB and diluted TSB (1:10) supplemented with

sodium chloride had significantly different hydrophobicity levels

(p <0.05) (Fig. 12). However, hydrophobicity levels of S. aureus

incubated at 25oC for 6 and 24 h were also influenced by the

addition of glucose in TSB and diluted TSB (1:10) (Fig. 11).

Hydrophobicity levels of S. aureus incubated at 25oC for 24 h in

diluted TSB (1:10) adjusted to pH of 6.0 were significantly

different (p <0.05) (Fig. 11). In particular, hydrophobicity of S.

aureus incubated at 37oC for 24 h in diluted TSB (1:10) was

influenced by all factors tested such as sodium chloride, glucose,

pH, temperature, incubation time, and nutrients (Fig. 12). Taken

together, biofilm formation, attachment, and hydrophobicity of the

pathogens differed depending on various environmental factors

and pathogen types.

Discussion

Biofilm formation, attachment, and cell hydrophobicity of L.

monocyteogenes, P. aeruginosa, and S. aureus under various

environmental conditions were evaluated. Several studies have

suggested that attachment and biofilm formation is modulated by

glucose present in the culture media (Stanley and Lazazzera,

2004; Pan et al., 2010). Our results demonstrated that biofilm

formation of pathogen was highly influenced by the addition of

Fig. 7 Hydrophobicity of Pseudomonas aeruginosa incubated at 25oC for 6 h in tryptic soy broth (TSB) (A) and in diluted TSB (1:10) (B), at 25oC for24 h in TSB (C) and diluted in TSB (1:10) (D). * means are significantly different compared to control (p <0.05).


glucose and sodium chloride compared to pH. Pan et al., (2010)

reported that L. monocytogenes generally forms higher-density

biofilms in TSBYE supplemented with 1% glucose at 37oC than

that at other concentrations, and that the optimal salt concentrations

for L. monocytogenes biofilm formation are 5% at 22.5oC and 2%

at 30 and 37oC. Similar to these results, we found that L.

monocytogenes produced biofilm at significantly higher levels in

all media supplemented with 1% glucose and 2% sodium chloride

than those in medium without supplementation (Fig. 1). We also

showed that a suspending medium at pH of 6.0 had a significant

effect on L. monocytogenes biofilm formation (Fig. 1). Duffy and

Sheridan (1997) found that the pH of the suspending medium

significantly affects the adhesion of L. monocytogenes NCTC

11994 cells to the membrane, with a greater number of microbial

cells adhering at lower pH values. Briandet et al. (1999a) also

reported that preculturing L. monocytogenes in an acid-

supplemented medium (pH=6.0) increases bacterial attachment to

stainless steel, confirming the influence of pH on biofilm

formation of L. monocytogenes. However, biofilm formation of L.

monocytogenes was not influenced by all levels of pH tested,

because difference was found only at pH of 6.0.

In the present study, S. aureus formed biofilms with higher

density compared to those of the control at 25oC when 2–7%

sodium chloride was added to TSB and when 0.25–5% glucose

was added to diluted TSB (1:10) (Fig. 9). Attachment of S. aureus

was influenced by adding sodium chloride (Fig. 10). Moretro et al.

(2003) demonstrated that S. aureus forms the thickest biofilm at

the highest sodium chloride concentration (5.4%) tested at 37oC.

The glucose concentration required for maximal biofilm formation

ranged between 0.3 and 1.3%, depending on the S. aureus strain.

Chaieb et al. (2007) demonstrated that the adherence of S.

epidermidis cells is inhibited at acidic pH levels and enhanced by

alkaline pH values. Our study demonstrated that the attachment of

S. aureus was inhibited at acidic pH levels, and that diluted TSB

Fig. 8 Hydrophobicity of Pseudomonas aeruginosa incubated at 37oC for 6 h in tryptic soy broth (TSB) (A) and in diluted TSB (1:10) (B), at 37oC for24 h in TSB (C) and in diluted TSB (1:10) (D). * means are significantly different compared to control (p <0.05).


(1:10) adjusted to pH of 6.4 resulted in high level of attachment

when S. aureus was attached on the polystyrene surface for 30

min and 6 h (Fig. 10).

Other studies have also investigated the attachment of P.

aeruginosa. Marchall et al. (1971) suggested that attachment of

Pseudomonas spp. is favored when the glucose concentration is

0.7% and is reduced when 1.4 or 2.1% glucose is present. Our

results showed that P. aeruginosa levels of attachment in TSB

supplemented with 0.25 and 1% glucose were higher than those of

the other bacteria (Fig. 6). In agreement with the results obtained

by Stanley (1983), adhesion of P. aeruginosa increased in the

presence of 10 mM NaCl or CaCl2, when P. aeruginosa attaches

to a batch of 304 stainless steel, and maximal adhesion occurred

at neutral pH. Vanhaecke et al. (1990) demonstrated that adhesion

of P. aeruginosa varies depending on the bacterial strain and

conditions; however, the influence of the NaCl concentration on

the extent of bacterial adhesion appeared to be minimal. In our

study, attachment of P. aeruginosa was not influenced by sodium

chloride (Fig. 6), although P. aeruginosa biofilm formation was

influenced by sodium chloride concentration (Fig. 5).

Petel et al. (2011) reported that biofilm formation by E. coli

O157:H7 strains 4688, 1918, and 5279 was significantly higher in

diluted TSB (1:10) than that in TSB or Luria-Bertani medium.

Similar results have been reported for E. coli, P. aeruginosa, and

L. monocytogenes under different environmental conditions

(Briandet et al., 1999b). However, in our study, biofilm formation

of pathogens in TSB on the surface of polystyrene was higher

than that in diluted TSB (1:10). Similarly, Mai and Conner (2007)

reported that the attachment of L. monocytogenes to stainless steel

and cultivated in BHI was significantly higher at 30 and 37oC

Fig. 9 Biofilm formation of Staphylococcus aureus on the surface of polystyrene incubated at 25oC for 24 h in tryptic soy broth (TSB) (A) and indiluted TSB (1:10) (B), at 37oC for 24 h in TSB (C) and in diluted TSB (1:10) (D). * means are significantly different compared to control (p <0.05).


compared to cultivation in a minimum medium. They also found

that attachment of L. monocytogenes is greater at higher

temperatures (30 and 37oC) than at lower temperatures (4, 20, and

42oC). Pan et al. (2010) demonstrated that L. monocytogenes

forms high density biofilms as temperature increases. In our study,

S. aureus and P. aeruginosa biofilm formation at 37oC was higher

than that at 25oC. However, L. monocytogenes biofilm formation

was not significantly affected by temperature.

Bacterial cell hydrophobicity could affect the attachment and

biofilm formation of pathogens. In our previous studies (unpublished

data), pathogens except for E. coli O157:H7 and B. cereus exhibited

a high degree of correlation between biofilm formation and cell

hydrophobicity for 24 h (r=0.9934). Takashi et al. (2010) also

reported that a high degree of correlation was observed between

adherence on PVC and cell hydrophobicity of L. monocytogenes

(ca. r=0.87). Many studies have indicated that bacterial cell

hydrophobicity is influenced by environmental conditions, such as

the temperature, growth media, and cultivation phase (Doelle et

al., 1982; Nikovskaya et al., 1989; Aono and Kobayashi, 1997;

Perez et al., 1998; Walker et al., 2005). Zikmanis et al. (2007)

reported that cell surface hydrophobicity values of Zymomonas

mobilis increase proportionally with increases in cultivation

temperature and concentration of the carbon source (glucose or

sucrose) in the medium. In the present study, hydrophobicity

levels of L. monocytogenes incubated in diluted TSB (1:10)

supplemented with glucose at 25oC for 6 and 24 h were significantly

different (Fig. 3). At 37oC, L. monocytogenes hydrophobicity was

influenced by glucose and sodium chloride when incubated for 6

and 24 h, respectively (Fig. 4). Levels of hydrophobicity of P.

aeruginosa and S. aureus were significantly different depending

on glucose concentration, although hydrophobicity varied depending

on the temperature, growth media, and cultivation phase (Figs. 7,

Fig. 10 Attachment of Staphylococcus aureus on the surface of polystyrene incubated for 30 min in tryptic soy broth (TSB) (A) and in diluted TSB(1:10) (B), for 6 h in TSB (C) and in diluted TSB (1:10) (D), * means are significantly different compared to control (p <0.05).


8, 11, and 12). Petel et al. (2011) demonstrated that the E. coli

strain O157:H7 for hydrophobicity was affected by the individual

strain and the growth phase. Each strain in the log phase (5 h,

37oC) was significantly more hydrophobic than the corresponding

strain in the stationary phase (18 h, 37oC). Jana et al. (1999)

reported that P. fluorescens hydrophobicity is dependent on

culture age, pH, and temperature; early-to mid-log exponential

phase cells were more hydrophobic than those in the stationary

phase. Maximum cell surface hydrophobicity of P. fluorescens

was observed at pH 7.0–7.5, with a decreasing trend at higher and

lower pHs. Chavant et al. (2002) demonstrated that hydrophobicity

levels of L. monocytogenes in the stationary phase at 37oC are

higher than those in bacteria at the exponential phase. L.

monocytogenes hydrophobicity in the exponential phase at 20oC is

higher than those in the stationary phase. In our study,

hydrophobicity levels of L. monocytogenes in the exponential

phase (6 h) at 25oC were higher than those in the stationary phase

(24 h), whereas hydrophobicity levels of L. monocytogenes in the

stationary phase (24 h) were higher at 37oC than those in the

exponential phase (6 h).

Biofilm formation, attachment, and cell hydrophobicity of the

tested pathogens were affected by environmental factors such as

glucose, sodium chloride, pH, temperature, incubation time, and

nutrients. In general, biofilm formation, attachment, and cell

hydrophobicity of the pathogens were significantly influenced by

the addition of glucose and sodium chloride. The biofilms of all

pathogens in TSB and at 37oC on the surface of polystyrene were

stronger than those in diluted TSB (1:10) and at 25oC. However,

the effects of environmental conditions on biofilm formation,

attachment, and cell hydrophobicity of pathogens varied depending

on the pathogen.

Fig. 11 Hydrophobicity of Staphylococcus aureus incubated at 25oC for 6 h in tryptic soy broth (TSB) (A) and in diluted TSB (1:10) (B), at 25oC for24 h in TSB (C) and in diluted TSB (1:10) (D). * means are significantly different compared to control (p <0.05).


Acknowledgment This research was supported by Basic Science Research

Program through the National Research Foundation of Korea (NRF) funded

by the Ministry of Education, Science and Technology (No. 2012-0004225).

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