1116 Journal of Food Protection, Vol. 77, No. 7, 2014, Pages 1116–1126 doi:10.4315/0362-028X.JFP-13-365 Copyright G, International Association for Food Protection

Thermodynamic Prediction of Growth Temperature Dependence in the Adhesion of Pseudomonas aeruginosa and Staphylococcus aureus to Stainless Steel and Polycarbonate MARWAN ABDALLAH,1,2 CORINNE BENOLIEL,2 CHARAFEDDINE JAMA,3 DJAMEL DRIDER,1 PASCAL DHULSTER,1 AND NOUR-EDDINE CHIHIB1* 1Laboratoire

de Proce´de´s Biologiques, Ge´nie Enzymatique et Microbien (ProBioGEM), IUT A/Polytech’Lille, Universite´ de Lille Science et Technologies Avenue Paul Langevin, F-59655 Villeneuve d’Ascq Cedex, France; 2Laboratoire SCIENTIS, Parc Biocitech - 102, Avenue Gaston Roussel, 93230 Romainville, France; and 3Laboratoire UMET, UMR-CNRS 8207, Ecole Nationale Supe´rieure de Chimie de Lille, Universite´ Lille 1, Avenue Dimitri Mendeleı¨ev, 59655 Villeneuve d’Ascq, France MS 13-365: Received 6 September 2013/Accepted 16 January 2014

ABSTRACT This study investigated the effect of growth temperature changes (20, 30, and 37uC) on the adhesion behavior of Pseudomonas aeruginosa and Staphylococcus aureus to stainless steel and polycarbonate. Adhesion assays were performed under static conditions at 20uC. In addition, the validity of the thermodynamic and extended Derjaguin, Landau, Verwey, and Overbeek theories as predictive tools of bacterial adhesion were studied. The surface properties of the bacterial cells and the substrates of attachment were characterized, and atomic force microscopy was used to analyze the surface topography. The results indicated that the highest adhesion rate of P. aeruginosa and S. aureus on both surfaces was observed when the cells were grown at 37uC. The bacterial adhesion to stainless steel was found to be two times higher than to polycarbonate for both bacteria, whatever the condition used. The present study underlined that the thermodynamic and the extended Derjaguin, Landau, Verwey, and Overbeek theories were able to partially predict the empirical results of P. aeruginosa adhesion. However, these theories failed to predict the adhesion behavior of S. aureus to both surfaces when the growth temperature was changed. The results of the microbial adhesion to solvent indicated that the adhesion rate to abiotic surfaces may correlate with the hydrophobicity of bacterial surfaces. The effect of surface topography on bacterial adhesion showed that surface roughness, even on the very low nanometer scale, has a significant effect on bacterial adhesion behavior.

The ability of bacteria to adhere to abiotic surfaces is a major concern in the food industry; biofilms may create a persistent source of contamination and may lead to food spoilage or food poisoning. Moreover, when contamination of food products occurs, evidence suggests that microorganisms on the surface of food processing equipment are a major source (9, 20, 37). These contaminants are mainly associated with water and raw foods. Food handlers have also been identified as a significant source of contaminants. Thus, microorganisms emerge in the food sector from different ecosystems, and these cells have been exposed to different conditions, such as temperature, water activity (aw), and pH (i.e., they have been exposed to specific background conditions) (35). Therefore, study of bacterial adhesion that takes into account the background exposure of the bacterial cells is important because it can help to reduce the microbiological risk associated with food contact surfaces. It is now established that bacterial adhesion to abiotic surfaces is influenced by various factors, such as microbial growth phase, culture conditions, temperature, ionic * Author for correspondence. Tel: z33 320417567; Fax: z33 328767356; E-mail: [email protected].

strength, and variability of strains (23, 25, 41, 43). Previous studies have also reported that the physicochemical properties of surfaces, such as hydrophobicity, play a crucial role in bacterial attachment to abiotic surfaces (12, 13, 22). However, other studies have indicated that physicochemical properties have only a minor role and that the correlations between surface properties and bacterial adhesion were poor (34). This discrepancy seems to be related to the method used to characterize the surface properties of bacterial cells. In fact, Hamadi and Latrache (11) failed to establish a correlation between the microbial adhesion to solvent and the contact angle outcomes. To predict bacterial adhesion, the Derjaguin, Verwey, Landau, and Overbeek (DLVO) theory has been used in several studies. According to this theory, as the bacterial cell approaches a surface of interest, the entire cell will be exposed to nonspecific physicochemical forces such as Lifshitz–van der Waals (LW) and electrostatic (EL) forces. The extended DLVO (XDLVO) theory added short-range Lewis acid-base (AB) interactions in microbial adhesion (4, 38). However, the validation of this theory as a predictive physicochemical model to study bacterial adhesion is still under investigation.

J. Food Prot., Vol. 77, No. 7

ENVIRONMENTAL AND THERMODYNAMIC PREDICTIONS OF BACTERIAL ADHESION

The present work was carried out on Pseudomonas aeruginosa and Staphylococcus aureus, which are involved in food spoilage and food poisoning. The purpose of the current work was to study the effect of growth temperature on bacterial adhesion to stainless steel and polycarbonate, two surfaces frequently used in food processing equipment. Adhesion assays were performed under static conditions at 20uC. In order to characterize the mechanisms of bacterial adhesion, cell surface properties were studied using contact angle measurements (CAMs) and microbial adhesion to organic solvent (MATS). In addition, CAMs and atomic force microscopy (AFM) were used to study the surface properties and topography of both stainless steel and polycarbonate, respectively. CAM outcomes were also used to predict bacterial adhesion using the LW-AB and the XDLVO theories. Subsequently, empirical adhesion data of bacteria cultivated on surfaces at different temperatures (20, 30, and 37uC) were compared with theoretical predictions. This study aimed to unravel the relationship between environmental factors and bacterial adhesion to surfaces. This data contributes to the understanding of, and therefore prevention of, bacterial adhesion to surfaces and subsequent biofilm formation. MATERIALS AND METHODS Bacterial strains and culture conditions. The bacterial strains used for this study were P. aeruginosa CIP 103467 and S. aureus CIP 4.83. The strains were stored at 280uC in tryptic soy broth (TSB; Biokar Diagnostics, Pantin, France) containing 40% (vol/vol) glycerol. To prepare precultures, 100 ml from frozen stock cultures was inoculated into 5 ml of TSB and then incubated at the culture temperature (i.e., 20, 30 or 37uC). The preculture at 20uC was incubated for 48 h, whereas those at 30 and 37uC were incubated for 24 h. The cultures used in each experiment were then prepared by inoculating 5 | 104 CFU/ml from the preculture broths into 50 ml of TSB in sterile 500-ml flasks. Cultures were incubated under shaking (160 rpm) at 20, 30, or 37uC and were stopped at the late exponential phase. Bacterial standardization and cell inoculum preparation. P. aeruginosa and S. aureus, grown at 20, 30, and 37uC as described previously, were harvested by centrifugation for 10 min at 3,500 | g (20uC). Bacteria were washed twice with 20 ml of 100 mM potassium phosphate buffer (PB; pH 7) and finally were resuspended in 20 ml of PB. The cells were dispersed by sonication at 37 kHz for 5 min at 25uC (Elmasonic S60H, Elma, Singen, Germany). Subsequently, bacteria were resuspended in the PB to a cell concentration of 1 | 108 CFU/ml by adjusting the optical density to OD620 nm ~ 0.110 ¡ 0.005 (108 CFU/ml) using an Ultrospec 1100 pro UV-visible light spectrophotometer (GE Healthcare, Waukesha, WI). Standardized cell suspensions were diluted 10-fold for use in the bacterial adhesion assays (107 CFU/ml). Slide preparation and adhesion assays. The stainless steel and polycarbonate surfaces were cleaned by soaking in ethanol 95% (Fluka, Sigma-Aldrich, St. Louis, MO) overnight to remove grease. Next, slides were rinsed in water and soaked in 500 ml of TDF4 detergent (5%; Franklab SA, Billancourt, France) for 20 min at 50uC under agitation. The slides were then thoroughly rinsed five times for 1 min with agitation in 500 ml of distilled water at room temperature to eliminate detergent, followed by three washes with ultrapure water (Milli-Q Academic, Millipore, Molsheim,

1117

France). The clean stainless steel slides were air dried and sterilized by autoclaving at 121uC for 15 min. Polycarbonate slides were sterilized for 10 min with absolute ethanol (Fluka, Sigma-Aldrich). The sterile slides were placed in a horizontal position in petri dishes. The upper face of each slide was covered with 3 ml of cell inoculum (107 CFU/ml) and incubated statically at 20uC for 60 min for the bacterial adhesion assays. After attachment, the coupons were removed using sterile forceps and were rinsed by gentle dipping into 30 ml of PB to remove excess liquid droplets and loosely attached cells. Cells were then stained for 10 min in the dark using acridine orange stain 0.01% (wt/vol), followed by gentle dipping in 30 ml of ultrapure water. The attached cells were quantified using epifluorescence microscopy (Nikon Optiphot-2 EFD3, Nikon Inc., Melville, NY). A total of 50 fields per coupon were scanned, and the fluorescent cells were enumerated. Counts were presented as number of bacteria in the microscopy field. The results present the average of three independent experiments, and two coupons were studied for each experiment. CAMs. The contact angles of the bacterial cell surfaces were measured using the sessile drop method. Bacterial suspensions, prepared as described above, were adjusted to an OD620 nm of 1.0 using either PB or ultrapure water. To evaluate the cell surface hydrophobicity and hydrophilicity, cells harvested by centrifugation were washed once with ultrapure water and were resuspended in the same water to an OD620 nm of 1.0. The cells suspended in ultrapure water or PB were vacuum filtered through a 0.45-mmpore-size nitrocellulose membrane filter (type HA, Millipore, Bedford, MA) to create a bacterial lawn. The bacterial cell filters were attached to glass slides using double-sided adhesive tape and were dried for 30 min at 37uC. The contact angles of water, diiodomethane, and formamide were measured immediately after drop deposition on the bacterial lawn, with a digital camera, using WinDrop software (20uC) (Digidrop goniometer, GBXInstruments, France). At least five drops of each probe liquid were deposited onto each filter. For stainless steel and polycarbonate surfaces, five drops of the probe liquid were also measured. CAM results present the average of the measurements taken on three independently bacterial or solid surfaces. MATS. The MATS method, described by Bellon-Fontaine et al. (3), based on the comparison of microbial cell affinity to both monopolar and apolar solvents, was used to determine the hydrophobicity and the electron donor (basic) or acceptor (acidic) properties of microbial cells. Experimentally, bacteria were suspended to an optical density of 0.9 at 405 nm (A0) (approximately 108 CFU/ml cell density) in 100 mM PB. A volume (2.4 ml) of each bacterial suspension was vortexed for 90 s with 0.4 ml of solvent. The mixture was allowed to stand for 15 min to ensure complete separation of the two phases. The optical density of the water phase was then measured using a spectrophotometer. The affinity of cells for each solvent was subsequently calculated by the following equation: Affinity~½1{ðA=A0 Þ

ð1Þ

where A0 is the absorbance measured at 405 nm of the bacterial suspension before mixing, and A is the absorbance after mixing. The following pairs of solvents, as described by Bellon-Fontaine et al. (3), were used: chloroform (an electron acceptor solvent), hexadecane (a nonpolar solvent), ethyl acetate (an electron donor solvent), and decane (a nonpolar solvent). Owing to the similar LW components of the surface tension in each pair of solvents, differences between the affinities to solvents would indicate the electron donor and electron acceptor character of the bacterial

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surface. The affinity of cells to hexadecane was used as a measure of cell surface hydrophobicity. Measurement of zeta potential. The electrical properties of bacteria were measured by microelectrophoresis using a Zeta Compact zetameter (CAD Instruments, Les Essarts-le-Roi, France), by tracking bacteria with a coupled device camera. The electrophoretic mobility of bacteria suspended in PB was converted to apparent zeta potentials according to the Helmotz-Smoluchowski equation (2). Bacteria were suspended in each 50 ml of PB to obtain approximately 70 bacteria per reading. The zeta potential of stainless steel and polycarbonate was measured using an electrokinetic analyzer (SurPASS, Anton Paar GmbH, Graz, Austria), as described elsewhere (14). The rectangular slides were placed in clamping cells, and the zeta potential was determined from the Smoluchowski equation by measuring the change in streaming current versus the applied differential pressure. For the electrolyte, 1 mM KCl solution was used, and 0.1 M HCl and 0.1 M NaOH were used to adjust the pH to 7. AFM. An atomic force microscope (Dimension 3100 microscope, Bruker, Santa Barbara, CA) was used to analyze the nanometer scale surface roughness of stainless steel and polycarbonate slides. The cantilever was a NCHV-A (Bruker), typically 125 mm with an apex curvature radius on the order of 10 nm, and the cantilever spring constant was 42 N/m. Root mean square roughness was determined over an area of 1 mm2 using WSXM software (Nanotec Electronica, Madrid, Spain). Surface energies of bacteria and substrata. The surface energy characteristics of the bacteria and materials were calculated according to Young’s equation (21, 40), expressed as: 0qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi pffiffiffiffiffiffiffiffiffiffiffiffiffi pffiffiffiffiffiffiffiffiffiffiffiffiffi1 LW z { cLW S cl cz c{ S cl S cl A ð2Þ cos h~{1z2@ z z cl cl cl where h is the contact angle and cl is the surface tension (mJ/m2) of the liquid used for the measurement. The subscript S refers to the solid surface or bacteria, and l refers to the liquid used for contact angle measurement. By using three different liquids with known cl, z { cLW l , cl , and cl values (water, formamide, and diiodomethane), the unknown surface tension components can be estimated (solid z z { LW { surfaces: cLW S , cS , and cS ; bacterial surfaces: cb , cb , and cb ). The bacterial and the substrata surface tensions are calculated using the following equation: pffiffiffiffiffiffiffiffiffiffiffiffiffi c~cLW zcAB ~cLW z2 cz c{ ð3Þ where c is the surface tension and cAB the polar component of the surface tension. LW-AB theory. In the thermodynamic theory related to the bacterial attachment (4), the derived free energies of adhesion do not account for a distance dependence of the interaction energy. According to this theory, the free energy of adhesion at contact (DGtot adh ) is the summation of these two components: LW AB DGtot adh ~DGadh zDGadh

ð4Þ

AB The LW DGLW adh and the AB DGadh interactions at contact between a bacterial surface (b) and a substratum surface (S) immersed in a liquid medium (l) can be calculated according to equations 5 and 6: qffiffiffiffiffiffiffiffi qffiffiffiffiffiffiffiffiqffiffiffiffiffiffiffiffi qffiffiffiffiffiffiffiffi LW LW DGLW cLW cLW ð5Þ adh ~{2 b { cl Sl { cl

DGAB adh ~2

qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi  qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi z z { {c{ { cz cz {c c S S b b {cl b |

pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffipffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi z { { { c{ c{ cz S {cl S {cl b {cl

ð6Þ

The XDLVO theory. According to the XDLVO theory, the interaction energy between the bacterial cell surface and the substratum (separated by a distance d) is the sum of LW (DGLW (d)), Lewis AB (DGAB (d)), and EL (DGEL (d)) interaction energies. The total XDLVO interaction energy is given as Bayoudh et al. (1). DGtot ðd Þ~DGLW ðd ÞzDGAB ðd ÞzDGEL (d)

ð7Þ

The interaction energies for each individual component, LW, AB, and EL, as a function of separation distance are given as in Bos et al. (4).  

A 2r ðdzrÞ dz2r {ln ð8Þ DGLW ðd Þ~{ 6 d ð1z2r Þ d where d is the separation distance, r is the radius of the bacterium, and A is the Hamaker constant, which can be calculated from equation 7 (4). A~12pd02 DGLW (d0 )

ð9Þ

DGLW adh

is calculated as described above, and d0 ~ 0.157 nm where is the minimum separation distance between the outermost cell surface and the substratum surface (41). The distance dependence of the AB interaction energies is given by Bos et al. (4). ðd0{d l Þ DGAB ðd Þ~2prDGAB adh exp

ð10Þ

DGAB adh

is calculated as described above (from the therwhere modynamic theory) and l is the correlation length of molecules in the liquid medium (estimated to be 0.6 nm for hydrophilic bacteria and 13 nm for hydrophobic bacteria) (39). EL interaction energies as a function of separation distance are also calculated according to Bos et al. (4).  DGEL ðd Þ~pee0 r f2b zf2S ( )   h i ð11Þ 2fb fs 1zeð{kdÞ ð{2kdÞ zln 1ze | 2 2 ln ð {kd Þ 1{e fb zfS where ee0 is the dielectric permittivity of the medium, fb and fs are the surface zeta potentials of the bacterial surface and collector surface in the surrounding liquid, respectively, and k is the reciprocal Debye length. Statistics. The results are presented as mean values and their standard error of mean. Data analysis was performed using Sigma Plot 11.0 (Systat Software Inc., San Jose, CA), using one-way analysis of variance (Tukey’s method) to determine the significance of differences. Results were considered significant at P , 0.05.

RESULTS Bacterial and substrata surfaces properties. No matter which theories are used to predict bacterial adhesion, LW, AB components, and surface charge are needed to calculate the LW, AB, and EL interaction energies between bacteria and surfaces. CAMs were then performed, and the data related to hd, hw, and hF were used to calculate the surface energy components of substrates and bacterial cells (Table 1).

2.0 1.1 0.8 1.0 1.9 2.3 0.3 0.5 37.1 50.8 55.1 46.9 43.2 54.2 47.1 43.4 1.8 1.7 1.1 1.6 3.1 1.9 1.2 0.7 ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ 6.9 19.9 18.4 8.4 9.8 25.7 13.3 1.1 0.1 0.5 0.2 0.2 0.3 0.3 0.6 0.1 ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ 0.2 2.2 2.1 0.5 0.5 2.6 3.8 0.1 2.6 3.1 1.1 1.6 3.1 1.7 2.1 0.6 ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ 52.1 46.4 41.3 34.2 51.4 64.3 11.8 7.2 1.6 0.7 0.4 0.8 0.8 0.5 0.9 0.5 ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ 0.6 2.0 2.2 1.6 2.4 1.1 0.4 0.2

29.3 30.9 36.7 38.5 34.7 28.5 33.7 43.3 ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ 64.1 29.3 18.2 60.9 61.4 76.5 33.8 33.5 1.3 1.5 1.4 1.8 2.4 1.5 2.1 1.4 ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ 2.8 1.2 0.6 1.5 2.8 1.5 1.7 2.3

51.3 30.1 29.8 60.7 50.4 54.9 60.6 79.3 ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ 58.7 55.7 45.6 42.5 50.9 59.7 50.9 55.5 Stainless steel Polycarbonate

Staphylococcus aureus

20 30 37 20 30 37 Pseudomonas aeruginosa

a

¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡

212.1 213.4 214.2 216.9 214.2 29.3 240.5 218.5

f c cAB cz c2 cLW hF hW hd T (uC)

TABLE 1. Bacterial and substratum surface characteristics a

Immersed in 100 mM phosphate buffer (PB; pH 7). T, bacterial growth temperature; hd, hF, and hW, contact angle measurements (in degrees) on lawns of bacterial cells and substrata immersed in PB; cLW, cz, c2, and cAB, the van der Waals, electron acceptor, electron donor, and polar components of surface tension (c; mJ/m2), respectively, of bacterial cells suspended in PB and substrata immersed in the same buffer; f, the zeta potential (mV) of bacterial and substrata surfaces.

ENVIRONMENTAL AND THERMODYNAMIC PREDICTIONS OF BACTERIAL ADHESION

1.0 0.8 1.2 0.6 0.7 1.6 0.7 1.3

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Surface properties of stainless steel and polycarbonate. The calculation of the surface tension indicated that stainless steel is more hydrophilic (47.1 mJ/m2) than polycarbonate (43.4 mJ/m2) (Table 1). In addition, the two surfaces presented a greater electron donor component than the electron acceptor component (c2, 11.8 and cz, 3.8 mJ/ m2 for the stainless steel and c2, 7.2 and cz, 0.1 mJ/m2 for the polycarbonate). Zeta potential measurement results indicate that the stainless steel had more than twofold greater negative charge than the polycarbonate (Table 1). Surface properties of bacteria grown at different temperatures. The results show that the growth temperature has a significant effect on the bacterial surface properties (Table 1). When the growth temperature increased from 20 to 37uC, our findings showed that LW component of P. aeruginosa surface tension increased from 29.3 to 36.7 mJ/m2 and the electron donor component decreased from 52.1 to 41.3 mJ/m2 (Table 1). The electron acceptor component was 0.2, 2.2, and 2.1 mJ/m2, respectively, when P. aeruginosa was grown at 20, 30, and 37uC (Table 1). The LW component of S. aureus surface tension decreased from 38.5 to 28.5 mJ/m2, whereas the electron donor component increased from 34.2 to 64.3 mJ/m2 when the growth temperature increased from 20 to 37uC (Table 1). When S. aureus was grown at 20 and 30uC, the electron acceptor component was 0.5 mJ/m2. This value increased to 2.6 mJ/m2 when S. aureus was cultivated at 37uC (Table 1). The results related to zeta potential measurements indicated that P. aeruginosa and S. aureus cells were negatively charged whatever the growth temperature. The results presented in Table 2 indicate that the growth temperature had no significant effect (P . 0.05) on the zeta potential (ca. 213 mV) of P. aeruginosa. However, the zeta potential of S. aureus cells decreased from 216.9 to 29.3 mV when growth temperature of this bacterium increased from 20 to 37uC (Table 1). Prediction of bacterial adhesion according to LWAB theory. Several theories have been proposed to predict bacterial adhesion to surfaces (4). The van Oss LW-AB theory was followed here because it was found to give consistent results with the microbial adhesion. In this theory, the surface free energy of adhesion (DGtot adh ) was divided in two parts: the LW acid and AB components (equation 2). From a thermodynamic point of view, adhesion or attraction between two surfaces occurs when DGtot adh is negative, and the adhesion is thermodynamically unfavorable when it is positive. The theoretical prediction underline that the adhesion of P. aeruginosa is favorable with negative values of DGtot adh whatever the conditions studied (Table 2). When the growth temperature increased from 20 to 37uC, the DGtot adh of this bacterium to stainless steel and to polycarbonate decreased from 22.7 to 26.5 mJ/m2 (P , 0.05) and from 23.5 to 29.7 (P , 0.05), respectively (Table 2). On the other hand, this theory predicted higher interactions with the stainless steel than the polycarbonate, whatever the growth temperature.

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TABLE 2. Interaction energy at contact between bacterial strains and stainless steel according to the LW-AB theory Stainless steel (mJ/m2) Strains

T (uC)

Pseudomonas aeruginosa

20 30 37 20 30 37

Staphylococcus aureus a

DGLW adh

21.7 22.1 23.2 23.6 22.8 21.6

¡ ¡ ¡ ¡ ¡ ¡

DGAB adh

0.3 0.2 0.1 0.2 0.2 0.1

21.1 21.6 23.2 29.1 20.5 7.2

¡ ¡ ¡ ¡ ¡ ¡

0.8 0.3 0.5 1.1 0.9 0.95

Polycarbonate (mJ/m2) DGtot adh

22.7 23.6 26.5 212.6 23.3 5.6

¡ ¡ ¡ ¡ ¡ ¡

DGLW adh

0.8 0.3 0.6 1.1 1.0 0.9

22.9 23.4 25.34 25.9 24.7 22.6

¡ ¡ ¡ ¡ ¡ ¡

DGtot adh

DGAB adh

0.5 0.2 0.2 0.3 0.3 0.2

20.5 22.6 24.4 212.8 0.1 12.3

¡ ¡ ¡ ¡ ¡ ¡

0.2 1.0 0.5 1.7 1.0 1.4

23.5 25.9 29.7 218.7 24.6 9.7

¡ ¡ ¡ ¡ ¡ ¡

0.5 1.1 0.4 1.8 1.1 1.3

AB tot Immersed in 100 mM phosphate buffer (pH 7). T, bacterial growth temperature; DGLW adh , DGadh , and DGadh , van der Waals, acid-base,

and thermodynamic interaction energy, respectively, at contact between bacterial and substrata surfaces. For S. aureus, the calculation of DGtot adh shows that the increase of growth temperature has a negative effect on its adhesion on both stainless steel and polycarbonate (Table 2). In addition, LW-AB theory predicted an important adhesion to polycarbonate in comparison to stainless steel when S. aureus is cultivated at 20 by contrast to 37uC (Table 2). When the growth temperature was 30uC, the DGtot adh between S. aureus and stainless steel or polycarbonate were similar (P . 0.05) (Table 2). Prediction of bacterial adhesion according to XDLVO theory. In the XDLVO theory, calculation of AB interactions between bacteria and surfaces varies when bacteria are either hydrophobic or hydrophilic. In order to assess the hydrophobicity and hydrophilicity of bacteria, the free energy of cohesion was calculated for bacteria immersed in water (Table 3). Under the conditions used, P. aeruginosa and S. aureus present a hydrophilic character with positive values of free energy of cohesion. When the growth temperature increased from 20 to 37uC, P. aeruginosa became more hydrophobic and the free energy of cohesion decreased from 34.9 to 12.6 mJ/m2 (Table 3). However, the hydrophilic character of S. aureus increased when growth temperature increased. The results indicated that the free energy of cohesion increased from 3.6 to 38.2 mJ/m2 when the growth temperature increased from 20 to 37uC (Table 3). The XDLVO theory relates to the origin of hydrophobic interactions in microbial adhesion and considers the fundamental noncovalent interactions: LW, EL, and Lewis AB forces (4). In this theory, the adhesion energies are calculated at the closest approach and as a function of the separation distance (Figs. 1A through 1F and 2A through 2F). P. aeruginosa and the XDLVO prediction. The results shown in Figure 1A through 1D reveal that DGLW and DGAB between P. aeruginosa and surfaces were negative whatever the conditions used, indicating attractive LW and AB interactions between P. aeruginosa and both stainless steel and polycarbonate. In addition, when growth temperature increased from 20 to 37uC, LW and AB interactions, between P. aeruginosa and both studied surfaces, increased significantly (P , 0.05) (Fig. 1A through 1D). Figure 1C and 1D shows that the AB interactions between P.

aeruginosa and surfaces, in a distance less than 3 nm, were much higher (two to six times higher) than LW interactions. Moreover, XDLVO predicted higher AB interactions with polycarbonate than stainless steel when P. aeruginosa was grown at 30 and 37uC. Figure 1E and 1F shows that the repulsive EL interactions between surfaces and P. aeruginosa were not significantly different with the growth temperature changes. The summation of WL, AB, and EL interactions as a function of separation distance predicted a greater adhesion of P. aeruginosa grown at 37uC to stainless steel and polycarbonate when compared with the cells grown at 30 and 20uC (P , 0.05) (Fig. 1G and 1H). Moreover, XDLVO predicted a greater adhesion to polycarbonate than to stainless steel when P. aeruginosa was cultivated at 30 and 37uC (P , 0.05). S. aureus and the XDLVO prediction. The results shown in Figure 2A and 2B reveal that the DGLW between S. aureus and surfaces are favorable with negative values, whatever the conditions of growth. In addition, the predicted results indicated that LW interactions between surfaces and S. aureus grown at 20uC are stronger than those grown at 30 and 37uC (Fig. 2A and 2B). The DGLW between S. aureus grown at 30 and 37uC are not significantly different whatever the surfaces used (P . 0.05). Attractive AB interactions between surfaces and S. aureus are found only when bacteria were cultivated at 20uC (Fig. 2C and 2D). However, XDLVO theory predicted repulsive AB interactions between S. aureus grown at 37uC and the studied surfaces. S. aureus cells grown at 37uC have higher repulsive AB interactions with polycarbonate than with stainless steel (P , 0.05). Nevertheless, the cells grown at 20uC showed higher attractive AB interactions with the polycarbonate than with the stainless steel. The theoretical prediction results show that the repulsive EL interactions between S. aureus and surfaces significantly decreased when growth temperature increased from 20 to 37uC (Fig. 2E and 2F). The summation of LW, AB, and EL interactions between S. aureus and surfaces reveals that the adhesion prediction followed the tendency of AB interactions results (Fig. 2G and 2H). Effect of growth temperature on the adhesion of P. aeruginosa and S. aureus to stainless steel and polycarbonate. In the current work, bacterial adhesion was

Staphylococcus aureus

T, growth temperature; hd, hF, and hW, contact angle measurement (degrees) on lawns of bacterial cells suspended in water; cLW, cz, c2, and cAB, the van der Waals, electron acceptor, electron donor, and polar components of surface tension (c; mJ/m2), respectively, of bacterial cells suspended in water; DGSWS, the free energy of cohesion (mJ/m2) between two identical surfaces immersed in water.

¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡ 20 30 37 20 30 37 Pseudomonas aeruginosa

60.2 57.0 44.0 42.4 50.9 58.9

0.7 0.6 0.7 16 0.6 1.0

54.7 35.3 32.5 62.7 52.9 54.4 ¡ ¡ ¡ ¡ ¡ ¡

0.3 0.8 0.8 0.6 0.3 0.9

67.7 33.2 18.4 62.6 59.8 72.4

¡ ¡ ¡ ¡ ¡ ¡

0.5 0.5 0.8 0.5 1.2 0.7

28.5 30.3 37.5 38.4 33.8 29.2

¡ ¡ ¡ ¡ ¡ ¡

0.4 0.4 0.3 0.8 0.3 0.6

50.1 43.7 38.6 30.6 43.7 57.6

1.0 1.1 0.9 1.5 2.0 1.7

0.4 2.0 2.0 0.5 0.2 1.4 ¡ ¡ ¡ ¡ ¡ ¡

0.1 0.2 0.1 0.1 0.1 0.1

9.09 18.68 17.55 8.21 5.71 17.9

¡ ¡ ¡ ¡ ¡ ¡

1.1 0.8 0.3 1.0 2.0 0.8

37.6 49.0 55.1 46.6 39.5 47.1

c cAB cz c2 cLW hF hW hd T (uC) Bacterium

TABLE 3. Cell surface hydrophobicity or hydrophilicity according to the thermodynamic theory a

a

34.9 20.8 12.6 3.6 26.2 38.2

¡ ¡ ¡ ¡ ¡ ¡

0.6 1.2 1.0 1.8 1.5 1.7

ENVIRONMENTAL AND THERMODYNAMIC PREDICTIONS OF BACTERIAL ADHESION

1.5 0.4 0.2 1.6 2.2 1.1

DGSWS

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performed on stainless steel and polycarbonate in order to study the correlation between the thermodynamic prediction and the empirical results. Our findings show that the bacterial adhesion on the two surfaces increased when the growth temperature increased (Fig. 3). The adhesion of P. aeruginosa on stainless steel increased significantly (by 1.9-fold) when the growth temperature increased from 20 to 37uC (P , 0.05) and by 1.6-fold when growth temperature increased from 30 to 37uC (P , 0.05) (Fig. 3A). Our results indicated also that the adhesion of P. aeruginosa onto polycarbonate significantly increased (by 1.8-fold) when growth temperature increased from 20 to 37uC (P , 0.05) and by 1.4-fold (P . 0.05) when the temperature increased from 30 to 37uC (Fig. 3B). These experimental results indicate that the adhesion rate of P. aeruginosa was two times higher on stainless steel than on polycarbonate whatever the growth temperature (Fig. 3). As seen in Figure 3, the adhesion of S. aureus to stainless steel and polycarbonate increased 1.6 and 1.2 times when growth temperature increased, respectively, from 20 to 37uC and from 30 to 37uC. In addition, the adhesion of S. aureus on stainless steel was two times higher than on polycarbonate, whatever the surface used (Fig. 3B). Bacterial surface properties according to MATS. In order to check the outcomes derived from the contact angle measurements, the MATS technique was used to characterize the bacterial surfaces properties of both P. aeruginosa and S. aureus. The results of Table 4 indicate that the hydrophobic character of P. aeruginosa and S. aureus increased when the growth temperature increased. The affinity of P. aeruginosa to hexadecane increased 1.8-fold (P , 0.05) when growth temperature increased from 20 to 37uC. P. aeruginosa grown at 30 and 37uC has the same affinity to hexadecane. The affinity of S. aureus cells to hexadecane increased about 2.1-fold (P , 0.05) when the growth temperature increased from 20 to 37uC and 1.4-fold (P , 0.05) when the temperature increased from 30 to 37uC. P. aeruginosa decreased the electron donor character from 0.35 to 0.18 (P , 0.05) when growth temperature increased from 20 to 37uC. The electron acceptor character increased from 0.02 to 0.14 (P , 0.05). S. aureus decreased the electron donor character from 0.35 to 0.51 (P , 0.05) and increased the electron acceptor character from 20.16 to 20.27 when the growth temperature increased from 20 to 37uC (Table 4). Surface topography of stainless steel and polycarbonate. The topography of stainless steel and polycarbonate was characterized using AFM, in order to study the relationship between the surface roughness and the bacterial adhesion. The results of AFM reveal that the two studied surfaces present a different surface topography (Fig. 4A through 4D). Our results indicated also that the surface of the stainless steel appears to be almost 10-fold rougher than the polycarbonate. The root mean square values were 20 and 2 nm for stainless steel and polycarbonate, respectively.

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FIGURE 1. Lifshitz–van der Waals (LW), acid-base (AB), electrostatic (EL), and XDLVO (tot) interactions between P. aeruginosa and stainless steel or polycarbonate as a function of separation distance. (A, C, E, and G) Interactions with stainless steel. (B, D, F, and H) Interactions with polycarbonate.

DISCUSSION The contamination of processing equipment surfaces and the cross-contamination of food products with foodborne pathogens is a major public health concern. To prevent infections related to these contaminants and to reduce the microbiological risk, more knowledge is needed on the influence of environmental conditions and bacterial ecological history at the first stage of biofilm formation (i.e., initial adhesion). When the growth temperature increased from 20 to 37uC, the ability of both P. aeruginosa and S. aureus to attach to stainless steel and polycarbonate surfaces increased. Our results seem to be consistent with what has been reported on the effect of growth temperature on the adhesion of P. aeruginosa (5), Listeria monocytogenes (10), and Escherichia coli to different abiotic surfaces (36). In addition, our work highlights the need to pay more attention to the ecological background and origins of the bacteria of interest, which significantly influences bacterial adhesion to abiotic surfaces.

To control biofilm formation in food processing, many studies have investigated the first step in biofilm formation, and different models have been proposed to describe and to predict bacterial adhesion to surfaces. However, it has been reported that the thermodynamic theory cannot fully explain initial bacterial adhesion to surfaces, which is consistent with our results. The inability may be the result of an inadequate description of EL interactions in the thermodynamic theory (32). Because this theory may not be able to fully explain microbial adhesion, we predicted that bacterial adhesion of cells, cultivated at different temperatures, would better follow the XDLVO theory. This model was found to be an effective and useful tool to predict and to explain the bacterial adhesion of different strains to abiotic surfaces (19, 31, 32). For P. aeruginosa, the XDLVO theory was able to correctly predict the adhesion of this bacterium on both studied surfaces when the growth temperature was increased. This correlation was mainly due to the increase of attractive LW and AB interactions with the rise of growth

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FIGURE 2. Lifshitz–van der Waals (LW), acid-base (AB), electrostatic (EL), and XDLVO (tot) interactions between S. aureus and stainless steel or polycarbonate as a function of separation distance. (A, C, E, and G) Interactions with stainless steel. (B, D, F, and H) Interactions with polycarbonate.

temperature. The effect of repulsive EL interactions on the total energy of XDLVO theory appeared to be minor compared to the other interactions. In fact, the high ionic strength of PB may decrease the magnitude and the effect of

EL interactions on the theoretical prediction (4). However, the XDLVO theory failed to predict the highest adhesion rate to stainless steel, whatever the growth temperature studied. Our results also show a higher absolute value, in a

FIGURE 3. Effect of growth temperature on the adhesion of P. aeruginosa and S. aureus to stainless steel and polycarbonate. (A) Adhesion of P. aeruginosa to stainless steel and polycarbonate; (B) adhesion of S. aureus to stainless steel and polycarbonate.

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TABLE 4. Effect of growth temperature on bacterial surface properties according to MATS a Bacterium

T (uC)

Pseudomonas aeruginosa

20 30 37 20 30 37

Staphylococcus aureus a

Chloroform

0.59 0.59 0.62 0.48 0.74 0.88

¡ ¡ ¡ ¡ ¡ ¡

0.02 0.04 0.02 0.01 0.01 0.02

Ethyl acetate

0.32 0.63 0.55 0.23 0.36 0.51

¡ ¡ ¡ ¡ ¡ ¡

0.02 0.04 0.02 0.03 0.01 0.03

Decane

0.30 0.52 0.41 0.39 0.67 0.78

¡ ¡ ¡ ¡ ¡ ¡

0.01 0.03 0.03 0.02 0.01 0.01

Hexadecane

0.24 0.40 0.42 0.13 0.27 0.37

¡ ¡ ¡ ¡ ¡ ¡

0.02 0.02 0.02 0.03 0.03 0.04

Electron donor

0.35 0.19 0.18 0.35 0.47 0.51

¡ ¡ ¡ ¡ ¡ ¡

0.02 0.06 0.03 0.02 0.03 0.05

Electron acceptor

0.02 0.11 0.14 20.16 20.31 20.27

¡ ¡ ¡ ¡ ¡ ¡

0.02 0.04 0.04 0.04 0.02 0.04

MATS, microbial adhesion to organic solvent; T, growth temperature. The difference between chloroform and hexadecane affinities of cells suspended in 100 mM phosphate buffer (PB; pH 7) determines the electron donor properties. The difference between ethyl acetate and decane affinities of cells suspended in 100 mM PB (pH 7) determines the electron acceptor properties.

distance less than 3 nm, of AB as compared with LW and EL interactions. This is in agreement with previous studies that showed that the AB interactions are the most important interactions in bacterial adhesion to surfaces (28, 30). The XDLVO theory predicted greater attractive AB interactions between P. aeruginosa and polycarbonate, in comparison to stainless steel; this was the theory’s main failure for this bacterium. For S. aureus, our experimental results show an increase in cell adhesion to stainless steel and polycarbonate with the rise of growth temperature. In this case the XDLVO theory completely failed to predict the adhesion of S. aureus to either surface and at any growth temperature studied. In fact, the XDLVO theory predicted the opposite of the empirical results obtained for the S. aureus adhesion. These results agree with studies that stated that in some cases the XDLVO theory is unable to predict bacterial adhesion to abiotic surfaces (6, 18, 28). This discrepancy was mainly FIGURE 4. The AFM characterization of stainless steel and polycarbonate. (A and C) Top and three-dimensional views of the stainless steel topography. (B and D) Top and three-dimensional views of the polycarbonate topography.

due to the influence of AB interactions on the XDLVO predictions of S. aureus adhesion. The calculation of AB interactions between bacteria and surfaces is based on electron acceptor and electron donor components of bacterial cells, which can be obtained from CAM outcomes. Previous studies have shown a lack of correlation between the outcomes of CAMs and other techniques, such as MATS, especially in the AB components (11). However, our results reveal that the electron donor property of bacterial surfaces using MATS correlated with the CAM results. These results are in agreement with the finding of Bellon-Fontaine et al. (3), who found a correlation between MATS and CAM outcomes. The decrease of the electron donor character and the increase of the electron acceptor character of P. aeruginosa when the growth temperature increased could explain the increase of adhesion onto both stainless steel and polycarbonate. This is promoted by the increase of attractive AB interactions

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between P. aeruginosa cells and the two surfaces. By contrast, the AB character of S. aureus, according to the MATS measurement, did not explain the differences found in the experimental results. The MATS result indicated that the increase in bacterial hydrophobicity with increasing bacterial growth temperature may have resulted in increasing of bacterial adhesion of P. aeruginosa and S. aureus. However, this result is not in agreement with what has been reported on the involvement of hydrophobicity in the adhesion of L. monocytogenes (26). In general, studies on the prediction of bacterial adhesion to abiotic surfaces were made in optimal culture conditions when strains are tested. However, bacteria in natural ecosystems, and or in man-made ones are subjected to various environmental stresses and this could influence the physicochemical properties of cell surfaces and then the behavior of bacterial adhesion. Presumably, intrinsic factors related to the cell envelope, such as adhesins, cell wall proteins, extracellular polymers, flagellar motility, pili are involved in bacterial attachment to surfaces (17). In turn, these structures are reportedly influenced by the growth temperature (7, 15, 24, 29). The discrepancy between empirical and theoretical results is probably due to the failure of the XDLVO to detect the molecular changes in bacterial surface that can take place with the change of growth conditions. This discrepancy could be also related to the surface roughness of abiotic surfaces. Although surface roughness is not included in the XDLVO theory, it may also have an effect on bacterial adhesion as previously reported (16, 33, 42). Mitik-Dineva et al. (27) underlined that the adhesion levels of P. aeruginosa and S. aureus appeared to be inversely correlated with surface roughness. However, our results indicated that stainless steel was 10 times rougher than polycarbonate and that bacterial adhesion was two times higher on stainless steel. Our results suggest that nanoscale surface roughness might exert a significant effect on the bacterial adhesion as previously described. Therefore, it should be considered as a parameter of primary interest alongside other well-recognized factors that control initial bacterial attachment. In conclusion, our results underlined that the bacterial ecological background in which the bacterium is found plays a role in the adhesion of bacteria to abiotic surfaces. Moreover, growth temperatures close to that of the human body seemed to increase the adhesion rate to food contact surfaces. This highlighted the important role that food handlers may play as a reservoir of potential biofilmforming pathogens such as S. aureus. Thus, food handlers should ensure personal hygiene and use the appropriate personal protective equipment in order to reduce this microbiological risk in the food area. The use of smooth surfaces in food processing equipment may also reduce the microbial contamination of surfaces. Our findings also underlined that physicochemical properties of bacteria and surfaces are equally important and are involved in bacterial adhesion. However, our results pointed out that neither the XDLVO nor the thermodynamic theory can fully predict experimental bacterial adhesion. More studies should consider the effect of environmental conditions and bacterial

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background on the bacterial adhesion to abiotic surfaces. It should be noted that other techniques such as AFM and chemical force microscopy have recently emerged as powerful approaches in bacterial adhesion studies (8, 10). Further experiments will focus on the quantification of bacterial adhesion forces using AFM in order to extend the knowledge of the mechanisms mediating bacterial adhesion to abiotic surfaces. ACKNOWLEDGMENTS The authors are grateful to French Agency for Research and Technology (ANRT) and SCIENTIS laboratory for the CIFRE grant supporting this work (CIFRE: 2010/0205).

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