Elaboration of highly hydrophobic polymeric surface--a potential strategy to reduce the adhesion of pathogenic bacteria?

Different polymeric surfaces have been modified in order to reach a high hydrophobic character, indeed the superhydrophobicity property. For this purpose, polypropylene and polystyrene have been treated by RF or µwaves CF4 plasma with different volumes, the results were compared according to the density of injected power. The effect of pretreatment such as mechanical abrasion or plasma activation was also studied. The modified surfaces were shown as hydrophobic, or even superhydrophobic depending of defects density. They were characterized by measurement of wettability and roughness at different scales, i.e. macroscopic, mesoscopic and atomic. It has been shown that a homogeneous surface at the macroscopic scale could be heterogeneous at lower mesoscopic scale. This was associated with the crystallinity of the material. The bioadhesion tests were performed with Gram positive and negative pathogenic strains: Listeria monocytogenes, Pseudomonas aeruginosa and Hafnia alvei. They have demonstrated an antibacterial efficiency of very hydrophobic and amorphous PS treated for all strains tested and a strain-dependent efficiency with modified PP surface being very heterogeneous at the mesoscopic scale. Thus, these biological results pointed out not only the respective role of the surface chemistry and topography in bacterial adhesion, but also the dependence on the peaks and valley distribution at bacteria dimension scale.

Possible overestimation of surface disinfection efficiency by assessment methods based on liquid sampling procedures as demonstrated by in situ quantification of spore viability.

The standard test methods used to assess the efficiency of a disinfectant applied to surfaces are often based on counting the microbial survivors sampled in a liquid, but total cell removal from surfaces is seldom achieved. One might therefore wonder whether evaluations of microbial survivors in liquid-sampled cells are representative of the levels of survivors in whole populations. The present study was thus designed to determine the "damaged/undamaged" status induced by a peracetic acid disinfection for Bacillus atrophaeus spores deposited on glass coupons directly on this substrate and to compare it to the status of spores collected in liquid by a sampling procedure. The method utilized to assess the viability of both surface-associated and liquid-sampled spores included fluorescence labeling with a combination of Syto 61 and Chemchrome V6 dyes and quantifications by analyzing the images acquired by confocal laser scanning microscopy. The principal result of the study was that the viability of spores sampled in the liquid was found to be poorer than that of surface-associated spores. For example, after 2 min of peracetic acid disinfection, less than 17% ± 5% of viable cells were detected among liquid-sampled cells compared to 79% ± 5% or 47% ± 4%, respectively, when the viability was evaluated on the surface after or without the sampling procedure. Moreover, assessments of the survivors collected in the liquid phase, evaluated using the microscopic method and standard plate counts, were well correlated. Evaluations based on the determination of survivors among the liquid-sampled cells can thus overestimate the efficiency of surface disinfection procedures.

The resistance of Bacillus atrophaeus spores to the bactericidal activity of peracetic acid is influenced by both the nature of the solid substrates and the mode of contamination.

AIMS: To evaluate the impact of the mode of contamination in relation with the nature of solid substrates on the resistance of spores of Bacillus atrophaeus -selected as surrogates of Bacillus anthracis- to a disinfectant, peracetic acid. METHODS AND RESULTS: Six materials confronted in urban and military environments were selected for their different structural and physicochemical properties. In parallel, two modes of contamination were examined, i.e. deposition and immersion. Deposition was used to simulate contamination by an aerosol and immersion by an extended contact with liquids. A pronounced difference in the biocontamination levels and spatial organization of spores was observed depending on the mode of contamination and the nature of the solid substrate considered, with consequences on decontamination. Contamination by immersion led to lower efficiency of peracetic acid decontamination than contamination by deposition. Infiltration of spores into porous materials after immersion is one reason. In contrast, the deposition mode aggregates cells at the surface of materials, explaining the similar disinfecting behaviour of porous and nonporous substrates when considering this inoculation route. CONCLUSIONS: The inoculation route was shown to be as influential a parameter as material characteristics (porosity and wettability) for decontamination efficacy. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide comparative information for the decontamination of B. atrophaeus spores in function of the mode of contamination and the nature of solid substrates.

Microbial inactivation using plasma-activated water obtained by gliding electric discharges.

AIM: To evaluate the microbial disinfection efficacy of a plasmachemical solution obtained by the activation of water with gliding electric discharges. METHODS AND RESULTS: Distilled water was activated for 5 min by a nonthermal quenched plasma of the glidarc type operating in humid air and at atmospheric pressure. The plasma-activated water (PAW) was then used to treat planktonic and adherent cells of Staphylococcus epidermidis, Leuconostoc mesenteroides (as models of Gram-positive bacteria), Hafnia alvei (a Gram-negative bacteria) and Saccharomyces cerevisiae (as a yeast model). The treatments were less efficient on adherent cells than on planktonic cells in the case of bacteria, but not of S. cerevisiae. Inactivation was more effective for bacteria than for the yeast. CONCLUSIONS: Significant reductions in microbial populations were achieved in all cases, demonstrating the effectiveness of this new approach to treat contaminated media. SIGNIFICANCE AND IMPACT OF THE STUDY: PAW is a promising solution with potential application to the decontamination of equipment and surfaces.

Fluorescence correlation spectroscopy to study diffusion and reaction of bacteriophages inside biofilms.

In the natural environment, most of the phages that target bacteria are thought to exist in biofilm ecosystems. The purpose of this study was to gain a clearer understanding of the reactivity of these viral particles when they come into contact with bacteria embedded in biofilms. Experimentally, we quantified lactococcal c2 phage diffusion and reaction through model biofilms using in situ fluorescence correlation spectroscopy with two-photon excitation. Correlation curves for fluorescently labeled c2 phage in nonreacting Stenotrophomonas maltophilia biofilms indicated that extracellular polymeric substances did not provide significant resistance to phage penetration and diffusion, even though penetration and diffusion were sometimes restricted because of the noncontractile tail of the viral particle. Fluctuations in the fluorescence intensity of the labeled phage were detected throughout the thickness of biofilms formed by c2-sensitive and c2-resistant strains of Lactococcus lactis but could never be correlated with time, revealing that the phage was immobile. This finding confirmed that recognition binding receptors for the viral particles were present on the resistant bacterial cell wall. Taken together, our results suggest that biofilms may act as "active" phage reservoirs that can entrap and amplify viral particles and protect them from harsh environments.

Surface properties and behaviour on abiotic surfaces of Staphylococcus carnosus, a genetically homogeneous species.

This work aimed to characterize the surface properties of Staphylococcus carnosus and the influence of different media on their ability to adhere and grow on industrial supports. As their colonization could be dependant of the strain, the genetic diversity of the strains was studied. The diversity of 13 strains analysed by pulsed-field gel electrophoresis revealed that the S. carnosus strains formed a homogeneous genetic group. Their surface properties, characterized by studying their affinity to solvents, were hydrophilic with a strong negative surface charge. The S. carnosus strain CIT 833 hardly adhered to polytetrafluoroethylene (PTFE) and stainless steel chips. Tryptic soy broth (TSB) was the most favourable medium for growth on stainless steel support while TSB/NaCl was better for growth on PTFE. Scanning electron microscopy (sem) showed that this strain weakly colonized both supports and did not form cell aggregates. Indeed, the strain did not synthesize polysaccharides. These results showed that S. carnosus adhered on different abiotic surfaces which are used in food factories but was not able to accumulate on these surfaces. The inability of S. carnosus to form biofilm could explain why S. carnosus is rarely isolated in meat processing environment.

Formation of biofilm by Staphylococcus xylosus.

The ability of 12 Staphylococcus xylosus strains to form biofilm was determined through the study of different criteria. Eleven out of the 12 strains were able to form biofilm, 10 preferentially on hydrophilic support (glass) and one, S. xylosus C2a, on both hydrophilic and hydrophobic (polystyrene) supports. The determination of bacterial surface properties showed that all strains were negatively charged with five strains moderately hydrophobic and seven hydrophilic. The bap and icaA genes, important for biofilm formation of some staphylococci, were searched. All strains were bap positive but icaA negative. Furthermore, S. xylosus strain C2a was studied on two supports widely used in the food industry, polytetrafluoroethylene (PTFE, hydrophobic) and stainless steel (hydrophilic) and appeared to adhere preferentially on stainless steel. Addition of 20 g/l of NaCl to Tryptic Soy Broth medium (TSB) did not improve significantly its adhesion but enhanced both bacterial growth and cell survival, which were optimum in this medium. Environmental scanning electron microscopy showed that S. xylosus C2a colonized the surface of stainless steel chips with intercellular spaces. The strain formed cell aggregates embedded in an amorphous polysaccharidic matrix. Indeed, synthesis of polysaccharides increased during growth on stainless steel chips in TSB.

Adsorption of a biosurfactant on surfaces to enhance the disinfection of surfaces contaminated with Listeria monocytogenes.

The effects of sodium hypochlorite (NaOCl) and peracetic acid/hydrogen peroxide (PAH) on the inactivation of adherent Listeria monocytogenes LO28 cells were examined. The surfaces tested were stainless steel and polytetrafluoroethylene (PTFE) conditioned or not with an anionic biosurfactant produced by Pseudomonas fluorescens. Dilution-neutralization methods were used to assess the effectiveness of sanitizer solutions on planktonic and adherent cells. Tests were performed on L. monocytogenes cultivated at 37 degrees Celsius (body temperature) or 20 degrees Celsius (ambient temperature). The results demonstrated that i) a total deficiency in nutrients induced by the incubation of cells in 0.15 M NaCl favored the action of NaOCl and PAH on planktonic cells; ii) by reducing the number of cells adhering to stainless steel, pre-conditioning of the surface with the biosurfactant reduced the level of contamination of the surface and thus favored the bactericidal activities of the disinfectants; and iii) the weak binding energies involved in the adsorption of the biosurfactant on PTFE surfaces resulted in there being no reduction by the polymer of the surface contamination. Furthermore, this study confirmed that adherent cells exhibited increased resistance to the actions of the disinfectants when compared to the resistance of planktonic cells.

Adhesion of Staphylococcus aureus and Staphylococcus epidermidis to the Episkin reconstructed epidermis model and to an inert 304 stainless steel substrate.

AIMS: The aim of this study was to evaluate the respective influence of the physicochemical interactions and the roughness involved in the first part of the biological substrate biocontamination. METHODS AND RESULTS: Therefore we compared the bioadhesion results obtained on the biological model substrate (Episkin) and on a commonly employed inert substrate (AISI 304 stainless steel), frequently used either in dermatology or in development of medical devices. The two studied strains presented different characteristics, both physicochemical and microbiological. Staphylococcus epidermidis, a relatively hydrophobic bacteria capable of exchanging interactions which are principally of the van der Waals type, adhered more to 304 steel than to the surface of reconstituted skin. As for S. aureus, an essentially basic, hydrophilic bacteria, was more adherent to Episkin (a bipolar, hydrophilic substrate) than to stainless steel (a unipolar, basic, hydrophilic substrate). CONCLUSIONS: In the absence of electrostatic interactions, the adhesion of substrate-dependent bacteria to the surface of reconstituted skin was dependent upon the balance between gamma(LW), gamma(+) and gamma(-). SIGNIFICANCE AND IMPACT OF THE STUDY: Consequently, so as to restrict microbial adhesion and reduce adhesive binding between micro-organisms and the surface of the skin, it would be preferable to render this substrate hydrophobic and apolar through the use of appropriate surface treatment.

Heterogeneity of diffusion inside microbial biofilms determined by fluorescence correlation spectroscopy under two-photon excitation.

Fluorescence correlation spectroscopy (FCS) under two-photon excitation was applied successfully to characterize the penetration and diffusion capabilities of fluorescent probes (latex beads and fluorescein isothiocyanate-dextran) of different size and electrical charge in two models of monomicrobial biofilms with low (Lactococcus lactis biofilm) or high (Stenotrophonas maltophilia biofilm) contents of extracellular polymeric substance (EPS). FCS measurements performed on each biofilm can show deviation from Brownian diffusion, depending on the local structure of the biofilm and the fluorophore size. In this case, we fitted the data to an anomalous diffusion model and determined apparent diffusion coefficients, which can be 50 times smaller than the values in aqueous solutions. This result was interpreted as steric hindrance of the diffusion of the fluorescent particles within the biofilm that can lead to a total inhibition as observed particularly in the mushroom-like structure of the S. maltophilia biofilm. Alternatively, mechanisms for the absence of FCS signal behavior were related to attractive electrostatic interactions between cationic particles and negatively charged bacteria or to specific interactions between dextrans and EPS of the biofilm matrix.

Adsorption of biosurfactant on solid surfaces and consequences regarding the bioadhesion of Listeria monocytogenes LO28.

AIMS: The influence of biosurfactant compounds produced by a strain of Pseudomonas fluorescens on the adhesion of Listeria monocytogenes LO28 to polytetrafluoroethylene (PTFE) and AISI 304 stainless steel surfaces was investigated. METHODS AND RESULTS: The biosurfactant was produced according to a simple, novel technique based on cultivation on nutrient agar. Adhesion studies were performed using L. monocytogenes cells cultured at 20 or 37 degrees C. CONCLUSIONS: A substrate-dependent behaviour of the LO28 strain (larger number of cells adhering to stainless steel than to PTFE), and a significant reduction (< 90%) in microbial adhesion levels through the prior adsorption of biosurfactants on stainless steel surfaces, which can be related to a change in the electron-donor characteristics of this substratum, was demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: The prior adsorption of biosurfactants on solid surfaces may constitute a new and effective means of combating the implantation of pathogenic micro-organisms in food processing plants.

Determination of the van der Waals, electron donor and electron acceptor surface tension components of static Gram-positive microbial biofilms.

A large number of studies have shown the influence of the physico-chemical properties of a surface on microbial adhesion phenomenon. In this study, we considered that the presence of a bacterial biofilm may be regarded as a "conditioning film" that may modify the physico-chemical characteristics of the support, and thus the adhesion capability of planktonic micro-organisms coming into contact with this substratum. In this context, we adapted a protocol for biofilm formation that allows, under our experimental conditions, contact angle measurements, the reference method to determine the energetic surface properties of a substratum. This made it possible to determine the van der Waals, electron acceptor and electron donor properties of static biofilms grown at 25 degrees C on stainless-steel slides with six Gram-positive bacteria isolated in dairy plants. A variance analysis indicated significant effects (P<0.05) of the bacterial strains and of the physiological state of the micro-organisms (planktonic or sessile) on the contact angles. To link the energetic properties of the six biofilms with direct adhesion experiments, we measured the affinity of fluorescent carboxylate-modified polystyrene beads for the different biofilm surfaces. The results correlated best with the electron-acceptor components of the biofilm surface energies, stressing the importance of Lewis acid-base interactions in adhesion mechanisms.

Physicochemical surface properties of five Listeria monocytogenes strains from a pork-processing environment in relation to serotypes, genotypes and growth temperature.

Physicochemical surface properties, related to electrostatic, van der Waals and Lewis acid-base interactions, of five Listeria monocytogenes strains isolated from pork-processing environments were determined after two subcultures at 37 degrees C and a final culture at three temperatures: 37, 10 and 4 degrees C. Three strains (Lm1, Lm114 and Lm191) were genetically related while two were unrelated (Lm25 and Lm74) according to ApaI-macrorestriction and pulsed-field gel electrophoresis (PFGE) typing. Listeria monocytogenes cell surfaces were generally negatively charged regardless of pH and tended to be hydrophilic due to a basic character. However, variable physicochemical surface properties of the five Listeria monocytogenes isolates were observed after growth at 37 degrees C. After growth at 10 degrees C, the three genetically related isolates exhibited similar surface properties and were slightly more hydrophilic and basic than the others. After growth at 4 degrees C, the five isolates displayed the same weak affinity for all kinds of solvents and low electrophoretic mobility values. A sharp decrease of temperature and subsequent growth of various Listeria monocytogenes strains resulted in loss of the physicochemical surface property variability, which may suggest the role of common chill adaptation mechanisms affecting surface properties.

Comparison of surface free energy between reconstructed human epidermis and in situ human skin.

BACKGROUND/AIMS: The objectives of this study were to characterise the Episkin(R) model of reconstructed epidermis histologically and energetic, and to compare the results with the data obtained by Mavon (9, 10) on different areas of skin on the human body and on different skin types. METHODS: Histologically, the two skin systems studied were stained using Goldner's trichome METHOD: Surface free energy was determined by the measurement of contact angles, using the posed drop METHOD: RESULTS: Histologically, the two skin systems demonstrated the same cellular organisation in stratified layers. With regard to surface free energy, Episkin, skin from the forehead, normal and hyper-seborrheic skins are hydrophilic surfaces, while skin from the forearm or of an oligo-seborrheic type tends to be hydrophobic. Episkin and skin from the forehead exhibit similar surface free energy profiles. It is impossible to state whether Episkin has characteristics closer to those of normal or hyper-seborrheic skin, but they differ significantly from those of oligo-seborrheic skin. CONCLUSION: The results obtained during this study justify the use of Episkin in a range of tests where the use of in situ human skin raises problems.

Listeria monocytogenes Scott A: cell surface charge, hydrophobicity, and electron donor and acceptor characteristics under different environmental growth conditions.

We determined the variations in the surface physicochemical properties of Listeria monocytogenes Scott A cells that occurred under various environmental conditions. The surface charges, the hydrophobicities, and the electron donor and acceptor characteristics of L. monocytogenes Scott A cells were compared after the organism was grown in different growth media and at different temperatures; to do this, we used microelectrophoresis and the microbial adhesion to solvents method. Supplementing the growth media with glucose or lactic acid affected the electrical, hydrophobic, and electron donor and acceptor properties of the cells, whereas the growth temperature (37, 20, 15, or 8 degrees C) primarily affected the electrical and electron donor and acceptor properties. The nonlinear effects of the growth temperature on the physicochemical properties of the cells were similar for cells cultivated in two different growth media, but bacteria cultivated in Trypticase soy broth supplemented with 6 g of yeast extract per liter (TSYE) were slightly more hydrophobic than cells cultivated in brain heart infusion medium (P < 0.05). Adhesion experiments conducted with L. monocytogenes Scott A cells cultivated in TSYE at 37, 20, 15, and 8 degrees C and then suspended in a sodium chloride solution (1.5 x 10(-1) or 1.5 x 10(-3) M NaCl) confirmed that the cell surface charge and the electron donor and acceptor properties of the cells had an influence on their attachment to stainless steel.

Effects of the growth procedure on the surface hydrophobicity of Listeria monocytogenes cells and their adhesion to stainless steel.

The aim of this study was to examine the physicochemical surface properties and the ability to adhere to stainless steel of three strains of Listeria monocytogenes after different cultivation procedures. To this end, bacteria were cultivated at 37 degrees C after storage at two frequently used temperatures (4 degrees C or -80 degrees C) and were then transferred into the liquid medium (trypticase soy broth supplemented with 6 g liter(-1) of yeast extract, pH 7.3) between one and four times. In addition, the influence of supplementing the growth medium with lactic acid was explored, this organic acid being representative of both the dairy and cured meat industries. The hydrophobic/hydrophilic and electron-acceptor/electron-donor characteristics of the strains were evaluated by the microbial adhesion to solvents method. Using this technique, we recorded an increase in the hydrophobic properties of one strain stored at 4 degrees C, with an increasing number of transfers in the media (P < 0.05). Another plant-isolated strain appeared more hydrophobic and stuck better to stainless steel when cells were stored at 4 degrees C rather than at -80 degrees C. Preculturing L. monocytogenes in a lactic acid-supplemented medium increased the affinity of microbial cells to solvents and the bacterial attachment to stainless steel (P < 0.05).

The role of the rodlet structure on the physicochemical properties of Aspergillus conidia.

Physico-chemical properties of Aspergillus conidia rely on their outer cell-wall rodlet layer. In A. fumigatus and A. nidulans, the rodlet structure is due to an hydrophobin encoded by homologous rodA genes. To evaluate the role of the rodlet structure on the physico-chemical properties of conidia, we compared hydrophobicity, Lewis acid-base (i.e. electron donor/acceptor) characteristics and electrostatic charge of hydrophobin-less (rodletless) mutant and wild-type conidia of A. fumigatus and A. nidulans. The results obtained by aqueous-solvent partitioning assays, microsphere adhesion assays and microelectrophoresis showed that the disruption of the rodA gene modifies surface properties of A. fumigatus and A. nidulans conidia, and confirmed that the rodlet layer plays a key role in their physico-chemical behaviour. The absence of this layer on A. fumigatus spores led to the appearance of weakly basic and acidic characteristics, and had a slight effect on the hydrophobicity of conidia. Whereas in A. nidulans, it induced a basic character, a marked decrease in hydrophobicity and in the polarization capacity (electronegativity) of conidia. These physico-chemical differences between A. fumigatus and A. nidulans rodletless conidia may be attributed to differences in the composition of the conidial outer cell-wall of the two species.

Cell surface characteristics of Lactobacillus casei subsp. casei, Lactobacillus paracasei subsp. paracasei, and Lactobacillus rhamnosus strains.

Hydrophilic and electrostatic cell surface properties of eight Lactobacillus strains were characterized by using the microbial adhesion to solvents method and microelectrophoresis, respectively. All strains appeared relatively hydrophilic. The strong microbial adhesion to chloroform, an acidic solvent, in comparison with microbial adhesion to hexadecane, an apolar n-alkane, demonstrated the particularity of lactobacilli to have an important electron donor and basic character and consequently their potential ability to generate Lewis acid-base interactions with a support. Regardless of their electrophoretic mobility (EM), strains were in general slightly negatively charged at alkaline pH. A pH-dependent behavior concerning cell surface charges was observed. The EM decreased progressively with more acidic pHs for the L. casei subsp. casei and L. paracasei subsp. paracasei strains until the isoelectric point (IEP), i.e., the pH value for which the EM is zero. On the other hand, the EM for the L. rhamnosus strains was stable from pH 8 to pH 3 to 4, at which point there was a shift near the IEP. Both L. casei subsp. casei and L. paracasei subsp. paracasei strains were characterized by an IEP of around 4, whereas L. rhamnosus strains possessed a markedly lower IEP of 2. The present study showed that the cell surface physicochemical properties of lactobacilli seem to be, at least in part and under certain experimental conditions, particular to the bacterial species. Such differences detected between species are likely to be accompanied by some particular changes in cell wall chemical composition.

Attachment of Salmonella choleraesuis choleraesuis to beef muscle and adipose tissues.

The attachment of Salmonella choleraesuis subsp. choleraesuis ATCC 15790 to beef muscle and adipose tissues was investigated. S. choleraesuis was found to adhere in higher numbers to muscle than to fat. The charge and the hydrophobicity of the surface of S. choleraesuis were evaluated by measurement of electrophoretic mobility, the contact angle with water, adhesion to hexadecane, and hydrophobic interaction chromatography. The overall negative charge of S. choleraesuis was masked by the high electrolyte concentration in the attachment medium (0.15 M phosphate-buffered saline). This bacterium was shown to possess a hydrophilic surface. Electrostatic interactions do not affect the attachment of S. choleraesuis to both lean and fat tissue, and there was no evidence for a role of hydrophobic interactions. However, the attachment of S. choleraesuis was reduced by 90% after mechanical removal of the flagella or after treatment of the bacteria with specific antiflagella serum. This reduction was attributed to a loss of bacterial mobility leading to a reduction in the number of cells reaching the tissue during the period of contact. Treatment of the tissue with a concentrated suspension of flagella or treatment of the bacteria with antisomatic serum (OMD) did not reduce the attachment of S. choleraesuis to tissues, indicating an absence of specific attachment sites for flagella or antigen O on the beef tissue surface.

Immobilization of fungal spores by adhesion.

Immobilization of conidiospores of Phanerochaete chrysosporium by adhesion was investigated in static and flow conditions on flat and on porous supports. Reducing the electrostatic repulsion between the spores and the support by adsorption of polycations on the support allows a better adhesion efficiency and a higher density of adhering spores and does not affect germination and growth. Formation of spore aggregates either in the suspension (high ionic strength) or on the support tends to decrease the surface coverage and to give an inhomogeneous distribution of adhering spores due to detachment of aggregates. The density of spores adhering from a flowing suspension is lower as compared with static conditions and does not exceed about 2% of surface coverage; this is due to the influence of tangential forces, to the short contact time with the surface, and to perturbation of the hydrodynamics along the surface by the previously immobilized spores. Obtaining a high coverage of the support by immobilized spores requires the absence of a tangential motion. (c) 1995 John Wiley & Sons, Inc.