Effect of Milk Proteins on Adhesion of Bacteria to Stainless Steel Surfaces

Stainless steel coupons were treated with skim milk and subsequently challenged with individual bacterial suspensions of Staphylococcus aureus, Pseudomonas fragi, Escherichia coli, Listeria monocytogenes, and Serratia marcescens. The numbers of attached bacteria were determined by direct epifluorescence microscopy and compared with the attachment levels on clean stainless steel with two different surface finishes. Skim milk was found to reduce adhesion of S. aureus, L. monocytogenes, and S. marcescens. P. fragi and E. coli attached in very small numbers to the clear surfaces, making the effect of any adsorbed protein layer difficult to assess. Individual milk proteins α-casein, β-casein, κ-casein, and α-lactalbumin were also found to reduce the adhesion of S. aureus and L. monocytogenes. The adhesion of bacteria to samples treated with milk dilutions up to 0.001% was investigated. X-ray photoelectron spectroscopy was used to determine the proportion of nitrogen in the adsorbed films. Attached bacterial numbers were inversely related to the relative atomic percentage of nitrogen on the surface. A comparison of two types of stainless steel surface, a 2B and a no. 8 mirror finish, indicated that the difference in these levels of surface roughness did not greatly affect bacterial attachment, and reduction in adhesion to a milk-treated surface was still observed. Cross-linking of adsorbed proteins partially reversed the inhibition of bacterial attachment, indicating that protein chain mobility and steric exclusion may be important in this phenomenon.     

Bacterial Adhesion at Synthetic Surfaces

A systematic investigation into the effect of surface chemistry on bacterial adhesion was carried out. In particular, a number of physicochemical factors important in defining the surface at the molecular level were assessed for their effect on the adhesion of Listeria monocytogenes, Salmonella typhimurium, Staphylococcus aureus, and Escherichia coli. The primary experiments involved the grafting of groups varying in hydrophilicity, hydrophobicity, chain length, and chemical functionality onto glass substrates such that the surfaces were homogeneous and densely packed with functional groups. All of the surfaces were found to be chemically well defined, and their measured surface energies varied from 15 to 41 mJ · m⁻². Protein adsorption experiments were performed with ³H-labelled bovine serum albumin and cytochrome c prior to bacterial attachment studies. Hydrophilic uncharged surfaces showed the greatest resistance to protein adsorption; however, our studies also showed that the effectiveness of poly(ethyleneoxide) (PEO) polymers was not simply a result of its hydrophilicity and molecular weight alone. The adsorption of the two proteins approximately correlated with short-term cell adhesion, and bacterial attachment for L. monocytogenes and E. coli also correlated with the chemistry of the underlying substrate. However, for S. aureus and S. typhimurium a different pattern of attachment occurred, suggesting a dissimilar mechanism of cell attachment, although high-molecular-weight PEO was still the least-cell-adsorbing surface. The implications of this for in vivo attachment of cells suggest that hydrophilic passivating groups may be the best method for preventing cell adsorption to synthetic substrates provided they can be grafted uniformly and in sufficient density at the surface.     

Chemical modification of polyvinyl chloride and silicone elastomer in inhibiting adhesion of Aeromonas hydrophila

Disease-causing bacteria of the genus Aeromonas are able to adhere to pipe materials, colonizing the surfaces and forming biofilms in water distribution systems. The aim of our research was to study how the modification of materials used commonly in the water industry can reduce bacterial cell attachment. Polyvinyl chloride and silicone elastomer surfaces were activated and modified with reactive organo-silanes by coupling or co-crosslinking silanes with the native material. Both the native and modified surfaces were tested using the bacterial strain Aeromonas hydrophila, which was isolated from the Polish water distribution system. The surface tension of both the native and modified surfaces was measured. To determine cell viability and bacterial adhesion two methods were used, namely plate count and luminometry. Results were expressed in colony-forming units (c.f.u.) and in relative light units (RLU) per cm². Almost all the chemically modified surfaces exhibited higher anti-adhesive and anti-microbial properties in comparison to the native surfaces. Among the modifying agents examined, poly[dimethylsiloxane-co-(N,N-dimethyl-N-n-octylammoniopropyl chloride) methylsiloxane)] terminated with hydroxydimethylsilyl groups (20 %) in silicone elastomer gave the most desirable results. The surface tension of this modifier, was comparable to the non-polar native surface. However, almost half of this value was due to the result of polar forces. In this case, in an adhesion analysis, only 1 RLU cm^?2 and less than 1 c.f.u. cm^?2 were noted. For the native gumosil, the results were 9,375 RLU cm^?2 and 2.5 × 10⁸ c.f.u. cm^?2, respectively. The antibacterial activity of active organo-silanes was associated only with the carrier surface because no antibacterial compounds were detected in liquid culture media, in concentrations that were able to inhibit cell growth.     

Atomic force microscopy and hydrodynamic characterization of the adhesion of <Emphasis Type="Italic">staphylococcus aureus</Emphasis> to hydrophilic and hydrophobic substrata at different pH values

Understanding the mechanism of the bacterial cell adhesion to solid surfaces is of great medical and industrial importance. Bacterial adhesion to inert surfaces, such as a catheter, and other indwelling devices can form biofilm, consequently cause severe morbidity and often fatal infections. Initial bacterial adhesion to the material surfaces is a complicated process that is affected by various physicochemical properties of both bacterial cells and substratum surfaces. The surface properties of the cells were characterized by the sessile drop technique. Moreover, the interfacial free energy of Staphylococcus aureus adhesion to the supporting materials was determined. The results showed that S. aureus examined at different pH levels could be considered hydrophilic. We noted hat the electron-donor character of S. aureus was important at intermediate pH (pH 5, pH 7, and pH 9) and it decreased at both limits acidic and basic conditions. In addition, the adhesion of Staphylococcus aureus ATCC 25923 to the hydrophilic glass and hydrophobic indium tin oxide (ITO)-coated glass surfaces at different pH values (2, 3, 5, 7, 9 and 11) was investigated using atomic force microscopy (AFM) and image analysis was assessed with the Mathlab^® program. The data analysis showed that cells (number of adhering cells to glass and ITO-coated glass surface) adhered strongly at acidic pH and weakly at alkaline pH. Also, S. aureus has the ability to attach to both hydrophobic and hydrophilic surfaces, but the adhesion was higher on hydrophobic surface.     

Nisin-activated hydrophobic and hydrophilic surfaces: assessment of peptide adsorption and antibacterial activity against some food pathogens

An effective antimicrobial packaging or food contact surface should be able to kill or inhibit micro-organisms that cause food-borne illnesses. Setting up such systems, by nisin adsorption on hydrophilic and hydrophobic surfaces, is still a matter of debate. For this purpose, nisin was adsorbed on two types of low-density polyethylene: the hydrophobic native film and the hydrophilic acrylic acid-treated surface. The antibacterial activity was compared for those two films and it was highly dependent on the nature of the surface and the nisin-adsorbed amount. The hydrophilic surfaces presented higher antibacterial activity and higher amount of nisin than the hydrophobic surfaces. The effectiveness of the activated surfaces was assessed against Listeria innocua and the food pathogens Listeria monocytogenes, Bacillus cereus, and Staphylococcus aureus. S. aureus was more sensitive than the three other test bacteria toward both nisin-functionalized films. Simulation tests to mimic refrigerated temperature showed that the films were effective at 20 and 4 °C with no significant difference between the two temperatures after 30 min of exposure to culture media.     

Surfactin reduces the adhesion of food-borne pathogenic bacteria to solid surfaces

Aims:  To investigate the effect of the biosurfactants surfactin and rhamnolipids on the adhesion of the food pathogens Listeria monocytogenes , Enterobacter sakazakii and Salmonella Enteritidis to stainless steel and polypropylene surfaces.
Methods and Results:  Quantification of bacterial adhesion was performed using the crystal violet staining technique. Preconditioning of surfaces with surfactin caused a reduction on the number of adhered cells of Ent. sakazakii and L. monocytogenes on stainless steel. The most significant result was obtained with L. monocytogenes where number of adhered cells was reduced by 10² CFU cm⁻². On polypropylene, surfactin showed a significant decrease on the adhesion of all strains. The adsorption of surfactin on polystyrene also reduces the adhesion of L. monocytogenes and Salm. Enteritidis growing cells. For short contact periods using nongrowing cells or longer contact periods with growing cells, surfactin was able to delay bacterial adhesion.
Conclusions:  The prior adsorption of surfactin to solid surfaces contributes on reducing colonization of the pathogenic bacteria.
Significance and Impact of the Study:  This is the first work investigating the effect of surfactin on the adhesion of the food pathogens L. monocytogenes , Ent. sakazakii and Salm. Enteritidis to polypropylene and stainless steel surfaces.     

Synthesis,characterization and in vitro activity of a surface-attached antimicrobial cationic peptide

Infection associated with implanted biomaterials is common and costly and such infections are extremely resistant to antibiotics and host defenses. Consequently, there is a need to develop surfaces which resist bacterial adhesion and colonization. The broad spectrum synthetic cationic peptide melimine has been covalently linked to a surface via two azide linkers, 4-azidobenzoic acid (ABA) or 4-fluoro-3-nitrophenyl azide (FNA), and the resulting surfaces characterized by X-ray photoelectron spectroscopy and contact angle measurements. The quantity of bound peptide was estimated by a modified Bradford assay. The antimicrobial efficacy of the two melimine-modified surfaces against Pseudomonas aeruginosa and Staphylococcus aureus was compared by scanning electron microscopy (SEM) and fluorescence microscopy. Attachment of melimine via ABA gave an approximately 4-fold greater quantity of melimine bound to the surface than attachment via FNA. Surfaces melimine-modified by either attachment strategy showed significantly reduced bacterial adhesion for both strains of bacteria. P. aeruginosa exposed to ABA–melimine and FNA–melimine surfaces showed marked changes in cell morphology when observed by SEM and a reduction of approximately 15-fold (p < 0.001) in the numbers of adherent bacteria compared to controls. For the ABA–melimine surface there was a 33% increase in cells showing damaged membranes (p = 0.0016) while for FNA–melimine there was no significant difference. For S. aureus there were reductions in bacterial adhesion of approximately 40-fold (p < 0.0001) and 5-fold (p = 0.008) for surfaces modified with melimine via ABA or FNA, respectively. There was an increase in cells showing damaged membranes on ABA–melimine surfaces of approximately 87% (p = 0.001) compared to controls, while for FNA–melimine there was no significant difference observed. The data presented in this study show that melimine has excellent potential for development as a broad spectrum antimicrobial coating for biomaterial surfaces. Further, it was observed that the efficacy of antimicrobial activity is related to the method of attachment.     

Carbohydrate Coating Reduces Adhesion of Biofilm-Forming Bacillus subtilis to Gold Surfaces

The growth of bacterial biofilms in pipes and food tanks causes severe problems in industry. Biofilms growing on medical implants or catheters are of great concern, as they can cause serious infections and decrease the functionality of the medical device. The prevention of bacterial adhesion—the first step in colonization and biofilm formation—is therefore very important. Current research comprises alterations in surface properties, the prevention of adhesin biosynthesis, inhibition with receptor analogs, or the development of anti-adhesive vaccines. We present a new approach that allows us to study bacterial adhesion with high sensitivity in real-time while testing several different surfaces in parallel. Using the cantilever-array technique we demonstrate that coating of gold surfaces with mono- or disaccharides results in a reduction of the bacterial adhesion of the biofilm-forming bacterium Bacillus subtilis NCIB 3610 to these gold surfaces. This reduction in bacterial adhesion is independent of the studied carbohydrate. Using several mutant strains, we investigate the underlying molecular interactions, and our results suggest that adhesion to gold surfaces is mediated by thiol groups present in proteins of the bacterial cell membrane or biofilm matrix proteins expressed at low levels by the wild-type strain. Furthermore, our data indicate that the adhesion of B. subtilis NCIB 3610 to carbohydrate-coated gold surfaces is facilitated by interactions between carbohydrates installed on the cantilever gold surface and an exopolysaccharide expressed by this strain. Understanding general and specific contributions of molecular interactions mediating bacterial adhesion will enable its prevention in the future.     

Fluorescence-Based Bacterial Overlay Method for Simultaneous In Situ Quantification of Surface-Attached Bacteria

For quantification of bacterial adherence to biomaterial surfaces or to other surfaces prone to biofouling, there is a need for methods that allow a comparative analysis of small material specimens. A new method for quantification of surface-attached biotinylated bacteria was established by in situ detection with fluorescence-labeled avidin-D. This method was evaluated utilizing a silicon wafer model system to monitor the influences of surface wettability and roughness on bacterial adhesion. Furthermore, the effects of protein preadsorption from serum, saliva, human serum albumin, and fibronectin were investigated. Streptococcus gordonii, Streptococcus mitis, and Staphylococcus aureus were chosen as model organisms because of their differing adhesion properties and their clinical relevance. To verify the results obtained by this new technique, scanning electron microscopy and agar replica plating were employed. Oxidized and poly(ethylene glycol)-modified silicon wafers were found to be more resistant to bacterial adhesion than wafers coated with hydrocarbon and fluorocarbon moieties. Roughening of the chemically modified surfaces resulted in an overall increase in bacterial attachment. Preadsorption of proteins affected bacterial adherence but did not fully abolish the influence of the original surface chemistry. However, in certain instances, mostly with saliva or serum, masking of the underlying surface chemistry became evident. The new bacterial overlay method allowed a reliable quantification of surface-attached bacteria and could hence be employed for measuring bacterial adherence on material specimens in a variety of applications.     

Characterisation and in vitro activities of surface attached dihydropyrrol-2-ones against Gram-negative and Gram-positive bacteria

Bacterial infection of biomedical devices is still a major barrier to their use. This is compounded by increasing antibiotic resistance. Here, the specific covalent attachment of a series of dihydropyrrol-2-one (DHP), analogues of bacterial quorum sensing inhibitors, to surfaces via a Michael-type addition reaction is described. Differences in efficiency of attachment related to the substituent groups were found by X-ray photoelectron spectroscopy. The physical characteristics of the surfaces were further explored by atomic force microscopy and contact angle measurements. The ability of these coatings to prevent the formation of a biofilm by Pseudomonas aeruginosa and Staphylococcus aureus was examined using confocal laser scanning microscopy and image analysis. The DHP-treated surfaces showed significant reductions in bacterial adhesion without increased killing for both strains of bacteria (p < 0.001). 5-Methylene-1-(prop-2-enoyl)-4-phenyl-dihydropyrrol-2-one was identified as having broad spectrum activity and consequently represents an excellent candidate for the development of novel surfaces for the prevention of biomedical device infections.     

High expression of a plectasin-derived peptide NZ2114 in Pichia pastoris and its pharmacodynamics, postantibiotic and synergy against Staphylococcus aureus

NZ2114, a new variant of plectasin, was overexpressed in Pichia pastoris X-33 via pPICZαA for the first time. The total secreted protein of fermentation supernatant reached 2,390 mg/l (29 °C) and 2,310 mg/l (25 °C), and the recombinant NZ2114 (rNZ2114) reached 860 mg/l (29 °C) and 1,309 mg/l (25 °C) at 96 h induction in a 5-l fermentor, respectively.The rNZ2114 was purified by cation exchange chromatography, and its yield was 583 mg/l with 94.8 % purity. The minimal inhibitory concentration (MIC) of rNZ2114 to four ATCC strains of Staphyloccocus aureus was evaluated from 0.028 to 0.90 μM. Meanwhile, it showed potent activity (0.11–0.90 μM) to 20 clinical isolates of MRSA. The rNZ2114 killed over 99.9 % of tested S. aureus (ATCC 25923 and ATCC 43300) in Mueller-Hinton medium within 6 h when treated with 4?×?MIC. The postantibiotic effect of rNZ2114 to S. aureus ATCC 25923 and ATCC 43300 was 18.6–45.6 and 1.7–3.5 h under 1×, 2×, and 4× MIC, respectively. The fractional inhibitory concentration index (FICI) indicated a synergistic effect between rNZ2114 and kanamycin, streptomycin, and vancomycin against S. aureus ATCC 25923 (FICI?=?0.125), and additivity between rNZ2114 and ampicillin, spectinomycin (FICI?=?0.625), respectively. To S. aureus ATCC 43300 [methicillin-resistant S. aureus (MRSA)], rNZ2114 showed a synergistic effect (FICI?=?0.125–0.3125) with kanamycin, ampicillin, streptomycin, and vancomycin, and antagonism with spectinomycin (FICI?=?8.0625). The rNZ2114 caused only less than 0.1 % hemolytic activity in the concentration of 128 μg/ml, and showed a good thermostability from 20 to 80 °C. In addition, it exhibited the highest activity at pH 8.0. These results suggested that large-scale production of NZ2114 is feasible using the P. pastoris expression system, and it could be a new potential antimicrobial agent for the prevention and treatment of S. aureus especially for MRSA infections.     

Development and characterization of essential oil component-based polymer films: a potential approach to reduce bacterial biofilm

The development of new polymeric materials aimed to control the bacterial biofilm appears to be an important practical approach. The goal of the present study was to prepare and characterize poly(ethylene-co-vinyl acetate) copolymer (EVA) films containing citronellol, eugenol, and linalool and evaluate their efficiency on growth and biofilm formation of Listeria monocytogenes, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa in monospecies and dual species. The results showed that the addition of oil components influenced the elastic modulus (15 % decrease), the tensile stress (30 % decrease), the elongation at break (10 % increase), and the contact angle values (10–20° decrease) while leaving the homogeneity of the surface unaltered. Among the polymeric films, EVA + citronellol and EVA + eugenol at 7 wt% had the best inhibitory effect. After 24–48 h of incubation, EVA + citronellol was more effective against the growth (30–60 % reduction) than EVA + eugenol (15–30 % inhibition). However, this inhibition decreased after 240 h of incubation. On the contrary, the biofilm evaluation revealed a strong inhibition trend also after prolonged incubation time: the amount of biomass per square centimeter formed on copolymer with oil components was significantly less (40–70 % decrease) than that on pure copolymer control for L. monocytogenes, S. aureus, and E. coli. When polymeric materials were simultaneously inoculated with combinations of S. aureus and E. coli, the biomass accumulated was higher for EVA + citronellol and lower for EVA + eugenol than that in monoculture biofilm. The findings were similar to the results obtained by 2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide assay that measures the metabolic activity of viable cells.     

Assessment of synergistic combination potential of probiotic and bacteriophage against antibiotic-resistant Staphylococcus aureus exposed to simulated intestinal conditions

This study was designed to evaluate the combined effect of probiotic Lactobacillus rhamnosus and bacteriophage SA11 on the control of antibiotic-sensitive Staphylococcus aureus (ASSA) and antibiotic-resistant S. aureus (ARSA) under the simulated intestinal conditions. The survivability of ASSA and ARSA were determined in the simulated phosphate-buffered saline (PBS)-, trypticase soy broth (TSB)-, and milk-based gastric juices adjusted to pH 2.0, 3.0, and 5.0 at 37 °C for 30 min. The inhibitory effect of bacteriophage SA11 and probiotic on the growth of ASSA and ARSA was evaluated in the simulated intestinal juices at 37 °C for 20 h. The least reductions in the numbers of ASSA and ARSA were observed in the milk-based gastric juices at pH 2.0 (<1 log). No significant changes in the teichoic acid-mediated sliding motility were observed for ASSA and ARSA after 30-min exposure to the simulated gastric juices (pH 2.0, 3.0, and 5.0), responsible for the enhanced bacterial attachment to the epithelial cells. The bacteriophage SA11 was stable down to pH 5.0 and up to 0.06 % bile salts. The bacteriophage SA11 combined with probiotic effectively inhibited the growth of ASSA and ARSA in the simulated intestinal conditions, showing more than 4 log reduction. The relative expression levels of adhesion-related genes (clfA, eno, and fnbA) and efflux-related genes (mdeA, norB, and norC) were less decreased in ARSA than in ASSA after exposure to the simulated gastrointestinal conditions. These results might shed light on the application of bacteriophage to control the ingested antibiotic-resistant foodborne pathogens in the intestinal tract.     

Antimicrobial activity of four cationic peptides immobilised to poly-hydroxyethylmethacrylate

The objective of this study was to immobilise and characterise a variety of antimicrobial peptides (AMPs) onto poly-hydroxyethylmethacrylate (pHEMA) surfaces to achieve an antibacterial effect. Four AMPs, viz. LL-37, melimine, lactoferricin and Mel-4 were immobilised on pHEMA by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) which assisted covalent attachment. Increasing concentrations of AMPs were immobilised to determine the effect on the adhesion of Pseudomonas aeruginosa and Staphylococcus aureus. The AMP immobilised pHEMAs were characterised by X-ray photoelectron spectroscopy (XPS) to determine the surface elemental composition and by amino acid analysis to determine the total amount of AMP attached. In vitro cytotoxicity of the immobilised pHEMA samples to mouse L929 cells was investigated. Melimine and Mel-4 when immobilised at the highest concentrations showed 3.1 ± 0.6 log and 1.3 ± 0.2 log inhibition against P. aeruginosa, and 3.9 ± 0.6 log and 2.4 ± 0.5 log inhibition against S. aureus, respectively. Immobilisation of LL-37 resulted in up to 2.6 ± 1.0 log inhibition against only P. aeruginosa, but no activity against S. aureus. LFc attachment showed no antibacterial activity. Upon XPS analysis, immobilised melimine, LL-37, LFc and Mel-4 had 1.57 ± 0.38%, 1.13 ± 1.36%, 0.66 ± 0.47% and 0.73 ± 0.32% amide nitrogen attached to pHEMA compared to 0.12 ± 0.14% in the untreated controls. Amino acid analysis determined that the total amount of AMP attachment to pHEMA was 44.3 ± 7.4 nmol, 3.8?±?0.2?nmol, 6.5 ± 0.6 nmol and 48.9 ± 2.3 nmol for the same peptides respectively. None of the AMP immobilised pHEMA surfaces showed any toxicity towards mouse L929 cells. The immobilisation of certain AMPs at nanomolar concentration to pHEMA is an effective option to develop a stable antimicrobial surface.     

<Emphasis Type="Italic">Escherichia coli,Pseudomonas aeruginosa</Emphasis>, and<Emphasis Type="Italic"> Staphylococcus aureus</Emphasis> Attachment Patterns on Glass Surfaces with Nanoscale Roughness

Attachment tendencies of Escherichia coli K12, Pseudomonas aeruginosa ATCC 9027, and Staphylococcus aureus CIP 68.5 onto glass surfaces of different degrees of nanometer-scale roughness have been studied. Contact-angle and surface-charge measurements, atomic force microscopy (AFM), scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM) were employed to characterize substrata and bacterial surfaces. Modification of the glass surface resulted in nanometer-scale changes in the surface topography, whereas the physicochemical characteristics of the surfaces remained almost constant. AFM analysis indicated that the overall surface roughness parameters were reduced by 60–70%. SEM, CLSM, and AFM analysis clearly demonstrates that although E. coli, P. aeruginosa and S. aureus present significantly different patterns of attachment, all of the species exhibited a greater propensity for adhesion to the “nano-smooth” surface. The bacteria responded to the surface modification with a remarkable change in cellular metabolic activity, as shown by the characteristic cell morphologies, production of extracellular polymeric substances, and an increase in the number of bacterial cells undergoing attachment.     

Development and validation of a high-throughput whole cell assay to investigate Staphylococcus aureus adhesion to host ligands

Staphylococcus aureus adhesion to the host''s skin and mucosae enables asymptomatic colonization and the establishment of infection. This process is facilitated by cell wall-anchored adhesins that bind to host ligands. Therapeutics targeting this process could provide significant clinical benefits; however, the development of anti-adhesives requires an in-depth knowledge of adhesion-associated factors and an assay amenable to high-throughput applications. Here, we describe the development of a sensitive and robust whole cell assay to enable the large-scale profiling of S. aureus adhesion to host ligands. To validate the assay, and to gain insight into cellular factors contributing to adhesion, we profiled a sequence-defined S. aureus transposon mutant library, identifying mutants with attenuated adhesion to human-derived fibronectin, keratin, and fibrinogen. Our screening approach was validated by the identification of known adhesion-related proteins, such as the housekeeping sortase responsible for covalently linking adhesins to the cell wall. In addition, we also identified genetic loci that could represent undescribed anti-adhesive targets. To compare and contrast the genetic requirements of adhesion to each host ligand, we generated a S. aureus Genetic Adhesion Network, which identified a core gene set involved in adhesion to all three host ligands, and unique genetic signatures. In summary, this assay will enable high-throughput chemical screens to identify anti-adhesives and our findings provide insight into the target space of such an approach.     

Effects of growth temperature on the adhesion ofPseudomonas aeruginosa ATCC 27853 to polystyrene

The aim of this work has been to analyse the effects of temperature on polystyrene adhesion ofPseudomonas aeruginosa ATCC 27853. The bacterial adhesion (expressed as percentage of hydrophobicity) has been measured during the cultivation of this strain at different temperature of growth (15, 30 and 47°C). Data obtained showed that an increase in temperature is a factor that increasing the virulence of the strain in terms of adhesion to polystyrene surfaces. This kind of experiments surely brings important information concerning the prevention of nosocomial infection.     

Variation in Bacterial ATP Level and Proton Motive Force Due to Adhesion to a Solid Surface

Bacterial adhesion to natural and man-made surfaces can be beneficial or detrimental, depending on the system at hand. Of vital importance is how the process of adhesion affects the bacterial metabolic activity. If activity is enhanced, this may help the cells colonize the surface, whereas if activity is reduced, it may inhibit colonization. Here, we report a study demonstrating that adhesion of both Escherichia coli and Bacillus brevis onto a glass surface resulted in enhanced metabolic activity, assessed through ATP measurements. Specifically, ATP levels were found to increase two to five times upon adhesion compared to ATP levels in corresponding planktonic cells. To explain this effect on ATP levels, we propose the hypothesis that bacteria can take advantage of a link between cellular bioenergetics (proton motive force and ATP formation) and the physiochemical charge regulation effect, which occurs as a surface containing ionizable functional groups (e.g., the bacterial cell surface) approaches another surface. As the bacterium approaches the surface, the charge regulation effect causes the charge and pH at the cell surface to vary as a function of separation distance. With negatively charged surfaces, this results in a decrease in pH at the cell surface, which enhances the proton motive force and ATP concentration. Calculations demonstrated that a change in pH across the cell membrane of only 0.2 to 0.5 units is sufficient to achieve the observed ATP increases. Similarly, the hypothesis indicates that positively charged surfaces will decrease metabolic activity, and results from studies of positively charged surfaces support this finding.Bacterial adhesion and biofilm formation are important to a wide array of fields, such as environmental, chemical, and biomedical engineering; food processing; materials science; marine science; and ecology and environmental sciences. A key consideration in the development of a biofilm is the initial interaction between the bacterium and the substrata and the way in which these interactions affect the metabolic activity of the cells. If the metabolic activity increases, this would help the cells colonize the surface, whereas if the metabolic activity decreases, then the cells may become inactive or die.There have been a number of studies that have examined the effects of attachment on bacterial metabolic activity. The first reported observation of this effect was made in 1943, where it was found that bacterial activity increased in the presence of glass surfaces, particularly with low nutrient concentrations (54). Since this first study, researchers have examined how various materials, including glass and polymer surfaces (10, 11, 24-26, 40, 44, 47), silicone surfaces (52), ceramic surfaces (32), dialysis membranes (18), activated carbon (7), clays (43), sands (31, 48), estuary particles (19), and ion exchange resins (46), affect the metabolic activity of attached bacteria.Different mechanisms have been examined in an attempt to explain how surfaces affect bacterial metabolic activity. For example, one study found that activity increased with bacterial hydrophobicity, presumably due to firmer adhesion on the surface (24). Other researchers found that bacterial activity both increased (11) and decreased (40) with decreasing hydrophobicity of the solid surface. A study of bacterial attachment to clays showed that most 2:1 clays enhanced respiration, while 1:1 clays had minimal effect (43). It has also been observed that positively charged surfaces can reduce cell viability (13, 26, 44, 47). Unfortunately, given these disparate results, the prediction of how a specific surface may affect bacterial metabolic activity remains elusive.Here, we propose a hypothesis on how surfaces may affect bacterial metabolic activity. This hypothesis is based on a link between the physiochemical charge regulation effect, which occurs as a surface with acid/base functional groups approaches another surface, and cellular bioenergetics, where ATP is produced from pH and electrostatic charge gradients (ΔpH and Δψ, respectively) set up across the bacterial cytoplasmic membrane, the sum of which is termed proton motive force (Δp). The charge regulation effect results in a variation in the cell surface pH as the cell approaches another surface, and the hypothesis we propose is that this variation in cell surface pH will affect ΔpH, ultimately affecting Δp, with a concomitant variation in the cellular ATP level.In order to explore this hypothesis, the objectives of the current study were to (i) demonstrate the direct link between bacterial adhesion to a surface and an increase in cellular ATP concentration and (ii) calculate the increase in Δp required to achieve the observed ATP increase. The gram-negative strain Escherichia coli K-12 and the gram-positive organism Bacillus brevis were the focus of this study. Two different solution chemistries were used, one containing phosphate buffer solution (PBS) (a phosphate-based buffer) and another containing piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) (a nitrogen- and sulfur-based buffer). Through the use of vials containing a fixed mass of one of three different-sized glass beads and also vials without glass beads as controls, various glass surface areas were available for cellular adhesion. These experiments were based on the concept that the total ATP concentration in vials with different surface areas for adhesion would vary as a function of surface area if adhesion affects ATP, whereas the total ATP concentration would be invariant with surface area if adhesion did not affect ATP. The number of adhered cells and total ATP per vial were quantified, allowing calculation of the ATP concentrations for both planktonic and adhered bacteria. To verify that bacterial adhesion, rather than just the presence of the glass beads, affected metabolic activity, an identical series of experiments was conducted with the nonionic surfactant Tween 80 present in solution. Tween 80 reduced bacterial adhesion to the glass beads and allowed the verification that the presence of the glass beads themselves did not have any effect on cellular ATP concentrations. Used in combination, these experiments allowed quantification of the effects of cell adhesion on the bacterial ATP concentration and Δp.     

Variations in carotenoid content and acyl chain composition in exponential,stationary and biofilm states of Staphylococcus aureus,and their influence on membrane biophysical properties

Bacteria are often found in close association with surfaces, resulting in the formation of biofilms. In Staphylococcus aureus (S. aureus), biofilms are implicated in the resilience of chronic infections, presenting a serious clinical problem world-wide. Here, S. aureus biofilms are grown under flow within clinical catheters at 37 °C. The lipid composition and biophysical properties of lipid extracts from these biofilms are compared with those from exponential growth and stationary phase cells. Biofilms show a reduction in iso and anteiso branching compensated by an increase in saturated fatty acids compared to stationary phase. A drastic reduction in carotenoid levels is also observed during biofilm formation. Thermotropic measurements of Laurdan GP and DPH polarization, show a reduction of lipid packing at 37 °C for biofilms compared to stationary phase. We studied the effects of carotenoid content on DMPG and DPPG model membranes showing trends in thermotropic behavior consistent with those observed in bacterial isolates, indicating that carotenoids participate in modulating lipid packing. Additionally, bending elastic constant (k_c) measurements using vesicle fluctuation analysis (VFA) show that the presence of carotenoids can increase membrane bending rigidity. The antimicrobial peptide Magainin H2 was less activity on liposomes composed of stationary phase compared to biofilms or exponential growth isolates. This study contributes to an understanding of how Staphylococcus aureus modulates the composition of its membrane lipids, and how those changes affect the biophysical properties of membranes, which in turn may play a role in its virulence and its resistance to different membrane-active antimicrobial agents.