The role of conditioning film formation in Pseudomonas aeruginosa PAO1 adhesion to inert surfaces in aquatic environments

Bacterial initial adhesion to inert surfaces in aquatic environments is highly dependent on the surface properties of the substratum, which can be altered significantly by the formation of conditioning films. In this study, the impact of conditioning films formed with extracellular polymeric substances (EPS) on bacterial adhesion was investigated. Adhesion of wild type Pseudomonas aeruginosa PAO1 to slides coated with model EPS components (alginate, humic substances, and bovine serum albumin (BSA)) was examined. Surface roughness of conditioning film coated slides was evaluated by atomic force microscopy (AFM), and its effect on the bacterial initial adhesion was not significant. X-ray photoelectron spectroscopy (XPS) studies were performed to determine the elemental surface compositions of bacterial cells and substrates. Results showed that bacterial adhesion to bare slides and slides coated with alginate and humic substances increased as ionic strength increased. Conversely, BSA coating enhanced bacterial adhesion at low ionic strength but hindered adhesion at higher ionic strength. It was concluded that forces other than hydrophobic and electrostatic interactions were involved in controlling bacterial adhesion to BSA coated surfaces. A steric model for polymer brushes that considers the combined influence of steric effects and DLVO interaction forces was shown to adequately describe the observed bacterial adhesion behaviors.     

Staphylococcus aureus-Fibronectin Interactions with and without Fibronectin-Binding Proteins and Their Role in Adhesion and Desorption

Adhesion and residence-time-dependent desorption of two Staphylococcus aureus strains with and without fibronectin (Fn) binding proteins (FnBPs) on Fn-coated glass were compared under flow conditions. To obtain a better understanding of the role of Fn-FnBP binding, the adsorption enthalpies of Fn with staphylococcal cell surfaces were determined using isothermal titration calorimetry (ITC). Interaction forces between staphylococci and Fn coatings were measured using atomic force microscopy (AFM). The strain with FnBPs adhered faster and initially stronger to an Fn coating than the strain without FnBPs, and its Fn adsorption enthalpies were higher. The initial desorption was high for both strains but decreased substantially within 2 s. These time scales of staphylococcal bond ageing were confirmed by AFM adhesion force measurement. After exposure of either Fn coating or staphylococcal cell surfaces to bovine serum albumin (BSA), the adhesion of both strains to Fn coatings was reduced, suggesting that BSA suppresses not only nonspecific but also specific Fn-FnBP interactions. Adhesion forces and adsorption enthalpies were only slightly affected by BSA adsorption. This implies that under the mild contact conditions of convective diffusion in a flow chamber, adsorbed BSA prevents specific interactions but does allow forced Fn-FnBP binding during AFM or stirring in ITC. The bond strength energies calculated from retraction force-distance curves from AFM were orders of magnitude higher than those calculated from desorption data, confirming that a penetrating Fn-coated AFM tip probes multiple adhesins in the outermost cell surface that remain hidden during mild landing of an organism on an Fn-coated substratum, like that during convective diffusional flow.     

Role of type 1 fimbriae and mannose in the development of Escherichia coli K12 biofilm: from initial cell adhesion to biofilm formation

The influence of type 1 fimbriae, mannose-sensitive structures, on biofilm development and maturation has been examined by the use of three isogenic Escherichia coli K12 strains: wild type, fimbriated, and non-fimbriated. Experiments with the three strains were done in minimal medium or Luria–Bertani broth supplemented with different concentrations of d-mannose. The investigation consisted of: (1) characterizing the bacterial surface of the three strains with respect to hydrophilicity and surface charge, (2) investigating the effect of type 1 fimbriae on bacterial adhesion rate and reversibility of initial adhesion on glass surfaces, and (3) verifying the role of type 1 fimbriae and exopolysaccharides (EPS) in biofilm maturation. The results suggest that type 1 fimbriae are not required for the initial bacterial adhesion on glass surfaces as the non-fimbriated cells had higher adhesion rates and irreversible deposition. Type 1 fimbriae, however, are critical for subsequent biofilm development. It was hypothesized that in the biofilm maturation step, the cells synthesize mannose-rich EPS, which functions as a ‘conditioning film’ that can be recognized by the type 1 fimbriae.     

Dental plaque formation

Dental plaque is a complex biofilm that accumulates on the hard tissues (teeth) in the oral cavity. Although over 500 bacterial species comprise plaque, colonization follows a regimented pattern with adhesion of initial colonizers to the enamel salivary pellicle followed by secondary colonization through interbacterial adhesion. A variety of adhesins and molecular interactions underlie these adhesive interactions and contribute to plaque development and ultimately to diseases such as caries and periodontal disease.     

Intermolecular forces and enthalpies in the adhesion of Streptococcus mutans and an antigen I/II-deficient mutant to laminin films

The antigen I/II family of surface proteins is expressed by most oral streptococci, including Streptococcus mutans, and mediates specific adhesion to, among other things, salivary films and extracellular matrix proteins. In this study we showed that antigen I/II-deficient S. mutans isogenic mutant IB03987 was nearly unable to adhere to laminin films under flow conditions due to a lack of specific interactions (0.8 x 10(6) and 1.1 x 10(6) cells cm(-2) at pH 5.8 and 6.8, respectively) compared with parent strain LT11 (21.8 x 10(6) and 26.1 x 10(6) cells cm(-2)). The adhesion of both the parent and mutant strains was slightly greater at pH 6.8 than at pH 5.8. In addition, atomic force microscopy (AFM) experiments demonstrated that the parent strain experienced less repulsion when it approached a laminin film than the mutant experienced. Upon retraction, combined specific and nonspecific adhesion forces were stronger for the parent strain (up to -5.0 and -4.9 nN at pH 5.8 and 6.8, respectively) than for the mutant (up to -1.5 and -2.1 nN), which was able to interact only through nonspecific interactions. Enthalpy was released upon adsorption of laminin to the surface of the parent strain but not upon adsorption of laminin to the surface of IB03987. A comparison of the adhesion forces in AFM with the adhesion forces reported for specific ligand-receptor complexes resulted in the conclusion that the number of antigen I/II binding sites for laminin on S. mutans LT11 is on the order of 6 x 10(4) sites per organism and that the sites are probably arranged along exterior surface structures, as visualized here by immunoelectron microscopy.     

Role of capsular colanic acid in adhesion of uropathogenic Escherichia coli

Urinary tract infections are the most common urologic disease in the United States and one of the most common bacterial infections of any organ system. Biofilms persist in the urinary tract and on catheter surfaces because biofilm microorganisms are resistant to host defense mechanisms and antibiotic therapy. The first step in the establishment of biofilm infections is bacterial adhesion; preventing bacterial adhesion represents a promising method of controlling biofilms. Evidence suggests that capsular polysaccharides play a role in adhesion and pathogenicity. This study focuses on the role of physiochemical and specific binding interactions during adhesion of colanic acid exopolysaccharide mutant strains. Bacterial adhesion was evaluated for isogenic uropathogenic Escherichia coli strains that differed in colanic acid expression. The atomic force microscope (AFM) was used to directly measure the reversible physiochemical and specific binding interactions between bacterial strains and various substrates as bacteria initially approach the interface. AFM results indicate that electrostatic interactions were not solely responsible for the repulsive forces between the colanic acid mutant strains and hydrophilic substrates. Moreover, hydrophobic interactions were not found to play a significant role in adhesion of the colanic acid mutant strains. Adhesion was also evaluated by parallel-plate flow cell studies in comparison to AFM force measurements to demonstrate that prolonged incubation times alter bacterial adhesion. Results from this study demonstrate that the capsular polysaccharide colanic acid does not enhance bacterial adhesion but rather blocks the establishment of specific binding as well as time-dependent interactions between uropathogenic E. coli and inert substrates.     

Role of Capsular Colanic Acid in Adhesion of Uropathogenic Escherichia coli

Urinary tract infections are the most common urologic disease in the United States and one of the most common bacterial infections of any organ system. Biofilms persist in the urinary tract and on catheter surfaces because biofilm microorganisms are resistant to host defense mechanisms and antibiotic therapy. The first step in the establishment of biofilm infections is bacterial adhesion; preventing bacterial adhesion represents a promising method of controlling biofilms. Evidence suggests that capsular polysaccharides play a role in adhesion and pathogenicity. This study focuses on the role of physiochemical and specific binding interactions during adhesion of colanic acid exopolysaccharide mutant strains. Bacterial adhesion was evaluated for isogenic uropathogenic Escherichia coli strains that differed in colanic acid expression. The atomic force microscope (AFM) was used to directly measure the reversible physiochemical and specific binding interactions between bacterial strains and various substrates as bacteria initially approach the interface. AFM results indicate that electrostatic interactions were not solely responsible for the repulsive forces between the colanic acid mutant strains and hydrophilic substrates. Moreover, hydrophobic interactions were not found to play a significant role in adhesion of the colanic acid mutant strains. Adhesion was also evaluated by parallel-plate flow cell studies in comparison to AFM force measurements to demonstrate that prolonged incubation times alter bacterial adhesion. Results from this study demonstrate that the capsular polysaccharide colanic acid does not enhance bacterial adhesion but rather blocks the establishment of specific binding as well as time-dependent interactions between uropathogenic E. coli and inert substrates.     

Electric field induced desorption of bacteria from a conditioning film covered substratum.

Desorption of three oral bacterial strains from a salivary conditioning film on an indium tin oxide electrode during application of a positive (bacterial adhesion to the anode) or a negative electric current was studied in a parallel plate flow chamber. Bacterial adhesion was from a flowing suspension of high ionic strength, after which the bacterial suspension was replaced by a low ionic strength solution without bacteria and currents ranging from -800 to +800 microA were applied. Streptococcus oralis J22 desorbed during application of a positive and negative electric current with a desorption probability that increased with increasing electric current. Two actinomyces strains, however, could not be stimulated to desorb by the electric currents applied. The desorption forces acting on adhering bacteria are electroosmotic in origin and working parallel to the electrode surface in case of a positive current, whereas they are electrophoretic and electrostatic in origin and working perpendicular to the surface in case of a negative current. By comparison of the effect of positive and negative electric currents, it can be concluded that parallel forces are more effective in stimulating bacterial desorption than perpendicular forces. The results of this study point to a new pathway of cleaning industrial and biomedical surfaces without the use of detergents or biocides.     

Using a multi-faceted approach to determine the changes in bacterial cell surface properties influenced by a biofilm lifestyle

Biofilm formation is a developmental process in which initial reversible adhesion is governed by physico-chemical forces, whilst irreversible adhesion is mediated by biological changes within a cell, such as the production of extracellular polymeric substances. Using two bacteria, E. coli MG1655 and B. cereus ATCC 10987, this study establishes that the surface of the bacterial cell also undergoes specific modifications, which result in biofilm formation and maintenance. Using various surface characterisation techniques and proteomics, an increase in the surface exposed proteins on E. coli cells during biofilm formation was demonstrated, along with an increase in hydrophobicity and a decrease in surface charge. For B. cereus, an increase in the surface polysaccharides during biofilm formation was found as well as a decrease in hydrophobicity and surface charge. This work therefore shows that surface modifications during biofilm formation occur and understanding these specific changes may lead to the formulation of effective biofilm control strategies in the future.     

Determination of the Shear Force at the Balance between Bacterial Attachment and Detachment in Weak-Adherence Systems, Using a Flow Displacement Chamber

We introduce a procedure for determining shear forces at the balance between attachment and detachment of bacteria under flow. This procedure can be applied to derive adhesion forces in weak-adherence systems, such as polymer brush coatings, which are currently at the center of attention for their control of bacterial adhesion and biofilm formation.     

变形链球菌对不同牙科充填材料的粘附及早期生物膜的形成

目的探讨变形链球菌对不同牙科充填材料的粘附和早期生物膜的形成.方法比较经放射性同位素3H-TDR(3H-胸腺嘧啶核苷)标记的变形链球菌对3种唾液包被的充填材料的粘附.采用蛋白质测量试剂盒定量分析其对唾液蛋白的吸附量;采用凝胶电泳和图像分析系统定量分析其对唾液白蛋白和α-淀粉酶的吸收率.结果各种材料对变形链球菌的粘附能力,对唾液蛋白的吸附能力均随着材料的不同而不同.Fuji IX对细菌的粘附量很高,但是对蛋白的吸附量却很低;而F2000对细菌的粘附量很低,对蛋白的吸附量却很高.结论在不同充填材料表面形成的生物膜是不同的,提示早期生物膜的形成具有一定的特异性.这种生物膜的差异对口腔微生态环境及龋病和/或牙周病的发展具有重要意义.     

Retention of the Gram-negative bacterium SW8 on surfaces under conditions relevant to the subsurface environment: Effects of conditioning films and substratum nature

Abstract Shallow (5–13 m) and deep (35–65 m) groundwaters were evaluated for their ability to generate conditioning films which affect bacterial adhesion to natural (sandstone, shale, andesite) and man-made substrata (polypropylene, stainless steel). Water contact angles indicated that all water samples produced conditioning films. Most films modified retention of the nonmotile Gram-negative bacterium SW8, but attachment of the organism did not correlate with water contact angles. Each borewater produced conditioning films with a characteristic attachment profile of SW8. Films adsorbed from standing borewaters often retained SW8 in different numbers than coatings derived from pumped bores. Groundwater chemistry was very heterogeneous and microbiological data indicated the presence of a diverse aerobic and anaerobic microbial community. These results indicate that conditioning films derived from dissolved compounds may play a significant role in controlling the interaction of bacteria with substrata in the subsurface.     

On the relative importance of specific and non-specific approaches to oral microbial adhesion

Abstract In this paper, it is suggested that specificity and non-specificity in (oral) microbial adhesion are different expressions for the same phenomena. It is argued that the same basic, physico-chemical forces are responsible for so-called 'non-specific' and 'specific' binding and that from a physico-chemical point of view the distinction between the two is an artificial one. Non-specific interactions arise from Van der Waals and electrostatic forces and hydrogen bonding, and originate from the entire cell. A specific bond consists of a combination of the same type of Van der Waals and electrostatic forces and hydrogen bonding, now originating from highly localized chemical groups, which together form a stereo-chemical combination. The absence or presence of specific receptor sites on microbial cell surfaces must therefore be reflected in the overall, non-specific surface properties of cells as well. This point is illustrated by showing that glucanbinding lectins on mutans streptococcal strains may determine the pH dependence of the zeta potentials of these cells. When studying microbial adhesion, a non-specific approach may be better suited to explain adhesion to inert substrata, whereas a specific approach may be preferred in case of adhesion to adsorbed protein films. Adhesion is, however, not as important in plaque formation in the human oral cavity as is retention, because low shear force periods. during which adhesion presumably occurs, are followed by high shear force periods, during which adhering cells must withstand these detachment forces. Evidence is provided that such detachment will be through cohesive failure in the pellicle mass, the properties of which are conditioned by the overall, non-specific substratum properties. Therefore, in vivo plaque formation may be more readily explained by a non-specific approach.     

On the relative importance of specific and non-specific approaches to oral microbial adhesion.

In this paper, it is suggested that specificity and non-specificity in (oral) microbial adhesion are different expressions for the same phenomena. It is argued that the same basic, physicochemical forces are responsible for so-called 'non-specific' and 'specific' binding and that from a physico-chemical point of view the distinction between the two is an artificial one. Non-specific interactions arise from Van der Waals and electrostatic forces and hydrogen bonding, and originate from the entire cell. A specific bond consists of a combination of the same type of Van der Waals and electrostatic forces and hydrogen bonding, now originating from highly localized chemical groups, which together form a stereochemical combination. The absence or presence of specific receptor sites on microbial cell surfaces must therefore be reflected in the overall, non-specific surface properties of cells as well. This point is illustrated by showing that glucan-binding lectins on mutans streptococcal strains may determine the pH dependence of the zeta potentials of these cells. When studying microbial adhesion, a non-specific approach may be better suited to explain adhesion to inert substrata, whereas a specific approach may be preferred in case of adhesion to adsorbed protein films. Adhesion is, however, not as important in plaque formation in the human oral cavity as is retention, because low shear force periods, during which adhesion presumably occurs, are followed by high shear force periods, during which adhering cells must withstand these detachment forces. Evidence is provided that such detachment will be through cohesive failure in the pellicle mass, the properties of which are conditioned by the overall, non-specific substratum properties. Therefore, in vivo plaque formation may be more readily explained by a non-specific approach.     

Dental plaque as a biofilm

Dental plaque is the diverse microbial community found on the tooth surface embedded in a matrix of polymers of bacterial and salivary origin. Once a tooth surface is cleaned, a conditioning film of proteins and glycoproteins is adsorbed rapidly to the tooth surface. Plaque formation involves the interaction between early bacterial colonisers and this film (the acquired enamel pellicle). To facilitate colonisation of the tooth surface, some receptors on salivary molecules are only exposed to bacteria once the molecule is adsorbed to a surface. Subsequently, secondary colonisers adhere to the already attached early colonisers (co-aggregation) through specific molecular interactions. These can involve protein-protein or carbohydrate-protein (lectin) interactions, and this process contributes to determining the pattern of bacterial succession. As the biofilm develops, gradients in biologically significant factors develop, and these permit the co-existence of species that would be incompatible with each other in a homogeneous environment. Dental plaque develops naturally, but it is also associated with two of the most prevalent diseases affecting industrialised societies (caries and periodontal diseases). Future strategies to control dental plaque will be targeted to interfering with the formation, structure and pattern of development of this biofilm.     

Developmental pathway for biofilm formation in curli-producing Escherichia coli strains: role of flagella, curli and colanic acid

This work was performed to establish a model describing bacterial surface structures involved in biofilm development, in curli-overproducing Escherichia coli K-12 strains, at 30°C, and in minimal growth medium. Using a genetic approach, in association with observations of sessile communities by light and electron microscopic techniques, the role of protein surface structures, such as flagella and curli, and saccharidic surface components, such as the E. coli exopolysaccharide, colanic acid, was determined. We show that, in the context of adherent ompR234 strains, (i) flagellar motility is not required for initial adhesion and biofilm development; (ii) both primary adhesion to inert surfaces and development of multilayered cell clusters require curli synthesis; (iii) curli display direct interactions with the substratum and form interbacterial bundles, allowing a cohesive and stable association of cells; and (iv) colanic acid does not appear critical for bacterial adhesion and further biofilm development but contributes to the biofilm architecture and allows for the formation of voluminous biofilms.     

The conditioning role of saliva in streptococcal attachment to hydroxyapatite surfaces

The solution properties of saliva and its role in conditioning both the substrate and the bacterial surface have been investigated with regard to the attachment of oral streptococci to hydroxyapatite surfaces. Saliva from eight subjects was used and the attachment of three organisms, Streptococcus mutans strains FA-1 (serotype b) and KPSK2 (serotype c) and S. sanguis T175-1, was studied. An adsorbed salivary layer on a hydroxyapatite surface substantially reduced the affinity of the organisms for the surface. Adsorbed saliva on the bacterial surfaces, however, tended to increase the organisms' affinity for saliva-coated apatite. The source of saliva was important in determining the extent of inhibition of attachment. The data indicated that the negatively charged and hydrophilic nature of salivary conditioning films was important in controlling bacterial adsorption to hydroxyapatite. The results also suggested that hydrophobic salivas could promote binding of the more hydrophobic bacteria known to be early colonizers of the teeth.     

Polysulfone and polyacrylate-based zwitterionic coatings for the prevention and easy removal of marine biofouling

A series of polysulfone and polyacrylate-based zwitterionic coatings were prepared on epoxy-primed aluminum substrata and characterized for their antifouling (AF) and fouling-release (FR) properties towards marine bacteria, microalgae and barnacles. The zwitterionic polymer coatings provided minimal resistance against bacterial biofilm retention and microalgal cell attachment, but facilitated good removal of attached microbial biomass by exposure to water-jet apparatus generated hydrodynamic shearing forces. Increasing the ion content of the coatings improved the AF properties, but required a stronger adhesive bond to the epoxy-primed aluminum substratum to prevent coating swelling and dissolution. Grafted poly(sulfobetaine) (gpSBMA), the most promising zwitterionic coating identified from microfouling evaluations, enabled the removal of four out of five barnacles reattached to its surface without incurring damage to their baseplates. This significant result indicated that gpSBMA relied predominately on its surface chemistry for its FR properties since it was very thin (~1–2 µm) relative to commercial coating standards (>200 µm).     

Separate oligosaccharide determinants mediate interactions of the low-molecular-weight salivary mucin with neutrophils and bacteria.

The low-molecular-weight human salivary mucin (MG2) coats oral surfaces, where it is in a prime location for governing cell adhesion. Since oligosaccharides form many of the interactive facets on mucin molecules, we examined MG2 glycosylation as it relates to the molecule's adhesive functions. Our previous study of MG2 oligosaccharide structures showed that the termini predominantly carry T, sialyl-T, Lewisx (Lex), sialyl Lex (sLex), lactosamine, and sialyl lactosamine determinants [Prakobphol, A., et al. (1998) Biochemistry 37, 4916-4927]. In addition, we showed that sLex determinants confer L-selectin ligand activity to this molecule. Here we studied adhesive interactions between MG2 and cells that traffic in the oral cavity: neutrophils and bacteria. Under flow conditions, neutrophils tethered to MG2-coated surfaces at forces between 1.25 and 2 dyn/cm2, i.e., comparable to the shear stress generated at the tooth surface by salivary flow ( approximately 0.8 dyn/cm2). MG2 was also found in association with neutrophils isolated from the oral cavity, evidence that the cells interact with this mucin in vivo. Since MG2 serves as an adhesion receptor for bacteria, the MG2 saccharides that serve this function were also identified. Seven of 18 oral bacteria strains that were tested adhered to MG2. Importantly, six of these seven strains adhered via T antigen, sialyl-T antigen, and/or lactosamine sequences. No adherence to Lex and sLex epitopes was detected in all the strains that were tested. Together, these results suggest that distinct subsets of MG2 saccharides function as ligands for neutrophil L-selectin and receptors for bacterial adhesion, a finding with interesting implications for both oral health and mucin function.