Mechanism Of Bacterial Adhesion Research Articles

Surface Science and Microbiology. The Strategy for Existence of Microorganisms. Adhesion to Solid Surfaces.

In natural and industrial environments, bacteria exist in adhering to solid surfaces such as metals, plastics, glass, soil particles, plant and animal tissues. Adherent bacteria are resistant to chemicals and heat compared with their planktonic cells, and may cause serious problems in bioprocess engineering, medicine and food processings. In order to clarify these observations and phenomena, it is necessary to study physicochemically the microstructure of solid surfaces, structure and properties of bacterial cell surfaces, mechanisms of bacterial adhesion and their correlations.

Investigation into the mechanism of bacterial adhesion to hydrogel-coated surfaces.

As a model for hydrogel-coated biomaterials, self-assembled monolayers of polyoxyethylene (POE) derivatives on sheets of polymeric biomaterials were prepared. The POE derivatives consisted of hydrophilic chains with different lengths and a long-chain alkyl group that served as an anchor function. The coatings obtained were analyzed with XPS and contact angle measurements showing hydrophilic chains of different lengths extending away from the surface. Bacterial adhesion was measured with a clinically relevant Klebsiella pneumoniae type strain and measurements reproduced 12 times. Bacterial adhesion decreased markedly with increasing hydrophilic chain length. Based upon these findings a new model for bacterial adhesion to hydrogel-coated surfaces is suggested: steric repulsion effects that increase with increasing chain length of grafted hydrophilic chains play an important role in bacterial adhesion to hydrogel-coated surfaces.

Floc stability and adhesion of green-fluorescent-protein-marked bacteria to flocs in activated sludge.

Wastewater is often treated using the activated sludge process. Flocculation and subsequent sedimentation of flocs are vital steps in this process that have direct influence on the quality of the effluent water from wastewater treatment plants. Since cells that remain free-living will decrease the quality of the effluent water it is important to understand the mechanisms of bacterial adhesion to flocs. The green fluorescent protein (GFP) was used as a cellular marker to study bacterial adhesion to activated sludge flocs in situ in sludge liquor. Cell surface hydrophobicity (CSH) was shown to be an important factor that determined the relative bacterial adhesion potential. High CSH correlated with high numbers of attached cells. However, the absolute adhesion of two test bacteria to different sludge flocs varied and could not be explained by the floc characteristics. Confocal laser scanning microscopy of GFP-marked cells showed their position in the floc matrix in situ. Hydrophobic cells attached not only on the surface but also within the floc, whereas hydrophilic cells did not. This indicates that cells may penetrate the flocs through channels and pores and increase the effective surface, which in turn makes the clarification of the wastewater effluent more efficient. The addition of polymers is common practice in wastewater treatment and was shown to increase bacterial adhesion to the flocs. A decrease in surface tension caused by addition of DMSO decreased adhesion, indicating the detrimental effect of surfactants on flocculation. An understanding of basic bacterial adhesion and aggregation mechanisms is important for the management and control of biotechnological wastewater treatment.

Concise review of mechanisms of bacterial adhesion to biomaterial surfaces.

This article reviews the mechanisms of bacterial adhesion to biomaterial surfaces and the factors affecting the adhesion. The process of bacterial adhesion includes an initial physicochemical interaction phase (phase one) and a late molecular and cellular interaction phase (phase two), which is a complicated process affected by many factors, including the characteristics of the bacteria themselves, the target material surface, and the environmental factors, such as the presence of serum proteins or bactericidal substances.

Laboratory methods for studies of bacterial adhesion

Prosthetic infection following total joint replacement can have catastrophic results both physically and psychologically for the patients, leading to complete failure of the arthroplasty, possible amputation, prolonged hospitalization, and even death. Bacterial adherence to biomaterial surfaces is an important step in the pathogenesis of prosthetic infection. The exact mechanism of prosthetic infection remains unclear. It is thought that certain bacteria, particularly coagulase-negative staphylococci, after adhering to biomaterial surfaces, secrete a layer polysaccharide, an extracellular substance (slime) and then form a biofilm (a biomass of bacteria and slime). The biofilm makes the embedded bacteria less accessible to the human defense system and significantly decreases antibiotic susceptibility. Before effective preventive or therapeutic measures can be achieved, the process, characteristics, or mechanism of bacterial adhesion to biomaterials have to be studied. In this review, the in-vitro experimental methods for bacterial adhesion will be discussed, which concentrates on (1) how to design a new in-vitro model of bacterial adhesion or biofilm formation, including the selection of bacteria, sample surface preparation, conducting bacterial adhesion or biofilm formation experiments, and the sample preparation for evaluation; and (2) methods for examining adhered bacteria and biofilm, which include microscopy for counting and morphological observation of adhered bacteria, viable bacterial counting methods, other direct or indirect bacterial counting methods, and the methods for evaluating biofilm.

Novel animal model for studying the molecular mechanisms of bacterial adhesion to bone-implanted metallic devices: role of fibronectin in Staphylococcus aureus adhesion.

Infection around metallic implants is a rare but severe complication of orthopaedic surgery. A novel animal model mimicking conditions of internal fixation devices was developed to evaluate the role of host proteins adsorbed on metallic devices in promoting adhesion and colonization of the material surfaces by Staphylococcus aureus. Small plates made of pure titanium were either fixed (three screws per plate) onto the iliac bones of guinea pigs or implanted into their subcutaneous space as controls. Five to 6 weeks after surgery, the plates and screws were removed from the previously killed animals, carefully rinsed in buffer, and tested in an in vitro assay of S. aureus adhesion to metallic surfaces. To evaluate the role of fibronectin in staphylococcal adhesion to explanted plates and screws, a mutant of S. aureus that is specifically defictive in fibronectin adhesion due to decreased expression of the fibronectin adhesin was compared with its isogenic parental strain. A significant reduction in adhesion of the fibronectin adhesin-defective mutant compared with the parental strain occurred on both the subcutaneously implanted and bone-implanted metallic plates. The results of this specific biological assay suggest that fibronectin is present on bone-implanted metallic devices and promotes attachment of S. aureus to their surfaces. This novel experimental model should help, to characterize several parameters of bacterial adhesion to metallic orthopaedic devices and to develop novel anti-adhesive strategies for preventing such infections.

The clinical use of contact lenses and collagen shields.

Contact lens wear before and after photorefractive or phototherapeutic keratectomy with excimer lasers has become an increasingly interesting subject. No negative side effects of contact lens wear prior to photorefractive keratectomy have yet been shown and refractive errors postoperatively do not seem to constitute a major practical problem. Normal side effects include the risk of bacterial infection and keratitis. The major pathogen in this context is still Pseudomonas aeruginosa, although increased insight into the mechanisms of bacterial adhesion and improvements in cleaning procedures now allow more effective preventive care of contact lenses themselves and of contact lens cases. Tear fluid research using leucotriene C4 as a possible indicator for subclinical inflammation has led to interesting results, and the importance of the arachidonic pathway and its metabolites has become more evident. These advances contribute considerably to the safe use of contact lenses in clinical ophthalmology, although major innovations such as the multifocal contact lenses have not reached the stage of perfection.

Reversibility and mechanism of bacterial adhesion

The reversibility and mechanisms of adhesion of various pseudomonads and coryneform bacteria having different hydrophobicities and negative cell surface charges on negatively charged Teflon and glass were studied. Adhesion at an ionic strength of 0.1 M was irreversible and corresponded to activation Gibbs energies for detachment higher than 5 kT for 19 out of 20 combinations of bacterial strains and surfaces. The data further demonstrate the importance of two groups of interactions: (i) the electrostatic and van der Waals interactions as described by the DLVO model, and (ii) the interactions between the outer cell surface macromolecules and the solids (steric interactions). At an ionic strength of 0.1 M, steric interactions control adhesion for all but two bacterium/substratum combinations tested. These interactions are attractive for seven moderately to strongly hydrophobic strains on Teflon and prevented detachment upon decreasing the ionic strength to less than 0.0001 M and also after applying shear forces. In contrast, steric interactions inhibited adhesion for more hydrophilic bacterium/substratum combinations for which detachment occurred upon reducing the ionic strength to less than 0.0001 M and/or after applying shear. The importance of the interactions included by the DLVO model is demonstrated by the following. (i) Two hydrophobic strains adhere irreversibly on glass by strong van der Waals attraction in a secondary DLVO minimum at an ionic strength of 0.1 M and detach when the ionic strength is reduced to less than 0.0001 M. (ii) Electrostatic repulsion inhibits deposition at lower ionic strength. The practical implications of these findings are discussed.

16] Kinetic analysis of microbial adhesion

Publisher Summary This chapter focuses on the kinetic analysis of microbial adhesion. Kinetic adsorption and desorption experiments provide a fundamental method for examining mechanisms of bacterial adhesion. Violations of assumptions (about cell–cell interactions, for example) are readily apparent, and yield information about the nature of the binding. Combining initial kinetic data with data gained from equilibrium experiments can also help to unravel complex adhesion mechanisms. Additional experiments assessing the effects of temperature and buffer changes on the kinetic constants provide information about the particular long-range and short-range forces and bacterial-surface constituents that are responsible for attachment. The kinetic approach allows investigators to identify any departure from ideal pseudo-first-order attachment kinetics that can help identify the cell structures responsible for attachment. Kinetic analysis also allows the separate investigation of low-affinity (long-range) association and stereospecific binding occurring at a short range. Systems in which the energy barrier between the long-range attachment and closer, stronger binding is large benefit greatly from an analysis of the first, reversible stage of adhesion because this is the most influential determinant of eventual adhesion. This stage is best studied kinetically.

Bacterial adsorption to fetuin and mucin pellicle.

The ability of Actinomyces viscosus (A. viscosus) WVU 627 to attach to hydroxyapatite (HA) beads treated with either bovine fetuin or bovine mucin was studied. All preparations used were blocked with bovine serum albumin prior to incubation with [3H] thymidine-labeled A.viscosus cells. The quantity of fetuin or mucin adsorbed on the HA beads was determined by use of [3H] fetuin or [3H] mucin. Approximately 6 micrograms protein of [3H] fetuin and 20 micrograms of protein of [3H] mucin adsorbed to 5 mg of HA beads at saturation, respectively. The presence of fetuin or mucin on HA beads promoted attachment of the organism. However, HA beads treated with human whole saliva as a positive control promoted A.viscosus attachment more effectively than HA beads treated with these glycoproteins. Attachment of two additional strains (B 236 and B 25) of A. viscosus to HA beads was also promoted by these glycoproteins. The number of A.viscosus cells which attached to fetuin-treated HA beads was dose-dependent, except for strain B 25. For all three A.viscosus strains tested, attachment to the experimental pellicle with mucin was dose-dependent. These findings suggest that the use of these newly developed bacterial adhesion assay systems may be effective for elucidating bacterial adhesion mechanisms.

Cellular and molecular mechanisms of bacterial adhesion to respiratory mucosa

Different bacterial species adhere avidly to respiratory mucus. Such adhesion, when followed by ciliary clearance, represents an important stage of the airway defense system. However, in pathological conditions, the mucociliary clearance may be severely reduced, and mucus-associated bacteria may multiply and infect the underlying epithelium. Only a few bacteria have been shown to adhere to ciliary membranes of functionally active ciliated cells. Therefore, the first way in which most of the respiratory pathogens associate with the airway epithelium is likely to be by their adhesion to mucus. Some bacteria also secrete products that may affect ciliary function and/or cause cell death and epithelial disruption. Respiratory pathogens that do not bind to normal ciliated cells may readily adhere to injured epithelial cells, or to the unmasked extracellular matrix. Furthermore, following injury, epithelial respiratory cells in the process of migration, in order to repair the wounds, may present receptors to which bacteria adhere. The adhesion to all of these epithelial receptors may contribute to the chronicity of many bacterial respiratory infections.

Monitoring microbiol adhesion and biofilm formation by attenuated total reflection/Fourier transform infrared spectroscopy

A major problem in accurately defining bacterial adhesion mechanisms and processes occurring in biofilms on surfaces is the lack of techniques that nondestructively provide on-line information about the microorganisms, their extracellular polymers, and metabolites. The attenuated total reflectance (ATR) technique of Fourier transform infrared spectroscopy (FT-IR) is ideally suited to monitor molecular interactions at the solution/internal reflection element (IRE) interface, and we report its application to biofilm research. Two methodologies were utilized to obtain the ATF/FT-IR spectra of living Caulobacter crescentus cells attached to germanium crystals. Initially, spectra of attached bacteria in high purity water produced molecular details of the attachment process without spectral interferences from components of the medium. A growth medium, utilized in the second method, allowed direct examination of the infrared absorption bands associated with the actively growing microorganisms on the surface of the IRE in the spectral region of 2000 to 1200 cm −1. Using the amide II band as a marker for biofilm biomass, the detection limit was determined to be approximately 5 × 10 5 cells·cm −2. These results proved that the ATR-FT/IR methodologies can be utilized to provide chemical information from bacteria and bacterial products located within approximately 1 μm of the surface without spectral interferences due to components of the medium.

Sub‐Minimum Inhibitory Concentrations of Ceftibuten Reduce Adherence of Escherichia coli to Human Cells and Induces Formation of Long Filaments

The minimal inhibitory concentrations (MICs) of an antibiotic are present for only a certain period of time, after which they become sub-inhibitory concentrations (sub-MICs). These sub-MICs are still active because they can interfere with the mechanism of bacterial adhesion, which is the first step in the sequence of events leading to infection. The purpose of the present study was to investigate the effects of sub-MICs of ceftibuten, a new third-generation cephalosporin, on the adhesion of Escherichia coli (E. coli) to human buccal cells. The degree of inhibition was maximal at 1/2 MIC and then gradually returned toward to the control values at 1/128 the MIC. The differences were statistically significant from 1/2 to 1/32 MIC. Since the MIC was 0.5 micrograms/ml, concentrations from 0.25 to 0.015 micrograms/ml significantly reduce bacterial adhesion. Ceftibuten also caused marked elongation of E. coli. These findings could help to explain the efficacy showed by ceftibuten in the treatment of respiratory and urinary tract infections when administered once daily.

Defense system in the biliary tract against bacterial infection.

Bacteria can invade the biliary tract by ascending from the duodenum and via the hematogenous route from the hepatic portal venous blood. The sphincter of Oddi, situated at the junction of the biliary tract and the upper gastrointestinal tract, forms an effective mechanical barrier to duodenal reflex and ascending bacterial infection. Conversely, Kupffer cells and the tight junctions between hepatocytes help prevent bacteria and toxic metabolites from entering the hepatobiliary system from the portal circulation. The continuous flushing action of bile and the bacteriostatic effects of bile salts keeps the biliary tract sterile under normal conditions. Secretory immunoglobulin A (sIgA), the predominant immunoglobulin in the bile, and mucus excreted by the biliary epithelium probably function as antiadherence factors, preventing microbial colonization. When barrier mechanisms break down, as in surgical or endoscopic sphincterotomy and with insertion of biliary stents, pathogenic bacteria enter the biliary system at high concentrations and take up residence on any foreign bodies. Intrabiliary pressure is a key factor in the development of cholangitis. Chronic biliary obstruction raises the intrabiliary pressure. This adversely influences the defensive mechanisms such as the tight junctions, Kupffer cell functions, bile flow, and sIgA production in the system, resulting in a higher incidence of septicemia and endotoxemia in these patients. Knowledge of biliary defense against infection is still quite primitive. Unclear are the roles of sIgA in the bile, mechanism of bacterial adhesion to the biliary epithelium, Kupffer cell function in biliary obstruction, and the antimicrobial activity of bile salts.

Cloning and expression of two different genes from Streptococcus dysgalactiae encoding fibronectin receptors.

Binding of bacteria to fibronectin has been implicated as a mechanism of bacterial adhesion to the host tissue. In this report we have analyzed the binding of a strain of Streptococcus dysgalactiae to fibronectin. The cells bind to a site in the NH2-terminal domain of the protein via trypsin-sensitive cell surface components. Furthermore, a lysate prepared by sonication of streptococcal cells contained fibronectin-binding proteins that inhibit the binding of the ligand to intact bacteria. When the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted to an Immobilon-P filter, and probed with 125I-labeled fibronectin, a 140-kDa fibronectin-binding protein was identified along with a number of smaller binding proteins. A genomic DNA library was constructed and screened for the expression of fibronectin-binding proteins. Two clones were isolated and shown to contain unrelated inserts by restriction mapping and cross-hybridization experiments. The two encoded proteins were also immunologically distinct although both bound to the same region of the fibronectin molecule, and both effectively inhibited the binding of 125I-fibronectin to bacterial cells. Immunological analyses showed that only one of the two proteins tentatively identified as fibronectin receptors was expressed in detectable quantities in the Streptococcus dysgalactiae strain under the culture conditions employed.

Role of the nematode surface coat in the adhesion of Clavibacter sp. to Anguina funesta and Anguina tritici

Clavibacter sp. (syn. Corynebacterium rathayi) adhered to both Anguina funesta (syn. Anguina agrostis) and Anguina tritici, but differences in the nature of adhesion were noted. Similar patterns of binding of the bacteria and of anti-wheat germ agglutinin antibody initially led us to believe that the mechanism of bacterial adhesion was related to the presence of wheat-germ agglutinin (WGA) on the outer cuticle of both species of nematodes and its complementary carbohydrate on the bacterial capsule. However, treatment of either species of nematode with sodium metaperiodate inhibited bacterial adhesion but not the binding of anti-WGA antibody. Bacterial adhesion, therefore, is not mediated by WGA on the nematodes' surface. Moreover, differences in patterns of bacterial adhesion to Anguina species, both before and after treatments with NaCl and detergents, suggest basic interspecific differences in the nature of adhesion. Electron microscopy confirmed the contribution of the nematodes' cuticular surface coat (SC) to the process of adhesion, but it is still not clear how the SC interacts with the bacterial capsule or which of its components are involved. While complete removal of the SC with periodate prevented bacterial adhesion, juveniles that naturally resisted bacterial adhesion did not lack a SC. One explanation could be that the SC of individuals, to which bacteria do not adhere naturally, lacks crucial components that cannot be defined by conventional EM.

Peptide analogs to a fibronectin receptor inhibit attachment of Staphylococcus aureus to fibronectin-containing substrates

Binding of cells of Staphylococcus aureus to fibronectin has been proposed as a mechanism of bacterial adhesion to host tissues. In this study, we have attempted to define the role of a recently identified fibronectin receptor in the adhesion of staphylococcal cells to fibronectin-containing substrates by using different receptor analogs as potential inhibitors of bacterial adherence. The results showed that synthetic peptides D1, D2, and D3, corresponding to variations of a repeated unit in the fibronectin-binding domain of the receptor, and ZZ-FR, a chimeric protein containing the fibronectin-binding domain of the receptor with the D1, D2, and D3 sequences, inhibited the attachment of staphylococcal cells to microtiter wells coated with intact fibronectin or with the 29-kilodalton amino-terminal fragment of fibronectin. The chimeric protein ZZ-FR also partially inhibited the adherence of staphylococci to human plasma clots formed in vitro but had no effect on bacterial adhesion to clots formed from fibronectin-depleted plasma. These data confirm previous reports suggesting that fibronectin may serve as a substrate for adhesion of staphylococcal cells and indicate that bacterial adhesion is mediated by the identified fibronectin receptor. Furthermore, analogs to the fibronectin receptor can be used to inhibit the adhesion of bacterial cells to these model substrates, and these analogs may be of clinical use.

59 PATTERNS OF HEp-2 CELL ADHERENCE OF DIARRHOEAGENIC ESCHERICHIA COLI

In 1979 Cravioto et al. showed that most enteropathogenic E. coli (EPEC) strains adhered to cultured HEp-2 cells whereas non-EPEC strains rarely adhered. Two patterns of adherence were described. Most EPEC adhered in localised microcolonies (LA) although a few displayed a diffuse pattern of adherence (DA). A new pattern of adhesion in which bacteria assume a characteristic ‘aggregative’ pattern (AA) both on the HEp-2 cell surface and between cells has recently been described in diarrhoeagenic E. coli of non-classical EPEC serotypes. To study mechanisms of adhesion we examined by electron microscopy the attachment to HEp-2 cells of strains exhibiting LA, DA and AA. LA was characterised by bacteria intimately attached to cuplike projections of the HEp-2 cell surface with associated disruption of the apical cell cytoskeleton and loss of cell surface microvilli; attachment closely resembling EPEC adhesion to intestinal mucosa. Strains exhibiting DA and AA did not show intimate attachment nor did they cause any alteration of cell surface architecture. A distinct halo surrounding bacteria and separating them from the HEp-2 cell surface was characteristic of AA strains. Specific staining showed DA to be capsule-mediated whereas AA was correlated with the production of specific rod-like fimbriae. These ultrastructural observations show that LA, DA and AA reflect different mechanisms of bacterial adhesion to HEp-2 cells and support preliminary evidence that AA is representative of a new class of diarrhoeagenic E. coli. Based on adherence properties, it would also appear that DA strains of classical EPEC serotypes may not be ‘true’ EPEC.

Mechanisms and clinical relevance of bacterial adhesion to polymers.

The mechanisms of bacterial adhesion to polymers with regard to their significance in the development of foreign-body infections are discussed. The morphological, physico-chemical and biological aspects are treated with special emphasis on the adhesion of coagulase-negative staphylococci to medical polymers. Strategies for the prevention of bacterial adhesion to biomaterials by developing antiadhesive polymers are given.

Antiphlogistine

Bacteria adhering to implanted medical devices can cause invasive microbial infections, of e.g. skin, lung or blood. In dentistry, Streptococcus gordonii is an early oral colonizer initiating dental biofilm formation and also being involved in life-threatening infective endocarditis. To treat oral biofilms, antibacterial mouth rinses are commonly used. Such initial biomaterial-bacteria interactions and the influence of antibacterial treatments are poorly understood and investigated here in situ by quartz crystal microbalance with dissipation monitoring (QCM-D). A saliva-coated titanium (Ti) biosensor is applied to analyze possible specific signal patterns indicating microbial binding mechanisms and bactericide-caused changes in bacterial film rigidity or cell leakage caused by a clinically relevant antibacterial agent (ABA), i.e., a mouth rinse comprising chlorhexidine (CHX) and cetylpyridinium chloride (CPC). Apparent missing mass effects during the formation of microscopically proven dense and vital bacterial films indicate punctual, specific binding of S. gordonii to the saliva-coated biosensor, compared to unspecific adhesion to pure Ti. Coincidentally to ABA-induced killing of surface-adhered bacteria, an increase of adsorbed dissipative mass can be sensed, contrary to the prior mass-loss. This suggests the acoustic sensing of the leakage of cellular content caused by bacterial cell wall rupturing and membrane damage upon the bactericidal attack. The results have significant implications for testing bacterial adhesion mechanisms and cellular integrity during interaction with antibacterial agents.