The influence of saliva on the hydrophobic surface properties of bacteria isolated from oral sites of macaque monkeys

Abstract The surface hydrophobicity of 64 bacterial strains isolated from discrete, intra-oral sites of monkeys ( Macaca fascicularis ) was determined by measuring their affinity for hexadecane. Bacteria were also exposed to monkey saliva which either increased or reduced the surface hydrophobicity of the cells. After exposure to saliva those bacteria isolated solely from the mucosal surfaces were significantly more hydrophobic than bacteria ( Streptococcus mutans and Actinomyces spp.) whose major habitat was the dentition. Streptococcus sanguis strains isolated from all intra-oral sites and among the early plaque formers were as hydrophobic as the organisms isolated only from the mucosal surfaces.     

Adherence ofStreptococcus gordonii HG 222 in the presence of saliva

The influence of the presence of saliva from different salivary glands on the adherence ofStreptococcus gordonii strain HG 222 to saliva-coated polystyrene surfaces was tested. In the presence of undiluted parotid saliva or diluted whole, submandibular and sublingual saliva the adherence of HG 222 was enhanced by the formation of small aggregates on the attachment surface. In the presence of undiluted whole, submandibular and sublingual saliva large aggregates were formed and the adherence to saliva-coated polystyrene surfaces was inhibited.Adherence in the presence of whole saliva compared to adherence in buffer was decreased when lower densities of bacterial suspension were used, although in this case in the presence of whole saliva smaller bacterial aggregates were formed.In conclusion, these results suggest that the presence of saliva in solution may both enhance and decrease the adherence ofS. gordonii HG 222 to saliva-coated polystyrene surfaces, partly depending on the size of bacterial aggregates that are formed in the presence of saliva.Abbreviations CHW saliva clarified human whole saliva - PAR saliva parotid saliva - SM saliva submandibular saliva - SL saliva sublingual saliva     

Electrolyte Effects on Attachment of an Estuarine Bacterium

The effect of electrolyte concentration on attachment of Vibrio alginolyticus to hydroxyapatite was determined. Bacterial affinity for attachment to the surface and surface capacity were derived from linearization of bacterial adsorption isotherms. At low concentrations (<0.1 M) the affinity of the bacteria for the surface increased with increasing ionic strength, in agreement with the D.L.V.O. theory of colloid interaction. At higher concentrations, bacterial affinity for the surface decreased with increasing concentration of cations and was not related to ionic strength changes in the medium. These results demonstrate a change in the mechanism by which salts affect bacterial attachment at salt concentrations above 0.1 M. The results are consistent with the relationship between the proportion of attached bacteria and salinity observed in previously published field studies. The results may also resolve differences between various attachment studies carried out in different ionic strength media, utilizing different bacteria, surfaces, and experimental methods.     

Adsorption from saliva to silica and hydroxyapatite surfaces and elution of salivary films by SDS and delmopinol

The adsorption of proteins from human whole saliva (HWS) onto silica and hydroxyapatite surfaces (HA) was followed by quartz crystal microbalance with dissipation (QCM-D) and ellipsometry. The influence of different surface properties and adsorption media (water and PBS) on the adsorption from saliva was studied. The viscoelastic properties of the salivary films formed on the solid surfaces were estimated by the use of the Voigt-based viscoelastic film model. Furthermore, the efficiency of SDS and delmopinol to elute the adsorbed salivary film from the surfaces was investigated at different surfactant concentrations. A biphasic kinetic regime for the adsorption from saliva on the silica and HA surfaces was observed, indicating the formation of a rigidly coupled first layer corresponding to an initial adsorption of small proteins and a more loosely bound second layer. The results further showed a higher adsorption from HWS onto the HA surfaces compared to the silica surfaces in both adsorption media (PBS and water). The adsorption in PBS led to higher adsorbed amounts on both surfaces as compared to water. SDS was found to be more efficient in removing the salivary film from both surfaces than delmopinol. The salivary film was found to be less tightly bound onto the silica surfaces since more of the salivary film could be removed with both SDS and delmopinol compared to that from the HA surface. When adsorption took place from PBS the salivary layer formed at both surfaces seemed to have a similar structure, with a high energy dissipation implying that a softer salivary layer is built up in PBS as opposed to that in water. Furthermore, the salivary layers adsorbed from water solutions onto the HA were found to be softer than those on silica.     

Bond Strengthening in Oral Bacterial Adhesion to Salivary Conditioning Films

Transition from reversible to irreversible bacterial adhesion is a highly relevant but poorly understood step in initial biofilm formation. We hypothesize that in oral biofilm formation, irreversible adhesion is caused by bond strengthening due to specific bacterial interactions with salivary conditioning films. Here, we compared the initial adhesion of six oral bacterial strains to salivary conditioning films with their adhesion to a bovine serum albumin (BSA) coating and related their adhesion to the strengthening of the binding forces measured with bacteria-coated atomic force microscopy cantilevers. All strains adhered in higher numbers to salivary conditioning films than to BSA coatings, and specific bacterial interactions with salivary conditioning films were accompanied by stronger initial adhesion forces. Bond strengthening occurred on a time scale of several tens of seconds and was slower for actinomyces than for streptococci. Nonspecific interactions between bacteria and BSA coatings strengthened twofold faster than their specific interactions with salivary conditioning films, likely because specific interactions require a closer approach of interacting surfaces with the removal of interfacial water and a more extensive rearrangement of surface structures. After bond strengthening, bacterial adhesion forces with a salivary conditioning film remained stronger than those with BSA coatings.     

Influence of substratum hydration and adsorbed macromolecules on bacterial attachment to surfaces.

The attachment of Pseudomonas fluorescens and an Acinetobacter sp. to hydrogel and polystyrene surfaces was investigated to evaluate the influence of adsorbed water and macromolecules on adhesion. With both organisms, there was a decrease in attachment numbers with increasing water content of the hydrogels. There was also a decrease in attachment with a decrease in water contact angle on untreated, tissue culture and sulfonated polystyrene surfaces; however, the attachment numbers were higher than would be expected on the basis of the hydrogel data. With P. fluorescens, attachment to untreated and tissue culture polystyrene was inhibited by bovine serum albumin, Escherichia coli lipopolysaccharide, and the supernatant from spent medium, both when the conditioning substances were added to the suspension of attaching cells and when they were preadsorbed onto the surfaces. Dextran inhibited attachment only when added to the bacterial suspension. Supernatants from centrifuged natural freshwater samples had no effect. Thus, hydration of a surface and the adsorption of macromolecules can reduce bacterial attachment; however, additional factors relating to the chemical composition of the substratum and polymeric stabilization of suspended cells can affect the adhesion interaction and resultant numbers of attached cells.     

The interaction between saliva and Actinobacillus actinomycetemcomitans influenced by the Zeta potential

The adhesion of Actinobacillus actinomycetemcomitans is a virulence factor in the aetiology of periodontitis and is determined by physico-chemical properties, e.g. surface charge and hydrophobicity, of the bacterial cell surface. Although oral surfaces are constantly coated with saliva, few studies have dealt with the binding of A. actinomycetemcomitans with saliva. In this report, the charge properties of A. actinomycetemcomitans have been studied through measurement of the zeta potential and the saliva-bacteria interaction investigated at different pH-values.At physiological conditions the zeta potential was negative, varying from -11 to -26 mV, for two laboratory and two fresh isolates of A. actinomycetemcomitans. Under these conditions, binding of the low-molecular-weight salivary mucin, lactoferrin, and S-IgA was confirmed using salivary samples and purified salivary fractions in liquid-phase and in ELISA. The iso-electric points of the laboratory and fresh clinical isolates of A. actinomycetemcomitans were determined at pH 4.6 and 3.8, respectively. At pH below the iso-electric point, giving positive values of the zeta potential, additional salivary protein species bound to A. actinomycetemcomitans, including the high-molecular-weight salivary mucin (MG1) and agglutinin. Binding of the low-molecular-weight salivary mucin (MG2), lactoferrin, and S-IgA, was hardly affected by this change in zeta potential. A salivary coating formed on the bacterium at pH 7 reduced the zeta potential of the laboratory strain Y4 greatly and an iso-electric point for the bacterium could not be determined. Overall, the study suggests that upon changes in environmental pH additional salivary attachment sites on the micro-organism are exposed.     

Influence of substratum hydration and adsorbed macromolecules on bacterial attachment to surfaces

The attachment of Pseudomonas fluorescens and an Acinetobacter sp. to hydrogel and polystyrene surfaces was investigated to evaluate the influence of adsorbed water and macromolecules on adhesion. With both organisms, there was a decrease in attachment numbers with increasing water content of the hydrogels. There was also a decrease in attachment with a decrease in water contact angle on untreated, tissue culture and sulfonated polystyrene surfaces; however, the attachment numbers were higher than would be expected on the basis of the hydrogel data. With P. fluorescens, attachment to untreated and tissue culture polystyrene was inhibited by bovine serum albumin, Escherichia coli lipopolysaccharide, and the supernatant from spent medium, both when the conditioning substances were added to the suspension of attaching cells and when they were preadsorbed onto the surfaces. Dextran inhibited attachment only when added to the bacterial suspension. Supernatants from centrifuged natural freshwater samples had no effect. Thus, hydration of a surface and the adsorption of macromolecules can reduce bacterial attachment; however, additional factors relating to the chemical composition of the substratum and polymeric stabilization of suspended cells can affect the adhesion interaction and resultant numbers of attached cells.     

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.     

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.     

The composition of enamel salivary films is different from the ones formed on dental materials

This study utilized two-dimensional gel electrophoresis (2DE) to illustrate the compositional differences between in vitro salivary conditioning films (denoted pellicles) formed on human enamel as well as on the dental materials titanium and poly(methyl methacrylate). The salivary pellicles were formed by immersing each surface in individual tubes containing small volumes of freshly collected whole saliva. Saliva remaining in the tubes after the pellicle formation for 2 h was visualized by means of 2DE and silver staining. The results showed that the protein patterns in 2DE of the liquid phase of saliva left after the exposure to the respective surfaces, regarding proteins <100 kDa in size, were different depending on the surface used. Several protein groups and/or individual proteins were shown to be distinct for each surface used.     

Analysis of bacterial detachment from substratum surfaces by the passage of air-liquid interfaces

A theoretical analysis of the detachment of bacteria adhering to substratum surfaces upon the passage of an air-liquid interface is given, together with experimental results for bacterial detachment in the absence and presence of a conditioning film on different substratum surfaces. Bacteria (Streptococcus sobrinus HG1025, Streptococcus oralis J22, Actinomyces naeslundii T14V-J1, Bacteroides fragilis 793E, and Pseudomonas aeruginosa 974K) were first allowed to adhere to hydrophilic glass and hydrophobic dimethyldichlorosilane (DDS)-coated glass in a parallel-plate flow chamber until a density of 4 x 10(6) cells cm(-2) was reached. For S. sobrinus HG1025, S. oralis J22, and A. naeslundii T14V-J1, the conditioning film consisted of adsorbed salivary components, while for B. fragilis 793E and P. aeruginosa 974K, the film consisted of adsorbed human plasma components. Subsequently, air bubbles were passed through the flow chamber and the bacterial detachment percentages were measured. For some experimental conditions, like with P. aeruginosa 974K adhering to DDS-coated glass and an air bubble moving at high velocity (i.e., 13.6 mm s(-1)), no bacteria detached upon passage of an air-liquid interface, while for others, detachment percentages between 80 and 90% were observed. The detachment percentage increased when the velocity of the passing air bubble decreased, regardless of the bacterial strain and substratum surface hydrophobicity involved. However, the variation in percentages of detachment by a passing air bubble depended greatly upon the strain and substratum surface involved. At low air bubble velocities the hydrophobicity of the substratum had no influence on the detachment, but at high air bubble velocities all bacterial strains were more efficiently detached from hydrophilic glass substrata. Furthermore, the presence of a conditioning film could either inhibit or stimulate detachment. The shape of the bacterial cell played a major role in detachment at high air bubble velocities, and spherical strains (i.e., streptococci) detached more efficiently than rod-shaped organisms. The present results demonstrate that methodologies to study bacterial adhesion which include contact with a moving air-liquid interface (i.e., rinsing and dipping) yield detachment of an unpredictable number of adhering microorganisms. Hence, results of studies based on such methodologies should be referred as "bacterial retention" rather than "bacterial adhesion".     

Determination of the cell surface hydrophobicity of oral bacteria using a modified hydrocarbon adherence method

Abstract Hydrophobic interactions between bacterial cell surfaces and colonisable substrates have been implicated in the mechanisms of bacterial adherence. However, current methods of assessing bacterial hydrophobicity as a function of adherence to liquid hydrocarbons (especially hexadecane) do not always produce accurate or reproducible results. Therefore, the present technique was developed using xylene. The hydrophobic surface properties of fresh and type strains of Bacteriodes gingivalis, Bacteriodes intermedius, Capnocytophaga spp., Streptococcus salivarius and Streptococcus sanguis suspended either in saliva ions buffer (SIB) or in saliva diluted in SIB were measured. In SIB the test strains were predominantly hydrophobic. The addition of saliva caused a significant reduction ( P < 0.05) in hydrophobicity compared to SIB alone, with 80% of the strains tested. Since oral bacteria will be suspended in saliva in vivo, it is concluded that bacteria in the oral cavity may be less hydrophobic than previous studies have suggested.     

Cell surface components of Streptococcus sanguis: relationship to aggregation, adherence, and hydrophobicity.

Cell surfaces of aggregation, adherence, and hydrophilic variants of Streptococcus sanguis were compared with cell surfaces of the parent strain with regard to their protein and antigenic constituents. Cell surface molecules were released by digestion with mutanolysin. Extraction with sodium dodecyl sulfate (SDS) urea, lithium diiodosalicylate, and boiling water did not solubilize any material which stained with AgNO3 in an SDS-polyacrylamide gel electrophoresis gel. The parent organism S. sanguis 12, which aggregates in saliva, adheres to saliva-coated hydroxyapatite and is hydrophobic, was found to possess a prominently staining 160,000 molecular weight (MW) protein. This protein was almost completely absent from strain 12na, a hydrophobic nonaggregating variant, and was completely absent from the hydrophilic nonaggregating strain 12L. Trypsinization of strain 12 resulted in the coincident loss of the 160,000-MW protein and the ability to aggregate in saliva. Trypsin treatment reduced but did not eliminate the hydrophobic character of the cells. Boiling destroyed their ability to aggregate, but did not alter their hydrophobicity. Cell wall digests of strain 12 contained a number of proteins which were absent from strains 12na and 12L. Mutanolysin digests of cell walls of the hydrophilic strains contained almost no material that was visible in a silver-stained SDS-polyacrylamide gel electrophoresis gel. Culture supernatants contained a number of proteins which were immunologically cross-reactive with cell surface proteins. The hydrophilic organisms released a number of 60,000- to 90,000-MW proteins not seen in culture supernatants from the parent strain.     

A Computational Fluid Dynamic Analysis of Peri-Bracket Salivary Flow Influencing the Microbial and Periodontal Parameters

Fixed vestibular appliances decrease the “self-cleansing” action of saliva and promote aggregation of dental plaque by disturbing the salivary flow field on tooth surfaces, leading to a higher prevalence of enamel demineralization and periodontal diseases. In the current study, we investigated the salivary dynamic characteristics of plaque retention and periodontal status around appliances during orthodontic treatment. By reconstructing lower central incisors and orthodontic appliances, we simulated saliva flow on the tooth surface and then characterized and quantified the salivary flow pattern surrounding the bracket and archwire. In parallel, we tested the total peri-bracket bacterial counts and periodontal status to assess interrelations. Our results demonstrate that orthodontic appliances disturb the salivary flow field on tooth surfaces and can lead to a decrease in salivary velocity and an increase in bacterial numbers. Local vortexes forming in the areas gingival to the bracket, together with the narrow space limitation, contributed to the periodontal inflammatory response. This study confirms that changes in salivary flow are an obvious predisposing factor for bacterial accumulation, and advances the ability to replicate, in vitro, the salivary characteristics of plaque retention and periodontal status around appliances during orthodontic treatment.     

Effects of DNP on the cell surface properties of marine bacteria and its implication for adhesion to surfaces

The effect of 2, 4-dinitrophenol (DNP) on the extracelluar polysaccharides (EPS), cell surface charge, and the hydrophobicity of six marine bacterial cultures was studied, and its influence on attachment of these bacteria to glass and polystyrene was evaluated. DNP treatment did not influence cell surface charge and EPS production, but had a significant effect on hydrophobicity of both hydrophilic (p = 0.05) and hydrophobic (p = 0.01) cultures. Significant reduction in the attachment of all the six cultures to glass (p = 0.02) and polystyrene (p = 0.03) was observed after DNP treatment. Moreover, hydrophobicity but not the cell surface charge or EPS production influenced bacterial cell attachment to glass and polystyrene. From this study, it was evident that DNP treatment influenced bacterial cell surface hydrophobicity, which in turn, reduced bacterial adhesion to surfaces.     

Laboratory studies on adhesion of microalgae to hard substrates

Adhesion of Chlorella vulgaris(chlorophyceae), Nitzschia amphibia(bacillariophceae) and Chroococcus minutus(cyanobacteria) to hydrophobic (perspex, titanium and stainless steel 316-L), hydrophilic (glass) and toxic (copper, aluminium brass and admiralty brass) substrata were studied in the laboratory. The influence of surface wettability, surface roughness, pH of the medium, culture age, culture density, cell viability and presence of organic and bacterial films on the adhesion of Nitzschia amphibia was also studied using titanium, stainless steel and glass surfaces. All three organisms attached more on titanium and stainless steel and less on copper and its alloys. The attachment varied significantly with respect to exposure time and different materials. The attachment was higher on rough surfaces when compared to smooth surfaces. Attachment was higher on pH 7 and above. The presence of organic film increased the attachment significantly when compared to control. The number of attached cells was found to be directly proportional to the culture density. Attachment by log phase cells was significantly higher when compared to stationary phase cells. Live cells attached more when compared to heat killed and formalin killed cells. Bacterial films of Pseudomonas putida increased the algal attachment significantly. %     

Characterisation of structures in salivary secretion film formation. An experimental study with atomic force microscopy

The purpose of the present study was to characterise the structure dynamics of pure salivary secretions retained on controlled surfaces with different surface energies in the early stage of salivary film formation. Germanium prisms prepared to have either low surface energy or medium surface energy were incubated in fresh secretions of either human parotid saliva (HPS) or human submandibular/sublingual saliva (HSMSLS) for 15, 90, and 180 min. After controlled rinsing with distilled water, the surfaces were air dried and thereafter imaged with atomic force microscopy (AFM). The amount of adsorbed material and the size of the structures detected increased with increased saliva exposure time. The film thicknesses varied from 10 to 150 nm, and both HPS and HSMSLS films contained structures with diameters varying from 40 nm to 2 microm. Some of these were clustered into special formations. The HPS films exhibited a more granular morphology than the HSMSLS films. Furthermore, branched lines were detected on the low surface energy germanium prisms incubated in saliva. The results indicate that exposure time, surface energy, and type of salivary secretion all are factors affecting the adsorption characteristics of salivary films.     

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.     

Novel molecules for intra-oral delivery of antimicrobials to prevent and treat oral infectious diseases

New molecules were designed for efficient intra-oral delivery of antimicrobials to prevent and treat oral infection. The salivary statherin fragment, which has high affinity for the tooth enamel, was used as a carrier peptide. This was linked through the side chain of the N-terminal residue to the C-terminus of a defensin-like 12-residue peptide to generate two bifunctional hybrid molecules, one with an ester linkage and the other with an anhydride bond between the carrier and the antimicrobial components. They were examined for their affinity to a HAP (hydroxyapatite) surface. The extent of the antimicrobial release in human whole saliva was determined using 13C-NMR spectroscopy. The candidacidal activity of the molecules was determined as a function of the antimicrobial release from the carrier peptide in human saliva. The hybrid-adsorbed HAP surface was examined against Candida albicans and Aggregatibacter actinomycetemcomitans using the fluorescence technique. The bifunctional molecules were tested on human erythrocytes, GECs (gingival epithelial cells) and GFCs (gingival fibroblast cells) for cytotoxicity. They were found to possess high affinity for the HAP mineral. In human whole saliva, a sustained antimicrobial release over a period of more than 40-60 h, and candidacidal activity consistent with the extent of hybrid dissociation were observed. Moreover, the bifunctional peptide-bound HAP surface was found to exhibit antimicrobial activity when suspended in clarified human saliva. The hybrid peptides did not show any toxic influence on human erythrocytes, GECs and GFCs. These novel hybrids could be safely used to deliver therapeutic agents intra-orally for the treatment and prevention of oral infectious diseases.