Oralis J22 Research Articles

Antagonistic interactions by a high H2 O2 -producing commensal streptococcus modulate caries development by Streptococcus mutans.

Dental caries (tooth-decay) is caused by biofilms harboring polymicrobial communities on teeth that leads to the onset of localized areas of enamel demineralization. Streptococcus mutans has been clinically associated with severe caries in childhood. Although commensal bacteria can combat S. mutans using self-generated antimicrobials such as hydrogen peroxide (H2 O2 ), constant sugar-rich diet consumption disrupts microbial homeostasis shifting toward cariogenic community. Recently, Streptococcus oralis subsp. tigurinus strain J22, an oral isolate, was identified as a uniquely potent H2 O2 producer. Here, we assess whether a high H2 O2 -producing commensal streptococcus can modulate the spatial organization and virulence of S. mutans within biofilms. Using an experimental biofilm model, we find that the presence of S. oralis J22 can effectively inhibit the clustering, accumulation, and spatial organization of S. mutans on ex vivo human tooth surface, resulting in significant reduction of enamel demineralization. Notably, the generation of H2 O2 via pyruvate oxidase (SpxB) from S. oralis J22 is not repressed by sugars (a common repressor in other mitis group streptococci), resulting in enhanced inhibition of S. mutans growth (vs. Streptococcus gordonii). We further investigate its impact on biofilm virulence using an in vivo rodent caries model under sugar-rich diet. Coinfection of S. mutans with S. oralis results in reduced caries development compared to either species infected alone, whereas coinfection with S. gordonii has negligible effects, suggesting that the presence of an efficient, high H2 O2 -producer can disrupt S. mutans virulence. This work demonstrates that oral isolates with unusual high H2 O2 production may be capable of modulating biofilm cariogenicity in vivo. The findings also highlight the importance of bacterial antagonistic interactions within polymicrobial communities in health and in disease-causing state.

The response of dual-species bacterial biofilm to 2% and 5% NaOCl mixed with etidronic acid: A laboratory real-time evaluation using optical coherence tomography.

AimThe addition of etidronic acid (HEDP) to sodium hypochlorite (NaOCl) could increase the antibiofilm potency of the irrigant, whilst maintaining the benefits of continuous chelation. Studies conducted so far have shown that mixing HEDP with NaOCl solutions of relatively low concentration does not compromise the antibiofilm efficacy of the irrigant. However, the working lifespan of NaOCl may decrease resulting in a reduction of its antibiofilm efficacy over time (efficiency). In this regard, continuous irrigant replenishment needs to be examined. This study investigated the response of a dual‐species biofilm when challenged with 2% and 5% NaOCl mixed with HEDP for a prolonged timespan and under steady laminar flow.MethodologyDual‐species biofilms comprised of Streptococcus oralis J22 and Actinomyces naeslundii T14V‐J1 were grown on human dentine discs in a constant depth film fermenter (CDFF) for 96 h. Biofilms were treated with 2% and 5% NaOCl, alone or mixed with HEDP. Irrigants were applied under steady laminar flow for 8 min. Biofilm response was evaluated by means of optical coherence tomography (OCT). Biofilm removal, biofilm disruption, rate of biofilm loss and disruption as well as bubble formation were assessed. One‐way anova, Wilcoxon's signed‐rank test and Kruskal–Wallis H test were performed for statistical analysis of the data. The level of significance was set at a ≤.05.ResultsIncreasing NaOCl concentration resulted in increased biofilm removal and disruption, higher rate of biofilm loss and disruption and increased bubble formation. Mixing HEDP with NaOCl caused a delay in the antibiofilm action of the latter, without compromising its antibiofilm efficacy.ConclusionsNaOCl concentration dictates the biofilm response irrespective of the presence of HEDP. The addition of HEDP resulted in a delay in the antibiofilm action of NaOCl. This delay affects the efficiency, but not the efficacy of the irrigant over time.

Chemical efficacy of several NaOCl concentrations on biofilms of different architecture: new insights on NaOCl working mechanisms.

AimTo investigate the anti‐biofilm efficacy and working mechanism of several NaOCl concentrations on dual‐species biofilms of different architecture as well as the changes induced on the architecture of the remaining biofilms.Methodology Streptococcus oralis J22 and Actinomyces naeslundii T14V‐J1 were co‐cultured under different growth conditions on saliva‐coated hydroxyapatite discs. A constant‐depth film fermenter (CDFF) was used to grow steady‐state, four‐day mature biofilms (dense architecture). Biofilms were grown under static conditions for 4 days within a confined space (less dense architecture). Twenty microlitres of buffer, 2‐, 5‐, and 10% NaOCl were applied statically on the biofilms for 60 s. Biofilm disruption and dissolution, as well as bubble formation, were evaluated with optical coherence tomography (OCT). The viscoelastic profile of the biofilms post‐treatment was assessed with low load compression testing (LLCT). The bacteria/extracellular polysaccharide (EPS) content of the biofilms was examined through confocal laser scanning microscopy (CLSM). OCT, LLCT and CLSM data were analysed through one‐way analysis of variance (ANOVA) and Tukey’s HSD post‐hoc test. Linear regression analysis was performed to test the correlation between bubble formation and NaOCl concentration. The level of significance was set at a < 0.05.ResultsThe experimental hypothesis according to which enhanced biofilm disruption, dissolution and bubble formation were anticipated with increasing NaOCl concentration was generally confirmed in both biofilm types. Distinct differences between the two biofilm types were noted with regard to NaOCl anti‐biofilm efficiency as well as the effect that the several NaOCl concentrations had on the viscoelasticity profile and the bacteria/EPS content. Along with the bubble generation patterns observed, these led to the formulation of a concentration and biofilm structure‐dependent theory of biofilm removal.ConclusionsBiofilm architecture seems to be an additional determining factor of the penetration capacity of NaOCl, and consequently of its anti‐biofilm efficiency.

Factors affecting the chemical efficacy of 2% sodium hypochlorite against oral steady-state dual-species biofilms: Exposure time and volume application.

AimTo study the influence of time and volume of 2% sodium hypochlorite (NaOCl) on biofilm removal and to investigate the changes induced on the biofilm architecture. Steady‐state, dual‐species biofilms of standardized thickness and a realistic contact surface area between biofilms and NaOCl were used.Methodology Streptococcus oralis J22 and Actinomyces naeslundii T14V‐J1 biofilms were grown on saliva‐coated hydroxyapatite discs within sample holders in the Constant Depth Film Fermenter (CDFF) for 96 h. Two per cent NaOCl was statically applied for three different time intervals (60, 120 and 300 s) and in two different volumes (20 and 40 μL) over the biofilm samples. The diffusion‐driven effects of time and volume on biofilm disruption and dissolution were assessed with Optical Coherence Tomography (OCT). Structural changes of the biofilms treated with 2% NaOCl were studied with Confocal Laser Scanning Microscopy (CLSM) and Low Load Compression Testing (LLCT). A two‐way analysis of variance (2‐way anova) was performed, enabling the effect of each independent variable as well as their interaction on the outcome measures.ResultsOptical coherence tomography revealed that by increasing the exposure time and volume of 2% NaOCl, both biofilm disruption and dissolution significantly increased. Analysis of the interaction between the two independent variables revealed that by increasing the volume of 2% NaOCl, significant biofilm dissolution could be achieved in less time. Examination of the architecture of the remaining biofilms corroborated the EPS‐lytic action of 2% NaOCl, especially when greater volumes were applied. The viscoelastic analysis of the 2% NaOCl‐treated biofilms revealed that the preceding application of higher volumes could impact their subsequent removal.ConclusionsTime and volume of 2% NaOCl application should be taken into account for maximizing the anti‐biofilm efficiency of the irrigant and devising targeted disinfecting regimes against remaining biofilms.

Chemical biofilm removal capacity of endodontic irrigants as a function of biofilm structure: optical coherence tomography, confocal microscopy and viscoelasticity determination as integrated assessment tools.

To investigate the influence of biofilm structure on the biofilm removal capacity of endodontic irrigants and to study changes in the architecture of the remaining biofilms. Streptococcus oralis J22 and Actinomyces naeslundii T14V-J1 were cocultured under different growth conditions on saliva-coated hydroxyapatite discs. A constant depth film fermenter (CDFF) was used to grow steady-state 4-day biofilms. Biofilms were grown under static conditions for 4 and 10days within a confined space. Twenty microlitres of 2% NaOCl, 2% Chlorhexidine (CHX), 17% Ethylene-diamine-tetra-acetic acid (EDTA) and buffer were applied statically on the biofilms for 60s. Biofilm removal was evaluated with optical coherence tomography (OCT). Post-treated biofilms were assessed via low load compression testing (LLCT) and Confocal laser scanning microscopy (CLSM). Optical coherence tomography data were analysed through a two-way analysis of variance (ANOVA). Low load compression testing and CLSM data were analysed through one-way ANOVA and Dunnett's post hoc test. The level of significance was set at a<0.05. The initial biofilm structure affected the biofilm removal capacity of the irrigants. NaOCl demonstrated the greatest chemical efficacy against the biofilms and was significantly more effective on the static than the CDFF biofilms (P<0.001). CHX was ineffective and caused a rearrangement of the biofilm structure. Ethylene-diamine-tetra-acetic acid exhibited a distinct removal effect only on the CDFF biofilms. Biofilm age influenced the structure of the remaining biofilms. The 4-day grown remaining biofilms had a significantly different viscoelastic pattern compared to the respective 10-day grown biofilms (P≤0.01), especially in the NaOCl-treated group. Confocal laser scanning microscopy analysis confirmed the CHX-induced biofilm structural rearrangement. Biofilm structure is an influential factor on the chemical efficacy of endodontic irrigants. Optical coherence tomography allows biofilm removal characteristics to be studied. NaOCl should remain the primary irrigant. Ethylene-diamine-tetra-acetic acid was effective against cell-rich/EPS-poor biofilms. Chlorhexidine did not remove biofilm, but rather rearranged its structure.

Efficacy of natural antimicrobials in toothpaste formulations against oral biofilms in vitro

ObjectivesTo evaluate the antimicrobial efficacies of two toothpaste formulations containing natural antimicrobials (herbal extracts and chitosan) against oral biofilms of different composition and maturational status. MethodsBacteria from a buffer suspension or fresh saliva were adhered for 2h to a salivary conditioning film and subsequently grown for 16h. Dual-species biofilms were prepared from Actinomyces naeslundii T14V-J1 and Streptococcus oralis J22, whilst multi-species biofilms were grown from freshly collected human saliva. Biofilms were exposed to 25wt% toothpaste supernatants. A chlorhexidine-containing mouthrinse and a buffer were used as positive- and negative-controls, respectively. Antibacterial efficacy was concluded from acute killing, bacterial removal, prevention of bacterial re-deposition and continued killing during re-deposition. ResultsThe herbal- and chitosan-based supernatants showed immediate killing of oral biofilm bacteria, comparable with chlorhexidine. Moreover, exposure of a biofilm to these supernatants or chlorhexidine, yielded ongoing killing of biofilm bacteria after exposure during re-deposition of bacteria to a matured 16h biofilm, but not to a much thinner initial biofilm formed by 2h adhesion only. This suggests that thicker, more matured biofilms can absorb and release oral antimicrobials. ConclusionsSupernatants based on herbal- and chitosan-based toothpastes have comparable immediate and ongoing antibacterial efficacies as chlorhexidine. Natural antimicrobials and chlorhexidine absorb in oral biofilms which contributes to their substantive action.

Antimicrobial and anti-adhesive potential of a biosurfactant Rufisan produced by Candida lipolytica UCP 0988

In the last years, researches developed with biosurfactants for application in the medical area have been revealing the promising biological activities of these biomolecules. In this work the antimicrobial and anti-adhesive properties of a biosurfactant Rufisan isolated from the yeast Candida lipolytica UCP 0988, growth in a medium supplemented with ground nut refinery residue was determined against several microorganisms. The biosurfactant was able to reduce the water surface tension from 70 to 25.3mN/m and showed a critical micelle concentration (CMC) of 0.03%. The biosurfactant was isolated after 72h of fermentation and was tested in concentrations varying from 0.75 to 12mg/l. The highest antimicrobial activities were observed against Streptococcus agalactiae, Streptococcus mutans, Streptococcus mutans NS, Streptococcus mutans HG, Streptococcus sanguis 12, Streptococcus oralis J22 at a concentration superior to the biosurfactant critical micelle concentration. Moreover, the biosurfactant showed anti-adhesive activity against most of the microorganisms tested. As far as we know, this is the first compilation of data on antimicrobial and anti-adhesive activities of a biosurfactant obtained from a Candida strain against such a broad group of microorganisms. The results obtained in this work showed that the biosurfactant from C. lipolytica is a potential antimicrobial and/or anti-adhesive agent for several biomedical applications.

Carbohydrate engineering of the recognition motifs in streptococcal co‐aggregation receptor polysaccharides

The cell wall polysaccharides of certain oral streptococci function as receptors for the lectin-like surface adhesins on other members of the oral biofilm community. Recognition of these receptor polysaccharides (RPS) depends on the presence of a host-like motif, either GalNAcbeta1-3Gal (Gn) or Galbeta1-3GalNAc (G), within the oligosaccharide repeating units of different RPS structural types. Type 2Gn RPS of Streptococcus gordonii 38 and type 2G RPS of Streptococcus oralis J22 are composed of heptasaccharide repeats that are identical except for their host-like motifs. In the current investigation, the genes for the glycosyltransferases that synthesize these motifs were identified by high-resolution nuclear magnetic resonance (NMR) analysis of genetically altered polysaccharides. RPS production was switched from type 2Gn to 2G by replacing wefC and wefD in the type 2Gn gene cluster of S. gordonii 38 with wefF and wefG from the type 2G cluster of S. oralis J22. Disruption of either wefC or wefF abolished cell surface RPS production. In contrast, disruption of wefD in the type 2Gn cluster or wefG in the type 2G cluster eliminated beta-GalNAc from the Gn motif or beta-Gal from the G motif, resulting in mutant polysaccharides with hexa- rather than heptasaccharide subunits. The mutant polysaccharides reacted like wild-type RPS with rabbit antibodies against type 2Gn or 2G RPS but were inactive as co-aggregation receptors. Additional mutant polysaccharides with GalNAcbeta1-3GalNAc or Galbeta1-3Gal recognition motifs were engineered by replacing wefC in the type 2Gn cluster with wefF or wefF in the type 2G cluster with wefC respectively. The reactions of these genetically modified polysaccharides as antigens and receptors provide further insight into the structural basis of RPS function.

Microbubble‐induced detachment of coadhering oral bacteria from salivary pellicles

The presence and maturity of the salivary pellicle influences microbial adhesion and its tenacity in the oral cavity, posing a challenge to different plaque-control systems. Some plaque-control systems rely on surface-tension forces arising from passing microbubbles sprayed over the pellicle. Passage of such bubbles is accompanied by a high fluid flow, but systematic studies are lacking on the contribution of fluid flow vs. microbubbles towards plaque removal. Therefore, the aim of this study was to determine the detachment efficacy of laminar fluid flow (wall shear rates 11,000-16,000 s(-1)), with and without microbubbles, towards the detachment of Actinomyces naeslundii T14V-J1 and Streptococcus oralis J22, and their coadhering aggregates, from salivary pellicles formed over 2 h or 16 h from reconstituted human whole saliva. Microbubbles in a fluid flow were more efficient at inducing single bacterial detachment, resulting in almost complete (97%) removal for S. oralis J22 and a 15-fold increase in A. naeslundii T14V-J1 removal as compared to the detachment caused by fluid flow alone. A. naeslundii was more difficult to remove and apparently formed the strongest bonds with high-molecular-weight proteins in 16-h pellicles. The detachment of coaggregates after 2 min left a substantial amount of adhered bacterial mass, including more than 60% of singly attached A. naeslundii on the pellicle surface, providing nucleation sites for the de novo adhesion of coadhering streptococci.

Influence of fluid shear and microbubbles on bacterial detachment from a surface.

Prevention of microbial adhesion and detachment of adhering microorganisms from surfaces is important in many environmental, industrial, and medical applications. Fluid shear is an obvious parameter for stimulating microbial detachment from surfaces, but recently it has been pointed out that a passing air-liquid interface also has potential in stimulating microbial detachment. In the present study, the ability of microbubbles to stimulate detachment of bacterial strains from a glass surface is compared with the effects of fluid flow. Adhesion and detachment of Actinomyces naeslundii T14V-J1, Streptococcus oralis J22, and their coadhering aggregates were studied on glass, mounted in a parallel plate flow chamber. High fluid wall shear rates (11,000 to 16,000 s(-1)) were established in a laminar flow regime in the absence and presence of microbubbles. Wall shear rates stimulated detachment ranging from 70% to 30% for S. oralis and A. naeslundii, respectively. Coadhering aggregates were detached up to 54%. The presence of microbubbles in the flow increased the detachment of A. naeslundii within 2 min of flow from 40% in the absence of microbubbles to 98%, while detachment of neither S. oralis nor coadhering aggregates was affected by the presence of microbubbles. In summary, extremely high fluid flows can be effective in stimulating microbial detachment, depending on the strain involved. The addition of microbubbles to the flow allows the detachment of tenaciously adhering bacteria not detached by flow alone, but not of adhering coaggregates.

Path-dependency of the interaction between coaggregating and between non-coaggregating oral bacterial pairs—a thermodynamic approach

Coaggregation, i.e. specific recognition between bacteria from different species, is a well-described phenomenon in the human oral cavity but remains physically poorly understood. With our study we aimed at elucidating some aspects of the mechanism of the coaggregation between the oral bacteria Streptococcus oralis J22 and Actinomyces naeslundii 147, in particular with respect to the driving force for coaggregation and its pathway-dependency. To that end, the macroscopic turbidity of the bacterial suspension, the morphology of the coaggregates, binding isotherms and heats of interaction were compared between the above-mentioned coaggregating bacterial pair and a non-coaggregating pair, Streptococcus sanguis PK1889 and A. naeslundii 147. The coaggregating pair forms large aggregates, which rapidly sediment from the suspension while the non-coaggregating pair forms only very small coaggregates that remain homogeneously suspended. Coaggregation is further characterized by a high affinity between the partner cells that bind to each other in a strong cooperative mode. The interactions between both pairs occur under the release of heat and are thus enthalpically favorable. More heat is released for the coaggregating than for the non-coaggregating pair. Adding the coaggregating bacteria in steps to each other leads to saturation of enthalpically favorable binding sites. This is observed when the streptococcus is added to the actinomyces as well as when the addition is done the other way around. It is concluded that the cooperativity of the coaggregation process is based on an increase of entropy. It is furthermore shown that the density of the coaggregates as well as the heat effect of formation of these coaggregates depend on the number of steps in which the partner cells are added to each other. Adding S. oralis J22 in three steps to A. naeslundii 147 results in the formation of denser coaggregates under the release of less heat, as compared to that of addition in one step. These differences point to a larger entropy increase when in a step-wise mixing the coaggregating bacteria are allowed to form more densely-packed coaggregates.

Enthalpy of interaction between coaggregating and non-coaggregating oral bacterial pairs—a microcalorimetric study

Bacterial adhesion and coaggregation are involved in the development of oral biofilms, called dental plaque. Although various techniques have already been used to study different aspects of these bacterial interactions, microcalorimetry has not yet been applied. This paper describes how isothermal reaction calorimetry can be employed to determine the enthalpy of coaggregation between two oral bacterial pairs. For most biological processes, the enthalpy tends to reach a minimum value, reflecting the most stable state, which is directly related to the heat content of the system. The calorimeter consists of four measuring units where reaction ampoules are filled with 1.5 ml of an Actinomyces naeslundii 147 suspension, while reference ampoules are filled with buffer only. After equilibration at 25 °C, 80 μl of a streptococcal suspension was titrated into the reaction ampoules. To study possible saturation of the binding sites on the actinomyces surface, three consecutive injections with streptococcal suspensions were done. Following each injection, a 20-μl aliquot was taken from the ampoule kept outside the calorimeter and the number of free ( S f) and bound ( S b) streptococci was determined microscopically. Experiments were carried out with a coaggregating streptococcal strain ( Streptococcus oralis J22) and a non-coaggregating strain ( Streptococcus sanguis PK1889), serving as a control. The coaggregation enthalpy was exothermic, that is, heat was released in the reaction ampoule upon coaggregation and the heat released by the coaggregating pair minus the heat released by the non-coaggregating pair yielded a coaggregation enthalpy of −0.015×10 −6 mJ/bound streptococcus for the first injection. Upon consecutive injections, the coaggregation enthalpy decreased to −0.0004×10 −6 mJ/bound streptococcus. Comparison with enthalpy changes reported for lectin–carbohydrate binding suggests that a huge number of binding sites are involved in the formation of one bacterial coaggregate.

Influence of dentifrices and dietary components in saliva on wettability of pellicle-coated enamel in vitro and in vivo.

In vitro salivary pellicles were found to be less hydrophobic by water contact angles than clinically formed pellicles. In this study, water contact angles were measured on enamel coated with pellicles adsorbed from reconstituted human whole saliva (RHWS) and after exposure to dentifrices or dietary components. In addition, adhesion of Streptococcus oralis J22 to pellicles formed from RHWS with minor amounts of milk added and after exposure to dentifrices was studied. Exposure of RHWS-pellicles to milk or salad oil yielded an increase in the hydrophobicity of in vitro pellicles to values observed in vivo, but a decrease was seen after exposure to a sugar solution. Pellicles formed from saliva with 0.4% milk added attracted less S. oralis cells than pellicles formed in the absence of milk components. Exposure of pellicles formed from saliva with milk added to various dentifrices had a variable effect on bacterial adhesion: markedly lower numbers of adhering S. oralis were found for a dentifrice with NaF, but exposure to dentifrices containing SnF2 or hexametaphosphate showed slightly increased adhesion. In summary, dietary components have influence on the hydrophobicity of enamel pellicles, while combinations of dietary components and dentifrices certainly influence the adhesiveness of the pellicles. The effects of dietary components on pellicle conditioning film should be taken into consideration in research on the development of ingredients to control intraoral surface chemistry and microbiology, as well as in the development of oral products.

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.

Reversibility of Bacterial Adhesion at an Electrode Surface

Deposition of four bacterial strains from a 1 mM potassium phosphate buffer (pH 7) to an indium tin oxide (ITO) electrode surface has been studied in a parallel plate flow chamber at three electrode potentials (−0.2, 0.1, and 0.5 V). Capacitance measurements demonstrated that the ITO surface was negatively charged with respect to the solution at the electric potentials applied, that is, bacteria deposited under repulsive electrostatic conditions. Initial deposition rates were independent of the electrode potential, except for Actinomyces naeslundii T14V-J1. Application of a more negative electrode potential yielded increased desorption of Streptococcus oralis J22, Staphylococcus epidermidis 3399, and A. naeslundii 147, whereas for A. naeslundii T14V-J1 desorption decreased. If a high (≥65 μA) cathodic current was applied by adjusting the potential between −0.4 and −0.5 V, adhering bacteria were stimulated to desorb with desorption probabilities increasing with increasing current density. Bacterial desorption could be described on the basis of the Derjaguin−Landau−Verwey−Overbeek theory assuming secondary minimum adhesion, except for A. naeslundii T14V-J1. When bacteria were forced to adhere in the primary minimum by application of a high (1.8 V) positive electrode potential during deposition, adhering bacteria could hardly be stimulated to desorb, indicating strong, irreversible adhesion. The deviating behavior of A. naeslundii T14V-J1 was attributed to direct contact between its relatively long surface appendages and the electrode surface.

Interaction of Two Oral Streptococcal Strains with Physicochemically Characterized Fluorosilane Diffusion Gradient Surfaces

Gradient surfaces have a defined variation in surface chemistry along their length that allow study of the influence of substratum wettability on bioadhesion phenomena along their length in relation with a controlled surface chemistry. (Tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-dimethylchlorosilane (MCFS) diffusion gradients were made on glass and characterized by advancing and receding water contact angles and scanning X-ray photoelectron spectroscopy. Model calculations demonstrated that the hydrophobic ends of these gradients were only 50% covered by MCFS, which could be confirmed by atomic force microscopy showing hydrophobic patches. Therewith, bacteria can interact with either hydrophobic or hydrophilic patches on the hydrophobic end of a diffusion gradient, while on the hydrophilic end there is no such choice. By use of a parallel plate flow chamber, the position-bound adhesion, including initial deposition rates and numbers of adhering bacteria after 3 h, of two different oral streptococcal strains was studied along the lengths of MCFS gradients. Streptococcus oralis J22 did not show any position-bound adhesion along the length of a gradient surface. The organism also had similar adhesion behavior on homogeneous, hydrophobic FEP-Teflon as on hydrophilic glass. Streptococcus sobrinus HG1025, however, adhered a-fold better to the hydrophobic end of a MCFS gradient than to its hydrophilic end, while also on homogeneous, hydrophobic FEP-Teflon adhesion was more extensive than on hydrophilic glass. When streptococci adhering along the length of a gradient were exposed to a passing liquid-air interface,no position-bound detachment was observed for any of the strains, but upon perfusion of the flow chamber with a detergent solution S. sobrinus HG1025 detached less from the hydrophobic end than from the hydrophilic end of the gradient. This study demonstrates, using MCFS diffusion gradients; that the sensitivity of bacterial strains to differences in substratum hydrophobicity, originating from a known chemical heterogeneity, is strain-dependent.

Characterization of a novel N‐acetylneuraminic acid‐specific Fusobacterium nucleatum PK1594 adhesin

Fusobacterium nucleatum has been identified as significantly associated with sites with active periodontal disease and, as a group, the oral fusobacteria coaggregate with members of all oral bacteria genera tested. Monoclonal antibodies were prepared and used in conjunction with other potential inhibitors, such as simple sugars and amino acids, to characterize coaggregation interactions, of F. nucleatum PK1594. Four unique monoclonal antibodies, 5H11, 14C7, 19F2 and 29C12, were obtained by their ability to inhibit coaggregation of F. nucleatum PK1594 with Actinomyces israelii PK16. They were also capable of inhibiting other coaggregations including Streptococcus oralis H1, S. oralis J22, Capnocytophaga ochracea ATCC33596, Prevotella denticola PK1277 and Prevotella intermedia PK1511. All of these interactions were completely inhibited by N-acetylneuraminic acid. Neither N-acetylneuraminic acid nor monoclonal antibody 5H11 had any inhibitory effect on other F. nucleatum PK1594 interactions, including all galactose-inhibitable coaggregations. The results indicate that F. nucleatum PK1594 expresses upon its surface a distinct type of adhesin that mediates coaggregation interactions that are inhibited by N-acetylneuraminic acid.

Influence of temperature on the co-adhesion of oral microbial pairs in saliva.

Coaggregation (interactions between two planktonic microorganisms) and co-adhesion (interactions between sessile and planktonic microorganisms) are believed to be important factors in the formation of dental plaque by many investigators, although others doubt whether coaggregation and co-adhesion occur in vivo. It is known that coaggregation and co-adhesion generally occur equally well in buffer as in saliva, but the influence of temperature on the co-adhesion of coaggregating oral microbial pairs in saliva is unknown. Therefore, co-adhesion of streptococci suspended in saliva to glass with adhering actinomyces present (1.0 x 10(6) cells cm-2) was studied in a parallel plate flow chamber in the temperature range from 22 degrees C to 40 degrees C. In the range from 22 degrees C up to 35 degrees C both pairs studied, Streptococcus oralis 34 with Actinomyces naeslundii 5951 and Streptococcus oralis J22 with A. naeslundii 5951, displayed similar co-adhesion kinetics and co-adhesion in a stationary end-point, but around and above 37 degrees C co-adhesion almost disappeared. Hence, we conclude that co-adhesion of coaggregating oral microbial pairs in saliva may be critically influenced by temperature, especially around the temperatures prevailing in the oral cavity.

A quantitative method to study co-adhesion of microorganisms in a parallel plate flow chamber. II: Analysis of the kinetics of co-adhesion

Recently (J. Microbiological Methods 20, 289–305, 1994), it was suggested that dental plaque formation involved both interspecies binding between planktonic microorganisms (‘coaggregation’) as well as between planktonic and sessile microorganisms (‘co-adhesion’) and that co-adhesion could be measured by analyzing the spatial arrangements of co-adhering microorganisms in a stationary end point state. Limited information is available, however, on the kinetics of coaggregation and co-adhesion. The aim of this work was to develop a quantitative method to study the kinetics of co-adhesion and to determine the effects of mass transport conditions, like shear and concentration, on the kinetics of co-adhesion. The kinetics of co-adhesion of the coaggregating and non-coaggregating streptococci ( S. oralis J22 and S. sanguis PK1889) to glass with adhering A. naeslundii T14V-J1 cells, have been studied in a parallel plate flow chamber using real time image analysis. Initial local deposition rates in the vicinity of the Actinomyces were similar as observed on other regions over the substratum surface (‘non-local deposition rates’) for the non-co-adhering pair, but were up to 19 times higher for the co-adhering pair. Thus, whereas we have previously shown that co-adhesion in a stationary end point state can be quantified by radial pair distribution functions, this paper demonstrates that the tendency of coaggregating strains to co-adhere is also reflected in the kinetics of co-adhesion. Local deposition rates increased with increasing streptococcal concentration for the coaggregating pair. However, if it is attempted to increase the local deposition rate by increasing the shear, it was found that local deposition rates decreased, most likely because the interaction time between the adhering actinomyces and the flowing streptococci becomes too short. The high deposition rates for the coaggregating pair could not be explained on the basis of convective-diffusional mass transport towards the substratum, but required accounting for collisions between adhering actinomyces and streptococci flowing parallel to the surface.

A quantitative method to study co-adhesion of microorganisms in a parallel plate flow chamber: basic principles of the analysis

Intermicrobial aggregation is described as one of the factors contributing to dental plaque formation. Intermicrobial aggregation is usually measured by mixing potential partners suspended in a liquid phase (‘coaggregation’). However, even if aggregation in the liquid phase occurs, adhesion of microorganisms to partners already adhering to a substratum surface may also occur (‘co-adhesion’). Coaggregation assays have been performed in order to measure coaggregation and to model co-adhesion, although it is not yet clear which the two prevails in vivo. Apart from being semi-quantitative (scores run from 0 to 4) it is questionable whether coaggregation assays really mimic co-adhesion. This study was designed to develop a method to quantitative assess co-adhesion of microbial pairs in order to gain a better understanding of the mechanisms governing co-adhesion. Co-adhesion of coaggregating and non-coaggregating partners ( S. oralis, S. sanguis and A. naeslundii) to glass has been studied in a parallel plate flow chamber using real time image analysis. The spatial arrangements of adhering bacteria were analyzed by radial pair distribution functions, revealing the relative density of adhering bacteria around adhering bacteria from the same (g 22(r)) or a partner strain (g 21(r)). Pair distribution functions g 21(r) of coaggregating pairs clearly reveal a preference of coaggregating streptococci ( S. oralis J22 and S. sanguis PK2951) to adhere around the actimomyces ( A. naeslundii PK213 or T14V-J1), which were used to coat the bare glass substratum. Besides, the distribution function g 21(r) showed differences in co-adhesion patterns for strains with the same coaggregation score. From the results presented in this paper it can be concluded that with a parallel plate flow chamber, co-adhesion can be quantified on a continuous scale under well controlled conditions, more closely resembling those occurring in vivo.