Optimization of an hydroxyapatite adhesion assay for Streptococcus sanguis

Abstract

Previous studies have compared the adhesion of [3H]thymidine-labeled Streptococcus sanguis to salivacoated hydroxyapatite (SHA) and buffer-coated hydroxyapatite (HA) beads. Although the hypotonic buffer used in these assays was adjusted to simulate saliva, it does not necessarily provide the optimal parameters for the quantitative estimate of adhesion under in vitro conditions. Optimization is necessary to provide the maximum sensitivity of the assay for detecting the effects of various salivas as well as for quantitating the effect of environmental growth conditions on the adhesion of S. sanguis to SHA and HA. A major distinction between the adhesion of S. sanguis to SHA and HA was observed when the bacterial concentration was varied. At high cell concentrations, the number of cells adhering to SHA was twice the number adhering to HA. Such differences were not detected at low cell concentrations. The optimal pH for the adsorption to both SHA and HA was 6. Changes in the ionic strength or addition of monoor divalent cations found in saliva had little effect on adhesion to HA. In contrast, high concentrations of monovalent cations inhibited adhesion to SHA. Anions such as carbonate, chloride, and sulfate did not have specific effects on adhesion, whereas acetate inhibited adhesion to both SHA and HA. Fluoride inhibited adhesion to both SHA and HA, suggesting an interaction between fluoride and hydroxyapatite. These results indicated that 2 mM phosphate buffer at a pH of 6 containing 5 mM KCl and 1 mM CaCl2 was the optimal buffer for studying the in vitro adhesion of S. sanguis to SHA.

An in vitro method to study the adherence of bacteria to saliva‐treated tooth enamel sections

“…Streptococcus species have been widely used in hydroxyapatite adherence studies and many reports conclude that S. satiguis demonstrates a high affinity for hydroxyapatite surfaces (1,3,6,12), whilst S. salivarius generally adheres less well (1,3,8,15). These results agree with the findings of the present study as far as the fresh strains of S. .sanguis and S, salivarius are concerned, although the converse was true for the type strains presumably due to the disruption of adherence patterns due to repeated subculture.…”

Actinobacillus actinomycetemcomitans fimbriae

“…Although further studies are necessary to confirm and extend these findings, the current findings suggest that the A. actinomycetemcomitans fimbriae are important mediators of adhesion to HA and SHA. The adsorption isotherms of the SP variants appear to be Linear compared to isotherms of other oral bacteria (6). This could be explained by the tendency ofthe aggregates to bind to a single receptor and thus saturation is not achieved.…”

“…We recognize that the SHA and HA models may not truly refiect the in vivo environment with which these organisms are associated and that cemental surfaces, perhaps coated with a gingival fluid or serum type of pellicle, may be more suitable. However, adhesion to HA and SHA allows a comparison to other known models and it is clear that A. actinomycetemcomitans has at least one order of magnitude less adhesion to these substrates than Streptococcus sanguis and other gram-positive bac- teria under comparable conditions (6). The attachment to gingival crevice epi-theUum, possibly a more important first step in the colonization of the gingival crevice, may involve entirely different mechanisms.…”

“…Adhesion of A. actinomycetemcomitans strains to HA and SHA was evaluated as described previously for streptococci (6). Briefly, bacteria were cultured aerobically in BHI containing ^H-thymidine (1 /iCi/ml), harvested and washed 3 times in buffered KCl (2 mM K2PO4, 5 mM KCl, 1 mM CaCh, pH 6.0) by centrifugation at 12,000 g for 10 min at 4°C.…”

Protein composition, adhesion, and agglutination properties of saliva in burning mouth syndrome

“…To measure saliva-induced agglutination, we used the spectrophotometric method described by RuNDEGREN (9) with Streptococcus mutans NCTC 10449 as the test microorganism. Saliva-induced adhesion was assayed with (methyl-^^H) thymidine-labeled S. mutans NCTC 10449, essentially according to EiFERT et al (10).…”