Relationship between Fluoride Concentration and Activity against Virulence Factors and Viability of a Cariogenic Biofilm: in vitro Study

Abstract

Despite widespread use of various concentrations of fluoride for the prevention of dental caries, the relationship between fluoride concentration and activity against cariogenic biofilms has not been much studied. Herein we investigated the relationship between fluoride concentration and activity against virulence factors and viability of Streptococcus mutans biofilms. S. mutans biofilms were formed on saliva-coated hydroxyapatite discs. The 70-hour-old biofilms were exposed to 0, 1, 3, 10, 30, 100, 300, 1,000 or 2,000 ppm F^-. The changes of virulence factors and viability of the biofilms were analyzed using biochemical methods and laser scanning confocal fluorescence microscopy. At 1-2,000 ppm F^-, the activity of fluoride against acid production, acid tolerance, and extracellular polysaccharide formation of S. mutans biofilms accurately followed a sigmoidal pattern of concentration dependence (R² = 0.94-0.99), with EC₅₀ values ranging from 3.07 to 24.7 ppm F^-. Generally, the activity of fluoride against the virulence factors was concentration-dependently augmented in 10-100 ppm F^- and did not increase further at concentrations higher than 100 ppm F^-. However, fluoride did not alter glucosyltransferase activity and viability of S. mutans biofilm cells in all concentrations tested. These results can provide a basis for the selection of appropriate fluoride concentrations that reduce the physiological ability of cariogenic biofilms.

Effect of 1-Minute Fluoride Treatment on Potential Virulence and Viability of a Cariogenic Biofilm

“…These findings indicate that 1-min fluoride treatment neither exerts bactericidal activity nor affects the bacterial biovolume of S. mutans biofilms. This confirmed the findings of a previous study that demonstrated that 2-hour fluoride treatment did not show biofilm bactericidal activity at 2,000 ppm F - [Pandit et al, 2013].…”

“…However, according to a previous study, even low concentrations (10 ppm F -) of fluoride affected the virulence properties and composition of S. mutans biofilms [Pandit et al, 2011b]. Furthermore, the half maximum effective concentration (EC 50 ) against the virulence properties of S. mutans biofilms ranged from 3.07 to 24.7 ppm F - [Pandit et al, 2013]. The difference in antivirulence concentration of fluoride between the present and previous studies can be attributed to differences in treatment duration.…”

“…Fluoride exerts anticaries activity by inhibiting tooth mineral dissolution and enhancing remineralization in carious lesions [ten Cate, 1999;Buzalaf et al, 2011]. Furthermore, many studies have demonstrated that fluoride can affect acidogenicity, aciduricity, and glucosyltransferase production of cariogenic bacteria in biofilms, especially S. mutans [Marquis et al, 2003;Pandit et al, 2013]. In addition, a recent study showed that fluoride inhibits the adherence of S. mutans to hydroxyapatite surfaces [Loskill et al, 2013].…”

Recovery of Acid Production in <b><i>Streptococcus mutans</i></b> Biofilms after Short-Term Fluoride Treatment

“…mutans UA159 (ATCC 700610; serotype c) biofilms were formed on saliva-coated hydroxyapatite (sHA) discs (2.93 cm 2 ; Clarkson Chromatography Products, Inc., South Williamsport, Pa., USA) placed in a vertical position in 24-well plates, as detailed elsewhere [Pandit et al, 2013]. Briefly, the sHA discs were prepared by incubation with filter-sterilized (0.22-μm low proteinbinding filter) human saliva for 1 h at 37 ° C. For biofilm formation, the sHA discs were transferred to a 24-well plate containing 1% sucrose (w/v) ultrafiltered (10 kDa molecular weight) tryptone yeast extract broth with S. mutans UA159 (2-5 × 10 6 colony-forming units, CFU/ml).…”

“…1 a). After treatment, the biofilms were dip-washed twice with salt solution (50 m M KCl + 1 m M MgCl 2 , pH 7.0) and incubated in filter-sterilized saliva (pH 7.3) for 0, 6, 12, 25, 50, or 100 min at 37 ° C. After incubation, biofilm pH drop assays were performed as described elsewhere [Belli et al, 1995;Pandit et al, 2013]. Briefly, the treated 46-hour-old biofilms were transferred to a 6-well plate containing salt solution.…”