Genetic and physiological effects of subinhibitory concentrations of oral antimicrobial agents on Streptococcus mutans biofilms.

Streptococcus mutans is the main etiological agent of dental caries because of its capacity to adhere to enamel structure and form biofilms. This study aimed to evaluate the effects of the anticariogenic agents - sodium fluoride (NaF) and chlorhexidine (CHX) - at levels below minimum inhibitory concentrations (sub-MICs) on the growth of planktonic cells and biofilms and on the expression of vicR and covR genes associated with the regulation of biofilm formation. MICs and minimum bactericidal concentrations (MBCs) of NaF and CHX were determined for S. mutans strains ATCC25175, UA159 and 3VF2. Growth curves were constructed for planktonic cells cultured in brain heart infusion (BHI) broth supplemented with NaF (0.125-0.75MIC) or CHX (0.25-0.75MIC). Biofilm formation assays were performed in microplates containing CHX or NaF at 0.5-1.0MIC and stained with violet crystal. Quantitative polymerase chain reaction determined the alterations in covR and vicR expression in cells exposed to antimicrobials at sub-MIC levels. NaF and CHX at sub-MIC levels affected the growth of planktonic cells of all three S. mutans strains, depending on the concentration tested. The biofilm formation in UA159 and 3VF2 was reduced by NaF at concentrations ≥0.5 MIC, while that of ATCC 25175 was reduced significantly irrespective of dose. In contrast, UA159 and 3VF2 biofilms were not affected by CHX at these levels, whereas those of ATCC 25175 were reduced significantly at all concentrations tested. Under sub-MIC conditions, CHX and (to a lesser degree) NaF increased vicR and covR expression in all three strains, although there were large differences between strains and treatment conditions employed. CHX and NaF at sub-MIC levels influence on the growth of S. mutans in planktonic and biofilm conditions and on transcript levels of biofilm-associated genes vicR and covR, in a dose-dependent manner.

Genotypic diversity and virulence traits of Streptococcus mutans in caries-free and caries-active individuals.

The present study evaluated the relationship between clonal diversity and some virulence traits of Streptococcus mutans isolated from eight caries-free and eight caries-active subjects. A total of 155 S. mutans isolates from caries-free subjects and 144 isolates from caries-active subjects were obtained from samples of saliva, dental plaque and tongue surface and identified by PCR. The isolates were submitted to arbitrarily primed (AP)-PCR (OPA-2 and OPA-13) and multilocus enzyme electrophoresis (MLEE) to establish the genotypic diversity. Production of water-insoluble glucan (WIG) (monitored by SDS-PAGE), final pH of cultures and the ability of bacterial cells to adhere to smooth glass in the presence of sucrose were measured. High and comparable abilities of MLEE and AP-PCR were found to distinguish S. mutans genotypes, using Simpson's index of discrimination (0.971 and 0.968, respectively). The results showed a significant difference (P < 0.01) in the number of genotypes when caries-free and caries-active groups were compared by both fingerprinting methods used. Final pH (P = 0.32) and the percentage of adherence to a glass surface (P = 0.62) did not show differences between the two groups; however, the intensities of WIG bands from the caries-active group were greater than those from the caries-free group (P < 0.01). In addition, WIG was positively correlated with the ability of S. mutans to adhere to a glass surface (r = 0.34, P = 0.02) from caries-active subjects. These data showed that AP-PCR analysis and MLEE are both effective methods for assessing the genetic relatedness of S. mutans. Using these techniques, it was found that there is a larger number of genotypes of S. mutans with increased ability to synthesize WIG in caries-active individuals.