Active detachment of Streptococcus mutans cells adhered to epon–hydroxylapatite surfaces coated with salivary proteins in vitro

Although the formation of biofilms has been much studied, detachment of adherent cells from biofilms has been relatively neglected. Recent results have shown that adherent Streptococcus mutans cells can actively detach from epon–hydroxylapatite (EHA) rods conditioned with hog gastric mucin. The mechanisms for adherence and detachment of Strep. mutans cells in this system was uncertain. In the present study, resting Strep. mutans cells were used to form a simple monolayer on EHA rods coated with saliva and salivary agglutinin (SAG). Preliminary experiments defined the variables for conditioning EHA with saliva and SAG and establishing the adherence of Strep. mutans to the conditioned surfaces. The results showed that salivary proteins including SAG adsorbed rapidly to EHA and that a relatively stable Strep. mutans NG8 monolayer was formed within 60 min of incubation. The monolayers were subsequently used for detachment studies. The results showed that adherent Strep. mutans cells detached in a temperature-dependent manner and responded to the addition of a preparation of surface protein-releasing enzyme (SPRE) obtained from Strep. mutans in a dose-dependent fashion. The effect of the exogenous SPRE on detachment could be abrogated by pronase treatment. Two putative SPRE-defective mutants (A and E) were generated by Tn917 mutagenesis. Both mutants possessed a single transposon insertion as demonstrated by Southern hybridization and appeared to be different from one another based on the hybridization patterns. Mutant A displayed an increased quantity of cell-surface antigen P1, an adhesin that interacts with SAG. At the same time mutant A was unable to release P1 and other high molecular-weight proteins from the cell surface. Mutant A detached at a significantly lower rate (21%) than the parent strain (37%) (p=0.05). SPRE prepared from mutant A was unable to release Strep. mutans NG8 adherent cells as compared to SPRE obtained from the wild-type cells. Collectively, these results suggest that the detachment of Strep. mutans adherent cells formed on salivary protein-coated EHA was an active process mediated by the action of SPRE.