Cell density modulates acid adaptation in Streptococcus mutans: Implications for survival in biofilms

Streptococcus mutans normally colonizes dental biofilms and is regularly ex posed to continual cycles of acidic pH during ingestion of fermentable diet ary carbohydrates. The ability of S. mutans to survive at low pH is an impo rtant virulence factor in the pathogenesis of dental caries. Despite a few studies of the acid adaptation mechanism of this organism, little work has focused on the acid tolerance of S. mutans growing in high-cell-density bio films. It is unknown whether biofilm growth, mode or high cell density affe cts acid adaptation by S. mutans. This study was initiated to examine the a cid tolerance response (ATR) of S. mutans biofilm cells and to determine th e effect of cell density on the induction of acid adaptation. S. mutans BM7 1 cells were first grown in broth cultures to examine acid adaptation assoc iated with growth phase, cell density, carbon starvation, and induction by culture filtrates. The cells were also grown in a chemostat-based biofilm f ermentor for biofilm formation. Adaptation of biofilm cells to low pH was e stablished in the chemostat by the acid generated from excess glucose metab olism, followed by a pH 3.5 acid shock for 3 h. Both biofilm and planktonic cells were removed to assay percentages of survival. The results showed th at S. mutans BM71 exhibited a log-phase ATR induced by low pH and a station ary-phase acid resistance induced by carbon starvation. Cell density was fo und to modulate acid adaptation in S. mutans log-phase cells, since pre-ada pted cells at a higher cell density or from a dense biofilm displayed signi ficantly higher resistance to the killing pH than the cells at it lower cel l density. The log-phase ATR could also be induced by a neutralized culture filtrate collected from a low-pH culture, suggesting that the culture filt rate contained an extracellular induction component(s) involved in acid ada ptation in S. mutans. Heat or proteinase treatment abolished the induction by the culture filtrate. The results also showed that mutants defective in the comC, -D, or -E genes, which encode a quorum sensing system essential f or cell density-dependent induction of genetic competence, had a diminished log-phase ATR. Addition of synthetic competence stimulating peptide (CSP) to the comC mutant restored the ATR. This study demonstrated that cell dens ity and biofilm growth mode modulated acid adaptation in S. mutans, suggest ing that optimal development of acid adaptation in this organism involves b oth low pH induction and cell-cell communication.