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

Abstract

Streptococcus mutans normally colonizes dental biofilms and is regularly exposed to continual cycles of acidic pH during ingestion of fermentable dietary carbohydrates. The ability of S. mutans to survive at low pH is an important 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 biofilms. It is unknown whether biofilm growth mode or high cell density affects acid adaptation by S. mutans. This study was initiated to examine the acid tolerance response (ATR) of S. mutans biofilm cells and to determine the effect of cell density on the induction of acid adaptation. S. mutans BM71 cells were first grown in broth cultures to examine acid adaptation associated with growth phase, cell density, carbon starvation, and induction by culture filtrates. The cells were also grown in a chemostat-based biofilm fermentor for biofilm formation. Adaptation of biofilm cells to low pH was established in the chemostat by the acid generated from excess glucose metabolism, 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 that S. mutans BM71 exhibited a log-phase ATR induced by low pH and a stationary-phase acid resistance induced by carbon starvation. Cell density was found to modulate acid adaptation in S. mutans log-phase cells, since pre-adapted cells at a higher cell density or from a dense biofilm displayed significantly higher resistance to the killing pH than the cells at a lower cell density. The log-phase ATR could also be induced by a neutralized culture filtrate collected from a low-pH culture, suggesting that the culture filtrate contained an extracellular induction component(s) involved in acid adaptation 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 for 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 density and biofilm growth mode modulated acid adaptation in S. mutans, suggesting that optimal development of acid adaptation in this organism involves both low pH induction and cell-cell communication.

FIG. 1

Effect of cell density on induction of acid adaptation in S. mutans BM71 log-phase cells. Cells were collected from pH 7.0 cultures and resuspended at various cell densities during the adaptation phase at pH 5.5 for 2 h and were then exposed to the killing pH of 3.5 for 3 h. Results are the mean ± SD of at least three independent cultures.

FIG. 2

Effects of cell density and adaptation time on induction of acid adaptation by S. mutans BM71. Cells were adapted by incubation in pH 5.5 TYG medium at low (OD₆₀₀ = 0.2) or high (OD₆₀₀ = 0.6) density for various times before exposure to the killing pH of 3.5 for 3 h. Results represent the mean number of surviving cells ± SD from at least three independent cultures.

FIG. 3

Survival kinetics of S. mutans BM71 exposed to killing pH of 3.5. Both adapted and unadapted stationary- and log-phase cultures were sampled at various time points after exposure to the killing pH of 3.5. The number of survivors was determined from at least three independent cultures.

FIG. 4

The neutralized culture filtrate collected from the chemostat at pH 5.2 ± 0.3 (mean ± standard deviation) was able to induce a log-phase ATR. The culture filtrate was diluted with fresh TYG medium (pH 7.5) to illustrate a dose-dependent effect. The results are the mean ± SD of at least three independent cultures.

FIG. 5

Effect of heat or proteinase treatment on induction of acid adaptation in S. mutans BM71 by pH-neutralized culture filtrates collected from pH 5.2 (adapted) and pH 7.0 (unadapted) cultures. The results are the mean ± SD of at least three independent cultures.

FIG. 6

Acid tolerance was assayed in mutants defective in comC, comD, and comE, the genes encoding a quorum sensing system essential for cell density-dependent induction of genetic competence in unadapted and adapted S. mutans log-phase grown cells. Addition of CSP into the culture of comC mutant partially restored the wild-type acid tolerance. The results are the mean ± SD of four independent cultures.

FIG. 7

The pH-adjusted culture filtrates collected from comC mutant (SMCC-L1) cultures that were maintained at pH 7.0 (filtrate 1) or pH 5.2 (filtrate 2) were added either individually or with synthetic CSP to unadapted SMCC-L1 cultures before exposure to the killing pH 3.5 for 3 h. The percentage of survivors ± SD was determined from four independent cultures.

FIG. 8

Effect of biofilm integrity on acid resistance of biofilms. Biofilms were either intact or dissociated from the surfaces by sonication. The biofilms were grown under the condition of either glucose or sucrose as a carbon source as described in Materials and Methods.

FIG. 9

Acid tolerance response of S. mutans BM71 biofilm cells. LPC, log-phase cells; PLC, planktonic cells; 12HB, 12-h biofilm; 1DB, 1-day biofilm; 5DB, 5-day biofilms. Results are the means ± standard deviation of four cultures.