Acid tolerance of biofilm cells of Streptococcus mutans

Abstract

Streptococcus mutans, a member of the dental plaque community, has been shown to be involved in the carious process. Cells of S. mutans induce an acid tolerance response (ATR) when exposed to sublethal pH values that enhances their survival at a lower pH. Mature biofilm cells are more resistant to acid stress than planktonic cells. We were interested in studying the acid tolerance and ATR-inducing ability of newly adhered biofilm cells of S. mutans. All experiments were carried out using flow-cell systems, with acid tolerance tested by exposing 3-h biofilm cells to pH 3.0 for 2 h and counting the number of survivors by plating on blood agar. Acid adaptability experiments were conducted by exposing biofilm cells to pH 5.5 for 3 h and then lowering the pH to 3.5 for 30 min. The viability of the cells was assessed by staining the cells with LIVE/DEAD BacLight viability stain. Three-hour biofilm cells of three different strains of S. mutans were between 820- and 70,000-fold more acid tolerant than corresponding planktonic cells. These strains also induced an ATR that enhanced the viability at pH 3.5. The presence of fluoride (0.5 M) inhibited the induction of an ATR, with 77% fewer viable cells at pH 3.5 as a consequence. Our data suggest that adhesion to a surface is an important step in the development of acid tolerance in biofilm cells and that different strains of S. mutans possess different degrees of acid tolerance and ability to induce an ATR.

FIG. 1.

Comparison of the percent survivors of planktonic and biofilm cultures of S. mutans H7 following exposure at different pH values for 2 h and control cells kept at pH 7.5. Survivors were plated on blood agar, and the values represent the means of three independent experiments. Symbols: ▴, biofilm cells; ▪, planktonic cells.

FIG. 2.

Effect of a 3-h preincubation in pH 5.5 MM4 medium on the subsequent survival at pH 3.5 for 30 min of biofilm cells of three strains of S. mutans. Control biofilms were maintained at pH 7.5. Values represent the percent viable cells as shown by LIVE/DEAD BacLight staining. Symbols: ▪, UA159;, H7; □, XAB5.

FIG. 3.

LIVE/DEAD BacLight stains of S. mutans UA159, H7, and XAB5. The control cells were kept at pH 7.5 and then exposed to pH 3.5 for 30 min. The adapted cells were exposed to pH 5.5 for 3 h and then exposed to pH 3.5 for 30 min. Viable cells are green due to staining with SYTO 9, and cells with damaged membranes are red due to staining with propidium iodine.

FIG. 4.

Effect of fluoride inhibition of glycolysis during the adaptation period at pH 5.5 on survival of S. mutans UA159 at pH 3.5 for 30 min. Values represent the percent viable cells as shown by LIVE/DEAD BacLight staining and plating on blood agar. Symbols: ▪, pH 5.5;, pH 5.5/3.5;, 0.5 M NaF, pH 5.5; □, 0.5 M NaF, pH 5.5/3.5.