Acid tolerance in early colonizers of oral biofilms

Abstract

Background: In caries, low pH drives selection and enrichment of acidogenic and aciduric bacteria in oral biofilms, and development of acid tolerance in early colonizers is thought to play a key role in this shift. Since previous studies have focussed on planktonic cells, the effect of biofilm growth as well as the role of a salivary pellicle on this process is largely unknown. We explored acid tolerance and acid tolerance response (ATR) induction in biofilm cells of both clinical and laboratory strains of three oral streptococcal species (Streptococcus gordonii, Streptococcus oralis and Streptococcus mutans) as well as two oral species of Actinomyces (A. naeslundii and A. odontolyticus) and examined the role of salivary proteins in acid tolerance development.

Methods: Biofilms were formed on surfaces in Ibidi® mini flow cells with or without a coating of salivary proteins and acid tolerance assessed by exposing them to a challenge known to kill non-acid tolerant cells (pH 3.5 for 30 min) followed by staining with LIVE/DEAD BacLight and confocal scanning laser microscopy. The ability to induce an ATR was assessed by exposing the biofilms to an adaptation pH (pH 5.5) for 2 hours prior to the low pH challenge.

Results: Biofilm formation significantly increased acid tolerance in all the clinical streptococcal strains (P < 0.05) whereas the laboratory strains varied in their response. In biofilms, S. oralis was much more acid tolerant than S. gordonii or S. mutans. A. naeslundii showed a significant increase in acid tolerance in biofilms compared to planktonic cells (P < 0.001) which was not seen for A. odontolyticus. All strains except S. oralis induced an ATR after pre-exposure to pH 5.5 (P < 0.05). The presence of a salivary pellicle enhanced both acid tolerance development and ATR induction in S. gordonii biofilms (P < 0.05) but did not affect the other bacteria to the same extent.

Conclusions: These findings suggest that factors such as surface contact, the presence of a salivary pellicle and sensing of environmental pH can contribute to the development of high levels of acid tolerance amongst early colonizers in oral biofilms which may be important in the initiation of caries.

Keywords: Acid tolerance response; Actinomyces; Pellicle; Salivary proteins; Streptococci.

Fig. 1

Comparison of the acid tolerance of different streptococcal (a) and Actinomyces (b) strains in planktonic culture and biofilms on uncoated surfaces. Acid tolerance was evaluated after an acid challenge (pH 3.5) followed by LIVE/DEAD® BacLight™ staining and CLSM, and expressed as % viability. The graphs show the mean and standard deviation of three independent biological replicates. *P < 0.05, **P < 0.001

Fig. 2

Comparison of the acid tolerance of biofilm cells of different streptococcal (a) and Actinomyces (b) strains on uncoated and salivary protein coated surfaces. Acid tolerance was evaluated after an acid challenge (pH 3.5) followed by LIVE/DEAD® BacLight™ staining and CLSM, and expressed as % viability. The graphs show the mean and standard deviation of three independent biological replicates. **P < 0.001

Fig. 3

Ability of different streptococcal (a) and Actinomyces (b) strains to induce an ATR during exposure to an adaptation pH (pH 5.5). Control cells were kept at pH 7.5. Increased acid tolerance (indicating induction of an ATR) was evaluated after an acid challenge (pH 3.5) followed by LIVE/DEAD® BacLight™ staining and CLSM, and expressed as % viability. The graphs show the mean and standard deviation of three independent biological replicates. **P < 0.001

Fig. 4

Effect of adhesion to salivary proteins on the ATR of different streptococcal (a) and Actinomyces (b) strains. Biofilm cells adhered to salivary protein coated or uncoated surfaces were exposed to an adaptation pH (pH 5.5). Increased acid tolerance (indicating induction of an ATR) was evaluated after an acid challenge (pH 3.5) followed by LIVE/DEAD® BacLight™ staining and CLSM, and expressed as % viability. The graphs show the mean and standard deviation of three independent biological replicates. *P < 0.05, **P < 0.001

Fig. 5

Example images of LIVE/DEAD® BacLight™ stained cells of S. gordonii CW and A. naeslundii CW viewed with CLSM. Control cells were kept at pH 7.5 and then exposed to acid challenge (pH 3.5). For ATR induction, cells were exposed to an adaptation pH (pH 5.5) for 2 h prior to exposure to pH 3.5. Viable cells (acid tolerant) appear green while dead cells (non-acid tolerant) appear red. Scale bar shows 20 μm