Abstract
BackgroundIn 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.MethodsBiofilms 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.ResultsBiofilm 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.ConclusionsThese 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.
Highlights
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
The clinical strains of S. gordonii (CW), S. mutans (B4B), and S. oralis (JD01) showed small but significant increases in acid tolerance in the biofilms compared to planktonic culture (2.1-fold, 4fold, and 1.1-fold increase respectively, P < 0.05, Fig. 1a)
Effect of adherence to a salivary pellicle on acid tolerance The surfaces to which bacteria adhere in the oral cavity are coated with a pellicle of saliva and we investigated whether salivary proteins could further enhance the surface-induced acid tolerance of the strains used in this study
Summary
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. Biofilm formation is initiated by interactions between bacteria and the acquired enamel pellicle, a thin film of proteins derived from saliva and gingival crevicular fluid [3] Colonizers such as Streptococcus and Actinomyces, can adhere to the tooth surface through non-specific interactions as well as specific binding of bacterial surface adhesins to salivary. Compensatory mechanisms, including buffering by saliva and the generation of alkaline end-products from salivary urea and arginine by bacteria, are able to counteract this perturbation and homeostasis is re-established [10] This has been termed the ‘dynamic stability stage’ by Nyvad and Takahashi [11] and under these conditions, the composition of the community remains relatively stable. Dental caries is thought to affect around 2.5 billion people worldwide [14] and estimates suggest that around 5% of the healthcare budgets of the Organisation for Economic Cooperation and Development (OECD) countries are consumed by the disease [15]
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