High Efficacy of eCSP in Inducing S. Mutans Cell Death

Objectives: Streptococcus mutans is indicated as the primary pathogen in dental caries. We have previously demonstrated that the Com quorum-sensing system in S. mutans mediates a variety of virulence characteristics. Elimination of competence-stimulating peptide (CSP) or its signal transduction system by deletion of the comC, D, or E genes results in attenuation in these virulence activities. However, adding higher-than-physiological-concentration exogenous competence-stimulating peptide (eCSP) in broth cultures induced species-specific S. mutans cell death. The objective of our investigation is to further assess the efficacy of eCSP in inducing S. mutans cell death in biofilms.

Methods: Sequential mixed biofilms were established by forming initial biofilms of early colonizers -- Streptococcus gordonii Challis and to Actinomyces naeslundii 12104 in 48-well plates. After 24h, S. mutans BM71 that containing a tetracycline-resistant plasmid is sequentially inoculated and incubated for additional 24h. The mixed biofilms were exposed to eCSP at concentrations of 0 and 200µM for 4 hours. S. mutans colony forming units representing species survival after eCSP treatment were counted on Mitis Salivarius agar plates supplemented with tetracycline. We also compared eCSP-induced S. mutans cell death in broth and in biofilms. S. mutans BM71 in log phase broth culture or in 24h biofilms was exposed to eCSP at concentrations of 0, 10, and 100µM for 4 hours and directly assessed under a confocal fluorescence microscope.

Results: eCSP dose-dependently eliminated S. mutans cells in both broth and biofilm monocultures. Significant eCSP-induced S. mutans cell death in sequentially formed mixed biofilm was also observed.

Conclusions: Our results indicate that eCSP induces S. mutans cell death at high efficacy in both broth cultures and in biofilms. Therefore, S. mutans eCSP may be utilized as novel strategy for caries prevention and/or treatment in future.