Streptococcus cristatus reduces cariogenicity of saliva-derived microcosms under pH-dependent conditions

Background: The study aims to investigate Streptococcus cristatus, an oral commensal bacterium, as a probiotic for dental caries prevention by modulating the oral microbiome. Methods: Saliva from four healthy donors was used to establish 24-h microcosm biofilms in an in vitro 96-well peg model. The preformed biofilms were then exposed to biofilm medium containing 0.2% sucrose (BM), with or without S. cristatus. They were grown for 48 h under two conditions: a constant pH-neutral regime (BM supplemented with 76 mM K₂HPO₄ and 15 mM KH₂PO₄, pH 7.0) or cariogenic pH-cycling regime (8 h pH-neutral and 16 h in BM containing 100 mM acetic acid, pH 5.5). Phosphate and acetate buffers were used to control pH. After 72 h, the biofilms were analyzed for biomass, lactic acid production, hydrogen peroxide (HP) concentrations, and microbial composition via 16S rRNA gene sequencing. Results: S. cristatus successfully integrated into 24-h preformed microcosm biofilms derived from individual saliva. Under pH-neutral conditions, it reduced biofilm biomass and lactate production while increasing hydrogen peroxide (HP) generation in a donor-dependent manner. Conversely, under cariogenic pH-cycling conditions, these inhibitory effects on biomass and lactate production were consistent across all donors, although HP was undetectable. Microbiome analysis revealed that S. cristatus increased species richness and mitigated the compositional shifts caused by pH-cycling. This was achieved by inhibiting Streptococcus salivarius/vestibularis across all donors, while promoting Streptococcus mitis group and Streptococcus anginosus in a donor-dependent manner. Conclusions: S. cristatus represents a promising microbiome modulator with the potential to substantially mitigate the cariogenicity of oral microcosms.