A collection of 113 Streptococcus strains from supragingival dental plaque of caries-free individuals were recently tested in vitro for direct antagonism of the dental caries pathogen Streptococcus mutans and for their capacity for arginine catabolism via the arginine deiminase system (ADS). To advance their evaluation as potential probiotics, 12 strains of commensal oral streptococci with various antagonistic and ADS potentials were assessed in a mouse model for oral (i.e., oral mucosal pellicles and saliva) and dental colonization under four diets (healthy or high-sucrose, with or without prebiotic arginine). Colonization by autochthonous bacteria was also monitored. One strain failed to colonize, whereas oral colonization by the other 11 strains varied by 3 log units. Dental colonization was high for five strains regardless of diet, six strains increased colonization with at least one high-sucrose diet, and added dietary arginine decreased dental colonization of two strains. Streptococcus sp. strain A12 (high in vitro ADS activity and antagonism) and two engineered mutants lacking the ADS (ΔarcADS) or pyruvate oxidase-mediated H2O2 production (ΔspxB) were tested for competition against S. mutans UA159. The A12 wild-type and ΔarcADS strains colonized only transiently, whereas the ΔspxB strain persisted, but without altering oral or dental colonization by S. mutans. In tests of four additional candidates, Streptococcus sanguinis BCC23 markedly attenuated S. mutans oral and dental colonization, enhanced colonization of autochthonous bacteria, and decreased the severity of smooth surface caries under highly cariogenic conditions. Results demonstrate the utility of the mouse model to evaluate potential probiotics, revealing little correlation between in vitro antagonism and competitiveness against S. mutans in vivo. IMPORTANCE Our results demonstrate that in vivo testing of potential oral probiotics can be accomplished and can yield information to facilitate the ultimate design and optimization of novel anticaries probiotics. We show that human oral commensals associated with dental health are an important source of potential probiotics that may be used to colonize patients under dietary conditions of highly various cariogenicity. Assessment of competitiveness against the dental caries pathogen Streptococcus mutans and impact on caries identified strains or genetic elements for further study. Results also uncovered strains that enhanced oral and dental colonization by autochthonous bacteria when challenged with S. mutans, suggesting cooperative interactions for future elucidation. Distinguishing a rare strain that effectively competes with S. mutans under conditions that promote caries further validates our systematic approach to more critically evaluating probiotics for use in humans.

Streptococcus sanguinis is an oral commensal and an etiological agent of infective endocarditis. Previous studies have identified the SsaACB manganese transporter as essential for endocarditis virulence; however, the significance of SsaACB in the oral environment has never been examined. Here we report that a ΔssaACB deletion mutant of strain SK36 exhibits reduced growth and manganese uptake under acidic conditions. Further studies revealed that these deficits resulted from the decreased activity of TmpA, shown in the accompanying paper to function as a ZIP‐family manganese transporter. Transcriptomic analysis of fermentor‐grown cultures of SK36 WT and ΔssaACB strains identified pH‐dependent changes related to carbon catabolite repression in both strains, though their magnitude was generally greater in the mutant. In strain VMC66, which possesses a MntH transporter, loss of SsaACB did not significantly alter growth or cellular manganese levels under the same conditions. Interestingly, there were only modest differences between SK36 and its ΔssaACB mutant in competition with Streptococcus mutans in vitro and in a murine oral colonization model. Our results suggest that the heterogeneity of the oral environment may provide a rationale for the variety of manganese transporters found in S. sanguinis.

Streptococcus sanguinis is an oral commensal and an etiological agent of infective endocarditis. Previous studies have identified the SsaACB manganese transporter as essential for endocarditis virulence; however, the significance of SsaACB in the oral environment has never been examined. Here we report that a ΔssaACB mutant of strain SK36 exhibits reduced growth and manganese uptake under acidic conditions. Further studies revealed that these deficits resulted from the decreased activity of TmpA, shown in the accompanying paper to function as a ZIP-family manganese transporter. Transcriptomic analysis of fermentor-grown cultures of SK36 WT and ΔssaACB strains identified pH-dependent changes related to carbon catabolite repression in both strains, though their magnitude was generally greater in the mutant. In strain VMC66, which possesses a MntH transporter, loss of SsaACB did not significantly alter growth or cellular manganese levels under the same conditions. Interestingly, there were only modest differences between SK36 and its ΔssaACB mutant in competition with Streptococcus mutans in vitro and in a murine oral colonization model. Our results suggest that the heterogeneity of the oral environment may provide a rationale for the variety of manganese transporters found in S. sanguinis and point to strategies for enhancing the safety of oral probiotics.