A Strain of Streptococcus mitis Inhibits Biofilm Formation of Caries Pathogens via Abundant Hydrogen Peroxide Production

Commensal oral streptococci that colonize supragingival biofilms deploy mechanisms to combat competitors within their niche. Here, we determined that Streptococcus mitis more effectively inhibited biofilm formation of Streptococcus mutans within a seven species panel. This phenotype was common amongst all assayed isolates of S. mutans , but was specific to a single strain of S. mitis , ATCC 49456. The growth inhibitory factor was not effectively carried in spent supernatants of S. mitis . However, we documented ATCC 49456 to accumulate 4-5 times more hydrogen peroxide (H ₂ O ₂ ) than other species tested, and 5-18 times more than other S. mitis strains assayed. The S. mutans biofilm formation inhibitory phenotype was reduced when grown in media containing catalase or with a S. mitis mutant of pyruvate oxidase ( spxB ; pox ), confirming that SpxB-dependent H ₂ O ₂ production was the main antagonistic factor. Addition of S. mitis within hours after S. mutans inoculation was effective at reducing biofilm biomass, but not for 24 h pre-formed biofilms. Transcriptome analysis revealed responses for both S. mitis and S. mutans , with several S. mutans differentially expressed genes following a gene expression pattern previously described, while others being unique to the interaction with S. mitis . Finally, we show that S. mitis also affected coculture biofilm formation of several other commensal streptococci. Our study shows that strains with abundant H ₂ O ₂ production are effective at inhibiting initial growth of caries pathogens like S. mutans , but are less effective at disrupting pre-formed biofilms and have the potential to influence the stability of other oral commensal strains.