Strain-Level Diversity Decouples Biofilm Architecture, Acidogenic and Aciduric Traits, and Antimicrobial Tolerance in Streptococcus mutans

Streptococcus mutans is a key contributor to dental caries, with its capacity to form structured biofilm microcolonies being a principal component of its cariogenic potential. Yet, most mechanistic studies rely on a limited number of laboratory strains and may not capture the functional diversity present across the species. Here, we characterized a panel of phenotypically and genomically diverse S. mutans isolates to determine how strain background influences biofilm architecture, extracellular matrix accumulation, acid-associated physiology, environmental responsiveness, and antimicrobial susceptibility. Quantitative high-resolution imaging revealed extensive heterogeneity in produced biofilm microcolony size, structure, and matrix composition, demonstrating that biofilm architecture is not a uniform species-level trait. Interestingly, the commonly used reference strain UA159 displayed an intermediate phenotype related to microcolony size and biofilm organization. Human saliva further altered biofilm structure and matrix accumulation in a strain-dependent manner rather than producing a standard species-wide response. Isolates also differed in growth and retained biofilm biomass under acidic conditions, while acid accumulation within mature biofilms varied independently of average microcolony volume, demonstrating that strains that produce larger microcolonies on average were not necessarily associated with greater acid accumulation. Susceptibility to the antiseptics chlorhexidine and cetylpyridinium chloride likewise differed among isolates and could not be predicted from formed biofilm architecture alone. Together, these findings demonstrate that disease-relevant traits commonly attributed to S. mutans are distributed unevenly and only partially coupled across strain backgrounds, with biofilm spatial organization failing to serve as a dominant phenotype linking acid accumulation, acid tolerance, and antimicrobial susceptibility.