Optimization and Evaluation of the 30S-S11 rRNA Gene for Taxonomic Profiling of Oral Streptococci

Dental caries is a multifactorial disease driven by interactions between the highly complex microbial biofilm community and host factors like diet, oral hygiene habits, and age. The oral streptococci are one of the most dominant members of the plaque biofilm and are implicated in disease but also in maintaining oral health. Current methods used for studying the supragingival plaque community commonly sequence portions of the16S rRNA gene, which often cannot taxonomically resolve members of the streptococcal community past the genus level due to their sequence similarity. The goal of this study was to design and evaluate a more reliable and cost-effective method to identify oral streptococci at the species level by applying a new locus, the 30S-S11 rRNA gene, for high-throughput amplicon sequencing. The study results demonstrate that the newly developed single-copy 30S-S11 gene locus resolved multiple amplicon sequence variants (ASVs) within numerous species, providing much improved taxonomic resolution over 16S rRNA V4. Moreover, the results reveal that different ASVs within a species were found to change in abundance at different stages of caries progression. These findings suggest that strains of a single species may perform distinct roles along a biochemical spectrum associated with health and disease. The improved identification of oral streptococcal species will provide a better understanding of the different ecological roles of oral streptococci and inform the design of novel oral probiotic formulations for prevention and treatment of dental caries. IMPORTANCE The microbiota associated with the initiation and progression of dental caries has yet to be fully characterized. Although much insight has been gained from 16S rRNA hypervariable region DNA sequencing, this approach has several limitations, including poor taxonomic resolution at the species level. This is particularly relevant for oral streptococci, which are abundant members of oral biofilm communities and major players in health and caries disease. Here, we develop a new method for taxonomic profiling of oral streptococci based on the 30S-S11 rRNA gene, which provides much improved resolution over 16S rRNA V4 (resolving 10 as opposed to 2 species). Importantly, 30S-S11 can resolve multiple amplicon sequence variants (ASVs) within species, providing an unprecedented insight into the ecological progression of caries. For example, our findings reveal multiple incidences of different ASVs within a species with contrasting associations with health or disease, a finding that has high relevance toward the informed design of prebiotic and probiotic therapy.