Bacterial composition and metabolomics of dental plaque from adolescents

Supragingival dental plaque samples were collected from 20 caries-free (CF; DMFT=0) and 20 caries (CA) Swedish adolescents. Fresh plaque samples were subjected to an ex vivo acid tolerance (AT) test where the proportion of bacteria resistant to an acid shock was evaluated through confocal microscopy and live/dead staining, and the metabolites produced were quantified by Nuclear Magnetic Resonance (NMR). In addition, DNA was extracted and the 16S rRNA gene was sequenced by Illumina sequencing, in order to characterize bacterial composition. There were no significant differences in the AT scores between CA and CF individuals. However, 7 out of 10 individuals with high acid tolerance scores were CA. Regarding bacterial composition, Abiotrophia, Prevotella and Veillonella were found at significantly higher levels in CA individuals (p=0.0085, 0.026 and 0.04 respectively) and Rothia and Corynebacterium at significantly higher levels in CF individuals (p=0.026 and 0.003, respectively). Streptococcus mutans was found at low frequencies and was absent in 60% of CA individuals. Random-forest predictive models indicate that at least 4 bacterial species or 9 genera are needed to distinguish CA from CF adolescents. Over 60 metabolites could be distinguished by NMR analysis, showing a significant clustering of organic acids with specific bacteria associated with CA and/or high AT individuals, being Scardovia wiggsiae the species with strongest associations. A significant clustering of ethanol and isopropanol with health-associated bacteria such as Rothia or Corynebacterium was also found. Accordingly, several relationships involving these compounds like the Ethanol:Lactate ratio were found to be significantly associated to acid tolerance and their predictive value for caries risk should be tested. We therefore propose that future caries risk studies should consider not only the use of multiple organisms as potential microbial biomarkers, but also their functional contribution (acidogenic pathway) and functional adaptation (acid tolerance).