Molecular investigations into the etiology of periodontal infections, with focus on oral spirochete bacteria

Abstract

Periodontal diseases such as periodontitis result from the disruption of healthy homeostatic balance within the resident oral microbial communities. This ‘dysbiosis’ results from the increased levels and activities of certain indigenous species of microorganism, referred to as pathogens or accessory pathogens. Approximately 50-60 species/species-level phylotypes of oral spirochete bacteria colonize the human oral cavity, all of which belong to the genus Treponema. Treponema denticola is a well-established periodontal-pathogen, which possesses numerous virulence-related activities. However, the distributions and etiological roles of other species and phylotypes of oral treponeme bacteria remain to be established. In addition, the establishment of oral treponeme communities during childhood remains poorly-understood. Here, I compared the genome biology of treponeme isolates from different species and species-level phylotypes. I also investigated the oral microbiota in supra- and subgingival plaque collected from children with a gingivitis or health status. Lastly, I analyzed the bacterial composition of subgingival plaque in canines with periodontitis and human patients with gingivitis or periodontitis, with a particular focus on the genus Treponema. In the first study, I utilized a combination of Oxford Nanopore Technologies minION and Illumina HiSeq sequencing platforms to obtain complete genome sequences for > 60 oral treponeme isolates spanning multiple species. Notably, my genome sequence data enabled the classification of ‘Treponema vincentii-like’ and ‘Treponema medium-like’ isolates into several distinct species/phylotypes. The distributions of genes encoding putative virulence-factors (including peptidases and cell-surface adhesins) displayed distinct species/phylotype-specific patterns, which may be indicative of their respective pathogenic activities. In the second study, I employed Illumina MiSeq sequencing of the hypervariable V3-V4 region of 16S rRNA gene amplicons to characterize the subgingival and supragingival microbiota within healthy juvenile subjects versus juvenile subjects with gingivitis. My results indicated that the gingivitis group had higher alpha‐diversities (amplicon sequence variant-diversity) than the healthy group; and that subgingival plaque had higher alpha‐diversities compared to paired supragingival plaque samples in both healthy and gingivitis groups. The data also showed a high interpersonal variability in oral microbiome composition. In the third study, I analyzed sequence data from a library of cloned treponeme pyrH (UMP kinase) gene sequences obtained from human subjects with gingivitis versus periodontitis. My analyses demonstrated that both groups commonly harboured diverse communities of oral treponemes, evidenced by the identification of numerous treponeme ‘pyrH genotypes’. Certain treponeme pyrH genotypes were associated with periodontitis. In the last study, I used an analogous Illumina MiSeq sequencing approach as well as an analogous clone library-based approach to characterize the bacterial composition of subgingival plaque in canines. Results showed that the canine subgingival microbiota contained many treponeme species/phylotypes that were highly-distinct from those that inhabited human oral niches. In conclusion, my results highlight the incredible species and genomic diversity present within oral microbial communities; and reveal that there is a complicated and nuanced relationship between Treponema taxa and the occurrence and the severity of periodontal diseases. (476 words)