The diversity of microbial life associated with periodontal health and disease

Abstract

Periodontal diseases are chronic inflammations of the tooth-supporting apparatus induced by microbial biofilm communities colonizing the dentogingival junction. The levels of certain bacterial ‘periodontopathogens’, e.g. Porphyromonas gingivalis, particular Synergistetes spp. or Treponema spp., have previously been shown to be associated with the occurrence and progression of periodontitis. General consensus is that Archaea are considerably less diverse than bacteria within subgingival niches. However, certain methanogenic archaea may also play role in periodontal disease. This thesis aims to characterize, in an ‘open-discovery’ fashion, the distributions of bacteria and archaea present in subgingival biofilms sampled from a variety of periodontal niches.

First, a small-scale study was performed on equine subgingival plaque (pooled, multi-site), to establish the applicability of our research methodology. 454 pyrosequencing of the V4-V6 hypervariable regions of the 16S ribosomal RNA (rRNA) genes was employed to analyze the subgingival bacterial profile of two periodontally-healthy horses. Additional 16S rRNA clone sequencing-based analyses were performed to further characterize taxonomy and phylogeny of the Synergistetes and Spirochaetes taxa present.

Second, 454 pyrosequencing was used to investigate the microbial profiles of pooled subgingival plaque from five human periodontal conditions, including aggressive periodontitis (AgP, n=10) and age-/gender-matched young healthy controls (YH, n=11); chronic periodontitis (CP, n=10); gingivitis (G, n=11), and age-/gender-matched healthy controls (H, n=12). Pyrosequencing data revealed that the prominent genera were Treponema, unclassified Synergistaceae, Anaerovorax and Porphyromonas. Operational taxonomic units (OTUs) corresponding to Treponema taxa were significantly more prevalent (p<0.001) and diverse within all three disease groups (AgP, CP, G) compared to corresponding healthy controls (YH and H).

Third, the profiles of archaea taxa present within subgingival plaque samples from the above five clinical categories were analyzed using Illumina (MiSeq) sequencing targeting the V3-V4 hypervariable regions of the 16S rRNA gene. More than 99.9% of the genus-level OTUs identified were assigned to the Methanobrevibacter genus. Low levels of Vadin CA11, Nitrospumilus and Marine group II taxa were also detected. No association could be found between any particular subgingival archaeal genus with specific periodontal status.

Fourth, phylogroup 1 and 2 Treponema present within subgingival niches were compared by analyzing pyrH and 16S rRNA gene sequences detectable within the same samples. A subsample of the AgP, CP, G and H subjects (n=18) were analyzed using a clone sequencing-based approach, using phylogroup 1/2 treponeme ‘pyrH-specific’ and spirochete full-length 16S rRNA specific PCR primer sets. Results indicated that Treponema medium, ‘Treponema vincentii’, ‘Treponema sp. IB’ and ‘Treponema sp. IC’-like sequences could be detected using phylogroup 1 pyrH primers, Treponema denticola-like sequences could be detected using phylogroup 2 pyrH primers, T. vincentii, T.spIB, T.spIC and T. denticola could be detected among treponemes from other phylotypes using 16S rRNA primer.

Taken together, my results demonstrated that Treponema OTUs were prevalent and diverse within subgingival biofilms and were associated with inflamed periodontal conditions. Further investigations are required to determine if the analysis of pyrH gene sequences represents an effective approach for surveying all oral treponeme populations. Archaea, predominantly the Methanobrevibacter, were readily detectable in subgingival plaque samples. (496 words)