Methods: 64 subgingival plaque samples from 32 subjects were selected for analysis. For each subject, 1 site with PPD≤3mm and 1 site with PPD≥6mm were chosen for sampling using sterile paper points. DNA was then extracted and the 16S rRNA gene was PCR-amplified with the universal primer pair D88 and E94. The amplified genes were then TOPO-cloned, sequenced and analyzed.
Results: 1,955 plasmid clones were sequenced, yielding 1,887 16S rRNA sequences of ca. 1,500bp suitable for analysis (920 sequences for deep pockets, and 967 for shallow sites). A total of 9 phyla and 67 genera were identified, with Firmicutes(69.9%), Proteobacteria(16.3%) and Fusobacteria(7.9%) as the 3 most abundant phyla. 318 operational taxonomic units(OTUs) were identified applying 98% sequence identity cutoff. 558 clones(30%) representing 189 OTUs corresponded to novel phylotypes. The remaining 70% clones (n=1,329) represented 129 known species, 73% (n=94) of which were uncultivable species. The estimated richness (Chao1 estimator) was 567.48 (95%CI: 478.62-705.49). The diversity (Shannon index) was 4.82 (95%CI: 4.76-4.88). 213 and 187 OTUs were identified in the deep and shallow subgingival communities, respectively. The Chao1 estimator was 381 (95%CI: 314-494) for deep and 280 (95%CI: 240-352) for shallow community. The diversity was 4.53(deep) and 4.46(shallow). Significant differences were found in the composition of the microbiota between the two communities using LibShuff (p<0.001).
Conclusions: Culture-independent 16S rRNA-based analysis gave significant insight into the diversity and richness of subgingival microbiota in a cohort without any oral hygiene intervention. The microbiota in deep sites was significantly more diverse and more complex than in shallow sites, indicating that host response may not be site-specific.