Microbiological characteristics of Sri Lankan tea-laborers without oral hygiene performance

Abstract

Objectives: (1) To survey the subgingival microbiota of Sri Lankan tea workers who had not performed oral hygiene ever, and (2) to compare the bacterial profiles present within deep versus shallow pockets using culture-independent 16S rRNA sequence analysis. 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.

This abstract is based on research that was funded entirely or partially by an outside source: Clinical Research Foundation, Brienz, Switzerland