Subgingival microbiota related to pocket probing depth in Sri-Lankan tea-laborers

Abstract

OBJECTIVE: To test the hypothesis of no difference between the subgingival microbiota in sites with various probing depths within the same subjects using a culture-independent, open-ended molecular approach. METHODS: 32 Sri Lankan tea laborers, who were documented to have no access to oral hygiene practices including toothbrushing for a period of 40 years, were included in the study. For each subject, 2 sites were chosen for sampling. The subgingival microbiota was sampled from a single shallow pocket (PPD≤3mm) and a single deep pocket (PPD≥6mm) using sterile paper points. Bacterial DNA was then extracted and near full-length 16S rRNA gene fragments were PCR-amplified using a ‘universal’ primer pair. The PCR-amplified 16S rRNA genes were ‘TOPO-cloned’, sequenced bidirectionally and analyzed using a variety of bioinformatic and statistical software. 16S rRNA gene clone libraries established for deep and shallow sites were systematically compared. RESULTS: 920 and 967 (non-chimeric) 16S rRNA sequences were obtained for the deep and shallow sites, respectively. Microbial communities within shallow pocket sites comprised: 9 phyla, equating to 221 operational taxonomic units (OTUs, 98% identity cut-off). Corresponding communities within deep pockets comprised: 7 phyla, 198 OTUs. 99 OTUs were common to both sites. The estimated OTU-richness for the deep-pocket and shallow-pocket communities were 398 and 316 OTUs, respectively (Chao1). At the genus level, significantly more Parvimonas, Serratia, Enterobacter, Staphylococcus, Eubacterium, Brevibacterium, Filifactor and Kluyvera were found in deep sites, while significantly more Granulicatella, Veillonella, Neisseria, Leptotrichia, Treponema and Shuttleworthia were found in shallow sites (p<0.01). Interestingly, Treponema was only identified in shallow sites. At the species level, Campylobacter rectus, Cedecea davisae and Actinomyces cardiffensis were identified only in deep sites. CONCLUSION: In this population with 40-years documented abstinence of oral hygiene, the microbiota within deep sites was notably different from that in shallow sites, contributing to the site-specificity of periodontitis.