Diversity of oral treponemes in patients with chronic periodontitis

Abstract

Numerous studies have demonstrated a strong association between periodontal disease and the presence of the oral spirochete Treponema denticola. Molecular analyses based on the sequence of 16S ribosomal RNA (rRNA) genes have revealed that over 60 different species or species-level phylotypes belonging to the genus Treponema are present within the oral cavity. However, the full extent of the diversity of oral Treponemes, as well as their possible pathogenic roles, remains to be established. Objectives: To gauge the diversity of Treponeme species within infected periodontal pockets of patients with chronic periodontitis. Methods: Subgingival plaque samples were collected from four separate diseased sites from three patients with chronic periodontitis. Total DNA was purified from the pooled samples from each patient, and near-full length regions of 16S rRNA genes were amplified by polymerase chain reaction (PCR) using previously-designed ‘treponeme specific' primers. PCR products were cloned into plasmids to form a library of treponeme-enriched 16S rRNA genes for each patient. 117 plasmids were sequenced in one or both directions to unambiguously identify the major species and phylotypes present in the three patients. Results: Fewer than 50% of plasmids sequenced contained Treponemal 16S rRNA genes. T. denticola. T. vincentii and T. socranskii were the dominant species detected, with several subspecies and novel phylotypes identified. T. medium, T. amylovorum, T. maltophilum and 3 novel species-level phylotypes of Treponema were also identified. Other species enriched using these ‘Treponeme-specific' primers belonged to the Olsenella, Atopobium, Cryptobacterium Flexistipes and Deferribacteraceae genera/families. Conclusions: Preliminary sequencing results from our Treponeme-enriched library of 16S rRNA genes from periodontitis patients have revealed significant diversity. We compare and contrast our results with those of related studies, and discuss the relative advantages and drawbacks associated with our ‘intensive and expensive' approach to characterizing oral treponeme populations.