Multilocus sequence analysis of phylogroup 1 and 2 oral treponeme strains

Abstract

More than 75 ‘species-level' phylotypes of spirochete bacteria belonging to the genus Treponema reside within the human oral cavity. The majority of these oral treponeme phylotypes correspond to as-yet uncultivated taxa, or strains of uncertain standing in taxonomy. Here, we analyze phylogenetic and taxonomic relationships between oral treponeme strains using a multilocus sequence analysis (MLSA) scheme based on the highly-conserved 16S rRNA, pyrH, recA and flaA genes. We utilize this MLSA scheme to analyze genetic data from a curated collection of oral treponeme strains (n=71) of diverse geographical origins. This comprises phylogroup 1 (n=23) and phylogroup 2 (n=48) treponeme strains; including all relevant ATCC reference strains. The taxonomy of all strains was confirmed or inferred via the analysis of ca. 1,450 bp 16S rRNA gene sequences using a combination of bioinformatic and phylogenetic approaches. Taxonomic and phylogenetic relationships between the respective treponeme strains were further investigated by analyzing individual and concatenated flaA (1,074 nt), recA (1,377 nt) and pyrH (696 nt) gene sequence datasets. Our data confirmed the species differentiation between Treponema denticola (n=41) and Treponema putidum (n=7) strains. Notably, our results clearly supported the differentiation of the 23 phylogroup 1 treponeme strains into 5 distinct ‘species-level' phylotypes. These respectively corresponded to ‘Treponema vincentii' (n=11), Treponema medium (n=1); ‘Treponema sinensis' (T. sp. IA; n=4); Treponema sp. IB (n=3); and Treponema sp. IC (n=4). In conclusion, our MLSA-based approach can be used to effectively discriminate oral treponeme taxa, confirm taxonomic assignment, and enable the delineation of species boundaries with high confidence.