Multilocus Sequence Analysis of Phylogroup 1 and 2 Oral Treponeme Strains
Authors: Huo Yong-Biao , Chan Yuki , Lacap-Bugler Donnabella C. , Mo Sisu , Woo Patrick C. Y. , Leung W. Keung , Watt Rory M. 
Published: November 2016

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 utilized 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 American Type Culture Collection 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-nucleotide [nt]),
recA
(1,377-nt), and
pyrH
(696-nt) gene sequence data sets. 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 five distinct “species-level” phylotypes. These respectively corresponded to “
Treponema vincentii
” (
n
= 11),
Treponema medium
(
n
= 1), “
Treponema sinensis
” (
Treponema
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.

IMPORTANCE
Periodontal diseases are caused by persistent polymicrobial biofilm infections of the gums and underlying tooth-supporting structures and have a complex and variable etiology. Although
Treponema denticola
is strongly associated with periodontal diseases, the etiological roles of other treponeme species/phylotypes are less well defined. This is due to a paucity of formal species descriptions and a poor understanding of genetic relationships between oral treponeme taxa. Our study directly addresses these issues. It represents one of the most comprehensive analyses of oral treponeme strains performed to date, including isolates from North America, Europe, and Asia. We envisage that our results will greatly facilitate future metagenomic efforts aimed at characterizing the clinical distributions of oral treponeme species/phylotypes, helping investigators to establish a more detailed understanding of their etiological roles in periodontal diseases and other infectious diseases. Our results are also directly relevant to various polymicrobial tissue infections in animals, which also involve treponeme populations.