Development of molecular approaches for the identification and characterization of oral treponeme bacteria

Abstract

Elevated populations of oral treponeme bacteria are associated with periodontal

diseases. Furthermore, genetic analyses of oral bacterial populations using 16S rRNAbased

approaches have previously shown that deeply-infected periodontal pockets

contain a large diversity of oral treponeme species (or species level phylotypes).

However, the diversity of treponeme populations present within ‘healthy’ dental

plaque has been relatively poorly investigated. In this thesis, I used a 16S rRNA-based

molecular approach to analyze the diversity of treponeme ‘species’ within human

subjects with chronic periodontitis, versus periodontally-healthy controls. I found that

similar levels of oral treponeme phylotype diversity were present in both subject

groups, but there were notable significant differences in the composition of treponeme

phylotypes present. Within both groups, oral phylogroup 1 treponemes had the highest

levels of phylotype diversity; phylogroup 2 treponemes had similar levels of diversity;

whilst phylogroup 6 treponemes were more abundant and exhibited greater phylotype

diversity in the healthy group. As has been previously found, T. denticola was

associated with periodontal disease.

Over the past few decades, numerous T. denticola strains have been isolated from

patients with periodontal diseases. However, their genetic diversity has never been

systematically analyzed. Here I describe the formulation of a multilocus sequence

analysis (MLSA) scheme for the characterization of T. denticola isolates. The

sequences of seven conserved genes (flaA, recA, pyrH, ppnK, dnaN, era and radC)

were determined within 16 diverse reference strains and clinical isolates of T.

denticola from China, Japan, Canada, the Netherlands and the USA. Results showed

that all 16 T. denticola strains were monophyletic, and formed at least 5 well-defined

clades, with OTK, ATCC 700768 and ATCC 700771 being the most diverse strains.

No geographical relationships could be established, but several strains isolated from

different continents appear to be very closely related. Whilst the use of the

concatenated seven-gene sequence dataset (6,769nt) had the highest straindiscriminatory

power, the concatenated sequence of the recA, pyrH and flaA genes

(3,150 nt) also had excellent strain-discriminatory abilities.

The recently-discovered oral phylogroup II treponeme, T. putidum shares many

biological similarities with T. denticola. Here, I used a small-scale MLSA-type

approach based on the recA and pyrH genes to systematically characterize five T.

putidum isolates. Results revealed that these T. putidum strains clustered together with

T. denticola ATCC 700768, and were well-separated from other T. denticola strains. T.

putidum cell morphology was investigated using electron microscopy and fluorescence

in situ hybridization (FISH) techniques. In addition, oral phylogroup II treponeme

populations present in a clinical sample of subgingival plaque were analyzed by

surveying the diversity of pyrH genotypes present. A diverse population of T.

denticola strains was revealed.

In conclusion, the MLSA approach developed in this study was shown to be a very

useful tool for the identification and characterization of oral treponeme isolates

belonging to phylogroup II (e.g. T. denticola, T. putidum). In future studies, this

powerful molecular approach may be further developed for the analysis of other oral

treponeme phylogroups, or for the analysis of spirochete populations within related

animal infections.