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. / published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy
| Identifer | oai:union.ndltd.org:HKU/oai:hub.hku.hk:10722/174459 |
| Date | January 2011 |
| Creators | Mo, Sisu., 莫思溯. |
| Contributors | Watt, RM, Jin, L |
| Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
| Source Sets | Hong Kong University Theses |
| Language | English |
| Detected Language | English |
| Type | PG_Thesis |
| Source | http://hub.hku.hk/bib/B47752798 |
| Rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works., Creative Commons: Attribution 3.0 Hong Kong License |
| Relation | HKU Theses Online (HKUTO) |
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