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

TitleDevelopment of molecular approaches for the identification and characterization of oral treponeme bacteria
Authors
Advisors
Advisor(s):Watt, RMJin, L
Issue Date2011
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Mo, S. [莫思溯]. (2011). Development of molecular approaches for the identification and characterization of oral treponeme bacteria. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b4775279
AbstractElevated 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.
DegreeDoctor of Philosophy
SubjectTreponema.
Dept/ProgramDentistry

 

DC FieldValueLanguage
dc.contributor.advisorWatt, RM-
dc.contributor.advisorJin, L-
dc.contributor.authorMo, Sisu.-
dc.contributor.author莫思溯.-
dc.date.issued2011-
dc.identifier.citationMo, S. [莫思溯]. (2011). Development of molecular approaches for the identification and characterization of oral treponeme bacteria. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b4775279-
dc.description.abstractElevated 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.-
dc.languageeng-
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)-
dc.relation.ispartofHKU Theses Online (HKUTO)-
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.-
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License-
dc.source.urihttp://hub.hku.hk/bib/B47752798-
dc.subject.lcshTreponema.-
dc.titleDevelopment of molecular approaches for the identification and characterization of oral treponeme bacteria-
dc.typePG_Thesis-
dc.identifier.hkulb4775279-
dc.description.thesisnameDoctor of Philosophy-
dc.description.thesislevelDoctoral-
dc.description.thesisdisciplineDentistry-
dc.description.naturepublished_or_final_version-
dc.identifier.doi10.5353/th_b4775279-
dc.date.hkucongregation2012-

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