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

Thesis (M.S.)--University of Toronto, 1998. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (M.S.)--University of Toronto, 1998. / Includes bibliographical references.

Periodontal disease encompasses a range of inflammatory infections that affect the human gingiva and underlying tissues that support the tooth. Periodontal disease, as typified by periodontitis, has a varied and highly complex polymicrobial etiology. Oral Treponema, as the only genus of spirochetes identified in the oral cavity, is one of the bacterial groups which have been found associated with the occurrence and severity of periodontal infections. Oral treponeme population has been demonstrated as a complex and diversified community. This study mainly investigated and compared the composition of treponeme and related bacterial populations present in the subgingival plaques from periodontitis subjects and periodontitis-free controls. In the first part of the study, the 16S rDNA clone library-based approach was introduced to systematically compare the diversity and composition of treponeme operational taxonomic units (OTUs) present within subgingival plaques collected from ten periodontitis subjects and ten periodontitis-free controls. The results clearly indicated that the subgingival plaque sampled from periodontitis subjects contained a significantly higher diversity and clonal abundance of oral treponeme OTUs than corresponding samples taken from the periodontitis-free controls. Community-based comparisons demonstrated that the periodontitis and control subjects contained quite distinct populations of treponeme OTUs in their corresponding periodontal niches. Most notably, the difference of treponeme populations with the diversity and clonal abundance increase in phylogroup 2 and decrease in phylogroup 6 was observed in periodontitis subjects compared to controls. Since the Spirochaetes/Synergistetes specific primer was used for 16S rDNA clone library construction, the Synergistetes sequences were also analyzed to investigate and compare the diversity and composition of members of the Synergistetes phylum in the subgingival plaques from periodontitis and control subjects. In agreement with previous studies, the results found Synergistetes clones were significantly more prevalent and more diverse in periodontitis subjects, and the presence of certain Synergistetes OTUs appeared associated with periodontitis. The major surface protein (Msp) was identified and investigated in several Treponema species, particularly phylogroup 1 and phylogroup 2 species, as a key virulence factor. In the second part of the study, the consensus primers, which cover the msp gene sequences present in a diverse set of phylogroup 1 and phylogroup 2 oral treponemes, were designed to systematically characterize the msp genotypes present in clinical treponeme communities in subgingival plaques collected from another ten periodontitis and ten periodontitis-free subjects. The results showed a great diverse of msp genotypes were identified in periodontal environment, particularly in periodontitis subgingival plaques, with majority of them have never been reported before. One of these genotypes, OTK-like NP5_2 appears to be associated with periodontitis. In the last part of the study, DNA aptamers were selected in vitro against the recombinant Msp fragment of T. denticola ATCC 35405 (rMsp405V). The preliminary study successfully selected aptamers with high binding affinity with the target protein as well as rMspNP5_2 which is the newly identified Msp homologue mentioned above. The characteristics of selected aptamers in terms of binding affinity (Kd) and predicted secondary structures were also reported. / published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

Thesis (M.S.P.H.)--University of Michigan, 1939.

Thesis (Ph. D.)--University of Rochester, 1997. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of British Columbia, 1998. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Rochester, 1997. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (M.S.P.H.)--University of Michigan, 1939.

Les intégrons sont des éléments génétiques d'ADN mobiles tout comme les transposons et les plasmides R. Ils sont grandement impliqués dans la dissémination par transfert horizontal de gènes de résistance qui se retrouvent sous forme de cassette. Une cassette est constituée d'un gène associé à un site de recombinaison spécifique nommé attC reconnu par une intégrase. L'intégrase est l'enzyme capable d'exciser des gènes sous forme de cassettes et de les intégrer dans son intégron au niveau du site attl. Il existe deux types d'intégrons : les intégrons de résistance et les intégrons chromosomiques. L'intégron chromosomique de Treponema denticola est le premier à avoir été identifié chez un spirochète et est le premier exemple où le gène codant pour l'intégrase est dans le même sens que les cassettes. De plus, cet intégron semble plus près des intégrons de résistance que les autres intégrons chromosomiques. Des expériences in vivo chez E. coli nous permettent de constater que des interactions entre l'intégron chromosomique de T. denticola et les intégrons de résistance sont possibles et viennent appuyer notre hypothèse que les intégrons chromosomiques sont à l'origine des intégrons de résistance.