| Summary: | Includes bibliographical references (pages 118-132). === Bacteroides thetaiotaomicron is an important human gut commensal organism that facilitates polysaccharide utilization, but can act as an opportunistic pathogen outside of this environment causing anaerobic bacteraemia and abscess formation. There is a growing resistance by B. thetaiotaomicron and other opportunistic pathogens to metronidazole, the leading drug of choice. This dissertation aimed to use physiological, molecular and biochemical analyses to elucidate the possible mechanism/s of metronidazole resistance in B. thetaiotaomicron, in order to extend the understanding of metronidazole resistance at a genetic level. A B. thetaiotaomicron VPI-5482 transposon-generated metronidazole resistant mutant, named B. thetaiotaomicron Tn MetR , that displayed a MIC = 8 μg/ml was isolated and characterized. Southern hybridization was used to identify the insertion of a single copy of the transposon in an intergenic region of a gene cluster involved in the uptake and catabolism of L-rhamnose. RNA hybridization studies confirmed that the genes in B. thetaiotaomicron Tn MetR were transcribed in the presence and absence of the substrate, L-rhamnose. In addition, five of the catabolic genes were expressed as an operon, rhaKlPAO. A monocistronic gene, rhaR, located downstream of the transposon insertion site had predicted amino acid sequence similarity to a group of AraC/XylS transcriptional regulators. Insertional inactivation of the coding sequence of this gene, using the pGERM suicide vector rendered this mutant, B. thetaiotaomicron rhaR", unable to utilize L-rhamnose. Introducing RhaR in this strain, on a plasmid, restored growth in the medium supplemented with L -rhamnose as the sole carbon source, confirming that rhaR is a positive regulator of the rhamnose operon. The transcriptional regulation of the rhamnose pathway was further examined by primer extension and two promoter sites, PrhaKIPAO and P rhaR were identified. The link between increased L·rhamnose metabolism and metronidazole resistance was further investigated by creating an overexpressor of the positive transcriptional regulator, named B. thetaiotaomicron (pL YLrhaR). The B. thetaiotaomicron Tn MetR and B. thetaiotaomicron (pL YLrhaR) exhibited resistance to metronidazole in medium with L-rhamnose as the sole carbon source, and displayed elevated levels of lactate dehydrogenase and decreased levels of pyruvate oxidoreductase activity. These two enzymes have been linked to the electron flux required for the intracellular anaerobic activation of metronidazole and this phenotype has previously been described in metronidazole resistant B. fragilis clinical isolates. This study demonstrated that the overexpression of the rhamnose pathway in B. thetaiotaomicron VPI-5482 resulted in metronidazole resistance and provides the first data to support this link.
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