Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / "May, 2001." Typescript. Vita. Includes bibliographical references (leaves 148-155). Also available on the Internet.

published_or_final_version / Microbiology / Master / Master of Medical Sciences

Backgroud: Clostridium difficile infection (CDI) is a global concern since the emergence of the hyper-virulent strain NAP1/BI/027. The incidence of CDI has been increasing and active surveillance of Clostridium difficile epidemiology is of paramount importance. In studying the epidemiology of CDI, PCR ribotyping is a very useful method in finding the genetic relatedness of outbreak and epidemic strains. Characterisation of Clostridium difficile isolates can help surveillance and active monitoring, hence reducing the chance of potential outbreaks. Aim: The aims of this study is to investigate the epidemiology of Clostridium difficile in Hong Kong in the year 2010, and that of a Clostridium difficile outbreak in 2011 by using capillary gel electrophoresis-based PCR ribotyping. Results: Among the 307 toxigenic isolates, 139 isolates (45.3%) were characterized as existing ribotypes. Ribotype 012 was the predominant ribotype (17.3%), followed by ribotype 002/0 (15.6%). A total of 144 isolates (46.9%) were characterized as new ribotypes. The remaining 24 isolates (7.8%) were characterized as of high resemblance of existing ribotypes. A total of 8/12 isolates (66.7%) of the outbreak strains were found to be ribotype 002/0. Conclusion: The predominant strains of Clostridium difficile in 2010 was ribotype 012. The 2011 outbreak in Kowloon Hospital was an outbreak of Clostridium difficile ribotype 002/0. Capillary gel electrophoresis-based PCR ribotyping was showed to be a useful tool in investigation of Clostridium difficile outbreaks and study of Clostridium difficile epidemiology. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

Introduction: Clostridium difficile infection (CDI) is the major bacterial agent causing hospital-acquired diarrhea. The incidence and severity of CDI has increased significantly in recent decades. The distribution of PCR ribotype varies among the countries. PCR ribotype 017 and 002 are the predominant ribotypes in China and Hong Kong respectively. Molecular characterization of C. difficile isolates is useful for outbreak investigation and surveillance. Both PCR ribotyping and Multilocus variable-number tandem-repeat analysis (MLVA) are reliable molecular tools for characterization of C. difficile from outbreak and sporadic case. MLVA allows subtyping of genetically related C. difficile isolates of the same ribotype by their distinctive MLVA patterns. The aim of this study was to characterize the outbreak and sporadic C. difficile isolates of PCR ribotype 002 and 017 by using capillary gel electrophoresis-based PCR ribotyping and MLVA. Their cytotoxicity, sporulation rate and germination efficiency were also investigated in the study. Results: A total of the 26 C. difficile isolates were identified by PCR ribotyping including fifteen isolates of PCR ribotype 002, eight isolates of ribotype 017, and three isolates of other ribotypes. The isolates of the same ribotypes were further sub-typed into outbreak and sporadic cluster. All isolates showed toxin-producing capability. The sporualtion rates of outbreak isolates of PCR ribotype 002 were significantly higher than that of sporadic isolates of PCR ribotype 002. Statistically higher sporulation rate was also observed in the outbreak isolates of PCR ribotype 017. The germination rate was also statistically higher in the outbreak isolates of PCR ribotype 002 than the sporadic cases. Conclusion: Capillary electrophoresis-based PCR ribotyping identified fifteen C. difficile PCR ribotype 002 and eight PCR ribotype 017 in this study. MLVA then refined these isolates into two corresponding sub-groups of outbreak and sporadic isolates. Higher sporulation and germination rates were observed in the outbreak isolates. Sporadic isolates demonstrated relatively lower sporulation and germination rates. The current evidences correlate the genotypic characterization to sporulation activity and germination efficiency. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

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Although progress has been made in the last decade in understanding the causes of colitis in horses, perhaps 60% of cases of fatal colitis in horses have no known cause. The role of type A Clostridium perfringens strains was evaluated in this study. Fecal samples from 55 horses (43 adults, 12 foals) with colitis were cultured for Clostridium difficile, Salmonella, and C. perfringens. Feces were also tested for C. difficile toxins A/B and C. perfringens toxins (alpha [CPA], beta2 [CPB2], enterotoxin [CPE]) using enzyme-linked immunosorbent assays (EIA). All fecal samples were negative for Salmonella. Clostridium perfringens and C. difficile were isolated from 40% and 5.4% of samples, respectively. No CPE was detected but 36.4% and 18.2% of animals were positive for CPA and CPB2 toxin, respectively. Subsequently, five C. perfringens isolates per fecal sample were genotyped and the supernatants of each of these isolates were evaluated for toxicity. None of the isolates were cpe, netB or tpeL positive, but atypical cpb2 and consensus cpb2 were identified in 13.6% and 3.6% isolates, respectively. All equine C. perfringens isolates showed mild toxicity effects compared to CPB producing C. perfringens positive control. Based on this study population, there was no evidence that C. perfringens had an important role in equine colitis. / Equine Guelph and Natural Sciences and Engineering Research Council

This thesis is an investigation of the binary toxin of Clostridium difficile. The aim was to improve the understanding of the role of the binary toxin in pathogenesis. Quantitative real-time PCR (qPCR) was used to study expression of cdtA encoding the binary toxin enzymatic domain, cdtR encoding the binary toxin regulator and tcdB encoding toxin B, in response to growth phase and antimicrobial treatments in 2 C. difficile strains. Validation of a set of stable reference genes was required prior to qPCR analysis of gene expression. A universal set of genes could not be identified and reference genes should be validated on a strain-specific basis. Significant increases or decreases in expression were observed in response to levofloxacin and enrofloxacin exposure. The 2 strains selected were from different ribotypes and did not always share expression patterns. Binary toxin loci were sequenced in and compared between 10 C. difficile strains. A non-sense mutation in the cdtR gene of a ribotype 078 strain was identified and found to be restricted to toxinotype V isolates. This mutation is predicted to result in a truncated, non-functional protein. Despite the mutation, cdtA expression was still detected by qPCR. Finally, an evaluation of commercial nucleic acid extraction kits was performed. All kits produced RNA of adequate quality and yield, however, RNA isolated using the the Roche MagNA Pure LC RNA Isolation Kit could not be analyzed using the Agilent Bioanalyzer. It could not properly assign RNA integrity numbers due to a failure to remove small RNAs which were interpreted as degradation. All kits were suitable for DNA extraction with the exception of the MagNA Pure LC DNA Isolation Kit III which produced sheared DNA. In conclusion, this study demonstrated that the binary toxin regulator isn’t necessary for toxin expression and suggests other regulators of expression exist. Binary toxin gene expression did not necessarily correlate with expression of tcdB and expression levels vary between strains. This study also highlighted how the heterogeneity of C. difficile complicates gene expression experiments and the need for assessment of nucleic acid extraction methods due to critical variations between established commercial systems.

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