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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(Uncorrected OCR) Abstract of the thesis entitled MOLECULAR BIOLOGY OF A CRYPTIC DEHALOGENASE FROM Burkholderia cepacia MBA4 Submitted by Laiju Sam For the degree of Doctor of Philosophy at The University of Hong Kong in July 2000 Burkholderia cepacia MBA4 has been previously shown to produce a single dehalogenase in batch culture condition. Other cryptic dehalogenases were detected in cells grown in continuous culture. In this study, one of the cryptic dehalogenases was cloned and characterised. This cryptic haloacid dehalogenase was designated Chd 1 and expressed constitutively in Escherichia coli. The structural gene, chdJ, was isolated from a 1.7-kb PstI fragment. This fragment. contains a functional promoter since the expression of chdl in E. coli is orientation independent. The nucleotide sequence of the fragment was determined and analysed and an open reading frame for 840 amino acids was identified. The nucleotide sequence of chdl did not show any homology with those of other dehalogenase genes. Comparison of the deduced amino acid sequence, however, showed significant homology, ranging from 42-50%, with the amino acid sequences of several other dehalogenases. Phylogenetic analysis indicated that Chd 1 is closely related to dehalogenase CI and dehalogenase IVa of Pseudomonas putida CBS3 and B. cepacia MBA4 respectively. 1 The recombinant Chdl had a native molecular weight of 58,000 daltons whereas the denatured molecular weight was found to be 27,000 daltons. Thus, the enzyme exists predominantly as a dimer. When the activity towards the two stereoisomers of 2-monochloropropionic acid were considered individually, the enzyme was found to be active towards the L-isomer only. The purified enzyme was most active towards monobromoacetic acid with specific activities of 5.44, 1.5, 4.56, and 1.95 J!mole of halide released/min/mg protein for monobromoacetic acid, 2-monobromopropionic acid, monochloroacetic acid and 2-monochloropropionic acid respectively. Maximum activity of the enzyme was observed at pH 6.5. The enzyme was found to be thermolabile. chdl was cloned in the T7 RNA polymerase driven pRSET A and Irc promoter based pPROEXHT expression vectors. However, no significant expression of chdl was obtained in these systems. Chd 1 differed from other dehalogenases in having a long leader sequence. This leader sequence contains a potential signal peptidase cleavage site. This is a property of periplasmic enzymes. In order to detect the unprocessed molecules of Chd 1, chdl was expressed in the temperature-sensitive E. coli strain IT 41 (lep9-mutant). This strain contains a mutation in the leader peptidase gene. The leader peptidase is inactive in this strain at the non-permissive temperature of o 42 C. Precursor molecules of Chd 1 was detected in cultures shifted to this non- permissive temperature. Periplasmic fractions isolated from E. coli harboring chdl were found to contain the active dehalogenase. 11 / abstract / toc / Botany / Doctoral / Doctor of Philosophy

published_or_final_version / Pathology / Master / Master of Philosophy

Bibliography: leaves 101-121. / vii, 122, [77] leaves, [21] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Reports the characterisation of mos genes and preliminary studies on the mos genes in R. leguminosarum br. viciae strain 1a. / Thesis (Ph.D.)--University of Adelaide, Dept. of Crop Protection, 1998?

Bibliography: leaves 135-166. / 166, [139] leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Provides insights into the structure/function relationships within the El Tor haemolysin (HlyA). Generates some constructs used to determine the three-dimensional structure of the protein and of the pore. The genetic organisation of the region flanking the hly locus suggests a pathogenicity island. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1998?

Corrigendum attached to back cover. / Includes bibliographical references (leaves 160-190). / x, 190, [20] leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Investigates the role of rhizopines in rhizobial competition for nodulation, and to isolate the rhizopine synthesis genes in Rhizobium leguminosarum bv. viciae. / Thesis (Ph.D.)--University of Adelaide, Dept. of Crop Protection, 1999

Bibliography: leaves 218-227. / 227 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / A series of potential receptors for biotin derivatives based on the 2,6-pyridinedicarboxamide motifs were investigated. / Thesis (Ph.D.)--University of Adelaide, Dept. of Chemistry, 2000?

Bibliography: leaves 135-166. / 166, [139] leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Provides insights into the structure/function relationships within the El Tor haemolysin (HlyA). Generates some constructs used to determine the three-dimensional structure of the protein and of the pore. The genetic organisation of the region flanking the hly locus suggests a pathogenicity island. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1998?

Corrigendum attached to back cover. / Includes bibliographical references (leaves 160-190). / x, 190, [20] leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Investigates the role of rhizopines in rhizobial competition for nodulation, and to isolate the rhizopine synthesis genes in Rhizobium leguminosarum bv. viciae. / Thesis (Ph.D.)--University of Adelaide, Dept. of Crop Protection, 1999