by Leung Wing-cheuk. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 118-133). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract (in English) --- p.ii -iii / Abstract (in Chinese) --- p.iv -v / Table of Contents --- p.vi / Contents of Chapter 1: Introduction --- p.vii -viii / Contents of Chapter 2: Materials & Methods --- p.ix / Contents of Chapter 3: Results --- p.x / Contents of Chapter 4: Discussion --- p.xi / List of Tables --- p.xii / List of Figures --- p.xiii / Chapter Chapter 1: --- Introduction --- p.1-40 / Chapter Chapter 2: --- Materials & Methods --- p.41-67 / Chapter Chapter 3: --- Results --- p.68-99 / Chapter Chapter 4: --- Discussion --- p.100-117 / References --- p.118-133

Introduction: The New Delhi metallo-β-lactamase (NDM-1) is an enzyme synthesized by bacterium which hydrolyses β-lactam antibiotics and inhibits their antibiotic effects. Its active site is highly diverse and the hydrolysis mechanism is not yet fully understood. So, there is currently no clinically useful inhibitor for this enzyme. Due to this β-lactam resistant characteristics and the ease of horizontal gene transfer between bacteria, NDM-1 producing bacteria poses a great threat to public health. Objective: Structural analysis on Zn-Zn distance in the active site of NDM-1 was carried out in order to gain a better insight on its hydrolysis mechanism. Method: Structures of NDM-1 were obtained from PDB database and were viewed on PyMOL. Out of 22 NDM-1 structures searched, 12 of them fulfilled the inclusion criterion and were included in this study. Zn-Zn distances were compared with different factors including pH and temperature during crystallization, presence of water in the active site and nature of ligand bound to active site. Results: A trend was observed when Zn-Zn distances were compared with pH. There was also a correlation between Zn-Zn distance and nature of ligand. Distance between the 2 zinc ions in the active site increased in acidic pH value of 6.5 or below and it favours substrate hydrolysis while neutral or alkaline pH shortened the distance between zinc ions leading to hydrolysis inhibition. Discussion: One NDM-1 structure can hydrolyse the substrate in the absence of water molecule. It suggested that there might be another alternative mechanism which does not involve water molecule as nucleophile. Future study can be carried out to verify this hypothesis. On the other hand, it was also suggested that a lower pH, likely to be a pH value of 6.5 or below, could lead to a relatively higher Zn-Zn distance, likely to be 4.00 Å or above. A Zn-Zn distance of 4.00 Å or above would facilitate the hydrolysis mechanism. In other words, a pH value lower than 6.5 might also promote the hydrolysis action against β-lactams. These give us a deeper understanding into the hydrolysis mechanism of NDM-1 and might be useful in future development of clinical inhibitor to tackle the public health threat brought by NDM-1. / published_or_final_version / Physiology / Master / Master of Medical Sciences

A novel and effective mechanism of forming resistance to beta-lactam antibiotics by producing hybrid CTX-M-type beta-lactamases through genetic recombination was reported recently, albeit sporadically. Its primary introductory factor is the horizontal resistant gene transfer that occurs within the 876 bp 〖bla〗_(CTX-M) gene in Escherichia coli isolates, followed by the extensive application of beta-lactam antimicrobials. 〖bla〗_(CTX-M) originated from the nonpathogenic commensal Kluyvera spp., but it has become widely distributed in pathogenic organisms by plasmid-mediated gene transfer. Beta-lactams are effective against both gram-positive and gram-negative bacteria. However, misusing or overusing of beta-lactams has caused a globally corresponding emergence of antimicrobial resistant strains because of the selective pressure. This study included a total of 1,441 E. coli isolates, including 1,101 from six categories of animals and 340 from humans of both sexes. In total 612 strains were detected as extended spectrum beta-lactamase (ESBL)-positive. By using molecular tests on 248 ESBL-producers, 14 non-duplicate strains harbouring hybrid CTX-M-type beta-lactamases were found in a subset of 170 animal strains. A total of three alleles for hybrid CTX-M-type beta-lactamases (n = 14) were found, namely, CTX-M-64 (n = 6), CTX-M-123 (n = 6), and CTX-M-132 (n = 2). Six 〖bla〗_(CTX-M-64) and one 〖bla〗_(CTX-M-132) genes were located on approximately 70 kb IncI2 plasmids, and five 〖bla 〗_(CTX-M-123) genes were carried by 110 kb IncI1 (plasmid ST108) plasmids. All the 〖bla〗_(hybrid CTX-M) genes were placed between ISEcp1 followed by a 45 bp spacer and a 47 bp spacer followed by a 344 bp orf477delta gene. Among the six CTX-M-64-harbouring isolates detected, three of them were ST1011, phylogroup E, from chickens; the remaining three were ST224, phylogroup B1; ST93 CC168, phylogroup A; and ST117, phylogroup F. Every CTX-M-64-harbouring isolate had the co-resistance trait against both ciprofloxacin and tetracycline. The CTX-M-123-harbouring isolates were ST1485, phylogroup E; ST2732, phylogroup D; ST10, phylogroup A; ST457, phylogroup F; and ST 1196, phylogroup B1. All six CTX-M-123-harbouring strains were co-resistant to trimethoprim-sulphamethoxazole and tetracycline, and all of their transconjugants or transformants inherited the ability to resist fosfomycin. Both CTX-M-132-harbouring strains came from phylogroup A. One strain was ST3103, and the other one with a non-transferable plasmid was ST746. This study unravels the molecular epidemiology of hybrid CTX-M-type beta-lactamase-harbouring E. coli isolates. It also compared the prevalence of these isolates among both animal and human strains. Although hybrid CTX-Mtype beta-lactamases were not found in 340 clinical urinary isolates, 14 hybrid CTX-M-type beta-lactamases were identified in 14 animals in just one year. Hence, this study sets off alarm bells for the public to become more concerned about the antimicrobial resistance situation among animals. Furthermore, the discovery of hybrid CTX-M-type beta-lactamase-harbouring isolates also indicates a novel mechanism of antimicrobial resistance, i.e., homologous recombination, and shows a new way to study antimicrobial resistance. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

by Yip Chi Wai. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (p. 94-113). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstract in Chinese.

Thesis (Ph. D.)--University of Chicago, Dept. of Biochemistry and Molecular Biology, August 2003. / Includes bibliographical references. Also available on the Internet.

Thesis (M. Phil.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 141-158).

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Microbiology / Master / Master of Philosophy