Char Tsui Shan. / Thesis submitted in: October 2000. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 92-114). / Abstracts in English and Chinese. / Abstract (in English) --- p.i / Abstract (in Chinese) --- p.iv / Acknowledgements --- p.vi / Table of Contents --- p.vii / Contents of Chapter 1: Introduction --- p.viii / Contents of Chapter 2: Materials & Methods --- p.viii / Contents of Chapter 3: Results --- p.x / Lists of Tables --- p.xi / List of Figures --- p.xii / Chapter Chapter 1: --- Introduction --- p.1-24 / Chapter Chapter 2: --- Material & Methods --- p.25 -41 / Chapter Chapter 3: --- Results --- p.42 -76 / Chapter Chapter 4: --- Discussion --- p.77 -89 / Chapter Chapter 5: --- Future Work --- p.90 -91 / References --- p.92 -107 / Chapter Chapter 1: --- Introduction / Chapter 1.1. --- Taxonomy of Enterococci --- p.1 / Chapter 1.2. --- Natural Habitat of Enterococci --- p.2 / Chapter 1.3. --- Characteristics of Enterococci --- p.2 / Chapter 1.4. --- Identification to Species Level of Enterococci --- p.2 / Chapter 1.5. --- Clinical Significance of Enterococci --- p.5 / Chapter 1.6 --- Clinical Management of Enterococcal Infections --- p.8 / Chapter 1.7. --- Antimicrobial Susceptibilities of Enterococci --- p.8 / Chapter 1.8. --- Antimicrobial Resistance of Enterococci --- p.11 / Chapter 1.9. --- Molecular typing of enterococci --- p.16 / Chapter 1.10. --- Epidemiological Studies of Enterococci --- p.19 / Chapter 1.11. --- Objectives --- p.21 / Chapter Chapter 2. --- Materials & Methods / Chapter 2.1. --- Collection of strains --- p.23 / Chapter 2.2. --- Quality control strains --- p.23 / Chapter 2.3. --- Identification of strains to genus level --- p.23 / Chapter 2.4. --- Identification of strains to species level --- p.24 -27 / Chapter 2.4.1. --- By commercially available biochemical method / Chapter 2.4.2. --- By molecular method 226}0ؤ multiplex PCR / Chapter 2.4.2.1. --- Source of primers / Chapter 2.4.2.2. --- Extraction of bacterial DNA / Chapter 2.4.2.3. --- Multiplex PCR / Chapter 2.4.2.4. --- Analysis of PCR products agarose gel electrophoresis / Chapter 2.4.3. --- By conventional biochemical methods --- p.27 / Chapter 2.5. --- Antimicrobial susceptibility testing --- p.27 -30 / Chapter 2.5.1. --- Preparation of antimicrobial agent stock solution / Chapter 2.5.2. --- Preparation of agar medium / Chapter 2.5.3. --- Preparation of inoculum and inoculation of plates / Chapter 2.5.4. --- Incubation and reading of plates / Chapter 2.6. --- Mechanisms of antibiotic resistance --- p.30 -35 / Chapter 2.6.1. --- Amplification of vancomycin resistance genes in enterococci by multiplex PCR / Chapter 2.6.1.1. --- Isolates / Chapter 2.6.1.2. --- Primers / Chapter 2.6.1.3. --- Extraction of bacterial DNA and PCR / Chapter 2.6.2. --- "Detection of gene coding for aminoglycoside-modifying enzyme (AAC6'-APH2"") by PCR" / Chapter 2.6.2.1. --- Isolates / Chapter 2.6.2.2. --- Primers / Chapter 2.6.2.3. --- Extraction of bacterial DNA and PCR / Chapter 2.6.3. --- Detection of β-lactamase in enterococci / Chapter 2.7. --- Molecular typing of enterococci by pulsed-field gel electrophoresis --- p.35 -37 / Chapter 2.7.1. --- Incorporation of chromosomal DNA into agarose plugs / Chapter 2.7.2. --- Digestion of chromosomal DNA in agarose plugs with restriction enzyme SmaI / Chapter 2.7.3. --- Pulsed-field gel electrophoresis / Chapter 2.7.4. --- Cluster analysis / Chapter 2.7.5. --- Data analysis / Chapter 2.8. --- Research plan --- p.37 / Chapter Chapter 3. --- Results / Chapter 3.1. --- Identification of enterococci by API 20 Strep and PCR --- p.39-46 / Chapter 3.2. --- Antimicrobial susceptibilities of enterococci --- p.47 -54 / Chapter 3.3. --- Detection of vancomycin resistance genes in enterococci by PCR --- p.55 -57 / Chapter 3.4. --- "Detection of gene coding for aminoglycoside-modifying enzyme (AAC6'- APH2"") by PCR" --- p.58 -60 / Chapter 3.5. --- Detection of β-lactamase in enterococci --- p.61 / Chapter 3.6. --- Resistance pattern of enterococci --- p.62 -64 / Chapter 3.7. --- Multiresistant enterococci investigated by pulsed-field gel electrophoresis

MSc., Faculty of Science, University of the Witwatersrand, 2011 / The increasing emergence of drug-resistant pathogens poses a major threat to public health. Although influenced by multiple factors, resistance is often associated with mutations in drug target-encoding or associated genes. The potential fitness cost of such resistance mutations is, in turn, a key determinant of the spread of drug-resistant strains. Rifampicin (RIF) is a frontline anti-tuberculosis agent that targets the rpoB-encoded β-subunit of the DNA-dependent RNA polymerase (RNAP). RIF resistance (RIFR) maps primarily to mutations in rpoB that might be expected to affect transcription and so the ability of the organism to cause disease. Accordingly, numerous studies have assessed the impact of RIFR on key fitness indicators in pathogens including Mycobacterium tuberculosis (MTB). In contrast, the specific consequences of RIFR for bacterial physiology remain poorly understood. Notably, previous studies of the effects of RIFR-associated rpoB mutations on mycobacterial physiology have been conducted using strains generated by RIF exposure, without accounting for the potential impact of second-site mutations that may compensate for fitness costs or contribute to drug resistance. In this study, site-directed mutagenesis and allelic exchange were employed to generate a panel of M. smegmatis (MSM) strains containing clinically-relevant RIFR-associated point mutations. Importantly, this methodology enables the introduction of rpoB mutations into defined strain backgrounds in the complete absence of RIF. Using this approach, we constructed “RIF naive” MSM rpoB mutant strains carrying either an S531L or H526Y mutation. The resulting mutants were 100-fold less susceptible to RIF than the isogenic, parental strain. Notably, the inclusion of selected efflux inhibitors in susceptibility assays had little impact on mutant susceptibility to RIF. In contrast, restoration of the wild-type allele returned the observed susceptibility to parental levels, thereby providing strong evidence of the sufficiency of a single rpoB mutation for clinical RIFR in mycobacteria. Competitive growth assays utilizing the S531L mutant and the parental strain exposed a growth defect for the S531L mutant. However, discriminating between wild-type and mutant rpoB strains proved a significant technical challenge, again highlighting the difficulties associated with inferring in vivo fitness from in vitro assays conducted under a limited number of different conditions. In summary, our results suggest the benefit of a deeper exploration of the physiological and fitness implications of RIFR-associated mutations. In addition, in coupling a system which enables an evaluation of the physiological consequences of drug resistance-associated mutations with evolutionary analyses, we provide preliminary evidence of the benefits of a multipronged approach to elucidating the physiological implications of drug resistance in MTB.

Anthony C.T. Liang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 73-76). / Abstract --- p.1 / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Introduction to antibiotics --- p.2 / Chapter 1.2 --- Use of antibiotics in antimicrobial chemotherapy --- p.3 / Chapter 1.3 --- Drug resistance in bacteria --- p.4 / Chapter 1.4 --- Genetic of infections drug resistance --- p.6 / Chapter 1.5 --- Clinical importance of drug resistance --- p.8 / Chapter 1.6 --- Resistance studies on a clinically- isolated Shigella Species --- p.9 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Culture media for bacteria growth --- p.10 / Chapter 2.2 --- Large scale plasmid preparation by CsCl density gradient centrifugation --- p.12 / Chapter 2.3 --- Minipreps of plasmid DNA by the alkaline lysis method --- p.14 / Chapter 2.4 --- Elution of DNA using the Geneclean Kit --- p.16 / Chapter 2.5 --- Transformation of plasmid DNA into E.coli DH5α --- p.17 / Chapter 2.6 --- Antibiotic sensitivity test and screening of resistance colonies --- p.19 / Chapter 2.7 --- Agarose electrophoresis of DNA --- p.20 / Chapter 2.8 --- Restriction and ligation --- p.21 / Chapter 2.9 --- Protocol for studying substrate profiles of aminoglycoside-modifying enzyme (AME) --- p.23 / Chapter 2.10 --- DNA sequencing using the T7 sequencing Kit from Pharmacia --- p.26 / Chapter Chapter 3 --- Antibiotic resistance studies on multiple resistant Shigella spp / Chapter 3.1 --- Introduction --- p.34 / Chapter 3.2 --- Conjugation and transformation experiment --- p.35 / Chapter 3.3 --- Extraction of plasmid DNA from Shigella 2731and transconjugant 14R525(2731) --- p.37 / Chapter 3.4 --- Discussion --- p.39 / Chapter Chapter 4 --- Cloning and characterization of beta-lactamase gene of Shigella2731 / Chapter 4.1 --- Introduction --- p.40 / Chapter 4.2 --- Cloning of the beta-lactam gene in Shigella2731 --- p.42 / Chapter 4.3 --- "Resistance pattern of El, E2, and S1" --- p.43 / Chapter 4.4 --- "Plasmid DNA extraction of El, E2, and S1" --- p.44 / Chapter 4.5 --- Restriction mapping of the plasmid pSFlOO --- p.47 / Chapter 4.6 --- Discussion --- p.51 / Chapter Chapter 5 --- Cloning and characterization of the aminoglycoside resistance gene / Chapter 5.1 --- Introduction --- p.53 / Chapter 5.2 --- Cloning of the aminoglycoside resistance genes --- p.56 / Chapter 5.3 --- Substrate profile studies on aminoglycoside- modifying enzyme (AME) activity on transformant G and S --- p.57 / Chapter 5.4 --- Subcloning of plamsid DNA from transformant S --- p.60 / Chapter 5.5 --- "DNA sequencing of fragments A, B, and C" --- p.65 / Chapter 5.6 --- Discussion --- p.68 / Chapter Chapter 6 --- Conclusion --- p.79 / Chapter Chapter 7 --- Reference --- p.73

by Chin Miu Ling, Nathalie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 130-143). / ABSTRACT --- p.i / ACKNOWLEDGEMENTS --- p.iii / LIST OF TABLES --- p.vii / LIST OF FIGURES --- p.x / INTRODUCTION --- p.1 / Chapter A --- Aminoglycosides --- p.1 / Chapter 1 --- Structure --- p.1 / Chapter 2 --- Classification --- p.1 / Chapter 3 --- Mode of action --- p.2 / Chapter 4 --- Types --- p.9 / Chapter B --- Mechanisms of aminoglycoside-resistance --- p.13 / Chapter C --- Aminoglycoside-modifying enzymes --- p.14 / Chapter 1 --- Classification --- p.19 / Chapter i --- Phosphotransferases --- p.19 / Chapter ii --- Adenylytransferases --- p.21 / Chapter iii --- Acetyltransferases --- p.22 / Chapter 2 --- Genes encoding AMEs --- p.24 / Chapter 3 --- Applications --- p.27 / Chapter D --- Prevalence of aminoglycoside-resistance --- p.28 / Chapter E --- Methods for the determination of aminoglycoside-modifying enzymes --- p.34 / Chapter 1 --- Examination of resistance phenotype --- p.35 / Chapter 2 --- Phosphocellulose paper binding assay --- p.38 / Chapter 3 --- Hybridization with specific gene probes --- p.39 / Chapter 4 --- Antibiotic inactivation --- p.44 / Chapter 5 --- High performance liquid chromatography (HPLC) --- p.44 / Chapter F --- Prevalence of aminoglycoside-modifying enzymes --- p.45 / Chapter G --- Objectives --- p.52 / MATERIALS AND METHODS --- p.53 / Materials --- p.53 / Chapter A --- Bacterial strains --- p.53 / Chapter 1 --- Standard strains --- p.53 / Chapter 2 --- Clinical isolates --- p.53 / Chapter B --- "Antibiotic, media, chemicals and instruments" --- p.55 / Methods --- p.55 / Chapter A --- Orevalence of aminoglycoside-resistance --- p.55 / Chapter B --- Susceptibility testing --- p.55 / Chapter C --- Characterization of aminoglycoside-modifying enzymes (AMEs) --- p.61 / Chapter 1 --- Extraction of enzymes --- p.61 / Chapter 2 --- Substrate profile analysis by the phosphocellulose paper binding assay --- p.62 / Chapter D --- Localization of resistance genes --- p.64 / Chapter 1 --- Genetic study --- p.64 / Chapter 2 --- Molecular studies --- p.67 / Chapter i --- Preparation of crude plasmid extracts --- p.68 / Chapter ii --- Agarose gel electrophoresis --- p.68 / Chapter E --- Plasmid profile analysis --- p.69 / Chapter F --- Plasmid fingerprinting --- p.69 / Chapter 1 --- Preparation of purified plasmid --- p.69 / Chapter 2 --- Restriction endonuclease digestion of plasmid DNA --- p.70 / Plan to achieve objectives --- p.71 / results --- p.73 / Chapter A --- Prevalence of aminoglycoside-resistant Gram-negative bacteria isolated in the Prince of Wales Hospital from 1989 to1992 --- p.73 / Chapter B --- "Susceptibility to 12 aminoglycosides of aminoglycoside-resistant E. coli, K pneumoniae and Ps. aeruginosa" --- p.78 / Chapter C --- "Aminoglycoside-modifying enzymes (AMEs) produced by E. coli, K pneumoniae and Ps. aeruginosa" --- p.88 / Chapter D --- Plasmid profile analysis --- p.93 / Chapter E --- Localization of aminoglycoside-resistance genes --- p.102 / discussion --- p.114 / Chapter A --- Aminoglycoside-resistance --- p.114 / Chapter B --- Mechanisms of aminoglycoside-resistance --- p.118 / Chapter C --- Genetic location of aminoglycoside-resistance and plasmid profiles --- p.122 / Chapter D --- Characterization of AMEs --- p.126 / Chapter E --- Areas for future research --- p.128 / references --- p.130 / appendix --- p.144

The global threat from antibiotic resistant organisms and the effect on human and animal health is now well acknowledged. One measure to control the emergence and dissemination of antibiotic resistant organisms is to monitor bacterial populations and examine the epidemiology of antibiotic resistance genes in naturally occurring bacteria. This study examines antibiotic resistant bacteria of human and animal origin and compares resistance gene transfer in the intestinal tract of animal models to that which occurs in vitro. / The study provides information about tetracycline resistance as it occurs in wild-type bacteria within the environment of the normal flora of an animal. The transfer of tetracycline resistance genes in vitro between E. coli isolates from different origins was found to be occurring at lower levels than that which occurred in vitro. The co-transfer of unselected spectinomycin, streptomycin and sulfadiazine resistance in animal models was also demonstrated. / The study has provided important information regarding the nature and epidemiology of antibiotic resistance in naturally occurring strains of E. coli and enterococci from Australia. This should form part of a larger study, which monitors commensal bacteria and collects data regarding antibiotic resistance in natural populations of bacteria. This evidence can then be used to reduce the levels of antibiotic resistance in the environment and reduce the risk to human and animal health. / Thesis (PhD)--University of South Australia, 2006.

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 6 uppsatser.

Thesis (M.S.)--University of Michigan, 1976.

Thesis (M.Sc. (Microbiology))--Mahidol University, 1978.

Thesis (Ph. D.)--University of Washington, 1996. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 1996. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.