by Ling Kin Wah, Thomas. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1991. / Includes bibliographical references (leaves 103-117). / ABSTRACT --- p.i / ACKNOWLEDGMENTS --- p.v / LIST OF ABBREVIATIONS --- p.vi / TABLE OF CONTENTS --- p.viii / LIST OF TABLES --- p.xv / LIST OF FIGURES --- p.xix / INTRODUCTION --- p.1 / LITERATURE REVIEW --- p.2 / Chapter 1. --- Structure of the bacterial cell envelope --- p.2 / Chapter 2 . --- The β-lactam antibiotics --- p.4 / Chapter 3. --- Mode of action of β-lactam antibiotics --- p.5 / Chapter 4. --- Penicillin-binding proteins (PBPs) --- p.6 / Chapter 5. --- Mechanisms of bacterial resistance to β-lactam antibiotics --- p.7 / Chapter 5.1 --- Non-enzymatic resistance --- p.7 / Chapter 5.1.1 --- Alteration in cell permeability --- p.8 / Chapter 5.1.2 --- Alteration of the target site --- p.9 / Chapter 5.1.3 --- Tolerance and persistence --- p.9 / Chapter 5.2 --- Enzyme-mediated resistance --- p.12 / Chapter 6. --- Transfer of resistance --- p.13 / Chapter 7. --- β-lactamases --- p.16 / Chapter 7.1 --- History --- p.16 / Chapter 7.2 --- Classification of β-lactamases --- p.17 / Chapter 7.2.1 --- Richmond and Sykes scheme --- p.17 / Chapter 7.2.2 --- Matthew scheme --- p.18 / Chapter 7.2.3 --- Bush scheme --- p.19 / Chapter 7.3 --- β-lactamases of Gram-negative bacteria --- p.19 / Chapter 7.3.1 --- Chromosomally-mediated β-lactamases --- p.19 / Chapter 7.3.2 --- Plasmid-mediated β-lactamases --- p.20 / Chapter 7.4 --- β-lactamase inhibitors --- p.25 / Chapter 7.5 --- Regulation of β-lactamase production --- p.28 / Chapter 7.5.1 --- β-lactamase induction --- p.28 / Chapter 7.5.2 --- Mutation to constitutive enzyme production --- p.29 / Chapter 7.5.3 --- β-lactam induced β-lactamase production --- p.30 / Chapter 8. --- Emergence of resistance due to production of β-lactamases --- p.31 / Chapter 8.1 --- Resistance in staphylococci --- p.32 / Chapter 8.2 --- Resistance in haemophili and gonococci --- p.33 / Chapter 8.3 --- Resistance in Enterobacteriaceae (non E. coli) --- p.34 / Chapter 8.4 --- Distribution of β-lactamases in E. coli --- p.35 / MATERIALS AND METHODS / Chapter 1. --- Bacterial strains --- p.38 / Chapter 1.1 --- Standard organisms --- p.38 / Chapter 1.2 --- Clinical isolates --- p.38 / Chapter 2. --- Antibiotics --- p.39 / Chapter 3. --- "Media, chemicals and culture conditions" --- p.39 / Chapter 4. --- Bacterial identification and viable bacterial count --- p.39 / Chapter 5. --- Antibiotic sensitivity testing --- p.40 / Chapter 5.1 --- Disk diffusion --- p.40 / Chapter 5.2 --- Determination of minimal inhibitory concentration (MIC) --- p.40 / Chapter 6. --- Plasmid analysis --- p.41 / Chapter 6.1 --- Transfer of drug resistance plasmids --- p.41 / Chapter 6. 2 --- Molecular studies of plasmids --- p.42 / Chapter 6.2.1 --- Extraction of plasmid DNA --- p.43 / Chapter 6.2.2 --- Agarose gel electrophoresis --- p.43 / Chapter 6.2.3 --- Molecular size determination --- p.44 / Chapter 7 . --- DNA hybridization --- p.44 / Chapter 7.1 --- DNA blotting --- p.44 / Chapter 7.1.1 --- Colony blotting --- p.45 / Chapter 7.1.2 --- Southern blotting --- p.45 / Chapter 7.2 --- Labeling of oligonucleotide probe --- p.46 / Chapter 7.3 --- Hybridization --- p.47 / Chapter 7.4 --- Autoradiography --- p.47 / Chapter 7.5 --- Re-use of blots --- p.48 / Chapter 8. --- Detection and screening for classification of β-lactamases --- p.48 / Chapter 8.1 --- Detection of β-lactamases --- p.48 / Chapter 8.1.1 --- Acidimetric --- p.48 / Chapter 8.1.2 --- Chromogenic substrate --- p.49 / Chapter 8.1.2.1 --- Whole cell --- p.49 / Chapter 8.1.2.2 --- Cell extract and filtrate --- p.49 / Chapter 8.2 --- Screening for classification of β-lactamases --- p.49 / Chapter 9. --- "Preparation, purification, qualitative and quantitative analyses of the β-lactamase from transconjugants TU117, TB117 and the recipient K12" --- p.51 / Chapter 9.1 --- Large scale preparation of enzyme --- p.51 / Chapter 9.2 --- Gel filtration --- p.52 / Chapter 9.3 --- Preparative isoelectric focusing (PIEF) --- p.53 / Chapter 9.4 --- Protein determination --- p.55 / Chapter 9.5 --- Qualitative analyses and characterization of β-lactamases --- p.56 / Chapter 9.5.1 --- Analytical isoelectric focusing --- p.56 / Chapter 9.5.1.1 --- Semi-quantitative determination of β-lactamases --- p.56 / Chapter 9.5.1.2 --- Polyacrylamide gel preparation --- p.57 / Chapter 9.5.1.3 --- Isoelectric focusing --- p.58 / Chapter 9.5.1.4 --- pH measurement --- p.58 / Chapter 9.5.1.5 --- Gel development and recording --- p.59 / Chapter 9.5.1.5.1 --- Nitrocefin staining --- p.59 / Chapter 9.5.1.5.2 --- Silver staining --- p.59 / Chapter 9.5.1.6 --- Isoelectric point (pI) determination --- p.60 / Chapter 9.5.2 --- Spectrophotometric assay of β-lactam substrates --- p.60 / Chapter 9.5.2.1 --- Absorption spectra of β-lactam antibiotics --- p.60 / Chapter 9.5.2.2 --- The molar extinction coefficient of β-lactam substrates --- p.60 / Chapter 9.5.2.3 --- Measurement of β-lactamase hydrolytic activities --- p.61 / Chapter 9.5.2.4 --- Determination of enzyme kinetics --- p.61 / Chapter 9.5.3 --- Molecular weight determination of proteins --- p.62 / Chapter 9.5.3.1 --- SDS-polyacrylamide gel preparation --- p.62 / Chapter 9.5.3.1.1 --- Resolving gel --- p.62 / Chapter 9.5.3.1.2 --- Stacking gel --- p.63 / Chapter 9.5.3.2 --- Electrophoresis --- p.63 / Chapter 9.5.3.3 --- Staining and recording --- p.64 / Chapter 9.5.3.4 --- Molecular weight determination --- p.64 / RESULTS / Chapter 1. --- Collection of organisms --- p.65 / Chapter 2 . --- Identification of organisms --- p.65 / Chapter 3. --- Antibiotic sensitivity testing --- p.66 / Chapter 4. --- Genetic and molecular studies of ampicillin- resistant plasmids --- p.68 / Chapter 4.1 --- Transfer of ampicillin-resistant factor --- p.68 / Chapter 4.1.1 --- E. coli K12 14R525 as recipient --- p.68 / Chapter 4.1.2 --- other Enterobacteriaceae --- p.68 / Chapter 4.2 --- Plasmid studies of E. coli --- p.69 / Chapter 5. --- Detection and identification of β-lactamases --- p.69 / Chapter 5.1 --- Analytical IEF --- p.70 / Chapter 5.2 --- DNA hybridization --- p.70 / Chapter 5.2.1 --- Colony blot hybridization --- p.70 / Chapter 5.2.2 --- Southern blot hybridization --- p.71 / Chapter 6. --- Characterization of TEM-1 producing E. coli --- p.71 / Chapter 6.1 --- Susceptibility testing --- p.71 / Chapter 6.2 --- Enzyme kinetic study --- p.72 / Chapter 6.2.1 --- Absorption spectra and molar extinction coefficient of β-lactam antibiotics --- p.72 / Chapter 6.2.2 --- Comparison of the substrate profiles --- p.73 / Chapter 6.3 --- Correlation of MICs to β-lactamase activities --- p.73 / Chapter 7. --- "Isolation, quantitation and characterization of β-lactamases isolated from three E. coli strains" --- p.74 / Chapter 7.1 --- Preparation of β-lactamases --- p.75 / Chapter 7. 2 --- Purification of β-lactamases --- p.76 / Chapter 7.2.1 --- Gel-filtration chromatography --- p.76 / Chapter 7.2.2 --- Preparative isoelectric focusing --- p.77 / Chapter 7.3 --- Characterization of the purified β-lactamases --- p.78 / Chapter 7.3.1 --- Isoelectric point --- p.78 / Chapter 7.3.2 --- Molecular weight assessment --- p.79 / Chapter 7.3.3 --- Enzyme kinetic study --- p.79 / DISCUSSION / Chapter 1. --- Epidemiology of ampici11in (or amoxycillin)- resistant E. coli --- p.81 / Chapter 2. --- Distribution of β-lactamases in ampicillin- resistant E. coli --- p.84 / Chapter 3. --- Correlation between level of resistance and β-lactamase activity --- p.86 / Chapter 4. --- Plasmid-mediated TEM-1 β-lactamase --- p.89 / Chapter 4.1 --- Transfer of resistance --- p.89 / Chapter 4 .2 --- Identification of β-lactamases by DNA hybridization --- p.91 / Chapter 5. --- Mechanism of high-level resistance --- p.93 / Chapter 5.1 --- Selection of resistant strains --- p.93 / Chapter 5.2 --- β-lactamases preparation and purification --- p.95 / Chapter 5.3 --- Hyperproduction of β-lactamase --- p.97 / SUMMARY AND CONCLUSIONS --- p.102 / REFERENCES --- p.103 / APPENDICES / Chapter 1. --- TABLES --- p.118 / Chapter 2. --- FIGURES --- p.153

Wong Yin Wai. / Thesis submitted in: June 2002. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 82-93). / Abstracts in English and Chinese. / Acknowledgement --- p.ii / Abstract --- p.iii / 摘要 --- p.v / Table of Content --- p.vi / List of Tables --- p.ix / List of Figures --- p.x / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Taxonomy of salmonellae --- p.1 / Chapter 1.2 --- Clinical significance --- p.2 / Chapter 1.3 --- Treatment of Salmonella infections --- p.4 / Chapter 1.4 --- Global and local prevalence of Salmonella --- p.5 / Chapter 1.5 --- Antimicrobial Susceptibilities --- p.8 / Chapter 1.5.1 --- Salmonella Typhimurium --- p.8 / Chapter 1.5.2 --- Other salmonellae --- p.9 / Chapter 1.5.3 --- Emergence of quinolone-resistant salmonellae --- p.9 / Chapter 1.6 --- Mechanisms of β-lactam resistance --- p.10 / Chapter 1.6.1 --- Enzymatic deactivation of β-lactam antibiotics --- p.10 / Chapter 1.6.2 --- Modifications of normal PBPs --- p.11 / Chapter 1.6.3 --- Alternative routes of peptidoglycan synthesis --- p.12 / Chapter 1.6.4 --- Impermeability and active efflux system --- p.12 / Chapter 1.7 --- Classification and nomenclature of β-lactamases --- p.13 / Chapter 1.7.1 --- Functional classification --- p.13 / Chapter 1.7.2 --- Molecular classification --- p.15 / Chapter 1.7.3 --- Nomenclature of β-lactamases --- p.16 / Chapter 1.8 --- β-Lactamases in salmonellae --- p.17 / Chapter 1.8.1 --- Salmonella Typhimurium --- p.17 / Chapter 1.8.2 --- Other salmonellae --- p.18 / Chapter 1.9 --- Methods for the characterization of β-lactamases --- p.18 / Chapter 1.9.1 --- Isoelectric focusing (IEF) --- p.19 / Chapter 1.9.2 --- β-Lactamase activity assays --- p.20 / Chapter 1.9.3 --- Hybridization with DNA probes --- p.20 / Chapter 1.9.4 --- Amplification of β-lactamase genes by polymerase chain reaction (PCR) --- p.21 / Chapter 1.9.5 --- Polymerase chain reaction - Single strand conformational polymorphism (PCR-SSCP) analysis --- p.23 / Chapter 1.9.6 --- Gene sequencing --- p.23 / Chapter 1.10 --- Objectives --- p.25 / Chapter 2 --- Materials and Methods --- p.26 / Chapter 2.1 --- Bacterial Strains --- p.26 / Chapter 2.1.1 --- Identification of salmonellae --- p.26 / Chapter 2.1.2 --- Antibiotics and chemicals used --- p.26 / Chapter 2.1.3 --- Antimicrobial susceptibility testing --- p.28 / Chapter 2.2 --- Localization of β-lactamase genes --- p.30 / Chapter 2.2.1 --- Transferability study --- p.30 / Chapter 2.3 --- Characterization of β-lactamases --- p.31 / Chapter 2.3.1 --- Extraction of crude β-lactamases --- p.31 / Chapter 2.3.2 --- Isoelectric focusing (IEF) --- p.31 / Chapter 2.4 --- Molecular characterization of β-lactamase genes --- p.33 / Chapter 2.4.1 --- Detection of TEM-type β-lactamase genes using polymerase chain reaction (PCR) --- p.33 / Chapter 2.4.2 --- Detection of OXA-type β-lactamase gene using PCR --- p.36 / Chapter 2.4.3 --- Detection of TEM mutations by polymerase chain reaction 一 single strand conformational polymorphism (PCR-SSCP) analysis --- p.37 / Chapter 2.4.4 --- Detection of OXA mutations by PCR-SSCP analysis --- p.38 / Chapter 2.4.5 --- Sequencing of β-lactamase genes --- p.39 / Chapter 2.4.5.1 --- Preparation of sequencing template --- p.39 / Chapter 2.4.5.2 --- Sequencing reaction --- p.39 / Chapter 2.4.5.3 --- Preparation of sequencing gel --- p.40 / Chapter 2.4.5.4 --- Silver staining of the sequencing gel --- p.41 / Chapter 2.5 --- Relatedness of ampicillin-resistant S. Typhimurium --- p.42 / Chapter 2.5.1 --- Pulsed field gel electrophoresis (PFGE) --- p.42 / Chapter 2.5.2 --- Cluster analysis --- p.44 / Chapter 3 --- Results --- p.46 / Chapter 3.1 --- Bacterial Strains --- p.46 / Chapter 3.1.1 --- Antimicrobial susceptibilities --- p.46 / Chapter 3.2 --- Characterization of β-lactamases by isoelectric focusing --- p.52 / Chapter 3.3 --- Characterization of β-lactamase genes --- p.53 / Chapter 3.3.1 --- Transferability of β-lactamase genes --- p.53 / Chapter 3.3.2 --- Detection of OXA-type β-lactamase gene by polymerase chain reaction (PCR) --- p.53 / Chapter 3.3.3 --- Detection of OXA-type mutations by polymerase chain reaction- single strand conformational polymorphism (PCR-SSCP) analysis --- p.56 / Chapter 3.3.4 --- Detection of TEM-type β-lactamase gene by PCR --- p.56 / Chapter 3.3.5 --- Detection of TEM-type mutations by PCR-SSCP analysis --- p.56 / Chapter 3.3.6 --- Sequencing of β-lactamase genes --- p.61 / Chapter 3.3.7 --- Pulsed-field gel electrophoresis --- p.64 / Chapter 4 --- Discussion --- p.67 / Chapter 4.1 --- Antimicrobial susceptibilities of S. Typhimurium in Hong Kong --- p.67 / Chapter 4.2 --- Transferability of resistance --- p.69 / Chapter 4.3 --- β-Lactamases of S. Typhimurium --- p.70 / Chapter 4.4 --- DNA sequence of β-lactamase genes --- p.72 / Chapter 4.5 --- Relatedness of ampicillin-resistant S. Typhimurium --- p.73 / Chapter 4.6 --- Methods for the characterization of β-lactamases --- p.75 / Chapter 4.7 --- Significance of this study --- p.78 / Chapter 4.8 --- Conclusions --- p.79 / Chapter 4.9 --- Further studies --- p.80 / References --- p.83

Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 5 uppsatser.

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