The Beta-lactamases of ampicillin-resistant, Escherichia coli.

• Other Authors: Ling, Kin Wah.
• Format: Others
• Language: English
• Published: Chinese University of Hong Kong 1991
• Subjects: Escherichia coli; Ampicillin Resistance
• Online Access: http://library.cuhk.edu.hk/record=b5887093; http://repository.lib.cuhk.edu.hk/en/item/cuhk-318906

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