Chio Weng Fan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 131-149). / Abstracts in English and Chinese. / Acknowledgments --- p.i / Abstract --- p.ii / Contents --- p.iv / List of tables --- p.ix / List of figures --- p.x / List of abbreviation --- p.xii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Biology of Staphylococci --- p.1 / Chapter 1.1.1 --- Taxonomy --- p.1 / Chapter 1.1.2 --- Characteristics of S. aureus --- p.2 / Chapter 1.2 --- Methicillin-resistant Staphylococcus aureus --- p.1 / Chapter 1.2.1 --- Description of MRSA --- p.7 / Chapter 1.2.2 --- Antibiotic Resistance of MRSA --- p.7 / Chapter 1.2.2.1 --- Resistance to β-lactam Antibiotics --- p.8 / Chapter 1.2.2.1.1 --- Production of β-lactamase --- p.8 / Chapter 1.2.2.1.2 --- Penicillin-binding Protein PBP2a --- p.8 / Chapter 1.2.2.1.3 --- Borderline Methicillin Resistance --- p.11 / Chapter 1.2.2.2 --- Resistance to Antibiotics other than β-lactams --- p.11 / Chapter 1.2.3 --- Epidemiology of MRSA --- p.15 / Chapter 1.2.3.1 --- Clinical Significance of MRSA --- p.15 / Chapter 1.2.3.1.1 --- Evolution of MRSA --- p.16 / Chapter 1.2.3.1.2 --- Epidemiology of MRSA Worldwide --- p.17 / Chapter 1.2.3.2 --- MRSA in Hong Kong --- p.21 / Chapter 1.3 --- Strain Typing for MRSA --- p.26 / Chapter 1.3.1 --- Phenotypic Methods --- p.27 / Chapter 1.3.1.1 --- Antibiotic Susceptibility Test --- p.27 / Chapter 1.3.1.2 --- Bacteriophage Typing --- p.28 / Chapter 1.3.1.3 --- Multilocus Enzyme Electrophoresis --- p.29 / Chapter 1.3.2 --- Genotypic Methods --- p.30 / Chapter 1.3.2.1 --- Analysis of Chromosomal DNA --- p.30 / Chapter 1.3.2.1.1 --- Pulsed-field Gel Electrophoresis (PFGE) --- p.30 / Chapter 1.3.2.1.2 --- Amplified Fragment Length Polymorphism (AFLP) --- p.31 / Chapter 1.3.2.2 --- Analysis of Gene Polymorphism --- p.32 / Chapter 1.3.2.2.1 --- PCR-Restriction Length Polymorphism (PCR-RFLP) --- p.32 / Chapter 1.3.2.2.2 --- Random Amplification of Polymorphic DNA (RAPD) --- p.33 / Chapter 1.3.2.2.3 --- Nucleotide Sequence Typing --- p.33 / Chapter 1.3.2.2.3.1 --- A Typing --- p.33 / Chapter 1.3.2.2.3.2 --- Multilocus Sequence Typing --- p.33 / Chapter 1.3.2.3 --- Hybridization by Southern Blotting --- p.34 / Chapter 1.3.2.3.1 --- MecA/Tn544 Probe Typing --- p.34 / Chapter 1.3.2.3.2 --- Binary Typing --- p.34 / Chapter 1.3.2.4 --- Analysis of Plasmid DNA --- p.35 / Chapter 1.4 --- Objectives of the Project --- p.37 / Chapter Chapter 2 --- Materials & Methods --- p.38 / Chapter 2.1 --- Bacterial Isolates & Culture Conditions --- p.38 / Chapter 2.1.1 --- Study Period & Sample Sources --- p.38 / Chapter 2.1.2 --- Selection of Bacterial Isolates --- p.38 / Chapter 2.1.3 --- Reference Strains --- p.38 / Chapter 2.1.4 --- Culture & Storage Conditions --- p.39 / Chapter 2.1.5 --- Identification of MRSA --- p.39 / Chapter 2.2 --- Antibiotic Susceptibility Testing --- p.40 / Chapter 2.3 --- PCR for MecA Gene --- p.43 / Chapter 2.3.1 --- DNA Preparation and Primer Design for mecA Gene --- p.43 / Chapter 2.3.2 --- PCR Amplification of mecA Gene --- p.45 / Chapter 2.3.2.1 --- Master Mix Preparation --- p.45 / Chapter 2.3.2.2 --- Polymerase Chain Reaction --- p.45 / Chapter 2.3.2.3 --- Analysis of PCR Products by Agarose Gel Electrophoresis --- p.45 / Chapter 2.3.2.4 --- Precautions to Avoid Cross-contamination --- p.46 / Chapter 2.4 --- Pulsed-field Gel Electrophoresis (PFGE) --- p.47 / Chapter 2.4.1 --- PFGE Protocol --- p.47 / Chapter 2.4.1.1 --- DNA Preparation for PFGE --- p.47 / Chapter 2.4.1.2 --- Restriction Enzyme Digestion --- p.48 / Chapter 2.4.1.3 --- Pulsed-field Gel Electrophoresis --- p.48 / Chapter 2.4.1.4 --- Standards & Markers for PFGE --- p.48 / Chapter 2.4.2 --- Optimization of PFGE --- p.49 / Chapter 2.4.3 --- Computer Analysis of PFGE Patterns --- p.49 / Chapter 2.5 --- Amplified Fragment Length Polymorphism (AFLP) --- p.52 / Chapter 2.5.1 --- Amplified Fragment Length Polymorphism Protocol --- p.52 / Chapter 2.5.1.1 --- DNA Preparation --- p.52 / Chapter 2.5.1.2 --- Enzyme Digestion & Ligation --- p.53 / Chapter 2.5.1.3 --- PCR for AFLP --- p.53 / Chapter 2.5.1.4 --- Gel Electrophoresis for AFLP --- p.54 / Chapter 2.5.1.5 --- Standards & Markers for AFLP --- p.55 / Chapter 2.5.2 --- Computer Analysis for AFLP Patterns --- p.55 / Chapter 2.6 --- Phage Typing --- p.58 / Chapter 2.6.1 --- Phages & Propagating Strains --- p.58 / Chapter 2.6.2 --- Phage Typing Protocol --- p.58 / Chapter 2.6.3 --- Data Analysis and Results Interpretation for Phage Tying --- p.62 / Chapter 2.7 --- Other Typing Methods --- p.63 / Chapter 2.7.1 --- PCR Restriction Fragment Length Polymorphism for the coa gene --- p.63 / Chapter 2.7.1.1 --- Primer Design --- p.63 / Chapter 2.7.1.2 --- DNA Preparation --- p.63 / Chapter 2.7.1.3 --- Optimization of PCR --- p.63 / Chapter 2.7.1.3.1 --- PCR Amplification --- p.64 / Chapter 2.7.1.3.2 --- Restriction Enzyme Digestion --- p.64 / Chapter 2.7.2 --- Multilocus Sequence Typing --- p.64 / Chapter 2.7.2.1 --- Preparation of Chromosomal DNA --- p.65 / Chapter 2.7.2.2 --- PCR for MLST Gene --- p.65 / Chapter 2.7.2.3 --- Purification of DNA for Sequencing --- p.67 / Chapter 2.7.2.4 --- PCR for Sequencing --- p.67 / Chapter 2.7.2.5 --- Sequencing by Automatic DNA Analyzer & Data Analysis --- p.68 / Chapter Chapter 3 --- Results --- p.69 / Chapter 3.1 --- Bacterial Isolates --- p.69 / Chapter 3.2 --- Antibiotic Susceptibility Testing --- p.72 / Chapter 3.3 --- PCR for MecA Gene --- p.78 / Chapter 3.4 --- Pulsed-field Gel Electrophoresis --- p.80 / Chapter 3.4.1 --- Optimization of PFGE --- p.80 / Chapter 3.4.2 --- Analysis of PFGE Profiles --- p.83 / Chapter 3.5 --- Amplified Fragment Length Polymorphism --- p.95 / Chapter 3.6 --- Phage Typing --- p.102 / Chapter 3.7 --- Other Typing Methods --- p.109 / Chapter 3.7.1 --- PCR Restriction Fragment Length Polymorphism for the Coa Gene --- p.109 / Chapter 3.7.1.1 --- Optimization of PCR conditions for the Coa Gene --- p.109 / Chapter 3.7.1.2 --- Analysis of MRSA by PCR-RFLP of the coa Gene --- p.109 / Chapter 3.7.2 --- Multilocus Sequence Typing --- p.115 / Chapter Chapter 4 --- Discussion --- p.116 / Chapter 4.1 --- Evaluation of Typing methods for MRSA --- p.116 / Chapter 4.1.1 --- Antibiotic Susceptibility Testing --- p.116 / Chapter 4.1.2 --- Phage Typing --- p.117 / Chapter 4.1.3 --- Pulsed-field Gel Electrophoresis --- p.118 / Chapter 4.1.4 --- Amplified Fragment Length Polymorphism --- p.119 / Chapter 4.1.5 --- PCR-Restriction Fragment Length Polymorphism for the Coa Gene --- p.120 / Chapter 4.1.6 --- Multilocus Sequence Typing --- p.121 / Chapter 4.1.7 --- Conclusion for Method Evaluation --- p.122 / Chapter 4.2 --- "Analysis of Results of Antibiotic Susceptibility Test, Phage Typing, PFGE, AFLP, PCR-RFLP (Coa) & MLST" --- p.124 / Chapter 4.2.1 --- Correlation between Methods --- p.124 / Chapter 4.2.2 --- Clinical Correlation --- p.125 / Chapter 4.2.3 --- Correlation between MRSA Isolates at PWH and Reference Strains --- p.128 / Chapter 4.3 --- Future Research --- p.129 / Chapter 4.4 --- Conclusion --- p.130 / Reference List --- p.131 / Appendix I Reagent & Material Lists for Methods --- p.150 / Appendix II Buffers & Media --- p.156 / Appendix III Coa Gene Sequences of Isolates from PWH and Reference Strains --- p.158 / Appendix IV Unique antibiotic resistance profiles --- p.165 / "Appendix V Details of MRSA iolates selected for AFLP, Phage typing and MLST" --- p.166