Molecular typing and characterisation of MRSA in Hong Kong.

Hung Ming Wai.Thesis (M.Phil.)--Chinese University of Hong Kong, 2004.Includes bibliographical references (leaves 146-169).Abstracts in English and Chinese.Abstract --- p.IAbstract (Chinese version) --- p.IIIAcknowledgments --- p.VContents --- p.VIList of Tables --- p.XIIList of Figures --- p.XIVList of Abbreviations & Symbols --- p.XVIIChapter Chapter 1 --- Introduction --- p.1Chapter 1.1 --- Methicillin-resistant Staphylococcus aureus --- p.1Chapter 1.1.1 --- Definition and Identification --- p.1Chapter 1.1.2 --- Antibiotic resistance --- p.2Chapter 1.1.2.1 --- Methicillin/oxacillin resistance --- p.2Chapter 1.1.2.2 --- Other antibiotic resistance --- p.2Chapter 1.2 --- Evolution of MRSA --- p.3Chapter 1.2.1 --- Staphylococcal Chromosome Cassette mec (SCCmec) --- p.3Chapter 1.2.2 --- The origin of mec element --- p.7Chapter 1.2.3 --- The acquisition of SCCmec: from MSSA to MRSA --- p.7Chapter 1.2.4 --- Five pandemic clones --- p.8Chapter 1.3 --- Epidemiology of MRSA --- p.10Chapter 1.3.1 --- MRSA Infections --- p.12Chapter 1.3.2 --- Community-acquired MRSA --- p.13Chapter 1.4 --- Staphylococcal Gene Regulators --- p.14Chapter 1.4.1 --- Accessory gene regulator (agr) --- p.14Chapter 1.4.1.1 --- Structure and functions of accessory gene regulator (agr) --- p.15Chapter 1.4.1.2 --- Autoinducing Peptide (AIP) --- p.16Chapter 1.4.1.3 --- Implication of agr I-IV --- p.18Chapter 1.4.2 --- Staphylococcal accessory regulator (sar) --- p.19Chapter 1.4.2.1 --- Structure and function of sar --- p.19Chapter 1.4.3 --- Difference in Functions of sar and agr --- p.20Chapter 1.4.4 --- "Other sar A homologues, SarS, R，T, U, and other Gene Regulators" --- p.22Chapter 1.5 --- Adhesin and toxin genes --- p.25Chapter 1.5.1 --- Adhesins --- p.25Chapter 1.5.2 --- Exotoxins --- p.26Chapter 1.6 --- Typing Methods --- p.29Chapter 1.6.1 --- Phenotypic typing method --- p.31Chapter 1.6.1.1 --- Antibiotic-susceptibility Test --- p.31Chapter 1.6.1.2 --- Bacteriophage Typing --- p.31Chapter 1.6.1.3 --- Serotyping --- p.32Chapter 1.6.1.4 --- Electrophoretic Protein Typing and Immunoblotting --- p.33Chapter 1.6.1.5 --- Multilocus Enzyme Electrophoresis --- p.34Chapter 1.6.1.6 --- Limitations of phenotyping --- p.34Chapter 1.6.2 --- Genotyping methods --- p.35Chapter 1.6.2.1 --- PCR Assays --- p.35Chapter 1.6.2.2 --- Pulsed-Field Gel Electrophoresis (PFGE) --- p.36Chapter 1.6.2.3 --- Multi-Locus Sequence Typing (MLST) --- p.37Chapter 1.6.2.4 --- Amplified Fragment Length Polymorphism (AFLP) --- p.38Chapter 1.6.2.5 --- Ribotyping --- p.38Chapter 1.6.2.6 --- Plasmid Typing --- p.39Chapter 1.6.2.7 --- Restriction Fragment Length Polymorphism --- p.39Chapter 1.6.2.8 --- Nucleic Acid Hybridization --- p.40Chapter 1.7 --- Objectives of the project --- p.41Chapter Chapter 2 --- Materials and Methods --- p.42Chapter 2.1 --- Bacterial Isolates & Culture conditions --- p.42Chapter 2.1.1 --- Bacterial Isolates --- p.43Chapter 2.1.2 --- Reference Strains --- p.43Chapter 2.1.3 --- Identification of MRSA --- p.44Chapter 2.2 --- Antibiotic Susceptibility Test --- p.45Chapter 2.3 --- Pulsed-Field Gel Electrophoresis (PFGE) --- p.47Chapter 2.3.1 --- DNA preparation --- p.47Chapter 2.3.2 --- Restriction digestion --- p.48Chapter 2.3.3 --- PFGE --- p.48Chapter 2.3.4 --- Analysis of band patterns --- p.48Chapter 2.4 --- Phage Typing --- p.50Chapter 2.4.1 --- Source of Phages & Propagating Strains --- p.50Chapter 2.4.2 --- Procedures of phage typing --- p.50Chapter 2.4.3 --- Routine check of the lytic strength of phages --- p.51Chapter 2.4.4 --- Evaluation of the reproducibility of phage typing --- p.51Chapter 2.5 --- Detection of SCCmec and mec Genes by Polymerase Chain Reaction (PCR) --- p.55Chapter 2.5.1 --- DNA preparation for PCR --- p.55Chapter 2.5.2 --- Master mix preparation and PCR conditions --- p.55Chapter 2.5.3 --- Electrophoresis of DNA Amplicors (PCR products) --- p.55Chapter 2.5.4 --- Detection of mecA gene by PCR --- p.56Chapter 2.5.5 --- Detection of SCCmec I-IV by PCR --- p.58Chapter 2.6 --- Multiplex Polymerase Chain Reaction --- p.60Chapter 2.6.1 --- Detection of agr I-IV --- p.60Chapter 2.6.2 --- Detection of Pyrogenic Toxin Genes --- p.62Chapter 2.6.2.1 --- Primer Mix Preparation for Multiplex PCR --- p.66Chapter 2.6.2.1.1 --- Sea-see Multiplex PCR --- p.66Chapter 2.6.2.1.2 --- Seg-sej Multiplex PCR --- p.66Chapter 2.6.2.1.3 --- "Eta, etb, tsst-1 for multiplex PCR" --- p.66Chapter 2.6.2.2 --- Multiplex Master Mix Preparation --- p.67Chapter 2.6.2.3 --- "PCR controls for pyrogenic toxins (sea-sej, eta-b, tsst-1)" --- p.67Chapter 2.6.2.4 --- Sequencing of PCR products --- p.68Chapter 2.6.3 --- PCR for adhesin and toxin genes --- p.69Chapter 2.6.3.1 --- "Multiplex Master mix preparation for lukE, fib, cna, icaD" --- p.71Chapter 2.6.3.2 --- "Multiplex Master mix preparation for fnbA,fnbB, hla, hlb, icaA" --- p.71Chapter 2.6.3.3 --- PCR controls for adhesin and toxin genes --- p.71Chapter 2.6.3.4 --- Size of PCR products resolved by gel electrophoresis --- p.72Chapter 2.6.3.5 --- Sequencing of PCR products --- p.72Chapter 2.7 --- Multi-locus sequence typing --- p.73Chapter 2.7.1 --- Bacterial Isolates --- p.73Chapter 2.7.2 --- Primer design --- p.73Chapter 2.7.3 --- PCR Master mix preparation --- p.73Chapter 2.7.4 --- PCR for MLST gene --- p.75Chapter 2.7.5 --- Sequencing of PCR products --- p.75Chapter 2.7.6 --- Data Analysis --- p.75Chapter 2.8 --- Sequence typing of Coa gene --- p.76Chapter 2.8.1 --- Bacterial Isolates --- p.76Chapter 2.8.2 --- Amplification of Coa Gene by PCR --- p.76Chapter 2.8.3 --- coa sequencing --- p.77Chapter 2.8.4 --- Sequence Analysis --- p.77Chapter Chapter 3 --- Results --- p.79Chapter 3.1 --- Bacterial Isolates --- p.79Chapter 3.2 --- PCR for mecA gene --- p.80Chapter 3.3 --- Antibiotics Susceptibility Test --- p.81Chapter 3.3.1 --- Antibiotic Resistance Profiles of MRSA --- p.82Chapter 3.4 --- Pulsed-field Gel Electrophoresis --- p.85Chapter 3.4.1 --- Optimization of PFGE and Inter-gel Variation --- p.85Chapter 3.4.2 --- Analysis of PFGE profiles --- p.87Chapter 3.4.3 --- PFGE patterns and antibiotic profiles --- p.93Chapter 3.5 --- Phage Typing --- p.96Chapter 3.6 --- SCCmec Typing --- p.98Chapter 3.6.1 --- PCR Optimization --- p.98Chapter 3.6.2 --- PCR and distribution of SCCmec types --- p.101Chapter 3.6.3 --- PFGE patterns and SCCmec types --- p.103Chapter 3.7 --- agr Typing --- p.104Chapter 3.7.1 --- Optimization of PCR --- p.104Chapter 3.7.2 --- PCR and Distribution of agr groups --- p.106Chapter 3.7.3 --- PFGE patterns and agr groups --- p.108Chapter 3.7.4 --- SCCmec types and agr groups --- p.108Chapter 3.8 --- "PCR for adhesin, pyrogenie and other toxin genes" --- p.110Chapter 3.8.1 --- Optimization of PCR --- p.110Chapter 3.8.2 --- "Distribution of adhesin, pyrogenic and other toxin genes" --- p.120Chapter 3.9 --- Multi-Locus Sequence Typing --- p.124Chapter 3.10 --- coa Sequence Typing --- p.126Chapter Chapter 4 --- Discussion --- p.128Chapter 4.1 --- Evaluation of the Typing methods for MRSA --- p.128Chapter 4.1.1 --- Antibiotic Susceptibility Test --- p.128Chapter 4.1.2 --- PFGE --- p.128Chapter 4.1.3 --- Phage Typing --- p.130Chapter 4.1.4 --- "PFGE, SCCmec typing and agr typing" --- p.131Chapter 4.1.5 --- MLST --- p.132Chapter 4.1.6 --- Sequencing typing of coa gene --- p.133Chapter 4.2 --- Characteristics of MRSA --- p.134Chapter 4.2.1 --- PCR for adhesin and toxin genes --- p.134Chapter 4.3 --- Key Characteristics of Hong Kong isolates --- p.137Chapter 4.4 --- Conclusion --- p.144Chapter 4 5 --- Future Research --- p.145Reference List --- p.146Appendix I - Materials/Reagents for Methods --- p.170Appendix II - Reagents Formula --- p.178Appendix III - coa sequence alignment --- p.180Appendix IV - Typing patterns and Characteristics of reference isolates --- p.193Appendix V - Miscellaneous --- p.195Appendix VI - Typing Patterns and Characteristics of PFGE type isolates --- p.20