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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.I / Abstract (Chinese version) --- p.III / Acknowledgments --- p.V / Contents --- p.VI / List of Tables --- p.XII / List of Figures --- p.XIV / List of Abbreviations & Symbols --- p.XVII / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Methicillin-resistant Staphylococcus aureus --- p.1 / Chapter 1.1.1 --- Definition and Identification --- p.1 / Chapter 1.1.2 --- Antibiotic resistance --- p.2 / Chapter 1.1.2.1 --- Methicillin/oxacillin resistance --- p.2 / Chapter 1.1.2.2 --- Other antibiotic resistance --- p.2 / Chapter 1.2 --- Evolution of MRSA --- p.3 / Chapter 1.2.1 --- Staphylococcal Chromosome Cassette mec (SCCmec) --- p.3 / Chapter 1.2.2 --- The origin of mec element --- p.7 / Chapter 1.2.3 --- The acquisition of SCCmec: from MSSA to MRSA --- p.7 / Chapter 1.2.4 --- Five pandemic clones --- p.8 / Chapter 1.3 --- Epidemiology of MRSA --- p.10 / Chapter 1.3.1 --- MRSA Infections --- p.12 / Chapter 1.3.2 --- Community-acquired MRSA --- p.13 / Chapter 1.4 --- Staphylococcal Gene Regulators --- p.14 / Chapter 1.4.1 --- Accessory gene regulator (agr) --- p.14 / Chapter 1.4.1.1 --- Structure and functions of accessory gene regulator (agr) --- p.15 / Chapter 1.4.1.2 --- Autoinducing Peptide (AIP) --- p.16 / Chapter 1.4.1.3 --- Implication of agr I-IV --- p.18 / Chapter 1.4.2 --- Staphylococcal accessory regulator (sar) --- p.19 / Chapter 1.4.2.1 --- Structure and function of sar --- p.19 / Chapter 1.4.3 --- Difference in Functions of sar and agr --- p.20 / Chapter 1.4.4 --- "Other sar A homologues, SarS, R,T, U, and other Gene Regulators" --- p.22 / Chapter 1.5 --- Adhesin and toxin genes --- p.25 / Chapter 1.5.1 --- Adhesins --- p.25 / Chapter 1.5.2 --- Exotoxins --- p.26 / Chapter 1.6 --- Typing Methods --- p.29 / Chapter 1.6.1 --- Phenotypic typing method --- p.31 / Chapter 1.6.1.1 --- Antibiotic-susceptibility Test --- p.31 / Chapter 1.6.1.2 --- Bacteriophage Typing --- p.31 / Chapter 1.6.1.3 --- Serotyping --- p.32 / Chapter 1.6.1.4 --- Electrophoretic Protein Typing and Immunoblotting --- p.33 / Chapter 1.6.1.5 --- Multilocus Enzyme Electrophoresis --- p.34 / Chapter 1.6.1.6 --- Limitations of phenotyping --- p.34 / Chapter 1.6.2 --- Genotyping methods --- p.35 / Chapter 1.6.2.1 --- PCR Assays --- p.35 / Chapter 1.6.2.2 --- Pulsed-Field Gel Electrophoresis (PFGE) --- p.36 / Chapter 1.6.2.3 --- Multi-Locus Sequence Typing (MLST) --- p.37 / Chapter 1.6.2.4 --- Amplified Fragment Length Polymorphism (AFLP) --- p.38 / Chapter 1.6.2.5 --- Ribotyping --- p.38 / Chapter 1.6.2.6 --- Plasmid Typing --- p.39 / Chapter 1.6.2.7 --- Restriction Fragment Length Polymorphism --- p.39 / Chapter 1.6.2.8 --- Nucleic Acid Hybridization --- p.40 / Chapter 1.7 --- Objectives of the project --- p.41 / Chapter Chapter 2 --- Materials and Methods --- p.42 / Chapter 2.1 --- Bacterial Isolates & Culture conditions --- p.42 / Chapter 2.1.1 --- Bacterial Isolates --- p.43 / Chapter 2.1.2 --- Reference Strains --- p.43 / Chapter 2.1.3 --- Identification of MRSA --- p.44 / Chapter 2.2 --- Antibiotic Susceptibility Test --- p.45 / Chapter 2.3 --- Pulsed-Field Gel Electrophoresis (PFGE) --- p.47 / Chapter 2.3.1 --- DNA preparation --- p.47 / Chapter 2.3.2 --- Restriction digestion --- p.48 / Chapter 2.3.3 --- PFGE --- p.48 / Chapter 2.3.4 --- Analysis of band patterns --- p.48 / Chapter 2.4 --- Phage Typing --- p.50 / Chapter 2.4.1 --- Source of Phages & Propagating Strains --- p.50 / Chapter 2.4.2 --- Procedures of phage typing --- p.50 / Chapter 2.4.3 --- Routine check of the lytic strength of phages --- p.51 / Chapter 2.4.4 --- Evaluation of the reproducibility of phage typing --- p.51 / Chapter 2.5 --- Detection of SCCmec and mec Genes by Polymerase Chain Reaction (PCR) --- p.55 / Chapter 2.5.1 --- DNA preparation for PCR --- p.55 / Chapter 2.5.2 --- Master mix preparation and PCR conditions --- p.55 / Chapter 2.5.3 --- Electrophoresis of DNA Amplicors (PCR products) --- p.55 / Chapter 2.5.4 --- Detection of mecA gene by PCR --- p.56 / Chapter 2.5.5 --- Detection of SCCmec I-IV by PCR --- p.58 / Chapter 2.6 --- Multiplex Polymerase Chain Reaction --- p.60 / Chapter 2.6.1 --- Detection of agr I-IV --- p.60 / Chapter 2.6.2 --- Detection of Pyrogenic Toxin Genes --- p.62 / Chapter 2.6.2.1 --- Primer Mix Preparation for Multiplex PCR --- p.66 / Chapter 2.6.2.1.1 --- Sea-see Multiplex PCR --- p.66 / Chapter 2.6.2.1.2 --- Seg-sej Multiplex PCR --- p.66 / Chapter 2.6.2.1.3 --- "Eta, etb, tsst-1 for multiplex PCR" --- p.66 / Chapter 2.6.2.2 --- Multiplex Master Mix Preparation --- p.67 / Chapter 2.6.2.3 --- "PCR controls for pyrogenic toxins (sea-sej, eta-b, tsst-1)" --- p.67 / Chapter 2.6.2.4 --- Sequencing of PCR products --- p.68 / Chapter 2.6.3 --- PCR for adhesin and toxin genes --- p.69 / Chapter 2.6.3.1 --- "Multiplex Master mix preparation for lukE, fib, cna, icaD" --- p.71 / Chapter 2.6.3.2 --- "Multiplex Master mix preparation for fnbA,fnbB, hla, hlb, icaA" --- p.71 / Chapter 2.6.3.3 --- PCR controls for adhesin and toxin genes --- p.71 / Chapter 2.6.3.4 --- Size of PCR products resolved by gel electrophoresis --- p.72 / Chapter 2.6.3.5 --- Sequencing of PCR products --- p.72 / Chapter 2.7 --- Multi-locus sequence typing --- p.73 / Chapter 2.7.1 --- Bacterial Isolates --- p.73 / Chapter 2.7.2 --- Primer design --- p.73 / Chapter 2.7.3 --- PCR Master mix preparation --- p.73 / Chapter 2.7.4 --- PCR for MLST gene --- p.75 / Chapter 2.7.5 --- Sequencing of PCR products --- p.75 / Chapter 2.7.6 --- Data Analysis --- p.75 / Chapter 2.8 --- Sequence typing of Coa gene --- p.76 / Chapter 2.8.1 --- Bacterial Isolates --- p.76 / Chapter 2.8.2 --- Amplification of Coa Gene by PCR --- p.76 / Chapter 2.8.3 --- coa sequencing --- p.77 / Chapter 2.8.4 --- Sequence Analysis --- p.77 / Chapter Chapter 3 --- Results --- p.79 / Chapter 3.1 --- Bacterial Isolates --- p.79 / Chapter 3.2 --- PCR for mecA gene --- p.80 / Chapter 3.3 --- Antibiotics Susceptibility Test --- p.81 / Chapter 3.3.1 --- Antibiotic Resistance Profiles of MRSA --- p.82 / Chapter 3.4 --- Pulsed-field Gel Electrophoresis --- p.85 / Chapter 3.4.1 --- Optimization of PFGE and Inter-gel Variation --- p.85 / Chapter 3.4.2 --- Analysis of PFGE profiles --- p.87 / Chapter 3.4.3 --- PFGE patterns and antibiotic profiles --- p.93 / Chapter 3.5 --- Phage Typing --- p.96 / Chapter 3.6 --- SCCmec Typing --- p.98 / Chapter 3.6.1 --- PCR Optimization --- p.98 / Chapter 3.6.2 --- PCR and distribution of SCCmec types --- p.101 / Chapter 3.6.3 --- PFGE patterns and SCCmec types --- p.103 / Chapter 3.7 --- agr Typing --- p.104 / Chapter 3.7.1 --- Optimization of PCR --- p.104 / Chapter 3.7.2 --- PCR and Distribution of agr groups --- p.106 / Chapter 3.7.3 --- PFGE patterns and agr groups --- p.108 / Chapter 3.7.4 --- SCCmec types and agr groups --- p.108 / Chapter 3.8 --- "PCR for adhesin, pyrogenie and other toxin genes" --- p.110 / Chapter 3.8.1 --- Optimization of PCR --- p.110 / Chapter 3.8.2 --- "Distribution of adhesin, pyrogenic and other toxin genes" --- p.120 / Chapter 3.9 --- Multi-Locus Sequence Typing --- p.124 / Chapter 3.10 --- coa Sequence Typing --- p.126 / Chapter Chapter 4 --- Discussion --- p.128 / Chapter 4.1 --- Evaluation of the Typing methods for MRSA --- p.128 / Chapter 4.1.1 --- Antibiotic Susceptibility Test --- p.128 / Chapter 4.1.2 --- PFGE --- p.128 / Chapter 4.1.3 --- Phage Typing --- p.130 / Chapter 4.1.4 --- "PFGE, SCCmec typing and agr typing" --- p.131 / Chapter 4.1.5 --- MLST --- p.132 / Chapter 4.1.6 --- Sequencing typing of coa gene --- p.133 / Chapter 4.2 --- Characteristics of MRSA --- p.134 / Chapter 4.2.1 --- PCR for adhesin and toxin genes --- p.134 / Chapter 4.3 --- Key Characteristics of Hong Kong isolates --- p.137 / Chapter 4.4 --- Conclusion --- p.144 / Chapter 4 5 --- Future Research --- p.145 / Reference List --- p.146 / Appendix I - Materials/Reagents for Methods --- p.170 / Appendix II - Reagents Formula --- p.178 / Appendix III - coa sequence alignment --- p.180 / Appendix IV - Typing patterns and Characteristics of reference isolates --- p.193 / Appendix V - Miscellaneous --- p.195 / Appendix VI - Typing Patterns and Characteristics of PFGE type isolates --- p.204

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_324630
Date January 2004
ContributorsHung, Ming Wai., Chinese University of Hong Kong Graduate School. Division of Microbiology.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xviii, 217 leaves : ill. (some col.) ; 30 cm.
CoverageHong Kong
RightsUse of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

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