Molecular identification and characterization of Streptococcus agalactiae in Hong Kong.

Cheuk Shing Ching.Thesis (M.Phil.)--Chinese University of Hong Kong, 2005.Includes bibliographical references (leaves 144-161).Abstracts in English and Chinese.ACKNOWLEDGMENTS --- p.I內容摘要 --- p.IIABSTRACT --- p.IVCONTENTS --- p.XILIST OF TABLES --- p.XILIST OF FIGURES --- p.XIABBREVIATIONS --- p.XIIChapter CHAPTER 1 --- INTRODUCTION --- p.1Chapter 1.1 --- Taxonomy of Streptococcus agalactiae --- p.1Chapter 1.2 --- Characteristics of Streptococcus agalactiae --- p.1Chapter 1.3 --- Epidemiology of GBS --- p.3Chapter 1.3.1 --- Risk groups --- p.3Chapter 1.3.1.1 --- Neonates --- p.3Chapter 1.3.1.2 --- Pregnant women --- p.5Chapter 1.3.1.3 --- Non-pregnant adult --- p.6Chapter 1.3.2 --- World wide distribution --- p.7Chapter 1.3.2.1 --- Serotypes --- p.7Chapter 1.3.2.2 --- Antibiotic susceptibility --- p.8Chapter 1.3.3 --- GBS diseases in Hong Kong --- p.10Chapter 1.4 --- Putative virulence factors and pathogenesis --- p.10Chapter 1.4.1 --- Capsular polysaccharide --- p.10Chapter 1.4.2 --- C5a peptidase --- p.11Chapter 1.4.3 --- β-haemolysin/cytolysin --- p.12Chapter 1.4.4 --- C protein and C a-like protein --- p.12Chapter 1.4.4.1 --- C protein --- p.12Chapter 1.4.4.1.1 --- C α protein --- p.13Chapter 1.4.4.1.2 --- Cβ protein --- p.14Chapter 1.4.4.2 --- C α-like protein --- p.15Chapter 1.4.5 --- Hyaluronate lyase --- p.16Chapter 1.4.6 --- CAMP factor --- p.17Chapter 1.4.7 --- Others --- p.17Chapter 1.5 --- Antibiotic resistance and resistance genes --- p.18Chapter 1.5.1 --- Macrolides --- p.18Chapter 1.5.2 --- Tetracyclines --- p.18Chapter 1.5.3 --- Aminoglycosides --- p.19Chapter 1.5.4 --- Fluoroquniolones --- p.20Chapter 1.5.5 --- Others --- p.20Chapter 1.6 --- Mobile genetic elements --- p.21Chapter 1.7 --- Typing methods --- p.22Chapter 1.7.1 --- Phenotypic methods --- p.23Chapter 1.7.1.1 --- Serotyping --- p.23Chapter 1.7.1.2 --- Multilocus enzyme electrophoresis (MLEE) --- p.23Chapter 1.7.2 --- Genotypic methods --- p.24Chapter 1.7.2.1 --- Restriction endonuclease analysis (REA) / restriction fragment-length polymorphism (RFLP) --- p.24Chapter 1.7.2.2 --- Pulsed-field gel electrophoresis (PFGE) --- p.25Chapter 1.7.2.3 --- Random amplified polymorphic DNA (RAPD) --- p.26Chapter 1.7.2.4 --- Sequencing --- p.26Chapter 1.8 --- Prevention --- p.29Chapter 1.8.1 --- Intrapartum antibiotic prophylaxis (IAP) --- p.29Chapter 1.8.2 --- GBS Vaccine --- p.33Chapter 1.9 --- Objectives --- p.34Chapter CHAPTER 2 --- METHODS AND MATERIALS --- p.35Chapter 2.1 --- Bacterial isolates --- p.35Chapter 2.2 --- Antibiotic susceptibility test --- p.37Chapter 2.2.1 --- Antibiotic preparation --- p.37Chapter 2.2.2 --- Microbroth dilution method --- p.39Chapter 2.2.2.1 --- Microtitre plate preparation --- p.39Chapter 2.2.2.2 --- Suspension preparation and inoculation --- p.39Chapter 2.2.2.3 --- End points determination --- p.40Chapter 2.2.3 --- Inducible lincomycin resistance determination --- p.40Chapter 2.3 --- Serotyping --- p.41Chapter 2.3.1 --- Preparation of antigens --- p.41Chapter 2.3.2 --- Typing of isolates --- p.42Chapter 2.4 --- Pulsed-field Gel Electrophoresis (PFGE) --- p.42Chapter 2.4.1 --- Preparation of DNA plug for PFGE --- p.43Chapter 2.4.2 --- Restriction enzyme digestion of GBS DNA --- p.43Chapter 2.4.3 --- Running of PFGE gel --- p.44Chapter 2.5 --- Molecular characterization --- p.44Chapter 2.5.1 --- Target genes --- p.44Chapter 2.5.2 --- DNA preparation --- p.51Chapter 2.5.3 --- Master mix preparation --- p.51Chapter 2.5.4 --- Polymerase chain reaction --- p.51Chapter 2.5.5 --- PCR product analysis by agarose gel electrophoresis --- p.52Chapter 2.5.6 --- DNA sequencing --- p.52Chapter 2.6 --- Data analysis --- p.53Chapter 2.6.1 --- PFGE and molecular characters analysis --- p.53Chapter 2.6.2 --- Sequences analysis --- p.53Chapter 2.7 --- Molecular identification by real-time PCR --- p.54Chapter 2.7.1 --- Bacterial strains --- p.54Chapter 2.7.2 --- DNA isolation for specimens --- p.56Chapter 2.7.3 --- Design of TaqMan primers and probes --- p.56Chapter 2.7.4 --- Cloning of target sequences --- p.59Chapter 2.7.5 --- Master mix of real-time PCR --- p.59Chapter 2.7.6 --- Specificity and detection limit --- p.60Chapter CHAPTER 3 --- RESULTS --- p.62Chapter 3.1 --- Serotype distribution of GBS --- p.62Chapter 3.1.1 --- Serotyping using antisera --- p.62Chapter 3.1.2 --- Serotyping by molecular method --- p.64Chapter 3.1.3 --- Molecular subtype of GBS serotype III --- p.66Chapter 3.1.4 --- Correlation of serotypes with diseases --- p.69Chapter 3.2 --- Antimicrobial susceptibility --- p.71Chapter 3.2.1 --- Phenotypic method --- p.71Chapter 3.2.2 --- Detection and distribution of resistance genes --- p.76Chapter 3.2.2.1 --- Tetracycline resistance --- p.76Chapter 3.2.2.2 --- Macrolide and lincosamide resistance --- p.77Chapter 3.2.2.3 --- Aminoglycoside resistance --- p.78Chapter 3.3 --- Molecular typing --- p.83Chapter 3.3.1 --- Pulsed-field gel electrophoresis (PFGE) --- p.83Chapter 3.3.2 --- Distribution of GBS surface protein genes profiles --- p.89Chapter 3.3.3 --- Distribution of mobile genetic elements --- p.92Chapter 3.4 --- "Analysis based on PFGE, surface protein genes, mobile genetic elements and antibiotic resistance genes" --- p.95Chapter 3.4.1 --- Intra-molecular serotype --- p.95Chapter 3.4.1.1 --- Molecular serotype Ia --- p.95Chapter 3.4.1.2 --- Molecular serotype Ib --- p.99Chapter 3.4.1.3 --- Molecular serotype II --- p.101Chapter 3.4.1.4 --- Molecular serotype III --- p.103Chapter 3.4.1.5 --- Molecular serotype V --- p.107Chapter 3.4.1.6 --- Molecular serotype VI --- p.110Chapter 3.4.1.7 --- "Molecular serotype IV, VII and VIII" --- p.110Chapter 3.4.1.8 --- Non-typeable isolate (NT) --- p.111Chapter 3.4.2 --- Analysis of Maternal and neonatal strains --- p.115Chapter 3.4.3 --- Comparison of GBS strains from Hong Kong to Australia and Korea --- p.118Chapter 3.5 --- Molecular identification of GBS by real-time PCR --- p.120Chapter 3.5.1 --- Specificity --- p.120Chapter 3.5.2 --- Detection limits --- p.122Chapter CHAPTER 4 --- DISCUSSION --- p.125Chapter 4.1 --- Laboratory methods for typing and characterization of GBS --- p.125Chapter 4.1.1 --- Serotyping by agglutination and molecular method --- p.125Chapter 4.1.2 --- Antibiotic susceptibility testing and resistance genes --- p.129Chapter 4.1.3 --- PFGE --- p.130Chapter 4.1.4 --- Surface protein genes --- p.131Chapter 4.1.5 --- Mobile genetic elements --- p.132Chapter 4.1.6 --- Real-time PCR --- p.133Chapter 4.2 --- Characterization of GBS in Hong Kong --- p.135Chapter 4.2.1 --- GBS in Hong Kong --- p.135Chapter 4.2.2 --- GBS from Australia and Korea --- p.141Chapter 4.3 --- Future research --- p.142Chapter 4.4 --- Conclusions --- p.143REFERENCES --- p.144APPENDIX I: MATERIALS AND REAGENTS --- p.162APPENDIX II: DENDROGRAMS --- p.16