by Koo Ching Irene. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 105-118). / Abstract also in Chinese. / Acknowledgments --- p.i / Abstract --- p.ii / Content --- p.iv / List of tables --- p.viii / List of figures --- p.x / Chapter Chapter 1: --- Introduction --- p.1 / Chapter A. --- Classification of salmonellae --- p.1 / Chapter B. --- Salmonellae enterica ser Enteritidis --- p.4 / Chapter C. --- Global increase in the prevalence of S. Enteritidis --- p.6 / Chapter D. --- Susceptibility of S. Enteritidis to antimicrobial agents --- p.13 / Chapter E. --- Methods for the epidemiological typing of S. Enteritidis --- p.17 / Chapter 1. --- Phenotypic methods --- p.17 / Chapter (1) --- Biotyping --- p.17 / Chapter (2) --- Antibiotic resistance pattern --- p.18 / Chapter (3) --- Phage typing --- p.18 / Chapter (4) --- Characterization of plasmids --- p.19 / Chapter a. --- Resistance plasmids --- p.20 / Chapter b. --- Transferability of plasmids --- p.20 / Chapter c. --- Incompatibility --- p.21 / Chapter 2. --- Molecular methods --- p.21 / Chapter (1) --- Plasmid analysis --- p.21 / Chapter a. --- Plasmid profile --- p.22 / Chapter b. --- Plasmid fingerprinting --- p.22 / Chapter (2) --- Chromosomal DNA fingerprinting --- p.24 / Chapter a. --- Restriction fragment length polymorphism (RFLP) of chromosomal DNA --- p.25 / Chapter b. --- Pulsed-field gel electrophoresis (PFGE) --- p.25 / Chapter c. --- Hybridization with specific gene probes --- p.26 / Chapter d. --- Ribotyping --- p.27 / Chapter e. --- Insertion sequence IS200 fingerprinting --- p.29 / Chapter (3) --- Polymerase chain reaction (PCR) --- p.30 / Chapter 3. --- Other --- p.32 / Chapter (1) --- Lipopoly saccharide (LPS) analysis --- p.32 / Chapter (2) --- "Whole cell protein profile analysis, fatty acid profile analysis, multilocus enzyme electrophoresis and Fourier-transform injfrared spectroscopy" --- p.33 / Chapter F. --- Epidemiology of S. Enteritidis in different parts of the world --- p.34 / Chapter G. --- Objectives --- p.35 / Chapter Chapter 2: --- Materials and Methods --- p.36 / Materials --- p.36 / Methods --- p.38 / Chapter A. --- Identification --- p.38 / Chapter 1. --- Biochemical tests --- p.38 / Chapter 2. --- Serotyping --- p.38 / Chapter B. --- Antimicrobial susceptibility testing --- p.39 / Chapter C. --- Characterization of β-lactamases --- p.39 / Chapter 1. --- Extraction of β-lactamases --- p.39 / Chapter 2. --- Determination of isoelectric points (pIs) --- p.41 / Chapter D. --- Characterization ofplasmids --- p.42 / Chapter 1. --- Genetic studies --- p.42 / Chapter (1) --- Transferability of resistance plasmids --- p.42 / Chapter (2) --- Mobilization of resistances --- p.43 / Chapter 2. --- Molecular studies --- p.44 / Chapter (1) --- Plasmid profile analysis --- p.44 / Chapter a. --- Plasmid extraction --- p.44 / Chapter b. --- Agarose gel electrophoresis --- p.45 / Chapter (2) --- Plasmid DNA fingerprinting --- p.45 / Chapter a. --- Preparation of pure plasmid DNA --- p.46 / Chapter b. --- Preparation of individual plasmid from strains harbouring more than one plasmid --- p.46 / Chapter c. --- Restriction endonuclease digestion of plasmid DNA --- p.47 / Chapter E. --- Total DNA fingerprinting --- p.47 / Chapter 1. --- Total DNA preparation --- p.48 / Chapter 2. --- Restriction endonuclease digestion of total DNA --- p.48 / Chapter 3. --- Ribotyping --- p.49 / Chapter (1) --- Restriction enzyme digestion of total DNA --- p.49 / Chapter (2) --- Transfer of DNA fragments to solid support --- p.49 / Chapter (3) --- Reverse transcription of rRNA into cDNA and labelling of cDNA --- p.50 / Chapter (4) --- Hybridization --- p.50 / Chapter (5) --- Detection of hybridized fragments --- p.51 / Chapter 4. --- AP-PCR (Arbitrary primed-PCR) --- p.51 / Chapter F. --- Experimental design --- p.52 / Chapter Chapter 3: --- Results --- p.54 / Chapter A. --- Prevalence of S. Enteritidis in the Prince of Wales Hospital --- p.54 / Chapter B. --- Antimicrobial susceptibilities --- p.58 / Chapter C. --- β-lactamases produced by β-lactam-resistant S. Enteritidis --- p.66 / Chapter D. --- Plasmid profile analysis --- p.66 / Chapter E. --- Characterization of resistance plasmids --- p.69 / Chapter F. --- Plasmid fingerprinting --- p.72 / Chapter G. --- Total DNA fingerprinting --- p.76 / Chapter H. --- Ribotyping --- p.82 / Chapter I. --- AP-PCR --- p.85 / Chapter J. --- "Correlation of plasmid analysis, total DNA fingerprinting, ribotyping and AP-PCR results" --- p.88 / Chapter Chapter 4: --- Discussion --- p.91 / Chapter A. --- Prevalence of S. Enteritidis --- p.91 / Chapter B. --- Susceptibilities of S. Enteritidis to antimicrobial agents --- p.92 / Chapter C. --- Evaluation of methods for epidemiological typing of S. Enteritidis --- p.94 / Chapter D. --- Molecular epidemiology of S. Enteritidis in Hong Kong --- p.99 / Chapter E. --- Areas for future research --- p.102 / References --- p.105 / Appendix --- p.119

A total of 842 single patient isolates of Salmonella spp. from the New Territories East Cluster hospitals, Hong Kong, were collected during 2002 and 2004. The most common Salmonella enterica serotype isolated was S. Enteritidis (29.7%, 250 of 842) followed by S. Typhimurium (13.7%, 115 of 842). The remaining 29.6% (249 of 842) belonged to 44 serotypes and 27.1% (228 of 842) were non-typeable. The majority of isolates were from patients aged two years or younger and were isolated during June to October of each of the three years. The susceptibilities to 19 antimicrobial agents of the 834 isolates that survived were tested. Resistant strains were investigated for [1] the mechanisms of resistance to fluoroquinolones and the third generation cephalosporins; [2] the genetic mechanisms of emergence of antibiotic-resistant salmonellae; and [3] their molecular epidemiology. / Less than half (46.9%, 391 of 834) of the isolates were susceptible to all the antimicrobial agents tested and 21.3% (178 of 834) were resistant to three and up to 14 in a total of 75 resistance patterns. Resistance to nalidixic acid increased from 18.9% (53 of 280) in 2002 to 36.6% (94 of 259) in 2004 (p <0.001) while reduced susceptibility and resistance to ciprofloxacin increased from 17.9% (50 of 280) to 39.4% (102 of 259) (p <0.001). All salmonellae remained susceptible to the third generation cephalosporins until 2003 when we isolated the first resistant isolate and two more in 2004. / No mutations in the quinolone resistance-determining region of target genes gyrA, gyrB, parC and parE were detectable in six of the 59 isolates that were resistant to 0.03 mg/l of ciprofloxacin and 14 that were susceptible to 0.03 mg/l of ciprofloxacin, all isolates being obtained in 2002. Forty-two isolates harboured one mutation, and one to eight harboured two to four mutations with those in positions Ser83 and/or Asp87 of the gyrA gene being the most common (89.8%, 53 of 59). No mutation was detected in the gyrB gene. A parC mutation at Ser80 was present only in strains with one or two gyrA mutation(s) while that at Thr57 could be present in strains without any other target gene mutations. A parE mutation (Ser458→Pro) was detected together with two gyrA and one parC mutations in only one isolate which was resistant to high concentrations of fluoroquinolones. Complementation experiments using a wild-type gyrA gene performed on isolates with gyrA gene mutations showed that mutations in gyrA contributed to fluoroquinolone-resistance. Only two among the 349 isolates that were obtained during 2002-2004 and resistant to 0.03 mg/l of ciprofloxacin harboured the qnr gene. / Of the three isolates that were resistant to the third generation cephalosporins, one, a S. Typhimurium, produced a beta-lactamase, CTX-M-9, of pI 8.1, and two, a S. Typhimurium and a S. Enteritidis, produced CTX-M-14, of pI 7.9. The blaCTX-M-9 gene was located on a class 1 integron on a 62 kb transferable plasmid and the blaCTX-M-14 gene was associated with the insertion sequence ISEcp1 and present on a 70 kb and a 92 kb transferable plasmid, respectively. This is the first report of a CTX-M-9 enzyme in S. Typhimurium in Hong Kong. (Abstract shortened by UMI.) / The MICs of nalidixic acid in the presence of 20 mg/l of Phe-Arg beta-naphthylamide (PAbetaN) for the 73 isolates that were tested for the presence of target gene mutations and S. Typhimurium ATCC 13311 were at least 4-fold lower than those of nalidixic acid in the absence of PAbetaN, indicating presence of an efflux system that could be inhibited by PAbetaN and of which nalidixic acid was a substrate. / Twenty-one isolates with different target gene mutations and fluoroquinolone susceptibilities were selected to investigate the effect of active efflux system, outer membrane permeability and target gene expression on fluoroquinolone-susceptibility. The amount of ciprofloxacin accumulated in the presence of carbonyl cyanide m-chloro-phenylhydrazone (CCCP) was significantly more than that in the absence of CCCP in 15 of these 21 strains, indicating presence of an efflux system that used proton motive force as energy. The amount of ciprofloxacin accumulated in 15 strains was significantly less than that in the standard strain (ATCC 13311) after the addition of CCCP, indicating that these strains were less permeable to ciprofloxacin than the standard strain. Real-time PCR experiments revealed that there were strains with overexpression of target genes as well as the acrB gene that codes for AcrB in the AcrAB-TolC efflux system. No aac(6')-Ib-cr was detected in our strains. / Jin, Yujuan. / "January 2008." / Adviser: M. L. Ling. / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4543. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (p. 195-219). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.