published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Dissemination of CTX-M type extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli (E. coli) is a serious health issue in Hong Kong. However, research knowledge concerning its dissemination mechanism and plasmid characteristics over time in health care setting is still limited. This study was conducted to characterize ESBL-producing E. coli from blood culture isolates and the epidemiology of the plasmids harboring CTX-M-14 collected from a healthcare region in Hong Kong from two periods of time. A total of 167 ESBL-producing E. coli in blood culture specimens were retrieved from period 1 (1996 - 1999, n = 50) and period 2 (2007 - 2008, n = 117). Antimicrobial susceptibilities were defined by disk diffusion method according to CLSI. Phylogenetic groups and CTX-M enzymes were detected among all the ESBL-producers. Clonal relatedness of the hosts was analyzed by pulsed-field gel electrophoresis and multi-locus sequence typing. A subset of 65 CTX-M-14-producing isolates was undergone for further plasmid characterization. Conjugation, PCR-based replicon typing, S1-PFGE, southern-blot hybridization, and genetic environment PCRs were performed. Plasmid PCR-restriction fragment length polymorphism (pRFLP), F-allele replicon sequence typing and variable region PCRs were studied in 54 F-plasmids obtained. Results showed that over half of the ESBL-positive isolates were non-susceptible to ciprofloxacin, cotrimoxazole and gentamicin. A surprisingly high number of CTX-M-type ESBL was carried by 98.2% (164/167) of the isolates. CTX-M-9 group (89.8%, 150/167) and CTX-M-14 (103/109) were predominantly found among both periods. Overall, nearly half (41.3%, 69/167) of the isolates belonged to 5 major clones. Clonal types undetermined-ST68 (n = 18) and O102-ST405 (n = 15) were dominant in period 1 while clonal types O25b-ST131 (n = 30), O15-ST69 (n = 5) and O12-ST12 (n = 3) emerged in period 2. Among a subset of 65 CTX-M-14 plasmids, most of them were transferable (84.6%, 55/65) with high frequency, similar plasmid sizes and genetic environment ISEcp1-blaCTX-M-14-IS903 (90.8%, 59/65). Replicon types of the CTX-M-14 encoding plasmids were FII (n = 48) or FII ± FIA/FIB types (n = 6), I1-I (n = 3), B/O (n = 2), K (n = 1) and undetermined (n = 4). Subtyping of 54 IncF plasmids by replicon sequence typing, pRFLP and PCR for marker genes (yac, malB, eitA, eitC and parAB) showed that 79.6% (43/54) of the plasmid subset exhibited identical or highly similar results with the completely sequenced plasmid, pHK01 (E. coli isolated from urine sample of a patient in Hong Kong, 2004). These 43 plasmids were originated from both period 1 (n = 11) and period 2 (n = 32). These pHK01-like plasmids were found to have spread to the major clones (ST68, ST405 and ST131) and multiple singleton isolates of all four phylogenetic groups. In conclusion, this study demonstrated the widespread dissemination of pHK01-like CTX-M-14 encoding plasmids among isolates of diverse genetic lineages over a decade. The dissemination was probably due to both clonal expansion and horizontal gene transfer of pHK01-like IncF plasmid. / published_or_final_version / Microbiology / Master / Master of Philosophy

published_or_final_version / abstract / Microbiology / Master / Master of Philosophy

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We have developed a novel TGF-β detection system, using genetically modified macrophages as cytokine biosensors, capable of detecting bioactive TGF-β in vivo and in vitro. Stimulation by TGF-β1 increases secretion of plasminogen activator inhibitor-1 (PAI-1) by activation of the promoter. An adenovirus containing the first 800bp of the PAI-1 promoter fused to the reporter gene β-galactosidase (Ad-PAI1800 βgal) was used to transfect primary cultures of rat bone marrow derived macrophages (BMDM). After transfection the BMDM were stimulated with TGF-β, 2 or 3 or left unstimulated. X-gal histochemical assay was performed 72-hours post-transfection to detect β-galactosidase expression. Macrophages transfected with Ad-PAI1800βgal and stimulated with TGF-β1, 2 or 3 showed a dose-dependent production of β-galacosidase. AdPAI1800βgal BMDM also expressed β-galactosidase when co-cultured with NR8383 rat alveolar macrophages transfected with recombinant adenovirus expressing active TGF-β1. Genetically modified macrophages could detect expression of TGF-β1 in vivo. AdPAI1800βgal transfected BMDM were co-injected with AdTGF-βl transfected NR8383 macrophages into the renal artery of rats with nephrotoxic nephritis (NTN). Isolated glomeruli showed X-gal positive macrophages on assay. AdPAI1800βgal BMDM were also injected into the renal artery of rats 7 days after the induction of Thy 1.1 nephritis, 100% of isolated glomeruli contained X-gal positive macrophages on assay. Signalling macrophages were capable of detecting TGF-β1 expression in sections of kidney tissue containing Ad-TGF-β1 transfected NR8383 macrophages. BMDM showing β-galactosidase were readily detected over the transfected glomeruli. Pre-treatment of Ad-PAI1800βgal transfected BMDM with IFN-γ prevented production of the reporter gene. However when stimulation with TGF-β occurred 20 hours after IFN-γ macrophage programmed unresponsiveness was abrogated. Macrophage responsiveness to TGF-β stimulation in Thy 1.1 nephritic kidneys was also prevented by pre-treatment with IFN-γ. This system demonstrates the use of genetically modified macrophages as both <i>in vivo </i>and <i>in vitro </i>sensors of the bioactive TGF-β providing a novel approach to assess cytokine activity.

Propranolol (PROP) is a β-blocker prescribed mainly to treat human cardiac diseases but with its wide usage it often makes its way into the aquatic environment. This study examined whether PROP alters developmental patterns and catecholamine (CA)-regulated processes in the zebrafish (Danio rerio) and if exposure during early life alters the stress response and behaviors of adults. The 48 h LC50 was 21.6 mg/L, well above environmental levels (0.00059 mg/L). Embryos/larvae continuously PROP-exposed had decreased and increased transcript levels of the β1-adrenoceptor at 1 dpf and 5 dpf, respectively. Stressed, PROP-exposed zebrafish had reduced testosterone and estradiol levels and exhibited less anxiety behaviours than control fish. Furthermore, adults previously PROP-exposed as embryos/larvae had decreased growth in terms of body length (0.0006 mg/L PROP) and mass (20 mg/L PROP). Changes in cholesterol and testosterone levels occurred in PROP-exposed fish. Thus PROP-exposure alters developmental patterns and CA-regulated process that are essential for normal behaviours and responses to stress, and at least some of these changes persist in the adult zebrafish.