Background Aminoglycosides are one of the clinically relevant antibiotics. Plasmid-encoded 16S rRNA methylase enzymes have emerged in clinical isolates of Gram-negative bacteria worldwide. The spread of these resistance determinants has become a great concern. Objectives The objectives of this study were to investigate the prevalence of 16S rRNA methylases and aminoglycoside modifying enzyme, AAC(3)-II in Escherichia coli isolated from human blood cultures and faecal samples of animals. E. coli isolates with unexplained aminoglycoside resistance phenotypes were investigated by detection of four aminoglycoside modifying enzymes, AAC(6’)-I, ANT(2”)-I, ANT(4’)-II and APH(3’)-VI. Methodology This study included 188 E. coli clinical isolates obtained from blood cultures of patients in one regional hospital between January 2004 and September 2010 and 81 E. coli isolates obtained from faecal samples of chickens, pigs, cattle, cats, dogs and rats between September 2008 and August 2011. All 269 E. coli isolates in this study were screened for the aac(3)-II gene and six 16S rRNA methylase genes(armA, rmtA, rmtB, rmtC, rmtD and rmtE)by two individual sets of multiplex PCR assays. A subset of 88E. coli isolates with aminoglycoside resistance phenotypes, which could not be explained by the genes detected, were subjected to detection of the aac(6’)-Ib, ant(2”)-Ia, ant(4’)-IIaand aph(3’)-Via genes by four individual PCR assays. The transfer of resistance of the rmtB gene was studied by conjugation experiments. The clonal relationship between rmtB-producing strains was investigated by pulsed-field gel electrophoresis. Results 67.6% (25/37) and 63.4% (26/41) of the Gen-R/Amk-NS group isolates from human and animal sources, respectively, were found to possess the aac(3)-IIgene. The aac(3)-IIgene was also found in 96.7% (146/151) Gen-R/Amk-S group human isolates. 21.6% (8/37) and 61%(25/41) of the Gen-R/Amk-NS isolates from human and animal sources, respectively, were found to possess the rmtB gene. The armA gene was found in one human and one animal isolates, which were both resistant to gentamicin and amikacin. No rmtA, rmtC, rmtD orrmtE genes were found in this study. Among 88E. coli isolates with unexplained aminoglycoside resistance phenotypes, the aac(6’)-Ib gene was found in51.2%(22/43) and 10% (4/40) of the Gen-R/Amk-NS group and the Gen-S/Amk-NS group, respectively. The ant(2”)-Ia gene was found in 11.6% (5/43) of the Gen-R/Amk-NS group E. coli isolates. No ant(4’)-IIa or aph(3’)-Via genes were found. No major PFGE cluster was observed among 32 rmtB-positive isolates by pulsed-field gel electrophoresis.In addition, amikacin resistance could be transferred by conjugation from 12rmtB-positive donors. Conclusion The present study showed that the rmtB gene was the most prevalent 16S rRNA methylase gene in both human and animal E. coli isolates. A high incidence of the aac(3)-IIgene was found among gentamicin-resistant strains. The spread of 16S rRNA methylases has aroused clinical concern and become a major therapeutic threat in the future. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

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

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a b s t r a c t Meat and meat products have been implicated in outbreaks of Escherichia coli O157:H7 in most parts of the world. In the Amathole District Municipality of the Eastern Cape Province of South Africa, a large number of households consume meat and meat products daily, although the microbiological quality of these types of food is questionable. The present study investigated the prevalence of E. coli O157:H7 isolated from selected meat and meat products (45 samples each of biltong, cold meat, mincemeat, and polony) sold in this area. Strains of E. coli O157:H7 were isolated by enrichment culture and confirmed by polymerase chain reaction (PCR). Also investigated were the antibiogram profiles of the E. coli O157:H7 isolates. Five (2.8%) out of 180 meat and meat products examined were positive for E. coli O157:H7 that carried the fliCH7, rfbEO157, and eaeA genes. Two of the E. coli O157:H7 isolates were resistant against all the eight antibiotics tested. To prevent E. coli O157:H7 infections, meat and meat products such as biltong, cold meat, mincemeat and polony should be properly handled, and packed in sterile polyvinyl wrappers.

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published_or_final_version / abstract / Microbiology / Master / Master of Philosophy

published_or_final_version / Botany / Master / Master of Philosophy