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

Norovirus (NoV) is a major cause of infectious human viral gastroenteritis. Detection is important to understanding the epidemiology of NoV and the viral dynamics of NoV infection which is poorly understood. In 2006, a marked increase in gastroenteritis outbreaks occurred worldwide. During this period, a total of 231 stool samples were obtained from patients with acute gastroenteritis from Australia and New Zealand. A total of 186 isolates of NoV were detected and sequenced to determine the genotype and relatedness to known epidemic NoV GII.4 variants. Two GII.4 variants, 2006a and 2006b, were identified in 61.8% and 11.3%, in these cases, respectively. Thus, the increase in NoV gastroenteritis in 2006 was linked to the emergence of two novel co-circulating GII.4 variants, 2006a and 2006b. During an outbreak in an aged-care facility, stool samples were collected from the onset of illness to cessation of viral excretion. Here, viral shedding peaked in the acute stage of illness and continued for an average of 28.7 days. The viral decay rate was 0.76 per day. Prolonged asymptomatic shedding of NoV was detected in the elderly. A quality control for the assessment of molecular based viral assays for NoV and other RNA viruses is necessary to meet current testing requirements. Available controls only monitor the RNA and DNA amplification steps. An MS2 bacteriophage BioBallTM with 100 pfu was evaluated and applied as a multi-purpose phage control. It was assessed as a quality control, in comparison to MS2 phage stock, to validate MS2 phage assays. Furthermore, MS2 BioBallTM was used as a process control for the molecular detection of RNA viruses. It validated every performed step, determined if the assay worked and its sensitivity. Thus, MS2 BioBallTM offered uniformity, stability and reproducibility across molecular based viral detection systems. Overall, this thesis provided valuable insight into the molecular epidemiology of NoV in the southern hemisphere and nature of NoV infections in the elderly. The MS2 BioBallTM provides standardisation and quality control of viral RNA assays. Understanding the genetic diversity and viral dynamics of NoV will be crucial to developing effective intervention and treatment strategies, and ultimately lead to reduced viral gastroenteritis worldwide.

Dichelobacter nodosus, an anaerobic bacterium, is the major transmissible agent of ovine footrot. The disease expresses as a virulent or benign lesion in the hoof. Virulence is related to the production of serine proteases, particularly a thermostable protease. Isolates of D. nodosus are characterised according to the type of protease produced (either heat-stable or heat-labile) and the electrophoretogram (zymogram) of the protease. This study reports on the use of the DNA-based typing techniques Pulsed-Field Gel Electrophoresis (PFGE) and Infrequent-Restriction-Site-PCR (IRS-PCR) to investigate the molecular epidemiology of D. nodosus, including a consideration of the relationship between genetic type, zymogram patterns and whole cell protein profiles. The aim of the project was to obtain a better understanding of D. nodosus strain diversity and dissemination in Australia and its relationship to virulence within the population. The overall intention was to use this information to assist in the long-term control of virulent footrot. Field isolates of D. nodosus from Western Australia (n = 735), New South Wales (n = 16), Victoria (n = 24) and South Australia (n = 21) were obtained and analysed. Both typing techniques that were used offered good differentiation between isolates for epidemiological purposes, and the results were in general agreement. PFGE provided slightly better discrimination between isolates, with 214 PFGE types (181 from Western Australia) compared to 94 IrsT types (77 from Western Australia). Within this diverse range of molecular types clonality was observed - with clones being defined as clusters of isolates having closely related PFGE types. The strains were categorised as genetically diverse, genetically similar or identified as the same strain. This diversity of genetic types was found overall, within flocks of sheep on a farm and within a single hoof where, on a number of occasions, multiple molecular types and zymogram types were found colonising a single hoof. One isolate that was experimentally inoculated into a flock of sheep produced six different genetic types when tested 12 months after the initial infection. This indicates that D. nodosus undergoes rapid genetic change, which means that follow-up epidemiological investigation of disease outbreaks and trace-backs need to be done as soon after infection as possible. The genetic differences appeared to be due to large insertions or deletions of DNA. Amongst sheep on some properties, isolates that had a different protease expression and virulence expression were found to have the same molecular type. Investigation of these isolates by SDS-PAGE showed that they also had the same whole cell protein profiles. Isolates from the same clonal groups also had the same protein profile, whereas genetically diverse isolates had different protein profiles. The lack of protein differences between isolates of the same molecular type, or within a clonal group, suggests that the differences in protease thermostability may be due to conformational changes in the protein, rather than to overall detectable genetic change and/or expression of different proteins. These results demonstrate that PFGE typing can be useful in predicting likely phenotypic expression of whole cell proteins. Further work is required to elucidate differences between virulent and benign strains of D. nodosus.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Thesis (M. Med. Sc.)--University of Hong Kong, 2005.

published_or_final_version / abstract / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Biological Sciences / Doctoral / Doctor of Philosophy