Antibiotic resistance is increasing worldwide, which is threatening the effectiveness of even the most potent and recent antibiotics. The successful treatment of disease is hampered due to the multidrug resistant (MDR) phenotype exhibited by the bacterial pathogens. Therefore, the aims of this thesis were to investigate MDR through several different approaches. Integrons are important contributors to the MDR profile of nosocomial isolates within Australia, therefore the incidence of integrons was assessed in a collection of 72 conjugative clinical plasmids isolated from E. coli, a cohort of 30 urinary tract infection (UTI) isolates and a cohort of four bacteria producing metallo-beta-lactamases (MBLs). Integrons were found in 63% (45/72) of the conjugative plasmids by polymerase chain reaction (PCR). Sequencing of gene cassette arrays revealed that cassettes of the dfr and aadA families were most common. Within the cohort of UTI bacteria, 37% (11/30) were positive for class 1 integrons, and the dfrA17-aadA5 gene cassette array was most common. The four MBL-producers contained the gene cassette blaIMP-4 found within a class 1 integron which was responsible for the MBL phenotype. An assay based on real-time PCR was also developed to measure the recombination activity of the integron integrase (IntI) enzymes. The existing method of IntI measurement, the in vivo conduction assay, was used as a basis for the development of the real-time PCR assay. Five 59-be from the gene cassettes aadB, orfA, sat2, dfrA1 and aacA4 were cloned as recognition sites used in the real-time PCR assay. IntI1 was the most active integrase and showed an activity of 2.31 ?? 10??-1 when recombining the aadB and orfA 59-be. The highest level of class 2 integrase activity was 2.00 ?? 10-1?? during recombination of the sat2 and dfrA1 59-be, while IntI3 showed its highest recombination frequency of 2.29 ?? 10-1?? when the aadB and orfA 59-be were used. Additionally, the real-time PCR assay was used assess the levels of IntI activity over time. Using this method, the level of recombination as time progressed remained stable at a level of 4.10 ?? 10-2????. MDR was also analysed in 37 Acinetobacter baumannii isolates which were collected from four hospitals in Sydney. Minimum inhibitory concentration (MIC) analysis to 25 antibiotics revealed that all isolates showed a reduced susceptibility to between five and 24 antibiotics. PCR was performed to detect the presence of resistance determinants. Class 1 integrons encoding resistance to aminoglycosides, antiseptics and disinfectants were found in 35 % (13/37) of the isolates. Aminoglycoside resistance genes including aphA1 (12/37), strA (1/37) and strB (22/39) were also found. Resistance to beta-lactams was also observed in all isolates, which correlated with the presence of the ampC and blaOXA-51-like genes. The insertion sequence ISAba1 which provides an alternative promoter leading to increased gene expression was found upstream of the ampC gene in 29 isolates; the same isolates also contained the identical insertion sequence upstream of the carbapenemase resistance gene blaOXA-23. These 29 isolates also possessed the tetracycline resistance gene tetB. All but one of these 29 isolates also contained the gene blaTEM-1. Resistance to quinolones and fluoroquinolones was attributed to the presence of a Ser83-Leu83 gyrA mutation present in 36 resistant isolates. Furthermore, a putative dihydrofolate resistance gene, folA, was found in all isolates. Repetitive extragenic palindromic PCR revealed the presence of seven clonal groups. Overall, this study demonstrated the widespread impact and dissemination of MDR within nosocomial settings in Australia. The use of new assays, such as the real-time PCR assay developed in this thesis, is essential to the understanding of dissemination of antibiotic resistance.
| Identifer | oai:union.ndltd.org:ADTP/272542 |
| Date | January 2009 |
| Creators | Mak, Jennifer Ka Yan, Biotechnology & Biomolecular Sciences, Faculty of Science, UNSW |
| Publisher | Awarded By:University of New South Wales. Biotechnology & Biomolecular Sciences |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://unsworks.unsw.edu.au/copyright, http://unsworks.unsw.edu.au/copyright |
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