Thesis (Ph.D.)--University of Wollongong, 2009. / Typescript. Includes bibliographical references: leaf 188-214.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

The utility of focal-plane-array Fourier transform infrared (FPA-FTIR) spectroscopy as a rapid method for the differentiation of antibiotic resistant foodborne pathogens was studied. / Optimum spectral acquisition and processing parameters as well as appropriate film thickness of bacterial films were empirically established for the discrimination between two Shigella species (S. flexneri and S sonnei) in order to optimize the scanning parameters of an FPA-FTIR spectrometer. A detailed study of the potential of FPA-FTIR spectroscopy for the discrimination between antibiotic resistant and sensitive strains from two Salmonella species (S. Typhimurium and S. Heidelberg) was subsequently undertaken. The results of these studies demonstrated that the infrared spectra recorded by an FPA-FTIR spectrometer contained sufficient information to differentiate between antibiotic resistant and sensitive strains of Salmonella. Accordingly, FPA-FTIR spectroscopy may potentially serve as a high-throughput technique for the identification of foodborne as well as antibiotic resistant bacteria. / Interpretation of the regions selected in relation to the different resistance mechanisms would require more detailed studies. However, the identification of specific biochemical markers based on such spectral interpretation is generally not feasible owing to the complexity of the FTIR spectra of microorganisms.

Infections caused by resistant pathogens fail to respond to treatment, resulting in increased costs due to prolonged illness and hospitalization. Determining the extent of resistance in animal populations is thus of great importance to public health. In this work, we first showed that asymptomatic populations of Salmonella in pigs present greater genotypic and phenotypic diversity than disease-associated populations. Second, we identified a clonal population structure associated with asymptomatic Salmonella found in the Canadian swine industry and we confirmed that food-producing pigs are a significant reservoir of Salmonella enterica, more particularly the clinically important serotype Typhimurium DT104. Finally, we identified the possible independent evolution of multidrug-resistance in serotypes Typhimurium, Derby and Heidelberg. Our work on asymptomatic Salmonella enterica stresses the importance of linking ecology and evolutionary biology to public health in order to understand and predict the response of pathogenic bacteria to selective pressure imposed by host immunity, whether naturally or artificially induced.

The use of antibiotics and antimicrobial drugs has played a large role in the elimination of many infectious diseases, however the wide spread use of such drugs has given rise to the phenomenon of antimicrobial resistance and has rendered antibiotics ineffective to a broad range of bacteria. The aim of the study was to ascertain the differences if any in the phenotypic and genotypic resistance profiles of K. pneumoniae isolated from a single tertiary hospital in two surveillance studies undertaken at different times, viz., 2001 and 2007 with special emphasis on ESBLs. A correlation with antibiotic use was also undertaken. ESBL positives were identified and phenotypic resistance profiles were generated based on the resistance profiles of individual isolates by means of their MIC data. The molecular detection of ESBLs was carried out using representative isolates and sequencing was based on the phenotypic expression of the most common ESBL genes. The data was summarized using median values and interquartile ranges. Antibiotic use and susceptibility in 2000 was compared to that in 2007 using a Wilcoxon signed rank test for paired data since the same drugs were tested in both years. Of the isolates that were tested, sequencing revealed that TEM – 1 was identified in all isolates and SHV-1 and SHV-2 were identified in 60 % in the isolates collected in 2000 and 77 % and 11 % respectively in the isolates collected in 2007. SHV – 11 was present in 67% of isolates from 2007 and 55% of those were in combination with SHV – 1. Sequencing also revealed CTXM-15 present in one of the isolates collected in 2007. There was 100% susceptibility to cefoxitin and only one isolate in 2007 showing an intermediate result to imipenem. No novel β-lactamases were identified in this study; however the decrease in susceptibility over time is proof of bacterial evolution. The variety of β-lactamases and diversity of plasmid profiles in these two small populations provides proof to the claim that dissemination of resistance in Klebsiella pneumonia is effortless. Statistical analysis showed an increase in resistance from the year 2000 to 2007 however the correlation between overall antibiotic use and the increase in resistance did not reach statistical significance. It was observed that resistance increased despite only a slight increase in the use of a few antibiotics to which we attributed co-carriage of resistance genes. / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, Westville, 2012.

Multi-drug resistance among bacterial pathogens can be mediated by a number of mechanisms, including multidrug efflux pumps. One such pump in Bacillus spp. is ykkCD, a heterodimer of the SMR family consisting of C and D subunits. Previous studies suggest that the expression of ykkCD is controlled by a putative riboswitch and that the antibiotic tetracycline binds to the riboswitch in vitro. Additional studies have shown that two derivatives of tetracycline also bind to the putative riboswitch. These findings now need to be validated by an in vivo study. In this study, the effects that tetracycline and its commercially available derivatives—doxycycline, minocycline, anhydrotetracycline, and oxytetracycline—have on the expression levels of the ykkCD gene in Bacillus subtilis were explored. The level of ykkCD expression was quantified using two different methods: (1) ykkCD protein levels was determined using a ykkCD RNA--galactosidase reporter gene construct and (2) ykkCD mRNA levels was quantified by quantitative RT-PCR. Although the findings from method (1) were inconclusive, upregulation was observed for tetracycline and minocycline, in agreement with the results of the previous binding studies. / Department of Chemistry

Access to abstract permanently restricted to Ball State community only / Access to thesis permanently restricted to Ball State community only / Department of Chemistry

This thesis examined molecular changes in the quinolone-resistance determining regions (QRDRs) of the topoisomerase genes, gyrA and parC of Neisseria gonorrhoeae and Neisseria meningitidis and their contribution to fluoroquinolone resistance (FQR). Initially models of FQR emergence were developed from analysis of resistant mutants generated in vitro. The effects of the nature and order of sequential changes in GyrA and ParC on FQR were explored by correlating QRDR changes with ciprofloxacin minimum inhibitory concentration (MIC) determinations. The in vitro models were validated by comparisons of QRDR changes and MICs in two populations of wild-type FQR N. gonorrhoeae over a wide MIC range (0.09 to 24??g/mL), and in a wild type FQR meningococcus. The in vitro activities of three newer quinolones with differential activity on GyrA and ParC were compared with that of ciprofloxacin. Key findings were that the initial QRDR changes always occurred in gyrA and were the predominant influence on phenotypic expression of FQR. QRDR alterations were acquired sequentially and two GyrA and two ParC changes represented the full complement of changes observed in gonococci and two GyrA and one ParC change those in meningococci. GyrA alterations at Ser-91 in gonococci and Thr???91 in meningococci were pivotal for the development of further resistance. ParC changes required the presence of two GyrA alterations for any major impact on FQR. ParC substitutions, Ser-87???Arg and Glu-91???Gly in gonococci and Cys- 85???Asp and Glu-91???Lys in meningococci led to the expression of the highest FQR levels. Examination of FQR in wild-type meningococci was necessarily restricted, but analyses using the broader MIC range available in in-vitro-derived FQR meningococci (0.09 to 16??g/mL) revealed the first ParC changes in N. meningitidis. The study also redefined QRDR boundaries and described novel mutations within them. The nature of sequence changes in GyrA and ParC in FQR Neisseria also affected the relative activities of the three newer quinolones. Trovafloxacin was the most active quinolone in vitro but MIC differences with ciprofloxacin were mutation-dependent. Grepafloxacin and moxifloxacin were only slightly more active than ciprofloxacin in the presence of multiple QRDR changes. This thesis provides a comprehensive analysis of the relationship between QRDR alterations and FQR in N. gonorrhoeae and offers insights into the potential for FQR development in N. meningitidis.

This thesis examined molecular changes in the quinolone-resistance determining regions (QRDRs) of the topoisomerase genes, gyrA and parC of Neisseria gonorrhoeae and Neisseria meningitidis and their contribution to fluoroquinolone resistance (FQR). Initially models of FQR emergence were developed from analysis of resistant mutants generated in vitro. The effects of the nature and order of sequential changes in GyrA and ParC on FQR were explored by correlating QRDR changes with ciprofloxacin minimum inhibitory concentration (MIC) determinations. The in vitro models were validated by comparisons of QRDR changes and MICs in two populations of wild-type FQR N. gonorrhoeae over a wide MIC range (0.09 to 24??g/mL), and in a wild type FQR meningococcus. The in vitro activities of three newer quinolones with differential activity on GyrA and ParC were compared with that of ciprofloxacin. Key findings were that the initial QRDR changes always occurred in gyrA and were the predominant influence on phenotypic expression of FQR. QRDR alterations were acquired sequentially and two GyrA and two ParC changes represented the full complement of changes observed in gonococci and two GyrA and one ParC change those in meningococci. GyrA alterations at Ser-91 in gonococci and Thr???91 in meningococci were pivotal for the development of further resistance. ParC changes required the presence of two GyrA alterations for any major impact on FQR. ParC substitutions, Ser-87???Arg and Glu-91???Gly in gonococci and Cys- 85???Asp and Glu-91???Lys in meningococci led to the expression of the highest FQR levels. Examination of FQR in wild-type meningococci was necessarily restricted, but analyses using the broader MIC range available in in-vitro-derived FQR meningococci (0.09 to 16??g/mL) revealed the first ParC changes in N. meningitidis. The study also redefined QRDR boundaries and described novel mutations within them. The nature of sequence changes in GyrA and ParC in FQR Neisseria also affected the relative activities of the three newer quinolones. Trovafloxacin was the most active quinolone in vitro but MIC differences with ciprofloxacin were mutation-dependent. Grepafloxacin and moxifloxacin were only slightly more active than ciprofloxacin in the presence of multiple QRDR changes. This thesis provides a comprehensive analysis of the relationship between QRDR alterations and FQR in N. gonorrhoeae and offers insights into the potential for FQR development in N. meningitidis.

An epidemic of staphylococcal infections occurred in New Zealand hospitals and communities from 1955-1963. The 'H', or 'Hospital Bug', a strain of Staphylococcus aureus characteristic of the epidemic, was resistant to the most commonly used antibiotics. Post-operative patients, the frail elderly and mothers and babies were particularly vulnerable to staphylococcal colonization and infection. This thesis places the H-Bug epidemic in its historical context, discussing the ways in which the government and health professionals responded to the rising incidence of staphylococcal infection, and the major effects of the epidemic on medical and hospital practice. It also examines the impact of persistent staphylococcal infection on women and families in the community. Primary sources provided the basis for this thesis. The H-Bug epidemic has gone largely unrecorded except in contemporary documents. Health Department files and Auckland Hospital Board records as well as newspaper clippings were important sources. The New Zealand epidemic was clearly linked to the global pandemic of antibiotic resistant staphylococcal infection, 1946-1966, through medical literature and archival documents. International medical journals, including the New Zealand Medical Journal, published numerous articles on the epidemiology of antibiotic-resistant staphylococcal infection, providing an excellent record of research, case studies, current opinion, and recommended practice. The most valuable contribution to an understanding of the impact and experience of the H-Bug epidemic was, however, provided by the nineteen people who agreed to be interviewed for the study. Interviewees included a wide variety of health professionals and women and their children, all of whom had personal experience or association with the epidemic. In this thesis it is argued that the main focus of the medical response was the prevention and control of hospital cross-infection, both to protect patients and to preserve the public perception of the hospital as a safe venue for care. Although the emergence of resistant strains of staphylococci was widely attributed to the misuse of antibiotics, this thesis contends that the Health Department was reluctant to impose restrictions on medical prescribing and that Health Department official and senior clinicians chose instead to modify hospital environments and clinical practice. Rooming-in was widely introduced to counter the epidemic despite the fact that a trial in 1959, at National Women's Hospital, did not demonstrate a reduction in infection rates among neonates. The concept endured, however, as it held strong appeal for hospital administrators hard pressed to keep wards adequately staffed with trained personnel. It was also supported by women and health professionals who were convinced of the benefits of a close mother-baby relationship from birth. The H-Bug epidemic was eventually resolved by the introduction of the methicillin antibiotics in the early 1960s. As a consequence, confidence in a pharmaceutical solution to infectious disease remained intact until the emergence of multiple antibiotic resistant organisms in the 1980s. The lessons of the H-Bug epidemic had been largely forgotten in the intervening years, ignored until New Zealand clinicians were reminded once again that antimicrobial resistance would inevitably accompany the indiscriminate use of antibiotics and inadequate attention to infection prevention and control.