Metronidazole resistance in clinical Bacteroides fragilis isolates from Groote Schuur Hospital, Cape Town, South Africa

Includes bibliographical references.Bacteroides fragilis, an anaerobic gut commensal and opportunistic pathogen, is a leading cause of anaerobic abscesses and bacteraemias. Treatment of infections is complicated by the emergence of resistance to several of the antibiotics, specifically metronidazole, used in the clinical setting. The aim of this thesis was to examine the levels of antibiotic resistance of 23 B. fragilis strains isolated at Groote Schuur Hospital, Cape Town, and determine the metronidazole (Mtz) resistance mechanisms present in order to evaluate the clinical risk of the spread of drug resistance. It also reports the identification and functional characterisation of a putative, novel nim gene in the B. fragilis 638R genome. Measurement of the minimum inhibitory concentration of the strains to antibiotics showed that 8% were highly resistant to imipenem and cefoxitin and 65% to tetracycline. All strains were sensitive to clindamycin. Two strains, B. fragilis GSH8 and GSH15 were Mtz resistant and strain GSH15 showed multidrug resistance to metronidazole, imipenem, cefoxitin and tetracycline. The genetic basis of the resistance to the various antibiotics was examined and could largely be attributed to the presence of previously published resistance genes. There were several exceptions to this which were investigated further at the genetic level with particular focus on imipenem and Mtz. In the cases of Mtz resistance, PCR screening did not detect any of the known nim genes but both strains showed increased lactate dehydrogenase (LDH) activity suggesting the involvement of the LDH/ pyruvate: ferredoxin oxidoreductase (PFOR) pathway. However, there was no reciprocal decrease in PFOR activity so the LDH/ PFOR pathway was not involved in the observed Mtz resistance. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) revealed that induction by exposure to sub-lethal doses of Mtz caused a slight increase in transcription of the efflux gene bmeB5. However, this was not statistically significant and still did not account for the observed Mtz resistance. A putative nim-like gene was identified on the B. fragilis 638R genome and was present in most of the strains examined. Phylogenetic and three dimensional analysis of the derived amino acid sequence revealed that the 638Rnim was more closely related to NimA from Deinococcus radiodurans (drNimA) than to any of the other nim genes. Heterologous overexpression of 638Rnim in an Escherichia coli Mtz sensitive mutant resulted in a 3-fold increase in Mtz resistance as compared to the control (6 vs 2 mg/L respectively). However, its possible role in Mtz resistance in B. fragilis could not be confirmed by overexpression or interruption of the gene in the homologous host. qRT-PCR showed that increased transcription of the gene approached statistically significant levels upon Mtz induction. This nim-like gene, therefore, warrants further investigation under a range of different induction conditions. The findings of this research provide useful information on the state of B. fragilis antibiotic resistance present in Groote Schuur Hospital and the incidence of the associated resistance genes. It also identifies and presents a preliminary characterisation of a putative novel nim gene as a basis for further functional studies on Mtz resistance mechanisms

Prevalence of gastrointestinal pathogenic bacteria in patients with diarrhoea attending Groote Schuur Hospital, Cape Town, South Africa

Background. Diarrhoea due to gastrointestinal infections is a significant problem facing the South African (SA) healthcare system. Infections can be acquired both from the community and from the hospital environment itself, the latter acting as a reservoir for potential pathogenic bacteria.Objectives. To examine the prevalence of a panel of potential diarrhoea-causing bacteria in patients attending a tertiary healthcare facility in Cape Town, SA.Methods. Polymerase chain reaction (PCR) primers specific for Clostridium difficile, Shigella spp., Salmonella spp., Klebsiella oxytoca, enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC/EHEC), Staphylococcus aureus, enterotoxigenic Bacteroides fragilis and Campylobacter spp. were used to screen total bacterial genomic DNA extracted from stool samples provided by 156 patients with diarrhoea attending Groote Schuur Hospital, Cape Town, SA.Results. C. difficile was the most frequently detected pathogen (16% of cases) in the 21 - 87-year-old patient range, but was not present in samples from the 16 - 20-year-old range. K. oxytoca (6%), EPEC/EHEC strains (9%) and S. aureus (6%) were also detected. The remaining pathogens were present at low frequencies (0 - 2.9%), and the occurrence of mixed infections was 5%. The majority of non-C. difficile-related diarrhoeas were community acquired.Conclusion. C. difficile was the main cause of infectious diarrhoea in the sampled patients, while K. oxytoca and EPEC/EHEC strains were present as relatively minor but potentially significant pathogens