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Title: Antibiotic resistance in Helicobacter pylori
Author: Chatsuwan, Tanittha
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2003
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Helicobacter pylori is a gram negative, microaerophilic bacteria that plays an important role in chronic gastritis and peptic ulcer disease. Multiple antimicrobial therapies including combinations of clarithromycin, metronidazole or amoxycillin and proton pump inhibitor have been used to eradicate H. pylori. Antimicrobial resistance in H. pylori has been associated with treatment failure. In this study, antimicrobial susceptibility patterns of amoxycillin, ciprofloxacin, clarithromycin, erythromycin, tetracycline and metronidazole were evaluated in 110 H. pylori strains isolated from 454 antral biopsies of patients undergoing endoscopy at the Royal Infirmary, Edinburgh. The MICs were determined by E-test. Resistance to clarithromycin and erythromycin was found in 8.2% (9/110) and 9.1% (10/110) respectively. Metronidazole resistance was demonstrated in 8 isolates (7.3%). Tetracycline resistance was found in one of the isolates (0.9%). Two isolates were resistant to ciprofloxacin (1.8%). Resistance to amoxycillin was not detected. Molecular mechanisms of fluoroquinolone, macrolide and metronidazole resistance in H. pylori were investigated. Resistant to fluoroquinolone has been associated with alterations in the Quinolone Resistance-Determining Region (QRDR) of gyrA gene. Mutation at position 91, leading to an amino acid change from Aspartic acid to Asparagine was found in 2 ciprofloxacin-resistant isolates. One isolate had a mutation at Asparagine-87 to Lysine. Mutations in the 23S rRNA conferring macrolide resistance were investigated. Mutations at position 2143 (A to G) were shown in seven of the ten macrolide-resistant isolates. Two of the seven isolates carried an additional T to C mutation at either position 2182 or 1934. Of the ten macrolide-resistant isolates, two had a single mutation at either position 2182 or 2195. Mutation at position 2182, however, has previously been identified not to be associated with macrolide resistance. The mutations at position at 1934 (T to C) and position 2195 (C to T) have not previously been reported. One of the ten isolates (MIC > 256 mg/L) had no alteration in the 23S rRNA. The results indicate that different mechanisms play a role in macrolide resistance in these H. pylori strains. Metronidazole resistance has been reported to be associated with mutations in the rdxA gene, encoding oxygen-insensitive nitroreductase. To investigate the role of rdxA, sequencing analysis of rdxA of metronidazole-resistant isolates was determined. The results showed that no particular amino acid substitution was associated with metronidazole resistance. One isolate contained a nonsense mutation, generating a stop codon. However, two metronidazole-sensitive strains had alterations in rdxA by insertions of a mini-IS605 sequence. These results suggest that alterations in rdxA are not the sole mechanism of metronidazole resistance and other mechanisms are required in the development of resistance. Since the prevalence rate of metronidazole resistance is variable, ranging from 11 to 70%, it is possible that some variation in reported resistance levels derives from difficulties in the method of sensitivity testing. To set a standard for susceptibility testing for metronidazole in H. pylori, the optimum conditions for sensitivity testing were evaluated. Activation of metronidazole requires an anaerobic environment. It was found that incubation under microaerophilic conditions elevated metronidazole MIC, suggesting that microaerophilic conditions cannot activate metronidazole to its active form. Pre-incubation of H. pylori in anaerobic conditions for 24 hours prior to incubation under microaerophilic conditions for 72 hours was found to be necessary to achieve accurate susceptibility results. This can explain why some centres report high levels of metronidazole resistance. To investigate the development of fluoroquinolone resistance in H. pylori, ciprofloxacin-resistant mutants were selected in vitro by exposing sensitive strains to serial increments of ciprofloxacin in Columbia blood agar plate. The QRDR of gyrA gene was analysed for mutations. Reduced susceptibility to ciprofloxacin was associated with either a single or double amino acid changes in gyrA gene. Mutations at position 85, 87 and 91 were found to be associated with ciprofloxacin resistance. The results also demonstrated that gyrA mutations are not sole contributors for the mechanism of ciprofloxacin resistance in H. pylori. As no parC gene has not yet been identified in H. pylori, any contribution from topoisomerase IV cannot be quantified. To investigate the DNA gyrase activity in H. pylori, gyrA and gyrB were separately cloned and overexpressed by using a T7 promoter vector, which contain a fusion tag of six histidine residues. GyrA and GyrB were purified by affinity chromatography using nickel-chelating resins. The ability of DNA gyrase to supercoil relaxed DNA was determined.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available