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Title: Fluoroquinolone resistance in Acinetobacter baumannii
Author: Hamouda, Ahmed
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2003
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Acinetobacter spp, particularly A. baumannii, are implicated in a wide spectrum of nosocomial infections, including bacteraemia, secondary meningitis, and urinary tract infections, but their most important role seems to be as agents of nosocomial pneumonia. Until 1988, the new fluorinated quinolones showed very good activity against Acinetobacter strains. However, reports of a high prevalence of fluoroquinolone resistance among Acinetobacter isolates have appeared. Therefore, understanding the mechanisms by which this phenomenon occurs in this pathogen is important. The two target proteins of the quinolones are the DNA gyrase (topoisomerase II) and topoisomerase IV. Studies with clinical isolates of A. baumannii have shown that mutations in the gyrA and parC genes were likely to be the major mechanisms of resistance. However, alteration in drug permeability or drug efflux could also contribute to quinolone resistance in A. baumannii. Initially, the purpose of this thesis is to compare the activities of the original fluoroquinolones (e.g. ciprofloxacin) with those of novel fluoroquinolones (e.g. moxifloxacin) against 47 A. baumannii isolates collected from five Scottish hospitals. MICs were determined by a standard agar dilution method and had shown that most of the new fluoroquinolones had better activity compared to ciprofloxacin. Nine clinical resistant isolates that exhibited decreased sensitivities to ciprofloxacin (MIC > 4mg/L) were investigated for their mechanisms of resistance. The mutations, in the gyrA and parC genes that might be responsible for resistance to ciprofloxacin were examined by PCR amplification, restriction fragment length polymorphism and sequencing. It was found that all but one isolate, for which the MIC of ciprofloxacin was 4mg/L, showed a change at Ser 83- Leu in the GyrA subunit. The remaining strain had a change at Gly 81-Cys in the GyrA subunit. Two strains for which the ciprofloxacin MICs were 64mg/L showed a double change of Ser 83-Leu or Gly 78- Cyst in Topoisomerase IV subunit C. The outer membrane profiles were also investigated and revealed that one isolate for which the ciprofloxacin MIC was 64mg/L lacked a 33KDa band. In vitro mutation studies, with challenges by ciprofloxacin and moxifloxacin, showed that all resistant mutants had a mutation in the gyrA gene where Serine has been substituted by Leucine at codon 83. However no parC mutations were found, even within highly resistant mutants. In addition the outer membrane profiles showed a deletion of 20 KDa band, and this might suggest that the pathway of resistance to fluoroquinolones is different between mutants and clinical isolates. By employing a biotin- and PCR- assisted capture method, a 500 bp of unknown segment of gyrB gene was amplified and sequenced, to establish if this gene was a contributory factor towards resistance.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available