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Title: The mechanisms of antibiotic resistance in clinical isolates of Acinetobacter, with special reference to carbapenem resistance
Author: Brown, Susan
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2000
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A total of 54 clinical isolates from Argentina, Turkey, Hong Kong, Spain and Singapore were investigated. Antimicrobial susceptibility testing detected resistance in all isolates to the majority of antimicrobials tested. A total of 69% demonstrated MIC values of imipenem above the recommended breakpoint value. A significantly lower level of resistance (16%) was observed to meropenem. A total of 61% of resistant isolates produced an unknown b-lactamase of pI 7.0 (main band). Inhibitor overlays of IEF gels demonstrated that this b-lactamase was a serine active-site enzyme that was able to bind imipenem. A modified bioassay was employed to detect imipenem hydrolysis, the rate of which was calculated as 0.75 nmoles min-1 ml-1 of sample, and the specific activity was 0.055 nmoles min-1 mg-1 of protein. Curing experiments with isolate 790 resulted in the loss of the b-lactamase of pI 7.0 and a subsequent reduction in the MIC value of imipenem to a susceptible level (16 mg/L to 0.5 mg/L). In addition, the modified microbiological assay detected an 11-fold decrease in the specific rate of imipenem hydrolysis with the cured strain (0.005 nmoles min-1 mg-1 of protein). These findings provided strong evidence that this b-lactamase (designated ARI-2) was associated with plasmid-mediated carbapenemase activity. Kinetic studies of ARI-2 following partial purification by anion exchange and gel filtration chromatography revealed that it was primarily a penicillinase. Weak hydrolysis of oxacillin and cloxacillin was also detected however, imipenem hydrolysis could not be demonstrated. The Mr of ARI-2 was estimated as 35.5 kd. Extraction of plasmid DNA of isolate 790 revealed the presence of a plasmid of approximately 40 kb in the parent but not in the cured strain which provided further evidence that the blaARI-2 gene was plasmid located. Hybridisation studied demonstrated some homology with the class D ARI-1 b-lactamase. Analysis of RFLP patterns of ARI-2-producing isolates from Argentina, Turkey, Hong Kong and Spain by PFGE indicated that horizontal transfer of the ARI-2 gene had occurred in strains from Argentina, but clonal spread was detected in the isolates from Turkey.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available