Characterisation of DNA gyrase and measurement of drug accumulation in clinical isolates of Acinetobacter baumannii resistant to fluoroquinolones

Twelve clinical isolates of Acinetobacter baumannii highly resistant to pefloxacin (MIC ≥ 32 mg/L) and to ciprofloxacin (MIC ≥ 16 mg/L), were studied. A susceptible isolate used as a reference (MIC of 0.032 and 0.25 mg/L for ciprofloxacin and pefloxacin, respectively) accumulated 85 mg of pefloxacin per litre of cell volume within 10 min, from a solution containing 10 mg/L of antibiotic. One resistant isolate accumulated the same amount of pefloxacin, while the 11 others accumulated between 40 and 70 mg/L of cell volume. The differences between reference and resistant isolates with respect to ciprofloxacin and sparfloxacin accumulation were less pronounced. There were no apparent differences in the outer membrane protein profiles of susceptible and resistant isolates. DNA gyrase was isolated from four A. baumannii and the minimum concentration of fluoroquinolones, required to inhibit gyrase-catalysed supercoiling of plasmid DNA was 5- to 80-fold higher for the resistant isolates than for the reference strain. Although most isolates showed some degree of reduced fluoroquinolone accumulation, a DNA gyrase mutation was more likely to be the main mechanism of the high level resistance encountered.