Role of AcrR and RamA in fluoroquinolone resistance in clinical Klebsiella pneumoniae isolates from Singapore

The MICs of ciprofloxacin for 33 clinical isolates of K. pneumoniae resistant to extended-spectrum cephalosporins from three hospitals in Singapore ranged from 0.25 to >128 mug/ml. Nineteen of the isolates were fluoroquinolone resistant according to the NCCLS guidelines. Strains for which the ciprofloxacin MIC was greater than or equal to0.5 mug/ml harbored a mutation in DNA gyrase A (Ser83--->Tyr, Leu, or Ile), and some had a secondary Asp87--->Asn mutation. Isolates for which the MIC was 16 mug/ml possessed an additional alteration in ParC (Ser80-->Ile, Trp, or Arg). Tolerance of the organic solvent cyclohexane was observed in 10 of the 19 fluoroquinolone-resistant strains; 3 of these were also pentane tolerant. Five of the 10 organic solvent-tolerant isolates overexpressed AcrA and also showed deletions within the acrR gene. Complementation of the mutated acrR gene with the wild-type gene decreased AcrA levels and produced a two- to fourfold reduction in the fluoroquinolone MICs. None of the organic solvent-tolerant clinical isolates overexpressed another efflux-related gene, acrE. While marA and soxS were not overexpressed, another marA homologue, ramA, was overexpressed in 3 of 10 organic solvent-tolerant isolates. These findings indicate that multiple target and nontarget gene changes contribute to fluoroquinolone resistance in K pneumoniae. Besides AcrR mutations, ramA overexpression (but not marA or soxS overexpression) was related to increased AcrAB efflux pump expression in this collection of isolates.