Topoisomerase targeting with and resistance to gemifloxacin in Staphylococcus aureus

Abstract

Gemifloxacin, a novel quinolone with potent activity against Staphylococcus aureus, was 8- to 16-fold more active against wild-type S. aureus than ciprofloxacin. The two- to fourfold increase in the MIC of gemifloxacin in genetically defined grlBA mutants and the twofold increase in a single gyrA mutant, supported by the low frequency of selection of resistant mutants at twice the MIC (7.4 x 10(-11) to 1.1 x 10(-10)), suggested similar targeting of the two enzymes by gemifloxacin. Dual mutations in both gyrase and topoisomerase IV caused a 64- to 128-fold increase in the MIC of gemifloxacin, similar to that seen with ciprofloxacin. Gemifloxacin also had similar activity in vitro against topoisomerase IV and gyrase purified from S. aureus (50% inhibitory concentrations of 0.25 and 0.31 micro g/ml, respectively). This activity was 10- to 20-fold higher than that of ciprofloxacin for topoisomerase IV and 33-fold higher than that for gyrase. In contrast to the in vitro findings, only topoisomerase IV mutants were selected in first-step mutants. Overexpression of the NorA efflux pump had a minimal effect on resistance to gemifloxacin, and a mutation in the promoter region of the gene for NorA was selected only in the sixth step of serial selection of mutants. Our data show that although gemifloxacin targets purified topoisomerase IV and gyrase similarly in vitro, topoisomerase IV is the preferred target in the bacteria. Selection of novel resistance mutations in grlA requires further expansion of quinolone-resistance-determining regions, and their study may provide increased insight into enzyme-quinolone interactions.

FIG. 1.

Decatenation of kDNA by wild-type GrlA and GrlB in the presence of gemifloxacin and ciprofloxacin. Assays were performed as described in Materials and Methods. The letter M indicates minicircles.

FIG. 2.

Decatenation of kDNA by GrlA (Ser80Phe) and wild-type GrlB in the presence of gemifloxacin and ciprofloxacin. The letter M indicates minicircles.

FIG. 3.

DNA supercoiling activity of gyrase in the presence of gemifloxacin and ciprofloxacin. R and S indicate relaxed and supercoiled DNA, respectively.