Mechanisms and Frequency of Resistance to Gatifloxacin in Comparison to AM-1121 and Ciprofloxacin in Staphylococcus aureus

Abstract

Gatifloxacin, an 8-methoxyfluoroquinolone, was found to be two-to fourfold more active against wild-type Staphylococcus aureus ISP794 than its desmethoxy derivative, AM-1121, and ciprofloxacin, another desmethoxy fluoroquinolone. Single grlBA mutations caused two-to fourfold increases in the MIC of gatifloxacin, and a single gyrase mutation was silent. Double mutations in gyrA and grlA or grlB caused a 32-fold increase in the MIC of gatifloxacin, in contrast to a 128-fold increase for ciprofloxacin and AM-1121. Overexpression of the NorA efflux pump had minimal effect on the MIC of gatifloxacin. The bactericidal activity of the three quinolones at four times the MIC differed only for a double mutant, with gatifloxacin exhibiting a killing pattern similar to that for ISP794, whereas ciprofloxacin and AM-1121 failed to show any killing. With gatifloxacin, selection of resistant mutants at twice the MIC was 100-to 1,000-fold less frequent than with the comparison quinolones, and mutants could rarely be selected at four times the MIC. The limit resistance in ISP74 was 512 times the MIC of gatifloxacin and 1,024 times the MICs of ciprofloxacin and AM-1121. Novel mutations in topoisomerase IV were selected in five of the six single-step mutants, three of which were shown to cause quinolone resistance by genetic studies. In conclusion, topoisomerase IV is the primary target of gatifloxacin. In contrast to comparison quinolones, mutations in both topoisomerase IV and gyrase are required for resistance to gatifloxacin by clinical breakpoints and do not abolish bactericidal effect, further supporting the benefit of the 8-methoxy substituent in gatifloxacin.

Quinolone Resistance Due to Reduced Target Enzyme Expression

“…The MICs of premafloxacin and ciprofloxacin were fourfold greater for mutant P18 than for wild-type strain ISP794 (Table 3); this difference in MICs was similar to the differences observed for other first-step mutants with parEC mutations selected with premafloxacin (20). The MICs of novobiocin (which was used to screen for some parE mutations [11,12,20,21,23,24]) and nalidixic acid (which was used to screen for some gyrA mutations [23]) were not different for the mutant and the wild type. Because other premafloxacin-selected, first-step mutants of S. aureus had had mutations in parE or parC (20), we first sequenced the quinolone resistance-determining regions of the parE and parC structural genes of mutant P18.…”

“…The PCR primers used to amplify a 652-bp internal fragment of parE together with the 378-bp upstream region and the conditions used for PCRs have been described previously (21). Cloning into the thermosensitive shuttle vector pCL52.1 and the allelic exchange procedure were performed as previously described (21).…”

“…The PCR primers used to amplify a 652-bp internal fragment of parE together with the 378-bp upstream region and the conditions used for PCRs have been described previously (21). Cloning into the thermosensitive shuttle vector pCL52.1 and the allelic exchange procedure were performed as previously described (21). The resultant colonies following the allelic exchange procedure were screened for susceptibility to tetracycline at a concentration of 3 g/ml and for resistance to premafloxacin and ciprofloxacin at concentrations of 0.008 and 0.25 g/ml, respectively.…”

Real-Time PCR Assay for Detection of Fluoroquinolone Resistance Associated with grlA Mutations in Staphylococcus aureus

“…The low level of resistance to ciprofloxacin and reduced susceptibility to the new fluoroquinolones suggested that this strain may have a single mutation at codon 80 of grlA. Therefore, these data corroborated those from others which have shown that a single grlA mutation is not sufficient to cause clinical resistance to levofloxacin and gatifloxacin but greatly decreased the susceptibility to these antimicrobial agents (9,30). …”

“…Indeed, 96% (49 of 51) of the fluoroquinolone-resistant S. aureus isolates tested in this study exhibited high-level resistance to ciprofloxacin (MIC, Ͼ8 g/ml). This high level of resistance is usually associated with mutations in grlA as well as in gyrA, gyrB, and/or grlB genes which generate cross-resistance to new fluoroquinolones (4,9,26). Among the 85 S. aureus isolates studied, 56 were resistant to oxacillin (MRSA strains).…”