a Eijkman-Winkler Institute for Clinical Microbiology, University Hospital Utrecht, Utrecht 3584CX, The Netherlands; b Institute for Medical Microbiology and Virology, Heinrich-Heine University, Düsseldorf 40225, Germany
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Abstract |
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Introduction |
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In Staphylococcus aureus, resistance to quinolones is primarily mediated through chromosomal point mutations in gyrA and gyrB (encoding subunits of DNA gyrase) and grlA and grlB (encoding subunits of DNA topoisomerase IV).5,6 Together these comprise the so-called quinolone-resistance-determining regions (QRDRs). Additionally, norA encodes an efflux pump contributing to reduced susceptibility. High levels of resistance can be achieved through the accumulation of mutations in QRDRs and the promoter region of norA.7 Earlier studies in Escherichia coli have demonstrated that the common mutations in gyrA have little or no effect on catalytic function of the gyrase.8 However, these studies in E. coli also detected rarer mutations in gyrA which did show a reduction in gyrase activity. By inference, it is therefore possible that some mutation types in the topoisomerase or gyrase of S. aureus may disadvantage the cell compared with wild-type organisms. For this study the hypothesis was made that strains of S. aureus with mutations in QRDRs giving rise to reduced susceptibility to ciprofloxacin would not be at a selective disadvantage relative to sensitive revertants spontaneously appearing in culture containing no antibiotic, and thus no detectable change in the culture composition would occur. Additional mutations may well arise in organisms grown with sub-MIC ciprofloxacin concentrations. Here we report the genotypic and phenotypic stability of quinolone resistance in S. aureus in an environment with or without a quinolone antibiotic selective pressure.
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Materials and methods |
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Organisms from cultures equating to 0, 20, 40, 80, 160, 200 and 500 generations of binary fission were studied. For each sample, dilutions of bacterial culture were made and approximately 75100 colonies were replica plated on to MuellerHinton agar plates containing dilutions of ciprofloxacin (provided by Bayer AG, Wüppertal, Germany) extending below and above the generation-zero MIC of the study isolates (1 and 128 mg/L, respectively). These experiments were done in duplicate using different sample dilutions. This allowed the screening of approximately 150200 colonies from each test culture for raised or lowered MICs compared with the generation-zero value. In the case of no detectable changes using this screening method, ten colonies from each of the four study strains were randomly selected from the replica plate containing the same antibiotic concentration as the study broth culture, and ciprofloxacin MICs were redetermined for each using an agar dilution method according to NCCLS recommended guidelines.9 Target DNA molecules were also sequenced with methodologies defined previously by our group.11
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Results and discussion |
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These data show that at least for these isolates clinical of S. aureus and ciprofloxacin the withdrawal of antibiotic from the environment does not reduce the proportion of resistant bacteria or levels of resistance. In addition, the characterized mutations within the QRDRs, in particular a single first-step mutation conferring low-level ciprofloxacin resistance in S. aureus NB1 and multiple mutations in different gene loci conferring high-level resistance in S. aureus NB126, remained stable in an antibiotic-free environment. Without a positive selection pressure, spontaneous reverse mutations occurring at any of the characterized mutated sites (presumably at a rate of approximately 106107 per generation) would not have been detected in this experiment. Furthermore, new mutations induced by a sub-MIC antibiotic selective pressure were detected, and rapidly stabilized within cultures. It is worth noting that there may be other as yet unknown factors operating in vivo which favour selection of susceptible revertants; investigation of this would require animal studies. Identical mutations are responsible for ciprofloxacin resistance in other unrelated strains of S. aureus. In addition, DNA gyrase and topoisomerase IV genetic and functional homologues have been characterized in a number of other Gram-positive and Gram-negative species, and it is likely that the conclusions of this study could be extrapolated to organisms other than S. aureus. Finally, stability of ciprofloxacin resistance and of the mutations in the QRDRs conferring it have been observed by our group in clonal lineages of S. aureus in the clinical environment over a period of years.12
Quinolones have undergone a renaissance in recent years and several newly developed compounds will soon be available for clinical use. Although these compounds may be more active, they all share the same topoisomerase and gyrase target sites, which are largely homologous among different microorganisms. It seems reasonable to assume that mutations conferring resistance to these new quinolones will behave in a similar manner, in terms of stability, to those characterized in this study. These studies strongly suggest that for quinolone antibiotics the immediate implementation of prudent prescribing practices may be the wisest strategy to limit emergence of resistance, since reversing a high incidence of quinolone resistance may not be achievable simply by withdrawing the antibiotics from clinical use.
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Notes |
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References |
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Received 16 June 1999; returned 28 September 1999; revised 9 November 1999; accepted 22 November 1999