In vitro induction and selection of fluoroquinolone-resistant mutants of Streptococcus pyogenes strains with multiple emm types

Abstract

To perform a systematic analysis of point mutations in the quinolone resistance determining regions (QRDRs) of the DNA gyrase and topoisomerase genes of emm type 6 and other emm types of Streptococcus pyogenes strains after in vitro exposure to stepwise increasing concentrations of levofloxacin. Twelve parent strains of S. pyogenes, each with a different emm type, were chosen for stepwise exposure to increasing levels of levofloxacin followed by selection of resistant mutants. The QRDRs of gyrA, gyrB, parC and parE correlating to mutants with increased MICs were analysed for point mutations. Multiple mutants with significantly increased MICs were generated from each strain. The amino acid substitutions identified were consistent regardless of emm type and were similar to the mechanisms of resistance reported in clinical isolates of S. pyogenes. The number of induction/selection cycles required for the emergence of key point mutations in gyrA and parC was variable among strains. For each parent-mutant set, when MIC increased, serine-81 of gyrA and serine-79 of parC were the primary targets for amino acid substitutions. No point mutations were found in the QRDRs of gyrB and parE in any of the resistant mutants sequenced. Despite its intrinsic polymorphism in the QRDR of parC, emm type 6 is not more likely to develop high-level resistance to fluoroquinolones when compared with other emm types. All emm types seem equally inducible to high-level fluoroquinolone resistance.