Emergence of high-level fluoroquinolone resistance in emm 6 Streptococcus pyogenes and in vitro resistance selection with ciprofloxacin, levofloxacin and moxifloxacin

Abstract

Objectives: To investigate the prevalence of fluoroquinolone resistance in Streptococcus pyogenes and its in vitro selection by ciprofloxacin and the respiratory fluoroquinolones, levofloxacin and moxifloxacin. Methods: S. pyogenes (n = 5851) recovered from pharyngitis and invasive infections during 2003-06 in Belgium were screened for fluoroquinolone non-susceptibility (ciprofloxacin MIC ≥2 mg/L) and further studied for mutations in the topoisomerase genes, reserpine-sensitive efflux, clonality by PFGE and emm typing. Fourteen well-characterized fluoroquinolone-non-susceptible or -susceptible isolates were exposed stepwise to increasing levels of ciprofloxacin, levofloxacin and moxifloxacin. Selected mutants with increased MICs were analysed for resistance mechanisms. Mutation frequencies at 2× and 4× MIC of moxifloxacin and levofloxacin were estimated for a clinical emm 6 parent strain carrying mutations in both parC and gyrA. Results: Prevalence of fluoroquinolone-non-susceptible S. pyogenes (n = 437; 7.47%) increased significantly from 2.08% and 5.08% to 13.11% during 2003-05 and decreased to 8.93% in 2006 (χ2 test; P≤0.001). emm 6 constituted 80.09% of the total fluoroquinolone-non-susceptible isolates. Of the 71 S. pyogenes sequenced, 70 harboured first-step parC or gyrA mutations correlating with ciprofloxacin MICs 2-8 mg/L. Reserpine-sensitive efflux was not observed. One emm 6 parC mutant (Ser79Ala) also showed a second-step mutation in gyrA (Ser81Tyr), with MICs of ciprofloxacin, levofloxacin and moxifloxacin of 32, 8 and 1 mg/L, respectively. Mean mutation frequencies under moxifloxacin selection were 500- to 30 000-fold higher for this strain than those for an emm 6 control strain. Selection of the emm 6 double mutant with moxifloxacin generated a mutant with a moxifloxacin MIC of 64 mg/L and a levofloxacin MIC of 128 mg/L, and an additional Asp83Tyr substitution in ParC. Conclusions: We report an emergence of levofloxacin and high-level ciprofloxacin resistance associated with a second-step gyrA mutation in a clinical emm 6 S. pyogenes. The observed high mutation frequency and in vitro selection of high-level resistance to the respiratory fluoroquinolones in the emm 6 double mutant is of concern. © The Author 2009. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.