Emergence of high-level fluoroquinolone resistance in emm6 Streptococcus pyogenes and in vitro resistance selection with ciprofloxacin, levofloxacin and moxifloxacin

To investigate the prevalence of fluoroquinolone resistance in Streptococcus pyogenes and its in vitro selection by ciprofloxacin and the respiratory fluoroquinolones, levofloxacin and moxifloxacin. 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 2x and 4x MIC of moxifloxacin and levofloxacin were estimated for a clinical emm6 parent strain carrying mutations in both parC and gyrA. 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 (chi(2) test; P <= 0.001). emm6 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 emm6parC 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 emm6 control strain. Selection of the emm6 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. We report an emergence of levofloxacin and high-level ciprofloxacin resistance associated with a second-step gyrA mutation in a clinical emm6 S. pyogenes. The observed high mutation frequency and in vitro selection of high-level resistance to the respiratory fluoroquinolones in the emm6 double mutant is of concern.