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We identified erm(A)-harbouring Streptococcus pyogenes that expressed three variant phenotypes: (1) low-level resistance to erythromycin (MICs 1–4 mg/L) but high azithromycin MICs in absolute terms (16–64 mg/L; n=6); (2) same as (1) but with a high clindamycin MIC (256 mg/L; n=1); and (3) high-level constitutive MLS (cMLS) resistance (n=1). Here we analysed the genetic basis of these novel phenotypes.The presence of erm(A) and the absence of macrolide/lincosamide resistance genes erm(B), mef and cfr were confirmed by PCR. erm(A), 23S rRNA, L4 and L22 genes were sequenced. Mutant erm(A) genes were cloned and electrotransformed into the macrolide-susceptible Escherichia coli AG100A. Clonality was determined by emm typing and PFGE. Effects of the identified mutations on free energy changes (ΔG) and putative configurations of the leader sequence were studied in silico.Point mutations (G98A, A137C, C140T and G205A) were observed in the erm(A) regulatory region of all eight erm(A)-harbouring S. pyogenes. Five and two isolates belonged to emm77 and emm89 clones, respectively, and one isolate was an emm1. E. coli transformed with mutant erm(A) harbouring G98A, A137C or C140T mutations (phenotypes 1 and 2) did not express high-level azithromycin or clindamycin resistance. However, cMLS resistance was clearly observed in transformants with erm(A) harbouring both A137C and G205A mutations (phenotype 3). In silico analysis showed that ΔG was minor except for the G205A mutation. Secondary structure predictions further showed that the A137C and G205A mutations together abolished the hairpin sequestering the ribosome-binding and initiation sites of the erm(A) gene, explaining the cMLS phenotype 3.We report point mutations in the erm(A) regulatory region leading to constitutive methylase expression and the presence of additional, as yet unidentified mechanisms mediating high-level azithromycin and clindamycin resistance in erm(A)-harbouring S. pyogenes.