In vitro selection of resistance to clindamycin related to alterations in the attenuator of the erm(TR) gene of Streptococcus pyogenes UCN1 induciblyresistant to erythromycin

A clinical isolate of Streptococcus pyogenes UCN1 intermediate to erythromy cin (MIC 1 mg/L) and susceptible to clindamycin (MIC 0.03 mg/L) harboured a n inducible erm(TR) gene encoding a ribosomal methylase. We have selected i n vitro, in the presence of concentrations of clindamycin ranging from 0.12 to 1 mg/L, one-step mutants that are highly resistant to this antibiotic ( MIC 64 mg/L) at a frequency of 10(-7). By contrast, in an erythromycin-susc eptible strain of S. pyogenes UCN5, mutants could be selected only by a low concentration of clindamycin (0.12 mg/L) at a frequency of 10(-9). Clindam ycin resistance in four of six S. pyogenes UCN1 mutants was associated with deletions of 163 and 6 bp, as well as a tandem duplication of 101 by in th e regulatory sequence of the erm(TR) gene. The role of these structural alt erations in clindamycin resistance was demonstrated by cloning the erm(TR) gene from the wild-type and mutant strains in Escherichia coli DB10, a muta nt susceptible to macrolides. Clindamycin resistance was expressed only whe n the erm(TR) gene was preceded by an altered attenuator. Mutations could l ead to the formation of mRNA secondary structures accounting for the access ibility of the ribosome-binding site and the initiation codon of the ErmTR methylase to the ribosomes, and subsequently for the translation of the erm (TR) transcripts. The easy selection in one step of mutants resistant to hi gh levels of clindamycin by concentrations of this antibiotic ranging from four to 40 times the MIC leads us to recommend caution in the use of clinda mycin therapy in group A Streptococcus infections.