The Genetic Environment of thecfrGene and the Presence of Other Mechanisms Account for the Very High Linezolid Resistance of Staphylococcus epidermidis Isolate 426-3147L

Abstract

ABSTRACTThe clinicalStaphylococcus epidermidisisolate 426-3147L exhibits an unusually high resistance to linezolid that exceeds 256 μg/ml. The presence of thecfrgene, encoding the RNA methyltransferase targeting an rRNA nucleotide located in the linezolid binding site, accounts for a significant fraction of resistance. The association ofcfrwith a multicopy plasmid is one of the factors that contribute to its elevated expression. Mapping of thecfrtranscription start sites identified the nativecfrpromoter. Furthermore, analysis of thecfrtranscripts inStaphylococcus epidermidis426-3147L showed that some of them originate from the upstream plasmid-derived promoters whose activity contributes to efficientcfrtranscription. The genetic environment of thecfrgene and its idiosyncratic transcription pattern result in increased activity of Cfr methyltransferase, leading to a high fraction of the ribosomes being methylated at A2503 of the 23S rRNA. Curing of theStaphylococcus epidermidis426-3147L isolate from thecfr-containing plasmid reduced the linezolid MIC to 64 μg/ml, indicating that other determinants contribute to resistance. Nucleotide sequence analysis revealed the presence of the C2534T mutation in two of the six 23S rRNA gene alleles as well as the presence of mutations in the genes of ribosomal proteins L3 and L4, which were previously implicated in linezolid resistance. Thus, the combination of resistance mechanisms operating through alteration of the drug target site appears to cause an unusually high level of linezolid resistance in the isolate.