Persistence of rRNA operon mutated copies and rapid re-emergence of linezolid resistance in Staphylococcus aureus

Abstract

Objectives: The G2576T mutation in domain V of 23S rRNA has been most often associated with the rare cases of linezolid resistance in Staphylococcus aureus. In a linezolid-susceptible S. aureus (A8761B) possessing a single mutated (G2576T) copy, originally derived from a resistant clinical isolate, we assessed the persistence of the mutation on further passage on antibiotic-free medium and the selection of resistance upon re-exposure of the susceptible strain to linezolid.

Methods: The stability of the mutant rRNA copy was tested through 40 serial passages on antibiotic-free medium. The re-emergence of linezolid-resistant mutants was examined after serial passage on successively increasing linezolid concentrations. The efficacy of novobiocin, at subinhibitory concentrations, to prevent or delay the emergence of resistant mutants was examined. Strain relatedness was confirmed by PFGE and domain V of individual rRNA copies was sequenced.

Results: After 40 passages in antibiotic-free medium, the linezolid MIC of derived strain A9584 remained stable at 2 mg/L and the G2576T mutation persisted in one 23S rRNA gene copy (copy number 2). Upon re-exposure of the strain to increasing concentrations of linezolid, linezolid resistance (MIC of 64 mg/L) emerged rapidly. In a representative derivative (A9753), the G2576T mutation was found in four of the five rRNA copies. All laboratory derivates were closely related by PFGE. When A9584 was applied to plates containing linezolid at 4 x MIC, resistant colonies emerged at a frequency of 8 x 10(-6). Novobiocin at 1/4 x MIC prevented the emergence of resistant colonies.

Conclusions: The persistence of the G2576T mutation in one rRNA operon copy in the absence of selective pressure suggests that the mutation has a minimal impact on the organism's fitness in vitro. Resistance to linezolid, associated with acquisition of multiple mutant copies, emerges rapidly upon re-exposure to linezolid. Novobiocin, predicted to interfere with gene conversion, may reduce the likelihood of rapid development of linezolid resistance.