Delayed development of linezolid resistance in Staphylococcus aureus following exposure to low levels of antimicrobial agents

Abstract

The development of resistance to linezolid (LZD) in gram-positive bacteria depends on the mutation of a single 23S rRNA gene, followed by homologous recombination and gene conversion of the other alleles. We sought to inhibit this process in Staphylococcus aureus using a range of antibacterial agents, including some that suppress recombination. A model for the rapid selection of LZD resistance was developed which allowed the selection of LZD-resistant mutants with G2576T mutations in all five copies of the 23S rRNA gene following only 5 days of subculture. The emergence of LZD-resistant isolates was delayed by exposing cultures to low concentrations of various classes of antibiotics. All antibiotic classes were effective in delaying the selection of LZD-resistant mutants and, with the exception of fusidic acid (FUS) and rifampin (RIF), prolonged the selection window from 5 to approximately 15 days. Inhibitors of DNA processing were no more effective than any other class of antibiotics at suppressing resistance development. However, the unrelated antimicrobials FUS and RIF were particularly effective at preventing the emergence of LZD resistance, prolonging the selection window from 5 to 25 days. The enhanced suppressive effect of FUS and RIF on the development of LZD resistance was lost in a recA-deficient host, suggesting that these drugs affect recA-dependent recombination. Furthermore, FUS and RIF were shown to be effective inhibitors of homologous recombination of a plasmid into the staphylococcal chromosome. We suggest that RIF or FUS in combination with LZD may have a role in preventing the emergence of LZD resistance.

FIG. 1.

Timeline for emergence of mutants resistant to LZD during subculture in the presence of LZD (4× the MIC) and the presence or absence of a second agent or agents (0.25× the IC₅₀). (A) Laboratory strain RN4220; (B) clinical strain EMRSA-15; (C) recA-deficient RN4220 derivative KB103; (D) KM183, an intermediate-level LZD^r RN4220 derivative with three G2576T 23S rRNA mutant copies.