Selection of Enterococcus faecium strains with stable and unstable resistance to the streptogramin RP 59500 using stepwise in vitro exposure

Therapy of vancomycin-resistant enterococcal infection is an increasing problem for many large medical centers. One promising agent for use against Enterococcus faecium is RP 59500 (Quinupristin/Dalfopristin). To assess the potential for emergence of resistant strains during clinical trials with this new compound, we collected and tested 11 vancomycin-resistant and three vancomycin-susceptible E. faecium strains. The strains were exposed to doubling dilutions of RP 59500, beginning at drug concentrations ranging from 0.25 to 16 μg/ml agar. A saturated swab with approximately 3 x 10⁷ organisms/ml was spread as a lawn on agar containing RP 59500 and incubated at 35°C for 48 h. Growth on the highest drug-containing plate was used to prepare the inoculum for the next series of resistance-selection experiments. After the final passage, the range of the highest RP 59500 plate concentration with growth was 32-512 μg/ml (initial resistance frequency ranging from 1 x 10^-6 to >1 x 10^-4). After the selection of resistant strains, the organisms were passed twice weekly on antimicrobial agent-free media to determine resistance stability in the absence of drug. Resistance in strains with an RP 59500 MIC of ≤16 μg/ml was stable after repeated passage for 4 weeks. These results indicate that stable resistance to RP 59500 can be selected in E. faecium when the organism is exposed to increasing drug concentrations only slightly above the minimum inhibitory concentration (MIC). This stable resistance is seen in strains exhibiting an MIC ≤16 μg/ml, and unstable in those with resistance where the RP 59500 MIC is less than eight times the original drug MIC. Our observation suggests more than one mechanism of resistance is likely present when stable resistance to RP 59500 develops.