Altered spectrum of multidrug resistance associated with a single point mutation in the Escherichia coli RND-type MDR efflux pump YhiV (MdtF)

Objectives: YhiV (MdtF) is an resistance nodulation division (RND) type eff lux pump in Escherichia coli with significant homology to AcrB but usually expressed at a low level in clinical isolates. When overexpressed the pump confers decreased susceptibility to a variety of substances including erythromycin and ethidium bromide (EtBr). We characterized two mutants of E. coli El 2 (A acrB A acrF) overexpressing yhiV that showed surprising differences in their spectrum of multidrug resistance (MDR). Methods: The two mutants obtained after repeated exposure of E. coli El 2 to levof loxacin were tested for antimicrobial susceptibility to a variety of agents and for intracellular accumulation of selected pump substrates. Gene expression was studied by quantitative RT-PCR, and yhiV was sequenced. Gene inactivation and replacement were done by phage X-based homologous recombination. Results: Mutant DKO20/1 overexpressed yhiV, showed a wild-type yhiV sequence and had >2-fold increased MICs of fluoroquinolones, novobiocin, macrolides/ketolides, EtBr, oxacillin and Phe-Arg-beta-naphthylamide (PA beta N, a putative eff lux pump inhibitor) compared with the El 2 parent. A second mutant, strain DKO1/17 that had the Val-610,Phe point mutation in YhiV differed from DKO20/1 by faster growth, >2-fold increased MICs of linezolid and tetracycline, but >2-fold decreased MICs of PA beta N, azithromycin and telithromycin. Inactivation of yhiV in DKO1 /17 and reintroduction of the wild-type and mutant yhiV sequence confirmed that the differing MICs of most of the drugs were associated with the observed single point mutation. Intracellular drug accumulation studies with linezolid and PA beta N were consistent with the MIC results. Conclusions: The region around amino acid Val-610 in YhiV appears to be involved in determining recognition and efficiency of export of a number of MDR eff lux pump substrates. This single point mutation in the periplasmic loop of the pump can increase resistance to a given drug such as a fluoroquinolone while decreasing resistance to another one.