JAC Advance Access originally published online on October 24, 2006
Journal of Antimicrobial Chemotherapy 2007 59(6):1216-1222; doi:10.1093/jac/dkl426
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Special section: Efflux |
Altered spectrum of multidrug resistance associated with a single point mutation in the Escherichia coli RND-type MDR efflux pump YhiV (MdtF)
1 Center for Infectious Diseases and Travel Medicine, University Hospital D-79106 Freiburg, Germany 2 Pharmacy Service, University Hospital D-79106 Freiburg, Germany 3 Department of Medicine, Albert-Ludwigs-University D-79106 Freiburg, Germany
Received 12 July 2006; returned 12 September 2006; accepted 25 September 2006
*Correspondence address. Medizinische Universitätsklinik, Hugstetter Strasse 55, D-79106 Freiburg, Germany. Tel: +49-761-270-1819; Fax: +49-761-270-1820; E-mail: kern{at}if-freiburg.de
Objectives: YhiV (MdtF) is an resistance nodulation division (RND) type efflux 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 E12 (
acrB 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 E12 to levofloxacin 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 
-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-ß-naphthylamide (PAßN, a putative efflux pump inhibitor) compared with the E12 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 PAßN, 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 PAßN 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 efflux 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.
Keywords: E. coli , fluoroquinolones , nosocomial pathogens
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