Altered spectrum of multidrug resistance associated with a single point mutation in the Escherichia coli RND-type MDR efflux pump YhiV (MdtF).
OBJECTIVE
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 (DeltaacrB DeltaacrF) 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 lambda-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 (PAbetaN, 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 PAbetaN, 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 PAbetaN 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.
Bohnert
Jürgen A
JA
Center for Infectious Diseases and Travel Medicine, University Hospital, Freiburg, Germany.
Schuster
Sabine
S
Fähnrich
Eva
E
Trittler
Rainer
R
Kern
Winfried V
WV
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000235:genetics*; D000081:Acetamides / Q000378:metabolism; D000483:Alleles; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000235:genetics; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics*; D064704:Levofloxacin; D000069349:Linezolid; D026901:Membrane Transport Proteins / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D015242:Ofloxacin / Q000494:pharmacology; D023303:Oxazolidinones / Q000378:metabolism; D010641:Phenotype; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000378:metabolism; D017354:Point Mutation / Q000235:genetics*; D012329:RNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018435:ATP Binding Cassette Transporter, Subfamily B; D000081:Acetamides; D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D018076:DNA, Complementary; D029968:Escherichia coli Proteins; C477833:MdtF protein, E coli; D026901:Membrane Transport Proteins; D023303:Oxazolidinones; D012329:RNA, Bacterial; D010649:Phenylalanine; D064704:Levofloxacin; C079641:phenylalanine-beta-naphthylamide; D015242:Ofloxacin; D000069349:Linezolid