Gene Cloning and Characterization of EfmA, a Multidrug Efflux Pump, from Enterococcus faecium

Abstract

A DNA fragment responsible for resistance to antimicrobial agents was cloned from chromosomal DNA of Enterococcus faecium FN-1, a clinically isolated strain. Escherichia coli KAM32, a drug-hypersusceptible mutant, was used as a host for gene cloning. Cells of E. coli KAM32 harboring a recombinant plasmid (pTFM8) carrying the DNA fragment became resistant to fluoroquinolones, macrolides, ethidium bromide, 4′,6-diamidino-2-phenylindole (DAPI) and tetraphenylphosphonium chloride (TPPCl). Three complete open reading frames (ORFs) were found in the DNA insert of pTFM8, and the deduced amino acid sequences of one of the ORFs showed high similarity to Mdt(A) from Lactococcus lactis. Mdt(A) is a multidrug efflux pump belonging to a major facilitator superfamily. We designated the ORF efmA. E. coli KAM32 cells harboring the efmA showed energy-dependent efflux of DAPI and TPP⁺. We also observed norfloxacin/H⁺ antiport due to EfmA. The mRNA expression of efmA was observed in E. faecium FN-1 grown without any exogenously added antimicrobial agents. Thus, we conclude that efmA is constitutively expressed under laboratory growth conditions and would contribute to intrinsic resistance against multiple antimicrobial agents in E. faecium FN-1.