Gene cloning and characterization of EfmA, a multidrug efflux pump, from Enterococcus faecium
Toshihiro Nishioka1, Wakano Ogawa, Teruo Kuroda
1Laboratory of Molecular Microbiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Japan.
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.
Insights
A novel gene, efmA, cloned from Enterococcus faecium, confers broad-spectrum antimicrobial resistance in E. coli. This gene encodes a multidrug efflux pump, contributing to intrinsic resistance in E. faecium.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Enterococcus faecium is a significant opportunistic pathogen.
- Intrinsic and acquired antimicrobial resistance are major clinical concerns.
- Multidrug efflux pumps play a crucial role in microbial resistance.
Purpose of the Study:
- To identify and characterize a DNA fragment from Enterococcus faecium FN-1 conferring antimicrobial resistance.
- To elucidate the mechanism of action of the identified resistance gene.
- To investigate the expression and contribution of the gene to intrinsic resistance in E. faecium.
Main Methods:
- Cloning of a DNA fragment from Enterococcus faecium into Escherichia coli.
- Phenotypic analysis of drug resistance in E. coli transformants.
- Sequence analysis of the cloned DNA fragment to identify open reading frames (ORFs).
- Functional characterization of the identified ORF (efmA) using efflux assays and substrate transport studies.
- Analysis of efmA mRNA expression in E. faecium.
Main Results:
- A DNA fragment from E. faecium FN-1 conferred resistance to fluoroquinolones, macrolides, ethidium bromide, DAPI, and TPPCl when cloned into E. coli.
- One ORF, designated efmA, was identified and showed similarity to known multidrug efflux pumps.
- EfmA mediated energy-dependent efflux of DAPI and TPP+, and norfloxacin/H+ antiport.
- efmA was constitutively expressed in E. faecium FN-1 under standard laboratory conditions.
Conclusions:
- The cloned gene efmA encodes a multidrug efflux pump.
- EfmA contributes to the intrinsic multidrug resistance of Enterococcus faecium.
- Constitutive expression of efmA under normal growth conditions suggests its role in inherent resistance mechanisms.
