BACTERIAL ANTIBIOTIC RESISTANCE IN AGRICULTURE

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Enterobacter cloacae is an important nosocomial pathogen which causes a variety of infections including bacteremia, lower respiratory tract infections, skin and soft tissue infections, urinary tract infections, endocarditis, intra-abdominal infections, septic arthritis, osteomyelitis, and ophthalmic infections. Recently, multidrug resistant E. cloacae have been reported with increasing frequency in clinical and environmental settings. These resistant bacteria circumvent toxic compounds, resulting in reduced efficacy in the treatment of infectious disease by antimicrobial agents. Thus, understanding the multidrug resistance mechanisms of this organism is very important in the efforts to reduce the conditions that make bacterial resistance to antimicrobial agents possible. To understand the resistance mechanisms in E. cloacae, we cloned a gene, designated emrF, which is responsible for multidrug resistance in Enterobacter cloacae using shot-gun cloning of chromosomal DNA. Host cells of E. coli KAM32 possessing the emrF gene showed elevated resistances to fosfomycin, rifampicin, ampicillin, amoxicilline, erythromycin and ethidium bromide. DNA sequencing and analysis revealed one open reading frame (ORF) encoding a novel protein of 120 amino acids. The deduced protein, EmrF, was predicted to have four transmembrane segments and found to be similar to the SMR family of multidrug efflux pumps using TMHMM and GenomeNet TBLASTN analyses. Furthermore, EmrF showed some conserved amino acid residues such as Tyr-40 and Tyr-60, and Tyr-40 which have previously been reported to mediate binding of some substrates. We found that EmrF mediated low level resistances to beta-lactams and ethidium, similar to the Qac subfamily; but EmrF did not show resistance to benzalkonium chloride. EmrF showed high resistance to fosfomycin and rifampicin. These antibiotics have not been reported as substrates of other SMR family members. We detected energy-dependent efflux of fosfomycin with everted membrane vesicles harboring EmrF, and found that EmrF is an H+-drug antiporter. We also observed ethidium efflux activity by comparing ethidium bromide accumulation in cells with and without EmrF. Our data indicate that EmrF is a novel H+-drug antiporter and a member of the SMR family of transporters. To our knowledge, this is the first report of a multidrug efflux pump in E. cloacae.