The mdt(A) gene, previously designated mef214, from Lactococcus lactis subsp. lactis plasmid pK214 encodes a protein [Mdt(A) (multiple drug transporter)] with 12 putative transmembrane segments (TMS) that contain typical motifs conserved among the efflux proteins of the major facilitator superfamily. However, it also has two C-motifs (conserved in the fifth TMS of the antiporters) and a putative ATP-binding site. Expression of the cloned mdt(A) gene decreased susceptibility to macrolides, lincosamides, streptogramins, and tetracyclines in L. lactis and Escherichia coli, but not in Enterococcus faecalis or in Staphylococcus aureus. Glucose-dependent efflux of erythromycin and tetracycline was demonstrated in L. lactis and in E. coli.Lactococci are important lactic acid bacteria used in the process of preparing fermented dairy products. Naturally found on plants and on parts of the body of cows, these bacteria are widely used as a starter culture in the dairy industry (37). Antibiotics used in animal husbandry have selected for antibiotic-resistant flora (44). Such resistant bacteria may contaminate milk and meat and persist in food made from their raw materials. Indeed, Lactococcus lactis subsp. lactis K214 isolated from a raw milk soft cheese has been shown to harbor a multiple antibiotic resistance plasmid (30). This plasmid, pK214, carries genes for chloramphenicol acetyltransferase (cat) and streptomycin adenylase (str), a tetracycline resistance gene [tet(S)], and a putative drug efflux gene previously named mef214 (30,38).Efflux proteins, membrane proteins distributed among gram-positive and gram-negative bacteria, are involved in transmembrane export of different substances such as heavy metals, organic solvents, dyes, disinfectants, and antibiotics (21,22,26,35). Some efflux proteins are specific to a single class of drugs, while others may transport a variety of chemically different compounds. These proteins have been broadly classified into two groups: the ATP-binding cassette (ABC) transporters (10, 28) and the secondary transporters (29). Drug efflux proteins in pathogens can mediate resistance causing therapeutic failures.Two multidrug transporters have been described in L. lactis. The first (LmrA) is a member of the ABC superfamily (3); the second (LmrP) is a proton-force-dependent transporter (4). Both transporters confer resistance to ethidium bromide, daunomycin, and tetraphenylphosphonium. Recent work shows resistance to macrolides, lincosamides, and streptogramins and tetracycline associated with LmrP expression (39).Characterization of mef214 demonstrated that it mediated multiple drug resistance; hence, it has been renamed mdt(A) for multiple drug transporter. Mdt(A) is a plasmid-specified protein which is unusual in that it is related to the major facilitator superfamily (MFS) and yet contains two motifs C highly conserved in antiporters (41) and a putative ATP-binding site. The mdt(A) gene was cloned and studied for phenotype and function.
MATERIALS AND METHODSBacterial strains, growth cond...
