Among 97 enterococci cultured from animals, gentamicin MICs were >2,000 g/ml for 9 isolates and between 250 and 1,024 g/ml for 6 isolates. For two isolates tested (gentamicin MICs, 256 and 512 g/ml, respectively), there was no in vitro synergy with penicillin plus gentamicin, resistance was transferable, and there was no hybridization with a probe specific for 6-aminoglycoside acetyltransferase-2؆-aminoglycoside phosphotransferase. The results of the study indicate the presence of a unique gentamicin resistance genotype in enterococci of animal origin.Reservoirs for antibiotic-resistant enterococci have not been completely determined. Animals, human food, and the inanimate environment have been suspected as sources for some resistant clinical isolates (1-4, 19, 23, 24, 32, 38). Evidence for a disseminated erythromycin resistance determinant mediated by Tn917-like sequences has been shown in enterococci isolated from pigs, chickens, and humans (32). More recently, glycopeptide-resistant strains (vancomycin-resistant enterococci) have been identified in the feces of animals and chicken carcasses (2,3,21,(23)(24)(25) as well as in sewage in Barcelona (37) and the United Kingdom (2). In the study described here we surveyed a sample of enterococci of animal origin for penicillin, glycopeptide, and aminoglycoside resistance.The enterococcal strains used in the study are listed in Table 1. Stool samples for culture were collected from 16 separate horses, six pigs, fecal cow slurry, and 11 separate chickens from four farms in southeastern Michigan. Antibiotics were not used as feed additives at any of the farms. Twenty-eight veterinary enterococcal isolates from 16 horses and 12 birds were from the University of Pennsylvania School of Veterinary Medicine. Food isolates were cultured from 29 whole frozen chicken carcasses (nine different brand names) sold in 17 supermarkets in southeastern Michigan. Other information on the animals was not available. Isolates were initially recovered on Columbia CNA with 5% sheep blood agar (Becton Dickinson Microbiology Systems, Cockeysville, Md.). For each culture, three enterococcal colonies were evaluated when different morphologic types occurred. Conventional biochemical tests were used to identify all isolates (10). DNA probes were used (8) for confirmation of the species of the Enterococcus faecium isolates that could not be differentiated by the typing system published by Facklam and Collins (10).Susceptibilities to ampicillin (Sigma Chemical Co.), gentamicin (Schering Corp., Bloomfield, N.J.), streptomycin (Sigma Chemical Co.), and vancomycin (Eli Lilly & Co., Indianapolis, Ind.) were determined by broth microdilution methods (20,28). Time-kill experiments, -lactamase detection, and DNA methods were as described elsewhere (14, 16, 25-27, 35, 36). A probe specific for the bifunctional 6Ј-aminoglycoside acetyltransferase-2Љ-aminoglycoside phosphotransferase (AAC6Ј-APH2Љ) enzyme in E. faecalis was used for localization of the gentamicin resistance determinant (13). Table 1 shows ...