A rabbit endocarditis model was utilized to evaluate the virulence conferred by the conjugative plasmid pADi with the following strains: Enterococcus faecalis plasmid-free FA2-2 and FA2-2 containing plasmids pADi (hemolysin and aggregation substance positive), pAM9O58 (insertional inactivation of hemolysin), and pAM944 or pAM947 (insertional inactivation of aggregation substance). All isolates were similar in ability to produce endocarditis. Mean vegetation weight was greater in animals inoculated with strains that produced aggregation substance (P < 0.01). Mortality was significantly increased in animals given FA2-2 containing pADI compared with those given all other strains (P < 0.01). These results suggest that the combination of hemolysin and aggregation substance is associated with increased mortality and that vegetation weight is associated with production of aggregation substance in experimental E. faecalis endocarditis.

We evaluated the molecular mechanism for resistance of 360 enterococci for which the gentamicin MICs were >128 g/ml. The aac(6)-Ie-aph(2؆)-Ia, aph(2؆)-Ic, and aph(2؆)-Id genes were identified by PCR in isolates from animals, food, and humans. The aph(2؆)-Ib gene was not identified in any of the isolates. Two Enterococcus faecalis isolates (MICs > 1,024 g/ml) from animals failed to generate a PCR product for any of the genes tested and likely contain a new unidentified aminoglycoside resistance gene. Pulsed-field gel electrophoresis (PFGE) analysis showed a diversity of strains. However, 1 human and 18 pork E. faecalis isolates from Michigan with the aac(6)-Ie-aph(2؆)-Ia gene had related PFGE patterns and 2 E. faecalis isolates from Oregon (1 human and 1 grocery store chicken isolate) had indistinguishable PFGE patterns. We found that when a gentamicin-resistant gene was present in resistant enterococci from animals, that gene was also present in enterococci isolated from food products of the same animal species. Although these data indicate much diversity among gentamicin-resistant enterococci, the data also suggest similarities in gentamicin resistance among enterococci isolated from humans, retail food, and farm animals from geographically diverse areas and provide evidence of the spread of gentamicin-resistant enterococci from animals to humans through the food supply.

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 ...

From 125 separate cloacal cultures from three turkey flocks fed virginiamycin, 104 Enterococcus faecium and 186Enterococcus faecalis isolates were obtained. As the turkeys aged, there was a higher percentage of quinupristin-dalfopristin-resistant E. faecium isolates, with isolates from the oldest flock being 100% resistant. There were no vancomycin-resistant enterococci. Results of pulsed-field gel electrophoresis (PFGE) indicated there were 11 PFGE types of E. faecalis and 7 PFGE types of E. faecium that were in more than one group of flock cultures.

Enterococcus gallinarum SF9117 is a veterinary isolate for which the MIC of gentamicin is 256 g/ml. Time-kill studies with a combination of ampicillin plus gentamicin failed to show synergism against SF9117. A probe representing aac(6)-aph(2؆) did not hybridize to DNA from SF9117. A 3.2-kb fragment from plasmid pYN134 of SF9117 was cloned and conferred resistance to gentamicin in Escherichia coli DH5␣. Nucleotide sequence analysis revealed the presence of a 918-bp open reading frame whose deduced amino acid sequence had a region with homology to the C-terminal domain of the bifunctional enzyme AAC(6)-APH(2؆). The gene is designated aph(2؆)-Ic, and its observed phosphotransferase activity is provisionally designated APH(2؆)-Ic. An intragenic probe hybridized to the genomic DNA from an Enterococcus faecium isolate from the peritoneal fluid of one patient and to the plasmid DNA of an Enterococcus faecalis isolate from the blood of another patient. An enterococcal isolate containing this novel resistance gene might not be readily detected in clinical laboratories that use gentamicin at 500 or 2,000 g/ml for screening for high-level resistance.