A six-step biochemical key is presented for the identification of all recognized Enterococcus spp. The key consists of 12 tests, but no more than 6 are needed for the most complicated identification. The reliability of the key has been evaluated with collection type strains and clinical and environmental isolates. This key has fewer tests than those reported in previous studies. There is no commercial kit that includes the whole set of tests. However, some of the tests are included in enzyme activity-based kits that could be used with the proposed key. The key is designed for use in routine applications, especially in environmental and clinical studies with a high number of isolates.

Aims: Scarce knowledge about the distribution of enterococci species in wastewaters limits any statement on their reliability as faecal indicators or the implications of antibiotic resistance transmission by these organisms through the water cycle. Enterococci have been involved in nosocomial infections and the spreading of antibiotic resistance through the food chain. The species distribution of enterococci and the presence of resistant strains to vancomycin and erythromycin were analysed in more than 400 raw and treated urban wastewaters, surface waters receiving these treated wastewaters and hospital wastewaters from three European countries. Methods and Results: A total of 9296 strains were isolated and biochemically phenotyped. The species identification was based on the comparison of biochemical profiles with those of more than 20 000 enterococci isolates from an international study. The prevalence of enterococcal isolates resistant to erythromycin (ERE) and vancomycin (VRE) was also analysed. ERE strains were present in a high proportion in all the studied samples. VRE strains were also isolated in all studied countries despite the time elapsed since the use of antimicrobial glycopeptides in animal production was banned in the European Union. Conclusions: Enterococcus faecalis and Ent. faecium were the most abundant species in all the studied wastewaters. All the studied wastewaters demonstrated high diversity and similar population structure and composition. ERE and VRE isolates were detected in most of the wastewaters. Significance and Impact of the Study: Urban and hospital wastewaters are useful targets for the evaluation of the prevalence of ERE and VRE isolates in the environment. It appears that these bacteria could pass through wastewater treatment plants and be transferred to surface waters.

Vancomycin-resistant enterococcci (VRE) in Europe are thought to have emerged partly due to the use of the glycopeptide avoparcin in animal husbandry. We compared the occurrence of VRE in geographical regions of Europe in which until 1997 large amounts of avoparcin were used (Spain, United Kingdom, and Denmark) with the occurrence of VRE in Sweden, where avoparcin was banned in 1986. We also studied the relatedness between VRE strains from different regions and habitats. In total, 2,580 samples were collected from humans, animals, and the environment (soil, sewage, recipient water). VRE resistant to 20 g/ml vancomycin were identified in 8.2% of the samples and were found most frequently in raw and treated urban sewage samples (means, 71% and 36% of the samples, respectively), pig manure (17%), and hospital sewage (16%). The proportions of VRE-positive sewage samples were similar in Sweden, Spain, and the United Kingdom, whereas pig feces and manure were more often positive in Spain than in Sweden (30% versus 1%). Most VRE were Enterococcus faecium carrying vanA, and computerized biochemical phenotyping of the isolates of different ecological origins showed a high degree of polyclonality. In conclusion, it seems that animal-associated VRE probably reflect the former use of avoparcin in animal production, whereas VRE in human-associated samples may be a result of antibiotic use in hospitals. Since there seems to be a reservoir of the resistance genes in all countries studied, precautions must be taken to limit the use of antibiotics and antibiotic-like feed additives.Enterococci are members of the normal intestinal microflora in humans and animals, and they are common in environments affected by animal and human fecal material. These organisms are not considered primary pathogens, but because of their ability to acquire high-level resistance to antimicrobial agents enterococci have emerged as nosocomial pathogens worldwide (30). Concern has especially been focused on enterococci that show high-level resistance to the glycopeptide antibiotic vancomycin (vancomycin-resistant enterococci [VRE]), which until recently has been the drug of last resort against multiresistant enterococci and against methicillin-resistant Staphylococcus aureus.In the United States, the incidence of bloodstream infection due to VRE increased from 0.4% in 1989 to 25.2% in 1999 (35), and between 15 and 24% of the hospitalized patients were reported to be colonized with VRE in 1998 (33). A later report indicated that 15% of all clinically relevant isolates of gram-positive cocci were VRE (17), whereas VRE of the VanA and VanB types have not been reported in animals so far. Thus, in the United States VRE are thought to have emerged and spread entirely as a result of the antibiotic use in hospitals. In Europe, infections caused by VRE remain a moderate problem in most countries. A European surveillance study found VRE in 7% of enterococcal isolates from blood cultures (13). On the other hand, healthy carriers of VRE in the community are relativ...

Aims: The changes in structure and composition of faecal coliforms and enterococcal populations in sewage from different treatment plants, and the elimination of vancomycin-and erythromycin-resistant enterococci (VRE and ERE, respectively) in these treatment plants was analysed to determine any selective reduction. Methods and Results: Faecal coliforms, enterococci, VRE, ERE and spores of sulphite-reducing bacteria were enumerated using standard methods. Samples were enriched where necessary in order to isolate antibiotic resistant strains. The structure and composition of these bacterial populations were determined by biochemical fingerprinting and clustering analysis. High diversity and similarity indexes were detected among all the bacterial populations in raw and treated sewage, independently of their origin and the treatment processes employed. Antibiotic resistant strains were detected in all sewage tested and no selective reduction was observed. Conclusions: The faecal coliforms and enterococci populations did not differ in the sewage samples studied. The vancomycin and erythromycin resistances of the enterococcal populations were similar in the sewage samples. Resistance to both antibiotics persisted after the treatment process independently of raw sewage flow, faecal origin or size of the human population contributing to sewage. However, sewage of mixed origin (human and animal) presented a lower similarity index for the two bacterial populations compared with that of the other human sewage analysed. Significance and Impact of the Study: Although a significant reduction in bacterial populations was observed, the persistence of VRE and ERE strains in the same proportions in sewage suggests that there is no selective elimination of bacterial populations during the treatment processes. The ability of antibiotic resistance strains to survive sewage treatment systems should be considered in certain water reuse programmes.

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