The ability of the ID 32E system to identify and discriminate 74 Escherichia coli O157 isolates among 106 E. coli non-O157 isolates was evaluated. The results showed atypical biochemical reactions but accurate identification at the species level and no unique biochemical profile numbers for E. coli O157, although these numbers were distinct from those of other serotypes.Verocytotoxigenic Escherichia coli (VTEC), a new public health problem worldwide, is represented by more than 100 serotypes that produce verocytotoxins, with two main serotypes, O157:H7 and O157:H(Ϫ). These last two serotypes, classified as enterohemorrhagic E. coli (EHEC), are implicated in hemorrhagic colitis, hemolytic-uremic syndrome, and thrombotic thrombocytopenic purpura. EHEC strains have been described as important and emergent food-borne pathogens (4, 18, 31). Other non-O157 VTEC serotypes, not deprived of pathogenicity, have been increasingly implicated in sporadic enteric diseases and outbreaks (18,31).Detection of E. coli O157:H7 is based on its recovery from samples and the presence of its virulence-associated factors (verocytotoxins) or the detection of its O157 antigens (3,30,31). Selective media for isolating O157:H7 strains rely on the fact that most of these strains display characteristic biochemical reactions (31): no ␤-glucuronidase activity and no D-sorbitol fermentation within 24 h at 37°C, except for some German or U.S. strains (10, 12). However, these last two biochemical features are not commonly used in routine clinical and food laboratories because of the simplicity of well-established commercial identification systems. The use of these systems on presumptive E. coli strains offers not only the advantage of confirming strains at the genus and/or species level but also the possibility to detect the presence of O157 EHEC by identifying profiles that are unique to these strains. For the Microscan dried conventional gram-negative identification panel (DadeMicroScan International, West Sacramento, Calif.), 90% of the tested E. coli O157:H7 strains shared two common biochemical profiles (1), whereas in the case of the API 20E (bioMérieux Vitek, Inc., Hazelwood, Mo.), 92% of the tested strains displayed the same profile (8).A new well-based identification system, ID 32E, was recently developed by bioMérieux Vitek for the identification of Enterobacteriaceae and other nonfastidious gram-negative bacteria, after an incubation period of 22 Ϯ 2 h at 37 Ϯ 1°C (17, 23). This system is an upgrading of the API 20E gallery and consists of 32 individual conic test wells (13 enzymatic, 3 biochemical, and 16 sugar utilization tests) in polystyrene trays (Table 1).A total of 180 serotyped E. coli strains were tested with the ID 32E system. These included 55 O157:H7 strains, 19 O157: H(Ϫ) strains confirmed by PCR on the rfb O157 locus (3), and 106 reference strains and laboratory isolates of non-O157:H7 or O157H(Ϫ) E. coli harboring (76 strains) or not harboring (30 strains) verocytotoxin genes, originating from patients, animals, and food...