An Escherichia coli O157:H7 strain isolated from a patient with hemorrhagic colitis was found to exhibit two slightly different colony morphology types on differential medium. Each morphological type, designated TT12A and TT12B, was isolated, and serological testing using various assays confirmed that both strains carried the O157 and the H7 antigens. Biochemical testing showed that the strains had identical profiles on AP120E analysis and, like typical O157:H7 strains, did not ferment sorbitol or exhibit -glucuronidase activity. Analysis with a multiplex PCR assay showed that TT12B did not carry the gene for either Shiga toxin 1 (Stx1) or Stx2, whereas these genes were present in TT12A and the toxins were produced. Apart from that, both strains carried the ؉93 gusA mutation, the cluster I ehxA gene for enterohemolysin, and the eae gene for ␥-intimin, which are all characteristics of the O157:H7 serotype. Phenotypic assays confirmed that both strains exhibited enterohemolysin activity and the attachment and effacing lesion on HeLa cells. Multilocus enzyme electrophoresis analysis showed that the strains are closely related genetically and belong in the same clonal group. Pulsed-field gel electrophoresis (PFGE) typing of XbaI-digested genomic DNA revealed that the two strains differed by two bands but shared 90% similarity and clustered in the same clade. All other non-Stxproducing O157:H7 strains examined clustered in a major clade that was distinct from that of Stx-producing O157:H7 strains. The findings that TT12B was identical to TT12A, except for Stx production, and its PFGE profile is also more closely related to that of Stx-producing O157:H7 strains suggest that TT12B was derived from TT12A by the loss of both stx genes.Serotype O157:H7 is the prototypic enterohemorrhagic Escherichia coli (EHEC) strain and the predominant serotype implicated in food-borne infections worldwide. The EHEC group is characterized by its unique virulence factors. These include the eae gene, which encodes intimin, a protein essential for the cellular attachment and effacing (A/E) lesion, and a 60-mDa plasmid that carries the ehxA gene, which encodes enterohemolysin. However, the production of Shiga toxins (Stx), particularly Stx1 and Stx2, encoded by the phage-borne stx1 and stx2 genes, is probably the most defining trait of EHEC, as these two toxins are most often implicated in disease in humans.Isogenic, non-Stx-producing variants of EHEC provide useful controls in studying the role of Stx in pathogenesis in animal models. The production of Stx can be eliminated from strains by inducing the Stx-encoding phages with sublethal dosages of UV or antibiotics, although prophage release often results in cell lysis (16). Alternatively, Gunzer et al. (12) deleted the Stx2-encoding sequences from a wild-type O157:H7 strain with a suicide vector and thus constructed a toxin deletion isogen that did not produce Stx. In addition, Karch et al. (14) reported spontaneous loss of Stx production by EHEC strains other than O157:H7 during routi...