Shiga toxin (Stx)-producing Escherichia coli (STEC) from patients with hemolytic-uremic syndrome (HUS), patients with diarrhea without HUS, or asymptomatic subjects were genotyped to assess associations between stx2 variants and clinical manifestations of infection. Neither stx2d nor stx2e was found in 268 STEC isolates from patients with HUS. Of 262 STEC isolates from patients with diarrhea, stx(2d) was found in 41 (15.6%; P<.000001), and stx2e was found in 12 (4.6%; P=.0004). The stx2c genotype frequency was similar among isolates from patients with HUS (3.7%) and diarrhea (5.0%). The frequencies of stx2c, stx2d, and stx2e among 96 STEC isolates from asymptomatic subjects were comparable to those among isolates from patients with diarrhea. None of the 626 STEC isolates contained stx2f. All stx2d-positive or stx2e-positive STEC isolates were eae negative and originated from subjects older than those with STEC isolates with stx2c. stx2c-positive STEC isolates can cause HUS, but the presence of stx2d or stx2e may predict a milder disease with a minimal risk of HUS.

Enterohemorrhagic Escherichia coli (EHEC) strains cause diarrhea and hemolytic uremic syndrome resulting from toxin-mediated microvascular endothelial injury. EHEC hemolysin (EHEC-Hly), a member of the RTX (repeats-in-toxin) family, is an EHEC virulence factor of increasingly recognized importance. The toxin exists as free EHEC-Hly and as EHEC-Hly associated with outer membrane vesicles (OMVs) released by EHEC during growth. Whereas the free toxin is lytic towards human endothelium, the biological effects of the OMV-associated EHEC-Hly on microvascular endothelial and intestinal epithelial cells, which are the major targets during EHEC infection, are unknown. Using microscopic, biochemical, flow cytometry and functional analyses of human brain microvascular endothelial cells (HBMEC) and Caco-2 cells we demonstrate that OMV-associated EHEC-Hly does not lyse the target cells but triggers their apoptosis. The OMV-associated toxin is internalized by HBMEC and Caco-2 cells via dynamin-dependent endocytosis of OMVs and trafficked with OMVs into endo-lysosomal compartments. Upon endosome acidification and subsequent pH drop, EHEC-Hly is separated from OMVs, escapes from the lysosomes, most probably via its pore-forming activity, and targets mitochondria. This results in decrease of the mitochondrial transmembrane potential and translocation of cytochrome c to the cytosol, indicating EHEC-Hly-mediated permeabilization of the mitochondrial membranes. Subsequent activation of caspase-9 and caspase-3 leads to apoptotic cell death as evidenced by DNA fragmentation and chromatin condensation in the intoxicated cells. The ability of OMV-associated EHEC-Hly to trigger the mitochondrial apoptotic pathway in human microvascular endothelial and intestinal epithelial cells indicates a novel mechanism of EHEC-Hly involvement in the pathogenesis of EHEC diseases. The OMV-mediated intracellular delivery represents a newly recognized mechanism for a bacterial toxin to enter host cells in order to target mitochondria.

By using sequence analysis of Shiga toxin 1 (Stx 1) genes from human and ovine Stx-producing Escherichia coli (STEC) strains, we identified an Stx1 variant in STEC of human origin that was identical to the Stx1 variant from ovine STEC, but demonstrated only 97.1 and 96.6% amino acid sequence identity in its A and B subunits, respectively, to the Stx1 encoded by bacteriophage 933J. We designated this variant "Stx1c" and developed stxB 1 restriction fragment length polymorphism and stx 1c -specific PCR strategies to determine the frequency and distribution of stx 1c among 212 STEC strains isolated from humans. During the past 20 years, Shiga toxin (Stx)-producing Escherichia coli (STEC) have emerged as important causes of diarrhea and the hemolytic-uremic syndrome (HUS) throughout the world (2,9,14,32,33). Stx are believed to be the cardinal virulence factors of STEC (20). Based on toxin neutralization assays (30) and sequence analysis of stx genes (11), two major toxin types, Stx1 and Stx2, have been assigned (11,20,30). Stx1 and Stx2 are not cross-neutralized by heterologous antisera in cell culture assays (30), and the structural genes encoding these toxins demonstrate approximately 55% overall nucleotide sequence identity (11). The Stx2 group has been shown to be highly heterogeneous, comprising, in addition to Stx2, several Stx2 variants that have been classified as Stx2c (29), Stx2d (24), Stx2e (34), and Stx2f (28). The substantial sequence heterogeneity observed between members of the Stx2 family (24,28,29,34) enabled the development of PCR techniques that differentiate stx 2 from its variants and identify the respective stx 2 alleles (7,24,28). This is of particular clinical importance, because STEC strains possessing different stx 2 variants appear to differ in their capacity to cause HUS (8). Information about the stx 2 allele of an infecting STEC strain has, therefore, considerable potential predictive value for the treating physician to assess the risk of HUS development in a patient that presents with STEC infection (8).In contrast to the Stx2 family, the Stx1 group appears to be more homogeneous. stx 1 genes carried in the genomes of bacteriophages H19B (6), H30 (18), and 933J (10) have identical nucleotide sequences (6, 10, 18) and differ by only three nucleotides in their A subunits, resulting in only one amino acid difference from the sequence of stx from Shigella dysenteriae type 1 (31). Paton et al. (21,22) reported three human STEC strains that possess slight variations in their stx 1 genes. Each of these stx 1 variants shared more than 99% nucleotide sequence identity with stx 1 from phage 933J and with stx from S. dysenteriae type 1 (21, 22). Stx1 encoded by these stx 1 variants differed by one and two amino acid residues in their A subunits from Stx of S. dysenteriae type 1 and from Stx1 encoded by phage 933, respectively, whereas their B subunits were identical to those of the latter two toxins (21,22). A considerably greater degree of sequence heterogeneity was observed in stx 1 genes of S...

The occurrence of Shiga toxin-producing Escherichia coli (STEC) was studied on four cattle farms. STEC were detected in 29-82% of the cattle. STEC with additional EHEC markers were detected on all farms. The occurrence of the complete virulence marker pattern (stx1 and/or stx2, eae, EHEC(hlyA), katP, espP) was correlated with the presence of known STEC serotypes. STEC O26:H11 and O165:H25 with the complete pattern of virulence markers were the most prevalent. STEC O157 (H7/H-) STEC O103:H2 and STEC O145:H- were found sporadically. Five clonal subgroups of the STEC O26:H11 isolates were identified by pulsed-field gel electrophoresis. STEC O26:H11 were present in three groups of cattle. This serotype was detected in a single group over the entire fattening period. Most STEC O26:H11 with the complete pattern of potential virulence markers were found in clinically healthy cattle. These animals may represent a risk factor for farmers and consumers.