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ABSTRACTIn May 2011, a large food-borne outbreak was traced to an unusual O104:H4 enteroaggregative Escherichia coli(EAEC) strain that produced Shiga toxin (Stx) type 2 (Stx2). We developed a mouse model to study the pathogenesis and treatment for this strain and examined the virulence of the isolate for Dutch belted rabbits. O104:H4 strain C227-11 was gavaged into C57BL/6 mice at 109to 1011CFU/animal. The infected animals were then given water with ampicillin (Amp; 5 g/liter) ad libitum. The C227-11-infected, Amp-treated C57BL/6 mice exhibited both morbidity and mortality. Kidneys from mice infected with C227-11 showed acute tubular necrosis, a finding seen in mice infected with typical Stx-producing E. coli. We provided anti-Stx2 antibody after infection and found that all of the antibody-treated mice gained more weight than untreated mice and, in another study, that all of the antibody-treated animals lived, whereas 3/8 phosphate-buffered saline-treated mice died. We further compared the pathogenesis of C227-11 with that of an Stx-negative (Stx-) O104:H4 isolate, C734-09, and an Stx2-phage-cured derivative of C227-11. Whereas C227-11-infected animals lost weight or gained less weight over the course of infection and died, mice infected with either of the Stx-isolates did not lose weight and only one mouse died. When the Stx-positive (Stx+) and Stx2-O104:H4 strains were compared in rabbits, greater morbidity and mortality were observed in rabbits infected with the Stx2+isolates than the Stx2-isolates. In conclusion, we describe two animal models for EAEC pathogenesis, and these studies show that Stx2 is responsible for most of the virulence observed in C227-11-infected mice and rabbits.

ABSTRACTWe compared the pathogenicity of intimin-negative non-O157:H7 Shiga toxin (Stx)-producing Escherichia coli(STEC) O91:H21 and O104:H21 strains with the pathogenicity of intimin-positive O157:H7 and O157:H−strains in neonatal pigs. We also examined the role of Stx2d-activatable genes and the large hemolysin-encoding plasmid of O91:H21 strain B2F1 in the pathogenesis of STEC disease in pigs. We found that all E. colistrains that made wild-type levels of Stx caused systemic illness and histological lesions in the brain and intestinal crypts, whereas none of the control Stx-negative E. colistrains evoked comparable central nervous system signs or intestinal lesions. By contrast, the absence of intimin, hemolysin, or motility had little impact on the overall pathogenesis of systemic disease during STEC infection. The most striking differences between pigs inoculated with non-O157 STEC strains and pigs inoculated with O157 STEC strains were the absence of attaching and effacing intestinal lesions in pigs inoculated with non-O157:H7 strains and the apparent association between the level of Stx2d-activatable toxin produced by an STEC strain and the severity of lesions.

ABSTRACTShiga toxin (Stx) types 1 and 2 are encoded within intact or defective temperate bacteriophages in Stx-producing Escherichia coli(STEC), and expression of these toxins is linked to bacteriophage induction. Among Stx2 variants, only stx2efrom one human STEC isolate has been reported to be carried within a toxin-converting phage. In this study, we examined the O91:H21 STEC isolate B2F1, which carries two functional alleles for the potent activatable Stx2 variant toxin, Stx2d, for the presence of Stx2d-converting bacteriophages. We first constructed mutants of B2F1 that produced one or the other Stx2d toxin and found that the mutant that produced only Stx2d1 made less toxin than the Stx2d2-producing mutant. Consistent with that result, the Stx2d1-producing mutant was attenuated in a streptomycin-treated mouse model of STEC infection. When the mutants were treated with mitomycin C to promote bacteriophage induction, Vero cell cytotoxicity was elevated only in extracts of the Stx2d1-producing mutant. Additionally, when mice were treated with ciprofloxacin, an antibiotic that induces the O157:H7 Stx2-converting phage, the animals were more susceptible to the Stx2d1-producing mutant. Moreover, an stx2d1-containing lysogen was isolated from plaques on strain DH5α that had been exposed to lysates of the mutant that produced Stx2d1 only, and supernatants from that lysogen transformed with a plasmid encoding RecA were cytotoxic when the lysogen was induced with mitomycin C. Finally, electron-microscopic examination of extracts from the Stx2d1-producing mutant showed hexagonal particles that resemble the prototypic Stx2-converting phage 933W. Together these observations provide strong evidence that expression of Stx2d1 is bacteriophage associated. We conclude that despite the sequence similarity of the stx2d1- and stx2d2-flanking regions in B2F1, Stx2d1 expression is repressed within the context of its toxin-converting phage while Stx2d2 expression is independent of phage induction.

Shiga toxin (Stx)-producing Escherichia coli(STEC) causes foodborne outbreaks of bloody diarrhea. There are two major types of immunologically distinct Stxs: Stx1a and Stx2a.