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In this review, we highlight recent work that has increased our understanding of the production and distribution of Shiga toxin in the environment. Specifically, we review studies that offer an expanded view of environmental reservoirs for Shiga toxin producing microbes in terrestrial and aquatic ecosystems. We then relate the abundance of Shiga toxin in the environment to work that demonstrates that the genetic mechanisms underlying the production of Shiga toxin genes are modified and embellished beyond the classical microbial gene regulatory paradigms in a manner that apparently "fine tunes" the trigger to modulate the amount of toxin produced. Last, we highlight several recent studies examining microbe/protist interactions that postulate an answer to the outstanding question of why microbes might harbor and express Shiga toxin genes in the environment.

Hemolytic uremic syndrome (HUS) is commonly associated with Shiga toxin (Stx)-producing Escherichia coli O157:H7 infection. This study examined patient samples for complement activation on leukocyte-platelet complexes and microparticles, as well as donor samples for Stx and lipopolysaccharide (O157LPS)-induced complement activation on platelet-leukocyte complexes and microparticles. Results, analyzed by flow cytometry, showed that whole blood from a child with HUS had surface-bound C3 on 30% of platelet-monocyte complexes compared with 14% after recovery and 12% in pediatric controls. Plasma samples from 12 HUS patients were analyzed for the presence of microparticles derived from platelets, monocytes, and neutrophils. Acute-phase samples exhibited high levels of platelet microparticles and, to a lesser extent, monocyte microparticles, both bearing C3 and C9. Levels decreased significantly at recovery. Stx or O157LPS incubated with donor whole blood increased the population of platelet-monocyte and platelet-neutrophil complexes with surface-bound C3 and C9, an effect enhanced by costimulation with Stx and O157LPS. Both Stx and O157LPS induced the release of C3- and C9-bearing microparticles from platelets and monocytes. Released microparticles were phagocytosed by neutrophils. The presence of complement on platelet-leukocyte complexes and microparticles derived from these cells suggests a role in the inflammatory and thrombogenic events that occur during HUS.

Aims: The effectiveness of four strains of Bifidobacteria against enterohemorrhagic Escherichia coli O157:H7 infection was studied using a Vero cell model., Methods and Results: E. coli O157 was inoculated on the Vero cell line before and after treatment with probiotic. The cytopathic effect (CPE) was evaluated during 24 h of incubation. The results indicated that Shiga toxin activity was inhibited by the probiotic. To prevent a Stx2 CPE, the probiotic needs one log more than the Stx1., Conclusion: The Vero cell assay, in particular, is a good model to evaluate the effect of Bifidobacteria inhibiting bacterial attachment because of soluble substances and the competitive aspect and could be used in a variety of foods like milk and yoghurt to protect pathogen bacteria., Significance and Impact of the Study: Probiotics could control pathogenic bacteria and Vero cell introduce as a model for evaluation of probiotics against pathogen bacteria., (© 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.)

Shiga toxins produced by Escherichia coli O157:H7 cause a wide spectrum of enteric diseases, such as lethal hemorrhagic colitis and hemolytic uremic syndrome. In this study, the B subunit protein of Shiga toxin type 1 (Stx1) was produced in the E. coli system, was further purified by Ni-column Affinity Chromatography method, and was then used as an immunogen to immunize laying hens for yolk immunoglobulin (IgY) production. Titers of IgY increased gradually with boosting vaccination and, finally, reached a level of 105, remaining steady over 1 year. Then the protective efficacy of IgY against Stx1 was evaluated by in vitro and in vivo experiments. It was shown that the anti-Stx1 IgY could effectively block the binding of Stx1 to the Hela cells and could protect BALB/c mice from toxin challenges. The data indicates the facility of using egg yolk IgY as a therapeutic intervention in cases of Shiga toxin intoxication.

Differences and similarities between Escherichia coli O157:H7 isolates from beef cattle and those from sporadic human outbreaks are not fully elucidated. Here, we compared 44 O157:H7 isolates of bovine and human origins (22 isolates of each) to better understand their cytotoxic potential. The Shiga toxin genes stx1, stx2, or both were detected in the 44 isolates, and all elicited Vero cell cytotoxicity. The greatest cytotoxicity was caused by bovine isolates having only stx2 and which represented the majority of such isolates (81.8%). However, no correlation was found between the level of stx gene transcription and cytotoxicity. All human and bovine isolates possessed variant type stx2 and stx2c, respectively, as determined by PCR-restriction fragment length polymorphism. Isolates harboring both stx1 and stx2 genes were much more frequent in human isolates (86.4%). The combination stx1-stx2c found in only four bovine isolates was less cytotoxic. It is clear that cytotoxicity alone cannot account for the apparent inability of O157:H7 bovine isolates to cause diseases in humans. We have found that stx1-stx2-containing or stx1-stx2c-containing isolates were less cytotoxic than several bovine isolates having only stx2c, suggesting that the stx gene combination or other virulence genes in specific genetic lineages may affect the disease outcome.

We found two Shiga toxin producing Escherichia coli O157:H7 strains isolated from humans carrying the stx(1) gene with an IS1203-like element (designated as IS1203v(1)). The IS1203v(1) was inserted into the coding region of the A subunit 7 bp upstream from the TGA termination codon, resulting in a loss of two amino acid residues (Ser-Ser) from its C terminus. Toxicity of the Stx1 was confirmed by Vero cell assay. IS1203v(1) hardly affected the stx(1) gene in either its expression or the toxicity of its product.

Shiga toxin 1 (Stx1) of enterohemorrhagic Escherichia coli O157:H7 was cloned, and four mutant Stx1s were constructed by site-directed mutagenesis with PCR. The wild-type and mutant Stx1s with amino acid replacements at positions 167 and 170 of the A subunit were purified by one-step affinity chromatography with commercially available Globotriose Fractogel, and the mutant Stxs were used for the immunization of mice. The mutant toxins were nontoxic to Vero cells in vitro and to mice in vivo and induced the immunoglobulin G antibody against the wild-type Stx1, which neutralized the cytotoxicity of Stx1. The induced antibody titers depended on the mutation at position 170 of the A subunit. The mice immunized with the mutant Stx1s were protected against a challenge of approximately 100 times the 50% lethal dose of the wild-type Stx1, suggesting that the mutant toxins are good candidates for toxoid vaccines for infection by Stx1-producing E. coli.

The effects of Escherichia coli O157:H7 (strains E30480 and PM601) and the associated verotoxins (VTs), VT1 and VT2, on stress-activated protein kinase and nuclear factor kappa B (NF-kappaB) signaling were investigated with Vero cells, which are extremely sensitive to the cytotoxic effects of E. coli O157:H7 in vitro. Cell-free supernatants prepared from E30480 and PM601 cultures and purified VT1 and VT2 stimulated a strong and prolonged (>4-h) activation of both c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase. However, JNK activity stimulated in response to E30480 supernatants was substantially reduced following pretreatment with anti-VT1 and anti-VT2 antibodies, while a VT1 and VT2 gene knockout mutant of PM601 was unable to stimulate JNK activity. E30480 supernatants also caused a sustained activation of NF-kappaB DNA binding, degradation of inhibitory kappa B alpha (IkappaBalpha), and an increase in inhibitory kappa B kinase alpha activity, although PM601 supernatants and VT1 and VT2 had no effect. However, preincubation with VTs prolonged the transient activation of NF-kappaB and IkappaBalpha degradation stimulated by either tumor necrosis factor alpha or interleukin 1beta, while preincubation with anti-VT antibodies prevented the prolonged loss of IkappaBalpha and partially reduced DNA binding in response to E30480 supernatants. These results strongly suggest that in Vero cells, VT plays an essential role in sustained JNK and NF-kappaB signaling in response to E. coli O157:H7 and that this action may underpin their cell-selective cytotoxic effects. These studies also suggest that another component released by strain E30480 contributes to the early activation of JNK and NF-kappaB.

This study examined whether macrophages are involved in the development of pathogenicity in Shiga-like toxin (SLT)-producing enterohemorrhagic Escherichia coil (EHEC) O157:H7. Macrophages were infected with the bacteria, after which the macrophage culture medium showed a clear increase in toxicity in rats in vivo as well as in rat aortic endothelial cells in vitro. The increased toxicity resulted mainly from a rapid increase in the concentrations of SLT type I (SLT-I) and type II (SLT-II) and partly from an increase in concentrations of the proinflammatory cytokines, tumor necrosis factor alpha (TNFalpha) and interleukin-1 (IL-1), in the culture medium. Most of the EHEC O157 added to the macrophage culture were quickly incorporated to form phagosomes, which then fused with lysosomes to become phagolysosomes. During this intracellular digestion process, the EHEC O157 remained alive for about 15 min, and continued synthesizing and secreting the toxins SLT-1 and SLT-II. The bacteria were then killed and digested in the phagolysosomes with significant amounts of the toxins retained. Subsequently, the contents of the phagolysosomes were exocytotically secreted from the macrophage cell membrane into the surrounding culture medium. Such a sequence of events in macrophages may occur in vivo, suggesting the active involvement of macrophages in the rapid increase in pathogenicity, such as seen in the onset of hemolytic-uremic syndrome (HUS) in patients infected with EHEC O157. The exocytotic secretion is considered to be one of the most basic cellular functions in macrophages.