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Background & Aims Shiga toxin (Stx)-producing Escherichia coli (eg, O157:H7) infection produces bloody diarrhea, while Stx inhibits protein synthesis and causes the life-threatening systemic complication of hemolytic uremic syndrome. The murine intestinal tract is resistant to O157:H7 and Stx, and human cells in culture fail to model the complex tissue responses to intestinal injury. We used genetically identical, human stem cell–derived intestinal tissues of varying complexity to study Stx toxicity in vitro and in vivo. Methods In vitro susceptibility to apical or basolateral exposure to Stx was assessed using human intestinal organoids (HIOs) derived from embryonic stem cells, or enteroids derived from multipotent intestinal stem cells. HIOs contain a lumen, with a single layer of differentiated epithelium surrounded by mesenchymal cells. Enteroids only contain epithelium. In vivo susceptibility was assessed using HIOs, with or without an enteric nervous system, transplanted into mice. Results Stx induced necrosis and apoptotic death in both epithelial and mesenchymal cells. Responses that require protein synthesis (cellular proliferation and wound repair) also were observed. Epithelial barrier function was maintained even after epithelial cell death was seen, and apical to basolateral translocation of Stx was seen. Tissue cross-talk, in which mesenchymal cell damage caused epithelial cell damage, was observed. Stx induced mesenchymal expression of the epithelial marker E-cadherin, the initial step in mesenchymal–epithelial transition. In vivo responses of HIO transplants injected with Stx mirrored those seen in vitro. Conclusions Intestinal tissue responses to protein synthesis inhibition by Stx are complex. Organoid models allow for an unprecedented examination of human tissue responses to a deadly toxin., Graphical abstract

Enterohemorrhagic Escherichia coli (EHEC) is a subtype of pathogenic E. coli that causes diarrhea or hemorrhagic colitis in humans, which can progresses to hemolytic uremic syndrome (HUS), a leading cause of acute renal failure in children, and morbidity and mortality in adults. Stool samples (n = 273) of patients (1 day-40 years old) suffered from bloody diarrhea and abdominal cramps, were examined bacteriologically and molecularly for the presence and pathogenicity of EHEC with phylogenetic analysis of the obtained stx1, stx2, and eaeA virulence genes' sequences. Overall, 71 (26.01%) E. coli isolates were identified as EHEC with the following serogroupes: O1:H11 (3), O128:H2 (9), O26:H11 (6), O157:H7 (3), O25:H2 (7), O145:H328 (2), O125:H6 (9), O86:H8 (5), O18:H15 (11) and untypable (16). The highest isolation rate were in samples belonged to infants below two years old (42.25%). Antimicrobial susceptibility testing showed that all isolates were highly sensitive to ciprofloxacin, nitrofurantoin, gentamycin, imipenem and vancomycin (100% each), however, they were resistant to ampicillin, cephalexin, penicillin and tetracycline (100% each). In-vitro pathogenicity testing of the isolates revealed that 67 (94.37%) isolates were positive for Congo red test, 47 (66.20%) isolates possessed P fimbriae (MRHA) and 17 (23.94%) possessed type 1 fimbriae (MSHA). Moreover, 46 (64.79%) isolates exhibited hemolysis and 42 (59.15%) isolates showed distinct cytopathic effect to Vero cells. Molecular detection of enterohemorrhagic E. coli (EHEC) pathotype virulence genes, confirmed the presence of stx1 gene in O157:H7 (MA2), O26:H11, O145:H328 and O125:H6 serogroups; stx2 gene in (O157:H7 (MA1), O128:H2 and O25:H2; while all serogroups except (O125:H6) carried the eaeA intimin virulence gene. A phylogenetic tree, based on the nucleotide sequences of toxin-encoding genes, demonstrates that Shiga toxin E. coli (STEC) isolates have considerable genetic variation and belong to various phylogenetic groupings.

Synthetic assembly of sugar moieties and amino acids in order to create “sugar-amino acid hybrid polymers” was accomplished by means of simple radical polymerization of carbohydrate monomers having an amino acid-modified polymerizable aglycon. Amines derived from globotriaoside and lactoside as glycoepitopes were condensed with known carbobenzyloxy derivatives, including Z-Gly, Z- l -Ala and Z-β-Ala, which had appropriate spacer ability and a chiral center to afford fully protected sugar-amino acid hybrid compounds in good yields. After deprotection followed by acryloylation, the water-soluble glycomonomers were polymerized with or without acrylamide in the presence of a radical initiator in water to give corresponding copolymers and homopolymers, which were shown by SEC analysis to have high molecular weights. Evaluation of the biological activities of the glycopolymers against Shiga toxins (Stxs) was carried out, and the results suggested that glycopolymers having highly clustered globotriaosyl residues had high affinity against Stx2 (KD = 2.7∼4.0 µM) even though other glycopolymers did not show any affinity or showed very weak binding affinity. When Stx1 was used for the same assay, all of the glycopolymers having globotriaosyl residues showed high affinity (KD = 0.30∼1.74 µM). Interestingly, couple of glycopolymers having lactosyl moieties had weaker binding affinity against Stx1. In addition, when cytotoxicity assays were carried out for both Stxs, glycopolymers having highly clustered globotriaosyl residues showed higher affinity than that of the copolymers, and only highly clustered-type glycopolymers displayed neutralization potency against Stx2.

Despite their general low incidence, Shiga toxin-producing Escherichia (E.) coli (STEC) infections are considered an important public health issue due to the severity of illness that can develop, particularly in young children. We report on two Austrian petting zoos, one in Tyrol (2015) and one in Vorarlberg (2016), which were identified as highly likely infection sources of STEC infections. The petting zoo related cases involved a case of hemolytic uremic syndrome (HUS) due to STEC O157:HNM in 2015 and an outbreak of STEC O157:H7 infections affecting five young children and two adults in 2016. The HUS case accounted for 2.8% of the 36 STEC O157:HNM/H7 infections notified in Austria in 2015 (5,9% of 17 HUS cases). The seven cases described for 2016 accounted for 4.0% of the 177 human STEC infections documented for Austria in 2016, and for 19.4% of the 36 STEC O157:HNM/H7 infections notified that year. The evaluation of the STEC infections described here clearly underlines the potential of sequence-based typing methods to offer suitable resolutions for public health applications. Furthermore, we give a state-of-the-art mini-review on the risks of petting zoos concerning exposure to the zoonotic hazard STEC and on proper measures of risk-prevention.

The life-threatening sequela of hemorrhagic colitis induced by Shiga toxins (Stx)-producing Escherichia coli (STEC) infections in humans is hemolytic uremic syndrome (HUS), the main cause of acute renal failure in early childhood. The key step in the pathogenesis of HUS is the appearance of Stx in the blood of infected patients because these powerful virulence factors are capable of inducing severe microangiopathic lesions in the kidney. During precocious toxemia, which occurs in patients before the onset of HUS during the intestinal phase, Stx bind to several different circulating cells. An early response of these cells might include the release of proinflammatory mediators associated with the development of HUS. Here, we show that primary human monocytes stimulated with Shiga toxin 1a (Stx1a) through the glycolipid receptor globotriaosylceramide released larger amounts of proinflammatory molecules (IL-1β, TNFα, IL-6, G-CSF, CXCL8, CCL2, CCL4) than Stx1a-treated neutrophils. The mediators (except IL-1β) are among the top six proinflammatory mediators found in the sera from patients with HUS in different studies. The molecules appear to be involved in different pathogenetic steps of HUS, i.e. sensitization of renal endothelial cells to the toxin actions (IL-1β, TNFα), activation of circulating monocytes and neutrophils (CXCL8, CCL2, CCL4) and increase in neutrophil counts in patients with poor prognosis (G-CSF). Hence, a role of circulating monocytes in the very early phases of the pathogenetic process culminating with HUS can be envisaged. Impairment of the events of precocious toxemia would prevent or reduce the risk of HUS in STEC-infected children.

Shiga toxin Escherichia coli (STEC) O157:H7 is an important zoonotic food borne pathogen causing gastroenteritis that may lead to life threatening hemorragic colitis (HC) and hemorrhagic uremic syndrome (HUS). 325 meat and tissue samples were tested for enumeration of O157:H7 strains using most probable number (MPN)-PCR targeting their specific genes flicH7 and rfbO157 followed by isolation, sereotyping and pathogenicity testing. The overall prevalence of O157:H7 was 41.3% (134/325) along the production and supply chain of pork (PSCP), being higher in supply chain (59%, 118/200) as compared to pig farms (12.8%, 16/125). Along the PSCP, the highest prevalence was found in slaughter houses (86.25%, 69/80) followed by wet- (53.3%, 32/60) and super-markets (28.3%, 17/60). The MPN values ranged from 3 to 1100 MPN/g in overall positive samples, being higher in slaughter houses followed by wet and super markets. Except from intestine and meat samples of slaughter house, the MPN was found higher in summer as compared to winter samples. Eight STEC O157:H7 isolated from meat and liver samples were tested in Balb/C mice for pathogenicity. After development of clinical signs and symptoms, 50-83.3% mortality was produced in the infected mice. Histopathological investigations revealed visible necrosis of intestinal epithelial cells, shedding of cellular debris in the intestine, while in the kidney, necrosis of renal cortical portion of tubular epithelial cells was observed. STEC O157:H7 is prevalent along PSCP around Hubei of China in different proportions being alarmingly higher in supply chain and markets which is a matter of concern for public health.

Verocytotoxin (VT)-producing Escherichia coli (VTEC) are a significant foodborne public health hazard, where most human infections are associated with six serogroups (O157, O26, O103, O145, O111 and O104). VTEC was the fourth most commonly reported zoonosis in the EU in 2015, with 5901 confirmed human cases. Ruminant animals, including cattle, are a major reservoir of VTEC. The consumption of VTEC-contaminated animal-derived foodstuffs, especially undercooked ground beef, is an important transmission route. To the best of our knowledge, there are few data available on the contamination of VTEC in meat products in Italy. During 2015 and 2016, 250 raw meat samples were collected from retail markets in southern Italy (Apulia) and analysed for the occurrence of vtx genes (vtx1/vtx2) at the Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata (IZS PB, Italy). In addition, the isolates were characterized by determining the presence of VTEC main virulence factors, the antimicrobial resistance profiles and the genetic relatedness by pulsed-field gel electrophoresis (PFGE). The results have shown that 8.4% (21/250) of the samples were positive for vtx genes in the preliminary screening step but VTEC strains were isolated from only 2% (5/250) of overall meat analysed samples, including raw ground beef, beef hamburger and beef carpaccio. 5 isolates displayed a multi-drug resistance phenotype. All VTEC strains were analysed by XbaI-PFGE and dendrogram revealed 5 distinct restriction profiles, indicating their relatively high genetic diversity. Although this study demonstrates a low prevalence of VTEC in raw beef marketed in southern Italy, the presence of potentially pathogenic E. coli strains points to the need for proper hygiene during meat production to reduce the risk of foodborne illness and transmission of multi-drug resistant organisms via foods to humans.

In exposure to outer pressure, microorganisms are capable of entry into the Viable But Non-Culturable (VBNC) state, and thus survive under various elimination processing. The survival microorganisms may yield negative results on culturing, and cause false negative for this golden standard methodology. In this study, a novel PMA-LAMP assay on the detection of Enterohemorrhage E. coli and shiga toxins has been developed and evaluated, with further application on a number of food borne E. coli strains. LAMP primers were designed on the target of rfbe for Enterohemorrhage E. coli and stx1with stx2 for shiga toxins. Via specific penetration through the damaged cell membrane of dead cells and intercalating into DNA, PMA could prevent DNA amplification of dead bacteria from LAMP, which enabled the differentiation of bacteria between VBNC state and dead state. The established PMA-LAMP showed significant advantage in rapidity, sensitivity and specificity, compared with regular PCR assay. The applicability had also been verified, demonstrating the PMA-LAMP was capable of detection on Enterohemorrhage E. coli and shiga toxins.

Immunotoxins are protein-based drugs consist of a target-specific binding domain and a cytotoxic domain to eliminate target cells. Such compounds are potentially therapeutic to combat diseases such as cancer. Generally, the B-subunit of Shiga toxin (STXB) receptor, globotriaosylceramide (Gb3), is expressed in high amounts on a number of human tumors cancer cells. In this study, we evaluated a new antitumor candidate called DT389-STXB chimeric protein, which genetically fused the DT to B-subunit of Shiga-like toxin (STXB). First a chimeric protein, encoding DT389-STXB was synthesized. The optimized chimeric protein expressed in E.coli BL21 (DE3) and confirmed by anti-His Western blot analysis. T47D, SKBR3, 4T1 and MCF7 cell lines were treated separately with purified DT389-STXB recombinant protein and functional activity of DT389-STXB was analyzed by the cell enzyme-linked immunosorbentassay (ELISA), MTT, ICC, Western blot and apoptosis tests. The results indicated that the recombinant DT389-STXB fusion protein with a molecular weight of 53 kDa was successfully expressed in E.coli BL21 (DE3) and the anti-His western-blot was used to confirm the presence of the protein. The DT389-STXB fusion protein attached to T47D, SKBR3 and 4T1 cell lines with the proper affinity and induced dose-dependent cytotoxicity against GB3-expressing cancer cells in vitro. Our results showed that DT389-STXB fusion protein may be a promising candidate for antitumor therapy agent against breast cancer; however, further studies are required to explore its efficacy in vivo for therapeutic applications.

The current work presents an overview of the use of phage display technology for the identification and characterization of potential neutralizing agents for Shiga toxins. The last major Shiga toxin-associated disease outbreak, which took place in Germany in 2011, showed the international community that Shiga toxins remain a serious threat to public health. This is also demonstrated by the lack of specific therapies against Shiga toxin-induced Hemolytic Uremic Syndrome (HUS). Since its inception, phage display technology has played a key role in the development of antigen-specific (poly)-peptides or antibody fragments with specific biological properties. Herein, we review the current literature regarding the application of phage display to identify novel neutralizing agents against Shiga toxins. We also briefly highlight reported discoveries of peptides and heavy chain antibodies (VHH fragments or nanobodies) that can neutralize the cellular damage caused by these potent toxins.

Here we report the first complete nucleotide sequence of a Shiga toxin (Stx) converting phage from a Shigella sonnei clinical isolate that harbors stx1 operon, first identified in the chromosome of Shigella dysenteriae type 1. The phage named Shigella phage 75/02 Stx displayed Podoviridae morphology. It proved to be transferable to Escherichia coli K-12 strains, and cytotoxicity of the lysogenized strains was demonstrated in Vero cell cultures. Genomic analysis revealed that the prophage genome is circular and its size is 60,875 nt that corresponds to 76 ORFs. The genome of Shigella phage 75/02 Stx shows a great degree of mosaic structure and its architecture is related to lambdoid phages. All the deduced proteins, including the 37 hypothetical proteins showed significant homologies to Stx phage proteins present in databases. The phage uniformly inserted into the ynfG oxidoreductase gene framed by phage integrase and antirepressor genes in parental S. sonnei and in the three lysogenized K-12 strains C600, DH5α and MG1655. The Stx1 prophage proved to be stable in its bacterial hosts and remained inducible.

The prevalence of potentially positive Shiga toxin-producing Escherichia coli (STEC) bovine hides and carcasses in three abattoirs in Costa Rica was estimated. Two export facilities (A and B) and one non-export establishment (C) were visited during the dry and rainy seasons of 2013. Swabs of hides pre-eviscerated and treated (180–220 peroxyacetic acid spray) carcasses were tested for the potential presence of STEC serogroups O26, O45, O103, O111, O121, and O145. The prevalence on hides during the rainy season was 86.7, 96.7 and 96.7% for facilities A, B, and C, respectively. During the dry season, the prevalence on hides was significantly lower in plants A and B (40% and 26.7%, respectively), but was marginally associated with the season in plant C (76.7%, P = 0.0523). The prevalence of non-O157 STEC markers on treated carcasses was low (0 to 3.3%), suggesting that all plants were effective in minimizing the target non-O157 STEC in beef destined for export and for domestic consumption.

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains are food- and waterborne pathogens that are often transmitted via beef products or fresh produce. STEC strains cause both sporadic infections and outbreaks, which may result in hemorrhagic colitis and hemolytic uremic syndrome. STEC strains may elaborate Stx1, Stx2, and/or subtypes of those toxins. Epidemiological evidence indicates that STEC that produce subtypes Stx2a, Stx2c, and/or Stx2d are more often associated with serious illness. The Stx2d subtype becomes more toxic to Vero cells after incubation with intestinal mucus or elastase, a process named "activation." Stx2d is not generally found in the E. coli serotypes most commonly connected to STEC outbreaks. However, STEC strains that are stx2d positive can be isolated from foods, an occurrence that gives rise to the question of whether those food isolates are potential human pathogens. In this study, we examined 14 STEC strains from fresh produce that were stx2d positive and found that they all produced the mucus-activatable Stx2d and that a subset of the strains tested were virulent in streptomycin-treated mice.

The aim of this study was to reveal phenotype/genotype characteristics of verotoxigenic Escherichia coli (VTEC) and multidrug resistant E. coli in food products of animal origin confiscated as illegal import at Austrian, German and Slovenian airports. VTEC isolates were obtained by using ISO guidelines 16654:2001 for O157 VTEC or ISO/ TS13136:2012 for non-O157 VTEC, with additional use of the RIDASCREEN® Verotoxin immunoassay. The testing of 1526 samples resulted in 15 VTEC isolates (1.0%) primarily isolated from hard cheese from Turkey and Balkan countries. Genotyping for virulence by using a miniaturized microarray identified a wide range of virulence determinants. One VTEC isolate (O26:H46) possessing intimin (eae) and all other essential genes of Locus of Enterocyte Effacement (LEE) was designated as enterohemorrhagic E. coli (EHEC). None of the other VTEC strains belonged to serogroups O157, O145, O111, O104 or O103. VTEC strains harbored either stx(1) (variants stx1(a) or stx(1c)) or st(x2) (variants stx(2a), stx(2b), stx(2a/d) or stx(2c/d)) genes. Pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) demonstrated high genetic diversity and identified three new sequence types (STs): 4505, 4506 and 4507. Food samples collected from the Vienna airport were also tested for E. coli quantities using the ISO 16649:2001, and for detection of multidrug resistant phenotypes and genotypes. The resulting 113 commensal E. coli isolates were first tested in a pre-screening against 6 selected antimicrobials to demonstrate multidrug resistance. The resulting 14 multidrug resistant (MDR) E. coli isolates, representing 0.9% of the samples, were subjected to further resistance phenotyping and to microarray analyses targeting genetic markers of antimicrobial resistance and virulence. Genotyping revealed various combinations of resistance determinants as well as the presence of class 1, class 2 integrons. The isolates harbored 6 to 11 antibiotic resistance genes as well as 1 to 14 virulence genes. In this panel of 14 MDR E. coli two strains proved to carry CTX-M type ESBLs, and one single isolate was identified as enteropathogenic E. coli (EPEC). In general, isolates carrying a high number of resistance determinants had lower number of virulence genes and vice versa. In conclusion, this first pilot study on the prevalence of VTEC and of MDR/ESBL E. coli in illegally imported food products of animal origin suggests that these strains could represent reservoirs for dissemination of potentially new types of pathogenic and MDR E. coli in Europe.

Enterohemorrhagic Escherichia coli (EHEC) strains are food borne pathogens with importance in public health. EHEC colonizes the large intestine and causes diarrhea, hemorrhagic colitis and in some cases, life-threatening hemolytic-uremic syndrome (HUS) due to the production of Shiga toxins (Stx). The lack of effective clinical treatment, sequelae after infection and mortality rate in humans supports the urgent need of prophylactic approaches, such as development of vaccines. Shedding from cattle, the main EHEC reservoir and considered the principal food contamination source, has prompted the development of licensed vaccines that reduce EHEC colonization in ruminants. Although murine models do not fully recapitulate human infection, they are commonly used to evaluate EHEC vaccines and the immune/protective responses elicited in the host. Mice susceptibility differs depending of the EHEC inoculums; therefore, displaying different mortality rates and Stx-mediated renal damage. Therefore, several experimental protocols have being pursued in this model to develop EHEC-specific vaccines. Recent candidate vaccines evaluated include those composed of virulence factors alone or as fused-subunits, DNA-based, attenuated bacteria and bacterial ghosts. In this review, we summarize progress in the design and testing of EHEC vaccines and the use of different strategies for the evaluation of novel EHEC vaccines in the murine model.

The purpose of this study was to investigate carriage and transfer of verocytotoxigenic Escherichia coli (VTEC) O157, O26, O111, O103 and O145 from fleece to dressed carcasses of 500 sheep, and to establish the virulence potential of recovered VTEC. Individual sheep were tracked and sampled (10 g fleece, full carcass swab) through the slaughter process. Samples were examined for the presence of verotoxin ( vt1 and vt2 ) genes using a duplex real-time PCR assay and positive samples were further screened for the presence of the above five serogroups by real-time PCR. VTEC cells were recovered from PCR positive samples by serogroup specific immunomagnetic separation and confirmed by serogroup specific latex agglutination and PCR. Isolates were subject to a virulence screen ( vt1 , vt2 , eaeA and hlyA ) by PCR and isolates carrying vt genes were examined by Pulsed-Field Gel Electrophoresis (PFGE). VTEC O26 was recovered from 5/500 (1.0%) fleece and 2/500 (0.4%) carcass samples. VTEC O157 was isolated from 4/500 (0.8%) fleece samples and 3/500 (0.6%) carcass samples. E. coli O103 was recovered from 84/500 (16.8%) fleece and 68/500 (13.6%) carcasses, but only one E. coli O103 isolate (0.2%) carried vt genes. E. coli O145 was recovered from one fleece sample, but did not carry vt genes. E. coli O111 was not detected in any samples. For the four serogroups recovered, the direct transfer from fleece to carcass was not observed with PFGE showing that VTEC O26 isolates from a matched fleece/carcass “pair” were not identical. This study shows that while VTEC O157 are being carried by sheep presented for slaughter in Ireland, other potentially clinically significant verotoxin producing strains (particularly VTEC O26) are emerging.

Enterohemorrhagic Escherichia coli cause approximately 1.5 million infections globally with 176,000 cases occurring in the United States annually from ingesting contaminated food, most frequently E. coli O157:H7 in ground beef or fresh produce. In severe cases, the painful prodromal hemorrhagic colitis is complicated by potentially lethal hemolytic uremic syndrome (HUS), particularly in children. Bacterial Shiga-like toxins (Stx1, Stx2) are primarily responsible for HUS and the kidney and neurologic damage that ensue. Small animal models are hampered by the inability to reproduce HUS with thrombotic microangiopathy, hemolytic anemia, and acute kidney injury. Earlier, we showed that nonhuman primates (Papio) recapitulated clinical HUS after Stx challenge and that novel therapeutic intervention rescued the animals. Here, we present detailed light and electron microscopic pathology examination of the kidneys from these Stx studies. Stx1 challenge resulted in more severe glomerular endothelial injury, whereas the glomerular injury after Stx2 also included prominent mesangiolysis and an eosinophilic inflammatory infiltration. Both toxins induced glomerular platelet-rich thrombi, interstitial hemorrhage, and tubular injury. Analysis of kidney and other organs for inflammation biomarkers showed a striking chemotactic profile, with extremely high mRNA levels for IL-8, monocyte chemoattractant protein 1, and macrophage inflammatory protein 1α and elevated urine chemokines at 48 hours after challenge. These observations give unique insight into the pathologic consequences of each toxin in a near human setting and present potential pathways for therapeutic intervention.

Cattle are the main asymptomatic reservoir of Escherichia coli O157:H7 which can cause illness to human. The objectives of the study were to measure the prevalence of E. coli O157:H7 on cattle slaughtered in Amman abattoir, detect virulence factors in the isolates, determine antibacterial resistance of the isolates, and know how the isolates are different or similar when compared to characterized isolates from developed countries. A total of 540 samples (feces, hide, and carcass) were tested for E. coli O157:H7 using the method of ISO 16654:(E). Conventional and multiplex PCR assays were used for serotype confirmation and virulence factor detection, respectively. Fifty E. coli O157:H7 isolates were identified and virulence factors eaeA and hlyA were present in all of the isolates. 60%, 12%, and 22% of the isolates harbored stx(1), stx(2), and stx(1) and stx(2), respectively. The prevalence rates of enterotoxigenic E. coli O157:H7 (n=47) were 8.3%, 10%, and 7.8% in feces, hides and carcasses, respectively. The antimicrobial profiles of the isolates showed an extensive resistance to erythromycin, neomycin and vancomycin and high sensitivity to ampicillin, ciprofloxacin, gentamicin, kanamycin and tetracycline.

Shiga toxin B-subunit (STxB) from Shigella dysenteriae targets in vivo antigen to cancer cells, dendritic cells (DC) and B cells, which preferentially express the globotriaosylceramide (Gb3) receptor. This pivotal role has encouraged scientists to investigate fusing STxB with other clinical antigens. Due to the challenges of obtaining a functional soluble form of the recombinant STxB, such as formation of inclusion bodies during protein expression, scientists tend to combine STxB with vaccine candidates rather than using their genetically fused forms. In this work, we fused HPV16 E7 as a vaccine candidate to the recombinantly-produced STxB. To minimize the formation of inclusion bodies, we investigated a number of conditions during the expression procedure. Then various strategies were used in order to obtain high yield of soluble recombinant protein from E. coli which included the use of different host strains, reduction of cultivation temperature, as well as using different concentrations of IPTG and different additives (Glycin, Triton X-100, ZnCl(2)). Our study demonstrated the importance of optimizing incubation parameters for recombinant protein expression in E. coli; also showed that the secretion production can be achieved over the course of a few hours when using additives such as glycine and Triton X-100. Interestingly, it was shown that when the culture mediums were supplemented by additives, there was an inverse ratio between time of induction (TOI) and the level of secreted protein at lower temperatures. This study determines the optimal conditions for high yield soluble E7-STxB expression and subsequently facilitates reaching a functionally soluble form of STxB-based vaccines, which can be considered as a potent vaccine candidate for cervical cancer.

Four hundred and fifty beef animal hides and a similar number of carcasses were screened for STEC in 3 beef abattoirs over a 12 month period using PCR and culture based methods. 67% (301/450) of hides and 27% (122/450) of carcasses were STEC PCR positive. Forty isolates representing 12 STEC serotypes (O5:H-, O13:H2, O26:H11, O33:H11, O55:H11, O113:H4, O128:H8, O136:H12, O138:H48, O150:H2, O168:H8 and ONT:H11) and 15 serotype–virulotype combinations were identified. This study provides much needed non-O157 STEC surveillance data and also provides further evidence of bovines as a source of clinically significant STEC as well as identifying 3 emerging serotypes O5:H- ( eae -β1), O13:H2 ( eae -ζ), and O150:H2 ( eae -ζ) that should be considered when developing beef testing procedures for non-O157 STEC.

The prevalence and characteristics of non-O157 Shiga toxin-producing Escherichia coli (STEC) in retail ground meat from the Washington D.C. area were investigated in this study. STEC from 480 ground beef and pork samples were identified using PCR screening followed by colony hybridization. The STEC isolates were serogrouped and examined for the presence of virulence genes (stx1, stx2, eae and hlyA), and antimicrobial susceptibility. PFGE was used to identify the clonal relationships of STEC isolates, and PCR-RFLP was employed to determine stx subtypes. In addition, the cytotoxicity of STEC isolates was determined using a Vero cell assay. STEC were identified in 12 (5.2%) of 231 ground pork and 13 (5.2%) of 249 ground beef samples. Among 32 STEC isolates recovered from the 25 samples, 12 (37.5%) carried stx2dact and 7 (21.9%) carried hlyA, but none carried eae. Nine isolates were identified as O91, and 17 (53.1%) isolates were resistant to two or more antimicrobials. Verotoxicity was detected in 26 (81.3%) of the STEC isolates. Thus, the retail ground meat was contaminated with a heterogeneous population of non-O157 STEC, some of which were potential human pathogens.

Conventional enzyme-linked immunosorbent assay (ELISA) is a gold standard for screening antibodies and testing for protein or antigen presence. A significant limitation of this assay resides in the fact that only one analyte can be assessed per microplate well. Here, we describe and investigate a new technology consisting of an automated ELISA system in which up to 10 analytes can be measured within one single well, thus improving productivity, accuracy, and repeatability by reducing the amount of human labor required. Another strength of the platform is that a user can load any necessary sets/subsets of beads to perform required assays, with improved flexibility compared to manufactured-loaded arrays for multiplex analysis. We also demonstrate that this system can be used to determine the pathogenicity (i.e., presence of Shiga toxins) and serotype (i.e., Escherichia coli O157) of E. coli isolates.

The purpose of this study was to investigate carriage and transfer of verocytotoxigenic Escherichia coli (VTEC) O157, O26, O111, O103 and O145 from faeces and hide to dressed carcasses of Irish cattle as well as establishing the virulence potential of VTEC carried by these cattle. Individual cattle was tracked and faecal samples, hide and carcass (pre-evisceration and post-wash) swabs were analysed for verotoxin (vt1 and vt2) genes using a duplex real-time PCR assay. Positive samples were screened for the five serogroups of interest by real-time PCR. Isolates were recovered from PCR positive samples using immunomagnetic separation and confirmed by latex agglutination and PCR. Isolates were subject to a virulence screen (vt1, vt2, eaeA and hlyA) by PCR. Isolates carrying vt genes were examined by Pulsed-Field Gel Electrophoresis (PFGE). Of the VTEC isolated, E. coli O157 was the most frequently recovered from hide (17.6%), faeces (2.3%) and pre-evisceration/post-wash carcass (0.7%) samples. VTEC O26 was isolated from 0.2% of hide swabs and 1.5% of faeces samples. VTEC O145 was isolated from 0.7% of faeces samples. VTEC O26 and VTEC O145 were not recovered from carcass swabs. Non-VTEC O103 was recovered from all sample types (27.1% hide, 8.5% faeces, 5.5% pre-evisceration carcass, 2.2% post-wash carcass), with 0.2% of hide swabs and 1.0% of faeces samples found to be positive for VTEC O103 isolates. E. coli O111 was not detected in any samples. For the four serogroups recovered, the direct transfer from hide to carcass was not observed. This study shows that while VTEC O157 are being carried by cattle presented for slaughter in Ireland, a number of other verotoxin producing strains are beginning to emerge.

We quantified translocation of Escherichia coli O157:H7 (ECOH) and non-O157:H7 verocytotoxigenic E. coli (STEC) into beef subprimals after brine injection and subsequently monitored their viability after cooking steaks cut therefrom. Beef subprimals were inoculated on the lean side with ca. 6.0 log CFU/g of a five-strain cocktail of rifampin-resistant ECOH or kanamycin-resistant STEC, and then passed once through an automatic brine-injector tenderizer, with the lean side facing upward. Brine solutions (9.9% ± 0.3% over fresh weight) consisted of 3.3% (wt/vol) of sodium tripolyphosphate and 3.3% (wt/vol) of sodium chloride, prepared both with (Lac(+), pH = 6.76) and without (Lac(-), pH = 8.02) a 25% (vol/vol) solution of a 60% potassium lactate-sodium diacetate syrup. For all samples injected with Lac(-) or Lac(+) brine, levels of ECOH or STEC recovered from the topmost 1 cm (i.e., segment 1) of a core sample obtained from tenderized subprimals ranged from ca. 4.7 to 6.3 log CFU/g; however, it was possible to recover ECOH or STEC from all six segments of all cores tested. Next, brine-injected steaks from tenderized subprimals were cooked on a commercial open-flame gas grill to internal endpoint temperatures of either 37.8 °C (100 °F), 48.8 °C (120 °F), 60 °C (140 °F), or 71.1 °C (160 °F). Regardless of brine formulation or temperature, cooking achieved reductions (expressed as log CFU per gram) of 0.3 to 4.1 of ECOH and 0.5 to 3.6 of STEC. However, fortuitous survivors were recovered even at 71.1 °C (160 °F) for ECOH and for STEC. Thus, ECOH and STEC behaved similarly, relative to translocation and thermal destruction: Tenderization via brine injection transferred both pathogens throughout subprimals and cooking highly contaminated, brine-injected steaks on a commercial gas grill at 71.1 °C (160 °F) did not kill all cells due, primarily, to nonuniform heating (i.e., cold spots) within the meat.

Shiga toxins (Stxs) are involved in the pathogenesis of hemolytic-uremic syndrome and other severe systemic complications following enterohemorrhagic Escherichia coli infection in humans. Passive immunotherapies using monoclonal antibodies have been shown to be effective for neutralizing the toxic effects of Stxs. However, animal-derived monoclonal antibodies are sometimes immunogenic and their production is both laborious and expensive. We here report the isolation of single-chain variable fragment antibodies against Stxs by screening a phage display library constructed from a naïve human repertoire. An antibody among the selected clones designated B22 bound to the binding subunits of both Stx-1 and Stx-2, and strongly neutralized the cytotoxicity of Stx-1. This is the first example of a monovalent antibody showing Stx-neutralizing activity. The B22 antibody is also completely naturally occurring in human, which reduces the possibility of adverse immunological effects, and can be easily produced using bacterial protein synthesis systems.

The ability of foodborne pathogens to survive in certain foods mainly depends on stress response mechanisms. Insight into molecular properties enabling pathogenic bacteria to survive in food is valuable for improvement of the control of pathogens during food processing. Raw milk cheeses are a potential source for human infections with Shiga toxin-producing Escherichia coli (STEC). In this review, we focused on the stress response mechanisms important for allowing STEC to survive raw milk cheese production processes. The major components and regulation pathways for general, acid, osmotic, and heat shock stress responses in E. coli and the implications of these responses for the survival of STEC in raw milk cheeses are discussed.

Shiga toxin is a member of AB toxin family and is composed of an A subunit which mediated toxicity and a homopentameric protein responsible for toxin binding and internalization into target cells. Another group of diarrheagenic Escherichia coli, enteroaggregative E. coli (EAEC) is a group of E. coli with aggregative adherence to epithelial cells, which play an important role in its pathogenesis. In the present investigation, the immune response of recombinant hybrid peptide composed of B subunit of Shiga toxin (StxB) and Aggregative Adherence Fimbriae (AAF) of EAEC (B-AAF/I, B-AAF/II) that elicited protective response was further characterized. The assessment of IgG subclasses (IgG1 and IgG2a) and cytokine production by these peptides indicated that although the hybrid peptides could induce immune response, but two adhesins behave differently in this regard. Lymphocyte proliferation assay and IFN-γ production were highly significant for B-AAF/II. Overall, based on the data obtained from this study it seems that mixed population of Th1-Th2 type of immune responses were induced by these hybrid peptides, which probably lead to observed protective response. In the present study, it is shown that the two hybrid peptides i.e. B-AAF/I and B-AAF/II, could be a promising strategy to make more effective and powerful vaccine.

The sequelae of infection with Escherichia coli O157:H7 include the potentially fatal haemolytic uraemic syndrome. The pathobiological process of E. coli O157:H7 is chiefly dependent on the production of Shiga-like toxins I and II (SLT-I and -II). Antibiotic treatment is currently refrained from since it may lead to enhanced release of SLTs from the bacterium. In this study, the potential utility of rifampicin in treating E. coli O157:H7 infections was assessed both in vitro and in vivo. Five strains of E. coli O157:H7 were tested by reverse transcriptase polymerase chain reaction (RT-PCR) for the transcription of the SLT-I- and SLT-II-encoding genes (stx1 and stx2, respectively). Treatment of bacterial strains with the rifampicin minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), or the MIC followed by the MBC led to the inhibition of stx1 and stx2 gene transcription. Treatment with the MIC or with the MIC followed by the MBC was also capable of limiting toxin release. SLT-I and SLT-II detection by reverse passive latex agglutination showed an effective decrease in toxin titres following treatment with the MIC of rifampicin or with the MIC followed by the MBC. Treatment of cultures with the MBC alone was not as effective in decreasing toxin titres. The efficacy of rifampicin in treating E. coli O157:H7-infected BALB/c mice was also assessed. Rifampicin treatment resulted in enhanced mouse survival and limited the weight loss of infected animals. In conclusion, both in vitro and in vivo tests showed that rifampicin may be useful in treating E. coli O157:H7 infection.

Escherichia coli O157:H7 is a human pathogen that has emerged from its less pathogenic progenitor, E. coli O55:H7, to form the EHEC 1 clade. In its emergence, E. coli O157:H7 formed three distinct clusters, each of which exists today. Sequencing and SNP analysis of Cluster 1 of this clade demonstrated constrained radiation from the cluster founder. Here we investigated the diversity of Cluster 2 strains by sequencing signature SNPs in six strains collected throughout Washington State. Our results suggest that successful Cluster 2 strains have radiated on only two branches from their founder; one of these two branches leads to Cluster 3. Constrained radiation appears to be a common theme among this pathogenic clade.

The prevalence of verocytotoxin producing Escherichia coli (VTEC), E. coli O157, and VTEC O157 in 474 swab samples from Danish beef carcasses was determined. The presence of E. coli O157 was determined by a culture method that included immunomagnetic separation (IMS) followed by real time PCR testing of isolates for verocytotoxin (vtx) genes. E. coli O157 was recovered from 4.2% of the carcass samples and VTEC O157 from 3.4% of the samples. All VTEC O157 contaminated carcasses were from bull calves and the VTEC O157 prevalence on bull calf carcasses was 7.3%. The VTEC O157 contaminated beef carcasses were sampled again after one week of cold storage, and 15 of the 16 carcasses were then VTEC O157 negative. The presence of VTEC was determined by a duplex real time PCR assay for vtx1 and vtx2 in DNA from enrichment cultures of swabs. In total 45.4% of the samples were VTEC positive. VTEC were isolated from 21% of 77 vtx-positive samples that were identified by replication of colonies on hydrophobic grid membrane filters followed by hybridisation with vtx specific DNA probes. Fourteen of the 16 VTEC isolates were non-O157 and these strains were negative for the virulence gene eae. A real time PCR assay for the E. coli O157 specific rfbE gene was developed. In total 22.4% of the enriched samples were positive for the O157 rfbE gene. The combined results of the vtx and rfbE real time PCR screening showed that 17.5% of the carcasses potentially were contaminated with VTEC O157. Screening of carcass swabs was expanded by real time PCR testing for eae in a subset of the samples. Of 244 samples, 25.4% were positive for both vtx and eae. The eae gene was detected in 81% of the vtx-positive samples and in 46% of 67 vtx-negative samples, indicating that bacteria harbouring eae are widespread on bovine carcasses. The present study shows that real time PCR screening of carcass samples for genes encoding virulence or other genetic markers is a reliable method for rapid identification of carcasses that potentially are contaminated with VTEC.

The examination of 2005 raw bovine (n = 950), caprine (n = 460) and ovine (n = 595) bulk milk samples collected throughout several regions in Greece for the presence of Escherichia coli serogroup O157 resulted in the isolation of 29 strains (1.4%) of which 21 were isolated from bovine (2.2%), 3 from caprine (0.7%) and 5 from ovine (0.8%) milk. Out of the 29 E. coli O157 isolates, only 12 (41.4%) could be classified as Shiga-toxigenic based on immunoassay and PCR results. All 12 Shiga-toxigenic E. coli serogroup O157 isolates belonged to the E. coli O157:H7 serotype. All except one of the 12 Shiga-toxin positive isolates were stx(2)-positive, five of which were also stx(1)-positive. The remaining isolate was positive only for the stx(1) gene. All stx-positive isolates (whether positive for stx(1), stx(2) or stx(1) and stx(2)) were also PCR-positive for the eae and ehxA genes. The remaining 17 E. coli O157 isolates (58.6%) were negative for the presence of the H7 flagellar gene by PCR, tested negative for Shiga-toxin production both by immunoassay and PCR, and among these, only four and three strains were PCR-positive for the eae and ehxA genes, respectively. All 29 E. coli O157 isolates displayed resistance to a wide range of antimicrobials, with the stx-positive isolates being, on average, resistant to a higher number of antibiotics than those which were stx-negative.

Sorting nexin 8 (SNX8) belongs to the sorting nexin protein family, whose members are involved in endocytosis and endosomal sorting and signaling. The function of SNX8 has so far been unknown. Here, we have investigated the role of SNX8 in intracellular transport of the bacterial toxin Shiga toxin (Stx) and the plant toxin ricin. After being endocytosed, these toxins are transported retrogradely from endosomes, via the Golgi apparatus and the endoplasmic reticulum (ER), into the cytosol, where they exert their toxic effect. Interestingly, our experiments show that SNX8 regulates the transport of Stx and ricin differently; siRNA-mediated knockdown of SNX8 significantly increased the Stx transport to the trans-Golgi network (TGN), whereas ricin transport was slightly inhibited. We also found that SNX8 colocalizes with early endosome antigen 1 (EEA1) and with retromer components, suggesting an endosomal localization of SNX8 and supporting our finding that SNX8 is involved in endosomal sorting.

The aim of this study was to develop a candidate vaccine against enterohemorrhagic Escherichia coli O157:H7. A ler deletion mutant derived from wild-type EHEC O157:H7 86-24 was constructed by use of suicide vector pCVD442. The bacteriophage encoding Shiga toxin (Stx) was excised by serial passage to produce a ler/stx deletion mutant, F25. Stx1 and Stx2 mutants were constructed by site-directed mutagenesis within the active center and membrane-spanning region of the toxin A subunit. Mutants stx1 and stx2 were then introduced into F25 to construct live attenuated candidate vaccine F105. The cytotoxicity of F25 was inactivated and that of F105 was significantly reduced in comparison with wild-type E. coli strain EDL933. Mice injected with candidate vaccine strains F25 and F105 gained weight and showed no clinical signs of disease. F25 and F105 reduced the colonization of wild-type O157:H7 in mouse intestine. Immunized pregnant mice were able to protect their suckling newborns from intragastric challenge with wild-type O157:H7. Immunized mice were protected against infection with wild-type O157:H7 and exhibited normal weight gain. Such attenuated vaccine strains may therefore have potential use as oral vaccines against O157:H7.

Escherichia coli O157 is often associated with hemorrhagic colitis and the hemolytic uremic syndrome (HUS). The verocytotoxins are considered to be the major virulence determinants. However, vt-negative E. coli O157 were recently isolated from patients with HUS. Several transmission routes to humans are described, but cattle feces are the primary source from which both the food supply and the environment become contaminated with E. coli O157. In a prevalence study performed on dairy, beef, mixed dairy/beef and veal farms in the summer of 2007, vt-negative isolates were detected on 11.8% (8/68) of the positive farms. From these eight farms, a total of 43 sorbitol-negative E. coli O157:H7 were collected. On five farms, only strains negative for the vt genes were present whereas both vt-negative and vt-positive strains could be detected on three other farms. Further characterization revealed that all isolates carried the eaeA and hlyA genes. Pulsed-field gel electrophoresis (PFGE) of all isolates resulted in nine different PFGE types and within the vt-negative strains, four different genotypes were identified, indicating that certain genetic clones are widespread over the cattle population.

Shiga toxin-producing Escherichia coli (STEC) are an important group of emerging pathogens, with ruminants recognised as their main natural reservoir. The aim of this work was to establish the prevalence of non-O157 STEC in free-ranging wild ruminants in the Extremadura region of Spain and to characterise them phenogenotypically. Faecal samples were collected from 243 wild ruminants, including Cervus elaphus, Capreolus capreolus, Dama dama and Ovis musimon and were examined for STEC using both phenotypic (Vero cells) and genotypic (PCR and PFGE) methods. Shiga toxin-producing Escherichia coli were isolated from 58 (23.9%) of the samples and a total of 65 isolates were characterised. A PCR method indicated that 11 (16.9%) strains carried the stx 1 gene, 44 (67.7%) carried the stx 2 gene and 10 (15.4%) carried both these genes. The ehxA gene was detected in 37 (57%) of the isolates but none contained either the eae or saa genes. The isolates were from a total of 12 ‘O’ serogroups, although 80% were restricted to the O2, O8, O128, O146, O166 and O174 serogroups. The most commonly isolated STEC bacteria, which were from the O146 serogroup, exhibited a high degree of polymorphism as indicated by PFGE. Shiga toxin-producing Escherichia coli isolates of serogroups O20, O25, O166, O171, O174 and O176 had not previously been found in wild ruminants. This is the first study to confirm that wild ruminants in Spain are a reservoir of STEC and are thus a potential source of human infection.

The hemolytic uremic syndrome (HUS) of childhood is, in its most common form, a non-immune microangiopathic hemolytic anemia. HUS typically follows an enteric infection with a Shiga toxin-producing Escherichia coli, usually belonging to serotype O157:H7. The antecedent infection is almost always manifested as non-bloody diarrhea. In about 80% of cases, the diarrhea becomes bloody between one and five days after the onset of diarrhea. The courses of acute gastrointestinal infections, and of HUS, in adults and children are similar. There are no convincing data that this easily recognizable form of microangiopathy is related to any inborn or acquired deficiency of ADAMTS13, and plasma therapies are not justified on either theoretical or empirical grounds. Similarly, there are no realistic animal data to support use of plasma exchange or infusion in children or adults with, or at risk for, HUS secondary to gastrointestinal infection with E. coli O157:H7. Best clinical practices involve rapid and accurate clinical and microbiological identification of infected patients, volume expansion, and support of the intestinal and extraintestinal complications that can ensue during acute enteric infection and associated HUS. Clinical clues include a sequence of events where the stool becomes bloody after a several-day interval of bloody diarrhea, considerable abdominal pain, five or more stools in the 24 h before presentation, pain on defection, and absence of fever at the time of presentation. Diagnosis should rely primarily on sorbitol MacConkey agar culture. Shiga toxin testing should not be used as the only screen to identify infected patients.

Verocytotoxin (VT)-producing Escherichia coli (VTEC) infection is associated with a spectrum of clinical manifestations that includes diarrhea, hemorrhagic colitis, and the hemolytic uremic syndrome (HUS). The occurrence of HUS in a minority of individuals in outbreaks of VTEC infection is a function of several pathogen and host factors. Pathogen factors include the inoculum size and serotype of the infecting strain, horizontally acquired genetic elements known as pathogenicity islands, and probably the VT type. Host factors that increase the risk of developing HUS include age, pre-existing immunity, gastric acidity, the use of antibiotics and anti-motility agents, and, probably, stress and genetic factors that modulate host response to infection, such as innate immunity and toxin receptor type, expression, and distribution. A better understanding of the pathogen and host determinants of HUS can aid in the development of more effective public health strategies to reduce the risk of developing HUS.

A longitudinal study was conducted on two dairy farms to investigate the pattern of shedding of verotoxin-producing Escherichia coli (VTEC) in goats. Faecal samples were taken from 20 goat kids once weekly during the first 4 weeks of life and then once every month for the next 5 months of life, and from 18 replacement animals and 15 adults once every month for 12 months. The proportion of samples containing VTEC was higher for replacement animals and adults (85.7% and 78.7%, respectively) than for goat kids (25.4%). About 90% of the VTEC colonies isolated from healthy goats belonged to five serogroups (O33, O76, O126, O146 and O166) but the most frequent serogroups of these isolates, except one, were different in the two herds studied. E. coli O157:H7 was found in three goat kids on only one occasion. None of the VTEC isolates, except the three E. coli O157:H7 isolates, was eae-positive. The patterns of shedding of VTEC in goat kids were variable, but, in contrast, most of the replacement animals and adults were persistent VTEC shedders. Our results show that isolates of VTEC O33, O76, O126, O146 and O166 are adapted for colonising the intestine of goats but that, in contrast, infection with VTEC O157:H7 in goats seems to be transient.

In this study, 100 raw meat samples were collected from 15 local Moroccan butcheries in five different areas of the city of Rabat during a period of 4 months. Overall, 7 of 15 butcheries from three areas of the city yielded strains of Escherichia coli O157. Single isolates from 9 (9%) of 100 raw meat samples were biochemically and serologically confirmed as E. coli O157. Using molecular techniques, two strains were positive for the Shiga toxin, with two additional strains containing an attaching-effacing gene. All potentially virulent serotypes isolated from these meat samples showed distinct pulsed-field gel electrophoresis profiles. Based on antibiotic susceptibility testing, more than 70% of the isolates were resistant to ampicillin and clavulanic acid-amoxicillin. Moreover, one strain was resistant to more than three antibiotics. Our study represents the first survey of E. coli O157 and related serotypes in raw meat products in Morocco.

The prevalence, serotypes and virulence profiles of Shiga toxin-producing Escherichia coli (STEC) were investigated in 205 healthy beef and dairy cattle, and 106 goats reared in the southeastern region of Minas Gerais State, Brazil. The prevalence of STEC was 57.5% (61/106) in goats, 39.2%, (40/102) in beef cattle and 17.5% (18/103) in dairy cattle. Among the 514 STEC isolates, 40 different serotypes were found and some of them were identified in a specific host. STEC isolates harboring stx1 corresponded to 15.6% (28/180), 26.7% (16/60) and 24.1% (66/274) in beef cattle, dairy cattle and goats, respectively. stx2 was found in 30% (54/180), 53.3% (32/60) and 34.7% (95/274) of beef and dairy cattle, and goats. stx1 plus stx2 sequences were harbored by 54.4% (98/180), 20% (12/60) and 41.2% (113/274) of beef cattle, dairy cattle and goats, respectively. The eae sequence was found in 15% (9/60) and 0.6% (1/180) of STEC isolates from dairy and beef cattle, respectively, and the toxB gene was found only in one O157:H7 strain isolated from beef cattle. Strains with the genetic profiles stx2 ehxA iha saa and stx1 stx2 ehxA iha saa were the most prevalent among STEC isolates from cattle. Profiles stx1 stx2 ehxA iha, stx2, and stx1 iha accounted for 75.5% (207 /274) of the STEC isolates from goats. While STEC strains carrying either stx2 alone or associated with stx1 were found more frequently in cattle, those harboring sequences stx1c and stx2d alone or associated with stx1c predominated in goats. Our data show a diversity of STEC strains in food-producing animals, most of them carrying genes linked to severe forms of human diseases.

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O176 has been determined. Component analysis together with 1H and 13C NMR spectroscopy was employed to elucidate the structure. Inter-residue correlations were determined by 1H, 1H NOESY and 1H, 13C heteronuclear multiple-bond correlation experiments. The PS is composed of tetrasaccharide repeating units with the following structure: [Formula: see text] Cross-peaks of low intensity from alpha-linked mannopyranosyl residues were present in the 1H, 1H TOCSY NMR spectra and further analysis of these showed that they originate from the terminal part of the polysaccharide. Consequently, the biological repeating unit has a 3-substituted N-acetyl-d-galactosamine residue at its reducing end. The repeating unit of the E. coli O176 O-antigen is similar to those from E. coli O17 and O77, thereby explaining the reported cross-reactivities between the strains, and identical to that of Salmonella cerro (O:6, 14, 18).

We have made adamantylGSLs by substituting the fatty acids of primarily, globotriaosyl ceramide(Gb(3)) and sulfogalactosyl ceramide(SGC), with the rigid alpha-adamantane hydrocarbon frame. These analogues have proven to be remarkably water-soluble but retain the receptor function of the parent membrane GSL. AdaGb(3) prevents the binding of verotoxins to target cells but increased pathology in vivo, likely due to the partitioning into receptor negative target cells to provide pseudo-receptors. Preincubation of HIV with adaGb(3) prevents cellular infection in vitro and viral-host cell fusion. Cellular accumulation of Gb(3) reduces HIV susceptibility in vitro, whereas lack of Gb(3) promotes infection, suggesting that Gb(3) expression could be a novel risk factor for HIV susceptibility. AdaGb(3) has proven to be a new inhibitor for the MDR1 drug pump (P-glycoprotein) and can reverse drug resistance in cell culture. AdaSGC is bound by hsp70/hsc70 within the N-terminal ATPase domain and inhibits chaperone function. When added to cells transfected with the DeltaF508 CFTR mutant, adaSGC was able to decrease ER degradation of this mutant protein, an hsc70 dependent process. Our finding that DeltaF508 CFTR expressing cells show reduced SGC biosynthesis suggests that SGC could be an additional natural regulator of the hsp70 chaperone ATPase cycle.

Multidrug resistance (MDR) via the ABC drug transporter (ABCB1), P-glycoprotein (P-gp/MDR1) overexpression, is a major obstacle in cancer chemotherapy. Many inhibitors reverse MDR but, like cyclosporin A (CsA), have significant toxicities. MDR1 is also a translocase that flips glucosylceramide inside the Golgi to enhance neutral glycosphingolipid (GSL) synthesis. We observed partial MDR1/globotriaosylceramide (Gb3) cell surface co-localization, and GSL removal depleted cell surface MDR1. MDR1 may therefore interact with GSLs. AdamantylGb3, a water-soluble Gb3 mimic, but not other GSL analogs, reversed MDR1-MDCK cell drug resistance. Cell surface MDR1 was up-regulated 1 h after treatment with CsA or adaGb3, but at 72 h, cell surface expression was lost. Intracellular MDR1 accumulated throughout, suggesting long term defects in plasma membrane MDR1 trafficking. AdaGb3 or CsA rapidly reduced rhodamine 123 cellular efflux. MDR1 also mediates gastrointestinal epithelial drug efflux, restricting oral bioavailability. Vinblastine apical-to-basal transport in polarized human intestinal C2BBe1 cells was significantly increased when adaGb3 was added to both sides, or to the apical side only, comparable with verapamil, a standard MDR1 inhibitor. Disulfide cross-linking of mutant MDR1s showed no binding of adaGb3 to the MDR1 verapamil/cyclosporin-binding site between surface proximal helices of transmembrane segments (TM) 6 and TM7, but rather to an adjacent site nearer the center of TM6 and the TM7 extracellular face, i.e. close to the bilayer leaflet interface. Verotoxin-mediated Gb3 endocytosis also up-regulated total MDR1 and inhibited drug efflux. Thus, a functional interplay between membrane Gb3 and MDR1 provides a more physiologically based approach to MDR1 regulation to increase the bioavailability of chemotherapeutic drugs.

The aim of this study was to characterize Shiga toxigenic Escherichia coli (STEC) by PCR using strains isolated from ham, beef, and cattle in Colombia. A total of 189 E. coli strains were tested for the presence of the uidA, stx1, and stx2 genes, and identification was confirmed by the automated PCR BAX system for E. coli O157:H7. Genes encoding Shiga-like toxins (stx) were found in eight (6.06%) of 132 strains previously isolated from minced beef; four (50%) of these strains yielded amplification products for both toxin genes (stx1 and stx2), and four (50%) yielded products only for the stx2 toxin. None of the strains analyzed were positive by PCR for the presence of the single base-pair mutation in the uidA gene from E. coli O157:H7; these results were confirmed by the BAX system analysis. A multiplex PCR assay was standardized for the three genes. Results from this study confirmed previous data about the low prevalence of E. coli O157:H7 and Shiga-like toxins in Colombia and is the first known report of the prevalence of non-O157 enterohemorrhagic E. coli in this country.

The prevalence of verocytotoxin-producing Escherichia coli (VTEC) O157 in 12–30-month-old beef finishing cattle in Scotland was determined using 1 g faeces samples enriched in buffered peptone water, followed by immunomagnetic separation (IMS) and isolation on sorbitol MacConkey agar with cefixime and tellurite supplement (CT-SMAC). A validated questionnaire was used to collect information that could be associated with the samples. Generalised Linear Models and Generalised Linear Mixed Models were used to identify factors associated with shedding both between and within groups. A total of 14,856 samples were collected from 952 farms, of which 1231 were positive for VTEC O157. Prevalence levels were calculated with 95% confidence intervals as follows: 7.9% (6.5%, 9.6%) of animals sampled were estimated to be shedding VTEC O157, while 22.8% (19.6%, 26.3%) of farms were estimated as having at least one animal shedding in the group sampled. The median percentage of animals shedding in positive groups was 25% (20%, 32%). An increased probability of a group containing a shedding animal was associated with larger numbers of finishing cattle, the presence of pigs on the farm, or the farm being classed as a dairy unit stocking beef animals. Farms that spread slurry on grazing land were more likely to have shedding animals, while those that spread manure were at lower risk. Groups with older animals were less likely to be identified as positive. There was no significant regional difference in group shedding probabilities, but the proportion of positive groups dropped over two successive years of the study. Higher mean levels of shedding in positive groups were associated with animals being housed rather than at pasture, and this effect was stronger in groups which had recently had a change in housing or diet. Farms with animals at pasture had lower mean prevalence where water was supplied from a natural source, as had farms with higher numbers of finishing cattle. There remained unexplained variability in mean prevalence levels on positive farms in different areas of Scotland.

Shiga-toxin-converting bacteriophages (Stx phages) are temperate phages of Escherichia coli, and can cause severe human disease. The spread of shiga toxins by Stx phages is directly linked to lysogen stability because toxins are only synthesized and released once the lytic cycle is initiated. Lysogens of Stx phages are known to be less stable than those of the related lambda phage; this is often described in terms of a 'hair-trigger' molecular switch from lysogeny to lysis. We have developed a mathematical model to examine whether known differences in operator regions and binding affinities between Stx phages and lambda phage can account for the lower stability of Stx lysogens. The Stx phage 933W has only two binding sites in its left operator region (compared to three in phage lambda), but this has a minimal effect on 933W lysogen stability. However, the relatively weak binding affinity between repressor molecules and the second binding site in the right operator is found to significantly reduce the stability of its lysogens, and may account for the hair-trigger nature of the switch. Reduced lysogen stability can lead to increased frequency of genetic recombination in bacterial genomes. The development of the mathematical model has considerable utility in understanding the behaviour and evolution of the molecular switch, with implications for phage-related diseases.

The targeted delivery of drugs to the cell interior can be accomplished by taking advantage of the various receptor-mediated endocytic pathways operating in a particular cell. Among these pathways, the retrograde trafficking pathway from endosomes to the Golgi apparatus, and endoplasmic reticulum is of special importance since it provides a route to deliver drugs bypassing the acid pH, hydrolytic environment of the lysosome. The existence of pathways for drug or antigen delivery to the endoplasmic reticulum and Golgi apparatus has been to a large extent an outcome of research on the trafficking of A/B type-bacterial or plant toxins such as Shiga toxin within the cell. The targeting properties of these toxins reside in their B subunit. In this article we present an overview of the multiplicity of pathways to deliver drugs intracellularly. We highlight the retrograde trafficking pathway illustrated by Shiga toxin and Shiga-like toxin, and the potential role of the B subunit of these toxins as carriers of drugs, antigens and imaging agents.

The aim of the present study was to investigate the occurrence of Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (STEC) in 'Castellano' cheese, a non-cooked and hard or semi-hard Spanish cheese made from ewe's milk. A total of 83 raw milk cheese samples with different ripening times (2.5, 6 and 12 months) were taken at 30 cheese factories. Samples were examined for the presence of STEC using in the first stage the Association of Official Analytical Chemists (AOAC) official method number 997.11, and then, in the second stage, isolates were tested for virulence genes using genotypic (PCR) methods. Three STEC strains were detected in two samples (2.4%) of 'Castellano' cheese, one with 2.5 and the other one with 12 month-ripening period. From those STEC isolates, two were identified as E. coli O14 and the third presented an O-specific polysaccharide not-groupable serologically (ONG). PCR showed that all isolates were characterized by harbouring the Shiga toxin (stx) stx1 gene and by the absence of the genes for stx2, eaeA, and ehxA virulence factors. This study revealed the potential of STEC to survive in long-ripened-hard cheeses.

Shiga toxigenic Escherichia coli (STEC) are important foodborne pathogens causing gastrointestinal disease worldwide. Bacterial attachment to food surfaces, such as stainless steel may lead to cross contamination of foods and subsequent foodborne disease. A variety of STEC isolates, including E. coli O157:H7/H- strains, were grown in planktonic (broth) and sessile (agar) culture, following which initial attachment to stainless steel was determined using epifluorescence microscopy. Experiments were performed to determine whether the number of bacteria attached to stainless steel differed between STEC strains and between the two modes of growth. No relationship was found between STEC strains and the number of bacteria attached to stainless steel. Five STEC strains, including one non-toxigenic O157 isolate, attached in significantly greater (p0.05) numbers to stainless steel following growth in planktonic culture compared to sessile culture. In contrast, two clinical strains of O157:H7 attached in significantly greater (p0.05) numbers following growth in sessile culture compared to planktonic culture. Thirteen out of twenty E. coli strains showed no significant difference (p0.05) in attachment when grown in planktonic or sessile culture. The change of interfacial free energy between the bacterial strains and stainless steel was calculated and the influence of free energy in attachment was determined. Although a significant variation (p0.05) in free energy values was found between STEC strains, no correlation was found between free energy values and bacterial counts on stainless steel. In addition, no correlation was also found between bacterial hydrophobicity and surface charge values or production of surface structures (type I fimbriae or flagella) (previously determined) with the number of bacteria attached to stainless steel. The results of this study suggest that different growth conditions (planktonic and sessile) can influence the attachment of STEC to stainless steel. Factors other than bacterial physicochemical properties and these surface structures may also influence STEC attachment to stainless steel.

Attachment of Shiga toxigenic Escherichia coli (STEC) to surfaces and the formation of biofilms may enhance persistence in a food processing environment and present a risk of contaminating products. Seven strains of STEC and three non-STEC strains were selected to compare two biofilm quantification methods; epifluorescence microscopy on stainless steel (SS) and a microtitre plate assay. The influence of prior growth in planktonic (nutrient broth) and sessile (nutrient agar) culture on biofilm production, as well as expression of surface structures and the possession of antigen 43 (encoded by agn43) on biofilm formation were also investigated. Biofilms were produced in diluted nutrient broth at 25 degrees C for 24 and 48 h. Curli expression was determined using congo red indicator agar, while the presence of agn43 was determined using polymerase chain reaction. No correlation was found between counts for epifluorescence microscopy on SS and the absorbance values obtained with the microtitre plate method for planktonic and sessile grown cultures. Different abilities of individual STEC strains to attach to SS and microtitre plates were found with some strains attaching better to each surface following growth in either planktonic or sessile culture. All O157 STEC strains had low biofilm counts on SS for planktonic and sessile grown cultures; however, one STEC O157:H- strain (EC516) had significantly greater (p0.05) biofilm production on microtitre plates compared to the other O157 STEC strains. EC516 and other STEC (O174:H21 and O91:H21) strains expressing curli fimbriae were found to produce significantly greater (p0.05) biofilms on microtitre plates compared to the non-curli expressing strains. No relationship was found between the production of type-I fimbriae, motility, agn43 and bacterial physicochemical properties (previously determined) and biofilm formation on SS or microtitre plates. Variations between the two biofilm determination methods may suggest that the biofilm production on microtitre plates may not be appropriate to represent other surfaces such as SS and that caution should be taken when selecting a method to quantify biofilm production on a surface.