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Hemolytic uremic syndrome (HUS) is a systemic sequelae from gastrointestinal infection with Shiga toxin (Stx) producing Escherichia coli (STEC) that can result in acute kidney injury, lasting renal disease, and death. Despite a window for intervention between hemorrhagic diarrhea and onset of HUS, no specific therapies exist to prevent or treat HUS following STEC infection. Furthermore, there is no way to predict which patients with STEC will develop HUS or any rapid way to determine which Stx variant is present. To address this, we have broadened the therpay to neutralize additional toxin variants. It contains a multimer of nanobodies derived from camelid heavy chain antibody fragments (VHHs). An improved V HH-based n eutralizing a gent (VNA2) is delivered intramuscularly as RNA combined with LION nanoparticles rather than mRNA, that replicates on administration (repRNA), resulting in a rapidly circulating VNA that can bind systemic toxin. The RNA/VNA2-Stx administered intramuscularly prevents toxicity and death in a mouse model of acute Stx toxicity., Competing Interests: The authors declare no conflict of interest.

Shiga toxin-producing Escherichia coli (STEC) is a zoonotic pathogen associated with severe disease. Cattle are recognized as the primary animal reservoir of STEC. This study reports the occurrence and characterization of STEC from dairy cows and evaluates the inhibition of adherence by cattle anti-STEC antibodies to the HEp-2 cell. From 151 samples, 30% (n = 45) were positive for stx by PCR screening (25.21% of dairy cows and 46.8% of growing calves). From these positive samples, 17 STEC isolates were characterized. In dairy cows, 3 out of 17 samples carried stx2, 3 out of 17 possessed stx1, and 2 out of 17 carried stx1/stx2. In growing cows, 8 out of 17 samples carried stx1 and 1 out of 17 carried stx1/stx2. Other virulence factors such as ehxA, saa, iha, cah, and eae were detected. The strains were typed into 3 E. coli O groups (O26, O91, and O130). The analysis of the HEp-2 adherence assays indicated that all serum from both cattle categories presented high levels of inhibition of adherence. Considering the severity of the symptoms caused by STEC in humans, searching for factors influencing the colonization of STEC in cattle would help identify strategies to avoid transmission and STEC infection., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Hemolytic Uremic Syndrome (HUS) associated with Shiga-toxigenic Escherichia coli (STEC) infections is the principal cause of acute renal injury in pediatric age groups. Shiga toxin type 2 (Stx2) has in vitro cytotoxic effects on kidney cells, including human glomerular endothelial (HGEC) and Vero cells. Neither a licensed vaccine nor effective therapy for HUS is available for humans. Recombinant antibodies against Stx2, produced in bacteria, appeared as the utmost tool to prevent HUS. Therefore, in this work, a recombinant FabF8:Stx2 was selected from a human Fab antibody library by phage display, characterized, and analyzed for its ability to neutralize the Stx activity from different STEC-Stx2 and Stx1/Stx2 producing strains in a gold standard Vero cell assay, and the Stx2 cytotoxic effects on primary cultures of HGEC. This recombinant Fab showed a dissociation constant of 13.8 nM and a half maximum effective concentration (EC 50 ) of 160 ng/mL to Stx2. Additionally, FabF8:Stx2 neutralized, in different percentages, the cytotoxic effects of Stx2 and Stx1/2 from different STEC strains on Vero cells. Moreover, it significantly prevented the deleterious effects of Stx2 in a dose-dependent manner (up to 83%) in HGEC and protected this cell up to 90% from apoptosis and necrosis. Therefore, this novel and simple anti-Stx2 biomolecule will allow further investigation as a new therapeutic option that could improve STEC and HUS patient outcomes.

Shiga toxin-producing Escherichia coli (STEC) produce a number of virulence factors that interfere with lymphocyte functions, including mitogen- and antigen-activated proliferation and pro-inflammatory cytokine synthesis. Here we describe how to isolate lymphocyte subsets from bovine peripheral blood as well as methods that we have used to study the effects of STEC products on lymphocyte proliferation and cytokine production. We also describe an assay that allows for the detection of association of a given protein with lymphocytes.

Many cattle are persistently colonized with Shiga toxin-producing Escherichia coli (STEC) and represent a major source of human infections with human-pathogenic STEC strains ( syn. enterohemorrhagic E. coli (EHEC)). Intervention strategies most effectively protecting humans best aim at the limitation of bovine STEC shedding. Mechanisms enabling STEC to persist in cattle are only partialy understood. Cattle were long believed to resist the detrimental effects of Shiga toxins (Stxs), potent cytotoxins acting as principal virulence factors in the pathogenesis of human EHEC-associated diseases. However, work by different groups, summarized in this review, has provided substantial evidence that different types of target cells for Stxs exist in cattle. Peripheral and intestinal lymphocytes express the Stx receptor globotriaosylceramide (Gb 3 syn. CD77) in vitro and in vivo in an activation-dependent fashion with Stx-binding isoforms expressed predominantly at early stages of the activation process. Subpopulations of colonic epithelial cells and macrophage-like cells, residing in the bovine mucosa in proximity to STEC colonies, are also targeted by Stxs. STEC-inoculated calves are depressed in mounting appropriate cellular immune responses which can be overcome by vaccination of the animals against Stxs early in life before encountering STEC. Considering Stx target cells and the resulting effects of Stxs in cattle, which significantly differ from effects implicated in human disease, may open promising opportunities to improve existing yet insufficient measures to limit STEC carriage and shedding by the principal reservoir host.

Typical haemolytic uraemic syndrome (STEC-HUS), caused by Shiga toxin (Stx)-producing Escherichia coli (STEC), is a serious, life-threating disease that mainly affects children. Bacteriological and genetic tests are commonly used in the routine laboratory diagnosis of STEC-HUS; however, serological methods have emerged as useful and reliable diagnostic tools, especially when bacterial isolation fails. In this study, we present the results of the serological investigation of 72 paediatric patients suspected for HUS, hospitalized during 2011-2019 at the Department of Pediatrics and Nephrology of Children's Hospitals in Poland. During the routine laboratory investigation STEC strains were isolated only from nine stool samples. However, serological investigations confirmed 45 cases of STEC infections in children with HUS. In this study, 22 (48·9%) paediatric patients were infected by E. coli serotype O26, 11 (24·4%) by serotype O145, 9 (20·0%) by serotype O157, and 3 (6·7%) by E. coli serotype O111. In the majority of these patients, in addition to a high level of IgA, IgG and IgM antibodies to lipopolysaccharide of particular E. coli serotypes, antibodies to recombinant proteins Tir, Stx2b and intimin were detected. Our results confirm that serological tests are useful in the diagnosis of STEC-HUS. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that serological analysis greatly complements bacterial isolation and helps in the diagnosis and confirmation of Shiga toxin (verotoxin)-producing Escherichia coli (STEC) infections. Serological tests can be performed to qualify the patient for the typical haemolytic uraemic syndrome (STEC-HUS). In Poland, STEC-HUS in children is mostly caused by the E. coli serotype O26, which indicates that there is an increasing number of non-O157 STEC infections associated with human diseases in Europe., (© 2020 The Society for Applied Microbiology.)

Haemolytic uraemic syndrome (HUS) remains a leading cause of paediatric acute kidney injury (AKI). Haemolytic uraemic syndrome is characterised by the triad of microangiopathic haemolytic anaemia, thrombocytopenia and AKI. In ~ 90% of cases, HUS is a consequence of infection with Shiga toxin-producing E. coli (STEC), most commonly serotype O157:H7. Acute mortality from STEC-HUS is now less than 5%; however, there is significant long-term renal morbidity in one third of survivors. Currently, no specific treatment exists for STEC-HUS. There is growing interest in the role of complement in the pathogenesis of STEC-HUS due to the discovery of inherited and acquired dysregulation of the alternative complement system in the closely related disorder, atypical HUS (aHUS). The treatment of aHUS has been revolutionised by the introduction of the anti-C5 monoclonal antibody, eculizumab. However, the role of complement and anti-complement therapy in STEC-HUS remains unclear. Herein, we review the current evidence of the role of complement in STEC-HUS focusing on the use of eculizumab in this disease.

Shiga toxin-producing Escherichia coli (STEC) strains are food-borne pathogens of public health concern. Despite ruminants are the most important reservoir, STEC human infections have also been attributed to pigs. We examined for the presence of STEC in 234 samples of swine caecal content collected during the year 2015 at Italian abattoirs in the framework of the harmonized monitoring of antimicrobial resistance (Decision 2013/652/EU). The presence of stx genes was detected in 122 (52.1%) samples, which were subsequently subjected to STEC isolation and characterization. The analysis of the 66 isolated STEC strains showed that the majority of the isolates (74.2%) possessed the stx2a gene subtype, in a few cases (16.7%) in combination with stx2b or stx2c. Only 25.8% of isolates possessed the stx2e subtype, typical of swine-adapted STEC. None of the isolates possessed the intimin-coding eae gene and the majority of them did not belong to serogroups commonly associated with human infections. The results of this study suggest that pigs can be considered as potential reservoir of certain STEC types., (© 2019 The Authors Veterinary Medicine and Science Published by John Wiley & Sons Ltd.)

Background: Hemolytic uremic syndrome (HUS) is a leading cause of acute kidney injury in children. Although international guidelines emphasize comprehensive evaluation and treatment with eculizumab, access to diagnostic and therapeutic facilities is limited in most developing countries. The burden of Shiga toxin-associated HUS in India is unclear; school-going children show high prevalence of anti-factor H (FH) antibodies. The aim of the consensus meeting was to formulate guidelines for the diagnosis and management of HUS in children, specific to the needs of the country., Methods: Four workgroups performed literature review and graded research studies addressing (i) investigations, biopsy, genetics, and differential diagnosis; (ii) Shiga toxin, pneumococcal, and infection-associated HUS; (iii) atypical HUS; and (iv) complement blockade. Consensus statements developed by the workgroups were discussed during a consensus meeting in March 2017., Results: An algorithm for classification and evaluation was developed. The management of Shiga toxin-associated HUS is supportive; prompt plasma exchanges (PEX) is the chief therapy in patients with atypical HUS. Experts recommend that patients with anti-FH-associated HUS be managed with a combination of PEX and immunosuppressive medications. Indications for eculizumab include incomplete remission with plasma therapy, life-threatening features, complications of PEX or vascular access, inherited defects in complement regulation, and recurrence of HUS in allografts. Priorities for capacity building in regional and national laboratories are highlighted., Conclusions: Limited diagnostic capabilities and lack of access to eculizumab prevent the implementation of international guidelines for HUS in most developing countries. We propose practice guidelines for India, which will perhaps be applicable to other developing countries.

Purpose: This study describes the epidemiology of Shiga toxin-producing Escherichia coli (STEC) infections in a population in the South East of England., Methods: From 1 November 2013 to 31 March 2017 participating diagnostic laboratories reported Shiga toxin gene (stx) positive real-time PCR results to local public health teams. Stx positive faecal samples/isolates were referred to the Gastrointestinal Bacteria Reference Unit (GBRU) for confirmation by culture and typing by whole genome sequencing (WGS). Key clinical information was collected by public health teams.Results/Key findings. Altogether, 548 faecal specimens (420 were non-travel associated) were stx positive locally, 535 were submitted to the GBRU. STEC were isolated from 42 %, confirmed by stx PCR in 21 % and 37 % were PCR negative. The most common non-travel associated STEC serogroups were O157, O26, O146 and O91. The annualized incidence of confirmed STEC infections (PCR or culture) was 5.8 per 100 000. The ratio of O157 to non-O157 STEC serogroups was 1:7. The annualized incidence of non-O157 haemolytic uraemic syndrome-associated Escherichia coli (HUSEC) strains was 0.4 per 100 000. Bloody diarrhoea was reported by 58 % of cases infected with E. coli O157, 33 % of cases infected with non-O157 HUSEC strains and 12 % of other lower risk non-O157 strains. Overall, 76 % of non-O157 HUSEC isolates possessed the eae virulence gene., Conclusions: HUSEC including serogroup O157 were uncommon and more likely to cause bloody diarrhoea than other STEC. The routine use of stx PCR testing can influence clinical management. Understanding the local epidemiology facilitates a proportionate public health response to STEC, based on clinical and microbiological characteristics including stx subtype(s).

Background and Objectives: Inherited complement hyperactivation is critical for the pathogenesis of atypical hemolytic uremic syndrome (HUS) but undetermined in postdiarrheal HUS. Our aim was to investigate complement activation and variants of complement genes, and their association with disease severity in children with Shiga toxin-associated HUS., Design, Setting, Participants, & Measurements: Determination of complement biomarkers levels and next-generation sequencing for the six susceptibility genes for atypical HUS were performed in 108 children with a clinical diagnosis of post-diarrheal HUS (75 Shiga toxin-positive, and 33 Shiga toxin-negative) and 80 French controls. As an independent control cohort, we analyzed the genotypes in 503 European individuals from the 1000 Genomes Project., Results: During the acute phase of HUS, plasma levels of C3 and sC5b-9 were increased, and half of patients had decreased membrane cofactor protein expression, which normalized after 2 weeks. Variants with minor allele frequency <1% were identified in 12 Shiga toxin-positive patients with HUS (12 out of 75, 16%), including pathogenic variants in four (four out of 75, 5%), with no significant differences compared with Shiga toxin-negative patients with HUS and controls. Pathogenic variants with minor allele frequency <0.1% were found in three Shiga toxin-positive patients with HUS (three out of 75, 4%) versus only four European controls (four out of 503, 0.8%) (odds ratio, 5.2; 95% confidence interval, 1.1 to 24; P =0.03). The genetic background did not significantly affect dialysis requirement, neurologic manifestations, and sC5b-9 level during the acute phase, and incident CKD during follow-up. However, the only patient who progressed to ESKD within 3 years carried a factor H pathogenic variant., Conclusions: Rare variants and complement activation biomarkers were not associated with severity of Shiga toxin-associated HUS. Only pathogenic variants with minor allele frequency <0.1% are more frequent in Shiga toxin-positive patients with HUS than in controls., (Copyright © 2019 by the American Society of Nephrology.)

Shiga toxin (Stx)-producing Escherichia coli (STEC) is the offending agent in post-diarrhea-associated hemolytic uremic syndrome (HUS), a disorder characterized by thrombocytopenia, microangiopathic hemolytic anemia, and acute kidney failure, with thrombi occluding the renal microvasculature. Endothelial dysfunction has been recognized as the trigger event in the development of microangiopathic processes. Glomerular endothelial cells are susceptible to the toxic effects of Stxs that, via nuclear factor kappa B (NF-κB) activation, induce the expression of genes encoding for adhesion molecules and chemokines, culminating in leukocyte adhesion and platelet thrombus formation on the activated endothelium. Complement activation via the alternative pathway has been seen in patients during the acute phase of STEC-associated HUS. Experimental evidence has highlighted the role of complement proteins in driving glomerular endothelium toward a thrombogenic phenotype. At the glomerular level, podocytes are also an important target of Stx-induced complement activation. Glomerular injury as a consequence of podocyte dysfunction and loss is thus a mechanism that might affect long-term renal outcomes in the disease. New approaches to targeting the complement system may be useful therapeutic options for patients with STEC-HUS.

Hemolytic uremic syndrome (HUS) is defined as a triad of noninmune microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury. The most frequent presentation is secondary to Shiga toxin (Stx)-producing Escherichia coli (STEC) infections, which is termed postdiarrheal, epidemiologic or Stx-HUS, considering that Stx is the necessary etiological factor. After ingestion, STEC colonize the intestine and produce Stx, which translocates across the intestinal epithelium. Once Stx enters the bloodstream, it interacts with renal endothelial and epithelial cells, and leukocytes. This review summarizes the current evidence about the involvement of inflammatory components as central pathogenic factors that could determine outcome of STEC infections. Intestinal inflammation may favor epithelial leakage and subsequent passage of Stx to the systemic circulation. Vascular damage triggered by Stx promotes not only release of thrombin and increased fibrin concentration but also production of cytokines and chemokines by endothelial cells. Recent evidence from animal models and patients strongly indicate that several immune cells types may participate in HUS physiopathology: neutrophils, through release of proteases and reactive oxygen species (ROS); monocytes/macrophages through secretion of cytokines and chemokines. In addition, high levels of Bb factor and soluble C5b-9 (sC5b-9) in plasma as well as complement factors adhered to platelet-leukocyte complexes, microparticles and microvesicles, suggest activation of the alternative pathway of complement. Thus, acute immune response secondary to STEC infection, the Stx stimulatory effect on different immune cells, and inflammatory stimulus secondary to endothelial damage all together converge to define a strong inflammatory status that worsens Stx toxicity and disease.

The early detection of Shiga toxin-producing Escherichia coli (STEC) is important for early diagnosis and preventing the spread of STEC. Although the confirmatory test for STEC should be based on the detection of Shiga toxin using molecular analysis, isolation permits additional characterization of STEC using a variety of methods, including O:H serotyping. The conventional slide agglutination O-antigen serogrouping used in many clinical laboratories is laborious and time-consuming. Surface plasmon resonance (SPR)-based immunosensors are commonly used to investigate a large variety of bio-interactions such as antibody/antigen, peptide/antibody, DNA/DNA, and antibody/bacteria interactions. SPR imaging (SPRi) is characterized by multiplexing capabilities for rapidly screening (approximately 100 to several hundred sensorgrams in parallel) molecules. SPRi-based O-antigen serogrouping method for STEC was recently developed by detecting the interactions between O-antigen-specific antibodies and bacterial cells themselves. The aim of this study was to evaluate its performance for E. coli serogrouping using clinical STEC isolates by comparing the results of slide agglutination tests. We tested a total of 188 isolates, including O26, O45, O91, O103, O111, O115, O121, O128, O145, O157, and O159. The overall sensitivity of SPRi-based O-antigen serogrouping was 98.9%. Only two O157 isolates were misidentified as nontypeable and O121. The detection limits of all serotypes were distributed between 1.1 × 10 6 and 17.6 × 10 6  CFU/ml. Pulsed-field gel electrophoresis (PFGE) revealed the heterogeneity of the examined isolates. In conclusion, SPRi is a useful method for the O-antigen serogrouping of STEC isolates, but the further evaluation of non-O157 minor serogroups is needed., (Copyright © 2018 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Cattle are a major reservoir for Shiga toxin-producing Escherichia coli (STEC) and harbor these bacteria in the intestinal tract. The prevalence, concentration, and STEC serogroup isolated in cattle varies between individuals. Hide removal at slaughter serves as a major point of carcass contamination and ultimately beef products. Certain STEC serogroups, such as O26, O45, O103, O111, O121, O145, and O157, containing the intestinal adherence factor intimin, pose a large economic burden to food producers because of testing and recalls. Human infection with STEC can cause illnesses ranging from diarrhea to hemorrhagic colitis and hemolytic uremic syndrome, and is commonly acquired through ingestion of contaminated foods, often beef products. Previously, most studies focused on O157 STEC, but there is growing recognition of the importance of non-O157 STEC serogroups. This review summarizes detection methods, prevalence, and methods for prediction of pathogenicity of non-O157 STEC from cattle hides and carcasses. A synthesis of procedures is outlined for general non-O157 STEC and targeted detection of specific STEC serogroups. Standardization of sample collection and processing procedures would allow for more robust comparisons among studies. Presence of non-O157 STEC isolated from cattle hides and carcasses and specific factors, such as point of sample collection and season, are summarized. Also, factors that might influence STEC survival on these surfaces, such as the microbial population on hides and microbial adherence genes, are raised as topics for future investigation. Finally, this review gives an overview on studies that have used genetic and cell-based methods to identify specific phenotypes of non-O157 STEC strains isolated from cattle to assess their risk to human health.

Strains of Shiga toxin-producing Escherichia coli (STEC) can cause the severe Hemolytic Uremic Syndrome (HUS). Shiga toxins are protein toxins that bind and kill microvascular cells, damaging vital organs. No specific therapeutics or vaccines have been licensed for use in humans yet. The most common route of infection is by consumption of dairy or farm products contaminated with STEC. Domestic cattle colonized by STEC strains represent the main reservoir, and thus a source of contamination. Outer Membrane Vesicles (OMV) obtained after detergent treatment of gram-negative bacteria have been used over the past decades for producing many licensed vaccines. These nanoparticles are not only multi-antigenic in nature but also potent immunopotentiators and immunomodulators. Formulations based on chemical-inactivated OMV (OMVi) obtained from a virulent STEC strain (O157:H7 serotype) were found to protect against pathogenicity in a murine model and to be immunogenic in calves. These initial studies suggest that STEC-derived OMV has a potential for the formulation of both human and veterinary vaccines.

Shiga toxin-producing Escherichia coli (STEC) is the major etiologic agent of hemolytic-uremic syndrome (HUS). The high rate of HUS emphasizes the urgency for the implementation of primary prevention strategies to reduce its public health impact. Argentina shows the highest rate of HUS worldwide, being E. coli O157 the predominant STEC-associated HUS serogroup (>70%), followed by E. coli O145 (>9%). To specifically detect these serogroups we aimed at developing highly specific monoclonal antibodies (mAbs) against the O-polysaccharide (O-PS) section of the lipopolysaccharide (LPS) of the dominant STEC-associated HUS serogroups in Argentina. The development of hybridomas secreting mAbs against O157 or O145 was carried out through a combined immunization strategy, involving adjuvated-bacterial immunizations followed by immunizations with recombinant O-PS-protein conjugates. We selected hybridoma clones that specifically recognized the engineered O-PS-protein conjugates of O157 or O145 serogroups. Indirect ELISA of heat-killed bacteria showed specific binding to O157 or O145 serogroups, respectively, while no cross-reactivity with other epidemiological important STEC strains, Brucella abortus, Salmonella group N or Yersinia enterocolitica O9 was observed. Western blot analysis showed specific recognition of the sought O-PS section of the LPS by all mAbs. Finally, the ability of the developed mAbs to bind the surface of whole bacteria cells was confirmed by flow cytometry, confocal microscopy and agglutination assays, indicating that these mAbs present an exceptional degree of specificity and relative affinity in the detection and identification of E. coli O157 and O145 serogroups. These mAbs may be of significant value for clinical diagnosis and food quality control applications. Thus, engineered O-PS specific moieties contained in the recombinant glycoconjugates used for combined immunization and hybridoma selection are an invaluable resource for the development of highly specific mAbs.

Latex agglutination tests (LAT) were developed and evaluated for the rapid detection of LPS antibodies against E. coli serogroup O157, O26, O104, O111 and O145. The latex tests have been proved to be sensitive, fast, easy-to-perform and cost-efficient tools for the screening serodiagnosis of VTEC infections causing haemolytic-uraemic syndrome., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Shiga toxin-producing Escherichia coli (STEC) is a known food pathogen, which main reservoir is the intestine of ruminants. The abundance of different STEC lineages in nature reflect a heterogeneity that is characterised by the differential expression of certain genotypic characteristics, which in turn are influenced by the environmental conditions to which the microorganism is exposed. Bacterial homeostasis and stress response are under the control of the alarmone guanosine tetraphosphate (ppGpp), which intrinsic levels varies across the E. coli species. In the present study, 50 STEC isolates from healthy sheep were evaluated regarding their ppGpp content, cytotoxicity and other relevant genetic and phenotypic characteristics. We found that the level of ppGpp and cytotoxicity varied considerably among the examined strains. Isolates that harboured the stx2 gene were the least cytotoxic and presented the highest levels of ppGpp. All stx2 isolates belonged to phylogroup A, while strains that carried stx1 or both stx1 and stx2 genes pertained to phylogroup B1. All but two stx2 isolates belonged to the stx2b subtype. Strains that belonged to phylogroup B1 displayed on average low levels of ppGpp and high cytotoxicity. Overall, there was a negative correlation between cytotoxicity and ppGpp.

Cattle are considered a reservoir of Shiga toxin-producing Escherichia coli (STEC). There is no information about the presence of antibodies against Shiga toxins in Brazilian bovine serum. Using ELISA, all sera tested showed antibodies against the two main STEC virulence factors; Stx1 and Stx2. Neutralizing antibodies against Stx1 and/or Stx2 were detected in all but one serum. In conclusion, our results indicated that these animals had been exposed to STEC producing both toxins., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Escherichia coli O157:H7 has frequently been associated with foodborne infections and is considered an adulterant in raw non-intact beef in the U.S. Shiga toxin-producing E. coli (STEC) belonging to serogroups O26, O45, O103, O111, O121, and O145 (known as the "big six" non-O157) were estimated to cause >70% of foodborne infections attributed to non-O157 serogroups in the U.S., as a result, these six serogroups have also been targeted by regulation in the U.S. The purpose of this study was to develop a rapid and high-throughput molecular method to group STEC isolates into seven clinically important serogroups (i.e., O157 and the "big six" non-O157 serogroups) targeted by regulation in the U.S. by interrogating single nucleotide polymorphisms (SNPs) in gnd. A collection of 195 STEC isolates, including isolates belonging to O157:H7 (n=18), O26 (n=21), O45 (n=19), O103 (n=24), O111 (n=24), O121 (n=23), O145 (n=21), and ten other STEC serogroups (n=45), was assembled and characterized by full gnd sequencing to identify informative SNPs for molecular serogrouping. A multiplex SNP typing assay was developed to interrogate twelve informative gnd SNPs by single base pair extension chemistry and used to characterize the STEC isolate collection assembled here. SNP types were assigned to each isolate by the assay and polymorphisms were confirmed with gnd sequence data. O-serogroup-specific SNP types were identified for each of the seven clinically important STEC serogroups, which allowed the differentiation of these seven STEC serogroups from other non-O157 STEC serogroups. Although serogroups of the "big six" non-O157 STEC and O157:H7 contained multiple SNP types per O-serogroup, there were no overlapping SNP types between serogroups. Our results demonstrate that molecular serogrouping of STEC isolates by interrogation of informative SNPs in gnd represents an alternative to traditional serogrouping by agglutination for rapid and high-throughput identification of clinically important STEC serogroups targeted by regulation for surveillance and epidemiological investigations., (Copyright © 2016. Published by Elsevier B.V.)

Background: Diarrhea-associated hemolytic uremic syndrome (HUS D+) caused by verotoxigenic E. coli strains (VTEC) is a major cause of acute kidney injury in children between 1 and 5 years of age. Because of the short presence of VTEC in the gastrointestinal tract as well as difficulties with the detection of the verotoxigenic strain, identification of HUS etiology might be challenging., Objectives: The aim of the study was to assess the clinical and diagnostic value of serological tests for specific antibodies against verotoxigenic strains of E. coli in patients with HUS., Material and Methods: Eight children aged 8 months - 7.1 years (mean 40 ± 29 months) with symptoms of acute kidney injury, hemolytic anemia and thrombocytopenia observed after hemorrhagic diarrhea were included to the study. VTEC presence was detected in a stool culture with subsequent analysis of the ability to produce verotoxin and the presence of VT1 and VT2 as well as intimin and enterohemolysin genes. In addition, the presence of specific IgA, IgM and IgG antibodies against E. coli serogroups O26, O103, O104, O111, O121, O145 and O157 was measured using ELISA., Results: In 3 subjects, VTEC O26, O157 and O104 serogroups were cultured in the stool and the specific IgA, IgM and IgG antibodies were detected. In 4 subjects, no VTEC strains were cultured, however, high titers of IgA, IgM and IgG antibodies against E. coli O26, O157 and O111 were detected. In a single patient, the negative results of bacteriological and serological analyses excluded VTEC etiology of HUS., Conclusions: A serological analysis of VTEC can confirm the result of stool culture for verotoxigenic E. coli strains and help to find the cause of HUS in case of negative results of a stool culture.

Hemolytic-uremic syndrome (HUS) is a thrombotic microangiopathy that is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and renal failure. Excess complement activation underlies atypical HUS and is evident in Shiga toxin-induced HUS (STEC-HUS). This Spotlight focuses on new knowledge of the role of Escherichia coli-derived toxins and polyphosphate in modulating complement and coagulation, and how they affect disease progression and response to treatment. Such new insights may impact on current and future choices of therapies for STEC-HUS., (© 2015 by The American Society of Hematology.)

Non-O157 Shiga toxin-producing Escherichia coli (STEC) strains are increasingly recognized as foodborne pathogens that trigger hemolytic uremic syndrome (HUS). The detection and isolation of these strains is important, but distinguishing their bacteriological profiles is difficult. A 2-year-old girl developed HUS with mild renal involvement 22 days after consuming barbecued meat. Clinical and laboratory findings gradually improved without specific treatment. Because neither enterohemorrhagic E. coli (EHEC) nor Shiga toxins were detected in stool cultures in a clinical laboratory and the patient tested negative for circulating antibodies to O157 lipopolysaccharide, the case was initially diagnosed as probable atypical HUS. Subsequent serodiagnostic microagglutination assay and polymerase chain reaction-based molecular testing, however, indicated the presence of the EHEC O121:H19 strain with stx2. Thus, to correctly diagnose and treat HUS, a system for detecting non-O157 STEC in a clinical setting is urgently needed., (© 2015 Japan Pediatric Society.)

Certain Shiga toxin-producing Escherichia coli (STEC) are virulent human pathogens that are most often acquired through contaminated food. The United States Department of Agriculture, Food Safety and Inspection Service has declared several serogroups of STEC as adulterants in non-intact raw beef products. Hence, sensitive and specific tests for the detection of these STEC are a necessity for implementation in food safety programs. E. coli serogroups are identified by their respective O-antigen moiety on the lipopolysaccharide (LPS) macromolecule. We propose that the development of O-antigen-specific immunological assays can facilitate simple and rapid discriminatory detection of STEC in beef. However, the resources (antigens and antibodies) required for such development are not readily available. To overcome this, we extracted and characterized LPS and O-antigen from six STEC strains. Using hot phenol extraction, we isolated the LPS component from each strain and purified it using a series of steps to eliminate proteins, nucleic acids, and lipid A antigens. Antigens and crude LPS extracts were characterized using gel electrophoresis, immunoblotting, and modified Western blotting with commercially available antibodies, thus assessing the serogroup specificity and sensitivity of available ligands as well. The results indicate that, while many commercially available antibodies bind LPS, their activities and specificities are highly variable, and often not as specific as those required for serogroup discrimination. This variability could be minimized by the production of antibodies specific for the O-antigen. Additionally, the antigens generated from this study provide a source of characterized LPS and O-antigen standards for six serogroups of STEC., (Published by Elsevier B.V.)

Foodborne pathogens continue to cause several outbreaks every year in many parts of the world. Among the bacterial pathogens involved, Shiga toxin-producing Escherichia coli, Campylobacter jejuni, and nontyphoidal Salmonella species cause a significant number of human infections worldwide, resulting in a huge annual economic burden that amounts to millions of dollars in health care costs. Human infections are primarily caused by the consumption of contaminated food. Vaccination of food-producing animals is an attractive, cost-effective strategy to lower the levels of these pathogens that will ultimately result in a safer food supply and fewer human infections. However, producers are often reluctant to routinely vaccinate animals against these pathogens since they do not cause any detectable clinical symptoms. This review highlights recent approaches used to develop effective food safety vaccines and the potential impact these vaccines might have on health care costs.

Currently, no simple assays are available for routine quantitative detection of Escherichia coli-produced Shiga toxin 2e (Stx2e) that causes porcine edema disease. Here, we present a novel quantitative detection method for Stx2e based on the measurement of Stx2e binding to the specific globotetraosylceramide (Gb4) receptor by ELISA (Gb4-ELISA). No cross-reactivity was found with the other Shiga toxins Stx1 and Stx2, indicating high specificity. When the recombinant Stx2e B subunit (Stx2eB) was used, the absorbance measured by Gb4-ELISA increased linearly with Stx2eB concentration in the range of 20-2,500 ng/ml. The Gb4-ELISA method can be easily performed, suggesting that it would be a useful diagnostic tool for porcine edema disease.

Hemolytic uremic syndrome is a rare disease, frequently responsible for renal insufficiency in children. Recent findings have led to renewed interest in this pathology. The discovery of new gene mutations in the atypical form of HUS and the experimental data suggesting the involvement of the complement pathway in the typical form, open new perspectives for treatment. This review summarizes the current state of knowledge on both typical and atypical hemolytic uremic syndrome pathophysiology and examines new perspectives for treatment., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Shiga toxin associated hemolytic uremic syndrome (Stx HUS), a thrombotic microangiopathy, is the most common cause of pediatric acute kidney injury but has no direct treatment. A better understanding of disease pathogenesis may help identify new therapeutic targets. For this reason, the role of complement is being actively studied while eculizumab, the C5 monoclonal antibody, is being used to treat Stx HUS but with conflicting results. A randomized controlled trial would help properly evaluate its use in Stx HUS while more research is required to fully evaluate the role complement plays in the disease pathogenesis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

During 2007-2010, 13 545 confirmed human verocytotoxin (VT)-producing Escherichia coli (VTEC) infections were reported in the European Union, including 777 haemolytic uraemic syndrome (HUS) cases. Clinical manifestations were reported for 53% of cases, 64% of which presented with diarrhoea alone and 10% with HUS. Isolates from 85% of cases were not fully serotyped and could not be classified on the basis of the Karmali seropathotype concept. There is no single or combination of phenotypic or genetic marker(s) that fully define 'pathogenic' VTEC. Isolates which contain the vtx2 (verocytotoxin 2) gene in combination with the eae (intimin-encoding) gene or aaiC (secreted protein of enteroaggregative E. coli) and aggR (plasmid-encoded regulator) genes have been associated with a higher risk of more severe illness. A molecular approach targeting genes encoding VT and other virulence determinants is thus proposed to allow an assessment of the potential severity of disease that may be associated with a given VTEC isolate.

Cattle are the most important reservoir for enterohemorrhagic Escherichia coli (EHEC), a subset of shigatoxigenic E. coli (STEC) capable of causing life-threatening infectious diseases in humans. In cattle, Shiga toxins (Stx) suppress the immune system thereby promoting long-term STEC shedding. First infections of animals at calves' age coincide with the lack of Stx-specific antibodies. We hypothesize that vaccination of calves against Shiga toxins prior to STEC infection may help to prevent the establishment of a persistent type of infection. The objectives of this study were to generate recombinant Shiga toxoids (rStx1mut & rStx2mut) by site-directed mutagenesis and to assess their immunomodulatory, antigenic, and immunogenic properties. Cultures of bovine primary immune cells were used as test systems. In ileal intraepithelial lymphocytes both, recombinant wild type Stx1 (rStx1WT) and rStx2WT significantly induced transcription of IL-4 mRNA. rStx1WT and rStx2WT reduced the expression of Stx-receptor CD77 (syn. Globotriaosylceramide, Gb3) on B and T cells from peripheral blood and of CD14 on monocyte-derived macrophages. At the same concentrations, rStx1mut and rStx2mut exhibited neither of these effects. Antibodies in sera of cattle naturally infected with STEC recognized the rStxmut toxoids equally well as the recombinant wild type toxins. Immunization of calves with rStx1mut plus rStx2mut led to induction of antibodies neutralizing Stx1 and Stx2. While keeping their antigenicity and immunogenicity recombinant Shiga toxoids are devoid of the immunosuppressive properties of the corresponding wild type toxins in cattle and candidate vaccines to mitigate long-term STEC shedding by the reservoir host.

Background: Stx toxin is a member of the AB5 family of bacterial toxins: the active A subunit has N-glycosidase activity against 28S rRNA, resulting in inhibition of protein synthesis in eukaryotic cells, and the pentamer ligand B subunits (StxB) bind to globotria(tetra)osylceramide receptors (Gb3/Gb4) on the cell membrane. Shiga toxin-producing Escherichia coli strains (STEC) may produce Stx1 and/or Stx2 and variants. Strains carrying Stx2 are considered more virulent and related to the majority of outbreaks, besides being usually associated with hemolytic uremic syndrome in humans. The development of tools for the detection and/or neutralization of these toxins is a turning point for early diagnosis and therapeutics. Antibodies are an excellent paradigm for the design of high-affinity, protein-based binding reagents used for these purposes., Methods and Findings: In this work, we developed two recombinant antibodies; scFv fragments from mouse hybridomas and Fab fragments by phage display technology using a human synthetic antibody library. Both fragments showed high binding affinity to Stx2, and they were able to bind specifically to the GKIEFSKYNEDDTF region of the Stx2 B subunit and to neutralize in vitro the cytotoxicity of the toxin up to 80%. Furthermore, the scFv fragments showed 79% sensitivity and 100% specificity in detecting STEC strains by ELISA., Conclusion: In this work, we developed and characterized two recombinant antibodies against Stx2, as promising tools to be used in diagnosis or therapeutic approaches against STEC, and for the first time, we showed a human monovalent molecule, produced in bacteria, able to neutralize the cytotoxicity of Stx2 in vitro.

Human infection with Shiga toxin-producing Escherichia coli (STEC) is a major cause of postdiarrheal hemolytic-uremic syndrome (HUS), a life-threatening condition characterized by hemolytic anemia, thrombocytopenia, and acute renal failure. E. coli O157:H7 is the dominant STEC serotype associated with HUS worldwide, although non-O157 STEC serogroups can cause a similar disease. The detection of anti-O157 E. coli lipopolysaccharide (LPS) antibodies in combination with stool culture and detection of free fecal Shiga toxin considerably improves the diagnosis of STEC infections. In the present study, we exploited a bacterial glycoengineering technology to develop recombinant glycoproteins consisting of the O157, O145, or O121 polysaccharide attached to a carrier protein as serogroup-specific antigens for the serological diagnosis of STEC-associated HUS. Our results demonstrate that using these antigens in indirect ELISAs (glyco-iELISAs), it is possible to clearly discriminate between STEC O157-, O145-, and O121-infected patients and healthy children, as well as to confirm the diagnosis in HUS patients for whom the classical diagnostic procedures failed. Interestingly, a specific IgM response was detected in almost all the analyzed samples, indicating that it is possible to detect the infection in the early stages of the disease. Additionally, in all the culture-positive HUS patients, the serotype identified by glyco-iELISAs was in accordance with the serotype of the isolated strain, indicating that these antigens are valuable not only for diagnosing HUS caused by the O157, O145, and O121 serogroups but also for serotyping and guiding the subsequent steps to confirm diagnosis., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Sheep constitute an important source of zoonotic pathogens as Shiga toxin-producing Escherichia coli (STEC). In this study, the prevalence, serotypes and virulence profiles of STEC were investigated among 130 healthy sheep from small and medium farms in southern Brazil. STEC was isolated from 65 (50%) of the tested animals and detected in all flocks. A total of 70 STEC isolates were characterized, and belonged to 23 different O:H serotypes, many of which associated with human disease, including hemolytic-uremic syndrome (HUS). Among the serotypes identified, O76:H19 and O65:H- were the most common, and O75:H14 and O169:H7 have not been previously reported in STEC strains. Most of the STEC isolates harbored only stx1, whereas the Stx2b subtype was the most common among those carrying stx2. Enterohemolysin (ehxA) and intimin (eae) genes were detected in 61 (87.1%) and four (5.7%) isolates, respectively. Genes encoding putative adhesins (saa, iha, lpfO113) and toxins (subAB and cdtV) were also observed. The majority of the isolates displayed virulence features related to pathogenesis of STEC, such as adherence to epithelial cells, high cytotoxicity and enterohemolytic activity. Ovine STEC isolates belonged mostly to phylogenetic group B1. PFGE revealed particular clones distributed in some farms, as well as variations in the degree of genetic similarity within serotypes examined. In conclusion, STEC are widely distributed in southern Brazilian sheep, and belonged mainly to serotypes that are not commonly reported in other regions, such as O76:H19 and O65:H-. A geographical variation in the distribution of STEC serotypes seems to occur in sheep., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Escherichia coli serogroups O5, O15, O26, O45, O55, O76, O91, O103, O104, O111, O113, O118, O121, O123, O128, O145, O146, O157, O165, O172, and O177 are the O-antigen forms of the most clinically relevant Shiga toxin-producing E. coli (STEC) serotypes. In this study, three multiplex PCR assays able to specifically detect these 21 serogroups were developed and validated. For this purpose, the O-antigen gene clusters of E. coli O5 and O76 were fully sequenced, their associated genes were identified on the basis of homology, and serogroup-specific primers were designed. After preliminary evaluation, these two primer pairs were proven to be highly specific and suitable for the development of PCR assays for O5 and O76 serogroup identification. Specific primers were also designed for serogroups O15, O45, O55, O91, O104, O113, O118, O123, O128, O146, O157, O165, O172, and O177 based on previously published sequences, and previously published specific primers for serogroups O26, O103, O111, O121, and O145 were also included. These 21 primer pairs were shown to be specific for their target serogroup when tested against E. coli type strains representing 169 known O-antigen forms of E. coli and Shigella and therefore suitable for being used in PCR assays for serogroup identification. In order to validate the three multiplex PCR assays, 22 E. coli strains belonging to the 21 covered serogroups and 18 E. coli strains belonging to other serogroups were screened in a double-blind test and their sensitivity was determined as 1 ng chromosomal DNA. The PCR assays developed in this study could be a faster, simpler, and less expensive strategy for serotyping of the most clinically relevant STEC strains in both clinical microbiology and public health laboratories, and so their development could benefit for clinical diagnosis, epidemiological investigations, surveillance, and control of STEC infections.

To help assess the clinical and public health risks associated with different Shiga toxin-producing Escherichia coli (STEC) strains, an empirical classification scheme was used to classify STEC into five "seropathotypes" (seropathotype A [high risk] to seropathotypes D and E [minimal risk]). This definition is of considerable value in cases of human infection but is also problematic because not all STEC infections are fully characterized and coupled to reliable clinical information. Outbreaks with emerging hybrid strains continuously challenge our understanding of virulence potential and may result in incorrect classification of specific pathotypes; an example is the hybrid strain that caused the 2011 outbreak in Germany, STEC/EAggEC O104:H4, which may deserve an alternative seropathotype designation. The integration of mobile virulence factors in the stepwise and parallel evolution of pathogenic lineages of STEC collides with the requirements of a good taxonomy, which separates elements of each group into subgroups that are mutually exclusive, unambiguous, and, together, include all possibilities. The concept of (sero)-pathotypes is therefore challenged, and the need to identify factors of STEC that absolutely predict the potential to cause human disease is obvious. Because the definition of hemolytic-uremic syndrome (HUS) is distinct, a basic and primary definition of HUS-associated E. coli (HUSEC) for first-line public health action is proposed: stx2 in a background of an eae- or aggR-positive E. coli followed by a second-line subtyping of stx genes that refines the definition of HUSEC to include only stx2a and stx2d. All other STEC strains are considered "low-risk" STEC.

Hemolytic uremic syndrome (HUS) is the major complication of gastrointestinal infections with enterohemorrhagic Escherichia coli (EHEC) and is mediated by the production of Shiga toxins (Stx). Although it has been previously reported that not only HUS patients but healthy children have anti-Stx antibodies, very little is known about how these infections impact on mucosal immune system to generate a specific immune response. This work aimed to evaluate the immune responses elicited after a single oral dose of EHEC in a mouse model of HUS at weaning. We found sequential activation of T and B lymphocytes together with an increased percentage of IgA-bearing B cells in Peyer's patches and mesenteric lymph nodes. We also found fecal anti-EHEC IgA and serum anti-Stx2 IgG in EHEC-inoculated mice. Besides, these mice were partially protected against an intravenous challenge with Stx2. These data demonstrate that one episode of EHEC infection is enough to induce activation in the gut-associated lymphoid tissue, especially the B cell compartment, and lead to the production of specific IgA in mucosal tissue and the generation of systemic protection against Stx2 in a percentage of intragastrically inoculated mice. These data also support the epidemiologic observation that a second episode of HUS is very rare.

Many pathogens produce the β-(1-6)-linked poly-N-acetylglucosamine (PNAG) surface polysaccharide that is being developed as a broadly protective antimicrobial vaccine. However, it is unknown whether systemically injected PNAG vaccines or antibodies would provide protective immunity against pathogens confined to the gastrointestinal tract such as Shiga toxin (Stx)-producing Escherichia coli (STEC), an important group of gastrointestinal (GI) pathogens for which effective immunotherapeutics are lacking. To ascertain whether systemic IgG antibody to PNAG impacts this infectious situation, a vaccine consisting of a synthetic nonamer of nonacetylated PNAG, 9GlcNH2, conjugated to the Shiga toxin 1b subunit (9GlcNH2-Stx1b) was produced. Rabbit antibodies raised to the conjugate vaccine were tested for bacterial killing and toxin neutralization in vitro and protection against infection in infant mice. Cell surface PNAG was detected on all 9 STEC isolates tested, representing 6 STEC serogroups, including E. coli O157:H7. Antibody to the 9GlcNH2-Stx1b conjugate neutralized Stx1 potently and Stx2 modestly. For O157:H7 and O104:H4 STEC strains, antibodies elicited by the 9GlcNH2-Stx1b conjugate possessed opsonic killing and bactericidal activity. Following intraperitoneal injection, antibodies to both PNAG and Stx were needed for infant mouse protection against O157 STEC. These antibodies also mediated protection against the Stx2-producing O104:H4 strain that was the cause of a recent outbreak in Germany, although sufficient doses of antibody to PNAG alone were protective against this strain in infant mice. Our observations suggest that vaccination against both PNAG and Stx, using a construct such as the 9GlcNH2-Stx1b conjugate vaccine, would be protective against a broad range of STEC serogroups. IMPORTANCE The presence of poly-N-acetylglucosamine (PNAG) on many pathogens presents an opportunity to target this one structure with a multispecies vaccine. Whether antibodies to PNAG can protect against pathogens confined to the gastrointestinal tract is not known. As Shiga toxin (Stx)-producing Escherichia coli (STEC) bacteria are serious causes of infection whose virulence is dependent on elaboration of Stx, we prepared a vaccine containing a synthetic nonamer of PNAG (9GlcNH2) conjugated to Shiga toxin 1b subunit (9GlcNH2-Stx1b) to evaluate bacterial killing, toxin neutralization, and protective efficacy in infant mice. All nine (100%) clinical strains of STEC from different serogroups expressed PNAG. Vaccine-induced antibody mediated in vitro killing of STEC and neutralization of both Stx1 and Stx2. Passive administration of antibody to the conjugate showed protection requiring immunity to both PNAG and Stx for O157 strains, although for an O104 strain, antibody to PNAG alone was protective. Immunity to PNAG may contribute to protection against STEC infections.

Shiga toxins are a family of genetically and structurally related toxins that are the primary virulence factors produced by the bacterial pathogens Shigella dysenteriae serotype 1 and certain Escherichia coli strains. The toxins are multifunctional proteins inducing protein biosynthesis inhibition, ribotoxic and ER stress responses, apoptosis, autophagy, and inflammatory cytokine and chemokine production. The regulated induction of inflammatory responses is key to minimizing damage upon injury or pathogen-mediated infections, requiring the concerted activation of multiple signaling pathways to control cytokine/chemokine expression. Activation of host cell signaling cascades is essential for Shiga toxin-mediated proinflammatory responses and the contribution of the toxins to virulence. Many studies have been reported defining the inflammatory response to Shiga toxins in vivo and in vitro, including production and secretion of tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), macrophage inflammatory protein-1α/β (MIP-1α/β), macrophage chemoattractant monocyte chemoattractant protein 1 (MCP-1), interleukin 8 (IL-8), interleukin 6 (IL-6), and Groβ. These cytokines and chemokines may contribute to damage in the colon and development of life threatening conditions such as acute renal failure (hemolytic uremic syndrome) and neurological abnormalities. In this review, we summarize recent findings in Shiga toxin-mediated inflammatory responses by different types of cells in vitro and in animal models. Signaling pathways involved in the inflammatory responses are briefly reviewed.

Shiga toxin-producing Escherichia coli (STEC) is a major cause of severe food-borne disease worldwide, and two Shiga toxins, Stx1 and Stx2, are primarily responsible for the serious disease consequence, hemolytic-uremic syndrome (HUS). Here we report identification of a panel of heavy-chain-only antibody (Ab) V(H) (VHH) domains that neutralize Stx1 and/or Stx2 in cell-based assays. VHH heterodimer toxin-neutralizing agents containing two linked Stx1-neutralizing VHHs or two Stx2-neutralizing VHHs were generally much more potent at Stx neutralization than a pool of the two-component monomers tested in cell-based assays and in vivo mouse models. We recently reported that clearance of toxins can be promoted by coadministering a VHH-based toxin-neutralizing agent with an antitag monoclonal antibody (MAb), called the "effector Ab," that indirectly decorates each toxin molecule with four Ab molecules. Decoration occurs because the Ab binds to a common epitopic tag present at two sites on each of the two VHH heterodimer molecules that bind to each toxin molecule. Here we show that coadministration of effector Ab substantially improved the efficacy of Stx toxin-neutralizing agents to prevent death or kidney damage in mice following challenge with Stx1 or Stx2. A single toxin-neutralizing agent consisting of a double-tagged VHH heterotrimer--one Stx1-specific VHH, one Stx2-specific VHH, and one Stx1/Stx2 cross-specific VHH--was effective in preventing all symptoms of intoxication from Stx1 and Stx2 when coadministered with effector Ab. Overall, the availability of simple, defined, recombinant proteins that provide cost-effective protection against HUS opens up new therapeutic approaches to managing disease.

Shiga toxin-producing Escherichia coli (STEC) food contaminations pose serious health concerns, and have been the subject of massive food recalls. STEC has been identified as the major cause of the life-threatening complication of hemolytic uremic syndrome (HUS). Besides supportive care, there currently are no therapeutics available. The use of antibiotics for combating pathogenic E. coli is not recommended because they have been shown to stimulate toxin production. Clearing Stx2 from the circulation could potentially lessen disease severity. In this study, we tested the in vivo neutralization of Stx2 in mice using monoclonal antibodies (mAbs). We measured the biologic half-life of Stx2 in mice and determined the distribution phase or t(1/2) α to be 3 min and the clearance phase or t(1/2) β to be 40 min. Neutralizing mAbs were capable of clearing Stx2 completely from intoxicated mouse blood within minutes. We also examined the persistence of these mAbs over time and showed that complete protection could be passively conferred to mice 4 weeks before exposure to Stx2. The advent of better diagnositic methods and the availability of a greater arsenal of therapeutic mAbs against Stx2 would greatly enhance treatment outcomes of life threatening E. coli infections.

Background: Shiga toxin-producing Escherichia coli (STEC) are frequent causes of severe human diseases ranging from diarrhea to hemolytic uremic syndrome. The existing strategy for detection of STEC relies on the unique sorbitol-negative fermentation property of the O157 strains, the most commonly identified serotype has been E. coli O157. It is becoming increasingly evident, however, that numerous non-O157 STEC serotypes also cause outbreaks and severe illnesses. It is necessary to have new methods that are capable of detecting all STEC strains., Methods and Findings: Here we describe the development of a sandwich ELISA assay for detecting both O157 and non-O157 STECs by incorporating a novel polyclonal antibody (pAb) against Stx2. The newly established immunoassay was capable of detecting Stx2a spiked in environmental samples with a limit of detection between 10 and 100 pg/mL in soil and between 100 and 500 pg/mL in feces. When applied to 36 bacterial strains isolated from human and environmental samples, this assay detected Stx2 in all strains that were confirmed to be stx2-positive by real-time PCR, demonstrating a 100% sensitivity and specificity., Conclusions: The sandwich ELISA developed in this study will enable any competent laboratory to identify and characterize Stx2-producing O157 and non-O157 strains in human and environmental samples, resulting in rapid diagnosis and patient care. The results of epitope mapping from this study will be useful for further development of a peptide-based antibody and vaccine.

Porcine edema disease (ED) is a communicable disease of shoats caused by infection with Shiga toxin (Stx)-producing Escherichia coli. Stx2e is classified as a 1A5B-type toxin and is a decisive virulence determinant of ED. The single A subunit of Stx2e possesses enzymatic activity and is accompanied by a pentamer of B subunits, which binds to the host receptor and delivers the A subunit into the cell. In the present study, we used a mouse model to evaluate the immunogenicity of 3 ED vaccine candidates: a non-toxic mutant holotoxin mStx2e and 2 Stx2eB-based fusion proteins, Stx2eA2B-His and Stx2eB-His. Systemic inoculation of mice with mStx2e- and the Stx2eB-derived antigens induced anti-Stx2e IgG responses that were fully and partially capable of neutralizing Stx2e cellular cytotoxicity, respectively. Intranasal immunization with mStx2e protected the mice from subsequent intraperitoneal challenge with a lethal dose of Stx2e, whereas immunization with Stx2eA2B-His and Stx2eB-His afforded partial protection. Analysis of serum cytokines revealed that mStx2e, but not the Stx2eB-based antigens, was capable of inducing a Th2-type immune response. These results suggest that although the Stx2eB-based antigens elicited an immune response to Stx2e, they did so through a different mechanism to the Th2-type response induced by mStx2e.

We prepared anti-Shiga toxin II variant (Stx2e) and anti-Stx2e B antibodies in chicken egg yolk and investigated their effectiveness in vitro and in vivo. Both Egg yolk antibodies (IgY) reduced the cytotoxic effects of Stx2e to Vero cells. The protective efficacy of anti-Stx2e IgY and anti-Stx2e B IgY was investigated in clean Kunming mice, which were experimentally induced with Stx2e. Peritoneal injection of anti-Stx2e IgY and anti-Stx2e B IgY apparently reduced the lethal effect of Stx2e to mice. These results indicate that both anti-Stx2e IgY and anti-Stx2e B IgY could protect mice from Stx2e challenge. Therefore, anti-Stx2e IgY or anti-Stx2e B IgY might be considered as potential candidate for therapeutics for porcine edema disease.

An increasing number of non-O157 Shiga toxin-producing Escherichia coli (STEC) infections and outbreaks have been reported. In this study, we evaluated the performance of ImmunoCard STAT!(®) (Meridian Bioscience, Inc., Cincinnati, OH, USA) as a method to screen stool specimens for STEC (O157 and non-O157). An in-house real-time PCR method was used as the "gold standard". We also evaluated the prevalence and clinical characteristics of STEC infections in the Alberta South West Zone. From July to November 2011, 819 stool specimens submitted for routine stool culture were tested. With our in-house real-time PCR, 7 O157:H7 and 10 non-O157 STEC isolates were identified for a total of 17 STECs. In comparison, ImmunoCard STAT!(®) identified a total of 6, resulting in a sensitivity and specificity of 35% and 99%, respectively (P < 0.05). Because of the low sensitivity, ImmunoCard STAT!(®) cannot be recommended as a routine screening test for STEC from enriched stool specimens. The rate of STEC positivity as detected by PCR was 2.08%, of which 0.86% was O157:H7 and 1.22% non-O157 STEC. Five of the 7 cases of STEC O157 infection experienced bloody diarrhea, and 1 developed hemolytic uremic syndrome., (Copyright © 2013 Elsevier Inc. All rights reserved.)

An antibody microarray was developed to detect the "top six" non-O157 serogroups, O26, O45, O103, O111, O121, and O145 of Shiga toxin-producing Escherichia coli (STEC), that have been declared as adulterant in meat by the Food Safety and Inspection Service of the United States Department of Agriculture. The sensitivity of the array was 10(5)CFU and the limit of detection of each serogroup in artificially inoculated ground beef was 1-10 CFU following 12h of enrichment. Optimal concentrations of antibodies for printing and labeling and bacterial dilutions for binding to the antibodies were assessed. The array utilized a minimal amount of antibodies and other reagents and may be utilized for screening of multiple target O groups of STEC in parallel, directly from enriched samples in less than 3h. Furthermore, the antibody array provides the flexibility to include other O serogroups of E. coli and may be adopted for high throughput screening. The method is potentially applicable to detect the pathogenic STEC O groups of E. coli in meat and other food, thus improving food safety and public health., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Xanthine oxidase (XO), also known as xanthine oxidoreductase, has long been considered an important host defense molecule in the intestine and in breastfed infants. Here, we present evidence that XO is released from and active in intestinal tissues and fluids in response to infection with enteropathogenic Escherichia coli (EPEC) and Shiga-toxigenic E. coli (STEC), also known as enterohemorrhagic E. coli (EHEC). XO is released into intestinal fluids in EPEC and STEC infection in a rabbit animal model. XO activity results in the generation of surprisingly high concentrations of uric acid in both cultured cell and animal models of infection. Hydrogen peroxide (H(2)O(2)) generated by XO activity triggered a chloride secretory response in intestinal cell monolayers within minutes but decreased transepithelial electrical resistance at 6 to 22 h. H(2)O(2) generated by XO activity was effective at killing laboratory strains of E. coli, commensal microbiotas, and anaerobes, but wild-type EPEC and STEC strains were 100 to 1,000 times more resistant to killing or growth inhibition by this pathway. Instead of killing pathogenic bacteria, physiologic concentrations of XO increased virulence by inducing the production of Shiga toxins from STEC strains. In vivo, exogenous XO plus the substrate hypoxanthine did not protect and instead worsened the outcome of STEC infection in the rabbit ligated intestinal loop model of infection. XO released during EPEC and STEC infection may serve as a virulence-inducing signal to the pathogen and not solely as a protective host defense.

Human infection by Shiga toxin producing Escherichia coli (STEC) is one of the most prevalent foodborne diseases. Shiga toxin type 2 (Stx2) is the major contributor to hemolytic-uremic syndrome (HUS) and other systemic complications caused by STEC. Although outbreaks of HUS due to the consumption of dairy products occur frequently, very few reports are available on assays for the detection of Stx2 in milk. In this study, we describe the development of five high-affinity monoclonal antibodies (dissociation constants below nM range) against Stx2 using a recombinant toxoid as an immunogen. These antibodies, designated Stx2-1, Stx2-2, Stx2-3, Stx2-4, and Stx2-5 are IgG1 or IgG2a heavy-chain subclass with kappa light-chains, did not cross-react with Stx1 and showed different preferences to variants of Stx2. Western blot analyses demonstrate that mAbs Stx2-2 and Stx2-5 bind both the A- and B-subunits, whereas the other 3 mAbs bind the A-subunit of Stx2a only. All antibodies bound stronger to the native than to the denatured Stx2a except the mAb Stx2-3, which bound equally well to both forms of the toxin. Of the five mAbs, Stx2-5 was capable of neutralizing Stx2a mediated cytotoxicity in Vero cells. Highly sensitive ELISA and immuno-PCR assays, capable of detecting 1 and 0.01 pg/mL of Stx2a in milk, were developed using mAb pair Stx2-1 and Stx2-2. Such assays are useful for routine diagnosis of Stx2 contamination in milk production process, thus reducing the risk of STEC outbreaks., (Published by Elsevier B.V.)