The Deleterious Effects of Shiga Toxin Type 2 Are Neutralized In Vitro by FabF8:Stx2 Recombinant Monoclonal Antibody.

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 (EC50) 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.

Interaction of Bovine Lymphocytes with Products of Shiga Toxin-Producing Escherichia coli.

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.

The Role of Escherichia coli Shiga Toxins in STEC Colonization of Cattle.

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 (Gb3syn. 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.

Antibody response to lipopolysaccharides and recombinant proteins of Shiga toxin (STX)-producing Escherichia coli (STEC) in children with haemolytic uraemic syndrome in Poland.

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.

Detection and isolation of Shiga Toxin-producing Escherichia coli (STEC) strains in caecal samples from pigs at slaughter in Italy.

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.

The epidemiology of Shiga toxin-producing Escherichia coli infections in the South East of England: November 2013-March 2017 and significance for clinical and public health.

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).

Hemolytic uremic syndrome in a developing country: Consensus guidelines.

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.

Complement Gene Variants and Shiga Toxin-Producing Escherichia coli-Associated Hemolytic Uremic Syndrome: Retrospective Genetic and Clinical Study.

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.

Shiga toxin triggers endothelial and podocyte injury: the role of complement activation.

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.

Eculizumab in the treatment of Shiga toxin haemolytic uraemic syndrome.

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.

OMV-based vaccine formulations against Shiga toxin producing Escherichia coli strains are both protective in mice and immunogenic in calves.

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.

Detection, Prevalence, and Pathogenicity of Non-O157 Shiga Toxin-Producing Escherichia coli from Cattle Hides and Carcasses.

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.

Evaluation of a surface plasmon resonance imaging-based multiplex O-antigen serogrouping for Escherichia coli using eleven major serotypes of Shiga -toxin-producing E. coli.

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 × 106 and 17.6 × 106 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.

Pathogenic role of inflammatory response during Shiga toxin-associated hemolytic uremic syndrome (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.

A recombinant O-polysaccharide-protein conjugate approach to develop highly specific monoclonal antibodies to Shiga toxin-producing Escherichia coli O157 and O145 serogroups.

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.

Development and evaluation of latex agglutination tests for the detection of human antibodies to the lipopolysaccharides of verocytotoxin-producing Escherichia coli (VTEC) serogroups O157 and non-O157.

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.

ppGpp and cytotoxicity diversity in Shiga toxin-producing Escherichia coli (STEC) isolates.

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.

Antibodies to Shiga toxins in Brazilian cattle.

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.