Search

Aim This study reports the presence of carbapenem-resistant Escherichia coli hybrid pathovars and its prevalence in 200 fresh-cut fruits from Accra. Methods and results Standard culture methods were used to quantify microbial indicators and E. coli on fresh-cut fruits retailed in formal and informal outlets in Accra. The Kirby–Bauer disc diffusion method was used to determine the antibiotic resistance profile of E. coli , while multiplex PCR was employed to identify the virulence and carbapenem-resistance genes. Escherichia coli prevalence in cut fruits was 17%, with pawpaw, watermelon, and mixed fruit having higher prevalence than pineapple. Of the 34 E. coli isolates from fresh-cut fruits, 44% showed broad resistance to beta-lactam antibiotics, while 5.9% showed carbapenem resistance. The study identified virulence genes associated with all E. coli isolates, including stx1, stx2, escV , and ipaH , of which 97% were hybrid pathovars bearing genes for Shiga toxin-producing E. coli /enteropathogenic E. coli /enteroinvasive E. coli. The carbapenemase gene, bla IMP, was associated with both carbapenem-resistant E. coli phenotypes identified. Conclusion Despite a low-carbapenem-resistance prevalence observed among E. coli isolates, hypervirulent hybrid strains of E. coli is present in fresh-cut fruits in the sampling area, posing a potential public health risk to fresh-cut fruit consumers. [ABSTRACT FROM AUTHOR]

Hemolytic-uremic syndrome (HUS) is a rare complication of an infection with Shiga toxin (Stx)-producing Escherichia coli (STEC-HUS), characterized by severe acute kidney injury, thrombocytopenia and microangiopathic hemolytic anemia, and specific therapy is still lacking. Thrombomodulin (TM) is a multi-domain transmembrane endothelial cell protein and its N-terminal domain has been implicated in the pathophysiology of some cases of HUS. Indeed, the administration of recombinant human TM (rhTM) may have efficacy in HUS. We used a Stx-based murine model of HUS to characterize the role of the N-terminal domain of TM. We show that mice lacking that domain (TMLed (−/−)) are more sensitive to Stx, with enhanced HUS progression seen at 4 days and increased mortality at 7 days post-HUS induction. In spite of these changes, renal function was less affected in surviving Stx-challenged TMLed (−/−) mice compared to their wild-type counterparts TMLed (+/+) at 7 days. Contrary to few clinical case reports from Japan, the administration of rhTM (0.06 mg/kg) to wild-type mice (C57BL/6J) with HUS did not protect against disease progression. This overall promising, but also contradictory body of evidence, requires further systematic preclinical and clinical investigations to clarify the role of TM in HUS as a potential therapeutic strategy. [ABSTRACT FROM AUTHOR]

Shiga toxin-producing Escherichia coli (STEC) is known as a crucial zoonotic food-borne pathogen. The main virulence factor of STEC is the production of Shiga toxins 1 and 2. A total of 400 poultry faecal samples were collected from different poultry farms in and around Tirupathi, Andhra Pradesh. The samples were cultured in selective and differential culture media, and the virulence genes of E. coli isolates were analyzed by PCR assay. Out of 400 poultry faecal samples, 170 isolates were isolated and confirmed as E. coli by different biochemical tests with an overall prevalence of 42.5%. Out of 170 E. coli isolates 154 (90.5%) were found positive by targeting uspA gene with an amplification of 884 bp. All the 154 PCR confirmed E. coli isolates subjected to multiplex PCR for the detection of virulent genes targeting eae A, stx 1 and stx 2 and obtained an amplification products, respectively at 384 bp, 180 bp and 254 bp. Out of 154 E. coli isolates, 54 (35.06%) isolates had eae A gene, 36 (23.37 %) had stx 1 gene, 41 (26.62 %) isolates had stx 2 gene, 16 (3.89 %) isolates had both eae A & stx 2, and 12 (7.79%) isolates had both stx 1 & stx 2. By avoiding contact with contaminated faeces and by maintaining proper hygienic measures at farm, contamination of poultry and poultry products may be avoided which in turn may safeguard the health of public. [ABSTRACT FROM AUTHOR]

Shiga toxin-producing Escherichia coli (STEC) causes acute intestinal infections and also causes acute renal failure, especially in children. Shiga toxins (Stx) occupy a central place in the pathogenesis of hemolytic uremic syndrome (HUS) in Escherichiosis. The presented work analyzes the effectiveness of laboratory diagnostics of enterohemorrhagic escherichiosis in patients at the stage of manifestation of HUS and/or acute renal failure using microbiological, immunological research methods and PCR analysis. The study used clinical material from 30 patients in the pediatric intensive care unit of the St. Vladimir Children’s City Clinical Hospital in Moscow with symptoms of HUS aged from 8 months to 5 years. Blood sera from 20 healthy donors were used as control. As a result of PCR analysis, stx2 DNA was detected in 23.3% of cases. Bacteriological research made it possible to sow a pure culture of Escherichia coli O157:H7 in only 3.3% of cases. Since the development of HUS begins in patients with acute intestinal infection caused by Shiga toxin-producing microorganisms starting from the 5th day of the disease, when antibiotic therapy is already carried out, the bacteria can be completely destroyed, which makes it difficult to identify them by bacteriological methods, as well as to detect genes encoding Shiga toxin in PCR analysis. Typically, patients with HUS are admitted to the intensive care unit 5–7 days after the onset of the disease, when class G immunoglobulins specific to the pathogen are already beginning to circulate in the blood. In this regard, the use of immunological tests can be effective to confirm the diagnosis of STEC infection. In our studies, enzyme immunoassay allowed us to detect antibodies to Stx2A in 63.3% and to Stx2B in 43.3% of patients. Using immunoblotting, antibodies to Stx2A were detected in all sera obtained from patients and in 66.7% of cases to Stx2B. Immunoblot analysis was characterized by higher sensitivity for detecting antibodies to Stx2, however, due to the presence of an immunological layer among healthy people, it is preferable to use ELISA analysis. In healthy donors with antibodies to Stx2, the antibody titer was significantly lower than in patients. Laboratory confirmation of the diagnosis of STEC infection is difficult when conducting microbiological and molecular genetic studies, which is confirmed in this work. The effectiveness of laboratory diagnostics can be expanded by performing an ELISA aimed at detecting antibodies to Stx2A.

Background: Hemolytic uremic syndrome (HUS) is an important cause of acute kidney injury in children. HUS is known as an acute disease followed by complete recovery, but patients may present with kidney abnormalities after long periods of time. This study evaluates the long-term outcome of Shiga toxin-producing Escherichia coli-associated HUS (STEC-HUS) in pediatric patients, 10 years after the acute phase of disease to identify risk factors for long-term sequelae. Methods: Over a 6-year period, 619 patients under 18 years of age with HUS (490 STEC-positive, 79%) were registered in Austria and Germany. Long-term follow-up data of 138 STEC-HUS-patients were available after 10 years for analysis. Results: A total of 66% (n = 91, 95% CI 0.57–0.73) of patients fully recovered showing no sequelae after 10 years. An additional 34% (n = 47, 95% CI 0.27–0.43) presented either with decreased glomerular filtration rate (24%), proteinuria (23%), hypertension (17%), or neurological symptoms (3%). Thirty had sequelae 1 year after STEC-HUS, and the rest presented abnormalities unprecedented at the 2-year (n = 2), 3-year (n = 3), 5-year (n = 3), or 10-year (n = 9) follow-up. A total of 17 patients (36.2%) without kidney abnormalities at the 1-year follow-up presented with either proteinuria, hypertension, or decreased eGFR in subsequent follow-up visits. Patients needing extracorporeal treatments during the acute phase were at higher risk of presenting symptoms after 10 years (p < 0.05). Conclusions: Patients with STEC-HUS should undergo regular follow-up, for a minimum of 10 years following their index presentation, due to the risk of long-term sequelae of their disease. An initial critical illness, marked by need of kidney replacement therapy or plasma treatment may help predict poor long-term outcome. [ABSTRACT FROM AUTHOR]

Typical hemolytic uremic syndrome (HUS) can occur as a severe systemic complication of infections with Shiga toxin (Stx)-producing Escherichia coli. Its pathology can be induced by Stx types, resulting in toxin-mediated damage to renal barriers, inflammation, and the development of acute kidney injury (AKI). Two sphingosine kinase (SphK) isozymes, SphK1 and SphK2, have been shown to be involved in barrier maintenance and renal inflammatory diseases. Therefore, we sought to determine their role in the pathogenesis of HUS. Experimental HUS was induced by the repeated administration of Stx2 in wild-type (WT) and SphK1 (SphK1−/−) or SphK2 (SphK2−/−) null mutant mice. Disease severity was evaluated by assessing clinical symptoms, renal injury and dysfunction, inflammatory status and sphingolipid levels on day 5 of HUS development. Renal inflammation and injury were found to be attenuated in the SphK2−/− mice, but exacerbated in the SphK1−/− mice compared to the WT mice. The divergent outcome appeared to be associated with oppositely altered sphingolipid levels. This study represents the first description of the distinct roles of SphK1−/− and SphK2−/− in the pathogenesis of HUS. The identification of sphingolipid metabolism as a potential target for HUS therapy represents a significant advance in the field of HUS research. [ABSTRACT FROM AUTHOR]

Foodborne diseases can be attributed not only to contamination with bacterial or fungal pathogens but also their associated toxins. Thus, to maintain food safety, innovative decontamination techniques for toxins are required. We previously demonstrated that an atmospheric-pressure dielectric-barrier discharge (APDBD) plasma generated by a roller conveyer plasma device is effective at inactivating bacteria and fungi in foods. Here, we have further examined whether the roller conveyer plasma device can be used to degrade toxins produced by foodborne bacterial pathogens, including aflatoxin, Shiga toxins (Stx1 and Stx2), enterotoxin B and cereulide. Each toxin was spotted onto an aluminum plate, allowed to dry, and then treated with APDBD plasma applied by the roller conveyer plasma device for different time periods. Assessments were conducted using a competitive enzyme-linked immunosorbent assay (ELISA) and liquid chromatography–tandem mass spectrometry (LC-MS/MS). The results demonstrate a significant time-dependent decrease in the levels of these toxins. ELISA showed that aflatoxin B1 concentrations were reduced from 308.6 µg/mL to 74.4 µg/mL within 1 min. For Shiga toxins, Stx1 decreased from 913.8 µg/mL to 65.1 µg/mL, and Stx2 from 2309.0 µg/mL to 187.6 µg/mL within the same time frame (1 min). Enterotoxin B levels dropped from 62.67 µg/mL to 1.74 µg/mL at 15 min, and 1.43 µg/mL at 30 min, but did not display a significant decrease within 5 min. LC-MS/MS analysis verified that cereulide was reduced to below the detection limit following 30 min of APDBD plasma treatment. Taken together, these findings highlight that a range of foodborne toxins can be degraded by a relatively short exposure to plasma generated by an APDBD using a roller conveyer device. This technology offers promising advancements in food safety, providing a novel method to alleviate toxin contamination in the food processing industry. [ABSTRACT FROM AUTHOR]

Background: Nutrition quality could be one of the reasons why, in the face of a Shiga toxin-producing enterohemorrhagic Escherichia coli outbreak, some patients experience more profound deleterious effects than others, including unfortunate deaths. Thus, the aim of this study was to determine whether high-fat and/or high-carbohydrate diets could negatively modulate the deleterious action of Shiga toxin 2 on ventral anterior and ventral lateral thalamic nuclei and the internal capsule, the neurological centers responsible for motor activity. Methods: Mice were fed a regular, high-fat, high-carbohydrate diet or a combination of both previous to the intravenous administration of Shiga toxin 2 or vehicle. Four days after intravenous administration, mice were subjected to behavioral tests and then sacrificed for histological and immunofluorescence assays to determine alterations in the neurovascular unit at the cellular and functional levels. Statistical analysis was performed using one-way analysis of variance followed by Bonferroni post hoc test. The criterion for significance was p = 0.0001 for all experiments. Results: The high-fat and the high-carbohydrate diets significantly heightened the deleterious effect of Stx2, while the combination of both diets yielded the worst results, including endothelial glycocalyx and oligodendrocyte alterations, astrocyte and microglial reactivity, neurodegeneration, and motor and sensitivity impairment. Conclusions: In view of the results presented here, poor nutrition could negatively influence patients affected by Stx2 at a neurological level. Systemic effects, however, cannot be ruled out.

Shiga toxin-producing Escherichia coli (STEC) are zoonotic enteric pathogens linked to human gastroenteritis worldwide. To aid the development of pathogen control efforts, the present study characterized the genotypic diversity and pathogenic potential of STEC recovered from sources near agricultural fields in Northwest Mexico. Samples were collected from irrigation river water and domestic animal feces in farms proximal to agricultural fields and were subjected to enrichment followed by immunomagnetic separation and plating on selective media for the recovery of the STEC isolates. Comparative genomic analyses indicated that the recovered STEC with the clinically relevant serotypes O157:H7, O8:H19, and O113:H21 had virulence genes repertoires associated with host cell adherence, iron uptake and effector protein secretion. Subsequent phenotypic characterization revealed multidrug resistance against aminoglycoside, carbapenem, cephalosporin, fluoroquinolone, penicillin, phenicol, and tetracycline, highlighting the need for improved surveillance on the use of antimicrobials. The present study indicated for the first time that river water in the agricultural Culiacan Valley in Mexico is a relevant key route of transmission for STEC O157 and non-O157 with a virulence potential. In addition, feces from domestic farm animals near surface waterways can act as potential point sources of contamination and transport of diverse STEC with clinically relevant genotypes.

The Shigatoxigenic Escherichia coli (STEC) are bacterial enteropathogens responsible for some intensive clinical syndromes such as bloody diarrhoea, hemorrhagic colitis, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, and renal failure. These pathotypes come under the Enterohemorrhagic Escherichia coli (EHEC) group. Monogastric farm animals such as pigs, horses, chickens, ducks, turkeys and aquatic animals like shellfish, fishes, and wild animals can act as major spillover hosts of STEC strains and could serve as the potential source of infection. The pathogen is notorious as a quickly emergent strain with acquired characteristics like different variants of Shigatoxin, many antibiotic degrading enzymes, Intimin, Enterohemolysin, Auto-agglutination Adhesins, Catalase-peroxidase, Zinc metalloprotease, Subtilase cytotoxin, tolerance to multiple adverse conditions, and biofilm formation. The bacteria are known for its long survival in different adverse physical-chemical conditions. The formation of biofilm is one of the major factors responsible for their persistence. Multidrug resistance is another related trait contributing to the high mortality rate of these strains. STEC strains are good candidates for studying the emergence of pathogens with acquired characteristics like genes. In this article, various virulent traits and multidrug resistance that enabled the strain to emerge as a serious public health menace were reviewed.

ABSTRACTShiga toxin-producing Escherichia coli (STEC) is widely distributed in the environment and is recognized for its association with severe illnesses in both animals and humans. As effective treatments and preventive measures remain limited, it becomes crucial to understand the mechanisms underlying STEC-induced diseases. Animal models allow analyzing the pathophysiology of diseases. However, current models do not accurately reproduce the full spectrum of diseases caused by STEC. In this study, we discussed the key characteristics and constraints of five animal models (mouse, rabbit, chicken, dog, and pig) used to study STEC infections.

Enterohemorrhagic E. coli (EHEC) is a group of pathogenic bacteria that is associated with worldwide human foodborne diarrheal illnesses and the development of hemolytic uremic syndrome, a potentially deadly condition associated with Shiga toxins (Stxs). Currently, approved vaccines for human prophylaxis against infection do not exist, and one barrier preventing the successful creation of EHEC vaccines is the absence of dependable animal models, including mice, which are naturally resistant to EHEC infection and do not manifest the characteristic signs of the illness. Our lab previously developed gold nanoparticle (AuNP)-based EHEC vaccines, and assessed their efficacy using Citrobacter rodentium, which is the mouse pathogen counterpart of EHEC, along with an Stx2d-producing strain that leads to more consistent disease kinetics in mice, including lethality. The purpose of this study was to continue evaluating these vaccines to increase protection. Here, we demonstrated that subcutaneous immunization of mice with AuNPs linked to the EHEC antigens EscC and intimin (Eae), either alone or simultaneously, elicits functional robust systemic humoral responses. Additionally, vaccination with both antigens together showed some efficacy against Stx2d-producing C. rodentium while AuNP-EscC successfully limited infection with non-Stx2d-producing C. rodentium. Overall, the collected results indicate that our AuNP vaccines have promising potential for preventing disease with EHEC, and that evaluation of novel vaccines using an appropriate animal model, like C. rodentium described here, could be the key to finally developing an effective EHEC vaccine that can progress into human clinical trials. [ABSTRACT FROM AUTHOR]

Shiga toxin (Stx), the main virulence factor of Shiga-toxin-producing E. coli (STEC), was first discovered in Shigella dysenteriae strains. While several other bacterial species have since been reported to produce Stx, STEC poses the most significant risk to human health due to its widespread prevalence across various animal hosts that have close contact with human populations. Based on its biochemical and molecular characteristics, Shiga toxin can be grouped into two types, Stx1 and Stx2, among which a variety of variants and subtypes have been identified in various bacteria and host species. Interestingly, the different Stx subtypes appear to vary in their host distribution characteristics and in the severity of diseases that they are associated with. As such, this review provides a comprehensive overview on the bacterial species that have been recorded to possess stx genes to date, with a specific focus on the various Stx subtype variants discovered in STEC, their prevalence in certain host species, and their disease-related characteristics. This review provides a better understanding of the Stx subtypes and highlights the need for rapid and accurate approaches to toxin subtyping for the proper evaluation of the health risks associated with Shiga-toxin-related bacterial food contamination and human infections. [ABSTRACT FROM AUTHOR]

The Shigatoxigenic Escherichia coli (STEC) are bacterial enteropathogens responsible for some intensive clinical syndromes such as bloody diarrhoea, hemorrhagic colitis, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, and renal failure. These pathotypes come under the Enterohemorrhagic Escherichia coli (EHEC) group. Monogastric farm animals such as pigs, horses, chickens, ducks, turkeys and aquatic animals like shellfish, fishes, and wild animals can act as major spillover hosts of STEC strains and could serve as the potential source of infection. The pathogen is notorious as a quickly emergent strain with acquired characteristics like different variants of Shigatoxin, many antibiotic degrading enzymes, Intimin, Enterohemolysin, Auto-agglutination Adhesins, Catalase-peroxidase, Zinc metalloprotease, Subtilase cytotoxin, tolerance to multiple adverse conditions, and biofilm formation. The bacteria are known for its long survival in different adverse physical-chemical conditions. The formation of biofilm is one of the major factors responsible for their persistence. Multidrug resistance is another related trait contributing to the high mortality rate of these strains. STEC strains are good candidates for studying the emergence of pathogens with acquired characteristics like genes. In this article, various virulent traits and multidrug resistance that enabled the strain to emerge as a serious public health menace were reviewed. [ABSTRACT FROM AUTHOR]

Shiga toxin-producing Escherichia coli (STEC) are zoonotic enteric pathogens linked to human gastroenteritis worldwide. To aid the development of pathogen control efforts, the present study characterized the genotypic diversity and pathogenic potential of STEC recovered from sources near agricultural fields in Northwest Mexico. Samples were collected from irrigation river water and domestic animal feces in farms proximal to agricultural fields and were subjected to enrichment followed by immunomagnetic separation and plating on selective media for the recovery of the STEC isolates. Comparative genomic analyses indicated that the recovered STEC with the clinically relevant serotypes O157:H7, O8:H19, and O113:H21 had virulence genes repertoires associated with host cell adherence, iron uptake and effector protein secretion. Subsequent phenotypic characterization revealed multidrug resistance against aminoglycoside, carbapenem, cephalosporin, fluoroquinolone, penicillin, phenicol, and tetracycline, highlighting the need for improved surveillance on the use of antimicrobials. The present study indicated for the first time that river water in the agricultural Culiacan Valley in Mexico is a relevant key route of transmission for STEC O157 and non-O157 with a virulence potential. In addition, feces from domestic farm animals near surface waterways can act as potential point sources of contamination and transport of diverse STEC with clinically relevant genotypes. [ABSTRACT FROM AUTHOR]

Background Shiga-toxin-producing Escherichia coli haemolytic uraemic syndrome affects ~100 United Kingdom children each year. Around half need dialysis, a quarter develop serious complications with long-term consequences and ~3% die. No effective intervention is known; however, some studies report eculizumab, effective in atypical haemolytic uraemic syndrome, may be effective. Objectives To determine whether the severity of Shiga-toxin-producing Escherichia coli haemolytic uraemic syndrome is less in those given eculizumab. Design Randomised, double-blind, placebo-controlled, parallel-group trial with internal pilot phase and nested mechanistic laboratory studies. Setting Paediatric nephrology units in 12 United Kingdom hospitals. Participants Children aged 6 months to < 19 years weighing ≥ 5 kg, with presumed Shiga-toxin-producing Escherichia coli haemolytic uraemic syndrome, including ‘injury’ or ‘failure’ category of the acute kidney injury paediatric risk/injury/failure/loss/end criteria. Intervention Participants were randomised in a 1 : 1 ratio to receive intravenous eculizumab or placebo on day 1 and 8. All received meningococcal vaccination and prophylactic antibiotics. Main outcome measures The primary outcome measure was a multidomain clinical severity score, reflecting morbidity until day 60. Secondary outcome measures included survival, duration of renal replacement therapy, persistent neurological defect (day 60) and presence of chronic kidney disease at 1 year. Mechanistic studies assessed complement activation and vascular endothelial growth factor profiles in plasma ± urine samples. In vitro cell co-culture work assessed the effect of Shiga toxin on endothelial cells. Results Thirty-six participants from 10 sites were randomised: 17 to eculizumab and 19 to placebo. The target sample size was 134 participants – recruitment stopped early due to low recruitment (factors included reduced incidence and limited out-of-hours research infrastructure) and the COVID-19 pandemic. The mean clinical severity score for participants randomised to eculizumab was 11.5 (standard deviation 8.4) compared to 14.6 (standard deviation 7.7) for participants randomised to placebo (adjusted mean difference: −2.5, 95% confidence interval −7.8 to 2.8, p = 0.3). Five participants (three eculizumab, two placebo) experienced an adverse event; there were seven serious adverse events in six participants (five eculizumab, one placebo). Urinary complement factor H and vascular endothelial growth factor levels were high initially and subsequently normalised. Shiga toxin caused a podocyte-dependent decrease in endothelial cell factor H levels. Conclusions and limitations There was no significant difference in mean clinical severity score between eculizumab and placebo groups – since the trial was underpowered, this cannot be interpreted as evidence of no effect. No significant safety concerns were observed. With further validation, the Eculizumab in Shiga-toxin-producing Escherichia coli Haemolytic Uraemic Syndrome clinical severity score may be an outcome measure for future trials. Our results imply that Shiga toxin causes complement-dependent glomerular endothelial cell injury through its action on podocytes and subsequent cellular cross-talk. Future work We will continue to investigate cross talk between podocytes and endothelial cells after exposure to Shiga toxin and further develop plasma/urine biomarkers for diagnosis of Shiga-toxin-producing Escherichia coli haemolytic uraemic syndrome. Trial registration This trial is registered as EudraCT-2016-000997-39 and ISRCTN89553116. Funding This award was funded by the National Institute for Health and Care Research (NIHR) Efficacy and Mechanism Evaluation (EME) programme (NIHR award ref: 14/48/43) and is published in full in Efficacy and Mechanism Evaluation; Vol. 11, No. 11. See the NIHR Funding and Awards website for further award information. Plain language summary Why did we do this study? Annually, approximately 100 United Kingdom children develop Shiga-toxin-producing Escherichia coli haemolytic uraemic syndrome after infection with a diarrhoea-causing bug. The bug makes a toxin (Shiga toxin) that damages blood vessels, especially in the kidneys. Half need dialysis (artificial kidney support), about a quarter suffer fits or a stroke and about 3% die. Most children fully recover, but about a quarter suffer permanent kidney or brain damage. There are no known effective treatments. Eculizumab, a medicine which blocks part of the immune system called complement, may work. What was the question? Does eculizumab reduce the severity of Shiga-toxin-producing Escherichia coli haemolytic uraemic syndrome? What did we do? We planned to recruit 134 children, but difficulties with recruitment and the COVID-19 pandemic meant the study was stopped early after 36 children had been recruited; 17 received eculizumab, 19 received a dummy medicine (placebo). We compared children in each group using a score that measured how their kidneys and other organs were affected. We studied samples of their blood and urine, and also how Shiga toxin damages kidney cells in the laboratory. What did we find? The severity of illness was similar in both groups; however, because we only studied a small number of children, we cannot be sure this means that eculizumab does not work. Eculizumab appeared to be safe in this condition. In the blood and urine samples, we saw evidence that complement is involved in the illness. We also discovered a new way that Shiga toxin damages kidney cells. What does this mean? We have been unable to show whether eculizumab is a worthwhile treatment for children with this condition. However, we have learnt lots about how the illness is caused and hope these results can be combined with other studies to give us a clearer answer. Scientific summary Background Shiga-toxin-producing Escherichia coli haemolytic uraemic syndrome affects around 100 UK children each year, following gastrointestinal infection with Shiga-toxin-producing E. coli. Around half of affected children will need dialysis, about a quarter develop serious complications with long-term consequences and about 3% die. All patients require long-term follow-up because of the risk of developing chronic kidney disease (CKD). No intervention has definitively been shown to reduce morbidity or mortality in this condition, and therefore treatment is supportive. Case reports and case series suggest that eculizumab (Soliris®, Alexion Pharmaceuticals, Boston, MA), an inhibitor of the complement system and an effective treatment for the related condition, atypical haemolytic uraemic syndrome, may be effective in this condition. Until very recently, there were no published data regarding the efficacy and safety of eculizumab in Shiga-toxin-producing E. coli haemolytic uraemic syndrome, and yet despite this its use has risen globally. A recent randomised phase 3 clinical trial reported comparison of eculizumab with placebo in 100 children with Shiga-toxin-producing E. coli haemolytic uraemic syndrome (Garnier A, Brochard K, Kwon T, Sellier-Leclerc A-L, Lahoche A, Allain Launay E, et al. Efficacy and safety of Eculizumab in pediatric patients affected by Shiga Toxin-Related Hemolytic and Uremic Syndrome: a randomized, placebo-controlled trial. J Am Soc Nephrol 2023;34:1561–73). Patients with severe multi-organ involvement were excluded. Four patients in the placebo group were withdrawn and subsequently received eculizumab. There was no difference between treatment groups in the proportion of children who required renal replacement therapy 48 hours after randomisation, In addition, no differences between groups were seen in the secondary outcome measures of extra-renal manifestations, duration of hospitalisation and mortality. During follow up, there was a slight difference in the proportion who exhibited renal sequelae at 12 months post randomisation (20 patients in the eculizumab group (43.48%) and 29 patients (64.44%) in the placebo group (P = 0.04). The authors concluded that eculizumab seemed to have no impact on the course of acute kidney injury and interpreted the 12-month follow-up data with caution. No data have been published which include the role of eculizumab in patients with severe manifestations of disease in a controlled setting. Objectives of the main trial The Eculizumab in Shiga-toxin-producing Escherichia coli Haemolytic Uraemic Syndrome (ECUSTEC) trial was designed to test the hypothesis that treatment with eculizumab reduces the severity of Shiga-toxin-producing E. coli haemolytic uraemic syndrome in children aged 6 months–< 19 years. We also wanted to assess the safety of eculizumab and test the hypothesis that treatment with eculizumab reduces the incidence of CKD following Shiga-toxin-producing E. coli haemolytic uraemic syndrome. Objectives of the mechanistic substudies The mechanistic component of the trial had the following objectives: to investigate the time course of systemic complement activation in Shiga-toxin-producing E. coli haemolytic uraemic syndrome and its relation to the severity of disease to determine whether thrombotic microangiopathy (TMA) in Shiga-toxin-producing E. coli haemolytic uraemic syndrome occurs via a Shiga-toxin-mediated reduction in podocyte vascular endothelial growth factor (VEGF) production, leading to loss of complement regulation to test whether neutrophils derived from patients with acute Shiga-toxin-producing E. coli haemolytic uraemic syndrome deliver Shiga toxin to podocytes to assess whether any genetic variations in patients with Shiga-toxin-producing E. coli haemolytic uraemic syndrome point to novel pathogenic mechanisms. Methods Design The trial was a randomised, double-blind, placebo-controlled, parallel-group trial of two doses of eculizumab in children (aged 6 months–< 19 years) with Shiga-toxin-producing E. coli Haemolytic Uraemic Syndrome. The trial had an internal pilot phase and included nested mechanistic laboratory studies and a cost-effectiveness evaluation, although the latter was not undertaken following the trial being stopped early. The mechanistic substudies were optional; all participants in the main trial were offered the opportunity to participate in the substudies, which involved providing blood and urine samples over the first 30 days of the trial. Participant blood and urine samples were used to explore the evidence for, and time course of, complement activation in this condition. Using both patient samples and an in vitro cell co-culture model, evidence was sought to support the hypothesis that Shiga toxin causes a glomerular TMA as a consequence of its effect on podocyte VEGF production. This included measurement of patient urine and plasma complement factor H (CFH) and VEGF and plasma complement activation products [by both enzyme-linked immunosorbent assay (ELISA) and a novel degradomics technique]. In the co-culture experiments, glomerular endothelial cells were exposed to Shiga toxin in the presence and absence of podocytes. Setting The trial was conducted in 12 sites in NHS hospital settings across the UK with the support of 88 Patient Identification Centres. Participants Informed consent was sought from parents/guardians of eligible children (those aged 6 months–< 19 years weighing ≥ 5 kg, with a clinical diagnosis of Shiga-toxin-producing E. coli haemolytic uraemic syndrome, including acute kidney injury (AKI) equivalent to the ‘injury’ or ‘failure’ category of the paediatric risk/injury/failure/loss/end criteria). Eligible young people aged 16–18 years provided their own consent for participation in the trial, with assent from younger children if appropriate (according to age). Screening and randomisation Screening began as soon as possible after a diagnosis of Shiga-toxin-producing E. coli haemolytic uraemic syndrome was suspected. Once eligibility was confirmed and informed consent obtained, the participants were commenced on prophylactic antibiotics, and unless contra-indicated or already administered, participants were also vaccinated against meningococcal infection. Participants were then randomised into the ECUSTEC trial via a secure online central randomisation system. Participants were randomised at the level of the individual in a 1 : 1 ratio to either eculizumab or placebo, which was commenced as soon as possible after randomisation. A minimisation algorithm was used to ensure balance in the treatment allocation over the following variables: centre, severity of AKI and hydration status. To avoid predictability in the randomisation, a random element was included in the minimisation algorithm, so that each patient had a probability (unspecified here), of being randomised to the opposite treatment that they would have otherwise received. Intervention Each participant received two intravenous infusion doses of either eculizumab (dose according to first two doses of induction regimen for atypical haemolytic uraemic syndrome) or placebo. The first dose was given as soon as possible after randomisation (designated day 1), with the second dose given 7 days later (i.e. on day 8). They also received vaccination against meningococcal disease and an 8-week course of antibiotic prophylaxis. The participants, parents/guardians, clinical staff and research teams were blind to randomised treatment allocation throughout the study. Outcome measures Primary The ECUSTEC clinical severity score (CSS), a purpose-developed, multidomain score comprising severity of AKI and extrarenal events. A single score is assigned at day 60 to reflect cumulative morbidity up until that point. The score ranges from 1 to 69 with higher scores indicating greater disease severity. Secondary Overall survival. Duration of renal replacement therapy (days). Duration of thrombocytopenia (number of consecutive days until platelet count > 150 × 109/l). Duration of haemolysis (number of days until lactate dehydrogenase within local centre reference range). Number of packed red blood cell transfusions required and volume (ml/kg). Duration markers of inflammation present (number of days until neutrophil cell count and C-reactive protein are in normal range for that centre). Persistent neurological defect at day 60 measured by structured expert assessment to include central nervous system examination, vision, hearing and neuropsychological assessment. CKD at 52 weeks (a composite end point of the presence of hypertension, albuminuria or estimated glomerular filtration rate (eGFR) < 90 ml/minute/1.73 m2 at 52 weeks). eGFR measurement using a centralised cystatin C assay at 52 weeks. Mechanistic studies Urine CFH levels. Urine VEGF levels. Presence of urine markers of podocyte damage (nephrin and Wilms tumour-1). Plasma VEGF and factor H levels. Plasma complement activation products (Bb, C3a, C4a and sC5b9 by ELISA and C3 and C4 activation markers by degradomics). Glomerular endothelial surface levels of factor H and C3d (a marker of complement activation) in a co-culture models of human conditionally immortalised podocytes and glomerular endothelial cells exposed to Shiga toxin. Whole exome sequencing and serum anti-factor H antibody levels. Results The target sample size was 134 participants, but recruitment was stopped early due to low recruitment and the impact of the COVID-19 pandemic. At the point the trial was stopped, 108 children had been screened for participation, of whom 87 were deemed eligible to participate. Thirty-six children were consented and randomised; 17 were randomised to eculizumab and 19 were randomised to placebo. One participant withdrew from the trial and one participant died. The majority of baseline data of the participants were comparable across the two groups; however, the participants in the placebo group were slightly older and consequently heavier than those in the eculizumab group. Reasons for slow recruitment included a fall in the incidence of Shiga-toxin-producing E. coli haemolytic uraemic syndrome during the trial period (up to a 37% reduction) and a lack of out-of-hours infrastructure for undertaking acute interventional clinical trials in children. The mean CSS at day 60 for participants randomised to eculizumab was 11.5 [standard deviation (SD) 8.4] compared to 14.6 (SD 7.7) for participants randomised to placebo [adjusted mean difference: −2.5, 95% confidence interval (CI) −7.8 to 2.8, p = 0.3]. Five participants (three eculizumab, two placebo) experienced an adverse event, and there were seven serious adverse events (SAEs) in six participants (five eculizumab, one placebo). None of the SAEs were considered related to the trial treatment. Mechanistic substudies Of the 36 participants recruited to the main trial, 32 consented to take part in the mechanistic studies and provided blood and/or urine samples. In anuric patients, only blood samples were collected. Urine factor H and vascular endothelial growth factor levels in serial samples The highest urine factor H levels were at day 1 (150 ng/ml), diminishing by day 4 (30 ng/ml), and completely normalising by day 30 (undetectable). The highest urine VEGF levels were at day 1 (average 1300 ng/ml) and by day 30 the levels were below 20 ng/ml. Markers of podocyte damage Western blots of urine cell pellets showed acute podocyte loss during active disease, which recovered by day 8. Plasma factor H and vascular endothelial growth factor levels No difference was seen for plasma levels of either factor H or VEGF at day 1 or day 30. Plasma degradomics analysis In a sample of five patients, N-termini consistent with complement C3 and C4 activation were much more abundant at day 1 compared with day 3. In vitro cell co-culture In response to Shiga toxin, there was a reduction in glomerular endothelial factor H levels, accompanied by evidence of complement activation (increased C3d levels) and this was critically dependent on the presence of podocytes. Shiga toxin had no effect when added to endothelial cells alone. Plasma complement activation products Mean plasma levels of Bb were elevated in both groups at baseline (4.38 mcg/ml in the eculizumab group and 10.38 mcg/ml in the placebo group, normal range 0.48–1.62 mcg/ml). They were also elevated at day 2 (5.91 mcg/ml in the eculizumab group and 4.09 mcg/ml in the placebo group) and day 4 (4.90 mcg/ml in the eculizumab group and 3.16 mcg/ml in the placebo group). At day 6 and day 8, Bb levels remained elevated in the placebo group (6.21 and 3.47 mcg/ml respectively) but were normal in the eculizumab group. In both groups, mean Bb levels were in the normal range at day 30. Mean plasma levels of C3a were elevated in both groups at baseline and at days 2 and 4. At days 6 and 8, mean levels remained elevated in the placebo group while mean levels in the eculizumab group were in the normal range. Levels were in the normal range for both groups by day 30. Mean plasma levels of C4a were elevated at all time points in both groups but fell significantly at day 30. Mean levels were 3852, 3026, 3423, 3067, 3425 and 1623 ng/ml at days 1, 2, 4, 6, 8 and 30 in the eculizumab group (normal range 110–699 ng/ml) and 3970, 3573, 2673, 4348, 2844 and 1992 ng/ml at the same respective time points in the placebo group. Mean plasma levels of sC5b9 were normal in both groups at all time points with the exception of day 4 and day 6 in the placebo group, which were elevated (487 and 514 ng/ml respectively, normal range 95–467 ng/ml). In the placebo group, a linear relationship was not established between CSS and baseline Bb (r = 0.43, p = 0.2); C3a (r = −0.16, p = 0.7); C4a (r = 0.15, p = 0.7) or sC5b9 (r = −0.17, p = 0.7). Similarly, a linear relationship was not established between CSS and the maximum value of Bb (r = 0.45, p = 0.1); C3a (r = 0.15, p = 0.6); C4a (r = 0.23, p = 0.4) or sC5b9 (r = −0.22, p = 0.5). Delivery of Shiga toxin to podocytes from patient-derived neutrophils Insufficient patient samples were obtained to complete this part of the work. Genetic variations in patients with Shiga-toxin-producing Escherichia coli haemolytic uraemic syndrome Data from whole exome sequencing have been obtained and analysis is ongoing. Conclusions In children with Shiga-toxin-producing E. coli haemolytic uraemic syndrome, the mean CSSs at day 60 were similar between those randomised to eculizumab and those randomised to placebo. However, since the trial was stopped early and did not recruit to the planned sample size, this cannot be interpreted as evidence of no effect. In order to deliver successful clinical trials of investigational medicinal products in acutely unwell children, a review of out-of-hours paediatric research infrastructure may be required. In the mechanistic substudies, we have established that urine factor H and VEGF levels are sensitive measures of early disease activity, and have demonstrated complement activation in patient serum using both ELISA and sophisticated proteomics technology. Urine factor H and VEGF levels and plasma degradomics for C3 and C4 proteins could all be further explored as new biomarkers of acute Shiga-toxin-producing E. coli haemolytic uraemic syndrome. Our co-culture cell work has demonstrated that podocyte cross-talk is responsible for factor H and complement activation levels on endothelial cells. Collectively this strongly supports the mechanistic hypothesis of a complement-mediated disease driven via the podocyte as the target cell. Trial registration This trial is registered as EudraCT2016-000997-39 and ISRCTN89553116. Funding This award was funded by the National Institute for Health and Care Research (NIHR) Efficacy and Mechanism Evaluation (EME) programme (NIHR award ref: 14/48/43) and is published in full in Efficacy and Mechanism Evaluation; Vol. 11, No. 11. See the NIHR Funding and Awards website for further award information.

Abstract Background Hemolysis is a cardinal feature of hemolytic uremic syndrome (HUS) and during hemolysis excess arginase 1 is released from red blood cells. Increased arginase activity leads to reduced L-arginine, as it is converted to urea and L-ornithine, and thereby reduced nitric oxide bioavailability, with secondary vascular injury. The objective of this study was to investigate arginase release in HUS patients and laboratory models and correlate arginase levels to hemolysis and kidney injury. Methods Two separate cohorts of patients (n = 47 in total) with HUS associated with Shiga toxin-producing enterohemorrhagic E. coli (EHEC) and pediatric controls (n = 35) were investigated. Two mouse models were used, in which mice were either challenged intragastrically with E. coli O157:H7 or injected intraperitoneally with Shiga toxin 2. An in vitro model of thrombotic microangiopathy was developed in which Shiga toxin 2- and E. coli O157 lipopolysaccharide-stimulated human blood cells combined with ADAMTS13-deficient plasma were perfused over glomerular endothelial cells. Two group statistical comparisons were performed using the Mann–Whitney test, multiple groups were compared using the Kruskal–Wallis test followed by Dunn’s procedure, the Wilcoxon signed rank test was used for paired data, or linear regression for continuous variables. Results HUS patients had excessively high plasma arginase 1 levels and activity (conversion of L-arginine to urea and L-ornithine) during the acute phase, compared to remission and controls. Arginase 1 levels correlated with lactate dehydrogenase activity, indicating hemolysis, as well as the need for dialysis treatment. Patients also exhibited high levels of plasma alpha-1-microglobulin, a heme scavenger. Both mouse models exhibited significantly elevated plasma arginase 1 levels and activity. Plasma arginase 1 levels correlated with lactate dehydrogenase activity, alpha-1-microglobulin and urea levels, the latter indicative of kidney dysfunction. In the in vitro model of thrombotic microangiopathy, bioactive arginase 1 was released and levels correlated to the degree of hemolysis. Conclusions Elevated red blood cell-derived arginase was demonstrated in HUS patients and in relevant in vivo and in vitro models. The excessively high arginase levels correlated to the degree of hemolysis and kidney dysfunction. Thus, arginase inhibition should be investigated in HUS.

Introduction. Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen associated with clinical cases of diarrhea in humans. Its main virulence factors are the Shiga toxins (Stx1 and Stx2). Cattle are the main reservoir of STEC, and many outbreaks in humans have been related to the consumption of undercooked ground beef contaminated with this pathogen. Objective. To determine the prevalence of STEC in ground beef commercialized in all the butcher shops of a township in the department of Quindío and to characterize the virulence genes of the strains found. Materials and methods. Thirty ground beef samples were taken in three different times; stx genes and other STEC virulence factors (eae, ehxA, saa) were detected by multiplex PCR. Results. The overall prevalence of STEC was 33.33 % (10/30 positive samples). We isolated eight non-O157 (LEE-negative) strains with four different genetic profiles: stx2 / stx2-ehxA-saa / stx1-stx2-ehxA-saa / stx1-saa. Conclusion. This is the first report on the prevalence of STEC in ground beef in a township in the department of Quindío.

Hemolytic-uremic syndrome (HUS) is a rare complication of an infection with Shiga toxin (Stx)-producing Escherichia coli (STEC-HUS), characterized by severe acute kidney injury, thrombocytopenia and microangiopathic hemolytic anemia, and specific therapy is still lacking. Thrombomodulin (TM) is a multi-domain transmembrane endothelial cell protein and its N-terminal domain has been implicated in the pathophysiology of some cases of HUS. Indeed, the administration of recombinant human TM (rhTM) may have efficacy in HUS. We used a Stx-based murine model of HUS to characterize the role of the N-terminal domain of TM. We show that mice lacking that domain (TMLed (−/−)) are more sensitive to Stx, with enhanced HUS progression seen at 4 days and increased mortality at 7 days post-HUS induction. In spite of these changes, renal function was less affected in surviving Stx-challenged TMLed (−/−) mice compared to their wild-type counterparts TMLed (+/+) at 7 days. Contrary to few clinical case reports from Japan, the administration of rhTM (0.06 mg/kg) to wild-type mice (C57BL/6J) with HUS did not protect against disease progression. This overall promising, but also contradictory body of evidence, requires further systematic preclinical and clinical investigations to clarify the role of TM in HUS as a potential therapeutic strategy.

Background: Hemolysis is a cardinal feature of hemolytic uremic syndrome (HUS) and during hemolysis excess arginase 1 is released from red blood cells. Increased arginase activity leads to reduced L-arginine, as it is converted to urea and L-ornithine, and thereby reduced nitric oxide bioavailability, with secondary vascular injury. The objective of this study was to investigate arginase release in HUS patients and laboratory models and correlate arginase levels to hemolysis and kidney injury. Methods: Two separate cohorts of patients (n = 47 in total) with HUS associated with Shiga toxin-producing enterohemorrhagic E. coli (EHEC) and pediatric controls (n = 35) were investigated. Two mouse models were used, in which mice were either challenged intragastrically with E. coli O157:H7 or injected intraperitoneally with Shiga toxin 2. An in vitro model of thrombotic microangiopathy was developed in which Shiga toxin 2- and E. coli O157 lipopolysaccharide-stimulated human blood cells combined with ADAMTS13-deficient plasma were perfused over glomerular endothelial cells. Two group statistical comparisons were performed using the Mann–Whitney test, multiple groups were compared using the Kruskal–Wallis test followed by Dunn's procedure, the Wilcoxon signed rank test was used for paired data, or linear regression for continuous variables. Results: HUS patients had excessively high plasma arginase 1 levels and activity (conversion of L-arginine to urea and L-ornithine) during the acute phase, compared to remission and controls. Arginase 1 levels correlated with lactate dehydrogenase activity, indicating hemolysis, as well as the need for dialysis treatment. Patients also exhibited high levels of plasma alpha-1-microglobulin, a heme scavenger. Both mouse models exhibited significantly elevated plasma arginase 1 levels and activity. Plasma arginase 1 levels correlated with lactate dehydrogenase activity, alpha-1-microglobulin and urea levels, the latter indicative of kidney dysfunction. In the in vitro model of thrombotic microangiopathy, bioactive arginase 1 was released and levels correlated to the degree of hemolysis. Conclusions: Elevated red blood cell-derived arginase was demonstrated in HUS patients and in relevant in vivo and in vitro models. The excessively high arginase levels correlated to the degree of hemolysis and kidney dysfunction. Thus, arginase inhibition should be investigated in HUS. [ABSTRACT FROM AUTHOR]

Hemolytic uremic syndrome (HUS) is an acute disease and the most common cause of childhood acute renal failure. HUS is characterized by a triad of symptoms: microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury. In most of the cases, HUS occurs as a result of infection caused by Shiga toxin-producing microbes: hemorrhagic Escherichia coli and Shigella dysenteriae type 1. They account for up to 90% of all cases of HUS. The remaining 10% of cases grouped under the general term atypical HUS represent a heterogeneous group of diseases with similar clinical signs. Emerging evidence suggests that in addition to E. coli and S. dysenteriae type 1, a variety of bacterial and viral infections can cause the development of HUS. In particular, infectious diseases act as the main cause of aHUS recurrence. The pathogenesis of most cases of atypical HUS is based on congenital or acquired defects of complement system. This review presents summarized data from recent studies, suggesting that complement dysregulation is a key pathogenetic factor in various types of infection-induced HUS. Separate links in the complement system are considered, the damage of which during bacterial and viral infections can lead to complement hyperactivation following by microvascular endothelial injury and development of acute renal failure. [ABSTRACT FROM AUTHOR]

Background: Enteropathogenic E. coli (EPEC) and Shiga toxin-producing E. coli (STEC) are categorized as attaching and effacing E. coli (AEEC) due to their eae gene. One of the essential causes of diarrhea in humans is AEEC, which affects birds, too, thereby being considered a zoonotic pathogen. Objectives: Our study aimed to determine AEEC and evaluate its antibiotic resistance and phylogroups. Methods: A total of 200 fecal samples were collected from pet birds referred to the Veterinary Medicine Hospital, University of Tehran. PCR methods were used to detect AEEC using uspA, eae, bfpA, stx1, and stx2 gene-specific primers. The antimicrobial susceptibility of the recovered isolates was determined by the agar disk diffusion and MIC methods. Their phylogroups were analyzed based on Clermont phylotyping methods. Results: Of 200 samples, we isolated 26 (13%) E. coli strains, 9 harbor eae genes. None of the ease-positive samples possessed the bfpA gene, but 4 had stx2, and 5 had stx1 and stx2 genes. Phylogenetic analysis identified the phylogenetic groups of all AEEC isolated strains but 2 (duck and cockatiel). Detected phylogroups include four B2 and three D. Based on our results, 7 out of 9 AEEC isolated strains showed multi-drug resistance. Conclusion: The discovery of common phylogroups of AEEC in pet birds (a common companion animal in Iran with intimate contact with their owners, especially children) and humans, as well as their resistance to a wide range of antibiotics used in human medicine, verifies AEEC as a serious public health threat. [ABSTRACT FROM AUTHOR]

Copyright of Biomédica: Revista del Instituto Nacional de Salud is the property of Instituto Nacional de Salud of Colombia and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Escherichia albertii is an emerging zoonotic foodborne pathogen. The clinical significance of this bacterium has increasingly been recognized worldwide. However, diagnostic method has not yet been established and its clinical manifestations are not fully understood. Here, we show that an Eacdt gene-based quantitative real-time PCR (qRT-PCR) developed in this study is 100% specific and sensitive when tested with 39 E. albertii and 36 non-E. albertii strains, respectively. Detection limit of the real-time PCR was 10 colony forming unit (CFU) and 1 pg of genomic DNA per PCR tube. When E. albertii was spiked with 4 × 100–106 CFU per mL to stool of healthy person, detection limit was 4.0 × 103 and 4.0 CFU per mL before and after enrichment culture, respectively. Moreover, the qRT-PCR was able to detect E. albertii in five children out of 246 (2%) but none from 142 adults suffering from gastroenteritis. All five E. albertii strains isolated carried eae and paa genes, however, only one strain harbored stx2f genes. Long-term shedding of stx2f gene-positive E. albertii in a child stool could be detected because of the qRT-PCR developed in this study which might have been missed if only conventional PCR and culture methods were employed. Furthermore, E. albertii isolated from siblings with diarrhea showed clonality by PFGE analysis. Taken together, these data suggest that the Eacdt gene-based qRT-PCR developed for the detection of E. albertii is useful and will assist in determining the real burden and clinical manifestation of E. albertii infections.

Illnesses associated with consuming infected milk and milk products are a widespread problem in low and middle-income countries. Shiga toxin-producing Escherichia coli (STEC) is a bacterium commonly found in raw milk and causes foodborne diseases ranging from mild diarrhea to severe hemorrhagic colitis and hemolytic uremic syndrome. This study aimed to investigate the virulence gene and antimicrobial resistance profiles of Shiga toxin-producing E. coli strains isolated from raw milk in dairy farms in and around Bahir Dar town. Raw milk samples (n = 128) collected from December 2021 to July 2022 were cultured, and E. coli strains were isolated using standard methods. Shiga toxin-producing E. coli strains were identified genotypically by the presence of the virulence markers using a single-plex polymerase chain reaction. The antibiotic susceptibility testing of Shiga toxin-producing E. coli isolates was done by the agar disk diffusion method. In total, 32 E. coli isolates were recovered from milk samples from lactating animals. PCR screening of these isolates resulted in 19 (59.3%) positives for Shiga toxin-producing E. coli. The stx2 gene was detected in 53% of cases, followed by stx1 (31%) and eae (16%. The STEC isolates were highly sensitive to ciprofloxacin (94.7%) and kanamycin (89.5%), while exhibiting significant resistance to amoxicillin (89.5%) and streptomycin (73.7%). The present study points out the occurrence of virulent and antibiotic-resistant Shiga toxin-producing E. coli strains in raw milk that could pose a potential risk to public health. Further analysis by whole genome sequencing is necessary for an in-depth assessment and understanding of their virulence and resistance factors. Moreover, large-scale studies are needed to identify the prevalence and potential risk factors and to prevent the spread of antibiotic-resistant STEC strains in the milk production chain.

Thrombotic microangiopathies (TMAs) have in common a terminal phenotype of microangiopathic hemolytic anemia with end-organ dysfunction. Thrombotic thrombocytopenic purpura results from von Willebrand factor multimerization, Shiga toxin-mediated hemolytic uremic syndrome causes toxin-induced endothelial dysfunction, while atypical hemolytic uremic syndrome results from complement system dysregulation. Drug-induced TMA, rheumatological disease-induced TMA, and renal-limited TMA exist in an intermediate space that represents secondary complement activation and may overlap with atypical hemolytic uremic syndrome clinically. The existence of TMA without microangiopathic hemolytic features, renal-limited TMA, represents an undiscovered syndrome that responds incompletely and inconsistently to complement blockade. Hematopoietic stem cell transplant-TMA represents another more resistant form of TMA with different therapeutic needs and clinical course. It has become apparent that TMA syndromes are an emerging field in nephrology, rheumatology, and hematology. Much work remains in genetics, molecular biology, and therapeutics to unravel the puzzle of the relationships and distinctions apparent between the different subclasses of TMA syndromes.

Hemolytic uremic syndrome (HUS) often causes neurologic symptoms, but they typically occur as a later symptom of the syndrome. In addition, the Shiga toxinproducing E. coli (STEC) which causes HUS rarely causes bacteremia. We present the case of a 10-year-old male with Smith-Magenis syndrome who was admitted to the hospital due to STEC gastroenteritis, who was initially improving with supportive care, and then subsequently developed fever and had multiple seizures which were different from his typical seizure semiology. Over the subsequent 48 hours he gradually developed microangiopathic hemolytic anemia consistent with HUS. His course was further complicated by E. coli bacteremia and oliguric renal failure requiring renal replacement therapy, depressed mental status, and difficult-to-control hypertension. This case demonstrates the importance of neurologic manifestations as a harbinger of developing HUS. [ABSTRACT FROM AUTHOR]

Typical hemolytic uremic syndrome (HUS) can occur as a severe systemic complication of infections with Shiga toxin (Stx)-producing Escherichia coli. Its pathology can be induced by Stx types, resulting in toxin-mediated damage to renal barriers, inflammation, and the development of acute kidney injury (AKI). Two sphingosine kinase (SphK) isozymes, SphK1 and SphK2, have been shown to be involved in barrier maintenance and renal inflammatory diseases. Therefore, we sought to determine their role in the pathogenesis of HUS. Experimental HUS was induced by the repeated administration of Stx2 in wild-type (WT) and SphK1 (SphK1−/−) or SphK2 (SphK2−/−) null mutant mice. Disease severity was evaluated by assessing clinical symptoms, renal injury and dysfunction, inflammatory status and sphingolipid levels on day 5 of HUS development. Renal inflammation and injury were found to be attenuated in the SphK2−/− mice, but exacerbated in the SphK1−/− mice compared to the WT mice. The divergent outcome appeared to be associated with oppositely altered sphingolipid levels. This study represents the first description of the distinct roles of SphK1−/− and SphK2−/− in the pathogenesis of HUS. The identification of sphingolipid metabolism as a potential target for HUS therapy represents a significant advance in the field of HUS research.

A growing body of evidence has underscored the role of horizontal gene transfer (HGT) in animal evolution. Previously, we discovered the horizontal transfer of the gene encoding the eukaryotic genotoxin cytolethal distending toxin B (cdtB) from the pea aphid Acyrthosiphon pisum secondary endosymbiont (APSE) phages to drosophilid and aphid nuclear genomes. Here, we report cdtB in the nuclear genome of the gall-forming "swede midge" Contarinia nasturtii (Diptera: Cecidomyiidae) via HGT. We searched all available gall midge genome sequences for evidence of APSE-to-insect HGT events and found five toxin genes (aip56, cdtB, lysozyme, rhs, and sltxB) transferred horizontally to cecidomyiid nuclear genomes. Surprisingly, phylogenetic analyses of HGT candidates indicated APSE phages were often not the ancestral donor lineage of the toxin gene to cecidomyiids. We used a phylogenetic signal statistic to test a transfer-by-proximity hypothesis for animal HGT, which suggested that microbe-to-insect HGT was more likely between taxa that share environments than those from different environments. Many of the toxins we found in midge genomes target eukaryotic cells, and catalytic residues important for toxin function are conserved in insect copies. This class of horizontally transferred, eukaryotic cell-targeting genes is potentially important in insect adaptation.

A growing body of evidence has underscored the role of horizontal gene transfer (HGT) in animal evolution. Previously, we discovered the horizontal transfer of the gene encoding the eukaryotic genotoxin cytolethal distending toxin B (cdtB) from the pea aphid Acyrthosiphon pisum secondary endosymbiont (APSE) phages to drosophilid and aphid nuclear genomes. Here, we report cdtB in the nuclear genome of the gall-forming "swede midge" Contarinia nasturtii (Diptera: Cecidomyiidae) via HGT. We searched all available gall midge genome sequences for evidence of APSE-to-insect HGT events and found five toxin genes (aip56, cdtB, lysozyme, rhs, and sltxB) transferred horizontally to cecidomyiid nuclear genomes. Surprisingly, phylogenetic analyses of HGT candidates indicated APSE phages were often not the ancestral donor lineage of the toxin gene to cecidomyiids. We used a phylogenetic signal statistic to test a transfer-by-proximity hypothesis for animal HGT, which suggested that microbe-to-insect HGT was more likely between taxa that share environments than those from different environments. Many of the toxins we found in midge genomes target eukaryotic cells, and catalytic residues important for toxin function are conserved in insect copies. This class of horizontally transferred, eukaryotic cell-targeting genes is potentially important in insect adaptation.

Shiga toxin-producing E. coli (STEC) are diarrheagenic strains that can cause bloody diarrhea and hemolytic-uremic syndrome. Their main virulence factor, the Shiga toxin (Stx), is encoded by phages integrated into the bacterial chromosome. Stx phages are widely diverse and carry many genes with limited or unknown function. As the toxin subtype Stx2a is associated with highly pathogenic strains, this study was mainly focused on the characterization of the stx flanking region of Stx2a phages. Of particular interest was a sialate O-acetylesterase (NanS-p), which has been described previously to be encoded downstream stx in some phage genomes and may confer a growth advantage for STEC. Complete DNA sequences of Stx2a phages and prophages were retrieved from the GenBank database, and the genomic regions from anti-terminator Q to holin S genes were bioinformatically analyzed. Predicted NanSp sequences from phages encoding other Stx subtypes were also studied. Additionally, expression of nanS-p was quantified by qPCR in strains selected from our laboratory collection. The analysis of Stx2a phage genomes showed that all carried the Q, stx2a, nanS-p and S genes, but with allele diversity and other sequence differences. In particular, sequence differences were detected in each of the three domains of NanS-p esterases encoded by Stx2a phages and other Stx phages; however, nanS-p was not identified in the Stx2e, Stx2f and Stx2g phages analyzed. The expression of nanS-p increased in most stx2a-positive strains under phage inducing conditions, as was previously shown for stx2a. As the present work showed diversity at the Q-S region among Stx phages, and particularly in the encoded NanS-p enzyme, future studies will be necessary to evaluate if NanS-p variants differ in their activity and to assess the impact of the absence of nanS-p in certain Stx phages.

ABSTRACT: Objectives: Shiga toxin–producing Escherichia coli (STEC) are globally significant foodborne pathogens. Dairy calves are a known reservoir of both O157 and non-O157 STEC. The objective of this study was to comprehensively evaluate the genomic attributes, diversity, virulence factors, and antimicrobial resistance gene (ARG) profiles of the STEC from preweaned and postweaned dairy calves in commercial dairy herds. Methods: In total, 31 non-O157 STEC were identified as part of a larger study focused on the pangenome of >1000 E. coli isolates from the faeces of preweaned and postweaned dairy calves on commercial dairy farms. These 31 genomes were sequenced on an Illumina NextSeq500 platform. Results: Based on the phylogenetic analyses, the STEC isolates were determined to be polyphyletic, with at least three phylogroups: A (32%), B1 (58%), and G (3%). These phylogroups represented at least 16 sequence types and 11 serogroups, including two of the ‘big six’ serogroups, O103 and O111. Several Shiga toxin gene subtypes were identified in the genomes, including stx1a, stx2a, stx2c, stx2d, and stx2g. Using the ResFinder database, the majority of the isolates (>50%) were determined to be multidrug-resistant strains because they harboured genes conferring resistance to three or more classes of antimicrobials, including some of human health significance (e.g., β-lactams, macrolides, and fosfomycin). Additionally, non-O157 STEC strain persistence and transmission within a farm was observed. Conclusion: Dairy calves are a reservoir of phylogenomically diverse multidrug-resistant non-O157 STEC. Information from this study may inform assessments of public health risk and guide preharvest prevention strategies focusing on STEC reservoirs.

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) is a group of pathogenic bacteria responsible for several foodborne-associated outbreaks of human diarrheal disease. Although EHEC is a major cause of morbidity as well as the condition known as hemolytic uremic syndrome, linked to the production of Shiga toxins (Stx), there are no licensed vaccines approved for human use. Fully assessing vaccine efficacy against EHEC infections is challenging because conventional adult mice are inherently resistant to EHEC infection and do not develop hallmark symptoms of the disease. Therefore, in this study, we utilized a strain of the murine pathogen Citrobacter rodentium that has been lysogenized to produce Stx2d, which consistently causes lethal infection and other measurable disease outcomes following oral challenge, to further evaluate the protective efficacy of our previously formulated EHEC gold nanoparticle (AuNP) vaccines. Using this model, along with a non-Stx2d-producing C. rodentium strain, we assessed the protection conferred by AuNPs conjugated to three EHEC antigens: EscC, LomW, and intimin (Eae). We demonstrated that intranasally immunizing mice with AuNP-Eae provides partial protection against mortality caused by Stx2d-producing C. rodentium and that AuNP-EscC and AuNP-Eae moderately reduce intestinal burden of the non-Stx2d-producing C. rodentium strain compared with adjuvant-only-treated mice. Additionally, the AuNP-protein vaccines induced antigen- and pathogen-specific serum IgG and fecal IgA against both EHEC and C. rodentium while in vitro functional assays indicate that the elicited antibodies are bactericidal and prevent adherence of C. rodentium to intestinal epithelial cells. We propose that C. rodentium murine data could translate into correlates of protection. IMPORTANCE Enterohemorrhagic Escherichia coli (EHEC) remains an important cause of diarrheal disease and complications worldwide, especially in children, yet there are no available vaccines for human use. Inadequate pre-clinical evaluation due to inconsistent animal models remains a major barrier to novel vaccine development. We demonstrate the usefulness of Stx2d-producing Citrobacter rodentium in assessing vaccine effectiveness because it more closely recapitulates human disease caused by EHEC.

AB5-type toxins are a group of secreted protein toxins that are central virulence factors for bacterial pathogens such as Shigella dysenteriae, Vibrio cholerae, Bordetella pertussis, and certain lineages of pathogenic Escherichia coli and Salmonella enterica. AB5 toxins are composed of an active (A) subunit that manipulates host cell biology in complex with a pentameric binding/delivery (B) subunit that mediates the toxin's entry into host cells and its subsequent intracellular trafficking. Broadly speaking, all known AB5-type toxins adopt similar structural architectures and employ similar mechanisms of binding, entering and trafficking within host cells. Despite this, there is a remarkable amount of diversity amongst AB5-type toxins; this includes different toxin families with unrelated activities, as well as variation within families that can have profound functional consequences. In this review, we discuss the diversity that exists amongst characterized AB5-type toxins, with an emphasis on the genetic and functional variability within AB5 toxin families, how this may have evolved, and its impact on human disease.

Enterohemorrhagic Escherichia coli (EHEC) are important pathogenic E. coli characterized by the production of Shiga toxin, intimin, and enterohemolysin, which are important virulence factors for pathogenesis. These virulence factors are the cause of enterocyte extinction and affect both intestinal cells and various extraintestinal cells such as lymphocytes, granulocytes, erythrocytes, and renal cells. The resultant severe bloody diarrhea and colonic infections are associated with life-threatening extraintestinal complications, leading to high morbidity and mortality worldwide. Although EHEC has been responsible for large outbreaks around the world, research into effective therapies to prevent and treat the infection in a timely manner and affordable diagnostic tests remains a challenge due to the complicated nature of the bacteria. This review addresses previous studies, the current status, and prospects regarding the severity, treatment, and diagnosis of EHEC with a focus on a new finding on the use of simple point of care diagnostic procedures.

Shiga toxin-producing Escherichia coli (STEC) is a foodborne zoonotic pathogen that causes diarrhea, hemorrhagic colitis (HC), and hemolytic uremic syndrome (HUS) worldwide. Since the infection can be asymptomatic, the circulation of STEC in some asymptomatic carriers, especially in healthy-food-related professionals, is not yet well understood. In this study, a total of 3987 anal swab samples from asymptomatic food handlers were collected, and ten swabs recovered STEC strains (0.251%). Of the ten STEC isolates, seven serotypes and eight sequence types (ST) were determined using whole genome sequencing (WGS). Two stx1 subtypes (stx1a and stx1c) and four stx2 subtypes (stx2a, stx2b, stx2d, and stx2e) were detected. Seven different insertion sites were found in fourteen Stx prophages, and the dmsB and yfhL were the newly identified insertion sites. The ten strains showed the variable Stx transcription levels after the mitomycin C induction. The whole-genome phylogeny indicated that the strains from the asymptomatic food handlers were genetically distant from the strains of HUS patients. The STEC isolates circulating in asymptomatic carriers might pose a low potential to cause disease. [ABSTRACT FROM AUTHOR]

Escherichia albertii is an emerging foodborne pathogen. To better understand the pathogenesis and health risk of this pathogen, comparative genomics and phenotypic characterization were applied to assess the pathogenicity potential of E. albertii strains isolated from wild birds in a major agricultural region in California. Shiga toxin genes stx2f were present in all avian strains. Pangenome analyses of 20 complete genomes revealed a total of 11,249 genes, of which nearly 80% were accessory genes. Both core gene-based phylogenetic and accessory gene-based relatedness analyses consistently grouped the three stx2f-positive clinical strains with the five avian strains carrying ST7971. Among the three Stx2f-converting prophage integration sites identified, ssrA was the most common one. Besides the locus of enterocyte effacement and type three secretion system, the high pathogenicity island, OI-122, and type six secretion systems were identified. Substantial strain variation in virulence gene repertoire, Shiga toxin production, and cytotoxicity were revealed. Six avian strains exhibited significantly higher cytotoxicity than that of stx2f-positive E. coli, and three of them exhibited a comparable level of cytotoxicity with that of enterohemorrhagic E. coli outbreak strains, suggesting that wild birds could serve as a reservoir of E. albertii strains with great potential to cause severe diseases in humans. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is considered as one of the leading causes of food-intoxication and on the other hand E. coli and particularly, Shiga toxin (ST) producing Escherichia coli (STEC) have emerged as important food-borne enteropathogens frequently associating serious to fatal disorders in humans and both species have shown to be resistant to a wide spectrum of antibiotics. The current study was performed to determine the prevalence of two common food-borne pathogens: Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli ) in white soft cheese being one of the most popular dairy products in Egypt. A total 150 samples of soft cheese were purchased from the market and were microbiologically tested. The S. aureus isolates were identified according to recommended biochemical tests, and polymerase chain reaction (PCR) targeting the nuc gene, as a species-specific marker gene for S. aureus. Meanwhile, E. coli isolates were identified based on biochemical tests, serological tests, haemolytic activity, and multiplex PCR. This has been done to assess capability of producing STs based on targeting stx1 and stx2 genes. Result revealed that S. aureus and E. coli were observed in 66.66% and 36% of samples, respectively. The antimicrobial resistance of isolates was phenotypically investigated against eleven different antibiotics and results showed the presence of a variable multidrug-resistance among isolates against selected antibiotics. These findings highlighted the importance of periodical screening of food-borne pathogens in artisanal products due to lack of strict hygienic measures in markets; the reason that facilitate the contamination of such artisanal products by several food-borne pathogens. [ABSTRACT FROM AUTHOR]

Introduction: Hemolytic-uremic syndrome (HUS) can occur as a systemic complication of infection with Shiga toxin (Stx)-producing Escherichia coli (STEC). Most well-known aspects of the pathophysiology are secondary to microthrombotic kidney disease including hemolytic anemia and thrombocytopenia. However, extrarenal manifestations, such as cardiac impairment, have also been reported. We have investigated whether these cardiac abnormalities can be reproduced in a murine animal model, in which administration of Stx, the main virulence factor of STEC, is used to induce HUS. Methods: Mice received either one high or multiple low doses of Stx to simulate the (clinically well-known) different disease courses. Cardiac function was evaluated by echocardiography and analyses of biomarkers in the plasma (troponin I and brain natriuretic peptide). Results: All Stx-challenged mice showed reduced cardiac output and depletion of intravascular volume indicated by a reduced end-diastolic volume and a higher hematocrit. Some mice exhibited myocardial injury (measured as increases in cTNI levels). A subset of mice challenged with either dosage regimen showed hyperkalemia with typical electrocardiographic abnormalities. Discussion: Myocardial injury, intravascular volume depletion, reduced cardiac output, and arrhythmias as a consequence of hyperkalemia may be prognosisrelevant disease manifestations of HUS, the significance of which should be further investigated in future preclinical and clinical studies. [ABSTRACT FROM AUTHOR]

Abstract Background Within the genus Escherichia, several monophyletic clades other than the traditionally defined species have been identified. Of these, cryptic clade I (C-I) appears to represent a subspecies of E. coli, but due to the difficulty in distinguishing it from E. coli sensu stricto, the population structure and virulence potential of C-I are unclear. Results We defined a set of true C-I strains (n = 465), including a Shiga toxin 2a (Stx2a)-producing isolate from a patient with bloody diarrhoea identified by the retrospective analyses using a C-I-specific detection system. Through genomic analysis of 804 isolates from the cryptic clades, including these C-I strains, we revealed their global population structures and the marked accumulation of virulence genes and antimicrobial resistance genes in C-I. In particular, half of the C-I strains contained hallmark virulence genes of Stx-producing E. coli (STEC) and/or enterotoxigenic E. coli (ETEC). We also found the host-specific distributions of virulence genes, which suggests bovines as the potential source of human infections caused by STEC- and STEC/ETEC hybrid-type C-I strains, as is known in STEC. Conclusions Our findings demonstrate the emergence of human intestinal pathogens in C-I lineage. To better understand the features of C-I strains and their infections, extensive surveillance and larger population studies of C-I strains are needed. The C-I-specific detection system developed in this study will be a powerful tool for screening and identifying C-I strains.

Enterohemorrhagic E. coli (EHEC) is a group of pathogenic bacteria that is associated with worldwide human foodborne diarrheal illnesses and the development of hemolytic uremic syndrome, a potentially deadly condition associated with Shiga toxins (Stxs). Currently, approved vaccines for human prophylaxis against infection do not exist, and one barrier preventing the successful creation of EHEC vaccines is the absence of dependable animal models, including mice, which are naturally resistant to EHEC infection and do not manifest the characteristic signs of the illness. Our lab previously developed gold nanoparticle (AuNP)-based EHEC vaccines, and assessed their efficacy using Citrobacter rodentium, which is the mouse pathogen counterpart of EHEC, along with an Stx2d-producing strain that leads to more consistent disease kinetics in mice, including lethality. The purpose of this study was to continue evaluating these vaccines to increase protection. Here, we demonstrated that subcutaneous immunization of mice with AuNPs linked to the EHEC antigens EscC and intimin (Eae), either alone or simultaneously, elicits functional robust systemic humoral responses. Additionally, vaccination with both antigens together showed some efficacy against Stx2d-producing C. rodentium while AuNP-EscC successfully limited infection with non-Stx2d-producing C. rodentium. Overall, the collected results indicate that our AuNP vaccines have promising potential for preventing disease with EHEC, and that evaluation of novel vaccines using an appropriate animal model, like C. rodentium described here, could be the key to finally developing an effective EHEC vaccine that can progress into human clinical trials.

Shiga toxin (Stx), the main virulence factor of Shiga-toxin-producing E. coli (STEC), was first discovered in Shigella dysenteriae strains. While several other bacterial species have since been reported to produce Stx, STEC poses the most significant risk to human health due to its widespread prevalence across various animal hosts that have close contact with human populations. Based on its biochemical and molecular characteristics, Shiga toxin can be grouped into two types, Stx1 and Stx2, among which a variety of variants and subtypes have been identified in various bacteria and host species. Interestingly, the different Stx subtypes appear to vary in their host distribution characteristics and in the severity of diseases that they are associated with. As such, this review provides a comprehensive overview on the bacterial species that have been recorded to possess stx genes to date, with a specific focus on the various Stx subtype variants discovered in STEC, their prevalence in certain host species, and their disease-related characteristics. This review provides a better understanding of the Stx subtypes and highlights the need for rapid and accurate approaches to toxin subtyping for the proper evaluation of the health risks associated with Shiga-toxin-related bacterial food contamination and human infections.

We report fatal meningitis in 2 neonates in France caused by Shiga toxin 1–producing Escherichia coli. Virulence factors capsular K1 antigen and salmochelin were present in both strains, potentially representing a new hybrid pathotype. Clinicians should remain aware of emerging pathotypes and design therapeutic strategies for neonatal E. coli infections.

IntroductionHemolytic–uremic syndrome (HUS) can occur as a systemic complication of infection with Shiga toxin (Stx)-producing Escherichia coli (STEC). Most well-known aspects of the pathophysiology are secondary to microthrombotic kidney disease including hemolytic anemia and thrombocytopenia. However, extrarenal manifestations, such as cardiac impairment, have also been reported. We have investigated whether these cardiac abnormalities can be reproduced in a murine animal model, in which administration of Stx, the main virulence factor of STEC, is used to induce HUS.MethodsMice received either one high or multiple low doses of Stx to simulate the (clinically well-known) different disease courses. Cardiac function was evaluated by echocardiography and analyses of biomarkers in the plasma (troponin I and brain natriuretic peptide).ResultsAll Stx-challenged mice showed reduced cardiac output and depletion of intravascular volume indicated by a reduced end-diastolic volume and a higher hematocrit. Some mice exhibited myocardial injury (measured as increases in cTNI levels). A subset of mice challenged with either dosage regimen showed hyperkalemia with typical electrocardiographic abnormalities.DiscussionMyocardial injury, intravascular volume depletion, reduced cardiac output, and arrhythmias as a consequence of hyperkalemia may be prognosis-relevant disease manifestations of HUS, the significance of which should be further investigated in future preclinical and clinical studies.

Shiga toxin type 2 (Stx2) is the primary virulence factor produced by Shiga toxin-producing enterohemorrhagic Escherichia coli (STEC), which causes epidemic outbreaks of gastrointestinal sickness and potentially fatal sequela hemolytic uremic syndrome (HUS). Most studies on Stx2-induced apoptosis have been performed with holotoxins, but the mechanism of how the A and B subunits of Stx2 cause apoptosis in cells is not clear. Here, we found that Stx2 A-subunit (Stx2A) induced mitochondrial damage, PINK1/Parkin-dependent mitophagy and apoptosis in Caco-2 cells. PINK1/Parkin-dependent mitophagy caused by Stx2A reduced apoptosis by decreasing the accumulation of reactive oxidative species (ROS). Mechanistically, Stx2A interacts with Tom20 on mitochondria to initiate the translocation of Bax to mitochondria, leading to mitochondrial damage and apoptosis. Overall, these data suggested that Stx2A induces mitochondrial damage, mitophagy and apoptosis via the interaction of Tom20 in Caco-2 cells and that mitophagy caused by Stx2A ameliorates apoptosis by eliminating damaged mitochondria. These findings provide evidence for the potential use of Tom20 inhibition as an anti-Shiga toxin therapy.

Seven serogroups of E. coli (Top seven E. coli) are frequently implicated in foodborne outbreaks in North America, largely due to their carriage of Shiga toxin genes (stx). This study aimed to profile resistance genes and virulence factors (VF), and their potential association with phylogeny and phenotypes of Top seven E. coli originating from cattle in Canada. 155 Top seven E. coli isolates previously characterized for heat and acid resistance and biofilm-forming ability were whole-genome sequenced and analyzed for phylogeny, VF, and stress resistance genes. The 155 E. coli strains belonged to six phylogroups: A (n = 32), B1 (n = 93), C (n = 3), D (n = 11), E (n = 15), and G (n = 1). Different phylogroups were clearly separated on the core genome tree, with strains of the same serotype closely clustered. The carriage of stx and the transmissible locus of stress tolerance (tLST), the extreme heat resistance marker, was mutually exclusive, in 33 and 15 genomes, respectively. A novel O84:H2 strain carrying stx1a was also identified. In total, 70, 41, and 32 VF, stress resistance genes and antibiotic resistance genes were identified. The stress resistance genes included those for metal (n = 29), biocides/acid (n = 4), and heat (n = 8) resistance. All heat resistance genes and most metal-resistance genes that were differentially distributed among the phylogroups were exclusively in phylogroup A. VF were least and most present in phylogroups A and D, respectively. No specific genes associated with acid resistance or biofilm formation phenotypes were identified. VF were more abundant (P