Eculizumab for Shiga-toxin-induced hemolytic uremic syndrome in adults with neurological involvement.

The role of eculizumab in treating Shiga-toxin-producing Escherichia coli (STEC) hemolytic uremic syndrome (HUS) patients with neurological involvement remains unclear. We describe two distinctly different STEC-HUS patients with neurologic involvement successfully managed with eculizumab, and perform a literature review of all published cases. Both patients had complete resolution of neurological symptoms after initiation of eculizumab. Eighty patients with STEC-HUS treated with eculizumab were identified in the literature, 68.7% had complete resolution of neurological symptoms. Based on our experience and literature review, three prevailing themes were noted: 1) Early eculizumab administration optimized neurological outcomes, 2) Symptom resolution may not be immediate, neurological symptoms may initially worsen before improvement, and 3) Plasma exchange yielded no benefit. Early administration of eculizumab may reverse neurotoxicity in patients with STEC-HUS.

Degradation of Toxins Derived from Foodborne Pathogens by Atmospheric-Pressure Dielectric-Barrier Discharge.

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.

Lung Ultrasound to Evaluate Fluid Status and Optimize Early Volume-Expansion Therapy in Children with Shiga Toxin-Producing Escherichia Coli-Haemolytic Uremic Syndrome: A Pilot Study.

Background: Shiga toxin-producing Escherichia coli-haemolytic uremic syndrome (STEC-HUS) can result in kidney and neurological complications. Early volume-expansion therapy has been shown to improve outcomes, but caution is required to avoid fluid overload. Lung ultrasound scanning (LUS) can be used to detect fluid overload and may be useful in monitoring hydration therapy. Methods: This prospective observational pilot study involved children with STEC-HUS who were recruited from a regional paediatric nephrology centre. B-line quantification by LUS was used to assess fluid status at the emergency department (ED) admission and correlated with the decrease in patient weight from the target weight. A control group of children on chronic dialysis therapy with episodes of symptomatic fluid overload was also enrolled in order to establish a B-line threshold indicative of severe lung congestion. Another cohort of "healthy" children, without renal or lung-related diseases, and without clinical signs of fluid overload was also enrolled in order to establish a B-line threshold indicative of euvolemia. Results: LUS assessment was performed in 10 children with STEC-HUS at ED admission, showing an average of three B-lines (range 0-10). LUS was also performed in 53 euvolemic children admitted to the ED not showing kidney and lung disease (healthy controls), showing a median value of two B-lines (range 0-7), not significantly different from children with STEC-HUS at admission (p = 0.92). Children with STEC-HUS with neurological involvement during the acute phase and those requiring dialysis presented a significantly lower number of B-lines at admission compared to patients with a good clinical course (p < 0.001). Patients with long-term renal impairment also presented a lower number of B-lines at disease onset (p = 0.03). Conclusions: LUS is a useful technique for monitoring intravenous hydration therapy in paediatric patients with STEC-HUS. A low number of B-lines at ED admission (<5 B-lines) was associated with worse short-term and long-term outcomes. Further studies are needed to determine the efficacy and safety of an LUS-guided strategy for reducing complications in children with STEC-HUS.

A fluorescence anisotropy-based competition assay to identify inhibitors against ricin and Shiga toxin ribosome interactions.

Ricin is one of the most toxic substances known and a type B biothreat agent. Shiga toxins (Stxs) produced by E. coli (STEC) and Shigella dysenteriae are foodborne pathogens. There is no effective therapy against ricin or STEC and there is an urgent need for inhibitors. Ricin toxin A subunit (RTA) and A1 subunit of Stx2a (Stx2A1) bind to the C-terminal domain (CTD) of the ribosomal P-stalk proteins to depurinate the sarcin/ricin loop. Modulation of toxin-ribosome interactions has not been explored as a strategy for inhibition. Therefore, development of assays that detect inhibitors targeting toxin-ribosome interactions remains a critical need. Here we describe a fluorescence anisotropy (FA)-based competitive binding assay using a BODIPY-TMR labeled 11-mer peptide (P11) derived from the P-stalk CTD to measure the binding affinity of peptides ranging from 3 to 11 amino acids for the P-stalk pocket of RTA and Stx2A1. Comparison of the affinity with the surface plasmon resonance (SPR) assay indicated that although the rank order was the same by both methods, the FA assay could differentiate better between peptides that show nonspecific interactions by SPR. The FA assay detects only interactions that compete with the labeled P11 and can validate inhibitor specificity and mechanism of action.

One problem, multiple potential targets: Where are we now in the development of small molecule inhibitors against Shiga toxin?

Shiga toxin-producing Escherichia coli (STEC) are a group of enteric pathogens which carry phage-encoded Shiga toxins (Stx). STEC infections begin with severe abdominal pain and non-bloody diarrhoea, which can progress to bloody diarrhoea after approximately 4-days post-infection. In high-risk groups such as children and the elderly, patients may develop haemolytic uremic syndrome (HUS). HUS is characterised by microangiopathic haemolytic anaemia, thrombocytopenia, and in severe disease acute renal failure. Traditional antibiotics have been linked with increased toxin production due to the activation of recA-mediated bacterial stress response, resulting in poorer patient outcomes. Therefore, treatment relies on supportive therapies. Antivirulence strategies have been explored as an alternative treatment for bacterial infections and blockers of virulence factors such as the Type III Secretion System. Recent improvements in the mechanistic understanding of the Stx pathway have led to the design of inhibitors to disrupt the pathway, leading to toxin-mediated ribosome damage. However, compounds have yet to progress beyond Phase III clinical trials successfully. This review explores the progress in developing small molecule inhibitors by collating lead compounds derived from in-silico and experimental approaches.

Enterohemorrhagic Escherichia coli: trigger for small vessel vasculitis.

We describe the case of a 19-year-old woman who presented with abdominal pain, vomiting, and a palpable purpuric rash. The patient subsequently developed dysentery and was found to have an infection from Shiga toxin-producing Escherichia coli. The patient also met diagnostic criteria for IgA vasculitis (also known as Henoch Schönlein purpura) but had negative immunofluorescence biopsies of the rash. The patient was treated with steroids and achieved recovery. To our knowledge, this is the first documented case of IgA vasculitis in the setting of an enterohemorrhagic E. coli infection. This case highlights an atypical presentation of IgA vasculitis and the need to include small vessel vasculitis as a differential diagnosis when treating patients of all ages.

Shiga toxin down-regulates ERG protein in endothelial cells and impairs angiogenesis.

BACKGROUND: Shiga toxin (Stx) can activate inflammatory signaling, leading to vascular dysfunction and promotion of a pro-thrombotic tissue microenvironment. Stx can trigger the development of the enterohemorrhagic (childhood) hemolytic uremic syndrome (eHUS), a triad of thrombocytopenia, hemolytic anemia, and acute kidney injury, often requiring dialysis. Additional features may include damage to other organs, including the gastrointestinal tract, pancreas, brain and cardiovascular system; death occurs in 2-5 %. eHUS is a thrombotic microangiopathy; thus, endothelial cell (EC) injury and platelet fibrin thrombus formation in glomerular arterioles and in the arterioles of other affected organs are likely. To elucidate mechanisms of this microangiopathy, we examined in human ECs the regulation of the platelet adhesion proteins P-selectin and von Willebrand factor (VWF), along with the downregulation of erythroblast-transformation-specific transcription factor (ERG) a key regulator of angiogenesis and megakaryocyte development. METHODS: VWF, P-selectin, and ERG levels were determined using immunofluorescence and Western blot in human umbilical endothelial cells (HUVECs). HUVECs were treated with tumor necrosis factor-alpha (TNF-α), Stx-1 or both, versus normal controls. Capillary morphogenesis on Matrigel was performed using HUVECs treated, for 22 h with TNF-α, Stx-1, or both, or treated 4 h with Stx-1 alone or in combination with TNF-α for 22 h. RESULTS: Stx-1 significantly reduced ERG and VWF expression on HUVECs, but upregulated P-selectin expression. ERG levels decreased with Stx-1 alone or in combination with TNF-α, in the nuclear, perinuclear and cytoplasmatic regions. Stx-1 reduced capillary morphogenesis, while Stx-1-TNF-α combined treatment reduced capillary morphogenesis still further. CONCLUSIONS: In the presence of Stx-1 or TNF-α or both treatments, ECs were activated, expressing higher levels of P-selectin and lower levels of VWF. Our findings, further, provide evidence that Stx-1 downregulates ERG, repressing angiogenesis in vitro.

γ-secretase facilitates retromer-mediated retrograde transport.

The retromer complex mediates retrograde transport of protein cargos from endosomes to the trans-Golgi network (TGN). γ-secretase is a multisubunit protease that cleaves the transmembrane domain of its target proteins. Mutations in genes encoding subunits of retromer or γ-secretase can cause familial Alzheimer disease (AD) and other degenerative neurological diseases. It has been reported that retromer interacts with γ-secretase, but the consequences of this interaction are not known. Here, we report that retromer-mediated retrograde protein trafficking in cultured human epithelial cells is impaired by inhibition of γ-secretase activity or by genetic elimination of γ-secretase. γ-secretase inhibitor XXI and knockout of PS1, the catalytic subunit of γ-secretase, inhibit endosome to TGN trafficking of retromer-dependent retrograde cargos, divalent metal transporter 1 isoform II (DMT1-II), cation-independent mannose-6-phosphate receptor (CIMPR), and shiga toxin. Trafficking of retromer-independent cargos, such as cholera toxin and a CIMPR mutant that does not bind to retromer was not affected by γ-secretase inhibition. XXI treatment and PS1 KO inhibit interaction of γ-secretase with retromer but do not inhibit the association of cargo with retromer or with γ-secretase in intact cells. Similarly, these treatments do not affect the level of Rab7-GTP, which regulates retromer-cargo interaction. These results suggest that the γ-secretase-retromer interaction facilitates retromer-mediated retrograde trafficking.

Characterization of Shiga Toxin-producing Escherichia coli Isolated from Cattle Around Ulaanbaatar City, Mongolia.

Shiga toxin-producing Escherichia coli (STEC) are associated with severe infections including hemorrhagic colitis and hemolytic uremic syndrome in humans. Ruminants are known as reservoirs of STEC; however, no data are available on STEC in ruminants in Mongolia, where more than 5 million cattle and 25 million sheep are raised. To disclose the existence and characteristics of STEC in Mongolia, in this study, we isolated and characterized STEC from cattle in Mongolia. We collected 350 rectal swabs of cattle from 30 farms near Ulaanbaatar city and isolated 45 STEC from 21 farms. Rectal swabs were precultured with modified Escherichia coli broth and then inoculated to Cefixime-Tellurite Sorbitol MacConkey agar plate and/or CHROMagar STEC agar plate for the isolation of STEC. The isolation ratios in each farm were from 0% to 40%. Multiplex PCR for the estimation of O- and H-serotypes identified 12 O-genotypes (Og-types) and 11 H-genotypes (Hg-types) from 45 isolates; however, Og-types of 19 isolates could not be determined. Stx gene subtyping by PCR identified 2 stx1 subtypes (1a and 1c) and 4 stx2 subtypes (2a, 2c, 2d, and 2g). Forty-five isolates were divided into 21 different groups based on the Og- and Hg-types, stx gene subtypes and the existence of virulence factors, ehxA, eae, and saa, which includes several major serotypes associated with human illness such as O26:H11 and O157:H7. The most dominant isolate, OgUT:H19 [stx1a (+), stx2a (+), ehxA (+) and saa (+)], was isolated from eight farms. This is the first report on the characterization of STEC in cattle in Mongolia, and the results suggest the importance of further monitoring of STEC contamination in the food chains as well as STEC infection in humans.

Genomic analysis of an outbreak of Shiga toxin-producing Escherichia coli O183:H18 in the United Kingdom, 2023.

In June 2023, UKHSA surveillance systems detected an outbreak of severe gastrointestinal symptoms caused by a rare serotype of Shiga toxin-producing Escherichia coli, STEC O183:H18. There were 26 cases aged 6 months to 74 years (42 % cases were aged 0-9 years), distributed across the UK with onset dates range between 22 May 2023 and 4 July 2023. The epidemiological and food chain investigations were inconclusive, although meat products made from beef mince were implicated as a potential vehicle. The outbreak strain belonged to sequence type (ST) 657 and harboured a Shiga toxin (stx) subtype stx2a located on a prophage that was unique in the UKHSA stx-encoding bacteriophage database. Plasmid encoded, putative virulence genes subA, ehxA, saa, iha, lpfA and iss were detected, however, the established STEC virulence genes involved in attachment to the gut mucosa (eae and aggR) were absent. The acquisition of stx across the global population structure of ST657 appeared to correspond with the presence of subA, ehxA, saa, iha, lpfA and iss. During the outbreak investigation, we used long read sequencing to characterise the plasmid and prophage content of this atypical STEC, to look for evidence to explain its recent emergence. Although we were unable to determine source and transmission route of the outbreak strain, the genomic analysis revealed potential clues as to how novel strains for STEC evolve. With the implementation of PCR capable of detecting all STEC, and genome sequencing for typing and virulence profiling, we have the tools to enable us to monitor the changing landscape of STEC. Improvements in the standardised collection of epidemiological data and trace-back strategies within the food industry, will ensure we have a surveillance system capable of alerting us to emerging threats to public health.

Increased resistance against tellurite is conferred by a mutation in the promoter region of uncommon tellurite resistance gene tehB in the ter-negative Shiga toxin-producing Escherichia coli O157:H7.

Resistance to potassium tellurite (PT) is an important indicator in isolating Shiga toxin-producing Escherichia coli (STEC) O157:H7 and other major STEC serogroups. Common resistance determinant genes are encoded in the ter gene cluster. We found an O157:H7 isolate that does not harbor ter but is resistant to PT. One nonsynonymous mutation was found in another PT resistance gene, tehA, through whole-genome sequence analyses. To elucidate the contribution of this mutation to PT resistance, complementation of tehA and the related gene tehB in isogenic strains and quantitative RT‒PCR were performed. The results indicated that the point mutation not only changed an amino acid of tehA, but also was positioned on a putative internal promoter of tehB and increased PT resistance by elevating tehB mRNA expression. Meanwhile, the amino acid change in tehA had negligible impact on the PT resistance. Comprehensive screening revealed that 2.3% of O157:H7 isolates in Japan did not harbor the ter gene cluster, but the same mutation in tehA was not found. These results suggested that PT resistance in E. coli can be enhanced through one mutational event even in ter-negative strains. IMPORTANCE: Selective agents are important for isolating Shiga toxin-producing Escherichia coli (STEC) because the undesirable growth of microflora should be inhibited. Potassium tellurite (PT) is a common selective agent for major STEC serotypes. In this study, we found a novel variant of PT resistance genes, tehAB, in STEC O157:H7. Molecular experiments clearly showed that one point mutation in a predicted internal promoter region of tehB upregulated the expression of the gene and consequently led to increased resistance to PT. Because tehAB genes are ubiquitous across E. coli, these results provide universal insight into PT resistance in this species.

Epidemiological Characteristics of Shiga Toxin-Producing Escherichia coli Responsible for Infections in the Polish Pediatric Population.

Shiga toxin-producing Escherichia coli (STEC) are zoonotic pathogens causing hemorrhagic colitis and hemolytic uremic syndrome (HUS) in children and the elderly. Stool samples were collected from 180 children hospitalized in five pediatric centers in Poland in 2018-2022. Direct stx1/stx2 gene detection by PCR in feces and E. coli isolates was performed. Antibiotic susceptibility was tested according to EUCAST v.12. Randomly selected isolates were serotyped with O157 antiserum and genotyped by pulsed-field gel electrophoresis (PFGE). A total of 44 E. coli isolates were confirmed as STEC by PCR. Among them, 84.4% were positive for stx2, and equally 6,8% for only stx1 and both stx1 and stx2 genes. The stx1 gene was also found in one Citrobacter freundii isolate. E. coli serotype O157 was present in 97.6% of the isolates. STEC infections most often occurred between June-October with a peak in July and August (51%). The highest, 77.8% of STEC isolates were found in the 1-5 years old group. No extended-spectrum ß-lactamases (ESBL) were found. Resistance only to amoxicillin/clavulanic acid (24.4%), piperacillin/tazobactam (3%), cefotaxime (6%), gentamicin (6%), ciprofloxacin (3%), azithromycin (3%), trimethoprim/sulfamethoxazole (24,2%) was detected. PFGE analysis showed 18 PFGE types with no clonal distribution. Eight isolates with A, B, and C PFGE types showed genetic relatedness in the type with no detection of transmission way of distribution. STEC strains pose a serious threat to human health, therefore demographic and epidemiological characteristics are crucial for their surveillance.

A challenging STEC strain isolation from patients' stools: an O166:H15 STEC strain with the stx2 gene.

Two patients with acute gastroenteritis tested positive for Shiga toxin-producing Escherichia coli (STEC) by polymerase chain reaction (PCR), and both strains carried the Shiga toxin 2 encoding gene. Since routine culture using CHROMagar STEC failed to recover these isolates, immunomagnetic separation (IMS) targeting the top six non-O157:H7 serotypes was used for isolate recovery. After two subsequent IMS runs, the STEC strains were isolated from trypticase soy broth with and without overnight enrichment for runs 1 and 2, respectively. Serotyping based on whole-genome sequencing revealed that both patients carried the strain O166:H15 STEC with the stx2 gene. Hence, the magnetic beads used in IMS appeared to have cross-reactivity with other E. coli serotypes. When the STEC isolates from both stools were cultured on CHROMagar STEC and sheep blood agar (BAP), two distinct colony sizes were apparent after overnight incubation. The small and large colonies were picked and separately cultured on both media, and colony growth was observed for 2 weeks at room temperature after an initial overnight incubation at 37°C. After 1 week, the colonies showed concentric ring structures with a darker center and a lighter surrounding on CHROMagar STEC and a "fried egg"-resembling structure with a raised circular center and a flat surrounding on BAP. Both colony types remained morphologically different on CHROMagar STEC throughout the 15 days. However, on BAP, their appearance was comparable by day 7. IMPORTANCE: Shiga toxin-producing E. coli (STEC) infections can lead to severe complications such as bloody diarrhea and hemolytic uremic syndrome (HUS), especially in young children and the elderly. Strains that carry the shiga toxin 2 gene (stx2), such as O157:H7, have been mostly linked with severe disease outcomes. In recent years, outbreaks caused by non-O157:H7 strains have increased. E. coli O166:H15 has been previously reported causing a gastroenteritis outbreak in 1996 as a non-STEC strain, however the O166:H15 serotype we recovered carried the stx2 gene. It was particularly challenging to isolate this strain from stools by culture. Consequently, we tested immunomagnetic separation for the STEC recovery, which was a novel approach on clinical stools. Virulence genes were included for the characterization of these isolates.

First Isolation of the Heteropathotype Shiga Toxin-Producing and Extra-Intestinal Pathogenic (STEC-ExPEC) E. coli O80:H2 in French Healthy Cattle: Genomic Characterization and Phylogenetic Position.

The emerging heteropathotype shigatoxigenic (STEC) and extra-intestinal pathogenic Escherichia coli (ExPEC) O80:H2 has been the second leading cause of pediatric HUS in France since the mid-2010s. In contrast with other highly pathogenic STEC serotypes, for which ruminants have clearly been identified as the main human infection source, this heteropathotype's reservoir remains unknown. In this context, we describe for the first time the isolation of seven STEC O80:H2 strains from healthy cattle on a single cattle farm in France. This study aimed at (i) characterizing the genome and (ii) investigating the phylogenetic positions of these O80:H2 STEC strains. The virulomes, resistomes, and phylogenetic positions of the seven bovine isolates were investigated using in silico typing tools, antimicrobial susceptibility testing and cgMLST analysis after short-read whole genome sequencing (WGS). One representative isolate (A13P112V1) was also subjected to long-read sequencing. The seven isolates possessed ExPEC-related virulence genes on a pR444_A-like mosaic plasmid, previously described in strain RDEx444 and known to confer multi-drug resistance. All isolates were clonally related and clustered with human clinical strains from France and Switzerland with a range of locus differences of only one to five. In conclusion, our findings suggest that healthy cattle in France could potentially act as a reservoir of the STEC-ExPEC O80:H2 pathotype.

Real-time PCR primers and probes for the detection of Shiga toxin genes, including novel subtypes.

Shiga toxin-producing Escherichia coli (STEC) are foodborne enteric pathogens. STEC are differentiated from other E. coli by detection of Shiga toxin (Stx) or its gene (stx). The established nomenclature of Stx identifies ten subtypes (Stx1a, Stx1c, Stxd, Stx2a to Stx2g). An additional nine subtypes have been reported and described (Stx1e, Stx2h to Stx2o). Many PCR protocols only detect a subset of Stx subtypes which limits their inclusivity. Here we describe a real-time PCR assay inclusive of the DNA sequences of representatives of all currently described Stx subtypes. A multiplex real-time PCR assay for detection of stx was developed using nine primers and four probes. Since the identification of STEC does not require differentiation of stx subtypes, the probes use the same fluorescent reporter to enable detection of multiple possible targets in a single reaction. The PCR mixture includes an internal positive control to detect inhibition of the reaction. Thus, the protocol can be performed on a two-channel real-time PCR platform. To reduce the biosafety risk inherent in the use of STEC cultures as process controls, the protocol also includes the option of a non-pathogenic E. coli transformant carrying a plasmid encoding the targeted fragment of the stx2a sequence. The inclusivity of the PCR was assessed against colonies of 137 STEC strains and one strain of Shigella dysenteriae, including strains carrying single copies of stx representing fourteen subtypes (stx1 a, c, d; stx2 a-j and o). Five additional subtypes (stx1e, 2k, 2l, 2m and 2n) were represented by E. coli transformed with plasmids encoding toxoid (enzymatically inactive A subunit) sequences. The exclusivity panel consisted of 70 bacteria, including 21 stx-negative E. coli. Suitability for food analysis was assessed with artificially inoculated ground beef, spinach, cheese, and apple cider. The real-time PCR generated positive results for all 19 stx subtypes, represented by colonies of STEC, S. dysenteriae and E. coli transformants carrying stx toxoid plasmids. Tests of exclusivity panel colonies were all negative. The real-time PCR detected the presence of stx in all inoculated food enrichments tested, and the presence of STEC was confirmed by isolation.

Genetic and virulence characteristics of hybrid Shiga toxin-producing and atypical enteropathogenic Escherichia coli strains isolated in South Korea.

Introduction: The predominant hybrid pathogenic E. coli, enterohemorrhagic E. coli (EHEC), combines characteristics of Shiga toxin-producing E. coli (STEC) and enteropathogenic E. coli (EPEC), contributing to global outbreaks with severe symptoms including fatal consequences. Since EHEC infection was designated as a notifiable disease in 2000 in South Korea, around 2000 cases have been reported, averaging approximately 90 cases annually. Aim: In this work, genome-based characteristic analysis and cell-based assay of hybrid STEC/aEPEC strains isolated from livestock feces, animal source foods, and water in South Korea was performed. Methods: To identify the virulence and antimicrobial resistance genes, determining the phylogenetic position of hybrid STEC/aEPEC strains isolated in South Korea, a combination of real-time PCR and whole-genome sequencing (WGS) was used. Additionally, to assess the virulence of the hybrid strains and compare them with genomic characterization, we performed a cell cytotoxicity and invasion assays. Results: The hybrid STEC/aEPEC strains harbored stx and eae genes, encoding Shiga toxins and E. coli attachment/effacement related protein of STEC and EPEC, respectively. Furthermore, all hybrid strains harbored plasmid-carried enterohemolysin(ehxCABD), a key virulence factor in prevalent pathogenic E. coli infections, such as diarrheal disease and hemolytic-uremic syndrome (HUS). Genome-wide phylogenetic analysis revealed a close association between all hybrid strains and specific EPEC strains, suggesting the potential acquisition of Stx phages during STEC/aEPEC hybrid formation. Some hybrid strains showed cytotoxic activity against HeLa cells and invasive properties against epithelial cells. Notably, all STEC/aEPEC hybrids with sequence type (ST) 1,034 (n = 11) exhibited higher invasiveness than those with E2348/69. This highlights the importance of investigating potential correlations between STs and virulence characteristics of E. coli hybrid strains. Conclusion: Through genome-based characterization, we confirmed that the hybrid STEC/aEPEC strains are likely EPEC strains that have acquired STEC virulence genes via phage. Furthermore, our results emphasize the potential increased danger to humans posed by hybrid STEC/aEPEC strains isolated in South Korea, containing both stx and eaeA, compared to STEC or EPEC alone.

Further Evaluation of Enterohemorrhagic Escherichia coli Gold Nanoparticle Vaccines Utilizing Citrobacter rodentium as the Model Organism.

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.

Detection of prolong excretion of Escherichia albertii in stool specimens of a 7-year-old child by a newly developed Eacdt gene-based quantitative real-time PCR method and molecular characterization of the isolates.

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.

Outcome 10 years after Shiga toxin-producing E. coli (STEC)-associated hemolytic uremic syndrome: importance of long-term follow-up.

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.

Pathogenomes and virulence profiles of representative big six non-O157 serogroup Shiga toxin-producing Escherichia coli.

Shiga toxin (Stx)-producing Escherichia coli (STEC) of non-O157:H7 serotypes are responsible for global and widespread human food-borne disease. Among these serogroups, O26, O45, O103, O111, O121, and O145 account for the majority of clinical infections and are colloquially referred to as the "Big Six." The "Big Six" strain panel we sequenced and analyzed in this study are reference type cultures comprised of six strains representing each of the non-O157 STEC serogroups curated and distributed by the American Type Culture Collection (ATCC) as a resource to the research community under panel number ATCC MP-9. The application of long- and short-read hybrid sequencing yielded closed chromosomes and a total of 14 plasmids of diverse functions. Through high-resolution comparative phylogenomics, we cataloged the shared and strain-specific virulence and resistance gene content and established the close relationship of serogroup O26 and O103 strains featuring flagellar H-type 11. Virulence phenotyping revealed statistically significant differences in the Stx-production capabilities that we found to be correlated to the strain's individual stx-status. Among the carried Stx1a, Stx2a, and Stx2d phages, the Stx2a phage is by far the most responsive upon RecA-mediated phage mobilization, and in consequence, stx2a + isolates produced the highest-level of toxin in this panel. The availability of high-quality closed genomes for this "Big Six" reference set, including carried plasmids, along with the recorded genomic virulence profiles and Stx-production phenotypes will provide a valuable foundation to further explore the plasticity in evolutionary trajectories in these emerging non-O157 STEC lineages, which are major culprits of human food-borne disease.