This study examines the in vitro antibacterial activity of palatal adipose tissue-derived mesenchymal stem cells (PAT-MSCs) and the expression of antimicrobial peptide LL-37, with a particular focus on the effect of three-dimensional (3D) nanofibrillar cellulose-based hydrogel, against Shiga toxin producing Escherichia coli (STEC) harboring stx1 and/or stx2 genes isolated from mastitis milk in Turkey. The antibacterial activity of conventionally cultured PAT-MSCs and 3D-cultured PAT-MSCs (PAT-MSCs-3D) was evaluated against STEC isolates and Escherichia coli ATCC 35150 using quantitative colony-forming unit (CFU) assay. The expression levels of antimicrobial peptide (AMP)-encoding genes were evaluated by quantitative real-time PCR, and AMP production was further validated by immunocytochemical staining. The results indicated that PAT-MSCs-3D exhibited significantly enhanced antibacterial efficacy, resulting in marked bacterial inhibition of all tested STEC strains, with bacterial reductions reaching up to 6-7 log under specific experimental conditions. Molecular and immunocytochemical analyses demonstrated increased expression of the antimicrobial peptide LL-37 in PAT-MSCs-3D compared to 2D cultures. Our results show that culturing PAT-MSCs in 3D conditions leads to a significant enhancement in their antimicrobial properties, which could be linked to the upregulation of LL-37.

The extensive application potential of nucleic acid aptamers in biosensing and therapeutic domains necessitates the development of cost-effective, time-efficient, and technically simpler synthesis protocols compared with conventional SELEX methods. In this study, an in silico-based de novo approach has been proposed to design an aptamer targeting the B subunits of Shiga-like toxin 2 (Stx2-B) protein, intended for use as a recognition element in biosensors for detecting Stx2. A single-stranded DNA library comprising 5000 sequences was generated and ranked based on their Gibbs free energy (ΔG) of the 2D structures. The top 100 sequences with the lowest ΔG values were modeled in 3D and individually docked with the Stx2 protein. Sequences exhibiting the highest docking scores were further analyzed through molecular dynamics simulations. Among the analyzed candidates, sequence A330 demonstrated the highest stability, rigidity, and compactness in complex with Stx2B, as indicated by root-mean-square deviation, root-mean-square fluctuation, radius of gyration, and hydrogen-bond analysis over 100 ns simulation trajectories. The strong binding affinity of A330 for the target (Kd = 459 ± 7 nM) was confirmed through circular dichroism and isothermal titration calorimetry analyses. This aptamer was subsequently employed as a recognition element in the fabrication of an electrochemical impedimetric aptasensor for Stx2 detection, utilizing a microscale interdigitated wave-type electrode. The developed sensor exhibited high sensitivity, a limit of detection of 4.63 pM, and a linear detection range of 10-400 pM (R2 = 0.99). The aptasensor's practical applicability was validated in milk samples, achieving a detection recovery of >93.06%. Overall, the proposed aptasensor shows strong potential for the sensitive and reliable monitoring of Shiga toxin-producing Escherichia coli contamination in real samples.

Leptospirosis is a globally prevalent zoonotic infection caused by pathogenic spirochaetes of the genus Leptospira Although typically associated with mild flu-like symptoms or Weil's disease (characterised by jaundice, renal failure and haemorrhagic manifestations), atypical and severe presentations can mimic other systemic disorders. A man in his 40s presented with fever, myalgia along with rapidly progressive jaundice, thrombocytopenia, microangiopathic haemolytic anaemia and acute kidney injury, preceded by a short episode of diarrhoea. The initial working diagnosis was haemolytic uraemic syndrome (HUS) secondary to Shiga toxin-producing Escherichia coli and he was treated with plasma exchange, transfusions and renal replacement therapy. However, stool examinations were negative for Shiga toxin and bacterial pathogens. Subsequent investigations confirmed leptospirosis. He improved significantly after treatment with doxycycline alongside supportive therapy. This case highlights the importance of considering leptospirosis as a differential diagnosis in patients with HUS, particularly in endemic regions, to avoid delays in targeted treatment.

Enterohemorrhagic Escherichia coli senses colonic oxygen availability to modulate its virulence and colonization.

TBC (Tre2/Bub2/Cdc16) domain-containing proteins constitute the widespread family of GTPase-activating proteins (GAPs). They interact with the Rab superfamily of small GTPases, stimulate GTP hydrolysis, and regulate vesicle trafficking. TBC1D17, involved in Shiga toxin trafficking, autophagy and glucose metabolism regulation, constitutes an example of GAP interacting with Rabs. Here we present the first crystal structures of the murine and human TBC domains of TBC1D17 proteins determined at 2.20 and 3.34 Å resolution, respectively. The TBC domain in both structures represents a heart-like shape. Our analyses revealed dimerization of the TBC domain through a fragment located near residues participating in GTP hydrolysis, a result we observed also in structures of closely related homologs. Furthermore, we tested Rab5a interactions with various fragments of TBC1D17. Interestingly, this protein contains an annotated, yet uncharacterized, Rab-binding domain (RBD) and our studies revealed strong interactions of Rab5a with TBC1D17 fragments containing RBD, while interactions with the TBC domain alone are much weaker. These results provide the first direct evidence for the critical role of the TBC1D17 RBD in interactions with Rab5a.

Carbohydrate–proteininteractions are essential for biologicalrecognition but often suffer from cross-reactivity. Multivalency canenhance the binding strength; however, it requires a precise spatialarrangement of carbohydrate ligands to match the protein binding sites.Controlling glycan presentation improves both avidity and selectivity,helping to reduce cross-reactivity. However, complex proteins, suchas AB5-type Shiga toxin (Stx), present additional challenges, as theB subunits form pentamers, and each subunit contains three nonequivalentglycan-binding sites. To address this, we developed oligoproline-basedcyclic nanoscaffolds and characterized them by using circular dichroismand ion-mobility spectrometry. Surface plasmon resonance analysisshowed that different glycan patterns on these scaffolds produceddistinct binding modes with the StxB pentamer. By coupling a fullytunable synthetic nanoscaffold platform with analytical methods capableof resolving complex binding behaviors, this work enables a deeperinvestigation of protein receptors and supports the design of moreselective multivalent biomolecules.

In January 2025, Santé publique France detected several cases of hemolytic uremic syndrome (HUS) in adults caused by a rare serotype of Shiga toxin-producing Escherichia coli, STEC O77g:H18. Epidemiological and traceability investigations linked the consumption of raw-milk cheese to these cases. The outbreak strain belonged to sequence type 69 (ST69) and harbored a Shiga toxin (stx) subtype stx2d located on a unique prophage. Putative virulence genes cdt-VABC, hlyE, fimH, and lpfA were detected; however, STEC virulence genes (eae and aggR) were absent. Phylogenetic analysis of E. coli O77g strains from public databases (Enterobase and NCBI) placed the outbreak strain within a cluster of clinical E. coli O77g:H18, ST69, stx2d strains isolated between 2012 and 2025, including one linked to a HUS case in France and three in Australia. This strain, distinct from O77g:H18 strains previously isolated from dairy products, is therefore not a new lineage. The closest cluster contains eight stx-negative strains isolated from poultry between 1990 and 2024 in Europe and North America. Differences in virulence gene composition between the "outbreak cluster" and the "poultry cluster" suggest a common ancestor that has diverged over time. The "outbreak cluster" is characterized by strains carrying an IncFIA/FIB plasmid encoding a putative enterotoxin gene (elt) absent in other E. coli strains in GenBank. We developed qPCR assays specific for the putative enterotoxin and O77g O-group. Combined with a PCR for stx2, these assays allow rapid screening of suspect unpasteurized cheese for the outbreak strain.IMPORTANCEThe emergence of hybrid pathotypes of Shiga toxin-producing Escherichia coli (STEC) has been of importance in recent years in public health. This outbreak, related to a rare serotype of STEC and involving an enterotoxigenic E. coli (ETEC)/STEC hybrid, highlights the necessity of phylogenetic analyses to investigate the origin of contamination with such strains. Our study suggests a common ancestor between the outbreak strain and strains isolated from poultry. We also provided molecular tools to target specifically this outbreak strain and help in future epidemiological investigations.

Introduction Hemolytic uremic syndrome (HUS) usually presents with a triad of symptoms: thrombocytopenia, hemolytic anaemia, and acute kidney injury. Currently, HUS is differentiated into HUS associated with Escherichia coli infection and Shiga toxin production, atypical HUS associated with complement system disorders, and secondary HUS associated with infection or drug-induced HUS, etc. During the differential diagnosis, thrombotic thrombocytopenic purpura (TTP), associated with reduced ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 motifs, member 13) activity or deficiency, should always be ruled out. Case description A 63-year-old woman with a history of ductal carcinoma of the pancreatic tail, surgically removed in February 2023, was undergoing adjuvant chemotherapy with gemcitabine. Additionally, patient had bronchial asthma, hypertension, and hypothyroidism treated with hormone replacement therapy. The patient was admitted to the emergency department due to severe anaemia requiring red blood cell transfusion and biochemical evidence of kidney damage. On admission, the patient reported weakness, intermittent shortness of breath, and palpitations, and denied nausea, vomiting, or bleeding symptoms. Laboratory tests revealed anaemia, thrombocytopenia, and markers of renal damage. Urinalysis revealed asymptomatic bacteruria, hematuria, and a small amount of protein. Abdominal ultrasound revealed post-resection changes of the pancreatic body and tail, a 19-mm hypoechoic right adrenal nodule, right renal cysts, and a non-dilated pelvicalyceal system. During hospitalisation, progressive deterioration of renal function was observed despite conservative treatment of acute kidney injury, as well as worsening blood pressure control despite gradual intensification of antihypertensive therapy. A suspicion was raised of the development of atypical hemolytic uremic syndrome during the course of adjuvant chemotherapy with gemcitabine. The ADAMTS13 activity was determined at 80.6%, and stool polymerase chain reaction for Shiga-toxigenic Escherichia coli was negative. Four therapeutic plasmapheresis sessions were performed, but they were ineffective. An application for eculizumab treatment was submitted under the drug program, which was rejected. Conclusions This case report illustrates a rare but severe complication of aHUS associated with gemcitabine treatment. Gemcitabine should be discontinued immediately if characteristic symptoms are observed. Plasmapheresis treatment was found to be ineffective in achieving significant clinical improvement, suggesting a potential need for complement inhibitor therapy, particularly eculizumab.

Enterohemorrhagic Escherichia coli (EHEC) is notable for the Shiga-like toxin and several secretion systems and virulence factors that facilitate intestinal colonization and pathogenesis. EHEC low infectious dose may be in part due to its Type VI secretion system (T6SS), which could have antibacterial activity and perhaps damage host cells, although its function and mechanism are unknown. The aim of this study was to understand T6SS activation in EHEC by replacing its unknown native promoter with two inducible promoters. We found that the structural components encoded in the tssB operon are expressed as part of a single polycistronic operon. Excitingly, in the other direction, the hcp3 region is formed of at least two separate operons, leading to incomplete T6SS activation and no secretion of T6SS substrates. Analysis of the region downstream of hcp3 revealed a transcriptional terminator, a promoter region, and multiple transcription factor binding sites; thereby, we designated it as h3R. This regulatory sequence represses the translation of vgrG3 but also contains a regulatory element that stabilizes the mRNA of hcp3, the gene located immediately upstream of vgrG3. Meanwhile, deletion of hns elevated T6SS mRNAs but was not sufficient for protein expression until further activated with DMEM or MM9, although not all proteins were expressed and protein secretion by other systems was modified. These data highlight the stringent regulatory control of the T6SS in EHEC and identify novel promoter regions that warrant further investigation to elucidate the role of this secretion system in bacterial competition and pathogenesis.IMPORTANCEEHEC harbors a T6SS, which in other bacteria play a role in intestinal colonization and pathogenesis. T6SS is repressed in vitro in EHEC, making its study difficult, but it has been suggested to be required for full pathogenesis of EHEC. Here, we aimed to activate the T6SS in vitro. We found that activation of the bidirectional promoter is not enough for full T6SS expression, as a regulatory sequence downstream, named h3R, tightly regulates its expression. Deletion of the master regulator hns was not enough for full activation, and additional activation was required to produce some, but not all, proteins, causing a loss of bacterial fitness. These findings bring us closer to a full understanding of the mechanisms that control its expression. We predict novel regulation sites that will be necessary to fully understand the activation mechanisms and, thus, the function of T6SS in EHEC and perhaps other bacteria.

Shiga toxin-producing Escherichia coli (STEC) are significant zoonotic pathogens worldwide, causing diseases ranging from diarrhea to hemolytic uremic syndrome (HUS). Ruminants, mainly cattle, are considered the primary reservoirs; however, the role of horses in the epidemiology of STEC remains poorly understood in Nigeria. A total of 231 rectal swabs were collected from horses across selected equine facilities in five Nigerian states. Samples were processed following standard procedures, and isolates were confirmed using the Analytical Profile Index (API 20E) kit. Antibiotic susceptibility testing (AST) was performed using the Kirby-Bauer disk diffusion method with a panel of nine antibiotics, and the isolated Escherichia (E.) coli were classified as sensitive or resistant according to CLSI guidelines. The Multiple Antibiotic Resistance (MAR) index was calculated to assess the risk of contamination. The E. coli isolates were screened for selected Shiga toxin genes (stx1, stx2 and eae) using polymerase chain reaction (PCR). Fourteen isolates (6%) were identified as Shiga toxin-producing E. coli; of these, 78.6% (11/14) expressed at least one virulence gene: 42.9% (6/14) carried stx2, 35.7% (5/14) carried eae, and no isolate carried stx1. All confirmed STEC isolates exhibited multidrug resistance (MDR). The isolates showed complete resistance (100%) to cefuroxime and ciprofloxacin, while all were susceptible to ofloxacin. MAR indices ranged from 0.2 to 0.8, with 77.8% of isolates exhibiting MAR indices ≥0.5. This study reports the detection of MDR STEC in horses in Nigeria, revealing high-level resistance to clinically important antibiotics and significant carriage of virulence genes. The high MAR indices indicate high-risk contamination sources. The detection of MDR STEC in this study underscores a significant health risk to horses, with potential for zoonotic transmission to humans and the environment.

Enterohemorrhagic HUS (eHUS), triggered by Shiga toxin (STX), is a thrombotic microangiopathy and major cause of acute kidney injury (AKI) in children. Of those hospitalized, ~65% require dialysis, while others may suffer seizures, neurocognitive deficits from cerebral ischemia, other organ failure ( ~ 30%), and death (3-5%). Long-term sequelae include chronic kidney disease, hypertension, insulin-dependent diabetes, and neurocognitive impairment. No therapeutic agents are effective and/or approved by the Food and Drug Administration, or similar bodies. We have identified two potential therapeutic targets-mannose-binding lectin-2 (MBL2) of the lectin complement pathway and clotting factor XIa (FXIa) of the intrinsic coagulation pathway. We previously reported the importance of MBL2 in a mouse model of Shiga Toxin-induced kidney injury, using 3F8, a mouse anti-human MBL2 antibody, and procoagulant activity in an endothelial monolayer model. We now report important roles for both clotting factor XI and MBL2 in this model, employing 3G3, a humanized anti-FXIa antibody, and 3F8 together. The antibody combination is superior to either antibody alone in reducing glomerular platelet deposition, and to 3F8 in reducing fibrin deposition. Only the combination is effective in lowering AKI, hemolysis, and weight loss. The antibody combination may have therapeutic application in affected children. Enterohemorrhagic hemolytic uremic syndrome causes acute renal failure in 65% of affected children, seizures and neurocognitive deficits from cerebral ischemic events in 3-5%, other organ system failure in 20-30%, and death in 3-5%. There presently exists no specific therapy, only supportive care. We have found in our mouse model of Shiga Toxin-induced kidney injury that monoclonal antibodies against the mannose binding lectin-2 (MBL2) of the lectin complement pathway, and factor XI/XIa of the contact coagulation pathway show clear benefit used singly, but even greater benefit in combination. The combination offers promise for an effective first specific therapy.

Non-O157 shiga toxin\u2013producing Escherichia coli (STEC) in Africa: a one health systematic review and meta-analysis of prevalence, antimicrobial resistance, and clinical outcomes

The Microbiological Quality of Ready-to-Eat Salad and Salad Components Collected from Retail and Catering Premises in England and Northern Ireland.

WCN26-4652 EARLY DIAGNOSIS AND PREVENTIVE MANAGEMENT OF SHIGA TOXIN–PRODUCING ESCHERICHIA COLI INFECTION IN CHILDREN: THE ITALIAN INTER-SOCIETY CONSENSUS FOR THE PREVENTION OF HEMOLYTIC UREMIC SYNDROME

In January 2025, the Escherichia coli National Reference Center of France detected an outbreak of hemolytic uremic syndrome (HUS) in adults, caused by Shiga toxin-producing E. coli negative for locus of enterocyte effacement (LEE). The outbreak included 18 confirmed cases of E. coli infection, 5 probable or possible cases detected by in-house specific PCR, and 2 additional cases from Scotland and Belgium. Whole-genome sequencing identified the outbreak strain as O77 g:K92:H18, belonging to phylogroup D; the strain harbored the Shiga toxin 2 gene variant stx2d-073-C165-02 and a 134-kb plasmid with enterotoxin genes (estb-STb2 and eltAB). Epidemiologic investigation implicated raw-milk cheese as the contamination source. The strain represents a singular hybrid pathotype of Shiga toxin-producing and enterotoxigenic E. coli, expressing a K92 capsule with known cross-immunogenicity to Neisseria meningitidis group C, which could explain the absence of pediatric cases. Related strains have been identified in international databases since 2005, suggesting global emergence.

Implementation of molecular screening for a more efficient Shiga toxin-producing Escherichia coli testing workflow.

Virulent phages MM-1 and MM-2 attack enterohemorrhagic Escherichia coli and Salmonella enterica strains.

Shiga toxin (Stx)-producing Escherichia coli (STEC) is a major cause of food-borne illnesses, and disease severity correlates with the production of Shiga toxins. While clinical symptoms such as bloody diarrhea and hemolytic uremic syndrome have been attributed to Stx, its contribution to bacterial fitness is not well understood. Here, we demonstrate that Stx2 enhances STEC colonization of the zebrafish gut by facilitating the partial displacement of gut resident microbes. Infection with Stx2-producing STEC strains or direct exposure of fish to purified Stx2 induces alterations in the zebrafish microbiome structure, impacting several bacterial phyla and genera, notably Pseudomonads. We show that Stx2 is sufficient to facilitate these changes by accelerating intestinal transit, leading to increased expulsion of select gut microbes, including resident Pseudomonas species. Additionally, prokinetic drug treatment causes similar changes in gut transit and expulsion of Pseudomonas. Collectively, these findings detail a novel mode of action of Stx2 on the host, and shed light on its contribution to bacterial fitness within the host intestine.

Phage-encoded Shiga toxin (Stx) released by Shiga toxin-producing E. coli (STEC) can kill multiple eukaryotic bacterial predators, including Acanthamoeba castellanii, Tetrahymena thermophila and Caenorhabditis elegans. However, the impact of Stx type, Stx amount, and the serogroup of the E. coli on the effectiveness of this exotoxin are poorly understood. These factors impact the severity of Stx-mediated disease in humans and therefore, by studying their role in modulating predator-prey interactions, we may gain insight into how these virulence factors evolved to contribute to human pathogenicity. Herein, we investigated the effects of these factors on predator killing by measuring the efficiency with which five different hemolytic uremic syndrome (HUS)-causing STEC strains consume and/or kill A. castellanii and C. elegans. These strains express various combinations of Stx types and amounts and O-antigens. We found that variations in Stx types and amounts significantly affect the ability of a given bacterial strain to kill predator A. castellanii and C. elegans, with higher Stx1 titers (HUSEC 31 vs. 19) and the presence of Stx2 alone (HUSEC 20) correlating with significantly greater predator killing. These attributes also affect STEC pathogenicity in humans, suggesting that ecological selective pressures for anti-predator defense inadvertently drive the evolution of strains with higher virulence potential in humans.

Antimicrobial resistance (AMR) in captive wildlife remains poorly characterized, particularly in zoological collections within the Middle East. This study investigated the occurrence, phenotypic resistance patterns, and genomic characteristics of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in clinically healthy captive ungulates at Al Ain Zoo, one of the largest zoological institutions located in an urban area in the United Arab Emirates (UAE). Fecal samples were collected non-invasively from 101 ungulates during routine husbandry and screened for ESBL-producing E. coli. Antimicrobial susceptibility testing (AST) was performed using the VITEK 2 system, and the identified ESBL-producing and multidrug resistant E. coli isolates underwent whole-genome sequencing for resistome, virulome, plasmid, and phylogenetic analyses. ESBL-producing E. coli were detected in 35 of 101 animals, corresponding to a carriage frequency of 34.7% (95% CI: 25.4-43.9%). Phenotypically, all isolates were resistant to β-lactam antibiotics, while resistance to fluoroquinolones and tetracycline was common. In contrast, susceptibility to aminoglycosides, imipenem, and polymyxin B was fully preserved. Based on exhibiting resistance to three or more antimicrobial classes, 26 of 35 isolates (74.3%) were classified as MDR. Genomic analysis revealed that all ESBL-producing E. coli harbored the bla CTX-M-15 gene, frequently accompanied by resistance determinants to quinolones (qnrS1), tetracyclines (tet(A)), sulfonamides, trimethoprim, and aminoglycosides. All AMR genes were located on conjugative plasmids, predominantly IncF-type backbones. The isolates exhibited a polyclonal population structure encompassing 18 multilocus sequence types and several small SNP-defined clusters, alongside a moderate virulence gene burden lacking Shiga toxin or classical extra-intestinal pathogenic markers. This study provides the first genomic baseline of ESBL-producing E. coli in zoo-housed ungulates in the UAE and Middle East region, highlighting the value of urban zoological collections as sentinel sites for integrated One Health AMR surveillance supporting animal health, conservation, and public health.