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.

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.

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.

Development of a Rapid and Sensitive CANARY Biosensor Assay for the Detection of Shiga Toxin 2 from Escherichia coli.

Shiga-toxin-producing Escherichia coli (STEC) causes a wide spectrum of diseases including hemorrhagic colitis and hemolytic uremic syndrome (HUS). The current Food Safety Inspection Service (FSIS) testing methods for STEC use the Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) protocol, which includes enrichment, cell plating, and genomic sequencing and takes time to complete, thus delaying diagnosis and treatment. We wanted to develop a rapid, sensitive, and potentially portable assay that can identify STEC by detecting Shiga toxin (Stx) using the CANARY (Cellular Analysis and Notification of Antigen Risks and Yields) B-cell based biosensor technology. Five potential biosensor cell lines were evaluated for their ability to detect Stx2. The results using the best biosensor cell line (T5) indicated that this biosensor was stable after reconstitution with assay buffer covered in foil at 4 °C for up to 10 days with an estimated limit of detection (LOD) of ≈0.1-0.2 ng/mL for days up to day 5 and ≈0.4 ng/mL on day 10. The assay detected a broad range of Stx2 subtypes, including Stx2a, Stx2b, Stx2c, Stx2d, and Stx2g but did not cross-react with closely related Stx1, abrin, or ricin. Additionally, this assay was able to detect Stx2 in culture supernatants of STEC grown in media with mitomycin C at 8 and 24 h post-inoculation. These results indicate that the STEC CANARY biosensor developed in this study is sensitive, reproducible, specific, rapid (≈3 min), and may be applicable for surveillance of the environment and food to protect public health.

Re-analysis of an outbreak of Shiga toxin-producing Escherichia coli O157:H7 associated with raw drinking milk using Nanopore sequencing.

The aim of this study was to compare Illumina and Oxford Nanopore Technology (ONT) sequencing data to quantify genetic variation to assess within-outbreak strain relatedness and characterise microevolutionary events in the accessory genomes of a cluster of 23 genetically and epidemiologically linked isolates related to an outbreak of Shiga toxin-producing Escherichia coli O157:H7 caused by the consumption of raw drinking milk. There were seven discrepant variants called between the two technologies, five were false-negative or false-positive variants in the Illumina data and two were false-negative calls in ONT data. After masking horizontally acquired sequences such as prophages, analysis of both short and long-read sequences revealed the 20 isolates linked to the outbreak in 2017 had a maximum SNP distance of one SNP between each other, and a maximum of five SNPs when including three additional strains identified in 2019. Analysis of the ONT data revealed a 47 kbp deletion event in a terminal compound prophage within one sample relative to the remaining samples, and a 0.65 Mbp large chromosomal rearrangement (inversion), within one sample relative to the remaining samples. Furthermore, we detected two bacteriophages encoding the highly pathogenic Shiga toxin (Stx) subtype, Stx2a. One was typical of Stx2a-phage in this sub-lineage (Ic), the other was atypical and inserted into a site usually occupied by Stx2c-encoding phage. Finally, we observed an increase in the size of the pO157 IncFIB plasmid (1.6 kbp) in isolates from 2019 compared to those from 2017, due to the duplication of insertion elements within the plasmids from the more recently isolated strains. The ability to characterize the accessory genome in this way is the first step to understanding the significance of these microevolutionary events and their impact on the genome plasticity and virulence between strains of this zoonotic, foodborne pathogen.

Shiga toxin-producing Escherichia coli isolated from hunted wild boar (Sus scrofa) in Switzerland.

INTRODUCTION: Shiga toxin-producing Escherichia (E.) coli (STEC) are zoonotic foodborne pathogens of significant public health importance. While ruminants are considered the main reservoir, wild animals are increasingly acknowledged as carriers and potential reservoirs of STEC. The aim of this study was to determine the occurrence of STEC in a total of 59 faecal samples of hunted wild boars (Sus scrofa) from two different regions in Switzerland (canton Thurgau in northern Switzerland and canton Ticino in southern Switzerland), and to characterise the isolates using a whole genome sequencing approach. After an enrichment step, Shiga-toxin encoding genes (stx) were detected by real-time PCR in 41 % (95 % confidence interval (95 %CI) 0,29 - 0,53) of the samples, and STEC were subsequently recovered from 22 % (95 %CI 0,13 - 0,34) of the same samples. Seven different serotypes and six different sequence types (STs) were found, with O146:H28 ST738 (n = 4) and O100:H20 ST2514 (n = 4) predominating. Subtyping of stx identified isolates with stx1c/stx2b (n = 1), stx2a (n = 1), stx2b (n = 6), and stx2e (n = 6). No isolate contained the eae gene, but all harboured additional virulence genes, most commonly astA (n = 10), hlyE (n = 9), and hra (n = 9). STEC O11:H5, O21:H21, and O146:H28 harboured virulence factors associated with extra-intestinal pathogenic E. coli (ExPEC), and STEC O100:H20 and O155:H26 possessed sta1 and/or stb and were STEC/enterotoxigenic E. coli (ETEC) hybrid pathotypes. Our results show that wild boars are carriers of STEC which may be distributed in the environment, possibly leading to the contamination of agricultural crops and water sources. The serogroups included STEC O146 which belongs to the most common non-O157 serogroups associated with human illness in Europe, with implications for public health. Since Stx2e-producing STEC have frequently been reported in swine and pork, STEC O100:H20 harbouring stx2e in faeces of wild boars may be relevant to free-range systems of pig farming because of the potential risk of transmission events at the wildlife-livestock interface.

INTRODUCTION: Les Escherichia (E.) coli producteurs de shiga-toxine (STEC) sont des agents pathogènes zoonotiques d'origine alimentaire qui revêtent une grande importance pour la santé publique. Alors que les ruminants sont considérés comme le principal réservoir, les animaux sauvages sont de plus en plus souvent reconnus comme porteurs et réservoirs potentiels de STEC. L'objectif de cette étude était de déterminer la présence de STEC dans un total de 59 échantillons fécaux de sangliers (Sus scrofa) chassés provenant de deux régions différentes de Suisse (canton de Thurgovie dans le nord de la Suisse et canton du Tessin dans le sud de la Suisse) et de caractériser les isolats en utilisant une approche de séquençage du génome entier. Après une étape d'enrichissement, les gènes codant pour la Shiga-toxine (stx) ont été détectés par PCR en temps réel dans 41% (intervalle de confiance à 95% (95%CI) 0,29 - 0,53) des échantillons, et les STEC ont ensuite été récupérés dans 22% (95%CI 0,13 - 0,34) des mêmes échantillons. Sept sérotypes différents et six types de séquence (ST) différents ont été trouvés, avec une prédominance de O146:H28 ST738 (n = 4) et O100:H20 ST2514 (n = 4). Le sous-typage des stx a permis d'identifier des isolats avec stx1c/stx2b (n = 1), stx2a (n = 1), stx2b (n = 6) et stx2e (n = 6). Aucun isolat ne contenait le gène eae, mais tous hébergeaient d'autres gènes de virulence, le plus souvent astA (n = 10), hlyE (n = 9) et hra (n = 9). Les STEC O11:H5, O21:H21 et O146:H28 présentaient des facteurs de virulence associés à des E. coli pathogènes extra-intestinaux (ExPEC), et les STEC O100:H20 et O155:H26 possédaient sta1 et/ou stb et étaient des pathotypes hybrides STEC/E. coli entérotoxinogène (ETEC).

The NMR structure of the Ea22 lysogenic developmental protein from lambda bacteriophage.

The ea22 gene resides in a relatively uncharacterized region of the lambda bacteriophage genome between the exo and xis genes and is among the earliest genes transcribed upon infection. In lambda and Shiga toxin-producing phages found in enterohemorrhagic E. coli (EHEC) associated with food poisoning, Ea22 favors a lysogenic over lytic developmental state. The Ea22 protein may be considered in terms of three domains: a short amino-terminal domain, a coiled-coiled domain, and a carboxy-terminal domain (CTD). While the full-length protein is tetrameric, the CTD is dimeric when expressed individually. Here, we report the NMR solution structure of the Ea22 CTD that is described by a mixed alpha-beta fold with a dimer interface reinforced by salt bridges. A conserved mobile loop may serve as a ligand for an unknown host protein that works with Ea22 to promote bacterial survival and the formation of new lysogens. From sequence and structural comparisons, the CTD distinguishes lambda Ea22 from homologs encoded by Shiga toxin-producing bacteriophages.

In Silico Analysis of Shiga Toxin-Producing Escherichia coli O157:H7 Strains from Presumptive Super- and Low-Shedder Cattle.

Cattle are the primary reservoir for STEC O157, with some shedding >104 CFU/g in feces, a phenomenon known as super-shedding (SS). The mechanism(s) responsible for SS are not understood but have been attributed to the environment, host, and pathogen. This study aimed to compare genetic characteristics of STEC O157 strains from cattle in the same commercial feedlot pens with SS or low-shedding (LS) status. Strains from SS (n = 35) and LS (n = 28) collected from 11 pens in three feedlots were analyzed for virulence genes, Shiga toxin-carrying bacteriophage insertion sites, and phylogenetic relationships. In silico analysis showed limited variation regarding virulence gene profiles. Stx-encoding prophage insertion sites mrlA and wrbA for stx1a and stx2a, respectively, were all occupied, but two isolates had fragments of the stx-carrying phage in mrlA and wrbA loci without stx1a and stx2a. All strains screened for lineage-specific polymorphism assay (LSPA-6) were 111111, lineage I. Of the isolates, 61 and 2 were clades 1 and 8, respectively. Phylogenetic analysis revealed that pens with more than one SS had multiple distantly related clusters of SS and LS isolates. Although virulence genes and lineage were largely similar within and across feedlots, multiple genetic origins of strains within a single feedlot pen illustrate challenges for on-farm control of STEC.

The NMR structure of the Orf63 lytic developmental protein from lambda bacteriophage.

The orf63 gene resides in a region of the lambda bacteriophage genome between the exo and xis genes and is among the earliest genes transcribed during infection. In lambda phage and Shiga toxin (Stx) producing phages found in enterohemorrhagic Escherichia coli (EHEC) associated with food poisoning, Orf63 expression reduces the host survival and hastens the period between infection and lysis thereby giving it pro-lytic qualities. The NMR structure of dimeric Orf63 reveals a fold consisting of two helices and one strand that all make extensive intermolecular contacts. Structure-based data mining failed to identify any Orf63 homolog beyond the family of temperate bacteriophages. A machine learning approach was used to design an amphipathic helical ligand that bound a hydrophobic cleft on Orf63 with micromolar affinity. This approach may open a new path towards designing therapeutics that antagonize the contributions of Stx phages in EHEC outbreaks.

Clinical and public health implications of increasing notifications of LEE-negative Shiga toxin-producing Escherichia coli in England, 2014-2022.

Introduction. Shiga toxin-producing Escherichia coli (STEC) belong to a diverse group of gastrointestinal pathogens. The pathogenic potential of STEC is enhanced by the presence of the pathogenicity island called the Locus of Enterocyte Effacement (LEE), including the intimin encoding gene eae.Gap statement. STEC serotypes O128:H2 (Clonal Complex [CC]25), O91:H14 (CC33), and O146:H21 (CC442) are consistently in the top five STEC serotypes isolated from patients reporting gastrointestinal symptoms in England. However, they are eae/LEE-negative and perceived to be a low risk to public health, and we know little about their microbiology and epidemiology.Aim. We analysed clinical outcomes and genome sequencing data linked to patients infected with LEE-negative STEC belonging to CC25 (O128:H2, O21:H2), CC33 (O91:H14) and, and CC442 (O146:H21, O174:H21) in England to assess the risk to public health.Results. There was an almost ten-fold increase between 2014 and 2022 in the detection of all STEC belonging to CC25, CC33 and CC442 (2014 n=38, 2022 n=336), and a total of 1417 cases. There was a higher proportion of female cases (55-70 %) and more adults than children, with patients aged between 20-40 and >70 most at risk across the different serotypes. Symptoms were consistent across the three dominant serotypes O91:H14 (CC33), O146:H21 (CC442) and O128:H2 (CC25) (diarrhoea >75 %; bloody diarrhoea 25-32 %; abdominal pain 64-72 %; nausea 37-45 %; vomiting 10-24 %; and fever 27-30 %). Phylogenetic analyses revealed multiple events of acquisition and loss of different stx-encoding prophage. Additional putative virulence genes were identified including iha, agn43 and subA.Conclusions. Continued monitoring and surveillance of LEE-negative STEC infections is essential due to the increasing burden of infectious intestinal disease, and the risk that highly pathogenic strains may emerge following acquisition of the Shiga toxin subtypes associated with the most severe clinical outcomes.

Eculizumab in severe Shiga toxin-mediated haemolytic uraemic syndrome.

Shiga toxin (Stx)-producing Escherichia coli-mediated haemolytic uraemic syndrome is a primary thrombotic microangiopathy, typified by the development of microangiopathic haemolytic anaemia, thrombocytopaenia and acute renal failure. It is a leading cause of acute renal failure in paediatrics, with a second peak in prevalence in adults over the age of 60. Presentations of Stx-producing E. coli-mediated haemolytic uraemic syndrome in young adults are rare. We present the case of a previously well female in her early 30s presenting with Stx-producing E. coli-mediated haemolytic uraemic syndrome with severe renal and neurological manifestations. Eculizumab was administered due to the severity of presentation and disease trajectory refractory to initial supportive therapy. A significant clinical and biochemical improvement was observed following eculizumab.

A colorimetric and SERS dual-mode aptasensor for the detection of Shiga toxin type II based on Mn/Fe-MIL(53)@AuNSs.

Shiga toxin type II (Stx2), the major virulence component of enterohemorrhagic Escherichia coli, is strongly associated with the life-threatening hemolytic uremic syndrome thus posing a substantial risk to food safety and human health. In this work, a dual-mode aptasensor with colorimetric and surface-enhanced Raman scattering was developed for Stx2 specific detection based on noble metal nanoparticles and Raman reporter loaded metal-organic framework (Mn/Fe-MIL(53)@AuNSs-MBA). The Mn/Fe-MIL(53)@AuNSs could catalyze the H2O2-mediated oxidation of 3,3',5,5'-tetramethylbenzidine (TMB), thereby enabling visual detection. Meanwhile, the SERS signal from MBA can be enhanced by the decorated AuNSs. Under optimal conditions, a linear range of 0.05-500 ng/mL with limit of detection (LOD) of 26 pg/mL was achieved in colorimetric mode and a linear range of 5-1000 ng/mL with LOD of 0.82 ng/mL in SERS mode, in which the dual-mode results complement each other, widening the linear range, increasing the accuracy and reliability of the detection. The method was further applied to the detection of Stx2 in milk with average recovery of 101.1 %, demonstrating its superior potential for bacterial toxin monitoring.

Volume expansion mitigates Shiga toxin-producing E. coli-hemolytic uremic syndrome in children.

BACKGROUND: Shiga toxin-producing E. coli-hemolytic uremic syndrome (STEC-HUS) is associated with high morbidity and relevant mortality. Previous small studies showed that volume expansion could improve the course and outcome of STEC-HUS. The aim of this single-center study was to evaluate the effect of volume expansion on the clinical course and outcome in STEC-HUS. METHODS: Data of pediatric patients with STEC-HUS were analyzed retrospectively. Course and outcome of patients treated with volume expansion (VE) from 2019 to 2022 (n = 38) were compared to historical controls (HC) from 2009 to 2018 (n = 111). RESULTS: Patients in the VE group had a significant relative median weight gain compared to HC (7.8% (3.4-11.3) vs. 1.2% (- 0.7-3.9), p < 0.0001) 48 h after admission. The need for dialysis was not reduced by VE (VE 21/38 (55.3%) vs. HC 64/111 (57.7%), p = 0.8). However, central nervous system involvement (impairment of consciousness, seizures, focal neurological deficits, and/or visual disturbances) was significantly reduced (VE 6/38 (15.8%) vs. HC 38/111 (34.2%), p = 0.039). None of the patients in the VE group died or developed chronic kidney disease (CKD) stage 5, whereas in the HC group, three patients died and three patients had CKD stage 5 at discharge. CONCLUSIONS: This study suggests that volume expansion may be associated with the mitigation of the acute course of STEC-HUS, especially severe neurological involvement and the development of CKD. Prospective trials should lead to standardized protocols for volume expansion in children with STEC-HUS.

Gastrointestinal involvement in STEC-associated hemolytic uremic syndrome: 10 years in a pediatric center.

BACKGROUND: The gastrointestinal (GI) tract represents one of the main targets of typical hemolytic uremic syndrome (HUS) in children. In this observational study, we tried to establish (1) the main features of GI complications during STEC-HUS and (2) the relationship between Escherichia coli serotypes and Shiga toxin (Stx) variants with hepatopancreatic involvement. METHODS: A total of 79 STEC-HUS patients were admitted to our pediatric nephrology department between January 2012 and June 2021. Evidence of intestinal, hepatobiliary, and pancreatic involvements was reported for each patient, alongside demographic, clinical, and laboratory features. Frequency of gastrointestinal complications across groups of patients infected by specific E. coli serotypes and Stx gene variants was evaluated. RESULTS: Six patients developed a bowel complication: two developed rectal prolapse, and four developed bowel perforation which resulted in death for three of them and in bowel stenosis in one patient. Acute pancreatitis was diagnosed in 13 patients. An isolated increase in pancreatic enzymes and/or liver transaminases was observed in 41 and 15 patients, respectively. Biliary sludge was detected in three, cholelithiasis in one. Forty-seven patients developed direct hyperbilirubinemia. Neither E. coli serotypes nor Shiga toxin variants correlated with hepatic or pancreatic involvement. CONCLUSIONS: During STEC-HUS, GI complications are common, ranging from self-limited elevation of laboratory markers to bowel perforation, a severe complication with a relevant impact on morbidity and mortality. Hepatopancreatic involvement is frequent, but usually short-lasting and self-limiting.

Efficacy of EHEC gold nanoparticle vaccines evaluated with the Shiga toxin-producing Citrobacter rodentium mouse model.

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.

A novel Shiga toxin 2a neutralizing antibody therapeutic with low immunogenicity and high efficacy.

Shiga toxin-producing Escherichia coli infections are difficult to treat due to the risk of antibiotic-induced stress upregulating the production of toxins, medical treatment is consequently limited to supportive care to prevent the development of hemolytic uremic syndrome (HUS). Here, we introduce a potentially therapeutic humanized mouse monoclonal antibody (Hu-mAb 2-5) targeting Stx2a, the most common Shiga toxin subtype identified from outbreaks. We demonstrate that Hu-mAb 2-5 has low immunogenicity in healthy adults ex vivo and high neutralizing efficacy in vivo, protecting mice from mortality and HUS-related tissue damage.

Combined usage of serodiagnosis and O antigen typing to isolate Shiga toxin-producing Escherichia coli O76:H7 from a hemolytic uremic syndrome case and genomic insights from the isolate.

IMPORTANCE: Hemolytic uremic syndrome (HUS) is a life-threatening disease caused by Shiga toxin-producing Escherichia coli (STEC) infection. The treatment approaches for STEC-mediated typical HUS and atypical HUS differ, underscoring the importance of rapid and accurate diagnosis. However, specific detection methods for STECs other than major serogroups, such as O157, O26, and O111, are limited. This study focuses on the utility of PCR-based O-serotyping, serum agglutination tests utilizing antibodies against the identified Og type, and isolation techniques employing antibody-conjugated immunomagnetic beads for STEC isolation. By employing these methods, we successfully isolated a STEC strain of a minor serotype, O76:H7, from a HUS patient.

Complex effects of the exo-xis region of the Shiga toxin-converting bacteriophage Φ24B genome on the phage development and the Escherichia coli host physiology.

Lambdoid bacteriophages are excellent models in studies on molecular aspects of virus-host interactions. However, some of them carry genes encoding toxins which are responsible for virulence of pathogenic strains of bacteria. Shiga toxin-converting bacteriophages (Stx phages) encode Shiga toxins that cause virulence of enterohemorrhagic Escherichia coli (EHEC), and their effective production depends on Stx prophage induction. The exo-xis region of the lambdoid phage genome consists of genes which are dispensable for the phage multiplication under laboratory conditions; however, they might modulate the virus development. Nevertheless, their exact effects on the phage and host physiology remained unclear. Here, we present results of complex studies on the role of the exo-xis region of bacteriophage Φ24B, one of Stx2b phages. Transcriptomic analyses, together with proteomic and metabolomic studies, provided the basis for understanding the functions of the exo-xis region. Genes from this region promoted lytic development of the phage over lysogenization. Moreover, expression of the host genes coding for DnaK, DnaJ, GrpE, and GroELS chaperones was impaired in the cells infected with the Δexo-xis phage mutant, relative to the wild-type virus, corroborating the conclusion about lytic development promotion by the exo-xis region. Proteomic and metabolomic analyses indicated also modulation of gad and nrf operons, and levels of amino acids and acylcarnitines, respectively. In conclusion, the exo-xis region controls phage propagation and host metabolism by influencing expression of different phage and bacterial genes, directing the virus to the lytic rather than lysogenic developmental mode.