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ABSTRACT Shiga toxin (Stx)-producing Escherichia coli (STEC) is a zoonotic pathogen with the ability to cause severe diseases like hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). Shiga toxin (Stx) is the key virulence factor in STEC and can be classified into two types, Stx1 and Stx2, and different subtypes. Stx2k is a newly reported Stx2 subtype in E. coli strains from diarrheal patients, animals, and raw meats exclusively in China so far. To understand the reservoir of Stx2k-producing E. coli (Stx2k-STEC), we investigated Stx2k-STEC strains in goat herds and examined their genetic characteristics using whole-genome sequencing. A total of 448 STEC strains were recovered from 2,896 goat fecal samples, and 37.95% (170/448) were Stx2k-STEC. Stx2k-STEC strains of serotype O93:H28 and sequence type 4038 (ST4038) were the most predominant and were detected over several years. Notably, 55% of Stx2k-STEC strains carried the heat-labile toxin (LT)-encoding gene (elt) defining enterotoxigenic E. coli (ETEC), thereby exhibiting the hybrid STEC/ETEC pathotype. Stx2k-converting prophage genomes clustered into four groups and exhibited high similarity within each group. Strains from patients, raw meat, sheep, and goats were intermixed distributed in the phylogenetic tree, indicating the risk for cross-species spread of Stx2k-STEC and pathogenic potential for humans. Further studies are required to investigate the Stx2k-STEC strains in other reservoirs and to understand the mechanism of persistence in these hosts. IMPORTANCE Strains of the recently reported Stx2k-STEC have been circulating in a variety of sources over time in China. Here, we show a high prevalence of Stx2k-STEC in goat herds. More than half of the strains were of the hybrid STEC/ETEC pathotype. Stx2k-STEC strains of predominant serotypes have been widespread in the goat herds over several years. Stx2k-converting prophages have exhibited a high level of similarity across geographical regions and time and might be maintained and transmitted horizontally. Given that goat-derived Stx2k-STEC strains share similar genetic backbones with patient-derived strains, the high prevalence of Stx2k-STEC in goats suggests that there is a risk of cross-species spread and that these strains may pose pathogenetic potential to humans. Our study thus highlights the need to monitor human Stx2k-STEC infections in this region and, by extension, in other geographic locations.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 (O157) is a major foodborne pathogen that causes severe illness in humans worldwide. The genome of O157 contains 177 genomic islands known as O islands (OIs), including Shiga toxin-converting phages (OI-45 and OI-93) and the locus for enterocyte effacement (LEE) pathogenicity island (OI-148). However, most genes in OIs are uncharacterized and code for unknown functions. In this study, we demonstrated, for the first time, that OI-9 encodes a novel transcriptional activator, Z0346 (named OvrB), which is required for bacterial adherence to host cells and LEE gene expression in O157. OvrB directly binds to the promoter region of LEE1 and activates the transcription of ler (encoding a master regulator of LEE genes), which in turn activates LEE1–5 genes to promote O157 adherence. Furthermore, mouse oral infection assays showed that OvrB promotes O157 colonization in the mouse intestine. Finally, OvrB is shown to be a widespread transcriptional activator of virulence genes in other enterohemorrhagic and enteropathogenic Escherichia coli serotypes. Our work significantly expands the understanding of bacterial virulence control and provides new evidence suggesting that horizontally transferred regulator genes mediate LEE gene expression.

Shiga toxin (Stx) is the key virulence factor in Shiga toxin producing Escherichia coli (STEC), which can cause diarrhea and hemorrhagic colitis with life-threatening complications. Stx comprises two toxin types, Stx1 and Stx2. Several Stx1/Stx2 subtypes have been identified in E. coli, which are variable in sequences, toxicity and host specificity. Here, we report the identification of a novel Stx2 subtype, designated Stx2k, in E. coli strains widely detected from diarrheal patients, animals, and raw meats in China over time. Stx2k exhibits varied cytotoxicity in vitro among individual strains. The Stx2k converting prophages displayed considerable heterogeneity in terms of insertion site, genetic content and structure. Whole genome analysis revealed that the stx2k-containing strains were genetically heterogeneous with diverse serotypes, sequence types, and virulence gene profiles. The nine stx2k-containing strains formed two major phylogenetic clusters closely with strains belonging to STEC, enterotoxigenic E. coli (ETEC), and STEC/ETEC hybrid. One stx2k-containing strain harbored one plasmid-encoded heat-stable enterotoxin sta gene and two identical copies of chromosome-encoded stb gene, exhibiting STEC/ETEC hybrid pathotype. Our finding enlarges the pool of Stx2 subtypes and highlights the extraordinary genomic plasticity of STEC strains. Given the wide distribution of the Stx2k-producing strains in diverse sources and their pathogenic potential, Stx2k should be taken into account in epidemiological surveillance of STEC infections and clinical diagnosis.

Regardless of the communal impact of Shiga toxins, till today neither a specific treatment nor licensed vaccine is available. Lactococcus lactis (L. lactis), generally regarded as safe organism, is well known to provide a valuable approach regarding the oral delivery of vaccines. This study was undertaken to evaluate the protective efficacy of Stx2a1 expressed in nisin‐inducible L. lactis, against Shiga toxins (Stx1, Stx2) in mouse model. Oral immunization of BALB/c mice with LL‐Stx2a1 elicited significant serum antibody titer with elevated fecal and serum IgA, along with minimized intestinal and kidney damage resulting in survival of immunized animals at 84% and 100% when challenged with 10 × LD50 of Escherichia coli O157 and Shigella dysenteriae toxins, respectively. HeLa cells incubated with immune sera and toxin mixture revealed high neutralizing capacity with 90% cell survivability against both the toxins. Mice immunized passively with both toxins and antibody mixture survived the observation period of 15 days, and the controls administered with sham sera and toxins were succumbed to death within 3 days. Our results revealed protective efficacy and toxin neutralization ability of LL‐Stx2a1, proposing it as an oral vaccine candidate against Shiga toxicity mediated by E. coli O157 and S. dysenteriae. [ABSTRACT FROM AUTHOR]

Hemolytic uremic syndrome (HUS), principally caused by shiga toxins (Stxs), is associated with Shiga toxin-producing Escherichia coli (STEC) infections. We previously reported Stx2 expression by host cells in vitro and in vivo. As the genes encoding the two Stx subunits are located in bacteriophage genomes, the aim of the current study was to evaluate the role of bacteriophage induction in HUS development in absence of an E. coli O157:H7 genomic background. Mice were inoculated with a non-pathogenic E. coli strain carrying the lysogenic bacteriophage 933W (C600Φ933W), and bacteriophage excision was induced by an antibiotic. The mice died 72 h after inoculation, having developed pathogenic damage typical of STEC infection. As well as renal and intestinal damage, markers of central nervous system (CNS) injury were observed, including aberrant immunolocalization of neuronal nuclei (NeuN) and increased expression of glial fibrillary acidic protein (GFAP). These results show that bacteriophage 933W without an E. coli O157:H7 background is capable of inducing the pathogenic damage associated with STEC infection. In addition, a novel mouse model was developed to evaluate therapeutic approaches focused on the bacteriophage as a new target.

Shiga toxin (Stx)-producing Escherichia coli (STEC) strain B2F1 produces Stx type 2d, a toxin that becomes more toxic towards Vero cells in the presence of intestinal mucus. STEC that make Stx2d are more pathogenic to streptomycin (Str)-treated mice than most STEC that produce Stx2a or Stx2c. However, purified Stx2d is only 2- or 7-fold more toxic by the intraperitoneal route than Stx2a or Stx2c, respectively. We hypothesized, therefore, that the toxicity differences among Stx2a, Stx2c, and Stx2d occur at the level of delivery from the intestine. To evaluate that hypothesis, we altered the toxin type produced by stx2d+ mouse virulent O91:H21 clinical isolate B2F1 to Stx2a or Stx2c. Because B2F1 encodes two copies of stx2d, we did these studies in a derivative of B2F1 in which stx2d1 was deleted. Although the strains were equivalently virulent to the Str-treated mice at the 1010 dose, the B2F1 strain that produced Stx2a was attenuated relative to the ones that produced Stx2d or Stx2c when administered at 103 CFU/mouse. We next compared the oral toxicities of purified Stx2a, Stx2c, and Stx2d. We found that purified Stx2d is more toxic than Stx2a or Stx2c upon oral administration at 4 µg/mouse. Taken together, these studies suggest that Stx2 toxins are most potent when delivered directly from the bacterium. Furthermore, because Stx2d and Stx2c have the identical amino acid composition in the toxin B subunit, our results indicate that the virulence difference between Stx2a and Stx2d and Stx2c resides in the B or binding subunit of the toxins.

Shigella strains are important agents of bacillary dysentery, and in recent years Shigella sonnei has emerged as the leading cause of shigellosis in industrialized and rapidly developing countries. More recently, several S. sonnei and Shigella flexneri strains producing Shiga toxin (Stx) have been reported from sporadic cases and from an outbreak in America. In the present study we aimed to shed light on the evolution of a recently identified Shiga toxin producing S. sonnei (STSS) isolated in Europe. Here we report the first completely assembled whole genome sequence of a multidrug resistant (MDR) Stx-producing S. sonnei (STSS) clinical strain and reveal its phylogenetic relations. STSS 75/02 proved to be resistant to ampicillin, streptomycin, tetracycline, chloramphenicol, thrimetoprim, and sulfomethoxazol. The genome of STSS 75/02 contains a 4,891,717 nt chromosome and seven plasmids including the 214 kb invasion plasmid (pInv) harboring type III secretion system genes and associated effectors. The chromosome harbors 23 prophage regions including the Stx1 converting prophage. The genome carries all virulence determinants necessary for an enteroinvasive lifestyle, as well as the Stx1 encoding gene cluster within an earlier described inducible converting prophage. In silico SNP genotyping of the assembled genome as well as 438 complete or draft S. sonnei genomes downloaded from NCBI GenBank revealed that S. sonnei 75/02 belongs to the more recently diverged global MDR lineage (IIIc). Targeted screening of 1131 next-generation sequencing projects taken from NCBI Short Read Archive of confirms that only a few S. sonnei isolates are Stx positive. Our results suggest that the acquisition of Stx phages could have occurred in different environments as independent events and that multiple horizontal transfers are responsible for the appearance of Stx phages in S. sonnei strains.

Here we report the first complete nucleotide sequence of a Shiga toxin (Stx) converting phage from a Shigella sonnei clinical isolate that harbors stx1 operon, first identified in the chromosome of Shigella dysenteriae type 1. The phage named Shigella phage 75/02 Stx displayed Podoviridae morphology. It proved to be transferable to Escherichia coli K-12 strains, and cytotoxicity of the lysogenized strains was demonstrated in Vero cell cultures. Genomic analysis revealed that the prophage genome is circular and its size is 60,875 nt that corresponds to 76 ORFs. The genome of Shigella phage 75/02 Stx shows a great degree of mosaic structure and its architecture is related to lambdoid phages. All the deduced proteins, including the 37 hypothetical proteins showed significant homologies to Stx phage proteins present in databases. The phage uniformly inserted into the ynfG oxidoreductase gene framed by phage integrase and antirepressor genes in parental S. sonnei and in the three lysogenized K-12 strains C600, DH5α and MG1655. The Stx1 prophage proved to be stable in its bacterial hosts and remained inducible. [ABSTRACT FROM AUTHOR]

Hemolytic uremic syndrome (HUS) is characterized by thrombotic microangiopathy of the glomerular microcirculation and other vascular beds. Its defining clinical phenotype is acute kidney injury, microangiopathic anemia, and thrombocytopenia. There are many etiologies of HUS including infection by Shiga toxin (Stx)-producing bacterial strains, medications, viral infections, malignancy, and mutations of genes coding for proteins involved in the alternative pathway of complement. In the aggregate, although HUS is a rare disease, it is one of the most common causes of acute kidney injury in previously healthy children and accounts for a sizable number of pediatric and adult patients who progress to end stage kidney disease. There has been great progress over the past 20 years in understanding the pathophysiology of HUS and its related disorders. There has been intense focus on vascular injury in HUS as the major mechanism of disease and target for effective therapies for this acute illness. In all forms of HUS, there is evidence of both systemic and intra-glomerular inflammation and perturbations in the immune system. Renewed investigation into these aspects of HUS may prove helpful in developing new interventions that can attenuate glomerular and tubular injury and improve clinical outcomes in patients with HUS

Shiga toxin-producing Escherichia coli (STEC) are important food-borne pathogens, with the main virulence factor of this bacterium being its capacity to secrete Shiga toxins (Stxs). Therefore, the use of certain antibiotics for the treatment of this infection, which induces the liberation of Stxs, is controversial. Reactive oxygen and nitrogen species are also involved in the pathogenesis of different diseases. The purpose of this study was to analyze the effects of antibiotics on biofilms of STEC and the relationships between cellular stress and the release of Stx. To this end, biofilms of reference and clinical strains were treated with antibiotics (ciprofloxacin, fosfomycin and rifaximin) and the production of oxidants, the antioxidant defense system and toxin release were evaluated. Ciprofloxacin altered the prooxidant-antioxidant balance, with a decrease of oxidant metabolites and an increase of superoxide dismutase and catalase activity, being associated with high-levels of Stx production. Furthermore, inhibition of oxidative stress by exogenous antioxidants was correlated with a reduction in the liberation of Stx, indicating the participation of this phenomenon in the release of this toxin. In contrast, fosfomycin and rifaximin produced less alteration with a minimal production of Stx. Our data show that treatment of biofilm-STEC with these antibiotics induces oxidative stress-mediated release of Stx. [ABSTRACT FROM AUTHOR]

Background: In 2011, there was an outbreak of Shiga toxin-producing Escherichia coli (STEC) infections in Japan. Approximately 62 % of patients with hemolytic-uremic syndrome also showed symptoms of encephalopathy. To determine the mechanisms of onset for encephalopathy during STEC infections, we conducted an in vitro study with glial cell lines and primary glial cells. Results: Shiga toxin 2 (Stx-2) in combination with lipopolysaccharide (LPS), or LPS alone activates nuclear factor-κB (NF-κB) signaling in glial cells. Similarly, Stx-2 in combination with LPS, or LPS alone increases expression levels of aquaporin 4 (AQP4) in glial cells. It is possible that overexpression of AQP4 results in a rapid and increased influx of osmotic water across the plasma membrane into cells, thereby inducing cell swelling and cerebral edema. Conclusions: We have showed that a combination of Stx-2 and LPS induced apoptosis of glial cells recently. Glial cells are indispensable for cerebral homeostasis; therefore, their dysfunction and death impairs cerebral homeostasis and results in encephalopathy. We postulate that the onset of encephalopathy in STEC infections occurs when Stx-2 attacks vascular endothelial cells of the blood-brain barrier, inducing their death. Stx-2 and LPS then attack the exposed glial cells that are no longer in contact with the endothelial cells. AQP4 is overexpressed in glial cells, resulting in their swelling and adversely affecting cerebral homeostasis. Once cerebral homeostasis is affected in such a way, encephalopathy is the likely result in STEC patients. [ABSTRACT FROM AUTHOR]

Highlights: [•] Role of complement regulatory proteins in the pathogenesis of Stx-associated HUS. [•] Shiga toxin 2 (Stx2) binds to the factor H (FH) family proteins FHR-1 and FHL-1. [•] FHR-1 binds to Stx2 via its C-terminal region (SCRs 3–5). [•] FHR-1 competes with FH for Stx2 binding leading to reduced cofactor activity of FH. [•] FHL-1 retains its cofactor activity in the fluid phase when bound to Stx2. [ABSTRACT FROM AUTHOR]

Shiga toxin (Stx)-producing Escherichia coli (STEC) is a zoonotic pathogen with the ability to cause severe diseases like hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). Shiga toxin (Stx) is the key virulence factor in STEC and can be classified into two types, Stx1 and Stx2, and different subtypes. Stx2k is a newly reported Stx2 subtype in E. coli strains from diarrheal patients, animals, and raw meats exclusively in China so far. To understand the reservoir of Stx2k-producing E. coli (Stx2k-STEC), we investigated Stx2k-STEC strains in goat herds and examined their genetic characteristics using whole-genome sequencing. A total of 448 STEC strains were recovered from 2,896 goat fecal samples, and 37.95% (170/448) were Stx2k-STEC. Stx2k-STEC strains of serotype O93:H28 and sequence type 4038 (ST4038) were the most predominant and were detected over several years. Notably, 55% of Stx2k-STEC strains carried the heat-labile toxin (LT)-encoding gene ( elt ) defining enterotoxigenic E. coli (ETEC), thereby exhibiting the hybrid STEC/ETEC pathotype. Stx2k-converting prophage genomes clustered into four groups and exhibited high similarity within each group. Strains from patients, raw meat, sheep, and goats were intermixed distributed in the phylogenetic tree, indicating the risk for cross-species spread of Stx2k-STEC and pathogenic potential for humans. Further studies are required to investigate the Stx2k-STEC strains in other reservoirs and to understand the mechanism of persistence in these hosts. IMPORTANCE Strains of the recently reported Stx2k-STEC have been circulating in a variety of sources over time in China. Here, we show a high prevalence of Stx2k-STEC in goat herds. More than half of the strains were of the hybrid STEC/ETEC pathotype. Stx2k-STEC strains of predominant serotypes have been widespread in the goat herds over several years. Stx2k-converting prophages have exhibited a high level of similarity across geographical regions and time and might be maintained and transmitted horizontally. Given that goat-derived Stx2k-STEC strains share similar genetic backbones with patient-derived strains, the high prevalence of Stx2k-STEC in goats suggests that there is a risk of cross-species spread and that these strains may pose pathogenetic potential to humans. Our study thus highlights the need to monitor human Stx2k-STEC infections in this region and, by extension, in other geographic locations.

Abstract: Toxins of Escherichia coli (STEC) causing Uremic Hemolytic Syndrome (UHS) generate oxidative stress in human blood with more production of nitric oxide (NO) than reactive oxygen species (ROS). Shiga toxin (Stx) together with the hemolysin (Hly) increased lipid oxidation, as evaluated by malondialdehyde MDA and oxidation of proteins. The addition of Ziziphus mistol Griseb extracts decreased NO, ROS, MDA and simultaneously caused an increase in the degradation of oxidized proteins to advanced oxidation protein products (AOPPs) in controls and samples with toxins. Furthermore, the nitrosylated proteins/AOPP ratio was reduced, due to the increase of AOPP. Z. mistol Griseb extracts exhibited a high proportion of polyphenols and flavonoids, with evident correlation with ferrous reduction antioxidant potential (FRAP). The plasma of eight children with UHS showed oxidative stress and NO stimulus, comparable to the effect of toxins during the assays in vitro. UHS children presented high levels of nitrosylated proteins respect to control children of similar age. Although the degradation of oxidized proteins to AOPP rose in UHS children, the nitrosylated proteins/AOPP rate increased as a consequence of the elevated nitrosative stress observed in these patients. [Copyright &y& Elsevier]

Abstract: Introduction: Endothelial expression of tissue factor (TF) may play a major role in (Stx)-related hemolytic uremic syndrome. We examined human umbilical vein endothelial cell (HUVEC) monolayers to determine the interaction between TF and TF pathway inhibitor (TFPI), hypothesizing that changes in TFPI modulate TF expression. Materials and Methods: We studied 1) cell surface expression of globotriasylceramide (Gb3, the receptor for Stx) with Stx-1 (10 pM), TNFα (20Ng/ml), or Stx-1 plus TNFα compared to control, 2) gene expression of TF and TFPI, 3) total cellular and cell surface antigenic TF and TFPI, 4) TFPI secretion into supernatant, and 5) factor Xa production. Results and Conclusions: Gb3 expression, negligible with control and Stx-1 alone, increased significantly with TNFα and with Stx-1 plus TNFα. TF mRNA increased 1.25±0.32- fold (N=9; p=0.041) with Stx-1 alone vs. 2.82±0.92-fold (N=13; p<0.0005) with TNFα alone. However, Stx-1 plus TNFα yielded a 6.51±3.48-fold increase (N=17; p<0.0005). TFPI mRNA decreased with TNFα (p<0.001) and Stx-1 plus TNFα (p<0.0005). Total cellular and cell surface TF antigen increased significantly with TNFα, but no further with Stx-1 plus TNFα. Total TFPI cellular and cell surface antigen levels, and TFPI secretion decreased significantly with Stx-1 plus TNFα. Median factor Xa production for Stx-1 plus TNFα vs TNFα alone increased (p<0.001) 3.24-fold. Our results indicate that a subinhibitory concentration of Stx-1 plus TNFα impairs TFPI gene expression, synthesis, cell-surface association, and secretion, leading to augmented functional TF. [Copyright &y& Elsevier]

Peptides binding to a Gb3 mimic were selected from 12-mer peptide library. The self-assembled monolayer (SAM) of a Gb3 mimic was formed on the gold surface, and biopanning was carried out with the phage display peptide library. After three rounds of biopanning, four individual sequences were obtained from 10 phage clones, and the selected peptides having the specific 7-mer sequence (FHENWPS) showed affinities to the Gb3 mimic as strong as to RCA120. Molecular dynamics calculations suggested that the peptides bound to the Gb3 mimic by hydrophobic interaction and hydrogen bonding formation, and the cooperative interactions played an important role in the recognition. The Stx-1 binding was inhibited by the peptides. [Copyright &y& Elsevier]

Shiga toxin 1 (Stx1) catalyses the removal of a specific adenine from 28S rRNA within ribosomes (RNA-N-glycosylase activity) and the removal of multiple adenines from DNA (DNA-glycosylase activity). For the in vitro activity the toxin requires activation by trypsin, urea and DTT which releases the enzymatically active A1 fragment. We show that activated Stx1 acts on DNA as a heat-stable enzyme. Moreover, heat-treatment of the pro-enzyme at acidic pH turns it into an enzymatically active species which efficiently depurinates DNA. Although the effect of this treatment is centred on the enzyme and not on DNA, we found no evidence for covalent modification of the holotoxin. We suggest that high temperatures and acidic buffer induce unfolding of the holotoxin allowing the substrate to gain access to the active site. Possible practical applications (rapid assay for Stx1 detection, use of the toxin for DNA sequencing) are discussed. [Copyright &y& Elsevier]

Apoptosis is regulated by several pathways, such as caspases, mitogen activated protein kinase (MAPK) and cAMP/cAMP-dependent protein kinase A (PKA) cascade. This study investigated the effect of β2-adrenoceptor activation on Shiga toxin (Stx)2-induced apoptosis in renal tubular cells and the contribution of these signalling pathways. Cultured human adenocarcinoma-derived tubular cells were exposed to Stx2 (64 pg/mL) for 2–24 hr following the addition of the β2-adrenoceptor agonist (terbutaline) to the incubation medium. Stx2-induced apoptosis and its amelioration by β2-adrenoceptor activation was confirmed using DNA degradation assays and by flow cytometry for annexin V, mitochondrial membrane potential and caspase(-3 and -7) activity. Exposure of cells to Stx2 for 24 hr increased the DNA fragmentation to 11.6±0.9%, compared to 3.3±0.2% in control cells (P<0.05) but was decreased to approximately 5–7% (P<0.05) in the presence of terbutaline. Furthermore, Stx2-stimulated apoptosis, detected by TUNEL, annexin V and mitochondrial potential, was inhibited by terbutaline (P<0.05) which was prevented by cAMP-PKA inhibitors and a β2-adrenoceptor antagonist. However, inhibition of Stx2-mediated caspase activity by terbutaline was partially blocked by cAMP-PKA inhibitors. On the other hand, p38MAPK inhibition by terbutaline prevented Stx2-induced apoptosis and caspase activity through a cAMP-independent pathway via β2-adrenoceptor. These data indicate that β2-adrenoceptor activation can inhibit Stx2-induced apoptosis of the cells, which may be caused by a reduction in caspase activity through cAMP-PKA activation and the p38MAPK pathway. [Copyright &y& Elsevier]

Shiga toxin 2-converting phage was isolated from Escherichia coli O157:H7 associated with an outbreak that occurred in Okayama, Japan in 1996 (M. Watarai, T. Sato, M. Kobayashi, T. Shimizu, S. Yamasaki, T. Tobe, C. Sasakawa and Y. Takeda, Infect. Immun. 61 (1998) 3210–3204). In this study, we analyzed the complete nucleotide sequence of Shiga toxin 2-converting phage, designated Stx2φ-I, and compared it with three recently reported Stx2-phage genomes. Stx2φ-I consisted of 61,765 bp, which included 166 open reading frames. When compared to 933W, VT2-Sakai and VT2-Sa phages, six characteristic regions (regions I–VI) were found in the Stx2 phage genomes although overall homology was more than 95% between these phages. Stx2φ-I exhibited remarkable differences in these regions as compared with VT-2 Sakai and VT2-Sa genes but not with 933W phage. Characteristic repeat sequences were found in regions I–IV where the genes responsible for the construction of head and tail are located. Regions V and VI, which are the most distinct portion in the entire phage genome were located in the upstream and downstream regions of the Stx2 operons that are responsible for the immunity and replication, and host lysis. These data indicated that Stx2φ-I is less homologous to VT2-Sakai and VT2-Sa phages, despite these three phages being found in the strains isolated at the almost same time in the same geographic region but closely related to 933W phage which was found in the E. coli O157 strain 933W isolated 14 years ago in a different geographic area. [Copyright &y& Elsevier]

Shiga toxin (Stx)-producing Escherichia coli (STEC) strain B2F1 produces Stx type 2d, a toxin that becomes more toxic towards Vero cells in the presence of intestinal mucus. STEC that make Stx2d are more pathogenic to streptomycin (Str)-treated mice than most STEC that produce Stx2a or Stx2c. However, purified Stx2d is only 2- or 7-fold more toxic by the intraperitoneal route than Stx2a or Stx2c, respectively. We hypothesized, therefore, that the toxicity differences among Stx2a, Stx2c, and Stx2d occur at the level of delivery from the intestine. To evaluate that hypothesis, we altered the toxin type produced by stx2d+ mouse virulent O91:H21 clinical isolate B2F1 to Stx2a or Stx2c. Because B2F1 encodes two copies of stx2d, we did these studies in a derivative of B2F1 in which stx2d1 was deleted. Although the strains were equivalently virulent to the Str-treated mice at the 1010 dose, the B2F1 strain that produced Stx2a was attenuated relative to the ones that produced Stx2d or Stx2c when administered at 103 CFU/mouse. We next compared the oral toxicities of purified Stx2a, Stx2c, and Stx2d. We found that purified Stx2d is more toxic than Stx2a or Stx2c upon oral administration at 4 µg/mouse. Taken together, these studies suggest that Stx2 toxins are most potent when delivered directly from the bacterium. Furthermore, because Stx2d and Stx2c have the identical amino acid composition in the toxin B subunit, our results indicate that the virulence difference between Stx2a and Stx2d and Stx2c resides in the B or binding subunit of the toxins. [ABSTRACT FROM AUTHOR]

Many of the current accredited methods for the molecular detection of Shiga toxin-producing Escherichia coli (STEC) in foods rely on a PCR-based screen for the pathotype-specific genetic markers stx and eae. Unfortunately, these methods can inaccurately conclude the presence of E. coli containing both stx and eae because of the inability of the methods to determine if the two genes originated from a single organism as opposed to a mixture of organisms. This study was undertaken to evaluate if a droplet digital PCR (ddPCR)-based method that does not require DNA isolation could reliably identify the presence of an STEC containing eae in beef samples by confirming that both genes reside within the same cell, even when present in a mixed culture. The ddPCR system used in this study, dd-Check STEC Solution (Bio-Rad), works without the need for DNA isolation by partitioning intact cells into emulsion droplets, where they are lysed, and subsequently undergo multiplexed endpoint PCR. This enables the assay to differentiate between samples where a single organism contains both stx and eae from samples in which stx and eae reside in different organisms. Comparisons were made between the dd-Check STEC Solution, the BAX System Real-Time PCR STEC assay suite (Hygiena), and the iQ-Check STEC PCR detection kit (Bio-Rad) using 37 unique simulations of E. coli contamination in ground beef. While no single platform was consistently superior at detecting eae and stx across all pathogens tested, the results indicated that the dd-Check STEC Solution has the potential to reduce the number of inaccurately identified samples when screening for E. coli with a stx + , eae + genotype because it can identify the co-existence of multiple virulence genes within a cell even when in the presence of a mixed microbial population containing identical genes. Ultimately, incorporation of this system could result in substantial cost savings by reducing the expenses incurred when product samples are incorrectly classified as containing E. coli with a stx + , eae + genotype. • Detects Shiga-toxin producing Escherichia coli (STEC) in the presence of complex food matrices such as ground beef • Detected genes for Shiga toxin (stx) and intimin (eae) from 47 unique E. coli strains (22 different O-groups) • Distinguishes co-localized genes within single cells from mixed populations • Results were comparable to two commercially available molecular screening platforms. [ABSTRACT FROM AUTHOR]

Shiga toxin (Stx) is the key virulence factor in Shiga toxin producing Escherichia coli (STEC), which can cause diarrhea and hemorrhagic colitis with life-threatening complications. Stx comprises two toxin types, Stx1 and Stx2. Several Stx1/Stx2 subtypes have been identified in E. coli , which are variable in sequences, toxicity and host specificity. Here, we report the identification of a novel Stx2 subtype, designated Stx2k, in E. coli strains widely detected from diarrheal patients, animals, and raw meats in China over time. Stx2k exhibits varied cytotoxicity in vitro among individual strains. The Stx2k converting prophages displayed considerable heterogeneity in terms of insertion site, genetic content and structure. Whole genome analysis revealed that the stx 2k -containing strains were genetically heterogeneous with diverse serotypes, sequence types, and virulence gene profiles. The nine stx 2k -containing strains formed two major phylogenetic clusters closely with strains belonging to STEC, enterotoxigenic E. coli (ETEC), and STEC/ETEC hybrid. One stx 2k -containing strain harbored one plasmid-encoded heat-stable enterotoxin sta gene and two identical copies of chromosome-encoded stb gene, exhibiting STEC/ETEC hybrid pathotype. Our finding enlarges the pool of Stx2 subtypes and highlights the extraordinary genomic plasticity of STEC strains. Given the wide distribution of the Stx2k-producing strains in diverse sources and their pathogenic potential, Stx2k should be taken into account in epidemiological surveillance of STEC infections and clinical diagnosis. [ABSTRACT FROM AUTHOR]

Hemolytic uremic syndrome (HUS), principally caused by shiga toxins (Stxs), is associated with Shiga toxin-producing Escherichia coli (STEC) infections. We previously reported Stx2 expression by host cells in vitro and in vivo . As the genes encoding the two Stx subunits are located in bacteriophage genomes, the aim of the current study was to evaluate the role of bacteriophage induction in HUS development in absence of an E. coli O157:H7 genomic background. Mice were inoculated with a non-pathogenic E. coli strain carrying the lysogenic bacteriophage 933W (C600Φ933W), and bacteriophage excision was induced by an antibiotic. The mice died 72 h after inoculation, having developed pathogenic damage typical of STEC infection. As well as renal and intestinal damage, markers of central nervous system (CNS) injury were observed, including aberrant immunolocalization of neuronal nuclei (NeuN) and increased expression of glial fibrillary acidic protein (GFAP). These results show that bacteriophage 933W without an E. coli O157:H7 background is capable of inducing the pathogenic damage associated with STEC infection. In addition, a novel mouse model was developed to evaluate therapeutic approaches focused on the bacteriophage as a new target.

Shigella strains are important agents of bacillary dysentery, and in recent years Shigella sonnei has emerged as the leading cause of shigellosis in industrialized and rapidly developing countries. More recently, several S. sonnei and Shigella flexneri strains producing Shiga toxin (Stx) have been reported from sporadic cases and from an outbreak in America. In the present study we aimed to shed light on the evolution of a recently identified Shiga toxin producing S. sonnei (STSS) isolated in Europe. Here we report the first completely assembled whole genome sequence of a multidrug resistant (MDR) Stx-producing S. sonnei (STSS) clinical strain and reveal its phylogenetic relations. STSS 75/02 proved to be resistant to ampicillin, streptomycin, tetracycline, chloramphenicol, thrimetoprim, and sulfomethoxazol. The genome of STSS 75/02 contains a 4,891,717 nt chromosome and seven plasmids including the 214 kb invasion plasmid (pInv) harboring type III secretion system genes and associated effectors. The chromosome harbors 23 prophage regions including the Stx1 converting prophage. The genome carries all virulence determinants necessary for an enteroinvasive lifestyle, as well as the Stx1 encoding gene cluster within an earlier described inducible converting prophage. In silico SNP genotyping of the assembled genome as well as 438 complete or draft S. sonnei genomes downloaded from NCBI GenBank revealed that S. sonnei 75/02 belongs to the more recently diverged global MDR lineage (IIIc). Targeted screening of 1131 next-generation sequencing projects taken from NCBI Short Read Archive of confirms that only a few S. sonnei isolates are Stx positive. Our results suggest that the acquisition of Stx phages could have occurred in different environments as independent events and that multiple horizontal transfers are responsible for the appearance of Stx phages in S. sonnei strains.

This review describes the epidemiology, pathophysiology, presentation, clinical causes, treatment, and long-term prognosis of pediatric patients who present with thrombotic microangiopathy. The focus is on hemolytic uremic syndrome and thrombotic thrombocytopenic purpura, the most common phenotypes of thrombotic microangiopathy., (Copyright © 2013 Elsevier Inc. All rights reserved.)