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Shiga toxin (Stx) is the key virulent factor in Shiga toxin-producing Escherichia coli (STEC). To date, three Stx1 subtypes and seven Stx2 subtypes have been described in E. coli , which differed in receptor preference and toxin potency. Here, we identified a novel Stx2 subtype designated Stx2h in E. coli strains isolated from wild marmots in the Qinghai-Tibetan plateau, China. Stx2h shares 91.9% nucleic acid sequence identity and 92.9% amino acid identity to the nearest Stx2 subtype. The expression of Stx2h in type strain STEC299 was inducible by mitomycin C, and culture supernatant from STEC299 was cytotoxic to Vero cells. The Stx2h converting prophage was unique in terms of insertion site and genetic composition. Whole genome-based phylo- and patho-genomic analysis revealed STEC299 was closer to other pathotypes of E. coli than STEC, and possesses virulence factors from other pathotypes. Our finding enlarges the pool of Stx2 subtypes and highlights the extraordinary genomic plasticity of E. coli strains. As the emergence of new Shiga toxin genotypes and new Stx-producing pathotypes pose a great threat to the public health, Stx2h should be further included in E. coli molecular typing, and in epidemiological surveillance of E. coli infections.

Shiga toxins (Stx) produced by Shiga toxin-producing Escherichia coli (STEC) and enterohemorrhagic E. coli (EHEC) are ribosome-inactivating AB 5 proteins that consist of one enzymatic active A-subunit (StxA) and a pentamer of non-covalently linked B-subunits (StxB). The description of Stx as an AB 5 protein and the observation that A-subunits without their corresponding B-subunits also intoxicate eukaryotic cells, led to the question whether A- and B-subunits are produced in the bacteria in a 1:5 ratio or whether the A-subunit of the clinically most prominent subtype Stx2a is transcribed in excess revealing free A-subunits released in the bacterial environment. The aim of this study was therefore, to investigate the genetic and protein-based background for this observation in six Stx2a-encoding STEC and EHEC wildtype strains. For this purpose, transcriptional analysis of the Stx2a subunit genes, stxA2a and stxB2a , was performed by quantitative real-time PCR in one foodborne O113:H21 STEC isolate (strain TS18/08) and five HUS-associated EHEC strains with the serotypes O157:H7/H − (HUSEC003, HUSEC004), O103:H − (HUSEC008), O26:H11 (HUSEC018), and O104:H4 (LB226692). Contrary to the hypothesis that the A- and B-subunit genes are expressed in a ratio of 1:5 comparable to the holotoxin structure or in a ratio of 1:1 based on the operon structure, the results showed that stxA2a was expressed 1.90 ± 0.55-times stronger than the gene encoding the B-subunit, possibly indicating the presence of free A-subunits. In addition, strain-specific differences regarding the mRNA fold-changes of the A-subunit gene were observed. By use of native polyacrylamide gel electrophoresis and subsequent Western blot analysis, those single A-subunits were indeed detected in the culture supernatants of all six strains. To investigate whether the transcription ratios between A- and B-subunits observed are in a similar range as the amount of subunit proteins present after translation, a quantitative ELISA specific for StxA2a and StxB2a was established. Quantification of the subunits on protein level by use of ELISA revealed that the subunit ratio of StxA2a:StxB2a is 1.10 ± 0.20 for the strains HUSEC003, HUSEC004 and HUSEC008, but 4.63 ± 0.31 for the strains TS18/08, LB226692, and HUSEC018. The results of this study demonstrated that on both, the transcriptional and the translational level, the established 1:5 subunit ratio is not present in all investigated strains. In addition, the ratios observed after translation indicate that in some strains StxA2a subunits are even produced in higher amounts than B-subunits.

Early determination of the Shiga toxin type of Shiga toxin-producing Escherichia coli (STEC) is crucial for guiding STEC-infected patients for proper and timely treatment and patient care. Most diagnostic microbiology laboratories rely on PCR assays to detect the presence of stx1 and/or stx2 and enzymatic immunoassays (EIA) to detect the presence of the Shiga toxins 1 and/or 2 in STEC-positive stool samples. Occasionally, the stool samples test positive for STEC by PCR assays but test negative for the presence of Shiga toxins. Insufficient toxin production under laboratory conditions is the main culprit of this discordance. To test whether EIA-based STEC detection could be improved, various clinical STEC strains were treated with mitomycin C, which is a commonly used inducer of Shiga toxin production. A dose-dependent increase in Shiga toxin production, in response to mitomycin C doses of up to 500 ng/mL, was observed without any bactericidal effects. Depending on the serotype, 5–50 times more Shiga toxin 2 was produced than Shiga toxin 1. Shiga toxin production was not induced by the mitomycin C treatment in certain STEC serotypes carrying the toxin subtypes stx1a, stx2a, 2b, 2f, or 2h. This diversity in toxin production indicates that other factors may determine toxin expression in certain STEC strains, which warrant further exploration.

Escherichia coli (EHEC) and Shigella dysenteriae serotype 1 are enterohemorrhagic bacteria that induce hemorrhagic colitis. This, in turn, may result in potentially lethal complications, such as hemolytic uremic syndrome (HUS), which is characterized by thrombocytopenia, acute renal failure, and neurological abnormalities. Both species of bacteria produce Shiga toxins (Stxs), a phage-encoded exotoxin inhibiting protein synthesis in host cells that are primarily responsible for bacterial virulence. Although most studies have focused on the pathogenic roles of Stxs as harmful substances capable of inducing cell death and as proinflammatory factors that sensitize the host target organs to damage, less is known about the interface between the commensalism of bacterial communities and the pathogenicity of the toxins. The gut contains more species of bacteria than any other organ, providing pathogenic bacteria that colonize the gut with a greater number of opportunities to encounter other bacterial species. Notably, the presence in the intestines of pathogenic EHEC producing Stxs associated with severe illness may have compounding effects on the diversity of the indigenous bacteria and bacterial communities in the gut. The present review focuses on studies describing the roles of Stxs in the complex interactions between pathogenic Shiga toxin-producing E. coli, the resident microbiome, and host tissues. The determination of these interactions may provide insights into the unresolved issues regarding these pathogens.

BackgroundHemolytic uremic syndrome (HUS) is a multisystemic disease. In a nationwide study, we characterized the incidence, clinical course, and prognosis of HUS caused by Shiga toxin (Stx)–producing Escherichia coli (STEC) strains with emphasis on risk factors, disease severity, and long-term outcome.MethodsThe data on pediatric HUS patients from 2000 to 2016 were collected from the medical records. STEC isolates from fecal cultures of HUS and non-HUS patients were collected from the same time period and characterized by whole genome sequencing analysis.ResultsFifty-eight out of 262 culture-positive cases developed verified (n = 58, 22%) STEC-HUS. Another 29 cases had probable STEC-HUS, the annual incidence of STEC-HUS being 0.5 per 100,000 children. Eleven different serogroups were detected, O157 being the most common (n = 37, 66%). Age under 3 years (OR 2.4), stx2 (OR 9.7), and stx2a (OR 16.6) were found to be risk factors for HUS. Fifty-five patients (63%) needed dialysis. Twenty-nine patients (33%) developed major neurological symptoms. Complete renal recovery was observed in 57 patients after a median 4.0 years of follow-up. Age under 3 years, leukocyte count over 20 × 109/L, and need for dialysis were predictive factors for poor renal outcome.ConclusionsAge under 3 years, stx2, and stx2a were risk factors for HUS in STEC-positive children. However, serogroup or stx types did not predict the renal outcome or major CNS symptoms.

Shiga toxin-producing Escherichia coli (STEC) cause substantial and costly illnesses. Leafy greens are the second most common source of foodborne STEC O157 outbreaks. We examined STEC outbreaks linked to leafy greens during 2009-2018 in the United States and Canada. We identified 40 outbreaks, 1,212 illnesses, 77 cases of hemolytic uremic syndrome, and 8 deaths. More outbreaks were linked to romaine lettuce (54%) than to any other type of leafy green. More outbreaks occurred in the fall (45%) and spring (28%) than in other seasons. Barriers in epidemiologic and traceback investigations complicated identification of the ultimate outbreak source. Research on the seasonality of leafy green outbreaks and vulnerability to STEC contamination and bacterial survival dynamics by leafy green type are warranted. Improvements in traceability of leafy greens are also needed. Federal and state health partners, researchers, the leafy green industry, and retailers can work together on interventions to reduce STEC contamination.

ABSTRACT Strictly lytic phages are considered powerful tools for biocontrol of foodborne pathogens. Safety issues needed to be addressed for the biocontrol of Shiga toxin-producing Escherichia coli (STEC) include: lysogenic conversion, Shiga toxin production through phage induction, and emergence/proliferation of bacteriophage insensitive mutants (BIMs). To address these issues, two new lytic phages, vB_EcoS_Ace (Ace) and vB_EcoM_Shy (Shy), were isolated and characterized for life cycle, genome sequence and annotation, pH stability and efficacy at controlling STEC growth. Ace was efficient in controlling host planktonic cells and did not stimulate the production of the Stx prophage or Shiga toxin. A single dose of phage did not lead to the selection of BIMs. However, when reintroduced, BIMs were detected after 24 h of incubation. The gain of resistance was associated with lower virulence, as a subset of BIMs failed to agglutinate with O157-specific antibody and were more sensitive to human serum complement. BIM's biofilm formation capacity and susceptibility to disinfectants was equal to that of the wild-type strain. Overall, this work demonstrated that phage Ace is a safe biocontrol agent against STEC contamination and that the burden of BIM emergence did not represent a greater risk in environmental persistence and human pathogenicity. The safety of using phages as biocontrol agents for STEC decontamination, from a human and environmental safety point of view.

Shiga toxin family members have recently been classified using a new nomenclature into three Stx1 subtypes (Stx1a, Stx1c, and Stx1d) and seven Stx2 subtypes (Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, Stx2f, and Stx2g). To develop screening methods for Stx genes, including all of these subtype genes, and Escherichia coli O26-, O111-, and O157-specific genes in laboratory investigations of Shiga toxin-producing E. coli (STEC) foodborne cases, we developed multiplex real-time PCR assays and evaluated their specificity and quantitative accuracy using STEC and non-STEC isolates, recombinant plasmids, and food enrichment cultures and by performing STEC spiking experiments with beef and sprout enrichment cultures. In addition, we evaluated the relationship between the recovery rates of the target strains by direct plating and immunomagnetic separation and the cycle threshold (CT) values of the real-time PCR assays for the Stx subtypes and STEC O26, O111, and O157 serogroups. All three stx1- and seven stx2-subtype genes were detected by real-time PCR with high sensitivity and specificity, and the quantitative accuracy of this assay was confirmed using control plasmids and STEC spiking experiments. The results of the STEC spiking experiments suggest that it is not routinely possible to isolate STEC from enrichment cultures with real-time PCR CT values greater than 30 by direct plating on MacConkey agar, although highly selective media and immunomagnetic beads were able to isolate the inoculated strains from the enrichment cultures. These data suggest that CT values obtained from the highly quantitative real-time PCR assays developed in this study provide useful information to develop effective isolation strategies for STEC from food samples. The real-time PCR assays developed here are expected to aid in investigations of infections or outbreaks caused by STEC harboring any of the stx-subtype genes in the new Stx nomenclature, as well as STEC O26, O111, and O157.

Shiga toxins (Stx) released by Stx-producing E. coli (STEC) are virulence factors that are most closely associated with hemolytic uremic syndrome (HUS), a life-threatening complication of intestinal infections by STEC. Stx have to enter into the circulatory system before they are delivered to target organs and cause damage. The presence of Stx in sera could be a risk indicator for HUS development. However, the detection of Stx, particularly Stx2, has been difficult due to the presence of Stx2-binding components in human serum. Here, we report new ELISA-based methods for the detection of Stx1 and Stx2 in human serum and the effect of guanidinium chloride on enhancing the sensitivity for the detection of Stx2. The recovery rate for Stx2 was 62% when Stx2-spiked serum samples were treated with guanidinium chloride at a concentration of 200 mM, in contrast to 17% without guanidinium chloride treatment. The effectiveness of guanidinium chloride treatment for the detection of Stx2 in human serum was validated using sera from STEC-infected patients. Coimmunoprecipitation results indicated a specific physical interaction between Stx2 and the human serum amyloid P component (HuSAP) in human serum samples. Our in vitro study demonstrated that the inhibition from HuSAP alone for the detection of Stx2 was only 20%, much less than 69.6% from human serum at Stx2 level 10 ng/mL, suggesting that there may be other factors that bind Stx2 in human serum. This study indicates that treatment of serum samples with guanidinium chloride may be useful for the early and sensitive detection of Stx2 in sera of STEC-infected patients, so preventive measures can be adopted in a timely manner.

Background. Shiga toxin (Stx) is the primary virulence factor of Stx-producing Escherichia coli (STEC). STEC can produce Stx1a and/or Stx2a, which are antigenically distinct. However, Stx2a-producing STEC are associated with more severe disease than strains producing both Stx1a and Stx2a. Methods and Results. To address the hypothesis that the reason for the association of Stx2a with more severe disease is because Stx2a crosses the intestinal barrier with greater efficiency that Stx1a, we covalently labeled Stx1a and Stx2a with Alexa Fluor 750 and determined the ex vivo fluorescent intensity of murine systemic organs after oral intoxication. Surprisingly, both Stxs exhibited similar dissemination patterns and accumulated in the kidneys. We next cointoxicated mice to determine whether Stx1a could impede Stx2a. Cointoxication resulted in increased survival and an extended mean time to death, compared with intoxication with Stx2a only. The survival benefit was dose dependent, with the greatest effect observed when 5 times more Stx1a than Stx2a was delivered, and was amplified when Stx1a was delivered 3 hours prior to Stx2a. Cointoxication with an Stx1a active site toxoid also reduced Stx2a toxicity. Conclusions. These studies suggest that Stx1a reduces Stx2a-mediated toxicity, a finding that may explain why STEC that produce only Stx2a are associated with more severe disease than strains producing Stx1a and Stx2a.