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Background: Camels harbouring multidrug-resistant Gram-negative bacteria are capable of transmitting various microorganisms to humans. This study aimed to determine the distribution of anti-microbial resistance among Escherichia coli (E. coli) isolated from the feces of apparently healthy camels in Egyptian abattoirs. Additionally, we sought to characterize Shiga toxin-producing E. coli (STEC) strains, assess their virulence potential, and investigate the possibility of camels spreading carbapenem- and colistin-resistant E. coli., Methods: 121 fecal swaps were collected from camels in different abattoirs in Egypt. Isolation and identification of E. coli were performed using conventional culture techniques and biochemical identification. All isolates obtained from the examined samples underwent genotyping through polymerase chain reaction (PCR) of the Shiga toxin-encoding genes (Stx1 and Stx2), the carbapenemase-encoding genes (bla KPC , bla OXA-48 , bla NDM , and bla VIM ), and the mcr genes for mcr-1 to mcr-5., Result: Bacteriological examination revealed 75 E. coli isolates. PCR results revealed that one strain (1.3%) tested positive for Stx1, and five (6.6%) were positive for Stx2. Among the total 75 strains of E. coli, the overall prevalence of carbapenemase-producing E. coli was 27, with 7 carrying bla OXA48 , 14 carrying bla NDM , and 6 carrying bla VIM . Notably, no strains were positive for bla KPC but a high prevalence rate of mcr genes were detected. mcr-1, mcr-2, mcr-3, and mcr-4 genes were detected among 3, 2, 21, and 3 strains, respectively., Conclusion: The results indicate that camels in Egypt may be a primary source of anti-microbial resistance (AMR) E. coli, which could potentially be transmitted directly to humans or through the food chain., (© 2024. The Author(s).)

Objectives: Shiga toxin-producing Escherichia coli (STEC) O157:H7 are zoonotic pathogens and transmission to humans occurs via contaminated food or contact with infected animals. The aim of this study was to describe the frequency, and distribution across the phylogeny, of antimicrobial resistance (AMR) determinants in STEC O157:H7 isolated from human cases in England., Methods: Short-read whole-genome sequencing data from 1473 isolates of STEC O157:H7 from all seven sub-lineages (Ia-Ic, IIa-IIc and I/II) were mapped to genes known to confer phenotypic resistance to 10 different classes of antibiotic. Long-read sequencing was used to determine the location and genomic architecture of the AMR determinants within phylogenetic clusters exhibiting multidrug resistance., Results: Overall, 216/1473 (14.7%) isolates had at least one AMR determinant, although the proportion of isolates exhibiting AMR varied by sub-lineage. The highest proportion of AMR determinants were detected in sub-lineages Ib (28/64, 43.7%), I/II (18/51, 35.3%) and IIc (122/440, 27.7%). In all sub-lineages, the most commonly detected AMR determinants conferred resistance to the aminoglycosides, tetracyclines and sulphonamides, while AMR determinants conferring resistance to fluroquinolones, macrolides and third-generation cephalosporins were rarely detected. Long-read sequencing analysis showed that the AMR determinants were co-located on the chromosome in sub-lineages Ib and lineage I/II, whereas those associated with sub-lineage IIc were encoded on the chromosome and/or large plasmids., Conclusions: AMR genes were unevenly distributed across the different sub-lineages of STEC O157:H7 and between different clades within the same sub-lineage. Long-read sequencing facilitates tracking the transmission of AMR at the pathogen and mobile genetic element level., (© Crown copyright 2023.)

After introduction of faecal multiplex PCR that includes targets for stx1 and stx2 genes, we found stx genes were detected in 120 specimens from 111 patients over a 31-month period from 2018-2020 from a total of 14,179 separate tests performed. The proportion of stx1 only vs stx2 only vs stx1 and stx2 was 35%, 22% and 42%, respectively. There were 54 specimens which were culture positive, with 33 different serotypes identified, the predominant serotype being O157:H7 (19%). Eighty-two patients had clinical data available; we found a high rate of fever (35%), bloody diarrhoea (34%), acute kidney injury (27%), hospital admission (80%) and detection of faecal co-pathogens (23%). Only one patient developed haemolytic uraemic syndrome. We found no significant association with stx genotype and any particular symptom or complication. We found a significant association of serotypes O157:H7 and O26:H11 with bloody stool, but no significant association with any other symptom or complication., (Crown Copyright © 2023. Published by Elsevier B.V. All rights reserved.)

Shiga toxin (Stxs)-producing enterohaemorrhagic Escherichia coli (EHEC) and Shigella dysenteriae serotype 1 are major causative agents of severe bloody diarrhea (known as hemorrhagic colitis) and hemolytic uremic syndrome (HUS) associated with extraintestinal complications such as acute renal failure and neurologic impairment in infected patients under 9 years of age. Extreme nephrotoxicity of Stxs in HUS patients is associated with severe outcomes, highlighting the need to develop technologies to detect low levels of the toxin in environmental or food samples. Currently, the conventional polymerase chain reaction (PCR) or immunoassay is the most broadly used assay to detect the toxin. However, these assays are laborious, time-consuming, and costly. More recently, numerous studies have described novel, highly sensitive, and portable methods for detecting Stxs from EHEC. To contextualize newly emerging Stxs detection methods, we briefly explain the basic principles of these methods, including lateral flow assays, optical detection, and electrical detection. We subsequently describe existing and newly emerging rapid detection technologies to identify and measure Stxs.

Background: Shiga toxin-producing Escherichia coli (STEC) is a zoonotic pathogen, that is transmitted from a variety of animals, especially cattle to humans via contaminated food, water, feaces or contact with infected environment or animals. The ability of STEC strains to cause gastrointestinal complications in human is due to the production of Shiga toxins (sxt). However, the transmission of multidrug-resistance STEC strains are linked with a severity of disease outcomes and horizontal spread of resistance genes in other pathogens. The result of this has emerged as a significant threat to public health, animal health, food safety, and the environment. Therefore, the purpose of this study is to investigate the antibiogram profile of enteric E. coli O157 isolated from food products and cattle faeces samples in Zagazig City, Al-Sharkia, Egypt, and to reveal the presence of Shiga toxin genes stx1 and stx2 as virulence factors in multidrug-resistant isolates. In addition to this, the partial 16S rRNA sequencing was used for the identification and genetic recoding of the obtained STEC isolates., Results: There was a total of sixty-five samples collected from different geographical regions at Zagazig City, Al-Sharkia-Egypt, which were divided into: 15 chicken meat (C), 10 luncheon (L), 10 hamburgers (H), and 30 cattle faeces (CF). From the sixty-five samples, only 10 samples (one from H, and 9 from CF) were identified as suspicious E. coli O157 with colourless colonies on sorbitol MacConkey agar media with Cefixime- Telurite supplement at the last step of most probable number (MPN) technique. Eight isolates (all from CF) were identified as multidrug-resistant (MDR) as they showed resistance to three antibiotics with multiple antibiotic resistance (MAR) index ≥ 0.23, which were assessed by standard Kirby-Bauer disc diffusion method. These eight isolates demonstrated complete resistance (100%) against amoxicillin/clavulanic acid, and high frequencies of resistance (90%, 70%, 60%,60%, and 40%) against cefoxitin, polymixin, erythromycin, ceftazidime, and piperacillin, respectively. Those eight MDR E. coli O157 underwent serological assay to confirm their serotype. Only two isolates (CF8, and CF13), both from CF, were showed strong agglutination with antisera O157 and H7, as well as resistance against 8 out of 13 of the used antibiotics with the highest MAR index (0.62). The presence of virulence genes Shiga toxins (stx1 and stx2) was assessed by PCR technique. CF8 was confirmed for carrying stx2, while CF13 was carrying both genes stx1, and stx2. Both isolates were identified by partial molecular 16S rRNA sequencing and have an accession number (Acc. No.) of LC666912, and LC666913 on gene bank. Phylogenetic analysis showed that CF8, and CF13 were highly homologous (98%) to E. coli H7 strain, and (100%) to E. coli DH7, respectively., Conclusion: The results of this study provides evidence for the occurrence of E. coli O157:H7 that carries Shiga toxins stx1 and/or stx2, with a high frequency of resistance to antibiotics commonly used in human and veterinary medicine, in Zagazig City, Al-Sharkia, Egypt. This has a high extent of public health risk posed by animal reservoirs and food products with respect to easy transmission causing outbreaks and transfer resistance genes to other pathogens in animal, human, and plants. Therefore, environmental, animal husbandry, and food product surveillance, as well as, clinical infection control, must be strengthened to avoid the extra spread of MDR pathogens, especially MDR STEC strains., (© 2023. The Author(s).)

Background: The low general toxicity against tumors expressing globotriaosylceramide (Gb3) and Shiga-like toxins produced by E. coli have been proposed as an anti-cancer therapy because of their specific target. This study aimed to determine the potency of the local strains of E. coli O157:H7 isolated from humans and cattle as a new breast cancer therapy by analyzing the cell cycle's inhibition and apoptosis induction., Material and Methods: Approximately 10 cultured T47D cells were subjected to Shiga-like toxin produced by four local isolates of E. coli O157:H7, including KL-48 (2) from humans, and SM-25 (1), SM-7 (1), DS-21 (4) from cattle. Using ATCC 43894 as a control, the treatment was observed for 24 h by two replications. In addition, a FITC-Annexin V and PI assay were used to observe apoptosis and necrosis effect, as well as to analyze the cell cycle using propidium iodide (PI) staining., Results: The results showed the toxicity effect of Shiga in the human T47 D cells line. The viability of the cells is subjected to Shiga-like toxins produced by KL-48 (2), SM7 (1), ATCC 43894, SM-25 (1), and DS-21 (4) isolates decreased with 15.20, 16.36, 22.17,  22.64, and 33.86%, in contrary to control of 94.36%. These were supported by the cells entering the late apoptosis of the cell cycle through each isolate with 67.66, 62.60, 63.68, 63.90, and 54.74%, and a control of 0.01%. Also, the necrosis cell for each treatment of 12.73, 19.3, 10.84, 10.53, and 4.86% was higher than the control of 5.51%. These were confirmed by the higher percentage of the cells treated with toxins of KL-48 (2), SM7(1), ATCC 43894, SM-25 (1), and DS-21 (4), which entered G0-G1 of the cell cycle phase with 66.41, 63.37, 61.52, 55.36, and 47.28%, respectively, than control of 40.69%. Additionally, the toxicity effect was supported by an increase in the cells entering the S and the G2-M phase of the cycle for each treatment., Conclusion: It is concluded that the Shiga-like toxin produced by E. coli O157:H7 local isolates can be developed as a drug against breast cancer based on its effect to arrest induction of the cell cycle and inducing apoptosis.

Enterohemorrhagic Escherichia coli (EHEC) is a subtype of pathogenic E. coli that causes diarrhea or hemorrhagic colitis in humans, which can progresses to hemolytic uremic syndrome (HUS), a leading cause of acute renal failure in children, and morbidity and mortality in adults. Stool samples (n = 273) of patients (1 day-40 years old) suffered from bloody diarrhea and abdominal cramps, were examined bacteriologically and molecularly for the presence and pathogenicity of EHEC with phylogenetic analysis of the obtained stx1, stx2, and eaeA virulence genes' sequences. Overall, 71 (26.01%) E. coli isolates were identified as EHEC with the following serogroupes: O1:H11 (3), O128:H2 (9), O26:H11 (6), O157:H7 (3), O25:H2 (7), O145:H328 (2), O125:H6 (9), O86:H8 (5), O18:H15 (11) and untypable (16). The highest isolation rate were in samples belonged to infants below two years old (42.25%). Antimicrobial susceptibility testing showed that all isolates were highly sensitive to ciprofloxacin, nitrofurantoin, gentamycin, imipenem and vancomycin (100% each), however, they were resistant to ampicillin, cephalexin, penicillin and tetracycline (100% each). In-vitro pathogenicity testing of the isolates revealed that 67 (94.37%) isolates were positive for Congo red test, 47 (66.20%) isolates possessed P fimbriae (MRHA) and 17 (23.94%) possessed type 1 fimbriae (MSHA). Moreover, 46 (64.79%) isolates exhibited hemolysis and 42 (59.15%) isolates showed distinct cytopathic effect to Vero cells. Molecular detection of enterohemorrhagic E. coli (EHEC) pathotype virulence genes, confirmed the presence of stx1 gene in O157:H7 (MA2), O26:H11, O145:H328 and O125:H6 serogroups; stx2 gene in (O157:H7 (MA1), O128:H2 and O25:H2; while all serogroups except (O125:H6) carried the eaeA intimin virulence gene. A phylogenetic tree, based on the nucleotide sequences of toxin-encoding genes, demonstrates that Shiga toxin E. coli (STEC) isolates have considerable genetic variation and belong to various phylogenetic groupings., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Objective: To analyze the results of an enhanced laboratory-surveillance protocol for bloody diarrhea aimed at identifying children with Shiga toxin-producing Escherichia coli (STEC) infection early in the course of the disease toward the early identification and management of patients with hemolytic uremic syndrome (HUS)., Study Design: The study (2010-2019) involved a referral population of 2.3 million children. Stool samples of patients with bloody diarrhea were screened for Shiga toxin (Stx) genes. Positive patients were rehydrated and monitored for hemoglobinuria until diarrhea resolved or STEC-HUS was diagnosed., Results: A total of 4767 children were screened; 214 (4.5%) were positive for either Stx1 (29.0%) or Stx2 (45.3%) or both Stx1+2 (25.7%); 34 patients (15.9%) developed STEC-HUS (0.71% of bloody diarrheas). Hemoglobinuria was present in all patients with HUS. Patients with Stx2 alone showed a greater risk of STEC-HUS (23.7% vs 12.7%) and none of the patients with Stx1 alone developed HUS. During the same period of time, 95 other patients were diagnosed STEC-HUS but were not captured by the screening program (26 had nonbloody diarrhea, 11 came from areas not covered by the screening program, and 58 had not been referred to the screening program, although they did meet the inclusion criteria). At HUS presentation, serum creatinine of patients identified by screening was significantly lower compared with that of the remaining patients (median 0.9 vs 1.51 mg/dL)., Conclusions: Nearly 1% of children with bloody diarrhea developed STEC-HUS, and its diagnosis was anticipated by the screening program for Stx. The screening of bloody diarrhea for Stx is recommended, and monitoring patients carrying Stx2 with urine dipstick for hemoglobinuria is suggested to identify the renal complication as early as possible., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Shiga toxins (Stxs) are classic bacterial toxins and major virulence factors of toxigenic Shigella dysenteriae and enterohemorrhagic Escherichia coli (EHEC). These toxins recognize a glycosphingolipid globotriaosylceramide (Gb3/CD77) as their receptor and inhibit protein synthesis in cells by cleaving 28S ribosomal RNA. They are the major cause of life-threatening complications such as hemolytic uremic syndrome (HUS), associated with severe cases of EHEC infection, which is the leading cause of acute kidney injury in children. The threat of Stxs is exacerbated by the lack of toxin inhibitors and effective treatment for HUS. Here, we briefly summarize the Stx structure, subtypes, in vitro and in vivo models, Gb3 expression and HUS, and then introduce recent studies using CRISPR-Cas9-mediated genome-wide screens to identify the host cell factors required for Stx action. We also summarize the latest progress in utilizing and engineering Stx components for biomedical applications.

Shiga toxin (Stx)-producing Escherichia coli (STEC) is an important foodborne pathogen with the ability to cause bloody diarrhea (BD) and hemolytic uremic syndrome (HUS). Little is known about enterohemolysin-encoded by ehxA . Here we investigated the prevalence and diversity of ehxA in 239 STEC isolates from human clinical samples. In total, 199 out of 239 isolates (83.26%) were ehxA positive, and ehxA was significantly overrepresented in isolates carrying stx 2a + stx 2c ( p < 0.001) and eae ( p < 0.001). The presence of ehxA was significantly associated with BD and serotype O157:H7. Five ehxA subtypes were identified, among which, ehxA subtypes B, C, and F were overrepresented in eae -positive isolates. All O157:H7 isolates carried ehxA subtype B, which was related to BD and HUS. Three ehxA groups were observed in the phylogenetic analysis, namely, group Ⅰ ( ehxA subtype A), group Ⅱ ( ehxA subtype B, C, and F), and group Ⅲ ( ehxA subtype D). Most BD- and HUS-associated isolates were clustered into ehxA group Ⅱ, while ehxA group Ⅰ was associated with non-bloody stool and individuals ≥10 years of age. The presence of ehxA + eae and ehxA + eae + stx 2 was significantly associated with HUS and O157:H7 isolates. In summary, this study showed a high prevalence and the considerable genetic diversity of ehxA among clinical STEC isolates. The ehxA genotypes (subtype B and phylogenetic group Ⅱ) could be used as risk predictors, as they were associated with severe clinical symptoms, such as BD and HUS. Furthermore, ehxA , together with stx and eae, can be used as a risk predictor for HUS in STEC infections.

Shiga toxin-producing E. coli (STEC) is a common foodborne pathogen in developed countries. STEC generates "attaching and effacing" (AE) lesions on colonic epithelium, characterized by effacement of microvilli and the formation of actin "pedestals" beneath intimately attached bacteria. In addition, STEC are lysogenized with a phage that, upon induction, can produce potent Shiga toxins (Stx), potentially leading to both hemorrhagic colitis and hemolytic uremic syndrome. Investigation of the pathogenesis of this disease has been challenging because STEC does not readily colonize conventional mice.Citrobacter rodentium (CR) is a related mouse pathogen that also generates AE lesions. Whereas CR does not produce Stx, a murine model for STEC utilizes CR lysogenized with an E. coli-derived Stx phage, generating CR(Φstx), which both colonizes conventional mice and readily gives rise to systemic disease. We present here key methods for the use of CR(Φstx) infection as a highly predictable murine model for infection and disease by STEC. Importantly, we detail CR(Φstx) inoculation by feeding, determination of pathogen colonization, production of phage and toxin, and assessment of intestinal and renal pathology. These methods provide a framework for studying STEC-mediated systemic disease that may aid in the development of efficacious therapeutics.

Shiga toxin-producing Escherichia coli (STEC) are usually found on food products due to contamination from the fecal origin, as their main environmental reservoir is considered to be the gut of ruminants. While this pathogen is far from the incidence of other well-known foodborne bacteria, the severity of STEC infections in humans has triggered global concerns as far as its incidence and control are concerned. Major control strategies for foodborne pathogens in food-related settings usually involve traditional sterilization/disinfection techniques. However, there is an increasing need for the development of further strategies to enhance the antimicrobial outcome, either on food-contact surfaces or directly in food matrices. Phages are considered to be a good alternative to control foodborne pathogens, with some phage-based products already cleared by the Food and Drug Administration (FDA) to be used in the food industry. In European countries, phage-based food decontaminants have already been used. Nevertheless, its broad use in the European Union is not yet possible due to the lack of specific guidelines for the approval of these products. Furthermore, some safety concerns remain to be addressed so that the regulatory requirements can be met. In this review, we present an overview of the main virulence factors of STEC and introduce phages as promising biocontrol agents for STEC control. We further present the regulatory constraints on the approval of phages for food applications and discuss safety concerns that are still impairing their use.

Background: Little is known on significance, diversity and characteristics of gut E. coli in goats despite their importance as food animals globally. We characterized the temporal dynamics in diversity of E. coli in fecal samples from a cohort of goat kids and adult meat goats on pasture over a one-year period. Isolates were characterized based on phylogenetic grouping, virulence genes; shiga toxins 1 and 2 (Stx1&Stx2) (STEC), intimin (eaeA), hemolysin (hly) and select important sero-groups (026, 045, 0103, 0126 and 0146) using molecular methods., Results: A total of 516 E. coli isolates were screened. Prevalence of virulence genes and STEC was 65 and 56% respectively. Prevalence of virulence genes and STEC was significantly higher in goat kids less than six months (76% /66%) than adults (48% /28%). Isolates with virulence profiles of two or more genes were also higher in young goat kids (50%) than adults (20%). Entero-pathogenic E. coli (EPEC-eaeA gene only) were mostly from pre-weaned goat kids while hly gene only isolates were significantly higher in adults. The stx1, stx2 and hly genes peaked around weaning (60, 63 and 52%) respectively. Goats kids were mostly hosts to group D (59%) while adults older than one year had B1 (75%) isolates. Group D isolates were most abundant at weaning (64%) and diarrhea samples (74%). Group B2 isolates overall (6%) were mostly detected around weaning (63%) while A isolates were 4% overall. Twenty-four isolates belonged to sero-groups 026, 0103 and 0146 with 70% of the isolates detected around weaning. Nineteen of these isolates were STEC with most harboring the stx1/stx2/hly/eae (25%) profile. Most belonged to O26 sero-group (75%) and phylogroup D (75%)., Conclusion: To our knowledge this is the first study to highlight longitudinal age related differences in E. coli phylogenetic diversity, abundance of virulence genes and select important sero-groups in goats. Differences detected suggest a possible role of age and weaning stress in influencing E. coli diversity in the gut of goats. The findings are relevant to both animal and public health to advise on further studies on caprine E. coli isolates as animal and human pathogens.

Introduction: Severe intestinal infections caused by V. cholerae, ETEC and EHEC have contributed to the mortality rate in developing countries. Vibrio Cholera, ETEC and EHEC bacterium with the production of CT, LT and Stx2 toxins respectively lead to severe watery and bloody diarrhea. This study aimed to investigate a trimeric vaccine candidate containing recombinant chimeric protein, encapsulate the protein in chitosan nanoparticles and assess its immunogenicity., Methods: The LSC recombinant gene was used. It is composed of LTB (L), STXB (S) and CTXB (C) subunits respectively. The LSC recombinant protein was expressed and purified and confirmed by western blotting. The purified protein was encapsulated in chitosan nanoparticles, and its size was measured. BalB/c mice were immunized in four groups through oral and injection methods by LSC protein. The antibody titer was then evaluated by ELISA, and finally, the challenge test of the toxins from all three bacteria was done on the immunized mouse., Results: After expression and purification LSC protein size of nanoparticles containing protein was measured at 104.6 nm. Nanoparticles were able to induce systemic and mucosal immune responses by generating a useful titer of IgG and IgA. The challenge results with LT, CT and Stx toxins showed that the LSC protein might partially neutralize the effect of toxins., Conclusion: LSC chimeric protein with the simultaneous three essential antigens have a protective effect against the toxins produced by ETEC, EHEC and Vibrio cholera bacteria and it can be used in vaccines to prevent Diarrhea caused by these three bacteria., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Temperate phage encoded Shiga toxin (Stx) kills the bacterivorous predator, Tetrahymena thermophila, providing Stx + Escherichia coli with a survival advantage over Stx - cells. Although bacterial death accompanies Stx release, since bacteria grow clonally the fitness benefits of predator killing accrue to the kin of the sacrificed organism, meaning Stx-mediated protist killing is a form of self-destructive cooperation. We show here that the fitness benefits of Stx production are not restricted to the kin of the phage-encoding bacteria. Instead, nearby "free loading" bacteria, irrespective of their genotype, also reap the benefit of Stx-mediated predator killing. This finding indicates that the phage-borne Stx exotoxin behaves as a public good. Stx is encoded by a mobile phage. We find that Stx-encoding phage can use susceptible bacteria in the population as surrogates to enhance toxin and phage production. Moreover, our findings also demonstrate that engulfment and concentration of Stx-encoding and susceptible Stx - bacteria in the Tetrahymena phagosome enhances the transfer of Stx-encoding temperate phage from the host to the susceptible bacteria. This transfer increases the population of cooperating bacteria within the community. Since these bacteria now encode Stx, the predation-stimulated increase in phage transfer increases the population of toxin encoding bacteria in the environment., (© 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Sequence type (ST) 33 of Shiga toxin-producing Escherichia coli (STEC) strain O91:H14 has been proposed as a potential domestic clone of STEC in Korea because of its high prevalence among human patients with mild diarrhea or asymptomatic carriers. Herein, the clonal diversity of 17 STEC O91:H14 isolates of ST33 during 2003 to 2014 was analyzed by pulsed-field gel electrophoresis, including 14 isolates from human patients and 3 from retail meats. Their virulence characteristics, acid resistance, and antimicrobial susceptibility were also determined. Our results showed that all isolates were clustered mainly into three different pulsotypes and were likely low pathogenic without antimicrobial resistance.

Background: Shiga toxins comprise a family of related proteins produced by bacteria Shigella dysenteriae and some strains of Escherichia coli that cause severe clinical manifestations. Severe Shiga toxin intoxication results in Haemolytic-Uremic Syndrome (HUS), up to 50% of HUS patients manifest some degree of renal failure and ~10% of such cases develop permanent renal failure or death., Objective: In present research work production of biologically active rStx from non-toxic rStxA and rStxB subunits were established that can be used in many biomedical applications., Methods: Purification of Shiga toxin from bacteria is a multistep time consuming process resulting in low yield. To overcome this problem, the rStxA and rStxB protein were separately cloned and expressed in E. coli host and purified through affinity chromatography. GST pull-down assay was performed for interaction study between rStxA and pentameric rStxB. The affinity between A and B subunits of reconstituted recombinant Shiga toxin (AB5) was determined by SPR. The biological activity of the toxin was confirmed in Vero cells and mouse lethality assay., Results: The yield of GST-StxA and His6X-StxB obtained after affinity chromatography was estimated to 2 and 5 mg/l, respectively. Samples analyzed in pull down assay revealed two bands of ~58 kDa (rStxA) and ~7.7 kDa (rStxB) on SDS-PAGE. Affinity was confirmed through SPR with KD of 0.85 pM. This rStx produced from 1:5 molar ratio found to be cytotoxic in Vero cell line and resulted lethality in mouse., Conclusions: Large scale production of rStx using the method can facilitate screening and evaluation of small molecule inhibitors for therapeutics development., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Glycosylation is a fundamental modification of proteins and membrane lipids. Toxins that utilize glycans as their receptors have served as powerful tools to identify key players in glycosylation processes. Here, we carried out Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9-mediated genome-wide loss-of-function screens using two related bacterial toxins, Shiga-like toxins (Stxs) 1 and 2, which use a specific glycolipid, globotriaosylceramide (Gb3), as receptors, and the plant toxin ricin, which recognizes a broad range of glycans. The Stxs screens identified major glycosyltransferases (GTs) and transporters involved in Gb3 biosynthesis, while the ricin screen identified GTs and transporters involved in N-linked protein glycosylation and fucosylation. The screens also identified lysosomal-associated protein transmembrane 4 alpha (LAPTM4A), a poorly characterized four-pass membrane protein, as a factor specifically required for Stxs. Mass spectrometry analysis of glycolipids and their precursors demonstrates that LAPTM4A knockout (KO) cells lack Gb3 biosynthesis. This requirement of LAPTM4A for Gb3 synthesis is not shared by its homolog lysosomal-associated protein transmembrane 4 beta (LAPTM4B), and switching the domains between them determined that the second luminal domain of LAPTM4A is required, potentially acting as a specific "activator" for the GT that synthesizes Gb3. These screens also revealed two Golgi proteins, Transmembrane protein 165 (TMEM165) and Transmembrane 9 superfamily member 2 (TM9SF2), as shared factors required for both Stxs and ricin. TMEM165 KO and TM9SF2 KO cells both showed a reduction in not only Gb3 but also other glycosphingolipids, suggesting that they are required for maintaining proper levels of glycosylation in general in the Golgi. In addition, TM9SF2 KO cells also showed defective endosomal trafficking. These studies reveal key Golgi proteins critical for regulating glycosylation and glycolipid synthesis and provide novel therapeutic targets for blocking Stxs and ricin toxicity., Competing Interests: The authors have declared that no competing interests exist.

Despite their general low incidence, Shiga toxin-producing Escherichia (E.) coli (STEC) infections are considered an important public health issue due to the severity of illness that can develop, particularly in young children. We report on two Austrian petting zoos, one in Tyrol (2015) and one in Vorarlberg (2016), which were identified as highly likely infection sources of STEC infections. The petting zoo related cases involved a case of hemolytic uremic syndrome (HUS) due to STEC O157:HNM in 2015 and an outbreak of STEC O157:H7 infections affecting five young children and two adults in 2016. The HUS case accounted for 2.8% of the 36 STEC O157:HNM/H7 infections notified in Austria in 2015 (5,9% of 17 HUS cases). The seven cases described for 2016 accounted for 4.0% of the 177 human STEC infections documented for Austria in 2016, and for 19.4% of the 36 STEC O157:HNM/H7 infections notified that year. The evaluation of the STEC infections described here clearly underlines the potential of sequence-based typing methods to offer suitable resolutions for public health applications. Furthermore, we give a state-of-the-art mini-review on the risks of petting zoos concerning exposure to the zoonotic hazard STEC and on proper measures of risk-prevention., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

A treasure trove of intracellular cancer drug targets remains hidden behind cell membranes. However, engineered pathogen-derived toxins such as Shiga toxins can deliver small or macromolecular drugs to specific intracellular organelles. After binding to ganglioglobotriaosylceramide (Gb3, CD77), the non-toxic subunit B (StxB) of the Shiga-holotoxin is endocytosed and delivers its payload by a unique retrograde trafficking pathway via the endoplasmic reticulum to the cytosol. This review provides an overview of biomedical applications of StxB-based drug delivery systems in targeted cancer diagnosis and therapy. Biotechnological production of the Stx-material is discussed from the perspective of developing efficacious and safe therapeutics., (Copyright © 2018. Published by Elsevier Inc.)

Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen, and serotype O157:H7 is typically associated with severe disease. Australia is unique in its STEC epidemiology, as severe cases are typically associated with non-O157 serogroups, and locally acquired O157 isolates are H-negative/nonmotile. The H-negative phenotype and reduced severity of disease compared to that associated with H7/motile strains are distinct features of Australian O157 strains, but the molecular mechanism behind this phenotype has not been reported. Accurate characterization of the H-negative phenotype is important in epidemiological surveillance of STEC. Serotyping is moving away from phenotype-based methods, as next generation sequencing allows rapid extrapolation of serotype through in silico detection of the O-antigen processing genes, wzx , wzy , wzm , and wzt , and the H-antigen gene, fliC The detection and genotyping of fliC alone is unable to determine the motility of the strain. Typically, most Australian O157:H-negative strains carry an H7 genotype yet phenotypically are nonmotile; thus, many are mischaracterized as H7 strains by in silico serotyping tools. Comparative genomic analysis of flagellar genes between Australian and international isolates was performed and an insertion at nucleotide (nt) 125 in the flgF gene was identified in H-negative isolates. Chi-square results showed that this insertion was significantly associated with the H-negative phenotype ( P < 0.0001). Phylogenetic analysis was also completed and showed that the Australian H-negative isolates with the insertion in flgF represent a clade within the O157 serogroup, distinct from O157:H7 serotypes. This study provides a genetic target for inferring the nonmotile phenotype of Australian O157 STEC, which increases the predictive value of in silico serotyping., (© Crown copyright 2018.)

Shiga toxin Escherichia coli (STEC) O157:H7 is an important zoonotic food borne pathogen causing gastroenteritis that may lead to life threatening hemorragic colitis (HC) and hemorrhagic uremic syndrome (HUS). 325 meat and tissue samples were tested for enumeration of O157:H7 strains using most probable number (MPN)-PCR targeting their specific genes flicH7 and rfbO157 followed by isolation, sereotyping and pathogenicity testing. The overall prevalence of O157:H7 was 41.3% (134/325) along the production and supply chain of pork (PSCP), being higher in supply chain (59%, 118/200) as compared to pig farms (12.8%, 16/125). Along the PSCP, the highest prevalence was found in slaughter houses (86.25%, 69/80) followed by wet- (53.3%, 32/60) and super-markets (28.3%, 17/60). The MPN values ranged from 3 to 1100 MPN/g in overall positive samples, being higher in slaughter houses followed by wet and super markets. Except from intestine and meat samples of slaughter house, the MPN was found higher in summer as compared to winter samples. Eight STEC O157:H7 isolated from meat and liver samples were tested in Balb/C mice for pathogenicity. After development of clinical signs and symptoms, 50-83.3% mortality was produced in the infected mice. Histopathological investigations revealed visible necrosis of intestinal epithelial cells, shedding of cellular debris in the intestine, while in the kidney, necrosis of renal cortical portion of tubular epithelial cells was observed. STEC O157:H7 is prevalent along PSCP around Hubei of China in different proportions being alarmingly higher in supply chain and markets which is a matter of concern for public health., (Copyright © 2017. Published by Elsevier Ltd.)

The rapid spreading of polymyxin E (colistin) resistance among bacterial strains through the horizontally transmissible mcr-1 and mcr-2 plasmids has become a serious concern. The emergence of these genes in Shiga toxin-producing Escherichia coli (STEC), a group of human pathogenic bacteria was even more worrisome, urging us to investigate the prevalence of mcr genes among STEC isolates. A total of 1000 STEC isolates, recovered from livestock, wildlife, produce and other environmental sources in a major production region for leafy vegetables in California during 2006-2014, were screened by PCR for the presence of plasmid-borne mcr-1 and mcr-2. All isolates tested yielded negative results, indicating if any, the occurrence rate of mcr-1/mcr-2 among STEC was very low in this agricultural region. This study provides valuable information such as sample size needed and methodologies for future surveillance programs of antimicrobial resistance.

Escherichia coli is one of the main etiological agents of neonatal calf diarrhea (NCD). The objective of this study was to assess the presence of virulence genes, genetic diversity, and antibiotic resistance mechanisms in E. coli associated with NCD in Uruguay. PCR was used to assess the presence of intimin, Shiga-like toxin, and stable and labile enterotoxin genes. Resistance to fluoroquinolones and oxyimino-cephalosporins was estimated on Müller-Hinton agar plates. Further antibiotic disc-diffusion tests were performed to assess bacterial multi-resistance. The presence of PMQR, ESBL, MCR-1, and integron genes was evaluated. Isolates were typed using ERIC-PCR, and 20 were selected for MLST, adhesion to Hep-2 cells, in vitro biofilm formation, and eukaryotic cytotoxicity. The prevalence of ETEC genes was lower than 3% in each case (estA and elt). Six isolates were EPEC (eae+) and 2 were EHEC/STEC (eae+/stx1+). The results of a diversity analysis showed high genetic heterogenicity among isolates. Additionally, different sequence types, including ST10, ST21, and ST69, were assigned to selected isolates. Thirty-six percent (96/264) of the isolates were fluoroquinolone-resistant, with 61/96 (63.5%) being multidrug-resistant. Additionally, 6 were oxyimino-cephalosporin-resistant. The qnrB, qnrS1, and bla CTX-M-14 genes were detected, whereas no isolates carried the mcr-1 gene. Isolates had the ability to adhere to Hep-2 cells and form biofilms. Only 1 isolate expressed toxins in vitro. E. coli from NCD cases in Uruguay are very diverse, potentially virulent, and may interact with eukaryotic cells. Zoonotic potential, together with resistance traits and the presence of horizontal transfer mechanisms, may play a significant role in infections caused by these microorganisms.

Verocytotoxin (VT)-producing Escherichia coli (VTEC) are a significant foodborne public health hazard, where most human infections are associated with six serogroups (O157, O26, O103, O145, O111 and O104). VTEC was the fourth most commonly reported zoonosis in the EU in 2015, with 5901 confirmed human cases. Ruminant animals, including cattle, are a major reservoir of VTEC. The consumption of VTEC-contaminated animal-derived foodstuffs, especially undercooked ground beef, is an important transmission route. To the best of our knowledge, there are few data available on the contamination of VTEC in meat products in Italy. During 2015 and 2016, 250 raw meat samples were collected from retail markets in southern Italy (Apulia) and analysed for the occurrence of vtx genes (vtx1/vtx2) at the Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata (IZS PB, Italy). In addition, the isolates were characterized by determining the presence of VTEC main virulence factors, the antimicrobial resistance profiles and the genetic relatedness by pulsed-field gel electrophoresis (PFGE). The results have shown that 8.4% (21/250) of the samples were positive for vtx genes in the preliminary screening step but VTEC strains were isolated from only 2% (5/250) of overall meat analysed samples, including raw ground beef, beef hamburger and beef carpaccio. 5 isolates displayed a multi-drug resistance phenotype. All VTEC strains were analysed by XbaI-PFGE and dendrogram revealed 5 distinct restriction profiles, indicating their relatively high genetic diversity. Although this study demonstrates a low prevalence of VTEC in raw beef marketed in southern Italy, the presence of potentially pathogenic E. coli strains points to the need for proper hygiene during meat production to reduce the risk of foodborne illness and transmission of multi-drug resistant organisms via foods to humans., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Shiga toxin producing Escherichia coli O157:H7 (STEC O157) is naturally found in the gastrointestinal tract of cattle and can cause severe disease in humans. There is limited understanding of the population dynamics and microevolution of STEC O157 at herd level. In this study, isolates from a closed beef herd of 23 cows were used to examine the population turnover in the herd. Of the nine STEC O157 clades previously described, clade 7 was found in 162 of the 169 isolates typed. Multiple locus variable number tandem repeat analysis (MLVA) differentiated 169 isolates into 33 unique MLVA types. Five predominant MLVA types were evident with most of the remaining types containing only a single isolate. MLVA data suggest that over time clonal replacement occurred within the herd. Genome sequencing of 18 selected isolates found that the isolates were divided into four lineages, representing four different 'clones' in the herd. Genome data confirmed clonal replacement over time and provided evidence of cross transmission of strains between cows. The findings enhanced our understanding of the population dynamics of STEC O157 in its natural host that will help developing effective control measures to prevent the spread of the pathogen to the human population., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

In exposure to outer pressure, microorganisms are capable of entry into the Viable But Non-Culturable (VBNC) state, and thus survive under various elimination processing. The survival microorganisms may yield negative results on culturing, and cause false negative for this golden standard methodology. In this study, a novel PMA-LAMP assay on the detection of Enterohemorrhage E. coli and shiga toxins has been developed and evaluated, with further application on a number of food borne E. coli strains. LAMP primers were designed on the target of rfbe for Enterohemorrhage E. coli and stx1with stx2 for shiga toxins. Via specific penetration through the damaged cell membrane of dead cells and intercalating into DNA, PMA could prevent DNA amplification of dead bacteria from LAMP, which enabled the differentiation of bacteria between VBNC state and dead state. The established PMA-LAMP showed significant advantage in rapidity, sensitivity and specificity, compared with regular PCR assay. The applicability had also been verified, demonstrating the PMA-LAMP was capable of detection on Enterohemorrhage E. coli and shiga toxins., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Aims: To evaluate the performance of the IQ-Check kits and the USDA Microbiology Laboratory Guidebook (MLG) methods for detection of the top seven Shiga toxin-producing Escherichia coli (STEC) (O157:H7, O26, O45, O103, O111, O121 and O145) in ground beef and both STEC and Salmonella in co-inoculated samples., Methods and Results: Ground beef samples inoculated with ~10 CFU of STEC or both STEC and Salmonella Typhimurium were stored at 4°C for 72 h, followed by screening with the IQ-Check and BAX System kit (MLG) methods that employ different enrichment media. STEC and S. Typhimurium were detected after 12 and 18 h and their presence was confirmed by colony isolation., Conclusions: Both methods were able to detect STEC in ground beef after 12 h of enrichment in samples inoculated with low levels of the pathogen. STEC and S. Typhimurium can be detected and isolated in co-inoculated ground beef samples., Significance and Impact of the Study: The IQ-Check methods are comparable to the MLG methods for detection of STEC and simultaneous detection of STEC and S. Typhimurium in seeded ground beef after a short enrichment time, thus the IQ-Check method can be useful for the food industry for rapid detection of these pathogens., (Published 2016. This article has been contributed to by US Government employees and their work is in the public domain in the USA.)

Objective: To determine the serotypes and drug resistance profiles of Shiga toxin-producing Escherichia coli (STEC) in animal stools from the Weishan area in Shandong Province, China. To provide the basis for further study. Methods: Five hundred animal stool samples (from pigs, cattle, sheep, dogs and birds) were collected from the Weishan area and STEC strains were isolated from these samples. Strains were serotyped by a serum agglutination test, and their drug resistance profiles were determined through antimicrobial sensitivity experiments. In this study, PCR was used to detect tetracycline resistance genes ( tetA , tetB , tetC , tetD ) and beta-lactam resistance genes ( blaSHV -1, blaCTX - M , blaTEM ). Results: Sixteen strains of STEC were isolated from animal stool samples. Thirteen strains were isolated from pig stool samples, two from bovine stool samples and one from a sheep stool sample. Two of the strains were identified as E. coli O157:H7, and other 14 strains were non-O157 STEC of different serotypes. Antimicrobial sensitivity experiments showed that 15 of the strains were multidrug resistant. The rates of resistance were as follows: nalidixic acid (12/16 strains), sulfisoxazole (11/16), trimethoprim and sulphame-thoxazole (11/16), doxycycline (9/16), azithromycin (9/16), tetracycline (9/16), chloramphenicol (8/16) and streptomycin (8/16). Therefore, nalidixic acid showed the highest rate of resistance among the strains, followed by trimethoprim and sulphame-thoxazole, and sulfisoxazole. Resistance to cefepime or imipenem was not detected. In total, three types of drug resistance genes ( tetA , tetB and tetC ) were detected among the 16 strains. Conclusion: The results showed that STEC strains isolated from animals in the Weishan area were of a range of serotypes. The 16 strains of STEC isolated from animal stools in this area were resistant to a number of antibiotics, with many strains displaying multidrug resistance.

Clinical outcomes of Shiga toxin (stx)-producing Escherichia coli infection are largely determined by virulence gene subtypes. This study used a polymerase chain reaction (PCR)-pyrosequencing assay to analyze single-nucleotide polymorphisms for subtyping three major virulence genes (stx 1 , stx 2 , eae) of pathogenic E. coli (O157, O26, O111, and O103) isolated from cattle over a 2-year interval (n = 465) and human clinical cases (n = 42) in western Canada. Most bovine isolates were PCR positive for at least one target virulence gene (367/465), whereas 100% of human isolates harbored eae in combination with at least one stx gene. Four Shiga toxin (1a, 2a, 2c, and 2e) and four eae (λ/γ1-eae, ɛ-eae, θ/γ2-eae, and β-eae) subtypes were identified in over 25 distinct virulence genotypes. Among cattle isolates, every serogroup, but O103, presented a dominant genotype (O157: stx 1a +stx 2a +λ/γ1-eae, O26: β-eae alone, and O111: stx 1a +θ/γ2-eae). Similar patterns were found in human isolates, although it was not possible to establish a clear genotypic association between the two sources. Many O157 and non-O157 cattle isolates lacked stx genes; the absence was greater in non-O157 (75/258) and O157:non-H7 (19/40) than in O157:H7 strains (1/164). In addition, there was a greater diversity of virulence genotypes of E. coli isolated from cattle than those of human diseases, which could be due to sample characteristics (e.g., source and clinical condition). However, the majority of cattle strains had virulence profiles identical to those of clinical cases. Consequently, determining the presence of certain stx (stx 1a and stx 2a ) and eae (λ/γ1-eae) subtypes known to cause human disease would be a valuable tool for risk assessment and prediction of disease outcome along the farm-to-fork continuum.

Shiga toxin-producing Escherichia coli (STEC) causes diarrhea and hemorrhagic colitis with life-threatening complications, such as hemolytic uremic syndrome. The aim of this study was to assess the molecular epidemiologic features of non-O157 STEC strains from different resources in China and illustrate the role of animal reservoirs or animal-derived foodstuffs in human STEC infections. A collection of 301 non-O157 STEC isolates from domestic and wild animals (i.e., cattle, goat, pig, yak, pika, and antelope), raw meats (i.e., beef, pork, mutton, chicken, and duck), diarrheal patients, and healthy carriers in different regions of China were selected in this study. Of the 301 analyzed STEC isolates, 67 serogroups, and 118 serotypes were identified; this included some predominant serogroups associated with human disease, such as O26, O45, O103, O111, and O121. Eighteen different combinations of stx subtypes were found. Eleven isolates carried the intimin gene eae , 93 isolates contained ehxA , and 73 isolates carried astA . The prevalence of other putative adhesion genes saa, paa, efa1 , and toxB was 28.90% (87), 6.98% (21), 2.31% (7), and 1% (3), respectively. The phylogenetic distribution of isolates was analyzed by multilocus sequence typing (MLST). Ninety-four sequence types were assigned across the 301 isolates. A subset of isolates recovered from yak and pika residing in the similar wild environments, Qinghai-Tibetan plateau, showed similar genetic profiles and more tendencies to cluster together. Isolates from goat and mutton exhibited close genetic relatedness with those from human-derived isolates, providing evidence that transmission may have occurred locally within intraspecies or interspecies, and importantly, from animal reservoirs, or raw meats to humans. Comparing isolates in this study with highly virulent strains by MLST, along with serotyping and virulence profiles, it is conceivable that some of isolates from goat, yak, or raw meats may have potential to cause human diseases.

Most Shiga toxin-producing Escherichia coli (STEC) strains associated with severe disease, such as hemolytic-uremic syndrome (HUS), carry large enterohemolysin-encoding (ehxA) plasmids, e.g., pO157 and pO103, that contribute to STEC clinical manifestations. Six ehxA subtypes (A through F) exist that phylogenetically cluster into eae-positive (B, C, F), a mix of eae-positive (E) and eae-negative (A), and a third, more distantly related, cluster of eae-negative (D) STEC strains. While subtype B, C, and F plasmids share a number of virulence traits that are distinct from those of subtype A, sequence data have not been available for subtype D and E plasmids. Here, we determined and compared the genetic composition of four subtype D and two subtype E plasmids to establish their evolutionary relatedness among ehxA subtypes and define their potential role in pathogenicity. We found that subtype D strains carry one exceptionally large plasmid (>200 kbp) that carries a variety of virulence genes that are associated with enterotoxigenic and enterohemorrhagic E. coli, which, quite possibly, enables these strains to cause disease despite being food isolates. Our data offer further support for the hypothesis that this subtype D plasmid represents a novel virulence plasmid, sharing very few genetic features with other plasmids; we conclude that these plasmids have evolved from a different evolutionary lineage than the plasmids carrying the other ehxA subtypes. In contrast, the 50-kbp plasmids of subtype E (pO145), although isolated from HUS outbreak strains, carried only few virulence-associated determinants, suggesting that the clinical presentation of subtype E strains is largely a result of chromosomally encoded virulence factors., Importance: Bacterial plasmids are known to be key agents of change in microbial populations, promoting the dissemination of various traits, such as drug resistance and virulence. This study determined the genetic makeup of virulence plasmids from rare enterohemolysin subtype D and E Shiga toxin-producing E. coli strains. We demonstrated that ehxA subtype D plasmids represent a novel E. coli virulence plasmid, and although subtype D plasmids were derived from nonclinical isolates, they encoded a variety of virulence determinants that are associated with pathogenic E. coli In contrast, subtype E plasmids, isolated from strains recovered from severely ill patients, carry only a few virulence determinants. The results of this study reemphasize the plasticity and vast diversity among E. coli plasmids. This work demonstrates that, although E. coli strains of certain serogroups may not be frequently associated with disease, they should not be underestimated in protecting human health and food safety., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Understanding the transmission dynamics of pathogens is essential to determine the epidemiology, ecology, and ways of controlling enterohemorrhagic Escherichia coli (EHEC) in animals and their environments. Our objective was to estimate the epidemiological fitness of common EHEC strains in cattle populations. For that purpose, we developed a Markov chain model to characterize the dynamics of 7 serogroups of enterohemorrhagic Escherichia coli (O26, O45, O103, O111, O121, O145, and O157) in cattle production environments based on a set of cross-sectional data on infection prevalence in 2 years in two U.S. states. The basic reproduction number (R0) was estimated using a Bayesian framework for each serogroup based on two criteria (using serogroup alone [the O-group data] and using O serogroup, Shiga toxin gene[s], and intimin [eae] gene together [the EHEC data]). In addition, correlations between external covariates (e.g., location, ambient temperature, dietary, and probiotic usage) and prevalence/R0 were quantified. R0 estimates varied substantially among different EHEC serogroups, with EHEC O157 having an R0 of >1 (∼1.5) and all six other EHEC serogroups having an R0 of less than 1. Using the O-group data substantially increased R0 estimates for the O26, O45, and O103 serogroups (R0 > 1) but not for the others. Different covariates had distinct influences on different serogroups: the coefficients for each covariate were different among serogroups. Our modeling and analysis of this system can be readily expanded to other pathogen systems in order to estimate the pathogen and external factors that influence spread of infectious agents., Importance: In this paper we describe a Bayesian modeling framework to estimate basic reproduction numbers of multiple serotypes of Shiga toxin-producing Escherichia coli according to a cross-sectional study. We then coupled a compartmental model to reconstruct the infection dynamics of these serotypes and quantify their risk in the population. We incorporated different sensitivity levels of detecting different serotypes and evaluated their potential influence on the estimation of basic reproduction numbers., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

This work were aimed to (a) determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Carum copticum essential oil (EO) against Escherichia. coli O157:H7 in vitro Trypticase Soy Broth, (TSB) and in ground beef; (b) evaluation of the effect of sub-inhibitory concentrations (sub-MICs) of EO on the growth of bacterium in TSB over 72 h (at 35 °C) and ground beef over 9 days (at 4 °C); and (c) investigation of gene expression involved in Shiga toxins production using relative quantitative real-time PCR method. The MIC in broth and ground beef medium were determined as 0.05 (v/v) and 1.75 % (v/w), respectively. In comparison with control cultures, the EO concentration of 0.03 % in broth caused reduction of colony counting as 1.93, 1.79, and 2.62 log10 CFU ml(-1) after 24, 48, and 72 h at 35 °C, and similarly EO (0.75 %) in ground beef resulted to reduction of colony counting as 1.03, 0.92, 1.48, and 2.12 log10 CFU g (-1) after 2, 5, 7, and 9 days at 4 °C, respectively. An increase and decrease in gene expression were observed as result of EO addition (0.03 %) to broth and (0.5 %) to ground beef was noticed, respectively.

The intracellular delivery of proteins with high efficiency in a receptor-specific manner is of great significance in molecular medicine and biotechnology, but remains a challenge. Herein, we present the development of a highly efficient and receptor-specific delivery platform for protein cargos by combining the receptor binding domain of Escherichia coli Shiga-like toxin and the translocation domain of Pseudomonas aeruginosa exotoxin A. We demonstrated the utility and efficiency of the delivery platform by showing a cytosolic delivery of diverse proteins both in vitro and in vivo in a receptor-specific manner. In particular, the delivery system was shown to be effective for targeting an intracellular protein and consequently suppressing the tumor growth in xenograft mice. The present platform can be widely used for intracellular delivery of diverse functional macromolecules with high efficiency in a receptor-specific manner. Biotechnol. Bioeng. 2016;113: 1639-1646. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

The role of antibiotics in the treatment of Shiga-like toxin (Stx)-producing E. coli infection is still controversial. This study investigated the effects of colistin on Vero cell cytotoxicity caused by the enterohemorrhagic EC O157:H7, and the effects of colistin on Stx and endotoxin release by EC O157:H7. Vero cells were incubated with supernatant collected from EC O157:H7 cultured for 18 h without (control) or with various concentrations of colistin. In the absence of colistin, Vero cell viability after 48 h was 29.1±6.5%. Under the same conditions, the overnight presence of colistin reduced cytotoxicity to Vero cells (viability: 97±3.5 to 56.5±14.4% for colistin concentrations ≥MIC). Sub-MIC concentrations of colistin also provided partial protection (viability: 38.8±12.5 to 36.6±14% for 0.125 and 0.06 mcg/ml colistin, respectively). Endotoxins contributed to the cytotoxic effects on Vero cells since lower but still significant protection was observed when colistin was added directly to the supernatant collected from cultures of untreated EC O157:H7. Colistin reduced Stx release in a concentration-dependent manner, also at sub-MIC concentrations. Coincubation of the supernatant from EC O157:H7 cultures with colistin markedly reduced the endotoxin concentration at all doses investigated. In conclusion, colistin protects Vero cells from EC O157:H7 at supra- and sub-MIC concentrations by inhibiting Stx release and binding endotoxins. Colistin might be a valuable treatment for clinically severe forms of EC O157:H7 infection.

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.

A novel procedure for the detection of specific DNA sequences has been developed. This procedure is based on the already known method employing PCR with appropriate primers and a sequence-specific DNA probe labeled with the fluorescent agent 6-carboxylfluorescein (FAM) at the 5' end and the fluorescence quencher BHQ-1 (black hole quencher) at the 3' end. However, instead of the detection of the fluorescence signal with the use of real-time PCR cyclers, fluorescence/luminescence spectrometers or fluorescence polarization readers, as in all previously-reported procedures, we propose visual observation of the fluorescence under UV light directly in the reaction tube. An example for the specific detection of the Shiga toxin-producing Escherichia coli (STEC) strains, by detecting Shiga toxin genes, is demonstrated. This method appears to be specific, simple, rapid and cost effective. It may be suitable for use in research laboratories, as well as in diagnostic units of medical institutions, even those equipped only with a thermocycler and a UV transilluminator, particularly if rapid identification of a pathogen is required.

Patterns of insertion sequence (IS)629, norV genotype, and Shiga toxin (Stx) genotype distribution were investigated amongst 203 enterohemorrhagic Escherichia coli O157 isolates collected in Yamaguchi Prefecture, Japan, between 2004 and 2013. A total of 114 IS629 patterns were identified; these were divided into eight IS groups (A-H). Ninety isolates carried an intact norV gene, whereas 113 isolates carried a norV with a 204-bp deletion. Other than one isolate from IS group G, all isolates with an intact norV belonged to groups A-F, whereas isolates with a mutant norV belonged to IS groups G and H. Seven stx genotypes were identified, and of those, stx1a/stx2a was predominant (n=105), followed by stx2c (n=32) and stx2a (n=27). The stx1a/stx2a genotype was associated with the mutant norV isolates, whereas isolates with an intact norV had the stx2c genotype. Therefore, certain combinations of IS type and stx genotype appear to be more frequent among O157 clades which may be useful for detection of predominant subtypes in the interest of public health.

Detailed laboratory characterization of Escherichia coli O157 is essential to inform epidemiological investigations. This study assessed the utility of whole-genome sequencing (WGS) for outbreak detection and epidemiological surveillance of E. coli O157, and the data were used to identify discernible associations between genotypes and clinical outcomes. One hundred five E. coli O157 strains isolated over a 5-year period from human fecal samples in Lothian, Scotland, were sequenced with the Ion Torrent Personal Genome Machine. A total of 8,721 variable sites in the core genome were identified among the 105 isolates; 47% of the single nucleotide polymorphisms (SNPs) were attributable to six "atypical" E. coli O157 strains and included recombinant regions. Phylogenetic analyses showed that WGS correlated well with the epidemiological data. Epidemiological links existed between cases whose isolates differed by three or fewer SNPs. WGS also correlated well with multilocus variable-number tandem repeat analysis (MLVA) typing data, with only three discordant results observed, all among isolates from cases not known to be epidemiologically related. WGS produced a better-supported, higher-resolution phylogeny than MLVA, confirming that the method is more suitable for epidemiological surveillance of E. coli O157. A combination of in silico analyses (VirulenceFinder, ResFinder, and local BLAST searches) were used to determine stx subtypes, multilocus sequence types (15 loci), and the presence of virulence and acquired antimicrobial resistance genes. There was a high level of correlation between the WGS data and our routine typing methods, although some discordant results were observed, mostly related to the limitation of short sequence read assembly. The data were used to identify sublineages and clades of E. coli O157, and when they were correlated with the clinical outcome data, they showed that one clade, Ic3, was significantly associated with severe disease. Together, the results show that WGS data can provide higher resolution of the relationships between E. coli O157 isolates than that provided by MLVA. The method has the potential to streamline the laboratory workflow and provide detailed information for the clinical management of patients and public health interventions., (Copyright © 2015, Holmes et al.)

Detection of Shiga toxins (Stx) is important for accurate diagnosis of Enterohemorrhagic Escherichia coli infection. In this study, we quantitatively analyzed Stx protein in nine patients' stool during an outbreak that occurred in Japan. Highly sensitive immunoassay (bead enzyme-linked immunosorbent assay (bead-ELISA)) revealed that the concentrations of toxins in stool of patients ranged from 0.71 to 10.44 ng/mL for Stx1 and 2.75 to 51.61 ng/mL for Stx2. To our knowledge, this is the first report that reveals the range of Stx protein concentrations in human stools.

Enterohemorrhagic Escherichia coli (EHEC) is the causative agent of bloody diarrhea and extraintestinal sequelae in humans, most importantly hemolytic-uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). Besides the bacteriophage-encoded Shiga toxin gene (stx), EHEC harbors the locus of enterocyte effacement (LEE), which confers the ability to cause attaching and effacing lesions. Currently, the vast majority of EHEC infections are caused by strains belonging to five O serogroups (the "big five"), which, in addition to O157, the most important, comprise O26, O103, O111, and O145. We hypothesize that these four non-O157 EHEC serotypes differ in their phylogenies. To test this hypothesis, we used multilocus sequence typing (MLST) to analyze a large collection of 250 isolates of these four O serogroups, which were isolated from diseased as well as healthy humans and cattle between 1952 and 2009. The majority of the EHEC isolates of O serogroups O26 and O111 clustered into one sequence type complex, STC29. Isolates of O103 clustered mainly in STC20, and most isolates of O145 were found within STC32. In addition to these EHEC strains, STC29 also included stx-negative E. coli strains, termed atypical enteropathogenic E. coli (aEPEC), yet another intestinal pathogenic E. coli group. The finding that aEPEC and EHEC isolates of non-O157 O serogroups share the same phylogeny suggests an ongoing microevolutionary scenario in which the phage-encoded Shiga toxin gene stx is transferred between aEPEC and EHEC. As a consequence, aEPEC strains of STC29 can be regarded as post- or pre-EHEC isolates. Therefore, STC29 incorporates phylogenetic information useful for unraveling the evolution of EHEC., (Copyright © 2015, Eichhorn et al.)

The aim of this study was to reveal phenotype/genotype characteristics of verotoxigenic Escherichia coli (VTEC) and multidrug resistant E. coli in food products of animal origin confiscated as illegal import at Austrian, German and Slovenian airports. VTEC isolates were obtained by using ISO guidelines 16654:2001 for O157 VTEC or ISO/ TS13136:2012 for non-O157 VTEC, with additional use of the RIDASCREEN® Verotoxin immunoassay. The testing of 1526 samples resulted in 15 VTEC isolates (1.0%) primarily isolated from hard cheese from Turkey and Balkan countries. Genotyping for virulence by using a miniaturized microarray identified a wide range of virulence determinants. One VTEC isolate (O26:H46) possessing intimin (eae) and all other essential genes of Locus of Enterocyte Effacement (LEE) was designated as enterohemorrhagic E. coli (EHEC). None of the other VTEC strains belonged to serogroups O157, O145, O111, O104 or O103. VTEC strains harbored either stx(1) (variants stx1(a) or stx(1c)) or st(x2) (variants stx(2a), stx(2b), stx(2a/d) or stx(2c/d)) genes. Pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) demonstrated high genetic diversity and identified three new sequence types (STs): 4505, 4506 and 4507. Food samples collected from the Vienna airport were also tested for E. coli quantities using the ISO 16649:2001, and for detection of multidrug resistant phenotypes and genotypes. The resulting 113 commensal E. coli isolates were first tested in a pre-screening against 6 selected antimicrobials to demonstrate multidrug resistance. The resulting 14 multidrug resistant (MDR) E. coli isolates, representing 0.9% of the samples, were subjected to further resistance phenotyping and to microarray analyses targeting genetic markers of antimicrobial resistance and virulence. Genotyping revealed various combinations of resistance determinants as well as the presence of class 1, class 2 integrons. The isolates harbored 6 to 11 antibiotic resistance genes as well as 1 to 14 virulence genes. In this panel of 14 MDR E. coli two strains proved to carry CTX-M type ESBLs, and one single isolate was identified as enteropathogenic E. coli (EPEC). In general, isolates carrying a high number of resistance determinants had lower number of virulence genes and vice versa. In conclusion, this first pilot study on the prevalence of VTEC and of MDR/ESBL E. coli in illegally imported food products of animal origin suggests that these strains could represent reservoirs for dissemination of potentially new types of pathogenic and MDR E. coli in Europe., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Unlabelled: In this study, a duplex qPCR assay was developed for the needs of the Irish fish industry to screen for the two major food-borne pathogens of fish, Listeria monocytogenes and Escherichia coli O157:H7. The assay can claim positive or negative results for two pathogens in one go in only 20 h including 16 h universal pre-enrichment and compared to traditional ISO approved plate culture methods the labour and the cost involved in testing of one sample is reduced to minimum. The highly specific genomic areas targeted for PCR amplification in the assay are the hly gene for listeriolysin O (LLO) of L. monocytogenes and the stx gene for Shiga-like toxin expressed by E. coli O157:H7. The detection limit of the assay is consistent with the consumer protection limits of 1 pg genomic DNA or 1 CFU 25 g(-1) fish meat (with enrichment) allowing the test to be considered as a substitute to standard plate culture methods., Significance and Impact of the Study: The study highlights a novel duplex qPCR for Listeria monocytogenes and Escherichia coli O157:H7 that could be used as an alternative to plate-based ISO or singleplex PCR methods while minimizing the costs. The assay uses rapid DNA extraction methods and locked nucleic acid probes. Sensitivity and specificity are 100 and 98·95% respectively. The potential for quantitative rage of the assay is 10(8) -10(1) CFU ml(-1) ., (© 2015 The Society for Applied Microbiology.)

This study evaluated the diagnostic performances of an ELISA method and a molecular method for the detection of verotoxin in faecal samples during an outbreak of haemolytic-uraemic syndrome (HUS) occurring in Apulia, Southern Italy. Two of the 16 faecal samples were positive for verotoxin when analysed by ELISA and resulted PCR positive for stx1, stx2, eaeA and serogroup O26. The other 14 faecal samples resulted negative with both tests. The detection of verotoxin in faecal samples by ELISA is a simple, sensitive, specific and rapid method (2 hours) of considerable utility for routine clinical testing laboratories without access to more specialized diagnostic procedures.

Leafy green vegetables, a popular and an indispensable ingredient of the daily menus of Egyptians' diets, currently presents a great concern in terms of microbiological hazards. To the best of our knowledge, this is the first report that provides scientific evidence for prevalence of shiga-toxigenic Escherichia coli (STEC) in leafy greens sold at open air local retail markets and superstores in the Egyptian environment. A total of 486 conventional and organic leafy green samples that are eaten raw were collected from different areas in Alexandria, evaluated for total E. coli counts (ECCs), and screened for E. coli O157:H7 using conventional and molecular methods. Recovery of E. coli (≥10(2)CFU/g) from all studied types of leafy greens was indicative of fecal contamination. Total ECCs in conventional samples ranged from 5.47 to 2.56 log CFU/g. Based on their inability to ferment sorbitol on CT-SMAC media, 26 presumptive E. coli O157 isolates were detected in 71.4% (270/378) of the studied conventional samples. From all studied organic samples, only 2 types (organic cabbage and parsley, 16.7%) were contaminated with presumptive E. coli O157. All 28 isolates were further serotyped as E. coli O157 by latex agglutination test, and biochemically confirmed as E. coli. Multiplex PCR assays confirmed the ability of 21.4% (6/28) of the E. coli O157 strains to produce shiga-toxins (Stxs), and their virulence markers were as follows: stx1, 66.6% (4/6); stx2, 50% (3/6); stx1/stx2, 16.7% (1/6); eaeA, 83.3% (5/6); and hlyA, 16.7% (1/6). Only 2 strains recovered from conventional and organic parsley could possibly be classified as E. coli O157:H7 based on the presence of stx-genes (either stx1 or stx2 or both). Results of the present research highlight that high E. coli loads, together with recovery of STEC O157 isolates could pose serious health risks to the produce consumers. This emphasizes the urgent need for health authorities to value and utilize the existing knowledge to identify strategies that reduce microbiological risks due to fecal contamination of agricultural products, and implement control measures at all stages of the food chain to specifically eliminate the presence of STEC O157 on the leafy green category., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Escherichia coli serogroup O111 is among the six most commonly reported non-O157:H7 Shiga toxin-producing E. coli (STEC), which are emerging as important foodborne pathogens. We have assembled a collection of environmental and clinical strains of E. coli O111 from diverse sources and investigated various genotypic and phenotypic characteristics of these strains to gain a better understanding of the epidemiology and biology of this serogroup. Sixty-three percent of the strains (24/38) were of H-type 8, which dominated the environmental- and outbreak-strains group, whereas the sporadic-case strains were more heterogeneous in H-type. All of the environmental and outbreak strains harbored the Shiga toxin 1 gene (stx1), eae, and ehx, and a subset of these also carried the Shiga toxin 2 gene (stx2). Only 9 of 16 sporadic-case strains produced stx1 and/or stx2, and these were mostly of H-type 8 and 10. Pulsed-field gel electrophoresis analysis revealed a cluster of environmental, outbreak, and sporadic illness strains with high phylogenetic similarity. Strains in this pulsogroup were all of the H8 type and STEC pathotype, and carried eae and ehx. Smaller clusters of highly similar STEC O111 strains included outbreak and sporadic illness strains isolated during different time periods or from different geographical locations. A distinct aggregative behavior was observed in the cultures of all environmental and outbreak STEC O111 strains, but not in those of sporadic-case strains. Among environmental and outbreaks strains, aggregation was positively correlated with production of curli fimbriae and RpoS function, and negatively with cellulose synthesis, while the nonaggregative behavior of sporadic-case strains correlated (positively) only with cellulose production. Our results indicate that STEC O111 strains sharing high genotypic similarity and important phenotypic traits with STEC O111 outbreak strains are present in the agricultural environment and may contribute to the burden of foodborne disease.

Enterohemorrhagic Escherichia coli (EHEC) is an important foodborne pathogen causing gastroenteritis and more severe complications, such as hemorrhagic colitis and hemolytic uremic syndrome. Pathology is most pronounced in the colon, but to date there is no direct clinical evidence showing EHEC binding to the colonic epithelium in patients. In this study, we investigated EHEC adherence to the human colon by using in vitro organ culture (IVOC) of colonic biopsy samples and polarized T84 colon carcinoma cells. We show for the first time that EHEC colonizes human colonic biopsy samples by forming typical attaching and effacing (A/E) lesions which are dependent on EHEC type III secretion (T3S) and binding of the outer membrane protein intimin to the translocated intimin receptor (Tir). A/E lesion formation was dependent on oxygen levels and suppressed under oxygen-rich culture conditions routinely used for IVOC. In contrast, EHEC adherence to polarized T84 cells occurred independently of T3S and intimin and did not involve Tir translocation into the host cell membrane. Colonization of neither biopsy samples nor T84 cells was significantly affected by expression of Shiga toxins. Our study suggests that EHEC colonizes and forms stable A/E lesions on the human colon, which are likely to contribute to intestinal pathology during infection. Furthermore, care needs to be taken when using cell culture models, as they might not reflect the in vivo situation., (Copyright © 2015, Lewis et al.)

The main pathogenic enterohemorrhagic Escherichia coli (EHEC) strains are defined as Shiga toxin (Stx)-producing E. coli (STEC) belonging to one of the following serotypes: O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28. Each of these five serotypes is known to be associated with a specific subtype of the intimin-encoding gene (eae). The objective of this study was to evaluate the prevalence of bovine carriers of these “top five” STEC in the four adult cattle categories slaughtered in France. Fecal samples were collected from 1,318 cattle, including 291 young dairy bulls, 296 young beef bulls, 337 dairy cows, and 394 beef cows. A total of 96 E. coli isolates, including 33 top five STEC and 63 atypical enteropathogenic E. coli (aEPEC) isolates, with the same genetic characteristics as the top five STEC strains except that they lacked an stx gene, were recovered from these samples.O157:H7 was the most frequently isolated STEC serotype. The prevalence of top five STEC (all serotypes included) was 4.5% in young dairy bulls, 2.4% in young beef bulls, 1.8% in dairy cows, and 1.0% in beef cows. It was significantly higher in young dairy bulls (P<0.05) than in the other 3 categories. The basis for these differences between categories remains to be elucidated. Moreover,simultaneous carriage of STEC O26:H11 and STEC O103:H2 was detected in one young dairy bull. Lastly, the prevalence of bovine carriers of the top five STEC, evaluated through a weighted arithmetic mean of the prevalence by categories, was estimated to 1.8% in slaughtered adult cattle in France.