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Sample records for shiga toxin stx2-encoding

  1. Prevalence of shiga toxin-producing Escherichia coli stx1, stx2, eaeA, and rfbE genes and survival of E. coli O157:H7 in manure from organic and low-input conventional dairy farms

    NARCIS (Netherlands)

    Franz, E.; Klerks, M.M.; Vos, de O.J.; Termorshuizen, A.J.; Bruggen, van A.H.C.

    2007-01-01

    Manure samples were collected from 16 organic (ORG) and 9 low-input conventional (LIC) Dutch dairy farms during August and September 2004 to determine the prevalence of the STEC virulence genes stx(1) (encoding Shiga toxin 1), stx(2) (encoding Shiga toxin 2), and eaeA (encoding intimin), as well as

  2. Shigella Sonnei and Shiga Toxin

    Centers for Disease Control (CDC) Podcasts

    2016-07-28

    Katherine Lamba, an infectious disease epidemiologist with the California Department of Public Health, discusses Shiga Toxin producing Shigella sonnei.  Created: 7/28/2016 by National Center for Emerging and Zoonotic Infectious Diseases (NCEZID).   Date Released: 7/28/2016.

  3. Shiga Toxin Therapeutics: Beyond Neutralization

    Directory of Open Access Journals (Sweden)

    Gregory Hall

    2017-09-01

    Full Text Available Ribotoxic Shiga toxins are the primary cause of hemolytic uremic syndrome (HUS in patients infected with Shiga toxin-producing enterohemorrhagic Escherichia coli (STEC, a pathogen class responsible for epidemic outbreaks of gastrointestinal disease around the globe. HUS is a leading cause of pediatric renal failure in otherwise healthy children, resulting in a mortality rate of 10% and a chronic morbidity rate near 25%. There are currently no available therapeutics to prevent or treat HUS in STEC patients despite decades of work elucidating the mechanisms of Shiga toxicity in sensitive cells. The preclinical development of toxin-targeted HUS therapies has been hindered by the sporadic, geographically dispersed nature of STEC outbreaks with HUS cases and the limited financial incentive for the commercial development of therapies for an acute disease with an inconsistent patient population. The following review considers potential therapeutic targeting of the downstream cellular impacts of Shiga toxicity, which include the unfolded protein response (UPR and the ribotoxic stress response (RSR. Outcomes of the UPR and RSR are relevant to other diseases with large global incidence and prevalence rates, thus reducing barriers to the development of commercial drugs that could improve STEC and HUS patient outcomes.

  4. Comparative genomics of Shiga toxin encoding bacteriophages

    Directory of Open Access Journals (Sweden)

    Smith Darren L

    2012-07-01

    Full Text Available Abstract Background Stx bacteriophages are responsible for driving the dissemination of Stx toxin genes (stx across their bacterial host range. Lysogens carrying Stx phages can cause severe, life-threatening disease and Stx toxin is an integral virulence factor. The Stx-bacteriophage vB_EcoP-24B, commonly referred to as Ф24B, is capable of multiply infecting a single bacterial host cell at a high frequency, with secondary infection increasing the rate at which subsequent bacteriophage infections can occur. This is biologically unusual, therefore determining the genomic content and context of Ф24B compared to other lambdoid Stx phages is important to understanding the factors controlling this phenomenon and determining whether they occur in other Stx phages. Results The genome of the Stx2 encoding phage, Ф24B was sequenced and annotated. The genomic organisation and general features are similar to other sequenced Stx bacteriophages induced from Enterohaemorrhagic Escherichia coli (EHEC, however Ф24B possesses significant regions of heterogeneity, with implications for phage biology and behaviour. The Ф24B genome was compared to other sequenced Stx phages and the archetypal lambdoid phage, lambda, using the Circos genome comparison tool and a PCR-based multi-loci comparison system. Conclusions The data support the hypothesis that Stx phages are mosaic, and recombination events between the host, phages and their remnants within the same infected bacterial cell will continue to drive the evolution of Stx phage variants and the subsequent dissemination of shigatoxigenic potential.

  5. NNDSS - Table II. Shiga toxin to Shigellosis

    Data.gov (United States)

    U.S. Department of Health & Human Services — NNDSS - Table II. Shiga toxin to Shigellosis - 2015. In this Table, provisional cases of selected notifiable diseases (≥1,000 cases reported during the preceding...

  6. NNDSS - Table II. Shiga toxin to Shigellosis

    Data.gov (United States)

    U.S. Department of Health & Human Services — NNDSS - Table II. Shiga toxin to Shigellosis - 2016. In this Table, provisional* cases of selected† notifiable diseases (≥1,000 cases reported during the preceding...

  7. Shiga toxin-producing Escherichia coli

    DEFF Research Database (Denmark)

    Pedersen, Rune Micha; Nielsen, Marc Trunjer Kusk; Möller, Sören

    2018-01-01

    OBJECTIVES: Shiga toxin-producing Escherichia coli (STEC) causes diarrhoeal disease, bloody diarrhoea and haemolytic uraemic syndrome. The aim of this study was to describe the incidence of STEC and the clinical features of STEC patients from a well-defined Danish population in which all fecal...

  8. Mechanism of Shiga Toxin Clustering on Membranes

    DEFF Research Database (Denmark)

    Pezeshkian, Weria; Gao, Haifei; Arumugam, Senthil

    2017-01-01

    The bacterial Shiga toxin interacts with its cellular receptor, the glycosphingolipid globotriaosylceramide (Gb3 or CD77), as a first step to entering target cells. Previous studies have shown that toxin molecules cluster on the plasma membrane, despite the apparent lack of direct interactions...... toxin molecules. By contrast, in coarse-grained computer simulations, a correlation was found between clustering and toxin nanoparticle-driven suppression of membrane fluctuations, and experimentally we observed that clustering required the toxin molecules to be tightly bound to the membrane surface....... The most likely interpretation of these findings is that a membrane fluctuation-induced force generates an effective attraction between toxin molecules. Such force would be of similar strength to the electrostatic force at separations around 1 nm, remain strong at distances up to the size of toxin...

  9. Emergence of Escherichia coli encoding Shiga toxin 2f in human Shiga toxin-producing E-coli (STEC) infections in the Netherlands, January 2008 to December 2011

    NARCIS (Netherlands)

    Friesema, I.; van der Zwaluw, K.; Schuurman, T.; Kooistra-Smid, M.; Franz, E.; van Duynhoven, Y.; van Pelt, W.

    2014-01-01

    The Shiga toxins of Shiga toxin-producing Escherichia coli (STEC) can be divided into Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2) with several sub-variants. Variant Stx(2f) is one of the latest described, but has been rarely associated with symptomatic human infections. In the enhanced STEC

  10. Lysogeny with Shiga Toxin 2-Encoding Bacteriophages Represses Type III Secretion in Enterohemorrhagic Escherichia coli

    Science.gov (United States)

    Xu, Xuefang; McAteer, Sean P.; Tree, Jai J.; Shaw, Darren J.; Wolfson, Eliza B. K.; Beatson, Scott A.; Roe, Andrew J.; Allison, Lesley J.; Chase-Topping, Margo E.; Mahajan, Arvind; Tozzoli, Rosangela; Woolhouse, Mark E. J.; Morabito, Stefano; Gally, David L.

    2012-01-01

    Lytic or lysogenic infections by bacteriophages drive the evolution of enteric bacteria. Enterohemorrhagic Escherichia coli (EHEC) have recently emerged as a significant zoonotic infection of humans with the main serotypes carried by ruminants. Typical EHEC strains are defined by the expression of a type III secretion (T3S) system, the production of Shiga toxins (Stx) and association with specific clinical symptoms. The genes for Stx are present on lambdoid bacteriophages integrated into the E. coli genome. Phage type (PT) 21/28 is the most prevalent strain type linked with human EHEC infections in the United Kingdom and is more likely to be associated with cattle shedding high levels of the organism than PT32 strains. In this study we have demonstrated that the majority (90%) of PT 21/28 strains contain both Stx2 and Stx2c phages, irrespective of source. This is in contrast to PT 32 strains for which only a minority of strains contain both Stx2 and 2c phages (28%). PT21/28 strains had a lower median level of T3S compared to PT32 strains and so the relationship between Stx phage lysogeny and T3S was investigated. Deletion of Stx2 phages from EHEC strains increased the level of T3S whereas lysogeny decreased T3S. This regulation was confirmed in an E. coli K12 background transduced with a marked Stx2 phage followed by measurement of a T3S reporter controlled by induced levels of the LEE-encoded regulator (Ler). The presence of an integrated Stx2 phage was shown to repress Ler induction of LEE1 and this regulation involved the CII phage regulator. This repression could be relieved by ectopic expression of a cognate CI regulator. A model is proposed in which Stx2-encoding bacteriophages regulate T3S to co-ordinate epithelial cell colonisation that is promoted by Stx and secreted effector proteins. PMID:22615557

  11. Detection of shiga toxins by lateral flow assay

    Science.gov (United States)

    Shiga toxin-producing Escherichia coli (STEC) produce Shiga toxins (Stxs) that can cause human disease and death. The contamination of food products with STEC represents a food safety problem that necessitates rapid and effective detection strategies to mitigate risk. In this manuscript we report ...

  12. Oxidative Stress in Shiga Toxin Production by Enterohemorrhagic Escherichia coli

    Directory of Open Access Journals (Sweden)

    Katarzyna Licznerska

    2016-01-01

    Full Text Available Virulence of enterohemorrhagic Escherichia coli (EHEC strains depends on production of Shiga toxins. These toxins are encoded in genomes of lambdoid bacteriophages (Shiga toxin-converting phages, present in EHEC cells as prophages. The genes coding for Shiga toxins are silent in lysogenic bacteria, and prophage induction is necessary for their efficient expression and toxin production. Under laboratory conditions, treatment with UV light or antibiotics interfering with DNA replication are commonly used to induce lambdoid prophages. Since such conditions are unlikely to occur in human intestine, various research groups searched for other factors or agents that might induce Shiga toxin-converting prophages. Among other conditions, it was reported that treatment with H2O2 caused induction of these prophages, though with efficiency significantly lower relative to UV-irradiation or mitomycin C treatment. A molecular mechanism of this phenomenon has been proposed. It appears that the oxidative stress represents natural conditions provoking induction of Shiga toxin-converting prophages as a consequence of H2O2 excretion by either neutrophils in infected humans or protist predators outside human body. Finally, the recently proposed biological role of Shiga toxin production is described in this paper, and the “bacterial altruism” and “Trojan Horse” hypotheses, which are connected to the oxidative stress, are discussed.

  13. 77 FR 9888 - Shiga Toxin-Producing Escherichia coli

    Science.gov (United States)

    2012-02-21

    ... Toxin-Producing Escherichia coli in Certain Raw Beef Products AGENCY: Food Safety and Inspection Service... toxin-producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, and O145). This new date..., that are contaminated with Shiga toxin-producing Escherichia coli (STEC) O26, O45, O103, O111, O121...

  14. Detection of Shiga Toxins by Lateral Flow Assay

    OpenAIRE

    Ching, Kathryn H.; He, Xiaohua; Stanker, Larry H.; Lin, Alice V.; McGarvey, Jeffery A.; Hnasko, Robert

    2015-01-01

    Shiga toxin-producing Escherichia coli (STEC) produce shiga toxins (Stxs) that can cause human disease and death. The contamination of food products with STEC represents a food safety problem that necessitates rapid and effective detection strategies to mitigate risk. In this manuscript, we report the development of a colorimetric lateral flow assay (LFA) for the rapid detection of Stxs in <10 min using a pair of monoclonal antibodies that bind epitopes common to Stx1 and six Stx2 variants...

  15. Literature Reference for Shiga and Shiga-like Toxin (Journal of Clinical Microbiology. 2007. 45(10): 3377–3380)

    Science.gov (United States)

    This procedure describes a rapid optical immunoassay for the detection of Stx-1 and Stx-2 using a commercially available kit for presumptive analysis of aerosol, solid, particulate, liquid and water samples contaminated with Shiga and Shiga-like toxin.

  16. 76 FR 58157 - Shiga Toxin-Producing Escherichia coli

    Science.gov (United States)

    2011-09-20

    ... disorders. Shiga toxin is the same toxin as is produced by Shigella, the bacteria that cause dysentery. In... addition, FSIS will conduct a for-cause food safety assessment (FSA) for every positive sample, as it does... conduct sample testing, follow-up testing and for-cause FSAs, the total cost to the Agency is about $454...

  17. Prevalence and antimicrobial sensitivity of Shiga-toxin-producing ...

    African Journals Online (AJOL)

    Nyambura

    provision of safe water, health education together with improvements in sanitation and personal hygiene ... available using a sterile well mouthed dry, leak proofs-plastic container (Neomedic Limited, China). Samples were .... and STEC O104 chromogenic agar media for detection of Shiga toxin-producing Escherichia.

  18. Prevalence and antimicrobial sensitivity of Shiga-toxin-producing ...

    African Journals Online (AJOL)

    Background: Escherichia coli is among the most common causes of diarrhoea in children below five years of age in developing countries. Diarrhoeal diseases rank the second most common cause of morbidity and mortality in developing countries. Here we report the magnitude of Shiga toxin-producing Escherichia coli ...

  19. Shiga Toxin (Stx) Gene Detection and Verotoxigenic Potentials of ...

    African Journals Online (AJOL)

    Non-0157 Escherichia coli, isolated from Nono (fermented fresh cow milk) sampled from four major Nigerian cities, namely, Abuja, Benin City, Lagos and Onitsha were investigated for the presence shiga toxins (stx1 and stx2) genes using PCR technique and for their verotoxigenic potentials using tissue culture assay on ...

  20. Differential cytotoxic actions of Shiga toxin 1 and Shiga toxin 2 on microvascular and macrovascular endothelial cells

    NARCIS (Netherlands)

    Bauwens, Andreas; Bielaszewska, Martina; Kemper, Björn; Langehanenberg, Patrik; von Bally, Gert; Reichelt, Rudolf; Mulac, Dennis; Humpf, Hans-Ulrich; Friedrich, Alexander W; Kim, Kwang S; Karch, Helge; Müthing, Johannes

    Shiga toxin (Stx)-mediated injury to vascular endothelial cells in the kidneys, brain and other organs underlies the pathogenesis of haemolytic uraemic syndrome (HUS) caused by enterohaemorrhagic Escherichia coli (EHEC). We present a direct and comprehensive comparison of cellular injury induced by

  1. A high-throughput, precipitating colorimetric sandwich ELISA microarray for shiga toxins

    Science.gov (United States)

    Shiga toxins 1 and 2 (Stx1 and Stx2) from Shiga toxin-producing E. coli (STEC) bacteria were simultaneously detected with a newly developed, high-throughput antibody microarray platform. The proteinaceous toxins were immobilized and sandwiched between biorecognition elements (monoclonal antibodies)...

  2. Detection of Shiga Toxins by Lateral Flow Assay

    Directory of Open Access Journals (Sweden)

    Kathryn H. Ching

    2015-04-01

    Full Text Available Shiga toxin-producing Escherichia coli (STEC produce shiga toxins (Stxs that can cause human disease and death. The contamination of food products with STEC represents a food safety problem that necessitates rapid and effective detection strategies to mitigate risk. In this manuscript, we report the development of a colorimetric lateral flow assay (LFA for the rapid detection of Stxs in <10 min using a pair of monoclonal antibodies that bind epitopes common to Stx1 and six Stx2 variants. This LFA provides a rapid and sensitive test for the detection of Stxs directly from STEC culture supernatants or at risk food samples with a 0.1 ng/mL limit of detection (LOD for Stx2a. This Stx LFA is applicable for use in the rapid evaluation of Stx production from cultured E. coli strains or as a tool to augment current methods as part of food safety testing.

  3. Detection of Shiga Toxins by Lateral Flow Assay

    Science.gov (United States)

    Ching, Kathryn H.; He, Xiaohua; Stanker, Larry H.; Lin, Alice V.; McGarvey, Jeffery A.; Hnasko, Robert

    2015-01-01

    Shiga toxin-producing Escherichia coli (STEC) produce shiga toxins (Stxs) that can cause human disease and death. The contamination of food products with STEC represents a food safety problem that necessitates rapid and effective detection strategies to mitigate risk. In this manuscript, we report the development of a colorimetric lateral flow assay (LFA) for the rapid detection of Stxs in detection of Stxs directly from STEC culture supernatants or at risk food samples with a 0.1 ng/mL limit of detection (LOD) for Stx2a. This Stx LFA is applicable for use in the rapid evaluation of Stx production from cultured E. coli strains or as a tool to augment current methods as part of food safety testing. PMID:25855129

  4. Enterohemorrhagic Escherichia coli infection stimulates Shiga toxin 1 macropinocytosis and transcytosis across intestinal epithelial cells.

    Science.gov (United States)

    Lukyanenko, Valeriy; Malyukova, Irina; Hubbard, Ann; Delannoy, Michael; Boedeker, Edgar; Zhu, Chengru; Cebotaru, Liudmila; Kovbasnjuk, Olga

    2011-11-01

    Gastrointestinal infection with Shiga toxins producing enterohemorrhagic Escherichia coli causes the spectrum of gastrointestinal and systemic complications, including hemorrhagic colitis and hemolytic uremic syndrome, which is fatal in ∼10% of patients. However, the molecular mechanisms of Stx endocytosis by enterocytes and the toxins cross the intestinal epithelium are largely uncharacterized. We have studied Shiga toxin 1 entry into enterohemorrhagic E. coli-infected intestinal epithelial cells and found that bacteria stimulate Shiga toxin 1 macropinocytosis through actin remodeling. This enterohemorrhagic E. coli-caused macropinocytosis occurs through a nonmuscle myosin II and cell division control 42 (Cdc42)-dependent mechanism. Macropinocytosis of Shiga toxin 1 is followed by its transcytosis to the basolateral environment, a step that is necessary for its systemic spread. Inhibition of Shiga toxin 1 macropinocytosis significantly decreases toxin uptake by intestinal epithelial cells and in this way provides an attractive, antibiotic-independent strategy for prevention of the harmful consequences of enterohemorrhagic E. coli infection.

  5. Mild Illness during Outbreak of Shiga Toxin-Producing Escherichia coli O157 Infections Associated with Agricultural Show, Australia.

    Science.gov (United States)

    Vasant, Bhakti R; Stafford, Russell J; Jennison, Amy V; Bennett, Sonya M; Bell, Robert J; Doyle, Christine J; Young, Jeannette R; Vlack, Susan A; Titmus, Paul; El Saadi, Debra; Jarvinen, Kari A J; Coward, Patricia; Barrett, Janine; Staples, Megan; Graham, Rikki M A; Smith, Helen V; Lambert, Stephen B

    2017-10-01

    During a large outbreak of Shiga toxin-producing Escherichia coli illness associated with an agricultural show in Australia, we used whole-genome sequencing to detect an IS1203v insertion in the Shiga toxin 2c subunit A gene of Shiga toxin-producing E. coli. Our study showed that clinical illness was mild, and hemolytic uremic syndrome was not detected.

  6. A Vero Cell Based Fluorescence Assay to Assess Relative Toxicities of Shiga Toxin 2 Subtypes from Escherichia coli

    Science.gov (United States)

    Shiga toxin-producing Escherichia coli is a leading cause worldwide of human gastroenteritis from food and waterborne sources. Shiga toxins 1 and 2 are important virulence factors linked to severe human illness. In particular, Shiga toxin 2 is composed of a diverse and heterogeneous group of subty...

  7. Lipid reorganization induced by Shiga toxin clustering on planar membranes.

    Directory of Open Access Journals (Sweden)

    Barbara Windschiegl

    Full Text Available The homopentameric B-subunit of bacterial protein Shiga toxin (STxB binds to the glycolipid Gb(3 in plasma membranes, which is the initial step for entering cells by a clathrin-independent mechanism. It has been suggested that protein clustering and lipid reorganization determine toxin uptake into cells. Here, we elucidated the molecular requirements for STxB induced Gb(3 clustering and for the proposed lipid reorganization in planar membranes. The influence of binding site III of the B-subunit as well as the Gb(3 lipid structure was investigated by means of high resolution methods such as fluorescence and scanning force microscopy. STxB was found to form protein clusters on homogenous 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC/cholesterol/Gb(3 (65:30:5 bilayers. In contrast, membranes composed of DOPC/cholesterol/sphingomyelin/Gb(3 (40:35:20:5 phase separate into a liquid ordered and liquid disordered phase. Dependent on the fatty acid composition of Gb(3, STxB-Gb(3 complexes organize within the liquid ordered phase upon protein binding. Our findings suggest that STxB is capable of forming a new membrane phase that is characterized by lipid compaction. The significance of this finding is discussed in the context of Shiga toxin-induced formation of endocytic membrane invaginations.

  8. A Structure-Function Analysis of Shiga-Like Toxin Type 2 of Enterohemorrhagic Escherichia Coli

    Science.gov (United States)

    1990-05-07

    implicated in bloody diarrhea and hemorrhagic colitis are classified as enterohemorrhagic E. coli ( EHEC ). Currently, the EHEC group consists of 3... EHEC with hemorrhagic colitis and the hemolytic uremic syndrome. Furthermore, recent reports have indicated that SLT- producing E. coli may play a...the Shiga toxin of Shigella dysenteriae I and the Shiga-like toxins of EHEC and in the identification of the toxin receptors, the biophysical

  9. Failure of manganese to protect from Shiga toxin.

    Directory of Open Access Journals (Sweden)

    Marsha A Gaston

    Full Text Available Shiga toxin (Stx, the main virulence factor of Shiga toxin producing Escherichia coli, is a major public health threat, causing hemorrhagic colitis and hemolytic uremic syndrome. Currently, there are no approved therapeutics for these infections; however manganese has been reported to provide protection from the Stx1 variant isolated from Shigella dysenteriae (Stx1-S both in vitro and in vivo. We investigated the efficacy of manganese protection from Stx1-S and the more potent Stx2a isoform, using experimental systems well-established for studying Stx: in vitro responses of Vero monkey kidney cells, and in vivo toxicity to CD-1 outbred mice. Manganese treatment at the reported therapeutic concentration was toxic to Vero cells in culture and to CD-1 mice. At lower manganese concentrations that were better tolerated, we observed no protection from Stx1-S or Stx2a toxicity. The ability of manganese to prevent the effects of Stx may be particular to certain cell lines, mouse strains, or may only be manifested at high, potentially toxic manganese concentrations.

  10. Shiga Toxin (Verotoxin)-Producing Escherichia coli in Japan.

    Science.gov (United States)

    Terajima, Jun; Iyoda, Sunao; Ohnishi, Makoto; Watanabe, Haruo

    2014-10-01

    A series of outbreaks of infection with Shiga toxin (verocytotoxin)-producing Escherichia coli or enterohemorrhagic E. coli (EHEC) O157:H7 occurred in Japan in 1996, the largest outbreak occurring in primary schools in Sakai City, Osaka Prefecture, where more than 7,500 cases were reported. Although the reason for the sudden increase in the number of reports of EHEC isolates in 1996 is not known, the number of reports has grown to more than 3,000 cases per year since 1996, from an average of 105 reports each year during the previous 5-year period (1991-1995). Despite control measures instituted since 1996, including designating Shiga toxin-producing E. coli infection as a notifiable disease, and nationwide surveillance effectively monitoring the disease, the number of reports remains high, around 3,800 cases per year. Serogroup O157 predominates over other EHEC serogroups, but isolation frequency of non-O157 EHEC has gone up slightly over the past few years. Non-O157 EHEC has recently caused outbreaks where consumption of a raw beef dish was the source of the infection, and some fatal cases occurred. Laboratory surveillance comprised prefectural and municipal public health institutes, and the National Institute of Infectious Diseases has contributed to finding not only multiprefectural outbreaks but recognizing sporadic cases that could have been missed as an outbreak without the aid of molecular subtyping of EHEC isolates. This short overview presents recent information on the surveillance of EHEC infections in Japan.

  11. Cationic Antimicrobial Peptides Inactivate Shiga Toxin-Encoding Bacteriophages

    Directory of Open Access Journals (Sweden)

    Manuel E. Del Cogliano

    2017-12-01

    Full Text Available Shiga toxin (Stx is the principal virulence factor during Shiga toxin-producing Escherichia coli (STEC infections. We have previously reported the inactivation of bacteriophage encoding Stx after treatment with chitosan, a linear polysaccharide polymer with cationic properties. Cationic antimicrobial peptides (cAMPs are short linear aminoacidic sequences, with a positive net charge, which display bactericidal or bacteriostatic activity against a wide range of bacterial species. They are promising novel antibiotics since they have shown bactericidal effects against multiresistant bacteria. To evaluate whether cationic properties are responsible for bacteriophage inactivation, we tested seven cationic peptides with proven antimicrobial activity as anti-bacteriophage agents, and one random sequence cationic peptide with no antimicrobial activity as a control. We observed bacteriophage inactivation after incubation with five cAMPs, but no inactivating activity was observed with the random sequence cationic peptide or with the non-alpha helical cAMP Omiganan. Finally, to confirm peptide-bacteriophage interaction, zeta potential was analyzed by following changes on bacteriophage surface charges after peptide incubation. According to our results we could propose that: (1 direct interaction of peptides with phage is a necessary step for bacteriophage inactivation, (2 cationic properties are necessary but not sufficient for bacteriophage inactivation, and (3 inactivation by cationic peptides could be sequence (or structure specific. Overall our data suggest that these peptides could be considered a new family of molecules potentially useful to decrease bacteriophage replication and Stx expression.

  12. Isolation, genotyping, and antimicrobial resistance of zoonotic shiga toxin-producing escherichia coli

    Science.gov (United States)

    Shiga toxin-producing Escherichia coli (STEC) is an enteric pathogen linked to outbreaks of human gastroenteritis with diverse clinical spectra. Traditional culture and isolation methods, including selective enrichment and differential plating, have enabled the effective recovery of STEC. Ruminants ...

  13. Two Shiga toxin 2 subtypes in a single Shiga toxin-producing Escherichia coli analyzed by RT-qPCR, MALDI-TOF-TOF-MS and top-down proteomic analysis

    Science.gov (United States)

    Shiga toxin-producing Escherichia coli (STEC) are increasingly linked to outbreaks of foodborne illness worldwide. Shiga toxin (Stx) is an AB5 toxin with an A-subunit and five identical B-subunits. Amino acid sequence differences between Stx types and subtypes result in differences in toxicity. I...

  14. Detection of Shiga toxin variants, virulence genes and the relationship to cytotoxicity in Shiga toxin-producing Escherichia coli (STEC) from domestic farm animals

    Science.gov (United States)

    Shiga toxin-producing Escherichia coli (STEC) can cause foodborne illnesses ranging from diarrhea to life-threating diseases such as hemorrhagic colitis, and hemolytic uremic syndrome in humans. In this study, we determined virulence genes, stx subtypes and we evaluated the cytotoxicity in STEC stra...

  15. Long-term outcomes of Shiga toxin hemolytic uremic syndrome.

    Science.gov (United States)

    Spinale, Joann M; Ruebner, Rebecca L; Copelovitch, Lawrence; Kaplan, Bernard S

    2013-11-01

    Shiga toxin-producing Escherichia coli (STEC) hemolytic uremic syndrome (HUS) is an important cause of acute kidney injury (AKI). The outcomes of STEC HUS have improved, and the acute mortality rate in children is 1-4%. About 70% of patients recover completely from the acute episode and the remainder have varying degrees of sequelae. Only a few retrospective studies have reviewed these patients over long periods. Methodological flaws include a lack of strict definitions, changing modes of treatment, ascertainment bias and loss of subjects to follow-up. The kidneys bear the brunt of the long-term damage: proteinuria (15-30% of cases); hypertension (5-15%); chronic kidney disease (CKD; 9-18%); and end-stage kidney disease (ESKD; 3%). A smaller number have extra-renal sequelae: colonic strictures, cholelithiasis, diabetes mellitus or brain injury. Most renal sequelae are minor abnormalities, such as treatable hypertension and/or variable proteinuria. Most of the patients who progress to ESKD do not recover normal renal function after the acute episode. Length of anuria (more than 10 days) and prolonged dialysis are the most important risk factors for a poor acute and long-term renal outcome. After the acute episode all patients must be followed for at least 5 years, and severely affected patients should be followed indefinitely if there is proteinuria, hypertension or a reduced glomerular filtration rate (GFR).

  16. Possible mistranslation of Shiga toxin from pathogenic Escherichia coli as measured by MALDI-TOF and Orbitrap mass spectrometry

    Science.gov (United States)

    RATIONALE: Shiga toxin-producing Escherichia coli (STEC) are often subjected to DNA damaging antibiotics during culturing in order to elicit the bacterial SOS response and up-regulation of bacteriophage-encoded proteins including Shiga toxin (Stx). However, such antibiotic exposure and stress may al...

  17. Altruism of Shiga toxin-producing Escherichia coli: recent hypothesis versus experimental results

    Directory of Open Access Journals (Sweden)

    Joanna M Los

    2013-01-01

    Full Text Available Shiga toxin-producing Escherichia coli (STEC may cause bloody diarrhea and hemorrhagic colitis, with subsequent systemic disease. Since genes coding for Shiga toxins (stx genes are located on lambdoid prophages, their effective production occurs only after prophage induction. Such induction and subsequent lytic development of Shiga toxin-converting bacteriophages results not only in production of toxic proteins, but also in the lysis (and thus, the death of the host cell. Therefore, one may ask the question: what is the benefit for bacteria to produce the toxin if they die due to phage production and subsequent cell lysis? Recently, a hypothesis was proposed (simultaneously but independently by two research groups that STEC may benefit from Shiga toxin production as a result of toxin-dependent killing of eukaryotic cells such as unicellular predators or human leukocytes. This hypothesis could make sense only if we assume that prophage induction (and production of the toxin occurs only in a small fraction of bacterial cells, thus, a few members of the population are sacrificed for the benefit of the rest, providing an example of ‘bacterial altruism’. However, various reports indicating that the frequency of spontaneous induction of Shiga toxin-converting prophages is higher than that of other lambdoid prophages might seem to contradict the for-mentioned model. On the other hand, analysis of recently published results, discussed here, indicated that the efficiency of prophage excision under conditions that may likely occur in the natural habitat of STEC is sufficiently low to ensure survival of a large fraction of the bacterial host. A molecular mechanism by which partial prophage induction may occur is proposed. We conclude that the published data supports the proposed model of bacterial ‘altruism’ where prophage induction occurs at a low enough frequency to render toxin production a positive selective force on the general STEC population.

  18. Detection of Shiga toxins genes by Multiplex PCR in clinical samples

    Directory of Open Access Journals (Sweden)

    2013-09-01

    Full Text Available Background: Different methods have been used for detection of shiga toxins; such as,  cell culture, ELISA, and RFPLA. However, all of these methods suffer from high cost, time-consumption and relatively low sensitivity. In this study we used Multiplex PCR method for detection of genes encoding shiga toxins. Material and Methods: In this study, 63 clinical samples were obtained from positive cultures of Shigella and E. coli O157, from Bahman 1391 until Ordibehesht 1392 in Mazandaran province. Initial confirmation of shiga toxins producing bacteria was performed by biochemical and serological methods. After DNA extraction, detection of stx1 and stx2 genes was accomplished by multiplex PCR.  For confirmation of the PCR amplicon, DNA sequencing was used. Antibiotic sensitivity tests were performed by disk diffusion method. Results:  Among the positive strains, 13 strains contained stx2 genes, 4 strains contained Stx/Stx1 genes and 4 strains harbored both Stx/Stx1 and Stx2. The DNA extracted from other Gram-negative bacteria was not protected by the relevant parts of these toxins. Sequencing of the amplified fragments indicated the correct toxin sequences.  The sensitivity for identification of Stx/Stx1 gene was 1.56 pg/ µl and for Stx2 was 1.08 pg/µl. The toxin positive strains were all sensitive to Cefixime, Gentamicin, Amikacin, Ceftriaxone, and Nitrofurantoin. Conclusion: This method is fast and accurate for detection of bacteria producing shiga toxin and can be used to identify different types of shiga toxin.

  19. Shiga toxin Mediated Neurologic Changes in Murine Model of Disease.

    Directory of Open Access Journals (Sweden)

    Suman Pradhan

    2016-09-01

    Full Text Available Seizures and neurologic involvement have been reported in patients infected with Shiga toxin (Stx producing E. coli, and hemolytic uremic syndrome (HUS with neurologic involvement is associated with more severe outcome. We investigated the extent of renal and neurologic damage in mice following injection of the highly potent form of Stx, Stx2a, and less potent Stx1. As observed in previous studies, Stx2a brought about moderate to acute tubular necrosis of proximal and distal tubules in the kidneys. Brain sections stained with hematoxylin and eosin (H&E appeared normal, although some red blood cell congestion was observed. Microglial cell responses to neural injury include up-regulation of surface-marker expression (e.g. Iba1 and stereotypical morphological changes. Mice injected with Stx2a showed increased Iba1 staining, mild morphological changes associated with microglial activation (thickening of processes, and increased microglial staining per unit area. Microglial changes were observed in the cortex, hippocampus, and amygdala regions, but not the nucleus. Magnetic resonance imaging (MRI of Stx2a-treated mice revealed no hyper-intensities in the brain, although magnetic resonance spectroscopy (MRS revealed significantly decreased levels of phosphocreatine in the thalamus. Less dramatic changes were observed following Stx1 challenge. Neither immortalized microvascular endothelial cells from the cerebral cortex of mice (bEnd.3 nor primary human brain microvascular endothelial cells were found to be susceptible to Stx1 or Stx2a. The lack of susceptibility to Stx for both cell types correlated with an absence of receptor expression. These studies indicate Stx causes subtle, but identifiable changes in the mouse brain.

  20. Shiga Toxin-Producing E. coli Infections Associated with Flour.

    Science.gov (United States)

    Crowe, Samuel J; Bottichio, Lyndsay; Shade, Lauren N; Whitney, Brooke M; Corral, Nereida; Melius, Beth; Arends, Katherine D; Donovan, Danielle; Stone, Jolianne; Allen, Krisandra; Rosner, Jessica; Beal, Jennifer; Whitlock, Laura; Blackstock, Anna; Wetherington, June; Newberry, Lisa A; Schroeder, Morgan N; Wagner, Darlene; Trees, Eija; Viazis, Stelios; Wise, Matthew E; Neil, Karen P

    2017-11-23

    In 2016, a multijurisdictional team investigated an outbreak of Shiga toxin-producing Escherichia coli (STEC) serogroup O121 and O26 infections linked to contaminated flour from a large domestic producer. A case was defined as infection with an outbreak strain in which illness onset was between December 21, 2015, and September 5, 2016. To identify exposures associated with the outbreak, outbreak cases were compared with non-STEC enteric illness cases, matched according to age group, sex, and state of residence. Products suspected to be related to the outbreak were collected for STEC testing, and a common point of contamination was sought. Whole-genome sequencing was performed on isolates from clinical and food samples. A total of 56 cases were identified in 24 states. Univariable exact conditional logistic-regression models of 22 matched sets showed that infection was significantly associated with the use of one brand of flour (odds ratio, 21.04; 95% confidence interval [CI], 4.69 to 94.37) and with tasting unbaked homemade dough or batter (odds ratio, 36.02; 95% CI, 4.63 to 280.17). Laboratory testing isolated the outbreak strains from flour samples, and whole-genome sequencing revealed that the isolates from clinical and food samples were closely related to one another genetically. Trace-back investigation identified a common flour-production facility. This investigation implicated raw flour as the source of an outbreak of STEC infections. Although it is a low-moisture food, raw flour can be a vehicle for foodborne pathogens.

  1. Inhibition of Shiga Toxin 2 (Stx2) in Apple Juices and its Resistance to Pasteurization

    Science.gov (United States)

    In the present study, we evaluated Shiga toxin (Stx2) activity in apple juices by measuring a decrease in dehydrogenase activity of Vero cells with the microculture tetrazolium (MTT) assay. Freshly prepared juice from Red Delicious apples and Golden Delicious apples inhibited the biological activity...

  2. An improved method for detection of Shiga toxin 2 in human serum

    Science.gov (United States)

    Shiga toxins (Stx) produced by Stx-producing Escherichia coli (STEC) are virulence factors that is most closely associated with hemolytic uremic syndrome (HUS), a life-threatening complication of intestinal infections by STEC. Stx have to enter into the circulation system before they can be delivere...

  3. Shiga toxin-producing serogroup O91 Escherichia coli strains isolated from food and environmental samples

    Science.gov (United States)

    Shiga toxin-producing Escherichia coli (STEC) strains of the O91: H21 serotype have caused severe infections, including hemolytic-uremic syndrome. Strains of the O91 serogroup have been isolated from food, animals, and the environment worldwide but are not well characterized. We used a microarray an...

  4. Antimicrobial Drug-Resistant Shiga Toxin-Producing Escherichia coli Infections, Michigan, USA.

    Science.gov (United States)

    Mukherjee, Sanjana; Mosci, Rebekah E; Anderson, Chase M; Snyder, Brian A; Collins, James; Rudrik, James T; Manning, Shannon D

    2017-09-01

    High frequencies of antimicrobial drug resistance were observed in O157 and non-O157 Shiga toxin-producing E. coli strains recovered from patients in Michigan during 2010-2014. Resistance was more common in non-O157 strains and independently associated with hospitalization, indicating that resistance could contribute to more severe disease outcomes.

  5. Human mannose-binding lectin inhibitor prevents Shiga toxin-induced renal injury

    DEFF Research Database (Denmark)

    Ozaki, Masayuki; Kang, Yulin; Tan, Ying Siow

    2016-01-01

    Hemolytic uremic syndrome caused by Shiga toxin-producing Escherichia coli (STEC HUS) is a worldwide endemic problem, and its pathophysiology is not fully elucidated. Here we tested whether the mannose-binding lectin (MBL2), an initiating factor of lectin complement pathway activation, plays a cr...

  6. Shiga toxin-producing E. coli and ruminant diets: A match made in heaven?

    Science.gov (United States)

    Shiga-toxin producing E. coli (STEC) are also known as enterohemorrhagic E. coli (EHEC), which are pathogenic bacteria that can cause serious illnesses in humans who consume contaminated foods or water. This pathogen is commonly associated with cattle as it can survive within the intestinal tract a...

  7. Classification of shiga toxin-producing escherichia coli (STEC) serotypes with hyperspectral microscope imagery

    Science.gov (United States)

    Non-O157:H7 Shiga toxin-producing Escherichia coli (STEC) strains such as O26, O45, O103, O111, O121 and O145 are recognized as serious outbreak to cause human illness due to their toxicity. Since a conventional microbiological method for cell counting is laborious and time-consuming process, optica...

  8. Rapid Detection of Escherichia coli O157 and Shiga Toxins by Lateral Flow Immunoassays

    Directory of Open Access Journals (Sweden)

    Jinliang Wang

    2016-03-01

    Full Text Available Shiga toxin-producing Escherichia coli O157:H7 (STEC cause food-borne illness that may be fatal. STEC strains enumerate two types of potent Shiga toxins (Stx1 and Stx2 that are responsible for causing diseases. It is important to detect the E. coli O157 and Shiga toxins in food to prevent outbreak of diseases. We describe the development of two multi-analyte antibody-based lateral flow immunoassays (LFIA; one for the detection of Stx1 and Stx2 and one for the detection of E. coli O157 that may be used simultaneously to detect pathogenic E. coli O157:H7. The LFIA strips were developed by conjugating nano colloidal gold particles with monoclonal antibodies against Stx1 and Stx2 and anti-lipid A antibodies to capture Shiga toxins and O157 antigen, respectively. Our results indicate that the LFIA for Stx is highly specific and detected Stx1 and Stx2 within three hours of induction of STEC with ciprofloxacin at 37 °C. The limit of detection for E. coli O157 LFIA was found to be 105 CFU/mL in ground beef spiked with the pathogen. The LFIAs are rapid, accurate and easy to use and do not require sophisticated equipment or trained personnel. Following the assay, colored bands on the membrane develop for end-point detection. The LFIAs may be used for screening STEC in food and the environment.

  9. Phylogeny and disease association of Shiga toxin-producing Escherichia coli O91

    NARCIS (Netherlands)

    Mellmann, Alexander; Fruth, Angelika; Friedrich, Alexander W; Wieler, Lothar H; Harmsen, Dag; Werber, Dirk; Middendorf, Barbara; Bielaszewska, Martina; Karch, Helge

    The diversity and relatedness of 100 Shiga toxin-producing Escherichia coli O91 isolates from different patients were examined by multilocus sequence typing. We identified 10 specific sequence types (ST) and 4 distinct clonal groups. ST442 was significantly associated with hemolytic uremic syndrome.

  10. Shiga toxin induces membrane reorganization and formation of long range lipid order

    DEFF Research Database (Denmark)

    Solovyeva, Vita; Johannes, Ludger; Simonsen, Adam Cohen

    2015-01-01

    Lateral variation of the in-plane orientation of lipids in a bilayer is referred to as texture. The influence of the protein Shiga toxin on orientational membrane texture was studied in phosphatidylcholine lipid bilayers using polarization two-photon fluorescence microscopy and atomic force micro...

  11. Rapid Detection of Escherichia coli O157 and Shiga Toxins by Lateral Flow Immunoassays.

    Science.gov (United States)

    Wang, Jinliang; Katani, Robab; Li, Lingling; Hegde, Narasimha; Roberts, Elisabeth L; Kapur, Vivek; DebRoy, Chitrita

    2016-03-25

    Shiga toxin-producing Escherichia coli O157:H7 (STEC) cause food-borne illness that may be fatal. STEC strains enumerate two types of potent Shiga toxins (Stx1 and Stx2) that are responsible for causing diseases. It is important to detect the E. coli O157 and Shiga toxins in food to prevent outbreak of diseases. We describe the development of two multi-analyte antibody-based lateral flow immunoassays (LFIA); one for the detection of Stx1 and Stx2 and one for the detection of E. coli O157 that may be used simultaneously to detect pathogenic E. coli O157:H7. The LFIA strips were developed by conjugating nano colloidal gold particles with monoclonal antibodies against Stx1 and Stx2 and anti-lipid A antibodies to capture Shiga toxins and O157 antigen, respectively. Our results indicate that the LFIA for Stx is highly specific and detected Stx1 and Stx2 within three hours of induction of STEC with ciprofloxacin at 37 °C. The limit of detection for E. coli O157 LFIA was found to be 10⁵ CFU/mL in ground beef spiked with the pathogen. The LFIAs are rapid, accurate and easy to use and do not require sophisticated equipment or trained personnel. Following the assay, colored bands on the membrane develop for end-point detection. The LFIAs may be used for screening STEC in food and the environment.

  12. Effect of environmental stresses on the survival and cytotoxicity of Shiga toxin-producing Escherichia coli

    Science.gov (United States)

    Shiga toxin producing Escherichia coli (STEC) causes serious illnesses leading to hospitalizations in the United States. Since bacteria exposed to sub-lethal treatment during food processing may exhibit enhanced virulence and resistance to subsequent processing, the present study attempts to investi...

  13. Translational errors in expression of Shiga toxin from pathogenic Escherichia coli as measured by MALDI-TOF-TOF and Orbitrap mass spectrometry

    Science.gov (United States)

    Introduction: Shiga toxin (Stx) is an AB5 toxin expressed by Shiga toxin-producing E. coli (STEC) and Shigella dysenteriae. The Stx holotoxin attaches to surface receptors of eukaryotic cells. After cellular envelopment, the toxin disrupts ribosomal protein synthesis causing cell death. Variations i...

  14. STEC

    African Journals Online (AJOL)

    USER

    2010-07-12

    ). STEC producing shiga toxin type 2 (Stx2, encoded by stx2 gene) appear to be more commonly responsible for serious complication such as HUS than those producing only shiga toxin type 1 (Stx 1, encoded by stx 1gene).

  15. Characteristics of Shiga Toxin-Producing Escherichia coli O157 in Slaughtered Reindeer from Northern Finland.

    Science.gov (United States)

    Zweifel, Claudio; Fierz, Lisa; Cernela, Nicole; Laaksonen, Sauli; Fredriksson-Ahomaa, Maria; Stephan, Roger

    2017-03-01

    Fecal samples collected from 470 slaughtered reindeer 6 to 7 months of age were screened by real-time PCR (after enrichment) for Shiga toxin genes (stx) and then for Escherichia coli serogroup O157. Shiga toxin genes were found frequently (>30% of samples), and serogroup O157 was detected in 20% of the stx-positive samples. From these samples, a total of 25 E. coli O157:H - isolates (nonmotile but PCR positive for fliC H7 ) were obtained. Twenty-four of these E. coli O157:H - isolates did not ferment sorbitol and originated from one geographic area. These 24 isolates belonged to the multilocus sequence type 11, typical for Shiga toxin-producing E. coli (STEC) O157:H7 and O157:H - , and harbored genes stx 1a , stx 2c , eae, and hlyA; the stx 2c subtype has been associated with high virulence. In contrast, one E. coli O157:H - isolate (multilocus sequence type 11) did ferment sorbitol, lacked Shiga toxin genes, but was positive for eae, hlyA, and sfpA. This isolate closely resembled an STEC that has lost its Shiga toxin genes. Additional examination revealed that reindeer can be colonized by various other STEC isolates; 21 non-O157 STEC isolates belonged to four multilocus sequence types, harbored stx 1a (8 isolates) or stx 2b (13 isolates), and in the stx 2b -positive isolates the recently described new allelic variants (subAB2-2 and subAB2-3) for subtilase cytotoxin were identified. Hence, slaughtered semidomesticated Finnish reindeer might constitute a little known reservoir for STEC O157:H7/H - and other serogroups, and the risk of direct or indirect transmission of these pathogens from reindeer to humans and domestic livestock must not be overlooked.

  16. Rapid detection of E. coli produced shiga-like toxins by lateral flow immunoassay in multiple food matrices

    Science.gov (United States)

    Shiga toxigenic E. coli (STEC) produce shiga-like toxins (Stx) that can cause human disease and death. The STEC serotype O157:H7 is a well-recognized foodborne contaminant and effective detection methods have been established. However, the emergence of non-O157 STEC strains has necessitated the deve...

  17. Shiga toxin-producing Escherichia coli in beef retail markets from Argentina

    OpenAIRE

    Brusa, Victoria; Aliverti, Virginia; Aliverti, Florencia; Ortega, Emanuel Eneas; de la Torre, Julian Horacio; Linares, Luciano Hector; Sanz, Marcelo Eduardo; Etcheverría, Analía Inés; Padola, Nora Lía; Galli, Lucía; Peral Garcia, Pilar; Copes, Julio Alberto; Leotta, Gerardo Anibal

    2016-01-01

    Shiga toxing-producing Escherichia coli (STEC) are foodborne pathogens that cause mild or serious diseases and can lead to people death. This study reports the prevalence and characteristics of STEC O157 and non-O157 in commercial ground beef and environmental samples, including meat table, knife, meat mincing machine and manipulator hands (n= 450) obtained from 90 retail markets over a nine-month period. The STEC isolates were serotyped and virulence genes as stx (Shiga toxin), rfbO157 (O157...

  18. A High-Throughput, Precipitating Colorimetric Sandwich ELISA Microarray for Shiga Toxins

    Directory of Open Access Journals (Sweden)

    Andrew Gehring

    2014-06-01

    Full Text Available Shiga toxins 1 and 2 (Stx1 and Stx2 from Shiga toxin-producing E. coli (STEC bacteria were simultaneously detected with a newly developed, high-throughput antibody microarray platform. The proteinaceous toxins were immobilized and sandwiched between biorecognition elements (monoclonal antibodies and pooled horseradish peroxidase (HRP-conjugated monoclonal antibodies. Following the reaction of HRP with the precipitating chromogenic substrate (metal enhanced 3,3-diaminobenzidine tetrahydrochloride or DAB, the formation of a colored product was quantitatively measured with an inexpensive flatbed page scanner. The colorimetric ELISA microarray was demonstrated to detect Stx1 and Stx2 at levels as low as ~4.5 ng/mL within ~2 h of total assay time with a narrow linear dynamic range of ~1–2 orders of magnitude and saturation levels well above background. Stx1 and/or Stx2 produced by various strains of STEC were also detected following the treatment of cultured cells with mitomycin C (a toxin-inducing antibiotic and/or B-PER (a cell-disrupting, protein extraction reagent. Semi-quantitative detection of Shiga toxins was demonstrated to be sporadic among various STEC strains following incubation with mitomycin C; however, further reaction with B-PER generally resulted in the detection of or increased detection of Stx1, relative to Stx2, produced by STECs inoculated into either axenic broth culture or culture broth containing ground beef.

  19. Characterization of a Shiga-like Toxin Converting Phage from an Escherichia coli Strain Responsible for Hemorrhagic Colitis in Humans

    Science.gov (United States)

    1985-04-30

    phage . VI. Initiate molecular epidemiologic studies in which strains of Esche- richia coli and other bacteria from clinical and natural sources are...or vibrio ···· strains that make Shiga-like toxin. Several bands exhibiting phage I’ homology were found in the E. coli normal flora #4 strain. H...MEDICAL CENTER WILFORD HALL AIR FORCE MEDICAL CENTER Title of Thesis: "Characterization of a Shiga-like Toxin Converting Phage from an Escherichia

  20. Locus of Adhesion and Autoaggregation (LAA), a pathogenicity island present in emerging Shiga Toxin?producing Escherichia coli strains

    OpenAIRE

    Montero, David A.; Velasco, Juliana; Del Canto, Felipe; Puente, Jose L.; Padola, Nora L.; Rasko, David A.; Farf?n, Mauricio; Salazar, Juan C.; Vidal, Roberto

    2017-01-01

    Shiga Toxin-producing Escherichia coli (STEC) are a group of foodborne pathogens associated with diarrhea, dysentery, hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). Shiga toxins are the major virulence factor of these pathogens, however adhesion and colonization to the human intestine is required for STEC pathogenesis. A subset of STEC strains carry the Locus of Enterocyte Effacement (LEE) pathogenicity island (PAI), which encodes genes that mediate the colonization of the huma...

  1. Effect of the Food Additives Sodium Citrate and Disodium Phosphate on Shiga Toxin-Producing Escherichia coli and Production of stx-Phages and Shiga toxin.

    Science.gov (United States)

    Lenzi, Lucas J; Lucchesi, Paula M A; Medico, Lucía; Burgán, Julia; Krüger, Alejandra

    2016-01-01

    Induction and propagation of bacteriophages along the food production chain can represent a significant risk when bacteriophages carry genes for potent toxins. The aim of this study was to evaluate the effect of different compounds used in the food industry on the growth of Shiga toxin-producing Escherichia coli (STEC) and the production of stx-phage particles and Shiga toxin. We tested the in vitro effect of lactic acid, acetic acid, citric acid, disodium phosphate, and sodium citrate on STEC growth. A bacteriostatic effect was observed in most of treated cultures. The exceptions were those treated with sodium citrate and disodium phosphate in which similar growth curves to the untreated control were observed, but with reduced OD600 values. Evaluation of phage production by plaque-based assays showed that cultures treated with sodium citrate and disodium phosphate released phages in similar o lower levels than untreated cultures. However, semi-quantification of Stx revealed higher levels of extracellular Stx in STEC cultures treated with 2.5% sodium citrate than in untreated cultures. Our results reinforce the importance to evaluate if additives and other treatments used to decrease bacterial contamination in food induce stx-phage and Stx production.

  2. Clinical Evaluation and Cost Analysis of Great Basin Shiga Toxin Direct Molecular Assay for Detection of Shiga Toxin-Producing Escherichia coli in Diarrheal Stool Specimens.

    Science.gov (United States)

    Faron, Matthew L; Ledeboer, Nathan A; Connolly, Jessica; Granato, Paul A; Alkins, Brenda R; Dien Bard, Jennifer; Daly, Judy A; Young, Stephen; Buchan, Blake W

    2017-02-01

    The Shiga Toxin Direct molecular assay (ST Direct) relies on nucleic acid amplification and solid array-based amplicon detection to identify Shiga toxin-producing Escherichia coli (STEC) in preserved stool specimens. Genes encoding Shiga toxin (stx 1 and stx 2 ), as well as the E. coli serotype O:157-specific marker rfbE, are simultaneously detected within 2 h. ST Direct was evaluated using 1,084 prospectively collected preserved stool specimens across five clinical centers. An additional 55 retrospectively collected, frozen specimens were included to increase the number of positive specimens evaluated. Results were compared to results from routine culture and an enzyme immunoassay (EIA) specific for the recovery and identification of STEC. ST Direct was found to be 93.2% sensitive and 99.3% specific for detection of stx 1 and stx 2 and 95.7% sensitive and 99.3% specific for detection of E. coli serotype O:157. All specimens with false-positive results were found to contain stx 1 or stx 2 or were found to be positive for serotype O:157 when analyzed using alternative molecular methods. All 4 false-negative stx 1 or stx 2 results were reported for frozen, retrospectively tested specimens. In all cases, the specimens tested positive for stx by an alternative FDA-cleared nucleic acid amplification test (NAAT) but were negative for stx 1 and stx 2 following nucleic acid sequence analysis. Based on these data, culture and EIA-based methods for detection of STEC are only 33% sensitive compared to molecular tests. A retrospective cost analysis demonstrated 59% of the cost of routine stool culture to be attributable to the identification of STEC. Taken together, these data suggest that ST Direct may provide a cost-effective, rapid molecular alternative to routine culture for the identification of STEC in preserved stool specimens. Copyright © 2017 American Society for Microbiology.

  3. Real-Time Fluorescence PCR Assays for Detection and Characterization of Shiga Toxin, Intimin, and Enterohemolysin Genes from Shiga Toxin-Producing Escherichia coli

    Science.gov (United States)

    Reischl, Udo; Youssef, Mohammad T.; Kilwinski, Jochen; Lehn, Norbert; Zhang, Wen Lan; Karch, Helge; Strockbine, Nancy A.

    2002-01-01

    PCR assays have proved useful for detecting and characterizing Shiga toxin-producing Escherichia coli (STEC). Recent advances in PCR technology have facilitated the development of real-time fluorescence PCR assays with greatly reduced amplification times and improved methods for the detection of amplified target sequences. We developed and evaluated two such assays for the LightCycler instrument: one that simultaneously detects the genes for Shiga toxins 1 and 2 (stx1 and stx2) and another that simultaneously detects the genes for intimin (eae) and enterohemolysin (E-hly). Amplification and sequence-specific detection of the two target genes were completed within 60 min. Findings from the testing of 431 STEC isolates of human and animal origin, 73 isolates of E. coli negative for stx genes, and 118 isolates of other bacterial species with the LightCycler PCR (LC-PCR) assays were compared with those obtained by conventional block cycler PCR analysis. The sensitivities and specificities of the LC-PCR assays were each 100% for the stx1, eae, and E-hly genes and 96 and 100%, respectively, for the stx2 gene. No stx2 genes were detected from 10 stx2f-positive isolates because of significant nucleotide differences in their primer annealing regions. Melting curve analyses of the amplified Shiga toxin genes revealed sequence variation within each of the tested genes that correlated with described and novel gene variants. The performance characteristics of the LC-PCR assays, such as their speed, detection method, and the potential subtyping information available from melting curve analyses, make them attractive alternatives to block cycler PCR assays for detecting and characterizing STEC strains. PMID:12089277

  4. Shiga Toxin-Producing Escherichia coli: a Single-Center, 11-Year Pediatric Experience

    Science.gov (United States)

    Schindler, Emily I.; Sellenriek, Patricia; Storch, Gregory A.; Tarr, Phillip I.

    2014-01-01

    The aim of this study was to identify the best practices for the detection of Shiga toxin-producing Escherichia coli (STEC) in children with diarrheal illness treated at a tertiary care center, i.e., sorbitol-MacConkey (SMAC) agar culture, enzyme immunoassay (EIA) for Shiga toxin, or the simultaneous use of both methods. STEC was detected in 100 of 14,997 stool specimens submitted for enteric culture (0.7%), with 65 cases of E. coli O157. Among E. coli O157 isolates, 57 (88%) were identified by both SMAC agar culture and EIA, 6 (9%) by SMAC agar culture alone, and 2 (3%) by EIA alone. Of the 62 individuals with diarrheal hemolytic uremic syndrome (HUS) seen at our institution during the study period, 16 (26%) had STEC isolated from cultures at our institution and 15 (24%) had STEC isolated at other institutions. No STEC was recovered in 31 cases (50%). Of the HUS cases in which STEC was isolated, 28 (90%) were attributable to E. coli O157 and 3 (10%) were attributable to non-O157 STEC. Consistent with previous studies, we have determined that a subset of E. coli O157 infections will not be detected if an agar-based method is excluded from the enteric culture workup; this has both clinical and public health implications. The best practice would be concomitant use of an agar-based method and a Shiga toxin EIA, but a Shiga toxin EIA should not be considered to be an adequate stand-alone test for detection of E. coli O157 in clinical samples. PMID:25078916

  5. Inactivation of shiga toxin-producing Escherichia coli in lean ground beef by gamma irradiation

    Science.gov (United States)

    Non-O157 serovars of Shiga Toxin-producing Escherichia coli (STEC) are now responsible for over 60% of STEC induced illnesses. The majority of illnesses caused by non-O157:H7 STEC have been due to serogroups O26, O121, O103, O45, O111, and O145, “the big/top six”, which are now considered adulterant...

  6. Virulence characteristics of Shiga toxin-producing Escherichia coli from raw meats and clinical samples.

    Science.gov (United States)

    Hoang Minh, Son; Kimura, Etsuko; Hoang Minh, Duc; Honjoh, Ken-ichi; Miyamoto, Takahisa

    2015-03-01

    Shiga toxin producing Escherichia coli (STEC) are dangerous foodborne pathogens. Foods are considered as important sources for STEC infection in human. In this study, STEC contamination of raw meats was investigated and the virulence factors of 120 clinical STEC strains characterized. STEC was detected in 4.4% of tested samples. Among 25 STEC strains from meats, five strains (20%) were positive for the eae gene, which encodes intimin, an important binding protein of pathogenic STEC. The remaining strains (80%) were eae-negative. However, 28% of them possessed the saa gene, which encodes STEC agglutinating adhesin. The ehxA gene encoding for enterohemolysin was found in 75% of the meat strains and the subAB gene, the product is of which subtilase cytotoxin, was found in 32% of these strains. The stx2a gene, a subtype of Shiga toxin gene (stx), was the most prevalent subtype among the identified meat STEC bacteria. None of the meat STEC was O157:H7 serotype. Nevertheless, 92% of them produced Shiga toxin (Stx). Among 120 clinical STEC strains, 30% and 70% strains harbored single and multiple stx subtypes, respectively. Most clinical STEC bacteria possessed eae (90.8%) and ehxA (96.7%) genes and 92.5% of them showed Stx productivity. Our study shows that some raw meat samples contain non-O157 STEC bacteria and some strains have virulence factors similar to those of clinical strains. © 2015 The Societies and Wiley Publishing Asia Pty Ltd.

  7. PCR Based Detection of Shiga Toxin Producing E. coli in Commercial Poultry and Related Environments

    Directory of Open Access Journals (Sweden)

    Homaira Anzum Himi

    2015-03-01

    Full Text Available Shiga toxin (Stx-producing E. coli (STEC is the most important foodborne pathogen which is the causal agent of mild diarrhea, bloody diarrhea, hemolytic-uremic syndrome (HUS in human. The present study was designed to determine the prevalence and identification of Shiga toxin (Stx-producing E. coli in poultry, detection of its source of infection in poultry and transmission pattern to human. For this purpose a total of 150 samples (cloacal swab-60, feed -15, water-15 and egg -60 were collected and analyzed in bacteriology laboratory by cultured in different bacteriological media followed by gram’s staining, biochemical tests and Polymerase Chain reaction (PCR. The PCR was performed by targeting 16s rRNA gene and shiga toxin producing gene in E. coli. Out of 150 collected samples, E. coli was found in 81 (54% samples. Presence of E. coli was 100% in both feed (n=15 and egg (n=60, whereas 10% in cloacal swab (n=6. Water samples were totally free of E. coli. The stx2 gene was detected in all samples whether all samples were negative for stx1 gene. The study revealed that, poultry feed acts as a source of E. coli infection in poultry, which may be transmitted to environment and human via meat or eggs. Antibiotic sensitivity test revealed that isolated bacteria were highly sensitive to Ciprofloxacin.

  8. Serine protease EspP from enterohemorrhagic Escherichia coli is sufficient to induce shiga toxin macropinocytosis in intestinal epithelium.

    Science.gov (United States)

    In, Julie; Lukyanenko, Valeriy; Foulke-Abel, Jennifer; Hubbard, Ann L; Delannoy, Michael; Hansen, Anne-Marie; Kaper, James B; Boisen, Nadia; Nataro, James P; Zhu, Chengru; Boedeker, Edgar C; Girón, Jorge A; Kovbasnjuk, Olga

    2013-01-01

    Life-threatening intestinal and systemic effects of the Shiga toxins produced by enterohemorrhagic Escherichia coli (EHEC) require toxin uptake and transcytosis across intestinal epithelial cells. We have recently demonstrated that EHEC infection of intestinal epithelial cells stimulates toxin macropinocytosis, an actin-dependent endocytic pathway. Host actin rearrangement necessary for EHEC attachment to enterocytes is mediated by the type 3 secretion system which functions as a molecular syringe to translocate bacterial effector proteins directly into host cells. Actin-dependent EHEC attachment also requires the outer membrane protein intimin, a major EHEC adhesin. Here, we investigate the role of type 3 secretion in actin turnover occurring during toxin macropinocytosis. Toxin macropinocytosis is independent of EHEC type 3 secretion and intimin attachment. EHEC soluble factors are sufficient to stimulate macropinocytosis and deliver toxin into enterocytes in vitro and in vivo; intact bacteria are not required. Intimin-negative enteroaggregative Escherichia coli (EAEC) O104:H4 robustly stimulate Shiga toxin macropinocytosis into intestinal epithelial cells. The apical macropinosomes formed in intestinal epithelial cells move through the cells and release their cargo at these cells' basolateral sides. Further analysis of EHEC secreted proteins shows that a serine protease EspP alone is able to stimulate host actin remodeling and toxin macropinocytosis. The observation that soluble factors, possibly serine proteases including EspP, from each of two genetically distinct toxin-producing strains, can stimulate Shiga toxin macropinocytosis and transcellular transcytosis alters current ideas concerning mechanisms whereby Shiga toxin interacts with human enterocytes. Mechanisms important for this macropinocytic pathway could suggest new potential therapeutic targets for Shiga toxin-induced disease.

  9. Serine protease EspP from enterohemorrhagic Escherichia coli is sufficient to induce shiga toxin macropinocytosis in intestinal epithelium.

    Directory of Open Access Journals (Sweden)

    Julie In

    Full Text Available Life-threatening intestinal and systemic effects of the Shiga toxins produced by enterohemorrhagic Escherichia coli (EHEC require toxin uptake and transcytosis across intestinal epithelial cells. We have recently demonstrated that EHEC infection of intestinal epithelial cells stimulates toxin macropinocytosis, an actin-dependent endocytic pathway. Host actin rearrangement necessary for EHEC attachment to enterocytes is mediated by the type 3 secretion system which functions as a molecular syringe to translocate bacterial effector proteins directly into host cells. Actin-dependent EHEC attachment also requires the outer membrane protein intimin, a major EHEC adhesin. Here, we investigate the role of type 3 secretion in actin turnover occurring during toxin macropinocytosis. Toxin macropinocytosis is independent of EHEC type 3 secretion and intimin attachment. EHEC soluble factors are sufficient to stimulate macropinocytosis and deliver toxin into enterocytes in vitro and in vivo; intact bacteria are not required. Intimin-negative enteroaggregative Escherichia coli (EAEC O104:H4 robustly stimulate Shiga toxin macropinocytosis into intestinal epithelial cells. The apical macropinosomes formed in intestinal epithelial cells move through the cells and release their cargo at these cells' basolateral sides. Further analysis of EHEC secreted proteins shows that a serine protease EspP alone is able to stimulate host actin remodeling and toxin macropinocytosis. The observation that soluble factors, possibly serine proteases including EspP, from each of two genetically distinct toxin-producing strains, can stimulate Shiga toxin macropinocytosis and transcellular transcytosis alters current ideas concerning mechanisms whereby Shiga toxin interacts with human enterocytes. Mechanisms important for this macropinocytic pathway could suggest new potential therapeutic targets for Shiga toxin-induced disease.

  10. Characterizing RecA-independent induction of Shiga toxin2-encoding phages by EDTA treatment.

    Directory of Open Access Journals (Sweden)

    Lejla Imamovic

    Full Text Available BACKGROUND: The bacteriophage life cycle has an important role in Shiga toxin (Stx expression. The induction of Shiga toxin-encoding phages (Stx phages increases toxin production as a result of replication of the phage genome, and phage lysis of the host cell also provides a means of Stx toxin to exit the cell. Previous studies suggested that prophage induction might also occur in the absence of SOS response, independently of RecA. METHODOLOGY/PRINCIPAL FINDINGS: The influence of EDTA on RecA-independent Stx2 phage induction was assessed, in laboratory lysogens and in EHEC strains carrying Stx2 phages in their genome, by Real-Time PCR. RecA-independent mechanisms described for phage λ induction (RcsA and DsrA were not involved in Stx2 phage induction. In addition, mutations in the pathway for the stress response of the bacterial envelope to EDTA did not contribute to Stx2 phage induction. The effect of EDTA on Stx phage induction is due to its chelating properties, which was also confirmed by the use of citrate, another chelating agent. Our results indicate that EDTA affects Stx2 phage induction by disruption of the bacterial outer membrane due to chelation of Mg(2+. In all the conditions evaluated, the pH value had a decisive role in Stx2 phage induction. CONCLUSIONS/SIGNIFICANCE: Chelating agents, such as EDTA and citrate, induce Stx phages, which raises concerns due to their frequent use in food and pharmaceutical products. This study contributes to our understanding of the phenomenon of induction and release of Stx phages as an important factor in the pathogenicity of Shiga toxin-producing Escherichia coli (STEC and in the emergence of new pathogenic strains.

  11. 78 FR 53017 - Changes to the Salmonella Verification Sampling Program: Analysis of Raw Beef for Shiga Toxin...

    Science.gov (United States)

    2013-08-28

    ... Toxin-Producin Escherichia coli and Salmonella AGENCY: Food Safety and Inspection Service, USDA. ACTION... Salmonella all raw beef samples that it collects for Shiga toxin-producing Escherichia coli (STEC) analysis....fsis.usda.gov/OPPDE/rdad/FRPubs/93-016F.pdf ). Among other things, the PR/HACCP rule set Salmonella...

  12. Comparative genomics and stx phage characterization of LEE-negative Shiga toxin-producing Escherichia coli

    Directory of Open Access Journals (Sweden)

    Susan Renee Steyert

    2012-11-01

    Full Text Available Infection by Escherichia coli and Shigella species are among the leading causes of death due to diarrheal disease in the world. Shiga toxin producing Escherichia coli (STEC that do not encode the locus of enterocyte effacement (LEE-negative STEC often possess Shiga toxin gene variants and have been isolated from humans and a variety of animal sources. In this study, we compare the genomes of nine LEE-negative STEC harboring various stx alleles with four complete reference LEE-positive STEC isolates. Compared to a representative collection of prototype E. coli and Shigella isolates representing each of the pathotypes, the whole genome phylogeny demonstrated that these isolates are diverse. Whole genome comparative analysis of the 13 genomes revealed that in addition to the absence of the LEE pathogenicity island, phage encoded genes including non-LEE encoded effectors, were absent from all nine LEE-negative STEC genomes. Several plasmid-encoded virulence factors reportedly identified in LEE-negative STEC isolates were identified in only a subset of the nine LEE-negative isolates further confirming the diversity of this group. In combination with whole genome analysis, we characterized the lambdoid phages harboring the various stx alleles and determined their genomic insertion sites. Although the integrase gene sequence corresponded with genomic location, it was not correlated with stx variant, further highlighting the mosaic nature of these phages. The transcription of these phages in different genomic backgrounds was examined. Expression of the Shiga toxin genes, stx1 and/or stx2, as well as the Q genes, were examined with quantitative reverse transcriptase polymerase chain reaction (qRT-PCR assays. A wide range of basal and induced toxin induction was observed. Overall, this is a first significant foray into the genome space of this unexplored group of emerging and divergent pathogens.

  13. New monoclonal antibodies against a novel subtype of Shiga toxin 1 produced by Enterobacter cloacae and their use in analysis of human serum

    Science.gov (United States)

    Shiga toxin (Stx) is a major virulence factor for several bacterial pathogens that cause potentially fatal illness, including Escherichia coli and Shigella spp. The continual emergence of new subtypes of Stxs presents challenges in clinical diagnosis of infections caused by Shiga toxin-producing org...

  14. Thermal inactivation of Escherichia coli 0157:H7 (ECOH) and non-0157 Shiga toxin-producing E.coli (STEC)in mechanically tenderized veal

    Science.gov (United States)

    We quantified thermal destruction of Shiga toxin-producing Escherichia coli O157:H7 (ECOH) and Shiga toxin-producing non-O157 E. coli (STEC) cells within mechanically tenderized veal cutlets following cooking on an electric skillet. For each of five trials, flattened veal cutlets (ca. 71.6 g; ca. 1/...

  15. Serine Protease EspP from Enterohemorrhagic Escherichia Coli Is Sufficient to Induce Shiga Toxin Macropinocytosis in Intestinal Epithelium

    OpenAIRE

    In, Julie; Lukyanenko, Valeriy; Foulke-Abel, Jennifer; Hubbard, Ann L.; Delannoy, Michael; Hansen, Anne-Marie; Kaper, James B.; Boisen, Nadia; Nataro, James P.; Zhu, Chengru; Boedeker, Edgar C.; Gir?n, Jorge A.; Kovbasnjuk, Olga

    2013-01-01

    Life-threatening intestinal and systemic effects of the Shiga toxins produced by enterohemorrhagic Escherichia coli (EHEC) require toxin uptake and transcytosis across intestinal epithelial cells. We have recently demonstrated that EHEC infection of intestinal epithelial cells stimulates toxin macropinocytosis, an actin-dependent endocytic pathway. Host actin rearrangement necessary for EHEC attachment to enterocytes is mediated by the type 3 secretion system which functions as a molecular sy...

  16. Sub-Lethal Dose of Shiga toxin 2 from Enterohemorrhagic Escherichia coli Affects Balance and Cerebellar Cythoarquitecture.

    Directory of Open Access Journals (Sweden)

    Luciana eD’Alessio

    2016-02-01

    Full Text Available Shiga toxin producing Escherichia coli may damage the central nervous system before or concomitantly to manifested hemolytic uremic syndrome symptoms. The cerebellum is frequently damaged during this syndrome, however the deleterious effects of Shiga toxin 2 has never been integrally reported by ultrastructural, physiological and behavioral means. The aim of this study was to determine the cerebellar compromise after intravenous administration of a sub-lethal dose of Shiga toxin 2 by measuring the cerebellar blood brain barrier permeability, behavioral task of cerebellar functionality (inclined plane test, and ultrastructural analysis (transmission electron microscope. Intravenous administration of vehicle (control group, sub-lethal dose of 0.5 ηg and 1 ηg of Stx2 per mouse were tested for behavioral and ultrastructural studies. A set of three independent experiments were performed for each study (n=6. Blood–Brain Barrier resulted damaged and consequently its permeability was significantly increased. Lower scores obtained in the inclined plane task denoted poor cerebellar functionality in comparison to their controls. The most significant lower score was obtained after 5 days of 1ηg of toxin administration. Transmission electron microscope micrographs from the Stx2-treated groups showed neurons with a progressive neurodegenerative condition in a dose dependent manner. As sub-lethal intravenous Shiga toxin 2 altered the blood brain barrier permeability in the cerebellum the toxin penetrated the cerebellar parenchyma and produced cell damaged with significant functional implications in the test balance.

  17. Functional capacity of Shiga-toxin promoter sequences in eukaryotic cells.

    Science.gov (United States)

    Bentancor, Leticia V; Bilen, Marcos F; Mejías, María P; Fernández-Brando, Romina J; Panek, Cecilia A; Ramos, Maria V; Fernández, Gabriela C; Isturiz, Martín; Ghiringhelli, Pablo D; Palermo, Marina S

    2013-01-01

    Shiga toxins (Stx) are the main virulence factors in enterohemorrhagic Escherichia coli (EHEC) infections, causing diarrhea and hemolytic uremic syndrome (HUS). The genes encoding for Shiga toxin-2 (Stx2) are located in a bacteriophage. The toxin is formed by a single A subunit and five B subunits, each of which has its own promoter sequence. We have previously reported the expression of the B subunit within the eukaryotic environment, probably driven by their own promoter. The aim of this work was to evaluate the ability of the eukaryotic machinery to recognize stx2 sequences as eukaryotic-like promoters. Vero cells were transfected with a plasmid encoding Stx2 under its own promoter. The cytotoxic effect on these cells was similar to that observed upon incubation with purified Stx2. In addition, we showed that Stx2 expression in Stx2-insensitive BHK eukaryotic cells induced drastic morphological and cytoskeletal changes. In order to directly evaluate the capacity of the wild promoter sequences of the A and B subunits to drive protein expression in mammalian cells, GFP was cloned under eukaryotic-like putative promoter sequences. GFP expression was observed in 293T cells transfected with these constructions. These results show a novel and alternative way to synthesize Stx2 that could contribute to the global understanding of EHEC infections with immediate impact on the development of treatments or vaccines against HUS.

  18. Sorbitol-fermenting Shiga toxin-producing Escherichia coli O157 in Austria.

    Science.gov (United States)

    Orth, Dorothea; Grif, Katharina; Zimmerhackl, Lothar Bernd; Würzner, Reinhard

    2009-01-01

    Infections with enterohemorrhagic Escherichia coli (EHEC) are the major cause of hemolytic uremic syndrome (HUS), the most common cause of acute renal failure in childhood. Shiga toxins are considered to be the most important virulence factor of EHEC strains. Non-sorbitol-fermenting EHEC O157:H7 is still the most prevalent serotype isolated worldwide; however, sorbitol-fermenting (SF) EHEC O157:H- (H- indicates nonmotility) strains are increasingly reported. Thirteen SF EHEC O157:H- strains (11 of human origin, two from animals) were detected in Austria between 2002 and 2008. Among the 11 human cases, seven suffered from HUS, two from diarrhea and the remaining two were asymptomatic. Seven of the cases were identified in patients living in or visiting (in one case) the province Salzburg, four were in patients from the province Vorarlberg. Three outbreaks with no more than three persons involved were detected, the other four cases occurred sporadically. The pulsed-field gel-electrophoresis banding patterns of the 13 SF EHEC O157:H- isolates were grouped into three distinct clusters (groups 1, 2 and 3). Strains of the three outbreaks were identical (except for one outbreak strain with one band difference) within each outbreak. In comparison, the Bavarian epidemic strain showed a pattern different from all SF O157:H- strains isolated in Austria. For effective detection of SF EHEC O157:H-, screening for Shiga toxins by ELISA and/or Shiga toxin genes by PCR is absolutely necessary; screening on the basis of phenotypic characteristics such as sorbitol-non-fermentation is not sufficient. Typing methods relying solely on investigation of O157 will detect these strains but should nevertheless also be avoided, so that the prevalent non-O157 strains causing HUS are not missed.

  19. Comparisons of Native and Chimeric Shiga Toxins Indicate that the Binding Subunit Dictates Degree of Toxicity

    Science.gov (United States)

    2014-03-17

    toxicity Africian green monkey kidney Cytotoxicity: (287) epithelial (Vero) Stxla lOX > Stx2a Human proximal tubule epithelial Cytotoxicity: Stx 1 a...sections from MAb protection/Stx2a i.g. intoxication study. (A) Moderate A TN ( •) of distal tubules was observed in kidneys of mice in Stx2a + TFTB 1...72 108. Hughes AK, Stricklett PK, Kohan DE. 1998. Cytotoxic effect of Shiga toxin-1 on human proximal tubule cells. Kidney Int 54:426-37 109. Hughes

  20. Survival of Shiga toxin-producing Escherichia coli O157:H7 in Minas frescal cheese

    OpenAIRE

    Frozi, Jesieli Braz; Domingues, Josiane Roberto; Esper, Luciana Maria Ramires; Corrêa da Rosa, Joel Maurício; Silva, Ana Luiza Sant’Anna da Costa; Gonzalez, Alice Gonçalves Martins

    2015-01-01

    Shiga toxin-producing Escherichia coli (STEC) O157:H7 strains (isolated by cattle’s faeces and a reference strain, EDL933), were inoculated into pasteurized milk (102 and 103 cells.mL–1) to prepare the Minas frescal cheese. As control was used uninfected milk. Physicochemical and microbiological analyses were performed to milk and elaborated cheese. The O157:H7 strains were quantified in the stages of cheese processing and during 0, 2, 4, 5, 7, 10 and 15 storage days at 8 °C ...

  1. Mass Spectrometry-Based Method of Detecting and Distinguishing Type 1 and Type 2 Shiga-Like Toxins in Human Serum

    Directory of Open Access Journals (Sweden)

    Christopher J. Silva

    2015-12-01

    Full Text Available Shiga-like toxins (verotoxins are responsible for the virulence associated with a variety of foodborne bacterial pathogens. Direct detection of toxins requires a specific and sensitive technique. In this study, we describe a mass spectrometry-based method of analyzing the tryptic decapeptides derived from the non-toxic B subunits. A gene encoding a single protein that yields a set of relevant peptides upon digestion with trypsin was designed. The 15N-labeled protein was prepared by growing the expressing bacteria in minimal medium supplemented with 15NH4Cl. Trypsin digestion of the 15N-labeled protein yields a set of 15N-labeled peptides for use as internal standards to identify and quantify Shiga or Shiga-like toxins. We determined that this approach can be used to detect, quantify and distinguish among the known Shiga toxins (Stx and Shiga-like toxins (Stx1 and Stx2 in the low attomole range (per injection in complex media, including human serum. Furthermore, Stx1a could be detected and distinguished from the newly identified Stx1e in complex media. As new Shiga-like toxins are identified, this approach can be readily modified to detect them. Since intact toxins are digested with trypsin prior to analysis, the handling of intact Shiga toxins is minimized. The analysis can be accomplished within 5 h.

  2. Annexin A1 and A2: roles in retrograde trafficking of Shiga toxin.

    Science.gov (United States)

    Tcatchoff, Lionel; Andersson, Sofia; Utskarpen, Audrun; Klokk, Tove Irene; Skånland, Sigrid S; Pust, Sascha; Gerke, Volker; Sandvig, Kirsten

    2012-01-01

    Annexins constitute a family of calcium and membrane binding proteins. As annexin A1 and A2 have previously been linked to various membrane trafficking events, we initiated this study to investigate the role of these annexins in the uptake and intracellular transport of the bacterial Shiga toxin (Stx) and the plant toxin ricin. Once endocytosed, both toxins are retrogradely transported from endosomes to the Golgi apparatus and the endoplasmic reticulum before being targeted to the cytosol where they inhibit protein synthesis. This study was performed to obtain new information both about toxin transport and the function of annexin A1 and annexin A2. Our data show that depletion of annexin A1 or A2 alters the retrograde transport of Stx but not ricin, without affecting toxin binding or internalization. Knockdown of annexin A1 increases Golgi transport of Stx, whereas knockdown of annexin A2 slightly decreases the same transport step. Interestingly, annexin A1 was found in proximity to cytoplasmic phospholipase A2 (cPLA(2)), and the basal as well as the increased Golgi transport of Stx upon annexin A1 knockdown is dependent on cPLA(2) activity. In conclusion, annexin A1 and A2 have different roles in Stx transport to the trans-Golgi network. The most prominent role is played by annexin A1 which normally works as a negative regulator of retrograde transport from the endosomes to the Golgi network, most likely by complex formation and inhibition of cPLA(2).

  3. Relevance of Biofilms in the Pathogenesis of Shiga-Toxin-Producing Escherichia coli Infection

    Directory of Open Access Journals (Sweden)

    Natalia Angel Villegas

    2013-01-01

    Full Text Available The present study was designed to determine the relationships among biofilm formation, cellular stress and release of Shiga toxin (Stx by three different clinical Shiga toxin-producing Escherichia coli (STEC strains. The biofilm formation was determined using crystal violet stain in tryptic soy broth or thioglycollate medium with the addition of sugars (glucose or mannose or hydrogen peroxide. The reactive oxygen species (ROSs were detected by the reduction of nitro blue tetrazolium and reactive nitrogen intermediates (RNI determined by the Griess assay. In addition, the activities of two antioxidant enzymes, superoxide dismutase (SOD and catalase (CAT, were studied. For the cytotoxicity studies, Vero cells were cultured with Stx released of STEC biofilms. The addition of sugars in both culture mediums resulted in an increase in biofilm biomass, with a decrease in ROS and RNI production, low levels of SOD and CAT activity, and minimal cytotoxic effects. However, under stressful conditions, an important increase in the antioxidant enzyme activity and high level of Stx production were observed. The disturbance in the prooxidant-antioxidant balance and its effect on the production and release of Stx evaluated under different conditions of biofilm formation may contribute to a better understanding of the relevance of biofilms in the pathogenesis of STEC infection.

  4. Cinnamon Oil Inhibits Shiga Toxin Type 2 Phage Induction and Shiga Toxin Type 2 Production in Escherichia coli O157:H7.

    Science.gov (United States)

    Sheng, Lina; Rasco, Barbara; Zhu, Mei-Jun

    2016-11-15

    This study evaluated the inhibitory effect of cinnamon oil against Escherichia coli O157:H7 Shiga toxin (Stx) production and further explored the underlying mechanisms. The MIC and minimum bactericidal concentration (MBC) of cinnamon oil against E. coli O157:H7 were 0.025% and 0.05% (vol/vol), respectively. Cinnamon oil significantly reduced Stx2 production and the stx 2 mRNA expression that is associated with diminished Vero cell cytotoxicity. Consistently, induction of the Stx-converting phage where the stx 2 gene is located, along with the total number of phages, decreased proportionally to cinnamon oil concentration. In line with decreased Stx2 phage induction, cinnamon oil at 0.75× and 1.0× MIC eliminated RecA, a key mediator of SOS response, polynucleotide phosphorylase (PNPase), and poly(A) polymerase (PAP I), which positively regulate Stx-converting phages, contributing to reduced Stx-converting phage induction and Stx production. Furthermore, cinnamon oil at 0.75× and 1.0× MIC strongly inhibited the qseBC and luxS expression associated with decreased AI-2 production, a universal quorum sensing signaling molecule. However, the expression of oxidative stress response genes oxyR, soxR, and rpoS was increased in response to cinnamon oil at 0.25× or 0.5× MIC, which may contribute to stunted bacterial growth and reduced Stx2 phage induction and Stx2 production due to the inhibitory effect of OxyR on prophage activation. Collectively, cinnamon oil inhibits Stx2 production and Stx2 phage induction in E. coli O157:H7 in multiple ways. This study reports the inhibitory effect of cinnamon oil on Shiga toxin 2 phage induction and Shiga toxin 2 production. Subinhibitory concentrations (concentrations below the MIC) of cinnamon oil reduced Stx2 production, stx 2 mRNA expression, and cytotoxicity on Vero cells. Subinhibitory concentrations of cinnamon oil also dramatically reduced both the Stx2 phage and total phage induction in E. coli O157:H7, which may be due to

  5. Ingested Shiga Toxin 2 (Stx2) Causes Histopathological Changes in Kidney, Spleen and Thymus Tissues and Mortality in Mice

    Science.gov (United States)

    The Shiga toxin (Stxs) producing bacterial strain, Escherichia coli O157:H7, colonizes the distal small intestine and the colon, initiating a very broad spectrum of illnesses such as hemolytic-uremic syndrome (HUS) characterized by microangiopathic hemolytic anemia, thrombocytopenia and acute renal ...

  6. Translocation and Fate of Shiga toxin-producting Escherichia coli in subprimals following blade tenderization and vacuum tumbling

    Science.gov (United States)

    Foodborne pathogens, such as Shiga toxin-producing Escherichia coli (STEC), a natural inhabitant on raw meat, may be transferred from the surface of the meat into the deeper layers of tissue following enhancement by blade tenderization and vacuum tumbling. Therefore, the consumption of enhanced beef...

  7. Rapid O serogroup identification of the six clinically relevant Shiga toxin-producing Escherichia coli by antibody microarray

    Science.gov (United States)

    Antibody array was developed for the detection of the top six non-O157 Shiga toxin-producing Escherichia coli O serogroups. Sensitivity of the array was 10**5 CFU, and the limit of detection of serogroups in ground beef was 1-10 CFU following 12 h of enrichment. The array utilized a minimal amount...

  8. Subtype-specific suppression of Shiga toxin 2 released from Escherichia coli upon exposure to protein synthesis inhibitors

    DEFF Research Database (Denmark)

    Pedersen, Malene Gantzhorn; Hansen, Claus; Riise, Erik

    2008-01-01

    Shiga toxins (Stx) are important virulence factors in the pathogenesis of severe disease including hemolytic-uremic syndrome, caused by Stx-producing Escherichia coli (STEC). STEC strains increase the release of Stx in vitro following the addition of fluoroquinolones, whereas protein synthesis...

  9. Biotinylation of environmentally isolated Shiga toxin-producing Escherichia coli (STEC) – specific bacteriophages for biosensor and biocontrol applications

    Science.gov (United States)

    Like common bacteriophages, Shiga toxin-producing Escherichia coli (STEC) bacteriophages are viruses that recognize and bind to specific bacterial host (STEC) for propagation. They co-exist with STEC hosts, which cause epidemic food and waterborne illnesses, but may act as host populations limiting ...

  10. Isolation and characterization of bacteriophages as potential agents against Shiga toxin – producing Escherichia coli (STEC) strains

    Science.gov (United States)

    Shigatoxin producing Escherichia coli (STEC) is a significant group of foodborne pathogens that can cause mild diarrhea to serious human illnesses. The gastrointestinal tracts of cattle and other ruminants are the primary reservoirs of STEC strains and may co-harbor bacteriophages as part of its ...

  11. Colonization of Enteroaggregative Escherichia coli and Shiga toxin-producing Escherichia coli in chickens and humans in southern Vietnam

    NARCIS (Netherlands)

    Trung, Nguyen Vinh; Nhung, Hoang Ngoc; Carrique-Mas, Juan J; Mai, Ho Huynh; Tuyen, Ha Thanh; Campbell, James; Nhung, Nguyen Thi; Van Minh, Pham; Wagenaar, Jaap A; Mai, Nguyen Thi Nhu; Hieu, Thai Quoc; Schultsz, Constance; Hoa, Ngo Thi

    2016-01-01

    BACKGROUND: Enteroaggregative (EAEC) and Shiga-toxin producing Escherichia coli (STEC) are a major cause of diarrhea worldwide. E. coli carrying both virulence factors characteristic for EAEC and STEC and producing extended-spectrum beta-lactamase caused severe and protracted disease during an

  12. Enhanced laboratory-based surveillance of Shiga-toxin-producing Escherichia coli O157 in The Netherlands.

    NARCIS (Netherlands)

    Duynhoven, Y.T.H.P. van; Jager, C.M. de; Heuvelink, A.E.; Zwaluw, W.K. van der; Maas, H.M.; Pelt, W. van; Wannet, W.J.B.

    2002-01-01

    The aim of this study was to analyse the results of a programme in the Netherlands for enhanced surveillance of Shiga-toxin-producing Escherichia coli (STEC) O157. In this programme, implemented in January 1999, all laboratories report positive cases to the public health services and submit isolates

  13. Colonization of Enteroaggregative Escherichia coli and Shiga toxin-producing Escherichia coli in chickens and humans in southern Vietnam

    NARCIS (Netherlands)

    Trung, Nguyen Vinh; Nhung, Hoang Ngoc; Carrique-Mas, Juan J.; Mai, Ho Huynh; Tuyen, Ha Thanh; Campbell, James; Nhung, Nguyen Thi; Minh, Van Pham; Wagenaar, Jaap A.; Mai, Nguyen Thi Nhu; Hieu, Thai Quoc; Schultsz, Constance; Hoa, Ngo Thi

    2016-01-01

    Background: Enteroaggregative (EAEC) and Shiga-toxin producing Escherichia coli (STEC) are a major cause of diarrhea worldwide. E. coli carrying both virulence factors characteristic for EAEC and STEC and producing extended-spectrum beta-lactamase caused severe and protracted disease during an

  14. Colonization of Enteroaggregative Escherichia coli and Shiga toxin-producing Escherichia coli in chickens and humans in southern Vietnam

    NARCIS (Netherlands)

    Trung, Nguyen Vinh; Nhung, Hoang Ngoc; Carrique-Mas, Juan J.; Mai, Ho Huynh; Tuyen, Ha Thanh; Campbell, James; Nhung, Nguyen Thi; van Minh, Pham; Wagenaar, Jaap A.; Mai, Nguyen Thi Nhu; Hieu, Thai Quoc; Schultsz, Constance; Hoa, Ngo Thi

    2016-01-01

    Enteroaggregative (EAEC) and Shiga-toxin producing Escherichia coli (STEC) are a major cause of diarrhea worldwide. E. coli carrying both virulence factors characteristic for EAEC and STEC and producing extended-spectrum beta-lactamase caused severe and protracted disease during an outbreak of E.

  15. Simultaneous direct detection of Shiga-toxin producing Escherichia coli (STEC) strains by gold nanoparticle optical sensing

    Science.gov (United States)

    Shiga-toxin producing Escherichia coli (STEC) strains (“Big Six” – O26, O45, O103, O111, O121, O145, and O157) represent significant groups of pathogens responsible for foodborne diseases. The objective of this study was to develop a colorimetric optical sensing assay that can simultaneously detect ...

  16. Assessing the Public Health Risk of Shiga-Toxin Producing Escherichia coli using a Rapid Diagnostic Screening Algorithm

    NARCIS (Netherlands)

    de Boer, Richard F.; Ferdous, Mithila; Ott, Alewijn; Scheper, Henk R.; Wisselink, Guido J.; Heck, Max E.; Rossen, John W.; Kooistra-Smid, Anna M. D.

    Shiga toxin-producing Escherichia coli (STEC) is an enteropathogen of public health concern because of its ability to cause serious illness and outbreaks. In this prospective study a diagnostic screening algorithm to categorise STEC infections into risk groups was evaluated. The algorithm consists

  17. Conditional function of autoaggregative protien cah and common cah mutations in Shiga toxin-producing Escherichia coli

    Science.gov (United States)

    Cah is a calcium-binding autotransporter protein involved in autoaggregation and biofilm formation. Although cah is widespread in Shiga toxin-producing Escherichia coli (STEC), we detected mutations in cah at a frequency of 31.3% in this pathogen. In STEC O157:H7 super-shedder strain SS17, a large d...

  18. Outbreak of Non-O157 Shiga Toxin-Producing Escherichia coli Infection from Consumption of Beef Sausage

    DEFF Research Database (Denmark)

    Ethelberg, S.; Smith, B.; Torpdahl, M.

    2009-01-01

    We describe an outbreak of Shiga toxin-producing Escherichia coli O26: H11 infection in 20 patients (median age, 2 years). The source of the infection was an organic fermented beef sausage. The source was discovered by using credit card information to obtain and compare customer transaction records...

  19. Classification of non-O157 shiga toxin-producing escherichia coli(STEC) serotypes with hyperspectral microscope imaging

    Science.gov (United States)

    Non-O157 Shiga toxin-producing Escherichia coli (STEC) strains such as O26, O45, O103, O111, O121 and O145 are recognized as serious outbreak to cause human illness due to their toxicity. A conventional microbiological method for cell counting is laborious and needs long time for the results. Since ...

  20. Mathematical modeling of growth of non-O157 Shiga Toxin-producing Escherichia coli in raw ground beef

    Science.gov (United States)

    The objective of this study was to investigate the growth of Shiga toxin-producing Escherichia coli (STEC, including serogroups O45, O103, O111, O121, and O145) in raw ground beef and to develop mathematical models to describe the bacterial growth under different temperature conditions. Three prima...

  1. Prevalence and characteristics of Shiga toxin-producing Escherichia coli in finishing pigs: implications on public health

    Science.gov (United States)

    Shiga toxin-producing Escherichia coli (STEC) are important food-borne pathogens, which can cause serious illnesses, including hemorrhagic colitis and hemolytic uremic syndrome. To examine if pigs are potential animal reservoirs for human STEC infections, we conducted a longitudinal cohort study in ...

  2. Carbon nanoparticles in lateral flow methods to detect genes encoding virulence factors of Shiga toxin-producing Escherichia coli

    NARCIS (Netherlands)

    Noguera, P.; Posthuma-Trumpie, G.A.; Tuil, van M.; Wal, van der F.J.; Boer, de A.; Moers, A.P.H.A.; Amerongen, van A.

    2011-01-01

    The use of carbon nanoparticles is shown for the detection and identification of different Shiga toxin-producing Escherichia coli virulence factors (vt1, vt2, eae and ehxA) and a 16S control (specific for E. coli) based on the use of lateral flow strips (nucleic acid lateral flow immunoassay,

  3. Prevalence of Farm and Slaughterhouse Workers Carrying Shiga Toxin-Producing Escherichia coli in Korea

    Science.gov (United States)

    Hong, Sahyun; Song, Seung Eun; Oh, Kyung Hwan; Kim, Seung Hak; Yoo, Seok ju; Lim, Hyun Sul; Park, Mi Sun

    2011-01-01

    Objectives The aim of this study was to investigate the distribution of Shiga toxin (Stx) gene-positive stool samples from dairy farmer and slaughterhouse workers in Gyeonggi-Do province. Methods A total of 621 samples from healthy farmers and 198 samples from slaughterhouse workers were screened by polymerase chain reaction (PCR) for Shiga toxigenic Escherichia coli (STEC) infection on stool samples. Results The PCR product of Stx-encoding genes was detected in 21 (3.4%) of 621 farmers and 15 (7.6%) of 198 slaughterhouse workers’ stool samples. Distribution of the Stx PCR positive workers by age increment revealed an increase in STEC infection with age increment in both workers. Distribution of the Stx PCR positive workers by working years revealed an increase in STEC infection with working years in farmers. Conclusion These results of the study show that slaughterhouse workers are at higher risk of STEC infection than farmers. In addition, slaughterhouse workers have a more potential source of food contamination of STEC and transmission. PMID:24159473

  4. Shiga toxin in enterohemorrhagic E.coli: regulation and novel anti-virulence strategies

    Science.gov (United States)

    Pacheco, Alline R.; Sperandio, Vanessa

    2012-01-01

    Enterohemorrhagic Escherichia coli (EHEC) are responsible for major outbreaks of bloody diarrhea and hemolytic uremic syndrome (HUS) throughout the world. The mortality associated with EHEC infections stems from the production and release of a potent Shiga toxin (Stx) by these bacteria. Stx induces cell death in endothelial cells, primarily in the urinary tract, causing HUS. Stx was first described in Shigella dysenteriae serotype I by Kiyoshi Shiga and was discovered later in EHEC. Multiple environmental cues regulate the expression of Stx, including temperature, growth phase, antibiotics, reactive oxygen species (ROS), and quorum sensing. Currently, there is no effective treatment or prophylaxis for HUS. Because antibiotics trigger Stx production and their use to treat EHEC infections is controversial, alternative therapeutic strategies have become the focus of intense research. One such strategy explores quorum sensing inhibitors as therapeutics. These inhibitors target quorum sensing regulation of Stx expression without interfering with bacterial growth, leading to the hypothesis that these inhibitors impose less selective pressure for bacteria to develop drug resistance. In this review, we discuss factors that regulate Stx production in EHEC, as well as novel strategies to prevent and/or minimize the development of HUS in infected subjects. PMID:22919672

  5. Shiga Toxin in Enterohemorrhagic E.coli: regulation and novel antivirulence strategies

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    Vanessa eSperandio

    2012-06-01

    Full Text Available Enterohemorrhagic Escherichia coli O157:H7 infects about 73,000 people annually in the USA and is a major cause of outbreaks of bloody diarrhea worldwide, and. In 5 to 7% of the cases, the person infected develops the potentially fatal sequelae hemolytic uremic syndrome (HUS, characterized by acute kidney failure. A hallmark of EHEC pathogenesis and cause of HUS is the production of Shiga toxin (Stx. Stx was first described by Kiyoshi Shiga in Shigella dysenterae serotype I and later discovered in EHEC, and it has been linked to HUS since 1983. Many factors regulate the production of Stx, including temperature, growth phase, antibiotics, reactive oxygen species and quorum sensing. Currently, there is no effective treatment or prophylaxis for HUS. Since the use of antibiotics is not advised to treat EHEC infections because it triggers Stx production, alternative antibacterial strategies need to be developed. Quorum sensing inhibitors represent a novel class of antibacterial compounds, which have the advantage of not interfering on bacterial growth, thereby without selective pressure that can lead to appearance of resistant strains. In this review, we discuss factors that regulate Stx production in EHEC as well as novel strategies to fight Stx and minimize development to HUS in EHEC-infected patients.

  6. Active Shiga-Like Toxin Produced by Some Aeromonas spp., Isolated in Mexico City.

    Science.gov (United States)

    Palma-Martínez, Ingrid; Guerrero-Mandujano, Andrea; Ruiz-Ruiz, Manuel J; Hernández-Cortez, Cecilia; Molina-López, José; Bocanegra-García, Virgilio; Castro-Escarpulli, Graciela

    2016-01-01

    Shiga-like toxins (Stx) represent a group of bacterial toxins involved in human and animal diseases. Stx is produced by enterohemorrhagic Escherichia coli, Shigella dysenteriae type 1, Citrobacter freundii , and Aeromonas spp.; Stx is an important cause of bloody diarrhea and hemolytic uremic syndrome (HUS). The aim of this study was to identify the stx 1 /stx 2 genes in clinical strains and outer membrane vesicles (OMVs) of Aeromonas spp., 66 strains were isolated from children who live in Mexico City, and Stx effects were evaluated in Vero cell cultures. The capacity to express active Stx1 and Stx2 toxins was determined in Vero cell cultures and the concentration of Stx was evaluated by 50% lethal dose (LD 50 ) assays, observing inhibition of damaged cells by specific monoclonal antibodies. The results obtained in this study support the hypothesis that the stx gene is another putative virulence factor of Aeromonas , and since this gene can be transferred horizontally through OMVs this genus should be included as a possible causal agents of gastroenteritis and it should be reported as part of standard health surveillance procedures. Furthermore, these results indicate that the Aeromonas genus might be a potential causative agent of HUS.

  7. Molecular mechanisms that mediate colonization of Shiga toxin-producing Escherichia coli strains.

    Science.gov (United States)

    Farfan, Mauricio J; Torres, Alfredo G

    2012-03-01

    Shiga toxin-producing Escherichia coli (STEC) is a group of pathogens which cause gastrointestinal disease in humans and have been associated with numerous food-borne outbreaks worldwide. The intimin adhesin has been considered for many years to be the only colonization factor in these strains. However, the rapid progress in whole-genome sequencing of different STEC serotypes has accelerated the discovery of other adhesins (fimbrial and afimbrial), which have emerged as important contributors to the intestinal colonization occurring during STEC infection. This review summarizes recent progress to identify and characterize, at the molecular level, novel adhesion and colonization factors in STEC strains, with an emphasis on their contribution to virulence traits, their host-pathogen interactions, the regulatory mechanisms controlling their expression, and their role as targets eliciting immune responses in the host.

  8. Nitrobenzylthioinosine (NBT), a nucleoside transport inhibitor, protects against Shiga toxin cytotoxicity in human microvascular endothelial cells.

    Science.gov (United States)

    Ohmi, K; Kiyokawa, N; Sekino, T; Suzuki, T; Mimori, K; Taguchi, T; Nakajima, H; Katagiri, Y U; Fujimoto, J; Nakao, H; Takeda, T

    2001-01-01

    Infections with Shiga toxin (Stx)-producing Escherichia coli (STEC) cause microvascular endothelial cell damage, resulting in hemorrhagic colitis and hemolytic uremic syndrome. The prevention of endothelial cell damage is therefore a crucial step in overcoming this disorder. Here, we report that nitrobenzylthioinosine (NBT), a nucleoside transport inhibitor, has a protective effect against the cytotoxicity of Stxs in human microvascular endothelial cells (HMVECs). The relative viability of cells treated with 1.5-15 pM of Stx1 was reduced to 10-20% of that without Stx1. However, the viability of cells treated with NBT (10-100 microM) remained higher than 80%, even in the presence of Stx1. NBT also protected against Stx1 cytotoxicity in sodium butyrate-treated hypersensitive HMVECs. The protective effect of NBT against Stx cytotoxicity may be due to the depletion of ATP in the cells, thereby inhibiting the entry of Stx1.

  9. Annexin A1 and A2: roles in retrograde trafficking of Shiga toxin.

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    Lionel Tcatchoff

    Full Text Available Annexins constitute a family of calcium and membrane binding proteins. As annexin A1 and A2 have previously been linked to various membrane trafficking events, we initiated this study to investigate the role of these annexins in the uptake and intracellular transport of the bacterial Shiga toxin (Stx and the plant toxin ricin. Once endocytosed, both toxins are retrogradely transported from endosomes to the Golgi apparatus and the endoplasmic reticulum before being targeted to the cytosol where they inhibit protein synthesis. This study was performed to obtain new information both about toxin transport and the function of annexin A1 and annexin A2. Our data show that depletion of annexin A1 or A2 alters the retrograde transport of Stx but not ricin, without affecting toxin binding or internalization. Knockdown of annexin A1 increases Golgi transport of Stx, whereas knockdown of annexin A2 slightly decreases the same transport step. Interestingly, annexin A1 was found in proximity to cytoplasmic phospholipase A2 (cPLA(2, and the basal as well as the increased Golgi transport of Stx upon annexin A1 knockdown is dependent on cPLA(2 activity. In conclusion, annexin A1 and A2 have different roles in Stx transport to the trans-Golgi network. The most prominent role is played by annexin A1 which normally works as a negative regulator of retrograde transport from the endosomes to the Golgi network, most likely by complex formation and inhibition of cPLA(2.

  10. Characterization of a Shiga-Like Toxin Converting Phage Isolated from an Escherichia coli Strain Responsible for Hemorrhagic Colitis in Humans

    Science.gov (United States)

    1985-01-01

    within the subcloned DNA of the phage . VI. Initiate molecular epidemiologic studies in which strains of Esche- richia col, and other bacteria from...B821/2/1. but not in shigella or vibrio strains that make Shiga-like toxin. Several bands exhibiting phage homology were found in the E. coli normal...oolooous bands, but Shioella and Vibrio strains that produce Shiga-iike toxin exhibited no detectable homology with labeled whole- phage DNA of the probe

  11. Strain-Level Discrimination of Shiga Toxin-Producing Escherichia coli in Spinach Using Metagenomic Sequencing.

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    Susan R Leonard

    Full Text Available Consumption of fresh bagged spinach contaminated with Shiga toxin-producing Escherichia coli (STEC has led to severe illness and death; however current culture-based methods to detect foodborne STEC are time consuming. Since not all STEC strains are considered pathogenic to humans, it is crucial to incorporate virulence characterization of STEC in the detection method. In this study, we assess the comprehensiveness of utilizing a shotgun metagenomics approach for detection and strain-level identification by spiking spinach with a variety of genomically disparate STEC strains at a low contamination level of 0.1 CFU/g. Molecular serotyping, virulence gene characterization, microbial community analysis, and E. coli core gene single nucleotide polymorphism (SNP analysis were performed on metagenomic sequence data from enriched samples. It was determined from bacterial community analysis that E. coli, which was classified at the phylogroup level, was a major component of the population in most samples. However, in over half the samples, molecular serotyping revealed the presence of indigenous E. coli which also contributed to the percent abundance of E. coli. Despite the presence of additional E. coli strains, the serotype and virulence genes of the spiked STEC, including correct Shiga toxin subtype, were detected in 94% of the samples with a total number of reads per sample averaging 2.4 million. Variation in STEC abundance and/or detection was observed in replicate spiked samples, indicating an effect from the indigenous microbiota during enrichment. SNP analysis of the metagenomic data correctly placed the spiked STEC in a phylogeny of related strains in cases where the indigenous E. coli did not predominate in the enriched sample. Also, for these samples, our analysis demonstrates that strain-level phylogenetic resolution is possible using shotgun metagenomic data for determining the genomic relatedness of a contaminating STEC strain to other

  12. Detection of Shiga Toxin-Producing Escherichia coli from Nonhuman Sources and Strain Typing.

    Science.gov (United States)

    Beutin, Lothar; Fach, Patrick

    2014-06-01

    Shiga toxin-producing Escherichia coli (STEC) strains are commonly found in the intestine of ruminant species of wild and domestic animals. Excretion of STEC with animal feces results in a broad contamination of food and the environment. Humans get infected with STEC through ingestion of contaminated food, by contact with the environment, and from STEC-excreting animals and humans. STEC strains can behave as human pathogens, and some of them, called enterohemorrhagic E. coli (EHEC), may cause hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS). Because of the diversity of STEC types, detection strategies for STEC and EHEC are based on the identification of Shiga toxins or the underlying genes. Cultural enrichment of STEC from test samples is needed for identification, and different protocols were developed for this purpose. Multiplex real-time PCR protocols (ISO/CEN TS13136 and USDA/FSIS MLG5B.01) have been developed to specifically identify EHEC by targeting the LEE (locus of enterocyte effacement)-encoded eae gene and genes for EHEC-associated O groups. The employment of more genetic markers (nle and CRISPR) is a future challenge for better identification of EHEC from any kinds of samples. The isolation of STEC or EHEC from a sample is required for confirmation, and different cultivation protocols and media for this purpose have been developed. Most STEC strains present in food, animals, and the environment are eae negative, but some of these strains can cause HC and HUS in humans as well. Phenotypic assays and molecular tools for typing EHEC and STEC strains are used to detect and characterize human pathogenic strains among members of the STEC group.

  13. Real-time isothermal detection of Shiga toxin-producing Escherichia coli using recombinase polymerase amplification.

    Science.gov (United States)

    Murinda, Shelton E; Ibekwe, A Mark; Zulkaffly, Syaizul; Cruz, Andrew; Park, Stanley; Razak, Nur; Paudzai, Farah Md; Ab Samad, Liana; Baquir, Khairul; Muthaiyah, Kokilah; Santiago, Brenna; Rusli, Amirul; Balkcom, Sean

    2014-07-01

    Shiga toxin-producing Escherichia coli (STEC) are a major family of foodborne pathogens of public health, zoonotic, and economic significance in the United States and worldwide. To date, there are no published reports on use of recombinase polymerase amplification (RPA) for STEC detection. The primary goal of this study was to assess the potential application of RPA in detection of STEC. This study focused on designing and evaluating RPA primers and fluorescent probes for isothermal (39°C) detection of STEC. Compatible sets of candidate primers and probes were designed for detection of Shiga toxin 1 and 2 (Stx1 and 2), respectively. The sets were evaluated for specificity and sensitivity against STEC (n=12) of various stx genotypes (stx1/stx2, stx1, or stx2, respectively), including non-Stx-producing E. coli (n=28) and other genera (n=7). The primers and probes that were designed targeted amplification of the subunit A moiety of stx1 and stx2. The assay detected STEC in real time (within 5-10 min at 39°C) with high sensitivity (93.5% vs. 90%; stx1 vs. stx2), specificity (99.1% vs. 100%; stx1 vs. stx2), and predictive value (97.9% for both stx1 vs. stx2). Limits of detection of ∼ 5-50 colony-forming units/mL were achieved in serially diluted cultures grown in brain heart infusion broth. This study successfully demonstrated for the first time that RPA can be used for isothermal real-time detection of STEC.

  14. Comparative Genomic and Phylogenetic Analysis of a Shiga Toxin Producing Shigella sonnei (STSS Strain

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    Domonkos Sváb

    2017-05-01

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

  15. Virulence genes, Shiga toxin subtypes, major O-serogroups, and phylogenetic background of Shiga toxin-producing Escherichia coli strains isolated from cattle in Iran.

    Science.gov (United States)

    Jajarmi, Maziar; Imani Fooladi, Abbas Ali; Badouei, Mahdi Askari; Ahmadi, Ali

    2017-08-01

    The aim of this study was to investigate the virulence potential of the isolated bovine STEC for humans in Iran. In this study a collection of STEC strains (n = 50) had been provided via four stages, including sampling from feces of cattle, E. coli isolation, molecular screening of Shiga toxin (stx) genes, and saving the STEC strains from various geographical areas in Iran. The STEC isolates were subjected to stx-subtyping, O-serogrouping, and phylo-grouping by conventional polymerase chain reaction (PCR). Occurrence of stx1 (52%) and stx2 (64%) was not significantly different (p = 0.1), and 16% of isolates carried both stx1 and stx2, simultaneously. In addition, 36% and 80% of the isolates were positive for eae and ehxA, respectively. Molecular subtyping showed that stx1a (52%), stx2a (44%), stx2c (44%), and stx2d (30%) were the most prevalent subtypes; two combinations stx2a/stx2c and stx2c/stx2d coexisted in 18% and 10% of STEC strains, respectively. Three important non-O157 serogroups, including O113 (20%), O26 (12%), and O111 (10%), were predominant, and none of the isolates belonged to O157. Importantly, one O26 isolate carried stx1, stx2, eae and ehxA and revealed highly virulent stx subtypes. Moreover, all the 21 serogrouped strains belonged to the B1 phylo-type. Our study highlights the significance of non-O157 STEC strains carrying highly pathogenic virulence genes in cattle population as the source of this pathogen in Iran. Since non-O157 STEC strains are not routinely tried in most diagnostic laboratories, majority of the STEC-associated human infections appear to be overlooked in the clinical settings. Copyright © 2017. Published by Elsevier Ltd.

  16. Virulence Potential of Activatable Shiga Toxin 2d-Producing Escherichia coli Isolates from Fresh Produce.

    Science.gov (United States)

    Melton-Celsa, Angela R; O'Brien, Alison D; Feng, Peter C H

    2015-11-01

    Shiga toxin (Stx)-producing Escherichia coli (STEC) strains are food- and waterborne pathogens that are often transmitted via beef products or fresh produce. STEC strains cause both sporadic infections and outbreaks, which may result in hemorrhagic colitis and hemolytic uremic syndrome. STEC strains may elaborate Stx1, Stx2, and/or subtypes of those toxins. Epidemiological evidence indicates that STEC that produce subtypes Stx2a, Stx2c, and/or Stx2d are more often associated with serious illness. The Stx2d subtype becomes more toxic to Vero cells after incubation with intestinal mucus or elastase, a process named "activation." Stx2d is not generally found in the E. coli serotypes most commonly connected to STEC outbreaks. However, STEC strains that are stx2d positive can be isolated from foods, an occurrence that gives rise to the question of whether those food isolates are potential human pathogens. In this study, we examined 14 STEC strains from fresh produce that were stx2d positive and found that they all produced the mucus-activatable Stx2d and that a subset of the strains tested were virulent in streptomycin-treated mice.

  17. Mouse in Vivo Neutralization of Escherichia coli Shiga Toxin 2 with Monoclonal Antibodies

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    Larry H. Stanker

    2013-10-01

    Full Text Available Shiga toxin-producing Escherichia coli (STEC food contaminations pose serious health concerns, and have been the subject of massive food recalls. STEC has been identified as the major cause of the life-threatening complication of hemolytic uremic syndrome (HUS. Besides supportive care, there currently are no therapeutics available. The use of antibiotics for combating pathogenic E. coli is not recommended because they have been shown to stimulate toxin production. Clearing Stx2 from the circulation could potentially lessen disease severity. In this study, we tested the in vivo neutralization of Stx2 in mice using monoclonal antibodies (mAbs. We measured the biologic half-life of Stx2 in mice and determined the distribution phase or t1/2 α to be 3 min and the clearance phase or t1/2 β to be 40 min. Neutralizing mAbs were capable of clearing Stx2 completely from intoxicated mouse blood within minutes. We also examined the persistence of these mAbs over time and showed that complete protection could be passively conferred to mice 4 weeks before exposure to Stx2. The advent of better diagnositic methods and the availability of a greater arsenal of therapeutic mAbs against Stx2 would greatly enhance treatment outcomes of life threatening E. coli infections.

  18. Specific egg yolk immunoglobulin as a new preventive approach for Shiga-toxin-mediated diseases.

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    Paola Neri

    Full Text Available Shiga toxins (Stxs are involved in the development of severe systemic complications associated with enterohemorrhagic Escherichia coli (EHEC infection. Various neutralizing agents against Stxs are under investigation for management of EHEC infection. In this study, we immunized chickens with formalin-inactivated Stx-1 or Stx-2, and obtained immunoglobulin Y (IgY from the egg yolk. Anti-Stx-1 IgY and anti-Stx-2 IgY recognized the corresponding Stx A subunit and polymeric but not monomeric B subunit. Anti-Stx-1 IgY and anti-Stx-2 IgY suppressed the cytotoxicity of Stx-1 and Stx-2 to HeLa 229 cells, without cross-suppressive activity. The suppressive activity of these IgY was abrogated by pre-incubation with the corresponding recombinant B subunit, which suggests that the antibodies directed to the polymeric B subunits were predominantly involved in the suppression. In vivo, the intraperitoneal or intravenous administration of these IgY rescued mice from death caused by intraperitoneal injection of the corresponding toxin at a lethal dose. Moreover, oral administration of anti-Stx-2 IgY reduced the mortality of mice infected intestinally with EHEC O157:H7. Our results therefore suggest that anti-Stx IgY antibodies may be considered as preventive agents for Stx-mediated diseases in EHEC infection.

  19. A Rapid and Sensitive Method to Measure the Functional Activity of Shiga Toxins in Human Serum

    Science.gov (United States)

    Arfilli, Valentina; Carnicelli, Domenica; Ardissino, Gianluigi; Torresani, Erminio; Scavia, Gaia; Brigotti, Maurizio

    2015-01-01

    Shiga toxins (Stx) have a definite role in the development of hemolytic uremic syndrome in children with hemorrhagic colitis caused by pathogenic Stx-producing Escherichia coli (STEC) strains. The dramatic effects of these toxins on the microvasculature of different organs, particularly of the kidney, are well known, whereas there is no consensus on the mechanism by which Stx reach the endothelia of target organs and/or indirectly injure these body sites. We hereby describe a quick (4 h), radioactive, Raji cell-based method designed for the detection of Stx in human sera. The assay monitors the translation impairment induced by these powerful inhibitors of protein synthesis, which are identified properly by neutralizing their activity with specific monoclonal antibodies. By this method, we detected for the first time the functional activity of Stx in sera of STEC-infected patients during hemorrhagic colitis. Recent research has pointed to a dynamic process of Stx-induced renal intoxication in which concurrent and interactive steps are involved. Our rapid and specific method could be useful for studying the kinetics of Stx during the natural course of STEC infection and the interplay between Stx activity in serum and Stx presence in different blood fractions (neutrophils, monocytes, platelets, leukocyte-platelet aggregates, microvesicles, lipoproteins). PMID:26556372

  20. Selective Evolution of Ligands by Exponential Enrichment to Identify RNA Aptamers against Shiga Toxins

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    Sreerupa Challa

    2014-01-01

    Full Text Available Infection with Shiga toxin- (Stx- producing E. coli causes life threatening hemolytic uremic syndrome (HUS, a leading cause of acute renal failure in children. Of the two antigenically distinct toxins, Stx1 and Stx2, Stx2 is more firmly linked with the development of HUS. In the present study, selective evolution of ligands by exponential enrichment (SELEX was used in an attempt to identify RNA aptamers against Stx1 and Stx2. After 5 rounds of selection, significant enrichment of aptamer pool was obtained against Stx2, but not against Stx1, using a RNA aptamer library containing 56 random nucleotides (N56. Characterization of individual aptamer sequences revealed that six unique RNA aptamers (mA/pC, mB/pA, mC, mD, pB, and pD recognized Stx2 in a filter binding assay. None of these aptamers bound Stx1. Aptamers mA/pC, mB/pA, mC, and mD, but not pB and pD, partially blocked binding of Alexa 488-labeled Stx2 with HeLa cells in a flow cytometry assay. However, none of the aptamers neutralized Stx2-mediated cytotoxicity and death of HeLa cells.

  1. Antibody Microarray for E. coli O157:H7 and Shiga Toxin in Microtiter Plates

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    Andrew G. Gehring

    2015-12-01

    Full Text Available Antibody microarray is a powerful analytical technique because of its inherent ability to simultaneously discriminate and measure numerous analytes, therefore making the technique conducive to both the multiplexed detection and identification of bacterial analytes (i.e., whole cells, as well as associated metabolites and/or toxins. We developed a sandwich fluorescent immunoassay combined with a high-throughput, multiwell plate microarray detection format. Inexpensive polystyrene plates were employed containing passively adsorbed, array-printed capture antibodies. During sample reaction, centrifugation was the only strategy found to significantly improve capture, and hence detection, of bacteria (pathogenic Escherichia coli O157:H7 to planar capture surfaces containing printed antibodies. Whereas several other sample incubation techniques (e.g., static vs. agitation had minimal effect. Immobilized bacteria were labeled with a red-orange-fluorescent dye (Alexa Fluor 555 conjugated antibody to allow for quantitative detection of the captured bacteria with a laser scanner. Shiga toxin 1 (Stx1 could be simultaneously detected along with the cells, but none of the agitation techniques employed during incubation improved detection of the relatively small biomolecule. Under optimal conditions, the assay had demonstrated limits of detection of ~5.8 × 105 cells/mL and 110 ng/mL for E. coli O157:H7 and Stx1, respectively, in a ~75 min total assay time.

  2. Quantitative Detection of Shiga Toxins Directly from Stool Specimens of Patients Associated with an Outbreak of Enterohemorrhagic Escherichia coli in Japan--Quantitative Shiga toxin detection from stool during EHEC outbreak.

    Science.gov (United States)

    Yamasaki, Eiki; Watahiki, Masanori; Isobe, Junko; Sata, Tetsutaro; Nair, G Balakrish; Kurazono, Hisao

    2015-10-27

    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.

  3. Effects of stresses on the growth and Cytotoxicity of Shiga-Toxin producing Escherichia coli in ground beef and spinach

    Science.gov (United States)

    The objectives of this study were to examine the effect of stresses on the growth and cytotoxicity of pathogenic Escherichia coli in beef and spinach. A mixture of three strains of Shiga toxin-producing E. coli (STEC) O157:H7 or four strains of non-O157 STEC O26:H11, O103:H1, O104:H4, and O145:NM wa...

  4. Shiga toxin-producing Escherichia coli O157:H7 in milk and milk products in Ogun State, Nigeria.

    Science.gov (United States)

    Ivbade, Akhigbe; Ojo, Olufemi Ernest; Dipeolu, Morenike Atinuke

    2014-01-01

    Shiga toxin producing Escherichia coli (STEC) O157 is a major cause of food-borne illnesses in humans. This study investigated the presence of STEC O157 in milk and milk products in Ogun State, Nigeria. Of a total of 202 samples 10 (5%) were positive for STEC O157 including 1 (2%) of 50 raw milk samples, 3 (6%) of 50 samples of fresh local cheese, 1 (2%) of 50 samples of fried local cheese and 5 (9.6%) of 52 fermented milk samples. There was no significant difference (p>0.05) in the prevalence of STEC O157 among the sample types. Of 10 isolates, shiga toxin 1 gene (stx1) was detected only in 2 samples (20%), shiga toxin 2 (stx2) was extracted only in 6 samples (60%), stx1 /stx2 in 2 samples (20.0%), intimin gene (eaeA) in 5 samples (50%), and enterohaemolysin (E-hlyA) gene was isolated in 7 (70%) samples. Rates of resistance of the STEC O157 isolates were: amoxicillin/clavulanic acid 100%, ampicillin 100%, chloramphenicol 60%, nalidixic acid 20%, norfloxacin 10%, streptomycin 30%, sulphamethoxazole/trimethprim 20%, and tetracycline 90%. The isolates were all susceptible to ciprofloxacin and neomycin. The presence of virulent multidrug resistant E. coli O157 strains in milk and milk products as revealed by this study unveils a risk of human exposure to these potentially fatal pathogens following consumption of contaminated products.

  5. Shiga toxin-producing Escherichia coli O157:H7 in milk and milk products in Ogun State, Nigeria

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    Akhigbe Ivbade

    2014-09-01

    Full Text Available Shiga toxin producing Escherichia coli (STEC O157 is a major cause of food-borne illnesses in humans. This study investigated the presence of STEC O157 in milk and milk products in Ogun State, Nigeria. Of a total of 202 samples 10 (5% were positive for STEC O157 including 1 (2% of 50 raw milk samples, 3 (6% of 50 samples of fresh local cheese, 1 (2% of 50 samples of fried local cheese and 5 (9.6% of 52 fermented milk samples. There was no significant difference (p>0.05 in the prevalence of STEC O157 among the sample types. Of 10 isolates, shiga toxin 1 gene (stx1 was detected only in 2 samples (20%, shiga toxin 2 (stx2 was extracted only in 6 samples (60%, stx1 /stx2 in 2 samples (20.0%, intimin gene (eaeA in 5 samples (50%, and enterohaemolysin (E-hlyA gene was isolated in 7 (70% samples. Rates of resistance of the STEC O157 isolates were: amoxicillin/clavulanic acid 100%, ampicillin 100%, chloramphenicol 60%, nalidixic acid 20%, norfloxacin 10%, streptomycin 30%, sulphamethoxazole/trimethprim 20%, and tetracycline 90%. The isolates were all susceptible to ciprofloxacin and neomycin. The presence of virulent multidrug resistant E. coli O157 strains in milk and milk products as revealed by this study unveils a risk of human exposure to these potentially fatal pathogens following consumption of contaminated products.

  6. PMA-LAMP for rapid detection of Escherichia coli and shiga toxins from viable but non-culturable state.

    Science.gov (United States)

    Yan, Muxia; Xu, Ling; Jiang, Hua; Zhou, Zhenwen; Zhou, Shishui; Zhang, Li

    2017-04-01

    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.

  7. Shiga toxin-producing Escherichia coli in beef retail markets from Argentina

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    Victoria eBrusa

    2013-01-01

    Full Text Available Shiga toxing-producing Escherichia coli (STEC are foodborne pathogens that cause mild or serious diseases and can lead to people death. This study reports the prevalence and characteristics of STEC O157 and non-O157 in commercial ground beef and environmental samples, including meat table, knife, meat mincing machine and manipulator hands (n= 450 obtained from 90 retail markets over a nine-month period. The STEC isolates were serotyped and virulence genes as stx (Shiga toxin, rfbO157 (O157 lipopolysaccharide, fliCH7 (H7 flagellin, eae (intimin, ehxA (enterohemolysin and saa (STEC autoagglutinating adhesin, were determined. STEC O157 were identified in 23 (25.5% beef samples and 16 (4.4% environmental samples, while STEC non-O157 were present in 47 (52.2% and 182 (50.5%, respectively. Among 54 strains isolated, 17 were STEC O157:H7 and 37 were STEC non-O157. The prevalent genotype for O157 was stx2/eae/ehxA/fliCH7 (83.4%, and for STEC non-O157 the most frequent ones were stx1/stx2/saa/ehxA (29.7%; stx2 (29.7%; and stx2/saa/ehxA (27%. None of the STEC non-O157 strains were eae-positive. Besides O157:H7, other 20 different serotypes were identified, being O8:H19, O178:H19 and O174:H28 the prevalent. Strains belonging to the same serotype could be isolated from different sources of the same retail market. Also, the same serotype could be detected in different stores. In conclusion, screening techniques are increasingly sensitive, but the isolation of STEC non-O157 is still a challenge. Moreover, with the results obtained from the present work, although more studies are needed, cross-contamination between meat and the environment could be suspected.

  8. Molecular basis of differential B-pentamer stability of Shiga toxins 1 and 2.

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    Deborah G Conrady

    2010-12-01

    Full Text Available Escherichia coli strain O157:H7 is a major cause of food poisoning that can result in severe diarrhea and, in some cases, renal failure. The pathogenesis of E. coli O157:H7 is in large part due to the production of Shiga toxin (Stx, an AB(5 toxin that consists of a ribosomal RNA-cleaving A-subunit surrounded by a pentamer of receptor-binding B subunits. There are two major isoforms, Stx1 and Stx2, which differ dramatically in potency despite having 57% sequence identity. Animal studies and epidemiological studies show Stx2 is associated with more severe disease. Although the molecular basis of this difference is unknown, data suggest it is associated with the B-subunit. Mass spectrometry studies have suggested differential B-pentamer stability between Stx1 and Stx2. We have examined the relative stability of the B-pentamers in solution. Analytical ultracentrifugation using purified B-subunits demonstrates that Stx2B, the more deadly isoform, shows decreased pentamer stability compared to Stx1B (EC(50 = 2.3 µM vs. EC(50 = 0.043 µM for Stx1B. X-ray crystal structures of Stx1B and Stx2B identified a glutamine in Stx2 (versus leucine in Stx1 within the otherwise strongly hydrophobic interface between B-subunits. Interchanging these residues switches the stability phenotype of the B-pentamers of Stx1 and Stx2, as demonstrated by analytical ultracentrifugation and circular dichroism. These studies demonstrate a profound difference in stability of the B-pentamers in Stx1 and Stx2, illustrate the mechanistic basis for this differential stability, and provide novel reagents to test the basis for differential pathogenicity of these toxins.

  9. Protozoan Predation of Escherichia coli O157:H7 Is Unaffected by the Carriage of Shiga Toxin-Encoding Bacteriophages.

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    Carrie E Schmidt

    Full Text Available Escherichia coli O157:H7 is a food-borne bacterium that causes hemorrhagic diarrhea and hemolytic uremic syndrome in humans. While cattle are a known source of E. coli O157:H7 exposure resulting in human infection, environmental reservoirs may also be important sources of infection for both cattle and humans. Bacteriophage-encoded Shiga toxins (Stx carried by E. coli O157:H7 may provide a selective advantage for survival of these bacteria in the environment, possibly through their toxic effects on grazing protozoa. To determine Stx effects on protozoan grazing, we co-cultured Paramecium caudatum, a common ciliate protozoon in cattle water sources, with multiple strains of Shiga-toxigenic E. coli O157:H7 and non-Shiga toxigenic cattle commensal E. coli. Over three days at ambient laboratory temperature, P. caudatum consistently reduced both E. coli O157:H7 and non-Shiga toxigenic E. coli populations by 1-3 log cfu. Furthermore, a wild-type strain of Shiga-toxigenic E. coli O157:H7 (EDL933 and isogenic mutants lacking the A subunit of Stx 2a, the entire Stx 2a-encoding bacteriophage, and/or the entire Stx 1-encoding bacteriophage were grazed with similar efficacy by both P. caudatum and Tetrahymena pyriformis (another ciliate protozoon. Therefore, our data provided no evidence of a protective effect of either Stx or the products of other bacteriophage genes on protozoan predation of E. coli. Further research is necessary to determine if the grazing activity of naturally-occurring protozoa in cattle water troughs can serve to decrease cattle exposure to E. coli O157:H7 and other Shiga-toxigenic E. coli.

  10. Survival of Shiga toxin-producing Escherichia coli O157:H7 in Minas frescal cheese

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    Jesieli Braz Frozi

    2015-03-01

    Full Text Available Shiga toxin-producing Escherichia coli (STEC O157:H7 strains (isolated by cattle’s faeces and a reference strain, EDL933, were inoculated into pasteurized milk (102 and 103 cells.mL–1 to prepare the Minas frescal cheese. As control was used uninfected milk. Physicochemical and microbiological analyses were performed to milk and elaborated cheese. The O157:H7 strains were quantified in the stages of cheese processing and during 0, 2, 4, 5, 7, 10 and 15 storage days at 8 °C onto Sorbitol MacConkey Agar supplemented with potassium tellurite and cefixime (CT-SMAC. O157:H7 was not present in the pasteurised milk prior to the artificial inoculation. At the end of the processing the cheese had 10 to 100 times more STEC O157:H7 than the initial inoculum. During the storage, the Minas frescal cheese exhibited the largest population increase on the 4th and 5th day when inoculated with 102 and 103 cells.mL–1, respectively. Additionally, viable cells were found up to the 10th and 15th day, according to the amount of initial inoculum. This number of cells is able to cause infection in humans, and therefore, Minas frescal cheese, even when stored under refrigeration, is a potential vehicle of disease caused by STEC O157:H7.

  11. Comparative analysis of the Shiga toxin converting bacteriophage first detected in Shigella sonnei.

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    Tóth, István; Sváb, Domonkos; Bálint, Balázs; Brown-Jaque, Maryury; Maróti, Gergely

    2016-01-01

    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.

  12. Shiga toxin-2 induces neutrophilia and neutrophil activation in a murine model of hemolytic uremic syndrome.

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    Fernández, G C; Rubel, C; Dran, G; Gómez, S; Isturiz, M A; Palermo, M S

    2000-06-01

    It has been demonstrated that infections due to Shiga toxins (Stx) producing Escherichia coli are the main cause of the hemolytic uremic syndrome (HUS). Although it is recognized that Stx damage the glomerular endothelium, clinical and experimental evidence suggests that the inflammatory response is able to potentiate Stx toxicity. Lipopolysaccharides (LPS) and neutrophils (PMN) represent two central components of inflammation during a gram-negative infection. In this regard, patients with high peripheral PMN counts at presentation have a poor prognosis. Since the murine model has been used to study LPS-Stx interactions, we analyzed the effects of Stx alone or in combination with LPS on the kinetics of neutrophil production and activation and their participation in renal damage. We observed a sustained neutrophilia after Stx2 injection. Moreover, these neutrophils showed increased expression of CD11b, enhanced cytotoxic capacity, and greater adhesive properties. Regarding the cooperative effects of LPS on Stx2 action, we demonstrated potentiation of neutrophilia and CD11b induction at early times by pretreatment with LPS. Finally, a positive correlation between neutrophil percentage and renal damage (assayed as plasmatic urea) firmly suggests a role for PMN in the pathogenesis of HUS.

  13. Shiga Toxin Glycosphingolipid Receptors in Human Caco-2 and HCT-8 Colon Epithelial Cell Lines.

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    Kouzel, Ivan U; Pohlentz, Gottfried; Schmitz, Julia S; Steil, Daniel; Humpf, Hans-Ulrich; Karch, Helge; Müthing, Johannes

    2017-10-25

    Shiga toxins (Stxs) released by enterohemorrhagic Escherichia coli (EHEC) into the human colon are the causative agents for fatal outcome of EHEC infections. Colon epithelial Caco-2 and HCT-8 cells are widely used for investigating Stx-mediated intestinal cytotoxicity. Only limited data are available regarding precise structures of their Stx receptor glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), and lipid raft association. In this study we identified Gb3Cer and Gb4Cer lipoforms of serum-free cultivated Caco-2 and HCT-8 cells, chiefly harboring ceramide moieties composed of sphingosine (d18:1) and C16:0, C22:0 or C24:0/C24:1 fatty acid. The most significant difference between the two cell lines was the prevalence of Gb3Cer with C16 fatty acid in HCT-8 and Gb4Cer with C22-C24 fatty acids in Caco-2 cells. Lipid compositional analysis of detergent-resistant membranes (DRMs), which were used as lipid raft -equivalents, indicated slightly higher relative content of Stx receptor Gb3Cer in DRMs of HCT-8 cells when compared to Caco-2 cells. Cytotoxicity assays revealed substantial sensitivity towards Stx2a for both cell lines, evidencing little higher susceptibility of Caco-2 cells versus HCT-8 cells. Collectively, Caco-2 and HCT-8 cells express a plethora of different receptor lipoforms and are susceptible towards Stx2a exhibiting somewhat lower sensitivity when compared to Vero cells.

  14. Effect of carvacrol on O157 and non-O157 Shiga toxin producing Escherichia coli

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    Alexandros Stratakos

    2017-06-01

    Full Text Available Shiga toxin Escherichia coli (STEC strains are important foodborne bacteria linked to diarrhea, enteritis, hemolytic-uremic syndrome and in some cases death. E. coli O157:H7 is the most common strain amongst STECs however non-O157 STECs have been connected with several outbreaks on an international level.  The use of natural plant extracts to reduce the risk from foodborne pathogens is gaining increasing importance. Therefore in this study, we tested the antibacterial effect of carvacrol, a major component of oregano essential oil, on E. coli serogroups O157, O26, O45, O103, O111, O121, O145 as well as serogroup O104 responsible for the massive outbreak in Germany in 2011. Carvacrol showed antibacterial effect on all strains tested. The relative electric conductivity was assessed in order to investigate the changes in membrane permeability and thus to investigate the antimicrobial mechanism of carvacrol. Results showed that the relative conductivity increased with increasing concentrations of carvacrol which showed that there was an increasing leakage of electrolytes due to disruption of the cell membrane. The data presented here revealed that carvacrol has the potential to be used as a natural antimicrobial against STECs.

  15. Antibiotic resistance and integrons in Shiga toxin-producing Escherichia coli (STEC

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    Rocío Colello

    2015-03-01

    Full Text Available Shiga toxin-producing Escherichia coli (STEC cause hemorrhagic colitis (HC and hemolytic-uremic syndrome in humans (HUS. Cattle are the main reservoir of STEC and transmission to humans occurs through contaminated food and water. Antibiotics are used in pig production systems to combat disease and improve productivity and play a key role in the dissemination of antibiotic resistance genes to the bacteria. Integrons have been identified in resistant bacteria allowing for the acquisition and dissemination of antibiotic resistance genes. STEC strains isolated from humans and animals have developed antibiotic resistance. In our laboratory, 21 non-157 STEC strains isolated from pigs were analyzed to detect class 1 and 2 integrons by PCR. Eight carried integrons, 7 of them harbored intl2. In another study 545 STEC strains were also analyzed for the presence of intl1 and intl2. Strains carrying intl1 belonged to isolates from environment (n = 1, chicken hamburger (n = 2, dairy calves (n = 4 and pigs (n = 8. Two strains isolated from pigs harbored intl2 and only one intl1/intl2, highlighting the presence of intl2 in pigs. The selection for multiresistant strains may contribute to the emergence of antibiotic resistant pathogens and facilitate the spreading of the mobile resistance elements to other bacteria.

  16. A family of indoles regulate virulence and Shiga toxin production in pathogenic E. coli.

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    Bettina Bommarius

    Full Text Available Enteropathogenic Escherichia coli (EPEC, enterohemorrhagic E. coli (EHEC and enteroaggregative E. coli (EAEC are intestinal pathogens that cause food and water-borne disease in humans. Using biochemical methods and NMR-based comparative metabolomics in conjunction with the nematode Caenorhabditis elegans, we developed a bioassay to identify secreted small molecules produced by these pathogens. We identified indole, indole-3-carboxaldehyde (ICA, and indole-3-acetic acid (IAA, as factors that only in combination are sufficient to kill C. elegans. Importantly, although lethal to C. elegans, these molecules downregulate several bacterial processes important for pathogenesis in mammals. These include motility, biofilm formation and production of Shiga toxins. Some pathogenic E. coli strains are known to contain a Locus of Enterocyte Effacement (LEE, which encodes virulence factors that cause "attaching and effacing" (A/E lesions in mammals, including formation of actin pedestals. We found that these indole derivatives also downregulate production of LEE virulence factors and inhibit pedestal formation on mammalian cells. Finally, upon oral administration, ICA inhibited virulence and promoted survival in a lethal mouse infection model. In summary, the C. elegans model in conjunction with metabolomics has facilitated identification of a family of indole derivatives that broadly regulate physiology in E. coli, and virulence in pathogenic strains. These molecules may enable development of new therapeutics that interfere with bacterial small-molecule signaling.

  17. Action of shiga toxin type-2 and subtilase cytotoxin on human microvascular endothelial cells.

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    María M Amaral

    Full Text Available The hemolytic uremic syndrome (HUS associated with diarrhea is a complication of Shiga toxin (Stx-producing Escherichia coli (STEC infection. In Argentina, HUS is endemic and responsible for acute and chronic renal failure in children younger than 5 years old. The human kidney is the most affected organ due to the presence of very Stx-sensitive cells, such as microvascular endothelial cells. Recently, Subtilase cytotoxin (SubAB was proposed as a new toxin that may contribute to HUS pathogenesis, although its action on human glomerular endothelial cells (HGEC has not been described yet. In this study, we compared the effects of SubAB with those caused by Stx2 on primary cultures of HGEC isolated from fragments of human pediatric renal cortex. HGEC were characterized as endothelial since they expressed von Willebrand factor (VWF and platelet/endothelial cell adhesion molecule 1 (PECAM-1. HGEC also expressed the globotriaosylceramide (Gb3 receptor for Stx2. Both, Stx2 and SubAB induced swelling and detachment of HGEC and the consequent decrease in cell viability in a time-dependent manner. Preincubation of HGEC with C-9 -a competitive inhibitor of Gb3 synthesis-protected HGEC from Stx2 but not from SubAB cytotoxic effects. Stx2 increased apoptosis in a time-dependent manner while SubAB increased apoptosis at 4 and 6 h but decreased at 24 h. The apoptosis induced by SubAB relative to Stx2 was higher at 4 and 6 h, but lower at 24 h. Furthermore, necrosis caused by Stx2 was significantly higher than that induced by SubAB at all the time points evaluated. Our data provide evidence for the first time how SubAB could cooperate with the development of endothelial damage characteristic of HUS pathogenesis.

  18. Serogroup-specific risk factors for Shiga toxin-producing Escherichia coli infection in Australia.

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    McPherson, Michelle; Lalor, Karin; Combs, Barry; Raupach, Jane; Stafford, Russell; Kirk, Martyn D

    2009-07-15

    Shiga toxin-producing Escherichia coli (STEC) is an important cause of foodborne illness. In Australia, risk factors for STEC infection have not been examined at a national level. We conducted a case-control study in 6 Australian jurisdictions from 2003 through 2007. A case patient was defined as a person from whom STEC was isolated or toxin production genes were detected in stool. Case patients were recruited from notifiable disease registers, and 3 control subjects frequency matched by age were selected from databases of controls. Using structured questionnaires, interviewers collected data on clinical illness, foods consumed, and exposures to potential environmental sources. We recruited 43 case patients infected with STEC serogroup O157, 71 case patients infected with non-O157 serogroups, and 304 control subjects. One patient infected with serogroup O157 and 7 infected with non-O157 serogroups developed hemolytic uremic syndrome. Compared with control subjects, case patients infected with STEC O157 were more likely to eat hamburgers, visit restaurants, have previously used antibiotics, or have family occupational exposure to red meat. Case patients infected with non-O157 STEC were more likely to eat sliced chicken meat or corned beef from a delicatessen, camp in the bush, eat catered meals, or have family occupational exposure to animals. Negative associations were observed for certain foods, particularly homegrown vegetables, fruits, or herbs. This study of risk factors for STEC infection by serogroup highlights risks associated with eating hamburgers and occupational handling of raw meat. To prevent infection, hamburgers must be cooked thoroughly, and people handling raw meat or who have close contact with animals must ensure adequate hygiene.

  19. Shiga toxin 1 interaction with enterocytes causes apical protein mistargeting through the depletion of intracellular galectin-3

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    Laiko, Marina; Murtazina, Rakhilya; Malyukova, Irina [Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); Zhu, Chengru; Boedeker, Edgar C. [Department of Medicine, University of New Mexico School of Medicine, Albuquerque, NM 87131 (United States); Gutsal, Oksana [Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); O' Malley, Robert; Cole, Robert N. [Department of Biochemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); Tarr, Phillip I. [Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110 (United States); Murray, Karen F. [Department of Pediatrics, Children' s Hospital and Regional Medical Center, Seattle, WA 98105 (United States); Kane, Anne [The Tufts New England Medical Center, Boston, MA 02111 (United States); Donowitz, Mark [Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); Kovbasnjuk, Olga, E-mail: okovbas1@jhmi.edu [Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States)

    2010-02-15

    Shiga toxins (Stx) 1 and 2 are responsible for intestinal and systemic sequelae of infection by enterohemorrhagic Escherichia coli (EHEC). However, the mechanisms through which enterocytes are damaged remain unclear. While secondary damage from ischemia and inflammation are postulated mechanisms for all intestinal effects, little evidence excludes roles for more primary toxin effects on intestinal epithelial cells. We now document direct pathologic effects of Stx on intestinal epithelial cells. We study a well-characterized rabbit model of EHEC infection, intestinal tissue and stool samples from EHEC-infected patients, and T84 intestinal epithelial cells treated with Stx1. Toxin uptake by intestinal epithelial cells in vitro and in vivo causes galectin-3 depletion from enterocytes by increasing the apical galectin-3 secretion. This Shiga toxin-mediated galectin-3 depletion impairs trafficking of several brush border structural proteins and transporters, including villin, dipeptidyl peptidase IV, and the sodium-proton exchanger 2, a major colonic sodium absorptive protein. The mistargeting of proteins responsible for the absorptive function might be a key event in Stx1-induced diarrhea. These observations provide new evidence that human enterocytes are directly damaged by Stx1. Conceivably, depletion of galectin-3 from enterocytes and subsequent apical protein mistargeting might even provide a means whereby other pathogens might alter intestinal epithelial absorption and produce diarrhea.

  20. Identification and Characterization of Novel Compounds Blocking Shiga Toxin Expression in Escherichia coli O157:H7

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    Alejandro Huerta-Uribe

    2016-11-01

    Full Text Available Infections caused by Shiga toxin-producing E. coli strains constitute a health problem, as they are problematic to treat. Shiga toxin (Stx production is a key virulence factor associated with the pathogenicity of enterohaemorrhagic E. coli (EHEC and can result in the development of haemolytic uremic syndrome in infected patients. The genes encoding Stx are located on temperate lysogenic phages integrated into the bacterial chromosome and expression of the toxin is generally coupled to phage induction through the SOS response. We aimed to find new compounds capable of blocking expression of Stx type 2 (Stx2 as this subtype of Stx is more strongly associated with human disease. High-throughput screening of a small-molecule library identified a lead compound that reduced Stx2 expression in a dose-dependent manner. We show that the optimised compound interferes with the SOS response by directly affecting the activity and oligomerisation of RecA, thus limiting phage activation and Stx2 expression. Our work suggests that RecA is highly susceptible to inhibition and that targeting this protein is a viable approach to limiting production of Stx2 by EHEC. This type of approach has the potential to limit production and transfer of other phage induced and transduced determinants.

  1. Molecular characterization and phylogeny of Shiga toxin-producing E. coli (STEC) from imported beef meat in Malaysia.

    Science.gov (United States)

    Abuelhassan, Nawal Nouridaim; Mutalib, Sahilah Abdul; Gimba, Fufa Ido; Yusoff, Wan Mohtar

    2016-09-01

    This study aimed at determining the presence and characterization of Escherichia coli and Shiga toxin-producing E. coli (STEC) from imported frozen beef meats. Seventy-four (74) frozen imported beef meat samples from two countries, India (42 samples) and Australia (32 samples), were collected and tested for E. coli. These samples were purchased from the frozen meat sections of five different supermarkets in different locations in Selangor, Malaysia, from April 2012 to October 2014. A total of 222 E. coli strains were isolated from the meat samples; 126 strains were isolated from country A (India), and 96 E. coli strains were from country of origin B (Australia), respectively. A total of 70 E. coli strains were identified and characterized. All E. coli strains were isolated into Fluorocult medium and identified using API 20E kit. All selected E. coli strains were characterized for Shiga toxin genes (stx1 and stx2). All biochemically identified E. coli in this study were further subjected to molecular detection through polymerase chain reaction (PCR) amplification and characterization using 16S ribosomal RNA (rRNA) gene of Shiga toxin-producing E. coli. Of the 70 E. coli strains, 11 strains were positive for both Shiga toxin genes (stx1 and stx2) and 11 (11/70) strains were positive for stx1 gene, while 25 (25/70) strains were positive for stx2 gene. The analysis of 16S rRNA gene of all the E. coli isolates in this study was successfully sequenced and analyzed, and based on sequence data obtained, a phylogenetic tree of the 16S rRNA gene was performed using Clustal W programme in MEGA 6.06 software. Phylogenetic tree showed that the E. coli isolates in our study cluster with the strain of E. coli isolated in other countries, which further confirm that the isolates of E. coli in this study are similar to those obtained in other studies. As a result, all the strains obtained in this study proved to be a strain of pathogenic E. coli, which may cause a serious outbreak

  2. Isolation and characteristics of Shiga toxin 2f-producing Escherichia coli among pigeons in Kyushu, Japan.

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    Koichi Murakami

    Full Text Available An increasing number of Shiga toxin 2f-producing Escherichia coli (STEC2f infections in humans are being reported in Europe, and pigeons have been suggested as a reservoir for the pathogen. In Japan, there is very little information regarding carriage of STEC2f by pigeons, prompting the need for further investigation. We collected 549 samples of pigeon droppings from 14 locations in Kyushu, Japan, to isolate STEC2f and to investigate characteristics of the isolates. Shiga toxin stx 2f gene fragments were detected by PCR in 16 (2.9% of the 549 dropping samples across four of the 14 locations. We obtained 23 STEC2f-isolates from seven of the original samples and from three pigeon dropping samples collected in an additional sampling experiment (from a total of seven locations across both sampling periods. Genotypic and phenotypic characteristics were then examined for selected isolates from each of 10 samples with pulsed-field gel electrophoresis profiles. Eight of the stx 2f gene fragments sequenced in this study were homologous to others that were identified in Europe. Some isolates also contained virulence-related genes, including lpfA O26, irp 2, and fyuA, and all of the 10 selected isolates maintained the eae, astA, and cdt genes. Moreover, five of the 10 selected isolates contained sfpA, a gene that is restricted to Shiga toxin-producing E. coli O165:H2 and sorbitol-fermenting Shiga toxin-producing E. coli O157:NM. We document serotypes O152:HNM, O128:HNM, and O145:H34 as STEC2f, which agrees with previous studies on pigeons and humans. Interestingly, O119:H21 was newly described as STEC2f. O145:H34, with sequence type 722, was described in a German study in humans and was also isolated in the current study. These results revealed that Japanese zoonotic STEC2f strains harboring several virulence-related factors may be of the same clonal complexes as some European strains. These findings provide useful information for public health

  3. A Topographical Atlas of Shiga Toxin 2e Receptor Distribution in the Tissues of Weaned Piglets

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    Daniel Steil

    2016-11-01

    Full Text Available Shiga toxin (Stx 2e of Stx-producing Escherichia coli (STEC is the primary virulence factor in the development of pig edema disease shortly after weaning. Stx2e binds to the globo-series glycosphingolipids (GSLs globotriaosylceramide (Gb3Cer, Galα1-4Galβ1-4Glcβ1-1Cer and globotetraosylceramide (Gb4Cer, GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1Cer, the latter acting as the preferential Stx2e receptor. We determined Stx receptor profiles of 25 different tissues of a male and a female weaned piglet using immunochemical solid phase binding assays combined with mass spectrometry. All probed tissues harbored GSL receptors, ranging from high (category I over moderate (category II to low content (category III. Examples of Gb4Cer expression in category I tissues are small intestinal ileum, kidney pelvis and whole blood, followed by colon, small intestinal duodenum and jejunum belonging to category II, and kidney cortex, cerebrum and cerebellum as members of category III organs holding true for both genders. Dominant Gb3Cer and Gb4Cer lipoforms were those with ceramides carrying constant sphingosine (d18:1 and a variable C16:0, C22:0 or C24:1/C24:0 fatty acid. From the mapping data, we created a topographical atlas for Stx2e receptors in piglet tissues and organs, which might be helpful to further investigations on the molecular and cellular mechanisms that underlie infections of Stx2e-producing STEC in pigs and their zoonotic potential for humans.

  4. Treatment and outcome of Shiga-toxin-associated hemolytic uremic syndrome (HUS).

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    Scheiring, Johanna; Andreoli, Sharon P; Zimmerhackl, Lothar Bernd

    2008-10-01

    Hemolytic uremic syndrome (HUS) is the most common cause of acute renal failure in childhood and the reason for chronic renal replacement therapy. It leads to significant morbidity and mortality during the acute phase. In addition to acute morbidity and mortality, long-term renal and extrarenal complications can occur in a substantial number of children years after the acute episode of HUS. The most common infectious agents causing HUS are enterohemorrhagic Escherichia coli (EHEC)-producing Shiga toxin (and belonging to the serotype O157:H7) and several non-O157:H7 serotypes. D(+) HUS is an acute disease characterized by prodromal diarrhea followed by acute renal failure. The classic clinical features of HUS include the triad of microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. HUS mortality is reported to be between 3% and 5%, and death due to HUS is nearly always associated with severe extrarenal disease, including severe central nervous system (CNS) involvement. Approximately two thirds of children with HUS require dialysis therapy, and about one third have milder renal involvement without the need for dialysis therapy. General management of acute renal failure includes appropriate fluid and electrolyte management, antihypertensive therapy if necessary, and initiation of renal replacement therapy when appropriate. The prognosis of HUS depends on several contributing factors. In general "classic" HUS, induced by EHEC, has an overall better outcome. Totally different is the prognosis in patients with atypical and particularly recurrent HUS. However, patients with severe disease should be screened for genetic disorders of the complement system or other underlying diseases.

  5. Taxonomy Meets Public Health: The Case of Shiga Toxin-Producing Escherichia coli.

    Science.gov (United States)

    Scheutz, Flemming

    2014-06-01

    To help assess the clinical and public health risks associated with different Shiga toxin-producing Escherichia coli (STEC) strains, an empirical classification scheme was used to classify STEC into five "seropathotypes" (seropathotype A [high risk] to seropathotypes D and E [minimal risk]). This definition is of considerable value in cases of human infection but is also problematic because not all STEC infections are fully characterized and coupled to reliable clinical information. Outbreaks with emerging hybrid strains continuously challenge our understanding of virulence potential and may result in incorrect classification of specific pathotypes; an example is the hybrid strain that caused the 2011 outbreak in Germany, STEC/EAggEC O104:H4, which may deserve an alternative seropathotype designation. The integration of mobile virulence factors in the stepwise and parallel evolution of pathogenic lineages of STEC collides with the requirements of a good taxonomy, which separates elements of each group into subgroups that are mutually exclusive, unambiguous, and, together, include all possibilities. The concept of (sero)-pathotypes is therefore challenged, and the need to identify factors of STEC that absolutely predict the potential to cause human disease is obvious. Because the definition of hemolytic-uremic syndrome (HUS) is distinct, a basic and primary definition of HUS-associated E. coli (HUSEC) for first-line public health action is proposed: stx2 in a background of an eae- or aggR-positive E. coli followed by a second-line subtyping of stx genes that refines the definition of HUSEC to include only stx2a and stx2d. All other STEC strains are considered "low-risk" STEC.

  6. Wild ungulates as disseminators of Shiga toxin-producing Escherichia coli in urban areas.

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    Alan B Franklin

    Full Text Available BACKGROUND: In 2008, children playing on a soccer field in Colorado were sickened with a strain of Shiga toxin-producing Escherichia coli (STEC O157:H7, which was ultimately linked to feces from wild Rocky Mountain elk. We addressed whether wild cervids were a potential source of STEC infections in humans and whether STEC was ubiquitous throughout wild cervid populations in Colorado. METHODOLOGY/PRINCIPAL FINDINGS: We collected 483 fecal samples from Rocky Mountain elk and mule deer in urban and non-urban areas. Samples testing positive for STEC were higher in urban (11.0% than non-urban (1.6% areas. Elk fecal samples in urban areas had a much higher probability of containing STEC, which increased in both urban and non-urban areas as maximum daily temperature increased. Of the STEC-positive samples, 25% contained stx1 strains, 34.3% contained stx2, and 13% contained both stx1 and stx2. Additionally, eaeA genes were detected in 54.1% of the positive samples. Serotypes O103, and O146 were found in elk and deer feces, which also have the potential to cause human illness. CONCLUSIONS/SIGNIFICANCE: The high incidence of stx2 strains combined with eaeA and E-hyl genes that we found in wild cervid feces is associated with severe human disease, such as hemolytic uremic syndrome. This is of concern because there is a very close physical interface between elk and humans in urban areas that we sampled. In addition, we found a strong relationship between ambient temperature and incidence of STEC in elk feces, suggesting a higher incidence of STEC in elk feces in public areas on warmer days, which in turn may increase the likelihood that people will come in contact with infected feces. These concerns also have implications to other urban areas where high densities of coexisting wild cervids and humans interact on a regular basis.

  7. Functional and phylogenetic analysis of ureD in Shiga toxin-producing Escherichia coli.

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    Steyert, Susan R; Rasko, David A; Kaper, James B

    2011-02-01

    Enterohemorrhagic Escherichia coli (EHEC) is a food-borne pathogen that can cause severe health complications and utilizes a much lower infectious dose than other E. coli pathotypes. Despite having an intact ure locus, ureDABCEFG, the majority of EHEC strains are phenotypically urease negative under tested conditions. Urease activity potentially assists with survival fitness by enhancing acid tolerance during passage through the stomach or by aiding with colonization in either human or animal reservoirs. Previously, in the EHEC O157:H7 Sakai strain, a point mutation in ureD, encoding a urease chaperone protein, was identified, resulting in a substitution of an amber stop codon for glutamine. This single nucleotide polymorphism (SNP) is observed in the majority of EHEC O157:H7 isolates and correlates with a negative urease phenotype in vitro. We demonstrate that the lack of urease activity in vitro is not solely due to the amber codon in ureD. Our analysis has identified two additional SNPs in ureD affecting amino acid positions 38 and 205, in both cases determining whether the encoded amino acid is leucine or proline. Phylogenetic analysis based on Ure protein sequences from a variety of urease-encoding bacteria demonstrates that the proline at position 38 is highly conserved among Gram-negative bacteria. Experiments reveal that the L38P substitution enhances urease enzyme activity; however, the L205P substitution does not. Multilocus sequence typing analysis for a variety of Shiga toxin-producing E. coli isolates combined with the ureD sequence reveals that except for a subset of the O157:H7 strains, neither the in vitro urease-positive phenotype nor the ureD sequence is phylogenetically restricted.

  8. Characterization of Urinary Tract Infection-Associated Shiga Toxin-Producing Escherichia coli

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    Toval, Francisco; Schiller, Roswitha; Meisen, Iris; Putze, Johannes; Kouzel, Ivan U.; Zhang, Wenlan; Karch, Helge; Bielaszewska, Martina; Mormann, Michael; Müthing, Johannes

    2014-01-01

    Enterohemorrhagic Escherichia coli (EHEC), a subgroup of Shiga toxin (Stx)-producing E. coli (STEC), is a leading cause of diarrhea and hemolytic-uremic syndrome (HUS) in humans. However, urinary tract infections (UTIs) caused by this microorganism but not associated with diarrhea have occasionally been reported. We geno- and phenotypically characterized three EHEC isolates obtained from the urine of hospitalized patients suffering from UTIs. These isolates carried typical EHEC virulence markers and belonged to HUS-associated E. coli (HUSEC) clones, but they lacked virulence markers typical of uropathogenic E. coli. One isolate exhibited a localized adherence (LA)-like pattern on T24 urinary bladder epithelial cells. Since the glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) are well-known receptors for Stx but also for P fimbriae, a major virulence factor of extraintestinal pathogenic E. coli (ExPEC), the expression of Gb3Cer and Gb4Cer by T24 cells and in murine urinary bladder tissue was examined by thin-layer chromatography and mass spectrometry. We provide data indicating that Stxs released by the EHEC isolates bind to Gb3Cer and Gb4Cer isolated from T24 cells, which were susceptible to Stx. All three EHEC isolates expressed stx genes upon growth in urine. Two strains were able to cause UTI in a murine infection model and could not be outcompeted in urine in vitro by typical uropathogenic E. coli isolates. Our results indicate that despite the lack of ExPEC virulence markers, EHEC variants may exhibit in certain suitable hosts, e.g., in hospital patients, a uropathogenic potential. The contribution of EHEC virulence factors to uropathogenesis remains to be further investigated. PMID:25156739

  9. Natural killer T (NKT cells accelerate Shiga toxin type 2 (Stx2 pathology in mice

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    Fumiko eObata

    2015-04-01

    Full Text Available Shiga toxin-producing Escherichia coli (STEC is a leading cause of childhood renal disease He-molytic Uremic Syndrome (HUS. The involvement of renal cytokines and chemokines is sus-pected to play a critical role in disease progression. In current article, we tested the hypothesis that NKT cells are involved in Stx2-induced pathology in vivo. To address this hypothesis we compared Stx2 toxicity in WT and CD1 knockout (KO mice. In CD1KO mice, which lack nat-ural killer T (NKT cells, Stx2-induced pathologies such as weight loss, renal failure, and death were delayed. In WT mice, Stx2-specific selective increase in urinary albumin occurs in later time points, and this was also delayed in NKT cell deficient mice. NKT cell-associated cytokines such as IL-2, IL-4, IFN-γ and IL-17 were detected in kidney lysates of Stx2-injected WT mice with the peak around 36 h after Stx2 injection. In CD1KO, there was a delay in the kinetics, and increases in these cytokines were observed 60 h post Stx2 injection. These data suggest that NKT cells accelerate Stx2-induced pathology in mouse kidneys. To determine the mechanism by which NKT cells promote Stx2-associated disease, in vitro studies were performed using murine renal cells. We found that murine glomerular endothelial cells and podocytes express functional CD1d molecules and can present exogenous antigen to NKT cells. Moreover, we observed the direct interaction between Stx2 and the receptor Gb3 on the surface of mouse renal cells by 3D STORM-TIRF which provides single molecule imaging. Collectively, these data suggest that Stx2 binds to Gb3 on renal cells and leads to aberrant CD1d-mediated NKT cell activation. Therefore, strategies targeting NKT cells could have a significant impact on Stx2-associated renal pathology in STEC disease.

  10. Natural killer T (NKT) cells accelerate Shiga toxin type 2 (Stx2) pathology in mice.

    Science.gov (United States)

    Obata, Fumiko; Subrahmanyam, Priyanka B; Vozenilek, Aimee E; Hippler, Lauren M; Jeffers, Tynae; Tongsuk, Methinee; Tiper, Irina; Saha, Progyaparamita; Jandhyala, Dakshina M; Kolling, Glynis L; Latinovic, Olga; Webb, Tonya J

    2015-01-01

    Shiga toxin-producing Escherichia coli (STEC) is a leading cause of childhood renal disease Hemolytic Uremic Syndrome (HUS). The involvement of renal cytokines and chemokines is suspected to play a critical role in disease progression. In current article, we tested the hypothesis that NKT cells are involved in Stx2-induced pathology in vivo. To address this hypothesis we compared Stx2 toxicity in WT and CD1 knockout (KO) mice. In CD1KO mice, which lack natural killer T (NKT) cells, Stx2-induced pathologies such as weight loss, renal failure, and death were delayed. In WT mice, Stx2-specific selective increase in urinary albumin occurs in later time points, and this was also delayed in NKT cell deficient mice. NKT cell-associated cytokines such as IL-2, IL-4, IFN-γ, and IL-17 were detected in kidney lysates of Stx2-injected WT mice with the peak around 36 h after Stx2 injection. In CD1KO, there was a delay in the kinetics, and increases in these cytokines were observed 60 h post Stx2 injection. These data suggest that NKT cells accelerate Stx2-induced pathology in mouse kidneys. To determine the mechanism by which NKT cells promote Stx2-associated disease, in vitro studies were performed using murine renal cells. We found that murine glomerular endothelial cells and podocytes express functional CD1d molecules and can present exogenous antigen to NKT cells. Moreover, we observed the direct interaction between Stx2 and the receptor Gb3 on the surface of mouse renal cells by 3D STORM-TIRF which provides single molecule imaging. Collectively, these data suggest that Stx2 binds to Gb3 on renal cells and leads to aberrant CD1d-mediated NKT cell activation. Therefore, strategies targeting NKT cells could have a significant impact on Stx2-associated renal pathology in STEC disease.

  11. Role of climate in the spread of shiga toxin-producing Escherichia coli infection among children

    Science.gov (United States)

    Acquaotta, Fiorella; Ardissino, Gianluigi; Fratianni, Simona; Perrone, Michela

    2017-09-01

    Haemolytic-uraemic syndrome (HUS) is a rare disease mainly affecting children that develops as a complication of shiga toxin-producing Escherichia coli (STEC) infection. It is characterised by acute kidney injury, platelet consumption and mechanical destruction of red blood cells (haemolysis). In order to test the working hypothesis that the spread of the infection is influenced by specific climatic conditions, we analysed all of the identified cases of infection occurring between June 2010 and December 2013 in four provinces of Lombardy, Italy (Milano, Monza Brianza, Varese and Brescia), in which a STEC surveillance system has been developed as part of a preventive programme. In the selected provinces, we recorded in few days a great number of cases and clusters which are unrelated for spatially distant or for the disease are caused by different STEC serotypes. In order to investigate a common factor that favoured the onset of infection, we have analysed in detail the weather conditions of the areas. The daily series of temperature, rain and relative humidity were studied to show the common climate peculiarities whilst the correlation coefficient and the principal component analysis (PCA) were used to point out the meteorological variable, maximum temperature, as the principal climate element in the onset of the infection. The use of distributed lag non-linear models (DLNM) and the climate indices characterising heat waves (HWs) has allowed to identify the weather conditions associated with STEC infection. The study highlighted a close temporal correlation between STEC infection in children and the number, duration and frequency of heat waves. In particular, if the maximum temperature is greater than 90th percentile, days classified as very hot, for 3 or more consecutive days, the risk of infection is increasing.

  12. Microalbuminuria and early renal response to lethal dose Shiga toxin type 2 in rats

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    Ochoa F

    2012-01-01

    Full Text Available Federico Ochoa1, Gisela Oltra1, Elizabeth Gerhardt1, Ricardo Hermes2, Lilian Cohen2, Alicia E Damiano3, Cristina Ibarra1, Nestor R Lago4, Elsa Zotta11Departamento de Fisiologia, Facultad de Medicina UBA, 2Laboratorio Análisis Clínicos, Hospital Juan A Fernández, 3Departamento de Ciencias Biológicas, Facultad de Farmacia y Bioquímica, UBA, Buenos Aires, Argentina; 4Departamento de Patología, Facultad de Medicina UBA, Buenos Aires, ArgentinaAbstract: In Argentina, hemolytic uremic syndrome (HUS constitutes the most frequent cause of acute renal failure in children. Approximately 2%–4% of patients die during the acute phase, and one-third of the 96% who survive are at risk of chronic renal sequelae. Little information is available about the direct effect of Shiga toxin type 2 (Stx2 on the onset of proteinuria and the evolution of toxin-mediated glomerular or tubular injury. In this work, rats were injected intraperitoneally with recombinant Escherichia coli culture supernatant containing Stx2 (sStx2; 20 µg/kg body weight to induce HUS. Functional, immunoblotting, and immunohistochemistry studies were carried out to determine alterations in slit diaphragm proteins and the proximal tubule endocytic system at 48 hours post-inoculation. We detected a significant increase in microalbuminuria, without changes in the proteinuria values compared to the control rats. In immunoperoxidase studies, the renal tubules and glomerular mesangium showed an increased expression of transforming growth factor ß1(TGF-ß1. The expression of megalin was decreased by immunoperoxidase and the cytoplasm showed a granular pattern of megalin expression by immunofluorescence techniques. Western blot analysis performed in the renal cortex from sStx2-treated and control rats using anti-nephrin and anti-podocalyxin antibodies showed a decreased expression of these proteins. We suggest that the alterations in slit diaphragm proteins and megalin expression could be related

  13. Differences in Shiga toxin and phage production among stx2g-positive STEC strains

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    Claudia Viviana Granobles Velandia

    2012-06-01

    Full Text Available Shigatoxigenic E. coli (STEC are characterized by the production of Shiga toxins (Stx encoded by temperate bacteriophages. Stx production is linked to the induction of the phage lytic cycle. Several stx variants have been described and differentially associated with the risk of developing severe illness.The variant named stx2g was first identified in a STEC strain isolated from the faeces of healthy cattle. Analysis of stx2g-positive strains isolated from humans, animals and environmental sources have shown that they have a close relationship. In this study, stx2g-positive STEC isolated from cattle were analyzed for phage and Stx production, with the aim to relate the results to differences observed in cytotoxicity.The presence of inducible phages was assessed by analyzing the bacterial growth/lysis curves and also by plaque assay. Bacterial growth curves in the absence of induction were similar for all isolates, however, notably differed among induced cultures. The two strains that clearly evidenced bacteriolysis under this condition also showed higher phage titers in plaque assays. However, only the phage plaques produced by one of these strains (FB 62 hybridized with a stx2-probe. Furthermore, the production of Stx was evaluated by EIA and Western immunoblotting in overnight supernatants. By EIA, we detected Stx only in supernatants of FB 62, with a higher signal with induced than in uninduced cultures. By immunoblotting, Stx2 could be detected after induction in all stx2g-positive isolates, but with lower amounts of Stx2B subunit in those supernatants where phages could not be detected.Taking into account all the results, several differences could be found among stx2g-positive strains. The strain with the highest cytotoxic titer showed higher levels of stx2-phages and toxin production by EIA, and the opposite was observed for strains that previously showed low cytotoxic titers, confirming that in stx2g-positive strains Stx production is

  14. Analisis Sekuen Probe Gena Shiga Like Toxin-2 dari Isolat Lokal Escherichia coli O157:H7 (PROBE SEQUANCE ANALYSIS OF SHIGA LIKE TOXIN-2 GEN FROM ESCHERICHIA COLI O157:H7 LOCAL ISOLATES

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    I Wayan Suardana

    2013-09-01

    Full Text Available A Shiga-like toxin producing Escherichia coli O157:H7 was detected in faecal samples of cattle, andhuman as well as in beef. The performance of agent indicated that it has been identified as harmful andoften life-threatening zoonotic agent. It is therefore important to analysed the genetic characteristic ofShiga toxin Escherichia coli (STEC and to develop a diagnostic probe in order  to optimalized of diagnostictest  for the agent. The  study was started by amplifiying  stx2 gene, purifying of PCR product, sequencingof stx2 gene, analyzing  of phylogenetic tree, and finally  by analyzing   of  diagnostic  probe candidate.Homology study showed that the genetic sequence of the local isolate of  E. coli O157:H7 i.e SM25(1isolated from cattle feces has  a genetic and fuctional similarity with  the control isolate i.e E. coli O157:H7ATCC 43894 originated from human.  Further study showed that a probe with  foreward primer  sequanceof 5’-AATTTATATGTGGCCGGGTTC-‘3 which were respectively designed as a PFS and PRS 176 bp product.Appeared to be potential candidate of diagnostic probe for the agent.

  15. The critical role of lipopolysaccharide in the upregulation of aquaporin 4 in glial cells treated with Shiga toxin.

    Science.gov (United States)

    Sugimoto, Naotoshi; Leu, Hue; Inoue, Natsumi; Shimizu, Masaki; Toma, Tomoko; Kuroda, Mondo; Saito, Takekatsu; Wada, Taizo; Yachie, Akihiro

    2015-09-18

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

  16. The Role of the Exo-Xis Region in Oxidative Stress-Mediated Induction of Shiga Toxin-Converting Prophages

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    Katarzyna Licznerska

    2016-01-01

    Full Text Available Previous studies indicated that these genetic elements could be involved in the regulation of lysogenization and prophage induction processes. The effects were dramatic in Shiga toxin-converting phage Φ24B after treatment with oxidative stress-inducing agent, hydrogen peroxide, while they were less pronounced in bacteriophage λ and in both phages irradiated with UV. The hydrogen peroxide-caused prophage induction was found to be RecA-dependent. Importantly, in hydrogen peroxide-treated E. coli cells lysogenic for either λ or Φ24B, deletion of the exo-xis region resulted in a significant decrease in the levels of expression of the S.O.S. regulon genes. Moreover, under these conditions, a dramatic decrease in the levels of expression of phage genes crucial for lytic development (particularly xis, exo, N, cro, O, Q, and R could be observed in Φ24B-, but not in λ-bearing cells. We conclude that genes located in the exo-xis region are necessary for efficient expression of both host S.O.S regulon in lysogenic bacteria and regulatory genes of Shiga toxin-converting bacteriophage Φ24B.

  17. Survey on the occurrence of shiga toxin producing strains of Escherichia coli (STEC) in drinking water from central Hessia

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    Stelz, A.; Zschoeck, M. [Untersuchungsamt Hessen, Giessen (Germany); Fels, K.; Alter, E. [Fachhochschule Giessen-Friedberg, Giessen (Germany)

    2001-07-01

    Consumption of unheated bovine food is said to be the main cause of outbreaks of enterohemorrhagic colitis (EC) and hemolytic syndrome (HUS) by E. coli O157 in man. Some cases were attributed to the ingestion of contaminated drinking water. Certain types of resources for drinking water supply are at risk of contamination with cattle faeces or sewage. In routine drinking water control E. coli is an index organism and must not be found in a sample of 100 ml. We investigated a total of 1700 drinking water samples from central Hessia (Germany) for shiga toxin producing strains of E. coli (STEC). A developed sensitive investigation scheme made it possible to detect up to 10 STEC per 100 ml water sample at least. ELISA was used for detection of shiga toxins 1 and 2, m-PCR for the genes encoding stx1, stx2, EHEC-hlyA and eaeA, plating on Enterohemolysin agar for detection of the enterohemolysin phenotype and slide agglutination technique for confirming E. coli O157. In 108 out of 1700 drinking water samples E. coli was detected, but none of the isolates belonged to serotype O157. ST1 and ST2 were not produced by the isolates. Thus, under normal circumstances drinking water in central Hessia seems to be no vehicle for STEC. (orig.)

  18. Locus of Adhesion and Autoaggregation (LAA), a pathogenicity island present in emerging Shiga Toxin-producing Escherichia coli strains.

    Science.gov (United States)

    Montero, David A; Velasco, Juliana; Del Canto, Felipe; Puente, Jose L; Padola, Nora L; Rasko, David A; Farfán, Mauricio; Salazar, Juan C; Vidal, Roberto

    2017-08-01

    Shiga Toxin-producing Escherichia coli (STEC) are a group of foodborne pathogens associated with diarrhea, dysentery, hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). Shiga toxins are the major virulence factor of these pathogens, however adhesion and colonization to the human intestine is required for STEC pathogenesis. A subset of STEC strains carry the Locus of Enterocyte Effacement (LEE) pathogenicity island (PAI), which encodes genes that mediate the colonization of the human intestine. While LEE-positive STEC strains have traditionally been associated with human disease, the burden of disease caused by STEC strains that lacks LEE (LEE-negative) has increased recently in several countries; however, in the absence of LEE, the molecular pathogenic mechanisms by STEC strains are unknown. Here we report a 86-kb mosaic PAI composed of four modules that encode 80 genes, including novel and known virulence factors associated with adherence and autoaggregation. Therefore, we named this PAI as Locus of Adhesion and Autoaggregation (LAA). Phylogenomic analysis using whole-genome sequences of STEC strains available in the NCBI database indicates that LAA PAI is exclusively present in a subset of emerging LEE-negative STEC strains, including strains isolated from HC and HUS cases. We suggest that the acquisition of this PAI is a recent evolutionary event, which may contribute to the emergence of these STEC.

  19. Comparison of a direct fecal Shiga-like toxin assay and sorbitol-MacConkey agar culture for laboratory diagnosis of enterohemorrhagic Escherichia coli infection.

    Science.gov (United States)

    Ritchie, M; Partington, S; Jessop, J; Kelly, M T

    1992-02-01

    A direct fecal Shiga-like toxin assay (DSLTA) was used to prospectively screen 9,449 unselected stool samples, received at the British Columbia Provincial Health Laboratories and the Metropolitan Laboratories of Vancouver, for Shiga-like toxin I and Shiga-like toxin II. The results were compared with results of routine stool culture on sorbitol-MacConkey agar (SMAC) for Escherichia coli O157:H7. Of 80 specimens positive by either method, 59 (74%) and 74 (93%) were positive by SMAC and DSLTA, respectively; 53 (66%) were positive by both methods, 21 (26%) were positive by DSLTA only, and 6 (7%) were positive by SMAC only. On further screening, Shiga-like toxin-producing E. coli were detected in 8 (38%) of the 21 stools positive by DSLTA only, including serotypes O157:H7 (1 stool), O26:K60 (5 stools), O128:K67 (1 stool), and O103:H2 (1 stool). For the remaining 13 stools in which no SLTEC was found but DSLTA was positive, clinical information revealed that 11 of 12 patients had diarrheal illnesses, and 4 of these 11 had bloody diarrhea or hemolytic-uremic syndrome. Stools positive only by SMAC were collected earlier in the illness than stools positive by DSLTA, suggesting that free fecal toxin levels may be too low to detect at this time. Overall we found that DSLTA detected 19% more positive specimens than SMAC and that Shiga-like toxin-producing E. coli serotypes other than E. coli O157:H7 are causing disease in the province of British Columbia, Canada.

  20. Real-time genomic investigation underlying the public health response to a Shiga toxin-producing Escherichia coli O26:H11 outbreak in a nursery

    NARCIS (Netherlands)

    Moran-Gilad, J; Rokney, A; Danino, D; Ferdous, M; Alsana, F; Baum, M; Dukhan, L; Agmon, V; Anuka, E; Valinsky, L; Yishay, R; Grotto, I; Rossen, J W A; Gdalevich, M

    2017-01-01

    Shiga toxin-producing Escherichia coli (STEC) is a significant cause of gastrointestinal infection and the haemolytic-uremic syndrome (HUS). STEC outbreaks are commonly associated with food but animal contact is increasingly being implicated in its transmission. We report an outbreak of STEC

  1. Occurrence and characterization of shiga toxin-producing Escherichia coli in raw meat, raw milk, and street vended juices in Bangladesh

    NARCIS (Netherlands)

    Islam, M.A.; Mondol, A.S.; Azmi, I.J.; Boer, de E.; Beumer, R.R.; Zwietering, M.H.; Heuvelink, A.E.; Talukder, K.A.

    2010-01-01

    The major objective of this study was to investigate the prevalence of Shiga toxin (Stx)–producing Escherichia coli (STEC) in different types of food samples and to compare their genetic relatedness with STEC strains previously isolated from animal sources in Bangladesh. We investigated a total of

  2. Investigation of environmental factors on the prevalence of free bacteriophages against Shiga toxin-producing Escherichia coli strains in produce pre-harvest environment in Salinas, California

    Science.gov (United States)

    Shiga toxin-producing E. coli (STEC) strains, commensal to gastrointestinal tracts of ruminants or other animals, have been associated with serious human illnesses and high mortality among immunocompromised populations. Along with the detection of STEC strains from fecal-contaminated environments su...

  3. Characterization of free lytic bacteriophages isolated from compost against O145 Shiga toxin-producing Escherichia coli (STEC) as a potential biocontrol agent

    Science.gov (United States)

    Shiga toxin-producing E. coli (STEC), one of the most prevalent foodborne pathogens, are notorious for hemolytic uremic syndrome (HUS) and causing high mortality among children and the elder population after infection. Besides O157 STEC, non-O157 STEC—particularly serogroup O145—is commonly associat...

  4. Mass spectrometry-based method of detecting and distinguishing type 1 and type 2 Shiga-like toxins in human serum

    Science.gov (United States)

    Shiga-like toxins (verotoxins) are a class of AB5 holotoxins that are responsible for the virulence associated with bacterial pathogens such as Shigella dysenteriae, shigatoxigenic and enterohemorrhagic strains of Escherichia coli (STEC and EHEC), and some Enterobacter strains. The actual expression...

  5. Molecular subtyping and distribution of the serine protease from shiga toxin-producing Escherichia coli among atypical enteropathogenic E. coli strains.

    Science.gov (United States)

    Cookson, Adrian L; Bennett, Jenny; Nicol, Carolyn; Thomson-Carter, Fiona; Attwood, Graeme T

    2009-04-01

    Atypical enteropathogenic Escherichia coli (aEPEC) and Shiga toxin-producing E. coli (STEC) were examined to determine the prevalence and sequence of espP, which encodes a serine protease. These analyses indicated shared espP sequence types between the two E. coli pathotypes and thus provide further insights into the evolution of aEPEC and STEC.

  6. Relationship between virulence gene profiles of atypical enteropathogenic Escherichia coli and Shiga toxin-producing E. coli isolates from cattle and sheep in New Zealand.

    Science.gov (United States)

    Cookson, Adrian L; Cao, Mingshu; Bennett, Jenny; Nicol, Carolyn; Thomson-Carter, Fiona; Attwood, Graeme T

    2010-06-01

    Virulence gene profiles of atypical enteropathogenic Escherichia coli (aEPEC) and Shiga toxin-producing E. coli (STEC) from cattle, sheep, and humans were examined to determine the relationship between pathotypes. Shared virulence factors (intimin, EHEC hemolysin, serine protease, and a type II secretion system) were identified, suggesting a dynamic evolutionary relationship between aEPEC and STEC.

  7. Mathematical modeling and numerical analysis of the growth of Non-O157 shiga toxin-producing Escherichia coli in spinach leaves

    Science.gov (United States)

    This study was conducted to investigate the growth of non-O157 Shiga toxin-producing Escherichia coli (STEC) in spinach leaves and to develop kinetic models to describe the bacterial growth. Six serogroups of non-O157 STEC, including O26, O45, O103, O111, O121, and O145, were used in the growth stu...

  8. The effect of deep frying or conventional oven cooking on inactivation of Shiga toxin-producing cells of Escherichia coli (STEC) in meatballs

    Science.gov (United States)

    We investigated the effects deep frying or oven cooking on inactivation of Shiga toxin-producing cells of Escherichia coli (STEC) in meatballs. A finely-ground veal and/or a beef-pork-veal mixture were inoculated (ca. 7.0 log CFU/g) with an eight-strain, genetically-marked cocktail of rifampicin-res...

  9. Carbon nanoparticles as detection labels in antibody microarrays. Detection of genes encoding virulence factors in Shiga toxin-producing Escherichia coli.

    NARCIS (Netherlands)

    Noguera, P.S.; Posthuma-Trumpie, G.A.; Tuil, Van M.; Wal, van der F.J.; Boer, De A.; Moers, A.P.H.A.; Amerongen, Van A.

    2011-01-01

    The present study demonstrates that carbon nanoparticles (CNPs) can be used as labels in microarrays. CNPs were used in nucleic acid microarray immunoassays (NAMIAs) for the detection of different Shiga toxin-producing Escherichia coli (STEC) virulence factors: four genes specific for STEC (vt1,

  10. Characterization of shiga toxin-producing Escherichia coli recovered from domestic animals to determine stx variants, virulence genes, and cytotoxicity in mammalian cells

    Science.gov (United States)

    Shiga toxin-producing Escherichia coli (STEC) can cause foodborne illnesses ranging from diarrhea to severe diseases such as hemorrhagic colitis (HC), and hemolytic uremic syndrome (HUS) in humans. In this study, we determined virulence genes, stx subtypes and we evaluated the cytotoxicity in mammal...

  11. Pectic oligosaccharide structure-function relationships: prebiotics, inhibitors of Escherichia coli O157:H7 adhesion and reduction of Shiga toxin cytotoxicity in HT29 cells

    Science.gov (United States)

    Shiga toxin (Stx)-producing, food-contaminating Escherichia coli (STEC) is a major health concern. Plant-derived pectin and pectic-oligosaccharides (POS) that are abundant in biomass have been considered as prebiotics and for the protection of humans from Stx intoxication. Five structurally differ...

  12. A translational murine model of sub-lethal intoxication with Shiga toxin 2 reveals novel ultrastructural findings in the brain striatum.

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    Carla Tironi-Farinati

    Full Text Available Infection by Shiga toxin-producing Escherichia coli causes hemorrhagic colitis, hemolytic uremic syndrome (HUS, acute renal failure, and also central nervous system complications in around 30% of the children affected. Besides, neurological deficits are one of the most unrepairable and untreatable outcomes of HUS. Study of the striatum is relevant because basal ganglia are one of the brain areas most commonly affected in patients that have suffered from HUS and since the deleterious effects of a sub-lethal dose of Shiga toxin have never been studied in the striatum, the purpose of this study was to attempt to simulate an infection by Shiga toxin-producing E. coli in a murine model. To this end, intravenous administration of a sub-lethal dose of Shiga toxin 2 (0.5 ηg per mouse was used and the correlation between neurological manifestations and ultrastructural changes in striatal brain cells was studied in detail. Neurological manifestations included significant motor behavior abnormalities in spontaneous motor activity, gait, pelvic elevation and hind limb activity eight days after administration of the toxin. Transmission electron microscopy revealed that the toxin caused early perivascular edema two days after administration, as well as significant damage in astrocytes four days after administration and significant damage in neurons and oligodendrocytes eight days after administration. Interrupted synapses and mast cell extravasation were also found eight days after administration of the toxin. We thus conclude that the chronological order of events observed in the striatum could explain the neurological disorders found eight days after administration of the toxin.

  13. Occurrence and quantification of Shiga toxin-producing Escherichia coli from food matrices

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    C. Sethulekshmi

    2018-02-01

    Full Text Available Aim: The objective of the study was to detect Shiga toxin-producing Escherichia coli (STEC and develop a quantitative polymerase chain reaction (qPCR assay to quantify the bacterial DNA present in different food matrices. Materials and Methods: A total of 758 samples were collected during a period from January 2015 to December 2016 from Kozhikode, Thrissur, and Alappuzha districts of Kerala. The samples consisted of raw milk (135, pasteurized milk (100, beef (132, buffalo meat (130, chevon (104, beef kheema (115, and beef sausage (42. All the samples collected were subjected to isolation and identification of STEC by conventional culture technique. Confirmation of virulence genes was carried out using PCR. For the quantification of STEC in different food matrices, a qPCR was standardized against stx1 gene of STEC by the construction of standard curve using SYBR green chemistry. Results: The overall occurrence of STEC in raw milk (n=135, beef (n=132, buffalo meat (n=130, chevon (n=104, and beef kheema (n=115 samples collected from Kozhikode, Thrissur, and Alappuzha districts of Kerala was 19.26%, 41.6%, 16.92%, 28.85%, and 41.74%, respectively. PCR revealed the presence of stx1 and stx2 genes in 88.46 and 83.64 and 30.77 and 40.00% of STEC isolates from raw milk and beef samples, respectively, while 100% of the STEC isolates from buffalo beef and beef kheema samples carried stx1 gene. Real-time qPCR assay was used to quantify the bacterial cells present in different food matrices. The standard curve was developed, and the slopes, intercept, and R2 of linear regression curves were -3.10, 34.24, and 0.99, respectively. Conclusion: The considerably high occurrence of STEC in the study confirms the importance of foods of animal origin as a vehicle of infection to humans. In the present study, on comparing the overall occurrence of STEC, the highest percentage of occurrence was reported in beef kheema samples. The study shows the need for rigid food

  14. Epidemiology of Shiga toxin producing Escherichia coli in Australia, 2000-2010

    Science.gov (United States)

    2012-01-01

    Background Shiga toxin-producing Escherichia coli (STEC) are an important cause of gastroenteritis in Australia and worldwide and can also result in serious sequelae such as haemolytic uraemic syndrome (HUS). In this paper we describe the epidemiology of STEC in Australia using the latest available data. Methods National and state notifications data, as well as data on serotypes, hospitalizations, mortality and outbreaks were examined. Results For the 11 year period 2000 to 2010, the overall annual Australian rate of all notified STEC illness was 0.4 cases per 100,000 per year. In total, there were 822 STEC infections notified in Australia over this period, with a low of 1 notification in the Australian Capital Territory (corresponding to a rate of 0.03 cases per 100,000/year) and a high of 413 notifications in South Australia (corresponding to a rate of 2.4 cases per 100,000/year), the state with the most comprehensive surveillance for STEC infection in the country. Nationally, 71.2% (504/708) of STEC infections underwent serotype testing between 2001 and 2009, and of these, 58.0% (225/388) were found to be O157 strains, with O111 (13.7%) and O26 (11.1%) strains also commonly associated with STEC infections. The notification rate for STEC O157 infections Australia wide between 2001-2009 was 0.12 cases per 100,000 per year. Over the same 9 year period there were 11 outbreaks caused by STEC, with these outbreaks generally being small in size and caused by a variety of serogroups. The overall annual rate of notified HUS in Australia between 2000 and 2010 was 0.07 cases per 100,000 per year. Both STEC infections and HUS cases showed a similar seasonal distribution, with a larger proportion of reported cases occurring in the summer months of December to February. Conclusions STEC infections in Australia have remained fairly steady over the past 11 years. Overall, the incidence and burden of disease due to STEC and HUS in Australia appears comparable or lower than

  15. Genotypes and phenotypes of Shiga toxin-producing Escherichia coli (STEC in Abeokuta, Southwestern Nigeria

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    Olowe OA

    2014-10-01

    Full Text Available Olugbenga Adekunle Olowe,1 Bukola W Aboderin,1,2 Olayinka O Idris,3 Victor O Mabayoje,4 Oluyinka O Opaleye,1 O Catherine Adekunle,1 Rita Ayanbolade Olowe,1 Paul Akinniyi Akinduti,5 Olusola Ojurongbe1 1Department of Medical Microbiology and Parasitology, College of Health Sciences, Osogbo, Osun State, Nigeria; 2Medical Microbiology Unit, Pathology Department, Federal Medical Centre, Abeokuta, Nigeria; 3Department of Microbiology, College of Sciences, Afe Babalola University, Ado Ekiti, Nigeria; 4Department of Haematology, College of Health Sciences, Ladoke Akintola University, Osogbo, Osun State, Nigeria; 5Department of Medical Microbiology and Parasitology, Olabisi Onabanjo University, Ago-Iwoye, Ogun State, Nigeria Purpose: To characterize the prevalence of hemolytic Shiga toxin-producing Escherichia coli (STEC with a multidrug-resistant pattern in different age groups in Abeokuta, Nigeria. Methods: Nonrepetitive E. coli isolates were collected from 202 subjects with or without evidence of diarrhea. Each isolate was biochemically identified and antimicrobial susceptibility testing was performed using the disk diffusion method. A sorbitol fermentation test of all the E. coli isolates was done and the minimum inhibitory concentration of suspected STEC was measured by the standard broth microdilution method to determine antibiotic resistance. The genotypes of stx1, stx2, and hlyA were determined by polymerase chain reaction assay. Results: The majority of subjects were aged ≥40 years (41.6% and were female (61.9%. Of the 202 subjects, 86.1% had STEC isolates (P<0.05. A high rate of STEC isolates resistant to amoxicillin (90.6%, cefotaxime (77.7%, and cefuroxime (75.7% was observed. Resistance to amoxicillin, gentamicin, and cefotaxime was demonstrated with a minimum inhibitory concentration >16 µg/mL in 13.9%, 11.4%, and 10.4% of the isolates, respectively. The prevalence of stx1, stx2, and hlyA was 13.9%, 6.9%, and 2.0%, respectively; 5.5% of

  16. Characterization and zoonotic impact of Shiga toxin producing Escherichia coli in some wild bird species

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    Hanaa Mohamed Fadel

    2017-09-01

    Full Text Available Aim: Wild birds are considered silent vectors of some zoonotic water and food borne pathogens of public health significance. Owing to the importance of Shiga toxin producing Escherichia coli (STEC as the most pathogenic among the emerging diarrheagenic E. coli groups that can infect man; the present study was designed to detect the occurrence of STEC among wild birds in Egypt. Materials and Methods: A total of 177 intestinal content swab samples originating from five wild bird species were investigated for the presence of E. coli and STEC by standard culture methods. Suspect STEC isolates were further characterized by serotyping, random amplified polymorphic DNA polymerase chain reaction (RAPD PCR, antimicrobial resistance pattern and PCR detection of stx1, stx2, and eae genes. Results: A total of 30 suspect STEC isolates from 30 positive birds' samples were detected and identified on STEC CHROMagar (semi-captive pigeons, 15; house crows, 8; cattle egrets, 3; moorhens, 2; and house teals, 2. 25 isolates were grouped into 13 serogroups (O:20, O:25, O:26, O:27, O:63, O:78, O:111, O:114, O:125, O:128, O:142, O:153, and O:158, while five were rough strains. The distribution of STEC virulence genes among wild birds was as follows: 16 birds carried stx1 gene only (nine pigeons [28.1%], six crows [7.1%], and one cattle egret [5.6%]. stx1 and stx2 genes together were detected in four birds (one cattle egret [5.6%], two moorhens [6.1%], and one house teal, [10%]. Only one pigeon (3.1% possessed the three alleles. Disk diffusion test results showed that cefixime was the most effective against STEC serotypes with (93.3% sensitivity, followed by gentamycin (56.7%, and amoxicillin (50%. On the other hand, all the recovered STEC isolates were resistant to cefotaxime, doxycycline, cephalothin, and sulfisoxazole. RAPD fingerprinting using primers OPA-2 and OPA-9 showed that STEC isolates were heterogeneous; they yielded 30 and 27 different clusters

  17. Detection of Shiga-like toxin producing Escherichia coli from raw milk cheeses produced in Wallonia [Belgium].

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    El-Lioui M.

    1999-01-01

    Full Text Available Shiga-like toxin Escherichia coli (STEC implicated in aqueous diarrhoea, haemorrhagic colitis and haemolytic uraemic syndrome, has become a serious health problem in various countries. In Belgium, all cases are sporadic and no outbreak has been detected so far. Cattle are thought to be a reservoir for E. coli O157:H7, and many foodborne diseases have been associated with the consumption of minced beef, beefburgers and raw milk. Recently, foodborne outbreaks were concerned with different unusual foods such as acidic products. Although some data suggest that STEC are not prevalent within dairy products, the aim of this work was to assess the prevalence of E. coli O157 and non-O157 STEC in raw milk cheeses produced in the southern part of Belgium (Wallonia. For this purpose, 153 frozen samples of soft and semi-soft cheeses made with raw cow, ewe and goat milk were analysed for the presence of E. coli O157 and STEC. By using a dynabeads immunomagnetic separation technique (Dynabeads anti-E. coli O157, Dynal followed by streaking onto sorbitol MacConckey agar, no sample was found contaminated by E. coli O157 serotype. By using polymerase chain reaction achieved from a loopful of confluent bacterial material growing onto MacConckey agar, the use of consensus primers detected stx genes in 11.1/ of the samples but Shiga-like toxin producing strains could be isolated only in five of them (3.3/. The isolation rate seems to be optimum for samples with a thermotolerant coliform count arround or below 102 cfu per g. The five Shiga-like toxin isolates were identified as belonging to the species Hafnia alvei or Enterobacter amnigenius without any accessory virulence factors needed to cause illness. Nevertheless, because of the ability of STEC to survive adverse conditions and the possibility for commensal non-pathogenic enteric bacteria to become pathogenic, raw milk cheeses are to be considered at risk for foodborne STEC contamination.

  18. Heterogeneity in Induction Level, Infection Ability, and Morphology of Shiga Toxin-Encoding Phages (Stx Phages) from Dairy and Human Shiga Toxin-Producing Escherichia coli O26:H11 Isolates

    Science.gov (United States)

    Bonanno, Ludivine; Petit, Marie-Agnès; Loukiadis, Estelle; Michel, Valérie

    2016-01-01

    Shiga toxin (Stx)-producing Escherichia coli (STEC) bacteria are foodborne pathogens responsible for diarrhea and hemolytic-uremic syndrome (HUS). Shiga toxin, the main STEC virulence factor, is encoded by the stx gene located in the genome of a bacteriophage inserted into the bacterial chromosome. The O26:H11 serotype is considered to be the second-most-significant HUS-causing serotype worldwide after O157:H7. STEC O26:H11 bacteria and their stx-negative counterparts have been detected in dairy products. They may convert from the one form to the other by loss or acquisition of Stx phages, potentially confounding food microbiological diagnostic methods based on stx gene detection. Here we investigated the diversity and mobility of Stx phages from human and dairy STEC O26:H11 strains. Evaluation of their rate of in vitro induction, occurring either spontaneously or in the presence of mitomycin C, showed that the Stx2 phages were more inducible overall than Stx1 phages. However, no correlation was found between the Stx phage levels produced and the origin of the strains tested or the phage insertion sites. Morphological analysis by electron microscopy showed that Stx phages from STEC O26:H11 displayed various shapes that were unrelated to Stx1 or Stx2 types. Finally, the levels of sensitivity of stx-negative E. coli O26:H11 to six Stx phages differed among the 17 strains tested and our attempts to convert them into STEC were unsuccessful, indicating that their lysogenization was a rare event. PMID:26826235

  19. Assessment of Shiga Toxin-Producing Escherichia coli O157 Illnesses Prevented by Recalls of Beef Products.

    Science.gov (United States)

    Seys, Scott A; Sampedro, Fernando; Hedberg, Craig W

    2015-09-01

    Beef product recall data from 2005 through 2012 associated with Shiga toxin-producing Escherichia coli (STEC) O157 contamination were used to develop quantitative models to estimate the number of illnesses prevented by recalls. The number of illnesses prevented was based on the number of illnesses that occurred relative to the number of pounds consumed, then extrapolated to the number of pounds of recalled product recovered. A simulation using a Program Evaluation and Review Technique (PERT) probability distribution with illness-related recalls estimated 204 (95% credible interval, 117-333) prevented STEC O157 illnesses from 2005 through 2012. Recalls not associated with illnesses had more recalled product recovered and prevented an estimated 83 additional STEC O157 illnesses. Accounting for underdiagnosis resulted in an estimated total of 7500 STEC O157 illnesses prevented over 8 years. This study demonstrates that recalls, although reactive in nature, are an important tool for averting further exposure and illnesses.

  20. Hemolytic uremic syndrome with mild renal involvement due to Shiga toxin-producing Escherichia coli (STEC O145 strain

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    Lucía Pérez

    Full Text Available Hemolytic uremic syndrome (HUS is a disorder characterized by the presence of the classic triad: microangiopathic hemolytic anemia, thrombocytopenia and acute renal injury. HUS without acute renal failure can be confused with other hematologic diseases. An infantile HUS caused by a Shiga-toxin-producing Escherichia coli (STEC O145 strain carrying genotype stx2, ehxA, eae subtype ?1 is herein reported. The infant did not require dialysis during the acute stage of HUS, evolved favorably, maintained normal blood pressure and normal renal function and had no recurrence until the last control. This could be due to several factors, such as the characteristics of infecting STEC strain and a reduction in host susceptibility to renal injury. This report highlights the regional participation of non-O157 STEC in childhood diseases and the importance of performing active surveillance for all forms of HUS.

  1. Prevalence and Characterization of Shiga Toxin-Producing Escherichia coli Isolated from Slaughtered Qurban Animal in Jakarta Province

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    Siti Gusti Ningrum

    2016-08-01

    Full Text Available This study was conducted to investigate the presence of shiga toxin producing Escherichia coli (STEC and the possibility of carrying rfbE gene and H7 flagellar on meat, liver, and stool samples collected from Jakarta Province of Indonesia. A total of 51 samples collected from meat, liver, and stool of slaughtered cattle from qurban festival were tested using conventional culture and multiplex PCR methods. STEC non O157 were detected in meat (5.3% and stool (8.3% with one isolate from stool carried H7 flagellar. However, all isolates were lacking of rfbE gene. In antimicrobial susceptibility tests, the STEC isolates showed antibiotic resistance to erythromycin and oxacillin. Overall, the result shows that meat and liver of this origin activity represents a potential risk to human health.

  2. Generation of divalent DNA vaccine based on p39 and shiga-like toxin 2 (Stx2 genes

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    Doosti Abbas

    2015-01-01

    Full Text Available The virulence factors such as shiga-like toxin (Stx and immunogenic P39 protein in Escherichia coli and Brucella melitensis are related to disease of digestive system in human worldwide. In the present study the stx2 and p39 genes were cloned into expression plasmid pEEF1D-FLAG (pcDNA 3.1+ as a divalent DNA vaccine candidate. The Enterohemorrhagic E. coli ATCC 3081 and smooth virulent B. melitensis strain M5 were obtained and cultured on specific media. Bacterial DNA was extracted from colonies and was used for p39 and stx2 genes amplification by PCR. The amplified products on 2% agarose gel electrophoresis were revealed 285 and 1220 bp fragments for stx2 and p39 genes, respectively. Each amplified genes were T/A cloned into pGEM-T easy vector and pGEM-T-stx2 and pGEM-T-p39 were produced. The stx2 and p39 genes were sub-cloned in linearized expression vector (pcDNA 3.1+ using HindIII, XhoI and XbaI restriction enzymes and pCDNA3-stx2-p39 was generated. This final construct was confirmed by PCR and enzymes digestion. The results were showed stx2 and p39 genes were sub-cloned, successfully into pcDNA 3.1+ to generate pcDNA 3.1+-stx2-p39 recombinant vector. According to these findings novel recombinant pcDNA 3.1+-stx2- p39 construct that was produced in this study could be useful as DNA vaccine candidate in animal models against shiga-like toxin producing E. coli and virulence B. melitensis strains in future studies.

  3. UV-Sensitivity of Shiga Toxin-Converting Bacteriophage Virions Φ24B, 933W, P22, P27 and P32

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    Sylwia Bloch

    2015-09-01

    Full Text Available Shiga toxin-converting bacteriophages (Stx phages are present as prophages in Shiga toxin-producing Escherichia coli (STEC strains. Theses phages can be transmitted to previously non-pathogenic E. coli cells making them potential producers of Shiga toxins, as they bear genes for these toxins in their genomes. Therefore, sensitivity of Stx phage virions to various conditions is important in both natural processes of spreading of these viruses and potential prophylactic control of appearance of novel pathogenic E. coli strains. In this report we provide evidence that virions of Stx phages are significantly more sensitive to UV irradiation than bacteriophage λ. Following UV irradiation of Stx virions at the dose of 50 J/m2, their infectivity dropped by 1–3 log10, depending on the kind of phage. Under these conditions, a considerable release of phage DNA from virions was observed, and electron microscopy analyses indicated a large proportion of partially damaged virions. Infection of E. coli cells with UV-irradiated Stx phages resulted in significantly decreased levels of expression of N and cro genes, crucial for lytic development. We conclude that inactivation of Stx virions caused by relatively low dose of UV light is due to damage of capsids that prevents effective infection of the host cells.

  4. UV-Sensitivity of Shiga Toxin-Converting Bacteriophage Virions Φ24B, 933W, P22, P27 and P32

    Science.gov (United States)

    Bloch, Sylwia; Nejman-Faleńczyk, Bożena; Topka, Gracja; Dydecka, Aleksandra; Licznerska, Katarzyna; Narajczyk, Magdalena; Necel, Agnieszka; Węgrzyn, Alicja; Węgrzyn, Grzegorz

    2015-01-01

    Shiga toxin-converting bacteriophages (Stx phages) are present as prophages in Shiga toxin-producing Escherichia coli (STEC) strains. Theses phages can be transmitted to previously non-pathogenic E. coli cells making them potential producers of Shiga toxins, as they bear genes for these toxins in their genomes. Therefore, sensitivity of Stx phage virions to various conditions is important in both natural processes of spreading of these viruses and potential prophylactic control of appearance of novel pathogenic E. coli strains. In this report we provide evidence that virions of Stx phages are significantly more sensitive to UV irradiation than bacteriophage λ. Following UV irradiation of Stx virions at the dose of 50 J/m2, their infectivity dropped by 1–3 log10, depending on the kind of phage. Under these conditions, a considerable release of phage DNA from virions was observed, and electron microscopy analyses indicated a large proportion of partially damaged virions. Infection of E. coli cells with UV-irradiated Stx phages resulted in significantly decreased levels of expression of N and cro genes, crucial for lytic development. We conclude that inactivation of Stx virions caused by relatively low dose of UV light is due to damage of capsids that prevents effective infection of the host cells. PMID:26402701

  5. N-chlorotaurine, a long-lived oxidant produced by human leukocytes, inactivates Shiga toxin of enterohemorrhagic Escherichia coli.

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    Christian Eitzinger

    Full Text Available N-chlorotaurine (NCT, the main representative of long-lived oxidants produced by granulocytes and monocytes, is known to exert broad-spectrum microbicidal activity. Here we show that NCT directly inactivates Shiga toxin 2 (Stx2, used as a model toxin secreted by enterohemorrhagic Escherichia coli (EHEC. Bacterial growth and Stx2 production were both inhibited by 2 mM NCT. The cytotoxic effect of Stx2 on Vero cells was removed by ≥5.5 mM NCT. Confocal microscopy and FACS analyses showed that the binding of Stx2 to human kidney glomerular endothelial cells was inhibited, and no NCT-treated Stx2 entered the cytosol. Mass spectrometry displayed oxidation of thio groups and aromatic amino acids of Stx2 by NCT. Therefore, long-lived oxidants may act as powerful tools of innate immunity against soluble virulence factors of pathogens. Moreover, inactivation of virulence factors may contribute to therapeutic success of NCT and novel analogs, which are in development as topical antiinfectives.

  6. Pro-Coagulant Endothelial Dysfunction Results from EHEC Shiga toxins and Host Damage-Associated Molecular Patterns

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    Chad L Mayer

    2015-04-01

    Full Text Available Hemolytic uremic syndrome (HUS from enterohemorrhagic E.coli infection (EHEC is a leading cause of kidney failure in otherwise healthy U.S. children. The bacterial Shiga toxins (Stx induce the characteristic coagulopathy of HUS, but the cell and organ damage during infection likely also releases inflammatory damage-associated molecular patterns (DAMPs that may exacerbate injury in concert with the toxins. To examine this, aortic and renal glomerular cell anticoagulant and barrier functions were studied after in vitro challenge with Stx1, Stx2 and DAMPs. There was significant loss of surface anticoagulant protein C pathway molecules, increased expression of prothrombotic PAR1 and reduced protein C activation capability by 15-27%. Histones nearly completely prevented activated protein C protection of endothelial cells from thrombin-induced permeability. In mice, lethal Stx2 challenge elevated plasma HMGB1 (day 2, 321±118%; p<0.01 and extracellular histones (day 3, 158±62%; p<0.01. Mice colonized with Stx2-expressing Citrobacter rodentium developed increased HMGB1 (day 5, 155±55%; p<0.01 and histones (day 3, 378±188%; p<0.01. Anti-histone antibody reduced both DAMPs to baseline, but was not sufficient to improve survival outcome or kidney function. Together, these data suggest a potential role for both Stx and DAMPs in producing endothelial injury and a prothrombotic environment.

  7. Shiga Toxin 1, as DNA Repair Inhibitor, Synergistically Potentiates the Activity of the Anticancer Drug, Mafosfamide, on Raji Cells

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    Piero Sestili

    2013-02-01

    Full Text Available Shiga toxin 1 (Stx1, produced by pathogenic Escherichia coli, targets a restricted subset of human cells, which possess the receptor globotriaosylceramide (Gb3Cer/CD77, causing hemolytic uremic syndrome. In spite of the high toxicity, Stx1 has been proposed in the treatment of Gb3Cer/CD77-expressing lymphoma. Here, we demonstrate in a Burkitt lymphoma cell model expressing this receptor, namely Raji cells, that Stx1, at quasi-non-toxic concentrations (0.05–0.1 pM, inhibits the repair of mafosfamide-induced DNA alkylating lesions, synergistically potentiating the cytotoxic activity of the anticancer drug. Conversely, human promyelocytic leukemia cells HL-60, which do not express Gb3Cer/CD77, were spared by the toxin as previously demonstrated for CD34+ human progenitor cells, and hence, in this cancer model, no additive nor synergistic effects were observed with the combined Stx1/mafosfamide treatment. Our findings suggest that Stx1 could be used to improve the mafosfamide-mediated purging of Gb3Cer/CD77+ tumor cells before autologous bone marrow transplantation.

  8. Vitamin B12 Uptake by the gut commensal bacteria bacteroides thetaiotaomicron limits the production of shiga toxin by enterohemorrhagic escherichia coli

    OpenAIRE

    Cordonnier, Charlotte; Le Bihan , Guillaume; Emond-Rheault , Jean-Guillaume; Garrivier, Annie; Harel, Josee; Jubelin, Grégory

    2016-01-01

    Enterohemorrhagic Escherichia coli (EHEC) are foodborne pathogens responsible for the development of bloody diarrhea and renal failure in humans. Many environmental factors have been shown to regulate the production of Shiga toxin 2 (Stx2), the main virulence factor of EHEC. Among them, soluble factors produced by human gut microbiota and in particular, by the predominant species Bacteroides thetaiotaomicron (B. thetaiotaomicron), inhibit Stx2 gene expression. In this study, we investigated t...

  9. Antibiotic Resistance and Virulence Potentials of Shiga Toxin-Producing Escherichia coli Isolates from Raw Meats of Slaughterhouses and Retail Markets in Korea.

    Science.gov (United States)

    Park, Hyun-Jung; Yoon, Jang Won; Heo, Eun-Jeong; Ko, Eun-Kyoung; Kim, Ki-Yeon; Kim, Young-Jo; Yoon, Hyang-Jin; Wee, Sung-Hwan; Park, Yong Ho; Moon, Jin San

    2015-09-01

    In this study, the prevalence of Shiga toxin-producing Escherichia coli (STEC) was investigated among raw meat or meat products from slaughterhouses and retail markets in South Korea, and their potential for antibiotic resistance and virulence was further analyzed. A total of 912 raw meats, including beef, pork, and chicken, were collected from 2008 to 2009. E. coli strains were frequently isolated in chicken meats (176/233, 75.9%), beef (102/217, 42.3%), and pork (109/235, 39.2%). Putative STEC isolates were further categorized, based on the presence or absence of the Shiga toxin (stx) genes, followed by standard O-serotyping. Polymerase chain reaction assays were used to detect the previously defined virulence genes in STEC, including Shiga toxins 1 and Shiga toxin 2 (stx1 and 2), enterohemolysin (ehxA), intimin (eaeA), STEC autoagglutination adhesion (saa), and subtilase cytotoxin (subAB). All carried both stx1 and eae genes, but none of them had the stx2, saa, or subAB genes. Six (50.0%) STEC isolates possessed the ehxA gene, which is known to be encoded by the 60-megadalton virulence plasmid. Our antibiogram profiling demonstrated that some STEC strains, particularly pork and chicken isolates, displayed a multiple drug-resistance phenotype. RPLA analysis revealed that all the stx1-positive STEC isolates produced Stx1 only at the undetectable level. Altogether, these results imply that the locus of enterocyte and effacement (LEE)-positive strains STEC are predominant among raw meats or meat products from slaughterhouses or retail markets in Korea.

  10. Detection and isolation of the "Top 7" Shiga toxin-producing Escherichia coli in ground beef: comparison of the Rapidfinder kits to the USDA microbiology laboratory guidebook method

    Science.gov (United States)

    Shiga toxin-producing E. coli (STEC) O157:H7 and serogroups O26, O45, O103, O111, O121, and O145 are often referred to as the “top 7” STEC, and these have been declared as adulterants in beef by the USDA Food Safety and Inspection Service (FSIS). The aim of this work was to compare the methods des...

  11. Homogenous, real-time duplex loop-mediated isothermal amplification using a single fluorophore-labeled primer and an intercalator dye: Its application to the simultaneous detection of Shiga toxin genes 1 and 2 in Shiga toxigenic Escherichia coli isolates.

    Science.gov (United States)

    Kouguchi, Yoshihiro; Fujiwara, Takako; Teramoto, Miki; Kuramoto, Mika

    2010-08-01

    We developed a completely homogeneous duplex loop-mediated isothermal amplification (LAMP) method. The present LAMP method employed a combination of a 6-carboxyfluorescein (FAM)-labeled primer (donor) for one target gene, a non-labeled primer for the other, and an intercalator ethidium bromide (EtBr) dye (acceptor) on the basis of fluorescence resonance energy transfer (FRET) between the FAM donor and EtBr acceptor. Measuring changes in fluorescence of FAM enabled the LAMP method to detect two different genes simultaneously. This method was used to detect Shiga toxin genes in Shiga toxigenic Escherichia coli isolates, demonstrating simultaneous detection of two different genes with rapidity and accuracy. Copyright 2010 Elsevier Ltd. All rights reserved.

  12. Upregulation of Shiga toxin receptor CD77/Gb3 and interleukin-1β expression in the brain of EHEC patients with hemolytic uremic syndrome and neurologic symptoms.

    Science.gov (United States)

    Hagel, Christian; Krasemann, Susanne; Löffler, Judith; Püschel, Klaus; Magnus, Tim; Glatzel, Markus

    2015-03-01

    In 2011, a large outbreak of Shiga toxin-producing enterohemorrhagic Escherichia coli (EHEC) infections occurred in northern Germany, which mainly affected adults. Out of 3842 patients, 104 experienced a complicated course comprising hemolytic uremic syndrome and neurological complications, including cognitive impairment, aphasia, seizures and coma. T2 hyperintensities on magnet resonance imaging (MRI) bilateral in the thalami and in the dorsal pons were found suggestive of a metabolic toxic effect. Five of the 104 patients died because of toxic heart failure. In the present study, the post-mortem neuropathological findings of the five EHEC patients are described. Histological investigation of 13 brain regions (frontal, temporal, occipital cortex, corpora mammillaria, thalamus, frontal operculum, corona radiata, gyrus angularis, pons, medulla oblongata, cerebellar vermis and cerebellar hemisphere) showed no thrombosis, ischemic changes or fresh infarctions. Further, no changes were found in electron microscopy. In comparison with five age-matched controls, slightly increased activation of microglia and a higher neuronal expression of interleukin-1β and of Shiga toxin receptor CD77/globotriaosylceramide 3 was observed. The findings were confirmed by Western blot analyses. It is suggested that CD77/globotriaosylceramide upregulation may be a consequence to Shiga toxin exposure, whereas increased interleukin-1β expression may point to activation of inflammatory cascades. © 2014 International Society of Neuropathology.

  13. Prevalence and characterization of Shiga Toxin-producing and enteropathogenic Escherichia coli in shellfish-harvesting areas and their watersheds

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    Balière eCharlotte

    2015-12-01

    Full Text Available During a two-year study, the presence of Shiga-toxin producing E. coli (STEC and enteropathogenic E. coli (EPEC was investigated in shellfish (n=238, seawater (n=12 and surface sediment (n=39 collected from three French coastal shellfish-harvesting areas and freshwaters (n=216 in their watersheds. PCR detection of Shiga toxin- (stx1/stx2 and intimin- (eae genes following enrichment from these samples revealed the presence of least one of the stx genes in 30.3% of shellfish batches, 85.9% of freshwater, 41.7% of seawater, and 28.2% of sediment samples, while the eae gene was observed in 74.8%, 100%, 100%, and 43.6% of shellfish batches, freshwater, seawater, and sediment samples, respectively. Twenty-eight STEC and 89 EPEC strains were isolated and analyzed in order to determine their serotype, phylogroup, and genetic relatedness and to evaluate the presence of the saa and ehxA genes encoding the STEC autoagglutinating adhesin and the enterohemolysin A, respectively. Finally, the ability to form biofilms and antimicrobial susceptibility were investigated for a selection of strains. Eighteen serotypes were identified among the STEC isolates and 57 among the EPEC isolates. A high diversity was observed within these strains, as 79 different PFGE patterns and 48 distinguishable sequence types were identified. Strains were found to belong mainly to phylogroups B1 and B2 and virulence was observed to be low as more than 85% of the strains possessed only stx1, stx2 or eae genes. One STEC and several EPEC strains belonged to three of the five highly pathogenic serogroups (i.e., O26, O103, and O145. The subset of strains tested for their capacity to form biofilms was mainly strongly to moderately adherent and more strains formed a strong biofilm at 18°C than at 30°C. Finally, more than 85% of analyzed strains were found to be sensitive to the 16 tested antibiotics. These data suggest the low risk of human infection by STEC if shellfish from these

  14. Promoter sequence of Shiga toxin 2 (Stx2) is recognized in vivo, leading to production of biologically active Stx2.

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    Bentancor, Leticia V; Mejías, Maria P; Pinto, Alípio; Bilen, Marcos F; Meiss, Roberto; Rodriguez-Galán, Maria C; Baez, Natalia; Pedrotti, Luciano P; Goldstein, Jorge; Ghiringhelli, Pablo D; Palermo, Marina S

    2013-10-01

    Shiga toxins (Stx) are the main agent responsible for the development of hemolytic-uremic syndrome (HUS), the most severe and life-threatening systemic complication of infection with enterohemorrhagic Escherichia coli (EHEC) strains. We previously described Stx2 expression by eukaryotic cells after they were transfected in vitro with the stx2 gene cloned into a prokaryotic plasmid (pStx2). The aim of this study was to evaluate whether mammalian cells were also able to express Stx2 in vivo after pStx2 injection. Mice were inoculated by hydrodynamics-based transfection (HBT) with pStx2. We studied the survival, percentage of polymorphonuclear leukocytes in plasma, plasma urea levels, and histology of the kidneys and the brains of mice. Mice displayed a lethal dose-related response to pStx2. Stx2 mRNA was recovered from the liver, and Stx2 cytotoxic activity was observed in plasma of mice injected with pStx2. Stx2 was detected by immunofluorescence in the brains of mice inoculated with pStx2, and markers of central nervous system (CNS) damage were observed, including increased expression of glial fibrillary acidic protein (GFAP) and fragmentation of NeuN in neurons. Moreover, anti-Stx2B-immunized mice were protected against pStx2 inoculation. Our results show that Stx2 is expressed in vivo from the wild stx2 gene, reproducing pathogenic damage induced by purified Stx2 or secondary to EHEC infection. Enterohemorrhagic Shiga toxin (Stx)-producing Escherichia coli (EHEC) infections are a serious public health problem, and Stx is the main pathogenic agent associated with typical hemolytic-uremic syndrome (HUS). In contrast to the detailed information describing the molecular basis for EHEC adherence to epithelial cells, very little is known about how Stx is released from bacteria in the gut, reaching its target tissues, mainly the kidney and central nervous system (CNS). In order to develop an efficient treatment for EHEC infections, it is necessary to understand the

  15. Proteomic View of Interactions of Shiga Toxin-Producing Escherichia coli with the Intestinal Environment in Gnotobiotic Piglets.

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    Rembert Pieper

    Full Text Available Shiga toxin (Stx-producing Escherichia coli cause severe intestinal infections involving colonization of epithelial Peyer's patches and formation of attachment/effacement (A/E lesions. These lesions trigger leukocyte infiltration followed by inflammation and intestinal hemorrhage. Systems biology, which explores the crosstalk of Stx-producing Escherichia coli with the in vivo host environment, may elucidate novel molecular pathogenesis aspects.Enterohemorrhagic E. coli strain 86-24 produces Shiga toxin-2 and belongs to the serotype O157:H7. Bacterial cells were scrapped from stationary phase cultures (the in vitro condition and used to infect gnotobiotic piglets via intestinal lavage. Bacterial cells isolated from the piglets' guts constituted the in vivo condition. Cell lysates were subjected to quantitative 2D gel and shotgun proteomic analyses, revealing metabolic shifts towards anaerobic energy generation, changes in carbon utilization, phosphate and ammonia starvation, and high activity of a glutamate decarboxylase acid resistance system in vivo. Increased abundance of pyridine nucleotide transhydrogenase (PntA and PntB suggested in vivo shortage of intracellular NADPH. Abundance changes of proteins implicated in lipopolysaccharide biosynthesis (LpxC, ArnA, the predicted acyltransferase L7029 and outer membrane (OM assembly (LptD, MlaA, MlaC suggested bacterial cell surface modulation in response to activated host defenses. Indeed, there was evidence for interactions of innate immunity-associated proteins secreted into the intestines (GP340, REG3-γ, resistin, lithostathine, and trefoil factor 3 with the bacterial cell envelope.Proteomic analysis afforded insights into system-wide adaptations of strain 86-24 to a hostile intestinal milieu, including responses to limited nutrients and cofactor supplies, intracellular acidification, and reactive nitrogen and oxygen species-mediated stress. Protein and lipopolysaccharide compositions of the OM

  16. Shiga toxin-producing Escherichia coli isolated from chicken meat in Iran: serogroups, virulence factors, and antimicrobial resistance properties.

    Science.gov (United States)

    Momtaz, Hassan; Jamshidi, Alireza

    2013-05-01

    The aim of the current study was to determine the virulence factors, serogroups, and antibiotic resistance properties of Shiga toxin-producing Escherichia coli isolated from chicken meat samples. A total of 422 chicken meat samples were collected from 5 townships of Iran. Specimens were immediately transferred to the laboratory in a cooler with an ice pack. Samples were cultured, and the positive culture samples were analyzed by PCR assays. Finally, the antimicrobial susceptibility test was performed using the disk diffusion method in Mueller-Hinton agar. According to the results, out of 422 samples, 146 (34.59%) were confirmed to be E. coli positive and among E. coli-positive samples, 51 (34.93%) and 31 (21.23%) were from attaching and effacing E. coli (AEEC) and enterohemorrhagic E. coli (EHEC) subgroups, respectively. All of the EHEC-positive samples had all stx1, eaeA, and ehly virulence genes, whereas only 5 (9.80%) of AEEC subgroup had all stx1, stx2, and eaeA genes. As the data revealed, O157 was the most prevalent and O111 was the least prevalent strains in the Shiga toxin-producing E. coli (STEC) population. Among STEC strains, sulI and blaSHV had the highest and lowest incidence rate, respectively. There was a high resistance to tetracycline (76.82%), followed by chloramphenicol (73.17%) and nitrofurantoin (63.41%), but there was low resistance to cephalotine (7.31%) antibiotics in isolated strains. Results shows that the PCR technique has a high performance for detection of serogroups, virulence genes, and antibiotic resistance genes in STEC strains. This study is the first prevalence report of detection of virulence genes, serogroups, and antibiotic resistance properties of STEC strains isolated from chicken meat samples in Iran. Based on the results, chicken meat is one of the main sources of STEC strains and its virulence factors in Iran, so an accurate meat inspection would reduce disease outbreaks.

  17. The Accessory Genome of Shiga Toxin-Producing Escherichia coli Defines a Persistent Colonization Type in Cattle.

    Science.gov (United States)

    Barth, Stefanie A; Menge, Christian; Eichhorn, Inga; Semmler, Torsten; Wieler, Lothar H; Pickard, Derek; Belka, Ariane; Berens, Christian; Geue, Lutz

    2016-09-01

    Shiga toxin-producing Escherichia coli (STEC) strains can colonize cattle for several months and may, thus, serve as gene reservoirs for the genesis of highly virulent zoonotic enterohemorrhagic E. coli (EHEC). Attempts to reduce the human risk for acquiring EHEC infections should include strategies to control such STEC strains persisting in cattle. We therefore aimed to identify genetic patterns associated with the STEC colonization type in the bovine host. We included 88 persistent colonizing STEC (STEC(per)) (shedding for ≥4 months) and 74 sporadically colonizing STEC (STEC(spo)) (shedding for ≤2 months) isolates from cattle and 16 bovine STEC isolates with unknown colonization types. Genoserotypes and multilocus sequence types (MLSTs) were determined, and the isolates were probed with a DNA microarray for virulence-associated genes (VAGs). All STEC(per) isolates belonged to only four genoserotypes (O26:H11, O156:H25, O165:H25, O182:H25), which formed three genetic clusters (ST21/396/1705, ST300/688, ST119). In contrast, STEC(spo) isolates were scattered among 28 genoserotypes and 30 MLSTs, with O157:H7 (ST11) and O6:H49 (ST1079) being the most prevalent. The microarray analysis identified 139 unique gene patterns that clustered with the genoserotypes and MLSTs of the strains. While the STEC(per) isolates possessed heterogeneous phylogenetic backgrounds, the accessory genome clustered these isolates together, separating them from the STEC(spo) isolates. Given the vast genetic heterogeneity of bovine STEC strains, defining the genetic patterns distinguishing STEC(per) from STEC(spo) isolates will facilitate the targeted design of new intervention strategies to counteract these zoonotic pathogens at the farm level. Ruminants, especially cattle, are sources of food-borne infections by Shiga toxin-producing Escherichia coli (STEC) in humans. Some STEC strains persist in cattle for longer periods of time, while others are detected only sporadically. Persisting

  18. Agitation down-regulates immunoglobulin binding protein EibG expression in Shiga toxin-producing Escherichia coli (STEC.

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    Thorsten Kuczius

    Full Text Available Shiga toxin (Stx-producing Escherichia coli (STEC carrying eibG synthesize Escherichia coli immunoglobulin binding protein (EibG. EibG nonspecifically binds to immunoglobulins and tends to aggregate in multimers but is poorly expressed in wild-type strains. To study synthesis of the proteins and their regulation in the pathogens, we identified natural growth conditions that increased EibG synthesis. EibG proteins as well as corresponding mRNA were highly expressed under static growth conditions while shearing stress created by agitation during growth repressed protein synthesis. Further regulation effects were driven by reduced oxygen tension, and pH up-regulated EibG expression, but to a lesser extent than growth conditions while decreased temperature down-regulated EibG. Bacteria with increased EibG expression during static growth conditions showed a distinct phenotype with chain formation and biofilm generation, which disappeared with motion. High and low EibG expression was reversible indicating a process with up- and down-regulation of the protein expression. Our findings indicate that shear stress represses EibG expression and might reduce bacterial attachments to cells and surfaces.

  19. Deteksi Gen Shiga Like Toxin 1 isolat escherichia Coli O157:H7 Asal Sapi Bali di Kuta Selatan, Badung

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    Dwi Lestari

    2015-08-01

    Full Text Available Salah satu bakteri pencemar adalah serotipe E. coli O157:H. Bakteri ini diketahui tumbuh dan berkembang dan menghasilkan toksin. Toksin yang dihasilkan oleh strain ini adalah verotoxin atau disebut sebagai shiga-like toxin (Stx.Penelitian ini menggunakan lima isolate E. coli O157:H7 yaitu FSKS 5 Pecatu, FSKS 17 Kutuh, FSKS 35 Ungasan, FSKS 44 Ungasan, dan FSKS 55 Jimbaran. Kegiatan penelitian diawali dengan kultivasi isolate melalui penumbuhan pada media Sorbitol MacConkey Agar (SMAC, dilanjutkan dengan konfirmasi E. coli O157 dengan uji latex agglutination test dan konfirmasi E. coli O157:H7 melalui pengujian dengan anti serum H7. Deteksi molekuler diawali dengan tahapan isolasi DNA genom, dilanjutkan dengan deteksi gen Stx 1 melalui metode Polimerase Chain Reaction (PCR, menggunakan primer LP 30 dan LP31. Hasil penelitian menunjukkan positif gen Stx 1 pada dua isolate yaitu FSKS 5 Pecatu dan FSKS 35 Ungasan yang dicirikan dengan terlihatnya band/pita dengan panjang produk 348 bp. Hasil penelitian ini menunjukkan bahwa isolate local FSKS 5 Pecatu dan FSKS 35 Ungasan, berpotensi sebagai agen zoonosis yang berpeluang besar menimbulkan diare berdarah.

  20. Soluble CD40 Ligand and Oxidative Response Are Reciprocally Stimulated during Shiga Toxin-Associated Hemolytic Uremic Syndrome

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    Maria J. Abrey Recalde

    2017-10-01

    Full Text Available Shiga toxin (Stx, produced by Escherichia coli, is the main pathogenic factor of diarrhea-associated hemolytic uremic syndrome (HUS, which is characterized by the obstruction of renal microvasculature by platelet-fibrin thrombi. It is well known that the oxidative imbalance generated by Stx induces platelet activation, contributing to thrombus formation. Moreover, activated platelets release soluble CD40 ligand (sCD40L, which in turn contributes to oxidative imbalance, triggering the release of reactive oxidative species (ROS on various cellular types. The aim of this work was to determine if the interaction between the oxidative response and platelet-derived sCD40L, as consequence of Stx-induced endothelium damage, participates in the pathogenic mechanism during HUS. Activated human glomerular endothelial cells (HGEC by Stx2 induced platelets to adhere to them. Although platelet adhesion did not contribute to endothelial damage, high levels of sCD40L were released to the medium. The release of sCD40L by activated platelets was inhibited by antioxidant treatment. Furthermore, we found increased levels of sCD40L in plasma from HUS patients, which were also able to trigger the respiratory burst in monocytes in a sCD40L-dependent manner. Thus, we concluded that platelet-derived sCD40L and the oxidative response are reciprocally stimulated during Stx2-associated HUS. This process may contribute to the evolution of glomerular occlusion and the microangiopathic lesions.

  1. Identification and molecular characterization of antimicrobial-resistant shiga toxin-producing Escherichia coli isolated from retail meat products.

    Science.gov (United States)

    Li, Ming-Cheng; Wang, Fang; Li, Fan

    2011-04-01

    Ten (2.7%) Shiga toxin-producing Escherichia coli (STEC) were isolated from 370 samples of raw minced beef, mutton, pork, and chicken from the Jilin region of China; and additional 10 E. coli O157:H7 isolates were previously isolated from different Jilin regions. Seventeen of the isolates were multiresistant, exhibiting resistance to ampicillin, ciprofloxacin, tetracycline, sulfamethoxazole-trimethoprim, gentamycin, and streptomycin. Class 1 integrons were detected in nine (45.0%) of the STEC isolates and consisted of serogroups O157, O62, O113, O149, and O70. Integrons containing amplicons of a 0.5-1.5 or 1.0 kb gene cassette were found in seven (77.8%) of the integron-containing isolates. Sequencing analysis revealed that these gene cassettes encode genes conferring resistance to trimethoprim (dfrA1) and streptomycin (aadA1). The 0.5 kb cassette described here was found to encode a putative transporter peptide in the STEC. Seventeen isolates contained plasmids with different bands, and transfer by conjugation between strains of E. coli demonstrated that class 1 integrons located on mobile plasmids could contribute to the emergence and dissemination of antimicrobial resistance to ampicillin, gentamycin, streptomycin, and sulfamethoxazole-trimethoprim amongst STEC. These data revealed the high prevalence of antimicrobial-resistant STEC isolates in Jilin's surrounding regions, providing important and useful surveillance information reflecting antimicrobial selection pressure. © Mary Ann Liebert, Inc.

  2. Detection of virulence factors and antimicrobial resistance patterns in shiga toxin-producing Escherichia coli isolates from sheep

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    Marcos R.A. Ferreira

    2015-09-01

    Full Text Available Abstract: In order to detect virulence factors in Shiga toxin-producing Escherichia coli (STEC isolates and investigate the antimicrobial resistance profile, rectal swabs were collected from healthy sheep of the races Santa Inês and Dorper. Of the 115 E. coli isolates obtained, 78.3% (90/115 were characterized as STEC, of which 52.2% (47/90 carried stx1 gene, 33.3% (30/90 stx2 and 14.5% (13/90 both genes. In search of virulence factors, 47.7% and 32.2% of the isolates carried the genes saa and cnf1. According to the analysis of the antimicrobial resistance profile, 83.3% (75/90 were resistant to at least one of the antibiotics tested. In phylogenetic classification grouped 24.4% (22/90 in group D (pathogenic, 32.2% (29/90 in group B1 (commensal and 43.3% (39/90 in group A (commensal. The presence of several virulence factors as well as the high number of multiresistant isolates found in this study support the statement that sheep are potential carriers of pathogens threatening public health.

  3. Spread and change in stress resistance of Shiga toxin-producing Escherichia coli O157 on fungal colonies.

    Science.gov (United States)

    Lee, Ken-Ichi; Kobayashi, Naoki; Watanabe, Maiko; Sugita-Konishi, Yoshiko; Tsubone, Hirokazu; Kumagai, Susumu; Hara-Kudo, Yukiko

    2014-11-01

    To elucidate the effect of fungal hyphae on the behaviour of Shiga toxin-producing Escherichia coli (STEC) O157, the spread and change in stress resistance of the bacterium were evaluated after coculture with 11 species of food-related fungi including fermentation starters. Spread distances of STEC O157 varied depending on the co-cultured fungal species, and the motile bacterial strain spread for longer distances than the non-motile strain. The population of STEC O157 increased when co-cultured on colonies of nine fungal species but decreased on colonies of Emericella nidulans and Aspergillus ochraceus. Confocal scanning microscopy visualization of green fluorescent protein-tagged STEC O157 on fungal hyphae revealed that the bacterium colonized in the water film that existed on and between hyphae. To investigate the physiological changes in STEC O157 caused by co-culturing with fungi, the bacterium was harvested after 7 days of co-culturing and tested for acid resistance. After co-culture with eight fungal species, STEC O157 showed greater acid resistance compared to those cultured without fungi. Our results indicate that fungal hyphae can spread the contamination of STEC O157 and can also enhance the stress resistance of the bacteria. © 2013 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  4. Molecular screening of bovine raw milk for the presence of Shiga toxin-producing Escherichia coli (STEC on dairy farms

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    Tatiane Vendramin

    2014-09-01

    Full Text Available Milkborne transmission of Shiga toxin- producing Escherichia coli (STEC has raised considerable concern due to recent outbreaks worldwide and poses a threat to public health. The aim of this study was to develop a sensitive and specific multiplex PCR assay to detect the presence of STEC in bovine raw milk. To identify E. coli (ATCC 25922 contamination, the gene uspA was used, and PCR sensitivity and specificity were accessed by testing diluted samples ranging from 2 to 2.0 × 10(6 CFU/mL. To detect STEC, the stx1 and stx2 genes were selected as targets. After reaction standardization, the multiplex assay was tested in raw milk collected from 101 cows on dairy farms. PCR assay for E. coli detection had a specificity of 100% and sensitivity of 79% (P<0.0001, with a lower detection limit of 2 CFU/mL. Multiplex PCR assay had 100% sensitivity for E. coli positive raw milk samples, and 31.1% were contaminated with STEC, 28.3% of stx2, and 1.9% of stx1. The multiplex PCR assay described in the present study can be employed to identify and screen E. coli harboring stx1 and stx2 genes in raw milk on dairy farms and in industries.

  5. Hemolytic uremic syndrome with mild renal involvement due to Shiga toxin-producing Escherichia coli (STEC) O145 strain.

    Science.gov (United States)

    Pérez, Lucía; Apezteguía, Lucía; Piñeyrúa, Cecilia; Dabezies, Agustín; Bianco, María N; Schelotto, Felipe; Varela, Gustavo

    2014-01-01

    Hemolytic uremic syndrome (HUS) is a disorder characterized by the presence of the classic triad: microangiopathic hemolytic anemia, thrombocytopenia and acute renal injury. HUS without acute renal failure can be confused with other hematologic diseases. An infantile HUS caused by a Shiga-toxin-producing Escherichia coli (STEC) O145 strain carrying genotype stx2, ehxA, eae subtype β1 is herein reported. The infant did not require dialysis during the acute stage of HUS, evolved favorably, maintained normal blood pressure and normal renal function and had no recurrence until the last control. This could be due to several factors, such as the characteristics of infecting STEC strain and a reduction in host susceptibility to renal injury. This report highlights the regional participation of non-O157 STEC in childhood diseases and the importance of performing active surveillance for all forms of HUS. Copyright © 2014 Asociación Colombiana de Psiquiatría. Publicado por Elsevier España. All rights reserved.

  6. Quantification of Shiga toxin 2-encoding bacteriophages, by real-time PCR and correlation with phage infectivity.

    Science.gov (United States)

    Imamovic, L; Serra-Moreno, R; Jofre, J; Muniesa, M

    2010-03-01

    To evaluate a qPCR-based protocol for the enumeration of Shiga toxin (Stx) 2 phages and to compare the results of qPCR with the number of infective Stx phage particles. An approach based on qPCR was applied to count Stx phages in five phage lysates of known titre. The number of viral particles from each phage lysate was determined by electron microscopy using latex spheres. The infectivity of the Stx phages was evaluated onto three bacterial host strains, by double agar layer assay and plaque blot hybridization. The number of phage particles detected by electron microscopy correlates with the number calculated by qPCR in all the phages assayed. The number of infectious phages was from 1 to 3 log10 units below the numbers obtained by qPCR and electron microscopy. The approach allows accurate quantification of Stx phages with a high recovery. The number of infectious phages is always below the number of phage particles detected by qPCR. The qPCR method is a good approach to enumerate Stx phages. However, these results should be carefully considered when related to the number of infectious phages for each lysate that could be applied in real samples, because values of infectious particles are always below the number of Stx phages detected by qPCR.

  7. Enteroaggregative Shiga toxin-producing Escherichia coli of serotype O104:H4 in Belgium and Luxembourg

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    K. De Rauw

    2014-09-01

    Full Text Available In 2011, a large outbreak of infections caused by Shiga toxin-producing Escherichia coli (STEC O104:H4 occurred in Germany. This exceptionally virulent strain combined virulence factors of enteroaggregative E. coli (EAggEC and STEC. After the outbreak only a few sporadic cases of infection with this rare serotype were reported, most of which were related to travel to the Middle East or North Africa. Here we describe two cases of enteroaggregative STEC (Agg-STEC O104:H4 infection that occurred in Belgium in 2012 and 2013 respectively. In both cases travel in a Mediterranean country preceded the infection. The first strain was isolated from the stool of a 42-year-old woman presenting bloody diarrhoea, who had travelled to Tunisia the week before. The second case involves a 14-year-old girl who, upon her return from Turkey to Belgium, suffered from an episode of bloody diarrhoea and haemolytic uraemic syndrome. Extended typing of the isolates with pulsed field gel electrophoresis revealed that the strains were closely related, though not exactly the same as the 2011 outbreak strain. This report supports the previously made hypothesis that Agg-STEC has a human reservoir and might be imported by travellers coming from an area where the pathogen is endemic. Furthermore, it emphasizes the concern that these bacteria may cause future outbreaks as evenly virulent O104:H4 isolates seem to be widespread.

  8. Risk of haemolytic uraemic syndrome caused by shiga-toxin-producing Escherichia coli infection in adult women in Japan.

    Science.gov (United States)

    Fujii, J; Mizoue, T; Kita, T; Kishimoto, H; Joh, K; Nakada, Y; Ugajin, S; Naya, Y; Nakamura, T; Tada, Y; Okabe, N; Maruyama, Y; Saitoh, K; Kurozawa, Y

    2016-04-01

    Shiga-toxin-producing Escherichia coli (STEC) infections usually cause haemolytic uraemic syndrome (HUS) equally in male and female children. This study investigated the localization of globotriaosylceramide (Gb3) in human brain and kidney tissues removed from forensic autopsy cases in Japan. A fatal case was used as a positive control in an outbreak of diarrhoeal disease caused by STEC O157:H7 in a kindergarten in Urawa in 1990. Positive immunodetection of Gb3 was significantly more frequent in female than in male distal and collecting renal tubules. To correlate this finding with a clinical outcome, a retrospective analysis of the predictors of renal failure in the 162 patients of two outbreaks in Japan was performed: one in Tochigi in 2002 and the other in Kagawa Prefecture in 2005. This study concludes renal failure, including HUS, was significantly associated with female sex, and the odds ratio was 4·06 compared to male patients in the two outbreaks. From 2006 to 2009 in Japan, the risk factor of HUS associated with STEC infection was analysed. The number of males and females and the proportion of females who developed HUS were calculated by age and year from 2006 to 2009. In 2006, 2007 and 2009 in adults aged >20 years, adult women were significantly more at risk of developing HUS in Japan.

  9. Inhibitory potential of strepthonium A against Shiga toxin production in enterohemorrhagic Escherichia coli (EHEC) strain EDL933.

    Science.gov (United States)

    Cheng, Cheng; Balasubramanian, Srikkanth; Fekete, Agnes; Krischke, Markus; Mueller, Martin J; Hentschel, Ute; Oelschlaeger, Tobias A; Abdelmohsen, Usama Ramadan

    2017-12-01

    The production of shiga toxin (Stx) is a critical step in the establishment and progress of enterohemorrhagic Escherichia coli (EHEC) infections. The possible release of Stx from dead and dying bacteria, and the risk of resistance development have restricted the usage of antibiotics against EHEC. The chlorinated quaternary ammonium compound, strepthonium A, was isolated from the culture of Streptomyces sp. SBT345 that was cultivated from the Mediterranean sponge Agelas oroides. The structure was elucidated and confirmed by spectroscopic analyses including 1D and 2D NMR, ESI-HRMS, as well as ESI-HRMS 2 . Strepthonium A follows Lipinski's rule of five with respect to its molecular weight, CLogP values and the number of hydrogen acceptors and donors. Verotoxin ELISA assay demonstrated that Strepthonium A reduced the Stx production in EHEC strain EDL933 at 80 μM concentration without growth inhibition. This study demonstrates the potential of strepthonium A in restraining the production of Stx in EHEC infections.

  10. Risk factors for Shiga toxin-producing Escherichia coli infections in preadolescent schoolchildren in Buenos Aires, Argentina.

    Science.gov (United States)

    Bentancor, Adriana Beatriz; Ameal, Luis A; Calviño, María F; Martinez, María C; Miccio, Luciano; Degregorio, Osvaldo J

    2012-05-14

    Shiga toxin-producing Escherichia coli (STEC) infections are the leading cause of hemolytic uremic syndrome (HUS). STEC is the most common cause of acute kidney disease, responsible for 20% of renal transplants in Argentina. In 2007, an epidemiological survey was conducted among 883 students from the fifth and sixth years of elementary education in the public schools of San Martin City, Buenos Aires, Argentina. Degree of exposure to the known risk factors previously detected in the region as primary causes of STEC infections was evaluated. Risk factors assessed included consumption of hamburgers, poor personal hygiene, and exposure to various types of drinking and recreational water. The study was designed to evaluate exposure to risk factors for STEC infections among different socioeconomic groups. Ninety-five percent of children surveyed reported consumption of hamburgers. Most of these hamburgers were precooked. Children of high and medium strata attended private swimming-pools, while children from the low stratum attended public pools. Only 30.2% of students washed their hands after going to the toilet and only 43.5% reported hand-washing before eating. Students demonstrated high levels of exposure to identified risk factors for STEC infections. Reduction of these risks will require cultural changes aimed at decreasing morbidity caused by food-borne infections. Institutional framework must provide the necessary resources to implement these changes and emphasize the importance of good personal hygiene. Health education must be implemented to increase food safety awareness of the consumers.

  11. Shiga toxin-producing E. coli (STEC) in swine: prevalence over the finishing period and characteristics of the STEC isolates.

    Science.gov (United States)

    Tseng, M; Fratamico, P M; Bagi, L; Manzinger, D; Funk, J A

    2015-02-01

    This descriptive longitudinal study was conducted to investigate the faecal shedding of Shiga toxin-producing E. coli (STEC) in finishing swine and to characterize the swine STEC isolates that were recovered. Three cohorts of finishing swine (n = 50/cohort; total 150 pigs) were included in the longitudinal study. Individual faecal samples were collected every 2 weeks (8 collections/pig) from the beginning (pig age 10 weeks) to the end (pig age 24 weeks) of the finishing period. STEC isolates were recovered in at least one sample from 65·3% (98/150) of the pigs, and the frequency distribution of first-time STEC detection during the finishing period resembled a point-source outbreak curve. Nineteen O:H serotypes were identified among the STEC isolates. Most STEC isolates (n = 148) belonged to serotype O59:H21 and carried the stx 2e gene. One O49:H21 STEC isolate carried the stx 2e and eae genes. High prevalence rates of STEC during the finishing period were observed, and STEC isolates in various non-O157 serogroups were recovered. These data enhance understanding of swine STEC epidemiology, and future research is needed to confirm whether or not swine STEC are of public health concern.

  12. The immunogenicity of fusion protein linking the carboxyl terminus of the B subunit of Shiga toxin 2 to the B subunit of E. coli heat-labile enterotoxin.

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    Ran, Xue Qin; Wang, Hong Zhen; Liu, Jin Juan; Li, Sheng; Wang, Jia Fu

    2008-02-05

    To augment the immunogenicity of the subunit B of Shiga toxin (Stx2e B) produced by Escherichia coli and protect piglets from edema disease in china, a fusion gene was constructed consisting of Stx2e B genetically linked at the N-terminus of the B subunit of heat-labile enterotoxin (LTB) in a translational fusion. After being induced with IPTG, the expressed fusion protein of Stx2e B-LTB was about 8.8% of total proteins, approximately 13 microg/ml of the bacteria culture. The Stx2e B-LTB fusion protein was found to be nontoxic to Vero cells at the dose higher than 1 microg/ml and to mice less than 100 microg/ml. Antibody titer against the fusion protein Stx2e B-LTB was 1:76,800, much higher than that of the recombinant Stx2e B protein (1:12,800) alone. All of the mice immunized with the Stx2e B-LTB fusion protein survived when challenged with a lethal dose (LD) of Stx2e toxin. The results showed that the poor immunogenicity of Stx2e B was overcome by conjugating the stx2e B to ltB. The immunogenicity of the constructed fusion protein Stx2e B-LTB in the present study was highly qualified to protect animals against Shiga toxin produced from Shiga toxin-producing Escherichia coli (STEC). The fusion protein of Stx2e B-LTB could be a candidate for a vaccine against edema disease and post-weaning diarrhea simultaneously in piglets.

  13. Detection of shiga-toxin producing E. coli (STEC) in leafy greens sold at local retail markets in Alexandria, Egypt.

    Science.gov (United States)

    Khalil, Rowaida K S; Gomaa, Mohamed A E; Khalil, Mahmoud I M

    2015-03-16

    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

  14. Phenotypic and genotypic characteristics of Shiga toxin-producing Escherichia coli isolated from surface waters and sediments in a Canadian urban-agricultural landscape

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    Stephanie eNadya

    2016-04-01

    Full Text Available A hydrophobic grid membrane filtration – Shiga toxin immunoblot method was used to examine the prevalence of Shiga toxin-producing Escherichia coli (STEC in four watersheds located in the Lower Mainland of British Columbia, Canada, a region characterized by rapid urbanization and intensive agricultural activity. STEC were recovered from 21.6, 23.2, 19.5 and 9.2 % of surface water samples collected monthly from five sites in each watershed over a period of one year. Overall prevalence was subject to seasonal variation however, ranging between 13.3 % during fall months and 34.3 % during winter months. STEC were also recovered from 23.8 % of sediment samples collected in one randomly selected site. One hundred distinct STEC isolates distributed among 29 definitive and 4 ambiguous or indeterminate serotypes were recovered from water and sediments, including isolates from Canadian priority serogroups O157 (3, O26 (4, O103 (5 and O111 (7. Forty seven isolates were further characterized by analysis of whole genome sequences to detect Shiga toxin gene (stx 1 and stx 2, intimin gene (eaeA allelic variants and acquired virulence factors. These analyses collectively showed that surface waters from the region support highly diverse STEC populations that include strains with virulence factors commonly associated with human pathotypes. The present work served to characterize the microbiological hazard implied by STEC to support future assessments of risks to public health arising from non-agricultural and agricultural uses of surface water resources in the region.

  15. Comparative genomic analysis of two novel sporadic Shiga toxin-producing Escherichia coli O104:H4 strains isolated 2011 in Germany.

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    Tietze, Erhard; Dabrowski, Piotr Wojciech; Prager, Rita; Radonic, Aleksandar; Fruth, Angelika; Auraß, Philipp; Nitsche, Andreas; Mielke, Martin; Flieger, Antje

    2015-01-01

    A large outbreak of gastrointestinal disease occurred in 2011 in Germany which resulted in almost 4000 patients with acute gastroenteritis or hemorrhagic colitis, 855 cases of a hemolytic uremic syndrome and 53 deaths. The pathogen was an uncommon, multiresistant Escherichia coli strain of serotype O104:H4 which expressed a Shiga toxin characteristic of enterohemorrhagic E. coli and in addition virulence factors common to enteroaggregative E. coli. During post-epidemic surveillance of Shiga toxin-producing E. coli (STEC) all but two of O104:H4 isolates were indistinguishable from the epidemic strain. Here we describe two novel STEC O104:H4 strains isolated in close spatiotemporal proximity to the outbreak which show a virulence gene panel, a Shiga toxin-mediated cytotoxicity towards Vero cells and aggregative adherence to Hep-2 cells comparable to the outbreak strain. They differ however both from the epidemic strain and from each other, by their antibiotic resistance phenotypes and some other features as determined by routine epidemiological subtyping methods. Whole genome sequencing of these two strains, of ten outbreak strain isolates originating from different time points of the outbreak and of one historical sporadic EHEC O104:H4 isolate was performed. Sequence analysis revealed a clear phylogenetic distance between the two variant strains and the outbreak strain finally identifying them as epidemiologically unrelated isolates from sporadic cases. These findings add to the knowledge about this emerging pathogen, illustrating a certain diversity within the bacterial core genome as well as loss and gain of accessory elements. Our results do also support the view that distinct new variants of STEC O104:H4 repeatedly might originate from yet unknown reservoirs, rather than that there would be a continuous diversification of a single epidemic strain established and circulating in Germany after the large outbreak in 2011.

  16. Conditional Function of Autoaggregative Protein Cah and Common cah Mutations in Shiga Toxin-Producing Escherichia coli.

    Science.gov (United States)

    Carter, Michelle Qiu; Brandl, Maria T; Kudva, Indira T; Katani, Robab; Moreau, Matthew R; Kapur, Vivek

    2018-01-01

    Cah is a calcium-binding autotransporter protein involved in autoaggregation and biofilm formation. Although cah is widespread in Shiga toxin-producing Escherichia coli (STEC), we detected mutations in cah at a frequency of 31.3% in this pathogen. In STEC O157:H7 supershedder strain SS17, a large deletion results in a smaller coding sequence, encoding a protein lacking the C-terminal 71 amino acids compared with Cah in STEC O157:H7 strain EDL933. We examined the function of Cah in biofilm formation and host colonization to better understand the selective pressures for cah mutations. EDL933-Cah played a conditional role in biofilm formation in vitro : it enhanced E. coli DH5α biofilm formation on glass surfaces under agitated culture conditions that prevented autoaggregation but inhibited biofilm formation under hydrostatic conditions that facilitated autoaggregation. This function appeared to be strain dependent since Cah-mediated biofilm formation was diminished when an EDL933 cah gene was expressed in SS17. Deletion of cah in EDL933 enhanced bacterial attachment to spinach leaves and altered the adherence pattern of EDL933 to bovine recto-anal junction squamous epithelial (RSE) cells. In contrast, in trans expression of EDL933 cah in SS17 increased its attachment to leaf surfaces, and in DH5α, it enhanced its adherence to RSE cells. Hence, the ecological function of Cah appears to be modulated by environmental conditions and other bacterial strain-specific properties. Considering the prevalence of cah in STEC and its role in attachment and biofilm formation, cah mutations might be selected in ecological niches in which inactivation of Cah would result in an increased fitness in STEC during colonization of plants or animal hosts. IMPORTANCE Shiga toxin-producing Escherichia coli (STEC) harbors genes encoding diverse adhesins, and many of these are known to play an important role in bacterial attachment and host colonization. We demonstrated here that the

  17. Comparison of Escherichia coli Isolates from humans, food, and farm and companion animals for presence of Shiga toxin-producing E. coli virulence markers.

    Science.gov (United States)

    Murinda, Shelton E; Nguyen, Lien T; Landers, Tippi L; Draughon, F Ann; Mathew, Alan G; Hogan, Joseph S; Smith, K Larry; Hancock, Dale D; Oliver, Stephen P

    2004-01-01

    The objective of this study was to characterize Escherichia coli isolates from dairy cows/feedlots, calves, mastitis, pigs, dogs, parrot, iguana, human disease, and food products for prevalence of Shiga toxin-producing E. coli (STEC) virulence markers. The rationale of the study was that, isolates of the same serotypes that were obtained from different sources and possessed the same marker profiles, could be cross-species transmissible. Multiplex polymerase chain reaction (PCR) was used to detect presence of genes encoding Shiga toxin 1 and 2 (stx1 and stx2), H7 flagella (flicC), enterohemolysin (hly) and intimin (eaeA) in E. coli isolates (n = 400). Shiga toxin-producing isolates were tested for production of Shiga toxins (Stx1 and Stx2 and enterohemolysin. Of the E. coli O157:H7/H- strains, 150 of 164 (mostly human, cattle, and food) isolates were stx+. Sixty-five percent of O157 STEC produced both Stx1 and Stx2; 32% and 0.7% produced Stx2 or Stx1, respectively. Ninety-eight percent of O157 STEC had sequences for genes encoding intimin and enterohemolysin. Five of 20 E. coli O111, 4 of 14 O128 and 4 of 10 O26 were stx+ . Five of 6 stx+ O26 and O111 produced Stx1, however, stx+ O128 were Stx-negative. Acid resistance (93.3%) and tellurite resistance (87.3%) were common attributes of O157 STEC, whereas, non-O157 stx+ strains exhibited 38.5% and 30.8% of the respective resistances. stx-positive isolates were mostly associated with humans and cattle, whereas, all isolates from mastitis (n = 105), and pigs, dogs, parrot and iguanas (n = 48) were stx-negative. Multiplex PCR was an effective tool for characterizing STEC pathogenic profiles and distinguished STEC O157:H7 from other STEC. Isolates from cattle and human disease shared similar toxigenic profiles, whereas isolates from other disease sources had few characteristics in common with the former isolates. These data suggest interspecies transmissibility of certain serotypes, in particular, STEC O157:H7, between

  18. Outbreak of Shiga toxin-producing Escherichia coli (STEC O157:H7 associated with romaine lettuce consumption, 2011.

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    Rachel B Slayton

    Full Text Available BACKGROUND: Shiga toxin-producing Escherichia coli (STEC O157:H7 is the causal agent for more than 96,000 cases of diarrheal illness and 3,200 infection-attributable hospitalizations annually in the United States. MATERIALS AND METHODS: We defined a confirmed case as a compatible illness in a person with the outbreak strain during 10/07/2011-11/30/2011. Investigation included hypothesis generation, a case-control study utilizing geographically-matched controls, and a case series investigation. Environmental inspections and tracebacks were conducted. RESULTS: We identified 58 cases in 10 states; 67% were hospitalized and 6.4% developed hemolytic uremic syndrome. Any romaine consumption was significantly associated with illness (matched Odds Ratio (mOR = 10.0, 95% Confidence Interval (CI = 2.1-97.0. Grocery Store Chain A salad bar was significantly associated with illness (mOR = 18.9, 95% CI = 4.5-176.8. Two separate traceback investigations for romaine lettuce converged on Farm A. Case series results indicate that cases (64.9% were more likely than the FoodNet population (47% to eat romaine lettuce (p-value = 0.013; 61.3% of cases reported consuming romaine lettuce from the Grocery Store Chain A salad bar. CONCLUSIONS: This multistate outbreak of STEC O157:H7 infections was associated with consumption of romaine lettuce. Traceback analysis determined that a single common lot of romaine lettuce harvested from Farm A was used to supply Grocery Store Chain A and a university campus linked to a case with the outbreak strain. An investigation at Farm A did not identify the source of contamination. Improved ability to trace produce from the growing fields to the point of consumption will allow more timely prevention and control measures to be implemented.

  19. Characterization of enteropathogenic and Shiga toxin-producing Escherichia coli in cattle and deer in a shared agroecosystem

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    Pallavi eSingh

    2015-04-01

    Full Text Available Shiga toxin-producing Escherichia coli (STEC is an important foodborne pathogen. Cattle are suggested to be an important reservoir for STEC; however, these pathogens have also been isolated from other livestock and wildlife. In this study we sought to investigate transmission of STEC, enterohemorrhagic E. coli (EHEC and enteropathogenic E. coli (EPEC between cattle and white-tailed deer in a shared agroecosystem. Cattle feces were collected from 100 animals in a Michigan dairy farm in July 2012, while 163 deer fecal samples were collected during two sampling periods (March and June. The locations of deer fecal pellets were recorded via geographic information system mapping and microsatellite multi-locus genotyping was used to link the fecal samples to individual deer at both time points. Following subculture to sorbitol MacConkey agar and STEC CHROMagar, the pathogens were characterized by serotyping, stx profiling, and PCR-based fingerprinting; multilocus sequence typing (MLST was performed on a subset. STEC and EHEC were cultured from 12% and 16% of cattle, respectively, and EPEC was found in 36%. Deer were significantly less likely to have a pathogen in March versus June where the frequency of STEC, EHEC, and EPEC was 1%, 6% and 22%, respectively. PCR fingerprinting and MLST clustered the cattle- and deer-derived strains together in a phylogenetic tree. Two STEC strains recovered from both animal species shared MLST and fingerprinting profiles, thereby providing evidence of interspecies transmission and highlighting the importance of wildlife species in pathogen shedding dynamics and persistence in the environment and cattle herds.

  20. Life on the outside: role of biofilms in environmental persistence of Shiga-toxin producing Escherichia coli

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    Philippe eVogeleer

    2014-07-01

    Full Text Available Escherichia coli is a heterogeneous species that can be part of the normal flora of humans but also include strains of medical importance. Among pathogenic members, Shiga-toxin producing E. coli (STEC are some of the more prominent pathogenic E. coli within the public sphere. STEC disease outbreaks are typically associated with contaminated beef, contaminated drinking water, and contaminated fresh produce. These water- and food-borne pathogens usually colonize cattle asymptomatically; cows will shed STEC in their feces and the subsequent fecal contamination of the environment and processing plants is a major concern for food and public safety. This is especially important because STEC can survive for prolonged periods of time outside its host in environments such as water, produce, and farm soil. Biofilms are hypothesized to be important for survival in the environment especially on produce, in rivers, and in processing plants. Several factors involved in biofilm formation such as curli, cellulose, poly-N-acetyl glucosamine, and colanic acid are involved in plant colonization and adherence to different surfaces often found in meat processing plants. In food processing plants, contamination of beef carcasses occurs at different stages of processing and this is often caused by the formation of STEC biofilms on the surface of several pieces of equipment associated with slaughtering and processing. Biofilms protect bacteria against several challenges, including biocides used in industrial processes. STEC biofilms are less sensitive than planktonic cells to several chemical sanitizers such as quaternary ammonium compounds, peroxyacetic acid, and chlorine compounds. Increased resistance to sanitizers by STEC growing in a biofilm is likely to be a source of contamination in the processing plant. This review focuses on the role of biofilm formation by STEC as a means of persistence outside their animal host and factors associated with biofilm formation.

  1. Shiga toxin-producing Escherichia coli strains isolated from dairy products - Genetic diversity and virulence gene profiles.

    Science.gov (United States)

    Douëllou, T; Delannoy, S; Ganet, S; Mariani-Kurkdjian, P; Fach, P; Loukiadis, E; Montel, Mc; Thevenot-Sergentet, D

    2016-09-02

    Shiga toxin-producing Escherichia coli (STEC) are widely recognized as pathogens causing food borne disease. Here we evaluate the genetic diversity of 197 strains, mainly STEC, from serotypes O157:H7, O26:H11, O103:H2, O111:H8 and O145:28 and compared strains recovered in dairy products against strains from human, meat and environment cases. For this purpose, we characterized a set of reference-collection STEC isolates from dairy products by PFGE DNA fingerprinting and a subset of these by virulence-gene profiling. PFGE profiles of restricted STEC total DNA showed high genomic variability (0.9976 on Simpson's discriminatory index), enabling all dairy isolates to be differentiated. High-throughput real-time PCR screening of STEC virulence genes were applied on the O157:H7 and O26:H11 STEC isolates from dairy products and human cases. The virulence gene profiles of dairy and human STEC strains were similar. Nevertheless, frequency-wise, stx1 was more prevalent among dairy O26:H11 isolates than in human cases ones (87% vs. 44%) while stx2 was more prevalent among O26:H11 human isolates (23% vs. 81%). For O157:H7 isolates, stx1 (0% vs. 39%), nleF (40% vs 94%) and Z6065 (40% vs 100%) were more prevalent among human than dairy strains. Our data point to differences between human and dairy strains but these differences were not sufficient to associate PFGE and virulence gene profiles to a putative lower pathogenicity of dairy strains based on their lower incidence in disease. Further comparison of whole-genome expression and virulence gene profiles should be investigated in cheese and intestinal tract samples. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Shiga Toxin-Producing Escherichia coli in Plateau Pika (Ochotona curzoniae) on the Qinghai-Tibetan Plateau, China.

    Science.gov (United States)

    Bai, Xiangning; Zhang, Wang; Tang, Xinyuan; Xin, Youquan; Xu, Yanmei; Sun, Hui; Luo, Xuelian; Pu, Ji; Xu, Jianguo; Xiong, Yanwen; Lu, Shan

    2016-01-01

    Shiga toxin-producing Escherichia coli (STEC) are an emerging group of zoonotic pathogens. Ruminants are the natural reservoir of STEC. In this study we determined the prevalence and characteristics of the STEC in plateau pika (Ochotona curzoniae) on the Qinghai-Tibetan Plateau, China. A total of 1116 pika samples, including 294 intestinal contents samples, 317 fecal samples, and 505 intestinal contents samples, were collected from May to August in the years 2012, 2013, and 2015, respectively. Twenty-one samples (1.88%) yielded at least one STEC isolate; in total, 22 STEC isolates were recovered. Thirteen different O serogroups and 14 serotypes were identified. One stx 1 subtype (stx 1a) and three stx 2 subtypes (stx 2a, stx 2b, and stx 2d) were present in the STEC isolates. Fifteen, fourteen, and three STEC isolates harbored the virulence genes ehxA, subA, and astA, respectively. Adherence-associated genes iha and saa were, respectively, present in 72.73 and 68.18% of the STEC isolates. Twenty antibiotics were active against all the STEC isolates; all strains were resistant to penicillin G, and some to cephalothin or streptomycin. The 22 STEC isolates were divided into 16 pulsed-field gel electrophoresis patterns and 12 sequence types. Plateau pikas may play a role in the ongoing circulation of STEC in the Qinghai-Tibetan plateau. This study provides the first report on STEC in plateau pikas and new information about STEC reservoirs in wildlife. Based on the serotypes, virulence gene profiles and multi-locus sequence typing (MLST) analysis, the majority of these pika STECs may pose a low public health risk.

  3. Prevalence and characteristics of Shiga toxin-producing Escherichia coli isolated from retail raw meats in China.

    Science.gov (United States)

    Bai, Xiangning; Wang, Hong; Xin, Youquan; Wei, Rongjie; Tang, Xinyuan; Zhao, Ailan; Sun, Hui; Zhang, Wang; Wang, Yan; Xu, Yanmei; Zhang, Zhengdong; Li, Qun; Xu, Jianguo; Xiong, Yanwen

    2015-05-04

    Shiga toxin-producing Escherichia coli (STEC) causes diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome in humans. Most human infections are attributed to consumption of STEC-contaminated foodstuffs of animal origin. In this study, we evaluated the prevalence of STEC from retail raw meats collected from two geographical regions in China. The results revealed that 166 out of 853 samples were stx-positive; 63 STEC isolates were recovered from 58 stx-positive samples including pork (4.4%, 14/318), beef (11.0%, 21/191), mutton (20.6%, 26/126), chicken (0.5%, 1/205), and duck (7.7%, 1/13). Twenty-six O serogroups and 33 O:H serotypes were identified. All three stx1 subtypes and five stx2 subtypes (2a to 2e) were found in the 63 STEC isolates, among which stx2e-positive STEC isolates were the most predominant (39.7%), followed by stx1c only (20.6%), stx1c+stx2b (14.3%), and stx1a only (9.5%). STEC isolates carried virulence genes eae (6.3%), ehxA (36.5%), katP (4.8%), astA (11.1%), and subA (36.5%). Of the four adherence-associated genes tested, toxB was absent, whereas saa, paa, and efa1 were present in 28, three, and one STEC isolates respectively. The STEC isolates were divided into 50 PFGE patterns and 33 sequence types. STEC from different sources and geographical regions were separated by PFGE and MLST. Our results revealed that there is a high genetic diversity of STEC in retail raw meats, some of which have potential to cause human diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Isolation of Shiga Toxin-Producing Escherichia coli from Ground Beef Using Multiple Combinations of Enrichment Broths and Selective Agars.

    Science.gov (United States)

    Brusa, Victoria; Piñeyro, Pablo E; Galli, Lucía; Linares, Luciano H; Ortega, Emanuel E; Padola, Nora L; Leotta, Gerardo A

    2016-03-01

    Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens, and beef cattle are recognized as the principal reservoir. The aims of this study were (1) to identify the most sensitive combination of selective enrichment broths and agars for STEC isolation in artificially inoculated ground beef samples, and (2) to evaluate the most efficient combination(s) of methods for naturally contaminated ground beef samples. A total of 192 ground beef samples were artificially inoculated with STEC and non-stx bacterial strains. A combination of four enrichment broths and three agars were evaluated for sensitivity, specificity, and positive predictive value for STEC isolation from experimentally inoculated samples. Enrichments with either modified tryptic soy broth (mTSB) containing 8 mg/L novobiocin (mTSB-8) or modified Escherichia coli (mEC) broth followed by isolation in MacConkey agar were the most sensitive combinations for STEC isolation of artificially inoculated samples. Independently, both enrichments media followed by isolation in MacConkey were used to evaluate ground beef samples from 43 retail stores, yielding 65.1% and 58.1% stx-positive samples by RT-PCR, respectively. No difference was observed in the isolate proportions between these two methods (8/25 [32%] and 8/28 [28.6%]). Identical serotypes and stx genotypes were observed in STEC strains isolated from the same samples by either method. In this study, no single enrichment protocol was sufficient to detect all STEC in artificially inoculated samples and had considerable variation in detection ability with naturally contaminated samples. Moreover, none of the single or combinations of multiple isolation agars used were capable of identifying all STEC serogroups in either artificially inoculated or naturally occurring STEC-contaminated ground beef. Therefore, it may be prudent to conclude that there is no single method or combination of isolation methods capable of identifying all STEC serogroups.

  5. Salt at concentrations relevant to meat processing enhances Shiga toxin 2 production in Escherichia coli O157:H7.

    Science.gov (United States)

    Harris, Shaun M; Yue, Wan-Fu; Olsen, Sarena A; Hu, Jia; Means, Warrie J; McCormick, Richard J; Du, Min; Zhu, Mei-Jun

    2012-10-15

    Escherichia coli (E. coli) O157:H7 remains a major food safety concern associated with meat, especially beef products. Shiga toxins (Stx) are key virulence factors produced by E. coli O157:H7 that are responsible for hemorrhagic colitis and Hemolytic Uremic Syndrome. Stx are heat stable and can be absorbed after oral ingestion. Despite the extensive study of E. coli O157:H7 survival during meat processing, little attention is paid to the production of Stx during meat processing. The objective of this study was to elucidate the effect of salt, an essential additive to processed meat, at concentrations relevant to meat processing (0%, 1%, 2%, 3%, W/V) on Stx2 production and Stx2 prophage induction by E. coli O157:H7 strains. For both E. coli O157:H7 86-24 and EDL933 strains, including 2% salt in LB broth decreased (Pproduction (as measured relative to Log(10)CFU) compared to that of the control (1% salt). Supplementing 3% salt decreased (Pproduction. Quantitative RT-PCR indicated that stx2 mRNA expression in culture media containing 2% salt was greatly increased (Pproduction and stx2 expression, the 2% salt group had highest lambdoid phage titer and stx2 prophage induction among all salt treatments. RecA is a key mediator of bacterial response to stress, which mediates prophage activation. Quantitative RT-PCR further indicated that recA mRNA expression was higher in both 2% and 3% salt than that of 0% and 1% salt treatments, indicating that stress was involved in enhanced Stx2 production. In conclusion, salt at the concentration used for meat processing enhances Stx production, a process linked to bacterial stress response and lambdoid prophage induction. Published by Elsevier B.V.

  6. Identification of genes expressed in cultures of E. coli lysogens carrying the Shiga toxin-encoding prophage Φ24B

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    Riley Laura M

    2012-03-01

    Full Text Available Abstract Background Shigatoxigenic E. coli are a global and emerging health concern. Shiga toxin, Stx, is encoded on the genome of temperate, lambdoid Stx phages. Genes essential for phage maintenance and replication are encoded on approximately 50% of the genome, while most of the remaining genes are of unknown function nor is it known if these annotated hypothetical genes are even expressed. It is hypothesized that many of the latter have been maintained due to positive selection pressure, and that some, expressed in the lysogen host, have a role in pathogenicity. This study used Change Mediated Antigen Technology (CMAT™ and 2D-PAGE, in combination with RT-qPCR, to identify Stx phage genes that are expressed in E. coli during the lysogenic cycle. Results Lysogen cultures propagated for 5-6 hours produced a high cell density with a low proportion of spontaneous prophage induction events. The expression of 26 phage genes was detected in these cultures by differential 2D-PAGE of expressed proteins and CMAT. Detailed analyses of 10 of these genes revealed that three were unequivocally expressed in the lysogen, two expressed from a known lysogenic cycle promoter and one uncoupled from the phage regulatory network. Conclusion Propagation of a lysogen culture in which no cells at all are undergoing spontaneous lysis is impossible. To overcome this, RT-qPCR was used to determine gene expression profiles associated with the growth phase of lysogens. This enabled the definitive identification of three lambdoid Stx phage genes that are expressed in the lysogen and seven that are expressed during lysis. Conservation of these genes in this phage genome, and other Stx phages where they have been identified as present, indicates their importance in the phage/lysogen life cycle, with possible implications for the biology and pathogenicity of the bacterial host.

  7. Characterization of enteropathogenic and Shiga toxin-producing Escherichia coli in cattle and deer in a shared agroecosystem.

    Science.gov (United States)

    Singh, Pallavi; Sha, Qiong; Lacher, David W; Del Valle, Jacquelyn; Mosci, Rebekah E; Moore, Jennifer A; Scribner, Kim T; Manning, Shannon D

    2015-01-01

    Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen. Cattle are suggested to be an important reservoir for STEC; however, these pathogens have also been isolated from other livestock and wildlife. In this study we sought to investigate transmission of STEC, enterohemorrhagic E. coli (EHEC) and enteropathogenic E. coli (EPEC) between cattle and white-tailed deer in a shared agroecosystem. Cattle feces were collected from 100 animals in a Michigan dairy farm in July 2012, while 163 deer fecal samples were collected during two sampling periods (March and June). The locations of deer fecal pellets were recorded via geographic information system mapping and microsatellite multi-locus genotyping was used to link the fecal samples to individual deer at both time points. Following subculture to sorbitol MacConkey agar and STEC CHROMagar, the pathogens were characterized by serotyping, stx profiling, and PCR-based fingerprinting; multilocus sequence typing (MLST) was performed on a subset. STEC and EHEC were cultured from 12 to 16% of cattle, respectively, and EPEC was found in 36%. Deer were significantly less likely to have a pathogen in March vs. June where the frequency of STEC, EHEC, and EPEC was 1, 6, and 22%, respectively. PCR fingerprinting and MLST clustered the cattle- and deer-derived strains together in a phylogenetic tree. Two STEC strains recovered from both animal species shared MLST and fingerprinting profiles, thereby providing evidence of interspecies transmission and highlighting the importance of wildlife species in pathogen shedding dynamics and persistence in the environment and cattle herds.

  8. Shiga toxin and lipopolysaccharide induce platelet-leukocyte aggregates and tissue factor release, a thrombotic mechanism in hemolytic uremic syndrome.

    Directory of Open Access Journals (Sweden)

    Anne-lie Ståhl

    Full Text Available BACKGROUND: Aggregates formed between leukocytes and platelets in the circulation lead to release of tissue factor (TF-bearing microparticles contributing to a prothrombotic state. As enterohemorrhagic Escherichia coli (EHEC may cause hemolytic uremic syndrome (HUS, in which microthrombi cause tissue damage, this study investigated whether the interaction between blood cells and EHEC virulence factors Shiga toxin (Stx and lipopolysaccharide (LPS led to release of TF. METHODOLOGY/PRINCIPAL FINDINGS: The interaction between Stx or LPS and blood cells induced platelet-leukocyte aggregate formation and tissue factor (TF release, as detected by flow cytometry in whole blood. O157LPS was more potent than other LPS serotypes. Aggregates formed mainly between monocytes and platelets and less so between neutrophils and platelets. Stimulated blood cells in complex expressed activation markers, and microparticles were released. Microparticles originated mainly from platelets and monocytes and expressed TF. TF-expressing microparticles, and functional TF in plasma, increased when blood cells were simultaneously exposed to the EHEC virulence factors and high shear stress. Stx and LPS in combination had a more pronounced effect on platelet-monocyte aggregate formation, and TF expression on these aggregates, than each virulence factor alone. Whole blood and plasma from HUS patients (n = 4 were analyzed. All patients had an increase in leukocyte-platelet aggregates, mainly between monocytes and platelets, on which TF was expressed during the acute phase of disease. Patients also exhibited an increase in microparticles, mainly originating from platelets and monocytes, bearing surface-bound TF, and functional TF was detected in their plasma. Blood cell aggregates, microparticles, and TF decreased upon recovery. CONCLUSIONS/SIGNIFICANCE: By triggering TF release in the circulation, Stx and LPS can induce a prothrombotic state contributing to the pathogenesis of

  9. Prevalence of Shiga toxin-producing Escherichia coli in food products of animal origin as determined by molecular methods.

    Science.gov (United States)

    Rantsiou, Kalliopi; Alessandria, Valentina; Cocolin, Luca

    2012-03-01

    In this study we report on the prevalence and distribution of Shiga toxin-producing Escherichia coli (STEC) in food products of animal origin, collected in the Piedmont region of Italy, as determined by a combination of quantitative PCR (qPCR) protocols, applied directly to the samples, and of culture-dependent isolation and subsequent molecular identification and characterization of isolates. The qPCR protocols were developed and optimized in this study and targeted the rpoB gene (as a marker for total E. coli) and the stx₁, stx₂ and eaeA genes (as markers for potentially virulent E.coli). They were then used to test for STEC in 101 food samples, before and after enrichment. A STEC prevalence of 42% (21/50) for dairy products and 70% (36/51) for meat products was obtained. A total of 54 STEC isolates were recovered from dairy and meat samples, resulting in a prevalence of 36% and 27% in dairy and meat products, respectively, by the culture method. A large number of strains carried the stx₂ gene (39 out of the 54 STEC strains) compared to strains that carried stx₁ (30 out of 54); only 11 out of 54 strains contained the eaeA gene, while 14 strains contained both stx₁ and stx₂. Eight of the 54 isolates belonged to the O157 serogroup, and none belonged to serogroups O26, O145, O111 or O103. Strains isolated from meat products were diverse, as determined by Enterobacterial repetitive intergenic consensus-PCR (ERIC), while those isolated from dairy products were more similar and grouped together by cluster analysis. The results of the qPCR approach showed a high prevalence of STEC in dairy and meat based products, mainly fermented, indicating a possible safety risk for these types of food commodities. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. Mouldy feed, mycotoxins and Shiga toxin - producing Escherichia coli colonization associated with Jejunal Hemorrhage Syndrome in beef cattle

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    Masson Luke

    2011-06-01

    Full Text Available Abstract Background Both O157 and non-O157 Shiga toxin - producing Escherichia coli (STECs cause serious human disease outbreaks through the consumption of contaminated foods. Cattle are considered the main reservoir but it is unclear how STECs affect mature animals. Neonatal calves are the susceptible age class for STEC infections causing severe enteritis. In an earlier study, we determined that mycotoxins and STECs were part of the disease complex for dairy cattle with Jejunal Hemorrhage Syndrome (JHS. For STECs to play a role in the development of JHS, we hypothesized that STEC colonization should also be evident in beef cattle with JHS. Aggressive medical and surgical therapies are effective for JHS, but rely on early recognition of clinical signs for optimal outcomes suggesting that novel approaches must be developed for managing this disease. The main objective of this study was to confirm that mouldy feeds, mycotoxins and STEC colonization were associated with the development of JHS in beef cattle. Results Beef cattle developed JHS after consuming feed containing several types of mycotoxigenic fungi including Fusarium poae, F. verticillioides, F. sporotrichioides, Penicillium roqueforti and Aspergillus fumigatus. Mixtures of STECs colonized the mucosa in the hemorrhaged tissues of the cattle and no other pathogen was identified. The STECs expressed Stx1 and Stx2, but more significantly, Stxs were also present in the blood collected from the lumen of the hemorrhaged jejunum. Feed extracts containing mycotoxins were toxic to enterocytes and 0.1% of a prebiotic, Celmanax Trademark, removed the cytotoxicity in vitro. The inclusion of a prebiotic in the care program for symptomatic beef calves was associated with 69% recovery. Conclusions The current study confirmed that STECs and mycotoxins are part of the disease complex for JHS in beef cattle. Mycotoxigenic fungi are only relevant in that they produce the mycotoxins deposited in the feed. A

  11. Colonization of Beef Cattle by Shiga Toxin-Producing Escherichia coli during the First Year of Life: A Cohort Study.

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    Raies A Mir

    Full Text Available Each year Shiga toxin-producing Escherichia coli (STEC are responsible for 2.8 million acute illnesses around the world and > 250,000 cases in the US. Lowering the prevalence of this pathogen in animal reservoirs has the potential to reduce STEC outbreaks in humans by controlling its entrance into the food chain. However, factors that modulate the colonization and persistence of STEC in beef cattle remain largely unidentified. This study evaluated if animal physiological factors such as age, breed, sex, and weight gain influenced the shedding of STEC in beef cattle. A cohort of beef calves (n = 260 from a multi-breed beef calf population was sampled every three months after birth to measure prevalence and concentration of STEC during the first year of life. Metagenomic analysis was also used to understand the association between the STEC colonization and the composition of gut microflora. This study identified that beef calves were more likely to shed STEC during the first 6 months and that STEC shedding decreased as the animal matured. Animal breed group, sex of the calf, and average weight gain were not significantly associated with STEC colonization. The metagenomic analysis revealed for the first time that STEC colonization was correlated with a lower diversity of gut microflora, which increases as the cattle matured. Given these findings, intervention strategies that segregate younger animals, more likely to be colonized by STEC from older animals that are ready to be harvested, could be investigated as a method to reduce zoonotic transmission of STEC from cattle to humans.

  12. Occurrence, genotyping, shiga toxin genes and associated risk factors of E. coli isolated from dairy farms, handlers and milk consumers.

    Science.gov (United States)

    Awadallah, M A; Ahmed, H A; Merwad, A M; Selim, M A

    2016-11-01

    The objectives of the current study were to determine the occurrence and genotypes of E. coli in dairy farms, workers and milk consumers and to evaluate risk factors associated with contamination of milk in dairy farms. Molecular characterization of shiga toxin associated genes and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) finger printing of E. coli from different sources were also studied. Paired milk samples and rectal swabs from 125 dairy cows, rectal swabs from 82 calves and hand swabs from 45 dairy workers from five dairy farms were collected. In addition, 100 stool samples from 70 diarrheic and 30 healthy humans were collected and examined for the presence of E. coli. E. coli was isolated from milk (22.4%), dairy cattle feces (33.6%), calf feces (35.4%), dairy worker hand swabs (11.1%) and stools of milk consumers (2%, from diarrheic patients only). Only stx1 was identified in seven of 12 E. coli O125 isolated from different sources. High genetic diversity was determined (Simpson's index of diversity, D = 1) and E. coli O125 isolates were classified into 12 distinct profiles, E1-E12. The dendrogram analysis showed that two main clusters were generated. Mastitis in dairy cows was considered a risk factor associated with contamination of the produced milk with E. coli. The isolation of E. coli from rectal swabs of dairy cows and calves poses a zoonotic risk through consumption of unpasteurized contaminated dairy milk. Educational awareness should be developed to address risks related to consumption of raw milk. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Shiga toxin-converting phages and the emergence of new pathogenic Escherichia coli: a world in motion

    Science.gov (United States)

    Tozzoli, Rosangela; Grande, Laura; Michelacci, Valeria; Ranieri, Paola; Maugliani, Antonella; Caprioli, Alfredo; Morabito, Stefano

    2014-01-01

    Shiga toxin (Stx)-producing Escherichia coli (STEC) are pathogenic E. coli causing diarrhea, hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). STEC are characterized by a constellation of virulence factors additional to Stx and have long been regarded as capable to cause HC and HUS when possessing the ability of inducing the attaching and effacing (A/E) lesion to the enterocyte, although strains isolated from such severe infections sometimes lack this virulence feature. Interestingly, the capability to cause the A/E lesion is shared with another E. coli pathogroup, the Enteropathogenic E. coli (EPEC). In the very recent times, a different type of STEC broke the scene causing a shift in the paradigm for HUS-associated STEC. In 2011, a STEC O104:H4 caused a large outbreak with more than 800 HUS and 50 deaths. Such a strain presented the adhesion determinants of Enteroaggregative E. coli (EAggEC). We investigated the possibility that, besides STEC and EAggEC, other pathogenic E. coli could be susceptible to infection with stx-phages. A panel of stx2-phages obtained from STEC isolated from human disease was used to infect experimentally E. coli strains representing all the known pathogenic types, including both diarrheagenic E. coli (DEC) and extra-intestinal pathogenic E. coli (ExPEC). We observed that all the E. coli pathogroups used in the infection experiments were susceptible to the infection. Our results suggest that the stx2-phages used may not have specificity for E. coli adapted to the intestinal environment, at least in the conditions used. Additionally, we could only observe transient lysogens suggesting that the event of stable stx2-phage acquisition occurs rarely. PMID:24999453

  14. Fate of Shiga toxin-producing and generic Escherichia coli during production and ripening of semihard raw milk cheese.

    Science.gov (United States)

    Peng, S; Hoffmann, W; Bockelmann, W; Hummerjohann, J; Stephan, R; Hammer, P

    2013-02-01

    The fate of 5 different Escherichia coli strains, including 3 Shiga toxin-producing E. coli (STEC) strains, was analyzed during the production and ripening of semihard raw milk cheese. The strains, which were previously isolated from raw milk cheese, were spiked into raw milk before cheese production at 2 different levels (approximately 10(1) and 10(3) cfu/mL, respectively). Two cheese types were produced, which differed in cooking temperatures (40 and 46°C). The cheeses were sampled during manufacture and the 16-wk ripening period. An increase in E. coli counts of approximately 3.5 log(10) cfu/g occurred from raw milk to fresh cheese at d 1, which was attributed to a concentration effect during cheese production and growth of the strains. During ripening over 16 wk, a slow, continuous decrease was observed for all strains. However, significant differences were found between the E. coli strains at the applied spiking levels, whereas the inactivation was similar in the 2 different cheese types. The 2 generic E. coli strains survived at higher counts than did the 3 STEC strains. Nevertheless, only 1 of the 3 STEC strains showed significantly weaker survival at both spiking levels and in both cheese types. Six of 16 cheeses made from raw milk at a low spiking level contained more than 10 cfu/g of STEC at the end of the 16-wk ripening process. After enrichment, STEC were detected in almost all cheeses at both spiking levels. Particularly because of the low infectious dose of highly pathogenic STEC, even low colony counts in raw milk cheese are a matter of concern. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  15. Simple method for Shiga toxin 2e purification by affinity chromatography via binding to the divinyl sulfone group.

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    Hideyuki Arimitsu

    Full Text Available Here we describe a simple affinity purification method for Shiga toxin 2e (Stx2e, a major causative factor of edema disease in swine. Escherichia coli strain MV1184 transformed with the expression plasmid pBSK-Stx2e produced Stx2e when cultivated in CAYE broth containing lincomycin. Stx2e bound to commercial D-galactose gel, containing α-D-galactose immobilized on agarose resin via a divinyl sulfone linker, and was eluted with phosphate-buffered saline containing 4.5 M MgCl2. A small amount of Stx2e bound to another commercial α-galactose-immobilized agarose resin, but not to β-galactose-immobilized resin. In addition, Stx2e bound to thiophilic adsorbent resin containing β-mercaptoethanol immobilized on agarose resin via a divinyl sulfone, and was purified in the same manner as from D-galactose gel, but the Stx2e sample contained some contamination. These results indicate that Stx2e bound to D-galactose gel mainly through the divinyl sulfone group on the resin and to a lesser extent through α-D-galactose. With these methods, the yields of Stx2e and attenuated mutant Stx2e (mStx2e from 1 L of culture were approximately 36 mg and 27.7 mg, respectively, and the binding capacity of the D-galactose gel and thiophilic adsorbent resin for Stx2e was at least 20 mg per 1 ml of resin. In addition, using chimeric toxins with prototype Stx2 which did not bind to thiophilic adsorbent resin and some types of mutant Stx2e and Stx2 which contained inserted mutations in the B subunits, we found that, at the least, asparagine (amino acid 17 of the B subunits was associated with Stx2e binding to the divinyl sulfone group. The mStx2e that was isolated exhibited vaccine effects in ICR mice, indicating that these methods are beneficial for large-scale preparation of Stx2e toxoid, which protects swine from edema disease.

  16. Decrease in Shiga toxin expression using a minimal inhibitory concentration of rifampicin followed by bactericidal gentamicin treatment enhances survival of Escherichia coli O157:H7-infected BALB/c mice

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    Abdelnoor Alexander M

    2011-09-01

    Full Text Available Abstract Background Treatment of Escherichia coli O157:H7 infections with antimicrobial agents is controversial due to an association with potentially fatal sequelae. The production of Shiga toxins is believed to be central to the pathogenesis of this organism. Therefore, decreasing the expression of these toxins prior to bacterial eradication may provide a safer course of therapy. Methods The utility of decreasing Shiga toxin gene expression in E. coli O157:H7 with rifampicin prior to bacterial eradication with gentamicin was evaluated in vitro using real-time reverse-transcription polymerase chain reaction. Toxin release from treated bacterial cells was assayed for with reverse passive latex agglutination. The effect of this treatment on the survival of E. coli O157:H7-infected BALB/c mice was also monitored. Results Transcription of Shiga toxin-encoding genes was considerably decreased as an effect of treating E. coli O157:H7 in vitro with the minimum inhibitory concentration (MIC of rifampicin followed by the minimum bactericidal concentration (MBC of gentamicin (> 99% decrease compared to treatment with gentamicin alone (50-75% decrease. The release of Shiga toxins from E. coli O157:H7 incubated with the MIC of rifampicin followed by addition of the MBC of gentamicin was decreased as well. On the other hand, the highest survival rate in BALB/c mice infected with E. coli O157:H7 was observed in those treated with the in vivo MIC equivalent dose of rifampicin followed by the in vivo MBC equivalent dose of gentamicin compared to mice treated with gentamicin or rifampicin alone. Conclusions The use of non-lethal expression-inhibitory doses of antimicrobial agents prior to bactericidal ones in treating E. coli O157:H7 infection is effective and may be potentially useful in human infections with this agent in addition to other Shiga toxin producing E. coli strains.

  17. Application of horse-radish peroxidase linked chemiluminescence to determine the production mechanism of Shiga-like toxins by E. coli O157:H7

    Science.gov (United States)

    Tu, Shu-I.; Uknalis, Joseph; Gehring, Andrew; He, Yiping

    2007-09-01

    A sandwiched immunoassay consisting of toxin capture by immunomagnetic beads (IMB) and toxin detection by horseradish peroxidase (HRP) linked chemiluminescence was used to follow the production of Shiga-like toxins (SLT) by E. coli O157:H7. The intensity of luminescence generated by the oxidation of luminol-liked compounds was used to represent the concentration of toxins produced. The time-course of SLT production by E. coli O157:H7 under different conditions was investigated. In pure culture, optimal generation of SLT showed a significant delay than the steady state of cell growth. In mixed cultures of SLT producing E. coli O157:H7 and non-SLT producing E. coli K-12 strain, the production of toxins was substantially decreased. However, the growth of E. coli O157:H7 was not affected by the presence of K-12 strain. This decrease in SLT production was also observed in radiation-sterile ground beef. In regular ground beef that might contain numerous other bacteria, the growth of E. coli O157:H7 in EC media was not significantly affected but the lowered production of SLT was observed. The results showed that mechanism of inducing SLT production was complex with both the growth time and growth environment could influence SLT production. The addition of homo-serine lactone to the growth media enhanced the production of SLT. Thus, possibly cell-cell communication may have a role in SLT production by E. coli O157:H7.

  18. IDENTIFIKASI ESCHERICHIA COLI O157:H7 SERTA DETEKSI GEN SHIGA LIKE TOXIN 1 DAN 2 ASAL FESES HEWAN, DAGING, DAN FESES MANUSIA

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    I Wayan Suardana

    2010-12-01

    Full Text Available Escherichia coli O157:H7 with the ability to produce shiga-like toxin was isolated from beef, cattle, chicken, and human feces. Due to its importance to human health, it is necessary to identify the genes encoding the production of shiga-like toxin, stx1 and stx2 respectively to further understand the pathogenesis. Isolation of E. coli was done on Eosin Methylene Blue Agar (EMBA, followed by identification on Sorbitol MacConkey Agar (SMAC, latex agglutination test, and H7 antiserum test, respectivelly. The existence of genes stx1 and stx2 in E. coli O157:H7 was confirmed molecularly using PCR method with specific primers LP 30/31 and LP 43/44, Stx2 (F/Stx2 (R respectively. Escherichia coli O157:H7 was isolated from 22 out of 344 samples (6,4%. Some isolates showed gene stx1 and stx2 was detected in two isolates as indicated by a 384 bp band (stx1 gene, 584 bp and 1588 bp bands (stx2 gene respectivelly. The results indicated that local isolates E. coli O157:H7 are potential as a zoonoses agent.

  19. Identifikasi Escherichia coli O157:H7 serta Deteksi Gen Shiga Like Toxin 1 dan 2 Asal Feses Hewan, Daging, dan Feses Manusia

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    I Wayan Suardana

    2016-04-01

    Full Text Available Escherichia coli O157:H7 with the ability to produce shiga-like toxin was isolated from beef, cattle,chicken, and human feces. Due to its importance to human health, it is necessary to identify the genesencoding the production of shiga-like toxin, stx1 and stx2 respectively to further understand the pathogenesis.Isolation of E. coli was done on Eosin Methylene Blue Agar (EMBA, followed by identification on SorbitolMacConkey Agar (SMAC, latex agglutination test, and H7 antiserum test, respectivelly. The existence ofgenes stx1 and stx2 in E. coli O157:H7 was confirmed molecularly using PCR method with specific primersLP 30/31 and LP 43/44, Stx2 (F/Stx2 (R respectively. Escherichia coli O157:H7 was isolated from 22 outof 344 samples (6,4%. Some isolates showed gene stx1 and stx2 was detected in two isolates as indicatedby a 384 bp band (stx1 gene, 584 bp and 1588 bp bands (stx2 gene respectivelly. The results indicatedthat local isolates E. coli O157:H7 are potential as a zoonoses agent.

  20. A secretome view of colonisation factors in Shiga toxin-encoding Escherichia coli (STEC): from enterohaemorrhagic E. coli (EHEC) to related enteropathotypes.

    Science.gov (United States)

    Monteiro, Ricardo; Ageorges, Valentin; Rojas-Lopez, Maricarmen; Schmidt, Herbert; Weiss, Agnes; Bertin, Yolande; Forano, Evelyne; Jubelin, Grégory; Henderson, Ian R; Livrelli, Valérie; Gobert, Alain P; Rosini, Roberto; Soriani, Marco; Desvaux, Mickaël

    2016-08-01

    Shiga toxin-encoding Escherichia coli (STEC) regroup strains that carry genes encoding Shiga toxin (Stx). Among intestinal pathogenic E. coli, enterohaemorrhagic E. coli (EHEC) constitute the major subgroup of virulent STEC. EHEC cause serious human disease such as haemorrhagic colitis and haemolytic-uremic syndrome. While EHEC have evolved from enteropathogenic E. coli, hybrids with enteroaggregative E. coli have recently emerged. Of note, some enteroinvasive E. coli also belong to the STEC group. While the LEE (locus of enterocyte effacement) is a key and prominent molecular determinant in the pathogenicity, neither all EHEC nor STEC contain the LEE, suggesting that they possess additional virulence and colonisation factors. Currently, nine protein secretion systems have been described in diderm-lipopolysaccharide bacteria (archetypal Gram-negative) and can be involved in the secretion of extracellular effectors, cell-surface proteins or assembly of cell-surface organelles, such as flagella or pili. In this review, we focus on the secretome of STEC and related enteropathotypes, which are relevant to the colonisation of biotic and abiotic surfaces. Considering the wealth of potential protein trafficking mechanisms, the different combinations of colonisation factors and modulation of their expression is further emphasised with regard to the ecophysiology of STEC. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  1. The inhibitory effect of colloidal bismuth hydroxide gel on Escherichia coli O157:H7 and on the activity of Shiga toxins.

    Science.gov (United States)

    Subils, Tomás; Casabonne, Cecilia; Balagué, Claudia

    2014-12-04

    Shiga toxin-producing Escherichia coli (STEC) is the causative agent of hemolytic uremic syndrome (HUS). Colloidal bismuth hydroxide gel (CBHG) is an anti-diarrheal and antisecretory compound, which does not inhibit gastrointestinal motility and reaches an in vivo gut concentration of 10.8 mg/ml of bismuth. Its action on bacteria has not been studied. We analyzed its inhibitory effects on STEC, as well as the deactivation of the Shiga toxin (Stx) and its ability to block the spread of genes encoding Stx. We determined a minimum inhibitory concentration and bactericidal concentration for the STEC O157:H7 strain (EDL933), with CBHG and Chobet® bismuth cream with pectin (CBCHP). We analyzed its effect on Stx by means of cytotoxicity assay and ELISA, as well as its effect on the free 933 W Stx phage. Effect on the EDL933 strain: CBHG: MIC 10 mg/ml of bismuth. MIC 6 mg/ml and MBC 15 mg/ml of bismuth. Effect on EDL933 virulence factors: significant decrease in active Stx and 933 W Stx phage titer. ELISA did not find significant differences with treatment. The results obtained may be useful in the development of new therapeutic strategies based on the use of CBHG to prevent or improve the prognosis of HUS, as it can be used to control STEC infections.

  2. Growth of Escherichia coli O157:H7, Non-O157 Shiga Toxin-Producing Escherichia coli , and Salmonella in Water and Hydroponic Fertilizer Solutions.

    Science.gov (United States)

    Shaw, Angela; Helterbran, Kara; Evans, Michael R; Currey, Christopher

    2016-12-01

    The desire for local, fresh produce year round is driving the growth of hydroponic growing systems in the United States. Many food crops, such as leafy greens and culinary herbs, grown within hydroponics systems have their root systems submerged in recirculating nutrient-dense fertilizer solutions from planting through harvest. If a foodborne pathogen were introduced into this water system, the risk of contamination to the entire crop would be high. Hence, this study was designed to determine whether Escherichia coli O157:H7, non-O157 Shiga toxin-producing E. coli , and Salmonella were able to survive and reproduce in two common hydroponic fertilizer solutions and in water or whether the bacteria would be killed or suppressed by the fertilizer solutions. All the pathogens grew by 1 to 6 log CFU/ml over a 24-h period, depending on the solution. E. coli O157:H7 reached higher levels in the fertilizer solution with plants (3.12 log CFU/ml), whereas non-O157 Shiga toxin-producing E. coli and Salmonella reached higher levels in the fertilizer solution without plants (1.36 to 3.77 log CFU/ml). The foodborne pathogens evaluated here survived for 24 h in the fertilizer solution, and populations grew more rapidly in these solutions than in plain water. Therefore, human pathogens entering the fertilizer solution tanks in hydroponic systems would be expected to rapidly propagate and spread throughout the system and potentially contaminate the entire crop.

  3. The effect of regions of interest and spectral pre-processing on the detection of non-O157 shiga-toxin producing escherichia coli serogroups on agar media by hyperspectral imaging

    Science.gov (United States)

    Food borne infection caused by Shiga toxin-producing Escherichia coli (STEC) is a major worldwide health concern. The best known STEC serotype is E. coli O157:H7, which can be easily identified when cultured on sorbitol-MacConkey (SMAC) agar. Recently, six non-O157 STEC serotypes have been found t...

  4. Immunoconcentration of Shiga toxin-producing Escherichia coli O157 from animal faeces and raw meats by using Dynabeads anti-E. coli O157 and the VIDAS system

    NARCIS (Netherlands)

    Islam, M.A.; Heuvelink, A.E.; Talukder, K.A.; Boer, de E.

    2006-01-01

    To identify the reservoirs and routes of transmission of Shiga toxin-producing Escherichia coli (STEC) O157, sensitive detection and isolation methods are necessary. The sensitivity of traditional culture methods can be improved significantly by the inclusion of an immunoconcentration step,

  5. Investigation of prevalence of free Shiga toxin-producing Escherichia coli (STEC)-specific bacteriophages and the correlation with STEC bacterial hosts in produce-growing area in Salinas, California

    Science.gov (United States)

    Shiga toxin-producing E. coli (STEC) strains, commensal to gastrointestinal tracts of ruminants or other animals, have been associated with serious human illnesses. Due to the natural habitats of STEC, the bacteriophages infectious against these bacteria are commonly isolated from fecal-contaminated...

  6. Isolation of bacteriophages against non-O157 and O157 Shiga toxin-producing Escherichia coli (STEC) from composting of non-fecal materials and the potential impact on produce safety

    Science.gov (United States)

    Composting is a complex process to produce fertilizers used to improve crop yields. A complete composting process usually confers bactericidal effect due to change of temperature and pH However, some produce outbreaks associated with Shiga toxin-producing E. coli (STEC) contamination were linked to ...

  7. Evaluation of the performance of the IQ-check kits and the USDA microbiology laboratory guidebook methods for detection of Shiga Toxin-Producing E. coli (STEC) and STEC and Salmonella simultaneously in ground beef

    Science.gov (United States)

    Aims: To evaluate the performance of the IQ-Check kits and the USDA Microbiology Laboratory Guidebook (MLG) methods for detection of the top 7 Shiga toxin-producing E. coli (STEC) (O157:H7, O26, O45, O103, O111, O121, and O145) in ground beef and both STEC and Salmonella in co-inoculated samples. M...

  8. Latex agglutination assays for detection and of non-O157 Shiga toxin-producing E. coli serogroups O26, O45, O103, O111, O121 and O145

    Science.gov (United States)

    Latex agglutination assays were developed for the top six non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups utilizing polyclonal antibodies. Rabbit antisera were affinity purified through Protein A/G columns and the isolated immunoglobulins (IgG) were covalently immobilized onto pol...

  9. Shiga toxin 2 subtypes of enterohemorrhagic E. coli O157:H- E32511 analyzed by RT-qPCR and top-down proteomics using MALDI-TOF-TOF-MS

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    We have measured the relative abundance of the B-subunits and mRNA transcripts of two Stx2 subtypes present in Shiga toxin-producing Escherichia coli (STEC) O157:H- strain E32511 using matrix-assisted laser desorption/ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-M...

  10. Shiga toxin-producing Escherichia coli in yaks (Bos grunniens from the Qinghai-Tibetan Plateau, China.

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    Xiangning Bai

    Full Text Available Shiga toxin (Stx-producing Escherichia coli (STEC are recognized as important human pathogens of public health concern. Many animals are the sources of STEC. In this study we determined the occurrence and characteristics of the STEC in yaks (Bos grunniens from the Qinghai-Tibetan plateau, China. A total of 728 yak fecal samples was collected from June to August, 2012 and was screened for the presence of the stx 1 and stx 2 genes by TaqMan real-time PCR after the sample was enriched in modified Tryptone Soya Broth. Of the 138 (18.96% stx 1 and/or stx 2-positive samples, 85 (61.59% were confirmed to have at least 1 STEC isolate present by culture isolation, from which 128 STEC isolates were recovered. All STEC isolates were serotyped, genotyped by pulsed-field gel electrophoresis (PFGE and characterized for the presence of 16 known virulence factors. Fifteen different O serogroups and 36 different O:H serotypes were identified in the 128 STEC isolates with 21 and 4 untypable for the O and H antigens respectively. One stx 1 subtype (stx 1a and 5 stx 2 subtypes (stx 2a, stx 2b, stx 2c, stx 2d and stx 2g were present in these STEC isolates. Apart from lpfA O157/OI-141, lpfA O157/OI-154, lpfA O113, katP and toxB which were all absent, other virulence factors screened (eaeA, iha, efa1, saa, paa, cnf1, cnf2, astA, subA, exhA and espP were variably present in the 128 STEC isolates. PFGE were successful for all except 5 isolates and separated them into 67 different PFGE patterns. For the 18 serotypes with 2 or more isolates, isolates of the same serotypes had the same or closely related PFGE patterns, demonstrating clonality of these serotypes. This study was the first report on occurrence and characteristics of STEC isolated from yaks (Bos grunniens from the Qinghai-Tibetan plateau, China, and extended the genetic diversity and reservoir host range of STEC.

  11. Molecular and Phylogenetic Characterization of Non-O157 Shiga Toxin-Producing Escherichia coli Strains in China

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    Xiangning Bai

    2016-11-01

    Full Text Available 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

  12. Colonization of Enteroaggregative Escherichia coli and Shiga toxin-producing Escherichia coli in chickens and humans in southern Vietnam.

    Science.gov (United States)

    Trung, Nguyen Vinh; Nhung, Hoang Ngoc; Carrique-Mas, Juan J; Mai, Ho Huynh; Tuyen, Ha Thanh; Campbell, James; Nhung, Nguyen Thi; Van Minh, Pham; Wagenaar, Jaap A; Mai, Nguyen Thi Nhu; Hieu, Thai Quoc; Schultsz, Constance; Hoa, Ngo Thi

    2016-09-09

    Enteroaggregative (EAEC) and Shiga-toxin producing Escherichia coli (STEC) are a major cause of diarrhea worldwide. E. coli carrying both virulence factors characteristic for EAEC and STEC and producing extended-spectrum beta-lactamase caused severe and protracted disease during an outbreak of E. coli O104:H4 in Europe in 2011. We assessed the opportunities for E. coli carrying the aggR and stx genes to emerge in 'backyard' farms in south-east Asia. Faecal samples collected from 204 chicken farms; 204 farmers and 306 age- and gender-matched individuals not exposed to poultry farming were plated on MacConkey agar plates with and without antimicrobials being supplemented. Sweep samples obtained from MacConkey agar plates without supplemented antimicrobials were screened by multiplex PCR for the detection of the stx1, stx2 and aggR genes. One chicken farm sample each (0.5 %) contained the stx1 and the aggR gene. Eleven (2.4 %) human faecal samples contained the stx1 gene, 2 samples (0.4 %) contained stx2 gene, and 31 (6.8 %) contained the aggR gene. From 46 PCR-positive samples, 205 E. coli isolates were tested for the presence of stx1, stx2, aggR, wzx O104 and fliC H4 genes. None of the isolates simultaneously contained the four genetic markers associated with E. coli O104:H4 epidemic strain (aggR, stx2, wzx O104 and fliC H4 ). Of 34 EAEC, 64.7 % were resistant to 3(rd)-generation cephalosporins. These results indicate that in southern Vietnam, the human population is a more likely reservoir of aggR and stx gene carrying E. coli than the chicken population. However, conditions for transmission of isolates and/or genes between human and animal reservoirs resulting in the emergence of highly virulent E. coli strains are still favorable, given the nature of'backyard' farms in Vietnam.

  13. Prevalence and characteristics of Shiga toxin-producing Escherichia coli in finishing pigs: Implications on public health.

    Science.gov (United States)

    Cha, Wonhee; Fratamico, Pina M; Ruth, Leah E; Bowman, Andrew S; Nolting, Jacqueline M; Manning, Shannon D; Funk, Julie A

    2018-01-02

    Shiga toxin-producing Escherichia coli (STEC) are important food-borne pathogens, which can cause serious illnesses, including hemorrhagic colitis and hemolytic uremic syndrome. To study the epidemiology of STEC in finishing pigs and examine the potential risks they pose for human STEC infections, we conducted a longitudinal cohort study in three finishing sites. Six cohorts of pigs (2 cohorts/site, 20 pigs/cohort) were randomly selected, and fecal samples (n=898) were collected every two weeks through their finishing period. Eighty-two pigs (68.3%) shed STEC at least once, and the proportion of STEC-positive pigs varied across sites (50-97.5%) and cohorts (15-100%). Clinically important serotypes, O157:H7 (stx 2c , eae) and O26:H11 (stx 1a , eae), were recovered from two pigs at sites C and A, respectively. The most common serotype isolated was O59:H21 (stx 2e ), which was particularly prevalent in site B as it was recovered from all STEC positive pigs (n=39). Each cohort showed different patterns of STEC shedding, which were associated with the prevalent serotype. The median shedding duration of STEC in pigs was 28days, consistent with our prior study. However, among pigs shedding O59:H21 at least once, pigs in cohort B2 had a significantly longer shedding duration of 42days (P<0.05) compared to other cohorts. Stx2e was the most commonly observed stx variant in finishing pigs (93.9%), in accordance with the previous studies. Stx2e has been reported to be significantly associated with edema disease in pigs, however, the pathogenicity in humans warrants further investigations. Nonetheless, our findings affirm that pigs are an important reservoir for human STEC infections, and that the circulating serotypes in a cohort and site management factors may significantly affect the prevalence of STEC. Molecular characterization of STEC isolates and epidemiological studies to identify risk factors for shedding in pigs are strongly warranted to further address the

  14. Whole genome sequencing for public health surveillance of Shiga Toxin-producing Escherichia coli other than serogroup O157

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    Marie Anne Chattaway

    2016-03-01

    Full Text Available Shiga toxin-producing Escherichia coli (STEC are considered to be a significant threat to public health due to the severity of gastrointestinal symptoms associated with human infection. In England STEC O157 is the most commonly detected STEC serogroup, however, the implementation of PCR at local hospital laboartories has resulted in an increase in the detection of STEC other than serogroup O157 (non-O157 STEC. The aim of this study was to evaluate the use of whole genome sequencing (WGS for routine public health surveillance of non-O157 STEC by comparing this approach to phenotypic serotyping and PCR for subtyping the stx-encoding genes. Of the 102 isolates where phenotypic and genotypic serotyping could be compared, 98 gave fully concordant results. The most common non-O157 STEC serogroups detected were O146 (22 and O26 (18. All but one of the 38 isolates that could not be phenotypically serotyped (designated O unidentifiable or O rough were serotyped using the WGS data. Of the 73 isolates where a flagella type was available by traditional phenotypic typing, all results matched the H-type derived from the WGS data. Of the 140 sequenced non-O157 isolates, 52 (37.1% harboured stx1 only, 42 (30.0% had stx2 only, 46 (32.9% carried stx1 and stx2. Of these, stx subtyping PCR results were available for 131 isolates and 121 of these had concordant results with the stx subtype derived from the WGS data. Non-specific primer binding during PCR amplification, due to the similarity of the stx2 subtype gene sequences was the most likely cause. The results of this study showed WGS provided a reliable and robust one-step process for characterisation of STEC. Deriving the full serotype from WGS data in real time has enabled us to report a higher level of strain discrimination while stx subtyping provides data on the pathogenic potential of each isolate, enabling us to predict clinical outcome of each case and to monitor the emergence of hyper-virulent strains.

  15. Prevalence and characterization of Listeria monocytogenes, Salmonella and Shiga toxin-producing Escherichia coli isolated from small Mexican retail markets of queso fresco.

    Science.gov (United States)

    Soto Beltran, Marcela; Gerba, Charles P; Porto Fett, Anna; Luchansky, John B; Chaidez, Cristobal

    2015-01-01

    Queso fresco (QF) is a handmade cheese consumed and produced in Latin America. In Mexico, QF production is associated with a microbiological risk. The aim of the study was to determine the incidence and characterization of Listeria monocytogenes, Salmonella spp., and Shiga toxin-producing Escherichia coli (STEC) in QF from retail markets of the north-western State of Sinaloa, Mexico, and to assess the effect of physicochemical parameters on Listeria presence. A total of 75 QF samples were obtained. L. monocytogenes, E. coli, and coliforms were detected in 9.3, 94, and 100%, respectively. Salmonella was not detected. STEC isolates showed virulence genes. Microbial loads were above the maximum values recommended by the Official Mexican Standards. Physicochemical parameters such as water activity (aw), moisture content, pH, and salinity played a role in Listeria prevalence in QF. Rigorous control in QF made in Culiacan, Mexico is needed to reduce the risk of foodborne pathogens.

  16. Survey of Shiga toxin-producing Escherichia coli O157:H7 in urban pigeons (Columba livia in the city of Napoli, Italy

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    Alessandro Fioretti

    2010-01-01

    Full Text Available Recently, several studies have demonstrated that pigeon is an important reservoir of Shiga toxin-producing E. coli O157:H7. The aim of this study was to evaluate the presence of this pathogen in urban pigeons in the city of Napoli. The sampling was carried out during the period November 2005/July 2006. The city was subdivided in 56 quadrants by Geographical Information System. Each quadrant was analysed three times. From each quadrant, 3 pigeons were analysed by cloacal swabs. A total of 504 cloacal swabs was obtained. We isolated four E. coli O157:H7 strains. By multiplex PCR, all strains carried eae and stx2 genes, whereas only one strain carried the stx1 gene. 2/4 isolated strains carried hly gene which is considered a hallmark of human pathogenic strains. Our results indicate that pigeon faces are a source of E. coli O157:H7 for birds, mammals and humans.

  17. Characterization of Shiga Toxin-Producing Escherichia coli Isolated from Ground Beef Collected in Different Socioeconomic Strata Markets in Buenos Aires, Argentina

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    Patricia Llorente

    2014-01-01

    Full Text Available Consumption of raw/undercooked ground beef is the most common route of transmission of Shiga toxin-producing E. coli (STEC. The aim of the study was to determine the STEC contamination level of the ground beef samples collected in 36 markets of different socioeconomic strata in Buenos Aires, Argentina, and the characterization of the isolated strains. Ninety-one out of 252 (36.1% samples were stx+. Fifty-seven STEC strains were recovered. Eleven STEC strains belonged to O157 serogroup, and 46 to non-O157 serogroups. Virulence markers of the 57 STEC were stx1, 5.3% (3/57; stx2, 86.0% (49/57; stx1/stx2, 8.8% (5/57; ehxA, 61.4% (35/57; eae, 26.3% (15/57; saa, 24.6% (14/57. Shiga toxin subtypes were stx2, 31.5% (17/54; stx2c-vhb, 24.1% (13/54; stx2c-vha, 20.4% (11/54; stx2/stx2c-vha, 14.8% (8/54; stx2/stx2c-vhb, 5.6% (3/54; stx2c-vha/vhb, 3.7% (2/54. Serotypes O178:H19 and O157:H7 were prevalent. Contamination rate of STEC in all strata was high, and the highest O157 contamination was observed at low strata at several sampling rounds. Persistence of STEC was not detected. Sixteen strains (28.1% were resistant to ampicillin, streptomycin, amikacin, or tetracycline. The STEC contamination level of ground beef could vary according to the sociocultural characteristics of the population.

  18. Prevalence of shiga toxin producing Escherichia coli, Salmonella enterica, and Listeria monocytogenes at public access watershed sites in a California Central Coast agricultural region.

    Science.gov (United States)

    Cooley, Michael B; Quiñones, Beatriz; Oryang, David; Mandrell, Robert E; Gorski, Lisa

    2014-01-01

    Produce contaminated with enteric pathogens is a major source of foodborne illness in the United States. Lakes, streams, rivers, and ponds were sampled with Moore swabs bi-monthly for over 2 years at 30 locations in the vicinity of a leafy green growing region on the Central California Coast and screened for Shiga toxin producing Escherichia coli (STEC), Salmonella enterica, and Listeria monocytogenes to evaluate the prevalence and persistence of pathogen subtypes. The prevalence of STEC from 1386 samples was 11%; 110 samples (8%) contained E. coli O157:H7 with the highest prevalence occurring close to cattle operations. Non-O157 STEC isolates represented major clinical O-types and 57% contained both shiga toxin types 1 and 2 and intimin. Multiple Locus Variable Number Tandem Repeat Analysis of STEC isolates indicated prevalent strains during the period of study. Notably, Salmonella was present at high levels throughout the sampling region with 65% prevalence in 1405 samples resulting in 996 isolates with slightly lower prevalence in late autumn. There were 2, 8, and 14 sites that were Salmonella-positive over 90, 80, and 70% of the time, respectively. The serotypes identified most often were 6,8:d:-, Typhimurium, and Give. Interestingly, analysis by Pulsed Field Gel Electrophoresis indicated persistence and transport of pulsotypes in the region over several years. In this original study of L. monocytogenes in the region prevalence was 43% of 1405 samples resulting in 635 individual isolates. Over 85% of the isolates belonged to serotype 4b with serotypes 1/2a, 1/2b, 3a, 4d with 4e representing the rest, and there were 12 and 2 sites that were positive over 50 and 80% of the time, respectively. Although surface water is not directly used for irrigation in this region, transport to the produce can occur by other means. This environmental survey assesses initial contamination levels toward an understanding of transport leading to produce recalls or outbreaks.

  19. Diversity of Shiga Toxin-Producing Escherichia coli (STEC) O26:H11 Strains Examined via stx Subtypes and Insertion Sites of Stx and EspK Bacteriophages

    Science.gov (United States)

    Bonanno, Ludivine; Loukiadis, Estelle; Mariani-Kurkdjian, Patricia; Oswald, Eric; Garnier, Lucille; Michel, Valérie

    2015-01-01

    Shiga toxin-producing Escherichia coli (STEC) is a food-borne pathogen that may be responsible for severe human infections. Only a limited number of serotypes, including O26:H11, are involved in the majority of serious cases and outbreaks. The main virulence factors, Shiga toxins (Stx), are encoded by bacteriophages. Seventy-four STEC O26:H11 strains of various origins (including human, dairy, and cattle) were characterized for their stx subtypes and Stx phage chromosomal insertion sites. The majority of food and cattle strains possessed the stx1a subtype, while human strains carried mainly stx1a or stx2a. The wrbA and yehV genes were the main Stx phage insertion sites in STEC O26:H11, followed distantly by yecE and sbcB. Interestingly, the occurrence of Stx phages inserted in the yecE gene was low in dairy strains. In most of the 29 stx-negative E. coli O26:H11 strains also studied here, these bacterial insertion sites were vacant. Multilocus sequence typing of 20 stx-positive or stx-negative E. coli O26:H11 strains showed that they were distributed into two phylogenetic groups defined by sequence type 21 (ST21) and ST29. Finally, an EspK-carrying phage was found inserted in the ssrA gene in the majority of the STEC O26:H11 strains but in only a minority of the stx-negative E. coli O26:H11 strains. The differences in the stx subtypes and Stx phage insertion sites observed in STEC O26:H11 according to their origin might reflect that strains circulating in cattle and foods are clonally distinct from those isolated from human patients. PMID:25819955

  20. Bad Phages in Good Bacteria: Role of the Mysterious orf63 of λ and Shiga Toxin-Converting Φ24B Bacteriophages

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    Aleksandra Dydecka

    2017-08-01

    Full Text Available Lambdoid bacteriophages form a group of viruses that shares a common schema of genome organization and lifecycle. Some of them can play crucial roles in creating the pathogenic profiles of Escherichia coli strains. For example, Shiga toxin-producing E. coli (STEC acquired stx genes, encoding Shiga toxins, via lambdoid prophages (Stx phages. The results obtained so far present the evidence for the relation between the exo-xis region of the phage genome and lambdoid phage development, however molecular mechanisms of activities of the exo-xis genes' products are still unknown. In view of this, we decided to determine the influence of the uncharacterized open reading frame orf63 of the exo-xis region on lambdoid phages development using recombinant prophages, λ and Stx phage Φ24B. We have demonstrated that orf63 codes for a folded protein, thus, it is a functional gene. NMR spectroscopy and analytical gel filtration were used to extend this observation further. From backbone chemical shifts, Orf63 is oligomeric in solution, likely a trimer and consistent with its small size (63 aa., is comprised of two helices, likely intertwined to form the oligomer. We observed that the deletion of phage orf63 does not impair the intracellular lambdoid phage lytic development, however delays the time and decreases the efficiency of prophage induction and in consequence results in increased survival of E. coli during phage lytic development. Additionally, the deletion of phage orf63 negatively influences expression of the major phage genes and open reading frames from the exo-xis region during prophage induction with hydrogen peroxide. We conclude, that lambdoid phage orf63 may have specific functions in the regulation of lambdoid phages development, especially at the stage of the lysis vs. lysogenization decision. Besides, orf63 probably participates in the regulation of the level of expression of essential phage genes and open reading frames from the exo

  1. Escherichia coli O104:H4 Pathogenesis: an Enteroaggregative E. coli/Shiga Toxin-Producing E. coli Explosive Cocktail of High Virulence.

    Science.gov (United States)

    Navarro-Garcia, Fernando

    2014-12-01

    A major outbreak caused by Escherichia coli of serotype O104:H4 spread throughout Europe in 2011. This large outbreak was caused by an unusual strain that is most similar to enteroaggregative E. coli (EAEC) of serotype O104:H4. A significant difference, however, is the presence of a prophage encoding the Shiga toxin, which is characteristic of enterohemorrhagic E. coli (EHEC) strains. This combination of genomic features, associating characteristics from both EAEC and EHEC, represents a new pathotype. The 2011 E. coli O104:H4 outbreak of hemorrhagic diarrhea in Germany is an example of the explosive cocktail of high virulence and resistance that can emerge in this species. A total of 46 deaths, 782 cases of hemolytic-uremic syndrome, and 3,128 cases of acute gastroenteritis were attributed to this new clone of EAEC/EHEC. In addition, recent identification in France of similar O104:H4 clones exhibiting the same virulence factors suggests that the EHEC O104:H4 pathogen has become endemically established in Europe after the end of the outbreak. EAEC strains of serotype O104:H4 contain a large set of virulence-associated genes regulated by the AggR transcription factor. They include, among other factors, the pAA plasmid genes encoding the aggregative adherence fimbriae, which anchor the bacterium to the intestinal mucosa (stacked-brick adherence pattern on epithelial cells). Furthermore, sequencing studies showed that horizontal genetic exchange allowed for the emergence of the highly virulent Shiga toxin-producing EAEC O104:H4 strain that caused the German outbreak. This article discusses the role these virulence factors could have in EAEC/EHEC O104:H4 pathogenesis.

  2. Development of a multiplex PCR assay for detection of Shiga toxin-producing Escherichia coli, enterohemorrhagic E. coli, and enteropathogenic E. coli strains.

    Science.gov (United States)

    Botkin, Douglas J; Galli, Lucía; Sankarapani, Vinoth; Soler, Michael; Rivas, Marta; Torres, Alfredo G

    2012-01-01

    Escherichia coli O157:H7 and other pathogenic E. coli strains are enteric pathogens associated with food safety threats and which remain a significant cause of morbidity and mortality worldwide. In the current study, we investigated whether enterohemorrhagic E. coli (EHEC), Shiga toxin-producing E. coli (STEC), and enteropathogenic E. coli (EPEC) strains can be rapidly and specifically differentiated with multiplex PCR (mPCR) utilizing selected biomarkers associated with each strain's respective virulence genotype. Primers were designed to amplify multiple intimin (eae) and long polar fimbriae (lpfA) variants, the bundle-forming pilus gene bfpA, and the Shiga toxin-encoding genes stx1 and stx2. We demonstrated consistent amplification of genes specific to the prototype EHEC O157:H7 EDL933 (lpfA1-3, lpfA2-2, stx1, stx2, and eae-γ) and EPEC O127:H6 E2348/69 (eae-α, lpfA1-1, and bfpA) strains using the optimized mPCR protocol with purified genomic DNA (gDNA). A screen of gDNA from isolates in a diarrheagenic E. coli collection revealed that the mPCR assay was successful in predicting the correct pathotype of EPEC and EHEC clones grouped in the distinctive phylogenetic disease clusters EPEC1 and EHEC1, and was able to differentiate EHEC1 from EHEC2 clusters. The assay detection threshold was 2 × 10(4) CFU per PCR reaction for EHEC and EPEC. mPCR was also used to screen Argentinean clinical samples from hemolytic uremic syndrome and diarrheal patients, resulting in 91% sensitivity and 84% specificity when compared to established molecular diagnostic procedures. In conclusion, our mPCR methodology permitted differentiation of EPEC, STEC and EHEC strains from other pathogenic E. coli; therefore, the assay becomes an additional tool for rapid diagnosis of these organisms.

  3. Fast detection of both O157 and non-O157 shiga-toxin producing Escherichia coli by real-time optical immunoassay.

    Science.gov (United States)

    Mondani, L; Delannoy, S; Mathey, R; Piat, F; Mercey, T; Slimani, S; Fach, P; Livache, T; Roupioz, Y

    2016-01-01

    Among bacterial pathogens involved in food-illnesses, seven serogroups (O26, O45, O103, O111, O121, O145 and O157) of Shiga-toxin producing Escherichia coli (STEC), are frequently identified. During such outbreak, and due to the perishable property of most foodstuff, the time laps for the identification of contaminated products and pathogens is thus critical to better circumvent their spread. Traditional detection methods using PCR or culture plating are time consuming and may present some limitations. In this study, we present a multiplexed immunoassay for the optical detection of most commonly enterohemorrhagic E. coli serogroups: O26, O45, O103, O111, O121, O145 and O157:H7 in a single device. The use of Surface Plasmon Resonance imaging not only enabled the label-free analysis of the samples but gave results in a real-time manner. A dedicated protocol was set up for the detection of both low contaminating bacterial concentrations of food samples (5 CFU per 25 g) and postenrichment aliquots. By combining one single device for the detection of O157 and non-O157 STEC in a label-free manner, this rapid approach may have an important economic and societal impact. This article presents a simple-to-operate immunoassay for the specific detection of Shiga-toxin producing Escherichia coli (STEC). This approach consists in the on-chip assay detection of viable cells on a specifically designed antibody microarray. By skipping any enrichment step and avoiding the use of labelling agent, this approach based on the Surface Plasmon Resonance imaging of the microarrays turns out to be much faster and more cost effective by comparison with standardized methods. © 2015 The Society for Applied Microbiology.

  4. Variation in the Distribution of Putative Virulence and Colonization Factors in Shiga Toxin-Producing Escherichia coli Isolated from Different Categories of Cattle

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    Analía I. Etcheverría

    2017-04-01

    Full Text Available Shiga toxin-producing Escherichia coli (STEC are pathogens of significant public health concern. Several studies have confirmed that cattle are the main reservoir of STEC in Argentina and other countries. Although Shiga toxins represent the primary virulence factors of STEC, the adherence and colonization of the gut are also important in the pathogenesis of the bacteria. The aim of this study was to analyze and to compare the presence of putative virulence factors codified in plasmid -katP, espP, subA, stcE- and adhesins involved in colonization of cattle -efa1, iha- in 255 native STEC strains isolated from different categories of cattle from different production systems. The most prevalent gene in all strains was espP, and the less prevalent was stcE. katP was highly detected in strains isolated from young and rearing calves (33.3%, while subA was predominant in those isolated from adults (71.21%. Strains from young calves showed the highest percentage of efa1 (72.46%, while iha showed a high distribution in strains from rearing calves and adults (87.04 and 98.48% respectively. It was observed that espP and iha were widely distributed throughout all strains, whereas katP, stcE, and efa1 were more associated with the presence of eae and subA with the eae-negative strains. A great proportion of eae-negative strains were isolated from adults -dairy and grazing farms- and from rearing calves -dairy and feedlot-, while mostly of the eae-positive strains were isolated from dairy young calves. Data exposed indicate a correlation between the category of the animal and the production systems with the presence or absence of several genes implicated in adherence and virulence of STEC.

  5. Inhibition of water absorption and selective damage to human colonic mucosa induced by Shiga toxin-2 are enhanced by Escherichia coli O157:H7 infection.

    Science.gov (United States)

    Albanese, Adriana; Gerhardt, Elizabeth; García, Hugo; Amigo, Natalia; Cataldi, Angel; Zotta, Elsa; Ibarra, Cristina

    2015-05-01

    Shiga toxin-producing Escherichia coli (STEC) strains are responsible for a variety of clinical syndromes including bloody and non-bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). Although multiple serotypes of STEC have been isolated from hemorrhagic colitis cases, E. coli O157:H7 is by far the most prevalent serotype associated with HUS. Shiga toxin is the major virulence factor of E. coli O157:H7 and is responsible for the more severe symptoms of the infection. However, the mechanisms involved in the pathogenesis of diarrhea mediated by Stx2 are not well known. In this study, we have determined the effects of E. coli O157:H7 strain 125/99 wild type (wt) on the human colonic mucosa mounted in an Ussing chamber. In response to 125/99wt, an inhibition of water absorption across human colonic mucosa was observed. Histological sections showed severe necrosis with detachment of the surface epithelium, mononuclear inflammatory infiltrate and loss of goblet cells after 1h of incubation with 125/99wt. These alterations were not observed with the isogenic mutant strain lacking stx2 or with the filter-sterilized culture supernatant from the 125/99wt strain. These results indicate that the cell damages in human colon are induced by Stx2, and that Stx2 production is increased by the interaction with bacterial cells. Identification of host cell-derived factors responsible for increasing Stx2 can lead to new strategies for modulating STEC infections. Copyright © 2015 Elsevier GmbH. All rights reserved.

  6. Dexamethasone rescues neurovascular unit integrity from cell damage caused by systemic administration of shiga toxin 2 and lipopolysaccharide in mice motor cortex.

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    Alipio Pinto

    Full Text Available Shiga toxin 2 (Stx2-producing Escherichia coli (STEC causes hemorrhagic colitis and hemolytic uremic syndrome (HUS that can lead to fatal encephalopathies. Neurological abnormalities may occur before or after the onset of systemic pathological symptoms and motor disorders are frequently observed in affected patients and in studies with animal models. As Stx2 succeeds in crossing the blood-brain barrier (BBB and invading the brain parenchyma, it is highly probable that the observed neurological alterations are based on the possibility that the toxin may trigger the impairment of the neurovascular unit and/or cell damage in the parenchyma. Also, lipopolysaccharide (LPS produced and secreted by enterohemorrhagic Escherichia coli (EHEC may aggravate the deleterious effects of Stx2 in the brain. Therefore, this study aimed to determine (i whether Stx2 affects the neurovascular unit and parenchymal cells, (ii whether the contribution of LPS aggravates these effects, and (iii whether an inflammatory event underlies the pathophysiological mechanisms that lead to the observed injury. The administration of a sub-lethal dose of Stx2 was employed to study in detail the motor cortex obtained from a translational murine model of encephalopathy. In the present paper we report that Stx2 damaged microvasculature, caused astrocyte reaction and neuronal degeneration, and that this was aggravated by LPS. Dexamethasone, an anti-inflammatory, reversed the pathologic effects and proved to be an important drug in the treatment of acute encephalopathies.

  7. VIABILIDADE DE Escherichia coli PRODUTORA DE TOXINA SHIGA (STEC NÃO-O157 EM QUEIJO TIPO MINAS FRESCAL. VIABILITY OF NON-O157 SHIGA TOXIN-PRODUCING Escherichia coli (STEC IN MINAS FRESCAL CHEESE

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    Tammy Priscila Chioda

    2009-07-01

    Full Text Available Escherichia coli, produtora de toxina Shiga (STEC, um patógeno emergente capaz de causar diarreia, colite hemorrágica e síndrome hemolítica urêmica em humanos, representa um grave problema de saúde pública em todo o mundo. O principal reservatório de STEC são os bovinos. STEC são transmitidas aos humanos, principalmente através de alimentos contaminados, destacando-se aqueles de origem bovina como carne, leite e seus derivados. O objetivo deste trabalho foi avaliar a viabilidade de STEC não-O157 em queijo minas frescal preparado com leite artificialmente contaminado com diferentes cepas dessas bactérias. Os queijos foram mantidos a 4°C e analisados no 1º, 2º, 4º, 6º e 10º dias de estocagem. As cepas de STEC mantiveram-se viáveis em 100% (32/32 dos queijos mantidos sob refrigeração por até dez dias. Os resultados mostram que o queijo minas pode ser veículo de transmissão de STEC. Recomenda-se a adoção de métodos higiênicos e sanitários desde a ordenha até o processo de produção do queijo para reduzir a possibilidade de contaminação com STEC.

    PALAVRAS-CHAVES: PCR, queijo minas, segurança alimentar, STEC.

    Shiga toxin-producing Escherichia coli (STEC an emergent foodborne pathogen that cause diarrhea, hemorrhagic colitis and haemolytic uremic syndrome in humans, represents a public health problem all over the world. Cattle are the main source of STEC. STEC are transmitted to humans by contaminated food, mainly those of bovine origin as meat and dairy products. This study aimed evaluates the non-O157 STEC viability of artificially inoculated in the milk used for the Minas Frescal cheese’s production. The cheese was kept at 4°C and analyzed at 1st, 2nd, 4th, 6th and 10th days after its production. 100% (32/32 of the cheese storad under refrigeration during 10 days had been the STEC strains viable. These results show that minas frescal cheese can transmit STEC. The adoption of good

  8. Immune Response in Calves Vaccinated with Type Three Secretion System Antigens and Shiga Toxin 2B Subunit of Escherichia coli O157:H7.

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    Luisina Martorelli

    Full Text Available Ruminants are the primary reservoir of Shiga-toxin producing Escherichia coli (STEC O157:H7 and the main source of infection for humans. The aim of this study was to assess the immunogenic properties of a candidate vaccine consisting on the recombinant proteins of E. coli O157:H7 IntiminC280, the carboxy-terminal fraction of Intimin γ, EspB and the fusion protein between the B subunit of Stx2 and Brucella Lumazine Synthase (BLS(BLS-Stx2B, in Holstein Fresian calves.To accomplish this goal we vaccinated calves with two doses of different vaccine formulations: 2 antigens (IntiminC280, EspB, 3 antigens (IntiminC280, EspB, BLS-Stx2B, BLS-Stx2B alone and a control non-vaccinated group. All antigens were expressed as recombinant proteins in E. coli. Specific IgG titres increased in vaccinated calves and the inclusion of BLS-Stx2B in the formulation seems to have a stimulatory effect on the humoral response to IntiminC280 and EspB after the booster. The neutralizing activity of antibodies against these two antigens was assessed in Red Blood Cell lysis assays and adherence to Hep-2 cells as a correlate of T3SS activity. Both sera from animals vaccinated with 2 or 3 antigens inhibited both virulence properties. Serological response to Stx2 was observed in animals vaccinated only with BLS-Stx2B and with 3 antigens and neutralization of Stx2 cytotoxicity was also observed in both groups. In conclusion, immunization of calves with BLS-Stx2B, IntiminC280 and EspB elicited a potent humoral response able to neutralize Shiga toxin 2 cytotoxity and the T3SS virulence properties in vitro. These results suggest that this formulation is a good candidate vaccine to reduce STEC shedding in cattle and needs to be further assessed in vivo.

  9. Immune Response in Calves Vaccinated with Type Three Secretion System Antigens and Shiga Toxin 2B Subunit of Escherichia coli O157:H7.

    Science.gov (United States)

    Martorelli, Luisina; Garbaccio, Sergio; Vilte, Daniel A; Albanese, Adriana A; Mejías, María P; Palermo, Marina S; Mercado, Elsa C; Ibarra, Cristina E; Cataldi, Angel A

    2017-01-01

    Ruminants are the primary reservoir of Shiga-toxin producing Escherichia coli (STEC) O157:H7 and the main source of infection for humans. The aim of this study was to assess the immunogenic properties of a candidate vaccine consisting on the recombinant proteins of E. coli O157:H7 IntiminC280, the carboxy-terminal fraction of Intimin γ, EspB and the fusion protein between the B subunit of Stx2 and Brucella Lumazine Synthase (BLS)(BLS-Stx2B), in Holstein Fresian calves.To accomplish this goal we vaccinated calves with two doses of different vaccine formulations: 2 antigens (IntiminC280, EspB), 3 antigens (IntiminC280, EspB, BLS-Stx2B), BLS-Stx2B alone and a control non-vaccinated group. All antigens were expressed as recombinant proteins in E. coli. Specific IgG titres increased in vaccinated calves and the inclusion of BLS-Stx2B in the formulation seems to have a stimulatory effect on the humoral response to IntiminC280 and EspB after the booster. The neutralizing activity of antibodies against these two antigens was assessed in Red Blood Cell lysis assays and adherence to Hep-2 cells as a correlate of T3SS activity. Both sera from animals vaccinated with 2 or 3 antigens inhibited both virulence properties. Serological response to Stx2 was observed in animals vaccinated only with BLS-Stx2B and with 3 antigens and neutralization of Stx2 cytotoxicity was also observed in both groups. In conclusion, immunization of calves with BLS-Stx2B, IntiminC280 and EspB elicited a potent humoral response able to neutralize Shiga toxin 2 cytotoxity and the T3SS virulence properties in vitro. These results suggest that this formulation is a good candidate vaccine to reduce STEC shedding in cattle and needs to be further assessed in vivo.

  10. From farm to table: follow-up of Shiga toxin-producing Escherichia coli throughout the pork production chain in Argentina

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    Rocío eColello

    2016-02-01

    Full Text Available Pigs are important reservoirs of Shiga toxin-producing Escherichia coli (STEC. The entrance of these strains into the food chain implies a risk to consumers because of the severity of hemolytic uremic syndrome. This study reports the prevalence and characterization of Shiga toxin-producing Escherichia coli (STEC throughout the pork production chain. From 764 samples, 31 (4.05% were stx positive by PCR screening. At farms, 2.86% of samples were stx positive; at slaughter, 4.08% of carcasses were stx positive and at boning rooms, 6% of samples were stx positive. These percentages decreased in pork meat ready for sale at sales markets (4.59%. From positive samples, 50 isolates could be characterized. At farms 37.5% of the isolates carried stx1/stx2 genes, 37.5% possessed stx2e and 25%, carried only stx2. At slaughter we detected 50% of isolates positive for stx2, 33% for stx2e and 16% for stx1/stx2. At boning rooms 59% of the isolates carried stx1/stx2, 14% stx2e and 5% stx1/stx2/stx2e. At retail markets 66% of isolates were positive for stx2, 17% stx2e and 17% stx1/stx2. For the other virulence factors, ehxA and saa were not detected and eae gene was detected in 12% of the isolates. Concerning putative adhesins, agn43 was detected in 72%, ehaA in 26%, aida in 8% and iha in 6% of isolates. The strains were typed into 14 E. coli O groups (O1, O2, O8, O15, O20, O35, O69, O78, O91, O121, O138, O142, O157, O180 and ten H groups (H9, H10, H16, H21, H26, H29, H30, H32, H45, H46. This study reports the prevalence and characterization of STEC strains through the chain pork suggesting the vertical transmission. STEC contamination originates in the farms and is transferred from pigs to carcasses in the slaughter process and increase in meat pork at boning rooms and sales markets. These results highlight the need to implement an integrated STEC control system based on good management practices on the farm and critical control point systems in the food chain.

  11. Quantification of plasmid DNA reference materials for Shiga toxin-producing Escherichia coli based on UV, HR-ICP-MS and digital PCR.

    Science.gov (United States)

    Liang, Wen; Xu, Li; Sui, Zhiwei; Li, Yan; Li, Lanying; Wen, Yanli; Li, Chunhua; Ren, Shuzhen; Liu, Gang

    2016-01-01

    The accuracy and metrology traceability of DNA quantification is becoming a critical theme in many fields, including diagnosis, forensic analysis, microorganism detection etc. Thus the research of DNA reference materials (RMs) and consistency of DNA quantification methods has attracted considerable research interest. In this work, we developed 3 plasmid candidate RMs, containing 3 target genes of Escherichia coli O157:H7 (E. coli O157:H7) and other Shiga toxin-producing Escherichia coli (STEC): stx1, stx2, and fliC (h7) respectively. Comprehensive investigation of the plasmid RMs was performed for their sequence, purity, homogeneity and stability, and then the concentration was quantified by three different methods: ultraviolet spectrophotometer (UV), high resolution inductively coupled plasma mass spectrometry (HR-ICP-MS) and digital PCR. As a routinely applied method for DNA analysis, UV was utilized for the quantification (OD260) and purity analysis for the plasmids. HR-ICP-MS quantified the plasmid DNA through analysing the phosphorus in DNA molecules. Digital PCR distributed the DNA samples onto a microarray chip containing thousands of reaction chambers, and quantified the DNA copy numbers by analysing the number of positive signals without any calibration curves needed. Based on the high purification of the DNA reference materials and the optimization of dPCR analysis, we successfully achieved good consistency between UV, HR-ICP-MS and dPCR, with relative deviations lower than 10 %. We then performed the co-quantification of 3 DNA RMs with three different methods together, and the uncertainties of their concentration were evaluated. Finally, the certified values and expanded uncertainties for 3 DNA RMs (pFliC, pStx1 and pStx2) were (1.60 ± 0.10) × 10(10) copies/μL, (1.53 ± 0.10) × 10(10) copies/μL and (1.70 ± 0.11) × 10(10) copies/μL respectively.Graphical abstractWe developed 3 plasmid candidate RMs, containing 3 target genes of

  12. Occurrence of virulent and antibiotic-resistant Shiga toxin-producing Escherichia coli in some food products and human stool in Egypt.

    Science.gov (United States)

    Hamed, Osman Mohamed; Sabry, Maha Ahmed; Hassanain, Nawal A; Hamza, Eman; Hegazi, Ahmed G; Salman, Marwa Badawy

    2017-10-01

    Shiga toxin-producing Escherichia coli (STEC) represent a severe public health issue worldwide, causing life-threatening diseases in the human gastrointestinal tract. This study aimed to determine the occurrence of virulent and antibiotic-resistant STEC in retail meat and milk products and human stool samples and to characterize the genes encoding for virulence and antibiotic resistance among the identified STEC isolates. A total of 260 food samples were randomly collected from retail markets in different localities of El Giza Governorate, Egypt. 50 stool specimens were obtained from children that had diarrhea at Embaba Fever Hospital. All collected samples were initially subjected to bacteriological examination and serotyping, and then subsequently, the isolates were exposed to polymerase chain reaction application and sequencing for the identification of the virulence-related genes. Finally, the virulent STEC isolates were tested for antibiotic susceptibility. Serotyping of the 76 biochemically identified isolates showed that 18 were STEC with a predominance of non-O157 (16) while 2 O157:K-serotype was detected only in one food and one human isolate. Molecular identification of the virulence genes illustrated that the minced meat showed the highest prevalence of STEC (8%) as compared to the other food products. In the humans, the O157 was the only serotype that expresses the Shiga toxin-associated gene ( eaeA ). Antibiotic susceptibility test displayed that 13 of the 17 food and human isolates (76.47%) were resistant to cephalothin (KF30). 9 of the 13 cephalothin-resistant isolates harbor the β lactamase ( bla TEM )-resistant gene. All isolates were sensitive to chloramphenicol, ciprofloxacin, amikacin, and gentamicin. DNA sequencing and phylogenetic analysis of the stx2- positive minced meat isolate revealed a high genetic relatedness with beef minced meat from the USA and Australia. This study showed the predominance of non-O157 among the identified isolates

  13. Occurrence of non-O157 Shiga toxin-producing Escherichia coli in dogs with diarrhea Ocorrência de Escherichia coli não-O157 Shigatoxigênica em cachorros com diarréia

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    Cleber Jacob Silva de Paula

    2008-09-01

    Full Text Available Shiga toxigenic Escherichia coli (STEC and Attaching and effacing E. coli (AEEC have been associated with diarrhea illness in dogs. From January to December 2006, 92 E. coli isolates from 25 diarrheic dogs were analyzed, by screening for the presence of Shiga toxin-producing (stx 1 and stx 2 and intimin (eae genes. Twelve isolates were detected by PCR to harbor the Shiga toxin genes (7 the stx 1 (7.6%; 5 the stx 2 (5.4%; and none both of them. Nine (9.8% of the E. coli isolates studied were eae positive non Shiga toxin-producing. Thirteen (62.0% isolates, carrying stx or eae gene, also showed a hemolysin production. The strains with virulence genes were also examined for resistance to 12 antimicrobial agents. Resistances to cephalothin (85.7%, streptomycin (81.0%, amoxicillin (71.4% and gentamicin (71.4% were predominantly observed.Escherichia coli Shiga toxigênica (STEC e E. coli Attaching- effacing (AEEC têm sido associadas à doença diarréica em cachorros. Entre janeiro e dezembro de 2006, 92 cepas de E. coli isoladas de 25 cachorros diarréicos foram examinadas. As cepas foram analisadas para a detecção dos genes produtores de Shiga toxina (stx 1 e stx 2 e da intimina (eae. Por meio de PCR foi observado que sete cepas (7,6% portavam o gene stx 1, cinco cepas (5,4% carregavam o gene stx 2 e nenhum cepa apresentou ambos os genes associados. Nove cepas de E. coli (9,8% apresentaram o gene eae isoladamente. Treze das cepas (62,0% que apresentaram os genes stx ou eae também apresentaram a produção de a hemolisina. As cepas que apresentaram genes de virulência foram também examinadas em relação à resistência a 12 agentes antimicrobianos. As resistências mais comuns foram para cefalotina (85,7%, estreptomicina (81,0%, amoxicilina (71,4% e gentamicina (71,4%.

  14. Seasonal prevalence of potentially positive non-O157 Shiga toxin-producing Escherichia coli (STEC) bovine hides and carcasses in Costa Rica.

    Science.gov (United States)

    Chaves, Byron D; Echeverry, Alejandro; García, Lyda G; Brashears, M Todd; Miller, Markus F; Brashears, Mindy M

    2015-12-01

    The prevalence of potentially positive Shiga toxin-producing Escherichia coli (STEC) bovine hides and carcasses in three abattoirs in Costa Rica was estimated. Two export facilities (A and B) and one non-export establishment (C) were visited during the dry and rainy seasons of 2013. Swabs of hides pre-eviscerated and treated (180-220 peroxyacetic acid spray) carcasses were tested for the potential presence of STEC serogroups O26, O45, O103, O111, O121, and O145. The prevalence on hides during the rainy season was 86.7, 96.7 and 96.7% for facilities A, B, and C, respectively. During the dry season, the prevalence on hides was significantly lower in plants A and B (40% and 26.7%, respectively), but was marginally associated with the season in plant C (76.7%, P=0.0523). The prevalence of non-O157 STEC markers on treated carcasses was low (0 to 3.3%), suggesting that all plants were effective in minimizing the target non-O157 STEC in beef destined for export and for domestic consumption. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Genetic characterization of Shiga toxin-producing Escherichia coli O26:H11 strains isolated from animal, food, and clinical samples

    Science.gov (United States)

    Krüger, Alejandra; Lucchesi, Paula M. A.; Sanso, A. Mariel; Etcheverría, Analía I.; Bustamante, Ana V.; Burgán, Julia; Fernández, Luciana; Fernández, Daniel; Leotta, Gerardo; Friedrich, Alexander W.; Padola, Nora L.; Rossen, John W. A.

    2015-01-01

    The Shiga-toxin producing Escherichia coli (STEC) may cause serious illness in human. Here we analyze O26:H11 strains known to be among the most reported STEC strains causing human infections. Genetic characterization of strains isolated from animal, food, and clinical specimens in Argentina showed that most carried either stx1a or stx2a subtypes. Interestingly, stx2a-positive O26:H11 rarely isolated from cattle in other countries showed to be an important proportion of O26:H11 strains circulating in cattle and food in our region. Seventeen percent of the isolates harbored more than one gene associated with antimicrobial resistance. In addition to stx, all strains contained the virulence genes eae-β, tir, efa, iha, espB, cif, espA, espF, espJ, nleA, nleB, nleC, and iss; and all except one contained ehxA, espP, and cba genes. On the other hand, toxB and espI genes were exclusively observed in stx2-positive isolates, whereas katP was only found in stx1a-positive isolates. Our results show that O26:H11 STEC strains circulating in Argentina, including those isolated from humans, cattle, and meat products, present a high pathogenic potential, and evidence that cattle can be a reservoir of O26:H11 strains harboring stx2a. PMID:26539413

  16. Microbial Safety and Sanitary Quality of Strawberry Primary Production in Belgium: Risk Factors for Salmonella and Shiga Toxin-Producing Escherichia coli Contamination

    Science.gov (United States)

    Delbeke, Stefanie; Ceuppens, Siele; Hessel, Claudia Titze; Castro, Irene; Jacxsens, Liesbeth; De Zutter, Lieven

    2015-01-01

    Strawberries are an important fruit in Belgium in both production and consumption, but little information is available about the presence of Salmonella and Shiga toxin-producing Escherichia coli (STEC) in these berries, the risk factors in agricultural production, and possible specific mitigation options. In 2012, a survey was undertaken of three soil and three soilless cultivation systems in Belgium. No Salmonella spp. were isolated. No STEC was detected in the strawberry samples (0 of 72), but STEC was detected by PCR in 11 of 78 irrigation water and 2 of 24 substrate samples. Culture isolates were obtained for 2 of 11 PCR-positive irrigation water samples and 2 of 2 substrate samples. Multivariable logistic regression analysis revealed elevated generic E. coli numbers (the odds ratio [OR] for a 1 log increase being 4.6) as the most important risk factor for STEC, together with the berry-picking season (elevated risk in summer). The presence of generic E. coli in the irrigation water (≥1 CFU per 100 ml) was mainly influenced by the type of irrigation water (collected rainfall water stored in ponds was more often contaminated than groundwater pumped from boreholes [OR = 5.8]) and the lack of prior treatment (untreated water versus water subjected to sand filtration prior to use [OR = 19.2]). The follow-up study in 2013 at one of the producer locations indicated cattle to be the most likely source of STEC contamination of the irrigation water. PMID:25636845

  17. Effectiveness of the Thermal Treatments Used for Curd Stretching in the Inactivation of Shiga Toxin-Producing O157 and O26 Escherichia coli

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    M. Trevisani

    2017-01-01

    Full Text Available The kneading treatment of the fresh curd in hot water is a critical control point in the manufacturing of mozzarella. Factors such as the ratio between hot water and curd mass, the rheological properties, and the mixing and kneading activity affect the processing time and the internal temperature of the curd. The aim of this study was to investigate the effect of thermal treatments on the fate of Shiga toxin-producing Escherichia coli (STEC. Nine curd samples (weight 160–270 g were artificially contaminated with O157 or O26 STEC and stretched in hot water (90–95°C for 5–10 min. Depending on the heating process and spinning, different nonisothermal profiles were recorded. Observed reductions of O157 and O26 STEC varied between 1.01 and more than 5.38 log⁡MPN (Most Probable Number/g at the end of the temperature treatments. Further, nonisothermal log-linear tail models were developed to compare observed reductions for O157 and O26 VTEC under variable temperature conditions. Results obtained showed that the comparison of predictions provided by the dynamic model with observations described well the linear inactivation pattern since nonsignificant differences were denoted at all profiles tested. The dynamic model developed can be useful to evaluate the effectiveness of the thermal treatments used in the manufacturing of mozzarella in the inactivation of STEC.

  18. Occurrence of non-O157 Shiga toxin-encoding Escherichia coli in artisanal mozzarella cheese in Brazil: risk factor associated with food workers

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    Patricia CARDOSO

    Full Text Available Abstract Mozzarella cheese is considered a safe food due to the high temperature treatment used in the traditional process of stretching into hot water; however, a post-process contamination during the cheese manufacture or during the processing (before distribution could occur. This study investigated the occurrence of Shiga toxin-encoding Escherichia coli (STEC strains in homemade-mozzarella cheese. In total, 59 raw milk cheese samples collected at a local producer in the Jequitinhonha Valley (Northeast of Minas Gerais, Brazil were submitted for microbiological analysis, and 38 (64.4% tested positive for E. coli. Then, 147 strains of E. coli were isolated from positive samples and screened by polymerase chain reaction for the presence of the genes encoding the following virulence factors: stx1 and stx 2 (verotoxin types 1 and 2 and eae (intimin. All the isolates were negative for the stx 2, 14 isolates (9.5% were positive for the stx1 gene, and 11 of them also harbored the eae gene. A food worker was probably involved in cheese contamination during the manufacture schedule. While the development of STEC infection in humans is not completely understood, all STEC- contaminated food can be considered potentially hazardous.

  19. Evaluation of applicability of DNA microarray-based characterization of bovine Shiga toxin-producing Escherichia coli isolates using whole genome sequence analysis.

    Science.gov (United States)

    Barth, Stefanie A; Menge, Christian; Eichhorn, Inga; Semmler, Torsten; Pickard, Derek; Geue, Lutz

    2017-09-01

    We assessed the ability of a commercial DNA microarray to characterize bovine Shiga toxin-producing Escherichia coli (STEC) isolates and evaluated the results using in silico hybridization of the microarray probes within whole genome sequencing scaffolds. From a total of 69,954 reactions (393 probes with 178 isolates), 68,706 (98.2%) gave identical results by DNA microarray and in silico probe hybridization. Results were more congruent when detecting the genoserotype (209 differing results from 19,758 in total; 1.1%) or antimicrobial resistance genes (AMRGs; 141 of 26,878; 0.5%) than when detecting virulence-associated genes (VAGs; 876 of 22,072; 4.0%). Owing to the limited coverage of O-antigens by the microarray, only 37.2% of the isolates could be genoserotyped. However, the microarray proved suitable to rapidly screen bovine STEC strains for the occurrence of high numbers of VAGs and AMRGs and is suitable for molecular surveillance workflows.

  20. Vitamin B12 Uptake by the Gut Commensal Bacteria Bacteroides thetaiotaomicron Limits the Production of Shiga Toxin by Enterohemorrhagic Escherichia coli

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    Charlotte Cordonnier

    2016-01-01

    Full Text Available Enterohemorrhagic Escherichia coli (EHEC are foodborne pathogens responsible for the development of bloody diarrhea and renal failure in humans. Many environmental factors have been shown to regulate the production of Shiga toxin 2 (Stx2, the main virulence factor of EHEC. Among them, soluble factors produced by human gut microbiota and in particular, by the predominant species Bacteroides thetaiotaomicron (B. thetaiotaomicron, inhibit Stx2 gene expression. In this study, we investigated the molecular mechanisms underlying the B. thetaiotaomicron-dependent inhibition of Stx2 production by EHEC. We determined that Stx2-regulating molecules are resistant to heat treatment but do not correspond to propionate and acetate, two short-chain fatty acids produced by B. thetaiotaomicron. Moreover, screening of a B. thetaiotaomicron mutant library identified seven mutants that do not inhibit Stx2 synthesis by EHEC. One mutant has impaired production of BtuB, an outer membrane receptor for vitamin B12. Together with restoration of Stx2 level after vitamin B12 supplementation, these data highlight vitamin B12 as a molecule produced by gut microbiota that modulates production of a key virulence factor of EHEC and consequently may affect the outcome of an infection.

  1. Vitamin B12 Uptake by the Gut Commensal Bacteria Bacteroides thetaiotaomicron Limits the Production of Shiga Toxin by Enterohemorrhagic Escherichia coli.

    Science.gov (United States)

    Cordonnier, Charlotte; Le Bihan, Guillaume; Emond-Rheault, Jean-Guillaume; Garrivier, Annie; Harel, Josée; Jubelin, Grégory

    2016-01-05

    Enterohemorrhagic Escherichia coli (EHEC) are foodborne pathogens responsible for the development of bloody diarrhea and renal failure in humans. Many environmental factors have been shown to regulate the production of Shiga toxin 2 (Stx2), the main virulence factor of EHEC. Among them, soluble factors produced by human gut microbiota and in particular, by the predominant species Bacteroides thetaiotaomicron (B. thetaiotaomicron), inhibit Stx2 gene expression. In this study, we investigated the molecular mechanisms underlying the B. thetaiotaomicron-dependent inhibition of Stx2 production by EHEC. We determined that Stx2-regulating molecules are resistant to heat treatment but do not correspond to propionate and acetate, two short-chain fatty acids produced by B. thetaiotaomicron. Moreover, screening of a B. thetaiotaomicron mutant library identified seven mutants that do not inhibit Stx2 synthesis by EHEC. One mutant has impaired production of BtuB, an outer membrane receptor for vitamin B12. Together with restoration of Stx2 level after vitamin B12 supplementation, these data highlight vitamin B12 as a molecule produced by gut microbiota that modulates production of a key virulence factor of EHEC and consequently may affect the outcome of an infection.

  2. Prevalence of Shiga toxin-producing and enteropathogenic Escherichia coli marker genes in diarrhoeic stools in a New Zealand catchment area.

    Science.gov (United States)

    Thomas, Rowan R; Brooks, Heather J L; O'Brien, Rory

    2017-01-01

    Shiga toxin-producing (STEC) and enteropathogenic (EPEC) Escherichia coli are gastrointestinal pathogens causing diarrhoeal and extraintestinal disease. Due to lack of EPEC screening and use of Sorbitol-MacConkey (SMAC) agar in faecal screening, the true prevalence of EPEC and non-O157 STEC in New Zealand diarrhoeal cases is unknown. Diarrhoeic stools sourced from Dunedin hospital were pre-enriched, DNA extracted with Chelex-100 resin and screened using a multiplex TaqMan quantitative PCR assay amplifying stx1, sxt2 and EPEC (eae) gene markers. Of the 522 diarrhoeic samples surveyed, 8 (1.53%) were PCR positive for stx1/stx2 and 23 (4.41%) were positive for eae. Six (75%) of the stx + samples were uncommon non-O157 serotypes, and the remainder were found to be positive for both O103 and O157 STEC somatic antigens. Results revealed shortcomings in current screening protocols for pathogenic E. coli; SMAC is not sufficiently discriminatory to detect emergent STEC serotypes and EPEC likely has an unappreciated role in cases of diarrhoea in New Zealand. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  3. Real-time genomic investigation underlying the public health response to a Shiga toxin-producing Escherichia coli O26:H11 outbreak in a nursery.

    Science.gov (United States)

    Moran-Gilad, J; Rokney, A; Danino, D; Ferdous, M; Alsana, F; Baum, M; Dukhan, L; Agmon, V; Anuka, E; Valinsky, L; Yishay, R; Grotto, I; Rossen, J W A; Gdalevich, M

    2017-10-01

    Shiga toxin-producing Escherichia coli (STEC) is a significant cause of gastrointestinal infection and the haemolytic-uremic syndrome (HUS). STEC outbreaks are commonly associated with food but animal contact is increasingly being implicated in its transmission. We report an outbreak of STEC affecting young infants at a nursery in a rural community (three HUS cases, one definite case, one probable case, three possible cases and five carriers, based on the combination of clinical, epidemiological and laboratory data) identified using culture-based and molecular techniques. The investigation identified repeated animal contact (animal farming and petting) as a likely source of STEC introduction followed by horizontal transmission. Whole genome sequencing (WGS) was used for real-time investigation of the incident and revealed a unique strain of STEC O26:H11 carrying stx2a and intimin. Following a public health intervention, no additional cases have occurred. This is the first STEC outbreak reported from Israel. WGS proved as a useful tool for rapid laboratory characterization and typing of the outbreak strain and informed the public health response at an early stage of this unusual outbreak.

  4. Risk Factors for Salmonella, Shiga Toxin-Producing Escherichia coli and Campylobacter Occurrence in Primary Production of Leafy Greens and Strawberries

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    Siele Ceuppens

    2015-08-01

    Full Text Available The microbiological sanitary quality and safety of leafy greens and strawberries were assessed in the primary production in Belgium, Brazil, Egypt, Norway and Spain by enumeration of Escherichia coli and detection of Salmonella, Shiga toxin-producing E. coli (STEC and Campylobacter. Water samples were more prone to containing pathogens (54 positives out of 950 analyses than soil (16/1186 and produce on the field (18/977 for leafy greens and 5/402 for strawberries. The prevalence of pathogens also varied markedly according to the sampling region. Flooding of fields increased the risk considerably, with odds ratio (OR 10.9 for Salmonella and 7.0 for STEC. A significant association between elevated numbers of generic E. coli and detection of pathogens (OR of 2.3 for STEC and 2.7 for Salmonella was established. Generic E. coli was found to be a suitable index organism for Salmonella and STEC, but to a lesser extent for Campylobacter. Guidelines on frequency of sampling and threshold values for E. coli in irrigation water may differ from region to region.

  5. DNA fingerprinting of Shiga-toxin producing Escherichia coli O157 based on Multiple-Locus Variable-Number Tandem-Repeats Analysis (MLVA

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    Vardund Traute

    2003-12-01

    Full Text Available Abstract Background The ability to react early to possible outbreaks of Escherichia coli O157:H7 and to trace possible sources relies on the availability of highly discriminatory and reliable techniques. The development of methods that are fast and has the potential for complete automation is needed for this important pathogen. Methods In all 73 isolates of shiga-toxin producing E. coli O157 (STEC were used in this study. The two available fully sequenced STEC genomes were scanned for tandem repeated stretches of DNA, which were evaluated as polymorphic markers for isolate identification. Results The 73 E. coli isolates displayed 47 distinct patterns and the MLVA assay was capable of high discrimination between the E. coli O157 strains. The assay was fast and all the steps can be automated. Conclusion The findings demonstrate a novel high discriminatory molecular typing method for the important pathogen E. coli O157 that is fast, robust and offers many advantages compared to current methods.

  6. Effect of gamma radiation on the reduction of Salmonella strains, Listeria monocytogenes, and Shiga toxin-producing Escherichia coli and sensory evaluation of minimally processed spinach (Tetragonia expansa).

    Science.gov (United States)

    Rezende, Ana Carolina B; Igarashi, Maria Crystina; Destro, Maria Teresa; Franco, Bernadette D G M; Landgraf, Mariza

    2014-10-01

    This study evaluated the effects of irradiation on the reduction of Shiga toxin-producing Escherichia coli (STEC), Salmonella strains, and Listeria monocytogenes, as well as on the sensory characteristics of minimally processed spinach. Spinach samples were inoculated with a cocktail of three strains each of STEC, Salmonella strains, and L. monocytogenes, separately, and were exposed to gamma radiation doses of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 kGy. Samples that were exposed to 0.0, 1.0, and 1.5 kGy and kept under refrigeration (4°C) for 12 days were submitted to sensory analysis. D10 -values ranged from 0.19 to 0.20 kGy for Salmonella and from 0.20 to 0.21 for L. monocytogenes; for STEC, the value was 0.17 kGy. Spinach showed good acceptability, even after exposure to 1.5 kGy. Because gamma radiation reduced the selected pathogens without causing significant changes in the quality of spinach leaves, it may be a useful method to improve safety in the fresh produce industry.

  7. Vitamin B12 Uptake by the Gut Commensal Bacteria Bacteroides thetaiotaomicron Limits the Production of Shiga Toxin by Enterohemorrhagic Escherichia coli

    Science.gov (United States)

    Cordonnier, Charlotte; Le Bihan, Guillaume; Emond-Rheault, Jean-Guillaume; Garrivier, Annie; Harel, Josée; Jubelin, Grégory

    2016-01-01

    Enterohemorrhagic Escherichia coli (EHEC) are foodborne pathogens responsible for the development of bloody diarrhea and renal failure in humans. Many environmental factors have been shown to regulate the production of Shiga toxin 2 (Stx2), the main virulence factor of EHEC. Among them, soluble factors produced by human gut microbiota and in particular, by the predominant species Bacteroides thetaiotaomicron (B. thetaiotaomicron), inhibit Stx2 gene expression. In this study, we investigated the molecular mechanisms underlying the B. thetaiotaomicron-dependent inhibition of Stx2 production by EHEC. We determined that Stx2-regulating molecules are resistant to heat treatment but do not correspond to propionate and acetate, two short-chain fatty acids produced by B. thetaiotaomicron. Moreover, screening of a B. thetaiotaomicron mutant library identified seven mutants that do not inhibit Stx2 synthesis by EHEC. One mutant has impaired production of BtuB, an outer membrane receptor for vitamin B12. Together with restoration of Stx2 level after vitamin B12 supplementation, these data highlight vitamin B12 as a molecule produced by gut microbiota that modulates production of a key virulence factor of EHEC and consequently may affect the outcome of an infection. PMID:26742075

  8. Assessment of Consumer Exposure to Salmonella spp., Campylobacter spp., and Shiga Toxin-Producing Escherichia coli in Meat Products at Retail in the City of Sao Paulo, Brazil.

    Science.gov (United States)

    Ristori, Christiane Asturiano; Rowlands, Ruth Estela Gravato; Martins, Cecília Geraldes; Barbosa, Maria Luisa; Dos Santos, Luis Fernando; Jakabi, Miyoko; de Melo Franco, Bernadette Dora Gombossy

    2017-08-01

    Meat products may be vehicles of bacterial pathogens to humans, and Salmonella spp., Campylobacter spp., and Shiga toxin-producing Escherichia coli (STEC) are the most relevant. The aim of this study was to generate data on prevalence of these three pathogens in 552 samples of meat products (hot dogs, pork sausages, raw ground beef, and raw chicken legs) sold at retail in the city of Sao Paulo, Brazil. Salmonella spp. was detected in 5.8% (32/552) of samples, comprising pork sausages 62.5% (20/32) and chicken legs 37.5% (12/32). The counts of Salmonella spp. were low, ranging from Salmonella Typhimurium (28.1%), Salmonella I 4,[5],12:i:- (15.6%), Salmonella Enteritidis (12.5%), Salmonella Derby, and Salmonella Brandenburg (9.4%). Campylobacter spp. was detected in 33 samples (6.0%), comprising chicken legs (82%) and ground beef (18%). All samples were negative for STEC. These results suggest that meat products when subjected to inadequate cooking and/or cross-contamination with other products ready for consumption can lead to occurrence of outbreaks, highlighting the risks associated with them.

  9. Association of clustered regularly interspaced short palindromic repeat (CRISPR) elements with specific serotypes and virulence potential of shiga toxin-producing Escherichia coli.

    Science.gov (United States)

    Toro, Magaly; Cao, Guojie; Ju, Wenting; Allard, Marc; Barrangou, Rodolphe; Zhao, Shaohua; Brown, Eric; Meng, Jianghong

    2014-02-01

    Shiga toxin-producing Escherichia coli (STEC) strains (n = 194) representing 43 serotypes and E. coli K-12 were examined for clustered regularly interspaced short palindromic repeat (CRISPR) arrays to study genetic relatedness among STEC serotypes. A subset of the strains (n = 81) was further analyzed for subtype I-E cas and virulence genes to determine a possible association of CRISPR elements with potential virulence. Four types of CRISPR arrays were identified. CRISPR1 and CRISPR2 were present in all strains tested; 1 strain also had both CRISPR3 and CRISPR4, whereas 193 strains displayed a short, combined array, CRISPR3-4. A total of 3,353 spacers were identified, representing 528 distinct spacers. The average length of a spacer was 32 bp. Approximately one-half of the spacers (54%) were unique and found mostly in strains of less common serotypes. Overall, CRISPR spacer contents correlated well with STEC serotypes, and identical arrays were shared between strains with the same H type (O26:H11, O103:H11, and O111:H11). There was no association identified between the presence of subtype I-E cas and virulence genes, but the total number of spacers had a negative correlation with potential pathogenicity (P CRISPR-cas system and potential virulence needs to be determined on a broader scale, and the biological link will need to be established.

  10. [Hygienic-sanitary quality in abattoirs from Tucuman province, Argentina. Detection, isolation and characterization of Shiga toxin-producing Escherichia coli].

    Science.gov (United States)

    Pérez Terrazzino, Gabriela B; Condorí, Marina S; López Campo, Alejandro; Vega, Silvia; Carbonari, Carolina; Chinen, Isabel; Rivas, Marta; de Castillo, Marta C; Jure, María A

    Cattle are the main reservoir of Shiga toxin-producing Escherichia coli (STEC), and the strategies to prevent the transmission of these microorganisms are concentrated in the slaughtering plant. The aim of this study was to evaluate the hygienic-sanitary quality and the frequency of detection of STEC in beef carcasses in abattoirs from Tucuman province. Two hundred and seventy four beef carcass sponges were processed; the count of generic E. coli was marginal in 9 (3,3%) of them. Escherichia coli O157 was isolated in 4 (1,4%) samples; 2 of which were characterized as stx 2c(vh-a) /eae/ehxA whereas the other 2 were non-toxigenic strains. Non-O157 E. coli ONT:H49, stx 2a /ehxA/saa was isolated from 1 sample (0,4%). In this work the quality of the analyzed product indicates that the good practices of manufacture are fulfilled in slaughtering facilities in Tucumán province. Copyright © 2017 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  11. Surveillance of Virulence Markers and Antibiotic Resistance of Shiga toxin Producing E.coli O157:H7 Strains from Meats Purchase in Shiraz

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    Mohammad Kargar

    2011-09-01

    Full Text Available Background: Shiga toxin Producing Escherichia coli O157:H7 is a common pathogen in cattle, which occasional causes some human disease. This bacterium can potentially contaminate meat and clinical cases of E.coli O157:H7 infections are often associated with consumption of undercooked ground beef. Methods: In this cross-sectional study 122 samples of ground meat were collected and after enrichment in specific culture media and evaluation sorbitol fermentation and their β-glucoronidase activity, the isolation of E.coli O157:H7 strains have been confirmed with specific antisera. Then virulence genes verotoxin, intimin and hemolysin with multiplex PCR and antibiotic resistance strains with disk diffusion method have been tested. Results: Out of specimens that have been supplied, 119 sorbitol negative colonies isolated which 3 strains O157:H7 (2.45% with specific antisera confirmed. Out of considered virulence genes, in two cases of these samples (1.64% the stx1 and eaeA genes were seen and also 2 isolated bacteria had resistance to erythromycin, tetracycline, ampicillin, penicillin, clindamicin, cefixime, novobiocin, and gentamicin antibiotics. Conclusion: As this organism lives in intestines of healthy cattle, preventive measures on cattle farms and during meat processing are necessary.

  12. Development of 11-Plex MOL-PCR Assay for the Rapid Screening of Samples for Shiga Toxin-Producing Escherichia coli

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    Travis A Woods

    2016-08-01

    Full Text Available Strains of Shiga toxin-producing Escherichia coli (STEC are a serious threat to the public health, with approximately half of the STEC related food-borne illnesses attributable to contaminated beef. We developed an assay that was able to screen samples for several important STEC associated serogroups (O26, O45, O103, O104, O111, O121, O145, O157 and three major virulence factors (eae, stx1, stx2 in a rapid and multiplexed format using the Multiplex oligonucleotide ligation-PCR (MOL-PCR assay chemistry. This assay detected unique STEC DNA signatures and was meant to be used on samples from various sources related to beef production, providing a multiplex and high-throughput complement to the multiplex PCR assays currently in use. Multiplex oligonucleotide ligation-PCR (MOL-PCR is a nucleic acid-based assay chemistry that relies on flow cytometry/image cytometry and multiplex microsphere arrays for the detection of nucleic acid-based signatures present in target agents. The STEC MOL-PCR assay provided greater than 90% analytical specificity across all sequence markers designed when tested against panels of DNA samples that represent different STEC serogroups and toxin gene profiles. This paper describes the development of the 11-plex assay and the results of its validation. This highly multiplexed, but more importantly dynamic and adaptable screening assay allows inclusion of additional signatures as they are identified in relation to public health. As the impact of STEC associated illness on public health is explored additional information on classification will be needed on single samples; thus, this assay can serve as the backbone for a complex screening system.

  13. Development of 11-Plex MOL-PCR Assay for the Rapid Screening of Samples for Shiga Toxin-Producing Escherichia coli.

    Science.gov (United States)

    Woods, Travis A; Mendez, Heather M; Ortega, Sandy; Shi, Xiaorong; Marx, David; Bai, Jianfa; Moxley, Rodney A; Nagaraja, T G; Graves, Steven W; Deshpande, Alina

    2016-01-01

    Strains of Shiga toxin-producing Escherichia coli (STEC) are a serious threat to the health, with approximately half of the STEC related food-borne illnesses attributable to contaminated beef. We developed an assay that was able to screen samples for several important STEC associated serogroups (O26, O45, O103, O104, O111, O121, O145, O157) and three major virulence factors (eae, stx 1 , stx 2) in a rapid and multiplexed format using the Multiplex oligonucleotide ligation-PCR (MOL-PCR) assay chemistry. This assay detected unique STEC DNA signatures and is meant to be used on samples from various sources related to beef production, providing a multiplex and high-throughput complement to the multiplex PCR assays currently in use. Multiplex oligonucleotide ligation-PCR (MOL-PCR) is a nucleic acid-based assay chemistry that relies on flow cytometry/image cytometry and multiplex microsphere arrays for the detection of nucleic acid-based signatures present in target agents. The STEC MOL-PCR assay provided greater than 90% analytical specificity across all sequence markers designed when tested against panels of DNA samples that represent different STEC serogroups and toxin gene profiles. This paper describes the development of the 11-plex assay and the results of its validation. This highly multiplexed, but more importantly dynamic and adaptable screening assay allows inclusion of additional signatures as they are identified in relation to public health. As the impact of STEC associated illness on public health is explored additional information on classification will be needed on single samples; thus, this assay can serve as the backbone for a complex screening system.

  14. Detection of E.Coli Strains Containing Shiga Toxin (Stx1/2 Gene in Diarrheal Specimens from Children Less than 5 Years Old by PCR Technique and Study of the Patterns of Antibiotic Resistance

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    MR Pourmand

    2009-10-01

    Full Text Available Introduction: Shiga toxin- producing Escherichia coli (STEC is an emerging bacterial pathogen in developing countries that causes several diseases such as diarrhea, hemorrhagic colitis (HC and hemolytic uremic syndrome (HUS, particularly in children. Aim of the research was detection of STEC in diarrheal specimens from under 5 year olds and study of the patterns of antibiotic resistance of these strains. Methods: In the study,300 fecal samples were collected from children with diarrhea referring to Ali Asghar Hospital. E.coli species were isolated by standard bacteriological and biochemical tests. Presence of shiga toxin genes (stx1/2 was investigated by PCR technique (Qiagen. Antibiogram test for strains containing the toxin gene was performed using 16 different antibiotic discs (MAST by disc diffusion agar (Kirby-Bauer method. Results: From 39 E.coli isolates, 9(23.1% strains were detected by PCR to contain stx1/2 gene. One strain was resistant to all 16 antibiotics. All the STEC strains were sensitive to meropenem (MRP, imipenem (IMI, gentamycin (GEN and nitrofurantoin (NI. 4(44.44% strains showed multi-drug resistant pattern. All these 4strains were resistant to cotrimoxazole(SxT. Also, 6(66.66% strains were resistant to at least one antibiotic. Conclusion: In Iran, shiga toxin- producing Escherichia coli (STEC may be a commonly bacterial pathogen causing diarrhea, particularly in children. Therefore, we should use new techniques for investigation of these strains. Increase in number of emerging and new strains that could be resistant to classic antibiotics such as cotrimoxazole may be foreseen. It is suggested that antibiotics prescription programs in treatment of diarrhea causing E.coli strains be updated.

  15. Shiga-Like Toxin 2 of Enterohemorrhagic Escherichia Coli (EHEC): Genetic Organization and Effects of Toxin in a Murine Model of EHEC Infection

    Science.gov (United States)

    1990-04-27

    FEBS Lett. 181:377-380. 100. Yanish-Perron, C , J. Viera, and J. Messing. 1985. Improved Ml3 phage cloning vectors: nucleotide sequences of M13 mp18 and pUC 19 vectors. Gene. 33: 103-119. ...aj., 1986). Sensitive bacterial hosts that are lysogenized by such toxin- converting phages acquire the capacity to produce the corresponding toxin...The production of SLT-l and SLT-II has been shown to be determined by specific phages in certain strains of EHEC serotypes isolated from humans

  16. Uremic Hemolitic Syndrome induced by Escherichia Coli producer of Shiga toxin in a pediatric patient with renal transplant

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    Ana Paula Ramírez

    2017-04-01

    Full Text Available El Síndrome Urémico Hemolítico (SUH secundario a la infección por Escherichia coli productora de toxina Shiga (STEC serotipo O157:H7 es una enfermedad endémica en Argentina según la Organización Panamericana de la Salud, siendo en nuestro país, y en los niños la primera causa de Injuria renal aguda (IRA y la segunda causa de Insuficiencia Renal Crónica (IRC. Esta entidad clínica y anátomo-patológica se caracteriza por presentación aguda de daño renal, anemia hemolítica microangiopática y trombocitopenia, que puede afectar otros parénquimas como intestino, sistema nervioso central, páncreas, corazón e hígado. Más del 90% de los pacientes afectados se recuperan en período agudo, aunque se han descrito secuelas renales a largo plazo hasta en el 25% de ellos. Según nuestro conocimiento aún no ha sido descrito en nuestro país casos de síndrome urémico hemolítico secundario a infección por Escherichia coli, productora de STEC luego del Trasplante Renal (TxR.

  17. Comparative Characterization of Shiga Toxin Type 2 and Subtilase Cytotoxin Effects on Human Renal Epithelial and Endothelial Cells Grown in Monolayer and Bilayer Conditions.

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    Romina S Álvarez

    Full Text Available Postdiarrheal hemolytic uremic syndrome (HUS affects children under 5 years old and is responsible for the development of acute and chronic renal failure, particularly in Argentina. This pathology is a complication of Shiga toxin (Stx-producing Escherichia coli infection and renal damage is attributed to Stx types 1 and 2 (Stx1, Stx2 produced by Escherichia coli O157:H7 and many other STEC serotypes. It has been reported the production of Subtilase cytotoxin (SubAB by non-O157 STEC isolated from cases of childhood diarrhea. Therefore, it is proposed that SubAB may contribute to HUS pathogenesis. The human kidney is the most affected organ because very Stx-sensitive cells express high amounts of biologically active receptor. In this study, we investigated the effects of Stx2 and SubAB on primary cultures of human glomerular endothelial cells (HGEC and on a human tubular epithelial cell line (HK-2 in monoculture and coculture conditions. We have established the coculture as a human renal proximal tubule model to study water absorption and cytotoxicity in the presence of Stx2 and SubAB. We obtained and characterized cocultures of HGEC and HK-2. Under basal conditions, HGEC monolayers exhibited the lowest electrical resistance (TEER and the highest water permeability, while the HGEC/HK-2 bilayers showed the highest TEER and the lowest water permeability. In addition, at times as short as 20-30 minutes, Stx2 and SubAB caused the inhibition of water absorption across HK-2 and HGEC monolayers and this effect was not related to a decrease in cell viability. However, toxins did not have inhibitory effects on water movement across HGEC/HK-2 bilayers. After 72 h, Stx2 inhibited the cell viability of HGEC and HK-2 monolayers, but these effects were attenuated in HGEC/HK-2 bilayers. On the other hand, SubAB cytotoxicity shows a tendency to be attenuated by the bilayers. Our data provide evidence about the different effects of these toxins on the bilayers

  18. Toxins

    Science.gov (United States)

    Toxins are substances created by plants and animals that are poisonous to humans. Toxins also include some medicines that are helpful in small doses, but poisonous in large amounts. Most toxins that cause problems ...

  19. Multicenter evaluation of the BD max enteric bacterial panel PCR assay for rapid detection of Salmonella spp., Shigella spp., Campylobacter spp. (C. jejuni and C. coli), and Shiga toxin 1 and 2 genes.

    Science.gov (United States)

    Harrington, S M; Buchan, B W; Doern, C; Fader, R; Ferraro, M J; Pillai, D R; Rychert, J; Doyle, L; Lainesse, A; Karchmer, T; Mortensen, J E

    2015-05-01

    Diarrhea due to enteric bacterial pathogens causes significant morbidity and mortality in the United States and worldwide. However, bacterial pathogens may be infrequently identified. Currently, culture and enzyme immunoassays (EIAs) are the primary methods used by clinical laboratories to detect enteric bacterial pathogens. We conducted a multicenter evaluation of the BD Max enteric bacterial panel (EBP) PCR assay in comparison to culture for the detection of Salmonella spp., Shigella spp., Campylobacter jejuni, and Campylobacter coli and an EIA for Shiga toxins 1 and 2. A total of 4,242 preserved or unpreserved stool specimens, including 3,457 specimens collected prospectively and 785 frozen, retrospective samples, were evaluated. Compared to culture or EIA, the positive percent agreement (PPA) and negative percent agreement (NPA) values for the BD Max EBP assay for all specimens combined were as follows: 97.1% and 99.2% for Salmonella spp., 99.1% and 99.7% for Shigella spp., 97.2% and 98.4% for C. jejuni and C. coli, and 97.4% and 99.3% for Shiga toxins, respectively. Discrepant results for prospective samples were resolved with alternate PCR assays and bidirectional sequencing of amplicons. Following discrepant analysis, PPA and NPA values were as follows: 97.3% and 99.8% for Salmonella spp., 99.2% and 100% for Shigella spp., 97.5% and 99.0% for C. jejuni and C. coli, and 100% and 99.7% for Shiga toxins, respectively. No differences in detection were observed for samples preserved in Cary-Blair medium and unpreserved samples. In this large, multicenter study, the BD Max EBP assay showed superior sensitivity compared to conventional methods and excellent specificity for the detection of enteric bacterial pathogens in stool specimens. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  20. Multiplex real-time PCR assay for detection of Escherichia coli O157:H7 and screening for non-O157 Shiga toxin-producing E. coli.

    Science.gov (United States)

    Li, Baoguang; Liu, Huanli; Wang, Weimin

    2017-11-09

    Shiga toxin-producing Escherichia coli (STEC), including E. coli O157:H7, are responsible for numerous foodborne outbreaks annually worldwide. E. coli O157:H7, as well as pathogenic non-O157:H7 STECs, can cause life-threating complications, such as bloody diarrhea (hemolytic colitis) and hemolytic-uremic syndrome (HUS). Previously, we developed a real-time PCR assay to detect E. coli O157:H7 in foods by targeting a unique putative fimbriae protein Z3276. To extend the detection spectrum of the assay, we report a multiplex real-time PCR assay to specifically detect E. coli O157:H7 and screen for non-O157 STEC by targeting Z3276 and Shiga toxin genes (stx1 and stx2). Also, an internal amplification control (IAC) was incorporated into the assay to monitor the amplification efficiency. The multiplex real-time PCR assay was developed using the Life Technology ABI 7500 System platform and the standard chemistry. The optimal amplification mixture of the assay contains 12.5 μl of 2 × Universal Master Mix (Life Technology), 200 nM forward and reverse primers, appropriate concentrations of four probes [(Z3276 (80 nM), stx1 (80 nM), stx2 (20 nM), and IAC (40 nM)], 2 μl of template DNA, and water (to make up to 25 μl in total volume). The amplification conditions of the assay were set as follows: activation of TaqMan at 95 °C for 10 min, then 40 cycles of denaturation at 95 °C for 10 s and annealing/extension at 60 °C for 60 s. The multiplex assay was optimized for amplification conditions. The limit of detection (LOD) for the multiplex assay was determined to be 200 fg of bacterial DNA, which is equivalent to 40 CFU per reaction which is similar to the LOD generated in single targeted PCRs. Inclusivity and exclusivity determinants were performed with 196 bacterial strains. All E. coli O157:H7 (n = 135) were detected as positive and all STEC strains (n = 33) were positive for stx1, or stx2, or stx1 and stx2 (Table 1). No cross reactivity was detected with Salmonella

  1. Persistence and reduction of Shiga toxin-producing Escherichia coli serotype O26:H11 in different types of raw fermented sausages.

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    Böhnlein, Christina; Kabisch, Jan; Müller-Herbst, Stefanie; Fiedler, Gregor; Franz, Charles M A P; Pichner, Rohtraud

    2017-11-16

    Fermented sausages have been identified as source of several outbreaks of Shiga toxin-producing Escherichia coli (STEC). Illnesses linked to non-O157 STEC serotypes appear to be on the rise worldwide, and serogroup O26 is the second most reported in Europe after O157. However, data on the behavior of serogroup O26 in food are rare, so that the aim of this study was to investigate the survival of STEC O26:H11 in different types of fermented sausages ("Teewurst", fast-ripened and long-fermented salami). Challenge studies were performed with an inoculation cocktail which consisted of three STEC O26:H11 strains isolated from human, cattle and food sources. In the short-ripened spreadable sausage type "Teewurst" STEC counts decreased by only 0.5 log 10 within 28days. In contrast, STEC reductions from 2.2 to 2.6 log 10 units were observed in the different salami products, while the most pronounced decrease of 1.0 log 10 unit within one day was detected in fast-ripened sausages with glucono delta-lactone (GdL). Moreover, numbers of the food-associated E. coli O26:H11 strain were significantly higher (psausages. Approximately 60% of all STEC isolates from GdL salami shared the genotypic virulence profile of the food-associated E. coli O26:H11 strain. In summary, hurdles of acidification and drying during salami ripening resulted in reductions of STEC O26:H11 counts. However, our results also indicate that STEC O26:H11 can persist in the environment of "Teewurst" and might therefore pose a risk to public health. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Prevalence, quantification and isolation of pathogenic shiga toxin Escherichia coli O157:H7 along the production and supply chain of pork around Hubei Province of China.

    Science.gov (United States)

    Khan, Sher Bahadar; Zou, Geng; Xiao, Ran; Cheng, Yuting; Rehman, Zia Ur; Ali, Sher; Memon, Atta Muhammad; Fahad, Shah; Ahmad, Irshad; Zhou, Rui

    2018-02-01

    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.

  3. Multiplex real-time PCR assays for detection of eight Shiga toxin-producing Escherichia coli in food samples by melting curve analysis.

    Science.gov (United States)

    Singh, Prashant; Mustapha, Azlin

    2015-12-23

    Shiga toxin-producing Escherichia coli (STEC) are pathogenic strains of E. coli that can cause bloody diarrhea and kidney failure. Seven STEC serogroups, O157, O26, O45, O103, O111, O121 and O145 are responsible for more than 71% of the total infections caused by this group of pathogens. All seven serogroups are currently considered as adulterants in non-intact beef products in the U.S. In this study, two multiplex melt curve real-time PCR assays with internal amplification controls (IACs) were standardized for the detection of eight STEC serogroups. The first multiplex assay targeted E. coli serogroups O145, O121, O104, and O157; while the second set detected E. coli serogroups O26, O45, O103 and O111. The applicability of the assays was tested using 11 different meat and produce samples. For food samples spiked with a cocktail of four STEC serogroups with a combined count of 10 CFU/25 g food, all targets of the multiplex assays were detected after an enrichment period of 6h. The assays also worked efficiently when 325 g of food samples were spiked with 10 CFU of STECs. The assays are not dependent on fluorescent-labeled probes or immunomagnetic beads, and can be used for the detection of eight STEC serogroups in less than 11h. Routine preliminary screening of STECs in food samples is performed by testing for the presence of STEC virulence genes. The assays developed in this study can be useful as a first- or second-tier test for the identification of the eight O serogroup-specific genes in suspected food samples. Copyright © 2015. Published by Elsevier B.V.

  4. Distribution Pattern of EcoR Phylogenetic Groups Among Shiga Toxin-Producing and Enteropathogenic Escherichia coli Isolated From Healthy Goats

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    Maziar Jajarmi

    2015-08-01

    Full Text Available Background: Escherichia coli can be categorized into four major phylogenetic groups (A, B1, B2, and D based on presence or absence of three markers including two genes (chuA and yjaA and an anonymous DNA fragment designated TSPE4.C2. Also, these groups are divided into seven phylogenetic subgroups A0, A1, B1, B22, B23, D1, and D2. Objectives: This study aimed to determine the distribution pattern of phylogenetic groups in Shiga Toxin-Producing Escherichia coli (STEC and Enteropathogenic Escherichia coli (EPEC isolated from asymptomatic goats in Kerman city, Iran. Materials and Methods: Two hundred and fifty fecal samples were obtained from healthy goats. All isolates were subjected to detection of phylogenetic markers chuA, yjaA and DNA fragment TspE4.C2 and virulence genes stx1, stx2 and eae. Results: In summary, among all isolates phylo-group B1 was the most prevalent (57.6% and other phylo-groups were A1 (20.4%, A0 (18.4%, D1 (2.8%, and B22 (0.8%. There was no isolate in B23 and D2 subgroups. Fifty samples (20% possessed at least one of the tested virulencegenes : stx1 (12%, stx1/stx2 (4%, eae (2.8%, stx1/eae (0.8%, and stx2 (0.4%. Thus, 41 (16.4% STEC, 7 (2.8% EPEC, and 2 (0.8% Enterohemorrhagic Escherichia coli (EHEC strains were isolated and allocated into four phylogenetic subgroups A0 (16%, A1 (12%, B1 (68%, and D1 (4%. Conclusions: Based on 250 fecal samples obtained from goats in industrial slaughterhouse of Kerman City, goats may be a potential reservoir of STEC in Kerman and B1 followed by A are the most prevalent phylogenetic groups among STEC and non-STEC isolates in this study.

  5. Mexican unpasteurised fresh cheeses are contaminated with Salmonella spp., non-O157 Shiga toxin producing Escherichia coli and potential uropathogenic E. coli strains: A public health risk.

    Science.gov (United States)

    Guzman-Hernandez, Rosa; Contreras-Rodriguez, Araceli; Hernandez-Velez, Rosa; Perez-Martinez, Iza; Lopez-Merino, Ahide; Zaidi, Mussaret B; Estrada-Garcia, Teresa

    2016-11-21

    Fresh cheeses are a main garnish of Mexican food. Consumption of artisanal fresh cheeses is very common and most of them are made from unpasteurised cow milk. A total of 52 fresh unpasteurised cheeses of five different types were purchased from a variety of suppliers from Tabasco, Mexico. Using the most probable number method, 67% and 63% of samples were positive for faecal coliforms and E. coli, respectively; revealing their low microbiological quality. General hygienic conditions and practices of traditional cheese manufacturers were poor; most establishments had unclean cement floors, all lacked windows and doors screens, and none of the food-handlers wore aprons, surgical masks or bouffant caps. After analysing all E. coli isolates (121 strains) for the presence of 26 virulence genes, results showed that 9 (17%) samples were contaminated with diarrheagenic E. coli strains, 8 harboured non-O157 Shiga toxin producing E. coli (STEC), and one sample contained both STEC and diffusely adherent E. coli strains. All STEC strains carried the stx1 gene. Potential uropathogenic E. coli (UPEC) strains were isolated from 15 (29%) samples; the most frequent gene combination was fimA-agn43. Two samples were contaminated with Salmonella. The results demonstrated that unpasteurised fresh cheeses produced in Tabasco are of poor microbiological quality and may frequently harbour foodborne pathogens. Food safety authorities in Mexico need to conduct more rigorous surveillance of fresh cheeses. Furthermore, simple and inexpensive measures as establishing programs emphasizing good hand milking practices and hygienic manufacturing procedures may have a major effect on improving the microbiological quality of these food items. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Disinfectant and Antimicrobial Susceptibility Profiles of the Big Six Non-O157 Shiga Toxin-Producing Escherichia coli Strains from Food Animals and Humans.

    Science.gov (United States)

    Beier, Ross C; Franz, Eelco; Bono, James L; Mandrell, Robert E; Fratamico, Pina M; Callaway, Todd R; Andrews, Kathleen; Poole, Toni L; Crippen, Tawni L; Sheffield, Cynthia L; Anderson, Robin C; Nisbet, David J

    2016-08-01

    The disinfectant and antimicrobial susceptibility profiles of 138 non-O157 Shiga toxin-producing Escherichia coli strains (STECs) from food animals and humans were determined. Antimicrobial resistance (AMR) was moderate (39.1% of strains) in response to 15 antimicrobial agents. Animal strains had a lower AMR prevalence (35.6%) than did human strains (43.9%) but a higher prevalence of the resistance profile GEN-KAN-TET. A decreasing prevalence of AMR was found among animal strains from serogroups O45 > O145 > O121 > O111 > O26 > O103 and among human strains from serogroups O145 > O103 > O26 > O111 > O121 > O45. One animal strain from serogroups O121 and O145 and one human strain from serogroup O26 had extensive drug resistance. A high prevalence of AMR in animal O45 and O121 strains and no resistance or a low prevalence of resistance in human strains from these serogroups suggests a source other than food animals for human exposure to these strains. Among the 24 disinfectants evaluated, all strains were susceptible to triclosan. Animal strains had a higher prevalence of resistance to chlorhexidine than did human strains. Both animal and human strains had a similar low prevalence of low-level benzalkonium chloride resistance, and animal and human strains had similar susceptibility profiles for most other disinfectants. Benzyldimethylammonium chlorides and C10AC were the primary active components in disinfectants DC&R and P-128, respectively, against non-O157 STECs. A disinfectant FS512 MIC ≥ 8 μg/ml was more prevalent among animal O121 strains (61.5%) than among human O121 strains (25%), which may also suggest a source of human exposure to STEC O121 other than food animals. Bacterial inhibition was not dependent solely on pH but was correlated with the presence of dissociated organic acid species and some undissociated acids.

  7. Microbial safety and sanitary quality of strawberry primary production in Belgium: risk factors for Salmonella and Shiga toxin-producing Escherichia coli contamination.

    Science.gov (United States)

    Delbeke, Stefanie; Ceuppens, Siele; Hessel, Claudia Titze; Castro, Irene; Jacxsens, Liesbeth; De Zutter, Lieven; Uyttendaele, Mieke

    2015-04-01

    Strawberries are an important fruit in Belgium in both production and consumption, but little information is available about the presence of Salmonella and Shiga toxin-producing Escherichia coli (STEC) in these berries, the risk factors in agricultural production, and possible specific mitigation options. In 2012, a survey was undertaken of three soil and three soilless cultivation systems in Belgium. No Salmonella spp. were isolated. No STEC was detected in the strawberry samples (0 of 72), but STEC was detected by PCR in 11 of 78 irrigation water and 2 of 24 substrate samples. Culture isolates were obtained for 2 of 11 PCR-positive irrigation water samples and 2 of 2 substrate samples. Multivariable logistic regression analysis revealed elevated generic E. coli numbers (the odds ratio [OR] for a 1 log increase being 4.6) as the most important risk factor for STEC, together with the berry-picking season (elevated risk in summer). The presence of generic E. coli in the irrigation water (≥1 CFU per 100 ml) was mainly influenced by the type of irrigation water (collected rainfall water stored in ponds was more often contaminated than groundwater pumped from boreholes [OR = 5.8]) and the lack of prior treatment (untreated water versus water subjected to sand filtration prior to use [OR = 19.2]). The follow-up study in 2013 at one of the producer locations indicated cattle to be the most likely source of STEC contamination of the irrigation water. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  8. Antibodies Directed against Shiga-Toxin Producing Escherichia coli Serotype O103 Type III Secreted Proteins Block Adherence of Heterologous STEC Serotypes to HEp-2 Cells.

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    Taseen S Desin

    Full Text Available Shiga toxin-producing Escherichia coli (STEC serotype O103 is a zoonotic pathogen that is capable of causing hemorrhagic colitis and hemolytic uremic syndrome (HUS in humans. The main animal reservoir for STEC is ruminants and hence reducing the levels of this pathogen in cattle could ultimately lower the risk of STEC infection in humans. During the process of infection, STECO103 uses a Type III Secretion System (T3SS to secrete effector proteins (T3SPs that result in the formation of attaching and effacing (A/E lesions. Vaccination of cattle with STEC serotype O157 T3SPs has previously been shown to be effective in reducing shedding of STECO157 in a serotype-specific manner. In this study, we tested the ability of rabbit polyclonal sera against individual STECO103 T3SPs to block adherence of the organism to HEp-2 cells. Our results demonstrate that pooled sera against EspA, EspB, EspF, NleA and Tir significantly lowered the adherence of STECO103 relative to pre-immune sera. Likewise, pooled anti-STECO103 sera were also able to block adherence by STECO157. Vaccination of mice with STECO103 recombinant proteins induced strong IgG antibody responses against EspA, EspB, NleA and Tir but not against EspF. However, the vaccine did not affect fecal shedding of STECO103 compared to the PBS vaccinated group over the duration of the experiment. Cross reactivity studies using sera against STECO103 recombinant proteins revealed a high degree of cross reactivity with STECO26 and STECO111 proteins implying that sera against STECO103 proteins could potentially provide neutralization of attachment to epithelial cells by heterologous STEC serotypes.

  9. Antibodies Directed against Shiga-Toxin Producing Escherichia coli Serotype O103 Type III Secreted Proteins Block Adherence of Heterologous STEC Serotypes to HEp-2 Cells.

    Science.gov (United States)

    Desin, Taseen S; Townsend, Hugh G; Potter, Andrew A

    2015-01-01

    Shiga toxin-producing Escherichia coli (STEC) serotype O103 is a zoonotic pathogen that is capable of causing hemorrhagic colitis and hemolytic uremic syndrome (HUS) in humans. The main animal reservoir for STEC is ruminants and hence reducing the levels of this pathogen in cattle could ultimately lower the risk of STEC infection in humans. During the process of infection, STECO103 uses a Type III Secretion System (T3SS) to secrete effector proteins (T3SPs) that result in the formation of attaching and effacing (A/E) lesions. Vaccination of cattle with STEC serotype O157 T3SPs has previously been shown to be effective in reducing shedding of STECO157 in a serotype-specific manner. In this study, we tested the ability of rabbit polyclonal sera against individual STECO103 T3SPs to block adherence of the organism to HEp-2 cells. Our results demonstrate that pooled sera against EspA, EspB, EspF, NleA and Tir significantly lowered the adherence of STECO103 relative to pre-immune sera. Likewise, pooled anti-STECO103 sera were also able to block adherence by STECO157. Vaccination of mice with STECO103 recombinant proteins induced strong IgG antibody responses against EspA, EspB, NleA and Tir but not against EspF. However, the vaccine did not affect fecal shedding of STECO103 compared to the PBS vaccinated group over the duration of the experiment. Cross reactivity studies using sera against STECO103 recombinant proteins revealed a high degree of cross reactivity with STECO26 and STECO111 proteins implying that sera against STECO103 proteins could potentially provide neutralization of attachment to epithelial cells by heterologous STEC serotypes.

  10. Circulating microRNAs in patients with Shiga-Toxin-producing E. coli O104:H4 induced hemolytic uremic syndrome.

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    Johan M Lorenzen

    Full Text Available BACKGROUND: In early May 2011, an outbreak of hemorrhagic colitis associated with hemolytic-uremic syndrome (HUS first developed in Northern Germany and spread to 15 other countries in Europe. The outbreak-strain O104:H4, which combined virulence factors of typical enteroaggregative and Shiga-Toxin-producing E. coli was associated with an unusual high rate of hemolytic uremic syndrome. Also an unexpected high rate of coma and seizures leading to mechanical ventilation and ICU treatment was observed. MicroRNAs are small ribonucleotides orchestrating gene expression. We tested whether circulating microRNAs in serum of HUS patients during the 2011 epidemics are altered in this patient cohort and related to clinical manifestations. METHODOLOGY/PRINCIPAL FINDINGS: We profiled microRNAs using RNA isolated from serum of patients and healthy age-matched controls. The results were validated in 38 patients at baseline, 29 patients during follow-up and 21 age-matched healthy controls by miRNA-specific quantitative RT-PCR. Circulating levels of miR-24, miR-126 were increased in HUS patients versus controls. There was no association between these microRNAs and renal function or the need for renal replacement therapy. In contrast, levels of miR-126 were associated with neurological symptoms at baseline and during follow-up. In addition, miR-126 (on admission and miR-24 (on admission and during follow-up were associated with platelet count. CONCLUSIONS/SIGNIFICANCE: Circulating microRNAs are strongly altered in this patient cohort and associated with neurological symptoms as well as platelet count.

  11. Feces of feedlot cattle contain a diversity of bacteriophages that lyse non-O157 Shiga toxin-producing Escherichia coli.

    Science.gov (United States)

    Wang, Jiaying; Niu, Yan D; Chen, Jinding; Anany, Hany; Ackermann, Hans-W; Johnson, Roger P; Ateba, Collins N; Stanford, Kim; McAllister, Tim A

    2015-07-01

    This study aimed to isolate and characterize bacteriophages that lyse non-O157 Shiga toxin-producing Escherichia coli (STEC) from cattle feces. Of 37 non-O157 STEC-infecting phages isolated, those targeting O26 (AXO26A, AYO26A, AYO26B), O103 (AXO103A, AYO103A), O111 (AXO111A, AYO111A), O121 (AXO121A, AXO121B), and O145 (AYO145A, AYO145B) were further characterized. Transmission electron microscopy showed that the 11 isolates belonged to 3 families and 6 genera: the families Myoviridae (types rV5, T4, ViI, O1), Siphoviridae (type T5), and Podoviridae (type T7). Genome size of the phages as determined by pulsed-field gel electrophoresis ranged from 38 to 177 kb. Excluding phages AXO26A, AYO103A, AYO145A, and AYO145B, all other phages were capable of lysing more than 1 clinically important strain from serogroups of O26, O91, O103, O111, O113, O121, and O128, but none exhibited infectivity across all serogroups. Moreover, phages AYO26A, AXO121A, and AXO121B were also able to lyse 4 common phage types of STEC O157:H7. Our findings show that a diversity of non-O157 STEC-infecting phages are harbored in bovine feces. Phages AYO26A, AYO26B, AXO103A, AXO111A, AYO111A, AXO121A, and AXO121B exhibited a broad host range against a number of serogroups of STEC and have potential for the biocontrol of STEC in the environment.

  12. Identification of Shiga toxin-producing (STEC) and enteropathogenic (EPEC) Escherichia coli in diarrhoeic calves and comparative genomics of O5 bovine and human STEC.

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    Fakih, I; Thiry, D; Duprez, J-N; Saulmont, M; Iguchi, A; Piérard, D; Jouant, L; Daube, G; Ogura, Y; Hayashi, T; Taminiau, B; Mainil, J G

    2017-04-01

    Escherichia coli producing Shiga toxins (Stx) and the attaching-effacing (AE) lesion (AE-STEC) are responsible for (bloody) diarrhoea in humans and calves while the enteropathogenic E. coli (EPEC) producing the AE lesion only cause non-bloody diarrhoea in all mammals. The purpose of this study was (i) to identify the pathotypes of enterohaemolysin-producing E. coli isolated between 2009 and 2013 on EHLY agar from less than 2 month-old diarrhoeic calves with a triplex PCR targeting the stx1, stx2, eae virulence genes; (ii) to serotype the positive isolates with PCR targeting the genes coding for ten most frequent and pathogenic human and calf STEC O serogroups; and (iii) to compare the MLSTypes and virulotypes of calf and human O5 AE-STEC after Whole Genome Sequencing using two server databases (www.genomicepidemiology.org). Of 233 isolates, 206 were triplex PCR-positive: 119 AE-STEC (58%), 78 EPEC (38%) and 9 STEC (4%); and the stx1+eae+ AE-STEC (49.5%) were the most frequent. Of them, 120 isolates (84% of AE-STEC, 23% of EPEC, 22% of STEC) tested positive with one O serogroup PCR: 57 for O26 (47.5%), 36 for O111 (30%), 10 for O103 (8%) and 8 for O5 (7%) serogroups. The analysis of the draft sequences of 15 O5 AE-STEC could not identify any difference correlated to the host. As a conclusion, (i) the AE-STEC associated with diarrhoea in young calves still belong to the same serogroups as previously (O5, O26, O111) but the O103 serogroup may be emerging, (ii) the O5 AE-STEC from calves and humans are genetically similar. Copyright © 2016. Published by Elsevier B.V.

  13. Molecular characterisation of human Shiga toxin-producing Escherichia coli O26 strains: results of an outbreak investigation, Romania, February to August 2016.

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    Usein, Codruţa-Romaniţa; Ciontea, Adriana Simona; Militaru, Cornelia Mãdãlina; Condei, Maria; Dinu, Sorin; Oprea, Mihaela; Cristea, Daniela; Michelacci, Valeria; Scavia, Gaia; Zota, Lavinia Cipriana; Zaharia, Alina; Morabito, Stefano

    2017-11-01

    IntroductionAt the beginning of 2016, an increase in paediatric haemolytic uremic syndrome (HUS) cases was observed in Romania. The microbiological investigations allowed isolation of Shiga toxin-producing Escherichia coli (STEC) O26 as the causative agent from most cases. Methods: An enhanced national surveillance of HUS and severe diarrhoea was established across the country following the identification of the first cases and was carried out until August 2016. A total of 15 strains were isolated from 10 HUS and five diarrhoea cases. Strains were characterised by virulence markers (i.e. stx type/subtype, eae , ehxA genes), phylogroup, genetic relatedness and clonality using PCR-based assays, PFGE and multilocus sequence typing (MLST). The first six strains were further characterised by whole genome sequencing (WGS). Results: Five PCR-defined genotypes were distinguished. All strains from HUS cases harboured stx2a and eae , with or without stx1a , while strains from diarrhoea cases carried exclusively stx1a and eae genes. PFGE resolved strains into multiple pulsotypes, compatible with a certain geographic segregation of the cases, and strains were assigned to phylogroup B1 and sequence type (ST) 21. WGS confirmed the results of conventional molecular methods, brought evidence of O26:H11 serotype, and complemented the virulence profiles. Discussion/conclusion: This first description of STEC O26 strains from cases in Romania showed that the isolates belonged to a diverse population. The virulence content of most strains highlighted a high risk for severe outcome in infected patients. Improving the national surveillance strategy for STEC infections in Romania needs to be further considered.

  14. Comparative Genomics of Recent Shiga Toxin-Producing Escherichia coli O104:H4: Short-Term Evolution of an Emerging Pathogen

    Science.gov (United States)

    Grad, Yonatan H.; Godfrey, Paul; Cerquiera, Gustavo C.; Mariani-Kurkdjian, Patricia; Gouali, Malika; Bingen, Edouard; Shea, Terrence P.; Haas, Brian J.; Griggs, Allison; Young, Sarah; Zeng, Qiandong; Lipsitch, Marc; Waldor, Matthew K.; Weill, François-Xavier; Wortman, Jennifer R.; Hanage, William P.

    2013-01-01

    ABSTRACT The large outbreak of diarrhea and hemolytic uremic syndrome (HUS) caused by Shiga toxin-producing Escherichia coli O104:H4 in Europe from May to July 2011 highlighted the potential of a rarely identified E. coli serogroup to cause severe disease. Prior to the outbreak, there were very few reports of disease caused by this pathogen and thus little known of its diversity and evolution. The identification of cases of HUS caused by E. coli O104:H4 in France and Turkey after the outbreak and with no clear epidemiological links raises questions about whether these sporadic cases are derived from the outbreak. Here, we report genome sequences of five independent isolates from these cases and results of a comparative analysis with historical and 2011 outbreak isolates. These analyses revealed that the five isolates are not derived from the outbreak strain; however, they are more closely related to the outbreak strain and each other than to isolates identified prior to the 2011 outbreak. Over the short time scale represented by these closely related organisms, the majority of genome variation is found within their mobile genetic elements: none of the nine O104:H4 isolates compared here contain the same set of plasmids, and their prophages and genomic islands also differ. Moreover, the presence of closely related HUS-associated E. coli O104:H4 isolates supports the contention that fully virulent O104:H4 isolates are widespread and emphasizes the possibility of future food-borne E. coli O104:H4 outbreaks. PMID:23341549

  15. Shiga toxin-producing Escherichia coli (STEC) O22:H8 isolated from cattle reduces E. coli O157:H7 adherence in vitro and in vivo.

    Science.gov (United States)

    Martorelli, L; Albanese, A; Vilte, D; Cantet, R; Bentancor, A; Zolezzi, G; Chinen, I; Ibarra, C; Rivas, M; Mercado, E C; Cataldi, A

    2017-09-01

    Shiga toxin-producing Escherichia coli (STEC) are a group of bacteria responsible for food-associated diseases. Clinical features include a wide range of symptoms such as diarrhea, hemorrhagic colitis and the hemolytic uremic syndrome (HUS), a life-threatening condition. Our group has observed that animals naturally colonized with STEC strains of unknown serotype were not efficiently colonized with E. coli O157:H7 after experimental infection. In order to assess the basis of the interference, three STEC strains were isolated from STEC persistently-colonized healthy cattle from a dairy farm in Buenos Aires, Argentina. The three isolated strains are E. coli O22:H8 and carry the stx1 and stx2d genes. The activatable activity of Stx2d was demonstrated in vitro. The three strains carry the adhesins iha, ehaA and lpf O113 . E. coli O22:H8 formed stronger biofilms in abiotic surface than E. coli O157:H7 (eae+, stx2+) and displayed a more adherent phenotype in vitro towards HeLa cells. Furthermore, when both serotypes were cultured together O22:H8 could reduce O157:H7 adherence in vitro. When calves were intragastrically pre-challenged with 10 8 CFU of a mixture of the three STEC strains and two days later challenged with the same dose of the strain E. coli O157:H7 438/99, the shedding of the pathogen was significantly reduced. These results suggest that E. coli O22:H8, a serotype rarely associated with human illness, might compete with O157:H7 at the bovine recto-anal junction, making non-O157 carrying-calves less susceptible to O157:H7 colonization and shedding of the bacteria to the environment. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Isolation and selection of coliphages as potential biocontrol agents of enterohemorrhagic and Shiga toxin-producing E. coli (EHEC and STEC) in cattle.

    Science.gov (United States)

    Dini, C; De Urraza, P J

    2010-09-01

    To isolate, characterize and select phages as potential biocontrol agents of enterohemorrhagic and Shiga toxin-producing Escherichia coli (EHEC and STEC) in cattle. Sixteen STEC and EHEC coliphages were isolated from bovine minced meat and stool samples and characterized with respect to their host range against STEC, EHEC and other Gram-negative pathogens; their morphology by electron microscopy; the presence of the stx1, stx2 and cI genes by means of PCR; RAPD and rep-PCR profiles; plaque formation; and acid resistance. Six isolates belonged to the Myoviridae and 10 to the Podoviridae families. The phages negative for stx and cI that formed large, well-defined plaques were all isolated using EHEC O157:H7 as host. Among them, only CA911 was a myophage and, together with CA933P, had the broadest host range for STEC and EHEC; the latter phage also infected Shigella and Pseudomonas. Isolates CA911, MFA933P and MFA45D differed in particle morphology and amplification patterns by RAPD and rep-PCR and showed the highest acidity tolerance. Myophage CA911 and podophages CA933P, MFA933P and MFA45D were chosen as the best candidates for biocontrol of STEC and EHEC in cattle. This work employs steps for a rational selection and characterization of bacteriophages as therapeutic agents. This report constitutes the first documentation of STEC and EHEC phages isolated in Argentina and proposes for the first time the use of rep-PCR as a complement of RAPD on DNA fingerprinting of phages. © 2010 The Authors. Journal compilation © 2010 The Society for Applied Microbiology.

  17. Genetic characterization of Shiga toxin-producing Escherichia coli (STEC) and atypical enteropathogenic Escherichia coli (EPEC) isolates from goat's milk and goat farm environment.

    Science.gov (United States)

    Álvarez-Suárez, María-Elena; Otero, Andrés; García-López, María-Luisa; Dahbi, Ghizlane; Blanco, Miguel; Mora, Azucena; Blanco, Jorge; Santos, Jesús A

    2016-11-07

    The aim of this study was to characterize a collection of 44 Shiga toxin-producing (STEC) and enteropathogenic Escherichia coli (EPEC) isolated from goat milk and goat farm environment. Of the 19 STEC isolates, five (26.3%) carried the stx1 gene, four (21.1%) the stx2 gene and 10 (52.6%) presented both stx genes. Six (31.6%) STEC strains were eae-positive and belonged to serotypes related to severe human disease (O157:H7 and O5:HNM). Another seven STEC strains were of serotype O146:H21 and three of serotype O166:H28, also linked to human disease. The STEC strains isolated from goat milk were of serotypes potentially pathogenic for humans. All the 25 EPEC isolates were considered atypical (aEPEC) and one aEPEC strain was of serotype O26:H11, a serotype frequently isolated in children with diarrhea. Multilocus sequence typing (MLST) was carried out with seven housekeeping genes and 23 sequence types (ST) were detected, 14 of them newly described. Twelve STs grouped STEC isolates and 11 STs grouped EPEC isolates. Genetic typing by pulsed field gel electrophoresis (PFGE) resulted in 38 patterns which grouped in 10 clusters. Well-defined groups were also observed for strains of pathogenic serotypes. In conclusion, strains of STEC and aEPEC belonging to serotypes related to severe human disease have been detected in goat milk and the goat farm environment. Ruminants are an important reservoir of STEC strains and the role of these animals as carriers of other pathogenic types of E. coli seems to be an emerging concern. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Genetic relatedness and novel sequence types of non-O157 Shiga toxin-producing Escherichia coli strains isolated in Argentina

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    Jimena Soledad Cadona

    2016-08-01

    Full Text Available Shiga toxin-producing Escherichia coli (STEC is a foodborne pathogen responsible for severe disease in humans such as hemolytic uremic syndrome (HUS and cattle, the principal reservoir. Identification of the clones/lineages is important as several characteristics, among them propensity to cause disease varies with STEC phylogenetic origin. At present, we do not know what STEC clones, especially of non-O157:H7, are circulating in Argentina. To fill this knowledge gap we assessed the genetic diversity of STEC strains isolated in Argentina from various sources, mostly cattle and food, using multilocus sequence typing (MLST. Our objectives were to determine the phylogenetic relationships among strains and to compare them with strains from different geographic origins, especially with those from clinical human cases, in order to evaluate their potential health risk. A total of 59 STEC isolates from 41 serotypes were characterized by MLST. Analysis using EcMLST database identified 38 sequence types (ST, 17 (45% of which were new STs detected in 18 serotypes. Fifteen out of 38 STs identified were grouped into 11 clonal groups (CGs and, 23 not grouped in any of the defined CGs. Different STs were found in the same serotype. Results highlighted a high degree of phylogenetic heterogeneity among Argentinean strains and they showed that several cattle and food isolates belonged to the same STs that are commonly associated with clinical human cases in several geographical areas. STEC is a significant public health concern. Argentina has the highest incidence of HUS in the world and this study provides the first data about which STEC clones are circulating. Data showed that most of them might pose a serious zoonotic risk and this information is important for developing public health initiatives. However, the actual potential risk will be defined by the virulence profiles, which may differ among isolates belonging to the same ST.

  19. The evolutionary divergence of Shiga toxin-producing Escherichia coli is reflected in clustered regularly interspaced short palindromic repeat (CRISPR) spacer composition.

    Science.gov (United States)

    Yin, Shuang; Jensen, Mark A; Bai, Jiawei; Debroy, Chitrita; Barrangou, Rodolphe; Dudley, Edward G

    2013-09-01

    The Shiga toxin-producing Escherichia coli (STEC) strains, including those of O157:H7 and the "big six" serogroups (i.e., serogroups O26, O45, O103, O111, O121, and O145), are a group of pathogens designated food adulterants in the United States. The relatively conserved nature of clustered regularly interspaced short palindromic repeats (CRISPRs) in phylogenetically related E. coli strains makes them potential subtyping markers for STEC detection, and a quantitative PCR (qPCR)-based assay was previously developed for O26:H11, O45:H2, O103:H2, O111:H8, O121:H19, O145:H28, and O157:H7 isolates. To better evaluate the sensitivity and specificity of this qPCR method, the CRISPR loci of 252 O157 and big-six STEC isolates were sequenced and analyzed along with 563 CRISPR1 and 624 CRISPR2 sequences available in GenBank. General conservation of spacer content and order was observed within each O157 and big-six serogroup, validating the qPCR method. Meanwhile, it was found that spacer deletion, the presence of an insertion sequence, and distinct alleles within a serogroup are sources of false-negative reactions. Conservation of CRISPR arrays among isolates expressing the same flagellar antigen, specifically, H7, H2, and H11, suggested that these isolates share an ancestor and provided an explanation for the false positives previously observed in the qPCR results. An analysis of spacer distribution across E. coli strains provided limited evidence for temporal spacer acquisition. Conversely, comparison of CRISPR sequences between strains along the stepwise evolution of O157:H7 from its O55:H7 ancestor revealed that, over this ∼7,000-year span, spacer deletion was the primary force generating CRISPR diversity.

  20. Distribution of pathogenicity islands OI-122, OI-43/48, and OI-57 and a high-pathogenicity island in Shiga toxin-producing Escherichia coli.

    Science.gov (United States)

    Ju, Wenting; Shen, Jinling; Toro, Magaly; Zhao, Shaohua; Meng, Jianghong

    2013-06-01

    Pathogenicity islands (PAIs) play an important role in Shiga toxin-producing Escherichia coli (STEC) pathogenicity. The distribution of PAIs OI-122, OI-43/48, and OI-57 and a high-pathogenicity island (HPI) were determined among 98 STEC strains assigned to seropathotypes (SPTs) A to E. PCR and PCR-restriction fragment length polymorphism assays were used to identify 14 virulence genes that belonged to the four PAIs and to subtype eae and stx genes, respectively. Phylogenetic trees were constructed based on the sequences of pagC among 34 STEC strains and iha among 67 diverse pathogenic E. coli, respectively. Statistical analysis demonstrated that the prevalences of OI-122 (55.82%) and OI-57 (82.35%) were significantly greater in SPTs (i.e., SPTs A, B, and C) that are frequently associated with severe disease than in other SPTs. terC (62.5%) and ureC (62.5%) in OI-43/48 were also significantly more prevalent in SPTs A, B, and C than in SPTs D and E. In addition, OI-122, OI-57, and OI-43/48 and their associated virulence genes (except iha) were found to be primarily associated with eae-positive STEC, whereas HPI occurred independently of the eae presence. The strong association of OI-122, OI-43/48, and OI-57 with eae-positive STEC suggests in part that different pathogenic mechanisms exist between eae-positive and eae-negative STEC strains. Virulence genes in PAIs that are associated with severe diseases can be used as potential markers to aid in identifying highly virulent STEC.

  1. OI-57, a genomic island of Escherichia coli O157, is present in other seropathotypes of Shiga toxin-producing E. coli associated with severe human disease.

    Science.gov (United States)

    Imamovic, Lejla; Tozzoli, Rosangela; Michelacci, Valeria; Minelli, Fabio; Marziano, Maria Luisa; Caprioli, Alfredo; Morabito, Stefano

    2010-11-01

    Strains of Shiga toxin-producing Escherichia coli (STEC) are a heterogeneous E. coli group that may cause severe disease in humans. STEC have been categorized into seropathotypes (SPTs) based on their phenotypic and molecular characteristics and the clinical features of the associated diseases. SPTs range from A to E, according to a decreasing rank of pathogenicity. To define the virulence gene asset ("virulome") characterizing the highly pathogenic SPTs, we used microarray hybridization to compare the whole genomes of STEC belonging to SPTs B, C, and D with that of STEC O157 (SPT A). The presence of the open reading frames (ORFs) associated with SPTs A and B was subsequently investigated by PCR in a larger panel of STEC and in other E. coli strains. A genomic island termed OI-57 was present in SPTs A and B but not in the other SPTs. OI-57 harbors the putative virulence gene adfO, encoding a factor enhancing the adhesivity of STEC O157, and ckf, encoding a putative killing factor for the bacterial cell. PCR analyses showed that OI-57 was present in its entirety in the majority of the STEC genomes examined, indicating that it represents a stable acquisition of the positive clonal lineages. OI-57 was also present in a high proportion of the human enteropathogenic E. coli genomes assayed, suggesting that it could be involved in the attaching-and-effacing colonization of the intestinal mucosa. In conclusion, OI-57 appears to be part of the virulome of pathogenic STEC and further studies are needed to elucidate its role in the pathogenesis of STEC infections.

  2. Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat.

    Science.gov (United States)

    Brusa, Victoria; Galli, Lucía; Linares, Luciano H; Ortega, Emanuel E; Lirón, Juan P; Leotta, Gerardo A

    2015-12-01

    Shiga toxin-producing Escherichia coli (STEC) are recognized as food-borne pathogens. We developed and validated two SYBR green PCR (SYBR-PCR) and a real-time multiplex PCR (RT-PCR) to detect stx1 and stx2 genes in meat samples, and compared these techniques in ground beef samples from retail stores. One set of primers and one hydrolysis probe were designed for each stx gene. For RT-PCR, an internal amplification control (IAC) was used. All PCR intra-laboratory validations were performed using pure strains and artificially contaminated ground beef samples. A total of 50 STEC and 30 non-STEC strains were used. Naturally contaminated ground beef samples (n=103) were obtained from retail stores and screened with SYBR-PCR and RT-PCR, and stx-positive samples were processed for STEC isolation. In the intra-laboratory validation, each PCR obtained a 1×10(2) CFU mL(-1) limit of detection and 100% inclusivity and exclusivity. The same results were obtained when different laboratory analysts in alternate days performed the assay. The level of agreement obtained with SYBR-PCR and RT-PCR was kappa=0.758 and 0.801 (P<0.001) for stx1 and stx2 gene detection, respectively. Two PCR strategies were developed and validated, and excellent performance with artificially contaminated ground beef samples was obtained. However, the efforts made to isolate STEC from retail store samples were not enough. Only 11 STEC strains were isolated from 35 stx-positive ground beef samples identically detected by all PCRs. The combination of molecular approaches based on the identification of a virulence genotypic profile of STEC must be considered to improve isolation. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Genetic Relatedness Among Shiga Toxin-Producing Escherichia coli Isolated Along the Animal Food Supply Chain and in Gastroenteritis Cases in Qatar Using Multilocus Sequence Typing.

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    Palanisamy, Srikanth; Chang, YuChen; Scaria, Joy; Penha Filho, Rafael Antonio Casarin; Peters, Kenlyn E; Doiphode, Sanjay H; Sultan, Ali; Mohammed, Hussni O

    2017-06-01

    Pathogenic Escherichia coli has been listed among the most important bacteria associated with foodborne illnesses around the world. We investigated the genetic relatedness among Shiga toxin-producing E. coli (STEC) isolated along the animal food supply chain and from humans diagnosed with gastroenteritis in Qatar. Samples were collected from different sources along the food supply chain and from patients admitted to the hospital with complaints of gastroenteritis. All samples were screened for the presence of E. coli O157:H7 and non-O157 STEC using a combination of bacterial enrichment and molecular detection techniques. A proportional sampling approach was used to select positive samples from each source for further multilocus sequence typing (MLST) analysis. Seven housekeeping genes described for STEC were amplified by polymerase chain reaction, sequenced, and analyzed by MLST. Isolates were characterized by allele composition, sequence type (ST) and assessed for epidemiologic relationship within and among different sources. Nei's genetic distance was calculated at the allele level between sample pools in each site downstream. E. coli O157:H7 occurred at a higher rate in slaughterhouse and retail samples than at the farm or in humans in our sampling. The ST171, an ST common to enterotoxigenic E. coli and atypical enteropathogenic E. coli, was the most common ST (15%) in the food supply chain. None of the genetic distances among the different sources was statistically significant. Enterohemorrhagic E. coli pathogenic strains are present along the supply chain at different levels and with varying relatedness. Clinical isolates were the most diverse, as expected, considering the polyclonal diversity in the human microbiota. The high occurrence of these food adulterants among the farm products suggests that implementation of sanitary measures at that level might reduce the risk of human exposure.

  4. Antibody Response to Shiga Toxins in Argentinean Children with Enteropathic Hemolytic Uremic Syndrome at Acute and Long-Term Follow-Up Periods

    Science.gov (United States)

    Fernández-Brando, Romina J.; Bentancor, Leticia V.; Mejías, María Pilar; Ramos, María Victoria; Exeni, Andrea; Exeni, Claudia; Laso, María del Carmen; Exeni, Ramón; Isturiz, Martín A.; Palermo, Marina S.

    2011-01-01

    Shiga toxin (Stx)-producing Escherichia coli (STEC) infection is associated with a broad spectrum of clinical manifestations that include diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). Systemic Stx toxemia is considered to be central to the genesis of HUS. Distinct methods have been used to evaluate anti-Stx response for immunodiagnostic or epidemiological analysis of HUS cases. The development of enzyme-linked immunosorbent assay (ELISA) and western blot (WB) assay to detect the presence of specific antibodies to Stx has introduced important advantages for serodiagnosis of HUS. However, application of these methods for seroepidemiological studies in Argentina has been limited. The aim of this work was to develop an ELISA to detect antibodies against the B subunit of Stx2, and a WB to evaluate antibodies against both subunits of Stx2 and Stx1, in order to analyze the pertinence and effectiveness of these techniques in the Argentinean population. We studied 72 normal healthy children (NHC) and 105 HUS patients of the urban pediatric population from the surrounding area of Buenos Aires city. Using the WB method we detected 67% of plasma from NHC reactive for Stx2, but only 8% for Stx1. These results are in agreement with the broad circulation of Stx2-expressing STEC in Argentina and the endemic behavior of HUS in this country. Moreover, the simultaneous evaluation by the two methods allowed us to differentiate acute HUS patients from NHC with a great specificity and accuracy, in order to confirm the HUS etiology when pathogenic bacteria were not isolated from stools. PMID:21559455

  5. Characterization of novel bacteriophage phiC119 capable of lysing multidrug-resistant Shiga toxin-producing Escherichia coli O157:H7

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    Luis Amarillas

    2016-09-01

    Full Text Available Background Shiga toxin-producing Escherichia coli (STEC is one of the most common and widely distributed foodborne pathogens that has been frequently implicated in gastrointestinal and urinary tract infections. Moreover, high rates of multiple antibiotic-resistant E. coli strains have been reported worldwide. Due to the emergence of antibiotic-resistant strains, bacteriophages are considered an attractive alternative to biocontrol pathogenic bacteria. Characterization is a preliminary step towards designing a phage for biocontrol. Methods In this study, we describe the characterization of a bacteriophage designated phiC119, which can infect and lyse several multidrug-resistant STEC strains and some Salmonella strains. The phage genome was screened to detect the stx-genes using PCR, morphological analysis, host range was determined, and genome sequencing were carried out, as well as an analysis of the cohesive ends and identification of the type of genetic material through enzymatic digestion of the genome. Results Analysis of the bacteriophage particles by transmission electron microscopy showed that it had an icosahedral head and a long tail, characteristic of the family Siphoviridae. The phage exhibits broad host range against multidrug-resistant and highly virulent E. coli isolates. One-step growth experiments revealed that the phiC119 phage presented a large burst size (210 PFU/cell and a latent period of 20 min. Based on genomic analysis, the phage contains a linear double-stranded DNA genome with a size of 47,319 bp. The phage encodes 75 putative proteins, but lysogeny and virulence genes were not found in the phiC119 genome. Conclusion These results suggest that phage phiC119 may be a good biological control agent. However, further studies are required to ensure its control of STEC and to confirm the safety of phage use.

  6. Prevalence and behavior of multidrug-resistant shiga toxin-producing Escherichia coli, enteropathogenic E. coli and enterotoxigenic E. coli on coriander.

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    Gómez-Aldapa, Carlos A; Segovia-Cruz, Jesús A; Cerna-Cortes, Jorge F; Rangel-Vargas, Esmeralda; Salas-Rangel, Laura P; Gutiérrez-Alcántara, Eduardo J; Castro-Rosas, Javier

    2016-10-01

    The prevalence and behavior of multidrug-resistant diarrheagenic Escherichia coli pathotypes on coriander was determined. One hundred coriander samples were collected from markets. Generic E. coli were determined using the most probable number procedure. Diarrheagenic E. coli pathotypes (DEPs) were identified using two multiplex polymerase chain reaction procedures. Susceptibility to sixteen antibiotics was tested for the isolated DEPs strains by standard test. The behavior of multidrug-resistant DEPs isolated from coriander was determined on coriander leaves and chopped coriander at 25°± 2 °C and 3°± 2 °C. Generic E. coli and DEPs were identified, respectively, in 43 and 7% of samples. Nine DEPs strains were isolated from positive coriander samples. The identified DEPs included Shiga toxin-producing E. coli (STEC, 4%) enterotoxigenic E. coli (ETEC, 2%) and enteropathogenic E. coli (EPEC, 1%). All isolated DEPs strains exhibited multi-resistance to antibiotics. On inoculated coriander leaves stored at 25°± 2 °C or 3°± 2 °C, no growth was observed for multidrug-resistant DEPs strains. However, multidrug-resistant DEPs strains grew in chopped coriander: after 24 h at 25° ± 2 °C, DEPs strains had grown to approximately 3 log CFU/g. However, at 3°± 2 °C the bacterial growth was inhibited. To the best of our knowledge, this is the first report of the presence and behavior of multidrug-resistant STEC, ETEC and EPEC on coriander and chopped coriander. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Real-time PCR and enzyme-linked fluorescent assay methods for detecting Shiga-toxin-producing Escherichia coli in mincemeat samples.

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    Stefan, A; Scaramagli, S; Bergami, R; Mazzini, C; Barbanera, M; Perelle, S; Fach, P

    2007-03-01

    This work aimed to compare real-time polymerase chain reaction (PCR) with the commercially available enzyme-linked fluorescent assay (ELFA) VIDAS ECOLI O157 for detecting Escherichia coli O157 in mincemeat. In addition, a PCR-based survey on Shiga-toxin-producing E. coli (STEC) in mincemeat collected in Italy is presented. Real-time PCR assays targeting the stx genes and a specific STEC O157 sequence (SILO157, a small inserted locus of STEC O157) were tested for their sensitivity on spiked mincemeat samples. After overnight enrichment, the presence of STEC cells could be clearly determined in the 25 g samples containing 10 bacterial cells, while the addition of five bacteria provided equivocal PCR results with Ct values very close to or above the threshold of 40. The PCR tests proved to be more sensitive than the ELFA-VIDAS ECOLI O157, whose detection level started from 50 bacterial cells/25 g of mincemeat. The occurrence of STEC in 106 mincemeat (bovine, veal) samples collected from September to November 2004 at five different points of sale in Italy (one point of sale in Arezzo, Tuscany, central Italy, two in Mantova, Lombardy, Northern Italy, and two in Bologna, Emilia-Romagna, upper-central Italy) was less than 1%. Contamination by the main STEC O-serogroups representing a major public health concern, including O26, O91, O111, O145, and O157, was not detected. This survey indicates that STEC present in these samples are probably not associated with pathogenesis in humans.

  8. Profile of Shiga toxin-producing Escherichia coli strains isolated from dogs and cats and genetic relationships with isolates from cattle, meat and humans.

    Science.gov (United States)

    Bentancor, A; Rumi, M V; Carbonari, C; Gerhardt, E; Larzábal, M; Vilte, D A; Pistone-Creydt, V; Chinen, I; Ibarra, C; Cataldi, A; Mercado, E C

    2012-05-04

    Pets can be reservoirs of Shiga toxin-producing Escherichia coli (STEC) strains. The aim of this study was to examine nine strains belonging to several serotypes (O91:H21, O91:H16, O178:H19, O8:H19, O22:H8, O22:HNT, ONT:H8), previously recovered from cats or dogs. To this end, we assessed a set of additional virulence genes (stx(2) subtype, subAB, ehxA, eae and saa), cytotoxic activity, and genetic relationships with strains isolated from cattle, meat and humans using pulsed-field gel electrophoresis (PFGE). Most of the isolates carried the stx(2) and/or stx(2vh-b) sequences, while only the O91:H21 isolate presented the mucus-activatable stx(2d) variant, as confirmed by sequencing the genes of subunits A and B. All the strains showed cytotoxic activity in cultured cells. One of the two O178:H19, selected for its high level of cytotoxicity in Vero cells, showed the ability to cause functional alterations in the human colon mucosa in vitro. None of the strains possessed the subAB, eae or saa genes and only the strains belonging to serotype O8:H19 carried the ehxA gene. The isolates shared 90-100% similarity by PFGE to epidemiologically unrelated strains of the corresponding serotypes recovered from cattle, meat or humans. Our results demonstrate that dogs and cats may have a role in the infection of humans by STEC, probably serving as a vehicle for bovine strains in the cycle of human infection, and thus emphasize the health risks for owners and their families. Copyright © 2011 Elsevier B.V. All rights reserved.

  9. Antimicrobial resistance in Shiga toxin-producing Escherichia coli serogroups O157 and O26 isolated from human cases of diarrhoeal disease in England, 2015.

    Science.gov (United States)

    Day, Martin; Doumith, Michel; Jenkins, Claire; Dallman, Timothy J; Hopkins, Katie L; Elson, Richard; Godbole, Gauri; Woodford, Neil

    2017-01-01

    Shiga toxin-producing Escherichia coli (STEC) are zoonotic and transmission to humans occurs via contaminated food or contact with infected animals. In this study, WGS data were used to predict antimicrobial resistance (AMR) in STEC from symptomatic human cases to assess the extent of transmission of antibiotic-resistant E. coli from animals to humans. WGS data from 430 isolates of STEC were mapped to genes known to be associated with phenotypic AMR. Susceptibility testing was performed by a breakpoint method on all viable isolates exhibiting resistance to at least one antimicrobial. 327/396 (82.6%) of STEC O157 and 22/34 (64.7%) of STEC O26 lacked identifiable resistance genes and were predicted to be fully susceptible to 11 diverse classes of antimicrobials. For the remaining 81 isolates, 74 were phenotypically tested and there was concordance between WGS-predicted resistance and expression of phenotypic resistance. The most common resistance profile was ampicillin, streptomycin, trimethoprim/sulphonamide and tetracycline occurring in 25 (5.8%) isolates. Resistance to other antimicrobials, including resistance to chloramphenicol (2.1%), resistance to azithromycin (0.2%) and reduced susceptibility to ciprofloxacin (2.6%), was less frequent. Three isolates were identified as ESBL producers. β-Lactams, trimethoprim/sulphonamides and tetracyclines account for the majority of therapeutic antimicrobials sold for veterinary use and this may be a risk factor for the presence of AMR in domestically acquired human clinical isolates of STEC. Isolates that were resistant to ampicillin, streptomycin, sulphonamide, tetracycline and azithromycin and had reduced susceptibility to ciprofloxacin were associated with cases who reported recent travel abroad. © Crown copyright 2016.

  10. Emerging types of Shiga toxin-producing E. coli (STEC O178 present in cattle, deer and humans from Argentina and Germany

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    Angelika eMiko

    2014-06-01

    Full Text Available More than 400 serotypes of Shiga toxin-producing Escherichia coli (STEC have been implicated in outbreaks and sporadic human diseases. In recent years STEC strains belonging to serogroup O178 have been commonly isolated from cattle and food of bovine origin in South America and Europe. In order to explore the significance of these STEC strains as potential human pathogens, 74 German and Argentinean E. coli O178 strains from animals, food and humans were characterized phenotypically and investigated for their serotypes, stx-genotypes and forty-three virulence-associated markers by a real-time PCR-microarray. The majority (n=66 of the O178 strains belonged to serotype O178:H19. The remaining strains divided into O178:H7 (n=6, O178:H10 (n=1 and O178:H16 (n=1. STEC O178:H19 strains were mainly isolated from cattle and food of bovine origin, but one strain was from a patient with hemolytic uremic syndrome (HUS. Genotyping of the STEC O178:H19 strains by pulsed-field gel electrophoresis revealed two major clusters of genetically highly related strains which differ in their stx-genotypes and non-Stx putative virulence traits, including adhesins, toxins and serine-proteases. Cluster A-strains including the HUS-strain (n=35 carried genes associated with severe disease in humans (stx2a, stx2d, ehxA, saa, subAB1, lpfAO113, terE combined with stx1a, espP, iha. Cluster B-strains (n=26 showed a limited repertoire of virulence genes (stx2c, pagC, lpfAO113, espP, iha. Among O178:H7 strains isolated from deer meat and patients with uncomplicated disease a new STEC variant was detected that is associated with the genotype stx1c/stx2b/ehxA/subAB2/espI/[terE]/espP/iha. None of the STEC O178 strains was positive for locus of enterocyte effacement (LEE- and nle-genes. Results indicate that STEC O178:H19 strains belong to the growing group of LEE-negative STEC that should be considered with respect to their potential to cause diseases in humans.

  11. Emerging types of Shiga toxin-producing E. coli (STEC) O178 present in cattle, deer, and humans from Argentina and Germany

    Science.gov (United States)

    Miko, Angelika; Rivas, Marta; Bentancor, Adriana; Delannoy, Sabine; Fach, Patrick; Beutin, Lothar

    2014-01-01

    More than 400 serotypes of Shiga toxin-producing Escherichia coli (STEC) have been implicated in outbreaks and sporadic human diseases. In recent years STEC strains belonging to serogroup O178 have been commonly isolated from cattle and food of bovine origin in South America and Europe. In order to explore the significance of these STEC strains as potential human pathogens, 74 German and Argentinean E. coli O178 strains from animals, food and humans were characterized phenotypically and investigated for their serotypes, stx-genotypes and 43 virulence-associated markers by a real-time PCR-microarray. The majority (n = 66) of the O178 strains belonged to serotype O178:H19. The remaining strains divided into O178:H7 (n = 6), O178:H10 (n = 1), and O178:H16 (n = 1). STEC O178:H19 strains were mainly isolated from cattle and food of bovine origin, but one strain was from a patient with hemolytic uremic syndrome (HUS). Genotyping of the STEC O178:H19 strains by pulsed-field gel electrophoresis revealed two major clusters of genetically highly related strains which differ in their stx-genotypes and non-Stx putative virulence traits, including adhesins, toxins, and serine-proteases. Cluster A-strains including the HUS-strain (n = 35) carried genes associated with severe disease in humans (stx2a, stx2d, ehxA, saa, subAB1, lpfAO113, terE combined with stx1a, espP, iha). Cluster B-strains (n = 26) showed a limited repertoire of virulence genes (stx2c, pagC, lpfAO113, espP, iha). Among O178:H7 strains isolated from deer meat and patients with uncomplicated disease a new STEC variant was detected that is associated with the genotype stx1c/stx2b/ehxA/subAB2/espI/[terE]/espP/iha. None of the STEC O178 strains was positive for locus of enterocyte effacement (LEE)- and nle-genes. Results indicate that STEC O178:H19 strains belong to the growing group of LEE-negative STEC that should be considered with respect to their potential to cause diseases in humans. PMID:24987616

  12. Postdiarrheal Shiga Toxin-Mediated Hemolytic Uremic Syndrome Similar to Septic Shock Síndrome urémico hemolítico símil shock séptico, posterior a diarrea mediada por toxina shiga

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    Patricia G. Valles

    2005-10-01

    Full Text Available The inflammatory response of host endothelial cells is included in the development of vascular damage observed in enterohemorrhagic Escherichia coli (EHEC infection, resulting in hemolytic uremic syndrome (HUS. The response to a non-conventional treatment for a group of D+ HUS (diarrhea positive HUS patients, with clinical hemodynamic parameters of septic shock was evaluated in this prospective study (1999-2003. Twelve children 2.8 ± 0.6 years old, with D+ HUS produced by E. coli infection with serological evidence of Shiga toxin, presenting severe unstable hemodynamic parameters and neurological dysfunction at onset, were studied. The protocol included fresh frozen plasma infusions, methylprednisolone pulses (10mg/k/day for three consecutive days and plasma exchange for five days, starting after admission to the intensive care unit (ICU. The twelve patients with increased pediatric risk of mortality (PRISM score: 18 ± 2 after admission to intensive care unit (ICU, required dialysis for 17.4 ± 4 days, mechanical ventilator assistance for 10 ± 1 days and early inotropic drugs support for 10.5 ± 1 days. Neurological dysfunction included generalized tonic-clonic seizures lasting for 5.4 ± 1 days, n:8. Focal seizures were present in the remaining patients. Dilated cardiomyopathy was present in 6 children. Eight children suffered hemorrhagic colitis. Nine patients survived. Within one year of the injury, neurological sequelae, Glasgow outcome scale (GOS 3 and 4, were present in two patients, chronic renal failure in one patient. We suggest that early introduction of this protocol could benefit D+ HUS patients with hemodynamic instability and neurological dysfunction at onset. Further studies are likely to elucidate the mechanisms involved in this early adverse clinical presentation of D+ HUS patients.La respuesta inflamatoria de la célula endotelial se incluye en el desarrollo del daño vascular observado en la infección por Escherichia coli

  13. Antimicrobial resistance patterns of Shiga toxin-producing Escherichia coli O157:H7 and O157:H7- from different origins.

    Science.gov (United States)

    Srinivasan, Velusamy; Nguyen, Lien T; Headrick, Susan I; Murinda, Shelton E; Oliver, Stephen P

    2007-01-01

    Shiga toxin-producing Escherichia coli (STEC) serotypes including O157:H7 (n = 129) from dairy cows, cull dairy cow feces, cider, salami, human feces, ground beef, bulk tank milk, bovine feces, and lettuce; and O157:H7- (n = 24) isolated from bovine dairy and bovine feedlot cows were evaluated for antimicrobial resistance against 26 antimicrobials and the presence of antimicrobial resistance genes (tetA, tetB, tetC, tetD, tetE, tetG, floR, cmlA, strA, strB, sulI, sulII, and ampC). All E. coli exhibited resistance to five or more antimicrobial agents, and the majority of isolates carried one or more target antimicrobial resistance gene(s) in different combinations. The majority of E. coli showed resistance to ampicillin, aztreonam, cefaclor, cephalothin, cinoxacin, and nalidixic acid, and all isolates were susceptible to chloramphenicol and florfenicol. Many STEC O157:H7 and O157:H7-isolates were susceptible to amikacin, carbenicillin, ceftriaxone, cefuroxime, ciprofloxacin, fosfomycin, moxalactam, norfloxacin, streptomycin, tobramycin, trimethoprim, and tetracycline. The majority of STEC O157:H7 (79.8%) and O157:H7- (91.7%) carried one or more antimicrobial resistance gene(s) regardless of whether phenotypically resistant or susceptible. Four tetracycline resistant STEC O157:H7 isolates carried both tetA and tetC. Other tetracycline resistance genes (tetB, tetD, tetE, and tetG) were not detected in any of the isolates. Among nine streptomycin resistant STEC O157:H7 isolates, eight carried strA-strB along with aadA, whereas the other isolate carried aadA alone. However, the majority of tetracycline and streptomycin susceptible STEC isolates also carried tetA and aadA genes, respectively. Most ampicillin resistant E. coli of both serotypes carried ampC genes. Among sulfonamide resistance genes, sulII was detected only in STEC O157:H7 (4 of 80 sulfonamide-resistant isolates) and sulI was detected in O157:H7- (1 of 16 sulfonamide resistant isolates). The emergence and

  14. Microbiological analysis of pre-packed sweet basil (Ocimum basilicum) and coriander (Coriandrum sativum) leaves for the presence of Salmonella spp. and Shiga toxin-producing E. coli.

    Science.gov (United States)

    Delbeke, Stefanie; Ceuppens, Siele; Jacxsens, Liesbeth; Uyttendaele, Mieke

    2015-09-02

    Enteric pathogens, such as Salmonella spp. and pathogenic Escherichia coli, have been detected and associated with food borne outbreaks from (imported) fresh leafy herbs. Screening on imported herbs from South East Asian countries has been described. However, limited information on prevalence of these pathogens is available from other sourcing regions. Therefore, fresh pre-packed basil and coriander leaves from a Belgian trading company were investigated for the presence of Salmonella spp., Shiga toxin-producing E. coli (STEC), generic E. coli and coliforms. In total 592 samples were collected originating from Belgium, Israel and Cyprus during 2013-2014. Multiplex PCR followed by further culture confirmation was used for the detection of Salmonella spp. and STEC, whereas the Petrifilm Select E. coli and VRBL-agar were used, respectively, for the enumeration of E. coli and coliforms. Salmonella was detected in 10 out of 592 samples (25g) (1.7%; 5 from basil and 5 from coriander), of which two samples were sourced from Israel and eight from Cyprus. The presence of STEC was suspected in 11 out of 592 samples (25g) (1.9%; 3 basil and 8 coriander), due to the detection of stx and eae genes, of which one sample originated from Belgium, four from Israel and six from Cyprus. No STEC was isolated by culture techniques, but in three samples a serotype (O26, O103 or O111) with its most likely associated eae-variant (β or θ) was detected by PCR. Generic E. coli was enumerated in 108 out of 592 samples, whereby 55, 32 and 13 samples respectively between 10-100, 100-1000 and 1000-10,000cfu/g and 8 samples exceeding 10,000cfu/g. Coliforms were enumerated in all herb samples at variable levels ranging from 1.6 to 7.5logcfu/g. Further statistics indicate that the E. coli class (categorized by level) was significantly correlated with the presence of Salmonella (p<0.001) or STEC (p=0.019), while coliform counts were significant correlated with Salmonella (p<0.001), but not with

  15. Isolation and characterization of non-O157 Shiga toxin-producing Escherichia coli from beef carcasses, cuts and trimmings of abattoirs in Argentina

    Science.gov (United States)

    Brusa, Victoria; Restovich, Viviana; Galli, Lucía; Teitelbaum, David; Signorini, Marcelo; Brasesco, Hebe; Londero, Alejandra; García, Diego; Padola, Nora Lía; Superno, Valeria; Sanz, Marcelo; Petroli, Sandra; Costa, Magdalena; Bruzzone, Mariana; Sucari, Adriana; Ferreghini, Marcela; Linares, Luciano; Suberbie, Germán; Rodríguez, Ricardo

    2017-01-01

    Several foods contaminated with Shiga toxin-producing Escherichia coli (STEC) are associated with human diseases. Some countries have established microbiological criteria for non-O157 STEC, thus, the absence of serogroups O26, O45, O103, O104, O111, O121, and O145 in sprouts from the European Union or ground beef and beef trimmings from the United States is mandatory. While in Argentina screening for O26, O103, O111, O145 and O121 in ground beef, ready-to-eat food, sausages and vegetables is mandatory, other countries have zero-tolerance for all STEC in chilled beef. The aim of this study was to provide data on the prevalence of non-O157 STEC isolated from beef processed in eight Argentinean cattle slaughterhouses producing beef for export and local markets, and to know the non-O157 STEC profiles through strain characterization and genotypic analysis. Samples (n = 15,965) from 3,205 beef carcasses, 9,570 cuts and 3,190 trimmings collected between March and September 2014 were processed in pools of five samples each. Pools of samples (n = 3,193) from 641 carcasses, 1,914 cuts and 638 trimming were analyzed for non-O157 STEC isolation according to ISO/CEN 13136:2012. Of these, 37 pools of carcasses (5.8%), 111 pools of cuts (5.8%) and 45 pools of trimmings (7.0%) were positive for non-O157 STEC. STEC strains (n = 200) were isolated from 193 pools of samples. The most prevalent serotypes were O174:H21, O185:H7, O8:H19, O178:H19 and O130:H11, and the most prevalent genotypes were stx2c(vh-b) and stx2a/saa/ehxA. O103:H21 strain was eae-positive and one O178:H19 strain was aggR/aaiC-positive. The prevalence of non-O157 STEC in beef carcasses reported here was low. None of the non-O157 STEC strains isolated corresponded to the non-O157 STEC serotypes and virulence profiles isolated from human cases in Argentina in the same study period. The application of microbiological criteria for each foodstuff should be determined by risk analysis in order to have a stringent

  16. Isolation and characterization of non-O157 Shiga toxin-producing Escherichia coli from beef carcasses, cuts and trimmings of abattoirs in Argentina.

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    Victoria Brusa

    Full Text Available Several foods contaminated with Shiga toxin-producing Escherichia coli (STEC are associated with human diseases. Some countries have established microbiological criteria for non-O157 STEC, thus, the absence of serogroups O26, O45, O103, O104, O111, O121, and O145 in sprouts from the European Union or ground beef and beef trimmings from the United States is mandatory. While in Argentina screening for O26, O103, O111, O145 and O121 in ground beef, ready-to-eat food, sausages and vegetables is mandatory, other countries have zero-tolerance for all STEC in chilled beef. The aim of this study was to provide data on the prevalence of non-O157 STEC isolated from beef processed in eight Argentinean cattle slaughterhouses producing beef for export and local markets, and to know the non-O157 STEC profiles through strain characterization and genotypic analysis. Samples (n = 15,965 from 3,205 beef carcasses, 9,570 cuts and 3,190 trimmings collected between March and September 2014 were processed in pools of five samples each. Pools of samples (n = 3,193 from 641 carcasses, 1,914 cuts and 638 trimming were analyzed for non-O157 STEC isolation according to ISO/CEN 13136:2012. Of these, 37 pools of carcasses (5.8%, 111 pools of cuts (5.8% and 45 pools of trimmings (7.0% were positive for non-O157 STEC. STEC strains (n = 200 were isolated from 193 pools of samples. The most prevalent serotypes were O174:H21, O185:H7, O8:H19, O178:H19 and O130:H11, and the most prevalent genotypes were stx2c(vh-b and stx2a/saa/ehxA. O103:H21 strain was eae-positive and one O178:H19 strain was aggR/aaiC-positive. The prevalence of non-O157 STEC in beef carcasses reported here was low. None of the non-O157 STEC strains isolated corresponded to the non-O157 STEC serotypes and virulence profiles isolated from human cases in Argentina in the same study period. The application of microbiological criteria for each foodstuff should be determined by risk analysis in order to have a

  17. Application of whole genome sequence data in analyzing the molecular epidemiology of Shiga toxin-producing Escherichia coli O157:H7/H.

    Science.gov (United States)

    Yokoyama, Eiji; Hirai, Shinichiro; Ishige, Taichiro; Murakami, Satoshi

    2018-01-02

    Seventeen clusters of Shiga toxin-producing Escherichia coli O157:H7/- (O157) strains, determined by cluster analysis of pulsed-field gel electrophoresis patterns, were analyzed using whole genome sequence (WGS) data to investigate this pathogen's molecular epidemiology. The 17 clusters included 136 strains containing strains from nine outbreaks, with each outbreak caused by a single source contaminated with the organism, as shown by epidemiological contact surveys. WGS data of these strains were used to identify single nucleotide polymorphisms (SNPs) by two methods: short read data were directly mapped to a reference genome (mapping derived SNPs) and common SNPs between the mapping derived SNPs and SNPs in assembled data of short read data (common SNPs). Among both SNPs, those that were detected in genes with a gap were excluded to remove ambiguous SNPs from further analysis. The effectiveness of both SNPs was investigated among all the concatenated SNPs that were detected (whole SNP set); SNPs were divided into three categories based on the genes in which they were located (i.e., backbone SNP set, O-island SNP set, and mobile element SNP set); and SNPs in non-coding regions (intergenic region SNP set). When SNPs from strains isolated from the nine single source derived outbreaks were analyzed using an unweighted pair group method with arithmetic mean tree (UPGMA) and a minimum spanning tree (MST), the maximum pair-wise distances of the backbone SNP set of the mapping derived SNPs were significantly smaller than those of the whole and intergenic region SNP set on both UPGMAs and MSTs. This significant difference was also observed when the backbone SNP set of the common SNPs were examined (Steel-Dwass test, P≤0.01). When the maximum pair-wise distances were compared between the mapping derived and common SNPs, significant differences were observed in those of the whole, mobile element, and intergenic region SNP set (Wilcoxon signed rank test, P≤0.01). When all

  18. [Detection and characterization of Shiga toxin-producing Escherichia coli in children treated at an inter-zonal pediatric hospital in the city of La Plata].

    Science.gov (United States)

    Oderiz, Sebastián; Leotta, Gerardo A; Galli, Lucía

    2018-01-11

    Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen that can cause watery diarrhea, bloody diarrhea (BD), and hemolytic uremic syndrome (HUS). The objective of this study was to determine the phenotypic and genotypic profiles of STEC strains isolated from children with BD and HUS treated at a pediatric hospital in the city of La Plata in the period 2006-2012, and to establish the clonal relationship of O157:H7 isolates by pulsed field electrophoresis. The percentage of positive samples was 4.9% and 39.2% in patients with BD and HUS, respectively. Seventy-seven STEC strains from 10 different serotypes were isolated, with 100% colony recovery, O157:H7 being the most frequent (71.4%) serotype, followed by O145:NM (15.6%). An average of 98.2% of O157:H7 isolates belonged to biotype C and were sensitive to all the antibiotics tested. All of them (100%) carried genotype stx 2 , eae, fliC H7 , ehxA, iha, efa, toxB, lpfA1-3 and lpfA2-2. When the clonal relationship of the O157:H7 strains was studied, a total of 42 patterns with at least 88% similarity were identified, and 6 clusters with identical profiles were established. The eae-negative isolates belonged to serotypes O59:H19, O102:H6, O174:NM and O174:H21. The strains O59:H19 and O174:H21 were positive for the aggR gene. This study shows that STEC of different serotypes and genotypes circulate in the city of La Plata and surroundings. Despite the genetic diversity observed between the O157:H7 isolates, some were indistinguishable by the subtyping techniques used. Copyright © 2017 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  19. Faecal carriage of extended-spectrum β-lactamase-producing Enterobacteriaceae and Shiga toxin-producing Escherichia coli in asymptomatic nursery children in Lower Saxony (Germany), 2014.

    Science.gov (United States)

    Harries, M; Dreesman, J; Rettenbacher-Riefler, S; Mertens, E

    2016-09-09

    Children may be at higher risk for carriage of antimicrobial-resistant bacteria because of higher usage of antimicrobials. They also have higher rates of Shiga toxin-producing Escherichia coli (STEC) infections than other population groups. Some infections, particularly in children, are asymptomatic, but still lead to the excretion of large numbers of bacteria and viruses that may cause clinical disease in other individuals. That is one reason why, in Lower Saxony as in other German federal states - asymptomatic carriers of STEC are excluded from nurseries and schools until three consecutive stool samples test negative in order to prevent secondary cases. The prevalence of children who are asymptomatic STEC carriers is unknown. But if it is high, this measure would have substantial socioeconomic effects on families. Infections with extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E) are an increasing problem for public health, especially for hospitals. However, there are no reliable estimates of the prevalence of asymptomatic ESBL-E carriers in Lower Saxony, as there is no mandatory requirement to report these carriers. In order to discuss the exclusion policies for children attending nurseries and ascertain a baseline of ESBL-E carriers, we conducted a cross-sectional study. The aim was to determine the prevalence of ESBL-E and STEC and identify risk factors for carriage in nursery children without diarrhoea (asymptomatic) aged 0-6 years in four selected districts in Northern Germany. During April-September 2014, we collected stool specimens with the support of voluntarily participating nurseries. We tested for STEC by PCR and for ESBL-E on chromogenic agar. Questionnaires answered by parents contained data on eating and drinking habits, outdoor activities, prior antibiotic treatment and animal contact for each participating child. We compared the epidemiological characteristics of ESBL-E carriers vs. non-carriers by using univariable analysis (P

  20. Development of three multiplex PCR assays targeting the 21 most clinically relevant serogroups associated with Shiga toxin-producing E. coli infection in humans.

    Science.gov (United States)

    Sánchez, Sergio; Llorente, María Teresa; Echeita, María Aurora; Herrera-León, Silvia

    2015-01-01

    Escherichia coli serogroups O5, O15, O26, O45, O55, O76, O91, O103, O104, O111, O113, O118, O121, O123, O128, O145, O146, O157, O165, O172, and O177 are the O-antigen forms of the most clinically relevant Shiga toxin-producing E. coli (STEC) serotypes. In this study, three multiplex PCR assays able to specifically detect these 21 serogroups were developed and validated. For this purpose, the O-antigen gene clusters of E. coli O5 and O76 were fully sequenced, their associated genes were identified on the basis of homology, and serogroup-specific primers were designed. After preliminary evaluation, these two primer pairs were proven to be highly specific and suitable for the development of PCR assays for O5 and O76 serogroup identification. Specific primers were also designed for serogroups O15, O45, O55, O91, O104, O113, O118, O123, O128, O146, O157, O165, O172, and O177 based on previously published sequences, and previously published specific primers for serogroups O26, O103, O111, O121, and O145 were also included. These 21 primer pairs were shown to be specific for their target serogroup when tested against E. coli type strains representing 169 known O-antigen forms of E. coli and Shigella and therefore suitable for being used in PCR assays for serogroup identification. In order to validate the three multiplex PCR assays, 22 E. coli strains belonging to the 21 covered serogroups and 18 E. coli strains belonging to other serogroups were screened in a double-blind test and their sensitivity was determined as 1 ng chromosomal DNA. The PCR assays developed in this study could be a faster, simpler, and less expensive strategy for serotyping of the most clinically relevant STEC strains in both clinical microbiology and public health laboratories, and so their development could benefit for clinical diagnosis, epidemiological investigations, surveillance, and control of STEC infections.

  1. Development of three multiplex PCR assays targeting the 21 most clinically relevant serogroups associated with Shiga toxin-producing E. coli infection in humans.

    Directory of Open Access Journals (Sweden)

    Sergio Sánchez

    Full Text Available Escherichia coli serogroups O5, O15, O26, O45, O55, O76, O91, O103, O104, O111, O113, O118, O121, O123, O128, O145, O146, O157, O165, O172, and O177 are the O-antigen forms of the most clinically relevant Shiga toxin-producing E. coli (STEC serotypes. In this study, three multiplex PCR assays able to specifically detect these 21 serogroups were developed and validated. For this purpose, the O-antigen gene clusters of E. coli O5 and O76 were fully sequenced, their associated genes were identified on the basis of homology, and serogroup-specific primers were designed. After preliminary evaluation, these two primer pairs were proven to be highly specific and suitable for the development of PCR assays for O5 and O76 serogroup identification. Specific primers were also designed for serogroups O15, O45, O55, O91, O104, O113, O118, O123, O128, O146, O157, O165, O172, and O177 based on previously published sequences, and previously published specific primers for serogroups O26, O103, O111, O121, and O145 were also included. These 21 primer pairs were shown to be specific for their target serogroup when tested against E. coli type strains representing 169 known O-antigen forms of E. coli and Shigella and therefore suitable for being used in PCR assays for serogroup identification. In order to validate the three multiplex PCR assays, 22 E. coli strains belonging to the 21 covered serogroups and 18 E. coli strains belonging to other serogroups were screened in a double-blind test and their sensitivity was determined as 1 ng chromosomal DNA. The PCR assays developed in this study could be a faster, simpler, and less expensive strategy for serotyping of the most clinically relevant STEC strains in both clinical microbiology and public health laboratories, and so their development could benefit for clinical diagnosis, epidemiological investigations, surveillance, and control of STEC infections.

  2. Genotypic Analyses of Shiga Toxin-Producing Escherichia coli O157 and Non-O157 Recovered from Feces of Domestic Animals on Rural Farms in Mexico

    Science.gov (United States)

    Amézquita-López, Bianca A.; Quiñones, Beatriz; Cooley, Michael B.; León-Félix, Josefina; Castro-del Campo, Nohelia; Mandrell, Robert E.; Jiménez, Maribel; Chaidez, Cristóbal

    2012-01-01

    Shiga toxin-producing Escherichia coli (STEC) are zoonotic enteric pathogens associated with human gastroenteritis worldwide. Cattle and small ruminants are important animal reservoirs of STEC. The present study investigated animal reservoirs for STEC in small rural farms in the Culiacan Valley, an important agricultural region located in Northwest Mexico. A total of 240 fecal samples from domestic animals were collected from five sampling sites in the Culiacan Valley and were subjected to an enrichment protocol followed by either direct plating or immunomagnetic separation before plating on selective media. Serotype O157:H7 isolates with the virulence genes stx2, eae, and ehxA were identified in 40% (26/65) of the recovered isolates from cattle, sheep and chicken feces. Pulse-field gel electrophoresis (PFGE) analysis grouped most O157:H7 isolates into two clusters with 98.6% homology. The use of multiple-locus variable-number tandem repeat analysis (MLVA) differentiated isolates that were indistinguishable by PFGE. Analysis of the allelic diversity of MLVA loci suggested that the O157:H7 isolates from this region were highly related. In contrast to O157:H7 isolates, a greater genotypic diversity was observed in the non-O157 isolates, resulting in 23 PFGE types and 14 MLVA types. The relevant non-O157 serotypes O8:H19, O75:H8, O111:H8 and O146:H21 represented 35.4% (23/65) of the recovered isolates. In particular, 18.5% (12/65) of all the isolates were serotype O75:H8, which was the most variable serotype by both PFGE and MLVA. The non-O157 isolates were predominantly recovered from sheep and were identified to harbor either one or two stx genes. Most non-O157 isolates were ehxA-positive (86.5%, 32/37) but only 10.8% (4/37) harbored eae. These findings indicate that zoonotic STEC with genotypes associated with human illness are present in animals on small farms within rural communities in the Culiacan Valley and emphasize the need for the development of control

  3. Antimicrobial Effects of Quillaja saponaria Extract Against Escherichia coli O157:H7 and the Emerging Non-O157 Shiga Toxin-Producing E. coli.

    Science.gov (United States)

    Sewlikar, Snigdha; D'Souza, Doris H

    2017-05-01

    Natural alternate methods to control the spread of Shiga toxin-producing Escherichia coli (STEC) are important to prevent foodborne outbreaks. Quillaja saponaria aqueous bark extracts (QE), cleared by the U.S. Food and Drug Administration as a natural flavorant, contain bioactive polyphenols, tannins, and tri-terpenoid saponins with anti-inflammatory and antimicrobial activity. The objective of this study was to determine the effects of commercial QE against E. coli O157:H7 and non-O157 strains over 16 h at 37 °C and RT. Overnight cultures of 4 E. coli O157:H7 strains and 6 non-O157 STECs in Tryptic Soy Broth (TSB) were washed and resuspended in phosphate-buffered saline (PBS, pH 7.2), and treated with QE and controls including citric acid (pH 3.75), sodium benzoate (0.1% w/w), acidified sodium benzoate (pH 3.75) or PBS for 6 h or 16 h. Recovered bacteria were enumerated after plating on Tryptic Soy Agar, from duplicate treatments, replicated thrice and the data were statistically analyzed. The 4 QE-treated E. coli O157:H7 strains from initial ∼7.5 log CFU had remaining counts between 6.79 and 3.5 log CFU after 16 h at RT. QE-treated non-O157 STECs showed lower reductions with remaining counts ranging from 6.81 to 4.55 log CFU after 16 h at RT.  Incubation at 37 °C caused reduction to nondetectable levels within 1 h, without any significant reduction in controls. Scanning electron microscopy studies revealed damaged cell membranes of treated bacteria after 1 h at 37 °C. QE shows potential to control the spread of STECs, and further research in model food systems is needed. © 2017 Institute of Food Technologists®.

  4. Reductions of Shiga toxin-producing Escherichia coli and Salmonella typhimurium on beef trim by lactic acid, levulinic acid, and sodium dodecyl sulfate treatments.

    Science.gov (United States)

    Zhao, Tong; Zhao, Ping; Chen, Dong; Jadeja, Ravirajsinh; Hung, Yen-Con; Doyle, Michael P

    2014-04-01

    Studies were done at 21 °C to determine the bactericidal activity of lactic acid, levulinic acid, and sodium dodecyl sulfate (SDS) applied individually and in combination on Shiga toxin-producing Escherichia coli (STEC) in pure culture and to compare the efficacy of lactic acid and levulinic acid plus SDS treatments applied by spray or immersion to inactivate STEC and Salmonella (10(7) CFU/cm2) on beef trim pieces (10 by 10 by 7.5 cm). Application of 3% lactic acid for 2 min to pure cultures was shown to reduce E. coli O26:H11, O45:H2, O111:H8, O103:H2, O121:H2, O145:NM, and O157:H7 populations by 2.1, 0.4, 0.3, 1.4, 0.3, 2.1, and 1.7 log CFU/ml, respectively. Treatment with 0.5% levulinic acid plus 0.05% SDS for 6 log/ml reduction). Beef surface temperature was found to affect the bactericidal activity of treatment with 3 % levulinic acid plus 2% SDS (LV-SDS). Treating cold (4 °C) beef trim with LV-SDS at 21, 62, or 81 °C for 30 s reduced E. coli O157:H7 by 1.0, 1.1, or 1.4 log CFU/cm2, respectively, whereas treating beef trim at 8 °C with LV-SDS at 12 °C for 0.1, 1, 3, or 5 min reduced E. coli O157:H7 by 1.4, 2.4, 2.5, or 3.3 log CFU/cm(2), respectively. Spray treatment of beef trim at 4 °C with 5 % lactic acid only reduced the E. coli O157:H7 population by 1.3 log CFU/cm2. Treating beef trim at 8 °C with LV-SDS for 1, 2, or 3 min reduced Salmonella Typhimurium by 2.1, 2.6, and >5.0 log CFU/cm2, respectively. Hand massaging the treated beef trim substantially reduced contamination of both pathogens, with no detectable E. coli O157:H7 or Salmonella Typhimurium (<5 CFU/cm2) on beef trim pieces treated with LV-SDS. Reduction of E. coli O157:H7 and Salmonella Typhimurium populations was enhanced, but bactericidal activity was affected by the meat temperature.

  5. Longitudinal study of two Irish dairy herds: Low numbers of of Shiga toxin-producing Escherichia coli O157 and O26 super-shedders identified

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    Brenda Patricia Murphy

    2016-11-01

    Full Text Available A 12-month longitudinal study was undertaken on two dairy herds to ascertain the Shiga-toxin producing Escherichia coli (STEC O157 and O26 shedding status of the animals and its impact (if any on raw milk. Cattle are a recognised reservoir for these organisms with associated public health and environmental implications. Animals shedding E. coli O157 at >10,000 CFU/g of faeces have been deemed super-shedders. There is a gap in the knowledge regarding super-shedding of other STEC serogroups. A cohort of 40 lactating cows from herds previously identified as positive for VTEC in a national surveillance project were sampled every second month between August, 2013 and July, 2014. Metadata on any potential super-shedders was documented including e.g. age of the animal, number of lactations and days in lactation, nutritional condition, somatic cell count and content of protein in milk to assess if any were associated with risk factors for super-shedding. Recto-anal mucosal swabs, raw milk, milk filters and water samples were procured for each herd. The swabs were examined for E. coli O157 and O26 using a quantitative real time PCR method. Counts (CFU swab-1 were obtained from a standard calibration curve that related real-time PCR cycle threshold (Ct values against the initial concentration of O157 or O26 in the samples. Results from Farm A: 305 animals were analysed; 15 E. coli O157 (5% were recovered, 13 were denoted STEC encoding either stx1 and/or stx2 virulence genes and 5 (2% STEC O26 were recovered. One super-shedder was identified shedding STEC O26 (stx1&2. Farm B: 224 animals were analysed; eight E. coli O157 (3.5% were recovered (seven were STEC and 9 (4% STEC O26 were recovered. Three super-shedders were identified, one was shedding STEC O157 (stx2 and two STEC O26 (stx2. Three encoded the adhering and effacement gene (eae and one isolate additionally encoded the haemolysin gene (hlyA. The results of this study show, low numbers of super

  6. Implementation of Whole Genome Sequencing (WGS for Identification and Characterization of Shiga Toxin-Producing Escherichia coli (STEC in the United States

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    Rebecca L Lindsey

    2016-05-01

    Full Text Available Shiga toxin-producing Escherichia coli (STEC is an important foodborne pathogen capable of causing severe disease in humans. Rapid and accurate identification and characterization techniques are essential during outbreak investigations. Current methods for characterization of STEC are expensive and time-consuming. With the advent of rapid and cheap whole genome sequencing (WGS benchtop sequencers, the potential exists to replace traditional workflows with WGS. The aim of this study was to validate tools to do reference identification and characterization from WGS for STEC in a single workflow within an easy to use commercially available software platform. Publically available serotype, virulence, and antimicrobial resistance databases were downloaded from the Center for Genomic Epidemiology (CGE (www.genomicepidemiology.org and integrated into a genotyping plug-in with in silico PCR tools to confirm some of the virulence genes detected from WGS data. Additionally, down sampling experiments on the WGS sequence data were performed to determine a threshold for sequence coverage needed to accurately predict serotype and virulence genes using the established workflow. The serotype database was tested on a total of 228 genomes and correctly predicted from WGS for 96.1% of O serogroups and 96.5% of H serogroups identified by conventional testing techniques. A total of 59 genomes were evaluated to determine the threshold of coverage to detect the different WGS targets, 40 were evaluated for serotype and virulence gene detection and 19 for the stx gene subtypes. For serotype, 95% of the O and 100% of the H serogroups were detected at > 40x and ≥ 30x coverage, respectively. For virulence targets and stx gene subtypes, nearly all genes were detected at > 40x, though some targets were 100% detectable from genomes with coverage ≥20x. The resistance detection tool was 97% concordant with phenotypic testing results. With isolates sequenced to > 40x

  7. Detection by hyperspectral imaging of shiga toxin-producing Escherichia coli serogroups O26, O45, O103, O111, O121, and O145 on rainbow agar.

    Science.gov (United States)

    Windham, William R; Yoon, Seung-Chul; Ladely, Scott R; Haley, Jennifer A; Heitschmidt, Jerry W; Lawrence, Kurt C; Park, Bosoon; Narrang, Neelam; Cray, William C

    2013-07-01

    The U.S. Department of Agriculture, Food Safety Inspection Service has determined that six non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, and O145) are adulterants in raw beef. Isolate and phenotypic discrimination of non-O157 STEC is problematic due to the lack of suitable agar media. The lack of distinct phenotypic color variation among non-O157serogroups cultured on chromogenic agar poses a challenge in selecting colonies for confirmation. In this study, visible and near-infrared hyperspectral imaging and chemometrics were used to detect and classify non-O157 STEC serogroups grown on Rainbow agar O157. The method was first developed by building spectral libraries for each serogroup obtained from ground-truth regions of interest representing the true identity of each pixel and thus each pure culture colony in the hyperspectral agar-plate image. The spectral library for the pure-culture non-O157 STEC consisted of 2,171 colonies, with spectra derived from 124,347 of pixels. The classification models for each serogroup were developed with a k nearest-neighbor classifier. The overall classification training accuracy at the colony level was 99%. The classifier was validated with ground beef enrichments artificially inoculated with 10, 50, and 100 CFU/ml STEC. The validation ground-truth regions of interest of the STEC target colonies consisted of 606 colonies, with 3,030 pixels of spectra. The overall classification accuracy was 98%. The average specificity of the method was 98% due to the low false-positive rate of 1.2%. The sensitivity ranged from 78 to 100% due to the false-negative rates of 22, 7, and 8% for O145, O45, and O26, respectively. This study showed the potential of visible and near-infrared hyperspectral imaging for detecting and classifying colonies of the six non-O157 STEC serogroups. The technique needs to be validated with bacterial cultures directly extracted from meat products and positive

  8. Sekuen Nukleotida Gene Shiga like toxin-2 dari Isolat Lokal Escherichia coli O157:H7 asal Hewan dan Manusia (NUCLEOTIDES SQUENCES OF SHIGA-LIKE TOXIN 2 GENES OF ESCHERICHIA COLI O157:H7 LOCAL ISOLATES ORIGINATED FROM ANIMALS AND HUMAN

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    I Wayan Suardana

    2017-04-01

    Full Text Available Animals/livestock, especially cattle, are known as the main reservoir of Escherichia coli O157: H7. As the only one of zoonotic E. coli, the pathogenicity of these bacteria is determined by its ability to produce one or more very potent cytotoxin known as Shiga-like toxin (Stx or verocytotoxin, particularly of the Stx2 type that is closely related to the incidence of hemolytic uremic syndrome (HUS in humans. This study analyzed the nucleotide sequences of stx2 gene between isolates from animals and humans in an effort to assess the potential zoonoses of the agent. The research activity was initiated by cultivating 20 isolates of E. coli O157:H7 collection based on result in the previous study i.e. 2 isolates originated from cattle feces, 2 isolates originated from beef, 2 isolates originated from chicken feces, 2 isolates originated from human feces, and 12 non-clinical isolates originated from human fecal who were suffering with renal failure. All isolates were confirmed on selective medium Sorbitol MacConkey Agar (SMAC followed by testing on aglutination O157 latex test, and H7 antisera. Molecular analysis of stx2 gene covering open reading frame (ORF of the stx2 gene was performed using the primer which was designed by researcher i.e. Stx2 (F/Stx2 (R. The results showed, there were 2 isolates i.e. KL-48 (2 originated from human feces and SM-25 (1 originated from cattle feces were positive for carrying a stx2 gene, which was marked by the 1587 bp PCR product. Analysis of sequencing showed both isolates had identical to stx2 nucleotide squences with E. phaga 933 as well as E. coli ATCC 933. These results indicate the both local isolates are potential as zoonotic agents with clinical effects similar to E. phaga 933 and E. coli ATCC 43894. ABSTRAK Hewan ternak khususnya sapi, dikenal sebagai reservoir utama Escherichia coli O157:H7. Sebagai satu-satunya serotipe E. coli yang bersifat zoonosis, patogenitas bakteri ini ditentukan oleh kemampuannya

  9. Mixed biofilm formation by Shiga toxin-producing Escherichia coli and Salmonella enterica serovar typhimurium enhanced bacterial resistance to sanitization due to extracellular polymeric substances

    Science.gov (United States)

    Shiga toxin–producing Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium are important foodborne pathogens capable of forming single-species biofilms or coexisting in multispecies biofilm communities. Bacterial biofilm cells are usually more resistant to sanitization than their pla...

  10. Serotypes, virulence genes and intimin types of Shiga toxin (verocytotoxin-producing Escherichia coli isolates from minced beef in Lugo (Spain from 1995 through 2003

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    Bernárdez María

    2007-03-01

    Full Text Available Abstract Background Shiga toxin-producing Escherichia coli (STEC have emerged as pathogens that can cause food-borne infections and severe and potentially fatal illnesses in humans, such as haemorrhagic colitis (HC and haemolytic uraemic syndrome (HUS. In Spain, like in many other countries, STEC strains have been frequently isolated from ruminants, and represent a significant cause of sporadic cases of human infection. In view of the lack of data on STEC isolated from food in Spain, the objectives of this study were to determine the level of microbiological contamination and the prevalence of STEC O157:H7 and non-O157 in a large sampling of minced beef collected from 30 local stores in Lugo city between 1995 and 2003. Also to establish if those STEC isolated from food possessed the same virulence profiles as STEC strains causing human infections. Results STEC were detected in 95 (12% of the 785 minced beef samples tested. STEC O157:H7 was isolated from eight (1.0% samples and non-O157 STEC from 90 (11% samples. Ninety-six STEC isolates were further characterized by PCR and serotyping. PCR showed that 28 (29% isolates carried stx1 genes, 49 (51% possessed stx2 genes, and 19 (20% both stx1 and stx2. Enterohemolysin (ehxA and intimin (eae virulence genes were detected in 43 (45% and in 25 (26% of the isolates, respectively. Typing of the eae variants detected four types: γ1 (nine isolates, β1 (eight isolates, ε1 (three isolates, and θ (two isolates. The majority (68% of STEC isolates belonged to serotypes previously detected in human STEC and 38% to serotypes associated with STEC isolated from patients with HUS. Ten new serotypes not previously described in raw beef products were also detected. The highly virulent seropathotypes O26:H11 stx1 eae-β1, O157:H7 stx1stx2 eae-γ1 and O157:H7 stx2eae-γ1, which are the most frequently observed among STEC causing human infections in Spain, were detected in 10 of the 96 STEC isolates. Furthermore

  11. Multilaboratory validation study of standardized multiple-locus variable-number tandem repeat analysis protocol for shiga toxin-producing Escherichia coli O157: a novel approach to normalize fragment size data between capillary electrophoresis platforms.

    Science.gov (United States)

    Hyytia-Trees, Eija; Lafon, Patricia; Vauterin, Paul; Ribot, Efrain M

    2010-02-01

    The PulseNet USA subtyping network recently established a standardized protocol for multiple-locus variable-number tandem repeat analysis (MLVA) to characterize Shiga toxin-producing Escherichia coli O157. To enable data comparisons from different laboratories in the same database, reproducibility and high quality of the data must be ensured. The aim of this study was to test the robustness and reproducibility of the proposed standardized protocol by subjecting it to a multilaboratory validation process and to address any discrepancies that may have arisen from the study. A set of 50 strains was tested in 10 PulseNet participating laboratories that used capillary electrophoresis instruments from two manufacturers. Six out of the 10 laboratories were able to generate correct MLVA types for 46 (92%) or more strains. The discrepancies in MLVA type assignment were caused mainly by difficulties in optimizing polymerase chain reactions that were attributed to technical inexperience of the staff and suboptimal quality of reagents and instrumentation. It was concluded that proper training of staff must be an integral part of technology transfer. The interlaboratory reproducibility of fragment sizing was excellent when the same capillary electrophoresis platform was used. However, sizing discrepancies of up to six base pairs for the same fragment were detected between the two platforms. These discrepancies were attributed to different dye and polymer chemistries employed by the manufacturers. A novel software script was developed to assign alleles based on two platform-specific (Beckman Coulter CEQ8000 and Applied Biosystems Genetic Analyzer 3130xl) look-up tables containing fragment size ranges for all alleles. The new allele assignment method was validated at the PulseNet central laboratory using a diverse set of 502 Shiga toxin-producing Escherichia coli O157 isolates. The validation confirmed that the script reliably assigned the same allele for the same fragment

  12. Antibodies derived from a toxoid MEFA (multiepitope fusion antigen) show neutralizing activities against heat-labile toxin (LT), heat-stable toxins (STa, STb), and Shiga toxin 2e (Stx2e) of porcine enterotoxigenic Escherichia coli (ETEC).

    Science.gov (United States)

    Rausch, Dana; Ruan, Xiaosai; Nandre, Rahul; Duan, Qiangde; Hashish, Emad; Casey, Thomas A; Zhang, Weiping

    2017-04-01

    Enterotoxigenic Escherichia coli (ETEC) strains are the main cause of diarrhea in pigs. Pig diarrhea especially post-weaning diarrhea remains one of the most important swine diseases. ETEC bacterial fimbriae including K88, F18, 987P, K99 and F41 promote bacterial attachment to intestinal epithelial cells and facilitate ETEC colonization in pig small intestine. ETEC enterotoxins including heat-labile toxin (LT) and heat-stable toxins type Ia (porcine-type STa) and type II (STb) stimulate fluid hyper-secretion, leading to watery diarrhea. Blocking bacteria colonization and/or neutralizing enterotoxicity of ETEC toxins are considered effective prevention against ETEC diarrhea. In this study, we applied the MEFA (multiepitope fusion antigen) strategy to create toxoid MEFAs that carried antigenic elements of ETEC toxins, and examined for broad antitoxin immunogenicity in a murine model. By embedding STa toxoid STa P12F (NTFYCCELCCNFACAGCY), a STb epitope (KKDLCEHY), and an epitope of Stx2e A subunit (QSYVSSLN) into the A1 peptide of a monomeric LT toxoid (LT R192G ), two toxoid MEFAs, 'LT R192G -STb-Stx2e-STa P12F ' and 'LT R192G -STb-Stx2e-3xSTa P12F ' which carried three copies of STa P12F , were constructed. Mice intraperitoneally immunized with each toxoid MEFA developed IgG antibodies to all four toxins. Induced antibodies showed in vitro neutralizing activities against LT, STa, STb and Stx2e toxins. Moreover, suckling piglets born by a gilt immunized with 'LT R192G -STb-Stx2e-3xSTa P12F ' were protected when challenged with ETEC strains, whereas piglets born by a control gilt developed diarrhea. Results from this study showed that the toxoid MEFA induced broadly antitoxin antibodies, and suggested potential application of the toxoid MEFA for developing a broad-spectrum vaccine against ETEC diarrhea in pigs. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Prevalence of Shiga toxin Producing Escherichia coli, Salmonella enterica and Listeria monocytogenes at Public Access Watershed Sites in a California Central Coast Agricultural Region

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    Michael B Cooley

    2014-03-01

    Full Text Available Produce contaminated with enteric pathogens is a major source of foodborne illness in the United States. Lakes, streams, rivers, and ponds were sampled with Moore swabs bi-monthly for over two years at 30 locations in the vicinity of a leafy green growing region on the Central California Coast and screened for Shiga toxin producing Escherichia coli (STEC, Salmonella enterica, and Listeria monocytogenes to evaluate the prevalence and persistence of pathogen subtypes. The prevalence of STEC from 1,386 samples was 11%; 110 samples (8% contained E. coli O157:H7 with the highest prevalence occurring close to cattle operations. Non-O157 STEC isolates represented major clinical O-types and 57% contained both shiga toxin types 1 and 2 and intimin. Multiple Locus Variable Number Tandem Repeat Analysis of STEC isolates indicated prevalent strains during the period of study. Notably, Salmonella was present at high levels throughout the sampling region with 65% prevalence in 1,405 samples resulting in 996 isolates with slightly lower prevalence in late autumn. There were 2, 8 and 14 sites that were Salmonella-positive over 90%, 80% and 70% of the time, respectively. The serotypes identified most often were 6,8:d:-, Typhimurium, and Give. Interestingly, analysis by Pulsed Field Gel Electrophoresis indicated persistence and transport of pulsotypes in the region over several years. In this original study of L. monocytogenes in the region prevalence was 43% of 1,405 samples resulting in 635 individual isolates. Over 85% of the isolates belonged to serotype 4b with serotypes 1/2a, 1/2b, 3a, 4d with 4e representing the rest, and there were 12 and 2 sites that were positive over 50% and 80% of the time, respectively. Although surface water is not directly used for irrigation in this region, transport to the produce can occur by other means. This environmental survey assesses initial contamination levels towards an understanding of transport leading to produce

  14. Rapid and simple method by combining FTA™ card DNA extraction with two set multiplex PCR for simultaneous detection of non-O157 Shiga toxin-producing Escherichia coli strains and virulence genes in food samples.

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    Kim, S A; Park, S H; Lee, S I; Ricke, S C

    2017-12-01

    The aim of this research was to optimize two multiplex polymerase chain reaction (PCR) assays that could simultaneously detect six non-O157 Shiga toxin-producing Escherichia coli (STEC) as well as the three virulence genes. We also investigated the potential of combining the FTA™ card-based DNA extraction with the multiplex PCR assays. Two multiplex PCR assays were optimized using six primer pairs for each non-O157 STEC serogroup and three primer pairs for virulence genes respectively. Each STEC strain specific primer pair only amplified 155, 238, 321, 438, 587 and 750 bp product for O26, O45, O103, O111, O121 and O145 respectively. Three virulence genes were successfully multiplexed: 375 bp for eae, 655 bp for stx1 and 477 bp for stx2. When two multiplex PCR assays were validated with ground beef samples, distinctive bands were also successfully produced. Since the two multiplex PCR examined here can be conducted under the same PCR conditions, the six non-O157 STEC and their virulence genes could be concurrently detected with one run on the thermocycler. In addition, all bands clearly appeared to be amplified by FTA card DNA extraction in the multiplex PCR assay from the ground beef sample, suggesting that an FTA card could be a viable sampling approach for rapid and simple DNA extraction to reduce time and labour and therefore may have practical use for the food industry. Two multiplex polymerase chain reaction (PCR) assays were optimized for discrimination of six non-O157 Shiga toxin-producing Escherichia coli (STEC) and identification of their major virulence genes within a single reaction, simultaneously. This study also determined the successful ability of the FTA™ card as an alternative to commercial DNA extraction method for conducting multiplex STEC PCR assays. The FTA™ card combined with multiplex PCR holds promise for the food industry by offering a simple and rapid DNA sample method for reducing time, cost and labour for detection of STEC in

  15. No evidence of the Shiga toxin-producing E. coli O104:H4 outbreak strain or enteroaggregative E. coli (EAEC found in cattle faeces in northern Germany, the hotspot of the 2011 HUS outbreak area

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    Wieler Lothar H

    2011-11-01

    Full Text Available Abstract Background Ruminants, in particular bovines, are the primary reservoir of Shiga toxin-producing E. coli (STEC, but whole genome analyses of the current German ESBL-producing O104:H4 outbreak strain of sequence type (ST 678 showed this strain to be highly similar to enteroaggregative E. coli (EAEC. Strains of the EAEC pathotype are basically adapted to the human host. To clarify whether in contrast to this paradigm, the O104:H4 outbreak strain and/or EAEC may also be able to colonize ruminants, we screened a total of 2.000 colonies from faecal samples of 100 cattle from 34 different farms - all located in the HUS outbreak region of Northern Germany - for genes associated with the O104:H4 HUS outbreak strain (stx2, terD, rfbO104, fliCH4, STEC (stx1, stx2, escV, EAEC (pAA, aggR, astA, and ESBL-production (blaCTX-M, blaTEM, blaSHV. Results The faecal samples contained neither the HUS outbreak strain nor any EAEC. As the current outbreak strain belongs to ST678 and displays an en-teroaggregative and ESBL-producing phenotype, we additionally screened selected strains for ST678 as well as the aggregative adhesion pattern in HEp-2 cells. However, we were unable to find any strains belonging to ST678 or showing an aggregative adhesion pattern. A high percentage of animals (28% shed STEC, corroborating previous knowl-edge and thereby proving the validity of our study. One of the STEC also harboured the LEE pathogenicity island. In addition, eleven animals shed ESBL-producing E. coli. Conclusions While we are aware of the limitations of our survey, our data support the theory, that, in contrast to other Shiga-toxin producing E. coli, cattle are not the reservoir for the O104:H4 outbreak strain or other EAEC, but that the outbreak strain seems to be adapted to humans or might have yet another reservoir, raising new questions about the epidemiology of STEC O104:H4.

  16. Development of a High Resolution Virulence Allelic Profiling (HReVAP Approach Based on the Accessory Genome of Escherichia coli to Characterize Shiga-toxin Producing E. coli (STEC

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    Valeria eMichelacci

    2016-02-01

    Full Text Available Shiga-toxin producing Escherichia coli (STEC strains possess a large accessory genome composed of virulence genes existing in multiple allelic variants, which sometimes segregate with specific STEC subpopulations. We analyzed the allelic variability of 91 virulence genes of STEC by Real Time PCR followed by melting curves analysis in 713 E. coli strains including 358 STEC. The 91 genes investigated were located on the LEE, OI-57 and OI-122 pathogenicity islands and displayed a total of 476 alleles in the study population. The combinations of the 91 alleles of each strain were termed allelic signatures and used to perform cluster analyses. We termed such an approach High Resolution Virulence Allelic Profiling (HReVAP and used it to investigate the phylogeny of STEC of multiple serogroups. The dendrograms obtained identified groups of STEC segregating approximately with the serogroups and allowed the identification of subpopulations within the single groups. The study of the allelic signatures provided further evidence of the coevolution of the LEE and OI-122, reflecting the occurrence of their acquisition through a single event. The HReVAP analysis represents a sensitive tool for studying the evolution of LEE-positive STEC.

  17. Efficacy of Urtoxazumab (TMA-15 Humanized Monoclonal Antibody Specific for Shiga Toxin 2 Against Post-Diarrheal Neurological Sequelae Caused by Escherichia coli O157:H7 Infection in the Neonatal Gnotobiotic Piglet Model

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    Rodney A. Moxley

    2017-01-01

    Full Text Available Enterohemorrhagic Escherichia coli (EHEC is the most common cause of hemorrhagic colitis and hemolytic uremic syndrome in human patients, with brain damage and dysfunction the main cause of acute death. We evaluated the efficacy of urtoxazumab (TMA-15, Teijin Pharma Limited, a humanized monoclonal antibody against Shiga toxin (Stx 2 for the prevention of brain damage, dysfunction, and death in a piglet EHEC infection model. Forty-five neonatal gnotobiotic piglets were inoculated orally with 3 × 109 colony-forming units of EHEC O157:H7 strain EDL933 (Stx1+, Stx2+ when 22–24 h old. At 24 h post-inoculation, piglets were intraperitoneally administered placebo or TMA-15 (0.3, 1.0 or 3.0 mg/kg body weight. Compared to placebo (n = 10, TMA-15 (n = 35 yielded a significantly greater probability of survival, length of survival, and weight gain (p <0.05. The efficacy of TMA-15 against brain lesions and death was 62.9% (p = 0.0004 and 71.4% (p = 0.0004, respectively. These results suggest that TMA-15 may potentially prevent or reduce vascular necrosis and infarction of the brain attributable to Stx2 in human patients acutely infected with EHEC. However, we do not infer that TMA-15 treatment will completely protect human patients infected with EHEC O157:H7 strains that produce both Stx1 and Stx2.

  18. Comparison of four enrichment broths for the detection of non-O157 Shiga-toxin-producing Escherichia coli O91, O103, O111, O119, O121, O145 and O165 from pure culture and food samples.

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    Kanki, M; Seto, K; Harada, T; Yonogi, S; Kumeda, Y

    2011-08-01

    We compared the efficiency of universal pre-enrichment broth (UPB), modified Escherichia coli broth containing novobiocin (mEC + n), modified Tryptic Soy Broth (mTSB) and mTSB with novobiocin (mTSB + n) for the enrichment of non-O157 Shiga-toxin-producing E. coli (STEC). Freeze-injured and control non-O157 STEC (O91, O103, O111, O119, O121, O145 and O165) strains were used to artificially contaminate beef and radish sprout samples, which were then cultivated in each of the four enrichment media. After incubation, STEC strains were detected by loop-mediated isothermal amplification (LAMP) and plating assays. Enrichment in mEC + n was least effective for facilitating the detection of uninjured STEC strains in radish sprouts, while mTSB + n was least effective for enriching freeze-injured non-O157 STEC strains from beef samples for detection by LAMP assay. UPB and mTSB were superior to mEC + n and mTSB + n for the enrichment of non-O157 STEC from food samples. The enrichment of non-O157 STEC was negatively affected by the addition of novobiocin to enrichment broths. Novobiocin should not be added to media used for the enrichment of non-O157 STEC in food when cell injury is anticipated. © 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.

  19. Prolonged fecal shedding of Shiga toxin-producing Escherichia coli among children attending day-care centers in Argentina Excreción prolongada de Escherichia coli productor de toxina Shiga en niños que concurren a jardines maternales de Argentina

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    E. Miliwebsky

    2007-06-01

    Full Text Available In this report we describe the detection and duration of fecal shedding of Shiga toxin-producing Escherichia coli (STEC O157 and non-O157 in symptomatic and asymptomatic cases during four events occurred among children in day-care centers in Argentina. In each event, the cases were identified among children, family contacts and staff members of the Institution. The isolates were characterized by pheno-genotyping and subtyping methods. The STEC fecal shedding was prolonged and intermittent. Strains O157:H7 (1st event; O26:H11 (2nd event; O26:H11 (3rd event and O145:NM (4th event were shed during 23-30, 37, 31 and 19 days, respectively. Considering the possibility of STEC intermittent long-term shedding, symptomatic and asymptomatic individuals should be excluded from the Institution until two consecutive stool cultures obtained at least 48 h apart, test negative.En el presente trabajo se describe la detección y el tiempo de excreción de Escherichia coli productor de toxina Shiga (STEC O157 y no-O157 en casos sintomáticos y asintomáticos durante cuatro eventos ocurridos en jardines maternales de Argentina. En cada evento se identificaron los casos entre los niños, sus familiares y el personal del jardín. Los aislamientos fueron caracterizados por técnicas feno-genotípicas y de subtipificación. La excreción de STEC fue, en general, prolongada e intermitente. Cepas STEC O157:H7 (1er evento; O26:H11 (2do evento; O26:H11 (3er evento y O145:NM (4to evento fueron excretadas durante 23-30, 37, 31 y 19 días, respectivamente. Dadas las características de la excreción, no debe permitirse el reingreso a la institución de todo niño o adulto con infección por STEC, sintomático o asintomático, hasta no tener dos coprocultivos negativos sucesivos, con intervalos de 48 horas entre ellos.

  20. Efecto citotóxico de la toxina shiga tipo 2 y su subunidad b en células epiteliales tubulares renales humanas en cultivo Cytotoxic effect of Shiga toxin type 2 and its B subunit on human renal tubular epithelial cell cultures

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    Virginia Pistone Creydt

    2005-04-01

    Full Text Available Escherichia coli enterohemorrágica productora de toxina Shiga (Stx causa diarrea acuosa, colitis hemorrágica y síndrome urémico hemolítico (SUH. En Argentina, el SUH es la principal causa de insuficiencia renal en niños. El objetivo de este trabajo fue estudiar la toxicidad de Stx tipo 2 (Stx2 y su subunidad B (Stx2B en células epiteliales tubulares renales humanas (CERH, en presencia y ausencia de factores inflamatorios. Los efectos citotóxicos se evaluaron como alteración de la funcionalidad del epitelio; daños histológicos; viabilidad celular; síntesis de proteínas y apoptosis celular. Los resultados muestran que Stx2 regula el pasaje de agua a través de CERH a tiempos menores de 1h de incubación. A tiempos mayores, hasta 72 hs, el estudio de la morfología, la viabilidad, la síntesis de proteínas y la apoptosis demostró que las CERH fueron sensibles a la acción citotóxica de Stx2 y Stx2B de una manera dosis y tiempo dependiente. Estos efectos fueron potenciados por lipopolisacáridos bacterianos (LPS, IL-1b, y butirato.Shiga toxin (Stx-producing E.coli causing watery diarrhea, hemorrhagic colitis and hemolytic-uremic syndrome (HUS. In Argentina, HUS is the most common cause of acute renal failure in children. The purpose of the present study was to examine the cytotoxicity of Stx type 2 (Stx2 and its B subunit (Stx2B on human renal tubular epithelial cells (HRTEC, in the presence and absence of inflammatory factors. Cytotoxic effects were assessed in terms of functionality of the epithelium, histological damage, cell viability, protein synthesis and cellular apoptosis. Results show that Stx2 regulates the passage of water through the HRTEC within an incubation period of 1h. Within longer periods, up to 72 hours, the study of morphology, viability, protein synthesis and apoptosis shows that HRTEC were sensitive to the cytotoxic action of Stx2 and Stx2B in a dose- and time-dependent manner. These effects were potentiated by

  1. Effectiveness of a Commercial Lactic Acid Bacteria Intervention Applied to Inhibit Shiga Toxin-Producing Escherichia coli on Refrigerated Vacuum-Aged Beef

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    Katie R. Kirsch

    2017-01-01

    Full Text Available Because of their antagonistic activity towards pathogenic and spoilage bacteria, some members of the lactic acid bacteria (LAB have been evaluated for use as food biopreservatives. The objectives of this study were to assess the antimicrobial utility of a commercial LAB intervention against O157 and non-O157 Shiga-toxigenic E. coli (STEC on intact beef strip loins during refrigerated vacuum aging and determine intervention efficacy as a function of mode of intervention application. Prerigor strip loins were inoculated with a cocktail (8.9±0.1 log10 CFU/ml of rifampicin-resistant (100.0 μg/ml; RifR O157 and non-O157 STEC. Inoculated loins were chilled to ≤4°C and treated with 8.7±0.1 log10 CFU/ml LAB intervention using either a pressurized tank air sprayer (conventional application or air-assisted electrostatic sprayer (ESS. Surviving STEC were enumerated on tryptic soy agar supplemented with 100.0 μg/ml rifampicin (TSAR to determine STEC inhibition as a function of intervention application method (conventional, ESS and refrigerated aging period (14, 28 days. Intervention application reduced STEC by 0.4 log10 CFU/cm2 (p0.05. Data indicate that the LAB biopreservative may assist beef safety protection when utilized within a multi-intervention beef harvest, fabrication, and aging process.

  2. Presence of Multidrug-Resistant Shiga Toxin-Producing Escherichia coli, Enteropathogenic E. coli and Enterotoxigenic E. coli, on Raw Nopalitos (Opuntia ficus-indica L.) and in Nopalitos Salads from Local Retail Markets in Mexico.

    Science.gov (United States)

    Gómez-Aldapa, Carlos A; Cerna-Cortes, Jorge F; Rangel-Vargas, Esmeralda; Torres-Vitela, Mdel Refugio; Villarruel-López, Angelica; Gutiérrez-Alcántara, Eduardo J; Castro-Rosas, Javier

    2016-05-01

    The presence of multidrug-resistant pathogenic bacteria in food is a significant public health concern. Diarrheagenic Escherichia coli pathotypes (DEPs) are foodborne bacteria. In Mexico, DEPs have been associated with diarrheal illness. There is no information about the presence of multidrug-resistant DEPs on fresh vegetables and in cooked vegetable salads in Mexico. "Nopalitos" (Opuntia ficus-indica L.) is a Cactacea extensively used as a fresh green vegetable throughout Mexico. The presence of generic E. coli and multidrug-resistant DEPs on raw whole and cut nopalitos and in nopalitos salad samples was determined. One hundred raw whole nopalitos (without prickles) samples, 100 raw nopalitos cut into small square samples, and 100 cooked nopalitos salad samples were collected from markets. Generic E. coli was determined using the most probable number procedures. DEPs were identified using two multiplex polymerase chain reaction procedures. Susceptibility to 16 antibiotics was tested for the isolated DEP strains by standard test. Of the 100 whole nopalitos samples, 100 cut nopalitos samples, and 100 nopalitos salad samples, generic E. coli and DEPs were identified, respectively, in 80% and 10%, 74% and 10%, and 64% and 8%. Eighty-two DEP strains were isolated from positive nopalitos samples. The identified DEPs included Shiga toxin-producing E. coli (STEC), enteropathogenic E. coli (EPEC), and enterotoxigenic E. coli (ETEC). All isolated strains exhibited resistance to at least six antibiotics. To the best of our knowledge, this is the first report of the presence of multidrug-resistant and antibiotic resistance profiles of STEC, ETEC, and EPEC on raw nopalitos and in nopalitos salads in Mexico.

  3. Isolation, Characterization and Antibiotic Resistance of Shiga Toxin-Producing Escherichia coli in Hamburger and Evolution of Virulence Genes stx1, stx2, eaeA and hly by Multiplex PCR

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    Mohammad Kargar

    2013-09-01

    Full Text Available Background & Objectives: Shiga toxin-producing Escherichia coli (STEC O157:H7 have emerged as pathogens that can cause food-borne infections and severe and potentially fatal illnesses in humans. E.coli O157:H7 colonizes the digestive tract of cattle and is transmitted to humans by food and water. The objectives of this study were to characterize the prevalence of E.coli O157:H7 isolates in hamburger in Shiraz and to test their antimicrobial sensitivity. Material & Methods: In this research, 428 samples of hamburger were collected from 7 main factories of meat products and enriched in TSB with novobiocin medium at 37ºC. Fermentation of sorbitol and lactose and activities of β- glucuronidase of separated bacteria were examined by using the SMAC and VRBA media and CHROMagar medium. Then isolation of E.coli O157:H7 was confirmed with the use of specific antisera; and with the multiplex PCR method, the presence of E.coli O157:H7 virulence genes – including stx1, stx2, eaeA, and hly – was analyzed. Finally, antibiotic resistance strains were tested with disk diffusion methods. Results: Out of all the examined samples, 264 (61.68% sorbitol-negative bacteria were separated in the CT-SMAC medium. After evaluation with specific antisera, the rate of the recognition of E.coli O157:H7 was 5 (1.17%. The stx1 and eaeA genes were diagnosed in 2 (0.47% cases of these samples. All the isolated bacteria were resistant to penicillin, clindamycin, and erythromycin antibiotics.Conclusion: The presence of STEC in animal products suggests that they may be a potential hazard for human health. A regular monitoring of STEC O157, mainly in hamburger, should be performed to prevent a possible consumer health threat.

  4. Risk of Escherichia coli O157:H7, Non-O157 Shiga Toxin-Producing Escherichia coli, and Campylobacter spp. in Food Animals and Their Products in Qatar.

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    Mohammed, Hussni O; Stipetic, Korana; Salem, Ahmed; McDonough, Patrick; Chang, Yung Fu; Sultan, Ali

    2015-10-01

    Escherichia coli O157:H7, non-O157 E. coli, and Campylobacter spp. are among the top-ranked pathogens that threaten the safety of food supply systems around the world. The associated risks and predisposing factors were investigated in a dynamic animal population using a repeat-cross-sectional study design. Animal and environmental samples were collected from dairy and camel farms, chicken processing plants, and abattoirs and analyzed for the presence of these pathogens using a combination of bacterial enrichment and real-time PCR tests without culture confirmation. Data on putative risk factors were also collected and analyzed. E. coli O157:H7 was detected by PCR at higher levels in sheep and camel feces than in cattle feces (odds ratios [OR], 6.8 and 21.1, respectively). Although the genes indicating E. coli O157:H7 were detected at a relatively higher rate (4.3%) in fecal samples from dairy cattle, they were less common in milk and udder swabs from the same animals (1 and 2%, respectively). Among the food adulterants, E. coli O103 was more common in cattle fecal samples, whereas O26 was more common in sheep feces and O45 in camel feces compared with cattle (OR, 2.6 and 3.1, respectively). The occurrence of E. coli in the targeted populations differed by the type of sample and season of the year. Campylobacter jejuni and Campylobacter coli were more common in sheep and camel feces than in cattle feces. Most of the survey and surveillance of E. coli focused on serogroup O157 as a potential foodborne hazard; however, based on the PCR results, non-O157 Shiga toxin-producing E. coli serotypes appeared to be more common, and efforts should be made to include them in food safety programs.

  5. Molecular Prediction of the O157:H-Negative Phenotype Prevalent in Australian Shiga Toxin-Producing Escherichia coli Cases Improves Concordance ofIn SilicoSerotyping with Phenotypic Motility.

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    Pintara, Alexander P; Guglielmino, Christine J D; Rathnayake, Irani U; Huygens, Flavia; Jennison, Amy V

    2018-04-01

    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 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.

  6. Different expression patterns of genes from the exo-xis region of bacteriophage λ and Shiga toxin-converting bacteriophage Ф24B following infection or prophage induction in Escherichia coli.

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    Bloch, Sylwia; Nejman-Faleńczyk, Bożena; Dydecka, Aleksandra; Łoś, Joanna M; Felczykowska, Agnieszka; Węgrzyn, Alicja; Węgrzyn, Grzegorz

    2014-01-01

    Lambdoid bacteriophages serve as useful models in microbiological and molecular studies on basic biological process. Moreover, this family of viruses plays an important role in pathogenesis of enterohemorrhagic Escherichia coli (EHEC) strains, as they are carriers of genes coding for Shiga toxins. Efficient expression of these genes requires lambdoid prophage induction and multiplication of the phage genome. Therefore, understanding the mechanisms regulating these processes appears essential for both basic knowledge and potential anti-EHEC applications. The exo-xis region, present in genomes of lambdoid bacteriophages, contains highly conserved genes of largely unknown functions. Recent report indicated that the Ea8.5 protein, encoded in this region, contains a newly discovered fused homeodomain/zinc-finger fold, suggesting its plausible regulatory role. Moreover, subsequent studies demonstrated that overexpression of the exo-xis region from a multicopy plasmid resulted in impaired lysogenization of E. coli and more effective induction of λ and Ф24B prophages. In this report, we demonstrate that after prophage induction, the increase in phage DNA content in the host cells is more efficient in E. coli bearing additional copies of the exo-xis region, while survival rate of such bacteria is lower, which corroborated previous observations. Importantly, by using quantitative real-time reverse transcription PCR, we have determined patterns of expressions of particular genes from this region. Unexpectedly, in both phages λ and Ф24B, these patterns were significantly different not only between conditions of the host cells infection by bacteriophages and prophage induction, but also between induction of prophages with various agents (mitomycin C and hydrogen peroxide). This may shed a new light on our understanding of regulation of lambdoid phage development, depending on the mode of lytic cycle initiation.

  7. The Gene tia, Harbored by the Subtilase-Encoding Pathogenicity Island, Is Involved in the Ability of Locus of Enterocyte Effacement-Negative Shiga Toxin-Producing Escherichia coli Strains To Invade Monolayers of Epithelial Cells.

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    Bondì, Roslen; Chiani, Paola; Michelacci, Valeria; Minelli, Fabio; Caprioli, Alfredo; Morabito, Stefano

    2017-12-01

    Locus of enterocyte effacement (LEE)-negative Shiga toxin (Stx)-producing Escherichia coli (STEC) strains are human pathogens that lack the LEE locus, a pathogenicity island (PAI) involved in the intimate adhesion of LEE-positive strains to the host gut epithelium. The mechanism used by LEE-negative STEC strains to colonize the host intestinal mucosa is still not clear. The cell invasion determinant tia , previously described in enterotoxigenic E. coli strains, has been identified in LEE-negative STEC strains that possess the subtilase-encoding pathogenicity island (SE-PAI). We evaluated the role of the gene tia , present in these LEE-negative STEC strains, in the invasion of monolayers of cultured cells. We observed that these strains were able to invade Caco-2 and HEp-2 cell monolayers and compared their invasion ability with that of a mutant strain in which the gene tia had been inactivated. Mutation of the gene tia resulted in a strong reduction of the invasive phenotype, and complementation of the tia mutation with a functional copy of the gene restored the invasion activity. Moreover, we show that the gene tia is overexpressed in bacteria actively invading cell monolayers, demonstrating that tia is involved in the ability to invade cultured monolayers of epithelial cells shown by SE-PAI-positive E. coli , including STEC, strains. However, the expression of the tia gene in the E. coli K-12 strain JM109 was not sufficient, in its own right, to confer to this strain the ability to invade cell monolayers, suggesting that at least another factor must be involved in the invasion ability displayed by the SE-PAI-positive strains. Copyright © 2017 American Society for Microbiology.

  8. Different expression patterns of genes from the exo-xis region of bacteriophage λ and Shiga toxin-converting bacteriophage Ф24B following infection or prophage induction in Escherichia coli.

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    Sylwia Bloch

    Full Text Available Lambdoid bacteriophages serve as useful models in microbiological and molecular studies on basic biological process. Moreover, this family of viruses plays an important role in pathogenesis of enterohemorrhagic Escherichia coli (EHEC strains, as they are carriers of genes coding for Shiga toxins. Efficient expression of these genes requires lambdoid prophage induction and multiplication of the phage genome. Therefore, understanding the mechanisms regulating these processes appears essential for both basic knowledge and potential anti-EHEC applications. The exo-xis region, present in genomes of lambdoid bacteriophages, contains highly conserved genes of largely unknown functions. Recent report indicated that the Ea8.5 protein, encoded in this region, contains a newly discovered fused homeodomain/zinc-finger fold, suggesting its plausible regulatory role. Moreover, subsequent studies demonstrated that overexpression of the exo-xis region from a multicopy plasmid resulted in impaired lysogenization of E. coli and more effective induction of λ and Ф24B prophages. In this report, we demonstrate that after prophage induction, the increase in phage DNA content in the host cells is more efficient in E. coli bearing additional copies of the exo-xis region, while survival rate of such bacteria is lower, which corroborated previous observations. Importantly, by using quantitative real-time reverse transcription PCR, we have determined patterns of expressions of particular genes from this region. Unexpectedly, in both phages λ and Ф24B, these patterns were significantly different not only between conditions of the host cells infection by bacteriophages and prophage induction, but also between induction of prophages with various agents (mitomycin C and hydrogen peroxide. This may shed a new light on our understanding of regulation of lambdoid phage development, depending on the mode of lytic cycle initiation.

  9. Prevalence of carriage of Shiga toxin-producing Escherichia coli serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 among slaughtered adult cattle in France.

    Science.gov (United States)

    Bibbal, Delphine; Loukiadis, Estelle; Kérourédan, Monique; Ferré, Franck; Dilasser, Françoise; Peytavin de Garam, Carine; Cartier, Philippe; Oswald, Eric; Gay, Emilie; Auvray, Frédéric; Brugère, Hubert

    2015-02-01

    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.

  10. Influence of primer sequences and DNA extraction method on detection of non-O157 Shiga toxin-producing Escherichia coli in ground beef by real-time PCR targeting the eae, stx, and serogroup-specific genes.

    Science.gov (United States)

    Wasilenko, Jamie L; Fratamico, Pina M; Narang, Neelam; Tillman, Glenn E; Ladely, Scott; Simmons, Mustafa; Cray, William C

    2012-11-01

    Non-O157 Shiga toxin-producing Escherichia coli (STEC) infections, particularly those caused by the "big six" or "top six" non-O157 serogroups (O26, O45, O103, O111, O121, and O145) can result in severe illness and complications. Because of their significant public health impact and the notable prevalence of STEC in cattle, methods for detection of the big six non-O157 STEC in ground beef have been established. Currently, the U.S. Department of Agriculture, Food Safety and Inspection Service detection methods for screening beef samples for non-O157 STEC target the stx(1), stx(2), and eae virulence genes, with the 16S rRNA gene as an internal control, in a real-time PCR multiplex assay. Further, the serogroup is determined by PCR targeting genes in the E. coli O-antigen gene clusters of the big six non-O157 serogroups. The method that we previously reported was improved so that additional stx variants, stx(1d), stx(2e), and stx(2g), are detected. Additionally, alignments of the primers targeting the eae gene were used to improve the detection assay so that eae subtypes that could potentially be of clinical significance would also be detected. Therefore, evaluation of alternative real-time PCR assay primers and probes for the stx and eae reactions was carried out in order to increase the stx and eae subtypes detected. Furthermore, a Tris-EDTA DNA extraction method was compared with a previously used procedure that was based on a commercially available reagent. The Tris-EDTA DNA extraction method significantly decreased the cycle threshold values for the stx assay (P primers and probes increased the subtypes detected to include stx(1d), stx(2e), and stx(2g), and sequence data showed that modification of the eae primer should allow the known eae subtypes to be detected.

  11. Prevalence of Carriage of Shiga Toxin-Producing Escherichia coli Serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 among Slaughtered Adult Cattle in France

    Science.gov (United States)

    Loukiadis, Estelle; Kérourédan, Monique; Ferré, Franck; Dilasser, Françoise; Peytavin de Garam, Carine; Cartier, Philippe; Oswald, Eric; Gay, Emilie; Auvray, Frédéric; Brugère, Hubert

    2014-01-01

    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. PMID:25527532

  12. Shiga (Vero-toxin producing Escherichia coli isolated from the hospital foods; virulence factors, o-serogroups and antimicrobial resistance properties

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    Reza Ranjbar

    2017-01-01

    Full Text Available Abstract Background According to the presence of the weak, diabetic and immunosuppressive patients in hospitals, hospital foods should have a high quality and safety. Cooking a lot of foods higher than daily requirement, storage of cooked foods in an inappropriate condition and presence of nurses and servants in distribution of food to patients are the main reasons caused contamination of hospital foods. Shiga toxigenic Escherichia coli is one of the common cause of food poisoning in hospitals. The present research was carried out to study the distribution of virulence factors, O-serogroups and antibiotic resistance properties in STEC strains recovered from Iranian hospital food samples. Methods Five-hundred and eighty raw and cooked food samples were collected and immediately transferred to the laboratory. E. coli-positive strains were subjected to PCR and disk diffusion method. Results Thirty-nine out of 580 (6.72% hospital food samples were contaminated with E. coli. Raw (20% and cooked meat (6% were the most commonly contaminated samples. Raw samples had the higher prevalence of E. coli (P <0.01. Samples which were collected in the summer season had the highest prevalence of bacteria (64.10%. Significant difference was seen between the prevalence of EHEC and AEEC subtypes (P <0.01. The most commonly detected virulence factors in both EHEC and AEEC subtypes were stx1 and eae. The most commonly detected serogroups were O26 (43.75% and O157 (25% and there were no positive results for O103, O145, O91, O113 and O128 serogroups. Aac (3-IV (100%, CITM (100% and tetA (62.50% were the most commonly detected antibiotic resistance genes. STEC strains harbored the highest levels of resistance against ampicillin (93.75%, gentamycin (93.75%, tetracycline (87.50% and ciprofloxacin (81.25%. All of the STEC strains were resistant to at least 3 antibiotics, while the prevalence of resistance against more than 12 antibiotics were 12.50%. Conclusions High

  13. Occurrence and characterization of Shiga toxin-producing Escherichia coli O157:H7 and other non-sorbitol-fermenting E. coli in cattle and humans in urban areas of Morogoro, Tanzania.

    Science.gov (United States)

    Lupindu, Athumani M; Olsen, John E; Ngowi, Helena A; Msoffe, Peter L M; Mtambo, Madundo M; Scheutz, Flemming; Dalsgaard, Anders

    2014-07-01

    Escherichia coli strains such as Shiga toxin-producing E. coli (STEC), enteropathogenic E. coli, enterotoxigenic, attaching, and effacing E. coli, and enteroinvasive E. coli cause diarrhea in humans. Although other serotypes exist, the most commonly reported STEC in outbreaks is O157:H7. A cross-sectional study was conducted to isolate and characterize non-sorbitol-fermenting (NSF) E. coli O157:H7 from urban and periurban livestock settings of Morogoro, Tanzania. Human stool, cattle feces, and soil and water samples were collected. Observations and questionnaire interview studies were used to gather information about cattle and manure management practices in the study area. E. coli were isolated on sorbitol MacConkey agar and characterized by conventional biochemical tests. Out of 1049 samples, 143 (13.7%) yielded NSF E. coli. Serological and antimicrobial tests and molecular typing were performed to NSF E. coli isolates. These procedures detected 10 (7%) pathogenic E. coli including STEC (n=7), enteropathogenic E. coli (EPEC) (n=2), and attaching and effacing E. coli (A/EEC) (n=1) strains. The STEC strains had the ability to produce VT1 and different VT2 toxin subtypes that caused cytopathic effects on Vero cells. The prevalence of STEC in cattle was 1.6%, out of which 0.9% was serotype O157:H7 and the overall prevalence of diarrheagenic E. coli in cattle was 2.2%. The serotypes O157:H7, O142:H34, O113:H21, O+:H-, O+:H16, and O25:H4 were identified. One ESBL-producing isolate showed the MLST type ST131. To our knowledge, this is the first finding in Tanzania of this recently emerged worldwide pandemic clonal group, causing widespread antimicrobial-resistant infections, and adds knowledge of the geographical distribution of ST131. Cattle manure was indiscriminately deposited within residential areas, and there was direct contact between humans and cattle feces during manure handling. Cattle and manure management practices expose humans, animals, and the environment

  14. Validación de una técnica de PCR múltiple para la detección de Escherichia coli productor de toxina Shiga Validation of a multiplex PCR for detection of Shiga toxin-producing Escherichia coli

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    G.A. Leotta

    2005-03-01

    Full Text Available La infección por Escherichia coli productor de toxina Shiga (STEC es causa de diarrea con o sin sangre, colitis hemorrágica y síndrome urémico hemolítico (SUH en humanos. El objetivo de este trabajo fue validar una técnica de PCR múltiple para el diagnóstico de STEC basado en la detección de los genes stx1, stx2 y rfbO157. La validación de la técnica se realizó en dos laboratorios independientes, en forma paralela. Se determinó rango de trabajo, selectividad y robustez. Se evaluó el desempeño de la técnica al combinar distintas concentraciones de dos cepas con diferentes factores de virulencia. El rango de trabajo dependió de la cepa analizada, los valores máximos y mínimos fueron 6,6 x 107 y 1,0 x 104 UFC/50 µl. El límite de detección fue de 1,0 x 104 UFC/50 µl y el límite de corte de 1,0 x 105 UFC/50 µl. La robustez fue óptima al modificar diferentes variables. Se obtuvo 100% de inclusividad, exclusividad, precisión analítica, valor predictivo positivo y valor predictivo negativo. No se observó interferencia al combinar distintas concentraciones de los factores de virulencia blanco de la reacción. La técnica validada es una alternativa apropiada para la detección y confirmación de STEC O157 y no-O157 a partir de cultivos bacterianos.Shiga toxin-producing Escherichia coli (STEC cause non-bloody or bloody diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS in humans. The aim of the present study was to validate a multiplex PCR for the STEC diagnosis based on the detection of stx1, stx2 and rfbO157 genes. The multiplex PCR validation was carried out in two independent laboratories in a parallel way. Work range, selectivity and robustness were established. The PCR performance was evaluated using different concentrations of two STEC strains harboring different target genes. The work range depended on the strain analyzed, the maximum and the minimum values were 6.6 x 107 and 1.0 x 104 CFU/50 µl. The

  15. Aislamiento de Escherichia coli productor de toxina Shiga durante un brote de gastroenteritis en un Jardín Maternal de la Ciudad de Mar del Plata Isolation of Shiga toxin-producing Escherichia coli strains during a gastrointestinal outbreak at a day care center in Mar del Plata City

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    D. Gomez

    2005-12-01

    considered risk factors. One case had Shiga toxin-producing Escherichia coli (STEC O103:H2 infection and other STEC O26:H11.The duration of shedding for the child with O26:H11 infection was 37 days. In the other symptomatic children, the pathogen was not recovered from fecal samples collected 6 or more days after the onset of the illness. This emphasizes that the collection of early samples is necessary to recover STEC strains. In order to prevent and control enteric diseases in day care facilities the following measures are necessary: optimal hygiene standards, early case reporting, and exclusion of those who remain culture-positive.

  16. Detección y caracterización de Escherichia coli productor de toxina Shiga a partir de casos clínicos y de alimentos en Uruguay Detection and characterization of Shiga toxin - producing Escherichia coli from clinical cases and food in Uruguay

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    G. Varela

    2008-06-01

    Full Text Available Establecimos la frecuencia de aislamiento de Escherichia coli productor de toxina Shiga (STEC a partir de muestras clínicas y de alimentos, así como las características fenotípicas y genotípicas de las cepas recuperadas. Se analizaron 198 muestras fecales de niños con diarrea sanguinolenta (DS, 14 muestras fecales de niños con síndrome urémico hemolítico (SUH y 220 muestras de carne picada. También se estudiaron 4 cepas STEC aisladas de alimentos embutidos. Se recuperó STEC de 3 (1,5% de los niños con DS, de 1 (7% niño con SUH y de 4 (1,8% de las muestras de carne picada. Todas las cepas fueron eae y ehxA positivas. Los serotipos detectados fueron: O157:H7 (9 cepas, O26:H11 (2 cepas, O111:NM (1 cepa y O145:HNT (1 cepa. Todas las cepas O157:H7 portaron el subtipo eae-g1; las cepas O26:H11 y O145:HNT portaron el subtipo eae-b1 y la cepa O111:NM portó el subtipo eae-g2/q. Las cepas STEC del mismo serogrupo mostraron alta diversidad genética. En Uruguay STEC no sería agente frecuente de diarrea con sangre en niños. Sin embargo, las cepas recuperadas presentaron los genes asociados con enfermedad severa y 2 de los 3 niños infectados con STEC evolucionaron a SUH. La carne picada y otros alimentos serían vehículos importantes de O157:H7.We have assessed the frequency of Shiga toxin-producing Escherichia coli (STEC in clinical and food samples as well as studied the genotypic and phenotypic characteristics of the recovered strains. One hundred ninety eight fecal samples from children with bloody diarrhea (BD, 14 from children with hemolytic uremic syndrome (HUS, 220 ground beef samples and 4 STEC isolates from other beef-derived products were analyzed. The STEC strains were isolated from 3 (1.5% children with bloody diarrhea, 1 (7% from a child with HUS and 4 (1.8% from ground beef samples. All strains were eae and ehxA positive. The serotypes found were: O157:H7 (9 strains, O26:H11 (2, O111: NM (1 and O145:HNT (1. All O157:H7 STEC

  17. A hemolytic-uremic syndrome-associated strain O113:H21 Shiga toxin-producing Escherichia coli specifically expresses a transcriptional module containing dicA and is related to gene network dysregulation in Caco-2 cells

    Science.gov (United States)

    Bando, Silvia Yumi; Iamashita, Priscila; Guth, Beatriz E.; dos Santos, Luis F.; Fujita, André; Abe, Cecilia M.; Ferreira, Leandro R.

    2017-01-01

    Shiga toxin-producing (Stx) Escherichia coli (STEC) O113:H21 strains are associated with human diarrhea and some of these strains may cause hemolytic uremic syndrome (HUS). The molecular mechanism underlying this capacity and the differential host cell response to HUS-causing strains are not yet completely understood. In Brazil O113:H21 strains are commonly found in cattle but, so far, were not isolated from HUS patients. Here we conducted comparative gene co-expression network (GCN) analyses of two O113:H21 STEC strains: EH41, reference strain, isolated from HUS patient in Australia, and Ec472/01, isolated from cattle feces in Brazil. These strains were cultured in fresh or in Caco-2 cell conditioned media. GCN analyses were also accomplished for cultured Caco-2 cells exposed to EH41 or Ec472/01. Differential transcriptome profiles for EH41 and Ec472/01 were not significantly changed by exposure to fresh or Caco-2 conditioned media. Conversely, global gene expression comparison of both strains cultured in conditioned medium revealed a gene set exclusively expressed in EH41, which includes the dicA putative virulence factor regulator. Network analysis showed that this set of genes constitutes an EH41 specific transcriptional module. PCR analysis in Ec472/01 and in other 10 Brazilian cattle-isolated STEC strains revealed absence of dicA in all these strains. The GCNs of Caco-2 cells exposed to EH41 or to Ec472/01 presented a major transcriptional module containing many hubs related to inflammatory response that was not found in the GCN of control cells. Moreover, EH41 seems to cause gene network dysregulation in Caco-2 as evidenced by the large number of genes with high positive and negative covariance interactions. EH41 grows slowly than Ec472/01 when cultured in Caco-2 conditioned medium and fitness-related genes are hypoexpressed in that strain. Therefore, EH41 virulence may be derived from its capacity for dysregulating enterocyte genome functioning and its

  18. Investigation of prevalence of free Shiga toxin-producing Escherichia coli (STEC)-specific bacteriophages and its correlation with STEC bacterial hosts in a produce-growing area in Salinas, California.

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    Liao, Yen-Te; Quintela, Irwin A; Nguyen, Kimberly; Salvador, Alexandra; Cooley, Michael B; Wu, Vivian C H

    2018-01-01

    Shiga toxin-producing E. coli (STEC) causes approximately 265,000 illnesses and 3,600 hospitalizations annually and is highly associated with animal contamination due to the natural reservoir of ruminant gastrointestinal tracts. Free STEC-specific bacteriophages against STEC strains are also commonly isolated from fecal-contaminated environment. Previous studies have evaluated the correlation between the prevalence of STEC-specific bacteriophages and STEC strains to improve animal-associated environment. However, the similar information regarding free STEC-specific bacteriophages prevalence in produce growing area is lacking. Thus, the objectives of this research were to determine the prevalence of STEC-specific phages, analyze potential effects of environmental factors on the prevalence of the phages, and study correlations between STEC-specific bacteriophages and the bacterial hosts in pre-harvest produce environment. Surface water from 20 samples sites was subjected to free bacteriophage isolation using host strains of both generic E. coli and STEC (O157, six non-O157 and one O179 strains) cocktails, and isolation of O157 and non-O157 STEC strains by use of culture methods combined with PCR-based confirmation. The weather data were obtained from weather station website. Free O145- and O179-specific bacteriophages were the two most frequently isolated bacteriophages among all (O45, O145, O157 and O179) in this study. The results showed June and July had relatively high prevalence of overall STEC-specific bacteriophages with minimum isolation of STEC strains. In addition, the bacteriophages were likely isolated in the area-around or within city-with predominant human impact, whereas the STEC bacterial isolates were commonly found in agriculture impact environment. Furthermore, there was a trend that the sample sites with positive of free STEC bacteriophage did not have the specific STEC bacterial hosts. The findings of the study enable us to understand the ecology

  19. Evaluation of a multiplex real-time PCR method for detecting shiga toxin-producing Escherichia coli in beef and comparison to the U.S. Department of Agriculture Food Safety and Inspection Service Microbiology laboratory guidebook method.

    Science.gov (United States)

    Fratamico, Pina M; Wasilenko, Jamie L; Garman, Bradley; Demarco, Daniel R; Varkey, Stephen; Jensen, Mark; Rhoden, Kyle; Tice, George

    2014-02-01

    The "top-six" non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, and O145) most frequently associated with outbreaks and cases of foodborne illnesses have been declared as adulterants in beef by the U.S. Department of Agriculture Food Safety and Inspection Service (FSIS). Regulatory testing in beef began in June 2012. The purpose of this study was to evaluate the DuPont BAX System method for detecting these top six STEC strains and strains of E. coli O157:H7. For STEC, the BAX System real-time STEC suite was evaluated, including a screening assay for the stx and eae virulence genes and two panel assays to identify the target serogroups: panel 1 detects O26, O111, and O121, and panel 2 detects O45, O103, O145. For E. coli O157:H7, the BAX System real-time PCR assay for this specific serotype was used. Sensitivity of each assay for the PCR targets was ≥1.23 × 10(3) CFU/ml in pure culture. Each assay was 100% inclusive for the strains tested (20 to 50 per assay), and no cross-reactivity with closely related strains was observed in any of the assays. The performance of the BAX System methods was compared with that of the FSIS Microbiology Laboratory Guidebook (MLG) methods for detection of the top six STEC and E. coli O157:H7 strains in ground beef and beef trim. Generally, results of the BAX System method were similar to those of the MLG methods for detecting non-O157 STEC and E. coli O157:H7. Reducing or eliminating novobiocin in modified tryptic soy broth (mTSB) may improve the detection of STEC O111 strains; one beef trim sample inoculated with STEC O111 produced a negative result when enriched in mTSB with 8 mg/liter novobiocin but was positive when enriched in mTSB without novobiocin. The results of this study indicate the feasibility of deploying a panel of real-time PCR assay configurations for the detection and monitoring of the top six STEC and E. coli O157:H7 strains in beef. The approach could easily be adapted

  20. An interlaboratory study on efficient detection of Shiga toxin-producing Escherichia coli O26, O103, O111, O121, O145, and O157 in food using real-time PCR assay and chromogenic agar.

    Science.gov (United States)

    Hara-Kudo, Yukiko; Konishi, Noriko; Ohtsuka, Kayoko; Iwabuchi, Kaori; Kikuchi, Rie; Isobe, Junko; Yamazaki, Takumiko; Suzuki, Fumie; Nagai, Yuhki; Yamada, Hiroko; Tanouchi, Atsuko; Mori, Tetsuya; Nakagawa, Hiroshi; Ueda, Yasufumi; Terajima, Jun

    2016-08-02

    To establish an efficient detection method for Shiga toxin (Stx)-producing Escherichia coli (STEC) O26, O103, O111, O121, O145, and O157 in food, an interlaboratory study using all the serogroups of detection targets was firstly conducted. We employed a series of tests including enrichment, real-time PCR assays, and concentration by immunomagnetic separation, followed by plating onto selective agar media (IMS-plating methods). This study was particularly focused on the efficiencies of real-time PCR assays in detecting stx and O-antigen genes of the six serogroups and of IMS-plating methods onto selective agar media including chromogenic agar. Ground beef and radish sprouts samples were inoculated with the six STEC serogroups either at 4-6CFU/25g (low levels) or at 22-29CFU/25g (high levels). The sensitivity of stx detection in ground beef at both levels of inoculation with all six STEC serogroups was 100%. The sensitivity of stx detection was also 100% in radish sprouts at high levels of inoculation with all six STEC serogroups, and 66.7%-91.7% at low levels of inoculation. The sensitivity of detection of O-antigen genes was 100% in both ground beef and radish sprouts at high inoculation levels, while at low inoculation levels, it was 95.8%-100% in ground beef and 66.7%-91.7% in radish sprouts. The sensitivity of detection with IMS-plating was either the same or lower than those of the real-time PCR assays targeting stx and O-antigen genes. The relationship between the results of IMS-plating methods and Ct values of real-time PCR assays were firstly analyzed in detail. Ct values in most samples that tested negative in the IMS-plating method were higher than the maximum Ct values in samples that tested positive in the IMS-plating method. This study indicates that all six STEC serogroups in food contaminated with more than 29CFU/25g were detected by real-time PCR assays targeting stx and O-antigen genes and IMS-plating onto selective agar media. Therefore, screening

  1. Development of a Robust Method for Isolation of Shiga Toxin-Positive Escherichia coli (STEC) from Fecal, Plant, Soil and Water Samples from a Leafy Greens Production Region in California

    Science.gov (United States)

    Cooley, Michael B.; Jay-Russell, Michele; Atwill, Edward R.; Carychao, Diana; Nguyen, Kimberly; Quiñones, Beatriz; Patel, Ronak; Walker, Samarpita; Swimley, Michelle; Pierre-Jerome, Edith; Gordus, Andrew G.; Mandrell, Robert E.

    2013-01-01

    During a 2.5-year survey of 33 farms and ranches in a major leafy greens production region in California, 13,650 produce, soil, livestock, wildlife, and water samples were tested for Shiga toxin (stx)-producing Escherichia coli (STEC). Overall, 357 and 1,912 samples were positive for E. coli O157:H7 (2.6%) or non-O157 STEC (14.0%), respectively. Isolates differentiated by O-typing ELISA and multilocus variable number tandem repeat analysis (MLVA) resulted in 697 O157:H7 and 3,256 non-O157 STEC isolates saved for further analysis. Cattle (7.1%), feral swine (4.7%), sediment (4.4%), and water (3.3%) samples were positive for E. coli O157:H7; 7/32 birds, 2/145 coyotes, 3/88 samples from elk also were positive. Non-O157 STEC were at approximately 5-fold higher incidence compared to O157 STEC: cattle (37.9%), feral swine (21.4%), birds (2.4%), small mammals (3.5%), deer or elk (8.3%), water (14.0%), sediment (12.3%), produce (0.3%) and soil adjacent to produce (0.6%). stx1, stx2 and stx1/stx2 genes were detected in 63%, 74% and 35% of STEC isolates, respectively. Subtilase, intimin and hemolysin genes were present in 28%, 25% and 79% of non-O157 STEC, respectively; 23% were of the “Top 6″ O-types. The initial method was modified twice during the study revealing evidence of culture bias based on differences in virulence and O-antigen profiles. MLVA typing revealed a diverse collection of O157 and non-O157 STEC strains isolated from multiple locations and sources and O157 STEC strains matching outbreak strains. These results emphasize the importance of multiple approaches for isolation of non-O157 STEC, that livestock and wildlife are common sources of potentially virulent STEC, and evidence of STEC persistence and movement in a leafy greens production environment. PMID:23762414

  2. Use of an Electrostatic Spraying System or the Sprayed Lethality in Container Method To Deliver Antimicrobial Agents onto the Surface of Beef Subprimals To Control Shiga Toxin-Producing Escherichia coli.

    Science.gov (United States)

    Stella, J Max; Luchansky, John B; Miller, Kelsey; Shoyer, Bradley A; Shane, Laura E; McGeary, Lianna; Osoria, Manuela; Stahler, Laura J; Sevart, Nicholas J; Phebus, Randall K; Thippareddi, Harshavardhan; Porto-Fett, Anna C S

    2017-08-01

    The efficacy of an electrostatic spraying system (ESS) and/or the sprayed lethality in container (SLIC) method to deliver antimicrobial agents onto the surface of beef subprimals to reduce levels of Shiga toxin-producing Escherichia coli (STEC) was evaluated. Beef subprimals were surface inoculated (lean side; ca. 5.8 log CFU per subprimal) with 2 mL of an eight-strain cocktail comprising single strains of rifampin-resistant (100 μg/mL) STEC (O26:H11, O45:H2, O103:H2, O104:H4, O111:H - , O121:H19, O145:NM, and O157:H7). Next, inoculated subprimals were surface treated with lauric arginate (LAE; 1%), peroxyacetic acid (PAA; 0.025%), or cetylpyridinium chloride (CPC; 0.4%) by passing each subprimal, with the inoculated lean side facing upward, through an ESS cabinet or via SLIC. Subprimals were then vacuum packaged and stored at 4°C. One set of subprimals was sampled after an additional 2 h, 3 days, or 7 days of refrigerated storage, whereas another set was retreated via SLIC after 3 days of storage with a different one of the three antimicrobial agents (e.g., a subprimal treated with LAE on day 0 was then treated with PAA or CPE on day 3). Retreated subprimals were sampled after 2 h or 4 days of additional storage at 4°C. A single initial application of LAE, PAA, or CPC via ESS or SLIC resulted in STEC reductions of ca. 0.3 to 1.3 log CFU per subprimal after 7 days of storage. However, when subprimals were initially treated with LAE, PAA, or CPC via ESS or SLIC and then separately retreated with a different one of these antimicrobial agents via SLIC on day 3, additional STEC reductions of 0.4 to 1.0 log CFU per subprimal were observed after an additional 4 days of storage. Application of LAE, PAA, or CPC, either alone or in combination, via ESS or SLIC is effective for reducing low levels (ca. 0.3 to 1.6 log CFU) of STEC that may be naturally present on the surface of beef subprimals.

  3. Phage Types and Genotypes of Shiga Toxin-Producing Escherichia coli O157:H7 Isolates from Humans and Animals in Spain: Identification and Characterization of Two Predominating Phage Types (PT2 and PT8)

    Science.gov (United States)

    Mora, Azucena; Blanco, Miguel; Blanco, Jesús E.; Alonso, M. Pilar; Dhabi, Ghizlane; Thomson-Carter, Fiona; Usera, Miguel A.; Bartolomé, Rosa; Prats, Guillermo; Blanco, Jorge

    2004-01-01

    Phage typing and DNA macrorestriction fragment analysis by pulsed-field electrophoresis (PFGE) were used for the epidemiological subtyping of a collection of Shiga toxin-producing Escherichia coli (STEC) O157:H7 strains isolated in Spain between 1980 and 1999. Phage typing distinguished a total of 18 phage types among 171 strains isolated from different sources (67 humans, 82 bovines, 12 ovines, and 10 beef products). However, five phage types, phage type 2 (PT2; 42 strains), PT8 (33 strains), PT14 (14 strains), PT21/28 (11 strains), and PT54 (16 strains), accounted for 68% of the study isolates. PT2 and PT8 were the most frequently found among strains from both humans (51%) and bovines (46%). Interestingly, we detected a significant association between PT2 and PT14 and the presence of acute pathologies. A group of 108 of the 171 strains were analyzed by PFGE, and 53 distinct XbaI macrorestriction patterns were identified, with 38 strains exhibiting unique PFGE patterns. In contrast, phage typing identified 15 different phage types. A total of 66 phage type-PFGE subtype combinations were identified among the 108 strains. PFGE subtyping differentiated between unrelated strains that exhibited the same phage type. The most common phage type-PFGE pattern combinations were PT2-PFGE type 1 (1 human and 11 bovine strains), PT8-PFGE type 8 (2 human, 6 bovine, and 1 beef product strains), PT2-PFGE subtype 4A (1 human, 3 bovine, and 1 beef product strains). Nine (29%) of 31 human strains showed phage type-PFGE pattern combinations that were detected among the bovine strains included in this study, and 26 (38%) of 68 bovine strains produced phage type-PFGE pattern combinations observed among human strains included in this study, confirming that cattle are a major reservoir of strains pathogenic for humans. PT2 and PT8 strains formed two groups which differed from each other in their motilities, stx genotypes, PFGE patterns, and the severity of the illnesses that they caused

  4. Actualización en el tratamiento del síndrome urémico hemolítico endémico: Patogénesis y tratamiento de la complicación sistémica más grave de las infecciones por Escherichia coli productor de toxina Shiga Update on the treatment of endemic hemolytic uremic syndrome: Pathogenesis and treatment of the most severe systemic complication of infections by Shiga toxin-producing Escherichia coli

    Directory of Open Access Journals (Sweden)

    Romina J. Fernández-Brando

    2011-08-01

    Full Text Available La forma típica o post-diarreica del síndrome urémico hemolítico (SUH es la complicación más grave de las infecciones por cepas de Escherichia coli productoras de toxina Shiga (STEC. En la Argentina el SUH es un problema crítico de salud pública, ya que representa la principal causa de falla renal aguda en la infancia, la segunda causa de falla renal crónica, y aporta el 20% de los casos de transplante renal durante la infancia y la adolescencia. A pesar de los avances en el conocimiento de su patogénesis, el único tratamiento actual de los pacientes con SUH es de sostén, y no existen terapias específicas ni preventivas. En la presente revisión expondremos los conocimientos básicos de los mecanismos patogénicos y discutiremos los enfoques terapéuticos tradicionales e innovadores, con especial foco en la situación nacional y los aportes hechos por grupos de la Argentina.The typical form of hemolytic uremic syndrome (HUS is the major complication of Shiga toxin-producing Escherichia coli (STEC infections. HUS is a critical health problem in Argentina since it is the main cause of acute renal failure in children and the second cause of chronic renal failure, giving account for 20% of renal transplants in children and adolescents in our country. In spite of the extensive research in the field, the mainstay of treatment for patients with HUS is supportive therapy, and there are no specific therapies preventing or ameliorating the disease course. In this review, we present the current knowledge about pathogenic mechanisms and discuss traditional and innovative therapeutic approaches, with special focus in national status and contributions made by Argentinean groups.

  5. Detección, aislamiento y caracterización de Escherichia coli productor de toxina Shiga a partir de carne molida fresca proveniente de carnicerías de Concepción, provincia de Tucumán Detection, isolation and characterization of Shiga toxin-producing Escherichia coli (STEC in fresh ground beef from butcher shops in Concepción, Tucumán Province

    Directory of Open Access Journals (Sweden)

    M. A. Jure

    2010-12-01

    Full Text Available Escherichia coli productor de toxina Shiga (STEC es un patógeno emergente transmitido por alimentos. Existen numerosos serotipos de STEC asociados a enfermedad en humanos, entre los cuales prevalece el serotipo O157:H7. La carne molida es el principal vehículo de transmisión. En la ciudad de Concepción, provincia de Tucumán, entre setiembre y diciembre de 2004 se diagnosticaron dos casos de síndrome urémico hemolítico (SUH. El objetivo de este trabajo fue detectar, aislar y caracterizar STEC O157 y no-O157 a partir de muestras de carne molida fresca obtenidas en las bocas de expendio. Entre los meses de setiembre y diciembre de 2004 se recolectaron 53 muestras de carne molida fresca en carnicerías de la ciudad de Concepción. Para la detección, el aislamiento y la caracterización de STEC O157:H7 se utilizó la metodología USDA-FSIS 2002. Para la detección de E. coli no-O157 se utilizaron dos técnicas de PCR; para el aislamiento y la caracterización se utilizó una metodología previamente validada en una etapa intralaboratorio. Siete muestras fueron positivas para el gen stx2, de las cuales 4 también fueron positivas para el gen rfbO157. Sin embargo, solo se aisló una cepa de E. coli O157:H7 biotipo C, portadora de los genes eae, stx2 y ehxA. El presente trabajo refleja la importancia de implementar técnicas que permitan detectar este grupo de patógenos emergentes a partir de productos cárnicos.Shiga toxin-producing Escherichia coli is an emerging foodborne pathogen. There are many STEC serotypes associated with human diseases, being the O157:H7 serotype the most prevalent. Ground beef is the main transmission vehicle. In Concepción city, Tucumán Province, between September and December 2004, two hemolytic uremic syndrome (HUS cases were diagnosed. The main objective of this work was to detect, isolate and characterize STEC O157 and non-O157 strains in fresh ground beef. Between September and December 2004, 53 fresh ground

  6. Detection of E. coli O157:H7 and Shigella dysenteriae toxins in clinical samples by PCR-ELISA

    Directory of Open Access Journals (Sweden)

    Jafar Amani

    2015-05-01

    Full Text Available Shiga toxin producing bacteria are potential causes of serious human disease such as hemorrhagic colitis, severe inflammations of ileocolonic regions of gastrointestinal tract, thrombocytopenia, septicemia, malignant disorders in urinary ducts, hemolytic uremic syndrome (HUS. Shiga toxin 1 (stx1, shiga toxin 2 (stx2, or a combination of both are responsible for most clinical symptoms of these diseases. A lot of methods have been developed so far to detect shiga toxins such as cell culture, ELISA, and RFPLA, but due to high costs and labor time in addition to low sensitivity, they have not received much attention. In this study, PCR-ELISA method was used to detect genes encoding shiga toxins1 and 2 (stx1 and stx2. To detect stx1 and stx2 genes, two primer pairs were designed for Multiplex-PCR then PCR-ELISA. PCR products (490 and 275, respectively were subsequently verified by sequencing. Sensitivity and specificity of PCR-ELISA method were determined by using genome serial dilution and Enterobacteria strains. PCR-ELISA method used in this study proved to be a rapid and precise approach to detect different types of shiga toxins and can be used to detect bacterial genes encoding shiga toxins.

  7. Occurrence of Shiga-toxigenic Escherichia coli in Stomoxys calcitrans (Diptera: Muscidae

    Directory of Open Access Journals (Sweden)

    Bruno Gomes de Castro

    Full Text Available This study aimed to verify the occurrence of Shiga toxin-producingEscherichia coli (STEC strains in three distinct anatomic parts of the stable fly Stomoxys calcitrans by multiplex polymerase chain reaction (PCR Multiplex. According to the results obtained,E. coli was identified in 19.5% of the stable flies. Shiga toxin genes were detected in 13% of the E. coli isolated, most frequently from the surface, followed by abdominal digestive tract and mouth apparatus of insects, respectively. This is the first study to detect presence of STEC in Stomoxys calcitrans in Brazil; it has also revealed the potential role of stable flies as carriers of pathogenic bacterial agents.

  8. Interplay between toxin transport and flotillin localization

    DEFF Research Database (Denmark)

    Pust, Sascha; Dyve, Anne Berit; Torgersen, Maria L

    2010-01-01

    The flotillin proteins are localized in lipid domains at the plasma membrane as well as in intracellular compartments. In the present study, we examined the importance of flotillin-1 and flotillin-2 for the uptake and transport of the bacterial Shiga toxin (Stx) and the plant toxin ricin and we...... for flotillin-1 or -2. However, the Golgi-dependent sulfation of both toxins was significantly reduced in flotillin knockdown cells. Interestingly, when the transport of ricin to the ER was investigated, we obtained an increased mannosylation of ricin in flotillin-1 and flotillin-2 knockdown cells. The toxicity...... of both toxins was twofold increased in flotillin-depleted cells. Since BFA (Brefeldin A) inhibits the toxicity even in flotillin knockdown cells, the retrograde toxin transport is apparently still Golgi-dependent. Thus, flotillin proteins regulate and facilitate the retrograde transport of Stx and ricin....

  9. Stool C difficile toxin

    Science.gov (United States)

    ... toxin; Colitis - toxin; Pseudomembranous - toxin; Necrotizing colitis - toxin; C difficile - toxin ... be analyzed. There are several ways to detect C difficile toxin in the stool sample. Enzyme immunoassay ( ...

  10. Prevalence and antimicrobial sensitivity of Shiga-toxin-producing ...

    African Journals Online (AJOL)

    Nyambura

    Background: Escherichia coli is among the most common causes of diarrhoea in children below five years of age in ... among the diarrhoeagenic E.coli (DEC) pathogenic strains which cause gastroenteritis cases ranging from ... faecal-oral route, through contaminated foods such as ground beef and unchlorinated water. A.

  11. Expression and purification of recombinant Shiga toxin 2B from ...

    African Journals Online (AJOL)

    sunny t

    (SDS-PAGE) and StxB2 yield was 450 µg ml-1 confirmed by Bradford assay. Recombinant Stx2B protein was produced in highly pure yield using HaloTag technology. Key words: Escherichia coli O157:H7, StxB gene, expression, HaloTag technology, purification. INTRODUCTION. Enterohemorrhagic Escherichia coli ...

  12. Expression and purification of recombinant Shiga toxin 2B from ...

    African Journals Online (AJOL)

    IMAC) column, followed by second step using HaloLink resin. The native Stx2B was obtained after column cleavage of halo-tag using HaloTEV protease. Maximum protein expression of Stx2B economically was obtained using 1 mM IPTG for 4 h at ...

  13. Bacteriophage-encoded shiga toxin gene in atypical bacterial host

    Directory of Open Access Journals (Sweden)

    Casas Veronica

    2011-07-01

    Full Text Available Abstract Background Contamination from fecal bacteria in recreational waters is a major health concern since bacteria capable of causing human disease can be found in animal feces. The Dog Beach area of Ocean Beach in San Diego, California is a beach prone to closures due to high levels of fecal indicator bacteria (FIB. A potential source of these FIB could be the canine feces left behind by owners who do not clean up after their pets. We tested this hypothesis by screening the DNA isolated from canine feces for the bacteriophage-encoded stx gene normally found in the virulent strains of the fecal bacterium Escherichia coli. Results Twenty canine fecal samples were collected, processed for total and bacterial fraction DNA, and screened by PCR for the stx gene. The stx gene was detected in the total and bacterial fraction DNA of one fecal sample. Bacterial isolates were then cultivated from the stx-positive fecal sample. Eighty nine of these canine fecal bacterial isolates were screened by PCR for the stx gene. The stx gene was detected in five of these isolates. Sequencing and phylogenetic analyses of 16S rRNA gene PCR products from the canine fecal bacterial isolates indicated that they were Enterococcus and not E. coli. Conclusions The bacteriophage-encoded stx gene was found in multiple species of bacteria cultivated from canine fecal samples gathered at the shoreline of the Dog Beach area of Ocean Beach in San Diego, California. The canine fecal bacteria carrying the stx gene were not the typical E. coli host and were instead identified through phylogenetic analyses as Enterococcus. This suggests a large degree of horizontal gene transfer of exotoxin genes in recreational waters.

  14. Shiga Toxin (Stx) Gene Detection and Verotoxigenic Potentials of ...

    African Journals Online (AJOL)

    DR-AMADI

    implicated severally as a very important food borne pathogen (Hussein, 2007). Many strains of E. coli have .... consisted of 2.5μl of PCR buffer (10mM Tris–. HCl pH 9, 50mM KCl, and 0.1% Triton X-100),. 2.5mM MgCl2 ... (GMEM) with addition of Hepes buffer as the growth medium. Growth medium was replaced with fresh ...

  15. Detection and Molecular Characterization of Sorbitol Negative Shiga Toxigenic Escherichia Coli in Chicken from Northwest of Iran

    Directory of Open Access Journals (Sweden)

    Aram Mokarizadeh

    2011-09-01

    Full Text Available AbstractShiga toxin-producing Escherichia coli (STEC are food-borne pathogens primarily associated with the consumption of contaminated ground beef and are an important food safety concern worldwide. STEC has been found to produce a family of related cytotoxins known as Shiga toxins (Stxs. Shiga toxins have been classified into two major classes, Stx1 and Stx2. A single strains of STEC can produce Stx1, Stx2 (or its variants or both. The aims of this project were to determine the prevalence and molecular characteristics of STEC isolates from chicken flocks in Northwest of Iran. A total of 350 fecal samples from 28 broiler farms were screened for the presence of STEC by conventional culture methods and polymerase chain reaction (PCR. All samples were initially subjected to phenotypical analysis using the Sorbitol MacConkey agar plate for the detection of the sorbitol negative E. coli, and then for genotypic analysis, by multiplex PCR for detection of stx1and stx2 genes. STEC were isolated from 14 (4 % chicken fecal samples. To our knowledge, this is the first report of isolation of STEC from poultry in Iran. To conclude, this work revealed the presence STEC strains harboring stx1 and stx2 gene in healthy chicken fecal samples in Northwest of Iran suggesting they can play as an important potential source of contamination for people working on broiler farms or are in contact with chicken carcasses at meat processing plants.

  16. Mode of Action of Shigella Toxin: Effects on Ribosome Structure and Function

    Science.gov (United States)

    1988-05-01

    Japanese that the deglycosylation mechanism is the correct, naturally occurring physiological means by which Shiga toxin inactivates ribosomes. If so, what ...1974. Sindrome hemolitico uremico: reporte de 60 casos asociados a una epidemia de enterocolitis hemorragica. Revista Colombiana de Ped. Puericult. 28

  17. Mechanism of inhibition of Shiga-toxigenicEscherichia coliSubAB cytotoxicity by steroids and diacylglycerol analogues.

    Science.gov (United States)

    Yahiro, Kinnosuke; Nagasawa, Sayaka; Ichimura, Kimitoshi; Takeuchi, Hiroki; Ogura, Kohei; Tsutsuki, Hiroyasu; Shimizu, Takeshi; Iyoda, Sunao; Ohnishi, Makoto; Iwase, Hirotaro; Moss, Joel; Noda, Masatoshi

    2018-12-01

    Shiga toxigenic Escherichia coli (STEC) are responsible for a worldwide foodborne disease, which is characterized by severe bloody diarrhea and hemolytic uremic syndrome (HUS). Subtilase cytotoxin (SubAB) is a novel AB 5 toxin, which is produced by Locus for Enterocyte Effacement (LEE)-negative STEC. Cleavage of the BiP protein by SubAB induces endoplasmic reticulum (ER) stress, followed by induction of cytotoxicity in vitro or lethal severe hemorrhagic inflammation in mice. Here we found that steroids and diacylglycerol (DAG) analogues (e.g., bryostatin 1, Ingenol-3-angelate) inhibited SubAB cytotoxicity. In addition, steroid-induced Bcl-xL expression was a key step in the inhibition of SubAB cytotoxicity. Bcl-xL knockdown increased SubAB-induced apoptosis in steroid-treated HeLa cells, whereas SubAB-induced cytotoxicity was suppressed in Bcl-xL overexpressing cells. In contrast, DAG analogues suppressed SubAB activity independent of Bcl-xL expression at early time points. Addition of Shiga toxin 2 (Stx2) with SubAB to cells enhanced cytotoxicity even in the presence of steroids. In contrast, DAG analogues suppressed cytotoxicity seen in the presence of both toxins. Here, we show the mechanism by which steroids and DAG analogues protect cells against SubAB toxin produced by LEE-negative STEC.

  18. Polyamine toxins

    DEFF Research Database (Denmark)

    Strømgaard, Kristian; Jensen, Lars S; Vogensen, Stine B

    2005-01-01

    Polyamine toxins, isolated from spiders and wasps, have been used as pharmacological tools for the study of ionotropic receptors, but their use have so far been hampered by their lack of selectivity. In this mini-review, we describe how careful synthetic modification of native polyamine toxins ha...

  19. Botulinum toxin

    Directory of Open Access Journals (Sweden)

    Nigam P

    2010-01-01

    Full Text Available Botulinum toxin, one of the most poisonous biological substances known, is a neurotoxin produced by the bacterium Clostridium botulinum. C. botulinum elaborates eight antigenically distinguishable exotoxins (A, B, C 1 , C 2 , D, E, F and G. All serotypes interfere with neural transmission by blocking the release of acetylcholine, the principal neurotransmitter at the neuromuscular junction, causing muscle paralysis. The weakness induced by injection with botulinum toxin A usually lasts about three months. Botulinum toxins now play a very significant role in the management of a wide variety of medical conditions, especially strabismus and focal dystonias, hemifacial spasm, and various spastic movement disorders, headaches, hypersalivation, hyperhidrosis, and some chronic conditions that respond only partially to medical treatment. The list of possible new indications is rapidly expanding. The cosmetological applications include correction of lines, creases and wrinkling all over the face, chin, neck, and chest to dermatological applications such as hyperhidrosis. Injections with botulinum toxin are generally well tolerated and side effects are few. A precise knowledge and understanding of the functional anatomy of the mimetic muscles is absolutely necessary to correctly use botulinum toxins in clinical practice.

  20. Multiplex detection of protein toxins using MALDI-TOF-TOF tandem mass spectrometry: application in unambiguous toxin detection from bioaerosol.

    Science.gov (United States)

    Alam, Syed Imteyaz; Kumar, Bhoj; Kamboj, Dev Vrat

    2012-12-04

    Protein toxins, such as botulinum neurotoxins (BoNTs), Clostridium perfringens epsilon toxin (ETX), staphylococcal enterotoxin B (SEB), shiga toxin (STX), and plant toxin ricin, are involved in a number of diseases and are considered as potential agents for bioterrorism and warfare. From a bioterrorism and warfare perspective, these agents are likely to cause maximum damage to a civilian or military population through an inhalational route of exposure and aerosol is considered the envisaged mode of delivery. Unambiguous detection of toxin from aerosol is of paramount importance, both for bringing mitigation protocols into operation and for implementation of effective medical countermeasures, in case a "biological cloud" is seen over a population. A multiplex, unambiguous, and qualitative detection of protein toxins is reported here using tandem mass spectrometry with MALDI-TOF-TOF. The methodology involving simple sample processing steps was demonstrated to identify toxins (ETX, Clostridium perfringes phospholipase C, and SEB) from blind spiked samples. The novel directed search approach using a list of unique peptides was used to identify toxins from a complex protein mixture. The bioinformatic analysis of seven protein toxins for elucidation of unique peptides with conservation status across all known sequences provides a high confidence for detecting toxins originating from any geographical location and source organism. Use of tandem MS data with peptide sequence information increases the specificity of the method. A prototype for generation of aerosol using a nebulizer and collection using a cyclone collector was used to provide a proof of concept for unambiguous detection of toxin from aerosol using precursor directed tandem mass spectrometry combined with protein database searching. ETX prototoxin could be detected from aerosol at 0.2 ppb concentration in aerosol.

  1. Role of host xanthine oxidase in infection due to enteropathogenic and Shiga-toxigenic Escherichia coli.

    Science.gov (United States)

    Crane, John K; Naeher, Tonniele M; Broome, Jacqueline E; Boedeker, Edgar C

    2013-04-01

    Xanthine oxidase (XO), also known as xanthine oxidoreductase, has long been considered an important host defense molecule in the intestine and in breastfed infants. Here, we present evidence that XO is released from and active in intestinal tissues and fluids in response to infection with enteropathogenic Escherichia coli (EPEC) and Shiga-toxigenic E. coli (STEC), also known as enterohemorrhagic E. coli (EHEC). XO is released into intestinal fluids in EPEC and STEC infection in a rabbit animal model. XO activity results in the generation of surprisingly high concentrations of uric acid in both cultured cell and animal models of infection. Hydrogen peroxide (H(2)O(2)) generated by XO activity triggered a chloride secretory response in intestinal cell monolayers within minutes but decreased transepithelial electrical resistance at 6 to 22 h. H(2)O(2) generated by XO activity was effective at killing laboratory strains of E. coli, commensal microbiotas, and anaerobes, but wild-type EPEC and STEC strains were 100 to 1,000 times more resistant to killing or growth inhibition by this pathway. Instead of killing pathogenic bacteria, physiologic concentrations of XO increased virulence by inducing the production of Shiga toxins from STEC strains. In vivo, exogenous XO plus the substrate hypoxanthine did not protect and instead worsened the outcome of STEC infection in the rabbit ligated intestinal loop model of infection. XO released during EPEC and STEC infection may serve as a virulence-inducing signal to the pathogen and not solely as a protective host defense.

  2. [Occurrence of Shiga toxigenic Escherichia coli strains in pigs and cattle at slaughterhouses in the Czech Republic in 2013].

    Science.gov (United States)

    Koláčková, Ivana; Házová, Klára; Skočková, Alena; Karpíšková, Renáta

    2014-06-01

    This study was performed in cooperation with the State Veterinary Administration (SVA) in order to monitor the occurrence of Shiga toxin-producing Escherichia coli isolates in swabs from the carcasses of pigs and cattle at slaughterhouses. From June to August 2013, SVA staff took 168 swabs from cattle and 318 from pigs at 157 different slaughters in the Czech Republic. Basic processing of the samples was carried out in the State Veterinary Institutes (SVIs) in Prague, Jihlava and Olomouc according to the methodical process coordinated by the National reference laboratory (NRL) for Escherichia coli (Czech Ministry of Agriculture). The procedure was based on the guideline ISO TS 13136. Out of the 486 swabs, twenty-two positive samples were detected. There were a total of 22 isolates of Shiga toxigenic E. coli (STEC) and 1 strain with the characteristic of enterohemorrhagic E. coli (EHEC). Genes typical for enteroaggregative E. coli (EAggEC) were not found in any of the isolates. Most STEC strains originated from pigs. The stx1 gene was detected twice (stx1a, stx1d) and the stx2 gene 13 times (12 times stx2e, once stx2a). Seven STEC isolates were detected from samples of cattle origin. One strain was stx1 (stx1a) -positive, the stx2 gene was found 6 times (4 stx2e, 1 stx2a and 1 stx2c). One isolate carried simultaneously both stx1a and stx2a. Each of the serogroups O91, O113 and O146 described as etiological agents of severe disease in humans were detected only once. None of these strains harbored additional virulence factors typical for strains causing serious illness. RESULTS of this study show the overall prevalence of Shiga toxigenic E. coli of 4.5 % and 0.2 % of enterohemorrhagic strains in the studied samples. Raw meat originating from local farms does not currently represent an important source of STEC for humans.

  3. Influence of yogurt fermentation and refrigerated storage on the stability of protein toxin contaminants.

    Science.gov (United States)

    Jackson, Lauren S; Triplett, Odbert A; Tolleson, William H

    2015-06-01

    Dairy products sold in a ready-to-eat form present the risk that adulterants persisting through manufacturing, storage, and distribution would reach consumers. Pathogenic microbes, including shigatoxigenic strains of Escherichia coli and the toxins they produce, are common food safety hazards associated with dairy products. Ricin and abrin are plant-derived ribosome-inactivating protein toxins related to the shiga-like toxins produced by E. coli. Limited information exists on the effects of manufacturing processes on the stabilities of these heat-resistant ribosome-inactivating proteins in the presence of foods. The goal of this study was to determine how typical yogurt manufacturing and storage processes influence ribosome-inactivating protein toxins. Ricin and abrin were added to skim or whole milk and batch pasteurized. Complete inactivation of both toxins was observed after 30 minutes at 85 °C. If the toxins were added after pasteurization, the levels of ricin and abrin in yogurt and their cytotoxic activities did not change significantly during fermentation or refrigerated storage for 4 weeks. The activities of ricin and abrin were inhibited by skim milk, nonfat yogurt, whole milk, and whole milk yogurt. The results showed minimal effects of the toxins on yogurt pH and %titratable acidity but inhibitory effects of yogurt on toxin activity. Published by Elsevier Ltd.

  4. Radiolabelling of cholera toxin

    International Nuclear Information System (INIS)

    Santos, R.G.; Neves, Nicoli M.J.; Abdalla, L.F.; Brandao, R.L.; Etchehebehere, L.; Lima, M.E. de; Nicoli, J.R.

    1999-01-01

    Binding of cholera toxin to ganglioside receptors of enterocyte microvilli catalyzes the activation of adenylate cyclase causing a rise in cAMP which final result is a copious diarrhea. Saccharomyces boulardii, a nonpathogenic yeast has been used to prevent diarrhea. Although the antidiarrheic properties of S. boulardii are widely recognized, this yeast has been used on empirical basis, and the mechanism of this protective effect is unknown. The addition of cholera toxin to S. boulardii induces the raising of cAMP that triggers the activation of neutral trehalase. This suggests that toxin specifically binding to cells, is internalized and active the protein phosphorylation cascade. Our objective is labeling the cholera toxin to verify the presence of binding sites on yeast cell surfaces for the cholera toxin. Cholera toxin was radiolabelled with Na 125 I by a chloramine-T method modified from Cuatrecasas and Griffiths et alii. The 125 I-Cholera toxin showed a specific radioactivity at about 1000 cpm/fmol toxin. Biological activity of labeled cholera toxin measured by trehalase activation was similar to the native toxin. (author)

  5. Radiolabelling of cholera toxin

    Energy Technology Data Exchange (ETDEWEB)

    Santos, R.G.; Neves, Nicoli M.J. [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN), Belo Horizonte, MG (Brazil); Abdalla, L.F.; Brandao, R.L.; Etchehebehere, L. [Ouro Preto Univ., MG (Brazil). Escola de Farmacia. Lab. de Fisiologia e Bioquimica de Microorganismos; Lima, M.E. de [Minas Gerais Univ., Belo Horizonte, MG (Brazil). Inst. de Ciencias Biologicas. Dept. de Bioquimica e Imunologia; Nicoli, J.R. [Minas Gerais Univ., Belo Horizonte, MG (Brazil). Inst. de Ciencias Biologicas. Dept. de Microbiologia

    1999-11-01

    Binding of cholera toxin to ganglioside receptors of enterocyte microvilli catalyzes the activation of adenylate cyclase causing a rise in cAMP which final result is a copious diarrhea. Saccharomyces boulardii, a nonpathogenic yeast has been used to prevent diarrhea. Although the antidiarrheic properties of S. boulardii are widely recognized, this yeast has been used on empirical basis, and the mechanism of this protective effect is unknown. The addition of cholera toxin to S. boulardii induces the raising of cAMP that triggers the activation of neutral trehalase. This suggests that toxin specifically binding to cells, is internalized and active the protein phosphorylation cascade. Our objective is labeling the cholera toxin to verify the presence of binding sites on yeast cell surfaces for the cholera toxin. Cholera toxin was radiolabelled with Na {sup 125} I by a chloramine-T method modified from Cuatrecasas and Griffiths et alii. The {sup 125} I-Cholera toxin showed a specific radioactivity at about 1000 cpm/fmol toxin. Biological activity of labeled cholera toxin measured by trehalase activation was similar to the native toxin. (author) 5 refs., 3 figs.; e-mail: nevesmj at urano.cdtn.br

  6. Recovery, characterization, and control of Shiga-toxin producing Escherichia coli(STEC) in meats

    Science.gov (United States)

    The objective of this presentation is to provide an overview of research conducted by the United States Department of Agriculture (USDA) at several locations at the Agricultural Research Service (ARS) in the Eastern United States. This research was conducted in collaboration with numerous national a...

  7. 77 FR 31975 - Shiga Toxin-Producing Escherichia coli in Certain Raw Beef Products

    Science.gov (United States)

    2012-05-31

    ... industry, collectively, is controlling for the presence of a designated food safety hazard in products... and certification of alternative methods for the microbiological analysis of food and beverages (Micro...

  8. Shiga Toxin Increases Formation of Clathrin-Coated Pits through Syk Kinase

    DEFF Research Database (Denmark)

    Utskarpen, Audrun; Massol, Ramiro; van Deurs, Bo

    2010-01-01

    with Stx results in an increase in the number of clathrin-coated profiles as determined by electron microscopy and on the number of structures containing the endocytic AP-2 adaptor at the plasma membrane determined by live-cell spinning disk confocal imaging. These responses to Stx require functional Syk...... activates signaling through the tyrosine kinase Syk. We previously described that Syk activity is important for Stx entry, but it remained unclear how this kinase modulates endocytosis of Stx. Here we characterized the effects of Stx and Syk on clathrin-coated pit formation. We found that acute treatment...... activity. We propose that a signaling pathway mediated by Syk and modulated by Stx leads to an increased number of endocytic clathrin-coated structures, thus providing a possible mechanism by which Stx enhances its own endocytosis....

  9. Development of an ELISA microarray assay for the sensitive and simultaneous detection of ten biodefense toxins.

    Energy Technology Data Exchange (ETDEWEB)

    Jenko, Kathryn; Zhang, Yanfeng; Kostenko, Yulia; Fan, Yongfeng; Garcia-Rodriguez, Consuelo; Lou, Jianlong; Marks, James D.; Varnum, Susan M.

    2014-10-21

    Plant and microbial toxins are considered bioterrorism threat agents because of their extreme toxicity and/or ease of availability. Additionally, some of these toxins are increasingly responsible for accidental food poisonings. The current study utilized an ELISA-based protein antibody microarray for the multiplexed detection of ten biothreat toxins, botulinum neurotoxins (BoNT) A, B, C, D, E, F, ricin, shiga toxins 1 and 2 (Stx), and staphylococcus enterotoxin B (SEB), in buffer and complex biological matrices. The multiplexed assay displayed a sensitivity of 1.3 pg/mL (BoNT/A, BoNT/B, SEB, Stx-1 and Stx-2), 3.3 pg/mL (BoNT/C, BoNT/E, BoNT/F) and 8.2 pg/mL (BoNT/D, ricin). All assays demonstrated high accuracy (75-120 percent recovery) and reproducibility (most coefficients of variation < 20%). Quantification curves for the ten toxins were also evaluated in clinical samples (serum, plasma, nasal fluid, saliva, stool, and urine) and environmental samples (apple juice, milk and baby food) with overall minimal matrix effects. The multiplex assays were highly specific, with little crossreactivity observed between the selected toxin antibodies. The results demonstrate a multiplex microarray that improves current immunoassay sensitivity for biological warfare agents in buffer, clinical, and environmental samples.

  10. Protection against Shiga-Toxigenic Escherichia coli by Non-Genetically Modified Organism Receptor Mimic Bacterial Ghosts.

    Science.gov (United States)

    Paton, Adrienne W; Chen, Austen Y; Wang, Hui; McAllister, Lauren J; Höggerl, Florian; Mayr, Ulrike Beate; Shewell, Lucy K; Jennings, Michael P; Morona, Renato; Lubitz, Werner; Paton, James C

    2015-09-01

    Shiga-toxigenic Escherichia coli (STEC) causes severe gastrointestinal infections in humans that may lead to life-threatening systemic sequelae, such as the hemolytic uremic syndrome (HUS). Rapid diagnosis of STEC infection early in the course of disease opens a window of opportunity for therapeutic intervention, for example, by administration of agents that neutralize Shiga toxin (Stx) in the gut lumen. We previously developed a recombinant bacterium that expresses a mimic of the Stx receptor globotriaosyl ceramide (Gb3) on its surface through modification of the lipopolysaccharide (A. W. Paton, R. Morona, and J. C. Paton, Nat Med 6:265-270, 2000, http://dx.doi.org/10.1038/73111). This construct was highly efficacious in vivo, protecting mice from otherwise fatal STEC disease, but the fact that it is a genetically modified organism (GMO) has been a barrier to clinical development. In the present study, we have overcome this issue by development of Gb3 receptor mimic bacterial ghosts (BGs) that are not classified as GMOs. Gb3-BGs neutralized Stx1 and Stx2 in vitro with high efficiency, whereas alternative Gb3-expressing non-GMO subbacterial particles (minicells and outer membrane blebs) were ineffective. Gb3-BGs were highly efficacious in a murine model of STEC disease. All mice (10/10) treated with Gb3-BGs survived challenge with a highly virulent O113:H21 STEC strain and showed no pathological signs of renal injury. In contrast, 6/10 mice treated with control BGs succumbed to STEC challenge, and survivors exhibited significant weight loss, neutrophilia, and histopathological evidence of renal damage. Thus, Gb3-BGs offer a non-GMO approach to treatment of STEC infection in humans, particularly in an outbreak setting. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  11. Microalgal toxin(s): characteristics and importance

    African Journals Online (AJOL)

    Prokaryotic and eukaryotic microalgae produce a wide array of compounds with biological activities. These include antibiotics, algicides, toxins, pharmaceutically active compounds and plant growth regulators. Toxic microalgae, in this sense, are common only among the cyanobacteria and dinoflagellates. The microalgal ...

  12. ESBL-producing Shiga-toxigenic E. coli (STEC) associated with piglet diarrhoea in India.

    Science.gov (United States)

    Mandakini, Rajkumari; Dutta, Tapan Kumar; Chingtham, Santhalembi; Roychoudhury, Parimal; Samanta, Indranil; Joardar, Sidhartha Narayan; Pachauau, Abigail R; Chandra, Rajesh

    2015-02-01

    The present study was conducted to investigate the prevalence of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli carrying other virulence genes associated with piglet diarrhoea. Faecal samples from the piglets with history of diarrhoea were processed for isolation of E. coli and were tested for their ability of ESBL production. ESBL encoding genes and other virulence genes were detected by specific PCR, and amplicons were sequenced. Ability of transferring the ESBL genes between the enteric bacteria was tested in in vitro HGT. A total of 170 E. coli was isolated, of which 43 (25.29 %) were confirmed as ESBL producer by double disc synergy test (DDST). Altogether, 6.47 and 2.94 % isolates were positive for bla TEM and bla CTX-M-15 genes, respectively, of which 2.35 % isolates were positive for both the genes and only 0.6 % isolate was positive for the bla CTX-M gene alone. The resistance traits could not be transferred to the recipient host. Based on PCR, 2 (1.18 %) and 1 (0.59 %) isolates were recorded as Shiga-toxin-producing E. coli (STEC) and enteropathogenic E. coli (EPEC), respectively. Both the STEC (one isolate positive for stx 2 and another positive for stx 2 and hlyA) isolates belonging to serogroup O2 and NT were also positive for the bla CTX-M-15 gene. This is the first report of ESBL-producing E. coli isolate possessing the STEC gene associated with piglet diarrhoea.

  13. Phage-mediated Shiga toxin (Stx) horizontal gene transfer and expression in non-Shiga toxigenic Enterobacter and Escherichia coli strains.

    Science.gov (United States)

    Khalil, Rowaida K S; Skinner, Craig; Patfield, Stephanie; He, Xiaohua

    2016-07-01

    Enterobacter cloacae M12X01451 strain recently identified from a clinical specimen produces a new Stx1 subtype (Stx1e) that was not neutralized by existing anti-Stx1 monoclonal antibodies. Acquisition of stx by Ent. cloacae is rare and origin/stability of stx1e in M12X01451 is not known. In this study, we confirmed the ability of Stx1a- and Stx1e-converting phages from an Escherichia coli O157:H7 strain RM8530 and M12X01451 respectively to infect several E. coli and Ent. cloacae strains. stx1e was detected in 97.5% and 72.5% of progenies of strains lysogenized by stx1e phage after 10 (T10) and 20 (T20) subcultures, versus 65% and 17.5% for stx1a gene. Infection of M12X01451 and RM8530 with each other's phages generated double lysogens containing both phages. stx1a was lost after T10, whereas the stx1e was maintained even after T20 in M12X01451 lysogens. In RM8530 lysogens, the acquired stx1e was retained with no mutations, but 20% of stx1a was lost after T20 ELISA and western blot analyses demonstrated that Stx1e was produced in all strains lysogenized by stx1e phage; however, Stx1a was not detected in any lysogenized strain. The study results highlight the potential risks of emerging Stx-producing strains via bacteriophages either in the human gastrointestinal tract or in food production environments, which are matters of great concern and may have serious impacts on human health. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  14. Botulinum Toxin for Rhinitis.

    Science.gov (United States)

    Ozcan, Cengiz; Ismi, Onur

    2016-08-01

    Rhinitis is a common clinical entity. Besides nasal obstruction, itching, and sneezing, one of the most important symptoms of rhinitis is nasal hypersecretion produced by nasal glands and exudate from the nasal vascular bed. Allergic rhinitis is an IgE-mediated inflammatory reaction of nasal mucosa after exposure to environmental allergens. Idiopathic rhinitis describes rhinitis symptoms that occur after non-allergic, noninfectious irritants. Specific allergen avoidance, topical nasal decongestants, nasal corticosteroids, immunotherapy, and sinonasal surgery are the main treatment options. Because the current treatment modalities are not enough for reducing rhinorrhea in some patients, novel treatment options are required to solve this problem. Botulinum toxin is an exotoxin generated by Clostridium botulinum. It disturbs the signal transmission at the neuromuscular and neuroglandular junction by inhibiting the acetylcholine release from the presynaptic nerve terminal. It has been widely used in neuromuscular, hypersecretory, and autonomic nerve system disorders. There have been a lot of published articles concerning the effect of this toxin on rhinitis symptoms. Based on the results of these reports, intranasal botulinum toxin A administration appears to be a safe and effective treatment method for decreasing rhinitis symptoms in rhinitis patients with a long-lasting effect. Botulinum toxin type A will be a good treatment option for the chronic rhinitis patients who are resistant to other treatment methods.

  15. Carbohydrate inhibitors of cholera toxin.

    Science.gov (United States)

    Kumar, Vajinder; Turnbull, W Bruce

    2018-01-01

    Cholera is a diarrheal disease caused by a protein toxin released by Vibrio cholera in the host's intestine. The toxin enters intestinal epithelial cells after binding to specific carbohydrates on the cell surface. Over recent years, considerable effort has been invested in developing inhibitors of toxin adhesion that mimic the carbohydrate ligand, with particular emphasis on exploiting the multivalency of the toxin to enhance activity. In this review we introduce the structural features of the toxin that have guided the design of diverse inhibitors and summarise recent developments in the field.

  16. Autoproteolytic Activation of Bacterial Toxins

    Directory of Open Access Journals (Sweden)

    Aimee Shen

    2010-05-01

    Full Text Available Protease domains within toxins typically act as the primary effector domain within target cells. By contrast, the primary function of the cysteine protease domain (CPD in Multifunctional Autoprocessing RTX-like (MARTX and Clostridium sp. glucosylating toxin families is to proteolytically cleave the toxin and release its cognate effector domains. The CPD becomes activated upon binding to the eukaryotic-specific small molecule, inositol hexakisphosphate (InsP6, which is found abundantly in the eukaryotic cytosol. This property allows the CPD to spatially and temporally regulate toxin activation, making it a prime candidate for developing anti-toxin therapeutics. In this review, we summarize recent findings related to defining the regulation of toxin function by the CPD and the development of inhibitors to prevent CPD-mediated activation of bacterial toxins.

  17. Shiga Toxigenic Escherichia coli in Iranian Pediatric Patients With and Without Diarrhea: O-Serogroups, Virulence Factors and Antimicrobial Resistance Properties

    Science.gov (United States)

    Dormanesh, Banafshe; Siroosbakhat, Soheila; Karimi Goudarzi, Peyman; Afsharkhas, Ladan

    2015-01-01

    Background: Shiga-toxigenic Escherichia coli is an important human pathogen cause of diarrhea, hemorrhagic colitis, hemolytic uremic syndrome and thrombotic thrombocytopenic purpura in humans is a significant public health. Objectives: The aim of this study was to determine the molecular characteristics and antimicrobial resistance properties of Shiga toxigenic Escherichia coli (STEC) strains with respect to their seasonal, age and geographical distributions in Iranian pediatric patients with and without diarrhea. Patients and Methods: Four hundred and eighty swab samples were taken from pediatric patients with and without diarrhea of four major provinces of Iran. Swab samples were immediately cultured and the positive culture samples were analyzed by the polymerase chain reaction (PCR) method. Finally, antimicrobial susceptibility testing was performed using the disk diffusion method in Mueller-Hinton agar. Results: In total, 118 out of 200 diarrheic stool samples (59%) and 77 out of 280 non-diarrheic stool samples (27.5%) were positive for E. coli. Samples taken from one to ten months old cases (73.33%) and those from Shiraz province (81.13%) were the most commonly infected. Samples taken in the summer season (91.66%) were the most commonly infected. A significant difference was shown between AEEC and EHEC strains of E. coli. The genes encoding Shiga toxins and intimin protein were the most commonly detected in all strains. O26 (33.33%), O111 (18.18%) and O91 (12.12%) serogroups had the highest incidence in patients with and without diarrhea. Prevalence of the genes that encode resistance against ampicillin (CITM), gentamicin (aac(3)-IV) and tetracycline (tetA) were 80.30%, 75.75% and 65.15%, respectively. The STEC strains harbored the highest levels of resistance against ampicillin (84.84%), gentamycin (78.78%), tetracycline (50%) and sulfamethoxazole (40.90%) antibiotics. We found that 55.08% of diarrheic and 1.29% of non-diarrheic E. coli isolates were

  18. H-NS Mutation-Mediated CRISPR-Cas Activation Inhibits Phage Release and Toxin Production ofEscherichia coliStx2 Phage Lysogen.

    Science.gov (United States)

    Fu, Qiang; Li, Shiyu; Wang, Zhaofei; Shan, Wenya; Ma, Jingjiao; Cheng, Yuqiang; Wang, Hengan; Yan, Yaxian; Sun, Jianhe

    2017-01-01

    Shiga toxin-converting bacteriophages (Stx phages) carry the stx gene and convert nonpathogenic bacterial strains into Shiga toxin-producing bacteria. There is limited understanding of the effect that an Escherichia coli ( E. coli ) clustered regularly interspaced short palindromic repeats (CRISPR)-Cas adaptive immune system has on Stx phage lysogen. We investigated heat-stable nucleoid-structuring (H-NS) mutation-mediated CRISPR-Cas activation and its effect on E. coli Stx2 phage lysogen. The Δ hns mutant (MG1655Δ hns ) of the E. coli K-12 strain MG1655 was obtained. The Δ hns mutant lysogen that was generated after Stx phage lysogenic infection had a repressed growth status and showed subdued group behavior, including biofilm formation and swarming motility, in comparison to the wild-type strain. The de-repression effect of the H-NS mutation on CRISPR-Cas activity was then verified. The results showed that cas gene expression was upregulated and the transformation efficiency of the wild-type CRISPR plasmids was decreased, which may indicate activation of the CRISPR-Cas system. Furthermore, the function of CRISPR-Cas on Stx2 phage lysogen was investigated by activating the CRISPR-Cas system, which contains an insertion of the protospacer regions of the Stx2 phage Min27. The phage release and toxin production of four lysogens harboring the engineered CRISPRs were investigated. Notably, in the supernatant of the Δ hns mutant lysogen harboring the Min27 spacer, both the progeny phage release and the toxin production were inhibited after mitomycin C induction. These observations demonstrate that the H-NS mutation-activated CRISPR-Cas system plays a role in modifying the effects of the Stx2 phage lysogen. Our findings indicated that H-NS mutation-mediated CRISPR-Cas activation in E. coli protects bacteria against Stx2 phage lysogeny by inhibiting the phage release and toxin production of the lysogen.

  19. Fusion of Cholera toxin B subunit (ctxB with Shigella dysenteriae type I toxin B subunit (stxB, Cloning and Expression that in E. coli

    Directory of Open Access Journals (Sweden)

    2012-12-01

    Full Text Available Background and Objective: Shiga toxin (STx is the main virulence factor in Shigella Dysenteriae type I and is composed of an enzymatic subunit STxA monomer and a receptor-binding STxB homopentamer. Shigella toxin B subunit (STxB is a non-toxic homopentameric protein responsible for toxin binding and internalization into target cells by interacting with glycolipid (Gb3. Cholera toxin B subunit (CTB has been known as a mucosal adjuvant for vaccines and genetic fusions of CTB with several hetroantigens such as stxB and can increase humoral and mucosal immunity response.Materials and Methods: In this study, after primer designing, the ctxB and stxB genes were amplified by PCR and cloned into the pGEM-T vector. The stxB gene with a nonfurin linker was fused to the ctB gene in the pGEM vector via the restriction enzyme method and thereafter the fused genes of ctB-stxB were subcloned in the pET28a(+ as an expression vector. The expressed chimeric protein was induced with IPTG and evaluated via the SDS.PAGE and Western blot techniques. Result: The pET28a (+/ctxB-stxB expression vector was confirmed by endonuclease digestion, PCR, and sequence analysis. The CTB-STB fusion protein was confirmed by the SDS-PAGE and Western-blot. Conclusion: The CTB-STB recombinant protein can be used as a new and desirable mucosal vaccine for Shigella Dysenteriae type I.

  20. Lymphocyte receptors for pertussis toxin

    Energy Technology Data Exchange (ETDEWEB)

    Clark, C.G.; Armstrong, G.D. (Univ. of Alberta, Edmonton (Canada))

    1990-12-01

    We have investigated human T-lymphocyte receptors for pertussis toxin by affinity isolation and photoaffinity labeling procedures. T lymphocytes were obtained from peripheral human blood, surface iodinated, and solubilized in Triton X-100. The iodinated mixture was then passed through pertussis toxin-agarose, and the fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoradiography of the fixed, dried gels revealed several bands in the pertussis toxin-bound fraction that were not observed in fractions obtained from histone or fetuin-agarose. Further investigations employed a photoaffinity labeling reagent, sulfosuccinimidyl 2-(p-azido-salicylamido)-1,3'-dithiopropionate, to identify pertussis toxin receptors in freshly isolated peripheral blood monocytic cells, T lymphocytes, and Jurkat cells. In all three cell systems, the pertussis toxin affinity probe specifically labeled a single protein species with an apparent molecular weight of 70,000 that was not observed when the procedure was performed in the presence of excess unmodified pertussis toxin. A protein comparable in molecular weight to the one detected by the photoaffinity labeling technique was also observed among the species that bound to pertussis toxin-agarose. The results suggest that pertussis toxin may bind to a 70,000-Da receptor in human T lymphocytes.

  1. Binding of ATP by pertussis toxin and isolated toxin subunits

    International Nuclear Information System (INIS)

    Hausman, S.Z.; Manclark, C.R.; Burns, D.L.

    1990-01-01

    The binding of ATP to pertussis toxin and its components, the A subunit and B oligomer, was investigated. Whereas, radiolabeled ATP bound to the B oligomer and pertussis toxin, no binding to the A subunit was observed. The binding of [ 3 H]ATP to pertussis toxin and the B oligomer was inhibited by nucleotides. The relative effectiveness of the nucleotides was shown to be ATP > GTP > CTP > TTP for pertussis toxin and ATP > GTP > TTP > CTP for the B oligomer. Phosphate ions inhibited the binding of [ 3 H]ATP to pertussis toxin in a competitive manner; however, the presence of phosphate ions was essential for binding of ATP to the B oligomer. The toxin substrate, NAD, did not affect the binding of [ 3 H]ATP to pertussis toxin, although the glycoprotein fetuin significantly decreased binding. These results suggest that the binding site for ATP is located on the B oligomer and is distinct from the enzymatically active site but may be located near the eukaryotic receptor binding site

  2. Binding of ATP by pertussis toxin and isolated toxin subunits

    Energy Technology Data Exchange (ETDEWEB)

    Hausman, S.Z.; Manclark, C.R.; Burns, D.L. (Center for Biologics Evaluation and Research, Bethesda, MD (USA))

    1990-07-03

    The binding of ATP to pertussis toxin and its components, the A subunit and B oligomer, was investigated. Whereas, radiolabeled ATP bound to the B oligomer and pertussis toxin, no binding to the A subunit was observed. The binding of ({sup 3}H)ATP to pertussis toxin and the B oligomer was inhibited by nucleotides. The relative effectiveness of the nucleotides was shown to be ATP > GTP > CTP > TTP for pertussis toxin and ATP > GTP > TTP > CTP for the B oligomer. Phosphate ions inhibited the binding of ({sup 3}H)ATP to pertussis toxin in a competitive manner; however, the presence of phosphate ions was essential for binding of ATP to the B oligomer. The toxin substrate, NAD, did not affect the binding of ({sup 3}H)ATP to pertussis toxin, although the glycoprotein fetuin significantly decreased binding. These results suggest that the binding site for ATP is located on the B oligomer and is distinct from the enzymatically active site but may be located near the eukaryotic receptor binding site.

  3. Captive wild birds as reservoirs of enteropathogenic E. coli (EPEC and Shiga-toxin producing E. coli (STEC

    Directory of Open Access Journals (Sweden)

    Lilian Aparecida Sanches

    Full Text Available ABSTRACT Psittacine birds have been identified as reservoirs of diarrheagenic Escherichia coli, a subset of pathogens associated with mortality of children in tropical countries. The role of other orders of birds as source of infection is unclear. The aim of this study was to perform the molecular diagnosis of infection with diarrheagenic E. coli in 10 different orders of captive wild birds in the state of São Paulo, Brazil. Fecal samples were analyzed from 516 birds belonging to 10 orders: Accipitriformes, Anseriformes, Columbiformes, Falconiformes, Galliformes, Passeriformes, Pelecaniformes, Piciformes, Psittaciformes and Strigiformes. After isolation, 401 E. coli strains were subjected to multiplex PCR system with amplification of genes eae and bfp (EPEC, stx1 and stx2 for STEC. The results of these tests revealed 23/401 (5.74% positive strains for eae gene, 16/401 positive strains for the bfp gene (3.99% and 3/401 positive for stx2 gene (0.75% distributed among the orders of Psittaciformes, Strigiformes and Columbiformes. None of strains were positive for stx1 gene. These data reveal the infection by STEC, typical and atypical EPEC in captive birds. The frequency of these pathotypes is low and restricted to few orders, but the data suggest the potential public health risk that these birds represent as reservoirs of diarrheagenic E. coli.

  4. Concerning Increase in Antimicrobial Resistance in Shiga Toxin-Producing Escherichia coli Isolated from Young Animals during 1980-2016.

    Science.gov (United States)

    Chirila, Flore; Tabaran, Alexandra; Fit, Nicodim; Nadas, George; Mihaiu, Marian; Tabaran, Flaviu; Cătoi, Cornel; Reget, Oana Lucia; Dan, Sorin Daniel

    2017-09-27

    This study was conducted in order to assess the antimicrobial resistance patterns of E. coli isolated from young animals affected between 1980 and 2016. The selected isolates for this study (n=175) carried stx 1 /stx 2 genes and the most prevalent type of pathogenic E. coli found belonged to serogroup O101, antigen (K99)-F41 positive. All STEC-positive isolates were tested for susceptibility to 11 antimicrobials. Multidrug resistance (MDR) increased from 11% during the 1980s to 40% between 2000 and 2016. Resistance to tetracycline and streptomycin was the most frequent co-resistance phenotype (37%). Co-resistance to tetracycline and sulfonamide was found in 21% of E. coli isolates, while the MDR pattern to tetracycline, sulfonamide, and streptomycin was observed in 12% of the strains tested. Only 8% of isolates were co-resistant to tetracycline, ampicillin, streptomycin, and sulfonamide. The most common resistance genes found were those encoding for tetracycline, sulphonamides, and streptomycin, with 54% (n=95) of the tested isolates containing at least one of the genes encoding tetracycline resistance. A total of 87% of E. coli that tested positive for tetracycline (tetA, tetB, and tetC) and sulphonamide (sul1) resistance genes were isolated between 2000 and 2016. A large number of isolates (n=21) carried int1 and a nucleotide sequence analysis revealed that all class 1 integron gene cassettes carried sul1, tet, and dfrA1 resistance genes. An increase was observed in the level of resistance to antimicrobials in Romania, highlighting the urgent need for a surveillance and prevention system for antimicrobial resistance in livestock in Eastern Europe.

  5. Occurrence of Escherichia coli, Campylobcter, Salmonella and Shiga-Toxin producing E. coli in Norwegian primary strawberry production

    OpenAIRE

    Johannessen, Gro Skøien; Eckner, Karl Fredrich; Heiberg, Nina; Monshaugen, Marte; Begum, Mumtaz; Økland, Marianne; Høgåsen, Helga Rachel

    2015-01-01

    The aim of this study was to investigate the bacteriological quality of strawberries at harvest and to study risk factors such as irrigation water, soil and picker’s hand cleanliness. Four farms were visited during the harvest season in 2012. Samples of strawberries, irrigation water, soil and hand swabs were collected and analyzed for E. coli, Campylobacter, Salmonella and STEC Although fecal indicators and pathogens were found in environmental samples, only one of 80 samples of strawberries...

  6. Isolation of shiga toxin-producing Escherichia coli O157:H7/NM from hamburger and chicken nugget

    Directory of Open Access Journals (Sweden)

    Ali Miri

    2014-01-01

    Material and Methods: From June 2013 to July 2013, a total of 190 hamburger (120 and chicken nugget (70, were collected from four randomly selected factories in Isfahan, Iran. They were evaluated for the presence of E. coli O157:H7/NM using microbiological culture and polymerase chain reaction. Statistical analysis was performed using SPSS software version 16.0 (SPSS Inc., Chicago o, IL, USA. Results: From a total of 190 samples analyzed four samples (2.1% were contaminated with E. coli O157. All of the E. coli O157 were isolated from hamburger samples (3.3% and chicken nugget samples were negative. Of four E. coli O157 isolated, only one sample was serotype E. coli O157:H7 and others were serotype E. coli O157:NM. Among four E. coli O157:H7/NM isolates, one strain was positive for all stx1, stx2, eaeA and ehxA genes. One strain was positive for stx2 gene. The other two were negative for these genes. All isolates (100% were resistant to one or more antimicrobial agents. Conclusions: The results of this study showed that hamburger could be a significant source of E. coli O157:H7 and E. coli O157:NM serotypes in Iran and multi-resistance was found in 27% of E. coli O157 strains and this is a major public health concern.

  7. Development of Recombinant Lactococcus lactis Displaying Albumin-Binding Domain Variants against Shiga Toxin 1 B Subunit

    Czech Academy of Sciences Publication Activity Database

    Zadravec, P.; Marečková, Lucie; Petroková, Hana; Hodnik, V.; Nanut, M. P.; Anderluh, G.; Strukelj, B.; Malý, Petr; Berlec, A.

    2016-01-01

    Roč. 11, č. 9 (2016), č. článku e0162625. E-ISSN 1932-6203 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:86652036 Keywords : escherichia-coli infections * controlled gene-expression * surface display * acid bacteria Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.806, year: 2016

  8. The first 30 years of Shiga toxin-producing Escherichia coli in cattle production: Incidence, preharvest ecology, and management

    Science.gov (United States)

    Of the 700 serotypes of Escherichia coli, most are commensal; however, some range from mildly to highly pathogenic and can cause death. The disease-causing enterovirulent E. coli are classified as: Enterotoxigenic E. coli (ETEC), Enteropathogenic E. coli (EPEC), Enteroinvasive E. coli (EIEC), and ...

  9. Radiation resistance of non-0157:H7 Shiga Toxin-Producing Escherichia coli suspended in refrigerated catfish fillet meat

    Science.gov (United States)

    Ionization (gamma) irradiation is a sustainable and important non-thermal treatment that has been very effective in controlling microorganisms and improving the safety and shelf life of foods. In the design of the food irradiation process, the knowledge of the radiation resistance of the target orga...

  10. The Regulation of Expression of the Stx2d Toxins in Shiga Toxin-producing Escherichia coli O91:H21 Strain B2F1

    Science.gov (United States)

    2002-01-01

    cytotoxicity assay (Lindgren, 1993). This latter result, combined with the lower toxicity of the mutant that produced Stx2d1 suggests that Stx2d1...Sato,K., Miyamoto,H., Yamamura,T., and Honda,T. (1999) Induction of prophages of enterohemorrhagic Escherichia coli O157:H7 with norfloxacin

  11. Food toxin detection with atomic force microscope

    Science.gov (United States)

    Externally introduced toxins or internal spoilage correlated pathogens and their metabolites are all potential sources of food toxins. To prevent and protect unsafe food, many food toxin detection techniques have been developed to detect various toxins for quality control. Although several routine m...

  12. Toxins: State of Journal Report, 2016

    Directory of Open Access Journals (Sweden)

    Vernon L. Tesh

    2015-12-01

    Full Text Available In the “Message from the Editor-in-Chief” posted on the Toxins website (see www.mdpi.com/journal/toxins/toxins-flyer.pdf, we wrote: “The editorial board and staff of Toxins are dedicated to providing a timely, peer-reviewed outlet for exciting, innovative primary research articles and concise, informative reviews from investigators in the myriad of disciplines contributing to our knowledge on toxins. [...

  13. Bio Warfare and Terrorism: Toxins and Other Mid-Spectrum Agents

    National Research Council Canada - National Science Library

    Madsen, James M

    2005-01-01

    ... counterparts are still by definition toxins. Related terms include phycotoxins (toxins from algae), mycotoxins (fungal toxins), phytotoxins (plant toxins), and venoms (toxins from animals, especially vertebrates...

  14. Effect of severe weather events on the shedding of Shiga toxigenic Escherichia coli in slaughter cattle and phenotype of serogroup O157 isolates.

    Science.gov (United States)

    Stanford, Kim; Reuter, Tim; Bach, Susan J; Chui, Linda; Ma, Angela; Conrad, Cheyenne C; Tostes, Renata; McAllister, Tim A

    2017-09-01

    High-event periods (HEPs) occur sporadically when beef carcasses and meat have episodes of acute contamination with Shiga toxin-producing Escherichia coli (STEC). In this study, severe weather events were investigated as catalysts for HEPs based on PCR and isolate prevalence of seven E. coli serogroups in slaughter cattle feces. Winter ambient temperatures with daily means 10.5oC warmer or 12.3°C colder than seasonal norms (-10.4°C) most altered STEC shedding. Fecal samples yielded increased proportions (P  10 min and one also had strong biofilm-forming potential. However, this isolate lacked eae and stx genes. Severe weather can influence STEC shedding, particularly of O157, and could possibly trigger HEPs. However, our data suggest that it is unlikely for isolates to carry virulence genes and possess phenotypes capable of evading post-harvest microbiological interventions. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  15. A novel mechanism of bacterial toxin transfer within host blood cell-derived microvesicles.

    Directory of Open Access Journals (Sweden)

    Anne-lie Ståhl

    2015-02-01

    Full Text Available Shiga toxin (Stx is the main virulence factor of enterohemorrhagic Escherichia coli, which are non-invasive strains that can lead to hemolytic uremic syndrome (HUS, associated with renal failure and death. Although bacteremia does not occur, bacterial virulence factors gain access to the circulation and are thereafter presumed to cause target organ damage. Stx was previously shown to circulate bound to blood cells but the mechanism by which it would potentially transfer to target organ cells has not been elucidated. Here we show that blood cell-derived microvesicles, shed during HUS, contain Stx and are found within patient renal cortical cells. The finding was reproduced in mice infected with Stx-producing Escherichia coli exhibiting Stx-containing blood cell-derived microvesicles in the circulation that reached the kidney where they were transferred into glomerular and peritubular capillary endothelial cells and further through their basement membranes followed by podocytes and tubular epithelial cells, respectively. In vitro studies demonstrated that blood cell-derived microvesicles containing Stx undergo endocytosis in glomerular endothelial cells leading to cell death secondary to inhibited protein synthesis. This study demonstrates a novel virulence mechanism whereby bacterial toxin is transferred within host blood cell-derived microvesicles in which it may evade the host immune system.

  16. Survey of environmental radiation dose rates in Kyoto and Shiga prefectures, Japan

    International Nuclear Information System (INIS)

    Minamia, Kazuyuki; Shimo, Michikuni; Oka, Mitsuaki; Ejiri, Kazutaka; Sugino, Masato; Minato, Susumu; Hosoda, Masahiro; Yamada, Junya; Fukushi, Masahiro

    2008-01-01

    We have measured environmental radiation dose rates in several Prefectures, such as Ai chi Prefecture, Gifu Prefecture, and Mie Prefecture, in central Japan. Recently, we measured the environmental radiation dose rates in Kyoto and Shiga Prefectures that are also located in central Japan with a car-borne survey system. At the time of measurement, Kyoto Prefecture (area: 4,613 km 2 ) had a total of 36 districts, and Shiga Prefecture (area: 3,387 km 2 ) a total of 26. Terrestrial gamma ray dose rates and secondary cosmic ray dose rates were measured by a 2 inches ψ x 2 inches NaI(Tl) scintillation counter and a handy-type altimeter (GPS eTrex Legend by Gamin), respectively. The following factors were taken into consideration the shielding effect of the car body, the effect of the road pavement, radon progeny borne by precipitation, and increases in tunnels and near the walls. Terrestrial gamma ray dose rates in Kyoto and Shiga Prefectures were estimated to be 51.7 ± 6.0 n Gy/h (district average: 52.4 ± 4.7 n Gy/h), 52.2 ± 10.5 n Gy/h (district average: 51.9 ± 8.1 n Gy/h), respectively. Secondary cosmic ray dose rates in Kyoto and Shiga Prefectures were 30.0 ± 0.6 n Gy/h (district average: 29.9 ±0.3 n Gy/h), 30.1 ± 0.3 n Gy/h (district average: 30.0 ± 0.2 n Gy/h), respectively. The environmental radiation dose rates due to the sum dose rates of terrestrial gamma ray and secondary cosmic ray in Kyoto and Shiga Prefectures were 81.7 ± 6.2 n Gy/h (district average: 82.3 ± 4.8 n Gy/h), 82.3 ± 10.6 n Gy/h (district average: 82.0 ± 8.1 n Gy/h), respectively. We confirmed that the environmental radiation dose rates in Kyoto and Shiga Prefectures mainly depended on the change of the terrestrial gamma ray dose rates, since the secondary cosmic ray dose rates had little change. Therefore, radiation dose-rate maps of the terrestrial gamma rays as well as maps of the environmental radiation dose-rate were drawn. (author)

  17. Biological Activities of Uric Acid in Infection Due to Enteropathogenic and Shiga-Toxigenic Escherichia coli

    OpenAIRE

    Crane, John K.; Broome, Jacqueline E.; Lis, Agnieszka

    2016-01-01

    In previous work, we identified xanthine oxidase (XO) as an important enzyme in the interaction between the host and enteropathogenic Escherichia coli (EPEC) and Shiga-toxigenic E. coli (STEC). Many of the biological effects of XO were due to the hydrogen peroxide produced by the enzyme. We wondered, however, if uric acid generated by XO also had biological effects in the gastrointestinal tract. Uric acid triggered inflammatory responses in the gut, including increased submucosal edema and re...

  18. Incorporation of a non-human glycan mediates human susceptibility to a bacterial toxin

    Energy Technology Data Exchange (ETDEWEB)

    Byres, Emma; Paton, Adrienne W.; Paton, James C.; Löfling, Jonas C.; Smith, David F.; Wilce, Matthew C.J.; Talbot, Ursula M.; Chong, Damien C.; Yu, Hai; Huang, Shengshu; Chen, Xi; Varki, Nissi M.; Varki, Ajit; Rossjohn, Jamie; Beddoe, Travis (Emory-MED); (UCD); (Adelaide); (Monash)

    2009-01-30

    AB{sub 5} toxins comprise an A subunit that corrupts essential eukaryotic cell functions, and pentameric B subunits that direct target-cell uptake after binding surface glycans. Subtilase cytotoxin (SubAB) is an AB{sub 5} toxin secreted by Shiga toxigenic Escherichia coli (STEC), which causes serious gastrointestinal disease in humans. SubAB causes haemolytic uraemic syndrome-like pathology in mice through SubA-mediated cleavage of BiP/GRP78, an essential endoplasmic reticulum chaperone. Here we show that SubB has a strong preference for glycans terminating in the sialic acid N-glycolylneuraminic acid (Neu5Gc), a monosaccharide not synthesized in humans. Structures of SubB-Neu5Gc complexes revealed the basis for this specificity, and mutagenesis of key SubB residues abrogated in vitro glycan recognition, cell binding and cytotoxicity. SubAB specificity for Neu5Gc was confirmed using mouse tissues with a human-like deficiency of Neu5Gc and human cell lines fed with Neu5Gc. Despite lack of Neu5Gc biosynthesis in humans, assimilation of dietary Neu5Gc creates high-affinity receptors on human gut epithelia and kidney vasculature. This, and the lack of Neu5Gc-containing body fluid competitors in humans, confers susceptibility to the gastrointestinal and systemic toxicities of SubAB. Ironically, foods rich in Neu5Gc are the most common source of STEC contamination. Thus a bacterial toxin's receptor is generated by metabolic incorporation of an exogenous factor derived from food.

  19. Hemolytic anemia caused by chemicals and toxins

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/000590.htm Hemolytic anemia caused by chemicals and toxins To use the sharing features on this page, please enable JavaScript. Hemolytic anemia caused by chemicals and toxins is a lack ...

  20. (AJST) DETECTION AND QUANTIFICATION OF TOXINS IN ...

    African Journals Online (AJOL)

    -, hepato- and neuro-toxins ..... risk assessment of cyanobacterial toxins. In: Hester,. R.E. and Harrison, R.M. (Eds.) Agricultural chemicals and the environment. Issues in Environmental. Science and Technology 5. The Royal Society of.

  1. Toxin synergism in snake venoms

    DEFF Research Database (Denmark)

    Laustsen, Andreas Hougaard

    2016-01-01

    Synergism between venom toxins exists for a range of snake species. Synergism can be derived from both intermolecular interactions and supramolecular interactions between venom components, and can be the result of toxins targeting the same protein, biochemical pathway or physiological process. Few...... simple systematic tools and methods for determining the presence of synergism exist, but include co-administration of venom components and assessment of Accumulated Toxicity Scores. A better understanding of how to investigate synergism in snake venoms may help unravel strategies for developing novel...

  2. Inhibition of cholera toxin and other AB toxins by polyphenolic compounds

    Science.gov (United States)

    All AB-type protein toxins have intracellular targets despite an initial extracellular location. These toxins use different methods to reach the cytosol and have different effects on the target cell. Broad-spectrum inhibitors against AB toxins are therefore hard to develop because the toxins use dif...

  3. Toxin yet not toxic: Botulinum toxin in dentistry

    Directory of Open Access Journals (Sweden)

    Archana M.S.

    2016-04-01

    Full Text Available Paracelsus contrasted poisons from nonpoisons, stating that “All things are poisons, and there is nothing that is harmless; the dose alone decides that something is a poison”. Living organisms, such as plants, animals, and microorganisms, constitute a huge source of pharmaceutically useful medicines and toxins. Depending on their source, toxins can be categorized as phytotoxins, mycotoxins, or zootoxins, which include venoms and bacterial toxins. Any toxin can be harmful or beneficial. Within the last 100 years, the perception of botulinum neurotoxin (BTX has evolved from that of a poison to a versatile clinical agent with various uses. BTX plays a key role in the management of many orofacial and dental disorders. Its indications are rapidly expanding, with ongoing trials for further applications. However, despite its clinical use, what BTX specifically does in each condition is still not clear. The main aim of this review is to describe some of the unclear aspects of this potentially useful agent, with a focus on the current research in dentistry.

  4. Aggregative adherence fimbriae I (AAF/I) mediate colonization of fresh produce and abiotic surface by Shiga toxigenic enteroaggregative Escherichia coli O104:H4

    Science.gov (United States)

    The Shiga toxigenic Escherichia coli O104:H4 bares the characteristics of both enterohemorrhagic (EHEC) and enteroaggregative (EAEC) E. coli. It produces plasmid encoded aggregative adherence fimbriae I (AAF/I) which mediate cell aggregation and biofilm formation in human intestine and promote Shiga...

  5. Risk Assessment of Shellfish Toxins

    Directory of Open Access Journals (Sweden)

    Rex Munday

    2013-11-01

    Full Text Available Complex secondary metabolites, some of which are highly toxic to mammals, are produced by many marine organisms. Some of these organisms are important food sources for marine animals and, when ingested, the toxins that they produce may be absorbed and stored in the tissues of the predators, which then become toxic to animals higher up the food chain. This is a particular problem with shellfish, and many cases of poisoning are reported in shellfish consumers each year. At present, there is no practicable means of preventing uptake of the toxins by shellfish or of removing them after harvesting. Assessment of the risk posed by such toxins is therefore required in order to determine levels that are unlikely to cause adverse effects in humans and to permit the establishment of regulatory limits in shellfish for human consumption. In the present review, the basic principles of risk assessment are described, and the progress made toward robust risk assessment of seafood toxins is discussed. While good progress has been made, it is clear that further toxicological studies are required before this goal is fully achieved.

  6. Inactivation of allergens and toxins.

    Science.gov (United States)

    Morandini, Piero

    2010-11-30

    Plants are replete with thousands of proteins and small molecules, many of which are species-specific, poisonous or dangerous. Over time humans have learned to avoid dangerous plants or inactivate many toxic components in food plants, but there is still room for ameliorating food crops (and plants in general) in terms of their allergens and toxins content, especially in their edible parts. Inactivation at the genetic rather than physical or chemical level has many advantages and classical genetic approaches have resulted in significant reduction of toxin content. The capacity, offered by genetic engineering, of turning off (inactivating) specific genes has opened up the possibility of altering the plant content in a far more precise manner than previously available. Different levels of intervention (genes coding for toxins/allergens or for enzymes, transporters or regulators involved in their metabolism) are possible and there are several tools for inactivating genes, both direct (using chemical and physical mutagens, insertion of transposons and other genetic elements) and indirect (antisense RNA, RNA interference, microRNA, eventually leading to gene silencing). Each level/strategy has specific advantages and disadvantages (speed, costs, selectivity, stability, reversibility, frequency of desired genotype and regulatory regime). Paradigmatic examples from classical and transgenic approaches are discussed to emphasize the need to revise the present regulatory process. Reducing the content of natural toxins is a trade-off process: the lesser the content of natural toxins, the higher the susceptibility of a plant to pests and therefore the stronger the need to protect plants. As a consequence, more specific pesticides like Bt are needed to substitute for general pesticides. Copyright © 2010 Elsevier B.V. All rights reserved.

  7. Exfoliative Toxins of Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Michal Bukowski

    2010-05-01

    Full Text Available Staphylococcus aureus is an important pathogen of humans and livestock. It causes a diverse array of diseases, ranging from relatively harmless localized skin infections to life-threatening systemic conditions. Among multiple virulence factors, staphylococci secrete several exotoxins directly associated with particular disease symptoms. These include toxic shock syndrome toxin 1 (TSST-1, enterotoxins, and exfoliative toxins (ETs. The latter are particularly interesting as the sole agents responsible for staphylococcal scalded skin syndrome (SSSS, a disease predominantly affecting infants and characterized by the loss of superficial skin layers, dehydration, and secondary infections. The molecular basis of the clinical symptoms of SSSS is well understood. ETs are serine proteases with high substrate specificity, which selectively recognize and hydrolyze desmosomal proteins in the skin. The fascinating road leading to the discovery of ETs as the agents responsible for SSSS and the characterization of the molecular mechanism of their action, including recent advances in the field, are reviewed in this article.

  8. [Questionnaire survey on medical care for male urethritis in community clinics in Shiga prefecture].

    Science.gov (United States)

    Yamashita, Hiroto; Araki, Isao; Kageyama, Susumu; Baba, Masato; Nakano, Etsuji; Okada, Yusaku

    2014-01-01

    Six regional medical associations in Shiga prefecture agreed to cooperate in an investigation of medical care for male gonococcal and chlamydial urethritis. In June 2011, we sent a questionnaire to 372 medical offices in Shiga prefecture, and analyzed replies of respondents. Ten urologists and 175 non-urologists responded to the survey (response rate 49.7%). Among 185 physicians, 52 (10 urologists and 42 nonurologists) have treated male patients with gonococcal and chlamydial urethritis. More than 20% (42/175) of non-urological clinics are involved in the medical management. At initial diagnosis for sexually transmitted male urethritis, all urologists select the nucleic acid amplification method (100%), whereas many non-urologists do not (35%). For the treatment of chlamydial urethritis, non-urologists select levofloxacin (LVFX, 52.8%) rather than azithromycin (AZM, 22.0%), whereas urologists use AZM (78.0%) mostly but do not use LVFX (0%) (p = 0.023). For the treatment of gonococcal urethritis, non-urologists prefer oral new quinolones (53.1%) compared to urologists (25.0%) (p = 0. 74). For cure judgment of gonoccocal and chlamydial urethritis, many non-urologists rely on the improvement of subjective symptoms (50 and 47%), but urologists do not (10 and 0%) (p = 0.022 and 0.026, respectively). As for recognition of the clinical guideline for sexually transmitted disease, most urologists (90%) know it, but few non-urologists (13%) do (p urethritis in Shiga prefecture. It is important to standardize the medical care for sexually transmitted male urethritis by familiarizing non-urological practitioners with the clinical guideline.

  9. Toxin production in Dinophysis and the fate of these toxins in marine mussels

    DEFF Research Database (Denmark)

    Nielsen, Lasse Tor

    Diarrhetic shellfish poisoning (DSP) poses a considerable threat to food safety and to the economy of shellfish fishers and farmers in many parts of the world. Thousands of DSP intoxications have been reported, and bivalve harvesting can sometimes be closed down several months in a row. The toxins....... acuta. I grew the two species in laboratory cultures at different irradiances (7-130 μmol photons m-2 s-1) and with different food availability. The results showed that irradiance had no effects on toxin profiles, and only limited effects of the cellular toxin contents. Rather, toxin production rates...... followed growth rates, thus giving stable toxin contents. Food availability also did not change the toxin profiles of either species, but starvation did increase the cellular contents of each of the toxins present. The observation that toxin production continued for several weeks after the ciliate food...

  10. Computational Studies of Snake Venom Toxins

    OpenAIRE

    Paola G. Ojeda; David Ramírez; Jans Alzate-Morales; Julio Caballero; Quentin Kaas; Wendy González

    2017-01-01

    Most snake venom toxins are proteins, and participate to envenomation through a diverse array of bioactivities, such as bleeding, inflammation, and pain, cytotoxic, cardiotoxic or neurotoxic effects. The venom of a single snake species contains hundreds of toxins, and the venoms of the 725 species of venomous snakes represent a large pool of potentially bioactive proteins. Despite considerable discovery efforts, most of the snake venom toxins are still uncharacterized. Modern bioinformatics t...

  11. Synthesis and biology of cyclic imine toxins, an emerging class of potent, globally distributed marine toxins.

    Science.gov (United States)

    Stivala, Craig E; Benoit, Evelyne; Aráoz, Rómulo; Servent, Denis; Novikov, Alexei; Molgó, Jordi; Zakarian, Armen

    2015-03-01

    From a small group of exotic compounds isolated only two decades ago, Cyclic Imine (CI) toxins have become a major class of marine toxins with global distribution. Their distinct chemical structure, biological mechanism of action, and intricate chemistry ensures that CI toxins will continue to be the subject of fascinating fundamental studies in the broad fields of chemistry, chemical biology, and toxicology. The worldwide occurrence of potent CI toxins in marine environments, their accumulation in shellfish, and chemical stability are important considerations in assessing risk factors for human health. This review article aims to provide an account of chemistry, biology, and toxicology of CI toxins from their discovery to the present day.

  12. Generality of toxins in defensive symbiosis: Ribosome-inactivating proteins and defense against parasitic wasps in Drosophila.

    Directory of Open Access Journals (Sweden)

    Matthew J Ballinger

    2017-07-01

    Full Text Available While it has become increasingly clear that multicellular organisms often harbor microbial symbionts that protect their hosts against natural enemies, the mechanistic underpinnings underlying most defensive symbioses are largely unknown. Spiroplasma bacteria are widespread associates of terrestrial arthropods, and include strains that protect diverse Drosophila flies against parasitic wasps and nematodes. Recent work implicated a ribosome-inactivating protein (RIP encoded by Spiroplasma, and related to Shiga-like toxins in enterohemorrhagic Escherichia coli, in defense against a virulent parasitic nematode in the woodland fly, Drosophila neotestacea. Here we test the generality of RIP-mediated protection by examining whether Spiroplasma RIPs also play a role in wasp protection, in D. melanogaster and D. neotestacea. We find strong evidence for a major role of RIPs, with ribosomal RNA (rRNA from the larval endoparasitic wasps, Leptopilina heterotoma and Leptopilina boulardi, exhibiting the hallmarks of RIP activity. In Spiroplasma-containing hosts, parasitic wasp ribosomes show abundant site-specific depurination in the α-sarcin/ricin loop of the 28S rRNA, with depurination occurring soon after wasp eggs hatch inside fly larvae. Interestingly, we found that the pupal ectoparasitic wasp, Pachycrepoideus vindemmiae, escapes protection by Spiroplasma, and its ribosomes do not show high levels of depurination. We also show that fly ribosomes show little evidence of targeting by RIPs. Finally, we find that the genome of D. neotestacea's defensive Spiroplasma encodes a diverse repertoire of RIP genes, which are differ in abundance. This work suggests that specificity of defensive symbionts against different natural enemies may be driven by the evolution of toxin repertoires, and that toxin diversity may play a role in shaping host-symbiont-enemy interactions.

  13. Generality of toxins in defensive symbiosis: Ribosome-inactivating proteins and defense against parasitic wasps in Drosophila.

    Science.gov (United States)

    Ballinger, Matthew J; Perlman, Steve J

    2017-07-01

    While it has become increasingly clear that multicellular organisms often harbor microbial symbionts that protect their hosts against natural enemies, the mechanistic underpinnings underlying most defensive symbioses are largely unknown. Spiroplasma bacteria are widespread associates of terrestrial arthropods, and include strains that protect diverse Drosophila flies against parasitic wasps and nematodes. Recent work implicated a ribosome-inactivating protein (RIP) encoded by Spiroplasma, and related to Shiga-like toxins in enterohemorrhagic Escherichia coli, in defense against a virulent parasitic nematode in the woodland fly, Drosophila neotestacea. Here we test the generality of RIP-mediated protection by examining whether Spiroplasma RIPs also play a role in wasp protection, in D. melanogaster and D. neotestacea. We find strong evidence for a major role of RIPs, with ribosomal RNA (rRNA) from the larval endoparasitic wasps, Leptopilina heterotoma and Leptopilina boulardi, exhibiting the hallmarks of RIP activity. In Spiroplasma-containing hosts, parasitic wasp ribosomes show abundant site-specific depurination in the α-sarcin/ricin loop of the 28S rRNA, with depurination occurring soon after wasp eggs hatch inside fly larvae. Interestingly, we found that the pupal ectoparasitic wasp, Pachycrepoideus vindemmiae, escapes protection by Spiroplasma, and its ribosomes do not show high levels of depurination. We also show that fly ribosomes show little evidence of targeting by RIPs. Finally, we find that the genome of D. neotestacea's defensive Spiroplasma encodes a diverse repertoire of RIP genes, which are differ in abundance. This work suggests that specificity of defensive symbionts against different natural enemies may be driven by the evolution of toxin repertoires, and that toxin diversity may play a role in shaping host-symbiont-enemy interactions.

  14. Highly affine and selective aptamers against cholera toxin as capture elements in magnetic bead-based sandwich ELAA.

    Science.gov (United States)

    Frohnmeyer, Esther; Frisch, Farina; Falke, Sven; Betzel, Christian; Fischer, Markus

    2018-03-10

    Aptamers are single-stranded DNA or RNA oligonucleotides, which have been emerging as recognition elements in disease diagnostics and food control, including the detection of bacterial toxins. In this study, we employed the semi-automated just in time-selection to identify aptamers that bind to cholera toxin (CT) with high affinity and specificity. CT is the main virulence factor of Vibrio cholerae and the causative agent of the eponymous disease. For the selected aptamers, dissociation constants in the low nanomolar range (23-56 nM) were determined by fluorescence-based affinity chromatography and cross-reactivity against related proteins was evaluated by direct enzyme-linked aptamer assay (ELAA). Aptamer CT916 has a dissociation constant of 48.5 ± 0.5 nM and shows negligible binding to Shiga-like toxin 1B, protein A and BSA. This aptamer was chosen to develop a sandwich ELAA for the detection of CT from binding buffer and local tap water. Amine-C6- or biotin-modified CT916 was coupled to magnetic beads to serve as the capture element. Using an anti-CT polyclonal antibody as the reporter, detection limits of 2.1 ng/ml in buffer and 2.4 ng/ml in tap water, with a wide log-linear dynamic range from 1 ng/ml to 1000 ng/ml and 500 ng/ml, respectively, were achieved. Copyright © 2018 Elsevier B.V. All rights reserved.

  15. Vth Pan American Symposium on Animal, Plant and Microbial Toxins

    National Research Council Canada - National Science Library

    Ownby, Charlotte

    1996-01-01

    ..., cardiotoxins, and antihemorrhagic factors. Presentations on plant and microbial toxins include work done on ricin, Clostridium perfringens enterotoxin, cone snail peptides, sea anemone toxins, proteinase inhibitors and maitotoxin...

  16. A Quantitative Electrochemiluminescence Assay for Clostridium perfringens alpha toxin

    National Research Council Canada - National Science Library

    Merrill, Gerald A; Rivera, Victor R; Neal, Dwayne D; Young, Charles; Poli, Mark A

    2006-01-01

    .... Biotinylated antibodies to C. perfringens alpha toxin bound to streptavidin paramagnetic beads specifically immunoadsorbed soluble sample alpha toxin which subsequently selectively immunoadsorbed ruthenium (Ru...

  17. Plant insecticidal toxins in ecological networks.

    Science.gov (United States)

    Ibanez, Sébastien; Gallet, Christiane; Després, Laurence

    2012-04-01

    Plant secondary metabolites play a key role in plant-insect interactions, whether constitutive or induced, C- or N-based. Anti-herbivore defences against insects can act as repellents, deterrents, growth inhibitors or cause direct mortality. In turn, insects have evolved a variety of strategies to act against plant toxins, e.g., avoidance, excretion, sequestration and degradation of the toxin, eventually leading to a co-evolutionary arms race between insects and plants and to co-diversification. Anti-herbivore defences also negatively impact mutualistic partners, possibly leading to an ecological cost of toxin production. However, in other cases toxins can also be used by plants involved in mutualistic interactions to exclude inadequate partners and to modify the cost/benefit ratio of mutualism to their advantage. When considering the whole community, toxins have an effect at many trophic levels. Aposematic insects sequester toxins to defend themselves against predators. Depending on the ecological context, toxins can either increase insects' vulnerability to parasitoids and entomopathogens or protect them, eventually leading to self-medication. We conclude that studying the community-level impacts of plant toxins can provide new insights into the synthesis between community and evolutionary ecology.

  18. Plant Insecticidal Toxins in Ecological Networks

    Directory of Open Access Journals (Sweden)

    Sébastien Ibanez

    2012-04-01

    Full Text Available Plant secondary metabolites play a key role in plant-insect interactions, whether constitutive or induced, C- or N-based. Anti-herbivore defences against insects can act as repellents, deterrents, growth inhibitors or cause direct mortality. In turn, insects have evolved a variety of strategies to act against plant toxins, e.g., avoidance, excretion, sequestration and degradation of the toxin, eventually leading to a co-evolutionary arms race between insects and plants and to co-diversification. Anti-herbivore defences also negatively impact mutualistic partners, possibly leading to an ecological cost of toxin production. However, in other cases toxins can also be used by plants involved in mutualistic interactions to exclude inadequate partners and to modify the cost/benefit ratio of mutualism to their advantage. When considering the whole community, toxins have an effect at many trophic levels. Aposematic insects sequester toxins to defend themselves against predators. Depending on the ecological context, toxins can either increase insects’ vulnerability to parasitoids and entomopathogens or protect them, eventually leading to self-medication. We conclude that studying the community-level impacts of plant toxins can provide new insights into the synthesis between community and evolutionary ecology.

  19. Toxin-Antitoxin Battle in Bacteria

    DEFF Research Database (Denmark)

    Cataudella, Ilaria

    This PhD thesis consists of three research projects revolving around the common thread of investigation of the properties and biological functions of Toxin-Antitoxin loci. Toxin-Antitoxin (TA) loci are transcriptionally regulated via an auto-inhibition mechanism called conditional cooperativity, ...

  20. Botulinum toxin — therapeutic effect in cosmetology

    OpenAIRE

    Morrison A.V.; Bocharova Y.M.; Morrison V.V.

    2016-01-01

    This review presents the data from published literatures and the research works conducted by the authors about mechanisms of action of botulinum toxin and its use in the practical medicine (particularly in dermatology and cosmetology). Indications and contraindications of botulinum toxin use in cosmetology are also considered in this work.

  1. Stealth and mimicry by deadly bacterial toxins

    DEFF Research Database (Denmark)

    Yates, S.P.; Jørgensen, Rene; Andersen, Gregers Rom

    2006-01-01

    Diphtheria toxin and exotoxin A are well-characterized members of the ADP-ribosyltransferase toxin family that serve as virulence factors in the pathogenic bacteria, Corynebacterium diphtheriae and Pseudomonas aeruginosa.  New high-resolution structural data of the Michaelis complex...

  2. Botulinum toxin for masseter hypertrophy.

    Science.gov (United States)

    Fedorowicz, Zbys; van Zuuren, Esther J; Schoones, Jan

    2013-09-09

    Benign masseter muscle hypertrophy is an uncommon clinical phenomenon of uncertain aetiology which is characterised by a soft swelling near the angle of the mandible. The swelling may on occasion be associated with facial pain and can be prominent enough to be considered cosmetically disfiguring. Varying degrees of success have been reported for some of the treatment options for masseter hypertrophy, which range from simple pharmacotherapy to more invasive surgical reduction. Injection of botulinum toxin type A into the masseter muscle is generally considered a less invasive modality and has been advocated for cosmetic sculpting of the lower face. Botulinum toxin type A is a powerful neurotoxin which is produced by the anaerobic organism Clostridium botulinum and when injected into a muscle causes interference with the neurotransmitter mechanism producing selective paralysis and subsequent atrophy of the muscle.This review is an update of a previously published Cochrane review. To assess the efficacy and safety of botulinum toxin type A compared to placebo or no treatment, for the management of benign bilateral masseter hypertrophy. We searched the following databases from inception to April 2013: the Cochrane Central Register of Controlled Trials (CENTRAL); MEDLINE (via PubMed); EMBASE (via embase.com); Web of Science; CINAHL; Academic Search Premier (via EBSCOhost); ScienceDirect; LILACS (via BIREME); PubMed Central and Google Scholar (from 1700 to 19 April 2013). We searched two bibliographic databases of regional journals (IndMED and Iranmedex) which were expected to contain relevant trials. We also searched reference lists of relevant articles and contacted investigators to identify additional published and unpublished studies. Randomised controlled trials (RCTs) and controlled clinical trials (CCTs) comparing intra-masseteric injections of botulinum toxin versus placebo administered for cosmetic facial sculpting in individuals of any age with bilateral benign

  3. Brown spider dermonecrotic toxin directly induces nephrotoxicity

    International Nuclear Information System (INIS)

    Chaim, Olga Meiri; Sade, Youssef Bacila; Bertoni da Silveira, Rafael; Toma, Leny; Kalapothakis, Evanguedes; Chavez-Olortegui, Carlos; Mangili, Oldemir Carlos; Gremski, Waldemiro; Dietrich, Carl Peter von; Nader, Helena B.; Sanches Veiga, Silvio

    2006-01-01

    Brown spider (Loxosceles genus) venom can induce dermonecrotic lesions at the bite site and systemic manifestations including fever, vomiting, convulsions, disseminated intravascular coagulation, hemolytic anemia and acute renal failure. The venom is composed of a mixture of proteins with several molecules biochemically and biologically well characterized. The mechanism by which the venom induces renal damage is unknown. By using mice exposed to Loxosceles intermedia recombinant dermonecrotic toxin (LiRecDT), we showed direct induction of renal injuries. Microscopic analysis of renal biopsies from dermonecrotic toxin-treated mice showed histological alterations including glomerular edema and tubular necrosis. Hyalinization of tubules with deposition of proteinaceous material in the tubule lumen, tubule epithelial cell vacuoles, tubular edema and epithelial cell lysis was also observed. Leukocytic infiltration was neither observed in the glomerulus nor the tubules. Renal vessels showed no sign of inflammatory response. Additionally, biochemical analyses showed such toxin-induced changes in renal function as urine alkalinization, hematuria and azotemia with elevation of blood urea nitrogen levels. Immunofluorescence with dermonecrotic toxin antibodies and confocal microscopy analysis showed deposition and direct binding of this toxin to renal intrinsic structures. By immunoblotting with a hyperimmune dermonecrotic toxin antiserum on renal lysates from toxin-treated mice, we detected a positive signal at the region of 33-35 kDa, which strengthens the idea that renal failure is directly induced by dermonecrotic toxin. Immunofluorescence reaction with dermonecrotic toxin antibodies revealed deposition and binding of this toxin directly in MDCK epithelial cells in culture. Similarly, dermonecrotic toxin treatment caused morphological alterations of MDCK cells including cytoplasmic vacuoles, blebs, evoked impaired spreading and detached cells from each other and from

  4. Pore-forming toxins in Cnidaria.

    Science.gov (United States)

    Podobnik, Marjetka; Anderluh, Gregor

    2017-12-01

    The ancient phylum of Cnidaria contains many aquatic species with peculiar lifestyle. In order to survive, these organisms have evolved attack and defense mechanisms that are enabled by specialized cells and highly developed venoms. Pore-forming toxins are an important part of their venomous arsenal. Along some other types, the most representative are examples of four protein families that are commonly found in other kingdoms of life: actinoporins, Cry-like proteins, aerolysin-like toxins and MACPF/CDC toxins. Some of the homologues of pore-forming toxins may serve other functions, such as in food digestion, development and response against pathogenic organisms. Due to their interesting physico-chemical properties, the cnidarian pore-forming toxins may also serve as tools in medical research and nanobiotechnological applications. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Studies of muscarinic neurotransmission with antimuscarinic toxins.

    Science.gov (United States)

    Potter, Lincoln T; Flynn, Donna D; Liang, Jing-Sheng; McCollum, Mark H

    2004-01-01

    M1 and M4 muscarinic receptors are the most prevalent receptors for acetylcholine in the brain, and m1-toxin1 and m4-toxin are the most specific ligands yet found for their extracellular faces. Both toxins are antagonists. These toxins and their derivatives with biotin, radioiodine and fluorophores are useful for studying M1- and M4-linked neurotransmission. We have used the rat striatum for many studies because this tissue express exceptionally high concentrations of both receptors, the striatum regulates movement, and movement is altered by antimuscarinic agents, M1-knockout and M4-knockout. These toxins and their derivatives may also be used for studies of M1 and M4 receptors in the hippocampus and cortex.

  6. Crystallization of isoelectrically homogeneous cholera toxin

    Energy Technology Data Exchange (ETDEWEB)

    Spangler, B.D.; Westbrook, E.M. (Argonne National Laboratory, IL (USA))

    1989-02-07

    Past difficulty in growing good crystals of cholera toxin has prevented the study of the crystal structure of this important protein. The authors have determined that failure of cholera toxin to crystallize well has been due to its heterogeneity. They have now succeeded in overcoming the problem by isolating a single isoelectric variant of this oligomeric protein (one A subunit and five B subunits). Cholera toxin purified by their procedure readily forms large single crystals. The crystal form has been described previously. They have recorded data from native crystals of cholera toxin to 3.0-{angstrom} resolution with our electronic area detectors. With these data, they have found the orientation of a 5-fold symmetry axis within these crystals, perpendicular to the screw dyad of the crystal. They are now determining the crystal structure of cholera toxin by a combination of multiple heavy-atom isomorphous replacement and density modification techniques, making use of rotational 5-fold averaging of the B subunits.

  7. Crystallization of isoelectrically homogeneous cholera toxin

    International Nuclear Information System (INIS)

    Spangler, B.D.; Westbrook, E.M.

    1989-01-01

    Past difficulty in growing good crystals of cholera toxin has prevented the study of the crystal structure of this important protein. The authors have determined that failure of cholera toxin to crystallize well has been due to its heterogeneity. They have now succeeded in overcoming the problem by isolating a single isoelectric variant of this oligomeric protein (one A subunit and five B subunits). Cholera toxin purified by their procedure readily forms large single crystals. The crystal form has been described previously. They have recorded data from native crystals of cholera toxin to 3.0-angstrom resolution with our electronic area detectors. With these data, they have found the orientation of a 5-fold symmetry axis within these crystals, perpendicular to the screw dyad of the crystal. They are now determining the crystal structure of cholera toxin by a combination of multiple heavy-atom isomorphous replacement and density modification techniques, making use of rotational 5-fold averaging of the B subunits

  8. Immunotoxins: The Role of the Toxin

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    David FitzGerald

    2013-08-01

    Full Text Available Immunotoxins are antibody-toxin bifunctional molecules that rely on intracellular toxin action to kill target cells. Target specificity is determined via the binding attributes of the chosen antibody. Mostly, but not exclusively, immunotoxins are purpose-built to kill cancer cells as part of novel treatment approaches. Other applications for immunotoxins include immune regulation and the treatment of viral or parasitic diseases. Here we discuss the utility of protein toxins, of both bacterial and plant origin, joined to antibodies for targeting cancer cells. Finally, while clinical goals are focused on the development of novel cancer treatments, much has been learned about toxin action and intracellular pathways. Thus toxins are considered both medicines for treating human disease and probes of cellular function.

  9. Botulinum toxin for the treatment of bruxism.

    Science.gov (United States)

    Tinastepe, Neslihan; Küçük, Burcu Bal; Oral, Koray

    2015-10-01

    Botulinum toxin, the most potent biological toxin, has been shown to be effective for a variety of disorders in several medical conditions, when used both therapeutically and cosmetically. In recent years, there has been a rising trend in the use of this pharmacological agent to control bruxing activity, despite its reported adverse effects. The aim of this review was to provide a brief overview to clarify the underlying essential ideas for the use of botulinum toxin in bruxism based on available scientific papers. An electronic literature search was performed to identify publications related to botulinum toxin and its use for bruxism in PubMed. Hand searching of relevant articles was also made to identify additional studies. Of the eleven identified studies, only two were randomized controlled trials, compared with the effectiveness of botulinum toxins on the reduction in the frequency of bruxism events and myofascial pain after injection. The authors of these studies concluded that botulinum toxin could be used as an effective treatment for reducing nocturnal bruxism and myofascial pain in patients with bruxism. Evidence-based research was limited on this topic. More randomized controlled studies are needed to confirm that botulinum toxin is safe and reliable for routine clinical use in bruxism.

  10. Computational Studies of Snake Venom Toxins.

    Science.gov (United States)

    Ojeda, Paola G; Ramírez, David; Alzate-Morales, Jans; Caballero, Julio; Kaas, Quentin; González, Wendy

    2017-12-22

    Most snake venom toxins are proteins, and participate to envenomation through a diverse array of bioactivities, such as bleeding, inflammation, and pain, cytotoxic, cardiotoxic or neurotoxic effects. The venom of a single snake species contains hundreds of toxins, and the venoms of the 725 species of venomous snakes represent a large pool of potentially bioactive proteins. Despite considerable discovery efforts, most of the snake venom toxins are still uncharacterized. Modern bioinformatics tools have been recently developed to mine snake venoms, helping focus experimental research on the most potentially interesting toxins. Some computational techniques predict toxin molecular targets, and the binding mode to these targets. This review gives an overview of current knowledge on the ~2200 sequences, and more than 400 three-dimensional structures of snake toxins deposited in public repositories, as well as of molecular modeling studies of the interaction between these toxins and their molecular targets. We also describe how modern bioinformatics have been used to study the snake venom protein phospholipase A2, the small basic myotoxin Crotamine, and the three-finger peptide Mambalgin.

  11. Computational Studies of Snake Venom Toxins

    Directory of Open Access Journals (Sweden)

    Paola G. Ojeda

    2017-12-01

    Full Text Available Most snake venom toxins are proteins, and participate to envenomation through a diverse array of bioactivities, such as bleeding, inflammation, and pain, cytotoxic, cardiotoxic or neurotoxic effects. The venom of a single snake species contains hundreds of toxins, and the venoms of the 725 species of venomous snakes represent a large pool of potentially bioactive proteins. Despite considerable discovery efforts, most of the snake venom toxins are still uncharacterized. Modern bioinformatics tools have been recently developed to mine snake venoms, helping focus experimental research on the most potentially interesting toxins. Some computational techniques predict toxin molecular targets, and the binding mode to these targets. This review gives an overview of current knowledge on the ~2200 sequences, and more than 400 three-dimensional structures of snake toxins deposited in public repositories, as well as of molecular modeling studies of the interaction between these toxins and their molecular targets. We also describe how modern bioinformatics have been used to study the snake venom protein phospholipase A2, the small basic myotoxin Crotamine, and the three-finger peptide Mambalgin.

  12. Engineering toxins for 21st century therapies.

    Science.gov (United States)

    Chaddock, John A; Acharya, K Ravi

    2011-04-01

    'Engineering Toxins for 21st Century Therapies' (9-10 September 2010) was part of the Royal Society International Seminar series held at the Kavli International Centre, UK. Participants were assembled from a range of disciplines (academic, industry, regulatory, public health) to discuss the future potential of toxin-based therapies. The meeting explored how the current structural and mechanistic knowledge of toxins could be used to engineer future toxin-based therapies. To date, significant progress has been made in the design of novel recombinant biologics based on domains of natural toxins, engineered to exhibit advantageous properties. The meeting concluded, firstly that future product development vitally required the appropriate combination of creativity and innovation that can come from the academic, biotechnology and pharma sectors. Second, that continued investigation into understanding the basic science of the toxins and their targets was essential in order to develop new opportunities for the existing products and to create new products with enhanced properties. Finally, it was concluded that the clinical potential for development of novel biologics based on toxin domains was evident. © 2011 The Authors Journal compilation © 2011 FEBS.

  13. Botulinum A toxin utilizations in obstetric palsy

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    Atakan Aydin

    2012-12-01

    Conclusion: We conclude that with the help of botulinum A toxin and physyotherapy, obstetrical palsy patient with cocontractions can significantly improve movements and may have less surgery. [Hand Microsurg 2012; 1(3.000: 89-94

  14. Bacterial toxins as pathogen weapons against phagocytes

    Directory of Open Access Journals (Sweden)

    Ana edo Vale

    2016-02-01

    Full Text Available Bacterial toxins are virulence factors that manipulate host cell functions and take over the control of vital processes of living organisms to favour microbial infection. Some toxins directly target innate immune cells, thereby annihilating a major branch of the host immune response. In this review we will focus on bacterial toxins that act from the extracellular milieu and hinder the function of macrophages and neutrophils. In particular, we will concentrate on toxins from Gram-positive and Gram-negative bacteria that manipulate cell signalling or induce cell death by either imposing direct damage to the host cells cytoplasmic membrane or enzymatically modifying key eukaryotic targets. Outcomes regarding pathogen dissemination, host damage and disease progression will be discussed.

  15. Updates on tetanus toxin: a fundamental approach

    Directory of Open Access Journals (Sweden)

    Md. Ahaduzzaman

    2015-03-01

    Full Text Available Clostridium tetani is an anaerobic bacterium that produces second most poisonous protein toxins than any other bacteria. Tetanus in animals is sporadic in nature but difficult to combat even by using antibiotics and antiserum. It is crucial to understand the fundamental mechanisms and signals that control toxin production for advance research and medicinal uses. This review was intended for better understanding the basic patho-physiology of tetanus and neurotoxins (TeNT among the audience of related field.

  16. Toxicological Perspective on Climate Change: Aquatic Toxins.

    Science.gov (United States)

    Botana, Luis M

    2016-04-18

    In recent years, our group and several others have been describing the presence of new, not previously reported, toxins of high toxicity in vectors that may reach the human food chain. These include tetrodotoxin in gastropods in the South of Europe, ciguatoxin in fish in the South of Spain, palytoxin in mussels in the Mediterranean Sea, pinnatoxin all over Europe, and okadaic acid in the south of the U.S. There seem to be new marine toxins appearing in areas that are heavy producers of seafood, and this is a cause of concern as most of these new toxins are not included in current legislation and monitoring programs. Along with the new toxins, new chemical analogues are being reported. The same phenomenom is being recorded in freshwater toxins, such as the wide appearance of cylindrospermopsin and the large worldwide increase of microcystin. The problem that this phenomenon, which may be linked to climate warming, poses for toxicologists is very important not only because there is a lack of chronic studies and an incomplete comprehension of the mechanism driving the production of these toxins but also because the lack of a legal framework for them allows many of these toxins to reach the market. In some cases, it is very difficult to control these toxins because there are not enough standards available, they are not always certified, and there is an insufficient understanding of the toxic equivalency factors of the different analogues in each group. All of these factors have been revealed and grouped through the massive increase in the use of LC-MS as a monitoring tool, legally demanded, creating more toxicological problems.

  17. Dinoflagellate Toxins Responsible for Ciguatera Food Poisoning

    Science.gov (United States)

    1991-03-30

    Virgin Gorda Prorocu’nt~um lirna 885 Little Lameshur Bay, St. John Prorocentrum lima 838 Unknown (cold water form) Prorocontrum lima 62, 105, 142... Prorocentrum concavum, Conference on Natural Toxins from Aquatic and Marine Environments. 4. Tindall, D.R. and Miller, D.M., (1987) Two potent tox-is fiom...NO. NO. N. ACCESSION NO. 11. TITLE (iclude Security Classification) DINOFLAGELLATE TOXINS RESPONSIBLE FOR CIGUATERA POISONING 12. PERSONAL AUTHOR(S

  18. Botulinum toxin for the treatment of strabismus.

    Science.gov (United States)

    Rowe, Fiona J; Noonan, Carmel P

    2017-03-02

    The use of botulinum toxin as an investigative and treatment modality for strabismus is well reported in the medical literature. However, it is unclear how effective it is in comparison to other treatment options for strabismus. The primary objective was to examine the efficacy of botulinum toxin therapy in the treatment of strabismus compared with alternative conservative or surgical treatment options. This review sought to ascertain those types of strabismus that particularly benefit from the use of botulinum toxin as a treatment option (such as small angle strabismus or strabismus with binocular potential, i.e. the potential to use both eyes together as a pair). The secondary objectives were to investigate the dose effect and complication rates associated with botulinum toxin. We searched CENTRAL (which contains the Cochrane Eyes and Vision Trials Register) (2016, Issue 6), Ovid MEDLINE, Ovid MEDLINE In-Process and Other Non-Indexed Citations, Ovid MEDLINE Daily, Ovid OLDMEDLINE (January 1946 to July 2016), Embase (January 1980 to July 2016), Latin American and Caribbean Literature on Health Sciences (LILACS) (January 1982 to July 2016), the ISRCTN registry (www.isrctn.com/editAdvancedSearch), ClinicalTrials.gov (www.clinicaltrials.gov), and the World Health Organization (WHO) International Clinical Trials Registry Platform (ICTRP) (www.who.int/ictrp/search/en). We did not use any date or language restrictions in the electronic searches for trials. We last searched the electronic databases on 11 July 2016. We handsearched the British and Irish Orthoptic Journal, Australian Orthoptic Journal, proceedings of the European Strabismological Association (ESA), International Strabismological Association (ISA) and International Orthoptic Association (IOA) (www.liv.ac.uk/orthoptics/research/search.htm) and American Academy of Paediatric Ophthalmology and Strabismus meetings (AAPOS). We contacted researchers who are active in this field for information about further

  19. [Botulinum toxin therapy for spasticity].

    Science.gov (United States)

    Masakado, Yoshihisa

    2014-09-01

    Botulinum toxin (BTX) administered as an adjunct to other interventions for spasticity can act as a useful and effective therapeutic tool for treating patients disabled by spasticity. Presence of other non-reflex motor disorders (muscle stiffness, shortness, and contracture) can complicate the clinical course and disturb rehabilitative process of patients with spasticity. Treatment of spasticity using BTX can improve paralysis by correcting muscular imbalance that follows these diseases. In patients with chronic severe spasticity, we also have to address unique and difficult-to-treat clinical conditions such as abnormal posture and movement disorders. The effectiveness of BTX in treating some of these conditions is discussed. Because patients with neurological disabilities can show complex dysfunctions, specific functional limitations, goals, and expected outcomes of treatment should be evaluated and discussed with the patient, family members, and caregivers, prior to initiating BTX therapy. BTX therapy might improve not only care, passive function, but also motor functions in these patients by supplementing intensive rehabilitation with repetitive transcranial magnetic stimulation, transcranial direct-current stimulation, peripheral electrical stimulation, muscle stretching, and other rehabilitation strategies.

  20. Affinity chromatography of tetanus toxin, tetanus toxoid, and botulinum A toxin on synaptosomes, and differentiation of their acceptors

    International Nuclear Information System (INIS)

    Habermann, E.

    1976-01-01

    125 I-labelled tetanus toxin and 125 I-labelled botulinum A neurotoxin are known to be specifically bound to brain synaptosomes. In order to discriminate between active toxin and inactive admixtures present in the starting material or arising during iodination, synaptosome columns were prepared using bromacetylcellulose and/or kieselgur (Celite) as carriers. Both types of columns adsorb the toxins from low ionic strength medium and release them if the pH and ionic strength are raised. Botulinum toxin was eluted with lower ionic strength than tetanus toxin, and could be freed from nontoxic admixtures. Analysis by affinity chromatography disclosed partially toxoided tetanus toxin in both labelled and unlabelled toxin samples. High concentrations of formaldehyde (0.5%) destroyed both toxicity and affinity to the synaptosomes of tetanus toxin. Low concentrations of formaldehyde (0.05%) yielded a derivative of low toxicity which was still, however less firmly, bound to synaptosomes. Tetanus and botulinum toxin differ by their acceptors. Whereas unlabelled botulinum toxin is unable to compete with labelled tetanus toxin, unlabelled tetanus toxin slightly competes with botulinum toxin. Both labelled toxins display anomalous binding behaviour in that they cannot be displaced completely even with a large excess of unlabelled toxin. (orig.) [de

  1. Serogroup-level resolution of the “Super-7” Shiga toxin-producing Escherichia coli using nanopore single-molecule DNA sequencing

    Science.gov (United States)

    DNA sequencing and other DNA-based methods, such as PCR, are now broadly used for detection and identification of bacterial foodborne pathogens. For the identification of foodborne bacterial pathogens, it is important to make taxonomic assignments to the species, or even subspecies level. Long-read ...

  2. Detection of verotoxin (Shiga-like toxin-producing and eae harboring Escherichia coli in some wild captive and domestic Equidae and Canidae

    Directory of Open Access Journals (Sweden)

    Koochakzadeh, A.

    2014-11-01

    Full Text Available The aim of this study was to investigate the prevalence of STEC and EPEC strains and E. coli O157 serogroup in some Equidae and Canidae. The fecal samples of 79 animals from 6 different species were evaluated for presence of these strains. All the Isolates were tested for virulence genes using multiplex-PCR. Non-sorbitol fermenting (NSF Escherichia coli isolates and positive strains for virulence factors were subjected to serogroupe specific PCR for rfb O157 gene. None of the STEC, EPEC and NFS strains in this study belonged to O157 serogroupe. While 36.64% of animals carried strains positive for one or more of the virulence factors tested, and 18.9% of animals harbored STEC strains (stx1, stx2 was not detected in this study. eae and Ehly positive strains were found in 3.79% and 22.7% of animals respectively. In conclusion, these species can act as a reservoir for EPEC and STEC strains. Also, since the study was conducted in some parts of Iran, a more accurate conclusion needs more distributed sampling. To our knowledge this is the first study which reports the faecal shedding of STEC and EPEC from wild captive Canidae and Equidae in Iran.

  3. Efficacy of antimicrobial compounds on surface decontamination of seven shiga toxin-producing Escherichia coli (STEC) and Salmonella inoculated onto fresh beef

    Science.gov (United States)

    Several antimicrobial compounds have been implemented in commercial meat processing plants for decontamination of pathogens on beef carcasses, but there are many commercially available, novel antimicrobial compounds that may be more effective and suitable to be implemented in beef processing pathoge...

  4. Evaluation of a multiplex real-time PCR method for detecting Shiga toxin-producing Escherichia coli in beef and comparison to the FSIS microbiology laboratory guidebook method

    Science.gov (United States)

    The “top-six” non-O157 STEC (O26, O45, O103, O111, O121, and O145) most frequently associated with outbreaks and cases of food-borne illnesses have been declared as adulterants in beef by the USDA Food Safety and Inspection Service (FSIS), and regulatory testing for these serogroups in beef began in...

  5. Genomic, proteomic and physiological characterization of a T5-like bacteriophage for control of Shiga toxin-producing Escherichia coli O157:H7.

    Directory of Open Access Journals (Sweden)

    Yan D Niu

    Full Text Available Despite multiple control measures, Escherichia coli O157:H7 (STEC O157:H7 continues to be responsible for many food borne outbreaks in North America and elsewhere. Bacteriophage therapy may prove useful for controlling this pathogen in the host, their environment and food. Bacteriophage vB_EcoS_AKFV33 (AKFV33, a T5-like phage of Siphoviridae lysed common phage types of STEC O157:H7 and not non-O157 E. coli. Moreover, STEC O157:H7 isolated from the same feedlot pen from which the phage was obtained, were highly susceptible to AKFV33. Adsorption rate constant and burst size were estimated to be 9.31 × 10(-9 ml/min and 350 PFU/infected cell, respectively. The genome of AKVF33 was 108,853 bp (38.95% G+C, containing 160 open reading frames (ORFs, 22 tRNA genes and 32 strong promoters recognized by host RNA polymerase. Of 12 ORFs without homologues to T5-like phages, 7 predicted novel proteins while others exhibited low identity (<60% to proteins in the National Centre for Biotechnology Information database. AKVF33 also lacked the L-shaped tail fiber protein typical of T5, but was predicted to have tail fibers comprised of 2 novel proteins with low identity (37-41% to tail fibers of E. coli phage phiEco32 of Podoviridae, a putative side tail fiber protein of a prophage from E. coli IAI39 and a conserved domain protein of E. coli MS196-1. The receptor-binding tail protein (pb5 shared an overall identify of 29-72% to that of other T5-like phages, with no region coding for more than 6 amino acids in common. Proteomic analysis identified 4 structural proteins corresponding to the capsid, major tail, tail fiber and pore-forming tail tip (pb2. The genome of AKFV33 lacked regions coding for known virulence factors, integration-related proteins or antibiotic resistance determinants. Phage AKFV33 is a unique, highly lytic STEC O157:H7-specific T5-like phage that may have considerable potential as a pre- and post-harvest biocontrol agent.