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      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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          Abstract

          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 stx 2c(vh-b) and stx 2a/ 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 monitoring system. Likewise, zero-tolerance intervention measures should be applied in beef, together with GMP and HACCP. Further, collaborative efforts for risk assessment, management and communication are extremely important to improve the safety of foodstuffs.

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          Association of genomic O island 122 of Escherichia coli EDL 933 with verocytotoxin-producing Escherichia coli seropathotypes that are linked to epidemic and/or serious disease.

          The distribution of EDL 933 O island 122 (OI-122) was investigated in 70 strains of Verocytotoxin-producing Escherichia coli (VTEC) of multiple serotypes that were classified into five "seropathotypes" (A through E) based on the reported occurrence of serotypes in human disease, in outbreaks, and/or in the hemolytic-uremic syndrome (HUS). Seropathotype A comprised 10 serotype O157:H7 and 3 serotype O157:NM strains. Seropathotype B (associated with outbreaks and HUS but less commonly than serotype O157:H7) comprised three strains each of serotypes O26:H11, O103:H2, O111:NM, O121:H19, and O145:NM. Seropathotype C comprised four strains each of serotypes O91:H21 and O113:H21 and eight strains of other serotypes that have been associated with sporadic HUS but not typically with outbreaks. Seropathotype D comprised 14 strains of serotypes that have been associated with diarrhea but not with outbreaks or HUS, and seropathotype E comprised animal VTEC strains of serotypes not implicated in human disease. All strains were tested for four EDL 933 OI-122 virulence genes (Z4321, Z4326, Z4332, and Z4333) by PCR. Negative PCRs were confirmed by Southern hybridization. Overall, 28 (40%) strains contained OI-122 (positive for all four virulence genes), 27 (38.6%) contained an "incomplete" OI-122 (positive for one to three genes), and 15 (21.4%) strains did not contain OI-122. The seropathotype distribution of complete OI-122 was as follows: 100% for seropathotype A, 60% for B, 36% for C, 15% for D, and 0% for E. The differences in the frequency of OI-122 between seropathotypes A, B, and C (associated with HUS) and seropathotypes D and E (not associated with HUS) and between seropathotypes A and B (associated with epidemic disease) and seropathotypes C, D, and E (not associated with epidemic disease) were highly significant (P < 0.0001).
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            Molecular analysis of the plasmid-encoded hemolysin of Escherichia coli O157:H7 strain EDL 933.

            In this study, we determined the nucleotide sequence of the 5.4-kb SalI restriction fragment of the recombinant plasmid pEO40-1, cloned from the large plasmid of enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain EDL 933. This revealed two open reading frames which shared approximately 60% homology to the hlyC and hlyA genes of the E. coli alpha-hemolysin (alpha-hly) operon. We termed these genes EHEC-hlyA and EHEC-hlyC to distinguish them from the alpha-hly genes. Preliminary sequence analysis indicated that another open reading frame homolog to the hlyB gene is located close to the 3' end of EHEC-hlyA. The predicted molecular masses of the EHEC-hlyA and EHEC-hlyC gene products were 107 and 19.9 kDa, respectively. The EHEC hemolysin protein (EHEC-Hly) was not secreted into the culture supernatant by the strain EDL 933. However, hemolytic activity was found in the broth culture supernatant after transforming EDL 933 with the recombinant plasmid pRSC6 carrying the hlyB and hlyD genes from the E. coli alpha-hemolysin operon. The EHEC hemolysin was precipitated and used as an antigen for immunoblot analysis. This demonstrated that 19 of 20 reconvalescent-phase serum samples from patients with hemolytic uremic syndrome reacted specifically with the antigen; conversely, only 1 of 20 control serum samples demonstrated reactivity. To investigate the prevalence of EHEC hemolysin genes in diarrheagenic E. coli, a PCR was developed to specifically detect EHEC-hlyA. All Shiga-like toxin-producing O157 strains and 12 of 25 Shiga-like toxin-producing non-O157 strains were PCR positive; strains of other categories of diarrheagenic E. coli were PCR negative. All PCR-positive strains hybridized with the CVD 419 probe. We found the CVD 419 probe to be identical to the 3.4-kb HindIII fragment of plasmid pEO40 carrying most of the EHEC-hlyA gene and a part of the putative EHEC-hlyB gene. In this study, the newly discovered EHEC hemolysin was shown to be responsible for the enterohemolytic phenotype and demonstrated to be related but not identical to alpha-hemolysin. The EHEC hemolysin appears to have clinical importance because it occurs in all O157 strains tested and is reactive to sera of patients with hemolytic uremic syndrome.
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              A New Family of Potent AB5 Cytotoxins Produced by Shiga Toxigenic Escherichia coli

              The Shiga toxigenic Escherichia coli (STEC) O113:H21 strain 98NK2, which was responsible for an outbreak of hemolytic uremic syndrome, secretes a highly potent and lethal subtilase cytotoxin that is unrelated to any bacterial toxin described to date. It is the prototype of a new family of AB5 toxins, comprising a single 35-kilodalton (kD) A subunit and a pentamer of 13-kD B subunits. The A subunit is a subtilase-like serine protease distantly related to the BA_2875 gene product of Bacillus anthracis. The B subunit is related to a putative exported protein from Yersinia pestis, and binds to a mimic of the ganglioside GM2. Subtilase cytotoxin is encoded by two closely linked, cotranscribed genes (subA and subB), which, in strain 98NK2, are located on a large, conjugative virulence plasmid. Homologues of the genes are present in 32 out of 68 other STEC strains tested. Intraperitoneal injection of purified subtilase cytotoxin was fatal for mice and resulted in extensive microvascular thrombosis, as well as necrosis in the brain, kidneys, and liver. Oral challenge of mice with E. coli K-12–expressing cloned subA and subB resulted in dramatic weight loss. These findings suggest that the toxin may contribute to the pathogenesis of human disease.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: InvestigationRole: MethodologyRole: ResourcesRole: SoftwareRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: InvestigationRole: Resources
                Role: InvestigationRole: MethodologyRole: ResourcesRole: ValidationRole: Writing – review & editing
                Role: ConceptualizationRole: InvestigationRole: ResourcesRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: ResourcesRole: SoftwareRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: InvestigationRole: Resources
                Role: InvestigationRole: MethodologyRole: ResourcesRole: ValidationRole: Writing – review & editing
                Role: ConceptualizationRole: InvestigationRole: Resources
                Role: InvestigationRole: Resources
                Role: ConceptualizationRole: InvestigationRole: Resources
                Role: InvestigationRole: Resources
                Role: ConceptualizationRole: InvestigationRole: Resources
                Role: Data curationRole: InvestigationRole: MethodologyRole: ResourcesRole: SoftwareRole: ValidationRole: Writing – review & editing
                Role: ConceptualizationRole: InvestigationRole: Resources
                Role: InvestigationRole: Resources
                Role: ConceptualizationRole: InvestigationRole: Resources
                Role: InvestigationRole: Resources
                Role: ConceptualizationRole: InvestigationRole: Resources
                Role: ConceptualizationRole: InvestigationRole: ResourcesRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SoftwareRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                22 August 2017
                2017
                : 12
                : 8
                : e0183248
                Affiliations
                [1 ] IGEVET—Instituto de Genética Veterinaria “Ing. Fernando N. Dulout” (UNLP-CONICET LA PLATA), Facultad de Ciencias Veterinarias UNLP, La Plata, Argentina
                [2 ] Laboratorio de Microbiología de Alimentos, Facultad de Ciencias Veterinarias UNLP, La Plata, Argentina
                [3 ] IPCVA–Instituto de Promoción de la Carne Vacuna Argentina, Ciudad Autónoma de Buenos Aires, Argentina
                [4 ] CONICET—EEA Rafaela, Instituto Nacional de Tecnología Agropecuaria (INTA), Santa Fe, Argentina
                [5 ] CIVETAN–Centro de Investigación Veterinaria Tandil (CONICET, CICPBA), Facultad de Ciencias Veterinarias, UNCPBA, Tandil, Argentina
                [6 ] Centro Estudios Infectológicos “Dr. Daniel Stamboulian”, División Alimentos, Ciudad Autónoma de Buenos Aires, Argentina
                [7 ] SENASA–Servicio Nacional de Sanidad y Calidad Agroalimentaria, Ciudad Autónoma de Buenos Aires, Argentina
                [8 ] Instituto de Economía (CICPES, INTA), Ciudad Autónoma de Buenos Aires, Argentina
                Cornell University, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0001-8707-8932
                Article
                PONE-D-17-15288
                10.1371/journal.pone.0183248
                5568767
                28829794
                9f988147-a6d1-4f83-9c2c-8058ad54f478
                © 2017 Brusa et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 19 April 2017
                : 1 August 2017
                Page count
                Figures: 0, Tables: 5, Pages: 16
                Funding
                The present study was supported by Argentine Beef Promotion Institute (IPCVA). The funder had a role in data collection. The funder had no role in study design, analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Agriculture
                Animal Products
                Meat
                Beef
                Biology and Life Sciences
                Nutrition
                Diet
                Food
                Meat
                Beef
                Medicine and Health Sciences
                Nutrition
                Diet
                Food
                Meat
                Beef
                Biology and Life Sciences
                Organisms
                Animals
                Vertebrates
                Amniotes
                Mammals
                Bovines
                Cattle
                Biology and Life Sciences
                Organisms
                Animals
                Vertebrates
                Amniotes
                Mammals
                Ruminants
                Cattle
                Medicine and Health Sciences
                Infectious Diseases
                Bacterial Diseases
                Hemolytic-Uremic Syndrome
                Biology and Life Sciences
                Anatomy
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                Medicine and Health Sciences
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                Medicine and Health Sciences
                Pathology and Laboratory Medicine
                Pathogens
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                People and places
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                South America
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                All relevant data are within the paper and its Supporting Information files.

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