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Sample records for o157 strains isolated

  1. Characterization of Escherichia coli O157:H7 strains isolated from supershedding cattle.

    PubMed

    Arthur, Terrance M; Ahmed, Rafiq; Chase-Topping, Margo; Kalchayanand, Norasak; Schmidt, John W; Bono, James L

    2013-07-01

    Previous reports have indicated that a small proportion of cattle shedding high levels of Escherichia coli O157:H7 is the main source for transmission of this organism between animals. Cattle achieving a fecal shedding status of 10(4) CFU of E. coli O157:H7/gram or greater are now referred to as supershedders. The aim of this study was to investigate the contribution of E. coli O157:H7 strain type to supershedding and to determine if supershedding was restricted to a specific set of E. coli O157:H7 strains. Fecal swabs (n = 5,086) were collected from cattle at feedlots or during harvest. Supershedders constituted 2.0% of the bovine population tested. Supershedder isolates were characterized by pulsed-field gel electrophoresis (PFGE), phage typing, lineage-specific polymorphism assay (LSPA), Stx-associated bacteriophage insertion (SBI) site determination, and variant analysis of Shiga toxin, tir, and antiterminator Q genes. Isolates representing 52 unique PFGE patterns, 19 phage types, and 12 SBI clusters were obtained from supershedding cattle, indicating that there is no clustering to E. coli O157:H7 genotypes responsible for supershedding. While being isolated directly from cattle, this strain set tended to have higher frequencies of traits associated with human clinical isolates than previously collected bovine isolates with respect to lineage and tir allele, but not for SBI cluster and Q type. We conclude that no exclusive genotype was identified that was common to all supershedder isolates.

  2. Isolation of a Citrobacter freundii strain which carries the Escherichia coli O157 antigen.

    PubMed Central

    Bettelheim, K A; Evangelidis, H; Pearce, J L; Sowers, E; Strockbine, N A

    1993-01-01

    A biochemically typical strain of Citrobacter freundii which carries the Escherichia coli O157 antigen is described. The significance of differentiating such strains from typical E. coli O157 strains is stressed. PMID:7681442

  3. Genetic features of human and bovine Escherichia coli O157:H7 strains isolated in Argentina.

    PubMed

    Pianciola, L; D'Astek, B A; Mazzeo, M; Chinen, I; Masana, M; Rivas, M

    2016-02-01

    Shiga toxin-producing Escherichia coli (STEC) are important food-borne pathogens associated with human diseases. In Argentina, O157:H7 is the dominant serotype in hemolytic uremic syndrome (HUS) cases. Previously, we have described the almost exclusive circulation of human E. coli O157 strains belonging to the hypervirulent clade 8 in Neuquén Province. The aim of the present study was to investigate, by a broad molecular characterization, if this particular distribution of E. coli O157 clades in Neuquén is similar to the situation in other regions of the country and if it may be originated in a similar profile in cattle, its main reservoir. Two-hundred and eighty O157 strains (54 bovine and 226 human) isolated between 2006 and 2008 in different regions of Argentina were studied. All strains harbored rfbO157, fliCH7, eae, and ehxA genes. The predominant genotype was stx2a/stx2c in human (76.1%) and bovine (55.5%) strains. All human isolates tested by Lineage-Specific Polymorphism Assay (LSPA-6), were lineage I/II; among bovine strains, 94.1% belonged to lineage I/II and 5.9% to lineage I. No LSPA-6 lineage II isolates were detected. Single nucleotide polymorphism (SNP) analysis has revealed the existence of nine clade phylogenetic groups. In our clinical strains collection, 87.6% belonged to the hypervirulent clade 8, and 12.4% were classified as clade 4/5. In bovine isolates, 59.3% strains were clade 8, 33.3% clade 4/5 and 7.4% clade 3. More than 80% of human strains showed the presence of 6 of the 7 virulence determinants described in the TW14359 O157 strain associated with the raw spinach outbreak in the U.S. in 2006. More than 80% of bovine strains showed the presence of 3 of these factors. The q933 allele, which has been related to high toxin production, was present in 98.2% of clinical strains and 75.9% of the bovine isolates. The molecular characterization of human STEC O157 strains allows us to conclude that the particular situation previously described

  4. Genetic features of human and bovine Escherichia coli O157:H7 strains isolated in Argentina.

    PubMed

    Pianciola, L; D'Astek, B A; Mazzeo, M; Chinen, I; Masana, M; Rivas, M

    2016-02-01

    Shiga toxin-producing Escherichia coli (STEC) are important food-borne pathogens associated with human diseases. In Argentina, O157:H7 is the dominant serotype in hemolytic uremic syndrome (HUS) cases. Previously, we have described the almost exclusive circulation of human E. coli O157 strains belonging to the hypervirulent clade 8 in Neuquén Province. The aim of the present study was to investigate, by a broad molecular characterization, if this particular distribution of E. coli O157 clades in Neuquén is similar to the situation in other regions of the country and if it may be originated in a similar profile in cattle, its main reservoir. Two-hundred and eighty O157 strains (54 bovine and 226 human) isolated between 2006 and 2008 in different regions of Argentina were studied. All strains harbored rfbO157, fliCH7, eae, and ehxA genes. The predominant genotype was stx2a/stx2c in human (76.1%) and bovine (55.5%) strains. All human isolates tested by Lineage-Specific Polymorphism Assay (LSPA-6), were lineage I/II; among bovine strains, 94.1% belonged to lineage I/II and 5.9% to lineage I. No LSPA-6 lineage II isolates were detected. Single nucleotide polymorphism (SNP) analysis has revealed the existence of nine clade phylogenetic groups. In our clinical strains collection, 87.6% belonged to the hypervirulent clade 8, and 12.4% were classified as clade 4/5. In bovine isolates, 59.3% strains were clade 8, 33.3% clade 4/5 and 7.4% clade 3. More than 80% of human strains showed the presence of 6 of the 7 virulence determinants described in the TW14359 O157 strain associated with the raw spinach outbreak in the U.S. in 2006. More than 80% of bovine strains showed the presence of 3 of these factors. The q933 allele, which has been related to high toxin production, was present in 98.2% of clinical strains and 75.9% of the bovine isolates. The molecular characterization of human STEC O157 strains allows us to conclude that the particular situation previously described

  5. Characterization of non-Shiga-toxin-producing Escherichia coli O157 strains isolated from dogs.

    PubMed

    Bentancor, A; Vilte, D A; Rumi, M V; Carbonari, C C; Chinen, I; Larzábal, M; Cataldi, A; Mercado, E C

    2010-01-01

    Shiga toxin-negative Escherichia coli O157 strains of various H types have been associated with diarrhea in children and are considered potentially pathogenic for humans. In this study, we describe non-Shiga toxin-producing E. coli O157 E. coli strains previously obtained from dogs in Argentina. Different E. coli phylogenetic lineages corresponding to flagellar types H16, H29 and H45 were identified. E. coli serotypes O157:H16 and O157:H45 contained intimin subtypes epsilon and alpha 1, respectively. Serotype O157:H45 carried the bfp gene encoding the bundle-forming pilus. Localized adherence-like patterns to HEp-2 cells were observed in O157:H16 strains, while O157:H45 adhered in a typical localized pattern. A total of eight different XbaI-pulse field electrophoresis patterns with more than 74 % similarity were identified among the nine E. coli O157:H16 strains. Our data emphasized the fact that dogs may harbor human pathogenic E. coli O157 which do not correspond to Shiga toxin-producing strains and whose potential human health hazard should not be underestimated. PMID:20461294

  6. Molecular Profiling of Escherichia coli O157:H7 and Non-O157 Strains Isolated from Humans and Cattle in Alberta, Canada

    PubMed Central

    Li, Vincent; Fach, Patrick; Delannoy, Sabine; Malejczyk, Katarzyna; Patterson-Fortin, Laura; Poon, Alan; King, Robin; Simmonds, Kimberley; Scott, Allison N.; Lee, Mao-Cheng

    2014-01-01

    Virulence markers in Shiga toxin-producing Escherichia coli (STEC) and their association with diseases remain largely unknown. This study determines the importance of 44 genetic markers for STEC (O157 and non-O157) from human clinical cases and their correlation to disease outcome. STEC isolated from a cattle surveillance program were also included. The virulence genes tested were present in almost all O157:H7 isolates but highly variable in non-O157 STEC isolates. Patient age was a significant determinant of clinical outcome. PMID:25540392

  7. Complete Genome Sequence of an Escherichia coli O157:H7 Strain Isolated from a Super-Shedder Steer.

    PubMed

    Teng, Lin; Ginn, Amber; Jeon, Soojin; Kang, Minyoung; Jeong, KwangCheol Casey

    2016-01-01

    We report here the complete genome sequence ofEscherichia coliO157:H7 strain JEONG-1266 isolated from a super- shedder steer in northwest Florida. Cattle are considered a primary reservoir ofE. coliO157:H7, and those cattle that excrete this pathogen in their feces at levels ≥10(4) CFU/g are known as super-shedders. PMID:27056233

  8. Detection and frequency of VT1, VT2 and eaeA genes in Escherichia coli O157 and O157:H7 strains isolated from cattle, cattle carcasses and abattoir environment in Istanbul.

    PubMed

    Yilmaz, Aysun; Gun, Huseyin; Ugur, Muammer; Turan, Nuri; Yilmaz, Huseyin

    2006-02-01

    The aim of this study was to detect VT1, VT2 and eaeA genes and to determine the frequency of these genes in Escherichia coli O157 and O157:H7 strains isolated from cattle, cattle carcasses and environmental samples of the 5 abattoirs located in Istanbul, Turkey. For this, the presence of VT1, VT2 and eaeA genes in 26 strains of E. coli O157:H7 and 6 strains of O157 was investigated by multiplex-PCR. The results have shown that eaeA gene was detected in all O157 and O157:H7 strains tested. Both VT2 and eaeA genes were detected in 4 (80%) of 5 strains of E. coli O157 and eaeA alone in 1 strain of O157. In 27 strains of O157:H7, 5 (18.5%) strains were found to be positive for VT1, VT2 and eaeA genes, 19 (70.3%) strains for both VT2 and eaeA and, 3 (11.1%) strains for only eaeA gene. Either VT1 alone or VT2 alone was not detected in any strains tested. eaeA gene alone in 2 strains, VT2-eaeA genes in 9 strains and VT1-VT2-eaeA genes in 2 strains were detected in 13 of E. coli O157:H7 strains isolated from cattle. eaeA alone in 1 strain, VT2-eaeA genes in 5 strains and VT1-VT2-eaeA genes in 2 strains were detected in 8 of E. coli O157:H7 strains isolated from carcasses. VT2-eaeA genes in 5 strains (isolated from hands, apron, knife and floor) and VT1-VT2-eaeA genes in 1 strain (isolated from knife) were also detected in 6 of E. coli O157:H7 strains isolated from environmental samples. This study reveals that most of the strains are found to be toxigenic and it is most likely that strains isolated from carcasses and abattoir environment originated from cattle feces. Therefore, HACCP systems are necessary from farm to table especially in the abattoirs to prevent contamination of meat and abattoir environment with intestinal content.

  9. Biofilm formation and sanitizer resistance of Escherichia coli O157:H7 strains isolated from "high event period" meat contamination.

    PubMed

    Wang, Rong; Kalchayanand, Norasak; King, David A; Luedtke, Brandon E; Bosilevac, Joseph M; Arthur, Terrance M

    2014-11-01

    In the meat industry, a "high event period" (HEP) is defined as a time period during which commercial meat plants experience a higher than usual rate of Escherichia coli O157:H7 contamination. Genetic analysis indicated that within a HEP, most of the E. coli O157:H7 strains belong to a singular dominant strain type. This was in disagreement with the current beef contamination model stating that contamination occurs when incoming pathogen load on animal hides, which consists of diverse strain types of E. coli O157:H7, exceeds the intervention capacity. Thus, we hypothesize that the HEP contamination may be due to certain in-plant colonized E. coli O157:H7 strains that are better able to survive sanitization through biofilm formation. To test our hypothesis, a collection of 45 E. coli O157:H7 strains isolated from HEP beef contamination incidents and a panel of 47 E. coli O157:H7 strains of diverse genetic backgrounds were compared for biofilm formation and sanitizer resistance. Biofilm formation was tested on 96-well polystyrene plates for 1 to 6 days. Biofilm cell survival and recovery growth after sanitization were compared between the two strain collections using common sanitizers, including quaternary ammonium chloride, chlorine, and sodium chlorite. No difference in "early stage" biofilms was observed between the two strain collections after incubation at 22 to 25°C for 1 or 2 days. However, the HEP strains demonstrated significantly higher potency of "mature" biofilm formation after incubation for 4 to 6 days. Biofilms of the HEP strains also exhibited significantly stronger resistance to sanitization. These data suggest that biofilm formation and sanitization resistance could have a role in HEP beef contamination by E. coli O157:H7, which highlights the importance of proper and complete sanitization of food contact surfaces and food processing equipment in commercial meat plants. PMID:25364934

  10. Isolation and Characterization of Verocytotoxin-Producing Escherichia coli O157 Strains from Dutch Cattle and Sheep

    PubMed Central

    Heuvelink, A. E.; van den Biggelaar, F. L. A. M.; de Boer, E.; Herbes, R. G.; Melchers, W. J. G.; Huis In ’t Veld, J. H. J.; Monnens, L. A. H.

    1998-01-01

    In the periods from July to November 1995 and 1996, fecal samples from Dutch cattle and sheep were collected at the main slaughterhouses of The Netherlands, located at different geographic sites. The samples were examined for the presence of verocytotoxin (VT)-producing Escherichia coli (VTEC) of serogroup O157. E. coli O157 strains could be isolated from 57 (10.6%) of 540 adult cattle, 2 (0.5%) of 397 veal calves, 2 (3.8%) of 52 ewes, and 2 (4.1%) of 49 lambs. Immunomagnetic separation with O157-specific-antibody-coated beads appeared to be significantly more sensitive than conventional plating for detection of the organism in feces. With the exception of two isolates from adult cattle which appeared to be negative for VT genes, all animal isolates were positive for both VT (VT1 and/or VT2) and E. coli attaching-and-effacing gene sequences, and therefore, they were regarded as potential human pathogens. Although genomic typing by pulsed-field gel electrophoresis revealed a wide variety of distinct restriction patterns, comparison of the 63 animal isolates with 33 fecal O157 VTEC strains previously isolated from humans with the diarrhea-associated form of the hemolytic-uremic syndrome by their phage types and VT genotypes showed a marked similarity between animal and human isolates: 30 (90.9%) of the 33 human isolates appeared to be of E. coli O157 strain types also isolated from cattle and sheep. It was concluded that Dutch cattle and sheep are an important reservoir of E. coli O157 strains that are potentially pathogenic for humans. PMID:9542902

  11. Characterisation of Shiga toxin-producing Escherichia coli O157 strains isolated from humans in Argentina, Australia and New Zealand

    PubMed Central

    Leotta, Gerardo A; Miliwebsky, Elizabeth S; Chinen, Isabel; Espinosa, Estela M; Azzopardi, Kristy; Tennant, Sharon M; Robins-Browne, Roy M; Rivas, Marta

    2008-01-01

    Background Shiga toxin-producing Escherichia coli (STEC) is an important cause of bloody diarrhoea (BD), non-bloody diarrhoea (NBD) and the haemolytic uraemic syndrome (HUS). In Argentina and New Zealand, the most prevalent STEC serotype is O157:H7, which is responsible for the majority of HUS cases. In Australia, on the other hand, STEC O157:H7 is associated with a minority of HUS cases. The main aims of this study were to compare the phenotypic and genotypic characteristics of STEC O157 strains isolated between 1993 and 1996 from humans in Argentina, Australia and New Zealand, and to establish their clonal relatedness. Results Seventy-three O157 STEC strains, isolated from HUS (n = 36), BD (n = 20), NBD (n = 10), or unspecified conditions (n = 7) in Argentina, Australia and New Zealand, were analysed. The strains were confirmed to be E. coli O157 by biochemical tests and serotyping. A multiplex polymerase chain reaction (PCR) was used to amplify the stx1, stx2 and rfbO157 genes and a genotyping method based on PCR-RFLP was used to determine stx1 and stx2 variants. This analysis revealed that the most frequent stx genotypes were stx2/stx2c (vh-a) (91%) in Argentina, stx2 (89%) in New Zealand, and stx1/stx2 (30%) in Australia. No stx1-postive strains were identified in Argentina or New Zealand. All strains harboured the eae gene and 72 strains produced enterohaemolysin (EHEC-Hly). The clonal relatedness of strains was investigated by phage typing and pulsed-field gel electrophoresis (PFGE). The most frequent phage types (PT) identified in Argentinian, Australian, and New Zealand strains were PT49 (n = 12), PT14 (n = 9), and PT2 (n = 15), respectively. Forty-six different patterns were obtained by XbaI-PFGE; 37 strains were grouped in 10 clusters and 36 strains showed unique patterns. Most clusters could be further subdivided by BlnI-PFGE. Conclusion STEC O157 strains isolated in Argentina, Australia, and New Zealand differed from each other in terms of stx

  12. Isolation and characterization of verocytotoxin-producing Escherichia coli O157 strains from Dutch cattle and sheep.

    PubMed

    Heuvelink, A E; van den Biggelaar, F L; de Boer, E; Herbes, R G; Melchers, W J; Huis in 't Veld, J H; Monnens, L A

    1998-04-01

    In the periods from July to November 1995 and 1996, fecal samples from Dutch cattle and sheep were collected at the main slaughterhouses of The Netherlands, located at different geographic sites. The samples were examined for the presence of verocytotoxin (VT)-producing Escherichia coli (VTEC) of serogroup 0157. E. coli O157 strains could be isolated from 57 (10.6%) of 540 adult cattle, 2 (0.5%) of 397 veal calves, 2 (3.8%) of 52 ewes, and 2 (4.1%) of 49 lambs. Immunomagnetic separation with O157-specific-antibody-coated beads appeared to be significantly more sensitive than conventional plating for detection of the organism in feces. With the exception of two isolates from adult cattle which appeared to be negative for VT genes, all animal isolates were positive for both VT (VT1 and/or VT2) and E. coli attaching-and-effacing gene sequences, and therefore, they were regarded as potential human pathogens. Although genomic typing by pulsed-field gel electrophoresis revealed a wide variety of distinct restriction patterns, comparison of the 63 animal isolates with 33 fecal O157 VTEC strains previously isolated from humans with the diarrhea-associated form of the hemolytic-uremic syndrome by their phage types and VT genotypes showed a marked similarity between animal and human isolates: 30 (90.9%) of the 33 human isolates appeared to be of E. coli O157 strain types also isolated from cattle and sheep. It was concluded that Dutch cattle and sheep are an important reservoir of E. coli O157 strains that are potentially pathogenic for humans. PMID:9542902

  13. [A selective nutrient medium for isolating clinical strains of Escherichia coli O157:H7].

    PubMed

    Sultanov, Z Z; Stepanova, E D; Kakulina, E A

    2000-01-01

    A dried selective culture medium, electrolyte-deficient sorbitol agar (EDS agar), for the isolation and preliminary identification of E. coli O157:H7 from clinical material has been developed. The medium is not inferior in its quality to analogous foreign media and requires no scarce ingredients for its manufacture.

  14. Characterization of Enterohemorrhagic Escherichia coli O111 and O157 Strains Isolated from Outbreak Patients in Japan

    PubMed Central

    Isobe, Junko; Kimata, Keiko; Shima, Tomoko; Kanatani, Jun-ichi; Shimizu, Miwako; Nagata, Akihiro; Kawakami, Keiko; Yamada, Mikiko; Izumiya, Hidemasa; Iyoda, Sunao; Morita-Ishihara, Tomoko; Mitobe, Jiro; Terajima, Jun; Ohnishi, Makoto; Sata, Tetsutaro

    2014-01-01

    In April and May 2011, there was a serious food-poisoning outbreak in Japan caused by enterohemorrhagic Escherichia coli (EHEC) strains O111:H8 and O157:H7 from raw beef dishes at branches of a barbecue restaurant. This outbreak involved 181 infected patients, including 34 hemolytic-uremic syndrome (HUS) cases (19%). Among the 34 HUS patients, 21 developed acute encephalopathy (AE) and 5 died. Patient stool specimens yielded E. coli O111 and O157 strains. We also detected both EHEC O111 stx2 and stx-negative E. coli O111 strains in a stock of meat block from the restaurant. Pulsed-field gel electrophoresis (PFGE) and multilocus variable-number tandem-repeat analysis (MLVA) showed that the stx-negative E. coli O111 isolates were closely related to EHEC O111 stx2 isolates. Although the EHEC O157 strains had diverse stx gene profiles (stx1, stx2, and stx1 stx2), the PFGE and MLVA analyses indicated that these isolates originated from a single clone. Deletion of the Stx2-converting prophage from the EHEC O111 stx2 isolates was frequently observed during in vitro growth, suggesting that strain conversion from an EHEC O111 stx2 to an stx-negative strain may have occurred during infection. PMID:24829231

  15. Phenotypic Diversity of Escherichia coli O157:H7 Strains Associated With the Plasmid O157

    PubMed Central

    Lim, Ji Youn; Hong, Joon Bae; Sheng, Haiqing; Shringi, Smriti; Kaul, Rajinder; Hovde, Carolyn J.

    2010-01-01

    Escherichia coli O157:H7, a food-borne pathogen, causes hemorrhagic colitis and the hemolytic-uremic syndrome. A putative virulence factor of E. coli O157:H7 is a 60-MDa plasmid (pO157) found in 99% of all clinical isolates and many bovine-derived strains. The well characterized E. coli O157:H7 Sakai strain (Sakai) and its pO157-cured derivative (Sakai-Cu) were compared for phenotypic differences. Sakai-Cu had enhanced survival in synthetic gastric fluid, did not colonize cattle as well as wild-type Sakai, and had unchanged growth rates and tolerance to salt and heat. These results are consistent with our previous findings with another E. coli O157:H7 disease outbreak isolate ATCC 43894 and its pO157-cured (43894-Cu). However, despite the essentially sequence identical pO157 in these strains, Sakai-Cu had changes in antibiotic susceptibility and motility that did not occur in the 43894-Cu strain. This unexpected result was systematically analyzed using phenotypic microarrays testing 1,920 conditions with Sakai, 43894, and the plasmid-cured mutants. The influence of the pO157 differed between strains on a wide number of growth/survival conditions. Relative expression of genes related to acid resistance (gadA, gadX, and rpoS) and flagella production (fliC and flhD) were tested using quantitative real-time PCR and gadA and rpoS expression differed between Sakai-Cu and 43894-Cu. The strain-specific differences in phenotype that resulted from the loss of essentially DNA-sequence identical pO157 were likely due to the chromosomal genetic diversity between strains. The O157:H7 serotype diversity was further highlighted by phenotypic microarray comparisons of the two outbreak strains with a genotype 6 bovine E. coli O157:H7 isolate, rarely associated with human disease. PMID:20571953

  16. Draft Genome Sequence of Shiga Toxin-Negative Escherichia coli O157:H7 Strain C1-057, Isolated from Feedlot Cattle.

    PubMed

    Yang, Hua; Carlson, Brandon; Geornaras, Ifigenia; Woerner, Dale; Sofos, John; Belk, Keith

    2016-01-01

    Escherichia coli O157:H7 is one of the major foodborne pathogens in the United States. We isolated a variant Shiga toxin-negative E. coli O157:H7 strain from feedlot cattle. We report here the draft genome sequence of this isolate, consisting of a chromosome of ~4.8 Mb and two plasmids of ~96 kb and ~14 kb. PMID:26941140

  17. High-Level Genotypic Variation and Antibiotic Sensitivity among Escherichia coli O157 Strains Isolated from Two Scottish Beef Cattle Farms

    PubMed Central

    Vali, Leila; Wisely, Karen A.; Pearce, Michael C.; Turner, Esther J.; Knight, Hazel I.; Smith, Alastair W.; Amyes, Sebastian G. B.

    2004-01-01

    Escherichia coli O157:H7 is a human pathogen that is carried and transmitted by cattle. Scotland is known to have one of the highest rates of E. coli O157 human infections in the world. Two hundred ninety-three isolates were obtained from naturally infected cattle and the environment on two farms in the Scottish Highlands. The isolates were typed by pulsed-field gel electrophoresis (PFGE) with XbaI restriction endonuclease enzyme, and 19 different variations in patterns were found. There was considerable genomic diversity within the E. coli O157 population on the two farms. The PFGE pattern of one of the observed subtypes matched exactly with that of a strain obtained from a Scottish patient with hemolytic-uremic syndrome. To examine the stability of an individual E. coli O157 strain, continuous subculturing of a strain was performed 110 times. No variation from the original PFGE pattern was observed. We found three indistinguishable subtypes of E. coli O157 on both study farms, suggesting common sources of infection. We also examined the antibiotic resistance of the isolated strains. Phenotypic studies demonstrated resistance of the strains to sulfamethoxazole (100%), chloramphenicol (3.07%), and at a lower rate, other antibiotics, indicating the preservation of antibiotic sensitivity in a rapidly changing population of E. coli O157. PMID:15466537

  18. Antimicrobial resistance of Shiga toxin (verotoxin)-producing Escherichia coli O157:H7 and non-O157 strains isolated from humans, cattle, sheep and food in Spain.

    PubMed

    Mora, Azucena; Blanco, Jesús E; Blanco, Miguel; Alonso, M Pilar; Dhabi, Ghizlane; Echeita, Aurora; González, Enrique A; Bernárdez, M Isabel; Blanco, Jorge

    2005-08-01

    A total of 722 Shiga toxin-producing Escherichia coli (STEC) isolates recovered from humans, cattle, ovines and food during the period from 1992 to 1999 in Spain were examined to determine antimicrobial resistance profiles and their association with serotypes, phage types and virulence genes. Fifty-eight (41%) out of 141 STEC O157:H7 strains and 240 (41%) out of 581 non-O157 STEC strains showed resistance to at least one of the 26 antimicrobial agents tested. STEC O157:H7 showed a higher percentage of resistant strains recovered from bovine (53%) and beef meat (57%) than from human (23%) and ovine (20%) sources, whereas the highest prevalence of antimicrobial resistance in non-O157 STEC was found among isolates recovered from beef meat (55%) and human patients (47%). Sulfisoxazole (36%) had the most common antimicrobial resistance, followed by tetracycline (32%), streptomycin (29%), ampicillin (10%), trimethoprim (8%), cotrimoxazole (8%), chloramphenicol (7%), kanamycin (7%), piperacillin (6%), and neomycin (5%). The multiple resistance pattern most often observed was that of streptomycin, sulfisoxazole, and tetracycline. Ten (7%) STEC O157:H7 and 71 (12%) non-O157 strains were resistant to five or more antimicrobial agents. Most strains showing resistance to five or more antimicrobial agents belonged to serotypes O4:H4 (4 strains), O8:H21 (3 strains), O20:H19 (6 strains), O26:H11 (8 strains eae-beta1), O111:H- (3 strains eae-gamma2), O118:H- (2 strains eae-beta1), O118:H16 (5 strains eae-beta1), O128:H- (2 strains), O145:H8 or O145:H- (2 strains eae-gamma1), O157:H7 (10 strains eae-gamma1), O171:H25 (3 strains), O177:H11 (5 strains eae-beta1), ONT:H- (3 strains/1 eae-beta1) and ONT:H21 (2 strains). Interestingly, most of these serotypes, i.e., those indicated in bold) were found among human STEC strains isolated from patients with hemolytic uremic-syndrome (HUS) reported in previous studies. We also detected, among non-O157 strains, an association between a higher

  19. PCR detection of Shiga toxins, enterohaemolysin and intimin virulence genes of Escherichia coli O157:H7 strains isolated from faeces of Anatolian water buffaloes in Turkey.

    PubMed

    Seker, E; Kuyucuoğlu, Y; Sareyyüpoğlu, B; Yardımcı, H

    2010-12-01

    The aim of this study was to detect Shiga toxins (stx1 and stx2), enterohaemolysin (EhlyA) and intimin (eaeA) virulence genes of 11 Escherichia coli O157:H7 strains isolated from faecal samples of 300 clinically healthy Anatolian water buffaloes by PCR. Multiplex PCR was used for the detection of stx1 and stx2, and singleplex PCRs were used for the detection of EhlyA and eaeA virulence genes respectively. A total of three (27.3%) strains were determined to harbour both of the stx1 and stx2 genes, of these, one (9.1%) only harboured these two genes alone, one (9.1%) also contained the EhlyA gene and one (9.1%) additionally contained the EhlyA and the eaeA genes. EhlyA gene was obtained from eight (72.7%) strains, six (54.5%) of these were alone. eaeA gene was positive in only one (9.1%) strain. Only one (9.1%) of the 11 E. coli O157:H7 strains harboured all the four virulence genes. Two (18.2%) of the isolates had none of the virulence genes. Enterohaemolysin was found to be the most common virulence factor. In conclusion, the virulence factors of E. coli O157:H7 strains isolated from the faeces of Anatolian water buffaloes were investigated and detected for the first time in Turkey.

  20. Prevalence, genetic characterization and virulence genes of sorbitol-fermenting Escherichia coli O157:H- and E. coli O157:H7 isolated from retail beef.

    PubMed

    Sallam, Khalid Ibrahim; Mohammed, Mahmoud Ahmed; Ahdy, Asmaa Mohammed; Tamura, Tomohiro

    2013-08-01

    Sorbitol-fermenting (SF) Escherichia coli O157:H- strains have emerged as important pathogens and have been associated with a higher incidence of progression to hemolytic-uremic syndrome (HUS) than non-sorbitol fermenting (NSF) E. coli O157:H7. The present study was carried out to determine the prevalence of SF E. coli O157:H- and NSF E. coli O157:H7 strains in retail beef products in Mansoura, Egypt. The contamination rates with rfbEO157-positive E. coli O157 strains were 26.7% (8/30), 10% (3/30) and 3.7% (1/27) in ground beef, beef burger, and fresh beef samples, respectively with an overall mean of 13.8% (12/87) among all meat products tested. SF E. coli O157:H- were the most dominant among the isolated O157 strains. Of the fifteen O157 strains isolated, 11 (73.3%) were SF E. coli O157:H-, while the remaining 4 (26.7%) were NSF E. coli O157:H7. The 11 SF O157H- strains were genetically positive for sfpA gene. Restriction fragment length polymorphism (RFLP) analysis for fliC gene demonstrated a similar pattern for both SF and NSF O157 isolates. PCR assays verified the existence of stx1 gene in 7 (46.7%) and stx2 gene in 13 (86.7%) of the 15 O157 strains isolated. Unexpectedly, two of the 15 O157 strains isolated were negative for Shiga toxin genes. The eae gene was identified in all of the 15 O157 strains except in one NSF O157:H7 strain. EHEC-hlyA gene was detected in 14 (93.3%) of the 15 O157 isolates, nonetheless only 11 strains showed enterohemolytic phenotype on blood agar. A combination of the four virulence genes, stx1, stx2, eae and EHEC-hlyA were detected in 7 (46.7%) strains, while six (40%) strains were positive for stx2, eae and hlyA genes. This is the first record for isolation of E. coli O157: H- in Egypt as well as in the African continent.

  1. Characterization of bacterial strains isolated from a beef-processing plant following cleaning and disinfection - Influence of isolated strains on biofilm formation by Sakaï and EDL 933 E. coli O157:H7.

    PubMed

    Marouani-Gadri, Nesrine; Augier, Gladys; Carpentier, Brigitte

    2009-07-31

    The objective of this study was to investigate the effects on Escherichia coli O157:H7 biofilm formation of bacteria isolated from meat site surfaces following cleaning and disinfection. We first isolated and identified, to the genus level, strains of the latter organisms. Samples were obtained by swabbing the surfaces of equipment or floors over areas ranging from 315 to 3200 cm(2) in a slaughter hall, a meat cutting room and a meat boning room of a meat-processing plant. The number of bacteria recovered from these surfaces ranged from <1 to> 10(5) CFU/cm(2). In the slaughter hall, stainless steel was in one case one of the most contaminated materials and in other cases one of the less contaminated. The same observation was made for conveyor belts made of polyvinyl chloride in the boning room. Dominant genera in the meat plant were Staphylococcus and Bacillus which were both 34% of the isolates from the slaughter hall and 14 and 4% respectively of the isolates from the cutting room. Randomly selected isolates of each of the genera recovered from the slaughter hall were cultured with E. coli O157:H7 in meat exudate at 15 degrees C to form dual-organism biofilms on polyurethane. In all cases but one, the isolates increased the numbers of attached E. coli O157:H7. The effects ranged from 0.37 to 1.11 for EDL 933 strain and from 0.19 to 1.38 log (CFU/cm(2)) for Sakaï strain. This is the first time that a resident microbiota of a meat-processing plant has been shown to have a favourable effect on E. coli O157:H7 colonization of a solid surface, which is of great interest from a food safety standpoint.

  2. Characterization of E. coli O157:H7 strains isolated from “High Event Period” beef trim contamination

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Introduction: A “High Event Period” (HEP) is defined as a time period in which commercial plants experience a higher than usual rate of E. coli O157:H7 contamination of beef trims. Our previous studies suggested that instead of being a direct result of bacteria on animal hides, in-plant biofilm for...

  3. Isolation and characterization of verocytotoxin-producing Escherichia coli O157 from slaughter pigs and poultry.

    PubMed

    Heuvelink, A E; Zwartkruis-Nahuis, J T; van den Biggelaar, F L; van Leeuwen, W J; de Boer, E

    1999-11-01

    Rectal contents and tonsils from Dutch slaughter pigs collected immediately after slaughter were examined for the presence of verocytotoxin (VT)-producing Escherichia coli (VTEC) of serogroup O157 (O157 VTEC). In addition, fresh fecal material from poultry layer flocks and turkey flocks collected on poultry farms was examined for the presence of O157 VTEC. E. coli O157 strains were isolated from two (1.4%) of 145 pigs. The strains were isolated from samples of rectal contents, all samples of tonsils being negative. While all 501 fecal samples from chicken flocks were found negative, E. coli O157 strains were isolated from six (1.3%) of 459 pooled fecal samples from turkey flocks. One of the porcine isolates and one of the turkey isolates contained the VT2 gene, the E. coli attaching-and-effacing gene, as well as the enterohemorrhagic E. coli hemolysin gene. Production of VT was confirmed by cytotoxicity tests on Vero cells. Based on these characteristics, the two stains were regarded as potentially pathogenic for humans. The porcine and the turkey isolate were further characterized as being of phage types 4 and 14, respectively. While biochemically typical of E. coli O157, the remaining six isolates were nonverocytotoxigenic and negative for both the E. coli attaching-and-effacing gene and the enterohemorrhagic E. coli hemolysin gene. All eight E. coli O157 isolates did not carry genes that encode E. coli heat-labile and heat-stable enterotoxins. It was concluded that pigs and poultry can be a source of O157 VTEC strains characteristic of those causing illness in man. The extent to which pigs and poultry play a role in the epidemiology of human O157 VTEC infection needs further research. PMID:10573393

  4. Immunomagnetic separation as a sensitive method for isolating Escherichia coli O157 from food samples.

    PubMed

    Wright, D J; Chapman, P A; Siddons, C A

    1994-08-01

    Minced beef samples inoculated with Escherichia coli O157 were cultured in buffered peptone water supplemented with vancomycin, cefsulodin and cefixime (BPW-VCC) and subcultured to cefixime tellurite sorbitol MacConkey (CT-SMAC) agar both directly and after immunomagnetic separation (IMS) of the organism with magnetic beads coated with an antibody against E. coli O157 (Dynabeads anti-E. coli O157, Dynal, Oslo). E. coli O157 was recovered from initial inocula of 200 organisms/g by direct subculture and 2 organisms/g by IMS. Twelve strains of E. coli O157 of different combinations of phage type, H antigen and toxin genotype were all recovered from initial inocula of two organisms/g by IMS. Non-specific binding of other organisms to the magnetic beads could be reduced by washing of the beads in PBS with Tween-20 0.002-0.005% E. coli O157 was not bound by magnetic coated with an unrelated antibody. During investigation of a dairy herd that was possibly linked to a small outbreak of infection with E. coli O157, the organism was isolated from 2 of 279 forestream milk samples from individual cattle; both isolates were made only by the IMS technique. IMS is rapid, technically simple, and a specific method for isolation of E. coli O157 and will be useful in epidemiological studies.

  5. Immunomagnetic separation as a sensitive method for isolating Escherichia coli O157 from food samples.

    PubMed Central

    Wright, D. J.; Chapman, P. A.; Siddons, C. A.

    1994-01-01

    Minced beef samples inoculated with Escherichia coli O157 were cultured in buffered peptone water supplemented with vancomycin, cefsulodin and cefixime (BPW-VCC) and subcultured to cefixime tellurite sorbitol MacConkey (CT-SMAC) agar both directly and after immunomagnetic separation (IMS) of the organism with magnetic beads coated with an antibody against E. coli O157 (Dynabeads anti-E. coli O157, Dynal, Oslo). E. coli O157 was recovered from initial inocula of 200 organisms/g by direct subculture and 2 organisms/g by IMS. Twelve strains of E. coli O157 of different combinations of phage type, H antigen and toxin genotype were all recovered from initial inocula of two organisms/g by IMS. Non-specific binding of other organisms to the magnetic beads could be reduced by washing of the beads in PBS with Tween-20 0.002-0.005% E. coli O157 was not bound by magnetic coated with an unrelated antibody. During investigation of a dairy herd that was possibly linked to a small outbreak of infection with E. coli O157, the organism was isolated from 2 of 279 forestream milk samples from individual cattle; both isolates were made only by the IMS technique. IMS is rapid, technically simple, and a specific method for isolation of E. coli O157 and will be useful in epidemiological studies. PMID:8062877

  6. Characterization of Shiga Toxigenic Escherichia coli O157 and Non-O157 Isolates from Ruminant Feces in Malaysia

    PubMed Central

    Perera, Asanthi; Clarke, Charles M.; Dykes, Gary A.; Fegan, Narelle

    2015-01-01

    Shiga toxigenic Escherichia coli (STEC) O157 and several other serogroups of non-O157 STEC are causative agents of severe disease in humans world-wide. The present study was conducted to characterize STEC O157 and non-O157 serogroups O26, O103, O111, O121, O45, and O145 in ruminants in Malaysia. A total of 136 ruminant feces samples were collected from 6 different farms in Peninsular Malaysia. Immunomagnetic beads were used to isolate E. coli O157 and non-O157 serogroups, while PCR was used for the detection and subtyping of STEC isolates. STEC O157:H7 was isolated from 6 (4%) feces samples and all isolates obtained carried stx2c,  eaeA-γ1, and ehxA. Non-O157 STEC was isolated from 2 (1.5%) feces samples with one isolate carrying stx1a, stx2a, stx2c, and ehxA and the other carrying stx1a alone. The presence of STEC O157 and non-O157 in a small percentage of ruminants in this study together with their virulence characteristics suggests that they may have limited impact on public health. PMID:26539484

  7. Characterization of Shiga Toxigenic Escherichia coli O157 and Non-O157 Isolates from Ruminant Feces in Malaysia.

    PubMed

    Perera, Asanthi; Clarke, Charles M; Dykes, Gary A; Fegan, Narelle

    2015-01-01

    Shiga toxigenic Escherichia coli (STEC) O157 and several other serogroups of non-O157 STEC are causative agents of severe disease in humans world-wide. The present study was conducted to characterize STEC O157 and non-O157 serogroups O26, O103, O111, O121, O45, and O145 in ruminants in Malaysia. A total of 136 ruminant feces samples were collected from 6 different farms in Peninsular Malaysia. Immunomagnetic beads were used to isolate E. coli O157 and non-O157 serogroups, while PCR was used for the detection and subtyping of STEC isolates. STEC O157:H7 was isolated from 6 (4%) feces samples and all isolates obtained carried stx 2c, eaeA-γ1, and ehxA. Non-O157 STEC was isolated from 2 (1.5%) feces samples with one isolate carrying stx 1a, stx 2a, stx 2c, and ehxA and the other carrying stx 1a alone. The presence of STEC O157 and non-O157 in a small percentage of ruminants in this study together with their virulence characteristics suggests that they may have limited impact on public health. PMID:26539484

  8. Virulence characterization and molecular subtyping of typical and atypical Escherichia coli O157:H7 and O157:H(-) isolated from fecal samples and beef carcasses in Mexico.

    PubMed

    Narváez-Bravo, Claudia; Echeverry, Alejandro; Miller, Markus F; Rodas-González, Argenis; Brashears, M Todd; Aslam, Mueen; Brashears, Mindy M

    2015-02-01

    The objective of the study was to characterize virulence genes and subtype Escherichia coli O157:H7 and O157:H( 2 ) isolates obtained from a vertically integrated feedlot slaughter plant in Mexico. A total of 1,695 samples were collected from feedlots, holding pens, colon contents, hides, and carcasses. E. coli O157:H7 detection and confirmation was carried out using conventional microbiology techniques, immunomagnetic separation, latex agglutination, and the BAX system. A total of 97 E. coli O157 strains were recovered and screened for key virulence and metabolic genes using multiplex and conventional PCR. Eighty-eight (91.72%) of the strains carried stx2, eae, and ehxA genes. Ten isolates (8.25%) were atypical sorbitol-fermenting strains, and nine were negative for the flicH7 gene and lacked eae, stx1, stx2, and ehxA. One sorbitol-positive strain carried stx2, eae, tir, toxB, and iha genes but was negative for stx1 and ehxA. Pulsed-field gel electrophoresis (PFGE) analysis yielded 49 different PFGE subtypes, showing a high genetic diversity; however, the majority of the typical isolates were closely related (80 to 90% cutoff). Atypical O157 isolates were not closely related within them or to typical E. coli O157:H7 isolates. Identical PFGE subtypes were found in samples obtained from colon contents, feedlots, holding pens, and carcasses. Isolation of a sorbitolfermenting E. coli O157 positive for a number of virulence genes is a novel finding in Mexico. These data showed that genetically similar strains of E. coli O157:H7 can be found at various stages of beef production and highlights the importance of preventing cross-contamination at the pre- and postharvest stages of processing.

  9. Genetic Relatedness and Novel Sequence Types of Non-O157 Shiga Toxin-Producing Escherichia coli Strains Isolated in Argentina.

    PubMed

    Cadona, Jimena S; Bustamante, Ana V; González, Juliana; Sanso, A Mariel

    2016-01-01

    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. PMID:27625995

  10. Genetic Relatedness and Novel Sequence Types of Non-O157 Shiga Toxin-Producing Escherichia coli Strains Isolated in Argentina

    PubMed Central

    Cadona, Jimena S.; Bustamante, Ana V.; González, Juliana; Sanso, A. Mariel

    2016-01-01

    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. PMID:27625995

  11. Genetic Relatedness and Novel Sequence Types of Non-O157 Shiga Toxin-Producing Escherichia coli Strains Isolated in Argentina

    PubMed Central

    Cadona, Jimena S.; Bustamante, Ana V.; González, Juliana; Sanso, A. Mariel

    2016-01-01

    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.

  12. Isolation and characterization of the Shiga toxin gene (stx)-bearing Escherichia coli O157 and non-O157 from retail meats in Shandong Province, China, and characterization of the O157-derived stx2 phages.

    PubMed

    Koitabashi, Tsutomu; Cui, Shan; Kamruzzaman, Muhammad; Nishibuchi, Mitsuaki

    2008-04-01

    Infection by Shiga toxin (Stx)-producing Escherichia coli of non-O157 and O157 serotypes are rare in China, but infection by O157 serotype was found in Shandong Province and three other provinces in China. To understand the reason for these rare infections and to determine the safety of retail meats in Shandong Province, we examined the distribution of Shiga toxin gene (stx)-bearing E. coli in retail meats and characterized the isolated stx-bearing strains. We used hybridization with DNA probes and isolated stx1- and/or stx2-positive E. coli from 31 (58%) of 53 retail meat samples, with beef showing the highest frequency (68%). Of 42 stx-positive isolates, none belonged to O157. Using the O157-specific immunomagnetic bead technique, we isolated E. coli O157 carrying the eae and stx2 genes from eight beef samples (26%). These strains produced little or no Stx2 and carried a unique q gene. Replication of the stx2 phages was detected in these strains, whereas stx2 phage replication was not detected in our previous study in which we examined similar stx2-bearing E. coli O157 strains from other Asian countries. Analysis of E. coli C600 lysogenized with the stx2 phages found in this study suggests that the lack of Stx2 production is due to changes in non-q gene region(s) of the phage genome or chromosomal mutation(s) in the host. Our data and reports by other workers suggest it is necessary to determine if various stx2-bearing E. coli O157 strains producing Stx2 to varying degrees are distributed in meats in various locations in China.

  13. Whole-genome sequence of Escherichia coli serotype O157:H7 strain EDL932 (ATCC 43894)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli serotype O157:H7 EDL 933 is a ground beef isolate associated with a 1983 hemorrhagic colitis outbreak. Considered the prototype O157:H7 strain, its derived genome sequence is a standard reference strain for comparative genomic studies of Shiga toxin-producing E. coli (STEC). Here we...

  14. Persistence of Escherichia coli O157 and non-O157 strains in agricultural soils

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin producing Escherichia coli O157 and non-O157 serogroups are known to cause serious diseases in human. However, research on the persistence of E. coli non-O157 serogroups in preharvest environment is limited. In the current study, we compared the survival behavior of E. coli O157 to that ...

  15. [Isolation and characterization of Escherichia coli O157 in bovine meat products and cattle in the province of Tucuman].

    PubMed

    Jure, María A; Condorí, Marina S; Pérez Terrazzino, Gabriela; Catalán, Mariana G; López Campo, Alejandro; Zolezzi, Gisella; Chinen, Isabel; Rivas, Marta; Castillo, Marta

    2015-01-01

    Escherichia coli O157 is an emergent pathogen associated with diarrhea, hemorrhagic colitis and hemolytic uremic syndrome. Meat products constitute an important transmission source of this microorganism. The aims of this study were to characterize E. coli O157 isolated from cattle and meat products collected from abattoirs and retail stores, to establish the clonal relatedness among regional isolates and to compare them with those in the national database. Between 2004 and 2013, 169 minced meat, 35 sausage and 216 carcass samples were analyzed. Thirteen E. coli O157 isolates were identified; 6 of which were O157:H7 and characterized as stx2c(vh-a)/eae/ehxA (n = 5) and stx2/eae/ehxA (n = 1). The 7 remaining isolates were non-toxigenic E. coli strains, and serotyped as O157:NT (n = 4), O157:NM (n = 1), O157:ND (n = 1) and O157:H16 (n = 1). The strains yielded different XbaI-PFGE patterns. Compared to the E. coli O157 isolates in the National Database, none of these patterns have been previously detected in strains of different origin in Argentina. PMID:26026230

  16. [Isolation and characterization of Escherichia coli O157 in bovine meat products and cattle in the province of Tucuman].

    PubMed

    Jure, María A; Condorí, Marina S; Pérez Terrazzino, Gabriela; Catalán, Mariana G; López Campo, Alejandro; Zolezzi, Gisella; Chinen, Isabel; Rivas, Marta; Castillo, Marta

    2015-01-01

    Escherichia coli O157 is an emergent pathogen associated with diarrhea, hemorrhagic colitis and hemolytic uremic syndrome. Meat products constitute an important transmission source of this microorganism. The aims of this study were to characterize E. coli O157 isolated from cattle and meat products collected from abattoirs and retail stores, to establish the clonal relatedness among regional isolates and to compare them with those in the national database. Between 2004 and 2013, 169 minced meat, 35 sausage and 216 carcass samples were analyzed. Thirteen E. coli O157 isolates were identified; 6 of which were O157:H7 and characterized as stx2c(vh-a)/eae/ehxA (n = 5) and stx2/eae/ehxA (n = 1). The 7 remaining isolates were non-toxigenic E. coli strains, and serotyped as O157:NT (n = 4), O157:NM (n = 1), O157:ND (n = 1) and O157:H16 (n = 1). The strains yielded different XbaI-PFGE patterns. Compared to the E. coli O157 isolates in the National Database, none of these patterns have been previously detected in strains of different origin in Argentina.

  17. Escherichia coli O157:H7 strains isolated from “High Event Period” beef contamination have strong biofilm forming ability and low sanitizer susceptibility which are associated with high pO157 plasmid copy number

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In the meat industry, a “High Event Period” (HEP) is defined as a time period when beef processing establishments experience an increased occurrence of product contamination by E. coli O157:H7. Our previous studies suggested that bacterial biofilm formation and sanitizer resistance might contribute...

  18. Genetically Marked Strains of Shiga Toxin-Producing O157:H7 and Non-O157 Escherichia coli: Tools for Detection and Modeling.

    PubMed

    Paoli, George C; Wijey, Chandi; Uhlich, Gaylen A

    2015-05-01

    Shiga toxin-producing E. coli (STEC) is an important group of foodborne pathogens in the United States and worldwide. Nearly half of STEC-induced diarrheal disease in the United States is caused by serotype O157:H7, while non-O157 STEC account for the remaining illnesses. Thus, the U.S. Department of Agriculture (USDA) Food Safety and Inspection Service has instituted regulatory testing of beef products and has a zero-tolerance policy for regulatory samples that test positive for STEC O157:H7 and six other non-O157 STEC (serogroups O26, O45, O103, O111, O121, and O145). In this study, positive control (PC) strains for the detection of STEC O157:H7 and the six USDA-regulated non-O157 STEC were constructed. To ensure that the food testing samples are not cross-contaminated by the PC sample, it is important that the STEC-PC strains are distinguishable from STEC isolated from test samples. The PC strains were constructed by integrating a unique DNA target sequence and a gene for spectinomycin (Sp) resistance into the chromosomes of the seven STEC strains. End-point and real-time PCR assays were developed for the specific detection of the PC strains and were tested using 93 strains of E. coli (38 STEC O157:H7, at least 6 strains of each of the USDA-regulated non-O157 STEC, and 2 commensal E. coli) and 51 strains of other bacteria (30 species from 20 genera). The PCR assays demonstrated high specificity for the unique target sequence. The target sequence was detectable by PCR after 10 culture passages (∼100 generations), demonstrating the stability of the integrated target sequence. In addition, the strains were tested for their potential use in modeling the growth of STEC. Plating the PC strains mixed with ground beef flora on modified rainbow agar containing Sp eliminated the growth of the background flora that grew on modified rainbow agar without Sp. Thus, these strains could be used to enumerate and model the growth of STEC in the presence of foodborne background

  19. Characterization of Shiga Toxin-Producing Escherichia coli O157 Isolates from Bovine Carcasses.

    PubMed

    Fontcuberta, M; Planell, R; Torrents, A; Sabaté, S; Gonzalez, R; Ramoneda, M; de Simón, M

    2016-08-01

    The main purpose of this study was to determine the prevalence of Escherichia coli O157 on bovine carcasses before and after chilling at a large slaughterhouse located in the city of Barcelona, Spain, to assess the effectiveness of dry chilling on reducing E. coli O157 contamination of carcasses. In addition, the study characterized the E. coli O157 strains isolated in terms of virulence factors, antibiotic susceptibility, and their genetic diversity. Individual bovine carcasses were sampled before (n = 300) and after (n = 300) chilling over an 8-month period. Positive samples for E. coli O157 were subjected to virulence screening by PCR (stx1, stx2, and eaeA genes and the fliCH7 gene), antimicrobial susceptibility testing, and molecular typing by pulsed-field gel electrophoresis. A total of 9.7% (29 of 300) of the nonrefrigerated carcasses examined and 2.3% (7 of 300) of the refrigerated carcasses were positive for E. coli O157. All the isolates were serotype O157:H7, 92% (33 of 36) carried the stx1, stx2, and eaeA genes, and 8% (3 of 36) carried the stx2 and eaeA genes. Antimicrobial susceptibility testing showed a high degree of resistance: 29 strains (81%) were resistant to at least 1 antimicrobial of the 12 antimicrobials tested; 69% (25 of 36) were resistant to 4 or more antimicrobials. Molecular typing by pulsed-field gel electrophoresis found a high diversity of genetic types, implying little cross-contamination in the slaughterhouse. This study confirms that E. coli O157:H7 is present on the carcasses slaughtered in Spain, although its prevalence is reduced by the dry chilling process used. The recovered isolates showed potential pathogenesis and a high degree of multidrug resistance, confirming the importance of bovine meat monitoring.

  20. Characterization of Shiga Toxin-Producing Escherichia coli O157 Isolates from Bovine Carcasses.

    PubMed

    Fontcuberta, M; Planell, R; Torrents, A; Sabaté, S; Gonzalez, R; Ramoneda, M; de Simón, M

    2016-08-01

    The main purpose of this study was to determine the prevalence of Escherichia coli O157 on bovine carcasses before and after chilling at a large slaughterhouse located in the city of Barcelona, Spain, to assess the effectiveness of dry chilling on reducing E. coli O157 contamination of carcasses. In addition, the study characterized the E. coli O157 strains isolated in terms of virulence factors, antibiotic susceptibility, and their genetic diversity. Individual bovine carcasses were sampled before (n = 300) and after (n = 300) chilling over an 8-month period. Positive samples for E. coli O157 were subjected to virulence screening by PCR (stx1, stx2, and eaeA genes and the fliCH7 gene), antimicrobial susceptibility testing, and molecular typing by pulsed-field gel electrophoresis. A total of 9.7% (29 of 300) of the nonrefrigerated carcasses examined and 2.3% (7 of 300) of the refrigerated carcasses were positive for E. coli O157. All the isolates were serotype O157:H7, 92% (33 of 36) carried the stx1, stx2, and eaeA genes, and 8% (3 of 36) carried the stx2 and eaeA genes. Antimicrobial susceptibility testing showed a high degree of resistance: 29 strains (81%) were resistant to at least 1 antimicrobial of the 12 antimicrobials tested; 69% (25 of 36) were resistant to 4 or more antimicrobials. Molecular typing by pulsed-field gel electrophoresis found a high diversity of genetic types, implying little cross-contamination in the slaughterhouse. This study confirms that E. coli O157:H7 is present on the carcasses slaughtered in Spain, although its prevalence is reduced by the dry chilling process used. The recovered isolates showed potential pathogenesis and a high degree of multidrug resistance, confirming the importance of bovine meat monitoring. PMID:27497130

  1. Molecular characterization of Verocytotoxigenic Escherichia coli O157:H7 isolates from Argentina by Multiple-Loci VNTR Analysis (MLVA)

    PubMed Central

    Bustamante, Ana V.; Lucchesi, Paula M.A.; Parma, Alberto E.

    2009-01-01

    The aim of this work was to adapt described MLVA protocols to the molecular typing and characterization of VTEC O157:H7 isolates from Argentina. Nine VNTR loci were amplified by PCR showing diversity values from 0.49 to 0.73. Nine MLVA profiles were observed and the cluster analysis indicated both unrelated and closely related VTEC O157:H7 strains. In spite of the limited number of isolates studied, the panel of VNTR used made it possible to perform a first approach of the high genetic diversity of native strains of O157:H7 by MLVA. PMID:24031443

  2. Escherichia coli O157:H7 bacteriophage Φ241 isolated from an industrial cucumber fermentation at high acidity and salinity

    PubMed Central

    Lu, Zhongjing; Breidt, Fred

    2015-01-01

    A novel phage, Φ241, specific for Escherichia coli O157:H7 was isolated from an industrial cucumber fermentation where both acidity (pH ≤ 3.7) and salinity (≥5% NaCl) were high. The phage belongs to the Myoviridae family. Its latent period was 15 min and average burst size was 53 phage particles per infected cell. The phage was able to lyse 48 E. coli O157:H7 strains, but none of the 18 non-O157 strains (including E. coli O104:H7) or the 2 O antigen-negative mutants of O157:H7 strain, 43895Δper (also lacking H7 antigen) and F12 (still expressing H7 antigen). However, the phage was able to lyse a per-complemented strain (43895ΔperComp) which expresses O157 antigen. These results indicated that phage Φ241 is specific for O157 antigen, and E. coli strains lacking O157 antigen were resistant to the phage infection, regardless of the presence or absence of H7 antigen. SDS-PAGE profile revealed at least 13 structural proteins of the phage. The phage DNA was resistant to many commonly used restriction endonucleases, suggesting the presence of modified nucleotides in the phage genome. At the multiplicity of infection of 10, 3, or 0.3, the phage caused a rapid cell lysis within 1 or 2 h, resulting in 3.5- or 4.5-log-unit reduction in cell concentration. The high lytic activity, specificity and tolerance to low pH and high salinity make phage Φ241 a potentially ideal biocontrol agent of E. coli O157:H7 in various foods. To our knowledge, this is the first report on E. coli O157:H7 phage isolated from high acidity and salinity environment. PMID:25741324

  3. Support vector machine applied to predict the zoonotic potential of E. coli O157 cattle isolates

    PubMed Central

    Lupolova, Nadejda; Dallman, Timothy J.; Gally, David L.

    2016-01-01

    Sequence analyses of pathogen genomes facilitate the tracking of disease outbreaks and allow relationships between strains to be reconstructed and virulence factors to be identified. However, these methods are generally used after an outbreak has happened. Here, we show that support vector machine analysis of bovine E. coli O157 isolate sequences can be applied to predict their zoonotic potential, identifying cattle strains more likely to be a serious threat to human health. Notably, only a minor subset (less than 10%) of bovine E. coli O157 isolates analyzed in our datasets were predicted to have the potential to cause human disease; this is despite the fact that the majority are within previously defined pathogenic lineages I or I/II and encode key virulence factors. The predictive capacity was retained when tested across datasets. The major differences between human and bovine E. coli O157 isolates were due to the relative abundances of hundreds of predicted prophage proteins. This finding has profound implications for public health management of disease because interventions in cattle, such a vaccination, can be targeted at herds carrying strains of high zoonotic potential. Machine-learning approaches should be applied broadly to further our understanding of pathogen biology. PMID:27647883

  4. Pheno-genotypic characterisation of Escherichia coli O157:H7 isolates from domestic and wild ruminants.

    PubMed

    Sánchez, S; Martínez, R; Rey, J; García, A; Blanco, J; Blanco, M; Blanco, J E; Mora, A; Herrera-León, S; Echeita, A; Alonso, J M

    2010-05-19

    Shiga toxin-producing Escherichia coli (STEC) O157:H7 represents a major public health concern worldwide, with ruminants recognised as their main natural reservoir. The aim of this work was to determine the phenotypic features and genetic relationships of 46 E. coli O157:H7 isolates obtained from sheep, cattle and deer faeces and from unpasteurised goat milk in Spain over a period of 11 years. Characterisation was performed by polymerase chain reaction (PCR), phage typing and pulsed-field gel electrophoresis (PFGE). An atypical E. coli O157:H7 strain (sorbitol-fermenting and beta-glucuronidase positive) originating from deer faeces was detected. Genes encoding Shiga toxins were detected in 69.6% of isolates, all of them carrying only the stx(2) gene. The isolates were from nine different phage types, although 67.4% were restricted to only three: PT14, PT34 and PT54. PT54 was the most prevalent phage type and contained isolates from cattle, sheep and deer. Majority of the isolates were from phage types previously found in strains associated with human infection. XbaI-PFGE identified 33 different types and 11 groups of closely related types (more than 85% similarity), one of which included 21 (45.7%) isolates originating from different animal species, including deer. These results indicate common origin or inter-species spread of genetically similar E. coli O157:H7 isolates and contribute to earlier investigations identifying deer as a natural source of E. coli O157:H7. The study also highlights the emergence of phenotypic variants of E. coli O157:H7, which may not be identified by routine culture methods or by biochemical tests used to characterise serotype O157:H7.

  5. REPETITIVE SEQUENCE BASED-PCR PROFILING OF ESCHERICHIA COLI O157 STRAINS FROM BEEF IN SOUTHERN THAILAND.

    PubMed

    Sukhumungoon, Pharanai; Tantadapan, Rujira; Rattanachuay, Pattamarat

    2016-01-01

    Beef and its products are potential vehicles of Escherichia coli O157, the most important serotype implicated in many large outbreaks of diarrheal infection in humans worldwide. There is a need for rapid detection of contaminated food in order to implement appropriate and effective control measures. In this study, repetitive sequence (rep)-PCR, using three different primers, BOXA1R, ERIC2 and (GTG)5, singly and in combinations, were employed to compare the genetic relatedness among E. coli O157 group with other diarrheagenic E. coli strains as controls. Although a combination of BOXA1R + ERIC2 + (GTG)5 primers generated a rep-PCR profile containing the highest number of amplicon bands among the DEC strains tested, dendrogram (at 80% similarity) exhibited the lowest DEC classification of 5 clusters, whereas that from BOXA1R or BOXA1R+ (GTG)5 rep-PCR profiling produced 8 clusters. Nevertheless, focusing E. coli O157 strains were grouped into 4 clusters irrespective of the rep-PCR profiles analyzed, and all 14 but two, PSU60 and PSU132, E. coli O157 strains isolated from beef in southern Thailand during 2012 to 2014 fell into a single cluster. Thus, rep-PCR profiling generated with BOXA1R or BOXA1R + (GTG)5 is sufficient for distinguishing among DEC strains, including E. coli O157 in southern Thailand. PMID:27086425

  6. Antimicrobial resistance in Escherichia coli O157 and non-O157 isolated from feces of domestic farm animals in Culiacan, Mexico

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Antimicrobial resistance in E. coli O157 and non-O157 strains is a matter of increasing concern, and the association with some virulence traits in the same bacteria remains unclear. Inappropriate antimicrobial use in human and animal therapy has been associated with selective pressure in enteric mi...

  7. Strain-Dependent Cellular Immune Responses in Cattle following Escherichia coli O157:H7 Colonization

    PubMed Central

    Corbishley, Alexander; Ahmad, Nur Indah; Hughes, Kirsty; Hutchings, Michael R.; McAteer, Sean P.; Connelley, Timothy K.; Brown, Helen; Gally, David L.

    2014-01-01

    Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic diarrhea and potentially fatal renal failure in humans. Ruminants are considered to be the primary reservoir for human infection. Vaccines that reduce shedding in cattle are only partially protective, and their underlying protective mechanisms are unknown. Studies investigating the response of cattle to colonization generally focus on humoral immunity, leaving the role of cellular immunity unclear. To inform future vaccine development, we studied the cellular immune responses of cattle during EHEC O157:H7 colonization. Calves were challenged either with a phage type 21/28 (PT21/28) strain possessing the Shiga toxin 2a (Stx2a) and Stx2c genes or with a PT32 strain possessing the Stx2c gene only. T-helper cell-associated transcripts at the terminal rectum were analyzed by reverse transcription-quantitative PCR (RT-qPCR). Induction of gamma interferon (IFN-γ) and T-bet was observed with peak expression of both genes at 7 days in PT32-challenged calves, while upregulation was delayed, peaking at 21 days, in PT21/28-challenged calves. Cells isolated from gastrointestinal lymph nodes demonstrated antigen-specific proliferation and IFN-γ release in response to type III secreted proteins (T3SPs); however, responsiveness was suppressed in cells isolated from PT32-challenged calves. Lymph node cells showed increased expression of the proliferation marker Ki67 in CD4+ T cells from PT21/28-challenged calves, NK cells from PT32-challenged calves, and CD8+ and γδ T cells from both PT21/28- and PT32-challenged calves following ex vivo restimulation with T3SPs. This study demonstrates that cattle mount cellular immune responses during colonization with EHEC O157:H7, the temporality of which is strain dependent, with further evidence of strain-specific immunomodulation. PMID:25267838

  8. Comparative pathogenicity of Escherichia coli O157 and intimin-negative non-O157 Shiga toxin-producing E coli strains in neonatal pigs.

    PubMed

    Dean-Nystrom, Evelyn A; Melton-Celsa, Angela R; Pohlenz, Joachim F L; Moon, Harley W; O'Brien, Alison D

    2003-11-01

    We compared the pathogenicity of intimin-negative non-O157:H7 Shiga toxin (Stx)-producing Escherichia coli (STEC) O91:H21 and O104:H21 strains with the pathogenicity of intimin-positive O157:H7 and O157:H(-) strains in neonatal pigs. We also examined the role of Stx2d-activatable genes and the large hemolysin-encoding plasmid of O91:H21 strain B2F1 in the pathogenesis of STEC disease in pigs. We found that all E. coli strains that made wild-type levels of Stx caused systemic illness and histological lesions in the brain and intestinal crypts, whereas none of the control Stx-negative E. coli strains evoked comparable central nervous system signs or intestinal lesions. By contrast, the absence of intimin, hemolysin, or motility had little impact on the overall pathogenesis of systemic disease during STEC infection. The most striking differences between pigs inoculated with non-O157 STEC strains and pigs inoculated with O157 STEC strains were the absence of attaching and effacing intestinal lesions in pigs inoculated with non-O157:H7 strains and the apparent association between the level of Stx2d-activatable toxin produced by an STEC strain and the severity of lesions. PMID:14573674

  9. First isolation of Escherichia coli O157:H7 from faecal and milk specimens from anatolian water buffaloes (Bubalus bubalus) in Turkey.

    PubMed

    Seker, E; Yardimci, H

    2008-12-01

    Three hundred rectal faecal samples and 213 raw milk samples obtained from the tanks and containers were examined using standard cultural methods. Escherichia coli O157:H7 was isolated from 11 (3.7%) of 300 faecal samples and 3 (1.4%) of 213 raw milk samples. It was determined that 8 (73%) of E. coli O157:H7 strains isolated from faecal samples originated from water buffaloes younger than 2 years of age and 3 (27%) from 2-year-old and older water buffaloes. This is the 1st isolation of Escherichia coli O157:H7 from faecal and milk samples of water buffaloes in Turkey.

  10. Comparative Genomic Analysis of Escherichia coli O157:H7 Isolated from Super-Shedder and Low-Shedder Cattle.

    PubMed

    Munns, Krysty D; Zaheer, Rahat; Xu, Yong; Stanford, Kim; Laing, Chad R; Gannon, Victor P J; Selinger, L Brent; McAllister, Tim A

    2016-01-01

    Cattle are the primary reservoir of the foodborne pathogen Escherichia coli O157:H7, with the concentration and frequency of E. coli O157:H7 shedding varying substantially among individual hosts. The term ''super-shedder" has been applied to cattle that shed ≥10(4) cfu E. coli O157:H7/g of feces. Super-shedders have been reported to be responsible for the majority of E. coli O157:H7 shed into the environment. The objective of this study was to determine if there are phenotypic and/or genotypic differences between E. coli O157:H7 isolates obtained from super-shedder compared to low-shedder cattle. From a total of 784 isolates, four were selected from low-shedder steers and six isolates from super-shedder steers (4.01-8.45 log cfu/g feces) for whole genome sequencing. Isolates were phage and clade typed, screened for substrate utilization, pH sensitivity, virulence gene profiles and Stx bacteriophage insertion (SBI) sites. A range of 89-2473 total single nucleotide polymorphisms (SNPs) were identified when sequenced strains were compared to E. coli O157:H7 strain Sakai. More non-synonymous SNP mutations were observed in low-shedder isolates. Pan-genomic and SNPs comparisons did not identify genetic segregation between super-shedder or low-shedder isolates. All super-shedder isolates and 3 of 4 of low-shedder isolates were typed as phage type 14a, SBI cluster 3 and SNP clade 2. Super-shedder isolates displayed increased utilization of galactitol, thymidine and 3-O-β-D-galactopyranosyl-D-arabinose when compared to low-shedder isolates, but no differences in SNPs were observed in genes encoding for proteins involved in the metabolism of these substrates. While genetic traits specific to super-shedder isolates were not identified in this study, differences in the level of gene expression or genes of unknown function may still contribute to some strains of E. coli O157:H7 reaching high densities within bovine feces.

  11. Comparative Genomic Analysis of Escherichia coli O157:H7 Isolated from Super-Shedder and Low-Shedder Cattle

    PubMed Central

    Munns, Krysty D.; Zaheer, Rahat; Xu, Yong; Stanford, Kim; Laing, Chad R.; Gannon, Victor P. J.; Selinger, L. Brent; McAllister, Tim A.

    2016-01-01

    Cattle are the primary reservoir of the foodborne pathogen Escherichia coli O157:H7, with the concentration and frequency of E. coli O157:H7 shedding varying substantially among individual hosts. The term ‘‘super-shedder” has been applied to cattle that shed ≥104 cfu E. coli O157:H7/g of feces. Super-shedders have been reported to be responsible for the majority of E. coli O157:H7 shed into the environment. The objective of this study was to determine if there are phenotypic and/or genotypic differences between E. coli O157:H7 isolates obtained from super-shedder compared to low-shedder cattle. From a total of 784 isolates, four were selected from low-shedder steers and six isolates from super-shedder steers (4.01–8.45 log cfu/g feces) for whole genome sequencing. Isolates were phage and clade typed, screened for substrate utilization, pH sensitivity, virulence gene profiles and Stx bacteriophage insertion (SBI) sites. A range of 89–2473 total single nucleotide polymorphisms (SNPs) were identified when sequenced strains were compared to E. coli O157:H7 strain Sakai. More non-synonymous SNP mutations were observed in low-shedder isolates. Pan-genomic and SNPs comparisons did not identify genetic segregation between super-shedder or low-shedder isolates. All super-shedder isolates and 3 of 4 of low-shedder isolates were typed as phage type 14a, SBI cluster 3 and SNP clade 2. Super-shedder isolates displayed increased utilization of galactitol, thymidine and 3-O-β-D-galactopyranosyl-D-arabinose when compared to low-shedder isolates, but no differences in SNPs were observed in genes encoding for proteins involved in the metabolism of these substrates. While genetic traits specific to super-shedder isolates were not identified in this study, differences in the level of gene expression or genes of unknown function may still contribute to some strains of E. coli O157:H7 reaching high densities within bovine feces. PMID:27018858

  12. Comparative Genomic Analysis of Escherichia coli O157:H7 Isolated from Super-Shedder and Low-Shedder Cattle.

    PubMed

    Munns, Krysty D; Zaheer, Rahat; Xu, Yong; Stanford, Kim; Laing, Chad R; Gannon, Victor P J; Selinger, L Brent; McAllister, Tim A

    2016-01-01

    Cattle are the primary reservoir of the foodborne pathogen Escherichia coli O157:H7, with the concentration and frequency of E. coli O157:H7 shedding varying substantially among individual hosts. The term ''super-shedder" has been applied to cattle that shed ≥10(4) cfu E. coli O157:H7/g of feces. Super-shedders have been reported to be responsible for the majority of E. coli O157:H7 shed into the environment. The objective of this study was to determine if there are phenotypic and/or genotypic differences between E. coli O157:H7 isolates obtained from super-shedder compared to low-shedder cattle. From a total of 784 isolates, four were selected from low-shedder steers and six isolates from super-shedder steers (4.01-8.45 log cfu/g feces) for whole genome sequencing. Isolates were phage and clade typed, screened for substrate utilization, pH sensitivity, virulence gene profiles and Stx bacteriophage insertion (SBI) sites. A range of 89-2473 total single nucleotide polymorphisms (SNPs) were identified when sequenced strains were compared to E. coli O157:H7 strain Sakai. More non-synonymous SNP mutations were observed in low-shedder isolates. Pan-genomic and SNPs comparisons did not identify genetic segregation between super-shedder or low-shedder isolates. All super-shedder isolates and 3 of 4 of low-shedder isolates were typed as phage type 14a, SBI cluster 3 and SNP clade 2. Super-shedder isolates displayed increased utilization of galactitol, thymidine and 3-O-β-D-galactopyranosyl-D-arabinose when compared to low-shedder isolates, but no differences in SNPs were observed in genes encoding for proteins involved in the metabolism of these substrates. While genetic traits specific to super-shedder isolates were not identified in this study, differences in the level of gene expression or genes of unknown function may still contribute to some strains of E. coli O157:H7 reaching high densities within bovine feces. PMID:27018858

  13. Restriction-Site-Specific PCR as a Rapid Test To Detect Enterohemorrhagic Escherichia coli O157:H7 Strains in Environmental Samples

    PubMed Central

    Kimura, Richard; Mandrell, Robert E.; Galland, John C.; Hyatt, Doreene; Riley, Lee W.

    2000-01-01

    Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important food-borne pathogen in industrialized countries. We developed a rapid and simple test for detecting E. coli O157:H7 using a method based on restriction site polymorphisms. Restriction-site-specific PCR (RSS-PCR) involves the amplification of DNA fragments using primers based on specific restriction enzyme recognition sequences, without the use of endonucleases, to generate a set of amplicons that yield “fingerprint” patterns when resolved electrophoretically on an agarose gel. The method was evaluated in a blinded study of E. coli isolates obtained from environmental samples collected at beef cattle feedyards. The 54 isolates were all initially identified by a commonly used polyclonal antibody test as belonging to O157:H7 serotype. They were retested by anti-O157 and anti-H7 monoclonal antibody enzyme-linked immunosorbent assay (ELISA). The RSS-PCR method identified all 28 isolates that were shown to be E. coli O157:H7 by the monoclonal antibody ELISA as belonging to the O157:H7 serotype. Of the remaining 26 ELISA-confirmed non-O157:H7 strains, the method classified 25 strains as non-O157:H7. The specificity of the RSS-PCR results correlated better with the monoclonal antibody ELISA than with the polyclonal antibody latex agglutination tests. The RSS-PCR method may be a useful test to distinguish E. coli O157:H7 from a large number of E. coli isolates from environmental samples. PMID:10831431

  14. Molecular and antimicrobial susceptibility analyses distinguish clinical from bovine Escherichia coli O157 strains.

    PubMed

    Vidovic, Sinisa; Tsoi, Sarah; Medihala, Prabhakara; Liu, Juxin; Wylie, John L; Levett, Paul N; Korber, Darren R

    2013-07-01

    A population-based study combining (i) antimicrobial, (ii) genetic, and (iii) virulence analyses with molecular evolutionary analyses revealed segregative characteristics distinguishing human clinical and bovine Escherichia coli O157 strains from western Canada. Human (n = 50) and bovine (n = 50) strains of E. coli O157 were collected from Saskatchewan and Manitoba in 2006 and were analyzed by using the six-marker lineage-specific polymorphism assay (LSPA6), antimicrobial susceptibility analysis, the colicin assay, plasmid and virulence profiling including the eae, ehxA, espA, iha, stx1, stx2, stx2c, stx2d, stx2d-activatable, stx2e, and stx2f virulence-associated genes, and structure analyses. Multivariate logistic regression and Fisher's exact test strongly suggested that antimicrobial susceptibility was the most distinctive characteristic (P = 0.00487) associated with human strains. Among all genetic, virulence, and antimicrobial determinants, resistance to tetracycline (P < 0.000) and to sulfisoxazole (P < 0.009) were the most strongly associated segregative characteristics of bovine E. coli O157 strains. Among 11 virulence-associated genes, stx2c showed the strongest association with E. coli O157 strains of bovine origin. LSPA6 genotyping showed the dominance of the lineage I genotype among clinical (90%) and bovine (70%) strains, indicating the importance of lineage I in O157 epidemiology and ecology. Population structure analysis revealed that the more-diverse bovine strains came from a unique group of strains characterized by a high degree of antimicrobial resistance and high frequencies of lineage II genotypes and stx2c variants. These findings imply that antimicrobial resistance generated among bovine strains of E. coli O157 has a large impact on the population of this human pathogen.

  15. PCR for the Specific Detection of an Escherichia coli O157:H7 Laboratory Control Strain.

    PubMed

    Knowles, Michael; Lambert, Dominic; Huszczynski, George; Gauthier, Martine; Blais, Burton W

    2015-09-01

    Control strains of bacterial pathogens such as Escherichia coli O157:H7 are commonly processed in parallel with test samples in food microbiology laboratories as a quality control measure to assure the satisfactory performance of materials used in the analytical procedure. Before positive findings can be reported for risk management purposes, analysts must have a means of verifying that pathogenic bacteria (e.g., E. coli O157:H7) recovered from test samples are not due to inadvertent contamination with the control strain routinely handled in the laboratory environment. Here, we report on the application of an in-house bioinformatic pipeline for the identification of unique genomic signature sequences in the development of specific oligonucleotide primers enabling the identification of a common positive control strain, E. coli O157:H7 (ATCC 35150), using a simple PCR procedure.

  16. Incidence and toxin production ability of Escherichia coli O157:H7 isolated from cattle trucks.

    PubMed

    Cuesta Alonso, E P; Gilliland, S E; Krehbiel, C R

    2007-10-01

    Twelve cattle trucks were analyzed for the presence of Escherichia coli O157:H7. Three of them had been washed prior to arrival, and the others had not. Seventy-five percent of the trailers were positive for the presence of this foodborne pathogen. A total of 54 cultures were isolated and identified as E. coli O157:H7, all from the trucks that had not been cleaned. Most of the cultures (96.4%) produced Shiga-like toxin (verotoxin). No E. coli O157:H7 was detected in cattle trucks that were cleaned before arrival at the cattle pens. The incidence of E. coli O157:H7 in transport trailers increases the potential risk of contamination of cattle and transmission from farms to feedlots and to packing plants. This contamination increases the potential of contamination of meat during harvest and the risk of foodborne illnesses.

  17. Antimicrobial Resistance Profiles in Escherichia coli O157 Isolates from Northern Colorado Dairies.

    PubMed

    McConnel, Craig S; Stenkamp-Strahm, Chloe M; Rao, Sangeeta; Linke, Lyndsey M; Magnuson, Roberta J; Hyatt, Doreene R

    2016-03-01

    Escherichia coli O157 (EcO157) infections can lead to serious disease and death in humans. Although the ecology of EcO157 is complex, ruminant animals serve as an important reservoir for human infection. Dairy cattle are unique because they may be a source of contamination for milk, meat, and manure-fertilized crops. Foodborne dairy pathogens such as EcO157 are of primary importance to public health. Antimicrobial resistance (AMR) is a complex phenomenon that complicates the treatment of serious bacterial infections and is of increasing concern. In the face of recommended use restrictions for antimicrobial agents in livestock operations, current AMR patterns in known foodborne pathogens should be documented. The objective of this study was to document AMR patterns in EcO157 isolates from dairies in northern Colorado using antimicrobial agents commonly found on dairies and representative of medically important antimicrobial drug classes. Seventy-five EcO157 isolates were recovered from three dairies. Six isolates were resistant to at least 1 of the 10 tested antimicrobial agents: four were resistant to streptomycin, sulfisoxazole, and tetracycline; one was resistant to streptomycin and tetracycline; and one was resistant to only tetracycline. All resistant isolates were from a single dairy. Overall, a low prevalence (8%) of AMR was observed among the 75 EcO157 isolates. No significant effects on AMR profiles due to virulence genes, parity, or previous antimicrobial treatments within the current lactation period were detected. The results of this study provide background information for future comparative studies investigating AMR trends. Future studies should include more participating farms and more samples and should control for potential confounding factors of AMR that may underlie individual farm variation. PMID:26939660

  18. Comparison of Six Chromogenic Agar Media for the Isolation of a Broad Variety of Non-O157 Shigatoxin-Producing Escherichia coli (STEC) Serogroups.

    PubMed

    Verhaegen, Bavo; De Reu, Koen; Heyndrickx, Marc; De Zutter, Lieven

    2015-06-17

    The isolation of non-O157 STEC from food samples has proved to be challenging. The selection of a suitable selective isolation agar remains problematic. The purpose of this study was to qualitatively and quantitatively evaluate six chromogenic agar media for the isolation of STEC: Tryptone Bile X-glucuronide agar (TBX), Rainbow® Agar O157 (RB), Rapid E. coli O157:H7 (RE), Modified MacConkey Agar (mMac), CHROMagarTM STEC (Chr ST) and chromIDTM EHEC (Chr ID). During this study, 45 E. coli strains were used, including 39 STEC strains belonging to 16 different O serogroups and 6 non-STEC E. coli. All E. coli strains were able to grow on TBX and RB, whereas one STEC strain was unable to grow on Chr ID and a number of other STEC strains did not grow on mMac, CHROMagar STEC and Rapid E. coli O157:H7. However, only the latter three agars were selective enough to completely inhibit the growth of the non-STEC E. coli. Our conclusion was that paired use of a more selective agar such as CHROMagar STEC together with a less selective agar like TBX or Chr ID might be the best solution for isolating non-O157 STEC from food.

  19. Prevalence of Stx phages in environments of a pig farm and lysogenic infection of the field E. coli O157 isolates with a recombinant converting Phage.

    PubMed

    Yan, Yaxian; Shi, Yibo; Cao, Dongmei; Meng, Xiangpeng; Xia, Luming; Sun, Jianhe

    2011-02-01

    The prevalence and nature of Shiga toxin (Stx)-producing Escherichia coli (STEC) and Stx phage were investigated in 720 swine fecal samples randomly collected from a commercial breeding pig farm in China over a 1-year surveillance period. Eight STEC O157 (1.1%), 33 STEC non-O157 (4.6%), and two stx-negative O157 (0.3%) isolates were identified. Fecal filtrates were screened directly for Stx phages using E. coli K-12 derivative strains MC1061 as indicator, yielding 15 Stx1 and 57 Stx2 phages. One Stx1 and eight Stx2 phages were obtained following norfloxacin induction of the eight field STEC O157 isolates. All Stx1 phages had hexagonal heads with long tails, while Stx2 phages had three different morphologies. Notably, most of field STEC O157 isolates released more free phages and Stx toxin after induction with ciprofloxacin. Furthermore, upon infection with the recombinant phage ΦMin27(Δstx::cat), E. coli laboratory strains produced both lysogenic and lytic phage, whereas two of the eight O157 STEC isolates produced only lysogens. The lysogens from laboratory strains produced infectious particles similar to ΦMin27. Similarly, the lysogens from the STEC O157 isolates released Stx phage too, although free ΦMin27(Δstx::cat) particles were not detected. Collectively, our results reveal that breeding pig farms could be important reservoirs for Stx phages and that residual antibacterial agents may enhance the release of Stx phages and the expression of Stx. PMID:20697714

  20. Prevalence of Stx phages in environments of a pig farm and lysogenic infection of the field E. coli O157 isolates with a recombinant converting Phage.

    PubMed

    Yan, Yaxian; Shi, Yibo; Cao, Dongmei; Meng, Xiangpeng; Xia, Luming; Sun, Jianhe

    2011-02-01

    The prevalence and nature of Shiga toxin (Stx)-producing Escherichia coli (STEC) and Stx phage were investigated in 720 swine fecal samples randomly collected from a commercial breeding pig farm in China over a 1-year surveillance period. Eight STEC O157 (1.1%), 33 STEC non-O157 (4.6%), and two stx-negative O157 (0.3%) isolates were identified. Fecal filtrates were screened directly for Stx phages using E. coli K-12 derivative strains MC1061 as indicator, yielding 15 Stx1 and 57 Stx2 phages. One Stx1 and eight Stx2 phages were obtained following norfloxacin induction of the eight field STEC O157 isolates. All Stx1 phages had hexagonal heads with long tails, while Stx2 phages had three different morphologies. Notably, most of field STEC O157 isolates released more free phages and Stx toxin after induction with ciprofloxacin. Furthermore, upon infection with the recombinant phage ΦMin27(Δstx::cat), E. coli laboratory strains produced both lysogenic and lytic phage, whereas two of the eight O157 STEC isolates produced only lysogens. The lysogens from laboratory strains produced infectious particles similar to ΦMin27. Similarly, the lysogens from the STEC O157 isolates released Stx phage too, although free ΦMin27(Δstx::cat) particles were not detected. Collectively, our results reveal that breeding pig farms could be important reservoirs for Stx phages and that residual antibacterial agents may enhance the release of Stx phages and the expression of Stx.

  1. Selective Enrichment with a Resuscitation Step for Isolation of Freeze-Injured Escherichia coli O157:H7 from Foods

    PubMed Central

    Hara-Kudo, Y.; Ikedo, M.; Kodaka, H.; Nakagawa, H.; Goto, K.; Masuda, T.; Konuma, H.; Kojima, T.; Kumagai, S.

    2000-01-01

    We studied injury of Escherichia coli O157:H7 cells in 11 food items during freeze storage and methods of isolating freeze-injured E. coli O157:H7 cells from foods. Food samples inoculated with E. coli O157:H7 were stored for 16 weeks at −20°C in a freezer. Noninjured and injured cells were counted by using tryptic soy agar and sorbitol MacConkey agar supplemented with cefixime and potassium tellurite. Large populations of E. coli O157:H7 cells were injured in salted cabbage, grated radish, seaweed, and tomato samples. In an experiment to detect E. coli O157:H7 in food samples artificially contaminated with freeze-injured E. coli O157:H7 cells, the organism was recovered most efficiently after the samples were incubated in modified E. coli broth without bile salts at 25°C for 2 h and then selectively enriched at 42°C for 18 h by adding bile salts and novobiocin. Our enrichment method was further evaluated by isolating E. coli O157:H7 from frozen foods inoculated with the organism prior to freezing. Two hours of resuscitation at 25°C in nonselective broth improved recovery of E. coli O157:H7 from frozen grated radishes and strawberries, demonstrating that the resuscitation step is very effective for isolating E. coli O157:H7 from frozen foods contaminated with injured E. coli O157:H7 cells. PMID:10877780

  2. stx genotype and molecular epidemiological analyses of Shiga toxin-producing Escherichia coli O157:H7/H- in human and cattle isolates.

    PubMed

    Kawano, K; Ono, H; Iwashita, O; Kurogi, M; Haga, T; Maeda, K; Goto, Y

    2012-02-01

    The relationship between human diseases caused by infection with Shiga toxin (Stx)-producing Escherichia coli (STEC) O157 strains and O157 strains isolated from cattle was investigated in an area where stockbreeding is prolific. For this purpose, the stx genotypes, the molecular epidemiological characteristics of 268 STEC O157 strains including 211 human-origin strains and 57 cattle-origin strains, and clinical manifestations of 210 STEC-infected people were analyzed. Of 211 human-origin strains, 92 strains (44%) were of the stx1/stx2 genotype, and 74 strains (35%) were of the stx2c genotype. Most of the people infected with stx2c genotype strains presented no symptoms or mild symptoms such as slight diarrhea, except for 3 patients with bloody diarrhea. Of the 57 cattle-origin strains, 27 strains (47%) were of the stx2c genotype and 17 strains (30%) were of the stx1/stx2 genotype. Pulsed-field gel electrophoresis (PFGE) and insertion sequence (IS) analysis demonstrated that 11 isolates (41%) of the 27 cattle isolates of the stx2c genotype had high homology (>95% identity) with human isolates. These results suggest that some genetic patterns of the stx2c genotype strains might be preserved in cattle or their surrounding environment for several years, and during these periods, they might have opportunities to infect people through various routes. Because of the mild virulence of the stx2c genotype strains, they seemed to be transmitted asymptomatically from cattle to humans and then spread from person to person. It may be a public health concern. Further, they occasionally cause severe symptoms in humans; therefore, caution is warranted for infections by stx2c genotype O157 strains, in addition to stx2-possessing genotype O157 strains.

  3. Dual-species biofilm formation by Escherichia coli O157:H7 and environmental bacteria isolated from fresh-cut processing facilities.

    PubMed

    Liu, Nancy T; Nou, Xiangwu; Lefcourt, Alan M; Shelton, Daniel R; Lo, Y Martin

    2014-02-01

    Biofilm formation is a mechanism adapted by many microorganisms that enhances the survival in stressful environments. In food processing facilities, foodborne bacterial pathogens, which many are poor biofilm formers, could potentially take advantage of this protective mechanism by interacting with other strong biofilm producers. The objective of this study was to determine the influence of bacteria native to fresh produce processing environments on the incorporation of Escherichia coli O157:H7 in biofilms. Bacteria strains representing 13 Gram-negative species isolated from two fresh produce processing facilities in a previous study were tested for forming dual-species biofilms with E. coli O157:H7. Strong biofilm producing strains of Burkholderia caryophylli and Ralstonia insidiosa exhibited 180% and 63% increase in biofilm biomass, and significant thickening of the biofilms (B. caryophylli not tested), when co-cultured with E. coli O157:H7. E. coli O157:H7 populations increased by approximately 1 log in dual-species biofilms formed with B. caryophylli or R. insidiosa. While only a subset of environmental isolates with strong biofilm formation abilities increased the presence of E. coli O157:H7 in biofilms, all tested E. coli O157:H7 exhibited higher incorporation in dual-species biofilms with R. insidiosa. These observations support the notion that E. coli O157:H7 and specific strong biofilm producing bacteria interact synergistically in biofilm formation, and suggest a route for increased survival potential of E. coli O157:H7 in fresh produce processing environments.

  4. Relationship between stx genotype and Stx2 expression level in Shiga toxin-producing Escherichia coli O157 strains.

    PubMed

    Kawano, Kimiko; Ono, Hidetoshi; Iwashita, Osamu; Kurogi, Mai; Haga, Takeshi; Maeda, Ken; Goto, Yoshitaka

    2012-07-01

    To determine the expression level of Shiga toxin (Stx) 2-related toxins (Stx2 and Stx2c) produced by each of 33 Stx-producing Escherichia coli (STEC) O157 strains, stx2 and stx2c mRNAs (stx2-related mRNA) were measured using real-time PCR with primers that recognize sequences common to stx2 and stx2c. The amount of Stx2 and Stx2c protein was measured using a reversed passive latex agglutination (RPLA) kit. Expression of stx2-related mRNA was significantly higher in STEC O157 strains carrying the stx2 gene (i.e., stx2, stx1/stx2, or stx2/stx2c) than in most strains that carried the stx2c gene but not the stx2 gene (i.e., stx2c or stx1/stx2c). RPLA might not measure the precise amount of each toxin variant; nevertheless, stx2-inclusive strains had 40-fold higher mean toxin titers than did strains that carried the stx2c gene but not the stx2 gene, with the exception of 1 stx2c strain. Interestingly, 1 stx2c strain that was isolated from a patient with severe hemorrhagic diarrhea had the highest stx2-related mRNA expression and the highest toxin titer of all 33 STEC O157 strains. Taken together, these findings indicated that measurement of stx2-related mRNA expression could reflect differences in production levels of toxins among STEC strains.

  5. Strain differences in fitness of Escherichia coli O157:H7 to resist protozoan predation and survival in soil.

    PubMed

    Ravva, Subbarao V; Sarreal, Chester Z; Mandrell, Robert E

    2014-01-01

    Escherichia coli O157:H7 (EcO157) associated with the 2006 spinach outbreak appears to have persisted as the organism was isolated, three months after the outbreak, from environmental samples in the produce production areas of the central coast of California. Survival in harsh environments may be linked to the inherent fitness characteristics of EcO157. This study evaluated the comparative fitness of outbreak-related clinical and environmental strains to resist protozoan predation and survive in soil from a spinach field in the general vicinity of isolation of strains genetically indistinguishable from the 2006 outbreak strains. Environmental strains from soil and feral pig feces survived longer (11 to 35 days for 90% decreases, D-value) with Vorticella microstoma and Colpoda aspera, isolated previously from dairy wastewater; these D-values correlated (P<0.05) negatively with protozoan growth. Similarly, strains from cow feces, feral pig feces, and bagged spinach survived significantly longer in soil compared to clinical isolates indistinguishable by 11-loci multi-locus variable-number tandem-repeat analysis. The curli-positive (C+) phenotype, a fitness trait linked with attachment in ruminant and human gut, decreased after exposure to protozoa, and in soils only C- cells remained after 7 days. The C+ phenotype correlated negatively with D-values of EcO157 exposed to soil (rs = -0.683; P = 0.036), Vorticella (rs = -0.465; P = 0.05) or Colpoda (rs = -0.750; P = 0.0001). In contrast, protozoan growth correlated positively with C+ phenotype (Vorticella, rs = 0.730, P = 0.0004; Colpoda, rs = 0.625, P = 0.006) suggesting a preference for consumption of C+ cells, although they grew on C- strains also. We speculate that the C- phenotype is a selective trait for survival and possibly transport of the pathogen in soil and water environments. PMID:25019377

  6. Strain differences in fitness of Escherichia coli O157:H7 to resist protozoan predation and survival in soil.

    PubMed

    Ravva, Subbarao V; Sarreal, Chester Z; Mandrell, Robert E

    2014-01-01

    Escherichia coli O157:H7 (EcO157) associated with the 2006 spinach outbreak appears to have persisted as the organism was isolated, three months after the outbreak, from environmental samples in the produce production areas of the central coast of California. Survival in harsh environments may be linked to the inherent fitness characteristics of EcO157. This study evaluated the comparative fitness of outbreak-related clinical and environmental strains to resist protozoan predation and survive in soil from a spinach field in the general vicinity of isolation of strains genetically indistinguishable from the 2006 outbreak strains. Environmental strains from soil and feral pig feces survived longer (11 to 35 days for 90% decreases, D-value) with Vorticella microstoma and Colpoda aspera, isolated previously from dairy wastewater; these D-values correlated (P<0.05) negatively with protozoan growth. Similarly, strains from cow feces, feral pig feces, and bagged spinach survived significantly longer in soil compared to clinical isolates indistinguishable by 11-loci multi-locus variable-number tandem-repeat analysis. The curli-positive (C+) phenotype, a fitness trait linked with attachment in ruminant and human gut, decreased after exposure to protozoa, and in soils only C- cells remained after 7 days. The C+ phenotype correlated negatively with D-values of EcO157 exposed to soil (rs = -0.683; P = 0.036), Vorticella (rs = -0.465; P = 0.05) or Colpoda (rs = -0.750; P = 0.0001). In contrast, protozoan growth correlated positively with C+ phenotype (Vorticella, rs = 0.730, P = 0.0004; Colpoda, rs = 0.625, P = 0.006) suggesting a preference for consumption of C+ cells, although they grew on C- strains also. We speculate that the C- phenotype is a selective trait for survival and possibly transport of the pathogen in soil and water environments.

  7. Clade 8 and Clade 6 Strains of Escherichia coli O157:H7 from Cattle in Argentina have Hypervirulent-Like Phenotypes.

    PubMed

    Amigo, Natalia; Mercado, Elsa; Bentancor, Adriana; Singh, Pallavi; Vilte, Daniel; Gerhardt, Elisabeth; Zotta, Elsa; Ibarra, Cristina; Manning, Shannon D; Larzábal, Mariano; Cataldi, Angel

    2015-01-01

    The hemolytic uremic syndrome (HUS) whose main causative agent is enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a disease that mainly affects children under 5 years of age. Argentina is the country with the highest incidence of HUS in the world. Cattle are a major reservoir and source of infection with E. coli O157:H7. To date, the epidemiological factors that contribute to its prevalence are poorly understood. Single nucleotide polymorphism (SNP) typing has helped to define nine E. coli O157:H7 clades and the clade 8 strains were associated with most of the cases of severe disease. In this study, eight randomly selected isolates of EHEC O157:H7 from cattle in Argentina were studied as well as two human isolates. Four of them were classified as clade 8 through the screening for 23 SNPs; the two human isolates grouped in this clade as well, while two strains were closely related to strains representing clade 6. To assess the pathogenicity of these strains, we assayed correlates of virulence. Shiga toxin production was determined by an ELISA kit. Four strains were high producers and one of these strains that belonged to a novel genotype showed high verocytotoxic activity in cultured cells. Also, these clade 8 and 6 strains showed high RBC lysis and adherence to epithelial cells. One of the clade 6 strains showed stronger inhibition of normal water absorption than E. coli O157:H7 EDL933 in human colonic explants. In addition, two of the strains showing high levels of Stx2 production and RBC lysis activity were associated with lethality and uremia in a mouse model. Consequently, circulation of such strains in cattle may partially contribute to the high incidence of HUS in Argentina.

  8. Clade 8 and Clade 6 Strains of Escherichia coli O157:H7 from Cattle in Argentina have Hypervirulent-Like Phenotypes

    PubMed Central

    Amigo, Natalia; Mercado, Elsa; Bentancor, Adriana; Singh, Pallavi; Vilte, Daniel; Gerhardt, Elisabeth; Zotta, Elsa; Ibarra, Cristina; Manning, Shannon D.; Larzábal, Mariano; Cataldi, Angel

    2015-01-01

    The hemolytic uremic syndrome (HUS) whose main causative agent is enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a disease that mainly affects children under 5 years of age. Argentina is the country with the highest incidence of HUS in the world. Cattle are a major reservoir and source of infection with E. coli O157:H7. To date, the epidemiological factors that contribute to its prevalence are poorly understood. Single nucleotide polymorphism (SNP) typing has helped to define nine E. coli O157:H7 clades and the clade 8 strains were associated with most of the cases of severe disease. In this study, eight randomly selected isolates of EHEC O157:H7 from cattle in Argentina were studied as well as two human isolates. Four of them were classified as clade 8 through the screening for 23 SNPs; the two human isolates grouped in this clade as well, while two strains were closely related to strains representing clade 6. To assess the pathogenicity of these strains, we assayed correlates of virulence. Shiga toxin production was determined by an ELISA kit. Four strains were high producers and one of these strains that belonged to a novel genotype showed high verocytotoxic activity in cultured cells. Also, these clade 8 and 6 strains showed high RBC lysis and adherence to epithelial cells. One of the clade 6 strains showed stronger inhibition of normal water absorption than E. coli O157:H7 EDL933 in human colonic explants. In addition, two of the strains showing high levels of Stx2 production and RBC lysis activity were associated with lethality and uremia in a mouse model. Consequently, circulation of such strains in cattle may partially contribute to the high incidence of HUS in Argentina. PMID:26030198

  9. Clade 8 and Clade 6 Strains of Escherichia coli O157:H7 from Cattle in Argentina have Hypervirulent-Like Phenotypes.

    PubMed

    Amigo, Natalia; Mercado, Elsa; Bentancor, Adriana; Singh, Pallavi; Vilte, Daniel; Gerhardt, Elisabeth; Zotta, Elsa; Ibarra, Cristina; Manning, Shannon D; Larzábal, Mariano; Cataldi, Angel

    2015-01-01

    The hemolytic uremic syndrome (HUS) whose main causative agent is enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a disease that mainly affects children under 5 years of age. Argentina is the country with the highest incidence of HUS in the world. Cattle are a major reservoir and source of infection with E. coli O157:H7. To date, the epidemiological factors that contribute to its prevalence are poorly understood. Single nucleotide polymorphism (SNP) typing has helped to define nine E. coli O157:H7 clades and the clade 8 strains were associated with most of the cases of severe disease. In this study, eight randomly selected isolates of EHEC O157:H7 from cattle in Argentina were studied as well as two human isolates. Four of them were classified as clade 8 through the screening for 23 SNPs; the two human isolates grouped in this clade as well, while two strains were closely related to strains representing clade 6. To assess the pathogenicity of these strains, we assayed correlates of virulence. Shiga toxin production was determined by an ELISA kit. Four strains were high producers and one of these strains that belonged to a novel genotype showed high verocytotoxic activity in cultured cells. Also, these clade 8 and 6 strains showed high RBC lysis and adherence to epithelial cells. One of the clade 6 strains showed stronger inhibition of normal water absorption than E. coli O157:H7 EDL933 in human colonic explants. In addition, two of the strains showing high levels of Stx2 production and RBC lysis activity were associated with lethality and uremia in a mouse model. Consequently, circulation of such strains in cattle may partially contribute to the high incidence of HUS in Argentina. PMID:26030198

  10. Draft Genome Sequences of Escherichia coli O157:H7 Strains Rafaela_II (Clade 8) and 7.1_Anguil (Clade 6) from Cattle in Argentina.

    PubMed

    Amadio, Ariel Fernando; Amigo, Natalia; Puebla, Andrea Fabiana; Farber, Marisa Diana; Cataldi, Angel Adrián

    2015-06-11

    Escherichia coli O157:H7 is a major etiologic agent of diseases in humans that cause diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome. Here, we report the draft genome sequences of two strains isolated from cattle that had high levels of Shiga toxin 2 and high lethality in mice.

  11. Draft Genome Sequences of Escherichia coli O157:H7 Strains Rafaela_II (Clade 8) and 7.1_Anguil (Clade 6) from Cattle in Argentina

    PubMed Central

    Amigo, Natalia; Puebla, Andrea Fabiana; Farber, Marisa Diana

    2015-01-01

    Escherichia coli O157:H7 is a major etiologic agent of diseases in humans that cause diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome. Here, we report the draft genome sequences of two strains isolated from cattle that had high levels of Shiga toxin 2 and high lethality in mice. PMID:26067964

  12. Draft Genome Sequences of Three European Laboratory Derivatives from Enterohemorrhagic Escherichia coli O157:H7 Strain EDL933, Including Two Plasmids

    PubMed Central

    Fellner, Lea; Huptas, Christopher; Simon, Svenja; Mühlig, Anna; Neuhaus, Klaus

    2016-01-01

    Escherichia coli O157:H7 EDL933, isolated in 1982 in the United States, was the first enterohemorrhagic E. coli (EHEC) strain sequenced. Unfortunately, European labs can no longer receive the original strain. We checked three European EDL933 derivatives and found major genetic deviations (deletions, inversions) in two strains. All EDL933 strains contain the cryptic EHEC-plasmid, not reported before. PMID:27056239

  13. [In vitro antibacterial activity of faropenem, a novel oral penem antibiotic, against enterohemorrhagic Escherichia coli O157 strains].

    PubMed

    Nasu, T; Okamoto, K; Nakanishi, T; Nishino, T

    1999-08-01

    Against enterohemorrhagic Escherichia coli (EHEC) O157 clinically isolated, the effects of faropenem (FRPM), a novel oral penem antibiotic, on the MICs, bactericidal activity, verotoxin (VT)-release, and lipopolysaccharide (LPS)-release were investigated in vitro and compared with those of other types of antibacterial agents. The MICs of FRPM in aerobic and anaerobic culture condition, were 0.78 and 0.39 microgram/ml, respectively. In aerobic condition, FRPM was more active than ampicillin, amoxicillin (AMPC), fosfomycin (FOM), kanamycin (KM), minocycline (MINO), and clarithromycin (CAM), but was slightly less active than cefdinir (CFDN), cefditoren (CDTR), and norfloxacin (NFLX) against O157 clinical isolates. In anaerobic condition, however, FRPM showed as strong activity as CFDN, CDTR, and NFLX. FOM, NFLX, and KM as well as the beta-lactams including FRPM indicated the powerful bactericidal activity against one strain of O157 clinical isolates. The effects of MINO and CAM were bacteriostatic. FOM and the beta-lactams including FRPM promoted verotoxin type 1 (VT1)-release, but rather suppressed verotoxin type 2 (VT2)-release from the same isolate. NFLX, however, promoted VT1-release and vast amount of VT2-release. In the case of KM, MINO, and CAM, the release suppression of both VT1 and VT2 was observed. FRPM, AMPC, and FOM had very weak activity on LPS-release, while CFDN, CDTR, and NFLX released a large amount of LPS from the strain. KM, MINO, and CAM had relatively weak activity. In these in vitro experiments, FRPM demonstrated the effective profile to the treatment for EHEC infection, except for the effect on VT1-release. These results suggest the possibility that FRPM shows good clinical efficacy for EHEC infection. PMID:10587879

  14. Novel sequence types of non-O157 Shiga toxin-producing Escherichia coli isolated from cattle.

    PubMed

    Isiko, J; Khaitsa, M; Bergholz, T M

    2015-06-01

    The objective of this study was to assess the genetic diversity of non-O157 Shiga toxin-producing Escherichia coli (STEC) isolates from cattle. Multi-locus sequence typing (MLST) was used to identify and compare the sequence types (STs) of 43 non-O157 STEC cattle isolates using the EcMLST database curated by the STEC Center at Michigan State University. For the 43 isolates, 19 STs were identified and 10 of those STs were novel compared to those in EcMLST. For the 43 isolates, 19 different serotypes were identified. STEC O22:H8, O174:H28 and O8:H19 were most common, and STEC O8 isolates were the most diverse, with seven different STs for isolates with that O group. STEC strains with O types identified in this study have been isolated from cattle by other researchers, as well as from cases of human gastroenteritis. Of the 10 novel STs identified, six were found to be closely related to previously identified STs, indicating that populations of non-O157 STEC in cattle are similar to those from other sources, including human clinical cases. Significance and impact of the study: The foodborne pathogen Shiga toxin-producing Escherichia coli (STEC) is a significant public health concern. One of the main reservoirs for STEC are cattle, which can directly or indirectly contribute to STEC in the food supply. The genetic subtype data presented here highlight the diversity of STEC that can be isolated from cattle. These results further our understanding of the ecology of STEC in the primary production environment, which is important for developing effective control measures to reduce this pathogen in the food supply.

  15. Genome Signatures of Escherichia coli O157:H7 Isolates from the Bovine Host Reservoir▿†

    PubMed Central

    Eppinger, Mark; Mammel, Mark K.; LeClerc, Joseph E.; Ravel, Jacques; Cebula, Thomas A.

    2011-01-01

    Cattle comprise a main reservoir of Shiga toxin-producing Escherichia coli O157:H7 (STEC). The significant differences in host prevalence, transmissibility, and virulence phenotypes among strains from bovine and human sources are of major interest to the public health community and livestock industry. Genomic analysis revealed divergence into three lineages: lineage I and lineage I/II strains are commonly associated with human disease, while lineage II strains are overrepresented in the asymptomatic bovine host reservoir. Growing evidence suggests that genotypic differences between these lineages, such as polymorphisms in Shiga toxin subtypes and synergistically acting virulence factors, are correlated with phenotypic differences in virulence, host ecology, and epidemiology. To assess the genomic plasticity on a genome-wide scale, we have sequenced the whole genome of strain EC869, a bovine-associated E. coli O157:H7 isolate. Comparative phylogenomic analysis of this key isolate enabled us to place accurately bovine lineage II strains within the genetically homogenous E. coli O157:H7 clade. Identification of polymorphic loci that are anchored both in the chromosomal backbone and horizontally acquired regions allowed us to associate bovine genotypes with altered virulence phenotypes and host prevalence. This study catalogued numerous novel lineage II-specific genome signatures, some of which appear to be associated intimately with the altered pathogenic potential and niche adaptation within the bovine rumen. The presented extended list of polymorphic markers is valuable in the development of a robust typing system critical for forensic, diagnostic, and epidemiological studies of this emerging human pathogen. PMID:21421787

  16. Insertions, Deletions, and Single-Nucleotide Polymorphisms at Rare Restriction Enzyme Sites Enhance Discriminatory Power of Polymorphic Amplified Typing Sequences, a Novel Strain Typing System for Escherichia coli O157:H7

    PubMed Central

    Kudva, Indira T.; Griffin, Robert W.; Murray, Megan; John, Manohar; Perna, Nicole T.; Barrett, Timothy J.; Calderwood, Stephen B.

    2004-01-01

    Polymorphic amplified typing sequences (PATS) for Escherichia coli O157:H7 (O157) was previously based on indels containing XbaI restriction enzyme sites occurring in O-island sequences of the O157 genome. This strain-typing system, referred to as XbaI-based PATS, typed every O157 isolate tested in a reproducible, rapid, straightforward, and easy-to-interpret manner and had technical advantages over pulsed-field gel electrophoresis (PFGE). However, the system was less discriminatory than PFGE and was unable to differentiate fully between unrelated isolates. To overcome this drawback, we enhanced PATS by using another infrequently cutting restriction enzyme, AvrII (also known as BlnI), to identify additional polymorphic regions that could increase the discriminatory ability of PATS typing. Referred to as AvrII-based PATS, the system identified seven new polymorphic regions in the O157 genome. Unlike XbaI, polymorphisms involving AvrII sites were caused by both indels and single-nucleotide polymorphisms occurring in O-island and backbone sequences of the O157 genome. AvrII-based PATS by itself provided poor discrimination of the O157 isolates tested. However, when primer pairs amplifying the seven polymorphic AvrII sites were combined with those amplifying the eight polymorphic XbaI sites (combined PATS), the discriminatory power of PATS was enhanced. Combined PATS matched related O157 isolates better than PFGE while differentiating between unrelated isolates. PATS typed every O157 isolate tested and directly targeted polymorphic sequences responsible for differences in the restriction digest patterns of O157 genomic DNA, utilizing PCR rather than relying on gel electrophoresis. This enabled PATS to resolve the ambiguity in PFGE typing, including that arising from the “more distantly related” and “untypeable” profiles. PMID:15184409

  17. Thermal tolerance of O157 and non-O157 Shiga toxigenic strains of Escherichia coli, Salmonella, and potential pathogen surrogates, in frankfurter batter and ground beef of varying fat levels.

    PubMed

    Vasan, Akhila; Geier, Renae; Ingham, Steve C; Ingham, Barbara H

    2014-09-01

    The non-O157 Shiga toxigenic Escherichia coli (STEC) serogroups most commonly associated with illness are O26, O45, O103, O111, O121, and O145. We compared the thermal tolerance (D55°C) of three or more strains of each of these six non-O157 STEC serogroups with five strains of O157:H7 STEC in 7% fat ground beef. D55°C was also determined for at least one heat-tolerant STEC strain per serogroup in 15 and 27% fat ground beef. D55°C of single-pathogen cocktails of O157 and non-O157 STEC, Salmonella, and potential pathogen surrogates, Pediococcus acidilactici and Staphylococcus carnosus, was determined in 7, 15, and 27% fat ground beef and in frankfurter batter. Samples (25 g) were heated for up to 120 min at 55°C, survivors were enumerated, and log CFU per gram was plotted versus time. There were significant differences in D55°C across all STEC strains heated in 7% fat ground beef (P < 0.05), but no non-O157 STEC strain had D55°C greater than the range observed for O157 STEC. D55°C was significantly different for strains within serogroups O45, O145, and O157 (P < 0.05). D55°C for non-O157 STEC strains in 15 and 27% fat ground beef were less than or equal to the range of D55°C for O157. D55°C for pathogen cocktails was not significantly different when measured in 7, 15, and 27% fat ground beef (P ≥ 0.05). D55°C of Salmonella in frankfurter batter was significantly less than for O157 and non-O157 STEC (P < 0.05). Thermal tolerance of pathogen cocktails in ground beef (7, 15, or 27% fat) and frankfurter batter was significantly less than for potential pathogen surrogates (P < 0.05). Results suggest that thermal processes in beef validated against E. coli O157:H7 have adequate lethality against non-O157 STEC, that thermal processes that target Salmonella destruction may not be adequate against STEC in some situations, and that the use of pathogen surrogates P. acidilactici and S. carnosus to validate thermal processing interventions in ground beef and

  18. Biotic and abiotic variables affecting internalization and fate of Escherichia coli O157:H7 isolates in leafy green roots.

    PubMed

    Erickson, Marilyn C; Webb, Cathy C; Davey, Lindsey E; Payton, Alison S; Flitcroft, Ian D; Doyle, Michael P

    2014-06-01

    Preharvest internalization of Escherichia coli O157:H7 into the roots of leafy greens is a food safety risk because the pathogen may be systemically transported to edible portions of the plant. In this study, both abiotic (degree of soil moisture) and biotic (E. coli O157:H7 exposure, presence of Shiga toxin genes, and type of leafy green) factors were examined to determine their potential effects on pathogen internalization into roots of leafy greens. Using field soil that should have an active indigenous microbial community, internalized populations in lettuce roots were 0.8 to 1.6 log CFU/g after exposure to soil containing E. coli O157:H7 at 5.6 to 6.1 log CFU/g. Internalization of E. coli O157:H7 into leafy green plant roots was higher when E. coli O157:H7 populations in soil were increased to 7 or 8 log CFU/g or when the soil was saturated with water. No differences were noted in the extent to which internalization of E. coli O157:H7 occurred in spinach, lettuce, or parsley roots; however, in saturated soil, maximum levels in parsley occurred later than did those in spinach or lettuce. Translocation of E. coli O157:H7 from roots to leaves was rare; therefore, decreases observed in root populations over time were likely the result of inactivation within the plant tissue. Shiga toxin-negative (nontoxigenic) E. coli O157:H7 isolates were more stable than were virulent isolates in soil, but the degree of internalization of E. coli O157:H7 into roots did not differ between isolate type. Therefore, these nontoxigenic isolates could be used as surrogates for virulent isolates in field trials involving internalization.

  19. Identification and Characterization of a Peculiar vtx2-Converting Phage Frequently Present in Verocytotoxin-Producing Escherichia coli O157 Isolated from Human Infections

    PubMed Central

    Grande, Laura; Michelacci, Valeria; Fioravanti, Rosa; Gally, David; Xu, Xuefang; La Ragione, Roberto; Anjum, Muna; Wu, Guanghui; Caprioli, Alfredo; Morabito, Stefano

    2014-01-01

    Certain verocytotoxin-producing Escherichia coli (VTEC) O157 phage types (PTs), such as PT8 and PT2, are associated with severe human infections, while others, such as PT21, seem to be restricted to cattle. In an attempt to delve into the mechanisms underlying such a differential distribution of PTs, we performed microarray comparison of human PT8 and animal PT21 VTEC O157 isolates. The main differences observed were in the vtx2-converting phages, with the PT21 strains bearing a phage identical to that present in the reference strain EDL933, BP933W, and all the PT8 isolates displaying lack of hybridization in some regions of the phage genome. We focused on the region spanning the gam and cII genes and developed a PCR tool to investigate the presence of PT8-like phages in a panel of VTEC O157 strains belonging to different PTs and determined that a vtx2 phage reacting with the primers deployed, which we named Φ8, was more frequent in VTEC O157 strains from human disease than in bovine strains. No differences were observed in the production of the VT2 mRNA when Φ8-positive strains were compared with VTEC O157 possessing BP933W. Nevertheless, we show that the gam-cII region of phage Φ8 might carry genetic determinants downregulating the transcription of the genes encoding the components of the type III secretion system borne on the locus of enterocyte effacement pathogenicity island. PMID:24799627

  20. Pulsed-field gel electrophoresis patterns of Escherichia coli O157 isolates from Kansas feedlots.

    PubMed

    Sargeant, J M; Shi, X; Sanderson, M W; Renter, D G; Nagaraja, T G

    2006-01-01

    This study investigated the prevalence and distribution of Escherichia coli O157 genetic types within and among feedlots using pulsed-field gel electrophoresis to separate XbaI-digested DNA. The study population consisted of 300 pens of cattle in 30 feedlots in Kansas that were sampled (feces, water, and water sediment) within a month of being shipped for slaughter. The prevalence of E. coli O157 was 8.5% in feces, 3.1% in water, and 4.5% in water sediment samples. A total of 424 E. coli O157 isolates were characterized by pulsed-field gel electrophoresis, and 139 subtypes (100% Dice similarity with no band differences) were identified. The majority of subtypes (70/139) was identified only once, but nine were identified 10 or more times. Identical subtypes were recovered from both feces and water tanks in 10 feedlots. The majority of subtypes were identified in only one feedlot, and the number of subtypes ranged from one to 23 within a feedlot and from one to seven within a pen. There were 10 feedlots with at least 15 positive samples. In these 10 feedlots, the most common subtype accounted for 16.9-78.6% of the isolates. Common subtypes differed among feedlots. In eight of the 10 feedlots, the most common subtype was identified in multiple pens. The results support a complex ecology for E. coli O157 in feedlot operations, with factors associated with exposure and transmission likely acting at a common level for multiple feedlots, within feedlots, and within pens of cattle.

  1. Comparative genomic analysis and adherence characteristics of supershedder strains of Escherichia coli O157:H7

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin-producing Escherichia coli O157:H7 (O157) is a zoonotic foodborne pathogen of major public health concern that results in considerable intestinal and extra-intestinal illness in humans. Asymptomatic cattle are the primary reservoir of O157 and harbor the pathogen at the terminal recto-an...

  2. Construction and characterization of outbreak Escherichia coli O157:H7 surrogate strains for use in field studies.

    PubMed

    Webb, Cathy C; Erickson, Marilyn C; Davey, Lindsey E; Payton, Alison S; Doyle, Michael P

    2014-11-01

    Escherichia coli O157:H7 has been the causative agent of many outbreaks associated with leafy green produce consumption. Elucidating the mechanism by which contamination occurs requires monitoring interactions between the pathogen and the plant under typical production conditions. Intentional introduction of virulent strains into fields is not an acceptable practice. As an alternative, attenuated strains of natural isolates have been used as surrogates of the virulent strains; however, the attachment properties and environmental stabilities of these attenuated isolates may differ from the unattenuated outbreak strains. In this study, the Shiga toxin (stx1, stx2, and/or stx2c) genes as well as the eae gene encoding intimin of two E. coli O157:H7 outbreak isolates, F4546 (1997 alfalfa sprout) and K4492 (2006 lettuce), were deleted. Individual gene deletions were confirmed by polymerase chain reaction (PCR) and DNA sequencing. The mutant strains did not produce Shiga toxin. The growth kinetics of these mutant strains under nutrient-rich and minimal conditions were identical to those of their wild-type strains. Attachment to the surface of lettuce leaves was comparable between wild-type/mutant pairs F4546/MD46 and K4492/MD47. Adherence to soil particles was also comparable between the virulent and surrogate pairs, although the F4546/MD46 pair exhibited statistically greater attachment than the K4492/MD47 pair (p≤0.05). Wild-type and mutant pairs F4546/MD46 and K4492/MD47 inoculated into wet or dry soils had statistically similar survival rates over the 7-day storage period at 20°C. A plasmid, pGFPuv, containing green fluorescent protein was transformed into each of the mutant strains, allowing for ease of identification and detection of surrogate strains on plant material or soil. These pGFPuv-containing surrogate strains will enable the investigation of pathogen interaction with plants and soil in the farm production environment where the virulent pathogen cannot

  3. Complete Genome Sequences of Escherichia coli O157:H7 Strains SRCC 1675 and 28RC, Which Vary in Acid Resistance

    PubMed Central

    Baranzoni, Gian Marco; Reichenberger, Erin R.; Kim, Gwang-Hee; Breidt, Frederick; Kay, Kathryn; Oh, Deog-Hwan

    2016-01-01

    The level of acid resistance among Escherichia coli O157:H7 strains varies, and strains with higher resistance to acid may have a lower infectious dose. The complete genome sequences belonging to two strains of Escherichia coli O157:H7 with different levels of acid resistance are presented here. PMID:27469964

  4. Complete genome sequences of Escherichia coli O157:H7 strains SRCC 1675 and 28RC that vary in acid resistance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The level of acid resistance among Escherichia coli O157:H7 strains varies, and strains with higher resistance to acid may have a lower infectious dose. The complete genome sequences belonging to two strains of Escherichia coli O157:H7 with different levels of acid resistance are presented....

  5. Complete Genome Sequences of Escherichia coli O157:H7 Strains SRCC 1675 and 28RC, Which Vary in Acid Resistance.

    PubMed

    Baranzoni, Gian Marco; Fratamico, Pina M; Reichenberger, Erin R; Kim, Gwang-Hee; Breidt, Frederick; Kay, Kathryn; Oh, Deog-Hwan

    2016-01-01

    The level of acid resistance among Escherichia coli O157:H7 strains varies, and strains with higher resistance to acid may have a lower infectious dose. The complete genome sequences belonging to two strains of Escherichia coli O157:H7 with different levels of acid resistance are presented here. PMID:27469964

  6. Comparative Analysis of Super-Shedder Strains of Escherichia coli O157:H7 Reveals Distinctive Genomic Features and a Strongly Aggregative Adherent Phenotype on Bovine Rectoanal Junction Squamous Epithelial Cells

    PubMed Central

    Cote, Rebecca; Katani, Robab; Moreau, Matthew R.; Kudva, Indira T.; Arthur, Terrance M.; DebRoy, Chitrita; Mwangi, Michael M.; Albert, Istvan; Raygoza Garay, Juan Antonio; Li, Lingling; Brandl, Maria T.; Carter, Michelle Q.; Kapur, Vivek

    2015-01-01

    Shiga toxin-producing Escherichia coli O157:H7 (O157) are significant foodborne pathogens and pose a serious threat to public health worldwide. The major reservoirs of O157 are asymptomatic cattle which harbor the organism in the terminal recto-anal junction (RAJ). Some colonized animals, referred to as “super-shedders” (SS), are known to shed O157 in exceptionally large numbers (>104 CFU/g of feces). Recent studies suggest that SS cattle play a major role in the prevalence and transmission of O157, but little is known about the molecular mechanisms associated with super-shedding. Whole genome sequence analysis of an SS O157 strain (SS17) revealed a genome of 5,523,849 bp chromosome with 5,430 open reading frames and two plasmids, pO157 and pSS17, of 94,645 bp and 37,446 bp, respectively. Comparative analyses showed that SS17 is clustered with spinach-associated O157 outbreak strains, and belongs to the lineage I/II, clade 8, D group, and genotype 1, a subgroup of O157 with predicted hyper-virulence. A large number of non-synonymous SNPs and other polymorphisms were identified in SS17 as compared with other O157 strains (EC4115, EDL933, Sakai, TW14359), including in key adherence- and virulence-related loci. Phenotypic analyses revealed a distinctive and strongly adherent aggregative phenotype of SS17 on bovine RAJ stratified squamous epithelial (RSE) cells that was conserved amongst other SS isolates. Molecular genetic and functional analyses of defined mutants of SS17 suggested that the strongly adherent aggregative phenotype amongst SS isolates is LEE-independent, and likely results from a novel mechanism. Taken together, our study provides a rational framework for investigating the molecular mechanisms associated with SS, and strong evidence that SS O157 isolates have distinctive features and use a LEE-independent mechanism for hyper-adherence to bovine rectal epithelial cells. PMID:25664460

  7. Correlation between geographic distance and genetic similarity in an international collection of bovine faecal Escherichia coli O157:H7 isolates.

    PubMed Central

    Davis, M. A.; Hancock, D. D.; Besser, T. E.; Rice, D. H.; Hovde, C. J.; Digiacomo, R.; Samadpour, M.; Call, D. R.

    2003-01-01

    Evidence from epidemiological and molecular studies of bovine Escherichia coli O157:H7 suggests that strains are frequently transmitted across wide geographic distances. To test this hypothesis, we compared the geographic and genetic distance of a set of international bovine Escherichia coli O157:H7 isolates using the Mantel correlation. For a measure of genetic relatedness, pulsed-field gel electrophoresis of six different restriction enzyme digests was used to generate an average Dice similarity coefficient for each isolate pair. Geographic distance was calculated using latitude and longitude data for isolate source locations. The Mantel correlation between genetic similarity and the logarithm of geographic distance in kilometers was -0.21 (P<0.001). The low magnitude of the Mantel correlation indicates that transmission over long distances is common. The occurrence of isolates from different continents on the same cluster of the dendrogram also supports the idea that Escherichia coli O157:H7 strains can be transferred with considerable frequency over global distances. PMID:14596534

  8. Genetically marked strains of Shiga toxin-producing O157:H7 and non-O157 Escherichia coli: Tools for detection and modelling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin-producing E. coli (STEC) are among the most important foodborne pathogens in the United States and worldwide. Nearly half of all STEC-induced diarrheal disease in the United States is caused by STEC O157:H7 while non-O157 STEC account for the remaining illnesses. Thus, the USDA Food Safe...

  9. Genetic Analysis for the Lack of Expression of the O157 Antigen in an O Rough:H7 Escherichia coli Strain

    PubMed Central

    Rump, Lydia V.; Feng, Peter C. H.; Fischer, Markus; Monday, Steven R.

    2010-01-01

    The O-antigen (rfb) operon and related genes of MA6, an O rough:H7 Shiga-toxigenic Escherichia coli strain, were examined to determine the cause of the lack of O157 expression. A 1,310-bp insertion, homologous to IS629, was observed within its gne gene. trans complementation with a functional gne gene from O157:H7 restored O157 antigen expression in MA6. PMID:19948859

  10. Multiple mechanisms responsible for strong Congo red-binding variants of Escherichia coli O157:H7 strains

    Technology Transfer Automated Retrieval System (TEKTRAN)

    High variability in the expression of csgD-dependent, biofilm-forming and adhesive properties is common among Shiga toxin-producing Escherichia coli (STEC). Although many strains of serotype O157:H7 form little biofilm, conversion to stronger biofilm phenotypes has been observed. In this study we sc...

  11. Genotypic characterization of Escherichia coli O157:H7 strains that cause diarrhea and hemolytic uremic syndrome in Neuquén, Argentina.

    PubMed

    Pianciola, Luis; Chinen, Isabel; Mazzeo, Melina; Miliwebsky, Elizabeth; González, Gladys; Müller, Constanza; Carbonari, Carolina; Navello, Mariano; Zitta, Eugenia; Rivas, Marta

    2014-05-01

    Shiga toxin-producing Escherichia coli (STEC) are important food-borne pathogens associated with cases of diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). E. coli O157:H7 is the dominant serotype in Argentina and also in Neuquén Province, in which HUS incidence is above the national average, with a maximum of 28.6 cases per 100,000 children less than 5 years old reported in 1998. The aim of this study was to characterize a collection of 70 STEC O157 strains isolated from patients with diarrhea and HUS treated in the province of Neuquén, Argentina, between 1998 and 2011. All strains harbored eae, ehxA, rfbO157, and fliCH7 genes, and stx2a/stx2c (78.7%) was the predominant genotype. A total of 64 (91.4%) STEC O157 strains belonged to the hypervirulent clade 8 tested using both 4 and 32 SNP typing schemes. The strains showed the highest values reported in the literature for 6 of the 7 virulence determinants described in the TW14359 O157 strain associated with the raw spinach outbreak in the U.S. in 2006. Clade 8 strains were strongly associated with two of them: ECSP_3286, factor encoding an outer membrane protein that facilitates the transport of the heme complex (P=0.001), and in particular extracellular factor ECSP_2870/2872, coding proteins related to adaptation to plant hosts (P=0.000004). The q933 allele, which has been related to high toxin production, was present in 97.1% of the strains studied for the anti-terminator Q gene. In summary, this study describes, for the first time in Argentina, the almost exclusive circulation of strains belonging to the hypervirulent clade 8, and also the presence of putative virulence factors in higher frequencies than those reported worldwide. These data may help to understand the causes of the particular epidemiological situation related to HUS in Neuquén Province.

  12. Molecular analysis of multidrug resistance in Shiga toxin-producing Escherichia coli O157:H7 isolated from meat and dairy products.

    PubMed

    Ahmed, Ashraf M; Shimamoto, Tadashi

    2015-01-16

    Shiga toxin-producing Escherichia coli (STEC) O157:H7 is an important food-borne pathogen that has been implicated in numerous disease outbreaks worldwide. Little is known about the extent and molecular basis of antimicrobial resistance in STEC O157:H7 of food origin. Therefore, the current study aimed to characterize the genetic basis of multidrug resistance in 54 STEC O157:H7 strains isolated from 1600 food samples (800 meat products and 800 dairy products) collected from different street venders, butchers, retail markets, and slaughterhouses in Egypt. Thirty-one of 54 (57.4%) isolates showed multidrug resistance phenotypes to at least three classes of antimicrobials. The highest incidence of antimicrobial resistance was to kanamycin (96.8%), followed by spectinomycin (93.6%), ampicillin (90.3%), streptomycin (87.1%), and tetracycline (80.6%). PCR and DNA sequencing were used to screen and characterize integrons and antibiotic resistance genes, and 29.6% and 5.6% of isolates were positive for class 1 and class 2 integrons, respectively. β-Lactamase-encoding genes were identified in 63.0% of isolates as follows: blaTEM₋₁ and blaTEM₋₅₂ in 35.2% and 1.9% isolates respectively; blaCMY₋₂ in 13.0% isolates; blaCTX-M in 5.6% isolates; blaSHV₋₁₂ in 5.6% isolates; and blaOXA₋₁ in 1.9% isolate. The plasmid-mediated quinolone resistance genes were identified in 13.0% of isolates as follows: qnrB, qnrS, and aac(6')-Ib-cr in 5.6%, 3.7%, and 3.7% isolates, respectively. Finally, the florfenicol resistance gene floR was identified in 7.4% of isolates. This study demonstrated that meat and dairy products are potential sources of multidrug resistant STEC O157:H7. To our knowledge, this is the first report of the occurrence of class 2 integrons, qnrB, qnrS, and aac(6')-Ib-cr in STEC O157:H7. PMID:25462925

  13. A novel transducible chimeric phage from Escherichia coli O157:H7 Sakai strain encoding Stx1 production.

    PubMed

    Sváb, Domonkos; Bálint, Balázs; Maróti, Gergely; Tóth, István

    2015-01-01

    Shiga toxin-producing Escherichia coli (STEC), and especially enterohaemorrhagic E. coli (EHEC) are important, highly virulent zoonotic and food-borne pathogens. The genes encoding their key virulence factors, the Shiga toxins, are distributed by converting bacteriophages, the Stx phages. In this study we isolated a new type of inducible Stx phage carrying the stx1 gene cluster from the prototypic EHEC O157:H7 Sakai strain. The phage showed Podoviridae morphology, and was capable of converting the E. coli K-12 MG1655 strain to Shiga toxin-producing phenotype. The majority of the phage genes originate from the stx2-encoding Sakai prophage Sp5, with major rearrangements in its genome. Beside certain minor recombinations, the genomic region originally containing the stx2 genes in Sp5 was replaced by a region containing six open reading frames from prophage Sp15 including stx1 genes. The rearranged genome, together with the carriage of stx1 genes, the morphology and the capability of lysogenic conversion represent a new type of recombinant Stx1 converting phage from the Sakai strain. PMID:25445656

  14. Genomic and molecular analysis of the hyperadherent phenotype of an Escherichia coli O157:H7 super shedder isolate

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga Toxin producing Escherichia coli (STEC) are a subtype of pathogenic E. coli and, in particular, STEC isolates of E. coli serotype O157:H7 are recognized as a major foodborne pathogen that can cause infections ranging from having simple intestinal discomfort to bloody diarrhea and life threaten...

  15. Escherichia coli O157:H7 bacteriophage (phi)241 isolated from an industrial cucumber fermentation at high acidity and salinity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A novel phage, (phi)241, specific for Escherichia coli O157:H7 was isolated from an industrial cucumber fermentation where both acidity (pH less than or equal to 3.7) and salinity (greater than or equal to 5% NaCl) were high. The phage belongs to the Myoviridae family. Its latent period was 15 min a...

  16. A comparison of immunomagnetic separation and direct culture for the isolation of verocytotoxin-producing Escherichia coli O157 from cases of bloody diarrhoea, non-bloody diarrhoea and asymptomatic contacts.

    PubMed

    Chapman, P A; Siddons, C A

    1996-04-01

    Enrichment culture in modified buffered peptone water followed by immunomagnetic separation (IMS) with magnetic beads coated with an antibody against Escherichia coli O157 was compared with direct culture on cefixime rhamnose sorbitol MacConkey agar (CR-SMAC) and cefixime tellurite sorbitol MacConkey agar (CT-SMAC) for the isolation of E. coli O157 from human faeces. In total, 690 samples were examined; E. coli O157 was isolated from 25 samples by IMS but from only 15 and 12 by direct culture on CT-SMAC and CR-SMAC, respectively. The difference in sensitivity of detection was at its most marked on screening repeat faecal samples from known cases and samples from asymptomatic contacts, when of 12 strains of E. coli O157 isolated by IMS, only five were isolated by direct culture. IMS is a sensitive and simple technique for the isolation of E. coli O157 from human faecal samples and should prove useful in elucidating further the epidemiology of this micro-organism.

  17. Phylogenetic classification of Escherichia coli O157:H7 strains of human and bovine origin using a novel set of nucleotide polymorphisms

    PubMed Central

    Clawson, Michael L; Keen, James E; Smith, Timothy PL; Durso, Lisa M; McDaneld, Tara G; Mandrell, Robert E; Davis, Margaret A; Bono, James L

    2009-01-01

    Background Cattle are a reservoir of Shiga toxin-producing Escherichia coli O157:H7 (STEC O157), and are known to harbor subtypes not typically found in clinically ill humans. Consequently, nucleotide polymorphisms previously discovered via strains originating from human outbreaks may be restricted in their ability to distinguish STEC O157 genetic subtypes present in cattle. The objectives of this study were firstly to identify nucleotide polymorphisms in a diverse sampling of human and bovine STEC O157 strains, secondly to classify strains of either bovine or human origin by polymorphism-derived genotypes, and finally to compare the genotype diversity with pulsed-field gel electrophoresis (PFGE), a method currently used for assessing STEC O157 diversity. Results High-throughput 454 sequencing of pooled STEC O157 strain DNAs from human clinical cases (n = 91) and cattle (n = 102) identified 16,218 putative polymorphisms. From those, 178 were selected primarily within genomic regions conserved across E. coli serotypes and genotyped in 261 STEC O157 strains. Forty-two unique genotypes were observed that are tagged by a minimal set of 32 polymorphisms. Phylogenetic trees of the genotypes are divided into clades that represent strains of cattle origin, or cattle and human origin. Although PFGE diversity surpassed genotype diversity overall, ten PFGE patterns each occurred with multiple strains having different genotypes. Conclusions Deep sequencing of pooled STEC O157 DNAs proved highly effective in polymorphism discovery. A polymorphism set has been identified that characterizes genetic diversity within STEC O157 strains of bovine origin, and a subset observed in human strains. The set may complement current techniques used to classify strains implicated in disease outbreaks. PMID:19463166

  18. [Isolation of enteropathogenic Escherichia coli O157:H16 identified in a diarrhea case in a child and his household contacts in La Pampa Province, Argentina].

    PubMed

    Silveyra, Ivana M; Pereyra, Adriana M; Alvarez, María G; Villagran, Mariana D; Baroni, Andrea B; Deza, Natalia; Carbonari, Claudia C; Miliwebsky, Elizabeth; Rivas, Marta

    2015-01-01

    Enteropathogenic Escherichia coli (EPEC) is a major causative agent of acute diarrhea in children in developing countries. This pathotype is divided into typical EPEC (tEPEC) and atypical EPEC (aEPEC), based on the presence of the bfp virulence factor associated with adhesion, encoded in the pEAF plasmid. In the present study, the isolation of aEPEC O157:H16 from a bloody diarrhea case in a child and his household contacts (mother, father and sister) is described. The strain was characterized as E. coli O157:H16 eae-ɛ-positive, sorbitol fermenter with β-glucuronidase activity, susceptible to all antimicrobials tested, and negative for virulence factors stx1, stx2, ehxA and bfp. XbaI-PFGE performed on all isolates showed the AREXHX01.1040 macrorestriction pattern, with 100% similarity. These results highlight the importance of epidemiological surveillance of E. coli O157-associated diarrhea cases identified in children and their family contacts, as well as the incorporation of molecular techniques that allow the detection of the different E. coli pathotypes.

  19. Phage types, virulence genes and PFGE profiles of Shiga toxin-producing Escherichia coli O157:H7 isolated from raw beef, soft cheese and vegetables in Lima (Peru).

    PubMed

    Mora, Azucena; León, Santana L; Blanco, Miguel; Blanco, Jesús E; López, Cecilia; Dahbi, Ghizlane; Echeita, Aurora; González, Enrique A; Blanco, Jorge

    2007-03-10

    The present study was conducted in Lima Metropolitana to evaluate the prevalence of Shiga toxin-producing Escherichia coli (STEC) O157:H7 in raw beef, raw ground beef, soft cheese and fresh vegetables, sampled at different markets in the city. Between October 2000 and February 2001, 407 food samples were collected from different markets in the 42 districts of Lima Metropolitana. Samples were assayed for E. coli O157 by selective enrichment in modified Tryptic Soy Broth containing novobiocin, followed by immunomagnetic separation (IMS) and plating onto sorbitol MacConkey agar supplemented with cefixime and potassium tellurite. Fifty (12.3%) of 407 food samples resulted positive for E. coli O157 isolation (23 of 102 ground beef; 15 of 102 beef meat; eight of 102 soft cheese and four of 101 fresh vegetables). Thirty-five E. coli O157 isolates were further analysed for the presence of virulence genes. All 35 were positive by PCR for O157 rfbE, fliCh7, eae-gamma1 and ehxA genes. In addition, genes encoding Shiga toxins were detected in 33 of 35 isolates, five isolates (14%) encoded stx(1), stx(2), and 28 (80%) stx2 only. The isolates were of seven different phage types (PT4, PT8, PT14, PT21, PT34, PT54, and PT87) with three phage types accounting for 80% of isolates: PT4 (15 isolates), PT14 (8 isolates), and PT21 (5 isolates). Interestingly, the majority (31 of 35; 89%) of E. coli O157:H7 isolates characterized in this study belonged mainly to the phage types previously found in STEC O157:H7 strains associated with severe human disease in Europe and Canada. Pulsed-field gel electrophoresis (PFGE) of 32 isolates revealed 14 XbaI-PFGE groups (I to XIV) of similarity >85%, with 23 (72%) isolates grouped in five clusters. Some isolates from different districts presented a high clonal relatedness. Thus, PFGE group VIII clustered eleven strains from nine different districts. The broad range of PFGE subtypes found in this study demonstrates the natural occurrence of many

  20. RcsB Contributes to the Distinct Stress Fitness among Escherichia coli O157:H7 Curli Variants of the 1993 Hamburger-Associated Outbreak Strains

    PubMed Central

    Parker, Craig T.; Louie, Jacqueline W.; Huynh, Steven; Fagerquist, Clifton K.; Mandrell, Robert E.

    2012-01-01

    Curli are adhesive fimbriae of Enterobactericaeae and are involved in surface attachment, cell aggregation, and biofilm formation. We reported previously that curli-producing (C+) variants of E. coli O157:H7 (EcO157) were much more acid sensitive than their corresponding curli-deficient (C−) variants; however, this difference was not linked to the curli fimbriae per se. Here, we investigated the underlying molecular basis of this phenotypic divergence. We identified large deletions in the rcsB gene of C+ variants isolated from the 1993 U.S. hamburger-associated outbreak strains. rcsB encodes the response regulator of the RcsCDB two-component signal transduction system, which regulates curli biogenesis negatively but acid resistance positively. Further comparison of stress fitness revealed that C+ variants were also significantly more sensitive to heat shock but were resistant to osmotic stress and oxidative damage, similar to C− variants. Transcriptomics analysis uncovered a large number of differentially expressed genes between the curli variants, characterized by enhanced expression in C+ variants of genes related to biofilm formation, virulence, catabolic activity, and nutrient uptake but marked decreases in transcription of genes related to various types of stress resistance. Supplying C+ variants with a functional rcsB restored resistance to heat shock and acid challenge in cells but blocked curli production, confirming that inactivation of RcsB in C+ variants was the basis of fitness segregation within the EcO157 population. This study provides an example of how genome instability of EcO157 promotes intrapopulation diversification, generating subpopulations carrying an array of distinct phenotypes that may confer the pathogen with survival advantages in diverse environments. PMID:22923406

  1. Multiple mechanisms responsible for strong Congo-red-binding variants of Escherichia coli O157:H7 strains.

    PubMed

    Chen, Chin-Yi; Nguyen, Ly-Huong T; Cottrell, Bryan J; Irwin, Peter L; Uhlich, Gaylen A

    2016-03-01

    High variability in the expression of csgD-dependent, biofilm-forming and adhesive properties is common among Shiga toxin-producing Escherichia coli. Although many strains of serotype O157:H7 form little biofilm, conversion to stronger biofilm phenotypes has been observed. In this study, we screened different strains of serotype O157:H7 for the emergence of strong Congo-red (CR) affinity/biofilm-forming properties and investigated the underlying genetic mechanisms. Two major mechanisms which conferred stronger biofilm phenotypes were identified: mutations (insertion, deletion, single nucleotide change) in rcsB region and stx-prophage excision from the mlrA site. Restoration of the native mlrA gene (due to prophage excision) resulted in strong biofilm properties to all variants. Whereas RcsB mutants showed weaker CR affinity and biofilm properties, it provided more possibilities for phenotypic presentations through heterogenic sequence mutations.

  2. Design and evaluation of two-stage multiplex real-time PCR method for detecting O157:H7 and non-O157 STEC strains from beef samples

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Introduction: E. coli O157:H7 was first recognized as a human pathogen in 1982 and until recently was the only E. coli strain mandated for testing by the USDA. In June 2012, the USDA declared six additional Shiga-toxin producing E. coli serogroups (O26, O45, O103, O111, O121, and O145) as adulterant...

  3. Reduction of Salmonella spp. and strains of Escherichia coli O157:H7 by gamma radiation of inoculated sprouts.

    PubMed

    Rajkowski, K T; Thayer, D W

    2000-07-01

    There have been several recent outbreaks of salmonellosis and infections with Escherichia coli O157:H7 linked to the consumption of raw sprouts. Use of ionizing radiation was investigated as a means to reduce or to totally inactivate these pathogens, if present, on the sprouts. The radiation D value, which is the amount of irradiation in kilograys for a 1-log reduction in cell numbers, for these pathogens was established using a minimum of five doses at 19 +/- 1 degrees C. Before inoculation, the sprouts were irradiated to 6 kGy to remove the background microflora. The sprouts were inoculated either with Salmonella spp. cocktails made with either meat or vegetable isolates or with E. coli O157:H7 cocktails made with either meat or vegetable isolates. The radiation D values for the Salmonella spp. cocktails on sprouts were 0.54 and 0.46 kGy, respectively, for the meat and vegetable isolates. The radiation D values for the E. coli O157:H7 cocktails on sprouts were 0.34 and 0.30 kGy, respectively, for the meat and vegetable isolates. Salmonella was not detected by enrichment culture on sprouts grown from alfalfa seeds naturally contaminated with Salmonella after the sprouts were irradiated to a dose of 0.5 kGy or greater. Ionizing radiation is a process that can be used to reduce the population of pathogens on sprouts.

  4. Improved selective and differential medium for isolation of Escherichia coli O157:H7.

    PubMed

    Park, Sang-Hyun; Ryu, Sangryeol; Kang, Dong-Hyun

    2011-01-01

    GMAC, a modified version of Sorbitol MacConkey medium (SMAC), was produced with a reduced quantity of selective agents and incorporated gentiobiose. GMAC supported a higher recovery rate of heat- or acid-injured Escherichia coli O157:H7 cells than SMAC with cefixime and tellurite (CT-SMAC), while differentiating E. coli O157:H7 from sorbitol-nonfermenting Hafnia alvei. PMID:20980579

  5. Improved Selective and Differential Medium for Isolation of Escherichia coli O157:H7▿

    PubMed Central

    Park, Sang-Hyun; Ryu, Sangryeol; Kang, Dong-Hyun

    2011-01-01

    GMAC, a modified version of Sorbitol MacConkey medium (SMAC), was produced with a reduced quantity of selective agents and incorporated gentiobiose. GMAC supported a higher recovery rate of heat- or acid-injured Escherichia coli O157:H7 cells than SMAC with cefixime and tellurite (CT-SMAC), while differentiating E. coli O157:H7 from sorbitol-nonfermenting Hafnia alvei. PMID:20980579

  6. Transport and Straining of E. coli O157:H7 in Saturated Porous Media

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The transport and deposition behavior of pathogenic Escherichia coli O157:H7 was studied under unfavorable electrostatic conditions in saturated quartz sands of various sizes (710, 360, 240, and 150 mm) and at several flow rates. At a given velocity, column effluent breakthrough values for E. coli t...

  7. Fitness of Outbreak and Environmental Strains of Escherichia coli O157:H7 in Aerosolizable Soil and Association of Clonal Variation in Stress Gene Regulation

    PubMed Central

    Ravva, Subbarao V.; Cooley, Michael B.; Sarreal, Chester Z.; Mandrell, Robert E.

    2014-01-01

    Airborne dust from feedlots is a potential mechanism of contamination of nearby vegetable crops with Escherichia coli O157:H7 (EcO157). We compared the fitness of clinical and environmental strains of EcO157 in <45 µm soil from a spinach farm. Differences in survival were observed among the 35 strains with D-values (days for 90% decreases) ranging from 1–12 days. Strains that survived longer, generally, were from environmental sources and lacked expression of curli, a protein associated with attachment and virulence. Furthermore, the proportion of curli-positive (C+) variants of EcO157 strains decreased with repeated soil exposure and the strains that were curli-negative (C−) remained C− post-soil exposure. Soil exposure altered expression of stress-response genes linked to fitness of EcO157, but significant clonal variation in expression was measured. Mutations were detected in the stress-related sigma factor, rpoS, with a greater percentage occurring in parental strains of clinical origin prior to soil exposure. We speculate that these mutations in rpoS may confer a differential expression of genes, associated with mechanisms of survival and/or virulence, and thus may influence the fitness of EcO157. PMID:25438010

  8. Wide Distribution of O157-Antigen Biosynthesis Gene Clusters in Escherichia coli

    PubMed Central

    Seto, Kazuko; Ooka, Tadasuke; Ogura, Yoshitoshi; Hayashi, Tetsuya; Osawa, Kayo; Osawa, Ro

    2011-01-01

    Most Escherichia coli O157-serogroup strains are classified as enterohemorrhagic E. coli (EHEC), which is known as an important food-borne pathogen for humans. They usually produce Shiga toxin (Stx) 1 and/or Stx2, and express H7-flagella antigen (or nonmotile). However, O157 strains that do not produce Stxs and express H antigens different from H7 are sometimes isolated from clinical and other sources. Multilocus sequence analysis revealed that these 21 O157:non-H7 strains tested in this study belong to multiple evolutionary lineages different from that of EHEC O157:H7 strains, suggesting a wide distribution of the gene set encoding the O157-antigen biosynthesis in multiple lineages. To gain insight into the gene organization and the sequence similarity of the O157-antigen biosynthesis gene clusters, we conducted genomic comparisons of the chromosomal regions (about 59 kb in each strain) covering the O-antigen gene cluster and its flanking regions between six O157:H7/non-H7 strains. Gene organization of the O157-antigen gene cluster was identical among O157:H7/non-H7 strains, but was divided into two distinct types at the nucleotide sequence level. Interestingly, distribution of the two types did not clearly follow the evolutionary lineages of the strains, suggesting that horizontal gene transfer of both types of O157-antigen gene clusters has occurred independently among E. coli strains. Additionally, detailed sequence comparison revealed that some positions of the repetitive extragenic palindromic (REP) sequences in the regions flanking the O-antigen gene clusters were coincident with possible recombination points. From these results, we conclude that the horizontal transfer of the O157-antigen gene clusters induced the emergence of multiple O157 lineages within E. coli and speculate that REP sequences may involve one of the driving forces for exchange and evolution of O-antigen loci. PMID:21876740

  9. Antagonistic effects of probiotic Escherichia coli Nissle 1917 on EHEC strains of serotype O104:H4 and O157:H7.

    PubMed

    Rund, Stefan A; Rohde, Holger; Sonnenborn, Ulrich; Oelschlaeger, Tobias A

    2013-01-01

    The largest EHEC outbreak up to now in Germany occurred in 2011. It was caused by the non-O157:H7 Shiga-toxinogenic enterohemorrhagic E. coli strain O104:H4. This strain encodes in addition to the Shiga toxin 2 (Stx2), responsible for the hemolytic uremic syndrome (HUS), several adhesins such as aggregative adherence fimbriae. Currently, there is no effective prophylaxis and treatment available for EHEC infections in humans. Especially antibiotics are not indicated for treatment as they may induce Stx production, thus worsening the symptoms. Alternative therapeutics are therefore desperately needed. We tested the probiotic Escherichia coli strain Nissle 1917 (EcN) for antagonistic effects on two O104:H4 EHEC isolates from the 2011 outbreak and on the classical O157:H7 EHEC strain EDL933. These tests included effects on adherence, growth, and Stx production in monoculture and co-culture together with EcN. The inoculum of each co-culture contained EcN and the respective EHEC strain either at a ratio of 1:1 or 10:1 (EcN:EHEC). Adhesion of EHEC strains to Caco-2 cells and to the mucin-producing LS-174T cells was reduced significantly in co-culture with EcN at the 1:1 ratio and very dramatically at the 10:1 ratio. This inhibitory effect of EcN on EHEC adherence was most likely not due to occupation of adhesion sites on the epithelial cells, because in monocultures EcN adhered with much lower bacterial numbers than the EHEC strains. Both EHEC strains of serotype O104:H4 showed reduced growth in the presence of EcN (10:1 ratio). EHEC strain EDL933 grew in co-culture with EcN only during the first 2h of incubation. Thereafter, EHEC counts declined. At 24h, the numbers of viable EDL933 was at or slightly below the numbers at the time of inoculation. The amount of Stx2 after 24h co-incubation with EcN (EcN:EHEC ratio 10:1) was for all 3 EHEC strains tested significantly reduced in comparison to EHEC monocultures. Obviously, EcN shows very efficient antagonistic activity on

  10. Use of ramification amplification assay for detection of Escherichia coli O157:H7 and other E. coli Shiga toxin-producing strains.

    PubMed

    Li, Fan; Zhao, Chunyan; Zhang, Wandi; Cui, Shenghui; Meng, Jianghong; Wu, Josephine; Zhang, David Y

    2005-12-01

    Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (STEC) strains are important human pathogens that are mainly transmitted through the food chain. These pathogens have a low infectious dose and may cause life-threatening illnesses. However, detection of this microorganism in contaminated food or a patient's stool specimens presents a diagnostic challenge because of the low copy number in the sample. Often, a more sensitive nucleic acid amplification method, such as PCR, is required for rapid detection of this microorganism. Ramification amplification (RAM) is a recently introduced isothermal DNA amplification technique that utilizes a circular probe for target detection and achieves exponential amplification through the mechanism of primer extension, strand displacement, and ramification. In this study, we synthesized a circular probe specific for the Shiga toxin 2 gene (stx(2)). Our results showed that as few as 10 copies of stx(2) could be detected, indicating that the RAM assay was as sensitive as conventional PCR. We further tested 33 isolates of E coli O157:H7, STEC, Shigella dysenteriae, and nonpathogenic E. coli by RAM assay. Results showed that all 27 STEC isolates containing the stx(2) gene were identified by RAM assay, while S. dysenteriae and nonpathogenic E. coli isolates were undetected. The RAM results were 100% in concordance with those of PCR. Because of its simplicity and isothermal amplification, the RAM assay could be a useful method for detecting STEC in food and human specimens.

  11. Multilocus sequence typing reveals a lack of diversity among Escherichia coli O157:H7 isolates that are distinct by pulsed-field gel electrophoresis.

    PubMed

    Noller, Anna C; McEllistrem, M Catherine; Stine, O Colin; Morris, J Glenn; Boxrud, David J; Dixon, Bruce; Harrison, Lee H

    2003-02-01

    Escherichia coli O157:H7 is a major cause of foodborne illness in the United States. Pulsed-field gel electrophoresis (PFGE) is the molecular epidemiologic method mostly commonly used to identify food-borne outbreaks. Although PFGE is a powerful epidemiologic tool, it has disadvantages that make a DNA sequence-based approach potentially attractive. Multilocus sequence typing (MLST) analyzes the internal fragments of housekeeping genes to establish genetic relatedness between isolates. We sequenced selected portions of seven housekeeping genes and two membrane protein genes (ompA and espA) of 77 isolates that were diverse by PFGE to determine whether there was sufficient sequence variation to be useful as an epidemiologic tool. There was no DNA sequence diversity in the sequenced portions of the seven housekeeping genes and espA. For ompA, all but five isolates had sequence identical to that of the reference strains. E. coli O157:H7 has a striking lack of genetic diversity in the genes we explored, even among isolates that are clearly distinct by PFGE. Other approaches to identify improved molecular subtyping methods for E. coli 0157:H7 are needed.

  12. Prevalence of O157:H7 and non-O157 E. coli in Iranian domestic sheep.

    PubMed

    Tahamtan, Yahya; Namavari, Mehdi

    2014-01-01

    The aim of the present study was the isolation of both E. coli O157 and non-O157 in sheep. Verotoxins (VT) 1, 2 and eae genes were tested for this propose. Sheep faces are an important source of Shiga toxin-producing Escherichia coli (STEC). Escherichia coli O157:H7 is a highly virulent food-borne pathogen and threat to public health. Rectal swab samples from sheep were collected during 2009-2010. Conventional plating and Polymerase Chain Reaction (PCR) were carried out according to virulence factors (Stx1, Stx2 and eaeA).There significant differences between prevalence of STEC and session were observed. It was at highest in spring and late summer. Six (3.92%) sheep carcasses were contaminated by E. coli O157:H7.Only six samples were positive by PCR specific for the VT2 gene and produced verocytotoxin VT2, whereas all isolates were negative for the presence of VT1 and eae virulence genes considered. Geographical variations and season may be influenced in the prevalence rate. The composition of the gastrointestinal flora may be changed by different diet and, therefore O157 STEC rate in sheep and lamb was different. Iranian sheep indicated as a natural host of E. coli O157 strains therefore, may be potentially pathogenic for humans. This is the first report of E. coli O157 detection from sheep in Iran.

  13. Comparison of O-antigen gene cluster from E. coli O157 from human and non-human strains

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin-containing Escherichia coli O157:H7 (STEC O157:H7) is an important zoonotic pathogen which can be transmitted to humans by ingestion of contaminated water or food. Cattle are a common reservoir for STEC O157:H7. This microorganism may cause diarrhea, bloody diarrhea, and hemolytic-uremic...

  14. Investigation of carbon storage regulation network (csr genes) and phenotypic differences between acid sensitive and resistant Escherichia coli O157:H7 strains

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Escherichia coli O157:H7 and related serotype strains have previously been shown to vary in acid resistance, however, little is known about strain specific mechanisms of acid resistance. We examined sensitive and resistant E. coli strains to determine the effects of growth in minimal and...

  15. Sheep as an important source of E. coli O157/O157:H7 in Turkey.

    PubMed

    Gencay, Yilmaz Emre

    2014-08-27

    Escherichia coli O157:H7 is a globally important foodborne pathogen and has been mainly associated with cattle as the reservoir. However, accumulating data shows the importance of sheep as an E. coli O157:H7 vehicle. The presence of E. coli O157/O157:H7 in recto-anal mucosal swap and carcass sponge samples of 100 sheep brought to the slaughterhouse in Kirikkale were analyzed over a year. Molecular characteristics (stx1, stx2, eaeA, hly, lpfA1-3, espA, eae-α1, eae-α2, eae-β, eae-β1, eae-β2, eae-γ1, eae-γ2/θ, stx1c, stx1d, stx2c, stx2d, stx2e, stx2f, stx2g, blaampC, tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), sul1, sul2, floR, cmlA, strA, strB and aadA) of 79 isolates were determined and minimum inhibitory concentrations of 20 different antibiotics were investigated. E. coli O157/O157:H7 was found in 18% of sheep included in the study and was more prevalent in yearlings than lambs and mature sheep, and male than female sheep, though none of the categories (season, sex or age range) had significant effect on prevalence. Furthermore, Shiga-toxigenic E. coli (STEC) O157:H7 was determined in 2% and 8% of sheep feces and carcasses, respectively. Additionally, lpfA1-3 and eae-γ1 were detected in all isolates. None of the isolates showed resistance against investigated antibiotics, even though 4 sorbitol fermenting E. coli O157 isolates were positive for tet(A), sul1 and aadA. This is the first study in Turkey that reveals the potential public health risk due to the contamination of sheep carcasses with potentially highly pathogenic STEC O157:H7 strains.

  16. Detection, Isolation, and Molecular Subtyping of Escherichia coli O157:H7 and Campylobacter jejuni Associated with a Large Waterborne Outbreak

    PubMed Central

    Bopp, Dianna J.; Sauders, Brian D.; Waring, Alfred L.; Ackelsberg, Joel; Dumas, Nellie; Braun-Howland, Ellen; Dziewulski, David; Wallace, Barbara J.; Kelly, Molly; Halse, Tanya; Musser, Kimberlee Aruda; Smith, Perry F.; Morse, Dale L.; Limberger, Ronald J.

    2003-01-01

    The largest reported outbreak of waterborne Escherichia coli O157:H7 in the United States occurred in upstate New York following a county fair in August 1999. Culture methods were used to isolate E. coli O157:H7 from specimens from 128 of 775 patients with suspected infections. Campylobacter jejuni was also isolated from stools of 44 persons who developed diarrheal illness after attending this fair. There was one case of a confirmed coinfection with E. coli O157:H7 and C. jejuni. Molecular detection of stx1 and stx2 Shiga toxin genes, immunomagnetic separation (IMS), and selective culture enrichment were utilized to detect and isolate E. coli O157:H7 from an unchlorinated well and its distribution points, a dry well, and a nearby septic tank. PCR for stx1 and stx2 was shown to provide a useful screen for toxin-producing E. coli O157:H7, and IMS subculture improved recovery. Pulsed-field gel electrophoresis (PFGE) was used to compare patient and environmental E. coli O157:H7 isolates. Among patient isolates, 117 of 128 (91.5%) were type 1 or 1a (three or fewer bands different). Among the water distribution system isolates, 13 of 19 (68%) were type 1 or 1a. Additionally, PFGE of C. jejuni isolates revealed that 29 of 35 (83%) had indistinguishable PFGE patterns. The PFGE results implicated the water distribution system as the main source of the E. coli O157:H7 outbreak. This investigation demonstrates the potential for outbreaks involving more than one pathogen and the importance of analyzing isolates from multiple patients and environmental samples to develop a better understanding of bacterial transmission during an outbreak. PMID:12517844

  17. Disinfectant and antimicrobial susceptibility profiles of the big six non-O157 Shiga toxin-producing Escherichia coli strains from food animals and humans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The disinfectant and antimicrobial susceptibility profiles of 144 non-O157 Shiga toxin-producing Escherichia coli (STECs) from food animals and humans were determined. An overall moderate prevalence of 38.9% antimicrobial resistance (AMR) was observed in these strains. Animal strains had a lower p...

  18. Studies to select appropriate nonpathogenic surrogate Escherichia coli strains for potential use in place of Escherichia coli O157:H7 and salmonella in pilot plant studiest.

    PubMed

    Eblen, Denise R; Annous, Bassam A; Sapers, Gerald M

    2005-02-01

    The response of a potential nonpathogenic surrogate organism to a particular treatment should closely mimic the response of the target pathogenic organism. In this study, growth characteristics (generation time, lag phase duration, and maximum population), pH at stationary phase, and survival characteristics (level of attachment and survival on apple surfaces, resistance to hydrogen peroxide decontamination treatments, and thermal resistance at 60 degrees C) of 15 nonpathogenic generic Escherichia coli strains and one nonpathogenic E. coli O157:H43 strain were compared with those of two E. coli O157:H7 strains and two Salmonella strains. Few differences in growth characteristics or pH at stationary phase were evident between nonpathogenic and pathogenic strains tested. However, considerably more separation among strains was seen following investigation of survival characteristics. E. coli ECRC 97.0152, which does not contain genes encoding for known virulence factors associated with E. coli O157:H7, appears to be a good surrogate candidate, with growth and survival characteristics similar to those of E. coli O157:H7 strains. The less heat-resistant surrogate strains E. coli NRRL B-766 and NRRL B-3054 and E. coli ATCC 11775, ATCC 25253, and ATCC 25922 may be used when attempting to model the heat resistance of Salmonella Montevideo G4639 and Salmonella Poona RM 2350, respectively. These surrogate strains may be useful for evaluating the efficacy of intervention steps in reducing populations of selected strains of E. coli O157:H7 and Salmonella in processing environments where these pathogens cannot be introduced.

  19. A Magnetic Nanoparticle Based Nucleic Acid Isolation and Purification Instrument for DNA Extraction of Escherichia Coli O157: H7.

    PubMed

    Chen, Yahui; Lin, Jianhan; Jiang, Qin; Chen, Qi; Zhang, Shengjun; Li, Li

    2016-03-01

    The objective of this study was to evaluate the performance of a nucleic acid isolation and purification instrument using Escherichia coli O157:H7 as the model. The instrument was developed with magnetic nanoparticles for efficiently capturing nucleic acids and an intelligent mechanical unit for automatically performing the whole nucleic acid extraction process. A commercial DNA extraction kit from Huier Nano Company was used as reference. Nucleic acids in 1 ml of E. coli O157: H7 at a concentration of 5 x 10(8) CFU/mL were extracted by using this instrument and the kit in parallel and then detected by an ultraviolet spectrophotometer to obtain A260 values and A260/A280 values for the determination of the extracted DNA's quantity and purity, respectively. The A260 values for the instrument and the kit were 0.78 and 0.61, respectively, and the A260/A280 values were 1.98 and 1.93. The coefficient of variations of these parallel tests ranged from 10.5% to 16.7%. The results indicated that this nucleic acid isolation and purification instrument could extract a comparable level of nucleic acid within 50 min compared to the commercial DNA extraction kit.

  20. Survival of a five-strain cocktail of Escherichia coli O157:H7 during the 60-day aging period of cheddar cheese made from unpasteurized milk.

    PubMed

    Schlesser, J E; Gerdes, R; Ravishankar, S; Madsen, K; Mowbray, J; Teo, A Y L

    2006-05-01

    The U.S. Food and Drug Administration Standard of Identity for Cheddar cheeses requires pasteurization of the milk, or as an alternative treatment, a minimum 60-day aging at > or =2 degrees C for cheeses made from unpasteurized milk, to reduce the number of viable pathogens that may be present to an acceptable risk. The objective of this study was to investigate the adequacy of the 60-day minimum aging to reduce the numbers of viable pathogens and evaluate milk subpasteurization heat treatment as a process to improve the safety of Cheddar cheeses made from unpasteurized milk. Cheddar cheese was made from unpasteurized milk inoculated with 10(1) to 10(5) CFU/ml of a five-strain cocktail of acid-tolerant Escherichia coli O157:H7. Samples were collected during the cheese manufacturing process. After pressing, the cheese blocks were packaged into plastic bags, vacuum sealed, and aged at 7 degrees C. After 1 week, the cheese blocks were cut into smaller-size uniform pieces and then vacuum sealed in clear plastic pouches. Samples were plated and enumerated for E. coli O157:H7. Populations of E. coli O157:H7 increased during the cheese-making operations. Population of E. coli O157:H7 in cheese aged for 60 and 120 days at 7 degrees C decreased less than 1 and 2 log, respectively. These studies confirm previous reports that show 60-day aging is inadequate to eliminate E. coli O157:H7 during cheese ripening. Subpasteurization heat-treatment runs were conducted at 148 degrees F (64.4 degrees C) for 17.5 s on milk inoculated with E. coli O157:H7 at 10(5) CFU/ml. These heat-treatment runs resulted in a 5-log E. coli O157: H7 reduction.

  1. Effect of molecules secreted by Lactobacillus acidophilus strain La-5 on Escherichia coli O157:H7 colonization.

    PubMed

    Medellin-Peña, Maira J; Griffiths, Mansel W

    2009-02-01

    The probiotic bacterium Lactobacillus acidophilus strain La-5 is a gut-colonizing microorganism that, when established, becomes an important part of the gastrointestinal (GI) tract microbiota. It has been shown to be effective against enterohemorrhagic Escherichia coli (EHEC) O157:H7 infection. We have previously shown that molecules released by probiotic strain La-5 influence the transcription of EHEC genes involved in colonization and quorum sensing. In this work, we report on the ability of these molecules to prevent the adherence of EHEC to epithelial cells and on its capacity to concentrate F-actin at adhesion sites. With a fluorescein-labeled phallotoxin, it was shown that La-5 cell-free spent medium (CFSM) fractions remarkably reduced attaching and effacing lesions in HeLa cells. We also observed a significant inhibition of bacterial adhesion to Hep-2 cells when they were treated with the same La-5 CFSM fractions. In order to observe if La-5 CFSM fractions exhibited the same effect in vivo, we studied the ability of luminescent EHEC constructs (LEE1::luxCDABE) to adhere to intestinal epithelial cells of specific-pathogen-free ICR mice following intragastric inoculation. Colonization of the GI tract by luminescent EHEC O157:H7 was monitored in real time with a slow-scan charge-coupled device camera. At the same time, fecal shedding of EHEC was studied. Following oral gavage of the La-5 active fraction, we observed a reduced amount of bioluminescence signal along with a decrease in fecal shedding by mice, indicating an effect on the ability of the organism to colonize the GI tract. Our results confirm past evidence of the possibility of blocking or interfering with EHEC's virulence by active molecules contained in the probiotic CFSM and identify novel therapeutic alternatives to antibiotic treatments in the fight against food-borne pathogens. PMID:19088323

  2. Lactobacillus rhamnosus strain GG prevents enterohemorrhagic Escherichia coli O157:H7-induced changes in epithelial barrier function.

    PubMed

    Johnson-Henry, K C; Donato, K A; Shen-Tu, G; Gordanpour, M; Sherman, P M

    2008-04-01

    Enterohemorrhagic Escherichia coli (EHEC) O157:H7 intimately attaches to intestinal epithelial monolayers and produces attaching and effacing (A/E) lesions. In addition, EHEC infection causes disruptions of intercellular tight junctions, leading to clinical sequelae that include acute diarrhea, hemorrhagic colitis, and the hemolytic-uremic syndrome. Current therapy remains supportive since antibiotic therapy increases the risk of systemic complications. This study focused on the potential therapeutic effect of an alternative form of therapy, probiotic Lactobacillus rhamnosus strain GG, to attenuate EHEC-induced changes in paracellular permeability in polarized MDCK-I and T84 epithelial cell monolayers. Changes in epithelial cell morphology, electrical resistance, dextran permeability, and distribution and expression of claudin-1 and ZO-1 were assessed using phase-contrast, immunofluorescence, and transmission electron microscopy and macromolecular flux. This study demonstrated that pretreatment of polarized MDCK-I and T84 cells with the probiotic L. rhamnosus GG reduced morphological changes and diminished the number of A/E lesions induced in response to EHEC O157:H7 infection. With probiotic pretreatment there was corresponding attenuation of the EHEC-induced drop in electrical resistance and the increase in barrier permeability assays. In addition, L. rhamnosus GG protected epithelial monolayers against EHEC-induced redistribution of the claudin-1 and ZO-1 tight junction proteins. In contrast to the effects seen with the live probiotic, heat-inactivated L. rhamnosus GG had no effect on EHEC binding and A/E lesion formation or on disruption of the barrier function. Collectively, these findings provide in vitro evidence that treatment with the probiotic L. rhamnosus strain GG could prove to be an effective management treatment for preventing injury of the epithelial cell barrier induced by A/E bacterial enteropathogens.

  3. Interactions of O157 and non-O157 Shiga toxin-producing Escherichia coli (STEC) recovered from bovine hide and carcass with human cells and abiotic surfaces.

    PubMed

    Matheus-Guimarães, Cecilia; Gonçalves, Evanilde Maria; Cabilio Guth, Beatriz E

    2014-03-01

    Different structures related to biofilm formation by Shiga toxin-producing Escherichia coli (STEC), particularly O157 strains, have been described, but there are few data regarding their involvement in non-O157 strains. The aim of this study was to determine the ability of 14 O157 and 8 non-O157 strains isolated from bovine hide and carcass to interact with biotic and abiotic surfaces and also to evaluate the role of different adhesins. Biofilm formation assays showed that four O157 and two non-O157 strains were able to adhere to glass, and that only one O157 strain adhered to polystyrene. Reverse transcriptase-polymerase chain reaction was carried out using biofilm-forming strains to determine the expression of antigen 43 (Ag43), curli, type 1 fimbriae, STEC autotransporter contributing to biofilm formation (Sab), calcium-binding antigen 43 homologue (Cah), and autotransporter protein of enterohemorrhagic E. coli (EhaA). Most of these structures were expressed under biofilm conditions. However, the lack of Ag43 in one non-O157 strain, as well as Cah and EhaA in two O157 strains, suggests that other adhesins are involved in biofilm formation in these strains. Despite the fact that adherence to HeLa cells was detected in 20 strains (91%), it was not possible to correlate biofilm formation with adherence patterns. Invasiveness in T84 and Caco-2 cells was observed in four and three O157 strains, respectively. Altogether, we showed that there are different sets of genes involved in the interactions of STEC with biotic and abiotic surfaces. Interestingly, one O157 strain that was able to form biofilm on both glass and polystyrene also adhered to and invaded human cells, indicating an important route for its persistence in the environment and interaction with the host. Additionally, the ability of non-O157 strains not carrying the LEE pathogenicity island to form biofilm highlights an industrial and health problem that cannot be neglected.

  4. Greater Diversity of Shiga Toxin-Encoding Bacteriophage Insertion Sites among Escherichia coli O157:H7 Isolates from Cattle than in Those from Humans▿

    PubMed Central

    Besser, Thomas E.; Shaikh, Nurmohammad; Holt, Nicholas J.; Tarr, Phillip I.; Konkel, Michael E.; Malik-Kale, Preeti; Walsh, Coilin W.; Whittam, Thomas S.; Bono, James L.

    2007-01-01

    Escherichia coli O157:H7, a zoonotic human pathogen for which domestic cattle are a reservoir host, produces a Shiga toxin(s) (Stx) encoded by bacteriophages. Chromosomal insertion sites of these bacteriophages define three principal genotypes (clusters 1 to 3) among clinical isolates of E. coli O157:H7. Stx-encoding bacteriophage insertion site genotypes of 282 clinical and 80 bovine isolates were evaluated. A total of 268 (95.0%) of the clinical isolates, but only 41 (51.3%) of the bovine isolates, belonged to cluster 1, 2, or 3 (P < 0.001). Thirteen additional genotypes were identified in isolates from both cattle and humans (four genotypes), from only cattle (seven genotypes), or from only humans (two genotypes). Two other markers previously associated with isolates from cattle or with clinical isolates showed similar associations with genotype groups within bovine isolates; the tir allele sp-1 and the Q933W allele were under- and overrepresented, respectively, among cluster 1 to 3 genotypes. Stx-encoding bacteriophage insertion site typing demonstrated that there is broad genetic diversity of E. coli O157:H7 in the bovine reservoir and that numerous genotypes are significantly underrepresented among clinical isolates, consistent with the possibility that there is reduced virulence or transmissibility to humans of some bovine E. coli O157:H7 genotypes. PMID:17142358

  5. Virulence characterization of non-O157 Shiga toxin-producing Escherichia coli isolates from food, humans and animals.

    PubMed

    Shen, Jinling; Rump, Lydia; Ju, Wenting; Shao, Jingdong; Zhao, Shaohua; Brown, Eric; Meng, Jianghong

    2015-09-01

    A total of 359 non-O157 STEC isolates from food, humans and animals were examined for serotypes, Shiga toxin subtypes and intimin subtypes. Isolates solely harboring stx2 from the three sources were selected for Vero cell cytotoxicity test. stx subtypes in eae negative isolates were more diverse than in eae positive isolates primarily carrying stx2a. Four eae subtypes (eaeβ,eaeε1,eaeγ1 and eaeγ2/θ) were observed and correlated with serotypes and flagella. Food isolates showed more diverse serotypes, virulence factors and cell cytotoxicities than human isolates. Some isolates from produce belonged to serotypes that have been implicated in human diseases, carried stx2a or/and stx2dact and exhibited high cell cytotoxicity similar to human isolates. This indicates that foods can be contaminated with potentially pathogenic STEC isolates that may cause human diseases. Given the increased produce consumption and growing burden of foodborne outbreaks due to produce, produce safety should be given great importance.

  6. Molecular Characterization of Human Atypical Sorbitol-Fermenting Enteropathogenic Escherichia coli O157 Reveals High Diversity.

    PubMed

    Kossow, Annelene; Zhang, Wenlan; Bielaszewska, Martina; Rhode, Sophie; Hansen, Kevin; Fruth, Angelika; Rüter, Christian; Karch, Helge; Mellmann, Alexander

    2016-05-01

    Alongside the well-characterized enterohemorrhagic Escherichia coli (EHEC) O157:H7, serogroup O157 comprises sorbitol-fermenting typical and atypical enteropathogenic E. coli (EPEC/aEPEC) strains that carry the intimin-encoding gene eae but not Shiga toxin-encoding genes (stx). Since little is known about these pathogens, we characterized 30 clinical isolates from patients with hemolytic uremic syndrome (HUS) or uncomplicated diarrhea with respect to their flagellin gene (fliC) type and multilocus sequence type (MLST). Moreover, we applied whole-genome sequencing (WGS) to determine the phylogenetic relationship with other eae-positive EHEC serotypes and the composition of the rfbO157 region. fliC typing resulted in five fliC types (H7, H16, H34, H39, and H45). Isolates of each fliC type shared a unique ST. In comparison to the 42 HUS-associated E. coli (HUSEC) strains, only the stx-negative isolates with fliCH7 shared their ST with EHEC O157:H7/H(-) strains. With the exception of one O157:H(-) fliCH16 isolate, HUS was exclusively associated with fliCH7. WGS corroborated the separation of the fliCH7 isolates, which were closely related to the EHEC O157:H7/H(-) isolates, and the diverse group of isolates exhibiting different fliC types, indicating independent evolution of the different serotypes. This was also supported by the heterogeneity within the rfbO157 region that exhibited extensive recombinations. The genotypic subtypes and distribution of clinical symptoms suggested that the stx-negative O157 strains with fliCH7 were originally EHEC strains that lost stx The remaining isolates form a distinct and diverse group of atypical EPEC isolates that do not possess the full spectrum of virulence genes, underlining the importance of identifying the H antigen for clinical risk assessment. PMID:26984976

  7. Detection and characterisation of O157:H7 and non-O157 Shiga toxin-producing Escherichia coli in wild boars.

    PubMed

    Sánchez, S; Martínez, R; García, A; Vidal, D; Blanco, J; Blanco, M; Blanco, J E; Mora, A; Herrera-León, S; Echeita, A; Alonso, J M; Rey, J

    2010-07-14

    The aim of this work was to determine the prevalence and characteristics of Escherichia coli O157:H7 and non-O157 Shiga toxin-producing E. coli (STEC) in free-ranging wild boars killed during the hunting season in southwest Spain. Faecal samples from 212 wild boars (Sus scrofa) were collected and examined for STEC. Characterisation of isolates was performed by PCR, serotyping, phage typing, and pulsed-field gel electrophoresis (PFGE). E. coli O157:H7 and non-O157 STEC were isolated from 7 (3.3%) and 11 (5.2%) animals, respectively, and the resulting 19 isolates were characterised. The PCR procedure indicated that 4 isolates carried the stx(1) gene, 12 carried the stx(2) gene, and 1 contained both of these genes. The ehxA, eae, and saa genes were detected in 13, 8, and 1 of the isolates, respectively. The eae-positive isolates comprised the types eae-gamma 1 and eae-zeta. The isolates belonged to 11 O:H serotypes, including 4 new serotypes not previously reported within STEC strains, and the majority of them were from serotypes previously associated with human infection. E. coli O157:H7 isolates belonged to phage types associated with severe human illness: PT14, PT34, and PT54. Indistinguishable PFGE types were found in E. coli O157:H7 isolates recovered from a wild boar and from a human patient with diarrhoea living in the same geographic area.

  8. Bison and bovine rectoanal junctions exhibit similar cellular architecture and Escherichia coli O157 adherence patterns

    PubMed Central

    2013-01-01

    Background Escherichia coli O157 (E. coli O157) has been isolated from bison retail meat, a fact that is important given that bison meat has been implicated in an E. coli O157-multistate outbreak. In addition, E. coli O157 has also been isolated from bison feces at slaughter and on farms. Cattle are well documented as E. coli O157 reservoirs, and the primary site of E. coli O157 persistence in such reservoirs is the rectoanal junction (RAJ), located at the distal end of the bovine gastrointestinal tract. Since bison and cattle share many genetic similarities manifested as common lineage, susceptibility to infection and the nature of immune responses to infectious agents, we decided to evaluate whether the RAJ of these animals were comparable both in terms of cellular architecture and as sites for adherence of E. coli O157. Specifically, we compared the histo-morphologies of the RAJ and evaluated the E. coli O157 adherence characteristics to the RAJ squamous epithelial (RSE) cells, from these two species. Results We found that the RAJ of both bison and cattle demonstrated similar distribution of epithelial cell markers villin, vimentin, cytokeratin, E-cadherin and N-cadherin. Interestingly, N-cadherin predominated in the stratified squamous epithelium reflecting its proliferative nature. E. coli O157 strains 86–24 SmR and EDL 933 adhered to RSE cells from both animals with similar diffuse and aggregative patterns, respectively. Conclusion Our observations further support the fact that bison are likely ‘wildlife’ reservoirs for E. coli O157, harboring these bacteria in their gastrointestinal tract. Our results also extend the utility of the RSE-cell assay, previously developed to elucidate E. coli O157-cattle RAJ interactions, to studies in bison, which are warranted to determine whether these observations in vitro correlate with those occurring in vivo at the RAJ within the bison gastrointestinal tract. PMID:24373611

  9. Influence of apple cultivars on inactivation of different strains of Escherichia coli O157:H7 in apple cider by UV irradiation.

    PubMed

    Basaran, N; Quintero-Ramos, A; Moake, M M; Churey, J J; Worobo, R W

    2004-10-01

    This study examined the effect of different apple cultivars upon the UV inactivation of Escherichia coli O157:H7 strains within unfiltered apple cider. Apple cider was prepared from eight different apple cultivars, inoculated with approximately 10(6) to 10(7) CFU of three strains of E. coli O157:H7 per ml (933, ATCC 43889, and ATCC 43895), and exposed to 14 mJ of UV irradiation per cm(2). Bacterial populations for treated and untreated samples were then enumerated by using nonselective media. E. coli O157:H7 ATCC 43889 showed the most sensitivity to this disinfection process with an average 6.63-log reduction compared to an average log reduction of 5.93 for both strains 933 and ATCC 43895. The highest log reduction seen, 7.19, occurred for strain ATCC 43889 in Rome cider. The same cider produced the lowest log reductions: 5.33 and 5.25 for strains 933 and ATCC 43895, respectively. Among the apple cultivars, an average log reduction range of 5.78 (Red Delicious) to 6.74 (Empire) was observed, with two statistically significant (alpha < or = 0.05) log reduction groups represented. Within the paired cultivar-strain analysis, five of eight ciders showed statistically significant (alpha < or = 0.05) differences in at least two of the E. coli strains used. Comparison of log reductions among the E. coli strains to the cider parameters of (o)Brix, pH, and malic acid content failed to show any statistically significant relationship (R(2) > or = 0.95). However, the results of this study indicate that regardless of the apple cultivar used, a minimum 5-log reduction is achieved for all of the strains of E. coli O157:H7 tested. PMID:15466551

  10. Influence of Apple Cultivars on Inactivation of Different Strains of Escherichia coli O157:H7 in Apple Cider by UV Irradiation

    PubMed Central

    Basaran, N.; Quintero-Ramos, A.; Moake, M. M.; Churey, J. J.; Worobo, R. W.

    2004-01-01

    This study examined the effect of different apple cultivars upon the UV inactivation of Escherichia coli O157:H7 strains within unfiltered apple cider. Apple cider was prepared from eight different apple cultivars, inoculated with approximately 106 to 107 CFU of three strains of E. coli O157:H7 per ml (933, ATCC 43889, and ATCC 43895), and exposed to 14 mJ of UV irradiation per cm2. Bacterial populations for treated and untreated samples were then enumerated by using nonselective media. E. coli O157:H7 ATCC 43889 showed the most sensitivity to this disinfection process with an average 6.63-log reduction compared to an average log reduction of 5.93 for both strains 933 and ATCC 43895. The highest log reduction seen, 7.19, occurred for strain ATCC 43889 in Rome cider. The same cider produced the lowest log reductions: 5.33 and 5.25 for strains 933 and ATCC 43895, respectively. Among the apple cultivars, an average log reduction range of 5.78 (Red Delicious) to 6.74 (Empire) was observed, with two statistically significant (α ≤ 0.05) log reduction groups represented. Within the paired cultivar-strain analysis, five of eight ciders showed statistically significant (α ≤ 0.05) differences in at least two of the E. coli strains used. Comparison of log reductions among the E. coli strains to the cider parameters of °Brix, pH, and malic acid content failed to show any statistically significant relationship (R2 ≥ 0.95). However, the results of this study indicate that regardless of the apple cultivar used, a minimum 5-log reduction is achieved for all of the strains of E. coli O157:H7 tested. PMID:15466551

  11. Isolation of Campylobacter from feral swine (Sus scrofa) on the ranch associated with the 2006 Escherichia coli O157:H7 spinach outbreak investigation in California

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We report the isolation of Campylobacter species from the same population of feral swine that was investigated in San Benito County, California during the 2006 spinach-related Escherichia coli O157:H7 outbreak. This is the first survey of Campylobacter in a free-ranging feral swine population in the...

  12. Acid Resistance and molecular characterization of Escherichia coli O157:H7 and different non-O157 Shiga toxin-producing E. coli serogroups

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to compare the acid resistance (AR) of seven non-O157 Shiga toxin-producing E. coli (STEC) strains belonging to serogroups O26, O45, O103, O104, O111, O121 and O145 with O157:H7 STEC isolated from various sources in 400 mM acetic acid solutions (AAS) at pH 3.2 and 30°...

  13. Acid resistance and molecular characterization of Escherichia coli O157:H7 and different Non-O157 shiga toxin-producing E. coli serogroups

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to compare the acid resistance (AR) of non-O157 Shiga toxin-producing Escherichia coli (STEC) strains belonging to serogroups O26, O45, O103, O104, O111, O121, and O145 with O157:H7 STEC isolated from various sources in 400 mM acetic acid solutions (AAS) at pH 3.2 and...

  14. Microbead-based immunoassay for simultaneous detection of Shiga toxins and isolation of Escherichia coli O157 in foods.

    PubMed

    Clotilde, Laurie M; Bernard, Clay; Hartman, Gary L; Lau, David K; Carter, J Mark

    2011-03-01

    Shiga toxin-producing Escherichia coli (STEC) is a significant foodborne pathogen with great economic consequences. There has been an increased food safety concern with this organism since outbreaks of human illnesses caused by this pathogen were first reported in 1982. Therefore, developing a reliable, sensitive, and rapid assay capable of detecting E. coli O157 and the main toxins produced by STEC (i.e., Shiga toxins 1 [Stx(1)] and 2 [Stx(2)]) will directly benefit regulatory agencies by minimizing analysis time. Here, we use Luminex technology to detect multiple analytes in a single 50-ml sample. Using commercially available monoclonal antibodies coupled to carboxylated magnetic microbeads, we developed an immunoassay capable of simultaneously serotyping E. coli O157 and detecting Stx(1) and/or Stx(2). The specificity and sensitivity of this immunoassay was tested against a collection of 34 E. coli isolates belonging to various O serogroups phenotypically different for Stx. The results were compared with microplate sandwich enzyme-linked immunosorbent assay (ELISA), and no cross-reactivity was observed for any of the monoclonal antibodies used. An increased sensitivity up to 1,000 times was observed in the microbead-based immunoassay when compared with the microplate sandwich ELISA. The results indicate that Luminex technology has the potential to simultaneously detect multiple targets without loss of specificity and/or sensitivity. A blind experiment was conducted with 48 samples of ground beef, lettuce, and milk spiked with ≤2 CFU/g E. coli. All the samples were correctly identified, with no false positives or false negatives. This microbead-based immunoassay could be extended to simultaneously detect additional foodborne pathogens and their toxic markers.

  15. Occurrence and survival of verocytotoxin-producing Escherichia coli O157 in raw cow's milk in The Netherlands.

    PubMed

    Heuvelink, A E; Bleumink, B; van den Biggelaar, F L; Te Giffel, M C; Beumer, R R; de Boer, E

    1998-12-01

    From May through November 1997, 1,011 samples of raw milk from bulk storage tanks were examined for the presence of verocytotoxin-producing Escherichia coli of serogroup O157 (O157 VTEC) by immunomagnetic separation following selective enrichment. The samples originated from 1,011 different dairy herds located throughout the Netherlands. O157 VTEC was not isolated from any of the milk samples examined. Additionally, survival of O157 VTEC in raw and UHT-sterilized cow's milk at 7 and 15 degrees C was studied, both in the absence and presence of an activated lactoperoxidase-thiocyanate-hydrogen peroxide system (LPS). Results indicated that the O157 VTEC strain tested was able to grow in raw milk at 7 degrees C as well as at 15 degrees C. Naturally occurring amounts of thiocyanate and hydrogen peroxide in the raw milk tested were not sufficient to activate the LPS. Although the LPS exhibited an antimicrobial activity against O157 VTEC in LPS-activated sterilized milk, O157 VTEC populations were not (or not as obviously) reduced in LPS-activated raw milk. Possibly background microflora were more sensitive to the LPS than the O157 VTEC test strain. It was concluded that raw milk contaminated with O157 VTEC will remain a hazard if kept at 7 or 15 degrees C. Effective pasteurization and avoiding postpasteurization contamination are necessary to ensure the safety of milk. PMID:9874335

  16. Virulence, Antimicrobial Resistance Properties and Phylogenetic Background of Non-H7 Enteropathogenic Escherichia coli O157

    PubMed Central

    Ferdous, Mithila; Kooistra-Smid, Anna M. D.; Zhou, Kai; Rossen, John W. A.; Friedrich, Alexander W.

    2016-01-01

    Escherichia coli (E.coli) O157 that do not produce Shiga toxin and do not possess flagellar antigen H7 are of diverse H serotypes. In this study, the antibiotic resistance properties, genotype of a set of virulence associated genes and the phylogenetic background of E. coli O157:non-H7 groups were compared. Whole genome sequencing was performed on fourteen O157:non-H7 isolates collected in the STEC-ID-net study. The genomes were compared with E. coli O157 genomes and a typical Enteropathogenic E. coli (tEPEC) genome downloaded from NCBI. Twenty-six (86%) of the analyzed genomes had the intimin encoding gene eae but of different types mostly correlating with their H types, e.g., H16, H26, H39, and H45 carried intimin type ε, β, κ, and α, respectively. They belonged to several E. coli phylogenetic groups, i.e., to phylogenetic group A, B1, B2, and D. Seven (50%) of our collected O157:non-H7 isolates were resistant to two or more antibiotics. Several mobile genetic elements, such as plasmids, insertion elements, and pathogenicity islands, carrying a set of virulence and resistance genes were found in the E. coli O157:non-H7 isolates. Core genome phylogenetic analysis showed that O157:non-H7 isolates probably evolved from different phylogenetic lineages and were distantly related to the E. coli O157:H7 lineage. We hypothesize that independent acquisition of mobile genetic elements by isolates of different lineages have contributed to the different molecular features of the O157:non-H7 strains. Although distantly related to the STEC O157, E. coli O157:non-H7 isolates from multiple genetic background could be considered as pathogen of concern for their diverse virulence and antibiotic resistance properties. PMID:27733849

  17. Growth of Stressed Strains of Four Non-O157 Shiga Toxin-Producing Escherichia coli Serogroups in Five Enrichment Broths.

    PubMed

    Verhaegen, Bavo; De Reu, Koen; Heyndrickx, Marc; Van Damme, Inge; De Zutter, Lieven

    2015-11-01

    The purpose of this study was to evaluate (i) the behavior of several strains of non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups (O26, O103, O111, and O145) exposed to different stress conditions and (ii) the growth dynamics of stressed and nonstressed non-O157 STEC cells in five enrichment media. STEC strains were exposed to acid, cold, and freeze stresses. Lethal and sublethal injuries were determined by plating in parallel on selective and nonselective agar media. Freeze stress (8 days, 20°C) caused the most lethal (95.3% ± 2.5%) injury, as well as the most sublethal (89.1% ± 8.8%) injury in the surviving population. Growth of stressed and nonstressed pure cultures of non-O157 STEC on modified tryptic soy broth, buffered peptone water (BPW), BPW with sodium pyruvate, Brila, and STEC enrichment broth (SEB) was determined using total viable counts. To compare growth capacities, growth after 7 and 24 h of enrichment was measured; lag phases and maximum growth rates were also calculated. In general, growth on BPW resulted in a short lag phase followed by a high maximum growth rate during the enrichment of all tested strains when using all three stress types. Furthermore, BPW ensured the highest STEC count after 7 h of growth. Supplementing the medium with sodium pyruvate did not improve the growth dynamics. The two selective media, Brila and SEB, were less efficient than BPW, but Brila's enrichment performance was remarkably better than that of SEB. This study shows that irrespective of the effect of background flora, BPW is still recommended for resuscitation of non-O157 STEC.

  18. Escherichia coli O157:H7: Animal Reservoir and Sources of Human Infection

    PubMed Central

    Ferens, Witold A.

    2011-01-01

    Abstract This review surveys the literature on carriage and transmission of enterohemorrhagic Escherichia coli (EHEC) O157:H7 in the context of virulence factors and sampling/culture technique. EHEC of the O157:H7 serotype are worldwide zoonotic pathogens responsible for the majority of severe cases of human EHEC disease. EHEC O157:H7 strains are carried primarily by healthy cattle and other ruminants, but most of the bovine strains are not transmitted to people, and do not exhibit virulence factors associated with human disease. Prevalence of EHEC O157:H7 is probably underestimated. Carriage of EHEC O157:H7 by individual animals is typically short-lived, but pen and farm prevalence of specific isolates may extend for months or years and some carriers, designated as supershedders, may harbor high intestinal numbers of the pathogen for extended periods. The prevalence of EHEC O157:H7 in cattle peaks in the summer and is higher in postweaned calves and heifers than in younger and older animals. Virulent strains of EHEC O157:H7 are rarely harbored by pigs or chickens, but are found in turkeys. The bacteria rarely occur in wildlife with the exception of deer and are only sporadically carried by domestic animals and synanthropic rodents and birds. EHEC O157:H7 occur in amphibian, fish, and invertebrate carriers, and can colonize plant surfaces and tissues via attachment mechanisms different from those mediating intestinal attachment. Strains of EHEC O157:H7 exhibit high genetic variability but typically a small number of genetic types predominate in groups of cattle and a farm environment. Transmission to people occurs primarily via ingestion of inadequately processed contaminated food or water and less frequently through contact with manure, animals, or infected people. PMID:21117940

  19. Isolation of Escherichia coli 0157:H7 strain from fecal samples of zoo animal.

    PubMed

    Mohammed Hamzah, Aseel; Mohammed Hussein, Aseel; Mahmoud Khalef, Jenan

    2013-01-01

    The isolation and characterization of Escherichia coli O157:H7 strains from 22 out of 174 fecal samples from petting zoo animals representing twenty-two different species (camel, lion, goats, zebra, bear, baboon monkey, Siberian monkey, deer, elk, llama, pony, horses, fox, kangaroo, wolf, porcupine, chickens, tiger, ostrich, hyena, dogs, and wildcats) were investigated. One petting Al-Zawraa zoological society of Baghdad was investigated for E. coli O157:H7 over a 16-month period that spanned two summer and two autumn seasons. Variation in the occurrence of E. coli O157:H7-positive petting zoo animals was observed, with animals being culture positive only in the summer months but not in the spring, autumn, or winter. E. coli O157:H7 isolates were distinguished by agglutination with E. coli O157:H7 latex reagent (Oxoid), identified among the isolates, which showed that multiple E. coli strains were isolated from one petting zoo animal, in which a single animal simultaneously shed multiple E. coli strains; E. coli O157:H7 was isolated only by selective enrichment culture of 2 g of petting zoo animal feces. In contrast, strains other than O157:H7 were cultured from feces of petting zoo animals without enrichment.

  20. Antibacterial effects of natural tenderizing enzymes on different strains of Escherichia coli O157:H7 and Listeria monocytogenes on beef.

    PubMed

    Eshamah, Hanan; Han, Inyee; Naas, Hesham; Acton, James; Dawson, Paul

    2014-04-01

    This study determined the efficacy of actinidin and papain on reducing Listeria monocytogenes and three mixed strains of Escherichia coli O157:H7 populations on beef. The average reduction of E. coli O157:H7 was greater than that of L. monocytogenes and higher concentrations of either protease yielded greater reduction in bacterial populations. For instance, actinidin at 700 mg/ml significantly (p≤0.05) reduced the population of L. monocytogenes by 1.49 log cfu/ml meat rinse after 3h at 25 & 35 °C, and by 1.45 log cfu/ml rinse after 24h at 5 °C, while the same actinidin concentration significantly reduced the populations of three mixed strains of E. coli O157:H7 by 1.81 log cfu/ml rinse after 3h at 25 & 35 °C, and 1.94 log cfu/ml rinse after 24h at 5 °C. These findings suggest that, in addition to improving the sensory attributes of beef, proteolytic enzymes can enhance meat safety when stored at suitable temperatures. PMID:24447905

  1. Mechanisms behind tailing in the pressure inactivation curve of a clinical isolate of Escherichia coli O157:H7.

    PubMed

    Noma, Seiji; Kajiyama, Daiki; Igura, Noriyuki; Shimoda, Mitsuya; Hayakawa, Isao

    2006-05-25

    The tailing in pressure inactivation curve of clinically isolated Escherichia coli O157:H7 was investigated. A typical tailing was observed after the treatment period for 30min when 10(7) CFU/ml of the cell suspension was subjected to pressure treatment at 300MPa and 25 degrees Celsius. There was no effect on the tailing profiles by the addition of pressure-killed cells and released cellular components. When cells survived at a tail portion were re-propagated (tail-culture) and subjected to second pressure treatment, the cells of the tail-culture exhibited eminently higher barotolerance compared to those of the original-culture, suggesting that the presence of genetically pressure-resistant subpopulation was responsible for the tailing. The cytoplasmic membrane of the tail-culture cells had higher stability to a pressure treatment at 100MPa for 10min than that of the original-culture, which was evidenced by lower permeability to ethidium bromide. The addition of non-ionic surfactants including 0.5microl/ml polyoxyethylene p-t-octylphenyl ester (Triton X-100) and 0.53mg/ml lauric sugar ester dramatically reduced the level of tailing and made the inactivation curve linear.

  2. Inactivation of escherichia coli O157:H7 in cattle drinking water by sodium caprylate.

    PubMed

    Amalaradjou, Mary Anne Roshni; Annamalai, Thirunavukkarasu; Marek, Patrick; Rezamand, Pedram; Schreiber, David; Hoagland, Thomas; Venkitanarayanan, Kumar

    2006-09-01

    Escherichia coli O157:H7 is an important foodborne pathogen. Cattle serve as one of the major reservoirs of E. coli O157:H7, excreting the pathogen in feces. Environmental persistence of E. coli O157:H7 is critical in its epidemiology on farms, and the pathogen has been isolated from cattle water troughs. Thus, there is a need for an effective method for killing E. coli O157:H7 in cattle drinking water. In this study, the efficacy of sodium caprylate for killing E. coli O157:H7 in cattle drinking water was investigated. A four-strain mixture of E. coli O157:H7 was inoculated (6.0 log CFU/ml) into 100-ml samples of well water containing 0, 75, 100, or 120 mM sodium caprylate. Water samples containing 1% (wt/vol) bovine feces or feed also were included. The samples were incubated at 21 or 8 degrees C for 21 days. Water samples were analyzed for viable E. coli O157:H7 on days 0, 1, 3, 5, and 7 and weekly thereafter. Triplicate samples of each treatment and control were included, and the study was repeated twice. The magnitude of E. coli O157:H7 inactivation in water significantly increased (P < 0.01) with increases in caprylate concentration and storage temperature. At 120 mM, sodium caprylate completely inactivated E. coli O157:H7 in all the samples after 1 to 20 days, depending on the treatments. The presence of feces or feed also had a significant effect (P < 0.01) on the antibacterial property of caprylate; the presence of feces decreased the antibacterial effect, whereas addition of feed enhanced the effect. These results indicate that sodium caprylate is effective in killing E. coli O157:H7 in cattle drinking water, but detailed cattle palatability studies of water containing caprylate are necessary.

  3. Support Vector Machine applied to predict the zoonotic potential of E. coli O157 cattle isolates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Methods based on sequence data analysis facilitate the tracking of disease outbreaks, allow relationships between strains to be reconstructed and virulence factors to be identified. However, these methods are used postfactum after an outbreak has happened. Here, we show that support vector machine a...

  4. Isolation of Multidrug-Resistant Escherichia coli O157 from Goats in the Somali Region of Ethiopia: A Cross-Sectional, Abattoir-Based Study.

    PubMed

    Dulo, Fitsum; Feleke, Aklilu; Szonyi, Barbara; Fries, Reinhard; Baumann, Maximilian P O; Grace, Delia

    2015-01-01

    Toxigenic Escherichia coli (E. coli) are an important cause of gastroenteritis in developing countries. In Ethiopia, gastroenteritis due to food-borne disease is a leading cause of death. Yet, there is no surveillance for E. coli O157 and little is known about the carriage of this pathogen in Ethiopia's livestock. This study aimed to assess the prevalence and levels of antimicrobial resistance of E. coli O157 in goat meat, feces, and environmental samples collected at a large abattoir in the Somali region of Ethiopia. The samples were enriched in modified tryptone broth containing novobiocin, and plated onto sorbitol MacConkey agar. Isolates were confirmed using indole test and latex agglutination. Antimicrobial susceptibility testing was conducted using the disk diffusion method. A total of 235 samples, including 93 goat carcass swabs, 93 cecal contents, 14 water, 20 hand, and 15 knife swabs were collected. Overall, six (2.5%) samples were contaminated with E. coli O157 of which two (2.1%) were isolated from cecal contents, three (3.2%) from carcass swabs, and one (7.1%) from water. All isolates were resistant to at least two of the 18 antimicrobials tested. Two isolates (33.3%) were resistant to more than five antimicrobials. Abattoir facilities and slaughter techniques were conducive to carcass contamination. This study highlights how poor hygiene and slaughter practice can result in contaminated meat, which is especially risky in Ethiopia because of the common practice of eating raw meat. We detect multi-resistance to drugs not used in goats, suggesting that drugs used to treat human infections may be the originators of antimicrobial resistance in livestock in this ecosystem. The isolation of multidrug-resistant E. coli O157 from goats from a remote pastoralist system highlights the need for global action on regulating and monitoring antimicrobial use in both human and animal populations. PMID:26561414

  5. Isolation of Multidrug-Resistant Escherichia coli O157 from Goats in the Somali Region of Ethiopia: A Cross-Sectional, Abattoir-Based Study

    PubMed Central

    Dulo, Fitsum; Feleke, Aklilu; Szonyi, Barbara; Fries, Reinhard; Baumann, Maximilian P. O.; Grace, Delia

    2015-01-01

    Toxigenic Escherichia coli (E. coli) are an important cause of gastroenteritis in developing countries. In Ethiopia, gastroenteritis due to food-borne disease is a leading cause of death. Yet, there is no surveillance for E. coli O157 and little is known about the carriage of this pathogen in Ethiopia’s livestock. This study aimed to assess the prevalence and levels of antimicrobial resistance of E. coli O157 in goat meat, feces, and environmental samples collected at a large abattoir in the Somali region of Ethiopia. The samples were enriched in modified tryptone broth containing novobiocin, and plated onto sorbitol MacConkey agar. Isolates were confirmed using indole test and latex agglutination. Antimicrobial susceptibility testing was conducted using the disk diffusion method. A total of 235 samples, including 93 goat carcass swabs, 93 cecal contents, 14 water, 20 hand, and 15 knife swabs were collected. Overall, six (2.5%) samples were contaminated with E. coli O157 of which two (2.1%) were isolated from cecal contents, three (3.2%) from carcass swabs, and one (7.1%) from water. All isolates were resistant to at least two of the 18 antimicrobials tested. Two isolates (33.3%) were resistant to more than five antimicrobials. Abattoir facilities and slaughter techniques were conducive to carcass contamination. This study highlights how poor hygiene and slaughter practice can result in contaminated meat, which is especially risky in Ethiopia because of the common practice of eating raw meat. We detect multi-resistance to drugs not used in goats, suggesting that drugs used to treat human infections may be the originators of antimicrobial resistance in livestock in this ecosystem. The isolation of multidrug-resistant E. coli O157 from goats from a remote pastoralist system highlights the need for global action on regulating and monitoring antimicrobial use in both human and animal populations. PMID:26561414

  6. Isolation of Multidrug-Resistant Escherichia coli O157 from Goats in the Somali Region of Ethiopia: A Cross-Sectional, Abattoir-Based Study.

    PubMed

    Dulo, Fitsum; Feleke, Aklilu; Szonyi, Barbara; Fries, Reinhard; Baumann, Maximilian P O; Grace, Delia

    2015-01-01

    Toxigenic Escherichia coli (E. coli) are an important cause of gastroenteritis in developing countries. In Ethiopia, gastroenteritis due to food-borne disease is a leading cause of death. Yet, there is no surveillance for E. coli O157 and little is known about the carriage of this pathogen in Ethiopia's livestock. This study aimed to assess the prevalence and levels of antimicrobial resistance of E. coli O157 in goat meat, feces, and environmental samples collected at a large abattoir in the Somali region of Ethiopia. The samples were enriched in modified tryptone broth containing novobiocin, and plated onto sorbitol MacConkey agar. Isolates were confirmed using indole test and latex agglutination. Antimicrobial susceptibility testing was conducted using the disk diffusion method. A total of 235 samples, including 93 goat carcass swabs, 93 cecal contents, 14 water, 20 hand, and 15 knife swabs were collected. Overall, six (2.5%) samples were contaminated with E. coli O157 of which two (2.1%) were isolated from cecal contents, three (3.2%) from carcass swabs, and one (7.1%) from water. All isolates were resistant to at least two of the 18 antimicrobials tested. Two isolates (33.3%) were resistant to more than five antimicrobials. Abattoir facilities and slaughter techniques were conducive to carcass contamination. This study highlights how poor hygiene and slaughter practice can result in contaminated meat, which is especially risky in Ethiopia because of the common practice of eating raw meat. We detect multi-resistance to drugs not used in goats, suggesting that drugs used to treat human infections may be the originators of antimicrobial resistance in livestock in this ecosystem. The isolation of multidrug-resistant E. coli O157 from goats from a remote pastoralist system highlights the need for global action on regulating and monitoring antimicrobial use in both human and animal populations.

  7. Escherichia coli O157 in ground beef from local retail markets in Pachuca, Mexico.

    PubMed

    Gómez-Aldapa, Carlos A; Díaz-Cruz, Claudio A; Cerna-Cortes, Jorge F; Torres-Vitela, M Del Refugio; Villarruel-López, Angelica; Rangel-Vargas, Esmeralda; Castro-Rosas, Javier

    2013-04-01

    Escherichia coli O157 strains have been recognized as pathogenic bacteria, of which raw beef is a known vehicle. An evaluation was done of the presence of E. coli O157 in ground beef from local retail markets in Pachuca, Hidalgo State, Mexico. A total of 120 ground beef samples (500 g) were tested for E. coli O157 by simultaneous application of the U. S. Department of Agriculture, Food Safety and Inspection Service (FSIS)'s Microbiology Laboratory Guidebook culture procedure 5.05, and two commercial kits, Reveal for E. coli O157:H7 and Visual Immunoprecipitate Assay (VIP) Gold for enterohemorrhagic E. coli. Two incubation times (8 and 20 h) were used with the commercial kits. Presence of stx1, stx2, and eaeA loci was determined by multiplex PCR. Of 360 subsamples (120 per procedure), 12 samples were found to be E. coli O157 positive by the FSIS culture method. With VIP, 73 subsamples were presumptive positive after 8 h of enrichment, and 60 were presumptive positive after 20 h of enrichment. Of these, only 6 (8 h) and 8 (20 h) subsamples were confirmed true positives with the FSIS method. With Reveal, 60 subsamples were presumptive positive after 8 h of enrichment and 50 were presumptive positive after 20 h of enrichment. Of these, only 6 (8 h) and 8 (20 h) subsamples were confirmed as true positives with the FSIS method. A total of 57 E. coli O157:H7 and 21 E. coli O157 strains were isolated. None of the O157 or O157:H7 strains had stx1 or stx2 loci, and only one had the eaeA locus. To our knowledge, this is the first report of the presence of E. coli O157 in commercial ground beef from Mexico, and the first report of isolation of a large number of stx-negative E. coli O157 and E. coli O157:H7 strains in Mexico.

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

    PubMed

    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.

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

    PubMed

    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. PMID:27497123

  10. Phenotypic antibiotic resistance of Escherichia coli and E. coli O157 isolated from water, sediment and biofilms in an agricultural watershed in British Columbia.

    PubMed

    Maal-Bared, Rasha; Bartlett, Karen H; Bowie, William R; Hall, Eric R

    2013-01-15

    This study examined the distribution of antibiotic resistant Escherichia coli and E. coli O157 isolated from water, sediment and biofilms in an intensive agricultural watershed (Elk Creek, British Columbia) between 2005 and 2007. It also examined physical and chemical water parameters associated with antibiotic resistance. Broth microdilution techniques were used to determine minimum inhibitory concentrations (MIC) for E. coli (n=214) and E. coli O157 (n=27) recovered isolates for ampicillin, cefotaxime, ciprofloxacin, nalidixic acid, streptomycin and tetracycline. Both E. coli and E. coli O157 isolates showed highest frequency of resistance to tetracycline, ampicillin, streptomycin and nalidixic acid; respectively. For E. coli, the highest frequency of resistance was observed at the most agriculturally-impacted site, while the lowest frequency of resistance was found at the headwaters. Sediment and river rock biofilms were the most likely to be associated with resistant E. coli, while water was the least likely. While seasonality (wet versus dry) had no relationship with resistance frequency, length of biofilm colonization of the substratum in the aquatic environment only affected resistance frequency to nalidixic acid and tetracycline. Multivariate logistic regressions showed that water depth, nutrient concentrations, temperature, dissolved oxygen and salinity had statistically significant associations with frequency of E. coli resistance to nalidixic acid, streptomycin, ampicillin and tetracycline. The results indicate that antibiotic resistant E. coli and E. coli O157 were prevalent in an agricultural stream. Since E. coli is adept at horizontal gene transfer and prevalent in biofilms and sediment, where ample opportunities for genetic exchange with potential environmental pathogens present themselves, resistant isolates may present a risk to ecosystem, wildlife and public health.

  11. Sequence of Colonization Determines the Composition of Mixed Biofilms by Escherichia coli O157:H7 and O111:H8 Strains.

    PubMed

    Wang, Rong; Kalchayanand, Norasak; Bono, James L

    2015-08-01

    Bacterial biofilms are one of the potential sources of cross-contamination in food processing environments. Shiga toxin-producing Escherichia coli (STEC) O157:H7 and O111:H8 are important foodborne pathogens capable of forming biofilms, and the coexistence of these two STEC serotypes has been detected in various food samples and in multiple commercial meat plants throughout the United States. Here, we investigated how the coexistence of these two STEC serotypes and their sequence of colonization could affect bacterial growth competition and mixed biofilm development. Our data showed that E. coli O157:H7 strains were able to maintain a higher cell percentage in mixed biofilms with the co-inoculated O111:H8 companion strains, even though the results of planktonic growth competition were strain dependent. On solid surfaces with preexisting biofilms, the sequence of colonization played a critical role in determining the composition of the mixed biofilms because early stage precolonization significantly affected the competition results between the E. coli O157:H7 and O111:H8 strains. The precolonizer of either serotype was able to outgrow the other serotype in both planktonic and biofilm phases. The competitive interactions among the various STEC serotypes would determine the composition and structure of the mixed biofilms as well as their potential risks to food safety and public health, which is largely influenced by the dominant strains in the mixtures. Thus, the analysis of mixed biofilms under various conditions would be of importance to determine the nature of mixed biofilms composed of multiple microorganisms and to help implement the most effective disinfection operations accordingly.

  12. Bison and bovine rectoanal junctions exhibit similar cellular architecture and Escherichia coli O157 adherence patterns

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli O157 (O157) is frequently isolated from bison retail meat, a fact that is important given that bison meat has also been implicated in an O157-multistate outbreak. In addition, O157 has also been isolated from bison feces at slaughter and on farms. Cattle are well documented as O15...

  13. Nutritional basis for colonization resistance by human commensal Escherichia coli strains HS and Nissle 1917 against E. coli O157:H7 in the mouse intestine.

    PubMed

    Maltby, Rosalie; Leatham-Jensen, Mary P; Gibson, Terri; Cohen, Paul S; Conway, Tyrrell

    2013-01-01

    Escherichia coli is a single species consisting of many biotypes, some of which are commensal colonizers of mammals and others that cause disease. Humans are colonized on average with five commensal biotypes, and it is widely thought that the commensals serve as a barrier to infection by pathogens. Previous studies showed that a combination of three pre-colonized commensal E. coli strains prevents colonization of E. coli O157:H7 in a mouse model (Leatham, et al., 2010, Infect Immun 77: 2876-7886). The commensal biotypes included E. coli HS, which is known to successfully colonize humans at high doses with no adverse effects, and E. coli Nissle 1917, a human commensal strain that is used in Europe as a preventative of traveler's diarrhea. We hypothesized that commensal biotypes could exert colonization resistance by consuming nutrients needed by E. coli O157:H7 to colonize, thus preventing this first step in infection. Here we report that to colonize streptomycin-treated mice E. coli HS consumes six of the twelve sugars tested and E. coli Nissle 1917 uses a complementary yet divergent set of seven sugars to colonize, thus establishing a nutritional basis for the ability of E. coli HS and Nissle 1917 to occupy distinct niches in the mouse intestine. Together these two commensals use the five sugars previously determined to be most important for colonization of E. coli EDL933, an O157:H7 strain. As predicted, the two commensals prevented E. coli EDL933 colonization. The results support a model in which invading pathogenic E. coli must compete with the gut microbiota to obtain the nutrients needed to colonize and establish infection; accordingly, the outcome of the challenge is determined by the aggregate capacity of the native microbiota to consume the nutrients required by the pathogen. PMID:23349773

  14. Nutritional basis for colonization resistance by human commensal Escherichia coli strains HS and Nissle 1917 against E. coli O157:H7 in the mouse intestine.

    PubMed

    Maltby, Rosalie; Leatham-Jensen, Mary P; Gibson, Terri; Cohen, Paul S; Conway, Tyrrell

    2013-01-01

    Escherichia coli is a single species consisting of many biotypes, some of which are commensal colonizers of mammals and others that cause disease. Humans are colonized on average with five commensal biotypes, and it is widely thought that the commensals serve as a barrier to infection by pathogens. Previous studies showed that a combination of three pre-colonized commensal E. coli strains prevents colonization of E. coli O157:H7 in a mouse model (Leatham, et al., 2010, Infect Immun 77: 2876-7886). The commensal biotypes included E. coli HS, which is known to successfully colonize humans at high doses with no adverse effects, and E. coli Nissle 1917, a human commensal strain that is used in Europe as a preventative of traveler's diarrhea. We hypothesized that commensal biotypes could exert colonization resistance by consuming nutrients needed by E. coli O157:H7 to colonize, thus preventing this first step in infection. Here we report that to colonize streptomycin-treated mice E. coli HS consumes six of the twelve sugars tested and E. coli Nissle 1917 uses a complementary yet divergent set of seven sugars to colonize, thus establishing a nutritional basis for the ability of E. coli HS and Nissle 1917 to occupy distinct niches in the mouse intestine. Together these two commensals use the five sugars previously determined to be most important for colonization of E. coli EDL933, an O157:H7 strain. As predicted, the two commensals prevented E. coli EDL933 colonization. The results support a model in which invading pathogenic E. coli must compete with the gut microbiota to obtain the nutrients needed to colonize and establish infection; accordingly, the outcome of the challenge is determined by the aggregate capacity of the native microbiota to consume the nutrients required by the pathogen.

  15. Prevalence of Escherichia coli O157:H7 in surface water near cattle feedlots.

    PubMed

    Tanaro, José D; Piaggio, Mercedes C; Galli, Lucía; Gasparovic, Alejandra M C; Procura, Francisco; Molina, Demián A; Vitón, Mauro; Zolezzi, Gisela; Rivas, Marta

    2014-12-01

    Between April 2009 and July 2011, 311 surface water samples in 48 cattle feedlots distributed in an area of about 67,000 km(2) were analyzed to examine the environmental dissemination of Escherichia coli O157:H7. Samples were taken inside and outside the pens, exposed and not exposed to runoff from corrals, near the feedlots. Two types of samples were defined: (1) exposed surface waters (ESW; n=251), downstream from cattle pens; and (2) nonexposed surface waters (NESW; n=60), upstream from cattle pens. By multiplex PCR, 177 (70.5%) ESW samples were rfb(O157)-positive, and 62 (24.7%) E. coli O157, and 32 (12.7%) Shiga toxin-producing E. coli (STEC) O157:H7 strains were isolated. In the NESW samples, 36 (60.0%) were rfb(O157)- positive, and 9 (15.0%) E. coli O157, and 6 (10.0%) STEC O157:H7 strains were isolated. These results showed that the environmental surface waters exposed to liquid discharges from intensive livestock operations tended to be contaminated with more STEC O157:H7 than NESW. However, no significant difference was found. This fact emphasizes the relevance of other horizontal routes of transmission, as the persistence of E. coli in the environment resulting from extensive livestock farming. By XbaI-PFGE, some patterns identified are included in the Argentine Database of E. coli O157, corresponding to strains isolated from hemolytic uremic syndrome and diarrhea cases, food, and animals, such as AREXHX01.0022, second prevalent pattern in Argentina, representing 5.5% of the total database. In the study area, characterized by the abundance of waterways, pathogens contained in feedlot runoff could reach recreational waters and also contaminate produce through irrigation, increasing the potential dissemination of STEC O157:H7 and the risk of human infections. The control of runoff systems from intensive livestock is necessary, but other alternatives should be explored to solve the problem of the presence of E. coli O157 in the aquatic rural environment

  16. Prevalence of Escherichia coli O157:H7 in surface water near cattle feedlots.

    PubMed

    Tanaro, José D; Piaggio, Mercedes C; Galli, Lucía; Gasparovic, Alejandra M C; Procura, Francisco; Molina, Demián A; Vitón, Mauro; Zolezzi, Gisela; Rivas, Marta

    2014-12-01

    Between April 2009 and July 2011, 311 surface water samples in 48 cattle feedlots distributed in an area of about 67,000 km(2) were analyzed to examine the environmental dissemination of Escherichia coli O157:H7. Samples were taken inside and outside the pens, exposed and not exposed to runoff from corrals, near the feedlots. Two types of samples were defined: (1) exposed surface waters (ESW; n=251), downstream from cattle pens; and (2) nonexposed surface waters (NESW; n=60), upstream from cattle pens. By multiplex PCR, 177 (70.5%) ESW samples were rfb(O157)-positive, and 62 (24.7%) E. coli O157, and 32 (12.7%) Shiga toxin-producing E. coli (STEC) O157:H7 strains were isolated. In the NESW samples, 36 (60.0%) were rfb(O157)- positive, and 9 (15.0%) E. coli O157, and 6 (10.0%) STEC O157:H7 strains were isolated. These results showed that the environmental surface waters exposed to liquid discharges from intensive livestock operations tended to be contaminated with more STEC O157:H7 than NESW. However, no significant difference was found. This fact emphasizes the relevance of other horizontal routes of transmission, as the persistence of E. coli in the environment resulting from extensive livestock farming. By XbaI-PFGE, some patterns identified are included in the Argentine Database of E. coli O157, corresponding to strains isolated from hemolytic uremic syndrome and diarrhea cases, food, and animals, such as AREXHX01.0022, second prevalent pattern in Argentina, representing 5.5% of the total database. In the study area, characterized by the abundance of waterways, pathogens contained in feedlot runoff could reach recreational waters and also contaminate produce through irrigation, increasing the potential dissemination of STEC O157:H7 and the risk of human infections. The control of runoff systems from intensive livestock is necessary, but other alternatives should be explored to solve the problem of the presence of E. coli O157 in the aquatic rural environment.

  17. Occurrence of Verocytotoxin-Producing Escherichia coli O157 on Dutch Dairy Farms

    PubMed Central

    Heuvelink, A. E.; van den Biggelaar, F. L. A. M.; Zwartkruis-Nahuis, J. T. M.; Herbes, R. G.; Huyben, R.; Nagelkerke, N.; Melchers, W. J. G.; Monnens, L. A. H.; de Boer, E.

    1998-01-01

    During the period from September 1996 through November 1996, 10 Dutch dairy farms were visited to collect fecal samples from all cattle present. The samples were examined for the presence of verocytotoxin (VT)-producing Escherichia coli (VTEC) of serogroup O157 (O157 VTEC) by immunomagnetic separation following selective enrichment. Cattle on 7 of the 10 dairy farms tested positive for O157 VTEC, with the proportion of cattle infected varying from 0.8 to 22.4%. On the seven farms positive for O157 VTEC, the excretion rate was highest in calves ages 4 to 12 months (21.2%). In a follow-up study, two O157 VTEC-positive farms and two O157 VTEC-negative farms identified in the prevalence study were revisited five times at intervals of approximately 3 months. Cattle on each farm tested positive at least once. The proportion of cattle infected varied from 0 to 61.0%. Excretion rates peaked in summer and were lowest in winter. Again, the highest prevalence was observed in calves ages 4 to 12 months (11.8%). O157 VTEC strains were also isolated from fecal samples from horses, ponies, and sheep and from milk filters and stable flies. O157 VTEC isolates were characterized by VT production and type, the presence of the E. coli attaching-and-effacing gene, phage type, and pulsed-field gel electrophoretic genotype. No overlapping strain types were identified among isolates from different farms except one. The predominance of a single type at each sampling suggests that horizontal transmission is an important factor in dissemination of O157 VTEC within a farm. The presence of more than one strain type, both simultaneously and over time, suggests that there was more than one source of O157 VTEC on the farms. Furthermore, this study demonstrated that the O157 VTEC status of a farm cannot be ascertained from a single visit testing a small number of cattle. PMID:9817858

  18. Occurrence of verocytotoxin-producing Escherichia coli O157 on Dutch dairy farms.

    PubMed

    Heuvelink, A E; van den Biggelaar, F L; Zwartkruis-Nahuis, J; Herbes, R G; Huyben, R; Nagelkerke, N; Melchers, W J; Monnens, L A; de Boer, E

    1998-12-01

    During the period from September 1996 through November 1996, 10 Dutch dairy farms were visited to collect fecal samples from all cattle present. The samples were examined for the presence of verocytotoxin (VT)-producing Escherichia coli (VTEC) of serogroup O157 (O157 VTEC) by immunomagnetic separation following selective enrichment. Cattle on 7 of the 10 dairy farms tested positive for O157 VTEC, with the proportion of cattle infected varying from 0.8 to 22.4%. On the seven farms positive for O157 VTEC, the excretion rate was highest in calves ages 4 to 12 months (21.2%). In a follow-up study, two O157 VTEC-positive farms and two O157 VTEC-negative farms identified in the prevalence study were revisited five times at intervals of approximately 3 months. Cattle on each farm tested positive at least once. The proportion of cattle infected varied from 0 to 61.0%. Excretion rates peaked in summer and were lowest in winter. Again, the highest prevalence was observed in calves ages 4 to 12 months (11.8%). O157 VTEC strains were also isolated from fecal samples from horses, ponies, and sheep and from milk filters and stable flies. O157 VTEC isolates were characterized by VT production and type, the presence of the E. coli attaching-and-effacing gene, phage type, and pulsed-field gel electrophoretic genotype. No overlapping strain types were identified among isolates from different farms except one. The predominance of a single type at each sampling suggests that horizontal transmission is an important factor in dissemination of O157 VTEC within a farm. The presence of more than one strain type, both simultaneously and over time, suggests that there was more than one source of O157 VTEC on the farms. Furthermore, this study demonstrated that the O157 VTEC status of a farm cannot be ascertained from a single visit testing a small number of cattle. PMID:9817858

  19. Surface properties of the Vero cytotoxin-producing Escherichia coli O157:H7.

    PubMed Central

    Sherman, P; Soni, R; Petric, M; Karmali, M

    1987-01-01

    Strains of Escherichia coli serotype O157:H7 are Vero cytotoxin-producing enteric pathogens which have been associated with sporadic cases and outbreaks of hemorrhagic colitis and with the hemolytic uremic syndrome in humans. In addition to toxin production, adherence of many pathogenic bacteria to intestinal mucosal surfaces is a critical primary step in the pathogenesis of diarrheal diseases. Although E. coli serotype O157:H7 organisms adhere to intestinal epithelia of orally infected animals in a pattern morphologically identical to that previously described in adherent, effacing E. coli infections, the mechanisms of bacterial adherence are not known. To determine the cell surface adhesins which mediate attachment of E. coli O157:H7 to epithelial surfaces, we evaluated the surface properties of these organisms. Five strains isolated from children with the hemolytic uremic syndrome were grown both in broth cultures and on agar media. Adherence and invasion of E. coli O157:H7 in Intestine 407 and HEp-2 epithelial cell lines was quantitated using an enteroinvasive E. coli strain (serotype O164:NM) as a control. Cell surface properties of E. coli O157:H7 were evaluated by agglutination of a series of erythrocytes, transmission electron microscopy, DEAE-ion-exchange chromatography, and hydrophobic interaction chromatography. E. coli O157:H7 strains adhered to but did not invade either Intestine 407 or HEp-2 cells. Homologous O157:H7 rabbit antiserum blocked attachment of bacteria to tissue culture cells, in contrast to heterologous antiserum and preimmune rabbit serum, which did not inhibit attachment of E. coli O157:H7. None of the five O15:H7 isolates mediated mannose-resistant hemagglutination under any of the in vitro culture conditions. One isolate mediated mannose-sensitive hemagglutination after serial passage in broth cultures. Pili and fibrillae were not visualized by electron microscopy on nonhemagglutinating organisms, but pili were demonstrated on the one

  20. Distribution of IS629 and stx genotypes among enterohemorrhagic Escherichia coli O157 isolates in Yamaguchi Prefecture, Japan, 2004-2013.

    PubMed

    Kameyama, Mitsuhiro; Tominaga, Kiyoshi; Yabata, Junko; Nomura, Yasuharu

    2015-11-01

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

  1. Isolation and molecular characterization of Salmonella enterica, Escherichia coli O157:H7 and Shigella spp. from meat and dairy products in Egypt.

    PubMed

    Ahmed, Ashraf M; Shimamoto, Tadashi

    2014-01-01

    Foodborne pathogens are a major threat to food safety, especially in developing countries where hygiene and sanitation facilities are often poor. Salmonella enterica, Escherichia coli O157:H7 and Shigella spp. are among the major causes of outbreaks of foodborne diseases. This large-scale study investigated the prevalence of these foodborne pathogens in meat (beef and chicken) and dairy products collected from street vendors, butchers, retail markets and slaughterhouses in Egypt. A total of 1600 food samples (800 meat products and 800 dairy products) were analyzed using culture and PCR based methods. S. enterica, E. coli O157:H7 and Shigella spp. were detected in 69 (4.3%), 54 (3.4%) and 27 (1.7%) samples respectively. S. enterica serovar Typhimurium, S. enterica serovar Enteritidis, S. enterica serovar Infantis and non-typable serovars were detected in 28 (1.8%), 22 (1.4%), 16 (1.0%) and 3 (0.1%) samples respectively. All E. coli O157:H7 isolates were positive for stx1 and/or stx2 virulence toxin genes. Shigella flexneri, Shigella sonnei and Shigella dysenteriae were detected in 18 (1.2%), 7 (0.4%) and 2 (0.1%) samples respectively. The incidences of S. enterica and Shigella spp. were higher in meat products (53; 6.6% and 16; 2.0%, respectively) than in dairy products (16; 2.0% and 11; 1.4%, respectively), while, E. coli O157:H7 was higher in dairy products (29; 3.6%) than in meat products (25; 3.1%). The incidence of foodborne pathogens in meat and dairy products was determined in a large-scale survey in Africa.

  2. Quantitative risk assessment of haemolytic and uremic syndrome linked to O157:H7 and non-O157:H7 Shiga-toxin producing Escherichia coli strains in raw milk soft cheeses.

    PubMed

    Perrin, Frédérique; Tenenhaus-Aziza, Fanny; Michel, Valérie; Miszczycha, Stéphane; Bel, Nadège; Sanaa, Moez

    2015-01-01

    Shiga-toxin producing Escherichia coli (STEC) strains may cause human infections ranging from simple diarrhea to Haemolytic Uremic Syndrome (HUS). The five main pathogenic serotypes of STEC (MPS-STEC) identified thus far in Europe are O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28. Because STEC strains can survive or grow during cheese making, particularly in soft cheeses, a stochastic quantitative microbial risk assessment model was developed to assess the risk of HUS associated with the five MPS-STEC in raw milk soft cheeses. A baseline scenario represents a theoretical worst-case scenario where no intervention was considered throughout the farm-to-fork continuum. The risk level assessed with this baseline scenario is the risk-based level. The impact of seven preharvest scenarios (vaccines, probiotic, milk farm sorting) on the risk-based level was expressed in terms of risk reduction. Impact of the preharvest intervention ranges from 76% to 98% of risk reduction with highest values predicted with scenarios combining a decrease of the number of cow shedding STEC and of the STEC concentration in feces. The impact of postharvest interventions on the risk-based level was also tested by applying five microbiological criteria (MC) at the end of ripening. The five MCs differ in terms of sample size, the number of samples that may yield a value larger than the microbiological limit, and the analysis methods. The risk reduction predicted varies from 25% to 96% by applying MCs without preharvest interventions and from 1% to 96% with combination of pre- and postharvest interventions.

  3. Vaccination of pregnant dams with intimin(O157) protects suckling piglets from Escherichia coli O157:H7 infection.

    PubMed

    Dean-Nystrom, Evelyn A; Gansheroff, Lisa J; Mills, Melody; Moon, Harley W; O'Brien, Alison D

    2002-05-01

    Cattle are important reservoirs of enterohemorrhagic Escherichia coli (EHEC) O157:H7 that cause disease in humans. Both dairy and beef cattle are asymptomatically and sporadically infected with EHEC. Our long-term goal is to develop an effective vaccine to prevent cattle from becoming infected and transmitting EHEC O157:H7 to humans. We used passive immunization of neonatal piglets (as a surrogate model) to determine if antibodies against EHEC O157 adhesin (intimin(O157)) inhibit EHEC colonization. Pregnant swine (dams) with serum anti-intimin titers of < or =100 were vaccinated twice with purified intimin(O157) or sham-vaccinated with sterile buffer. Intimin(O157)-specific antibody titers in colostrum and serum of dams were increased after parenteral vaccination with intimin(O157). Neonatal piglets were allowed to suckle vaccinated or sham-vaccinated dams for up to 8 h before they were inoculated with 10(6) CFU of a Shiga toxin-negative (for humane reasons) strain of EHEC O157:H7. Piglets were necropsied at 2 to 10 days after inoculation, and intestinal samples were collected for determination of bacteriological counts and histopathological analysis. Piglets that ingested colostrum containing intimin(O157)-specific antibodies from vaccinated dams, but not those nursing sham-vaccinated dams, were protected from EHEC O157:H7 colonization and intestinal damage. These results establish intimin(O157) as a viable candidate for an EHEC O157:H7 antitransmission vaccine.

  4. Vaccination of pregnant dams with intimin(O157) protects suckling piglets from Escherichia coli O157:H7 infection.

    PubMed

    Dean-Nystrom, Evelyn A; Gansheroff, Lisa J; Mills, Melody; Moon, Harley W; O'Brien, Alison D

    2002-05-01

    Cattle are important reservoirs of enterohemorrhagic Escherichia coli (EHEC) O157:H7 that cause disease in humans. Both dairy and beef cattle are asymptomatically and sporadically infected with EHEC. Our long-term goal is to develop an effective vaccine to prevent cattle from becoming infected and transmitting EHEC O157:H7 to humans. We used passive immunization of neonatal piglets (as a surrogate model) to determine if antibodies against EHEC O157 adhesin (intimin(O157)) inhibit EHEC colonization. Pregnant swine (dams) with serum anti-intimin titers of < or =100 were vaccinated twice with purified intimin(O157) or sham-vaccinated with sterile buffer. Intimin(O157)-specific antibody titers in colostrum and serum of dams were increased after parenteral vaccination with intimin(O157). Neonatal piglets were allowed to suckle vaccinated or sham-vaccinated dams for up to 8 h before they were inoculated with 10(6) CFU of a Shiga toxin-negative (for humane reasons) strain of EHEC O157:H7. Piglets were necropsied at 2 to 10 days after inoculation, and intestinal samples were collected for determination of bacteriological counts and histopathological analysis. Piglets that ingested colostrum containing intimin(O157)-specific antibodies from vaccinated dams, but not those nursing sham-vaccinated dams, were protected from EHEC O157:H7 colonization and intestinal damage. These results establish intimin(O157) as a viable candidate for an EHEC O157:H7 antitransmission vaccine. PMID:11953378

  5. Detection and Prevalence of Verotoxin-Producing Escherichia coli O157 and Non-O157 Serotypes in a Canadian Watershed

    PubMed Central

    Johnson, R. P.; Holtslander, B.; Mazzocco, A.; Roche, S.; Thomas, J. L.; Pollari, F.

    2014-01-01

    Verotoxin-producing Escherichia coli (VTEC) strains are the cause of food-borne and waterborne illnesses around the world. Traditionally, surveillance of the human population as well as the environment has focused on the detection of E. coli O157:H7. Recently, increasing recognition of non-O157 VTEC strains as human pathogens and the German O104:H4 food-borne outbreak have illustrated the importance of considering the broader group of VTEC organisms from a public health perspective. This study presents the results of a comparison of three methods for the detection of VTEC in surface water, highlighting the efficacy of a direct VT immunoblotting method without broth enrichment for detection and isolation of O157 and non-O157 VTEC strains. The direct immunoblot method eliminates the need for an enrichment step or the use of immunomagnetic separation. This method was developed after 4 years of detecting low frequencies (1%) of E. coli O157:H7 in surface water in a Canadian watershed, situated within one of the FoodNet Canada integrated surveillance sites. By the direct immunoblot method, VTEC prevalence estimates ranged from 11 to 35% for this watershed, and E. coli O157:H7 prevalence increased to 4% (due to improved method sensitivity). This direct testing method provides an efficient means to enhance our understanding of the prevalence and types of VTEC in the environment. This study employed a rapid evidence assessment (REA) approach to frame the watershed findings with watershed E. coli O157:H7 prevalences reported in the literature since 1990 and the knowledge gap with respect to VTEC detection in surface waters. PMID:24487525

  6. Enumeration of sublethally injured Escherichia coli O157:H7 ATCC 43895 and Escherichia coli strain B-41560 using selective agar overlays versus commercial methods.

    PubMed

    Smith, Amanda R; Ellison, Alysha L; Robinson, Amanda L; Drake, Maryanne; McDowell, Susan A; Mitchell, James K; Gerard, Patrick D; Heckler, Rachel A; McKillip, John L

    2013-04-01

    Quality control procedures during food processing may involve direct inoculation of food samples onto appropriate selective media for subsequent enumeration. However, sublethally injured bacteria often fail to grow, enabling them to evade detection and intervention measures and ultimately threaten the health of consumers. This study compares traditional selective and nonselective agar-based overlays versus two commercial systems (Petrifilm and Easygel) for recovery of injured E. coli B-41560 and O157:H7 strains. Bacteria were propagated in tryptic soy broth (TSB), ground beef slurry, and infant milk formula to a density of 10(6) to 10(8) CFU/ml and then were stressed for 6 min either in lactic acid (pH 4.5) or heat shocked for 3 min at 60°C. Samples were pour plated in basal layers of either tryptic soy agar (TSA), sorbitol MacConkey agar (SMAC), or violet red bile agar (VRB) and were resuscitated for 4 h prior to addition of agar overlays. Other stressed bacteria were plated directly onto Petrifilm and Easygel. Results indicate that selective and nonselective agar overlays recovered significantly higher numbers (greater than 1 log) of acid- and heat-injured E. coli O157:H7 from TSB, ground beef, and infant milk formula compared with direct plating onto selective media, Petrifilm, or Easygel, while no significant differences among these media combinations were observed for stressed E. coli B-41560. Nonstressed bacteria from TSB and ground beef were also recovered at densities significantly higher in nonselective TSA-TSA and in VRB-VRB and SMAC-SMAC compared with Petrifilm and Easygel. These data underscore the need to implement food safety measures that address sublethally injured pathogens such as E. coli O157:H7 in order to avoid underestimation of true densities for target pathogens.

  7. The effect of oxidative stress on gene expression of Shiga toxin-producing Escherichia coli (STEC) O157:H7 and non-O157 serotypes.

    PubMed

    Mei, Gui-Ying; Tang, Joshua; Carey, Christine; Bach, Susan; Kostrzynska, Magdalena

    2015-12-23

    Understanding the survival mechanisms used by Shiga toxin-producing Escherichia coli (STEC), including O157:H7 and non-O157 serotypes, is important for minimizing contamination of fresh produce and occurrence of foodborne outbreaks. Recent outbreaks linked to leafy green vegetables and sprouted seeds have prompted researchers to focus on investigating decontamination strategies. Several studies showed that hydrogen peroxide (H2O2) treatment has been effective in reducing pathogens on fresh produce. As such, the effect of hydrogen peroxide on stress-associated and virulence gene expression in six STEC isolates was investigated in this study. Logarithmic phase cells of E. coli O157:H7 (EDL933) and non-O157 serotypes, including E. coli O26:H11 (EC20070549), O103:H2 (EC19970811), O104:H4 (NML#11-3088), O111:NM (EC20070546) and O145:NM (EC19970355) were exposed to 2.5mM H2O2 for 40 min and gene expression was evaluated using quantitative real-time PCR. Different patterns of gene expression were observed in E. coli O157:H7 and non-O157 serotypes. Particularly, Shiga toxin gene stx2 was upregulated in O157:H7, but not in O104:H4. Moreover, stx1 was significantly upregulated in STEC O157:H7, but only slightly upregulated Stx1-positive non-O157 serotypes. However genes related to motility (fliC) and intimin gene (eae) were downregulated in most strains. Stress-associated sodA gene encoding manganese superoxide dismutase was significantly upregulated in all serotypes. The dps gene coding for non-specific DNA binding protein was upregulated in O145:NM, O111:NM, O103:H2 and O26:H11. However genes related to cold shock (cspC) and acid resistance (gadW) were significantly downregulated in all strains tested. The results of this study provide a basic understanding of the oxidative stress impact on survival and virulence of non-O157 serotype STEC strains.

  8. Verotoxinogenic Escherichia coli (VTEC) O157:H7 – A Nationwide Swedish Survey of Bovine Faeces

    PubMed Central

    Albihn, A; Eriksson, E; Wallen, C; Aspán, A

    2003-01-01

    In the autumn of 1995 the first outbreaks of enterohemorrhagic Escherichia coli O157:H7 including ca 100 human cases were reported in Sweden. From outbreaks in other countries it is known that cattle may carry these bacteria and in many cases is the source of infection. Therefore, the present study was performed to survey the Swedish bovine population for the presence of verotoxin-producing E. coli (VTEC) of serotype O157:H7. Individual faecal samples were collected at the 16 main Swedish abattoirs from April 1996 to August 1997. Of 3071 faecal samples, VTEC O157 were found in 37 samples indicating a prevalence of 1.2% (CI95% 0.8–1.6). All 37 isolates carried genes encoding for verotoxin (VT1 and/or VT2), intimin, EHEC-haemolysin and flagellin H7 as determined by PCR. Another 3 strains were of serotype O157:H7 but did not produce verotoxins. The 37 VTEC O157:H7 strains were further characterised by phage typing and pulsed-field gel electrophoresis. The results clearly show that VTEC O157:H7 is established in the Swedish bovine population and indicate that the prevalence of cattle carrying VTEC O157:H7 is correlated to the overall geographical distribution of cattle in Sweden. Results of this study have formed the basis for specific measures recommended to Swedish cattle farmers, and furthermore, a permanent monitoring programme was launched for VTEC O157:H7 in Swedish cattle at slaughter. PMID:14650543

  9. A statewide outbreak of Escherichia coli O157:H7 infections in Washington State.

    PubMed

    Ostroff, S M; Griffin, P M; Tauxe, R V; Shipman, L D; Greene, K D; Wells, J G; Lewis, J H; Blake, P A; Kobayashi, J M

    1990-08-01

    In November 1986, a statewide outbreak of Escherichia coli O157:H7 infections in Washington State was identified after a physician in an eastern Washington community hospitalized three patients with hemorrhagic colitis which progressed to thrombotic thrombocytopenic purpura. Epidemiologic investigation identified 37 cases in this community and linked the illnesses to a local restaurant which had served ground beef that was the suspected initial vehicle of transmission. The plasmid profile and toxin production pattern (Shiga-like toxin II alone) of the outbreak strain provided a unique strain marker. E. coli O157:H7 infections caused by this strain were simultaneously seen in other parts of the state among nursing home residents and in patients with the hemolytic-uremic syndrome, and an increase in sporadic cases of hemorrhagic colitis was noted at a Seattle health maintenance organization. It is suspected that a contaminated product, probably ground beef distributed statewide, was the common source. Tracing of this meat led to farms where rectal swabs from six (1%) of 539 cattle tested yielded E. coli O157:H7, although the plasmids and toxin production patterns of these isolates differed from the human outbreak strain. Introduction of a single strain of E. coli O157:H7 has the potential to cause widespread concurrent outbreaks. Such outbreaks are likely to escape recognition until heightened screening and surveillance for E. coli O157:H7 is established.

  10. Acid Resistance and Molecular Characterization of Escherichia coli O157:H7 and Different Non-O157 Shiga Toxin-Producing E. coli Serogroups.

    PubMed

    Kim, Gwang-Hee; Breidt, Frederick; Fratamico, Pina; Oh, Deog-Hwan

    2015-10-01

    The objective of this study was to compare the acid resistance (AR) of non-O157 Shiga toxin-producing Escherichia coli (STEC) strains belonging to serogroups O26, O45, O103, O104, O111, O121, and O145 with O157:H7 STEC isolated from various sources in 400 mM acetic acid solutions (AAS) at pH 3.2 and 30 °C for 25 min with or without glutamic acid. Furthermore, the molecular subgrouping of the STEC strains was analyzed with the repetitive sequence-based PCR (rep-PCR) method using a DiversiLab(TM) system. Results for a total of 52 strains ranged from 0.31 to 5.45 log reduction CFU/mL in the absence of glutamic acid and 0.02 to 0.33 CFU/mL in the presence of glutamic acid except for B447 (O26:H11), B452 (O45:H2), and B466 (O104:H4) strains. Strains belonging to serogroups O111, O121, and O103 showed higher AR than serotype O157:H7 strains in the absence of glutamic acid. All STEC O157:H7 strains exhibited a comparable DNA pattern with more than 95% similarity in the rep-PCR results, as did the strains belonging to serogroups O111 and O121. Surprisingly, the DNA pattern of B458 (O103:H2) was similar to that of O157:H7 strains with 82% similarity, and strain B458 strain showed the highest AR to AAS among the O103 strains with 0.44 log reduction CFU/mL without glutamic acid. In conclusion, STEC serotypes isolated from different sources exhibited diverse AR and genetic subtyping patterns. Results indicated that some non-O157 STEC strains may have higher AR than STEC O157:H7 strains under specific acidic conditions, and the addition of glutamic acid provided enhanced protection against exposure to AAS.

  11. Reduction of Adherence of E. coli O157:H7 to HEp-2 Cells and to Bovine Large Intestinal Mucosal Explants by Colicinogenic E. coli

    PubMed Central

    Etcheverría, A. I.; Arroyo, G. H.; Alzola, R.; Parma, A. E.

    2011-01-01

    Enterohemorrhagic E. coli strains (EHEC) had emerged as foodborne pathogens and cause in human diarrhea and hemolytic-uremic syndrome. Because of the widespread distribution of EHEC serotypes and O157 and non-O157 in cattle population, its control will require interventions at the farm level such as the administration of probiotics that produce inhibitory metabolites. E. coli O157:H7 shows tissue tropisms for the gastrointestinal tract (GIT) of cattle. The aim of this study was to test the ability of a colicinogenic E. coli (isolated from bovine) to reduce the adherence of E. coli O157:H7 to HEp-2 cells and to GIT of cattle. We inoculated HEp-2 cells and bovine colon explants with both kinds of strains. Colicinogenic E. coli was able to reduce the adherence of E. coli O157:H7 to HEp-2 cells and to bovine tissues. PMID:23724308

  12. Relevance of nontoxigenic strains as surrogates for Escherichia coli O157:H7 in groundwater contamination potential: role of temperature and cell acclimation time.

    PubMed

    Castro, Felipe D; Tufenkji, Nathalie

    2007-06-15

    Nontoxigenic bacteria are commonly used as indicators for predicting the contamination potential of pathogens in natural or engineered aqueous environments. In this study, column transport experiments were used to examine the relevance of two nontoxigenic strains of Escherichia coli O157:H7 as potential surrogates for the well-known pathogen. Experiments conducted at 11 degrees C indicate that only one of the nontoxigenic strains may be an appropriate surrogate for predicting the migration potential of the pathogen at low solution ionic strengths. Results of various bacterial characterization methods indicate that differences in cell attachment could qualitatively, but convincingly, be related to differences in cell surface charge. Additional experiments conducted at 22 degrees C reveal the influence of temperature on bacterial cell surface charge and cell attachmentto sand. The role of cell acclimation time to an artificial groundwater solution is also examined, showing little change in the degree of cell attachment over a period of several weeks.

  13. Fecal carriage of Escherichia coli O157:H7 and carcass contamination in cattle at slaughter in northern Italy.

    PubMed

    Alonso, Silvia; Mora, Azucena; Blanco, Miguel; Blanco, Jesús E; Dahbi, Ghizlane; Ferreiro, María T; López, Cecilia; Alberghini, Leonardo; Albonetti, Sabrina; Echeita, Aurora; Trevisani, Marcello; Blanco, Jorge

    2007-06-01

    Feedlot cattle slaughtered at a large abattoir in northern Italy during 2002 were examined for intestinal carriage and carcass contamination with Escherichia coli O157:H7. Carcass samples were taken following the excision method described in the Decision 471/2001/EC, and fecal material was taken from the colon of the calves after evisceration. Bacteria were isolated and identified according to the MFLP-80 and MFLP-90 procedures (Food Directorate's Health Canada's). Eighty-eight non-sorbitol-fermenting E. coli O157:H7 isolates were obtained from 12 of the 45 calves examined. In particular, E. coli O157:H7 isolates were found in 11 (24%) fecal and five (11%) carcass samples. PCR analysis showed that all 11 fecal samples and five carcass samples carried eae-gamma1-positive E. coli O157:H7 isolates. In addition, genes encoding Shigatoxins were detected in O157:H7 isolates from nine and two of those 11 fecal and five carcasses, respectively. A representative group of 32 E. coli O157:H7 isolates was analyzed by phage typing and DNA macrorestriction fragment analysis (PFGE). Five phage types (PT8, PT32v, PT32, PT54, and PT not typable) and seven (I-VII) distinct restriction patterns of similarity >85% were detected. Up to three different O157:H7 strains in an individual fecal sample and up to four from the same animal could be isolated. These findings provide evidence of the epidemiological importance of subtyping more than one isolate from the same sample. Phage typing together with PFGE proved to be very useful tools to detect cross-contamination among carcasses and should therefore be included in HACCP programs at abattoirs. The results showed that the same PFGE-phage type E. coli O157:H7 profile was detected in the fecal and carcass samples from an animal, and also in two more carcasses corresponding to two animals slaughtered the same day.

  14. Effects of environmental parameters on the dual-species biofilms formed by Escherichia coli O157:H7 and Ralstonia insidiosa, a strong biofilm producer isolated from a fresh-cut produce processing plant.

    PubMed

    Liu, Nancy T; Nou, Xiangwu; Bauchan, Gary R; Murphy, Charles; Lefcourt, Alan M; Shelton, Daniel R; Lo, Y Martin

    2015-01-01

    Biofilm-forming bacteria resident to food processing facilities are a food safety concern due to the potential of biofilms to harbor foodborne bacterial pathogens. When cultured together, Ralstonia insidiosa, a strong biofilm former frequently isolated from produce processing environments, has been shown to promote the incorporation of Escherichia coli O157:H7 into dual-species biofilms. In this study, interactions between E. coli O157:H7 and R. insidiosa were examined under different incubating conditions. Under static culture conditions, the incorporation of E. coli O157:H7 into biofilms with R. insidiosa was not significantly affected by either low incubating temperature (10°C) or by limited nutrient availability. Greater enhancement of E. coli O157:H7 incorporation in dual-species biofilms was observed by using a continuous culture system with limited nutrient availability. Under the continuous culture conditions used in this study, E coli O157:H7 cells showed a strong tendency of colocalizing with R. insidiosa on a glass surface at the early stage of biofilm formation. As the biofilms matured, E coli O157:H7 cells were mostly found at the bottom layer of the dual-species biofilms, suggesting an effective protection by R. insidiosa in the mature biofilms.

  15. Escherichia coli O157:H7 Lacking the qseBC-Encoded Quorum-Sensing System Outcompetes the Parental Strain in Colonization of Cattle Intestines

    PubMed Central

    Casey, T. A.

    2014-01-01

    The qseBC-encoded quorum-sensing system regulates the motility of Escherichia coli O157:H7 in response to bacterial autoinducer 3 (AI-3) and the mammalian stress hormones epinephrine (E) and norepinephrine (NE). The qseC gene encodes a sensory kinase that autophosphorylates in response to AI-3, E, or NE and subsequently phosphorylates its cognate response regulator QseB. In the absence of QseC, QseB downregulates bacterial motility and virulence in animal models. In this study, we found that 8- to 10-month-old calves orally inoculated with a mixture of E. coli O157:H7 and its isogenic qseBC mutant showed significantly higher fecal shedding of the qseBC mutant. In vitro analysis revealed similar growth profiles and motilities of the qseBC mutant and the parental strain in the presence or absence of NE. The magnitudes of the response to NE and expression of flagellar genes flhD and fliC were also similar for the qseBC mutant and the parental strain. The expression of ler (a positive regulator of the locus of enterocyte effacement [LEE]), the ler-regulated espA gene, and the csgA gene (encoding curli fimbriae) was increased in the qseBC mutant compared to the parental strain. On the other hand, growth, motility, and transcription of flhD, fliC, ler, espA, and csgA were significantly reduced in the qseBC mutant complemented with a plasmid-cloned copy of the qseBC genes. Thus, in vitro motility and gene expression data indicate that the near-parental level of motility, ability to respond to NE, and enhanced expression of LEE and curli genes might in part be responsible for increased colonization and fecal shedding of the qseBC mutant in calves. PMID:24413602

  16. Prevalence and antimicrobial resistance of porcine O157 and non-O157 Shiga toxin-producing Escherichia coli from India.

    PubMed

    Rajkhowa, Swaraj; Sarma, Dilip Kumar

    2014-08-01

    The aims of this study were to determine the prevalence of Shiga toxin-producing Escherichia coli (STEC) strains in pigs as a possible STEC reservoir in India as well as to characterize the STEC strains and to determine the antimicrobial resistance pattern of the strains. A total of 782 E. coli isolates from clinically healthy (n = 473) and diarrhoeic piglets (309) belonging to major pig-producing states of India were screened by the polymerase chain reaction (PCR) assay for the presence of virulence genes characteristic for STEC, that is, Shiga toxin-producing gene(s) (stx1, stx2), intimin (eae), enterohemolysin (hlyA) and STEC autoagglutinating adhesin (Saa). Overall STEC were detected in 113 (14.4%) piglets, and the prevalence of E. coli O157 and non-O157 STEC were 4 (0.5%) and 109 (13.9%), respectively. None of the O157 STEC isolates carried gene encoding for H7 antigen (fliCh7). The various combinations of virulence genes present in the strains studied were stx1 in 4.6%, stx1 in combination with stx2 gene in 5.1%, stx1 in combination with stx2 and ehxA in 0.6%, stx1 in combination with stx2 and eae in 0.2% and stx2 alone in 3.7%. All STEC isolates were found negative for STEC autoagglutinating adhesin (Saa). The number of STEC isolates which showed resistance to antimicrobials such as ampicillin, tetracycline, streptomycin, lincomycin, nalidixic acid, sulfadiazine, penicillin, gentamicin, kanamycin and ceftriaxone were 100, 99, 98, 97, 95, 94, 92, 88, 85 and 85, respectively. Ninety-seven isolates showed resistance to more than 2 antimicrobials, and 8 resistance groups (R1 to R8) were observed. This study demonstrates that pigs in India harbour both O157 and non-O157 STEC, and this may pose serious public health problems in future.

  17. Novel real-time PCR method to detect Escherichia coli O157:H7 in raw milk cheese and raw ground meat.

    PubMed

    Miszczycha, Stéphane D; Ganet, Sarah; Duniere, Lysiane; Rozand, Christine; Loukiadis, Estelle; Thevenot-Sergentet, Delphine

    2012-08-01

    Raw milk, raw milk cheeses, and raw ground meat have been implicated in Escherichia coli O157:H7 outbreaks. Developing methods to detect these bacteria in raw milk and meat products is a major challenge for food safety. The aim of our study was to develop a real-time PCR assay to detect E. coli O157:H7 in raw milk cheeses and raw ground meat. Well-known primers targeting a mutation at position +93 of the uidA gene in E. coli O157:H7 were chosen, and a specific TaqMan-minor groove binder probe was designed. This probe targets another mutation, at position +191 of the uidA gene in E. coli O157:H7. The first step in the study was to evaluate the specificity of this probe with 156 different O157:H7/NM strains and 48 non-O157:H7/NM strains of E. coli. The sensitivity of the method was evaluated by pre- and postinoculation of cheeses and meat enrichments with different E. coli O157:H7 strains. All the E. coli O157:H7 isolates tested were positive, and none of the other bacteria were detected. Our results indicate that this method is sensitive enough to detect 10(2) E. coli O157:H7 isolates per ml of cheese or meat enrichment broth (24 h at 41.5° C) and is more sensitive than the International Organization for Standardization reference method. We can conclude that this new real-time PCR protocol is a useful tool for rapid, specific, and sensitive detection of E. coli O157:H7 in raw milk and raw ground meat products.

  18. Improvement of biomolecular methods for the identification and typing of Escherichia coli O157:H7 isolated from raw meat.

    PubMed

    Paris, A; Bonardi, S; Bacci, C; Boni, E; Salmi, F; Bassi, L; Brindani, F

    2010-06-01

    The aim of the study was to evaluate the sensitivity of two m-PCR methods for the quantitative determination of E. coli O157:H7 in foodstuffs. Genomic serotyping was carried out on bacterial cultures, and the necessary time was optimized to increase the resolution of the method. Subsequently, artificial contamination trials using meat were conducted to assess method accuracy in foodstuffs and pursue the genetic typing of pathogens. Measurement thresholds were shown to range between 10(5) and 10(6) CFU/mL, but were reduced by four logarithmic cycles in 80% of samples. Relative to the meat contamination trials, serotypes were identified after 24 hours, corresponding to 10 CFU/mL inoculum, with higher rates seen when m-TSB was used for enrichment. Inoculated samples were found to contain three virulence factors (hlyA, eaeA, and stx1).

  19. Detection of Escherichia coli O157:H7 in the beef marketed in Malaysia.

    PubMed

    Radu, S; Abdul Mutalib, S; Rusul, G; Ahmad, Z; Morigaki, T; Asai, N; Kim, Y B; Okuda, J; Nishibuchi, M

    1998-03-01

    Twelve strains of Escherichia coli O157:H7 were isolated from 9 of 25 beef samples purchased from retail stores in Malaysia. These strains produced Shiga toxin 2 with or without Shiga toxin 1 and had the eae gene and a 60-MDa plasmid. The antibiograms and the profiles of the arbitrarily primed PCR of the strains were diverse, suggesting that the strains may have originated from diverse sources. PMID:9501454

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

    PubMed

    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. PMID:25273960

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

    PubMed

    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.

  2. Outbreak of Escherichia coli O157 infection associated with a music festival.

    PubMed

    Crampin, M; Willshaw, G; Hancock, R; Djuretic, T; Elstob, C; Rouse, A; Cheasty, T; Stuart, J

    1999-04-01

    Seven persons who attended the Glastonbury Music Festival were infected with Vero cytotoxin-producing Escherichia coli O157 and an eighth person had serological evidence of infection. Cases were reported from different parts of England. Patients were interviewed by telephone about clinical symptoms, festival attendance, camping details, food history, water exposure, and contact with mud and animals. The interviews identified no common food source, differing use of water sources and widely dispersed camping sites. Escherichia coli O157 strains from seven persons and from a cow belonging to a herd that had previously grazed the site all belonged to phage type 2 and possessed genes for Vero cytotoxin 2. Drug resistance and DNA-based tests showed that six patients were infected with strains indistinguishable from each other and from the bovine isolate. The most likely vehicle of infection was mud contaminated with Escherichia coli O157 from infected cattle. PMID:10385018

  3. Outbreak of Escherichia coli O157 infection associated with a music festival.

    PubMed

    Crampin, M; Willshaw, G; Hancock, R; Djuretic, T; Elstob, C; Rouse, A; Cheasty, T; Stuart, J

    1999-04-01

    Seven persons who attended the Glastonbury Music Festival were infected with Vero cytotoxin-producing Escherichia coli O157 and an eighth person had serological evidence of infection. Cases were reported from different parts of England. Patients were interviewed by telephone about clinical symptoms, festival attendance, camping details, food history, water exposure, and contact with mud and animals. The interviews identified no common food source, differing use of water sources and widely dispersed camping sites. Escherichia coli O157 strains from seven persons and from a cow belonging to a herd that had previously grazed the site all belonged to phage type 2 and possessed genes for Vero cytotoxin 2. Drug resistance and DNA-based tests showed that six patients were infected with strains indistinguishable from each other and from the bovine isolate. The most likely vehicle of infection was mud contaminated with Escherichia coli O157 from infected cattle.

  4. Viability of a multi-strain mixture of Listeria monocytogenes, Salmonella typhimurium, or Escherichia coli O157:H7 inoculated into the batter or onto the surface of a soudjouk-style fermented semi-dry sausage

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Multi-strain mixtures of Listeria monocytogenes, Salmonella typhimurium, or Escherichia coli O157:H7 were separately inoculated either into soudjouk batter or onto the surface of slices of commercial soudjouk to levels of ca. 6.0 log10 CFU per gram or slice, respectively. After fermentation and dryi...

  5. Pathogenesis of Escherichia coli O157:H7 strain 86-24 following oral infection of BALB/c mice with an intact commensal flora

    PubMed Central

    Mohawk, Krystle L.; Melton-Celsa, Angela R.; Zangari, Tonia; Carroll, Erica E.; O’Brien, Alison D.

    2010-01-01

    Escherichia coli O157:H7 is a food-borne pathogen that can cause hemorrhagic colitis and, occasionally, hemolytic uremic syndrome, a sequela of infection that can result in renal failure and death. Here we sought to model the pathogenesis of orally-administered E. coli O157:H7 in BALB/c mice with an intact intestinal flora. First, we defined the optimal dose that permitted sustained fecal shedding of E. coli O157:H7 over 7 days (~109 colony-forming units). Next, we monitored the load of E. coli O157:H7 in intestinal sections over time and observed that the cecum was consistently the tissue with the highest E. coli O157:H7 recovery. We then followed the expression of two key E. coli O157:H7 virulence factors, the adhesin intimin and Shiga toxin type 2, and detected both proteins early in infection when bacterial burdens were highest. Additionally, we noted that during infection, animals lost weight and ~30% died. Moribund animals also exhibited elevated levels of blood urea nitrogen, and, on necropsy, showed evidence of renal tubular damage. We conclude that conventional mice inoculated orally with high doses of E. coli O157:H7 can be used to model both intestinal colonization and subsequent development of certain extraintestinal manifestations of E. coli O157:H7 disease. PMID:20096770

  6. RcsB contributes to the distinct stress fitness between Escherichia coli O157:H7 curli variants of 1993 hamburger-associated outbreak strains

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Curli are adhesive fimbriae of Enterobactericaeae and are involved in surface attachment, cell aggregation and biofilm formation. We previously reported that natural curli variants of E. coli O157:H7 (EcO157) displayed distinct acid resistance; however, this difference was not linked to the curli fi...

  7. Comparative evaluation of biofilm formation and tolerance to a chemical shock of pathogenic and nonpathogenic Escherichia coli O157:H7 strains.

    PubMed

    Marouani-Gadri, N; Chassaing, D; Carpentier, B

    2009-01-01

    Seven Escherichia coli O157:H7 strains, three pathogenic (including epidemic EDL933 and Sakai) and four nonpathogenic (including mutants of EDL933 and Sakai), were compared to find a model strain to avoid the use of European third-class biological agents in biofilm studies. Comparison was performed on attached populations reached at the end of growth in eight environmental conditions defined as the combinations of three two-level factors: (i) culture medium composed of meat exudate and glucose-supplemented minimal salts medium (MSM), (ii) growth temperatures of 15 and 25 degrees C, and (iii) materials of stainless steel and polyurethane. The influence of each of these four factors (strain, medium, temperature, and material) often depended on the level of at least one of the others. Exudate produced attached populations that were larger than or similar to those obtained with MSM, except for EDL933 mutant at 15 degrees C. When exudate led to larger populations than did MSM, the highest differences of up to 1.8 log CFU/cm2 were observed with the nonepidemic strains grown at 25 degrees C. Populations of these strains were not significantly different in any of the conditions studied, but they were different from the epidemic strains in some conditions. No nonpathogenic mutant was representative of its parental strain. Furthermore, the Sakai mutant biofilm was significantly more reduced than its parental strain was after chemical shock. It is therefore not possible to find a surrogate of either EDL933 or Sakai, and it is advisable that main results be validated on a pathogenic strain whenever nonpathogenic strains are used.

  8. The CsgA and Lpp Proteins of an Escherichia coli O157:H7 Strain Affect HEp-2 Cell Invasion, Motility, and Biofilm Formation ▿

    PubMed Central

    Uhlich, Gaylen A.; Gunther, Nereus W.; Bayles, Darrell O.; Mosier, Derek A.

    2009-01-01

    In Escherichia coli O157:H7 strain ATCC 43895, a guanine-to-thymine transversion in the csgD promoter created strain 43895OR. Strain 43895OR produces an abundant extracellular matrix rich in curli fibers, forms biofilms on solid surfaces, invades cultured epithelial cells, and is more virulent in mice than strain 43895. In this study we compared the formic acid-soluble proteins expressed by strains 43895OR and 43895 using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and identified two differentially expressed proteins. A 17-kDa protein unique to strain 43895OR was identified from matrix-assisted laser desorption ionization-time of flight analysis combined with mass spectrometry (MS) and tandem MS (MS/MS) as the curli subunit encoded by csgA. A <10-kDa protein, more highly expressed in strain 43895, was identified as the Lpp lipoprotein. Mutants of strain 43895OR with disruption of lpp, csgA, or both lpp and csgA were created and tested for changes in phenotype and function. The results of this study show that both Lpp and CsgA contribute to the observed colony morphology, Congo red binding, motility, and biofilm formation. We also show that both CsgA and Lpp are required by strain 43895OR for the invasion of cultured HEp-2 cells. These studies suggest that in strain 43895OR, the murein lipoprotein Lpp indirectly regulates CsgA expression through the CpxAR system by a posttranscriptional mechanism. PMID:19179421

  9. Enhanced surface colonization by Escherichia coli O157:H7 in biofilms formed by an Acinetobacter calcoaceticus isolate from meat-processing environments.

    PubMed

    Habimana, Olivier; Heir, Even; Langsrud, Solveig; Asli, Anette Wold; Møretrø, Trond

    2010-07-01

    A meat factory commensal bacterium, Acinetobacter calcoaceticus, affected the spatial distribution of Escherichia coli O157:H7 surface colonization. The biovolume of E. coli O157:H7 was 400-fold higher (1.2 x 10(6) microm(3)) in a dynamic cocultured biofilm than in a monoculture (3.0 x 10(3) microm(3)), and E. coli O157:H7 colonized spaces between A. calcoaceticus cell clusters.

  10. 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).

    PubMed

    Mora, Azucena; Blanco, Miguel; Blanco, Jesús E; Alonso, M Pilar; Dhabi, Ghizlane; Thomson-Carter, Fiona; Usera, Miguel A; Bartolomé, Rosa; Prats, Guillermo; Blanco, Jorge

    2004-09-01

    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.

  11. Absence of internalization of Escherichia coli O157:H7 into germinating tissue of field-grown leafy greens.

    PubMed

    Erickson, Marilyn C; Webb, Cathy C; Díaz-Pérez, Juan Carlos; Davey, Lindsey E; Payton, Alison S; Flitcroft, Ian D; Phatak, Sharad C; Doyle, Michael P

    2014-02-01

    Both growth chamber and field studies were conducted to investigate the potential for Escherichia coli O157:H7 to be internalized into leafy green tissue when seeds were germinated in contaminated soil. Internalized E. coli O157:H7 was detected by enrichment in both spinach (Spinacia oleracea L.) and lettuce (Lactuca sativa L.) seedlings when seeds were germinated within the growth chamber in autoclaved and nonautoclaved soil, respectively, contaminated with E. coli O157:H7 at 2.0 and 3.8 log CFU/g, respectively. Internalized E. coli O157:H7 populations could be detected by enumeration within leafy green tissues either by increasing the pathogen levels in the soil or by autoclaving the soil. Attempts to maximize the exposure of seed to E. coli O157:H7 by increasing the mobility of the microbe either through soil with a higher moisture content or through directly soaking the seeds in an E. coli O157:H7 inoculum did not increase the degree of internalization. Based on responses obtained in growth chamber studies, internalization of E. coli O157:H7 surrogates (natural isolates of Shiga toxin-negative E. coli O157:H7 or recombinant [stx- and eae-negative] outbreak strains of E. coli O157:H7) occurred to a slightly lesser degree than did internalization of the virulent outbreak strains of E. coli O157:H7. The apparent lack of internalized E. coli O157:H7 when spinach and lettuce were germinated from seed in contaminated soil (ca. 3 to 5 log CFU/g) in the field and the limited occurrence of surface contamination on the seedlings suggest that competition from indigenous soil bacteria and environmental stresses were greater in the field than in the growth chamber. On the rare occasion that soil contamination with E. coli O157:H7 exceeded 5 log CFU/g in a commercial field, this pathogen probably would not be internalized into germinating leafy greens and/or would not still be present at the time of harvest.

  12. Absence of internalization of Escherichia coli O157:H7 into germinating tissue of field-grown leafy greens.

    PubMed

    Erickson, Marilyn C; Webb, Cathy C; Díaz-Pérez, Juan Carlos; Davey, Lindsey E; Payton, Alison S; Flitcroft, Ian D; Phatak, Sharad C; Doyle, Michael P

    2014-02-01

    Both growth chamber and field studies were conducted to investigate the potential for Escherichia coli O157:H7 to be internalized into leafy green tissue when seeds were germinated in contaminated soil. Internalized E. coli O157:H7 was detected by enrichment in both spinach (Spinacia oleracea L.) and lettuce (Lactuca sativa L.) seedlings when seeds were germinated within the growth chamber in autoclaved and nonautoclaved soil, respectively, contaminated with E. coli O157:H7 at 2.0 and 3.8 log CFU/g, respectively. Internalized E. coli O157:H7 populations could be detected by enumeration within leafy green tissues either by increasing the pathogen levels in the soil or by autoclaving the soil. Attempts to maximize the exposure of seed to E. coli O157:H7 by increasing the mobility of the microbe either through soil with a higher moisture content or through directly soaking the seeds in an E. coli O157:H7 inoculum did not increase the degree of internalization. Based on responses obtained in growth chamber studies, internalization of E. coli O157:H7 surrogates (natural isolates of Shiga toxin-negative E. coli O157:H7 or recombinant [stx- and eae-negative] outbreak strains of E. coli O157:H7) occurred to a slightly lesser degree than did internalization of the virulent outbreak strains of E. coli O157:H7. The apparent lack of internalized E. coli O157:H7 when spinach and lettuce were germinated from seed in contaminated soil (ca. 3 to 5 log CFU/g) in the field and the limited occurrence of surface contamination on the seedlings suggest that competition from indigenous soil bacteria and environmental stresses were greater in the field than in the growth chamber. On the rare occasion that soil contamination with E. coli O157:H7 exceeded 5 log CFU/g in a commercial field, this pathogen probably would not be internalized into germinating leafy greens and/or would not still be present at the time of harvest. PMID:24490912

  13. Whole-Genome Sequencing for National Surveillance of Shiga Toxin–Producing Escherichia coli O157

    PubMed Central

    Dallman, Timothy J.; Byrne, Lisa; Ashton, Philip M.; Cowley, Lauren A.; Perry, Neil T.; Adak, Goutam; Petrovska, Liljana; Ellis, Richard J.; Elson, Richard; Underwood, Anthony; Green, Jonathan; Hanage, William P.; Jenkins, Claire; Grant, Kathie; Wain, John

    2015-01-01

    Background. National surveillance of gastrointestinal pathogens, such as Shiga toxin–producing Escherichia coli O157 (STEC O157), is key to rapidly identifying linked cases in the distributed food network to facilitate public health interventions. In this study, we used whole-genome sequencing (WGS) as a tool to inform national surveillance of STEC O157 in terms of identifying linked cases and clusters and guiding epidemiological investigation. Methods. We retrospectively analyzed 334 isolates randomly sampled from 1002 strains of STEC O157 received by the Gastrointestinal Bacteria Reference Unit at Public Health England, Colindale, in 2012. The genetic distance between each isolate, as estimated by WGS, was calculated and phylogenetic methods were used to place strains in an evolutionary context. Results. Estimates of linked clusters representing STEC O157 outbreaks in England and Wales increased by 2-fold when WGS was used instead of traditional typing techniques. The previously unidentified clusters were often widely geographically distributed and small in size. Phylogenetic analysis facilitated identification of temporally distinct cases sharing common exposures and delineating those that shared epidemiological and temporal links. Comparison with multi locus variable number tandem repeat analysis (MLVA) showed that although MLVA is as sensitive as WGS, WGS provides a more timely resolution to outbreak clustering. Conclusions. WGS has come of age as a molecular typing tool to inform national surveillance of STEC O157; it can be used in real time to provide the highest strain-level resolution for outbreak investigation. WGS allows linked cases to be identified with unprecedented specificity and sensitivity that will facilitate targeted and appropriate public health investigations. PMID:25888672

  14. Production and characterization of a monoclonal antibody specific for enterohemorrhagic Escherichia coli of serotypes O157:H7 and O26:H11.

    PubMed Central

    Padhye, N V; Doyle, M P

    1991-01-01

    A monoclonal antibody (MAb 4E8C12) specific for Escherichia coli O157:H7 and O26:H11 was produced by immunizing BALB/c mice with a rough strain of E. coli O157:H7. The antibody reacted strongly by a direct enzyme-linked immunosorbent assay with each of 36 strains of E. coli O157:H7. No cross-reactivity was observed with strains of Salmonella spp., Yersinia enterocolitica, Shigella dysenteriae, Proteus spp., Escherichia hermanii, Klebsiella pneumoniae, Campylobacter jejuni, Serratia marcescens, Citrobacter spp., Enterobacter cloacae, Hafnia alvei, Aeromonas hydrophila, and all except five strains of E. coli other than serotype O157:H7 (including strains of serotype O157 but not H7). The E. coli strains (all of serotype O26:H11) that reacted with the antibody were enterohemorrhagic E. coli (EHEC) that were isolated from patients with hemolytic uremic syndrome or hemorrhagic colitis and produced verotoxin similar to that of E. coli O157:H7. MAb 4E8C12 belongs to the subclass immunoglobulin G2a and has a kappa light chain. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins of E. coli of different serotypes followed by Western immunoblot analysis revealed that MAb 4E8C12 reacted specifically with two proteins of EHEC strains of serotypes O157:H7 and O26:H11 with apparent molecular weights of 5,000 to 6,000. These proteins appeared to be markers specific for EHEC strains of serotypes O157:H7 and O26:H11. This MAb, because of its specificity, may be a useful reagent of an immunoassay for the rapid detection of these types of EHEC isolates in clinical and food specimens. Images PMID:1993773

  15. Prevalence of Escherichia coli O157:H7 in Children with Bloody Diarrhea Referring to Abuzar Teaching Hospital, Ahvaz, Iran

    PubMed Central

    Khosravi, Azar Dokht; Sheikh, Ahmad Farajzadeh; Ahmadzadeh, Ali; Shamsizadeh, Ahmad

    2016-01-01

    Introduction Escherichia coli O157: H7 are recognized as important aetiological agents of diarrhea in children, particularly in developed countries. Aim The aim of the study was to determine the rates of detection of E. coli O157: H7strains among children in Ahvaz, Iran. Materials and Methods From June 2010 to December 2010, 137 diarrheal stool samples of children were collected. E.coli was identified by standard microbiological techniques. O157 or O157:H7 subtypes discerned by serological tests. Results Of the 137 E. coli isolates, enteropathogens were found in 53 (38.7%) of the patients as follow: Shigella spp. (75.5%), EPEC (enteropathogenic E. coli) (16.9%), Campylobacter spp. (3.8%) and Salmonella spp. (3.8%). None of the isolated E. coli was O157:H7 serotype. Conclusion This shows that non-O157:H7 E. coli are the major cause of paediatric infections in this region of Iran. PMID:26894066

  16. Escherichia coli O157:H7/NM prevalence in raw beef, camel, sheep, goat, and water buffalo meat in Fars and Khuzestan provinces, Iran.

    PubMed

    Rahimi, Ebrahim; Kazemeini, Hamid Reza; Salajegheh, Mohammad

    2012-01-01

    Enterohemorrhagic Escherichia coli (EHEC) of the O157:H7 serotype is a worldwide zoonotic pathogen responsible for the majority of severe cases of human EHEC disease. The aim of the present study was to investigate the prevalence of E. coli O157: H7/NM in raw meat samples from two provinces of Iran. During a period from March 2010 to March 2011. Two hundred and ninety five raw meat samples were collected from beef (n= 85), camel, (n= 50), sheep (n= 62), goat (n= 60), and water buffalo (n=38). Fourteen (4.7%) of the 295 samples were positive for E. coli O157. The highest prevalence of E. coli O157 was found in beef samples (8.2%), followed by water buffalo (5.3%), sheep (4.8%), camel (2.0%), and goat (1.7%). Of fourteen E. coli O157 isolates, only one was determined to be serotype O157: H7 while 13 were determined as serotype O157: NM. Of the 14 E. coli O157:H7/NM isolates, one, four, two, and one strains were positive for stx1, stx2, eaeA and ehlyA genes, respectively. The prevalence of this organism varied between seasons with the highest prevalence of E. coli O157 occurring in summer (9.3%). The results of this study showed that beef and water buffalo meat are a significant source for human EHEC E. coli O157:H7/NM infection in Iran. The data reported in this study provides some useful baseline in formation for future research such as molecular or epidemiologic works.

  17. Growth media simulating ileal and colonic environments affect the intracellular proteome and carbon fluxes of enterohemorrhagic Escherichia coli O157:H7 strain EDL933.

    PubMed

    Polzin, Sabrina; Huber, Claudia; Eylert, Eva; Elsenhans, Ines; Eisenreich, Wolfgang; Schmidt, Herbert

    2013-06-01

    In this study, the intracellular proteome of Escherichia coli O157:H7 strain EDL933 was analyzed by two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) spectrometry after growth in simulated ileal environment media (SIEM) and simulated colonic environment media (SCEM) under aerobic and microaerobic conditions. Differentially expressed intracellular proteins were identified and allocated to functional protein groups. Moreover, metabolic fluxes were analyzed by isotopologue profiling with [U-(13)C(6)]glucose as a tracer. The results of this study show that EDL933 responds with differential expression of a complex network of proteins and metabolic pathways, reflecting the high metabolic adaptability of the strain. Growth in SIEM and SCEM is obviously facilitated by the upregulation of nucleotide biosynthesis pathway proteins and could be impaired by exposition to 50 µM 6-mercaptopurine under aerobic conditions. Notably, various stress and virulence factors, including Shiga toxin, were expressed without having contact with a human host.

  18. Determination of Antimicrobial Activity of Sorrel (Hibiscus sabdariffa) on Esherichia coli O157:H7 Isolated from Food, Veterinary, and Clinical Samples

    PubMed Central

    Fullerton, Marjorie; Khatiwada, Janak; Johnson, Jacqueline U.; Davis, Shurrita

    2011-01-01

    Abstract The use of medicinal plants as natural antimicrobial agents is gaining popularity. Sorrel (Hibiscus sabdariffa) is widely used for the treatment of diseases. The objective of this study was to investigate the antimicrobial activity of sorrel on Escherichia coli O157:H7 isolates from food, veterinary, and clinical samples. Phenolics of the calyces were extracted from 10 g of ground, freeze-dried samples using 100 mL of 80% aqueous methanol. Concentrations of 10%, 5%, and 2.5% methanol extract of sorrel were investigated for its antimicrobial activity. Inhibition zones were indicated by a lack of microbial growth due to inhibitory concentrations of sorrel diffused into semisolid culture medium beneath the sorrel-impregnated disk. The results of this experiment showed that the most potent sorrel concentration was 10%, then 5%, and finally 2.5%. The overall mean zone of inhibition for the sorrel extract was 12.66 mm for 10%, 10.75 mm for 5%, and 8.9 mm for 2.5%. The highest inhibition zones (11.16 mm) were observed in veterinary samples, and the lowest (10.57 mm) in the food samples. There were significant (P<.05) differences among mean zones of inhibition found in the food, veterinary, and clinical sources. Based on the source of samples and concentration of sorrel extract, the lowest mean inhibition was 7.00±0.04 mm from clinical samples, and the highest was 15.37±0.61 mm from a food source. These findings indicated that sorrel was effective at all levels in inhibiting E. coli O157:H7; thus it possesses antimicrobial activity and hold great promise as an antimicrobial agent. PMID:21548802

  19. Determination of antimicrobial activity of sorrel (Hibiscus sabdariffa) on Escherichia coli O157:H7 isolated from food, veterinary, and clinical samples.

    PubMed

    Fullerton, Marjorie; Khatiwada, Janak; Johnson, Jacqueline U; Davis, Shurrita; Williams, Leonard L

    2011-09-01

    The use of medicinal plants as natural antimicrobial agents is gaining popularity. Sorrel (Hibiscus sabdariffa) is widely used for the treatment of diseases. The objective of this study was to investigate the antimicrobial activity of sorrel on Escherichia coli O157:H7 isolates from food, veterinary, and clinical samples. Phenolics of the calyces were extracted from 10 g of ground, freeze-dried samples using 100 mL of 80% aqueous methanol. Concentrations of 10%, 5%, and 2.5% methanol extract of sorrel were investigated for its antimicrobial activity. Inhibition zones were indicated by a lack of microbial growth due to inhibitory concentrations of sorrel diffused into semisolid culture medium beneath the sorrel-impregnated disk. The results of this experiment showed that the most potent sorrel concentration was 10%, then 5%, and finally 2.5%. The overall mean zone of inhibition for the sorrel extract was 12.66 mm for 10%, 10.75 mm for 5%, and 8.9 mm for 2.5%. The highest inhibition zones (11.16 mm) were observed in veterinary samples, and the lowest (10.57 mm) in the food samples. There were significant (P<.05) differences among mean zones of inhibition found in the food, veterinary, and clinical sources. Based on the source of samples and concentration of sorrel extract, the lowest mean inhibition was 7.00±0.04 mm from clinical samples, and the highest was 15.37±0.61 mm from a food source. These findings indicated that sorrel was effective at all levels in inhibiting E. coli O157:H7; thus it possesses antimicrobial activity and hold great promise as an antimicrobial agent.

  20. Truncated enterohemorrhagic Escherichia coli (EHEC) O157:H7 intimin (EaeA) fusion proteins promote adherence of EHEC strains to HEp-2 cells.

    PubMed Central

    McKee, M L; O'Brien, A D

    1996-01-01

    Intimin, the product of the eaeA gene in enterohemorrhagic Escherichia coli O157:H7 (EHEC), is required for intimate adherence of these organisms to tissue culture cells and formation of the attaching and effacing lesion in the gnotobiotic pig. Because of the importance of intimin in the pathogenesis of EHEC O157:H7 infection in this animal model, we began a structure-function analysis of EaeA. For this purpose, we constructed amino-terminal fusions of the intimin protein with six histidine residues to form two independent fusions. The longer fusion, RIHisEae, contained 900 of the 935 predicted amino acids and included all but the extreme amino terminus. The second fusion, RVHdHisEae, consisted of the carboxyl two-thirds of the protein. Purified extracts of either construct enhanced binding of wild-type 86-24 to HEp-2 cells and conferred HEp-2 cell adherence on 86-24eaeDelta10, an eaeA deletion mutant, and B2F1, an EHEC O91:1-121 eaeA mutant strain. When 86-24eaeDelta10 was transformed with either of the plasmids encoding the intimin fusion proteins, the transformant behaved like the wild-type parent strain and displayed localized adherence to HEp-2 cells, with positive fluorescent-actin staining. In addition, polyclonal antisera raised against RIHisEae reacted with both fusion constructs and recognized an outer membrane protein of the same mass as intimin (97 kDa) in EHEC and enteropathogenic E. coli but not E. coli K-12. The intimin-specific antisera also blocked adherence of EHEC to HEp-2 cells. Thus, intimin (i) is a 97-kDa outer membrane protein in EHEC that serves as a requisite adhesin for attachment of the bacteria to epithelial cells, even when the protein is truncated by one-third at its amino terminus and (ii) can be added exogenously to specifically facilitate HEp-2 cell adherence of EHEC but not E. coli K-12. PMID:8675331

  1. Topological data analysis of Escherichia coli O157:H7 and non-O157 survival in soils.

    PubMed

    Ibekwe, Abasiofiok M; Ma, Jincai; Crowley, David E; Yang, Ching-Hong; Johnson, Alexis M; Petrossian, Tanya C; Lum, Pek Y

    2014-01-01

    Shiga toxin-producing E. coli O157:H7 and non-O157 have been implicated in many foodborne illnesses caused by the consumption of contaminated fresh produce. However, data on their persistence in soils are limited due to the complexity in datasets generated from different environmental variables and bacterial taxa. There is a continuing need to distinguish the various environmental variables and different bacterial groups to understand the relationships among these factors and the pathogen survival. Using an approach called Topological Data Analysis (TDA); we reconstructed the relationship structure of E. coli O157 and non-O157 survival in 32 soils (16 organic and 16 conventionally managed soils) from California (CA) and Arizona (AZ) with a multi-resolution output. In our study, we took a community approach based on total soil microbiome to study community level survival and examining the network of the community as a whole and the relationship between its topology and biological processes. TDA produces a geometric representation of complex data sets. Network analysis showed that Shiga toxin negative strain E. coli O157:H7 4554 survived significantly longer in comparison to E. coli O157:H7 EDL 933, while the survival time of E. coli O157:NM was comparable to that of E. coli O157:H7 EDL 933 in all of the tested soils. Two non-O157 strains, E. coli O26:H11 and E. coli O103:H2 survived much longer than E. coli O91:H21 and the three strains of E. coli O157. We show that there are complex interactions between E. coli strain survival, microbial community structures, and soil parameters.

  2. Topological data analysis of Escherichia coli O157:H7 and non-O157 survival in soils

    PubMed Central

    Ibekwe, Abasiofiok M.; Ma, Jincai; Crowley, David E.; Yang, Ching-Hong; Johnson, Alexis M.; Petrossian, Tanya C.; Lum, Pek Y.

    2014-01-01

    Shiga toxin-producing E. coli O157:H7 and non-O157 have been implicated in many foodborne illnesses caused by the consumption of contaminated fresh produce. However, data on their persistence in soils are limited due to the complexity in datasets generated from different environmental variables and bacterial taxa. There is a continuing need to distinguish the various environmental variables and different bacterial groups to understand the relationships among these factors and the pathogen survival. Using an approach called Topological Data Analysis (TDA); we reconstructed the relationship structure of E. coli O157 and non-O157 survival in 32 soils (16 organic and 16 conventionally managed soils) from California (CA) and Arizona (AZ) with a multi-resolution output. In our study, we took a community approach based on total soil microbiome to study community level survival and examining the network of the community as a whole and the relationship between its topology and biological processes. TDA produces a geometric representation of complex data sets. Network analysis showed that Shiga toxin negative strain E. coli O157:H7 4554 survived significantly longer in comparison to E. coli O157:H7 EDL 933, while the survival time of E. coli O157:NM was comparable to that of E. coli O157:H7 EDL 933 in all of the tested soils. Two non-O157 strains, E. coli O26:H11 and E. coli O103:H2 survived much longer than E. coli O91:H21 and the three strains of E. coli O157. We show that there are complex interactions between E. coli strain survival, microbial community structures, and soil parameters. PMID:25250242

  3. Transcriptomic analysis of Escherichia coli O157:H7 and K-12 cultures exposed to inorganic and organic acids in stationary phase reveals acidulant- and strain-specific acid tolerance responses.

    PubMed

    King, Thea; Lucchini, Sacha; Hinton, Jay C D; Gobius, Kari

    2010-10-01

    The food-borne pathogen Escherichia coli O157:H7 is commonly exposed to organic acid in processed and preserved foods, allowing adaptation and the development of tolerance to pH levels otherwise lethal. Since little is known about the molecular basis of adaptation of E. coli to organic acids, we studied K-12 MG1655 and O157:H7 Sakai during exposure to acetic, lactic, and hydrochloric acid at pH 5.5. This is the first analysis of the pH-dependent transcriptomic response of stationary-phase E. coli. Thirty-four genes and three intergenic regions were upregulated by both strains during exposure to all acids. This universal acid response included genes involved in oxidative, envelope, and cold stress resistance and iron and manganese uptake, as well as 10 genes of unknown function. Acidulant- and strain-specific responses were also revealed. The acidulant-specific response reflects differences in the modes of microbial inactivation, even between weak organic acids. The two strains exhibited similar responses to lactic and hydrochloric acid, while the response to acetic acid was distinct. Acidulant-dependent differences between the strains involved induction of genes involved in the heat shock response, osmoregulation, inorganic ion and nucleotide transport and metabolism, translation, and energy production. E. coli O157:H7-specific acid-inducible genes were identified, suggesting that the enterohemorrhagic E. coli strain possesses additional molecular mechanisms contributing to acid resistance that are absent in K-12. While E. coli K-12 was most resistant to lactic and hydrochloric acid, O157:H7 may have a greater ability to survive in more complex acidic environments, such as those encountered in the host and during food processing.

  4. Escherichia coli O26 in feedlot cattle: fecal prevalence, isolation, characterization, and effects of an E. coli O157 vaccine and a direct-fed microbial.

    PubMed

    Paddock, Zac D; Renter, David G; Cull, Charley A; Shi, Xiarong; Bai, Jianfa; Nagaraja, Tiruvoor G

    2014-03-01

    Escherichia coli O26 is second only to O157 in causing foodborne, Shiga toxin-producing E. coli (STEC) infections. Our objectives were to determine fecal prevalence and characteristics of E. coli O26 in commercial feedlot cattle (17,148) that were enrolled in a study to evaluate an E. coli O157:H7 siderophore receptor and porin (SRP(®)) vaccine (VAC) and a direct-fed microbial (DFM; 10(6) colony-forming units [CFU]/animal/day of Lactobacillus acidophilus and 10(9) CFU/animal/day of Propionibacterium freudenreichii). Cattle were randomly allocated to 40 pens within 10 complete blocks; pens were randomly assigned to control, VAC, DFM, or VAC+DFM treatments. Vaccine was administered on days 0 and 21, and DFM was fed throughout the study. Pen-floor fecal samples (30/pen) were collected weekly for the last 4 study weeks. Samples were enriched in E. coli broth and subjected to a multiplex polymerase chain reaction (PCR) designed to detect O26-specific wzx gene and four major virulence genes (stx1, stx2, eae, and ehxA) and to a culture-based procedure that involved immunomagnetic separation and plating on MacConkey agar. Ten presumptive E. coli colonies were randomly picked, pooled, and tested by the multiplex PCR. Pooled colonies positive for O26 serogroup were streaked on sorbose MacConkey agar, and 10 randomly picked colonies per sample were tested individually by the multiplex PCR. The overall prevalence of E. coli O26 was higher (p<0.001) by the culture-based method compared to the PCR assay (22.7 versus 10.5%). The interventions (VAC and or DFM) had no impact on fecal shedding of O26. Serogroup O26 was recovered in pure culture from 23.9% (260 of 1089) of O26 PCR-positive pooled colonies. Only 7 of the 260 isolates were positive for the stx gene and 90.1% of the isolates possessed an eaeβ gene that codes for intimin subtype β, but not the bfpA gene, which codes for bundle-forming pilus. Therefore, the majority of the O26 recovered from feedlot cattle feces was

  5. Fate of enterohemorrhagic Escherichia coli O157:H7 in apple cider with and without preservatives.

    PubMed

    Zhao, T; Doyle, M P; Besser, R E

    1993-08-01

    A strain of enterohemorrhagic Escherichia coli serotype O157:H7 isolated from a patient in an apple cider-related outbreak was used to study the fate of E. coli O157:H7 in six different lots of unpasteurized apple cider. In addition, the efficacy of two preservatives, 0.1% sodium benzoate and 0.1% potassium sorbate, used separately and in combination was evaluated for antimicrobial effects on the bacterium. Studies were done at 8 or 25 degrees C with ciders having pH values of 3.6 to 4.0. The results revealed that E. coli O157:H7 populations increased slightly (ca. 1 log10 CFU/ml) and then remained stable for approximately 12 days in lots inoculated with an initial population of 10(5) E. coli O157:H7 organisms per ml and held at 8 degrees C. The bacterium survived from 10 to 31 days or 2 to 3 days at 8 or 25 degrees C, respectively, depending on the lot. Potassium sorbate had minimal effect on E. coli O157:H7 populations, with survivors detected for 15 to 20 days or 1 to 3 days at 8 or 25 degrees C, respectively. In contrast, survivors in cider containing sodium benzoate were detected for only 2 to 10 days or less than 1 to 2 days at 8 or 25 degrees C, respectively. The highest rates of inactivation occurred in the presence of a combination of 0.1% sodium benzoate and 0.1% potassium sorbate. The use of 0.1% sodium benzoate, an approved preservative used by some cider processors, will substantially increase the safety of apple cider in terms of E. coli O157:H7, in addition to suppressing the growth of yeasts and molds. PMID:8368839

  6. Epidemiological studies on Escherichia coli O157:H7 in Egyptian sheep.

    PubMed

    Kamel, Mohammed; Abo El-Hassan, Diea G; El-Sayed, Amr

    2015-08-01

    In the present work, the epidemiological role of apparently healthy sheep in transmission of Escherichia coli O157:H7 in different seasons was investigated. Fecal samples (convenience sampling) of apparently healthy farmed sheep (three farms, n = 70) and from 15 wandering flocks fed on city wastes (n = 80) in the Giza governorate were examined. The samples were collected in spring under mild weather conditions and during hot summer to be compared. Out of the 150 animals, 13 (8.7%) were E. coli O157 shedders. The 13 ovine sorbitol-negative E. coli O157 were characterized by different PCR sets. The eae gene was detected in 11 isolate (85%), stx1 in 3 isolates (23%), stx2 in 8 isolates (62%), and finally the hlyA in 11 isolate (85%). Among the 13 isolates, 2 strains (15%) were positive for eae, stx1, stx2, and hlyA as gene combination, one isolate (8%) for eae, stx1, and hlyA, 5 isolates (38%) for eae, stx2, and hlyA, 1 isolate (8%) for eae and stx2, 2 isolates (15%) contained eae and hlyA, 1 isolate (8%) contained hlyA only, and finally, 1 isolate (8%) did not contain any of these genes. None of the isolates showed the gene combination eae stx1, stx1 hlyA, or stx2 hlyA. The results indicated significant association of unfavorable weather and management conditions on O157:H7 shedding while the age or sex did not play any role in this process.

  7. Early attachment sites for Shiga-toxigenic Escherichia coli O157:H7 in experimentally inoculated weaned calves.

    PubMed

    Dean-Nystrom, Evelyn A; Stoffregen, William C; Bosworth, Brad T; Moon, Harley W; Pohlenz, Joachim F

    2008-10-01

    Weaned 3- to 4-month-old calves were fasted for 48 h, inoculated with 10(10) CFU of Shiga toxin-positive Escherichia coli (STEC) O157:H7 strain 86-24 (STEC O157) or STEC O91:H21 strain B2F1 (STEC O91), Shiga toxin-negative E. coli O157:H7 strain 87-23 (Stx(-) O157), or a nonpathogenic control E. coli strain, necropsied 4 days postinoculation, and examined bacteriologically and histologically. Some calves were treated with dexamethasone (DEX) for 5 days (3 days before, on the day of, and 1 day after inoculation). STEC O157 bacteria were recovered from feces, intestines, or gall bladders of 74% (40/55) of calves 4 days after they were inoculated with STEC O157. Colon and cecum were sites from which inoculum-type bacteria were most often recovered. Histologic lesions of attaching-and-effacing (A/E) O157(+) bacteria were observed in 69% (38/55) of the STEC O157-inoculated calves. Rectum, ileocecal valve, and distal colon were sites most likely to contain A/E O157(+) bacteria. Fecal and intestinal levels of STEC O157 bacteria were significantly higher and A/E O157(+) bacteria were more common in DEX-treated calves than in nontreated calves inoculated with STEC O157. Fecal STEC O157 levels were significantly higher than Stx(-) O157, STEC O91, or control E. coli; only STEC O157 cells were recovered from tissues. Identifying the rectum, ileocecal valve, and distal colon as early STEC O157 colonization sites and finding that DEX treatment enhances the susceptibility of weaned calves to STEC O157 colonization will facilitate the identification and evaluation of interventions aimed at reducing STEC O157 infection in cattle. PMID:18723644

  8. Early Attachment Sites for Shiga-Toxigenic Escherichia coli O157:H7 in Experimentally Inoculated Weaned Calves▿

    PubMed Central

    Dean-Nystrom, Evelyn A.; Stoffregen, William C.; Bosworth, Brad T.; Moon, Harley W.; Pohlenz, Joachim F.

    2008-01-01

    Weaned 3- to 4-month-old calves were fasted for 48 h, inoculated with 1010 CFU of Shiga toxin-positive Escherichia coli (STEC) O157:H7 strain 86-24 (STEC O157) or STEC O91:H21 strain B2F1 (STEC O91), Shiga toxin-negative E. coli O157:H7 strain 87-23 (Stx− O157), or a nonpathogenic control E. coli strain, necropsied 4 days postinoculation, and examined bacteriologically and histologically. Some calves were treated with dexamethasone (DEX) for 5 days (3 days before, on the day of, and 1 day after inoculation). STEC O157 bacteria were recovered from feces, intestines, or gall bladders of 74% (40/55) of calves 4 days after they were inoculated with STEC O157. Colon and cecum were sites from which inoculum-type bacteria were most often recovered. Histologic lesions of attaching-and-effacing (A/E) O157+ bacteria were observed in 69% (38/55) of the STEC O157-inoculated calves. Rectum, ileocecal valve, and distal colon were sites most likely to contain A/E O157+ bacteria. Fecal and intestinal levels of STEC O157 bacteria were significantly higher and A/E O157+ bacteria were more common in DEX-treated calves than in nontreated calves inoculated with STEC O157. Fecal STEC O157 levels were significantly higher than Stx− O157, STEC O91, or control E. coli; only STEC O157 cells were recovered from tissues. Identifying the rectum, ileocecal valve, and distal colon as early STEC O157 colonization sites and finding that DEX treatment enhances the susceptibility of weaned calves to STEC O157 colonization will facilitate the identification and evaluation of interventions aimed at reducing STEC O157 infection in cattle. PMID:18723644

  9. [Escherichia coli O157:H7 detection in fresh ground beef and hamburgers].

    PubMed

    Marzocca, M A; Marucci, P L; Sica, M G; Alvarez, E E

    2006-01-01

    Escherichia coli O157:H7 is an emergent pathogen associated with food transmitted diseases. In 1982, Escherichia coli O157:H7 was for the first time identified as the cause of two hemorrhagic colitis outbreaks in the United States. It is now well known that most cases of hemolytic uremic syndrome are caused by these bacteria. The objective of this work was to detect the microorganism in fresh ground beef and hamburgers. From April 2003 to August 2004 samples were taken at sale points of our supermarket chain, totalling 37 and 43, respectively. These samples were processed using the EC selective enrichment broth containing novobiocin, then followed by the application of an immunocapture method (TECRA E. COLI O157 IMMUNOCAPTURE ECOICM 20), and later isolation in MacConkey sorbitol agar with cefixime and potassium tellurite, in a chromogenic medium. The suspected strains were genotypically characterized by PCR detection of the stx1, stx2, eaeA, and EHEC-hlyA genes, and by a colony blot hybridization assay. Serotyping, antimicrobial susceptibility patterns, and production of Stx by a specific cytotoxicity assay on Vero cells were also determined. E coli O157:H7 was isolated in only one fresh ground beef sample (2,7%), identified as gene eae (+)/ stx2/EHEC-hlyA.

  10. [Escherichia coli O157:H7 detection in fresh ground beef and hamburgers].

    PubMed

    Marzocca, M A; Marucci, P L; Sica, M G; Alvarez, E E

    2006-01-01

    Escherichia coli O157:H7 is an emergent pathogen associated with food transmitted diseases. In 1982, Escherichia coli O157:H7 was for the first time identified as the cause of two hemorrhagic colitis outbreaks in the United States. It is now well known that most cases of hemolytic uremic syndrome are caused by these bacteria. The objective of this work was to detect the microorganism in fresh ground beef and hamburgers. From April 2003 to August 2004 samples were taken at sale points of our supermarket chain, totalling 37 and 43, respectively. These samples were processed using the EC selective enrichment broth containing novobiocin, then followed by the application of an immunocapture method (TECRA E. COLI O157 IMMUNOCAPTURE ECOICM 20), and later isolation in MacConkey sorbitol agar with cefixime and potassium tellurite, in a chromogenic medium. The suspected strains were genotypically characterized by PCR detection of the stx1, stx2, eaeA, and EHEC-hlyA genes, and by a colony blot hybridization assay. Serotyping, antimicrobial susceptibility patterns, and production of Stx by a specific cytotoxicity assay on Vero cells were also determined. E coli O157:H7 was isolated in only one fresh ground beef sample (2,7%), identified as gene eae (+)/ stx2/EHEC-hlyA. PMID:16784134

  11. Leakage of Intracellular UV Materials of High Hydrostatic Pressure-Injured Escherichia Coli O157:H7 Strains in Tomato Juice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effect of high hydrostatic pressure (HHP) treatment on inactivation, injury and recovery of Salmonella Enteritidis and Escherichia coli O157:H7 cocktail inoculated in tomato juice (pH 4.1) and phosphate buffer saline (PBS. pH 7.2) at 8.0 log CFU/ml and treated at 350, 400, 450 MPa for 20 min at ...

  12. Sequence of colonization determines the composition of mixed biofilms by Escherichia coli O157:H7 and O111:H8 strains

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacterial biofilms are one of the potential sources of cross-contamination in food processing environments. Shiga-toxin producing Escherichia coli (STEC) O157:H7 and O111:H8 are important foodborne pathogens capable of forming biofilms, and the coexistence of these two STEC serotypes has been detec...

  13. Escherichia coli O157:H7 lacking qseBC encoded quorum sensing system outcompetes the parent strain in colonization of cattle intestine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The qseBC encoded quorum-sensing system (QS) regulates motility of enterohemorrhagic Escherichia coli (EHEC) O157:H7 in response to bacterial autoinducer-3 (AI-3) and mammalian stress hormones epinephrine (E) and norepinephrine (NE). The qseC gene encodes a sensory kinase that post-autophosphorylati...

  14. Serotype O157:H7 Escherichia coli from bovine and meat sources.

    PubMed

    Dorn, C R; Angrick, E J

    1991-06-01

    Serotype O157:H7 Escherichia coli strains from several different bovine and meat (beef) sources were studied to determine the diversity of their virulence properties and to compare their plasmid characteristics. Eighteen strains from cattle feces, 2 from water buffalo feces, 3 from beef samples, and 2 from feces of human hemolytic uremic syndrome cases were examined. All of these strains hybridized with the CVD419 DNA probe which identifies serotype O157:H7 and many other serotypes of verocytotoxin-producing E. coli. Of 15 bovine strains that hybridized with two verocytotoxin DNA probes, 8 hybridized with both verocytotoxin 1 (VT1) and VT2 probes, 5 hybridized with only the VT2 probe, and 2 hybridized with only the VT1 probe. This distribution was similar to that reported for O157:H7 E. coli isolated from humans. All three beef isolates hybridized with both VT1 and VT2 probes. All strains that hybridized with the VT probes were positive in the verocytotoxin assay, and all probe-negative strains were negative in the assay. All the strains possessed large plasmids with molecular sizes ranging from 53 to 64 MDa. Fifteen of the 20 cattle and water buffalo strains had one or more additional small plasmids. Restriction patterns resulting from HindIII, SmaI, and BamHI digestions of the large plasmids were used to compare all possible pairs of five different single plasmid-bearing strains from different countries (Egypt, England, and the United States). The restriction patterns of these strains were distinct, and the mean coefficients of similarity for these comparisons ranged from 71 to 91%, indicating a moderate degree of genetic diversity. This diversity and the presence of multiple plasmids in many bovine and human O157:H7 strains reinforce the usefulness of plasmid analysis in future studies. Only four of the 20 bovine strains and 1 of the 3 beef strains possessed the capability for adherence to HEp-2 and Intestine 407 cells in the presence of mannose, indicating that

  15. Incidence and Tracking of Escherichia coli O157:H7 in a Major Produce Production Region in California

    PubMed Central

    Cooley, Michael; Carychao, Diana; Crawford-Miksza, Leta; Jay, Michele T.; Myers, Carol; Rose, Christopher; Keys, Christine; Farrar, Jeff; Mandrell, Robert E.

    2007-01-01

    Fresh vegetables have become associated with outbreaks caused by Escherichia coli O157:H7 (EcO157). Between 1995–2006, 22 produce outbreaks were documented in the United States, with nearly half traced to lettuce or spinach grown in California. Outbreaks between 2002 and 2006 induced investigations of possible sources of pre-harvest contamination on implicated farms in the Salinas and San Juan valleys of California, and a survey of the Salinas watershed. EcO157 was isolated at least once from 15 of 22 different watershed sites over a 19 month period. The incidence of EcO157 increased significantly when heavy rain caused an increased flow rate in the rivers. Approximately 1000 EcO157 isolates obtained from cultures of>100 individual samples were typed using Multi-Locus Variable-number-tandem-repeat Analysis (MLVA) to assist in identifying potential fate and transport of EcO157 in this region. A subset of these environmental isolates were typed by Pulse Field Gel Electrophoresis (PFGE) in order to make comparisons with human clinical isolates associated with outbreak and sporadic illness. Recurrence of identical and closely related EcO157 strains from specific locations in the Salinas and San Juan valleys suggests that transport of the pathogen is usually restricted. In a preliminary study, EcO157 was detected in water at multiple locations in a low-flow creek only within 135 meters of a point source. However, possible transport up to 32 km was detected during periods of higher water flow associated with flooding. During the 2006 baby spinach outbreak investigation, transport was also detected where water was unlikely to be involved. These results indicate that contamination of the environment is a dynamic process involving multiple sources and methods of transport. Intensive studies of the sources, incidence, fate and transport of EcO157 near produce production are required to determine the mechanisms of pre-harvest contamination and potential risks for human

  16. Verotoxin-producing Escherichia coli in Spain: prevalence, serotypes, and virulence genes of O157:H7 and non-O157 VTEC in ruminants, raw beef products, and humans.

    PubMed

    Blanco, Jorge; Blanco, Miguel; Blanco, Jesus E; Mora, Azucena; González, Enrique A; Bernárdez, Maria I; Alonso, Maria P; Coira, Amparo; Rodriguez, Asuncion; Rey, Joaquin; Alonso, Juan M; Usera, Miguel A

    2003-04-01

    In Spain, as in many other countries, verotoxin-producing Escherichia coli (VTEC) strains have been frequently isolated from cattle, sheep, and foods. VTEC strains have caused seven outbreaks in Spain (six caused by E. coli O157:H7 and one by E. coli O111:H- [nonmotile]) in recent years. An analysis of the serotypes indicated serological diversity. Among the strains isolated from humans, serotypes O26:H11, O111:H-, and O157:H7 were found to be more prevalent. The most frequently detected serotypes in cattle were O20:H19, O22:H8, O26:H11, O77:H41, O105:H18, O113:H21, O157:H7, O171:H2, and OUT (O untypeable):H19. Different VTEC serotypes (e.g., O5:H-, O6:H10, O91:H-, O117:H-, O128:H-, O128:H2, O146:H8, O146:H21, O156:H-, and OUT:H21) were found more frequently in sheep. These observations suggest a host serotype specificity for some VTEC. Numerous bovine and ovine VTEC serotypes detected in Spain were associated with human illnesses, confirming that ruminants are important reservoirs of pathogenic VTEC. VTEC can produce one or two toxins (VT1 and VT2) that cause human illnesses. These toxins are different proteins encoded by different genes. Another virulence factor expressed by VTEC is the protein intimin that is responsible for intimate attachment of VTEC and effacing lesions in the intestinal mucosa. This virulence factor is encoded by the chromosomal gene eae. The eae gene was found at a much less frequency in bovine (17%) and ovine (5%) than in human (45%) non-O157 VTEC strains. This may support the evidence that the eae gene contributes significantly to the virulence of human VTEC strains and that many animal non-O157 VTEC strains are less pathogenic to humans.

  17. Verotoxin-producing Escherichia coli in Spain: prevalence, serotypes, and virulence genes of O157:H7 and non-O157 VTEC in ruminants, raw beef products, and humans.

    PubMed

    Blanco, Jorge; Blanco, Miguel; Blanco, Jesus E; Mora, Azucena; González, Enrique A; Bernárdez, Maria I; Alonso, Maria P; Coira, Amparo; Rodriguez, Asuncion; Rey, Joaquin; Alonso, Juan M; Usera, Miguel A

    2003-04-01

    In Spain, as in many other countries, verotoxin-producing Escherichia coli (VTEC) strains have been frequently isolated from cattle, sheep, and foods. VTEC strains have caused seven outbreaks in Spain (six caused by E. coli O157:H7 and one by E. coli O111:H- [nonmotile]) in recent years. An analysis of the serotypes indicated serological diversity. Among the strains isolated from humans, serotypes O26:H11, O111:H-, and O157:H7 were found to be more prevalent. The most frequently detected serotypes in cattle were O20:H19, O22:H8, O26:H11, O77:H41, O105:H18, O113:H21, O157:H7, O171:H2, and OUT (O untypeable):H19. Different VTEC serotypes (e.g., O5:H-, O6:H10, O91:H-, O117:H-, O128:H-, O128:H2, O146:H8, O146:H21, O156:H-, and OUT:H21) were found more frequently in sheep. These observations suggest a host serotype specificity for some VTEC. Numerous bovine and ovine VTEC serotypes detected in Spain were associated with human illnesses, confirming that ruminants are important reservoirs of pathogenic VTEC. VTEC can produce one or two toxins (VT1 and VT2) that cause human illnesses. These toxins are different proteins encoded by different genes. Another virulence factor expressed by VTEC is the protein intimin that is responsible for intimate attachment of VTEC and effacing lesions in the intestinal mucosa. This virulence factor is encoded by the chromosomal gene eae. The eae gene was found at a much less frequency in bovine (17%) and ovine (5%) than in human (45%) non-O157 VTEC strains. This may support the evidence that the eae gene contributes significantly to the virulence of human VTEC strains and that many animal non-O157 VTEC strains are less pathogenic to humans. PMID:12671177

  18. Infrequent internalization of Escherichia coli O157:H7 into field-grown leafy greens.

    PubMed

    Erickson, Marilyn C; Webb, Cathy C; Diaz-Perez, Juan Carlos; Phatak, Sharad C; Silvoy, John J; Davey, Lindsey; Payton, Alison S; Liao, Jean; Ma, Li; Doyle, Michael P

    2010-03-01

    Several sources of contamination of fresh produce by Escherichia coli O157:H7 (O157) have been identified and include contaminated irrigation water and improperly composted animal waste; however, field studies evaluating the potential for internalization of O157 into leafy greens from these sources have not been conducted. Irrigation water inoculated with green fluorescent plasmid-labeled Shiga toxin-negative strains (50 ml of 10(2), 10(4), or 10(6) CFU of O157 per ml) was applied to soil at the base of spinach plants of different maturities in one field trial. In a second trial, contaminated compost (1.8 kg of 10(3) or 10(5) CFU of O157 per g) was applied to field plots (0.25 by 3.0 m) prior to transplantation of spinach, lettuce, or parsley plants. E. coli O157:H7 persisted in the soil up to harvest (day 76 posttransplantation) following application of contaminated irrigation water; however, internalized O157 was not detected in any spinach leaves or in roots exposed to O157 during the early or late growing season. Internalized O157 was detected in root samples collected 7 days after plants were contaminated in mid-season, with 5 of 30 samples testing positive for O157 by enrichment; however, O157 was not detected by enrichment in surface-disinfected roots on days 14 or 22. Roots and leaves from transplanted spinach, lettuce, and parsley did not internalize O157 for up to 50 days in the second trial. These results indicate that internalization of O157 via plant roots in the field is rare and when it does occur, O157 does not persist 7 days later.

  19. Genetic diversity and antimicrobial resistance among isolates of Escherichia coli O157: H7 from feces and hides of super-shedders and low-shedding pen-mates in two commercial beef feedlots

    PubMed Central

    2012-01-01

    Background Cattle shedding at least 104 CFU Escherichia coli O157:H7/g feces are described as super-shedders and have been shown to increase transmission of E. coli O157:H7 to other cattle in feedlots. This study investigated relationships among fecal isolates from super-shedders (n = 162), perineal hide swab isolates (PS) from super-shedders (n = 137) and fecal isolates from low-shedder (< 104 CFU/g feces) pen-mates (n = 496) using pulsed-field gel electrophoresis (PFGE). A subsample of these fecal isolates (n = 474) was tested for antimicrobial resistance. Isolates of E. coli O157:H7 were obtained from cattle in pens (avg. 181 head) at 2 commercial feedlots in southern Alberta with each steer sampled at entry to the feedlot and prior to slaughter. Results Only 1 steer maintained super-shedder status at both samplings, although approximately 30% of super-shedders in sampling 1 had low-shedder status at sampling 2. A total of 85 restriction endonuclease digestion clusters (REPC; 90% or greater similarity) and 86 unique isolates (< 90% similarity) were detected, with the predominant REPC (30% of isolates) being isolated from cattle in all feedlot pens, although it was not associated with shedding status (super- or low-shedder; P = 0.94). Only 2/21 super-shedders had fecal isolates in the same REPC at both samplings. Fecal and PS isolates from individual super-shedders generally belonged to different REPCs, although fecal isolates of E. coli O157:H7 from super- and low-shedders showed greater similarity (P < 0.001) than those from PS. For 77% of super-shedders, PFGE profiles of super-shedder fecal and PS isolates were distinct from all low-shedder fecal isolates collected in the same pen. A low level of antimicrobial resistance (3.7%) was detected and prevalence of antimicrobial resistance did not differ among super- and low-shedder isolates (P = 0.69), although all super-shedder isolates with antimicrobial resistance (n = 3) were

  20. E. coli O157 on Scottish cattle farms: Evidence of local spread and persistence using repeat cross-sectional data

    PubMed Central

    2014-01-01

    Background Escherichia coli (E. coli) O157 is a virulent zoonotic strain of enterohaemorrhagic E. coli. In Scotland (1998-2008) the annual reported rate of human infection is 4.4 per 100,000 population which is consistently higher than other regions of the UK and abroad. Cattle are the primary reservoir. Thus understanding infection dynamics in cattle is paramount to reducing human infections. A large database was created for farms sampled in two cross-sectional surveys carried out in Scotland (1998 - 2004). A statistical model was generated to identify risk factors for the presence of E. coli O157 on farms. Specific hypotheses were tested regarding the presence of E. coli O157 on local farms and the farms previous status. Pulsed-field gel electrophoresis (PFGE) profiles were further examined to ascertain whether local spread or persistence of strains could be inferred. Results The presence of an E. coli O157 positive local farm (average distance: 5.96km) in the Highlands, North East and South West, farm size and the number of cattle moved onto the farm 8 weeks prior to sampling were significant risk factors for the presence of E. coli O157 on farms. Previous status of a farm was not a significant predictor of current status (p = 0.398). Farms within the same sampling cluster were significantly more likely to be the same PFGE type (p < 0.001), implicating spread of strains between local farms. Isolates with identical PFGE types were observed to persist across the two surveys, including 3 that were identified on the same farm, suggesting an environmental reservoir. PFGE types that were persistent were more likely to have been observed in human clinical infections in Scotland (p < 0.001) from the same time frame. Conclusions The results of this study demonstrate the spread of E. coli O157 between local farms and highlight the potential link between persistent cattle strains and human clinical infections in Scotland. This novel insight into the epidemiology of

  1. Geographic Divergence of Bovine and Human Shiga Toxin–Producing Escherichia coli O157:H7 Genotypes, New Zealand1

    PubMed Central

    Cookson, Adrian L.; Campbell, Donald M.; Duncan, Gail E.; Prattley, Deborah; Carter, Philip; Besser, Thomas E.; Shringi, Smriti; Hathaway, Steve; Marshall, Jonathan C.; French, Nigel P.

    2014-01-01

    Shiga toxin-producing Escherichia coli (STEC) O157:H7 is a zoonotic pathogen of public health concern worldwide. To compare the local and large-scale geographic distributions of genotypes of STEC O157:H7 isolates obtained from various bovine and human sources during 2008–2011, we used pulsed-field gel electrophoresis and Shiga toxin–encoding bacteriophage insertion (SBI) typing. Using multivariate methods, we compared isolates from the North and South Islands of New Zealand with isolates from Australia and the United States. The STEC O157:H7 population structure differed substantially between the 2 islands and showed evidence of finer scale spatial structuring, which is consistent with highly localized transmission rather than disseminated foodborne outbreaks. The distribution of SBI types differed markedly among isolates from New Zealand, Australia, and the United States. Our findings also provide evidence for the historic introduction into New Zealand of a subset of globally circulating STEC O157:H7 strains that have continued to evolve and be transmitted locally between cattle and humans. PMID:25568924

  2. Effect of spinach cultivar and bacterial adherence factors on survival of Escherichia coli O157:H7 on spinach leaves.

    PubMed

    Macarisin, Dumitru; Patel, Jitendra; Bauchan, Gary; Giron, Jorge A; Ravishankar, Sadhana

    2013-11-01

    Similar to phytopathogens, human bacterial pathogens have been shown to colonize the plant phylloplane. In addition to environmental factors, such as temperature, UV, relative humidity, etc., the plant cultivar and, specifically, the leaf blade morphological characteristics may affect the persistence of enteropathogens on leafy greens. This study was conducted to evaluate the effect of cultivar-dependent leaf topography and the role of strain phenotypic characteristics on Escherichia coli O157:H7 persistence on organic spinach. Spinach cultivars Emilia, Lazio, Space, and Waitiki were experimentally inoculated with the foodborne E. coli O157:H7 isolate EDL933 and its isogenic mutants deficient in cellulose, curli, or both curli and cellulose production. Leaves of 6-week-old plants were inoculated with 6.5 log CFU per leaf in a biosafety level 2 growth chamber. At 0, 1, 7, and 14 days, E. coli O157:H7 populations were determined by plating on selective medium and verified by laser scanning confocal microscopy. Leaf morphology (blade roughness and stoma density) was evaluated by low-temperature and variable-pressure scanning electron microscopy. E. coli O157:H7 persistence on spinach was significantly affected by cultivar and strain phenotypic characteristics, specifically, the expression of curli. Leaf blade roughness and stoma density influenced the persistence of E. coli O157:H7 on spinach. Cultivar Waitiki, which had the greatest leaf roughness, supported significantly higher E. coli O157:H7 populations than the other cultivars. These two morphological characteristics of spinach cultivars should be taken into consideration in developing intervention strategies to enhance the microbial safety of leafy greens.

  3. Assessment of Virulence Factors Characteristic of Human Escherichia coli Pathotypes and Antimicrobial Resistance in O157:H7 and Non-O157:H7 Isolates from Livestock in Spain

    PubMed Central

    Cabal, A.; Gómez-Barrero, S.; Porrero, C.; Bárcena, C.; López, G.; Cantón, R.; Gortázar, C.; Domínguez, L.

    2013-01-01

    The distribution of virulence factors (VFs) typical of diarrheagenic Escherichia coli and the antimicrobial resistance (AMR) profiles were assessed in 780 isolates from healthy pigs, broilers, and cattle from Spain. VF distribution was broader than expected, although at low prevalence for most genes, with AMR being linked mainly to host species. PMID:23603685

  4. Assessment of virulence factors characteristic of human Escherichia coli pathotypes and antimicrobial resistance in O157:H7 and non-O157:H7 isolates from livestock in Spain.

    PubMed

    Cabal, A; Gómez-Barrero, S; Porrero, C; Bárcena, C; López, G; Cantón, R; Gortázar, C; Domínguez, L; Álvarez, J

    2013-07-01

    The distribution of virulence factors (VFs) typical of diarrheagenic Escherichia coli and the antimicrobial resistance (AMR) profiles were assessed in 780 isolates from healthy pigs, broilers, and cattle from Spain. VF distribution was broader than expected, although at low prevalence for most genes, with AMR being linked mainly to host species.

  5. [Survival of VTEC O157 and non-O157 in water troughs and bovine feces].

    PubMed

    Polifroni, Rosana; Etcheverría, Analía I; Arroyo, Guillermo H; Padola, Nora L

    2014-01-01

    Verotoxin-producing Escherichia coli (VTEC) is the etiologic agent of hemolytic-uremic syndrome (HUS), which typically affects children ranging in age from six months to five years old. Transmission is produced by consumption of contaminated food, by direct contact with animals or the environment and from person to person. In previous studies we determined that the environment of a dairy farm is a non-animal reservoir; thus, we proposed to study the survival of 4 VTEC isolates (O20:H19; O91:H21; O157:H7 and O178:H19) in sterile water troughs and bovine feces by viable bacteria count and detection of virulence genes by PCR. It was demonstrated that the survival of different VTEC isolates (O157 and non-O157) varied in terms of their own characteristics as well as of the environmental conditions where they were found. The main differences between isolates were their survival time and the maximal counts reached. The competitive and adaptive characteristics of some isolates increase the infection risk for people that are visiting or working on a farm, as well as the risk for reinfection of the animals and food contamination.

  6. Foodborne transmission of sorbitol-fermenting Escherichia coli O157:[H7] via ground beef: an outbreak in northern France, 2011.

    PubMed

    King, L A; Loukiadis, E; Mariani-Kurkdjian, P; Haeghebaert, S; Weill, F-X; Baliere, C; Ganet, S; Gouali, M; Vaillant, V; Pihier, N; Callon, H; Novo, R; Gaillot, O; Thevenot-Sergentet, D; Bingen, E; Chaud, P; de Valk, H

    2014-12-01

    Sorbitol-fermenting Escherichia coli O157:[H7] is a particularly virulent clone of E. coli O157:H7 associated with a higher incidence of haemolytic uraemic syndrome and a higher case fatality rate. Many fundamental aspects of its epidemiology remain to be elucidated, including its reservoir and transmission routes and vehicles. We describe an outbreak of sorbitol-fermenting E. coli O157:[H7] that occurred in France in 2011. Eighteen cases of paediatric haemolytic uraemic syndrome with symptom onset between 6 June and 15 July 2011 were identified among children aged 6 months to 10 years residing in northern France. A strain of sorbitol-fermenting E. coli O157:[H7] stx2a eae was isolated from ten cases. Epidemiological, microbiological and trace-back investigations identified multiply-contaminated frozen ground beef products bought in a supermarket chain as the outbreak vehicle. Strains with three distinct pulsotypes that were isolated from patients, ground beef preparations recovered from patients' freezers and from stored production samples taken at the production plant were indistinguishable upon molecular comparison. This investigation documents microbiologically confirmed foodborne transmission of sorbitol-fermenting of E. coli O157 via beef and could additionally provide evidence of a reservoir in cattle for this pathogen.

  7. Shiga toxin-producing Escherichia coli O157, O26 and O111 in cattle faeces and hides in Italy

    PubMed Central

    Bonardi, S.; Alpigiani, I.; Tozzoli, R.; Vismarra, A.; Zecca, V.; Greppi, C.; Bacci, C.; Bruini, I.; Brindani, F.

    2015-01-01

    Introduction Ruminants are regarded as the natural reservoir for Shiga toxin-producing Escherichia coli (STEC), especially of serogroup O157. Materials and methods During 2011 and 2012, 320 samples (160 faecal samples from the rectum and 160 hide samples from the brisket area) were collected from 160 cattle at slaughter in Northern Italy during warm months (May to October). Cattle were reared in different farms and their age at slaughter ranged between nine months and 15 years, most of them being culled cattle (median age: six years; average age: 4.6 years). Samples were tested by immunomagnetic-separation technique for E coli O157 and O26 and by a screening PCR for stx genes followed by cultural detection of STEC. The virulence genes stx1, stx2, eae, and e-hlyA were detected and among stx2-positive isolates the presence of the stx2a and stx2c variants was investigated. Results Twenty-one of 160 cattle (13.1 per cent; 95 per cent CI 8.3 to 19.4 per cent) were found to be faecal carriers of STEC. STEC O157 was found in 10 (6.3 per cent) samples, STEC O26 in six (3.8 per cent) and STEC O111 in one (0.6 per cent). Four isolates (2.5 per cent) were O not determined (OND). Six out of 160 (3.8 per cent; 95 per cent CI 1.4 to 8.0 per cent) hide samples were positive for STEC; four hides (2.5 per cent) were contaminated by STEC O157 and two (1.3 per cent) by STEC O26. In three cattle (1.9 per cent) STEC from both faeces and hides were detected. Among STEC O157, 87.5 per cent of them carried the stx2c gene and 12.5 per cent carried both stx1 and stx2c genes. No O157 isolate harboured stx2a variant. STEC O26 and O111 carried the stx1 gene only. One OND strain carried both the stx2a and stx2c genes. Conclusions This study shows that STEC O157 from cattle can harbour the stx2c variant, which is associated with haemolytic uraemic syndrome in humans, and that cattle hides may be a source of human pathogenic STEC O157 and O26 in the slaughterhouse environment. PMID:26392887

  8. Utility of Whole-Genome Sequencing of Escherichia coli O157 for Outbreak Detection and Epidemiological Surveillance

    PubMed Central

    Allison, Lesley; Ward, Melissa; Dallman, Timothy J.; Clark, Richard; Fawkes, Angie; Murphy, Lee; Hanson, Mary

    2015-01-01

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

  9. Utility of Whole-Genome Sequencing of Escherichia coli O157 for Outbreak Detection and Epidemiological Surveillance.

    PubMed

    Holmes, Anne; Allison, Lesley; Ward, Melissa; Dallman, Timothy J; Clark, Richard; Fawkes, Angie; Murphy, Lee; Hanson, Mary

    2015-11-01

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

  10. A community outbreak of Vero cytotoxin producing Escherichia coli O157 infection linked to a small farm dairy.

    PubMed

    Clark, A; Morton, S; Wright, P; Corkish, J; Bolton, F J; Russell, J

    1997-12-12

    A community outbreak of infection with Vero cytotoxin producing Escherichia coli O157 (VTEC 0157) occurred in a small area of north west England in 1996. An outbreak control team was established to investigate the outbreak and implement control measures. Nine people developed symptomatic infections with VTEC O157, and a further three were found to be excreting the bacteria. All were infected with the same genotype of VTEC O157. Three children under 5 years of age and one adult were admitted to hospital. One child developed haemolytic uraemic syndrome. All cases recovered. All primary cases had consumed milk from a particular farm dairy. No other common foods were identified. The farm dairy had a faulty pasteuriser and the potential for post pasteurisation contamination existed. VTEC O157 was isolated from a milk sock specimen and from two cows, but these strains differed from that infecting the cases. All local doctors and the public were alerted and advised about preventative measures. Distribution of unpasteurised milk from the farm was discontinued as was the sale of pasteurised milk when the faulty pasteuriser was discovered. A replacement pasteuriser was installed and checked before milk was released for human consumption. No conclusive evidence of the origin of this outbreak was found, but the farm was the most probable source. The investigations raised concerns about the distribution of VTEC O157 colonised dairy cattle, the natural history of such colonisation, the effectiveness of pasteurisation with respect to the elimination of VTEC O157, and the effectiveness of current legislation for the prevention and control of milkborne infection.

  11. Differential Virulence of Clinical and Bovine-Biased Enterohemorrhagic Escherichia coli O157:H7 Genotypes in Piglet and Dutch Belted Rabbit Models

    PubMed Central

    Shringi, Smriti; García, Alexis; Lahmers, Kevin K.; Potter, Kathleen A.; Muthupalani, Sureshkumar; Swennes, Alton G.; Hovde, Carolyn J.; Call, Douglas R.; Fox, James G.

    2012-01-01

    Enterohemorrhagic Escherichia coli O157:H7 (EHEC O157) is an important cause of food and waterborne illness in the developed countries. Cattle are a reservoir host of EHEC O157 and a major source of human exposure through contaminated meat products. Shiga toxins (Stxs) are an important pathogenicity trait of EHEC O157. The insertion sites of the Stx-encoding bacteriophages differentiate EHEC O157 isolates into genogroups commonly isolated from cattle but rarely from sick humans (bovine-biased genotypes [BBG]) and those commonly isolated from both cattle and human patients (clinical genotypes [CG]). Since BBG and CG share the cardinal virulence factors of EHEC O157 and are carried by cattle at similar prevalences, the infrequent occurrence of BBG among human disease isolates suggests that they may be less virulent than CG. We compared the virulence potentials of human and bovine isolates of CG and BBG in newborn conventional pig and weaned Dutch Belted rabbit models. CG-challenged piglets experienced severe disease accompanied by early and high mortality compared to BBG-challenged piglets. Similarly, CG-challenged rabbits were likely to develop lesions in kidney and intestine compared with the BBG-challenged rabbits. The CG strains used in this study carried stx2 and produced significantly higher amounts of Stx, whereas the BBG strains carried the stx2c gene variant only. These results suggest that BBG are less virulent than CG and that this difference in virulence potential is associated with the Stx2 subtype(s) carried and/or the amount of Stx produced. PMID:22025512

  12. Inactivation of different strains of Escherichia coli O157:H7 in various apple ciders treated with dimethyl dicarbonate (DMDC) and sulfur dioxide (SO2) as an alternative method.

    PubMed

    Basaran-Akgul, N; Churey, J J; Basaran, P; Worobo, R W

    2009-02-01

    Escherichia coli has been identified as the causative agent in numerous foodborne illness outbreaks associated with the consumption of fresh apple cider. Apple cider has a pH which is normally below 4.0 and would not be considered a medium capable of supporting the growth of foodborne pathogens. The association of unpasteurized apple cider with foodborne illness due to E. coli O157:H7 has however, led to increased interest in potential alternative methods to produce pathogen free cider. Apple cider was prepared from eight different apple cultivars, inoculated with approximately 10(6)-10(7) CFU of three strains of E. coli O157:H7 per ml (933, ATCC 43889, and ATCC 43895) and tested to determine the effectiveness of sulfur dioxide (SO(2)) and dimethyl dicarbonate (DMDC). Bacterial populations for treated and untreated samples were then enumerated by using non-selective media. Eight different ciders were treated with DMDC (125 and 250 ppm) and SO(2) (25, 50, 75, 100 ppm). Greater than a 5-log reduction was achieved at room temperature with 250 ppm of DMDC and 50 ppm of SO(2) after the incubation time of 6h and 24h, respectively. Addition of DMDC and/or SO(2) may offer an inexpensive alternative to thermal pasteurization for the production of safe apple cider for small apple cider producers. PMID:19028298

  13. High-Quality Draft Genome Sequences for Five Non-O157 Shiga Toxin-Producing Escherichia coli Strains Generated with PacBio Sequencing and Optical Maps

    PubMed Central

    Rowe, Lori; Garcia-Toledo, Lisley; Loparev, Vladimir; Knipe, Kristen; Stripling, Devon; Martin, Haley; Trees, Eija; Juieng, Phalasy; Batra, Dhwani; Strockbine, Nancy

    2016-01-01

    Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen. We report here the high-quality draft whole-genome sequences of five STEC strains isolated from clinical cases in the United States. This report is for STEC of serotypes O55:H7, O79:H7, O91:H14, O153:H2, and O156:H25. PMID:27365352

  14. Effect of Lactobacillus Strains on Intestinal Microflora and Mucosa Immunity in Escherichia coli O157:H7-Induced Diarrhea in Mice.

    PubMed

    Bian, Xin; Wang, Ting-Ting; Xu, Min; Evivie, Smith Etareri; Luo, Guang-Wen; Liang, Hong-Zhang; Yu, Shang-Fu; Huo, Gui-Cheng

    2016-07-01

    This study investigated the effects of KLDS 1.8701 and AD1 administrations by gavage on intestinal microflora and mucosal immunity in diarrhea mice infected by Escherichia coli O157:H7 compared to normal mice. The levels of E. coli, Enterobacteria, and Enterococcus decreased significantly (P < 0.05), while viable counts of Lactobacilli and Bifidobacterium increased in diarrhea mice. Moreover, KLDS 1.8701 and AD1 improved secretion of secretory immunoglobulin A and enhanced the levels of interferon-γ and interleukin. Results indicate that KLDS 1.8701 and AD1 could effectively alleviate diarrhea in mice via modulation of intestinal microflora and improve the function of immune system. The study on the effect of KLDS1.8701 and AD1 supplementation in human flora-associated animal models was recommended. PMID:27025726

  15. Proteomic and phenotypic analysis of triclosan tolerant verocytotoxigenic Escherichia coli O157:H19.

    PubMed

    Sheridan, A; Lenahan, M; Condell, O; Bonilla-Santiago, R; Sergeant, K; Renaut, J; Duffy, G; Fanning, S; Nally, J E; Burgess, C M

    2013-03-27

    Triclosan is a biocidal active agent commonly used in domestic and industrial formulations. Currently, there is limited understanding of the mechanisms involved in triclosan tolerance in Escherichia coli O157. The aim of this study was to identify the differences between a triclosan susceptible E. coli O157:H19 isolate (minimum inhibitory concentration; MIC 6.25 μg/ml) and its triclosan tolerant mutant (MIC>8000 μg/ml) at a proteomic and phenotypic level. Two dimensional DIGE was used to identify differences in protein expression between the reference strain and triclosan tolerant mutant in the presence and absence of triclosan. DIGE analysis indicates the proteome of the reference E. coli O157:H19 was significantly different to its triclosan tolerant mutant. Significant changes in protein expression levels in the triclosan tolerant mutant included the known triclosan target FabI which encodes enoyl reductase, outer membrane proteins and the filament structural protein of flagella, FliC. Phenotypic studies showed that the triclosan tolerant mutant MIC decreased in the presence of efflux inhibitor phenyl-arginine-β-naphthylamide and biofilm formation was increased in the mutant strain. The data generated indicates that enhanced triclosan tolerance is a result of multiple mechanisms which act together to achieve high-level resistance, rather than mutation of FabI alone.

  16. Gram-negative bacterial isolates from fresh-cut processing plants enhance the presence of Escherichia Coli O157:H7 in dual-species biofilms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Biofilms formed by resident microflora may provide a microenvironment for foodborne bacterial pathogens to survive and cause cross-contamination in fresh-cut processing and handling facilities. The objective of this study is to determine the impact of individual bacteria strains isolated from two l...

  17. Internalization of Escherichia Coli O157:H7 by Bovine Rectal Epithelial Cells

    PubMed Central

    Sheng, Haiqing; Wang, Jing; Lim, Ji Youn; Davitt, Christine; Minnich, Scott A.; Hovde, Carolyn J.

    2011-01-01

    Escherichia coli O157:H7 (O157) causes human diarrheal disease and healthy cattle are its primary reservoir. O157 colonize the bovine epithelial mucosa at the recto-anal junction (RAJ). Previous studies show that O157 at this site are not eliminated by aggressive interventions including applications of O157-specific lytic bacteriophages and other bactericidal agents. We hypothesize that some O157 at the RAJ mucosa are protected from these killing agents by host cell internalization. To test this hypothesis, rectal biopsies from O157 culture positive and negative cattle were analyzed by fluorescent microscopy and subjected to gentamicin protection assays. GFP-labeled bacteria were found located deep within the tissue crypts and a small number of O157 were recovered from rectal biopsies after gentamicin treatment. Primary bovine rectal epithelial (PBRE) cell cultures were incubated with O157 and subjected to gentamicin protection assays. Strains ATCC 43895, 43894, Sakai, and WSU180 entered the PBRE cells with different levels of efficiency ranging from 0.18 to 19.38% of the inocula. Intracellular bacteria were confirmed to be within membrane-bounded vacuoles by electron microscopy. Cytochalasin D curtailed internalization of O157 indicating internalization was dependent on eukaryotic microfilament assembly. Strain ATCC 43895 exhibited the highest efficiency of internalization and survived for at least 24 h within PBRE cells. Deletion mutation of intimin or its receptor in ATCC 43895 did not reduce bacterial internalization. This strain produced more biofilm than the others tested. Retrospective analysis of cattle challenged with two O157 strains, showed ATCC 43895, the most efficient at host cell internalization, was most persistent. PMID:21687423

  18. Relationship between pathogenicity for humans and stx genotype in Shiga toxin-producing Escherichia coli serotype O157.

    PubMed

    Kawano, K; Okada, M; Haga, T; Maeda, K; Goto, Y

    2008-03-01

    To examine the reason why people infected with Shiga toxin (Stx) producing Escherichia coli (STEC) O157 strains develop varying clinical manifestations, 65 STEC O157 isolates originating from 64 different occurrences of infection in Miyazaki Prefecture in 2001-2003 and their 79 infected individuals were analyzed by stx genotyping, quantitative analysis of reversed passive latex agglutination (RPLA), genomic DNA analysis using pulsed-field gel electrophoresis (PFGE), and clinical manifestations. The isolates were found to carry the following stx genes: stx2vha alone (60.0%), stx1/stx2 (27.7%), stx1/stx2vha (6.1%), stx2 alone (3.1%), and stx2/stx2vha (3.1%). No strain carried the stx1 gene alone. STEC strains carrying stx2 were more frequently associated with clinical manifestations of hemolytic-uremic syndrome (HUS) or bloody diarrhea than those carrying stx2vha. Clusters of PFGE banding patterns were correlated well with the stx genotypes. We conclude that stx genotype is one of the important factors of clinical outcome of STEC O157 infection and that pathogenicity for humans was higher in the stx2 genotype strains than in the stx2vha genotype strains, as reported previously by other researchers. Further, we newly found that four clusters identified by PFGE using restriction enzyme XbaI, stx genotypes and clinical manifestations were well correlated with each other.

  19. Non-O157 Shiga Toxin-Producing E. coli Associated with Muscle Foods

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli strains that produce Shiga toxins, referred to as Shiga toxin-producing E. coli (STEC) or verotoxigenic E. coli (VTEC), cause hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). E. coli O157:H7 is the most common cause of STEC infection; however, numerous non-O157 STECs b...

  20. Antibacterial activity of cinnamaldehyde and Sporan against Escherichia coli O157:H7 and Salmonella

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The in vitro antimicrobial effect of cinnamaldehyde and Sporan in combination with acetic acid against E. coli O157:H7 and Salmonella was investigated. A five strain cocktail of E. coli O157:H7 and Salmonella were inoculated in Luria-Bertoni broth (LB broth, 7 log CFU ml-1) containing cinnamaldehyde...

  1. Identification of Escherichia coli O157 by Using a Novel Colorimetric Detection Method with DNA Microarrays

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin-producing Escherichia coli O157:H7 is a leading cause of foodborne illness worldwide. To evaluate better methods to rapidly detect and genotype E. coli O157 strains, the present study evaluated the use of ampliPHOX, a novel colorimetric detection method based on photopolymerization, for...

  2. Characterization of the pathogenome and phylogenomic classification of enteropathogenic Escherichia coli of the O157:non-H7 serotypes

    PubMed Central

    Sanjar, Fatemeh; Rusconi, Brigida; Hazen, Tracy H.; Koenig, Sara S.K.; Mammel, Mark K.; Feng, Peter C.H.; Rasko, David A.; Eppinger, Mark

    2015-01-01

    Escherichia coli of the O157 serogroup are comprised of a diverse collection of more than 100 O157:non-H7 serotypes that are found in the environment, animal reservoir and infected patients and some have been linked to severe outbreaks of human disease. Among these, the enteropathogenic E. coli O157:non-H7 serotypes carry virulence factors that are hallmarks of enterohemorrhagic E. coli, such as causing attaching and effacing lesions during human gastrointestinal tract infections. Given the shared virulence gene pool between O157:H7 and O157:non-H7 serotypes, our objective was to examine the prevalence of virulence traits of O157:non-H7 serotypes within and across their H-serotype and when compared to other E. coli pathovars. We sequenced six O157:non-H7 genomes complemented by four genomes from public repositories in an effort to determine their virulence state and genetic relatedness to the highly pathogenic enterohemorrhagic O157:H7 lineage and its ancestral O55:H7 serotype. Whole-genome-based phylogenomic analysis and molecular typing is indicative of a non-monophyletic origin of the heterogeneous O157:non-H7 serotypes that are only distantly related to the O157:H7 serotype. The availability of multiple genomes enables robust phylogenomic placement of these strains into their evolutionary context, and the assessment of the pathogenic potential of the O157:non-H7 strains in causing human disease. PMID:25962987

  3. Probing genomic diversity and evolution of Escherichia coli O157 by single nucleotide polymorphisms

    PubMed Central

    Zhang, Wei; Qi, Weihong; Albert, Thomas J.; Motiwala, Alifiya S.; Alland, David; Hyytia-Trees, Eija K.; Ribot, Efrain M.; Fields, Patricia I.; Whittam, Thomas S.; Swaminathan, Bala

    2006-01-01

    Infections by Shiga toxin-producing Escherichia coli O157:H7 (STEC O157) are the predominant cause of bloody diarrhea and hemolytic uremic syndrome in the United States. In silico comparison of the two complete STEC O157 genomes (Sakai and EDL933) revealed a strikingly high level of sequence identity in orthologous protein-coding genes, limiting the use of nucleotide sequences to study the evolution and epidemiology of this bacterial pathogen. To systematically examine single nucleotide polymorphisms (SNPs) at a genome scale, we designed comparative genome sequencing microarrays and analyzed 1199 chromosomal genes (a total of 1,167,948 bp) and 92,721 bp of the large virulence plasmid (pO157) of eleven outbreak-associated STEC O157 strains. We discovered 906 SNPs in 523 chromosomal genes and observed a high level of DNA polymorphisms among the pO157 plasmids. Based on a uniform rate of synonymous substitution for Escherichia coli and Salmonella enterica (4.7 × 10−9 per site per year), we estimate that the most recent common ancestor of the contemporary β-glucuronidase-negative, non-sorbitolfermenting STEC O157 strains existed ca. 40 thousand years ago. The phylogeny of the STEC O157 strains based on the informative synonymous SNPs was compared to the maximum parsimony trees inferred from pulsed-field gel electrophoresis and multilocus variable numbers of tandem repeats analysis. The topological discrepancies indicate that, in contrast to the synonymous mutations, parts of STEC O157 genomes have evolved through different mechanisms with highly variable divergence rates. The SNP loci reported here will provide useful genetic markers for developing high-throughput methods for fine-resolution genotyping of STEC O157. Functional characterization of nucleotide polymorphisms should shed new insights on the evolution, epidemiology, and pathogenesis of STEC O157 and related pathogens. PMID:16606700

  4. Detection of Escherichia Coli O157:H7 in Fecal Samples in Meat Goats

    ERIC Educational Resources Information Center

    Mobley, Ray; Madden, Uford; Brooks-Walter, Alexis

    2004-01-01

    Studies have reported the isolation of Escherichia coli (E. coli)O157:H7 from pork, lamb and poultry products, and from other animals including deer, horses, dogs, birds and humans. There is limited or no information on the presence of the organism in goats. The objectives of this study were to determine if E. coli O157:H7 was naturally occurring…

  5. Insight into Shiga toxin genes encoded by Escherichia coli O157 from whole genome sequencing

    PubMed Central

    Ashton, Philip M.; Perry, Neil; Ellis, Richard; Petrovska, Liljana; Wain, John; Grant, Kathie A.; Jenkins, Claire

    2015-01-01

    The ability of Shiga toxin-producing Escherichia coli (STEC) to cause severe illness in humans is determined by multiple host factors and bacterial characteristics, including Shiga toxin (Stx) subtype. Given the link between Stx2a subtype and disease severity, we sought to identify the stx subtypes present in whole genome sequences (WGS) of 444 isolates of STEC O157. Difficulties in assembling the stx genes in some strains were overcome by using two complementary bioinformatics methods: mapping and de novo assembly. We compared the WGS analysis with the results obtained using a PCR approach and investigated the diversity within and between the subtypes. All strains of STEC O157 in this study had stx1a, stx2a or stx2c or a combination of these three genes. There was over 99% (442/444) concordance between PCR and WGS. When common source strains were excluded, 236/349 strains of STEC O157 had multiple copies of different Stx subtypes and 54 had multiple copies of the same Stx subtype. Of those strains harbouring multiple copies of the same Stx subtype, 33 had variants between the alleles while 21 had identical copies. Strains harbouring Stx2a only were most commonly found to have multiple alleles of the same subtype (42%). Both the PCR and WGS approach to stx subtyping provided a good level of sensitivity and specificity. In addition, the WGS data also showed there were a significant proportion of strains harbouring multiple alleles of the same Stx subtype associated with clinical disease in England. PMID:25737808

  6. Insight into Shiga toxin genes encoded by Escherichia coli O157 from whole genome sequencing.

    PubMed

    Ashton, Philip M; Perry, Neil; Ellis, Richard; Petrovska, Liljana; Wain, John; Grant, Kathie A; Jenkins, Claire; Dallman, Tim J

    2015-01-01

    The ability of Shiga toxin-producing Escherichia coli (STEC) to cause severe illness in humans is determined by multiple host factors and bacterial characteristics, including Shiga toxin (Stx) subtype. Given the link between Stx2a subtype and disease severity, we sought to identify the stx subtypes present in whole genome sequences (WGS) of 444 isolates of STEC O157. Difficulties in assembling the stx genes in some strains were overcome by using two complementary bioinformatics methods: mapping and de novo assembly. We compared the WGS analysis with the results obtained using a PCR approach and investigated the diversity within and between the subtypes. All strains of STEC O157 in this study had stx1a, stx2a or stx2c or a combination of these three genes. There was over 99% (442/444) concordance between PCR and WGS. When common source strains were excluded, 236/349 strains of STEC O157 had multiple copies of different Stx subtypes and 54 had multiple copies of the same Stx subtype. Of those strains harbouring multiple copies of the same Stx subtype, 33 had variants between the alleles while 21 had identical copies. Strains harbouring Stx2a only were most commonly found to have multiple alleles of the same subtype (42%). Both the PCR and WGS approach to stx subtyping provided a good level of sensitivity and specificity. In addition, the WGS data also showed there were a significant proportion of strains harbouring multiple alleles of the same Stx subtype associated with clinical disease in England.

  7. [Investigation of verotoxigenic Escherichia coli O157:H7 incidence in gastroenteritis patients].

    PubMed

    Erdoğan, Haluk; Levent, Belkıs; Erdoğan, Aşkın; Güleşen, Revasiye; Arslan, Hande

    2011-07-01

    Escherichia coli O157:H7 is the most common serotype among verotoxigenic E.coli (VTEC) strains that cause haemolytic uremic syndrome. Although sporadic VTEC cases originating from Turkey and small outbreaks have been reported from our country, VTEC has not been routinely investigated in most of the diagnostic microbiology laboratories in Turkey and studies related to this topic are limited. In this study, the incidence of E.coli O157:H7 in patients who were admitted to Alanya Research and Application Hospital of Baskent University with the complaints of acute gastroenteritis between September 2005 and September 2008, was investigated. Stool samples collected from 1815 diarrheal patients (of them 50.5% were male; 49.3% were ? 5 years old; 10.2% were tourists) were evaluated initially by direct microscopy and then inoculated to hectoen enteric agar, EMB agar, Skirrow agar and cefixime tellurite sorbitol MacConkey (CT-SMC) agar media for cultivation. The sorbitol-negative colonies which were compatible with E.coli according to the conventional methods were tested with E.coli polyvalent and 0157 and H7 monovalent antisera and agglutination positive strains were also investigated for verotoxin production in Vero cell cultures. VTEC RPLA toxin detection kit (Oxoid, UK) was used for further identification of toxin type of verotoxin positive strains. Fecal leukocytes were detected in 41.3% of the samples in direct microscopy, while 27% (173/639) of the samples were also found positive for amoeba antigen, 6% (24/396) for rotavirus antigen, 1.2% (22/1815) for Salmonella spp., 0.6% (11/1815) for Shigella spp., 0.2% (4/1815) for Giardia trophozoites and 0.06% (1/1815) for Campylobacter jejuni. The isolation rate of sorbitol-negative E.coli strains was %0.8 (14/1815), and two of them were identified as E.coli O157:H7 by monovalent antisera, and both of them were determined as verotoxin-producers in cell culture. Verotoxin types of those isolates were found as verotoxin 1 in one

  8. Dynamic of Mutational Events in Variable Number Tandem Repeats of Escherichia coli O157:H7

    PubMed Central

    Bustamante, A. V.; Sanso, A. M.; Segura, D. O.; Parma, A. E.; Lucchesi, P. M. A.

    2013-01-01

    VNTRs regions have been successfully used for bacterial subtyping; however, the hypervariability in VNTR loci is problematic when trying to predict the relationships among isolates. Since few studies have examined the mutation rate of these markers, our aim was to estimate mutation rates of VNTRs specific for verotoxigenic E. coli O157:H7. The knowledge of VNTR mutational rates and the factors affecting them would make MLVA more effective for epidemiological or microbial forensic investigations. For this purpose, we analyzed nine loci performing parallel, serial passage experiments (PSPEs) on 9 O157:H7 strains. The combined 9 PSPE population rates for the 8 mutating loci ranged from 4.4 × 10−05 to 1.8 × 10−03 mutations/generation, and the combined 8-loci mutation rate was of 2.5 × 10−03 mutations/generation. Mutations involved complete repeat units, with only one point mutation detected. A similar proportion between single and multiple repeat changes was detected. Of the 56 repeat mutations, 59% were insertions and 41% were deletions, and 72% of the mutation events corresponded to O157-10 locus. For alleles with up to 13 UR, a constant and low mutation rate was observed; meanwhile longer alleles were associated with higher and variable mutation rates. Our results are useful to interpret data from microevolution and population epidemiology studies and particularly point out that the inclusion or not of O157-10 locus or, alternatively, a differential weighting data according to the mutation rates of loci must be evaluated in relation with the objectives of the proposed study. PMID:24093095

  9. A Novel Approach to Investigate Internalization of Escherichia coli O157:H7 in Lettuce and Spinach

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The chromosomal integration of the green fluorescent protein (gfp) gene was successfully accomplished into four nalidixic acid resistant E. coli strains: two O157:H7 strains from produce outbreaks, 4407 and 5279, one O157:H7 strain from a beef-associated outbreak, 86-24h11, and a non-pathogenic comm...

  10. Genomic anatomy of Escherichia coli O157:H7 outbreaks.

    PubMed

    Eppinger, Mark; Mammel, Mark K; Leclerc, Joseph E; Ravel, Jacques; Cebula, Thomas A

    2011-12-13

    The rapid emergence of Escherichia coli O157:H7 from an unknown strain in 1982 to the dominant hemorrhagic E. coli serotype in the United States and the cause of widespread outbreaks of human food-borne illness highlights a need to evaluate critically the extent to which genomic plasticity of this important enteric pathogen contributes to its pathogenic potential and its evolution as well as its adaptation in different ecological niches. Aimed at a better understanding of the evolution of the E. coli O157:H7 pathogenome, the present study presents the high-quality sequencing and comparative phylogenomic analysis of a comprehensive panel of 25 E. coli O157:H7 strains associated with three nearly simultaneous food-borne outbreaks of human disease in the United States. Here we present a population genetic analysis of more than 200 related strains recovered from patients, contaminated produce, and zoonotic sources. High-resolution phylogenomic approaches allow the dynamics of pathogenome evolution to be followed at a high level of phylogenetic accuracy and resolution. SNP discovery and study of genome architecture and prophage content identified numerous biomarkers to assess the extent of genetic diversity within a set of clinical and environmental strains. A total of 1,225 SNPs were identified in the present study and are now available for typing of the E. coli O157:H7 lineage. These data should prove useful for the development of a refined phylogenomic framework for forensic, diagnostic, and epidemiological studies to define better risk in response to novel and emerging E. coli O157:H7 resistance and virulence phenotypes. PMID:22135463

  11. Genomic anatomy of Escherichia coli O157:H7 outbreaks

    PubMed Central

    Eppinger, Mark; Mammel, Mark K.; Leclerc, Joseph E.; Ravel, Jacques; Cebula, Thomas A.

    2011-01-01

    The rapid emergence of Escherichia coli O157:H7 from an unknown strain in 1982 to the dominant hemorrhagic E. coli serotype in the United States and the cause of widespread outbreaks of human food-borne illness highlights a need to evaluate critically the extent to which genomic plasticity of this important enteric pathogen contributes to its pathogenic potential and its evolution as well as its adaptation in different ecological niches. Aimed at a better understanding of the evolution of the E. coli O157:H7 pathogenome, the present study presents the high-quality sequencing and comparative phylogenomic analysis of a comprehensive panel of 25 E. coli O157:H7 strains associated with three nearly simultaneous food-borne outbreaks of human disease in the United States. Here we present a population genetic analysis of more than 200 related strains recovered from patients, contaminated produce, and zoonotic sources. High-resolution phylogenomic approaches allow the dynamics of pathogenome evolution to be followed at a high level of phylogenetic accuracy and resolution. SNP discovery and study of genome architecture and prophage content identified numerous biomarkers to assess the extent of genetic diversity within a set of clinical and environmental strains. A total of 1,225 SNPs were identified in the present study and are now available for typing of the E. coli O157:H7 lineage. These data should prove useful for the development of a refined phylogenomic framework for forensic, diagnostic, and epidemiological studies to define better risk in response to novel and emerging E. coli O157:H7 resistance and virulence phenotypes. PMID:22135463

  12. Phytic Acid and Sodium Chloride Show Marked Synergistic Bactericidal Effects against Nonadapted and Acid-Adapted Escherichia coli O157:H7 Strains

    PubMed Central

    Kim, Nam Hee

    2015-01-01

    The synergistic antimicrobial effects of phytic acid (PA), a natural extract from rice bran, plus sodium chloride against Escherichia coli O157:H7 were examined. Exposure to NaCl alone at concentrations up to 36% (wt/wt) for 5 min did not reduce bacterial populations. The bactericidal effects of PA alone were much greater than those of other organic acids (acetic, citric, lactic, and malic acids) under the same experimental conditions (P < 0.05). Combining PA and NaCl under conditions that yielded negligible effects when each was used alone led to marked synergistic effects. For example, whereas 0.4% PA or 3 or 4% NaCl alone had little or no effect on cell viability, combining the two completely inactivated both nonadapted and acid-adapted cells, reducing their numbers to unrecoverable levels (>7-log CFU/ml reduction). Flow cytometry confirmed that PA disrupted the cell membrane to a greater extent than did other organic acids, although the cells remained viable. The combination of PA and NaCl induced complete disintegration of the cell membrane. By comparison, none of the other organic acids acted synergistically with NaCl, and neither did NaCl-HCl solutions at the same pH values as the test solutions of PA plus NaCl. These results suggest that PA has great potential as an effective bacterial membrane-permeabilizing agent, and we show that the combination is a promising alternative to conventional chemical disinfectants. These findings provide new insight into the utility of natural compounds as novel antimicrobial agents and increase our understanding of the mechanisms underlying the antibacterial activity of PA. PMID:26637600

  13. Phytic Acid and Sodium Chloride Show Marked Synergistic Bactericidal Effects against Nonadapted and Acid-Adapted Escherichia coli O157:H7 Strains.

    PubMed

    Kim, Nam Hee; Rhee, Min Suk

    2015-12-04

    The synergistic antimicrobial effects of phytic acid (PA), a natural extract from rice bran, plus sodium chloride against Escherichia coli O157:H7 were examined. Exposure to NaCl alone at concentrations up to 36% (wt/wt) for 5 min did not reduce bacterial populations. The bactericidal effects of PA alone were much greater than those of other organic acids (acetic, citric, lactic, and malic acids) under the same experimental conditions (P < 0.05). Combining PA and NaCl under conditions that yielded negligible effects when each was used alone led to marked synergistic effects. For example, whereas 0.4% PA or 3 or 4% NaCl alone had little or no effect on cell viability, combining the two completely inactivated both nonadapted and acid-adapted cells, reducing their numbers to unrecoverable levels (>7-log CFU/ml reduction). Flow cytometry confirmed that PA disrupted the cell membrane to a greater extent than did other organic acids, although the cells remained viable. The combination of PA and NaCl induced complete disintegration of the cell membrane. By comparison, none of the other organic acids acted synergistically with NaCl, and neither did NaCl-HCl solutions at the same pH values as the test solutions of PA plus NaCl. These results suggest that PA has great potential as an effective bacterial membrane-permeabilizing agent, and we show that the combination is a promising alternative to conventional chemical disinfectants. These findings provide new insight into the utility of natural compounds as novel antimicrobial agents and increase our understanding of the mechanisms underlying the antibacterial activity of PA.

  14. Role of glycoside hydrolase genes in sinigrin degradation by E. coli O157:H7.

    PubMed

    Cordeiro, Roniele P; Doria, Juan H; Zhanel, George G; Sparling, Richard; Holley, Richard A

    2015-07-16

    This work examined Escherichia coli O157:H7 strain 02-0304 for putative genes responsible for sinigrin hydrolysis. Sinigrin is a glucosinolate present in Oriental mustard (Brassica juncea), and its hydrolysis is mediated in plants by the enzyme myrosinase. Sinigrin hydrolysis by plant or bacterial myrosinase yields allyl isothiocyanate (AITC) which is bactericidal. In silico analysis using public databases found sequence similarity between plant myrosinase and enzymes encoded by genes from β-glucosidase families in E. coli O157:H7. Specifically, 6-phospho-β-glucosidase encoded by the genes bglA and ascB (family 1), and chbF (family 4) present in E. coli O157:H7 showed the highest similarity. Polymerase chain reaction (PCR) confirmed the presence of bglA, ascB, and chbF in the clinical E. coli strain tested. Disruption of these genes in wild-type E. coli O157:H7 strain 02-0304 using lambda-red replacement created single and double mutants. The relative importance of each gene in the hydrolysis of sinigrin by E. coli O157:H7 was also assessed by comparing gene expression and sinigrin degradation rates among the E. coli O157:H7 wild-type strain and its mutants. The results suggested that the genes bglA and ascB play a substantial role in the degradation of sinigrin by E. coli O157:H7 strain 02-0304.

  15. Role of glycoside hydrolase genes in sinigrin degradation by E. coli O157:H7.

    PubMed

    Cordeiro, Roniele P; Doria, Juan H; Zhanel, George G; Sparling, Richard; Holley, Richard A

    2015-07-16

    This work examined Escherichia coli O157:H7 strain 02-0304 for putative genes responsible for sinigrin hydrolysis. Sinigrin is a glucosinolate present in Oriental mustard (Brassica juncea), and its hydrolysis is mediated in plants by the enzyme myrosinase. Sinigrin hydrolysis by plant or bacterial myrosinase yields allyl isothiocyanate (AITC) which is bactericidal. In silico analysis using public databases found sequence similarity between plant myrosinase and enzymes encoded by genes from β-glucosidase families in E. coli O157:H7. Specifically, 6-phospho-β-glucosidase encoded by the genes bglA and ascB (family 1), and chbF (family 4) present in E. coli O157:H7 showed the highest similarity. Polymerase chain reaction (PCR) confirmed the presence of bglA, ascB, and chbF in the clinical E. coli strain tested. Disruption of these genes in wild-type E. coli O157:H7 strain 02-0304 using lambda-red replacement created single and double mutants. The relative importance of each gene in the hydrolysis of sinigrin by E. coli O157:H7 was also assessed by comparing gene expression and sinigrin degradation rates among the E. coli O157:H7 wild-type strain and its mutants. The results suggested that the genes bglA and ascB play a substantial role in the degradation of sinigrin by E. coli O157:H7 strain 02-0304. PMID:25897994

  16. Health Risk of Escherichia coli O157:H7 in Drinking Water and Meat and Meat Products and Vegetables to Diarrhoeic Confirmed and Non-Confirmed HIV/AIDS Patients

    NASA Astrophysics Data System (ADS)

    Abong`O, B. O.; Momba, M. N. B.; Rodda, N.

    The current study explored the health risk of E. coli O157:H7 to diarrhoeic confirmed and non-confirmed HIV/AIDS patients due to their exposure to presumed ingestion of water, meat products and vegetables ostensibly contaminated with E. coli O157:H7. Strains of E. coli O157:H7 were isolated by enrichment culture and on Cefixime-Telurite Sorbitol MacConkey agar. Average counts of presumptive E. coli O157 were used for dose-response assessment. Probability of infection to confirmed and non-confirmed HIV/AIDS patients was 20 and 27% from meat and meat products, 21% and 15% from vegetables and 100% due to ingestion of 1500 mL person-1 day-1 of water. Drinking water had higher probability of transmitting E. coli O157:H7 infections than meat and meat products and vegetables. Probability of E. coli O157:H7 infections were high for confirmed HIV/AIDS patients than for non-confirmed patients. Water and foods consumed by HIV/AIDS patients should be safe of any microbial contaminants, these waters and foods should as well be investigated for other enteric pathogens to establish their safety.

  17. Public Health Investigation of Two Outbreaks of Shiga Toxin-Producing Escherichia coli O157 Associated with Consumption of Watercress.

    PubMed

    Jenkins, Claire; Dallman, Timothy J; Launders, Naomi; Willis, Caroline; Byrne, Lisa; Jorgensen, Frieda; Eppinger, Mark; Adak, Goutam K; Aird, Heather; Elviss, Nicola; Grant, Kathie A; Morgan, Dilys; McLauchlin, Jim

    2015-06-15

    An increase in the number of cases of Shiga toxin-producing Escherichia coli (STEC) O157 phage type 2 (PT2) in England in September 2013 was epidemiologically linked to watercress consumption. Whole-genome sequencing (WGS) identified a phylogenetically related cluster of 22 cases (outbreak 1). The isolates comprising this cluster were not closely related to any other United Kingdom strain in the Public Health England WGS database, suggesting a possible imported source. A second outbreak of STEC O157 PT2 (outbreak 2) was identified epidemiologically following the detection of outbreak 1. Isolates associated with outbreak 2 were phylogenetically distinct from those in outbreak 1. Epidemiologically unrelated isolates on the same branch as the outbreak 2 cluster included those from human cases in England with domestically acquired infection and United Kingdom domestic cattle. Environmental sampling using PCR resulted in the isolation of STEC O157 PT2 from irrigation water at one implicated watercress farm, and WGS showed this isolate belonged to the same phylogenetic cluster as outbreak 2 isolates. Cattle were in close proximity to the watercress bed and were potentially the source of the second outbreak. Transfer of STEC from the field to the watercress bed may have occurred through wildlife entering the watercress farm or via runoff water. During this complex outbreak investigation, epidemiological studies, comprehensive testing of environmental samples, and the use of novel molecular methods proved invaluable in demonstrating that two simultaneous outbreaks of STEC O157 PT2 were both linked to the consumption of watercress but were associated with different sources of contamination.

  18. Public Health Investigation of Two Outbreaks of Shiga Toxin-Producing Escherichia coli O157 Associated with Consumption of Watercress.

    PubMed

    Jenkins, Claire; Dallman, Timothy J; Launders, Naomi; Willis, Caroline; Byrne, Lisa; Jorgensen, Frieda; Eppinger, Mark; Adak, Goutam K; Aird, Heather; Elviss, Nicola; Grant, Kathie A; Morgan, Dilys; McLauchlin, Jim

    2015-06-15

    An increase in the number of cases of Shiga toxin-producing Escherichia coli (STEC) O157 phage type 2 (PT2) in England in September 2013 was epidemiologically linked to watercress consumption. Whole-genome sequencing (WGS) identified a phylogenetically related cluster of 22 cases (outbreak 1). The isolates comprising this cluster were not closely related to any other United Kingdom strain in the Public Health England WGS database, suggesting a possible imported source. A second outbreak of STEC O157 PT2 (outbreak 2) was identified epidemiologically following the detection of outbreak 1. Isolates associated with outbreak 2 were phylogenetically distinct from those in outbreak 1. Epidemiologically unrelated isolates on the same branch as the outbreak 2 cluster included those from human cases in England with domestically acquired infection and United Kingdom domestic cattle. Environmental sampling using PCR resulted in the isolation of STEC O157 PT2 from irrigation water at one implicated watercress farm, and WGS showed this isolate belonged to the same phylogenetic cluster as outbreak 2 isolates. Cattle were in close proximity to the watercress bed and were potentially the source of the second outbreak. Transfer of STEC from the field to the watercress bed may have occurred through wildlife entering the watercress farm or via runoff water. During this complex outbreak investigation, epidemiological studies, comprehensive testing of environmental samples, and the use of novel molecular methods proved invaluable in demonstrating that two simultaneous outbreaks of STEC O157 PT2 were both linked to the consumption of watercress but were associated with different sources of contamination. PMID:25841005

  19. Enumeration of Escherichia coli O157:H7 in Outbreak-Associated Beef Patties.

    PubMed

    Gill, Alexander; Huszczynski, George

    2016-07-01

    An outbreak of five cases of Escherichia coli O157 infection that occurred in Canada in 2012 was linked to frozen beef patties seasoned with garlic and peppercorn. Unopened retail packs of beef patties from the implicated production lot were recovered and analyzed to enumerate E. coli O157, other E. coli strains, and total coliforms. E. coli O157 was not recovered by direct enumeration on selective agar media. E. coli O157 in the samples was estimated at 3.1 most probable number per 140 g of beef patty, other E. coli was 11 CFU/g, and coliforms were 120 CFU/g. These results indicate that the presence of E. coli O157 in ground beef at levels below 0.1 CFU/g may cause outbreaks. However, the roles of temperature abuse, undercooking, and crosscontamination in amplifying the risk are unknown.

  20. Enumeration of Escherichia coli O157:H7 in Outbreak-Associated Beef Patties.

    PubMed

    Gill, Alexander; Huszczynski, George

    2016-07-01

    An outbreak of five cases of Escherichia coli O157 infection that occurred in Canada in 2012 was linked to frozen beef patties seasoned with garlic and peppercorn. Unopened retail packs of beef patties from the implicated production lot were recovered and analyzed to enumerate E. coli O157, other E. coli strains, and total coliforms. E. coli O157 was not recovered by direct enumeration on selective agar media. E. coli O157 in the samples was estimated at 3.1 most probable number per 140 g of beef patty, other E. coli was 11 CFU/g, and coliforms were 120 CFU/g. These results indicate that the presence of E. coli O157 in ground beef at levels below 0.1 CFU/g may cause outbreaks. However, the roles of temperature abuse, undercooking, and crosscontamination in amplifying the risk are unknown. PMID:27357049

  1. Enhanced detection sensitivity of Escherichia coli O157:H7 using surface-modified gold nanorods

    PubMed Central

    Ramasamy, Mohankandhasamy; Yi, Dong Kee; An, Seong Soo A

    2015-01-01

    Escherichia coli O157:H7 (O157) is a Gram negative and highly virulent bacteria found in food and water sources, and is a leading cause of chronic diseases worldwide. Diagnosis and prevention from the infection require simple and rapid analysis methods for the detection of pathogens, including O157. Endogenous membrane peroxidase, an enzyme present on the surface of O157, was used for the colorimetric detection of bacteria by catalytic oxidation of the peroxidase substrate. In this study, we have analyzed the impact of the synthesized bare gold nanorods (AuNRs) and silica-coated AuNRs on the growth of E. coli O157. Along with the membrane peroxidase activity of O157, other bacteria strains were analyzed. Different concentrations of nanorods were used to analyze the growth responses, enzymatic changes, and morphological alterations of bacteria by measuring optical density, 3,3′,5,5′-tetramethylbenzidine assay, flow cytometry analysis, and microscopy studies. The results revealed that O157 showed higher and continuous membrane peroxidase activity than other bacteria. Furthermore, O157 treated with bare AuNRs showed a decreased growth rate in comparison with the bacteria with surface modified AuNRs. Interestingly, silica-coated AuNRs favored the growth of bacteria and also increased membrane peroxidase activity. This result can be particularly important for the enzymatic analysis of surface treated AuNRs in various microbiological applicants. PMID:26347081

  2. Enhanced detection sensitivity of Escherichia coli O157:H7 using surface-modified gold nanorods.

    PubMed

    Ramasamy, Mohankandhasamy; Yi, Dong Kee; An, Seong Soo A

    2015-01-01

    Escherichia coli O157:H7 (O157) is a Gram negative and highly virulent bacteria found in food and water sources, and is a leading cause of chronic diseases worldwide. Diagnosis and prevention from the infection require simple and rapid analysis methods for the detection of pathogens, including O157. Endogenous membrane peroxidase, an enzyme present on the surface of O157, was used for the colorimetric detection of bacteria by catalytic oxidation of the peroxidase substrate. In this study, we have analyzed the impact of the synthesized bare gold nanorods (AuNRs) and silica-coated AuNRs on the growth of E. coli O157. Along with the membrane peroxidase activity of O157, other bacteria strains were analyzed. Different concentrations of nanorods were used to analyze the growth responses, enzymatic changes, and morphological alterations of bacteria by measuring optical density, 3,3',5,5'-tetramethylbenzidine assay, flow cytometry analysis, and microscopy studies. The results revealed that O157 showed higher and continuous membrane peroxidase activity than other bacteria. Furthermore, O157 treated with bare AuNRs showed a decreased growth rate in comparison with the bacteria with surface modified AuNRs. Interestingly, silica-coated AuNRs favored the growth of bacteria and also increased membrane peroxidase activity. This result can be particularly important for the enzymatic analysis of surface treated AuNRs in various microbiological applicants. PMID:26347081

  3. Comparative study on the high pressure inactivation behavior of the Shiga toxin-producing Escherichia coli O104:H4 and O157:H7 outbreak strains and a non-pathogenic surrogate.

    PubMed

    Reineke, Kai; Sevenich, Robert; Hertwig, Christian; Janßen, Traute; Fröhling, Antje; Knorr, Dietrich; Wieler, Lothar H; Schlüter, Oliver

    2015-04-01

    Enterohemorrhagic Escherichia coli strains cause each year thousands of illnesses, which are sometimes accompanied by the hemolytic uremic syndrome, like in the 2011 outbreak in Germany. For preservation thermal pasteurization is commonly used, which can cause unwanted quality changes. To prevent this high pressure treatment is a potential alternative. Within this study, the 2011 outbreak strain O104:H4, an O157:H7 and a non-pathogenic strain (DSM1116) were tested. The cells were treated in buffer (pH 7 and pH 5) and carrot juice (pH 5.1) in a pressure temperature range of 0.1-500 MPa and 20-70 °C. Flow cytometry was used to investigate the pressure impact on cell structures of the strain DSM1116. Both pathogenic strains had a much higher resistance in buffer and carrot juice than the non-pathogenic surrogate. Further, strains cultivated and treated at a lower pH-value showed higher pressure stability, presumably due to variations in the membrane composition. This was confirmed for the strain DSM1116 by flow cytometry. Cells cultivated and treated at pH 5 had a stronger ability to retain their membrane potential but showed higher rates of membrane permeabilization at pressures <200 MPa compared to cells cultivated and treated at pH 7. These cells had the lowest membrane permeabilization rate at around 125 MPa, possibly denoting that variations in the fatty acid composition and membrane fluidity contribute to this stabilization phenomenon.

  4. Comparative study on the high pressure inactivation behavior of the Shiga toxin-producing Escherichia coli O104:H4 and O157:H7 outbreak strains and a non-pathogenic surrogate.

    PubMed

    Reineke, Kai; Sevenich, Robert; Hertwig, Christian; Janßen, Traute; Fröhling, Antje; Knorr, Dietrich; Wieler, Lothar H; Schlüter, Oliver

    2015-04-01

    Enterohemorrhagic Escherichia coli strains cause each year thousands of illnesses, which are sometimes accompanied by the hemolytic uremic syndrome, like in the 2011 outbreak in Germany. For preservation thermal pasteurization is commonly used, which can cause unwanted quality changes. To prevent this high pressure treatment is a potential alternative. Within this study, the 2011 outbreak strain O104:H4, an O157:H7 and a non-pathogenic strain (DSM1116) were tested. The cells were treated in buffer (pH 7 and pH 5) and carrot juice (pH 5.1) in a pressure temperature range of 0.1-500 MPa and 20-70 °C. Flow cytometry was used to investigate the pressure impact on cell structures of the strain DSM1116. Both pathogenic strains had a much higher resistance in buffer and carrot juice than the non-pathogenic surrogate. Further, strains cultivated and treated at a lower pH-value showed higher pressure stability, presumably due to variations in the membrane composition. This was confirmed for the strain DSM1116 by flow cytometry. Cells cultivated and treated at pH 5 had a stronger ability to retain their membrane potential but showed higher rates of membrane permeabilization at pressures <200 MPa compared to cells cultivated and treated at pH 7. These cells had the lowest membrane permeabilization rate at around 125 MPa, possibly denoting that variations in the fatty acid composition and membrane fluidity contribute to this stabilization phenomenon. PMID:25475283

  5. Efficacy of levulinic acid-sodium dodecyl sulfate against Encephalitozoon intestinalis, Escherichia coli O157:H7, and Cryptosporidium parvum.

    PubMed

    Ortega, Ynes R; Torres, Maria P; Tatum, Jessica M

    2011-01-01

    Foodborne parasites are characterized as being highly resistant to sanitizers used by the food industry. In 2009, a study reported the effectiveness of levulinic acid in combination with sodium dodecyl sulfate (SDS) in killing foodborne bacteria. Because of their innocuous properties, we studied the effects of levulinic acid and SDS at various concentrations appropriate for use in foods, on the viability of Cryptosporidium parvum and Encephalitozoon intestinalis. The viability of Cryptosporidium and E. intestinalis was determined by in vitro cultivation using the HCT-8 and RK-13 cell lines, respectively. Two Escherichia coli O157:H7 isolates were also used in the present study: strain 932 (a human isolate from a 1992 Oregon meat outbreak) and strain E 0018 (isolated from calf feces). Different concentrations and combinations of levulinic acid and SDS were tested for their ability to reduce infectivity of C. parvum oocysts (10(5)), E. intestinalis spores (10(6)), and E. coli O157:H7 (10(7)/ml) when in suspension. Microsporidian spores were treated for 30 and 60 min at 20 ± 2°C. None of the combinations of levulinic acid and SDS were effective at inactivating the spores or oocysts. When Cryptosporidium oocysts were treated with higher concentrations (3% levulinic acid-2% SDS and 2% levulinic acid-1% SDS) for 30, 60, and 120 min, viability was unaffected. E. coli O157:H7, used as a control, was highly sensitive to the various concentrations and exposure times tested. SDS and levulinic acid alone had very limited effect on E. coli O157:H7 viability, but in combination they were highly effective at 30 and 60 min of incubation. In conclusion, Cryptosporidium and microsporidia are not inactivated when treated for various periods of time with 2% levulinic acid-1% SDS or 3% levulinic acid-2% SDS at 20°C, suggesting that this novel sanitizer cannot be used to eliminate parasitic contaminants in foods. PMID:21219777

  6. Molecular detection of nine clinically important non-O157 Escherichia coli serogroups from raw sheep meat in Chaharmahal-va-Bakhtiari province, Iran.

    PubMed

    Tahmasby, Hossein; Mehrabiyan, Samaneh; Tajbakhsh, Elahe; Farahmandi, Sharmin; Monji, Hadi; Farahmandi, Keyvan

    2014-08-01

    STEC isolates and also stx-negative Escherichia coli isolates from sheep meat from the Chaharmahal-va-Bakhtiari province, Iran were analyzed for nine clinically important non-O157 serotypes by PCR. A total of 90 E. coli isolates were tested. Stx-positive and eae-positive E. coli isolates did not belong to the nine most clinically relevant non-O157 STECs. Of the 80 non-STEC isolates, two belonged to the O103 and two belonged to the O128 groups. Stx-negative E. coli O103 and O128 strains isolated have potential in acquiring stx genes and continuing into the digestive system of consumers. Further studies are needed to analyze virulence characteristics of these E. coli strains to determine their potential role in causing disease in humans. For the sake of public health, it is important to monitor and investigate non-O157 diarrheagenic E. coli strains in meat in order to control and prevent them.

  7. Comparison of eight different agars for the recovery of clinically relevant non-O157 Shiga toxin-producing Escherichia coli from baby spinach, cilantro, alfalfa sprouts and raw milk.

    PubMed

    Kase, Julie A; Maounounen-Laasri, Anna; Son, Insook; Lin, Andrew; Hammack, Thomas S

    2015-04-01

    The FDA Bacteriological Analytical Manual (BAM) Chapter 4a recommends several agars for isolating non-O157 Shiga toxin-producing Escherichia coli (STEC); not all have been thoroughly tested for recovering STECs from food. Using E. coli strains representing ten clinically relevant O serogroups (O26, O45, O91, O103, O104, O111, O113, O121, O128, O145) in artificially-contaminated fresh produce--bagged baby spinach, alfalfa sprouts, cilantro, and raw milk--we evaluated the performance of 8 different agars. Performance was highly dependent upon strain used and the presence of inhibitors, but not necessarily dependent on food matrix. Tellurite resistant-negative strains, O91:-, O103:H6, O104:H21, O113:H21, and O128, grew poorly on CHROMagar STEC, Rainbow agar O157, and a modified Rainbow O157 (mRB) agar. Although adding washed sheep's blood to CHROMagar STEC and mRB agars improved overall performance; however, this also reversed the inhibition of non-target bacteria provided by original formulations. Variable colony coloration made selecting colonies from Rainbow agar O157 and mRB agars difficult. Study results support a strategy using inclusive agars (e.g. L-EMB, SHIBAM) in combination with selective agars (R & F E. coli O157:H7, CHROMagar STEC) to allow for recovery of the most STECs while increasing the probability of recovering STEC in high bacterial count matrices. PMID:25475297

  8. Comparison of eight different agars for the recovery of clinically relevant non-O157 Shiga toxin-producing Escherichia coli from baby spinach, cilantro, alfalfa sprouts and raw milk.

    PubMed

    Kase, Julie A; Maounounen-Laasri, Anna; Son, Insook; Lin, Andrew; Hammack, Thomas S

    2015-04-01

    The FDA Bacteriological Analytical Manual (BAM) Chapter 4a recommends several agars for isolating non-O157 Shiga toxin-producing Escherichia coli (STEC); not all have been thoroughly tested for recovering STECs from food. Using E. coli strains representing ten clinically relevant O serogroups (O26, O45, O91, O103, O104, O111, O113, O121, O128, O145) in artificially-contaminated fresh produce--bagged baby spinach, alfalfa sprouts, cilantro, and raw milk--we evaluated the performance of 8 different agars. Performance was highly dependent upon strain used and the presence of inhibitors, but not necessarily dependent on food matrix. Tellurite resistant-negative strains, O91:-, O103:H6, O104:H21, O113:H21, and O128, grew poorly on CHROMagar STEC, Rainbow agar O157, and a modified Rainbow O157 (mRB) agar. Although adding washed sheep's blood to CHROMagar STEC and mRB agars improved overall performance; however, this also reversed the inhibition of non-target bacteria provided by original formulations. Variable colony coloration made selecting colonies from Rainbow agar O157 and mRB agars difficult. Study results support a strategy using inclusive agars (e.g. L-EMB, SHIBAM) in combination with selective agars (R & F E. coli O157:H7, CHROMagar STEC) to allow for recovery of the most STECs while increasing the probability of recovering STEC in high bacterial count matrices.

  9. Whole Genome Sequencing for Public Health Surveillance of Shiga Toxin-Producing Escherichia coli Other than Serogroup O157

    PubMed Central

    Chattaway, Marie A.; Dallman, Timothy J.; Gentle, Amy; Wright, Michael J.; Long, Sophie E.; Ashton, Philip M.; Perry, Neil T.; Jenkins, Claire

    2016-01-01

    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 laboratories has resulted in an increase in the detection of 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. Of the 10 discordant results, 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 characterization 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. PMID:26973632

  10. Detection, Virulence Gene Assessment and Antibiotic Resistance Pattern of O157 Enterohemorrhagic Escherichia coli in Tabriz, Iran

    PubMed Central

    Akhi, Mohammad Taghi; Ostadgavahi, Ali Toloue; Ghotaslou, Reza; Asgharzadeh, Mohammad; Pirzadeh, Tahereh; Sorayaei Sowmesarayi, Vida; Memar, Mohammad Yousef

    2015-01-01

    Background: Shiga toxin-producing Escherichia coli (STEC) is a food-borne pathogen and infection with this organism causes illnesses such as bloody diarrhea, hemorrhagic colitis and hemolytic-uremic syndrome. Objectives: Considering the lack of any information about the prevalence rate and the antibiotic resistance pattern of O157:H7 serotype in Tabriz, finding answers to the above mentioned subjects was among the goals of this study. Materials and Methods: Two hundred E. coli strains from diarrheal or non-diarrheal stools of outpatients and hospitalized cases in Tabriz Imam Reza hospital were isolated between September and December 2014 using MacConkey agar and standard biochemical tests and then cultured on sorbitol MacConkey agar. The sorbitol-negative isolates were confirmed as the O157 serotype using O157 antisera. A multiplex polymerase chain reaction (PCR) method was used for the detection of stx-1, stx-2, eae, and mdh genes and the antibiotic resistance pattern of these isolates was determined using Kirby-Bauer method and clinical and laboratory standards institute (CLSI) standards. Results: Of the isolates 11 (5.5%) were sorbitol-negative, which were later analyzed by multiplex PCR and the results revealed that 2 (18.18%) isolates contained the stx-1 gene, 10 (90.91%) contained the stx-2 gene, and 5 (45.45%) contained the eae gene. The stx-2 and eae genes were the most commonly encountered virulence factors. All or most of the isolates were susceptible to ceftazidime (100%), gentamicin (100%), ciprofloxacin (100%), nalidixic acid (90.9%), trimetoprim sulfamethoxazole (90.9%), chloramphenicol (90.9%), ampicillin (81.8%), and cephalothin (72.7%). On the contrary, moderate susceptibility of the isolates to doxycycline (54.5%) was observed. Conclusions: Due to the low frequency of STEC O157 and the high susceptibility rates of the isolates to the tested antibiotics in this study, STEC O157 has not become a major problem in Tabriz yet, but comprehensive

  11. Molecular characterization of Escherichia coli O157:H7 hide contamination routes: feedlot to harvest.

    PubMed

    Childs, K D; Simpson, C A; Warren-Serna, W; Bellenger, G; Centrella, B; Bowling, R A; Ruby, J; Stefanek, J; Vote, D J; Choat, T; Scanga, J A; Sofos, J N; Smith, G C; Belk, K E

    2006-06-01

    This study was conducted to identify the origin of Escherichia coli O157:H7 contamination on steer hides at the time of harvest. Samples were collected from the feedlot, transport trailers, and packing plant holding pens and from the colons and hides of feedlot steers. A total of 50 hide samples were positive for E. coli O157:H7 in two geographical locations: the Midwest (25 positive hides) and Southwest (25 positive hides). Hide samples were screened, and the presence of E. coli O157: H7 was confirmed. E. coli O157:H7 isolates were fingerprinted by pulsed-field gel electrophoresis and subjected to multiplex PCR procedures for amplification of E. coli O157:H7 genes stx1, stx2, eaeA, fliC, rfbEO157, and hlyA. Feedlot water trough, pen floor, feed bunk, loading chute, truck trailer side wall and floor, packing plant holding pen floor and side rail, and packing plant cattle drinking water samples were positive for E. coli O157:H7. Pulsed-field gel electrophoresis banding patterns were analyzed after classifying isolates according to the marker genes present and according to packing plant. In this study, hide samples positive for E. coli O157:H7 were traced to other E. coli O157:H7-positive hide, colon, feedlot pen floor fecal, packing plant holding pen drinking water, and transport trailer side wall samples. Links were found between packing plant side rails, feedlot loading chutes, and feedlot pens and between truck trailer, different feedlots, and colons of multiple cattle. This study is the first in which genotypic matches have been made between E. coli O157:H7 isolates obtained from transport trailer side walls and those from cattle hide samples within the packing plant. PMID:16786841

  12. Genetic diversity of Shiga toxin-producing Escherichia coli O157 : H7 recovered from human and food sources.

    PubMed

    Elhadidy, Mohamed; Elkhatib, Walid F; Elfadl, Eman A Abo; Verstraete, Karen; Denayer, Sarah; Barbau-Piednoir, Elodie; De Zutter, Lieven; Verhaegen, Bavo; De Rauw, Klara; Piérard, Denis; De Reu, Koen; Heyndrickx, Marc

    2015-01-01

    The aim of this study was to identify an epidemiological association between Shiga toxin-producing Escherichia coli O157 : H7 strains associated with human infection and with food sources. Frequency distributions of different genetic markers of E. coli O157 : H7 strains recovered from human and food sources were compared using molecular assays to identify E. coli O157 : H7 genotypes associated with variation in pathogenic potential and host specificity. Genotypic characterization included: lineage-specific polymorphism assay (LSPA-6), clade typing, tir (A255T) polymorphism, Shiga toxin-encoding bacteriophage insertion site analysis and variant analysis of Shiga toxin 2 gene (stx2a and stx2c) and antiterminator Q genes (Q933 and Q21). The intermediate lineage (LI/II) dominated among both food and human strains. Compared to other clades, clades 7 and 8 were more frequent among food and human strains, respectively. The tir (255T) polymorphism occurred more frequently among human strains than food strains. Q21 and Q933 + Q21 were found at significantly higher frequencies among food and human strains, respectively. Moreover, stx2a and stx2a+c were detected at significantly higher frequencies among human strains compared to food strains. Bivariate analysis revealed significant concordance (P<0.05) between the LSPA-6 assay and the other typing methods. Multivariable regression analysis suggested that tir (255T) was the most distinctive genotype that can be used to detect bacterial clones with potential risk for human illness from food sources. This study supported previous reports of the existence of diversity in genetic markers among different isolation sources by including E. coli O157 : H7 strains from both food and human sources. This might enable tracking genotypes with potential risk for human illness from food sources.

  13. [Biological characters of enterohemorrhagic Escherichia coli isolates from diarrhea patients in Saitama (1990-1992)].

    PubMed

    Yamada, F; Kurazono, T; Yamaguchi, M; Ohzeki, Y; Okuyama, Y

    1994-12-01

    A total of 16 strains of Enterohemorrhagic Escherichia coli (EHEC) isolated from diarrea patients in Saitama from 1990 to 1992 were tested for their serotype, verotoxin production, biochemical characteristics, antibiotics sensitivity and plasmid profiles. By serotype analysis, 14 strains from two outbreaks and 12 sporadic cases were classified as type O157:H7, one as O111:H-(not motility) and one as O128:H2. Typing of verotoxin by gene analysis using Polymerase Chain Reaction (PCR) showed that 9 of O157:H7 strains including two cases from outbreaks and O128:H2 have VT1 and VT2 genes, other O157:H7 have the VT2 gene and O111:H-has only the VT1 gene. Biochemical characteristic analysis indicated two strains of O157:H7 type from outbreaks were biotype II and the rest of O157:H7 were biotype I. One of the O157:H7 strain from a sporadic case showed positive for urease production. According to sensitivity tests against antibiotics, out of the O157:H7 group, one strain was resistant against ABPC, one against SM and two strains resistant to SM-TC. For plasmid profiles, all strains had 94 Kb plasmids and several smaller sizes of plasmids. But 5 strains of O157:H7 had 94 Kb plasmid only.

  14. Defects in polynucleotide phosphorylase impairs virulence in Escherichia coli O157:H7.

    PubMed

    Hu, Jia; Zhu, Mei-Jun

    2015-01-01

    Polynucleotide phosphorylase (PNPase) is reported to regulate virulence in Salmonella, Yersinia sp. and Campylobacter jejuni, yet its role in Escherichia coli O157:H7 has not been investigated. To gain insights into its roles in E. coli O157:H7 virulence, pnp deletion mutants were generated and the major virulence factors were compared to their parental wild type strains. Deletion of pnp in E. coli O157:H7 dramatically decreased stx2 mRNA expression and Stx2 protein production, and impaired lambdoid prophage activation in E. coli O157:H7. Quantitative PCR further confirmed that the Stx2 phage lytic growth was repressed by pnp deletion. Consistent with reduced Stx2 production and Stx2 phage activation, the transcriptional levels of genes involved in phage lysis and replication were down-regulated. In addition, disruption of pnp in E. coli O157:H7 decreased its adhesion to intestinal epithelial cells as well as cattle colonic explant tissues. On the other hand, PNPase inactivation in E. coli O157:H7 enhanced Tir protein content and the transcription of type three secretion system components, including genes encoding intimin, Tir, and EspB as well as locus of enterocyte and effacement positive regulator, Ler. Collectively, data indicate that PNPase has pleiotropic effects on the virulence of E. coli O157:H7. PMID:26347717

  15. Enumeration of Escherichia coli O157 in Outbreak-Associated Gouda Cheese Made with Raw Milk.

    PubMed

    Gill, Alexander; Oudit, Denise

    2015-09-01

    In this article, we discuss the enumerative analysis for Escherichia coli O157 in two raw milk Gouda cheese products (A and B), implicated in an outbreak of 29 cases of E. coli O157:H7 illness that occurred across Canada in 2013. Samples were enumerated for E. coli O157 by most probable number (MPN) over a period of 30 to 60 days after the end of the outbreak. Samples (55.55 g) of product A (n = 14) were analyzed at 146 to 180 days postproduction. E. coli O157 was isolated from six samples at 19.9 to 44.6 MPN/kg. The E. coli O157 concentration of product A estimated from the results of all 14 samples was 9.5 MPN/kg. Samples (55.55 g) of product B (n = 20) were analyzed at 133 to 149 days postproduction. E. coli O157 was isolated from four samples at 19.9 MPN/kg. The E. coli O157 concentration of product B estimated from the results of all 20 samples was 3.7 MPN/kg. Analysis of a 305-g sample of product A (n = 1) stored at 4°C until 306 days postproduction revealed that the E. coli O157 concentration had declined to 3.6 MPN/kg. E. coli O157 could not be isolated from 555-g samples of product B (n = 5) after 280 days postproduction. The physicochemical parameters (pH, water activity, percent moisture, and percent salt) of both cheese products were found to be in the normal range for this type of product. The results of this study demonstrate that E. coli O157 could not replicate during storage at 4°C in the products tested but was capable of survival following aging and prolonged storage. This indicates that, if contaminated, the minimum 60-day aging period, which is required for raw milk Gouda cheeses, is not sufficient in all cases to ensure that the product does not contain viable cells of E. coli O157. The results also indicate that samples sizes greater than 100 g may be required to reliably detect E. coli O157 in cheese products associated with outbreaks.

  16. KatP contributes to OxyR-regulated hydrogen peroxide resistance in Escherichia coli serotype O157:H7

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli K12 defends against peroxide mediated oxidative damage using two catalases, hydroperoxidase I (katG) and hydroperoxidase II (katE) and the peroxiredoxin, alkyl hydroperoxide reductase (ahpC). In E. coli O157:H7 strain ATCC 43895 (EDL933), plasmid pO157 encodes for an additional cata...

  17. Effects of the ergot alkaloids dihydroergotamine, ergonovine, and ergotamine on growth of Escherichia coli O157:H7 in vitro

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A series of experiments were conducted to evaluate the effects of ergot alkaloids (dihydroergotamine, ergonovine, and ergotamine) on E. coli O157:H7 in both pure and mixed ruminal fluid culture. Alkaloids were added to solutions of E. coli O157:H7 strains 933 (pure and ruminal cultures) and 6058 (r...

  18. Enhancing the thermal destruction of Escherichia coli O157:H7 in ground beef patties by trans-cinnamaldehyde

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study investigated the effect of trans-cinnamaldehyde, an active ingredient in cinnamon, for inactivating E. coli O157:H7 in undercooked ground beef patties. A five-strain mixture of E. coli O157:H7 was inoculated into ground beef (90% lean and 10% fat) at approximately 7.0 log CFU/g, followed ...

  19. Non-O157 Shiga toxin-producing Escherichia coli: prevalence associated with meat animals and controlling interventions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This paper reviews the current state of knowledge of non-O157 STEC in products of meat animals. There is a wide range in pathogenicity of STEC strains. Potential regulation in meat products is currently focused on the group of six O groups the CDC indicates accounts of 71% of non-O157 STEC illness...

  20. The Escherichia coli O157:H7 bovine rumen fluid proteome reflects adaptive bacterial responses

    PubMed Central

    2014-01-01

    Background To obtain insights into Escherichia coli O157:H7 (O157) survival mechanisms in the bovine rumen, we defined the growth characteristics and proteome of O157 cultured in rumen fluid (RF; pH 6.0-7.2 and low volatile fatty acid content) obtained from rumen-fistulated cattle fed low protein content “maintenance diet” under diverse in vitro conditions. Results Bottom-up proteomics (LC-MS/MS) of whole cell-lysates of O157 cultured under anaerobic conditions in filter-sterilized RF (fRF; devoid of normal ruminal microbiota) and nutrient-depleted and filtered RF (dRF) resulted in an anaerobic O157 fRF-and dRF-proteome comprising 35 proteins functionally associated with cell structure, motility, transport, metabolism and regulation, but interestingly, not with O157 virulence. Shotgun proteomics-based analysis using isobaric tags for relative and absolute quantitation used to further study differential protein expression in unfiltered RF (uRF; RF containing normal rumen microbial flora) complemented these results. Conclusions Our results indicate that in the rumen, the first anatomical compartment encountered by this human pathogen within the cattle gastrointestinal tract (GIT), O157 initiates a program of specific gene expression that enables it to adapt to the in vivo environment, and successfully transit to its colonization sites in the bovine GIT. Further experiments in vitro using uRF from animals fed different diets and with additional O157 strains, and in vivo using rumen-fistulated cattle will provide a comprehensive understanding of the adaptive mechanisms involved, and help direct evolution of novel modalities for blocking O157 infection of cattle. PMID:24559513

  1. Inactivation of Escherichia coli O157:H7 attached to spinach harvester blade using bacteriophage.

    PubMed

    Patel, Jitendra; Sharma, Manan; Millner, Patricia; Calaway, Todd; Singh, Manpreet

    2011-04-01

    Outbreaks associated with leafy greens have focused attention on the transfer of human pathogens to these commodities during harvest with commercial equipment. Attachment of Escherichia coli O157:H7 on new or rusty spinach harvester blades immersed in spinach extract or 10% tryptic soy broth (TSB) was investigated. Bacteriophages specific for E. coli O157:H7 were evaluated to kill cells attached to blade. A cocktail of five nalidixic acid-resistant E. coli O157:H7 isolates was transferred to 25 mL of spinach extract or 10% TSB. A piece of sterilized spinach harvester blade (2×1") was placed in above spinach extract or 10% TSB and incubated at room (22 °C) or dynamic (30 °C day, 20 °C night) temperatures. E. coli O157:H7 populations attached to blade during incubation in spinach extract or 10% TSB were determined. When inoculated at 1 log CFU/mL, E. coli O157:H7 attachment to blades after 24 and 48 h incubation at dynamic temperature (6.09 and 6.37 log CFU/mL) was significantly higher than when incubated at 22 °C (4.84 and 5.68 log CFU/mL), respectively. After 48 h incubation, two blades were sprayed on each side with a cocktail of E. coli O157-specific bacteriophages before scraping the blade, and subsequent plating on Sorbitol MacConkey media-nalidixic acid. Application of bacteriophages reduced E. coli O157:H7 populations by 4.5 log CFU on blades after 2 h of phage treatment. Our study demonstrates that E. coli O157:H7 can attach to and proliferate on spinach harvester blades under static and dynamic temperature conditions, and bacteriophages are able to reduce E. coli O157:H7 populations adhered to blades. PMID:21453119

  2. Inactivation of Escherichia coli O157:H7 attached to spinach harvester blade using bacteriophage.

    PubMed

    Patel, Jitendra; Sharma, Manan; Millner, Patricia; Calaway, Todd; Singh, Manpreet

    2011-04-01

    Outbreaks associated with leafy greens have focused attention on the transfer of human pathogens to these commodities during harvest with commercial equipment. Attachment of Escherichia coli O157:H7 on new or rusty spinach harvester blades immersed in spinach extract or 10% tryptic soy broth (TSB) was investigated. Bacteriophages specific for E. coli O157:H7 were evaluated to kill cells attached to blade. A cocktail of five nalidixic acid-resistant E. coli O157:H7 isolates was transferred to 25 mL of spinach extract or 10% TSB. A piece of sterilized spinach harvester blade (2×1") was placed in above spinach extract or 10% TSB and incubated at room (22 °C) or dynamic (30 °C day, 20 °C night) temperatures. E. coli O157:H7 populations attached to blade during incubation in spinach extract or 10% TSB were determined. When inoculated at 1 log CFU/mL, E. coli O157:H7 attachment to blades after 24 and 48 h incubation at dynamic temperature (6.09 and 6.37 log CFU/mL) was significantly higher than when incubated at 22 °C (4.84 and 5.68 log CFU/mL), respectively. After 48 h incubation, two blades were sprayed on each side with a cocktail of E. coli O157-specific bacteriophages before scraping the blade, and subsequent plating on Sorbitol MacConkey media-nalidixic acid. Application of bacteriophages reduced E. coli O157:H7 populations by 4.5 log CFU on blades after 2 h of phage treatment. Our study demonstrates that E. coli O157:H7 can attach to and proliferate on spinach harvester blades under static and dynamic temperature conditions, and bacteriophages are able to reduce E. coli O157:H7 populations adhered to blades.

  3. Displacement of Escherichia coli O157:H7 from rumen medium containing prebiotic sugars.

    PubMed

    de Vaux, Albane; Morrison, Mark; Hutkins, Robert W

    2002-02-01

    A fed-batch, anaerobic culture system was developed to assess the behavior of Escherichia coli O157:H7 in a rumen-like environment. Fermentation medium consisted of either 50% (vol/vol) raw or sterile rumen fluid and 50% phosphate buffer. Additional rumen fluid was added twice per day, and samples were removed three times per day to simulate the exiting of digesta and microbes from the rumen environment under typical feeding regimens. With both types of medium, anaerobic and enteric bacteria reached 10(10) and 10(4) cells/ml, respectively, and were maintained at these levels for at least 5 days. When a rifampin-resistant strain of E. coli O157:H7 was inoculated into medium containing raw rumen fluid, growth did not occur. In contrast, when this strain was added to sterile rumen fluid medium, cell densities increased from 10(6) to 10(9) CFU/ml within 24 h. Most strains of E. coli O157:H7 are unable to ferment sorbitol; therefore, we assessed whether the addition of sorbitol as the only added carbohydrate could be used to competitively exclude E. coli O157:H7 from the culture system. When inoculated into raw rumen broth containing 3 g of sorbitol per liter, E. coli O157:H7 was displaced within 72 h. The addition of other competitive sugars, such as L-arabinose, trehalose, and rhamnose, to rumen medium gave similar results. However, whenever E. coli O157:H7 was grown in sterile rumen broth containing sorbitol, sorbitol-positive mutants appeared. These results suggest that a robust population of commensal ruminal microflora is required to invoke competitive exclusion of E. coli O157:H7 by the addition of "nonfermentable" sugars and that this approach may be effective as a preharvest strategy for reducing carriage of E. coli O157:H7 in the rumen.

  4. Inactivation of Escherichia coli O157:H7 in simulated human gastric fluid.

    PubMed

    Tamplin, Mark L

    2005-01-01

    Human disease caused by Escherichia coli O157:H7 is a function of the number of cells that are present at potential sites of infection and host susceptibility. Such infectious doses are a result, in part, of the quantity of cells that are ingested and that survive human host defenses, such as the low-pH environment of the stomach. To more fully understand the kinetics of E. coli O157:H7 survival in gastric fluid, individual E. coli O157:H7 strains were suspended in various media (i.e., saline, cooked ground beef [CGB], and CGB containing a commercial antacid product [CGB+A]), mixed at various proportions with simulated human gastric fluid (SGF), and then incubated at 37 degrees C for up to 4 h. The highest inactivation rate among nine E. coli O157:H7 strains was observed in saline. Specifically, the average survival rates in 100:1 and 10:1 proportions of SGF-saline were -1.344 +/- 0.564 and -0.997 +/- 0.388 log(10) CFU/h, respectively. In contrast, the average inactivation rate for 10 E. coli O157:H7 strains suspended in 10:1 SGF-CGB was -0.081 +/- 0.068, a rate that was 12-fold lower than that observed for SGF-saline. In comparison, the average inactivation rate for Shigella flexneri strain 5348 in 100:1 and 10:1 SGF-saline was -8.784 and -17.310, respectively. These latter inactivation rates were 7- to 17-fold higher than those for E. coli O157:H7 strains in SGF-saline and were 4-fold higher than those for E. coli O157:H7 strains in SGF-CGB. The survival rate of E. coli O157:H7 strain GFP80EC increased as the dose of antacid increased from one-half to twice the prescribed dose. A similar trend was observed for the matrix pH over the range of pH 1.6 to 5.7, indicating that pH is a primary factor affecting E. coli O157:H7 survival in SGF-CGB+A. These results can be used in risk assessment to define dose-response relationships for E. coli O157:H7 and to evaluate potential surrogate organisms.

  5. Genotypic analyses of shiga toxin-producing Escherichia coli O157 and non-O157 recovered from feces of domestic animals on rural farms in Mexico.

    PubMed

    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

  6. Enumeration of verocytotoxigenic Escherichia coli (VTEC) O157 and O26 in milk by quantitative PCR.

    PubMed

    Mancusi, Rocco; Trevisani, Marcello

    2014-08-01

    Quantitative real-time polymerase chain reaction (qPCR) can be a convenient alternative to the Most Probable Number (MPN) methods to count VTEC in milk. The number of VTEC is normally very low in milk; therefore with the aim of increasing the method sensitivity a qPCR protocol that relies on preliminary enrichment was developed. The growth pattern of six VTEC strains (serogroups O157 and O26) was studied using enrichment in Buffered Peptone Water (BPW) with or without acriflavine for 4-24h. Milk samples were inoculated with these strains over a five Log concentration range between 0.24-0.50 and 4.24-4.50 Log CFU/ml. DNA was extracted from the enriched samples in duplicate and each extract was analysed in duplicate by qPCR using pairs of primers specific for the serogroups O157 and O26. When samples were pre-enriched in BPW at 37°C for 8h, the relationship between threshold cycles (CT values) and VTEC Log numbers was linear over a five Log concentration range. The regression of PCR threshold cycle numbers on VTEC Log CFU/ml had a slope coefficient equal to -3.10 (R(2)=0.96) which is indicative of a 10-fold difference of the gene copy numbers between samples (with a 100 ± 10% PCR efficiency). The same 10-fold proportion used for inoculating the milk samples with VTEC was observed, therefore, also in the enriched samples at 8h. A comparison of the CT values of milk samples and controls revealed that the strains inoculated in milk grew with 3 Log increments in the 8h enrichment period. Regression lines that fitted the qPCR and MPN data revealed that the error of the qPCR estimates is lower than the error of the estimated MPN (r=0.982, R(2)=0.965 vs. r=0.967, R(2)=0.935). The growth rates of VTEC strains isolated from milk should be comparatively assessed before qPCR estimates based on the regression model are considered valid. Comparative assessment of the growth rates can be done using spectrophotometric measurements of standardized cultures of isolates and

  7. Lytic bacteriophages reduce Escherichia coli O157

    PubMed Central

    Ferguson, Sean; Roberts, Cheryl; Handy, Eric; Sharma, Manan

    2013-01-01

    The role of lytic bacteriophages in preventing cross contamination of produce has not been evaluated. A cocktail of three lytic phages specific for E. coli O157:H7 (EcoShield™) or a control (phosphate buffered saline, PBS) was applied to lettuce by either; (1) immersion of lettuce in 500 ml of EcoShield™ 8.3 log PFU/ml or 9.8 log PFU/ml for up to 2 min before inoculation with E. coli O157:H7; (2) spray-application of EcoShield™ (9.3 log PFU/ml) to lettuce after inoculation with E. coli O157:H7 (4.10 CFU/cm2) following exposure to 50 μg/ml chlorine for 30 sec. After immersion studies, lettuce was spot-inoculated with E. coli O157:H7 (2.38 CFU/cm2). Phage-treated, inoculated lettuce pieces were stored at 4°C for and analyzed for E. coli O157:H7 populations for up to 7 d. Immersion of lettuce in 9.8 log PFU/ml EcoShield™ for 2 min significantly (p < 0.05) reduced E. coli O157:H7 populations after 24 h when stored at 4°C compared with controls. Immersion of lettuce in suspensions containing high concentrations of EcoShield™ (9.8 log PFU/ml) resulted in the deposition of high concentrations (7.8 log log PFU/cm2) of bacteriophages on the surface of fresh cut lettuce, potentially contributing to the efficacy of the lytic phages on lettuce. Spraying phages on to inoculated fresh cut lettuce after being washed in hypochlorite solution was significantly more effective in reducing E. coli O157:H7 populations (2.22 log CFU/cm2) on day 0 compared with control treatments (4.10 log CFU/cm2). Both immersion and spray treatments provided protection from E. coli O157:H7 contamination on lettuce, but spray application of lytic bacteriophages to lettuce was more effective in immediately reducing E. coli O157:H7 populations fresh cut lettuce. PMID:23819106

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

    PubMed

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

    2016-09-01

    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.

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

    PubMed

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

    2016-09-01

    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. PMID:27257743

  10. [Pollution of EHEC O157:H7 in six types of food in Henan Province].

    PubMed

    Zhang, Ding; Liao, Xingguang; Zhang, Xiuli; Li, Li

    2003-09-01

    In order to investigate the distribution of contamination of EHEC O157:H7 in 6 types of food in Henan Province and characterize the effect of seasonal factors on distribution of O157:H7 contamination so as to prevent and control food contamination, Henan Province was divided into five sampling areas according to the natural geographical features. Samples were taken randomly in summer and winter sales links. After selective culture of increasing reproduction of O157:H7, it was screened by immuno-gold reagent, and pathogenic bacterium were isolated after accumulation of immunomagnetic. Then, it was identified by the bioMérieux VITEK32 AMS system, GNI+ and sero-reaction. Results showed that 28 EHEC O157:H7 were isolated in 1463 samples among which the positive rate of raw meat and fresh vegetable was 3.3% and 3.2% respectively (the highest of all), and EHEC O157:H7 was not found in yogurt. The positive rate in summer (2.5%) was obviously higher than that in winter (1.1%). It could be concluded that the contamination of EHEC O157:H7 in food was serious. Its positive rate was positively correlated with the epidemic of infective diarrhea. Therefore, attention should be given by relevant departments concerned and spervision and testing should be strengthened in livestock and farmyard bird slaughtering link, vegetable production link and sales link for better prevention of the breaking out and epidemic of food born diseases.

  11. Light Scattering Sensor for Direct Identification of Colonies of Escherichia coli Serogroups O26, O45, O103, O111, O121, O145 and O157

    PubMed Central

    Tang, Yanjie; Kim, Huisung; Singh, Atul K.; Aroonnual, Amornrat; Bae, Euiwon; Rajwa, Bartek; Fratamico, Pina M.; Bhunia, Arun K.

    2014-01-01

    Background Shiga-toxin producing Escherichia coli (STEC) have emerged as important foodborne pathogens, among which seven serogroups (O26, O45, O103, O111, O121, O145, O157) are most frequently implicated in human infection. The aim was to determine if a light scattering sensor can be used to rapidly identify the colonies of STEC serogroups on selective agar plates. Methodology/Principal Findings Initially, a total of 37 STEC strains representing seven serovars were grown on four different selective agar media, including sorbitol MacConkey (SMAC), Rainbow Agar O157, BBL CHROMagarO157, and R&F E. coli O157:H7, as well as nonselective Brain Heart Infusion agar. The colonies were scanned by an automated light scattering sensor, known as BARDOT (BActerial Rapid Detection using Optical scattering Technology), to acquire scatter patterns of STEC serogroups, and the scatter patterns were analyzed using an image classifier. Among all of the selective media tested, both SMAC and Rainbow provided the best differentiation results allowing multi-class classification of all serovars with an average accuracy of more than 90% after 10–12 h of growth, even though the colony appearance was indistinguishable at that early stage of growth. SMAC was chosen for exhaustive scatter image library development, and 36 additional strains of O157:H7 and 11 non-O157 serovars were examined, with each serogroup producing unique differential scatter patterns. Colony scatter images were also tested with samples derived from pure and mixed cultures, as well as experimentally inoculated food samples. BARDOT accurately detected O157 and O26 serovars from a mixed culture and also from inoculated lettuce and ground beef (10-h broth enrichment +12-h on-plate incubation) in the presence of natural background microbiota in less than 24 h. Conclusions BARDOT could potentially be used as a screening tool during isolation of the most important STEC serovars on selective agar plates from food samples in

  12. A tandem duplication of a 5-bp sequence in the rcsB gene confers biofilm-producing phenotype in Escherichia coli O157:H7 strain 86-24

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Biofilm formation, which is an important bacterial survival and virulence attribute, is controlled by intricate regulatory networks. Enterohemorrhagic Escherichia coli O157:H7 is an important foodborne pathogen because infections with this agent could lead to hemorrhagic colitis, kidney dysfunction,...

  13. Ecology of Escherichia coli O157:H7 in commercial dairies in southern Alberta.

    PubMed

    Stanford, K; Croy, D; Bach, S J; Wallins, G L; Zahiroddini, H; McAllister, T A

    2005-12-01

    Shedding of Escherichia coli O157:H7 was monitored monthly over a 1-yr period by collecting pooled fecal pats (FECAL) and manila ropes orally accessed for 4 h (ROPE) from multiple pens of cattle in 5 commercial dairies in southern Alberta, Canada. Using immunomagnetic separation, E. coli O157:H7 was isolated from cows on 4 of the dairies and from 13.5% of FECAL and 1.1% of ROPE samples. Pulsed-field gel electrophoresis of XbaI- and SpeI-digested bacterial DNA of the 65 isolates produced 23 unique restriction endonuclease digestion patterns, although 92% of the isolates belonged to 3 restriction endonuclease digestion pattern clusters sharing a minimum 90% homology. Collection of positive isolates was 15 times more likely from June through September. Across dairies, peak somatic cell count occurred in July, August, September, and November. The likelihood of positive isolates was 2.6 times higher in calves and heifers compared with mature cows. This study indicates that ROPE would be of little value for the detection of E. coli O157:H7 in dairy herds unless oral contact with ROPE could be increased in mature animals. Additionally, mitigation strategies for E. coli O157:H7 should be targeted to the months of July, August, and September and toward immature animals for maximum impact. All farms displayed unique combinations of seasonality of shedding and diversity of E. coli O157:H7 subtypes. The fact that seasonal prevalence of E. coli O157:H7 largely coincided with peak somatic cell count within climatically controlled dairy barns suggests that similar environmental factors may be enhancing fecal shedding E. coli O157:H7 and the incidence of mastitis.

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

    PubMed Central

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

    2016-01-01

    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. PMID:27023604

  15. Low prevalence of Escherichia coli O157:H7 in horses in Ohio, USA.

    PubMed

    Lengacher, Brandy; Kline, Terence R; Harpster, Laura; Williams, Michele L; Lejeune, Jeffrey T

    2010-11-01

    Manure from draft animals deposited in fields during vegetable and fruit production may serve as a potential source of preharvest pathogen contamination of foods. To better quantify this risk, we determined the prevalence of Escherichia coli O157:H7 in horses. Between June and September 2009, freshly voided fecal samples were collected from horses stabled on 242 separate premises in Ohio, USA. Overall, the prevalence of E. coli O157:H7 was 1 of 242 (0.4% prevalence, 95% confidence interval [CI] = 0.01 to 2.28). E. coli O157:H7 was recovered from none of the 107 equine fecal samples (0% prevalence, 95% CI = 0.00 to 3.39) that originated from locations without ruminant presence, and only 1 of the 135 horse fecal samples (0.7% prevalence, 95% CI = 0.02 to 4.06) from sites where ruminants were also present. The lone positive sample was collected from a horse that was costabled with a goat. Subsequent sampling at that location identified indistinguishable subtypes of E. coli O157:H7 present in the cohoused goat, in the environment, insects, sheep, and other goats housed in an adjacent field. E. coli O157:H7 was not isolated from the five subsequent samples from this horse. These data indicate that E. coli O157:H7 carriage by horses is an uncommon event.

  16. Meat Science and Muscle Biology Symposium: Development of bacteriophage treatments to reduce Escherichia coli O157:H7 contamination of beef products and produce.

    PubMed

    Hong, Y; Pan, Y; Ebner, P D

    2014-04-01

    Escherichia coli O157:H7 remains a foodborne pathogen of concern with infections associated with products ranging from ground beef to produce to processed foods. We previously demonstrated that phage-based technologies could reduce foodborne pathogen colonization in live animals. Here, we examined if a 3-phage cocktail could reduce E. coli O157:H7 in experimentally contaminated ground beef, spinach, and cheese. The 3 phages were chosen from our E. coli O157:H7 phage library based on their distinct origins of isolation, lytic ranges, and rapid growth (40- to 50-min life cycle). Two phages belonged to the Myoviridae family and the other phage belonged to the Siphoviridae family. The phage cocktail was added to ground beef, spinach leaves, and cheese slices contaminated with E. coli O157:H7 (10(7) cfu) at a multiplicity of infection of 1. Phage treatment reduced (P < 0.05) the concentrations of E. coli O157:H7 by 1.97 log10 cfu/mL in ground beef when stored at room temperature (24 °C) for 24 h, 0.48 log10 cfu/mL at refrigeration (4 °C), and 0.56 log10 cfu/mL in undercooked condition (internal temperature of 46 °C). Likewise, phage treatment reduced (P < 0.05) E. coli O157:H7 by 3.28, 2.88, and 2.77 log10 cfu/mL in spinach when stored at room temperature for 24, 48, and 72 h, respectively. Phage treatment, however, did not reduce E. coli O157:H7 concentrations in contaminated cheese. Additionally, 3 phage-resistant E. coli O157:H7 strains (309-PR [phage resistant] 1, 309-PR4, and 502-PR5) were isolated and characterized to test if phage resistance could limit long-term use of phages as biocontrol agents. Growth kinetics and adsorption assays indicated that phage resistance in strains 309-PR4 and 502-PR5 was mediated, at least in part, by prevention of phage adsorption. Phage resistance in strain 309-PR1 was the result of limited phage proliferation. Phage resistance was stably maintained in vitro throughout a 4-d subculture period in the absence of phage. No

  17. Meat Science and Muscle Biology Symposium: Development of bacteriophage treatments to reduce Escherichia coli O157:H7 contamination of beef products and produce.

    PubMed

    Hong, Y; Pan, Y; Ebner, P D

    2014-04-01

    Escherichia coli O157:H7 remains a foodborne pathogen of concern with infections associated with products ranging from ground beef to produce to processed foods. We previously demonstrated that phage-based technologies could reduce foodborne pathogen colonization in live animals. Here, we examined if a 3-phage cocktail could reduce E. coli O157:H7 in experimentally contaminated ground beef, spinach, and cheese. The 3 phages were chosen from our E. coli O157:H7 phage library based on their distinct origins of isolation, lytic ranges, and rapid growth (40- to 50-min life cycle). Two phages belonged to the Myoviridae family and the other phage belonged to the Siphoviridae family. The phage cocktail was added to ground beef, spinach leaves, and cheese slices contaminated with E. coli O157:H7 (10(7) cfu) at a multiplicity of infection of 1. Phage treatment reduced (P < 0.05) the concentrations of E. coli O157:H7 by 1.97 log10 cfu/mL in ground beef when stored at room temperature (24 °C) for 24 h, 0.48 log10 cfu/mL at refrigeration (4 °C), and 0.56 log10 cfu/mL in undercooked condition (internal temperature of 46 °C). Likewise, phage treatment reduced (P < 0.05) E. coli O157:H7 by 3.28, 2.88, and 2.77 log10 cfu/mL in spinach when stored at room temperature for 24, 48, and 72 h, respectively. Phage treatment, however, did not reduce E. coli O157:H7 concentrations in contaminated cheese. Additionally, 3 phage-resistant E. coli O157:H7 strains (309-PR [phage resistant] 1, 309-PR4, and 502-PR5) were isolated and characterized to test if phage resistance could limit long-term use of phages as biocontrol agents. Growth kinetics and adsorption assays indicated that phage resistance in strains 309-PR4 and 502-PR5 was mediated, at least in part, by prevention of phage adsorption. Phage resistance in strain 309-PR1 was the result of limited phage proliferation. Phage resistance was stably maintained in vitro throughout a 4-d subculture period in the absence of phage. No

  18. Variable colonization of chickens perorally inoculated with Escherichia coli O157:H7 and subsequent contamination of eggs.

    PubMed Central

    Schoeni, J L; Doyle, M P

    1994-01-01

    Challenging 1-day-old White Leghorn chicks perorally with 2.6 x 10(1) to 2.6 x 10(5) Escherichia coli O157:H7 bacteria per chick resulted in cecal colonization at all levels. Two of six chicks inoculated with only 2.6 x 10(1) E. coli O157:H7 bacteria carried 10(3) to 10(4) E. coli O157:H7 bacteria per g of cecal tissue when sacrificed 3 months postinoculation. E. coli O157:H7 colonization persisted at least 10 to 11 months when chicks were administered 10(8) E. coli O157:H7 bacteria. Eggs from five hens that were fecal shedders of E. coli O157:H7 until the termination of the study (10 to 11 months) were assayed for E. coli O157:H7. The organism was isolated from the shells of 14 of 101 (13.9%) eggs but not from the yolks and whites. Considering that chicks can be readily colonized by small populations of E. coli O157:H7 and continue to be long-term shedders, it is possible that chickens and hen eggs can serve as vehicles of this human pathogen. PMID:8085831

  19. Highly Virulent Non-O157 Enterohemorrhagic Escherichia coli (EHEC) Serotypes Reflect Similar Phylogenetic Lineages, Providing New Insights into the Evolution of EHEC

    PubMed Central

    Eichhorn, Inga; Heidemanns, Katrin; Semmler, Torsten; Kinnemann, Bianca; Mellmann, Alexander; Harmsen, Dag; Anjum, Muna F.; Schmidt, Herbert; Fruth, Angelika; Valentin-Weigand, Peter; Heesemann, Jürgen; Suerbaum, Sebastian; Karch, Helge

    2015-01-01

    Enterohemorrhagic Escherichia coli (EHEC) is the causative agent of bloody diarrhea and extraintestinal sequelae in humans, most importantly hemolytic-uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). Besides the bacteriophage-encoded Shiga toxin gene (stx), EHEC harbors the locus of enterocyte effacement (LEE), which confers the ability to cause attaching and effacing lesions. Currently, the vast majority of EHEC infections are caused by strains belonging to five O serogroups (the “big five”), which, in addition to O157, the most important, comprise O26, O103, O111, and O145. We hypothesize that these four non-O157 EHEC serotypes differ in their phylogenies. To test this hypothesis, we used multilocus sequence typing (MLST) to analyze a large collection of 250 isolates of these four O serogroups, which were isolated from diseased as well as healthy humans and cattle between 1952 and 2009. The majority of the EHEC isolates of O serogroups O26 and O111 clustered into one sequence type complex, STC29. Isolates of O103 clustered mainly in STC20, and most isolates of O145 were found within STC32. In addition to these EHEC strains, STC29 also included stx-negative E. coli strains, termed atypical enteropathogenic E. coli (aEPEC), yet another intestinal pathogenic E. coli group. The finding that aEPEC and EHEC isolates of non-O157 O serogroups share the same phylogeny suggests an ongoing microevolutionary scenario in which the phage-encoded Shiga toxin gene stx is transferred between aEPEC and EHEC. As a consequence, aEPEC strains of STC29 can be regarded as post- or pre-EHEC isolates. Therefore, STC29 incorporates phylogenetic information useful for unraveling the evolution of EHEC. PMID:26231647

  20. Highly Virulent Non-O157 Enterohemorrhagic Escherichia coli (EHEC) Serotypes Reflect Similar Phylogenetic Lineages, Providing New Insights into the Evolution of EHEC.

    PubMed

    Eichhorn, Inga; Heidemanns, Katrin; Semmler, Torsten; Kinnemann, Bianca; Mellmann, Alexander; Harmsen, Dag; Anjum, Muna F; Schmidt, Herbert; Fruth, Angelika; Valentin-Weigand, Peter; Heesemann, Jürgen; Suerbaum, Sebastian; Karch, Helge; Wieler, Lothar H

    2015-10-01

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

  1. Efficacy of integrated treatment of UV light and low dose gamma irradiation on Escherichia coli O157:H7 and Salmonella enterica on grape tomatoes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Efficacy of integrated treatment of UVC and low dose Gamma irradiation to inactivate mixed Strains of Escherichia coli O157:H7 and Salmonella enterica inoculated on whole Grape tomatoes was evaluated. A mixed bacterial cocktail composed of a three strain mixture of E. coli O157:H7 (C9490, E02128 an...

  2. Escherichia coli O157:H7.

    PubMed

    Mead, P S; Griffin, P M

    1998-10-10

    Escherichia coli O157 was first identified as a human pathogen in 1982. One of several Shiga toxin-producing serotypes known to cause human illness, the organism probably evolved through horizontal acquisition of genes for Shiga toxins and other virulence factors. E. coli O157 is found regularly in the faeces of healthy cattle, and is transmitted to humans through contaminated food, water, and direct contact with infected people or animals. Human infection is associated with a wide range of clinical illness, including asymptomatic shedding, non-bloody diarrhoea, haemorrhagic colitis, haemolytic uraemic syndrome, and death. Since laboratory practices vary, physicians need to know whether laboratories in their area routinely test for E. coli O157 in stool specimens. Treatment with antimicrobial agents remains controversial: some studies suggest that treatment may precipitate haemolytic uraemic syndrome, and other studies suggest no effect or even a protective effect. Physicians can help to prevent E. coli O157 infections by counselling patients about the hazards of consuming undercooked ground meat or unpasteurised milk products and juices, and about the importance of handwashing to prevent the spread of diarrhoeal illness, and by informing public-health authorities when they see unusual numbers of cases of bloody diarrhoea or haemolytic uraemic syndrome.

  3. Survival of O157:H7 and non-O157 serogroups of Escherichia coli in bovine rumen fluid and bile salts.

    PubMed

    Free, Angela L; Duoss, Heather A; Bergeron, Leeanne V; Shields-Menard, Sara A; Ward, Emily; Callaway, Todd R; Carroll, Jeffery A; Schmidt, Ty B; Donaldson, Janet R

    2012-11-01

    While Shiga toxin-producing Escherichia coli (STEC) reside asymptomatically within ruminants, particularly cattle, these strains pose a serious health risk to humans. Research related to STEC has historically focused upon O157:H7. However, with an increase in foodborne outbreaks of non-O157 origin and recent changes in testing for non-O157 by the U.S. Department of Agriculture Food Safety and Inspection Service (USDA-FSIS), there is now a critical need to understand the biological activity of non-O157 serogroups. The focus of this study was to determine whether variations exist in the ability of different serotypes of STEC to survive within bovine rumen fluid medium and bile salts. The results of this study demonstrated through viable plate count analysis that the five serotypes tested (O157:H7, O111:H8, O103:K.:H8, O145:H28, and O26:H11) were capable of growing in rumen fluid medium. However, the concentrations of the serotypes O103:K.:H8 and O26:H11 after 24 h were significantly less (p < 0.05) than that observed for the other serotypes tested. A significant decrease (p = 0.03) in the survival of O103:K.:H8 in 50 mg/mL of bovine bile salts in comparison to the other STEC strains tested was also observed. Collectively, these data suggest that non-O157 serogroups of E. coli respond differently to the environment of the bovine gastrointestinal tract. Further research is needed to elucidate how these differential physiological variations correlate with alterations in colonization success within ruminants and how they may impact human illnesses.

  4. Escherichia coli O157:H7 genetic diversity in bovine fecal samples.

    PubMed

    Jacob, M E; Almes, K M; Shi, X; Sargeant, J M; Nagaraja, T G

    2011-07-01

    Escherichia coli O157:H7 causes foodborne illness in humans; cattle are considered a primary reservoir for the organism, and transmission is often through contaminated food products or water. The objective of this study was to determine the genetic diversity of E. coli O157:H7 within a single individual bovine fecal sample based on pulsed-field gel electrophoresis (PFGE) typing. Fecal samples (n=601) were collected from dairy and beef cattle at three separate facilities, and E. coli O157:H7 was isolated by enrichment, immunomagnetic separation, and plating on selective medium. The prevalence of E. coli O157:H7 was 46 (7.7%) of 601. From each positive fecal sample, up to 10 putative colonies were tested, and isolates from samples with at least seven positive colonies were subtyped using PFGE and tested for six major virulence genes by multiplex PCR. A total of 254 E. coli O157:H7 isolates from 27 samples met these criteria and were included in PFGE analysis. Fifteen PFGE subtypes (<100% Dice similarity) were detected among the 254 isolates, and there were no common subtypes between the three locations. Seven (26%) of 27 fecal samples had E. coli O157:H7 isolates with different PFGE subtypes (mean=2.1) within the same sample. The virulence gene profiles of different isolates from the same sample were always identical, regardless of the number of PFGE types. The results of this study suggest that determining the PFGE pattern of a single isolate from a bovine sample may not be sufficient when comparing isolates from feces, hides, or carcasses, because multiple PFGE subtypes are present. PMID:21740722

  5. Prolonged and mixed non-O157 Escherichia coli infection in an Australian household.

    PubMed

    Staples, M; Graham, R M A; Doyle, C J; Smith, H V; Jennison, A V

    2012-05-01

    An Australian family was identified through a Public Health follow up on a Shiga-toxigenic Escherichia coli (STEC) positive bloody diarrhoea case, with three of the four family members experiencing either symptomatic or asymptomatic STEC shedding. Bacterial isolates were submitted to stx sequence sub-typing, multi-locus variable number tandem repeat analysis (MLVA), multi-locus sequence typing (MLST) and binary typing. The analysis revealed that there were multiple strains of STEC being shed by the family members, with similar virulence gene profiles and the same serogroup but differing in their MLVA and MLST profiles. This study illustrates the potentially complicated nature of non-O157 STEC infections and the importance of molecular epidemiology in understanding disease clusters.

  6. Is Shiga Toxin-Negative Escherichia coli O157:H7 Enteropathogenic or Enterohemorrhagic Escherichia coli? Comprehensive Molecular Analysis Using Whole-Genome Sequencing

    PubMed Central

    Ferdous, Mithila; Zhou, Kai; Mellmann, Alexander; Morabito, Stefano; Croughs, Peter D.; de Boer, Richard F.; Kooistra-Smid, Anna M. D.; Friedrich, Alexander W.

    2015-01-01

    The ability of Escherichia coli O157:H7 to induce cellular damage leading to disease in humans is related to numerous virulence factors, most notably the stx gene, encoding Shiga toxin (Stx) and carried by a bacteriophage. Loss of the Stx-encoding bacteriophage may occur during infection or culturing of the strain. Here, we collected stx-positive and stx-negative variants of E. coli O157:H7/NM (nonmotile) isolates from patients with gastrointestinal complaints. Isolates were characterized by whole-genome sequencing (WGS), and their virulence properties and phylogenetic relationship were determined. Because of the presence of the eae gene but lack of the bfpA gene, the stx-negative isolates were considered atypical enteropathogenic E. coli (aEPEC). However, they had phenotypic characteristics similar to those of the Shiga toxin-producing E. coli (STEC) isolates and belonged to the same sequence type, ST11. Furthermore, EPEC and STEC isolates shared similar virulence genes, the locus of enterocyte effacement region, and plasmids. Core genome phylogenetic analysis using a gene-by-gene typing approach showed that the sorbitol-fermenting (SF) stx-negative isolates clustered together with an SF STEC isolate and that one non-sorbitol-fermenting (NSF) stx-negative isolate clustered together with NSF STEC isolates. Therefore, these stx-negative isolates were thought either to have lost the Stx phage or to be a progenitor of STEC O157:H7/NM. As detection of STEC infections is often based solely on the identification of the presence of stx genes, these may be misdiagnosed in routine laboratories. Therefore, an improved diagnostic approach is required to manage identification, strategies for treatment, and prevention of transmission of these potentially pathogenic strains. PMID:26311863

  7. Shiga toxin-producing Escherichia coli O157:H7 from extensive cattle of the fighting bulls breed.

    PubMed

    Sánchez, S; Martínez, R; García, A; Blanco, J; Echeita, A; Hermoso de Mendoza, J; Rey, J; Alonso, J M

    2010-04-01

    Shiga toxin-producing Escherichia coli (STEC) O157:H7 represents a major public health concern worldwide, with cattle recognized as their main natural reservoir. The aim of this work was to determine the prevalence and the pheno-genotypic characteristics of STEC O157:H7 in a herd with 268 cattle of the fighting bulls breed (De Lidia breed) managed under extensive conditions in the South-West of Spain. Rectal-anal swabs of all animals were collected and examined for STEC O157:H7 by performing an immunomagnetic concentration and separation procedure combined with PCR, and the resulting isolates were characterized by both phenotypic and genotypic methods. Overall, STEC O157:H7 was isolated from seven animals (2.6%) in the herd. The PCR procedure indicated that all seven isolates displayed stx(2), eae-gamma1, ehxA, O157 rfbE, and fliCh7 genes. They belonged to phage types 4 (one isolate) and 42 (two isolates), and four isolates reacted with typing phages but did not conform to a recognized pattern. Among the seven isolates there were five indistinguishable PFGE patterns and other two which differed only in < or =2 restriction fragments, supporting the existence of horizontal transmission among animals in the herd. The present study demonstrates that cattle managed under extensive conditions in Spain can excrete STEC O157:H7 with their faeces. To our knowledge this is the first isolation of this pathogen from De Lidia cattle.

  8. Effect of Bifidobacterium thermacidophilum probiotic feeding on enterohemorrhagic Escherichia coli O157:H7 infection in BALB/c mice.

    PubMed

    Gagnon, Mélanie; Kheadr, Ehab E; Dabour, Nassra; Richard, Denis; Fliss, Ismaïl

    2006-08-15

    The effectiveness of Bifidobacterium thermacidophilum RBL 71 as a probiotic against enterohemorrhagic Escherichia coli O157:H7 infection was studied using a murine model. BALB/c mice were fed the probiotic for 7 days before or after single challenge with E. coli O157:H7. Fecal B. thermacidophilum RBL 71 and E. coli O157:H7 counts obtained by selective culturing methods were assessed for 1 week before and after infection while feed intake, body weight and composition were monitored during 1 week after infection. Histology of gut tissue (jejunum, ileum and colon) and production of fecal IgA antibodies and serum IgG+IgM antibodies to E. coli O157:H7 were analyzed until 1 and 2 weeks post-infection, respectively. The pathogenicity of E. coli O157:H7, marked by body weight loss and intestinal histopathological changes in the infected group, was significantly reduced in the B. thermacidophilum-treated group. Feeding B. thermacidophilum RBL 71 for 7 days before infection resulted in greater post-challenge feed intake and weight gain and lower fecal levels of E. coli O157:H7. Post-infection levels of anti-E. coli O157:H7-specific IgA in feces and IgG+IgM in serum were higher in mice fed bifidobacteria. Intestinal injuries were also attenuated and reaction of the lymphoid component in the mucosa of the ileum was greater in the bifidobacteria-fed group. A lesser degree of protection against E. coli O157:H7 infection was observed when bifidobacteria were given during the 7 days after E. coli O157:H7 infection. These results demonstrate that feeding the probiotic B. thermacidophilum RBL 71 to mice can reduce the severity of E. coli O157:H7 infection, and suggest that this strain represents a good candidate for the prevention of enteric infections in human.

  9. Surrogate selection for Escherichia coli O157:H7 based on cryotolerance and attachment to romaine lettuce.

    PubMed

    Kim, Jin Kyung; Harrison, Mark A

    2009-07-01

    Using nonpathogenic surrogates in place of pathogens when evaluating commercial food processing operations offers safety advantages, but their usefulness may be limited if they do not behave in the same manner in challenge situations. Nonpathogenic Escherichia coli strains were compared with E. coli O157:H7 based on cryotolerance, cell surface characteristics (hydrophobicity, zeta potential, and morphology), and attachment to lettuce. Populations for all strains were reduced less than 1 log CFU/ml over 7 days of storage at -18 degrees C. After 1 day of storage, the survival rate for E. coli ATCC 25922 was 44.3%, similar to that of E. coli O157:H7 (49%). No capsule was produced by any of the strains. E. coli O157:H7 expressed curli at both 20 and 37 degrees C, whereas E. coli ATCC 25922 expressed curli only when grown at 20 degrees C. Hydrophobicity of E. coli ATCC 25922 was 53.5%, similar to that of E. coli O157:H7 (56.2%). The zeta potentials of nonpathogenic E. coli and E. coli O157:H7 cells were -4.95 to -10.92 mV. The zeta potential of E. coli ATCC 25922 was not significantly different (P > 0.05) from that of E. coli O157:H7 at 37 degrees C and was the closest value to that of E. coli O157:H7 at 20 degrees C. E. coli ATCC 25922 exhibited the greatest attachment to lettuce among the surrogates and was not significantly different from E. coli O157:H7 (P > 0.05). Based on cryotolerance and cell surface characteristics, E. coli ATCC 25922 is a useful surrogate for E. coli O157:H7 for studies involving attachment to fresh produce.

  10. Viability of multi-strain mixtures of Listeria monocytogenes, Salmonella typhimurium, or Escherichia coli O157:H7 inoculated into the batter or onto the surface of a soudjouk-style fermented semi-dry sausage.

    PubMed

    Porto-Fett, A C S; Hwang, C-A; Call, J E; Juneja, V K; Ingham, S C; Ingham, B H; Luchansky, J B

    2008-09-01

    The fate of Listeria monocytogenes, Salmonella typhimurium, or Escherichia coli O157:H7 were separately monitored both in and on soudjouk. Fermentation and drying alone reduced numbers of L. monocytogenes by 0.07 and 0.74 log(10)CFU/g for sausages fermented to pH 5.3 and 4.8, respectively, whereas numbers of S. typhimurium and E. coli O157:H7 were reduced by 1.52 and 3.51 log(10)CFU/g and 0.03 and 1.11 log(10)CFU/g, respectively. When sausages fermented to pH 5.3 or 4.8 were stored at 4, 10, or 21 degrees C, numbers of L. monocytogenes, S. typhimurium, and E. coli O157:H7 decreased by an additional 0.08-1.80, 0.88-3.74, and 0.68-3.17 log(10)CFU/g, respectively, within 30 days. Storage for 90 days of commercially manufactured soudjouk that was sliced and then surface inoculated with L. monocytogenes, S. typhimurium, and E. coli O157:H7 generated average D-values of ca. 10.1, 7.6, and 5.9 days at 4 degrees C; 6.4, 4.3, and 2.9 days at 10 degrees C; 1.4, 0.9, and 1.6 days at 21 degrees C; and 0.9, 1.4, and 0.25 days at 30 degrees C. Overall, fermentation to pH 4.8 and storage at 21 degrees C was the most effective treatment for reducing numbers of L. monocytogenes (2.54 log(10)CFU/g reduction), S. typhimurium (> or =5.23 log(10)CFU/g reduction), and E. coli O157:H7 (3.48 log(10)CFU/g reduction). In summary, soudjouk-style sausage does not provide a favorable environment for outgrowth/survival of these three pathogens.

  11. Sequence variability of P2-like prophage genomes carrying the cytolethal distending toxin V operon in Escherichia coli O157.

    PubMed

    Sváb, Domonkos; Horváth, Balázs; Maróti, Gergely; Dobrindt, Ulrich; Tóth, István

    2013-08-01

    Cytolethal distending toxins (CDT) are potent cytotoxins of several Gram-negative pathogenic bacteria, including Escherichia coli, in which five types (CDT-I to CDT-V) have been identified so far. CDT-V is frequently associated with Shiga-toxigenic E. coli (STEC), enterohemorrhagic E. coli (EHEC) O157 strains, and strains not fitting any established pathotypes. In this study, we were the first to sequence and annotate a 31.2-kb-long, noninducible P2-like prophage carrying the cdt-V operon from an stx- and eae-negative E. coli O157:H43 strain of bovine origin. The cdt-V operon is integrated in the place of the tin and old phage immunity genes (termed the TO region) of the prophage, and the prophage itself is integrated into the bacterial chromosome between the housekeeping genes cpxP and fieF. The presence of P2-like genes (n = 20) was investigated in a further five CDT-V-positive bovine E. coli O157 strains of various serotypes, three EHEC O157:NM strains, four strains expressing other variants of CDT, and eight CDT-negative strains. All but one CDT-V-positive atypical O157 strain uniformly carried all the investigated genomic regions of P2-like phages, while the EHEC O157 strains missed three regions and the CDT-V-negative strains carried only a few P2-like sequences. Our results suggest that P2-like phages play a role in the dissemination of cdt-V between E. coli O157 strains and that after integration into the bacterial chromosome, they adapted to the respective hosts and became temperate.

  12. Sequence Variability of P2-Like Prophage Genomes Carrying the Cytolethal Distending Toxin V Operon in Escherichia coli O157

    PubMed Central

    Sváb, Domonkos; Horváth, Balázs; Maróti, Gergely; Dobrindt, Ulrich

    2013-01-01

    Cytolethal distending toxins (CDT) are potent cytotoxins of several Gram-negative pathogenic bacteria, including Escherichia coli, in which five types (CDT-I to CDT-V) have been identified so far. CDT-V is frequently associated with Shiga-toxigenic E. coli (STEC), enterohemorrhagic E. coli (EHEC) O157 strains, and strains not fitting any established pathotypes. In this study, we were the first to sequence and annotate a 31.2-kb-long, noninducible P2-like prophage carrying the cdt-V operon from an stx- and eae-negative E. coli O157:H43 strain of bovine origin. The cdt-V operon is integrated in the place of the tin and old phage immunity genes (termed the TO region) of the prophage, and the prophage itself is integrated into the bacterial chromosome between the housekeeping genes cpxP and fieF. The presence of P2-like genes (n = 20) was investigated in a further five CDT-V-positive bovine E. coli O157 strains of various serotypes, three EHEC O157:NM strains, four strains expressing other variants of CDT, and eight CDT-negative strains. All but one CDT-V-positive atypical O157 strain uniformly carried all the investigated genomic regions of P2-like phages, while the EHEC O157 strains missed three regions and the CDT-V-negative strains carried only a few P2-like sequences. Our results suggest that P2-like phages play a role in the dissemination of cdt-V between E. coli O157 strains and that after integration into the bacterial chromosome, they adapted to the respective hosts and became temperate. PMID:23770900

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

    PubMed

    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.

  14. Environmental sources and transmission of Escherichia coli O157 in feedlot cattle.

    PubMed Central

    Van Donkersgoed, J; Berg, J; Potter, A; Hancock, D; Besser, T; Rice, D; LeJeune, J; Klashinsky, S

    2001-01-01

    A study was conducted in 2 feedlots in southern Alberta to identify environmental sources and management factors associated with the prevalence and transmission of Escherichia coli O157:H7. Escherichia coli O157:H7 was isolated in preslaughter pens of cattle from feces (0.8%), feedbunks (1.7%), water troughs (12%), and incoming water supplies (4.5%), but not from fresh total mixed rations. Fresh total mixed rations did not support the growth of E. coli O157:H7 and E. coli from bovine feces following experimental inoculation. Within a feedlot, the feces, water troughs, and feedbunks shared a few indistinguishable subtypes of E. coli O157:H7. A few subtypes were repeatedly isolated in the same feedlot, and the 2 feedlots shared a few indistinguishable subtypes. The prevalence of E. coli O157:H7 in water troughs of preslaughter cattle in 1 feedlot was associated with season, maximum climatic temperatures the week before sampling; total precipitation the week before sampling, and coliform and E. coli counts in the water trough. PMID:11565371

  15. Effect of Neem (Azadirachta indica) on the Survival of Escherichia coli O157:H7 in Dairy Manure.

    PubMed

    Ravva, Subbarao V; Korn, Anna

    2015-07-01

    Escherichia coli O157:H7 (EcO157) shed in cattle manure can survive for extended periods of time and intervention strategies to control this pathogen at the source are critical as produce crops are often grown in proximity to animal raising operations. This study evaluated whether neem (Azadirachta indica), known for its antimicrobial and insecticidal properties, can be used to amend manure to control EcO157. The influence of neem materials (leaf, bark, and oil) on the survival of an apple juice outbreak strain of EcO157 in dairy manure was monitored. Neem leaf and bark supplements eliminated the pathogen in less than 10 d with a D-value (days for 90% elimination) of 1.3 d. In contrast, nearly 4 log CFU EcO157/g remained after 10 d in neem-free manure control. The ethyl acetate extractable fraction of neem leaves was inhibitory to the growth of EcO157 in LB broth. Azadirachtin, a neem product with insect antifeedant properties, failed to inhibit EcO157. Application of inexpensive neem supplements to control pathogens in manure and possibly in produce fields may be an option for controlling the transfer of foodborne pathogens from farm to fork. PMID:26184255

  16. Effect of Neem (Azadirachta indica) on the Survival of Escherichia coli O157:H7 in Dairy Manure.

    PubMed

    Ravva, Subbarao V; Korn, Anna

    2015-07-10

    Escherichia coli O157:H7 (EcO157) shed in cattle manure can survive for extended periods of time and intervention strategies to control this pathogen at the source are critical as produce crops are often grown in proximity to animal raising operations. This study evaluated whether neem (Azadirachta indica), known for its antimicrobial and insecticidal properties, can be used to amend manure to control EcO157. The influence of neem materials (leaf, bark, and oil) on the survival of an apple juice outbreak strain of EcO157 in dairy manure was monitored. Neem leaf and bark supplements eliminated the pathogen in less than 10 d with a D-value (days for 90% elimination) of 1.3 d. In contrast, nearly 4 log CFU EcO157/g remained after 10 d in neem-free manure control. The ethyl acetate extractable fraction of neem leaves was inhibitory to the growth of EcO157 in LB broth. Azadirachtin, a neem product with insect antifeedant properties, failed to inhibit EcO157. Application of inexpensive neem supplements to control pathogens in manure and possibly in produce fields may be an option for controlling the transfer of foodborne pathogens from farm to fork.

  17. Inhibiting the Growth of Escherichia coli O157:H7 in Beef, Pork, and Chicken Meat using a Bacteriophage

    PubMed Central

    Seo, Jina; Seo, Dong Joo; Oh, Hyejin; Jeon, Su Been; Oh, Mi-Hwa; Choi, Changsun

    2016-01-01

    This study aimed to inhibit Escherichia coli (E. coli) O157:H7 artificially contaminated in fresh meat using bacteriophage. Among 14 bacteriophages, the highly lytic bacteriophage BPECO19 strain was selected to inhibit E. coli O157:H7 in artificially contaminated meat samples. Bacteriophage BPECO19 significantly reduced E. coli O157:H7 bacterial load in vitro in a multiplicity of infection (MOI)-dependent manner. E. coli O157:H7 was completely inhibited only in 10 min in vitro by the treatment of 10,000 MOI BPECO19. The treatment of BPECO19 at 100,000 MOI completely reduced 5 Log CFU/cm2 E. coli O157:H7 bacterial load in beef and pork at 4 and 8h, respectively. In chicken meat, a 4.65 log reduction of E. coli O157:H7 was observed at 4 h by 100,000 MOI. The treatment of single bacteriophage BPECO19 was an effective method to control E. coli O157:H7 in meat samples. PMID:27194926

  18. Inhibiting the Growth of Escherichia coli O157:H7 in Beef, Pork, and Chicken Meat using a Bacteriophage.

    PubMed

    Seo, Jina; Seo, Dong Joo; Oh, Hyejin; Jeon, Su Been; Oh, Mi-Hwa; Choi, Changsun

    2016-01-01

    This study aimed to inhibit Escherichia coli (E. coli) O157:H7 artificially contaminated in fresh meat using bacteriophage. Among 14 bacteriophages, the highly lytic bacteriophage BPECO19 strain was selected to inhibit E. coli O157:H7 in artificially contaminated meat samples. Bacteriophage BPECO19 significantly reduced E. coli O157:H7 bacterial load in vitro in a multiplicity of infection (MOI)-dependent manner. E. coli O157:H7 was completely inhibited only in 10 min in vitro by the treatment of 10,000 MOI BPECO19. The treatment of BPECO19 at 100,000 MOI completely reduced 5 Log CFU/cm(2) E. coli O157:H7 bacterial load in beef and pork at 4 and 8h, respectively. In chicken meat, a 4.65 log reduction of E. coli O157:H7 was observed at 4 h by 100,000 MOI. The treatment of single bacteriophage BPECO19 was an effective method to control E. coli O157:H7 in meat samples.

  19. Inhibiting the Growth of Escherichia coli O157:H7 in Beef, Pork, and Chicken Meat using a Bacteriophage.

    PubMed

    Seo, Jina; Seo, Dong Joo; Oh, Hyejin; Jeon, Su Been; Oh, Mi-Hwa; Choi, Changsun

    2016-01-01

    This study aimed to inhibit Escherichia coli (E. coli) O157:H7 artificially contaminated in fresh meat using bacteriophage. Among 14 bacteriophages, the highly lytic bacteriophage BPECO19 strain was selected to inhibit E. coli O157:H7 in artificially contaminated meat samples. Bacteriophage BPECO19 significantly reduced E. coli O157:H7 bacterial load in vitro in a multiplicity of infection (MOI)-dependent manner. E. coli O157:H7 was completely inhibited only in 10 min in vitro by the treatment of 10,000 MOI BPECO19. The treatment of BPECO19 at 100,000 MOI completely reduced 5 Log CFU/cm(2) E. coli O157:H7 bacterial load in beef and pork at 4 and 8h, respectively. In chicken meat, a 4.65 log reduction of E. coli O157:H7 was observed at 4 h by 100,000 MOI. The treatment of single bacteriophage BPECO19 was an effective method to control E. coli O157:H7 in meat samples. PMID:27194926

  20. Effect of Neem (Azadirachta indica) on the Survival of Escherichia coli O157:H7 in Dairy Manure

    PubMed Central

    Ravva, Subbarao V.; Korn, Anna

    2015-01-01

    Escherichia coli O157:H7 (EcO157) shed in cattle manure can survive for extended periods of time and intervention strategies to control this pathogen at the source are critical as produce crops are often grown in proximity to animal raising operations. This study evaluated whether neem (Azadirachta indica), known for its antimicrobial and insecticidal properties, can be used to amend manure to control EcO157. The influence of neem materials (leaf, bark, and oil) on the survival of an apple juice outbreak strain of EcO157 in dairy manure was monitored. Neem leaf and bark supplements eliminated the pathogen in less than 10 d with a D-value (days for 90% elimination) of 1.3 d. In contrast, nearly 4 log CFU EcO157/g remained after 10 d in neem-free manure control. The ethyl acetate extractable fraction of neem leaves was inhibitory to the growth of EcO157 in LB broth. Azadirachtin, a neem product with insect antifeedant properties, failed to inhibit EcO157. Application of inexpensive neem supplements to control pathogens in manure and possibly in produce fields may be an option for controlling the transfer of foodborne pathogens from farm to fork. PMID:26184255

  1. Inactivation of Escherichia coli O157:H7 by essential oil from Cinnamomum zeylanicum.

    PubMed

    Senhaji, Ouafae; Faid, Mohamed; Kalalou, Ichraq

    2007-04-01

    Escherichia coli O157:H7 is a pathogen strain, which causes hemorrhagic colitis, hemolytic uremic syndrome and thrombotic thrombocytopenic purpura in humans. The control of bacterial cells in foods is an important factor to reduce foodborne diseases due to E. coli O157:H7. Assays to inactivate E. coli O157:H7 were carried out by using the cinnamon oil obtained by steam distillation for 6 hours. When E. coli O157:H7 cells were incubated at 37 degrees C for 2 hours in the presence of 0.025% of the essential oil from cinnamon, a dramatic decrease was observed in the viable counts (from 10(7) to 3.10(4) CFU/mL-1). In the presence of 0.05% of the oil, most of cells were killed after 30 min, suggesting that the antimicrobial activity of essential oil is bactericidal against E. coli. The minimal inhibitory concentration of the essential oil from cinnamon was around 625 ppm against E. coli O157:H7 and E. coli ATCC 25921, around 1250 ppm against E. coli ATCC25922 and around 2500 ppm against E. coli ATCC11105.

  2. A novel approach to investigate the uptake and internalization of Escherichia coli O157:H7 in spinach cultivated in soil and hydroponic media

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Internalization of E. coli O157:H7 into spinach plants through root uptake is a potential route of contamination. A Tn7-based plasmid vector was used to insert the green fluorescent protein (gfp) gene into the attTn7 site in the E. coli chromosome. Three gfp-labeled E. coli inocula, O157:H7 strains ...

  3. Serodiagnosis Using Microagglutination Assay during the Food-Poisoning Outbreak in Japan Caused by Consumption of Raw Beef Contaminated with Enterohemorrhagic Escherichia coli O111 and O157

    PubMed Central

    Isobe, Junko; Shima, Tomoko; Kanatani, Jun-ichi; Kimata, Keiko; Shimizu, Miwako; Kobayashi, Naoto; Tanaka, Tomoko; Iyoda, Sunao; Ohnishi, Makoto; Sata, Tetsutaro

    2014-01-01

    A microagglutination (MA) assay to identify antibodies to Escherichia coli O111 and O157 was conducted in sera collected from 60 patients during a food-poisoning outbreak affecting 181 patients in Japan which was caused by the consumption of contaminated raw beef. Enterohemorrhagic E. coli (EHEC) O111:H8 and/or O157:H7 was isolated from the stools of some of the patients, but the total rate of positivity for antibodies to O111 (45/60, 75.0%) was significantly higher than that for antibodies to O157 (10/60, 16.7%). The MA titers of antibodies to O111 measured in patients with hemolytic-uremic syndrome and bloody diarrhea were higher than those measured in patients with only diarrhea. In patients from whose stool no isolates of E. coli O111 and O157 were obtained, the positive antibody detection rates were 12/19 (63.2%) for O111 and 2/19 (10.5%) for O157, and the MA titers of antibodies to O111 measured were higher than those to O157. Similarly, the MA titers of antibodies to O111 were significantly higher than those to O157, regardless of the other groups, including groups O111, O111 and O157, and O157. These serodiagnosis results suggest that EHEC O111:H8 stx2 played a primary role in the pathogenesis of this outbreak. Furthermore, our findings suggest that the isolates from the patients' stool specimens were not always the major causative pathogen in patients with multiple EHEC infections, because the sera from patients from whose stools only O157 was isolated were positive for antibodies to O111. Measuring antibodies to E. coli O antigen is helpful especially in cases with multiple EHEC infections, even with a non-O157 serotype. PMID:24452161

  4. Biofilm formation and sanitizer resistance of Escherichia coli 0157:H7 strains isolated from "High Event Period" meat contamination

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In the meat industry, a “High Event Period” (HEP) is defined as a time period during which commercial meat plants experience a higher than usual rate of E. coli O157:H7 contamination. Genetic analysis indicated that within a HEP, most of the E. coli O157:H7 strains belong to a singular dominant str...

  5. Reduction of Escherichia coli O157:H7 in cattle drinking-water by trans-cinnamaldehyde.

    PubMed

    Charles, Anu Susan; Baskaran, Sangeetha Ananda; Murcott, Christine; Schreiber, David; Hoagland, Thomas; Venkitanarayanan, Kumar

    2008-12-01

    Cattle serve as a major reservoir of E. coli O157:H7 and excrete the pathogen in feces. Environmental persistence of E. coli O157:H7 plays a vital role in its epidemiology on farms, and cattle water troughs are a demonstrated long-term reservoir of E. coli O157:H7 for animals. The objective of this study was to investigate the potential of low concentrations of trans-cinnamaldehyde for killing E. coli O157:H7 in cattle drinking-water. A five-strain mixture of E. coli O157:H7 was inoculated (at approximately 8.0 log colony-forming units [CFU]/mL) into 100 mL samples of well water containing 0, 0.03, 0.05, 0.07, or 0.1% trans-cinnamaldehyde. Additionally, water samples containing (1% w/v) bovine feces or feed were also included. The samples were incubated at 21 degrees , 8 degrees , or 4 degrees C for 7 days and tested for viable E. coli O157:H7 on days 0, 1, 3, 5, and 7. Triplicate samples of each treatment and control were included and the study was replicated twice. All concentrations of trans-cinnamaldehyde were effective in killing E. coli O157:H7 in water, but the magnitude of killing significantly increased with increase in trans-cinnamaldehyde concentration and storage temperature (p < 0.05). The presence of feed or feces in water decreased the antibacterial effect of trans-cinnamaldehyde on E. coli O157:H7 (p < 0.05). This study indicated that trans-cinnamaldehyde is effective in killing E. coli O157:H7 in cattle drinking-water, but detailed palatability studies on cattle intake of water containing the antimicrobial are needed.

  6. Non-O157 Shiga toxin-producing Escherichia coli: detection and characterization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli strains that produce Shiga toxins, referred to as Shiga toxin-producing E. coli (STEC) or verotoxigenic E. coli (VTEC) are important food-borne pathogens that cause hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). E. coli O157:H7 is a common cause of STEC infection; ho...

  7. E. COLI O157:H7: HOW CAN WE USE MICROBIAL ECOLOGY TO REDUCE IN CATTLE?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    More than 76 million citizens are sickened each year by consuming foods contaminated with pathogenic bacteria. The most important food-borne pathogen in cattle remains E. coli O157:H7 and its closely related enterohemorrhagic E. coli strains (EHEC) cause more than 90,000 illnesses each year in the ...

  8. Assessment of commercial chromogenic solid media for the detection of non-O157 Shiga toxin-producing Escherichia coli (STEC).

    PubMed

    Zelyas, Nathan; Poon, Alan; Patterson-Fortin, Laura; Johnson, Roger P; Lee, Winki; Chui, Linda

    2016-07-01

    Detection of Shiga toxin-producing Escherichia coli (STEC) has evolved significantly since the introduction of sorbitol-MacConkey agar. This study compares four chromogenic media (CHROMagar™ STEC, Rainbow® O157 agar, CHROMagar™ O157, and Colorex® O157) in their identification of non-O157 STEC. When 161 non-O157 STEC were directly inoculated onto each medium, detection rates on CHROMagar™ STEC, Rainbow® O157 agar, CHROMagar™ O157 and Colorex® O157 were 90%, 70%, 3.7% and 6.8%, respectively. Tellurite minimal inhibitory concentrations (MICs) correlated with growth on CHROMagar™ STEC as 20 of 22 isolates with poor or no growth had MICs ≤1μg/mL. Stool spiking experiments revealed that CHROMagar™ STEC had the highest recovery of the six most common non-O157 STEC, ranging from 30% (in mucoid stool) to 98% (in watery stool). When using clinical stool samples, CHROMagar™ STEC had a sensitivity, specificity, positive predictive value, and negative predictive value of 84.6%, 87%, 13.9%, and 99.6%, respectively.

  9. Factors contributing to the emergence of Escherichia coli O157 in Africa.

    PubMed

    Effler, E; Isaäcson, M; Arntzen, L; Heenan, R; Canter, P; Barrett, T; Lee, L; Mambo, C; Levine, W; Zaidi, A; Griffin, P M

    2001-01-01

    In 1992, a large outbreak of bloody diarrhea caused by Escherichia coli O157 infections occurred in southern Africa. In Swaziland, 40,912 physician visits for diarrhea in persons ages >5 years were reported during October through November 1992. This was a sevenfold increase over the same period during 1990-91. The attack rate was 42% among 778 residents we surveyed. Female gender and consuming beef and untreated water were significant risks for illness. E. coli O157:NM was recovered from seven affected foci in Swaziland and South Africa; 27 of 31 patient and environmental isolates had indistinguishable pulsed-field gel electrophoresis patterns. Compared with previous years, a fivefold increase in cattle deaths occurred in October 1992. The first heavy rains fell that same month (36 mm), following 3 months of drought. Drought, carriage of E. coli O157 by cattle, and heavy rains with contamination of surface water appear to be important factors contributing to this outbreak.

  10. Characterization of Unexpected Growth of Escherichia coli O157:H7 by Modeling

    PubMed Central

    Cornu, Marie; Delignette-Muller, Marie Laure; Flandrois, Jean-Pierre

    1999-01-01

    Modeling of batch kinetics in minimal synthetic medium was used to characterize Escherichia coli O157:H7 growth, which appeared to be different from the exponential growth expected in minimal synthetic medium and observed for E. coli K-12. The turbidimetric kinetics of 14 of the 15 O157:H7 strains tested (93%) were nonexponential, whereas 25 of the 36 other E. coli strains tested (70%) exhibited exponential kinetics. Moreover, the anomaly was almost corrected when the minimal medium was supplemented with methionine. These observations were confirmed with two reference strains by using plate count monitoring. In mixed cultures, E. coli K-12 had a positive effect on E. coli O157:H7 and corrected its growth anomaly. This demonstrated that commensalism occurred, as the growth curve for E. coli K-12 was not affected. The interaction could be explained by an exchange of methionine, as the effect of E. coli K-12 on E. coli O157:H7 appeared to be similar to the effect of methionine. PMID:10583983

  11. Curli fimbriae are conditionally required in Escherichia coli O157:H7 for initial attachment and biofilm formation.

    PubMed

    Carter, Michelle Qiu; Louie, Jacqueline W; Feng, Doris; Zhong, Wayne; Brandl, Maria T

    2016-08-01

    Several species of enteric pathogens produce curli fimbriae, which may affect their interaction with surfaces and other microbes in nonhost environments. Here we used two Escherichia coli O157:H7 outbreak strains with distinct genotypes to understand the role of curli in surface attachment and biofilm formation in several systems relevant to fresh produce production and processing. Curli significantly enhanced the initial attachment of E. coli O157:H7 to spinach leaves and stainless steel surfaces by 5-fold. Curli was also required for E. coli O157:H7 biofilm formation on stainless steel and enhanced biofilm production on glass by 19-27 fold in LB no-salt broth. However, this contribution was not observed when cells were grown in sterile spinach lysates. Furthermore, both strains of E. coli O157:H7 produced minimal biofilms on polypropylene in LB no-salt broth but considerable amounts in spinach lysates. Under the latter conditions, curli appeared to slightly increase biofilm production. Importantly, curli played an essential role in the formation of mixed biofilm by E. coli O157:H7 and plant-associated microorganisms in spinach leaf washes, as revealed by confocal microscopy. Little or no E. coli O157:H7 biofilms were detected at 4 °C, supporting the importance of temperature control in postharvest and produce processing environments.

  12. Perspectives on super-shedding of Escherichia coli O157:H7 by cattle.

    PubMed

    Munns, Krysty D; Selinger, L Brent; Stanford, Kim; Guan, Leluo; Callaway, Todd R; McAllister, Tim A

    2015-02-01

    Escherichia coli O157:H7 is a foodborne pathogen that causes illness in humans worldwide. Cattle are the primary reservoir of this bacterium, with the concentration and frequency of E. coli O157:H7 shedding varying greatly among individuals. The term "super-shedder" has been applied to cattle that shed concentrations of E. coli O157:H7 ≥ 10⁴ colony-forming units/g feces. Super-shedders have been reported to have a substantial impact on the prevalence and transmission of E. coli O157:H7 in the environment. The specific factors responsible for super-shedding are unknown, but are presumably mediated by characteristics of the bacterium, animal host, and environment. Super-shedding is sporadic and inconsistent, suggesting that biofilms of E. coli O157:H7 colonizing the intestinal epithelium in cattle are intermittently released into feces. Phenotypic and genotypic differences have been noted in E. coli O157:H7 recovered from super-shedders as compared to low-shedding cattle, including differences in phage type (PT21/28), carbon utilization, degree of clonal relatedness, tir polymorphisms, and differences in the presence of stx2a and stx2c, as well as antiterminator Q gene alleles. There is also some evidence to support that the native fecal microbiome is distinct between super-shedders and low-shedders and that low-shedders have higher levels of lytic phage within feces. Consequently, conditions within the host may determine whether E. coli O157:H7 can proliferate sufficiently for the host to obtain super-shedding status. Targeting super-shedders for mitigation of E. coli O157:H7 has been proposed as a means of reducing the incidence and spread of this pathogen to the environment. If super-shedders could be easily identified, strategies such as bacteriophage therapy, probiotics, vaccination, or dietary inclusion of plant secondary compounds could be specifically targeted at this subpopulation. Evidence that super-shedder isolates share a commonality with isolates

  13. Use of clustered regularly interspaced short palindromic repeat sequence polymorphisms for specific detection of enterohemorrhagic Escherichia coli strains of serotypes O26:H11, O45:H2, O103:H2, O111:H8, O121:H19, O145:H28, and O157:H7 by real-time PCR.

    PubMed

    Delannoy, Sabine; Beutin, Lothar; Fach, Patrick

    2012-12-01

    We explored the genetic diversity of the clustered regularly interspaced short palindromic repeat (CRISPR) regions of enterohemorrhagic Escherichia coli (EHEC) to design simplex real-time PCR assays for each of the seven most important EHEC serotypes worldwide. A panel of 958 E. coli strains investigated for their CRISPR loci by high-throughput real-time PCR showed that CRISPR polymorphisms in E. coli strongly correlated with both O:H serotypes and the presence of EHEC virulence factors (stx and eae genes). The CRISPR sequences chosen for simplex real-time PCR amplification of EHEC strains belonging to the top 7 EHEC serogroups differentiated clearly between EHEC and non-EHEC strains. Specificity estimates for the CRISPR PCR assays varied from 97.5% to 100%. Sensitivity estimates for the assays ranged from 95.7% to 100%. The assays targeting EHEC O145:H28, O103:H2, and O45:H2 displayed 100% sensitivity. The combined usage of two simplex PCR assays targeting different sequences of the O26 CRISPR locus allowed detection of EHEC O26:H11 with 100% sensitivity. By combining two simplex PCR assays targeting different sequences of the EHEC O157 CRISPR locus, EHEC O157:H7 was detected with 99.56% sensitivity. EHEC O111:H8 and EHEC O121:H19 were detected with 95.9% and 95.7% sensitivity, respectively. This study demonstrates that the identification of EHEC serotype-specific CRISPR sequences is more specific than the mere identification of O-antigen gene sequences, as is used in current PCR protocols for detection of EHEC strains.

  14. Whole Genome Sequencing for Genomics-Guided Investigations of Escherichia coli O157:H7 Outbreaks.

    PubMed

    Rusconi, Brigida; Sanjar, Fatemeh; Koenig, Sara S K; Mammel, Mark K; Tarr, Phillip I; Eppinger, Mark

    2016-01-01

    Multi isolate whole genome sequencing (WGS) and typing for outbreak investigations has become a reality in the post-genomics era. We applied this technology to strains from Escherichia coli O157:H7 outbreaks. These include isolates from seven North America outbreaks, as well as multiple isolates from the same patient and from different infected individuals in the same household. Customized high-resolution bioinformatics sequence typing strategies were developed to assess the core genome and mobilome plasticity. Sequence typing was performed using an in-house single nucleotide polymorphism (SNP) discovery and validation pipeline. Discriminatory power becomes of particular importance for the investigation of isolates from outbreaks in which macrogenomic techniques such as pulse-field gel electrophoresis or multiple locus variable number tandem repeat analysis do not differentiate closely related organisms. We also characterized differences in the phage inventory, allowing us to identify plasticity among outbreak strains that is not detectable at the core genome level. Our comprehensive analysis of the mobilome identified multiple plasmids that have not previously been associated with this lineage. Applied phylogenomics approaches provide strong molecular evidence for exceptionally little heterogeneity of strains within outbreaks and demonstrate the value of intra-cluster comparisons, rather than basing the analysis on archetypal reference strains. Next generation sequencing and whole genome typing strategies provide the technological foundation for genomic epidemiology outbreak investigation utilizing its significantly higher sample throughput, cost efficiency, and phylogenetic relatedness accuracy. These phylogenomics approaches have major public health relevance in translating information from the sequence-based survey to support timely and informed countermeasures. Polymorphisms identified in this work offer robust phylogenetic signals that index both short- and

  15. Whole Genome Sequencing for Genomics-Guided Investigations of Escherichia coli O157:H7 Outbreaks

    PubMed Central

    Rusconi, Brigida; Sanjar, Fatemeh; Koenig, Sara S. K.; Mammel, Mark K.; Tarr, Phillip I.; Eppinger, Mark

    2016-01-01

    Multi isolate whole genome sequencing (WGS) and typing for outbreak investigations has become a reality in the post-genomics era. We applied this technology to strains from Escherichia coli O157:H7 outbreaks. These include isolates from seven North America outbreaks, as well as multiple isolates from the same patient and from different infected individuals in the same household. Customized high-resolution bioinformatics sequence typing strategies were developed to assess the core genome and mobilome plasticity. Sequence typing was performed using an in-house single nucleotide polymorphism (SNP) discovery and validation pipeline. Discriminatory power becomes of particular importance for the investigation of isolates from outbreaks in which macrogenomic techniques such as pulse-field gel electrophoresis or multiple locus variable number tandem repeat analysis do not differentiate closely related organisms. We also characterized differences in the phage inventory, allowing us to identify plasticity among outbreak strains that is not detectable at the core genome level. Our comprehensive analysis of the mobilome identified multiple plasmids that have not previously been associated with this lineage. Applied phylogenomics approaches provide strong molecular evidence for exceptionally little heterogeneity of strains within outbreaks and demonstrate the value of intra-cluster comparisons, rather than basing the analysis on archetypal reference strains. Next generation sequencing and whole genome typing strategies provide the technological foundation for genomic epidemiology outbreak investigation utilizing its significantly higher sample throughput, cost efficiency, and phylogenetic relatedness accuracy. These phylogenomics approaches have major public health relevance in translating information from the sequence-based survey to support timely and informed countermeasures. Polymorphisms identified in this work offer robust phylogenetic signals that index both short- and

  16. Survival mechanism of Escherichia coli O157:H7 against combined treatment with acetic acid and sodium chloride.

    PubMed

    Lee, Sun-Young; Kang, Dong-Hyun

    2016-05-01

    The combination of salt and acid is commonly used in the production of many foods, including pickles and fermented foods. However, in our previous studies, the addition of salt significantly reduced the inhibitory effect of acetic acid on Escherichia coli O157:H7 in laboratory media and pickled cucumbers. Therefore, this study was conducted to determine the mechanism by which salt confers resistance against acetic acid in E. coli O157:H7. The addition of high concentrations (up to 9% or 15% [w/v]) of salt increased the resistance of E. coli O157:H7 to acetic acid treatment. Combined treatment with acetic acid and salt showed varying results among different bacterial strains (an antagonistic effect for E. coli O157:H7 and Shigella and a synergistic effect for Listeria monocytogenes and Staphylococcus aureus). The addition of salt increased the cytoplasmic pH of E. coli O157:H7, but decreased the cytoplasmic pH of L. monocytogenes and S. aureus on treatment with acetic acid. Therefore, the addition of salt increases the acid resistance of E. coli O157:H7 possibly by increasing its acid resistance response and consequently preventing the acidification of its cytoplasm by organic acids.

  17. Bifidobacterium spp. influences the production of autoinducer-2 and biofilm formation by Escherichia coli O157:H7.

    PubMed

    Kim, Younghoon; Lee, Jae Won; Kang, Seo-Gu; Oh, Sejong; Griffiths, Mansel W

    2012-10-01

    The effect of Bifidobacterium spp. on the production of quorum-sensing (QS) signals and biofilm formation by enterohemorrhagic Escherichia coli (EHEC) O157:H7 was investigated. In an AI-2 bioassay, cell extracts of Bifidobacterium longum ATCC 15707 resulted in a 98-fold reduction in AI-2 activity in EHEC O157:H7 as well as in the Vibrio harveyi reporter strain, even though they did not inhibit the growth of EHEC O157:H7. In addition, they resulted in a 36% reduction in biofilm formation by the organism. Consistently, the virulence of EHEC O157:H7 was significantly attenuated by the presence of cell extracts of B. longum ATCC 15707 in the Caenorhabditis elegans nematode in vivo model. By a proteome analysis using two dimensional electrophoresis (2-DE), we determined that seven proteins including formation of iron-sulfur protein (NifU), thiol:disulfide interchange protein (DsbA), and flagellar P-ring protein (FlgI) were differentially regulated in the EHEC O157:H7 when supplemented with cell extracts of B. longum ATCC 15707. Taken together, these findings propose a novel function of a dairy adjunct in repressing the virulence of EHEC O157:H7.

  18. The role of heightened surveillance in an outbreak of Escherichia coli O157.H7.

    PubMed Central

    Roberts, C. L.; Mshar, P. A.; Cartter, M. L.; Hadler, J. L.; Sosin, D. M.; Hayes, P. S.; Barrett, T. J.

    1995-01-01

    After instituting laboratory screening for Escherichia coli O157.H7, a Connecticut hospital isolated the organism from four persons in September 1993. As a result, an outbreak of E. coli O157.H7 associated with a country club was detected. The club had served hamburger from the same shipment at two picnics. Attendees of two picnics were interviewed, stool cultures were obtained from symptomatic persons, and the remaining hamburger was cultured. Twenty (22%) of 89 persons who ate hamburger became ill, compared with 1 of 60 who did not eat hamburger (relative risk = 13.5, 95% confidence interval 3.2-56.3). Among persons who ate hamburgers, illness was strongly associated with eating hamburger that was not thoroughly cooked (P < 0.001). All 20 samples from 5 remaining boxes of patties yielded E. coli O157.H7. Isolates from hamburger and case-patients were indistinguishable by pulsed-field gel electrophoresis. Heightened surveillance can rapidly identify outbreaks and may mitigate their impact. However, continued review of food safety issues is necessary if E. coli O157.H7 outbreaks are to be prevented. PMID:8557076

  19. Detecting and genotyping Escherichia coli O157:H7 using multiplexed PCR and nucleic acid microarrays.

    PubMed

    Call, D R; Brockman, F J; Chandler, D P

    2001-07-20

    Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance. As an alternative to conventional technologies, we examined the sensitivity and specificity of nucleic acid microarrays for detecting and genotyping E. coli O157:H7. The array was composed of oligonucleotide probes (25-30 mer) complementary to four virulence loci (intimin, Shiga-like toxins I and II, and hemolysin A). Target DNA was amplified from whole cells or from purified DNA via single or multiplexed polymerase chain reaction (PCR), and PCR products were hybridized to the array without further modification or purification. The array was 32-fold more sensitive than gel electrophoresis and capable of detecting amplification products from < 1 cell equivalent of genomic DNA (1 fg). Immunomagnetic capture, PCR and a microarray were subsequently used to detect 55 CFU ml(-1) (E. coli O157:H7) from chicken rinsate without the aid of pre-enrichment. Four isolates of E. coli O157:H7 and one isolate of O91:H2, for which genotypic data were available, were unambiguously genotyped with this array. Glass-based microarrays are relatively simple to construct and provide a rapid and sensitive means to detect multiplexed PCR products; the system is amenable to automation.

  20. Detecting and Genotyping Escherichia coli O157:H7 using multiplexed PCR and nucleic acid microarrays

    SciTech Connect

    Call, Douglas R.; Brockman, Fred J. ); Chandler, Darrell P.

    2000-12-01

    Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance. As an alternative to conventional technologies, we examined the sensitivity and specificity of nucleic acid microarrays for detecting and genotyping E. coli O157:H7. The array was composed of oligonucleotide probes (25-30 mer) complementary to four virulence loci (intimin, Shiga-like toxins I and II, and hemolysin A). Target DNA was amplified from whole cells or from purified DNA via single or multiplexed polymerase chain reaction (PCR), and PCR products were hybridized to the array without further modification or purification. The array was 32-fold more sensitive than gel electrophoresis and capable of detecting amplification products from < 1 cell equivalent of genomic DNA (1 fg). Immunomagnetic capture, PCR and a microarray were subsequently used to detect 55 CFU ml-1 (E. coli O157:H7) from chicken rinsate without the aid of pre-enrichment. Four isolates of E. coli O157:H7 and one isolate of O91:H2, for which genotypic data were available, were unambiguously genotyped with this array. Glass based microarrays are relatively simple to construct and provide a rapid and sensitive means to detect multiplexed PCR products and the system is amenable to automation.

  1. Detecting and genotyping Escherichia coli O157:H7 using multiplexed PCR and nucleic acid microarrays

    SciTech Connect

    Call, Douglas R.; Brockman, Fred J.; Chandler, Darrell P.

    2001-07-05

    Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance. As an alternative to conventional technologies, we examined the sensitivity and specificity of nucleic acid microarrays for detecting and genotyping E. coli O157:H7. The array was composed of oligonucleotide probes (25-30 mer) complementary to four virulence loci (intimin, Shiga-like toxins I and II, and hemolysin A). Target DNA was amplified from whole cells or from purified DNA via single or multiplexed polymerase chain reaction (PCR), and PCR products were hybridized to the array without further modification or purification. The array was 32-fold more sensitive than gel electrophoresis and capable of detecting amplification products from < 1 cell equivalent of genomic DNA (1 fg). Immunomagnetic capture, PCR and a microarray were subsequently used to detect 55 CFUs ml-1 (E. coli O157:H7) from chicken rinsate without the aid of pre-enrichment. Four isolates of E. coli O157:H7 and one isolate of O91:H2, for which genotypic data were available, were unambiguously genotyped with this array. Glass based microarrays are relatively simple to construct and provide a rapid and sensitive means to detect multiplexed PCR products and the system is amenable to automation.

  2. Evaluation of lactic acid as an initial and secondary subprimal intervention for Escherichia coli O157:H7, non-O157 Shiga toxin-producing E. coli, and a nonpathogenic E. coli surrogate for E. coli O157:H7.

    PubMed

    Pittman, C I; Geornaras, I; Woerner, D R; Nightingale, K K; Sofos, J N; Goodridge, L; Belk, K E

    2012-09-01

    Lactic acid can reduce microbial contamination on beef carcass surfaces when used as a food safety intervention, but effectiveness when applied to the surface of chilled beef subprimal sections is not well documented. Studies characterizing bacterial reduction on subprimals after lactic acid treatment would be useful for validations of hazard analysis critical control point (HACCP) systems. The objective of this study was to validate initial use of lactic acid as a subprimal intervention during beef fabrication followed by a secondary application to vacuum-packaged product that was applied at industry operating parameters. Chilled beef subprimal sections (100 cm(2)) were either left uninoculated or were inoculated with 6 log CFU/cm(2) of a 5-strain mixture of Escherichia coli O157:H7, a 12-strain mixture of non-O157 Shiga toxin-producing E. coli (STEC), or a 5-strain mixture of nonpathogenic (biotype I) E. coli that are considered surrogates for E. coli O157:H7. Uninoculated and inoculated subprimal sections received only an initial or an initial and a second "rework" application of lactic acid in a custombuilt spray cabinet at 1 of 16 application parameters. After the initial spray, total inoculum counts were reduced from 6.0 log CFU/cm(2) to 3.6, 4.4, and 4.4 log CFU/cm(2) for the E. coli surrogates, E. coli O157:H7, and non-O157 STEC inoculation groups, respectively. After the second (rework) application, total inoculum counts were 2.6, 3.2, and 3.6 log CFU/cm(2) for the E. coli surrogates, E. coli O157:H7, and non-O157 STEC inoculation groups, respectively. Both the initial and secondary lactic acid treatments effectively reduced counts of pathogenic and nonpathogenic strains of E. coli and natural microflora on beef subprimals. These data will be useful to the meat industry as part of the HACCP validation process.

  3. Validation of the ANSR(®) E. coli O157:H7 Method for Detection of E. coli O157:H7.

    PubMed

    Viator, Ryan; Alles, Susan; Le, Quynh-Nhi; Hosking, Edan; Meister, Evan; Pinkava, Lisa; Tovar, Eric; Mozola, Mark; Rice, Jennifer

    2016-05-01

    A performance validation of the ANSR(®) for E. coli O157:H7 method was conducted in selected food matrixes. This assay uses selective nicking enzyme amplification technology to amplify target genes. Samples are enriched for 12-24 h and then lysed. The assay is completed within 40 min using real-time detection in a combination incubator/fluorescence detector and software. When 44 distinct strains of Escherichia coli O157:H7 and 6 strains of E. coli O157:NM were tested for inclusivity, all 50 strains produced positive results. In exclusivity testing, 57 strains representing 33 species of closely related Gram-negative bacteria belonging to the Enterobacteriaceae family, including 11 non-H7 O157 strains and shiga toxin-producing E. coli other than O157:H7, were evaluated. All 57 nontarget strains generated negative ANSR assay results. Using 80% lean ground beef and beef trim (approximately 20% fat), ANSR method performance was compared to the U.S. Department of Agriculture, Food Safety and Inspection Service Microbiology Laboratory Guidebook reference culture procedure. ANSR performance with baby spinach and sprout irrigation water was measured against the U.S. Food and Drug Administration Bacteriological Analytical Manual reference method. ANSR method performance was not statistically different to that of the reference methods using two different enrichment options. For ground beef and beef trim, the standard enrichment in modified Tryptone Soya Broth can be analyzed using the ANSR assay with a 1:10 dilution of the enrichment in phosphate-buffered saline and produces equivalent results to the reference method. Additionally, in most matrixes tested (exception is spinach which required 24 h enrichment) the assay offers great efficiency and flexibility over the reference method with a 12-24 h single-step enrichment. Equivalent results were observed at both time points (12 and 24 h) to reference methods. Small changes to the assay parameters minimally affected ANSR

  4. The endophytic lifestyle of Escherichia coli O157:H7: quantification and internal localization in roots.

    PubMed

    Wright, Kathryn M; Chapman, Sean; McGeachy, Kara; Humphris, Sonia; Campbell, Emma; Toth, Ian K; Holden, Nicola J

    2013-04-01

    The foodborne pathogen Escherichia coli O157:H7 is increasingly associated with fresh produce (fruit and vegetables). Bacterial colonization of fresh produce plants can occur to high levels on the external tissue but bacteria have also been detected within plant tissue. However, questions remain about the extent of internalization, its molecular basis, and internal location of the bacteria. We have determined the extent of internalization of E. coli O157:H7 in live spinach and lettuce plants and used high-resolution microscopy to examine colony formation in roots and pathways to internalization. E. coli O157:H7 was found within internal tissue of both produce species. Colonization occurred within the apoplast between plant cells. Furthermore, colonies were detected inside the cell wall of epidermal and cortical cells of spinach and Nicotiana benthamiana roots. Internal colonization of epidermal cells resembled that of the phytopathogen Pectobacterium atrosepticum on potato. In contrast, only sporadic cells of the laboratory strain of E. coli K-12 were found on spinach, with no internal bacteria evident. The data extend previous findings that internal colonization of plants appears to be limited to a specific group of plant-interacting bacteria, including E. coli O157:H7, and demonstrates its ability to invade the cells of living plants. PMID:23506361

  5. Effect of high pressure processing on the survival of Shiga toxin-producing Escherichia coli (Big Six vs. O157:H7) in ground beef.

    PubMed

    Hsu, HsinYun; Sheen, Shiowshuh; Sites, Joseph; Cassidy, Jennifer; Scullen, Butch; Sommers, Christopher

    2015-06-01

    High pressure processing (HPP) is a safe and effective technology for improving food safety. Non-O157:H7 Shiga Toxin-producing Escherichia coli (STEC) have been increasingly implicated in foodborne illness outbreaks and recalls, and the USDA Food Safety Inspection Service (FSIS) has designated them as adulterants in meat (e.g. ground beef). In this study we compared the inactivation of multi-isolate cocktails of E. coli O157:H7 versus the non-O157:H7 STEC "Big Six" (i.e. O26, O45, O103, O111, O121, and O145) in ground beef (83% lean) using HPP at refrigeration temperature (4-7 °C). A >5-log CFU/g inactivation of both the Big Six and O157:H7 cocktails were observed at 450 MPa for 15 min. In general, the Big Six cocktail was found more sensitive to pressure stress (p < 0.05). In contrast, HPP treatment at 250 MPa (30 min) inactivated only 2.3 log of the Big Six versus 1.0 log of O157:H7. HPP treatment at 350 MPa (30 min) inactivated 4.7 log of the Big Six vs. 3.2 log of O157:H7. Multiple-cycle HPP cycles (250 or 350 MPa, three 5 min treatments) did not result in a 5 log reduction of the non-O157:H7 or O157:H7 STEC. Our results indicate that HPP inactivation parameters which are effective for O157:H7 STEC can be used for the non-O157:H7 Big Six isolates in ground beef.

  6. Escherichia coli O157:H7 diarrhoea associated with well water and infected cattle on an Ontario farm.

    PubMed Central

    Jackson, S. G.; Goodbrand, R. B.; Johnson, R. P.; Odorico, V. G.; Alves, D.; Rahn, K.; Wilson, J. B.; Welch, M. K.; Khakhria, R.

    1998-01-01

    A 16-month old female child living on an Ontario dairy farm was taken to hospital suffering from bloody diarrhoea. Escherichia coli O157:H7 was isolated from her stool. Initial tests of well water samples were negative for E. coli by standard methods but culture of selected coliform colonies on sorbitol-MacConkey agar led to isolation of E. coli O157:H7. E. coli O157:H7 was also isolated from 63% of cattle on the farm. The E. coli O157:H7 isolates from the child, the water and the cattle were phage type 14, produced verotoxins 1 and 2, and were highly related on analysis by pulsed field gel electrophoresis. The child did not have known direct contact with the cattle and did not consume unpasteurized milk. Hydrogeological investigation revealed the design and location of the well would allow manure-contaminated surface water to flow into the well. This investigation demonstrates that cattle farm well water is a potential source of E. coli O157:H7 which may not be identified by standard screening for E. coli in water. PMID:9528813

  7. Acid and alcohol tolerance of Escherichia coli O157:H7 in pulque, a typical Mexican beverage.

    PubMed

    Gómez-Aldapa, Carlos A; Díaz-Cruz, Claudio A; Villarruel-López, Angelica; Del Refugio Torres-Vitela, M; Rangel-Vargas, Esmeralda; Castro-Rosas, Javier

    2012-03-01

    Pulque is a traditional Mexican fermented alcoholic beverage produced from the nectar of maguey agave plants. No data exist on the behavior of Escherichia coli O157:H7 in agave nectar and pulque. An initial trial was done of the behavior of E. coli O157:H7 during fermentation of nectar from a single producer, a nectar mixture from different producers and "seed" pulque. A second trial simulating artisanal pulque production was done by contaminating fresh nectar with a cocktail of three E. coli O157:H7 strains, storing at 16 ° and 22 °C for 14 h, adding seed pulque and fermenting until pulque was formed. A third trial used pulque from the second trial stored at 22 °C as seed to ferment fresh nectar at 22 °C for 48 h (fermentation cycle). This procedure was repeated for an additional two fermentation cycles. During incubation at 16 ° or 22 °C in the first trial, the E. coli O157:H7 strains multiplied in both the single producer nectar and nectar mixture, reaching maximum concentration at 12h. E. coli O157:H7 cell concentration then decreased slowly, although it survived at least 72 h in both fermented nectars. E. coli O157:H7 did not multiply in the seed pulque but did survive at least 72 h. In the second trial, the numbers of E. coli O157:H7 increased approximately 1.5 log CFU/ml at 22 °C and 1.2 log CFU/ml at 16 °C after 14 h. After seed pulque was added, E. coli O157:H7 concentration decreased to approximately 2 log CFU/ml, and then remained constant until pulque was produced. In the third trial, the E. coli O157:H7 cells multiplied and survived during at least three nectar fermentation cycles. The results suggest that E. coli O157:H7 can develop acid and alcohol tolerance in pulque, and constitutes a public health risk for pulque consumers.

  8. Insertion/deletion-based approach for the detection of Escherichia coli O157:H7 in freshwater environments.

    PubMed

    Wong, Shirley Y; Paschos, Athanasios; Gupta, Radhey S; Schellhorn, Herb E

    2014-10-01

    Enterohemorrhagic Escherichia coli O157:H7 is responsible for many outbreaks of gastrointestinal illness and hemolytic uremic syndrome worldwide. Monitoring this pathogen in food and water supplies is an important public health issue. Highly conserved genetic markers, which are characteristic for specific strains, can provide direct identification of target pathogens. In this study, we examined a new detection strategy for pathogenic strains of E. coli O157:H7 serotype based on a conserved signature insertion/deletion (CSI) located in the ybiX gene using TaqMan-probe-based quantitative PCR (qPCR). The qPCR assay was linear from 1.0 × 10(2) to 1.0 × 10(7) genome copies and was specific to O157:H7 when tested against a panel of 15 non-O157:H7 E. coli. The assay also maintained detection sensitivity in the presence of competing E. coli K-12, heterologous nontarget DNA spiked in at a 1000-fold and 800-fold excess of target DNA, respectively, demonstrating the assay's ability to detect E. coli O157:H7 in the presence of high levels of background DNA. This study thus validates the use of strain-specific CSIs as a new class of diagnostic marker for pathogen detection. PMID:25166281

  9. Escherichia coli O157:H7, Campylobacter jejuni, and Salmonella Prevalence in cull dairy cows marketed in northeastern Ohio.

    PubMed

    Dodson, Kathryn; LeJeune, Jeffrey

    2005-05-01

    Preharvest management factors are predicted to impact the prevalence of foodborne pathogens in cattle sent to slaughter. We simultaneously examined the prevalence and antibiotic resistance patterns of Campylobacter jejuni, Salmonella, and Escherichia coli O157:H7 isolated from cull dairy cattle at two livestock auctions in northeastern Ohio. Between April and September 2002, a total of 1,026 fecal samples were collected. C. jejuni was isolated from 48 of 686 (7%) fecal samples, Salmonella was isolated from 39 of 585 (6.7%) samples, and E. coli O157:H7 was isolated from 21 of 1,026 (2.1%) samples. Of the 585 samples tested for all three pathogens, at least one pathogen was identified in 86 of 585 (15%) samples. One sample was positive for both E. coli O157:H7 and C. jejuni, and five samples yielded both C. jejuni and Salmonella. Size of herd of origin could be traced for 75 to 85% of samples collected. Salmonella was isolated at higher frequencies from herds larger than 60 cattle than from smaller herds (9.0 versus 3.5%, P = 0.02). In contrast, size of herd of origin did not significantly affect the E. coli O157:H7 and C. jejuni prevalence. Approximately 90% of Salmonella and E. coli O157:H7 isolates were pansensitive to a panel of 16 antibiotics. Thirty-six percent of C. jejuni isolates were resistant to tetracycline. In this study, antibiotic resistance among the foodborne pathogens isolated from cull diary cattle was rare. Although size of dairy herd of origin was positively associated with Salmonella prevalence, herd size was not strongly associated with E. coli O157:H7 and C. jejuni prevalence in market dairy cattle. These results can be used to assess the food safety risks associated with the slaughter of cull dairy cattle.

  10. A multistate outbreak of Escherichia coli O157:H7 infections linked to alfalfa sprouts grown from contaminated seeds.

    PubMed Central

    Breuer, T.; Benkel, D. H.; Shapiro, R. L.; Hall, W. N.; Winnett, M. M.; Linn, M. J.; Neimann, J.; Barrett, T. J.; Dietrich, S.; Downes, F. P.; Toney, D. M.; Pearson, J. L.; Rolka, H.; Slutsker, L.; Griffin, P. M.

    2001-01-01

    A multistate outbreak of Escherichia coli O157:H7 infections occurred in the United States in June and July 1997. Two concurrent outbreaks were investigated through independent case-control studies in Michigan and Virginia and by subtyping isolates with pulsed-field gel electrophoresis (PFGE). Isolates from 85 persons were indistinguishable by PFGE. Alfalfa sprouts were the only exposure associated with E. coli O157:H7 infection in both Michigan and Virginia. Seeds used for sprouting were traced back to one common lot harvested in Idaho. New subtyping tools such as PFGE used in this investigation are essential to link isolated infections to a single outbreak. PMID:11747724

  11. Association of prophage antiterminator Q alleles and susceptibility to food-processing treatments applied to Escherichia coli O157 in laboratory media.

    PubMed

    Malone, Aaron S; Yousef, Ahmed E; LeJeune, Jeffrey T

    2007-11-01

    Resistance of Escherichia coli O157 to inactivation by high-pressure processing, heat, and UV and gamma radiation was associated with the allele of the prophage-encoded antiterminator Q gene present upstream of the Shiga toxin gene stx2. Increased processing may be required to kill certain strains of E. coli O157, and the choice of strains used as surrogate markers for processing efficiency is critical.

  12. Transcriptomic Response of Escherichia coli O157:H7 to Oxidative Stress▿ †

    PubMed Central

    Wang, Siyun; Deng, Kaiping; Zaremba, Sam; Deng, Xiangyu; Lin, Chiahui; Wang, Qian; Tortorello, Mary Lou; Zhang, Wei

    2009-01-01

    Chlorinated water is commonly used in industrial operations to wash and sanitize fresh-cut, minimally processed produce. Here we compared 42 human outbreak strains that represented nine distinct Escherichia coli O157:H7 genetic lineages (or clades) for their relative resistance to chlorine treatment. A quantitative measurement of resistance was made by comparing the extension of the lag phase during growth of each strain under exposure to sublethal concentrations of sodium hypochlorite in Luria-Bertani or brain heart infusion broth. Strains in clade 8 showed significantly (P < 0.05) higher resistance to chlorine than strains from other clades of E. coli O157:H7. To further explore how E. coli O157:H7 responds to oxidative stress at transcriptional levels, we analyzed the global gene expression profiles of two strains, TW14359 (clade 8; associated with the 2006 spinach outbreak) and Sakai (clade 1; associated with the 1996 radish sprout outbreak), under sodium hypochlorite or hydrogen peroxide treatment. We found over 380 genes were differentially expressed (more than twofold; P < 0.05) after exposure to low levels of chlorine or hydrogen peroxide. Significantly upregulated genes included several regulatory genes responsive to oxidative stress, genes encoding putative oxidoreductases, and genes associated with cysteine biosynthesis, iron-sulfur cluster assembly, and antibiotic resistance. Identification of E. coli O157:H7 strains with enhanced resistance to chlorine decontamination and analysis of their transcriptomic response to oxidative stress may improve our basic understanding of the survival strategy of this human enteric pathogen on fresh produce during minimal processing. PMID:19666735

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

    PubMed

    Schmidt, Carrie E; Shringi, Smriti; Besser, Thomas E

    2016-01-01

    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. PMID:26824472

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

    PubMed Central

    Schmidt, Carrie E.; Shringi, Smriti; Besser, Thomas E.

    2016-01-01

    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. PMID:26824472

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

    PubMed

    Schmidt, Carrie E; Shringi, Smriti; Besser, Thomas E

    2016-01-01

    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.

  16. Inactivation of Escherichia coli JM109, DH5alpha, and O157:H7 suspended in Butterfield's Phosphate Buffer by gamma irradiation.

    PubMed

    Sommers, C H; Rajkowski, K T

    2008-03-01

    Food irradiation is a safe and effective method for inactivation of pathogenic bacteria, including Escherichia coli O157:H7, in meat, leafy greens, and complex ready-to-eat foods without affecting food product quality. Determining the radiation dose needed to inactivate E. coli O157:H7 in foods and the validation of new irradiation technologies are often performed through inoculation of model systems or food products with cocktails of the target bacterium, or use of single well-characterized isolates. In this study, the radiation resistance of 4 E. coli strains, 2 DNA repair deficient strains used for cloning and recombinant DNA technology (JM109 and DH5alpha) and 2 strains of serotype O157:H7 (C9490 and ATCC 35150), were determined. The D-10 values for C9490, ATCC 35150, JM109, and DH5alpha stationary phase cells suspended in Butterfield's Phosphate Buffer and irradiated at 4 degrees C were 229 (+/- 9.00), 257 (+/- 7.00), 61.2 (+/- 10.4), and 51.2 (+/- 8.82) Gy, respectively. The results of this study indicate that the extreme radiation sensitivity of JM109 and DH5alpha makes them unsuitable for use as surrogate microorganisms for pathogenic E. coli in the field of food irradiation research. Use of E. coli JM109 and DH5alpha, which carry mutations of the recA and gyrA genes required for efficient DNA repair and replication, is not appropriate for determination of radiation inactivation kinetics and validation of radiation processing equipment.

  17. Naturally resident and exogenously applied T4-like and T5-like bacteriophages can reduce Escherichia coli O157:H7 levels in sheep guts

    PubMed Central

    Raya, Raul R; Oot, Rebecca A; Moore-Maley, Ben; Wieland, Serena; Callaway, Todd R; Kutter, Elizabeth M

    2011-01-01

    In preparing sheep for an in vivo Escherichia coli O157:H7 eradication trial, we found that 20/39 members of a single flock were naturally colonized by O157:H7-infecting phages. Characterization showed these were all one phage type (subsequently named CEV2) infecting 15/16 O157:H7, 7/72 ECOR and common lab strains. Further characterization by PFGE (genome∼120 kb), restriction enzyme digest (DNA appears unmodified), receptor studies (FhuA but not TonB is required for infection) and sequencing (>95% nucleotide identity) showed it is a close relative of the classically studied coliphage T5. Unlike T5, CEV2 infects O157:H7 in vitro, both aerobically and anaerobically, rapidly adsorbing and killing, but resistant mutants regrew within 24 h. When used together with T4-like CEV1 (MOI ∼2 per phage), bacterial killing was longer lasting. CEV2 did not reproduce when co-infecting the same cell as CEV1, presumably succumbing to CEV1's ability to shut off transcription of cytosine-containing DNA. In vivo sheep trials to remove resident O157:H7 showed that a cocktail of CEV2 and CEV1 (∼1011 total PFU) applied once orally was more effective (>99.9% reduction) than CEV1 alone (∼99%) compared to the untreated phage-free control. Those sheep naturally carrying CEV2, receiving no additional phage treatment, had the lowest O157:H7 levels (∼99.99% reduction). These data suggest that phage cocktails are more effective than individual phage in removing O157:H7 that have taken residence if the phage work in concert with one another and that naturally resident O157:H7-infecting phages may prevent O157:H7 gut colonization and be one explanation for the transient O157:H7 colonization in ruminants. PMID:21687531

  18. Shiga toxin-producing Escherichia coli in Central Greece: prevalence and virulence genes of O157:H7 and non-O157 in animal feces, vegetables, and humans.

    PubMed

    Pinaka, O; Pournaras, S; Mouchtouri, V; Plakokefalos, E; Katsiaflaka, A; Kolokythopoulou, F; Barboutsi, E; Bitsolas, N; Hadjichristodoulou, C

    2013-11-01

    In Greece, Shiga toxin-producing Escherichia coli (STEC) have only been sporadically reported. The objective of this study was to estimate the prevalence of STEC and Escherichia coli O157:H7 in farm animals, vegetables, and humans in Greece. A total number of 1,010 fecal samples were collected from farm animals (sheep, goats, cattle, chickens, pigs), 667 diarrheal samples from humans, and 60 from vegetables, which were cultured in specific media for STEC isolates. Enzyme-linked immunosorbent assay (ELISA) was used to detect toxin-producing colonies, which, subsequently, were subjected to a multiplex polymerase chain reaction (PCR) for stx1, stx2, eae, rfbE O157, and fliC h7 genes. Eighty isolates (7.9 %) from animal samples were found to produce Shiga toxin by ELISA, while by PCR, O157 STEC isolates were detected from 8 (0.8 %) samples and non-O157 STEC isolates from 43 (4.2 %) samples. STEC isolates were recovered mainly from sheep and goats, rarely from cattle, and not from pigs and chickens, suggesting that small ruminants constitute a potential risk for human infections. However, only three human specimens (0.4 %) were positive for the detection of Shiga toxins and all were PCR-negative. Similarly, all 60 vegetable samples were negative for toxin production and for toxin genes, but three samples (two roman rockets and one spinach) were positive by PCR for rfbE O157 and fliC h7 genes. These findings indicate that sheep, goats, cattle, and leafy vegetables can be a reservoir of STEC and Escherichia coli O157:H7 isolates in Greece, which are still rarely detected among humans. PMID:23677425

  19. Molecular analysis of shiga toxin-producing Escherichia coli strains isolated from hemolytic-uremic syndrome patients and dairy samples in France.

    PubMed

    Pradel, Nathalie; Bertin, Yolande; Martin, Christine; Livrelli, Valérie

    2008-04-01

    Shiga toxin-producing Escherichia coli (STEC) has been associated with food-borne diseases ranging from uncomplicated diarrhea to hemolytic-uremic syndrome (HUS). While most outbreaks are associated with E. coli O157:H7, about half of the sporadic cases may be due to non-O157:H7 serotypes. To assess the pathogenicity of STEC isolated from dairy foods in France, 40 strains isolated from 1,130 raw-milk and cheese samples were compared with 15 STEC strains isolated from patients suffering from severe disease. The presence of genes encoding Shiga toxins (stx(1), stx(2), and variants), intimin (eae and variants), adhesins (bfp, efa1), enterohemolysin (ehxA), serine protease (espP), and catalase-peroxidase (katP) was determined by PCR and/or hybridization. Plasmid profiling, ribotyping, and pulsed-field gel electrophoresis (PFGE) were used to further compare the strains at the molecular level. A new stx(2) variant, stx(2-CH013), associated with an O91:H10 clinical isolate was identified. The presence of the stx(2), eae, and katP genes, together with a combination of several stx(2) variants, was clearly associated with human-pathogenic strains. In contrast, dairy food STEC strains were characterized by a predominance of stx(1), with a minority of isolates harboring eae, espP, and/or katP. These associations may help to differentiate less virulent STEC strains from those more likely to cause disease in humans. Only one dairy O5 isolate had a virulence gene panel identical to that of an HUS-associated strain. However, the ribotype and PFGE profiles were not identical. In conclusion, most STEC strains isolated from dairy products in France showed characteristics different from those of strains isolated from patients.

  20. Genome-Wide Transposon Mutagenesis Reveals a Role for pO157 Genes in Biofilm Development in Escherichia coli O157:H7 EDL933▿

    PubMed Central

    Puttamreddy, Supraja; Cornick, Nancy A.; Minion, F. Chris

    2010-01-01

    Enterohemorrhagic Escherichia coli O157:H7, a world-wide human food-borne pathogen, causes mild to severe diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. The ability of this pathogen to persist in the environment contributes to its dissemination to a wide range of foods and food processing surfaces. Biofilms are thought to be involved in persistence, but the process of biofilm formation is complex and poorly understood in E. coli O157:H7. To better understand the genetics of this process, a mini-Tn5 transposon insertion library was constructed in strain EDL933 and screened for biofilm-negative mutants using a microtiter plate assay. Ninety-five of 11,000 independent insertions (0.86%) were biofilm negative, and transposon insertions were located in 51 distinct genes/intergenic regions that must be involved either directly or indirectly in biofilm formation. All of the 51 biofilm-negative mutants showed reduced biofilm formation on both hydrophilic and hydrophobic surfaces. Thirty-six genes were unique to this study, including genes on the virulence plasmid pO157. The type V secreted autotransporter serine protease EspP and the enterohemolysin translocator EhxD were found to be directly involved in biofilm formation. In addition, EhxD and EspP were also important for adherence to T84 intestinal epithelial cells, suggesting a role for these genes in tissue interactions in vivo. PMID:20351142

  1. Cross contamination of Escherichia coli O157:H7 between lettuce and wash water during home-scale washing.

    PubMed

    Jensen, Dane A; Friedrich, Loretta M; Harris, Linda J; Danyluk, Michelle D; Schaffner, Donald W

    2015-04-01

    Lettuce and leafy greens have been implicated in multiple foodborne disease outbreaks. This study quantifies cross contamination between lettuce pieces in a small-scale home environment. A five-strain cocktail of relevant Escherichia coli O157:H7 strains was used. Bacterial transfer between single inoculated lettuce leaf pieces to 10 non-inoculated lettuce leaf pieces that were washed in a stainless steel bowl of water for 30 s, 1 min, 2 min, and 5 min was quantified. Regardless of washing time, the wash water became contaminated with 90-99% of bacteria originally present on the inoculated lettuce leaf piece. The E. coli O157:H7 concentration on initially inoculated leaf pieces was reduced ∼ 2 log CFU. Each initially uncontaminated lettuce leaf piece had ∼ 1% of the E. coli O157:H7 from the inoculated lettuce piece transferred to it after washing, with more transfer occurring during the shortest (30 s) and longest (5 min) wash times. In all cases the log percent transfer rates were essentially normally distributed. In all scenarios, most of the E. coli O157:H7 (90-99%) transferred from the inoculated lettuce pieces to the wash water. Washing with plain tap water reduces levels of E. coli O157:H7 on the inoculated lettuce leaf pieces, but also spreads contamination to previously uncontaminated leaf pieces. PMID:25475312

  2. Cross contamination of Escherichia coli O157:H7 between lettuce and wash water during home-scale washing.

    PubMed

    Jensen, Dane A; Friedrich, Loretta M; Harris, Linda J; Danyluk, Michelle D; Schaffner, Donald W

    2015-04-01

    Lettuce and leafy greens have been implicated in multiple foodborne disease outbreaks. This study quantifies cross contamination between lettuce pieces in a small-scale home environment. A five-strain cocktail of relevant Escherichia coli O157:H7 strains was used. Bacterial transfer between single inoculated lettuce leaf pieces to 10 non-inoculated lettuce leaf pieces that were washed in a stainless steel bowl of water for 30 s, 1 min, 2 min, and 5 min was quantified. Regardless of washing time, the wash water became contaminated with 90-99% of bacteria originally present on the inoculated lettuce leaf piece. The E. coli O157:H7 concentration on initially inoculated leaf pieces was reduced ∼ 2 log CFU. Each initially uncontaminated lettuce leaf piece had ∼ 1% of the E. coli O157:H7 from the inoculated lettuce piece transferred to it after washing, with more transfer occurring during the shortest (30 s) and longest (5 min) wash times. In all cases the log percent transfer rates were essentially normally distributed. In all scenarios, most of the E. coli O157:H7 (90-99%) transferred from the inoculated lettuce pieces to the wash water. Washing with plain tap water reduces levels of E. coli O157:H7 on the inoculated lettuce leaf pieces, but also spreads contamination to previously uncontaminated leaf pieces.

  3. Analysis of Escherichia coli O157:H7 Survival in Ovine or Bovine Manure and Manure Slurry

    PubMed Central

    Kudva, Indira T.; Blanch, Kathryn; Hovde, Carolyn J.

    1998-01-01

    Farm animal manure or manure slurry may disseminate, transmit, or propagate Escherichia coli O157:H7. In this study, the survival and growth of E. coli O157:H7 in ovine or bovine feces under various experimental and environmental conditions were determined. A manure pile collected from experimentally inoculated sheep was incubated outside under fluctuating environmental conditions. E. coli O157:H7 survived in the manure for 21 months, and the concentrations of bacteria recovered ranged from <102 to 106 CFU/g at different times over the course of the experiment. The DNA fingerprints of E. coli O157:H7 isolated at month 1 and month 12 were identical or very similar. A second E. coli O157:H7-positive ovine manure pile, which was periodically aerated by mixing, remained culture positive for 4 months. An E. coli O157:H7-positive bovine manure pile was culture positive for 47 days. In the laboratory, E. coli O157:H7 was inoculated into feces, untreated slurry, or treated slurry and incubated at −20, 4, 23, 37, 45, and 70°C. E. coli O157:H7 survived best in manure incubated without aeration at temperatures below 23°C, but it usually survived for shorter periods of time than it survived in manure held in the environment. The bacterium survived at least 100 days in bovine manure frozen at −20°C or in ovine manure incubated at 4 or 10°C for 100 days, but under all other conditions the length of time that it survived ranged from 24 h to 40 days. In addition, we found that the Shiga toxin type 1 and 2 genes in E. coli O157:H7 had little or no influence on bacterial survival in manure or manure slurry. The long-term survival of E. coli O157:H7 in manure emphasizes the need for appropriate farm waste management to curtail environmental spread of this bacterium. This study also highlights the difficulties in extrapolating laboratory data to on-farm conditions. PMID:9726855

  4. Specific detection of live Escherichia coli O157:H7 using tetracysteine-tagged PP01 bacteriophage.

    PubMed

    Wu, Lina; Song, Yiyi; Luan, Tian; Ma, Ling; Su, Liuqin; Wang, Shuo; Yan, Xiaomei

    2016-12-15

    Sensitive and rapid detection of Escherichia coli O157:H7, one of the most notorious bacterial pathogens, is urgently needed for public health protection. Yet, the existing methods are either lack of speed or limited in discriminating viable and dead cells. Using a recombinant bacteriophage, here we report the development of a rapid and sensitive method for live E. coli O157:H7 detection. First, the wild-type PP01 phage was engineered with a tetracysteine (TC)-tag fused with the small outer capsid (SOC) protein. Then, this PP01-TC phage was used to inoculate bacterial sample for 30min. Specific infection and rapid replication of PP01-TC phage in viable E. coli O157:H7 host cell yields a large number of progeny phages with capsids displaying TC tags that can be fluorescently labeled by a membrane permeable biarsenical dye (FlAsH). The bright green fluorescence of single E. coli O157:H7 cells can be readily detected by flow cytometry (FCM) and fluorescence microscopy. High specificity of the assay was verified with seven other bacterial strains. Practical application in E. coli O157:H7 detection in drinks was successfully demonstrated with artificially contaminated 100% apple juice. In less than three hours (including sample preconcentration) and with 40mL of sample volume, as low as 1cfu/mL E. coli O157:H7 can be detected in the presence of large excess of other nontarget bacteria via fluorescence microscopic measurement. The as-developed TC-PP01-FlAsH approach shows a great potential in the safeguard of liquid food products by providing rapid, sensitive, and specific detection of live E. coli O157:H7.

  5. Predictors and risk factors for the intestinal shedding of Escherichia coli O157 among working donkeys (Equus asinus) in Nigeria

    PubMed Central

    Jedial, Jesse T.; Shittu, Aminu; Tambuwal, Faruk M.; Abubakar, Mikail B.; Garba, Muhammed K.; Kwaga, Jacob P.; Fasina, Folorunso O.

    2015-01-01

    Objectives Escherichia coli are an important group of bacteria in the normal gastrointestinal system but can sometimes cause infections in domestic animals and man. Donkeys are routinely used as multipurpose animal but details of burdens of potentially infectious bacteria associated with it are limited. The prevalence and associations between intestinal shedding of E. coli O157 and animal characteristics and management factors were studied among 240 randomly selected working donkeys in north-western Nigeria. Design Four local government areas, of Sokoto State in north-western Nigeria were recruited in this study. A multistage randomised cluster design was used to select subjects and donkey owners within selected zones. Confirmation of infection was based on bacterial culture, isolation and biochemical test for E. coli O157 from faecal samples. Results Of the total bacteria isolated, 203 of the 329 (61.70 per cent) were E. coli, 76 of which was E. coli serotype O157. A multivariable logistic regression model was used to examine the relation between intestinal shedding of E. coli O157 and selected variables. The analysis yielded five potential predictors of shedding: soft faeces in donkeys, Akaza and Fari ecotypes of donkey were positive predictors while maize straw as feed and sampling during the cold dry period were negative predictors. Conclusions This study concludes that controlling intestinal shedding of E. coli O157 among working donkeys in Nigeria is possible using the identified predictors in planning appropriate interventions to reduced human risk of infection. PMID:26392892

  6. Long polar fimbriae of enterohemorrhagic Escherichia coli O157:H7 bind to extracellular matrix proteins.

    PubMed

    Farfan, Mauricio J; Cantero, Lidia; Vidal, Roberto; Botkin, Douglas J; Torres, Alfredo G

    2011-09-01

    Adherence to intestinal cells is a key process in infection caused by enterohemorrhagic Escherichia coli (EHEC). Several adhesion factors that mediate the binding of EHEC to intestinal cells have been described, but the receptors involved in their recognition are not fully characterized. Extracellular matrix (ECM) proteins might act as receptors involved in the recognition of enteric pathogens, including EHEC. In this study, we sought to characterize the binding of EHEC O157:H7 to ECM proteins commonly present in the intestine. We found that EHEC prototype strains as well as other clinical isolates adhered more abundantly to surfaces coated with fibronectin, laminin, and collagen IV. Further characterization of this phenotype, by using antiserum raised against the LpfA1 putative major fimbrial subunit and by addition of mannose, showed that a reduced binding of EHEC to ECM proteins was observed in a long polar fimbria (lpf) mutant. We also found that the two regulators, H-NS and Ler, had an effect in EHEC Lpf-mediated binding to ECM, supporting the roles of these tightly regulated fimbriae as adherence factors. Purified Lpf major subunit bound to all of the ECM proteins tested. Finally, increased bacterial adherence was observed when T84 cells, preincubated with ECM proteins, were infected with EHEC. Taken together, these findings suggest that the interaction of Lpf and ECM proteins contributes to the EHEC colonization of the gastrointestinal tract.

  7. An outbreak of Escherichia coli O157:H7 infections associated with leaf lettuce consumption.

    PubMed

    Ackers, M L; Mahon, B E; Leahy, E; Goode, B; Damrow, T; Hayes, P S; Bibb, W F; Rice, D H; Barrett, T J; Hutwagner, L; Griffin, P M; Slutsker, L

    1998-06-01

    In July 1995, 40 Montana residents were identified with laboratory-confirmed Escherichia coli O157:H7 infection; 52 residents had bloody diarrhea without laboratory confirmation. The median age of those with laboratory-confirmed cases was 42 years (range, 4- 86); 58% were female. Thirteen patients were hospitalized, and 1 developed hemolytic-uremic syndrome. A case-control study showed that 19 (70%) of 27 patients but only 8 (17%) of 46 controls reported eating purchased (not home-grown) leaf lettuce before illness (matched odds ratio, 25.3; 95% confidence interval, 3.9-1065.6). Pulsed-field gel electrophoresis identified a common strain among 22 of 23 isolates tested. Implicated lettuce was traced to two sources: a local Montana farm and six farms in Washington State that shipped under the same label. This outbreak highlights the increasing importance of fresh produce as a vehicle in foodborne illness. Sanitary growing and handling procedures are necessary to prevent these infections.

  8. SAS molecular tests Escherichia coli O157 detection kit. Performance tested method 031203.

    PubMed

    Bapanpally, Chandra; Montier, Laura; Khan, Shah; Kasra, Akif; Brunelle, Sharon L

    2014-01-01

    The SAS Molecular tests Escherichia coli O157 Detection method, a loop-mediated isothermal amplification method, performed as well as or better than the U.S. Department of Agriculture, Food Safety Inspection Service Microbiology Laboratory Guidebook and the U.S. Food and Drug Administration Bacteriological Analytical Manual reference methods for ground beef, beef trim, bagged mixed lettuce, and fresh spinach. Ground beef (30% fat, 25 g test portion) was validated for 7-8 h enrichment, leafy greens were validated in a 6-7 h enrichment, and ground beef (30% fat, 375 g composite test portion) and beef trim (375 g composite test portion) were validated in a 16-20 h enrichment. The method performance for meat and leafy green matrixes was also shown to be acceptable under conditions of co-enrichment with Salmonella. Thus, after a short co-enrichment step, ground beef, beef trim, lettuce, and spinach can be tested for both Salmonella and E. coli O157. The SAS Molecular tests Salmonella Detection Kit was validated using the same test portions as for the SAS Molecular tests E. coli O157 Detection Kit and those results are presented in a separate report. Inclusivity and exclusivity testing revealed no false negatives and no false positives among the 50 E. coli 0157 strains, including H7 and non-motile strains, and 30 non-E. coli O157 strains examined. Finally, the method was shown to be robust when variations to DNA extract hold time and DNA volume were varied. The method comparison and robustness data suggest a full 7 h enrichment time should be used for 25 g ground beef test portions.

  9. Difference between Escherichia coli O157:H7 and non-pathogenic E. coli: survival and growth in seasonings.

    PubMed

    Yokoigawa, K; Takikawa, A; Kawai, H

    1999-01-01

    We examined the survival and growth of Escherichia coli O157:H7 cells incubated with several seasonings, in comparison with those of non-pathogenic E. coli. The cells were incubated at 25 degrees C for 24 h with several concentrations of NaCl, sucrose, soy sauce, worcester sauce and tomato ketchup, and their survival ratios were determined. The E. coli O157:H7 strains showed relatively higher survival ratios in 0.5-1.0 M sucrose, 25% soy sauce and 12.5-50% worcester sauce than the non-pathogenic strains, but slightly lower survival ratios in 0.5-2.0 M NaCl. A noteworthy difference between E. coli O157:H7 and the non-pathogenic strains was that incubation in the presence of 12.5% soy sauce allowed the growth of E. coli O157:H7 strains but reduced the viable cell numbers of non-pathogenic E. coli strains. PMID:16232665

  10. Transcriptomic analysis of triclosan-susceptible and -tolerant Escherichia coli O157:H19 in response to triclosan exposure.

    PubMed

    Lenahan, Mary; Sheridan, Áine; Morris, Dermot; Duffy, Geraldine; Fanning, Séamus; Burgess, Catherine M

    2014-04-01

    Triclosan is an active agent that is commonly found in biocide formulations which are used by the food industry to control microbial contamination. The aim of this study was to use microarray analysis to compare gene expression between a triclosan-susceptible Escherichia coli O157:H19 isolate (minimum inhibitory concentration [MIC] 6.25 μg/ml) and its in vitro generated triclosan-tolerant mutant (MIC >8,000 μg/ml). Gene expression profiling was performed on the wild-type and mutant isogenic pairs after 30 min exposure to the parent MIC for triclosan and an untreated control. Microarray analysis was carried out using the Affymetrix GeneChip E. coli Genome 2.0 Array, and differential expression of genes was analyzed using the pumaDE method in Bioconductor R software. Wild-type gene expression was found to be significantly different from the triclosan-tolerant mutant for a large number of genes, even in the absence of triclosan exposure. Significant differences were observed in the expression of a number of pathway genes involved in metabolism, transport, and chemotaxis. In particular, gene expression in the triclosan-tolerant mutant was highly up-regulated for 33 of 38 genes belonging to the flagellar assembly pathway. The presence of extended flagella in the mutant isolate was confirmed visually by transmission electron microscopy, although no significant difference was observed in the motility of the parent and mutant at low levels of triclosan. Data from this study show that at a transcriptomic level, a triclosan-tolerant E. coli O157:H19 mutant is significantly different from the wild-type strain in a number of different pathways, providing an increased understanding of triclosan tolerance.

  11. Capture of Escherichia coli O157:H7 Using Immunomagnetic Beads of Different Size and Antibody Conjugating Chemistry

    PubMed Central

    Tu, Shu-I; Reed, Sue; Gehring, Andrew; He, Yiping; Paoli, George

    2009-01-01

    Immunomagnetic beads (IMB) were synthesized using anti-Escherichia coli O157 antibodies and magnetic beads of two different sizes (1 μm and 2.6 to 2.8 μm) that contained a streptavidin coating, activated carboxyl groups or tosylated surfaces. The synthesized IMB, together with a commercially available IMB, were used to capture different strains of E. coli O157:H7 and E. coli O157:NM. The E. coli capture was measured by the time resolved fluorescence (TRF) intensity using a sandwich assay which we have previously demonstrated of having a sensitivity of 1 CFU/g after 4.5 hour enrichment [1]. The analyses of measured TRF intensity and determined antibody surface concentration indicated that larger beads provided higher response signals than smaller beads and were more effective in capturing the target of interest in pure culture and ground beef. In addition, while each type of IMB showed different favorable capture of E. coli O157:H7, streptavidin-coated IMB elicited the highest response, on average. Streptavidin-coated IMB also provided an economic benefit, costing less than $0.50 per assay. The results could be used to guide the proper choice of IMB for applications in developing detection processes for E. coli O157:H7. PMID:22399935

  12. E. coli O157 phage type 21/28 outbreak in North Cumbria associated with pasteurized milk.

    PubMed

    Goh, S; Newman, C; Knowles, M; Bolton, F J; Hollyoak, V; Richards, S; Daley, P; Counter, D; Smith, H R; Keppie, N

    2002-12-01

    In March 1999, a large community outbreak of Escherichia coli O157 infection occurred in North Cumbria. A total of 114 individuals were reported to the Outbreak Control Team (OCT); 88 had laboratory confirmed E. coli O157. Twenty-eight (32%) of the confirmed cases were admitted to hospital, including three children (3.4%) with haemolytic uraemic syndrome. There were no deaths. A case-control study found that illness was strongly associated with drinking pasteurized milk from a local farm (P = <0.0001) on single variable analysis. Microbiological investigations at the farm revealed E. coli O157 phage type (PT) 21/28 VT 2 which was indistinguishable from the human isolates by pulsed field gel electrophoresis. At the time of occurrence this was the largest E. coli O157 outbreak in England and Wales and the first E. coli O157 PT 21/28 VT 2 outbreak associated with pasteurized milk. This outbreak highlights lessons to be learnt regarding on-farm pasteurization.

  13. Chromogenic agar medium for detection and isolation of Escherichia coli serogroups O26, O45, O103, O111, O121, and O145 from fresh beef and cattle feces.

    PubMed

    Kalchayanand, Norasak; Arthur, Terrance M; Bosilevac, Joseph M; Wells, James E; Wheeler, Tommy L

    2013-02-01

    Non-O157 Shiga toxin-producing Escherichia coli (STEC) strains are clinically important foodborne pathogens. Unlike E. coli O157:H7, these foodborne pathogens have no unique biochemical characteristics to readily distinguish them from other E. coli strains growing on plating media. In this study, a chromogenic agar medium was developed in order to differentiate among non-O157 STEC strains of serogroups O26, O45, O103, O111, O121, and O145 on a single agar medium. The ability of this chromogenic agar medium to select and distinguish among these pathogens is based on a combination of utilization of carbohydrates, b -galactosidase activity, and resistance to selective agents. The agar medium in combination with immunomagnetic separation was evaluated and successfully allowed for the detection and isolation of these six serogroups from artificially contaminated fresh beef. The agar medium in combination with immunomagnetic separation also allowed successful detection and isolation of naturally occurring non-O157 STEC strains present in cattle feces. Thirty-five strains of the top six non-O157 STEC serogroups were isolated from 1,897 fecal samples collected from 271 feedlot cattle. This chromogenic agar medium could help significantly in routine screening for the top six non-O157 STEC serogroups from beef cattle and other food.

  14. An outbreak of Vero cytotoxin producing Escherichia coli O157 infection associated with takeaway sandwiches.

    PubMed

    McDonnell, R J; Rampling, A; Crook, S; Cockcroft, P M; Wilshaw, G A; Cheasty, T; Stuart, J

    1997-12-12

    An outbreak of food poisoning due to Escherichia coli O157 phage type 2 Vero cytotoxin 2 affected 26 people in southern counties of England in May and June 1995. The organism was isolated from faecal specimens from 23 patients, 16 of whom lived in Dorset and seven in Hampshire. Isolates were indistinguishable by phage typing, Vero cytotoxin gene typing, restriction fragment length polymorphism, and pulsed field gel electrophoresis. Three associated cases, linked epidemiologically to the outbreak, were confirmed serologically by detection of antibodies to E. coli O157 lipopolysaccharide. Twenty-two of the 26 patients were adults: four were admitted to hospital with haemorrhagic colitis. Four cases were children: two were admitted to hospital with haemolytic uraemic syndrome (HUS). There were no deaths. Although E. coli O157 was not isolated from any food samples, illness was associated with having eaten cold meats in sandwiches bought from two sandwich producers, in Weymouth and in Portsmouth. Both shops were supplied by the same wholesaler, who kept no records and obtained cooked meats from several sources in packs that did not carry adequate identification marks. It was, therefore, impossible to trace back to the original producer or to investigate further to determine the origin of contamination with E. coli O157. To protect the public health it is essential that all wholesale packs of ready-to-eat food carry date codes and the producer's identification mark. Detailed record keeping should be part of hazard analysis critical control point (HACCP) systems and should be maintained throughout the chain of distribution from the producer to retail outlets.

  15. A conductance method for the identification of Escherichia coli O157:H7 using bacteriophage AR1.

    PubMed

    Chang, Tsung C; Ding, Hwia C; Chen, Shiowwen

    2002-01-01

    The feasibility of using a specific phage (AR1) in conjunction with a conductance method for the identification of Escherichia coli O157:H7 was evaluated. The multiplication of strains of E. coli O157:H7 was inhibited by AR1; therefore, a time point (detection time, DT) at which an accelerating change in conductance in the culture broth was not obtained. Bacterial strains were subcultured on sorbitol-MacConkey agar and incubated at 35 degrees C for 24 h, and the ability of the bacteria to ferment sorbitol was recorded. An aliquot of 0.5 ml of the bacterial suspension (10(7) CFU/ml) and 0.5 ml of the phage suspension (10(8) PFU/ml) were added to the conductance tube of a Malthus analyzer containing 5 ml of culture broth. The tubes were incubated at 35 degrees C, and conductance changes in the tubes were continuously monitored at 6-min intervals for 24 h by the instrument. A positive reaction was defined as an E. coli strain that could not utilize sorbitol and caused no conductance change (i.e., no DT) within an incubation period of 24 h. Of the 41 strains of E. coli O157:H7 tested, all produced positive reactions. When a total of 155 strains of non-O157:H7 E. coli were tested, 14 did not have a DT within 24 h. However, among these 14 strains, 13 were sorbitol fermenters, and the remaining one was a nonfermenter. Therefore, by definition, only one strain produced a false-positive reaction. The sensitivity and specificity of the present method were 100% (41 of 41) and 99.4% (154 of 155), respectively. The present method incorporating conductimetric measurement and phage AR1 for the identification of E. coli O157:H7 was simple and capable of automation.

  16. Use of Potential Probiotic Lactic Acid Bacteria (LAB) Biofilms for the Control of Listeria monocytogenes, Salmonella Typhimurium, and Escherichia coli O157:H7 Biofilms Formation

    PubMed Central

    Gómez, Natacha C.; Ramiro, Juan M. P.; Quecan, Beatriz X. V.; de Melo Franco, Bernadette D. G.

    2016-01-01

    Use of probiotic biofilms can be an alternative approach for reducing the formation of pathogenic biofilms in food industries. The aims of this study were (i) to evaluate the probiotic properties of bacteriocinogenic (Lactococcus lactis VB69, L. lactis VB94, Lactobacillus sakei MBSa1, and Lactobacillus curvatus MBSa3) and non-bacteriocinogenic (L. lactis 368, Lactobacillus helveticus 354, Lactobacillus casei 40, and Weissela viridescens 113) lactic acid bacteria (LAB) isolated from Brazilian’s foods and (ii) to develop protective biofilms with these strains and test them for exclusion of Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella Typhimurium. LAB were tested for survival in acid and bile salt conditions, surface properties, biosurfactant production, β-galactosidase and gelatinase activity, antibiotic resistance and presence of virulence genes. Most strains survived exposure to pH 2 and 4% bile salts. The highest percentages of auto-aggregation were obtained after 24 h of incubation. Sixty-seven percentage auto-aggregation value was observed in W. viridescens 113 and Lactobacillus curvatus MBSa3 exhibited the highest co-aggregation (69% with Listeria monocytogenes and 74.6% with E. coli O157:H7), while the lowest co-aggregation was exhibited by W. viridescens 113 (53.4% with Listeria monocytogenes and 38% with E. coli O157:H7). Tests for hemolytic activity, bacterial cell adherence with xylene, and drop collapse confirmed the biosurfactant-producing ability of most strains. Only one strain (L. lactis 368) produced β-galactosidase. All strains were negative for virulence genes cob, ccf, cylLL, cylLs, cyllM, cylB, cylA and efaAfs and gelatinase production. The antibiotic susceptibility tests indicated that the MIC for ciprofloxacin, clindamycin, gentamicin, kanamycin, and streptomycin did not exceed the epidemiological cut-off suggested by the European Food Safety Authority. Some strains were resistant to one or more antibiotics and

  17. Use of Potential Probiotic Lactic Acid Bacteria (LAB) Biofilms for the Control of Listeria monocytogenes, Salmonella Typhimurium, and Escherichia coli O157:H7 Biofilms Formation.

    PubMed

    Gómez, Natacha C; Ramiro, Juan M P; Quecan, Beatriz X V; de Melo Franco, Bernadette D G

    2016-01-01

    Use of probiotic biofilms can be an alternative approach for reducing the formation of pathogenic biofilms in food industries. The aims of this study were (i) to evaluate the probiotic properties of bacteriocinogenic (Lactococcus lactis VB69, L. lactis VB94, Lactobacillus sakei MBSa1, and Lactobacillus curvatus MBSa3) and non-bacteriocinogenic (L. lactis 368, Lactobacillus helveticus 354, Lactobacillus casei 40, and Weissela viridescens 113) lactic acid bacteria (LAB) isolated from Brazilian's foods and (ii) to develop protective biofilms with these strains and test them for exclusion of Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella Typhimurium. LAB were tested for survival in acid and bile salt conditions, surface properties, biosurfactant production, β-galactosidase and gelatinase activity, antibiotic resistance and presence of virulence genes. Most strains survived exposure to pH 2 and 4% bile salts. The highest percentages of auto-aggregation were obtained after 24 h of incubation. Sixty-seven percentage auto-aggregation value was observed in W. viridescens 113 and Lactobacillus curvatus MBSa3 exhibited the highest co-aggregation (69% with Listeria monocytogenes and 74.6% with E. coli O157:H7), while the lowest co-aggregation was exhibited by W. viridescens 113 (53.4% with Listeria monocytogenes and 38% with E. coli O157:H7). Tests for hemolytic activity, bacterial cell adherence with xylene, and drop collapse confirmed the biosurfactant-producing ability of most strains. Only one strain (L. lactis 368) produced β-galactosidase. All strains were negative for virulence genes cob, ccf, cylLL, cylLs, cyllM, cylB, cylA and efaAfs and gelatinase production. The antibiotic susceptibility tests indicated that the MIC for ciprofloxacin, clindamycin, gentamicin, kanamycin, and streptomycin did not exceed the epidemiological cut-off suggested by the European Food Safety Authority. Some strains were resistant to one or more antibiotics and resistance

  18. Saltelli Global Sensitivity Analysis and Simulation Modelling to Identify Intervention Strategies to Reduce the Prevalence of Escherichia coli O157 Contaminated Beef Carcasses

    PubMed Central

    Brookes, Victoria J.; Jordan, David; Davis, Stephen; Ward, Michael P.; Heller, Jane

    2015-01-01

    Introduction Strains of Shiga-toxin producing Escherichia coli O157 (STEC O157) are important foodborne pathogens in humans, and outbreaks of illness have been associated with consumption of undercooked beef. Here, we determine the most effective intervention strategies to reduce the prevalence of STEC O157 contaminated beef carcasses using a modelling approach. Method A computational model simulated events and processes in the beef harvest chain. Information from empirical studies was used to parameterise the model. Variance-based global sensitivity analysis (GSA) using the Saltelli method identified variables with the greatest influence on the prevalence of STEC O157 contaminated carcasses. Following a baseline scenario (no interventions), a series of simulations systematically introduced and tested interventions based on influential variables identified by repeated Saltelli GSA, to determine the most effective intervention strategy. Results Transfer of STEC O157 from hide or gastro-intestinal tract to carcass (improved abattoir hygiene) had the greatest influence on the prevalence of contaminated carcases. Due to interactions between inputs (identified by Saltelli GSA), combinations of interventions based on improved abattoir hygiene achieved a greater reduction in maximum prevalence than would be expected from an additive effect of single interventions. The most effective combination was improved abattoir hygiene with vaccination, which achieved a greater than ten-fold decrease in maximum prevalence compared to the baseline scenario. Conclusion Study results suggest that effective interventions to reduce the prevalence of STEC O157 contaminated carcasses should initially be based on improved abattoir hygiene. However, the effect of improved abattoir hygiene on the distribution of STEC O157 concentration on carcasses is an important information gap—further empirical research is required to determine whether reduced prevalence of contaminated carcasses is

  19. Standardized Escherichia coli O157:H7 Exposure Studies in Cattle Provide Evidence that Bovine Factors Do Not Drive Increased Summertime Colonization.

    PubMed

    Sheng, Haiqing; Shringi, Smriti; Baker, Katherine N K; Minnich, Scott A; Hovde, Carolyn J; Besser, Thomas E

    2016-02-01

    The increased summertime prevalence of cattle carriage of enterohemorrhagic Shiga toxin-producing Escherichia coli O157:H7 (STEC O157) is associated with the increased summertime incidence of human infection. The mechanism driving the seasonality of STEC O157 carriage among cattle is unknown. We conducted experimental challenge trials to distinguish whether factors extrinsic or intrinsic to cattle underlie the seasonality of STEC O157 colonization. Holstein steers (n = 20) exposed to ambient environmental conditions were challenged with a standardized pool of STEC O157 strains four times at 6-month intervals. The densities and durations of rectoanal junction mucosa (RAJ) colonization with STEC O157 were compared by season (winter versus summer), dose (10(9) CFU versus 10(7) CFU), and route of challenge (oral versus rectal). Following summer challenges, the RAJ STEC O157 colonization density was significantly lower (P = 0.016) and the duration was shorter (P = 0.052) than for winter challenges, a seasonal pattern opposite to that observed naturally. Colonization was unaffected by the challenge route, indicating that passage through the gastrointestinal microbiome did not significantly affect the infectious dose to the RAJ. A 2-log reduction of the challenge doses in the second-year trials was accompanied by similarly reduced RAJ colonization in both seasons (P < 0.001). These results refute the hypothesis that cattle are predisposed to STEC O157 colonization during the summer months, either due to intrinsic factors or indirectly due to gastrointestinal tract microbiome effects. Instead, the data support the hypothesis that the increased summertime STEC O157 colonization results from increased seasonal oral exposure to this pathogen. PMID:26607594

  20. Using Comparative Genomics for Inquiry-Based Learning to Dissect Virulence of Escherichia coli O157:H7 and Yersinia pestis

    PubMed Central

    Baumler, David J.; Banta, Lois M.; Hung, Kai F.; Schwarz, Jodi A.; Cabot, Eric L.; Glasner, Jeremy D.; Perna, Nicole T.

    2012-01-01

    Genomics and bioinformatics are topics of increasing interest in undergraduate biological science curricula. Many existing exercises focus on gene annotation and analysis of a single genome. In this paper, we present two educational modules designed to enable students to learn and apply fundamental concepts in comparative genomics using examples related to bacterial pathogenesis. Students first examine alignments of genomes of Escherichia coli O157:H7 strains isolated from three food-poisoning outbreaks using the multiple-genome alignment tool Mauve. Students investigate conservation of virulence factors using the Mauve viewer and by browsing annotations available at the A Systematic Annotation Package for Community Analysis of Genomes database. In the second module, students use an alignment of five Yersinia pestis genomes to analyze single-nucleotide polymorphisms of three genes to classify strains into biovar groups. Students are then given sequences of bacterial DNA amplified from the teeth of corpses from the first and second pandemics of the bubonic plague and asked to classify these new samples. Learning-assessment results reveal student improvement in self-efficacy and content knowledge, as well as students' ability to use BLAST to identify genomic islands and conduct analyses of virulence factors from E. coli O157:H7 or Y. pestis. Each of these educational modules offers educators new ready-to-implement resources for integrating comparative genomic topics into their curricula. PMID:22383620

  1. Anaerobic Conditions Promote Expression of Sfp Fimbriae and Adherence of Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:NM to Human Intestinal Epithelial Cells▿

    PubMed Central

    Müsken, Anne; Bielaszewska, Martina; Greune, Lilo; Schweppe, Christian H.; Müthing, Johannes; Schmidt, Herbert; Schmidt, M. Alexander; Karch, Helge; Zhang, Wenlan

    2008-01-01

    The sfp gene cluster, unique to sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:NM strains, encodes fimbriae that mediate mannose-resistant hemagglutination in laboratory E. coli strains but are not expressed in wild-type SF EHEC O157:NM strains under standard laboratory conditions. We investigated whether Sfp fimbriae are expressed under conditions that mimic the intestinal environment and whether they contribute to the adherence of SF EHEC O157:NM strains to human intestinal epithelial cells. The transcription of sfpA (encoding the major fimbrial subunit) was upregulated in all strains investigated, and all expressed SfpA and possessed fimbriae that reacted with an anti-SfpA antibody when the strains were grown on solid media under anaerobic conditions. Sfp expression was absent under aerobic conditions and in liquid media. Sfp upregulation under anaerobic conditions was significantly higher on blood agar and a medium simulating the colonic environment than on a medium simulating the ileal environment (P < 0.05). The induction of Sfp fimbriae in SF E. coli O157:NM strains correlates with increased adherence to Caco-2 and HCT-8 cells. Our data indicate that the expression of Sfp fimbriae in SF E. coli O157:NM strains is induced under conditions resembling those of the natural site of infection and that Sfp fimbriae may contribute to the adherence of the organisms to human intestinal epithelium. PMID:18083855

  2. Effect of curli expression and hydrophobicity of E. coli O157:H7 on attachment to fresh produce surfaces

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aim: To investigate the effect of curli expression on cell hydrophobicity, biofilm formation, and attachment to cut and intact fresh produce surfaces. Methods and Results: Five E. coli O157:H7 strains were evaluated for curli expression, hydrophobicity, biofilm formation, and attachment of E. co...

  3. Curli fimbriae are conditionally required in Escherichia coli O157:H7 for initial attachment and biofilm formation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several species of enteric pathogens produce curli fimbriae, which may affect their interaction with surfaces and other microbes in nonhost environments. Here we used two E. coli O157:H7 outbreak strains with distinct genotypes to understand the role of curli in surface attachment and biofilm format...

  4. BEHAVIOR OF ESCHERICHIA COLI O157:H7, LISTERIA MONOCYTOGENES, AND SALMONELLA TYPHIMURIUM IN TEEWURST, A RAW SPREADABLE SAUSAGE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The fate of Listeria monocytogenes, Salmonella Typhimurium, or Escherichia coli O157:H7 were separately monitored both in and on teewurst, a traditional raw and spreadable sausage of Germanic origin. Multi-strain cocktails of each pathogen (ca. 5.0 log CFU/g) were used to separately inoculate teewur...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  6. Occurrence of non-O157 Shiga toxin-producing Escherichia coli in two commercial swine farms in the Eastern Cape Province, South Africa.

    PubMed

    Iwu, Chinwe Juliana; Iweriebor, Benson Chuks; Obi, Larry Chikwelu; Okoh, Anthony Ifeanyi

    2016-02-01

    Shiga toxin-producing Escherichia coli (STEC) is one of the most significant causes of food-borne infections capable of causing serious health complications in humans. Even though ruminants are known to be the major reservoirs of STEC, other non-ruminant food producing animals may also harbour pathogenic E. coli strains. In this study, we investigated the prevalence of E. coli serogroups O26, O111, O121, O145, and O157 and their associated virulence genes (stx1, stx2, eae, and ehxA) in swine faecal samples obtained from the two major commercial farms located in the Nkonkobe Municipality, Eastern Cape, South Africa. The proportions of serogroups detected were O26; 35 (7%), O145; 14 (2.8%), and O157:H7; 43 (8.6%) of the total animals sampled. Out of the 500 animals sampled, 22 isolates of E. coli (1.4%) tested positive for the stx2 gene, and 7 of these isolates belonged to E. coli O26 serogroup, while the remaining 15 most likely belonged to serogroups untargeted in this study. Other virulence genes (stx1, eae, and ehxA) that we screened for were not detected. These findings reveal that pigs within the Eastern Cape Province of South Africa can harbour Shiga toxin-producing E. coli.

  7. Occurrence of non-O157 Shiga toxin-producing Escherichia coli in two commercial swine farms in the Eastern Cape Province, South Africa.

    PubMed

    Iwu, Chinwe Juliana; Iweriebor, Benson Chuks; Obi, Larry Chikwelu; Okoh, Anthony Ifeanyi

    2016-02-01

    Shiga toxin-producing Escherichia coli (STEC) is one of the most significant causes of food-borne infections capable of causing serious health complications in humans. Even though ruminants are known to be the major reservoirs of STEC, other non-ruminant food producing animals may also harbour pathogenic E. coli strains. In this study, we investigated the prevalence of E. coli serogroups O26, O111, O121, O145, and O157 and their associated virulence genes (stx1, stx2, eae, and ehxA) in swine faecal samples obtained from the two major commercial farms located in the Nkonkobe Municipality, Eastern Cape, South Africa. The proportions of serogroups detected were O26; 35 (7%), O145; 14 (2.8%), and O157:H7; 43 (8.6%) of the total animals sampled. Out of the 500 animals sampled, 22 isolates of E. coli (1.4%) tested positive for the stx2 gene, and 7 of these isolates belonged to E. coli O26 serogroup, while the remaining 15 most likely belonged to serogroups untargeted in this study. Other virulence genes (stx1, eae, and ehxA) that we screened for were not detected. These findings reveal that pigs within the Eastern Cape Province of South Africa can harbour Shiga toxin-producing E. coli. PMID:26851595

  8. Short communication: survival of Escherichia coli O157:H7 in dairy cattle drinking water.

    PubMed

    Rice, E W; Johnson, C H

    2000-09-01

    Cattle drinking water from two dairy farms was used in a study to determine the survival characteristics of the bacterial pathogen Escherichia coli O157:H7 and wild-type E. coli. The E. coli O157:H7 inoculum consisted of a consortium of isolates obtained from dairy cattle. Fresh manure was used as the source for the wild-type E. coli. In the water source from farm 1 the pathogens were present at both 5 and 15 degrees C during the 16-d duration of the study. In the water source from farm 2, the pathogens were detected at 5 degrees C through d 8 and through d 4 at 15 degrees C. The fecal indicator, wild-type E. coli, was always present when the pathogens were present.

  9. Increase in activity of essential oil components carvacrol and thymol against Escherichia coli O157:H7 by addition of food stabilizers.

    PubMed

    Burt, Sara A; Vlielander, René; Haagsman, Henk P; Veldhuizen, Edwin J A

    2005-05-01

    The major components of oregano and thyme essential oils that had previously been shown to inhibit Escherichia coli O157:H7 were determined by high-performance liquid chromatography with UV detection and liquid chromatographic tandem mass spectrometry. The MICs and MBCs of carvacrol, thymol, p-cymene, and gamma-terpinene against a strain of E. coli O157: H7 phage type 34 isolated from bovine feces were determined by microdilution assay. The constituents were then tested in checkerboard assays to detect possible interactions. Carvacrol and thymol displayed bacteriostatic and bactericidal properties with MICs of 1.2 mmol/liter and were additive in combination. p-Cymene and gamma-terpinene displayed no measurable antibacterial activity up to 50 mmol/liter, and neither influenced the activity of carvacrol or thymol. Growth curves in the presence of nonlethal concentrations of carvacrol with the addition of agar (0.05%, wt/vol) or carrageenan (0.125%, wt/vol) as stabilizer were produced by optical density measurement. The stabilizers agar and carrageenan both significantly improved the effectiveness of carvacrol in broth, possibly because of a delay in the separation of the hydrophobic substrate from the aqueous phase of the medium. When carvacrol was dissolved in ethanol before addition to broth, stabilizers were not needed. Carvacrol and thymol, particularly when used in combination with a stabilizer or in an ethanol solution, may be effective in reducing the number or preventing growth of E. coli O157:H7 in liquid foods. PMID:15895722

  10. Clinical Escherichia coli strains carrying stx genes: stx variants and stx-positive virulence profiles.

    PubMed

    Eklund, Marjut; Leino, Kirsikka; Siitonen, Anja

    2002-12-01

    Altogether, 173 Shiga toxin-producing Escherichia coli (STEC) serotype O157 (n = 111) and non-O157 (n = 62) isolates from 170 subjects were screened by PCR-restriction fragment length polymorphism for eight different stx genes. The results were compiled according to serotypes, phage types of O157, production of Stx toxin and enterohemolysin, and the presence of eae. The stx genes occurred in 11 combinations; the most common were stx(2) with stx(2c) (42%), stx(2) alone (21%), and stx(1) alone (16%). Of the O157 strains, 64% carried stx(2) with stx(2c) versus 2% of the non-O157 strains (P < 0.001). In the non-O157 strains, the prevailing gene was stx(1) (99% versus 1% in O157 strains; P < 0.001). In addition, one strain (O Rough:H4:stx(2c)) which has not previously been described as associated with hemolytic-uremic syndrome (HUS) was found. Ten stx-positive virulence profiles were responsible for 71% of all STEC infections. Of these profiles, five accounted for 71% of the 21 strains isolated from 20 patients with HUS or thrombotic thrombocytopenic purpura (TTP). The strains having the virulence profile that caused mainly HUS or TTP or bloody diarrhea produced Stx with titers of >/=1:128 (90%) more commonly than did other strains (51%; P < 0.001). These strains were also more commonly enterohemolytic (98% versus 68% for other strains; P < 0.001) and possessed the eae gene (100%) more commonly than did other strains (74%; P < 0.001). A particular virulence profile, O157:H7:PT2:stx(2):stx(2c):eae:Ehly, was significantly more frequently associated with HUS and bloody diarrhea than were other profiles (P = 0.02) and also caused the deaths of two children. In this study, the risk factors for severe symptoms were an age of <5 years and infection by the strain of O157:H7:PT2 mentioned above.

  11. A longitudinal study of Escherichia coli strains isolated from captive mammals, birds, and reptiles in Trinidad.

    PubMed

    Gopee, N V; Adesiyun, A A; Caesar, K

    2000-09-01

    A longitudinal study was conducted of the prevalence and characteristics of Escherichia coli in mammals, birds, and reptiles housed at the Emperor Valley Zoo, Trinidad. During a 6-mo study period, swabs were obtained from fecal samples that were randomly collected from the enclosures of animals from these three taxonomic groups every 3 wk. With snakes, both cloacal and fecal swabs were obtained. Fecal and cloacal swabs were cultured for E. coli on eosin methylene blue agar. The production of mucoid colonies and hemolytic colonies and non-sorbitol fermenter status were identified. The occurrence of O157 strains was determined amongst E. coli isolates that were non-sorbitol fermenters, and the disc diffusion method was used to determine the antibiograms of isolates. The frequency of E. coli isolation was significantly higher in mammals compared with birds and reptiles. Overall, the frequencies of isolation of E. coli from omnivores. herbivores, and carnivores, 87.2%, 70.0%, and 57.3%, respectively, regardless of animal class, were significantly different. Most (99.6%) of the E. coli isolates tested for antibiotic sensitivity exhibited resistance to one or more of the eight antimicrobial agents used. The possession of phenotypic virulence markers by the E. coli isolates studied and the generally high resistance to antimicrobial agents may have health implications for the zoological collection.

  12. Evaluation of Escherichia coli biotype 1 as a surrogate for Escherichia coli O157:H7 for cooking, fermentation, freezing, and refrigerated storage in meat processes.

    PubMed

    Keeling, Carisa; Niebuhr, Steven E; Acuff, Gary R; Dickson, James S

    2009-04-01

    Five Escherichia coli biotype I isolates were compared with E. coli O157:H7 under four common meat processing conditions. The processes that were evaluated were freezing, refrigerating, fermentation, and thermal inactivation. For each study, at least one surrogate organism was not statistically different when compared with E. coli O157:H7. However, the four studies did not consistently show the same isolate as having this agreement. The three studies that involved temperature as a method of controlling or reducing the E. coli population all had at least one possible surrogate in common. In the fermentation study, only one isolate (BAA-1429) showed no statistical difference when compared with E. coli O157:H7. However, the population reductions that were observed indicated the isolates BAA-1427 and BAA-1431 would overestimate the surviving E. coli O157:H7 population in a fermented summer sausage. When all of the data from all of the surrogates were examined, it was found that isolates BAA-1427, BAA-1429, and BAA-1430 would be good surrogates for all four of the processes that were examined in this study. There was no statistical difference noted between these three isolates and E. coli O157:H7 in the refrigeration study. These isolates resulted in smaller population reductions than did E. coli O157:H7 in the frozen, fermentation, and thermal inactivation studies. This would indicate that these isolates would overpredict the E. coli O157:H7 population in these three instances. This overprediction results in an additional margin of safety when using E. coli biotype 1 as a surrogate.

  13. Evaluation of Escherichia coli biotype 1 as a surrogate for Escherichia coli O157:H7 for cooking, fermentation, freezing, and refrigerated storage in meat processes.

    PubMed

    Keeling, Carisa; Niebuhr, Steven E; Acuff, Gary R; Dickson, James S

    2009-04-01

    Five Escherichia coli biotype I isolates were compared with E. coli O157:H7 under four common meat processing conditions. The processes that were evaluated were freezing, refrigerating, fermentation, and thermal inactivation. For each study, at least one surrogate organism was not statistically different when compared with E. coli O157:H7. However, the four studies did not consistently show the same isolate as having this agreement. The three studies that involved temperature as a method of controlling or reducing the E. coli population all had at least one possible surrogate in common. In the fermentation study, only one isolate (BAA-1429) showed no statistical difference when compared with E. coli O157:H7. However, the population reductions that were observed indicated the isolates BAA-1427 and BAA-1431 would overestimate the surviving E. coli O157:H7 population in a fermented summer sausage. When all of the data from all of the surrogates were examined, it was found that isolates BAA-1427, BAA-1429, and BAA-1430 would be good surrogates for all four of the processes that were examined in this study. There was no statistical difference noted between these three isolates and E. coli O157:H7 in the refrigeration study. These isolates resulted in smaller population reductions than did E. coli O157:H7 in the frozen, fermentation, and thermal inactivation studies. This would indicate that these isolates would overpredict the E. coli O157:H7 population in these three instances. This overprediction results in an additional margin of safety when using E. coli biotype 1 as a surrogate. PMID:19435219

  14. 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.

    PubMed

    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

  15. 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

    PubMed Central

    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

  16. 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.

    PubMed

    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.

  17. Computing TaqMan probes for multiplex PCR detection of E. coli O157 serotypes in water.

    PubMed

    Ram, Siya; Shanker, Rishi

    2005-01-01

    Diarrheagenic E. coli strains contribute to water related diseases in urban and rural environment in developing and developed world. E. coli pathotype and pathogenicity varies due to complex multifactorial mechanism involving a large number of virulence factors. Rapid assessment of the virulence pattern of E. coli isolates is possible by Real-Time PCR probes like TaqMan. For designing TaqMan probes and primers for multiplex PCR selected E. coli gene sequences: stx1, stx2, hlyA, chuA, eae, lacZ, lamB and fimA were retrieved from NCBI's GenBank database. The alignment of the multiple sequences and analysis of conserved sequences was carried out using ClustalW and BLAST programs. The primers and Taqmen probes were designed using Beacon Designer software version 2.1 for two multiplexed PCR assays. In silico PCR simulation of these assays showed PCR products for stx2 (248bp) stx1 (102 bp), lacZ (228bp) and lamB (86 bp) in multiplex #1 and eae (200bp), chuA (147 bp), hlyA (141bp) and fimA (79 bp) in multiplex #2, respectively. These multiplexed PCR amplification products and probes can be used to identify and confirm presence of O157:H7/ H7-, O157:H43/45 and O26:H-/H11 serotypes. In conclusion, multiplex Real-Time Polymerase Chain Reaction oligomers and TaqMan probes designed and validated in silico will be helpful in management of water quality and outbreaks, by improving specificity and minimizing time needed for in vitro verification work.

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

    NASA Astrophysics Data System (ADS)

    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.

  19. High efficiency generalized transduction in Escherichia coli O157:H7.

    PubMed

    Marinus, Martin G; Poteete, Anthony R

    2013-01-01

    Genetic manipulation in enterohemorrhagic E. coli O157:H7 is currently restricted to recombineering, a method that utilizes the recombination system of bacteriophage lambda, to introduce gene replacements and base changes inter alia into the genome. Bacteriophage 933W is a prophage in E. coli O157:H7 strain EDL933, which encodes the genes ( stx2AB) for the production of Shiga toxin which is the basis for the potentially fatal Hemolytic Uremic Syndrome in infected humans. We replaced the stx2AB genes with a kanamycin cassette using recombineering. After induction of the prophage by ultra-violet light, we found that bacteriophage lysates were capable of transducing to wildtype, point mutations in the lactose, arabinose and maltose genes. The lysates could also transduce tetracycline resistant cassettes. Bacteriophage 933W is also efficient at transducing markers in E. coli K-12. Co-transduction experiments indicated that the maximal amount of transferred DNA was likely the size of the bacteriophage genome, 61 kB. All tested transductants, in both E. coli K-12 and O157:H7, were kanamycin-sensitive indicating that the transducing particles contained host DNA.

  20. Gene expression induced in Escherichia coli O157:H7 upon exposure to model apple juice.

    PubMed

    Bergholz, Teresa M; Vanaja, Sivapriya Kailasan; Whittam, Thomas S

    2009-06-01

    Escherichia coli O157:H7 has caused serious outbreaks of food-borne illness via transmission in a variety of food vehicles, including unpasteurized apple juice, dried salami, and spinach. To understand how this pathogen responds to the multiple stresses of the food environment, we compared global transcription patterns before and after exposure to model apple juice. Transcriptomes of mid-exponential- and stationary-phase cells were evaluated after 10 min in model apple juice (pH 3.5) using microarrays probing 4,886 open reading frames. A total of 331 genes were significantly induced upon exposure of cells to model apple juice, including genes involved in the acid, osmotic, and oxidative stress responses as well as the envelope stress response. Acid and osmotic stress response genes, including asr, osmC, osmB, and osmY, were significantly induced in response to model apple juice. Multiple envelope stress responses were activated as evidenced by increased expression of CpxR and Rcs phosphorelay-controlled genes. Genes controlled by CpxR (cpxP, degP, and htpX) were significantly induced 2- to 15-fold upon exposure to apple juice. Inactivation of CpxRA resulted in a significant decrease in survival of O157:H7 in model apple juice compared to the isogenic parent strain. Of the 331 genes induced in model apple juice, 104 are O157-specific genes, including those encoding type three secretion effectors (espJ, espB, espM2, espL3, and espZ). Elucidating the response of O157:H7 to acidic foods provides insight into how this pathogen is able to survive in food matrices and how exposure to foods influences subsequent transmission and virulence.

  1. Dietary choice affects Shiga toxin-producing Escherichia coli (STEC) O157:H7 colonization and disease.

    PubMed

    Zumbrun, Steven D; Melton-Celsa, Angela R; Smith, Mark A; Gilbreath, Jeremy J; Merrell, D Scott; O'Brien, Alison D

    2013-06-01

    The likelihood that a single individual infected with the Shiga toxin (Stx)-producing, food-borne pathogen Escherichia coli O157:H7 will develop a life-threatening sequela called the hemolytic uremic syndrome is unpredictable. We reasoned that conditions that enhance Stx binding and uptake within the gut after E. coli O157:H7 infection should result in greater disease severity. Because the receptor for Stx, globotriaosylceramide, is up-regulated in the presence of butyrate in vitro, we asked whether a high fiber diet (HFD) that reportedly enhances butyrate production by normal gut flora can influence the outcome of an E. coli O157 infection in mice. To address that question, groups of BALB/c mice were fed high (10%) or low (2%) fiber diets and infected with E. coli O157:H7 strain 86-24 (Stx2+). Mice fed an HFD exhibited a 10- to 100-fold increase in colonization, lost 15% more body weight, exhibited signs of morbidity, and had 25% greater mortality relative to the low fiber diet (LFD)-fed group. Additionally, sections of intestinal tissue from HFD-fed mice bound more Stx1 and expressed more globotriaosylceramide than did such sections from LFD-fed mice. Furthermore, the gut microbiota of HFD-fed mice compared with LFD-fed mice contained reduced levels of native Escherichia species, organisms that might protect the gut from colonization by incoming E. coli O157:H7. Taken together, these results suggest that susceptibility to infection and subsequent disease after ingestion of E. coli O157:H7 may depend, at least in part, on individual diet and/or the capacity of the commensal flora to produce butyrate.

  2. Inactivation of Escherichia coli O157:H7 in apple juice and apple cider by trans-cinnamaldehyde.

    PubMed

    Baskaran, Sangeetha Ananda; Amalaradjou, Mary Anne Roshni; Hoagland, Thomas; Venkitanarayanan, Kumar

    2010-06-30

    This study investigated the antimicrobial effect of low concentrations of trans-cinnamaldehyde (TC) on Escherichia coli O157:H7 in apple juice and apple cider. A five-strain mixture of E. coli O157:H7 was inoculated into apple juice or cider at approximately 6.0 log CFU/ml, followed by the addition of TC (0%v/v, 0.025%v/v, 0.075%v/v and 0.125%v/v). The inoculated apple juice samples were incubated at 23 degrees C and 4 degrees C for 21 days, whereas the cider samples were stored only at 4 degrees C. The pH of apple juice and cider, and E. coli O157:H7 counts were determined on days 0, 1, 3, 5, 7, 14 and 21. TC was effective (P<0.05) in inactivating E. coli O157:H7 in apple juice and apple cider. At 23 degrees C, 0.125 and 0.075%v/v TC completely inactivated E. coli O157:H7 in apple juice (negative by enrichment) on days 1 and 3, respectively. At 4 degrees C, 0.125 and 0.075%v/v TC decreased the pathogen counts in the juice and cider to undetectable levels on days 3 and 5, respectively. Results indicate that low concentrations of TC could be used as an effective antimicrobial to inactivate E. coli O157:H7 in apple juice and apple cider.

  3. Survival of Escherichia coli O157:H7 in dry fermented sausages containing micro-encapsulated probiotic lactic acid bacteria.

    PubMed

    Muthukumarasamy, Parthiban; Holley, Richard A

    2007-02-01

    Escherichia coli O157:H7 is capable of surviving the rigorous processing steps during the manufacture of dry fermented sausages. The effect of adding two probiotic organisms, Lactobacillus reuteri and Bifidobacterium longum as co-cultures with the meat starter cultures Pediococcus pentosaceus and Staphylococcus carnosus on the viability of E. coli O157:H7 in dry fermented sausages was studied. A 5 strain cocktail of E. coli O157:H7 was added at 7.4 log cfu/g to the sausage batter and challenged with either or both Lb. reuteri or B. longum before or after they were micro-encapsulated. Sausages were fermented at < or = 26 degrees C and 88% relative humidity (RH) followed by drying at 75% RH and 13 degrees C for 25 d. The pH, water activity (aw), protein, moisture, and numbers of all inoculated organisms were monitored during processing. The pH and aw decreased from 5.7 and 0.98 to 4.9 and 0.88 at the end of fermentation and drying, respectively. These processes reduced E. coli O157:H7 by 1.0 and 0.7 log cfu/g at the end of fermentation and drying, respectively. Unencapsulated Lb. reuteri with or without B. longum reduced E. coli O157:H7 by 3.0 log cfu/g and B. longum caused a 1.9 log cfu/g reduction. While micro-encapsulation increased survival of Lb. reuteri and B. longum, it reduced their inhibitory action against E. coli O157:H7. PMID:16943098

  4. Effectiveness of Active Packaging on Control of Escherichia Coli O157:H7 and Total Aerobic Bacteria on Iceberg Lettuce.

    PubMed

    Lu, Haixia; Zhu, Junli; Li, Jianrong; Chen, Jinru

    2015-06-01

    Contaminated leafy green vegetables have been linked to several outbreaks of human gastrointestinal infections. Antimicrobial interventions that are adoptable by the fresh produce industry for control of pathogen contamination are in great demand. This study was undertaken to evaluate the efficacy of sustained active packaging on control of Escherichia coli O157:H7 and total aerobic bacteria on lettuce. Commercial Iceberg lettuce was inoculated with a 3-strain mixture of E. coli O157:H7 at 10(2) or 10(4) CFU/g. The contaminated lettuce and un-inoculated controls were placed respectively in 5 different active packaging structures. Traditional, nonactive packaging structure was included as controls. Packaged lettuce was stored at 4, 10, or 22 °C for 3 wk and sampled weekly for the population of E. coli O157:H7 and total aerobic bacteria. Results showed that packaging structures with ClO2 generator, CO2 generator, or one of the O2 scavengers effectively controlled the growth of E. coli O157:H7 and total aerobic bacteria under all storage conditions. Packaging structure with the ClO2 generator was most effective and no E. coli O157:H7 was detected in samples packaged in this structure except for those that were inoculated with 4 log CFU/g of E. coli O157:H7 and stored at 22 °C. Packaging structures with an oxygen scavenger and the allyl isothiocyanate generator were mostly ineffective in control of the growth of the bacteria on Iceberg lettuce. The research suggests that some of the packaging structures evaluated in the study can be used to control the presence of foodborne pathogens on leafy green vegetables.

  5. Bacteriophage 2851 Is a Prototype Phage for Dissemination of the Shiga Toxin Variant Gene 2c in Escherichia coli O157:H7▿ †

    PubMed Central

    Strauch, Eckhard; Hammerl, Jens Andre; Konietzny, Antje; Schneiker-Bekel, Susanne; Arnold, Walter; Goesmann, Alexander; Pühler, Alfred; Beutin, Lothar

    2008-01-01

    The production of Shiga toxin (Stx) (verocytotoxin) is a major virulence factor of Escherichia coli O157:H7 strains (Shiga toxin-producing E. coli [STEC] O157). Two types of Shiga toxins, designated Stx1 and Stx2, are produced in STEC O157. Variants of the Stx2 type (Stx2, Stx2c) are associated with high virulences of these strains for humans. A bacteriophage designated 2851 from a human STEC O157 encoding the Stx2c variant was described previously. Nucleotide sequence analysis of the phage 2851 genome revealed 75 predicted coding sequences and indicated a mosaic structure typical for lambdoid phages. Analyses of free phages and K-12 phage 2851 lysogens revealed that upon excision from the bacterial chromosome, the loss of a phage-encoded IS629 element leads to fusion of phage antA and antB genes, with the generation of a recombined antAB gene encoding a strong antirepressor. In wild-type E. coli O157 as well as in K-12 strains, phage 2851 was found to be integrated in the sbcB locus. Additionally, phage 2851 carries an open reading frame which encodes an OspB-like type III effector similar to that found in Shigella spp. Investigation of 39 stx2c E. coli O157 strains revealed that all except 1 were positive for most phage 2851-specific genes and possessed a prophage with the same border sequences integrated into the sbcB locus. Phage 2851-specific sequences were absent from most stx2c-negative E. coli O157 strains, and we suggest that phage 2851-like phages contributed significantly to the dissemination of the Stx2c variant toxin within this group of E. coli. PMID:18824528

  6. Factors contributing to the emergence of Escherichia coli O157 in Africa.

    PubMed Central

    Effler, E.; Isaäcson, M.; Arntzen, L.; Heenan, R.; Canter, P.; Barrett, T.; Lee, L.; Mambo, C.; Levine, W.; Zaidi, A.; Griffin, P. M.

    2001-01-01

    In 1992, a large outbreak of bloody diarrhea caused by Escherichia coli O157 infections occurred in southern Africa. In Swaziland, 40,912 physician visits for diarrhea in persons ages >5 years were reported during October through November 1992. This was a sevenfold increase over the same period during 1990-91. The attack rate was 42% among 778 residents we surveyed. Female gender and consuming beef and untreated water were significant risks for illness. E. coli O157:NM was recovered from seven affected foci in Swaziland and South Africa; 27 of 31 patient and environmental isolates had indistinguishable pulsed-field gel electrophoresis patterns. Compared with previous years, a fivefold increase in cattle deaths occurred in October 1992. The first heavy rains fell that same month (36 mm), following 3 months of drought. Drought, carriage of E. coli O157 by cattle, and heavy rains with contamination of surface water appear to be important factors contributing to this outbreak. PMID:11747693

  7. Vero cytotoxin-producing Escherichia coli O157 gastroenteritis in farm visitors, North Wales.

    PubMed

    Payne, Christopher J I; Petrovic, Marko; Roberts, Richard J; Paul, Ashish; Linnane, Eithne; Walker, Mark; Kirby, David; Burgess, Anthony; Smith, Robert M M; Cheasty, Thomas; Willshaw, Geraldine; Salmon, Roland L

    2003-05-01

    An outbreak of Vero cytotoxin-producing Escherichia coli O157 (VTEC O157) gastroenteritis in visitors to an open farm in North Wales resulted in 17 primary and 7 secondary cases of illness. E. coli O157 Vero cytotoxin type 2, phage type 2 was isolated from 23 human cases and environmental animal fecal samples. A case-control study of 16 primary case-patients and 36 controls (all children) showed a significant association with attendance on the 2nd day of a festival, eating ice cream or cotton candy (candy floss), and contact with cows or goats. On multivariable analysis, only the association between illness and ice cream (odds ratio [OR]=11.99, 95% confidence interval [CI] 1.04 to 137.76) and cotton candy (OR=51.90, 95% CI 2.77 to 970.67) remained significant. In addition to supervised handwashing, we recommend that foods on open farms only be eaten in dedicated clean areas and that sticky foods be discouraged. PMID:12737734

  8. Vero Cytotoxin–Producing Escherichia coli O157 Gastroenteritis in Farm Visitors, North Wales

    PubMed Central

    Petrovic, Marko; Roberts, Richard J.; Paul, Ashish; Linnane, Eithne; Walker, Mark; Kirby, David; Burgess, Anthony; Smith, Robert M.M.; Cheasty, Thomas; Willshaw, Geraldine; Salmon, Roland L.

    2003-01-01

    An outbreak of Vero cytotoxin–producing Escherichia coli O157 (VTEC O157) gastroenteritis in visitors to an open farm in North Wales resulted in 17 primary and 7 secondary cases of illness. E. coli O157 Vero cytotoxin type 2, phage type 2 was isolated from 23 human cases and environmental animal fecal samples. A case-control study of 16 primary case-patients and 36 controls (all children) showed a significant association with attendance on the 2nd day of a festival, eating ice cream or cotton candy (candy floss), and contact with cows or goats. On multivariable analysis, only the association between illness and ice cream (odds ratio [OR]=11.99, 95% confidence interval [CI] 1.04 to 137.76) and cotton candy (OR=51.90, 95% CI 2.77 to 970.67) remained significant. In addition to supervised handwashing, we recommend that foods on open farms only be eaten in dedicated clean areas and that sticky foods be discouraged. PMID:12737734

  9. Escherichia coli O157:H7 Outbreak Associated with Restaurant Beef Grinding.

    PubMed

    Torso, Lauren M; Voorhees, Ronald E; Forest, Stephen A; Gordon, Andrew Z; Silvestri, Sharon A; Kissler, Bonnie; Schlackman, Jessica; Sandt, Carol H; Toma, Paul; Bachert, Joel; Mertz, Kristen J; Harrison, Lee H

    2015-07-01

    Escherichia coli O157:H7 is a common cause of foodborne illness in the United States. Beef ground at establishments regulated by the U.S. Department of Agriculture, Food Safety and Inspection Service is routinely tested for E. coli O157:H7. Prior to December 2013, boxed beef product (wholesale cuts of beef, such as beef loin, packaged into bags and boxed for shipping) was not always tested for this pathogen. Downstream processors or retailers may grind the product; and, if the ground beef is not cooked to the recommended temperature, pathogens on the exterior of the beef introduced to the interior through grinding may survive. On 18 October 2013, the Allegheny County Health Department identified two E. coli O157:H7 cases, both of whom were food handlers at restaurant A, a restaurant that ground locally produced boxed beef for hamburgers on site. Case finding was conducted through public messaging, employee surveys, and disease surveillance. All potential cases were interviewed using a standard questionnaire. A confirmed case was defined as laboratory-confirmed E. coli O157:H7 with exposure to restaurant A. A probable case was defined as a patient with compatible symptoms and exposure to restaurant A but without laboratory confirmation. All human and food isolates were characterized by pulsed-field gel electrophoresis and multilocus variable-number tandem repeat analysis. The analysis identified 14 confirmed and 10 probable cases of E. coli; 18 nonintact ground beef samples tested positive for E. coli O157:H7. Nine confirmed cases were restaurant A employees. All confirmed cases recalled eating a restaurant A hamburger in the 10 days before illness onset; most cases reported consuming medium to rare hamburgers. Multiple pulsed-field gel electrophoresis and multilocus variable-number tandem repeat analysis patterns were identified among both the human and ground beef isolates, and the patient isolates matched those found in ground beef samples. Restaurant A

  10. Escherichia coli O157:H7 Outbreak Associated with Restaurant Beef Grinding.

    PubMed

    Torso, Lauren M; Voorhees, Ronald E; Forest, Stephen A; Gordon, Andrew Z; Silvestri, Sharon A; Kissler, Bonnie; Schlackman, Jessica; Sandt, Carol H; Toma, Paul; Bachert, Joel; Mertz, Kristen J; Harrison, Lee H

    2015-07-01

    Escherichia coli O157:H7 is a common cause of foodborne illness in the United States. Beef ground at establishments regulated by the U.S. Department of Agriculture, Food Safety and Inspection Service is routinely tested for E. coli O157:H7. Prior to December 2013, boxed beef product (wholesale cuts of beef, such as beef loin, packaged into bags and boxed for shipping) was not always tested for this pathogen. Downstream processors or retailers may grind the product; and, if the ground beef is not cooked to the recommended temperature, pathogens on the exterior of the beef introduced to the interior through grinding may survive. On 18 October 2013, the Allegheny County Health Department identified two E. coli O157:H7 cases, both of whom were food handlers at restaurant A, a restaurant that ground locally produced boxed beef for hamburgers on site. Case finding was conducted through public messaging, employee surveys, and disease surveillance. All potential cases were interviewed using a standard questionnaire. A confirmed case was defined as laboratory-confirmed E. coli O157:H7 with exposure to restaurant A. A probable case was defined as a patient with compatible symptoms and exposure to restaurant A but without laboratory confirmation. All human and food isolates were characterized by pulsed-field gel electrophoresis and multilocus variable-number tandem repeat analysis. The analysis identified 14 confirmed and 10 probable cases of E. coli; 18 nonintact ground beef samples tested positive for E. coli O157:H7. Nine confirmed cases were restaurant A employees. All confirmed cases recalled eating a restaurant A hamburger in the 10 days before illness onset; most cases reported consuming medium to rare hamburgers. Multiple pulsed-field gel electrophoresis and multilocus variable-number tandem repeat analysis patterns were identified among both the human and ground beef isolates, and the patient isolates matched those found in ground beef samples. Restaurant A

  11. Multistate outbreak of Escherichia coli O157:H7 infections associated with in-store sampling of an aged raw-milk Gouda cheese, 2010.

    PubMed

    McCollum, J T; Williams, N J; Beam, S W; Cosgrove, S; Ettestad, P J; Ghosh, T S; Kimura, A C; Nguyen, L; Stroika, S G; Vogt, R L; Watkins, A K; Weiss, J R; Williams, I T; Cronquist, A B

    2012-10-01

    In 2010, 41 patients ill with Escherichia coli O157:H7 isolates determined to be indistinguishable by pulsed-field gel electrophoresis were identified among residents of five Southwestern U.S. states. A majority of patients reported consuming complimentary samples of aged raw-milk Gouda cheese at national warehouse chain store locations; sampling Gouda cheese was significantly associated with illness (odds ratio, 9.0; 95 % confidence interval, 1.7 to 47). Several Gouda samples yielded the O157:H7 outbreak strain, confirming the food vehicle and source of infections. Implicated retail food-sampling operations were inconsistently regulated among affected states, and sanitation deficiencies were common among sampling venues. Inspection of the cheese manufacturer indicated deficient sanitation practices and insufficient cheese curing times. Policymakers should continue to reexamine the adequacy and enforcement of existing rules intended to ensure the safety of raw-milk cheeses and retail food sampling. Additional research is necessary to clarify the food safety hazards posed to patrons who consume free food samples while shopping.

  12. Detection of viable Escherichia coli O157:H7 in ground beef by propidium monoazide real-time PCR.

    PubMed

    Liu, Yarui; Mustapha, Azlin

    2014-01-17

    Escherichia coli O157:H7 associated with food has caused many serious public health problems in recent years. However, only viable cells of this pathogen can cause infections, and false-positive detection caused by dead cells can lead to unnecessary product recalls. The objective of this study was to develop and optimize a method that combines propidium monoazide (PMA) staining with real-time PCR to detect only viable cells of E. coli O157:H7 in ground beef. PMA is a DNA intercalating dye that can penetrate compromised membranes of dead cells and bind to cellular DNA, preventing its amplification via a subsequent PCR. Three strains of E. coli O157:H7 (505B, G5310 and C7927) at concentrations of 10(0) to 10(8)CFU/mL were used as live cells. Dead cells were obtained by heating cell suspensions at 85°C for 15 min. Suspensions were treated with PMA and the optimized assay was applied to artificially contaminated ground beef with two different fat contents (10% and 27%). DNA was extracted and amplified by TaqMan® real-time PCR assay targeting the uidA gene for detection of E. coli O157:H7. Plasmid pUC19 was added as an internal amplification control (IAC). A treatment of 25 μM PMA with a 10-min light exposure on ice was sufficient to eliminate DNA from 10(8) dead E. coli O157