<p>Though pathogenic strains represent a small portion of the total variety of existing <em>Escherichia coli </em>strains, they contribute extensively to human morbidity and mortality. Disease outbreaks caused by enterohaemorrhagic <em>E. coli</em> of the serotype O157:H7 and the “Big Six” serotypes (i.e., O26, O45, O103, O111, O121 and O145) have driven the development of assays for pathogen detection. From culture-based assays requiring several days for confirmation of target organisms, to quantitative PCR (qPCR) tests that provide pathogen identification in several hours’ time, the sensitivity, specificity and speed of bacterial diagnostics have seen improvements that increased the efficacy of assays used to detect pathogens at clinically relevant levels. One relatively unexplored field of diagnostics is the use of conserved signature insertion/deletions (CSIs) as stable genetic markers for pathogen detection. This thesis presents two qPCR assays that target an <em>E. coli</em> O157:H7-specific insertion in a CSI. In a more preliminary study, an EvaGreen-based qPCR assay was developed that had a detection limit of 16 <em>E. coli</em> O157:H7 genome equivalents. An improved format of the O157:H7-specific CSI assay, using TaqMan probes, was later established. TaqMan probes are sequence-specific, while DNA-intercalating EvaGreen dye is sequence-independent. Though the TaqMan probe-based assay had a higher detection limit of 100 genome equivalents, the assay maintained detection sensitivity in presence of genetically similar (<em>E. coli</em> K-12) and dissimilar (fish sperm) DNA in excess amounts (1000-fold and 800-fold excess of target DNA, respectively), demonstrating its potential for pathogen detection in environmental samples where the presence of background flora may influence detection. These assays thus represent an exploration into the use of CSIs as diagnostic tools. This thesis also provides a guide for future developments of pathogen detection using CSIs, such as those that may be present in toxigenic species of Cyanobacteria and human pathogens, including <em>Vibrio</em> and <em>Campylobacter</em>.</p> / Master of Science (MSc)

Fresh produce is increasingly associated with foodborne outbreaks. In order to develop effective intervention and measures to reduce microbial risks, it is essential to attain a better understand the mechanisms of attachment of foodborne pathogens to fruits and vegetables. Using lettuce as a model, the attachment of Escherichia coli O157:H7 to produce surfaces was studied. Strains expressing various extracellular proteins (curli, O157-antigen, and intimin) known to influence attachment of E. coli to intestinal cells were evaluated for their physicochemical properties and ability to adhere to cut edge and whole leaf lettuce. Escherichia coli O157:H7 strains included: 0018, 43894 and 43895 (curli producing and non-producing); 86-24 (WT), F-12 (O157-antigen negative), pRFBE (O-antigen replaced on plasmid); and 86-24, 86-24Ã eae10 (intimin negative). The eleven strains were surveyed for their hydrophobicity and cell charge using hydrophobic interaction chromatography (HIC) and electrostatic interaction chromatography (ESIC) techniques. Iceberg lettuce squares (2 x 2 cm) were inoculated with E. coli O157:H7 strains separately (7.0 log CFU/square) and dried in a laminar flow hood. Lettuce was sampled before (unrinsed) and after being rinsed twice with sterile de-ionized water (rinsed). Strips (2 mm wide) of each cut edge of the lettuce were aseptically removed. Cut-edge and whole-leaf samples were homogenized and spiral plated onto Luria-Bertani agar, supplemented with nalidixic acid (50ppm), to assess levels of bacteria remaining on the lettuce leaf after rinsing. The rinse steps were not effective in significantly removing bacteria from lettuce (p>0.05). Curli-producing and non-producing strains preferentially attached to cut edge versus the whole leaf portions of lettuce (p<0.05); however the 86-24 strains showed no preference for attachment. With the exception of 0018 curli-producing and non-producing strains, presence/absence of extracellular proteins surveyed did not influence attachment of E. coli O157:H7 to either cut edge or whole leaf lettuce. There was significantly greater attachment of the curli-producing 0018 strain over the curli non-producing 0018 strain to cut and whole lettuce surfaces (p<0.05). Production of curli and O-polysaccharide significantly increased (p<0.05) the cell's overall hydrophobicity of the cell; however this did not affect attachment (p<0.05). The overall cell charge of all strains was negative; however, charge did not affect attachment of E. coli O157:H7 to lettuce. The presence of extracellular appendages (curli, O157-antigen, intimin) as well as hydrophobicity and cell charge properties had no affect on attachment of the cell to lettuce. / Ph. D.

The antibacterial efficacy of hydrogen peroxide treatments in four fruit juices was determined. Preservative free apple cider, white grape, and purple grape juice were inoculated with ~ 6.4 log CFU/ml of a five strain, acid adapted, nalidixic acid resistant E. coli O157:H7 cocktail. Orange juice was inoculated with a comparable Salmonella spp. cocktail. In the first study, 0.017% and 0.012% H₂O₂ was added in combination with 0.1% and 0.3% of the dominant organic acid (OA) to 4°C and 25°C juices, with samples taken each day for 21 days. H₂O₂ was a significant factor in all juices (p < 0.05) except white grape (lack of data), and both 0.017% H₂O₂ treatments reduced counts in apple cider, orange juice, and white grape to undetectable numbers within 48 hrs as cultured on tryptone soy agar + 0.05% nalidixic acid (TSAN). Treatments in purple grape juice were less effective overall, and more dependent on OA concentration (p < 0.001) than H₂O₂. There were instances where bacterial survival in apple cider, purple grape, and orange juice continued for 21 days after treatment, and sometimes outlasted the control. These occurrences were dependent on temperature (25°C) and H₂O₂ (0.012%), but not on OA. However, OA concentration was a significant factor (p < 0.05) overall in apple cider and purple grape juice, but not in orange juice. In the second study, 0.015% and 0.03% H₂O₂ was added to 10, 25, and 40°C apple cider and orange juice inoculated with 6.4 log CFU/ml E. coli O157:H7 and Salmonella spp. respectively. Only 0.03% H₂O₂ was effective in reducing counts to undetectable numbers in both juices. However, both temperature and H₂O₂ were significant factors (p < 0.0001) in bacterial destruction, with 0.03% H₂O₂ at 40°C giving undetectable numbers at ≤ 3 and ≤ 6 hours in orange juice and apple cider respectively. It has been demonstrated that at ~ ≥ 0.017%, H₂O₂ can provide a 5 log reduction of these pathogens in fruit juice. Increasing temperature and organic acid concentration can improve its rate of effectiveness in certain juices. However, sensory concerns may negate its use in some products. / Master of Science

Cinnamic acid (CA) is a naturally occurring organic acid that is found in some fruits and a number of spices. CA has antimicrobial activity against certain spoilage microorganisms and pathogenic bacteria. However, the acid is poorly soluble in water. Cyclodextrin molecules have a hydrophobic cavity that allows them to serve as a host for insoluble molecules in aqueous matrices. This study was conducted to determine if the aqueous solubility of cinnamic acid could be improved via complexation with α- or β-cyclodextrins, and if these complexes could be used to control bacterial pathogens in juices. Based upon phase solubility analysis, α-cyclodextrin was chosen as the host molecule for the remainder of this study. In complex with α-cyclodextrin, the solubility of cinnamic acid increased from approximately 400 mg/L to 3800 mg/L. Prepared cinnamic acid complexed with α-cyclodextrin was aseptically added (400 mg/L and 1000 mg/L) to orange juice inoculated with a Salmonella enterica (7 log CFU/mL) and apple cider inoculated with Escherichia coli O157:H7 (7 log CFU/mL). Cider and orange juice samples were extracted on day 0 and at 24 h intervals for seven days and spread plated onto Tryptic Soy Agar. Cinnamic acid was effective for reducing populations of both bacterial pathogens in juice. Populations of E. coli O157:H7 in the apple cider were significantly reduced after 7 days at 25.6 ± 0.42°C at concentrations of 400 mg/L (5-log CFU/mL reduction) and 1000 mg/L (6-log CFU/mL reduction) cyclodextrin-cinnamic acid. S. enterica counts were also reduced in orange juice at 4° C treated with 400 mg/L (2.7-log CFU/mL reduction) and 1000 mg/L (3.2-log CFU/mL reduction) complexed cinnamic acid. The much improved solubility of this compound provides food processors with greater flexibility in using cinnamic acid in their product formulations. / Master of Science

Contaminated wheat flour has been identified as the probable vehicle of a multi-state outbreak of Escherichia coli O157:H7 associated with ready-to-bake cookie dough. Several cookie dough manufacturers are currently using heat-treated flour for ready-to-bake products, although data on thermal inactivation of foodborne pathogens in wheat flour remains scarce. The objective of this research was to first determine appropriate methods and parameters for bacterial inoculation and thermal treatment of wheat flour, and to subsequently determine the population reductions of E. coli O157:H7 and Salmonella Agona in artificially contaminated wheat flour following thermal treatment for 1, 5, 15 or 30 minutes at 55, 60, 65 or 70°C in a shaking water bath. Flour samples (aw = 0.55) in sterile plastic bags were individually inoculated (~109 CFU/g), pulsified to distribute cultures, and pressed to a uniform thickness (1mm) prior to heat treatment. Following treatment, samples were rapidly cooled and diluted with peptone water; then plated onto Tryptic Soy Agar (TSA) and incubated at 37°C for 24 h prior to enumeration. The minimum heat treatments required for a 5-log reduction in microbial populations (~ 109CFU/g to ~ 104CFU/g) were 5 minutes at 70°C and 30 minutes at 70°C for E. coli O157:H7 and S. Agona, respectively. This research supports the hypothesis that the microbiological safety of ready-to-bake products may be improved by the use of heat-treated flour. / Master of Science in Life Sciences

Les Escherichia coli entérohémorragiques (EHEC) sont responsables de toxi-infections alimentaires conduisant à des colites hémorragiques pouvant se compliquer d’un syndrome hémolytique et urémique. Une fois arrivés dans l’intestin, les EHEC adhèrent aux cellules épithéliales en causant des lésions d’attachement-effacement. Le système de sécrétion de type III et les protéines effectrices requis pour ce phénotype sont codés majoritairement par le locus d’effacement des entérocytes (LEE), constitué de plusieurs opérons (LEE1-5). Notre étude a permis de clarifier une des cascades de régulation contrôlant l’expression du LEE. Par des analyses en qRT-PCR et des immuno précipitations de la chromatine, nous avons déterminé que les régulateurs GadE et GadX sont des répresseurs indirects de l’expression du LEE. GadE active l’expression de gadX, et GadX réprime l’expression de ler, codant pour le principal activateur des opérons LEE2-5. De plus, GadE réprime aussi l’expression des opérons LEE4 et LEE5 indépendamment de Ler. En retour, Ler réprime l’expression de gadE et de gadX. Le monoxyde d’azote (NO) est un effecteur majeur de la réponse immune innée, produit en particulier par les cellules épithéliales intestinales. Il avait été montré que le NO réprime l’expression du LEE et active celle de gadE et de gadX. Notre étude a permis d’identifier le régulateur clé responsable de ces régulations, NsrR. NsrR réprime indirectement l’expression de gadE et gadX et active l’expression des opérons LEE1, LEE4 et LEE5 en se fixant sur leurs promoteurs respectifs. En présence de NO, NsrR devient inactif. Ainsi, le NO réprime directement l’expression du LEE en supprimant la fixation de NsrR aux promoteurs du LEE1, LEE4 et LEE5, et indirectement en activant l’expression de gadE et donc de gadX. Un modèle de régulation intégrant l’ensemble de ces résultats est proposé. D’autre part, nous avons identifié et caractérisé une nouvelle phosphodiestérase spécifique des EHEC les plus pathogènes, VmpA. Par son activité d’hydrolyse du di-GMPc, VmpA contrôle la mobilité bactérienne, la formation de biofilm, et probablement l’expression du LEE, mais aurait aussi un rôle plus général dans la physiologie des EHEC. / Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen causing hemorrhagic colitis and Hemolytic and Uremic Syndrome (HUS). After reaching the gut, EHEC adhere to the epithelial intestinal cells causing attachment/effacement lesions (A/E lesions). The locus of enterocyte effacement (LEE) encodes for a type three secretion system and several effector proteins required for A/E lesions. The LEE is composed of five main operons (LEE1-5). In this work we identified the molecular mechanisms of one of the regulatory cascades controlling LEE expression. Using qRT-PCR and chromatin immunoprécipitation we determined that GadE and GadX are two indirect repressors of LEE expression. GadE activates gadX expression, and GadX represses ler expression, the latter encoding the main activator of LEE2-5 operons. Moreover, GadE also represses LEE4 and LEE5 expression independently of Ler. In turn, Ler represses gadE and gadX expression. Nitric oxide (NO) is a crucial effector of the innate immune response, in part produced by intestinal epithelial cells. It has been shown previously that NO represses LEE and activates gadE and gadX expression. In this study we identified the key regulator responsible for these regulations: NsrR. NsrR represses indirectly gadE and gadX expression and activates LEE1, LEE4 and LEE5 expression by binding to their respective promoter. In the presence of NO, NsrR is inactivated. Thus, NO directly represses LEE expression by relieving NsrR binding to the LEE1, LEE4 and LEE5 promoters, and indirectly by activating gadE and gadX expression. A regulatory model is proposed based on these results.In addition, we identified and characterized a new phosphodiesterase which is specific for the most virulent EHEC strains: VmpA. By degrading c-di-GMPc, VmpA controls motility, biofilms formation, and probably LEE expression. It would also have a global effect on EHEC physiology.

Les Escherichia coli entérohémorragiques (EHEC) sont des E. coli producteurs de Shiga-toxines (STEC) représentant le quatrième agent responsable de toxi-infections alimentaires en Europe. La contamination par ces pathogènes résulte principalement de l’ingestion de produits alimentaires contaminés par les fèces de bovins, dont le tube digestif apparait comme le principal réservoir naturel des EHEC. Ces pathogènes survivent dans le tractus digestif du ruminant, qui est porteur sain, et semblent bien adaptés à l’ensemble de cet écosystème complexe. Réduire le portage animal est une stratégie de choix afin de limiter les toxi-infections humaines à EHEC. L’objectif de cette thèse était d’approfondir les connaissances sur la physiologie et l’écologie des EHEC dans le tube digestif du bovin, une étape primordiale pour proposer, à terme, différentes stratégies visant à limiter le portage. L’analyse du transcriptome de la souche EHEC O157:H7 de référence EDL933 a permis l’identification de voies métaboliques utilisées par les EHEC dans différents compartiments du tube digestif de l’animal. Certains sucres, dont ceux issus de la couche de mucus intestinal, et acides aminés ainsi que l’éthanolamine semblent représenter des substrats importants pour la survie des EHEC tout au long du tube digestif du bovin. Cette étude transcriptomique a également mis en évidence l’activation, par la souche EHEC, de nombreux systèmes de résistance à différents stress rencontrés dans le tube digestif bovin, dont les systèmes toxines/anti-toxines. L’activation de ces systèmes et la capacité à former des biofilms ont également été observées chez une souche STEC O157:H7 d’origine bovine, la souche MC2, dans des conditions mimant une persistance dans l’environnement. La caractérisation génomique et phénotypique permet de considérer cette souche comme pathogène et des études réalisées in vitro et in vivo ont indiqué que la souche MC2 était capable de persister dans le tube digestif du bovin mais aussi dans l’environnement de l’élevage. L’inoculation expérimentale de bovins par la souche MC2 a permis de mettre au point le premier modèle animal reproductible de portage et d’excrétion des STEC O157:H7 décrit en France. Ce modèle pourra être utilisé pour tester in vivo l’effet d’additifs alimentaires, tels que les probiotiques, afin de réduire le portage et l’excrétion de souches EHEC par les bovins, et donc limiter la contamination de l’Homme. / Enterohaemorrhagic Escherichia coli (EHEC) are Shiga-toxin producing E. coli (STEC) which represent the fourth pathogen leading to foodborne illness in Europe. Contamination by these pathogens results mainly from the ingestion of food contaminated by feces of bovine, for which the digestive tract appears as the main natural reservoir of EHEC. These pathogens survive in the digestive tract of ruminants, which is healthy carriers, and seem well-adapted to this complex ecosystem. Reducing animal carriage is a strategy of choice to limit EHEC human infections. The aim of this thesis was to increase our knowledge on the physiology and ecology of EHEC in the digestive tract of bovine, a key step to propose, ultimately, different strategies to limit the carriage. Transcriptome analysis of the EHEC O157:H7 reference strain EDL933 allowed the identification of metabolic pathways used by EHEC in different compartments of the digestive tract of the animal. Some carbohydrates, including those from the intestinal mucus layer, and amino acids as well as ethanolamine appear to be important substrates for the survival of EHEC throughout the bovine digestive tract. This transcriptomic study also revealed the activation, by the EHEC strain, of several stress resistance systems encountered in the bovine digestive tract, including toxin/anti-toxin systems. The activation of these systems and the ability to form biofilms have also been observed in a bovine STEC O157:H7 strain, MC2 strain, under conditions mimicking persistence in the environment. Genomic and phenotypic characterization allows this strain to be considered as pathogenic and in vitro and in vivo studies indicated that the MC2 strain was able to persist in the bovine digestive tract but also in the farm environment. The experimental inoculation of bovines with the MC2 strain led to the development, for the first time in France, of a reproducible animal model of carriage and excretion of STEC O157:H7. This model could be used to test in vivo the effect of food additives, such as probiotics, in order to reduce the carriage and excretion of EHEC strains by bovines, and thus limit the contamination of humans.

Master of Science / Food Science / Kelly J.K. Getty / James L. Marsden / Escherichia coli O157:H7 outbreaks have been linked to leafy green produce and bagged spinach. The objective of this study was to evaluate a Photohydroionization (PHI) panel (novel advanced oxidation technology) and varying concentrations of lactic acid washes for controlling E. coli O157:H7 on baby spinach. Leaves were dip inoculated with a five-strain cocktail of E. coli O157:H7 inoculum having a concentration between 5-6 log CFU/ml. Leaves were submerged in inoculum for 30 s and dried for 1 h. Non-inoculated and inoculated leaves were washed for 30 s in food grade lactic acid diluted to concentrations of 0.5, 1.0, or 2.0% and allowed to dry for 10 min. For PHI treatment, leaves were placed under the PHI panel and treated for 1, 2, or 5 min on both sides for total treatment times of 2, 4 or 10 min. Following treatments, leaves were either sealed in low-density polyethylene bags or enumerated. Samples were enumerated at 0, 3, 7, 10, and 14 days following inoculation. Ten gram samples were diluted with sterile peptone and stomached for one min, and then 0.1 ml was plated onto sorbitol MacConkey agar with cefixime and tellurite plates that were incubated at 37°C for 24 h. For lactic acid treatments, E. coli O157:H7 populations were different (P < 0.05) compared to the control. There was no difference (P > 0.05) due to sampling time so sampling times where pooled together for each lactic acid concentration of 0.5, 1.0, and 2.0% and resulted in 2.01, 2.78, and 3.67 log CFU/g reductions, respectively. Leaves treated with 1.0% and 2.0% lactic acid had color degradation and were organoleptically unacceptable by day 14. When leaves were treated with PHI for 1, 2, or 5 min per side, E. coli O157:H7 populations were reduced 1.6, 1.49, or 1.95 log CFU/g, respectively. Leaves treated with PHI were not different from one another, but were different (P < 0.05) from the positive control. No color change occurred in leaves treated with PHI. The PHI panel and lactic acid washes of 0.5% or higher are effective in reducing E. coli O157:H7 in baby spinach.

E. coli O157:H7 é um patógeno de importância em alimentos, tendo sido envolvido, nos últimos anos, em surtos de grandes proporções, principalmente por produtos cárneos. Entretanto sua ocorrência em alimentos, particularmente em carne crua, é baixa e poderia, eventualmente, ser atribuída à atividade antagônica expressa por outros microrganismos presentes. Assim sendo, foi avaliada a interferência de bactérias que fazem parte da microbiota normal de carne sobre a multiplicação de E. coli O157:H7 em carne bovina moída mantida em refrigeração e em temperatura ambiente. Com essa finalidade, foram realizados testes de desafio (\"challenge tests\") em porções de 25 g de carne bovina moída inoculadas com diferentes concentrações de E. coli O157:H7 (101, 103 e 106 CFC/g), desafiadas com diferentes inóculos de E. coli não patogênica, Pseudomonas putida e Leuconostoc spp. As cepas de Pseudamonas putida e de Leuconostoc spp., isoladas de carne, foram selecionadas em função de atividade inibitória contra E. calí O157:H7 observada \"in vitro\". Para o monitoramento de E. coli O157:H7, foram utilizados o método convencional, ou seja, plaqueamento em ágar Mac Conkey-sorbitol e identificação de colônias (testes bioquímicos e sorológicos), bem como um método considerado rápido, empregando o Petrifilm&#8482; Kit-HEC. De maneira geral, não foram observadas interferências significativas da presença de diferentes inóculos de E. coli não patogênica, P. putida e Leuconostoc spp., sobre a multiplicação de diferentes inóculos de E. coli O157:H7 à temperatura ambiente e à temperatura de refrigeração. Paralelamente, o Petrifilm&#8482; Kit-HEC revelou um alto índice de correlação com o ágar Mac Conkey-sorbitol (97,2%), com contagens da mesma ordem de grandeza. Os experimentos à temperatura ambiente revelaram um maior índice de correlação (99,0%), quando comparados àqueles à temperatura de refrigeração (94,9%). Aparentemente, a baixa ocorrência de E. coli O157:H7 em alimentos, particularmente em carne bovina crua, não pode ser atribuída à atividade antagônica de alguns microrganismos presentes. / Escherichia coli O157:H7 is a foodborne pathogen of increasing importance, since it has been involved in several threatening outbreaks, most of them associated with meat products. Though, it is possible that the low occurrence af E. coli O157:H7 in food, particularly in meat, may be due to antagonistic effects af other microorganisms present. Therefore, the influence of some bacteria isolated from meat, over E. coli O157:H7 in meat samples stored at chill and room temperatures was evaluated. For that purpose, studies were performed on 25 g of ground beef inoculated with different spiking levels of E. coli O157:H7 (101, 103 and 106 CFC/g), challenged with different spiking levels of non pathogenic E. coli, Pseudomonas putida or Leuconostoc spp. The Ps. putida and Leuconostoc spp. strains were selected based on deferred antagonism observed against E. coli O157:H7. Multiplication was monitored by means of cultural methods, employing sorbitol Mac Conkey agar and additional identification tests, and the rapid method Petrifilm&#8482; Kit-HEC. No significant influence of non pathogenic E. coli, Pseudomonas putida and Leuconostoc spp. over the multiplication of E. coli O157:H7 was observed. Results on Petrifilm&#8482; Kit-HEC showed high correlation with results on sorbitol Mac Conkey agar (97,2%). Experiments performed with meat kept at room temperatures resulted in higher correlation values (99,0%), when compared to those of meat kept at chill temperatures (94,9%). Apparently, the low occurrence of E. coli O157:H7 in food, particularly in raw meat, can\'t be attributed to antagonistic effects of other bacteria from natural microflora.

As Escherichia (E.) coli produtoras de toxinas Shiga (STEC) compõem um dos mais importantes grupos de patógenos alimentares do mundo. Dentre as STEC, a E. coli O157:H7 tem sido a mais amplamente estudada, uma vez que pode causar diarréia sanguinolenta, anemia hemolítica, síndrome urêmica hemolítica (HUS) e púrpura trombótica trombocitopênica (TTP), sendo que a carne bovina tem sido um dos principais veículos desse microrganismo. O objetivo deste estudo foi investigar a presença de E. coli O157:H7 em amostras de carne moída coletadas no Estado do Rio Grande do Sul (RS), sul do Brasil. Para tanto, 95 amostras de carne moída foram coletadas em diferentes municípios do RS. Dentre essas amostras, três isolados foram identificadas como prováveis E. coli O157:H7, segundo os testes recomendados pelo USDA/FSIS. Nesses métodos as cepas cresceram em meio TSB adicionado de novobiocina e casaminoácido, foram positivas para o teste de “screening” utilizando anticorpos específicos, desenvolveram colônias típicas nos meios SMAC (MacConkey sorbitol) e SMAC-CT (Cefixina-telurito), após terem sido submetidas à separação imunomagnética (IMS), aglutinaram o anti-soro para a E. coli O157 e não apresentaram atividade de ß-glucoronidase. Após a caracterização genotípica por PCR Multiplex, investigando genes de virulência (rfbO157, stx1 e stx2), no Laboratório de referência para a vigilância regional de HUS e diarréias sanguinolentas no cone sul, do Ministério da Saúde da Argentina (INEI-ANLIS), os resultados apontaram que os três isolados foram negativos para os fatores de virulência, não produziram Shiga toxinas, não sendo classificados como E. coli 0157:H7. Cabe ressaltar que a caracterização genotípica dessas cepas dificilmente é realizada em indústrias de alimentos, e mesmo resultados falso-positivos para E. coli O157, como os demonstrados nesse trabalho, poderiam afetar significativamente o comércio nacional e internacional de carne bovina brasileira. / The Shiga toxin-producing Escherichia coli (STEC) is one of the most important food pathogen groups worldwide. Among the STEC, E. coli O157:H7 has been the most widely studied, once it may cause bloody or nonbloody diarrhea, hemolytic anemia, hemolytic uremic syndrome (HUS), and thrombotic thrombocytopenic purpura (TTP), being beef one of the main carriers of this microorganism. The aim of the present study was to investigate the presence of E. coli O157:H7 in ground beef samples collected in the State of Rio Grande do Sul (RS), Southern Brazil. Thus, 95 ground beef samples were collected in different cities of RS. Among the samples, three isolates were identified as probable E. coli O157:H7, according to tests recommended by USDA/FSIS. In these methods, the strains grew in TSB medium added to novobiocine and casamino acid, were positive in the screening test using specific antibodies, developed typical colonies in SMAC (MacConkey sorbitol) and SMAC-CT (Cefixime tellurite) media, after being subjected to Immunomagnetic Separation (IMS), agglutinated the antiserum to E. coli O157 and did not show ß-glucoronidase activity. After the genotypic characterization by PCR Multiplex, investigating virulence genes (rfbO157, stx1 and stx2), at the Reference laboratory for regional surveillance of HUS and bloody diarrheas in the Southern Cone, from the Ministry of Health of Argentina (INEI-ANLIS), the results demonstrated that the three isolates were negative for the virulence factors, did not produce Shiga toxins, not being classified as E. coli 0157:H7. It is worth mentioning that the genotypic characterization of these strains is hardly performed in food industries, and even false-positive results for E. coli O157, as the ones presented in this study, could significantly affect the national and international trade of Brazilian beef.