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™ 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™ 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™ 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™ 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.

The main objective of this study was to determine the pressure induced inactivation mechanism of pressure-resistant Escherichia coli O157:H7 933 and Staphylococcus aureus 485 in a low acid food. Firstly, inactivation curves of pathogens were obtained at 200 to 400 MPa at 40&ordm / C in peptone water and carrot juice. First-order and Weibull models were fitted and Weibull model described the inactivation curves of both pathogens more accurately than first-order model, revealing that food systems could exhibit either protective or sensitizing effect on microorganisms. Carrot juice had a protective effect on E. coli O157:H7 whereas it had a sensitizing effect on S. aureus, due to the naturally occurring constituents or phytoalexins in carrot roots that could have a toxic effect. Secondly, scanning electron microscopy (SEM) and fluorescent microscopy images of studied pathogens were taken. Developed software was used to analyze SEM images to calculate the change in the view area and volume of cells. Membrane integrity of pressurized cells was also examined using fluorescent microscopy images. The increase in average values of the view area and volume of both pathogens was significant for the highest pressure levels studied. The increase in volume and the view area could be explained by the modification of membrane properties, i.e., disruption or increase in permeability, lack of membrane integrity, denaturation of membrane-bound proteins and pressure-induced phase transition of membrane lipid bilayer. The change in volume and the view area of microorganisms added another dimension to the understanding of inactivation mechanisms of microbial cells by HHP.

Access to abstract permanently restricted to Ball State community only / Access to thesis permanently restricted to Ball State community only / Department of Biology

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.

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.

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™ 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™ 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™ 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™ 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.

Les bactéries pathogènes Escherichia coli entérohémorrhagiques (EHEC) O157:H7 causent des toxi-infections sévères chez l’humain. Les biofilms des EHEC rendent difficile leur contrôle dans les environnements favorisant leur persistance. Certaines souches O157:H7 dont celle de référence Sakai, possèdent une capacité accrue à s'autoagréger et former des biofilms. L’étude a visé à identifier les gènes impliqués dans l’autoagrégation et la formation de biofilm chez la souche Sakai, et vérifier l’association des facteurs identifiés avec d’autres EHEC O157:H7 du même phénotype. Avec une banque de mutants Tn10 de la souche Sakai, des mutants non-autoagglutinants et non-formateurs de biofilms des gènes csgB et csgG furent sélectionnés et caractérisés. Ces mutants formaient significativement moins de biofilms et d´autoagrégats que Sakai, et ne produisaient plus curli. La complémentation des mutants restaurait le phénotype sauvage. De plus, des gènes responsables de la biogenèse de curli csgA, csgB et csgG étaient significativement surexprimés chez Sakai, comparativement avec la souche EDL933 non-autoagglutinant, qui forme moins de biofilm. Parmi les souches d’E. coli 0157:H7 on distingue deux groupes d’isolats: Sakai-like et EDL933-like selon leur production de curli, et leur capacité à former des autoagrégats et biofilms. Nos résultats suggèrent qu’une surproduction de fibres de curli dans un sous-ensemble de souches O157:H7 pourrait être responsable de leur phénotype particulier d'autoagrégation et de formation de biofilms forts. Le projet permet de mieux cerner le mécanisme de formation de biofilm dans EHEC et renforce l’hypothèse que le curli est une cible intéressante pour contrer la persistance des EHEC en environnements naturels et industriels. / Enterohemorrhagic E. coli (EHEC) O157:H7 is an important foodborne pathogen that causes severe toxi-infections in humans. These bacteria have a higher capacity to form biofilm, impeding the control of the contamination in different environments and allowing their persistence. Some E. coli O157:H7 strains, including the Sakai reference strain, display a distinctive ability to autoaggregate and form strong biofilms. The aim of this work was to identify the genes involved in autoaggregation and biofilm formation in Sakai strain, and to verify the association between identified factors and the same phenotype in other EHEC O157: H7. We found that csgA, csgB and csgG curli genes were significantly overexpressed in strain Sakai compared to strain EDL933, a low biofilm-former and non-autoaglutinating strain. Sakai csgB and csgG Tn10 mutants formed significantly less biofilm and autoaggregation than the wild-type strain, and lost the curli-producing phenotype. Complementation restored the strong autoaggregation and biofilm formation phenotype, and the curliated morphotype of Sakai. In addition, E. coli O157: H7 isolates tested for curli formation, Sakai-like strains were curli-producing, whereas EDL933-like strains were non-curliated. These results suggest that overproduction of extracellular curli fibers in a subset of E. coli O157: H7 strains may be responsible for their particular phenotype of autoaggregation and strong biofilm formation. The project provides a better understanding of the mechanism of biofilm formation in EHEC, as well as reinforcing the hypothesis that curli fibers are an attractive target to counter the persistence of these bacteria in natural and industrial environments.

Within the last decade, pork consumption has steadily increased and continues to be the most consumed meat globally. However, pathogenic bacterial strains resistant to antibiotics have also been increasingly found in pig farms, animals, and the environment. Bacterial food poisoning cases due to Salmonella spp. and Escherichia coli (E. coli) O157:H7 appear to be linked with a variety of pork products. The meat industry has recognized that research is needed to combat the multi-drug resistance in foodborne pathogens with alternative methods of control. This study evaluated the effects of both E. coli- and Salmonella-specific lytic bacteriophages and lactic acid (LA) on E. coli O157:H7, Salmonella Enteritidis, Salmonella Montevideo, and Salmonella Heidelberg growth in raw pork loins ready for marination. The efficacy of the treatments was determined after 1 h of application and marination. Lytic bacteriophage 5% significantly (PSalmonella spp. population by 2.30 log CFU/cm2 when compared with the initial surface attachment. Moreover, the combined treatment of LA 2.5% + phage 5% significantly (PSalmonella population by 2.35 log CFU/cm2 after 1 hour of attachment. In the post-tenderization surface samples, the combination of both phage and LA showed (PP>0.05) when analyzing the translocation of Salmonella spp. on pork loins. Similar treatment efficacy results were observed in the application of E. coli O157:H7 on pork. Following antimicrobial treatments, both control and treated loin samples were enumerated after 1 h at 4°C. Both the lytic bacteriophage 5% and the combination of lytic bacteriophage 5% with lactic acid 2.5% had a significant reduction of E. coli O157:H7 on surface attachment after 1 h of treatment application. Lytic bacteriophage 5% and Lactic acid 2.5% significantly (P < 0.05) reduced the surface bacterial population by 1.89 log CFU/cm2. Lytic bacteriophage 5% alone significantly (P < 0.05) reduced the surface bacterial population by 1.90 log CFU/cm2 when compared with the initial surface attachment groups. Moreover, in the post-tenderization surface samples, lytic bacteriophage 5% and the combination of lytic bacteriophage 5% with lactic acid 2.5% were the only treatments that had a significant reduction (P < 0.05) when compared with the control group. Interestingly, lactic acid 2.5% was the only treatment that had a significant reduction (P < 0.05) of 0.76 log CFU/cm2 when analyzing the translocation of pathogens on pork chops.

No description available.