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.

Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2017-03-14T17:16:12Z No. of bitstreams: 1 Lopes, Patricia Regina Kraschinski [Dissertação, 2016].pdf: 1353027 bytes, checksum: d3bd0a8823abd0576a16231891f91a13 (MD5) / Made available in DSpace on 2017-03-14T17:16:12Z (GMT). No. of bitstreams: 1 Lopes, Patricia Regina Kraschinski [Dissertação, 2016].pdf: 1353027 bytes, checksum: d3bd0a8823abd0576a16231891f91a13 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O objetivo deste estudo foi avaliar a qualidade microbiológica do leite bovino em diferentes etapas da cadeia produtiva de uma cooperativa de leite do Estado do Rio de Janeiro, e investigar a presença de STEC e STEC O157 no leite e nas fezes dos animais ordenhados. Foram coletadas 100 amostras de leite nas diferentes etapas da cadeia produtiva, sendo 54 amostras do galão de cada propriedade rural, 25 amostras de leite do tanque de refrigeração comunitário, 14 amostras dos carros-tanque isotérmicos e 7 amostras do tanque de resfriamento da indústria bem como 10 amostras de leite pasteurizado. Foram também avaliadas 63 amostras de swab retal do animal ordenhado e 35 amostras de leite recém ordenhado. Para conferir a eficiência da pasteurização foram realizados os testes de peroxidase e fosfatase alcalina. A maioria das amostras (75,9%) de leite cru coletadas nos galões dos produtores apresentou condições microbiológicas satisfatórias, contagem bacteriana total (CBT) < 5,5 log UFC/mL. No entanto, 72% das amostras de leite coletadas no tanque de refrigeração comunitário, 100% das amostras coletadas nos carros-tanque isotérmicos e 100% das amostras coletadas nos tanques de refrigeração da indústria apresentaram condições microbiológicas insatisfatórias. Todas as amostras de leite pasteurizado foram aprovadas quanto a CBT, além de apresentarem a enzina peroxidase ativa e a enzima fosfatase alcalina inativa. No entanto, 80% destas amostras foram classificadas em condições microbiológicas insatisfatórias por apresentarem coliformes totais e E. coli acima dos padrões estabelecidos pela legislação brasileira. Das 100 amostras de leite cru analisadas, 65 apresentaram o gene stx, sendo 33 (61,1%) amostras do galão do produtor, 13 (52%) amostras do tanque de refrigeração comunitário, 14 (100%) amostras dos carros-tanque isotérmicos e cinco (71,4%) amostras dos tanques de resfriamento da indústria. Cinquenta e seis (88,9%) amostras fecais e 17 (48,6%) amostras de leite recém-ordenhado apresentaram o gene stx. Com exceção de uma amostra, todas as amostras de leite recém ordenhado provinham de um animal carreador do gene stx. Foram isolados STEC O157:H7 em 11,9% das amostras de fezes bovinas stxpositivas. Todas as cepas STEC O157:H7 isoladas apresentaram os genes stx2c, eae , tir , espA , espB , ler, iha, astA, EHEC-hlyA e espP. A qualidade microbiológica dos produtos lácteos está diretamente relacionada à qualidade da matéria-prima, com isso, é necessário a adequação dos produtores rurais quanto à implementação de Boas Práticas Agropecuárias, além da implementação das Boas Práticas de Fabricação pela indústria de laticínios / The aim of this study was to evaluate the microbiological quality of cow's milk in different stages of the production chain of a cooperative state milk Rio de Janeiro, and to investigate the presence of STEC and STEC O157 on themilk and the faeces of milked animals. 100 milk samples at different stages of the production chain were collected, 54 samples of a gallon of each rural property, 25 milk samples of Community coolant tank, 14 samples of insulated tank cars and 7 samples of the industrial cooling tank; and 10 samples of pasteurized milk. It was also evaluated 63 samples of rectal swab of milking animals and 35 sample of freshly milked milk. To check the effectiveness of the pasteurization tests peroxidase and alkaline phosphatase were performed. The majority of samples (75.9%) of raw milk collected in gallons producers had satisfactory microbiological conditions, total bacterial count (TBC) < 5.5 log CFU/mL. However, 72% of milk samples collected in the Community coolant tank, 100% of the samples collected in the isothermal tank cars and 100% of the samples collected in the industrial cooling tanks had unsatisfactory microbiological conditions. All (100%) samples of pasteurized milk were approved as the TBC, besides having the peroxidase enzyme is of not presenting the enzyme alkaline phosphatase. However, 80% of these samples were classified as unsatisfactory microbiological conditions for presenting total coliforms and E. coli above the standards set by law. Of 100 raw milk samples analyzed, 65 showed the stx gene, 33 (61.1%) of the producer gallon, 13 (52%) of the community coolant tank, 14 (100%) of the isothermal tank cars and five (71.4%) of cooling tanks industry. Fifty-six (88.9%) and 17 faecal samples (48.6%) of freshly milked milk samples showed the stx gene. With the exception of one sample, all milk samples freshly milked, came from a carrier animal stx gene. They were isolated STEC O157: H7 in 11.9% of samples stx-positive cattle dung. All strains STEC O157: H7 isolated genes showed stx2c, eae , tir , espA , espB , read, iha, astA, EHEC-hlyA and espP. The microbiological quality of dairy products is directly related to the quality of the raw material, thus, the adequacy of farmers as the implementation of Best Practices of Agricultural is required in addition to the implementation of Good Manufacturing Practices by the dairy industry