This study investigates the analyses and clustering of Campylobacter spp., Listeria monocytogenes and Shiga toxin-producing Escherichia coli (STEC) at Livsmedelsverket. Livsmedelsverket is a control authority in Sweden. They work with eating habits, what food contains and safe food and good drinking water, where outbreak investigations of the above-mentioned bacterial types is a part of the work. For the investigations Livsmedelsverket uses a pipeline that is written in the programming language Python. The purpose of this project is to add identification of virulence genes and analysis of the STEC bacterium to the script. But also to develop the existing method to be able to cluster more isolates without losing information, enable the user to adjust parameters in the pipeline and write an ethical analysis to the work that is done. Our study shows the analysis and clustering of the three different types of bacteria, clustering of the samples from the analysis, both adaptively and statically, and that it can determine serotype, sequence type and virulence genes. We therefore conclude that STEC can be added to outbreak investigations at Livsmedelsverkets in-house pipeline. The clustering method has also been modified to be able to use more of the information given from the samples with the restriction of having lower accuracy.

Verschiedene ELISA zur Erkennung von Stx1 und Stx2 in Rinderkotproben wurden auf ihrer Anwendbarkeit hin verglichen. Der ELISA nach RICHTER et al. (1997) unter Anwendung von Hydatidenflüssigkeiten von Echinococcus granulosus als Beschichtungssubstanz und monoklonalen Antikörper als Bindungsmolekül stellte sich als die praktikabelste Lösung im Vergleich mit dem ELISA mit monoklonalen Antikörpern (RANDALL et al., 1997) und einem ELISA mit kommerziellem Gb3 heraus. Der ELISA nach RICHTER et al. (1997) wurde gegen den klassischen Vero-Zell- Neutralisationstest validiert. Zu diesem Zweck wurden 100 E. coli-Feldisolate mit beiden Tests ausgewertet. Die zur Quantifizierung von Ergebnissen angewendete Coulter-Methode (Ermittlung der Überlebensraten der Vero-Zellen mit Hilfe des Z2-Partikelzählers) erwies sich als geeignet, während zwei Färbungsmethoden (KV- und MTT-Färbungsmethode) aufgrund von Einstellungsproblemen bei der spektralfotometrischen Messungen nicht zu auswertbaren Ergebnissen geführt hatten. Die Ergebnisse des ELISA und des Vero-Zell-Neutralisationstest wurden in einer „Two- Graphic Receiver-Operating Characteristic“ (TG-ROC)-Analyse verglichen. Die Indexwerte 0,020 und 0,040 wurden als „cut-off“ für den Stx1- bzw. Stx2-ELISA anhand dieser Analyse 79 festgelegt. Die geschätze Sensitivität und Spezifität für den Stx1-ELISA betrugen 100% (pu = 100%; po = 100%), für den Stx2-ELISA betrug die Sensitivität 100% (pu = 100%; po = 100%) und die Spezifität 94% (pu = 88,4%; po = 99,7%). Nach diesen Ergebnissen ist der ELISA ein geeigneter Test und kann den Vero-Zell-Neutralisationstest ersetzen. Der validierte ELISA wurde in einem Feldversuch parallel mit der MK1/MK2-PCR (KARCH und MEYER, 1989) eingesetzt. In einem Zeitraum vom 15.01.97 bis 31.12.99 wurden 1030 Rinderkotproben mit beiden Tests auf das Vorkommen von STEC untersucht. Die PCR wies stx-Gene bei 500 (49%) der Proben nach. Der ELISA wies nur 307 (30%) Stx-positive nach. Bei 264 positiven und 487 negativen Ergebnissen stimmten die Resultate zu 751 Proben (73%) überein. Der berechnete Kappa-Wert war 0,452. Der Kappa-Wert spricht für eine mäßige Übereinstimmung beider Tests. Diesen Ergebnissen nach sollte die PCR als „pre-screening“-Test favorisiert werden, obwohl eine Kombination beider Tests zum Stx-Nachweis am besten geeignet wäre.

Enterohemorrhagic E. coli (EHEC) infection is a leading cause of acute kidney failure in otherwise healthy children, and a leading cause of foodborne illness with an outsized economic impact from outbreaks. EHEC secrete two Shiga-like toxins (Stx1 and Stx2) which are AB5 holotoxins that inhibit protein synthesis in cells expressing the toxin receptor Gb3. Infection with EHEC typically begins with a diarrheal prodrome that can progress in 5-15% of cases to hemolytic uremic syndrome (HUS), a clinical diagnosis characterized by thrombocytopenia, hemolytic anemia, and thrombotic microangiopathy. Historically, strains of EHEC expressing Stx2 have been associated with more severe disease. We hypothesized that tissue injury due to the toxins leads to the release of damage-associated molecular patterns (DAMPs), which act through inflammatory receptors to promote the endothelial dysfunction that drives this disease alongside the inciting Shiga toxins. Here we demonstrate that two well-characterized DAMPs, extracellular histones and HMGB1, are produced in two different mouse models when Stx2 is present; one model represents challenge with the toxin alone, and the second model introduces toxin through secretion with a lysogenized bacterium, C. rodentium, mimicking EHEC colonization. We investigate whether Stx1, Stx2, or histones affect the endothelial expression of well-characterized members of the protein C pathway, namely the endothelial protein C receptor (ECPR), protease-activated receptor 1 (PAR1), and thrombomodulin (TM), on human aortic (HAEC) and human renal glomerular endothelial cells (HRGEC). We show that Stx and/or histones reduce endothelial expression of these anti-coagulant molecules and histones dramatically increase expression of pro-thrombotic PAR-1. These changes lead to physiologically important decreases in activated protein C (APC), a critical anti-coagulant and cytoprotective molecule. Finally, we demonstrate that histones exacerbate thrombin's barrier-disruptive effects on the endothelium, and prevent APC's protective effects. These data provide novel mechanistic insight into the endothelial dysfunction that characterizes HUS and also provide a new perspective on systemic consequences of the bacterial Shiga toxins that might drive organ injury in susceptible patients.

No description available.

A Quantitative Microbial Risk Assessment (QMRA) has been undertaken to utilize research on Shiga-toxin Escherichia coli (STEC) contamination in beef for the benefit of public health. The QMRA operates as a 2nd order Monte Carlo simulation to create stochastic mathematical models that incorporate all of the key components of STEC contamination from farm to fork. The resulting model is able to identify knowledge gaps, public health risks, and simulate theoretical changes in the beef system. However, high variability in processing plant intervention literature has prompted a meta-analysis to determine informed estimates of intervention effectiveness for QMRA parameterization. Meta-analysis derived least-squares means bacterial log reductions for acetic acid, lactic acid, steam vacuum, and water wash interventions on carcass surfaces (n=249) were 1.44 [95% CI: 0.73 – 2.15], 2.07 [1.48 – 2.65], 3.09 [2.46 – 3.73], and 1.90 [1.33 – 2.47] log CFU/cm2, respectively. Least-squares means log reductions for acetic acid, lactic acid, sodium hydroxide, and water wash on hide surfaces (n=47) were 2.21 [1.36 – 3.05], 3.02 [2.16 – 3.88], 3.66 [2.60 – 4.72], and 0.08 [-0.94 – 1.11] log CFU/cm2, respectively. Meta-regressions showed that temperature, duration of application, microbial starting concentration, extra water washes, inoculation type, organism type, sample method, surface type, and antimicrobial concentrations were all significant predictors of intervention effectiveness. Finally, after observing authors use substituted values for samples found below a detection limit in primary plant intervention literature, simulations were run to assess the impact of substitution on a random-effects meta-analysis. Simulation results show that substitution practices artificially decrease effectiveness estimates and increase heterogeneity. / Master of Science

Globalmente ocorrem cerca de 800.000 mortes de crianças menores de cinco anos associadas à diarreia, principalmente na África subsaariana, sul da Ásia e América Latina. Dentre os patógenos causadores de diarreia, Escherichia coli diarreiogênica (DEC) é o agente etiológico bacteriano mais comum, incluindo E. coli enteropatogênica (EPEC) e E. coli produtora da toxina de Shiga e seu subgrupo enterohemorrágica (STEC/EHEC). Os dados epidemiológicos indicam a importância do diagnóstico precoce e sua realização em locais com pouca infraestrutura. Desta forma o objetivo deste trabalho foi o desenvolvimento de um teste rápido, sensível e específico para o diagnóstico de EPEC e STEC/EHEC. Primeiramente, foram definidas diferentes condições do cultivo bacteriano: Dulbecco\'s modified Eagle\'s (DMEM), DMEM contendo 1% de triptona e DMEM pré-condicionado para o cultivo dos isolados de EPEC/EHEC e avaliação da produção/secreção das proteínas secretadas EspA e EspB, utilizando anticorpos monoclonais (MAb) e policlonais (PAb) anti-EspA ou anti-EspB por ELISA indireto. Para a avaliação da liberação das toxinas de Shiga para o sobrenadante do cultivo bacteriano de STEC/EHEC, foram testados diferentes condições de tratamento, o cultivo bacteriano foi tratado com Triton X-100 e o sedimento foi tratado com tampão de lise B-PER utilizando MAb e PAb anti-Stx1 ou anti-Stx2 por ELISA de captura. Subsequentemente, foi desenvolvido e avaliado o teste de aglutinação em látex para a detecção de EspB em isolados de EPEC/EHEC, e Stx1 e Stx2 em isolados de STEC/EHEC. EspB foi definida como biomarcador, o MAb anti-EspB como ferramenta para o diagnóstico de EPEC/EHEC, e a condição ideal para a produção/secreção de EspB foi o cultivo em DMEM. Para o diagnóstico de STEC/EHEC a condição ideal para liberação das toxinas Stx foi o tratamento do cultivo com Triton X-100. Tanto o ELISA, como a aglutinação em látex apresentaram sensibilidades e especificidades exigidas para testes diagnósticos de doenças negligenciadas em países em desenvolvimento e os testes de aglutinação em látex para a detecção destes patógenos foram precisos, rápidos e fáceis de executar, sendo portanto promissores para a utilização em laboratórios com mínima infraestrutura. / There are 800,000 deaths associated with diarrhea worldwide in children under five, and these are mainly in sub-Saharan Africa, Southeast Asia and Latin America. Among the causative pathogens of diarrhea, diarrheagenic Escherichia coli (DEC) is the most common bacterial etiological agent, including enteropathogenic E. coli (EPEC) and Shiga toxin-producing E. coli and its subgroup enterohemorrhagic E. coli (STEC/EHEC). Epidemiological data indicate the importance of early diagnosis and its realization in places with limited resources. Therefore, the objective of this work was to develop a rapid, sensitive and specific test for the diagnosis of EPEC and STEC/EHEC. First, different bacterial growth conditions were evaluated: Dulbecco\'s modified Eagle\'s medium (DMEM) or DMEM containing 1% tryptone, and DMEM pre-conditioned with EPEC/EHEC isolates. The production/secretion of the secreted proteins EspA and EspB was determined by indirect ELISA utilizing anti-EspA or anti-EspB monoclonal (MAb) and polyclonal (PAb) antibodies. Different treatments were tested for their effect on the release of Shiga toxins into the medium of STEC/EHEC bacterial cultures. The bacterial culture supernatant was treated with Triton X-100, and the sediment was treated with B-PER lysis buffer. The toxins release was determined by capture ELISA using anti-Stx1 or anti-Stx2 MAb and PAb. Subsequently, a latex agglutination test was developed and evaluated for the detection of EspB in EPEC/EHEC isolates and of Stx1 and Stx2 in STEC/EHEC isolates. EspB was defined as the biomarker and anti-EspB MAb as the tool for the diagnosis of EPEC/EHEC. The ideal conditions for the production/secretion of EspB were cultivation in DMEM. For the diagnosis of STEC/EHEC, the ideal conditions for the release of Stx were Triton X-100 treatment. ELISA as well as latex agglutination showed the sensitivities and specificities required for diagnostic tests of neglected diseases in developing countries. The latex agglutination test for the detection of these pathogens was precise, rapid and easy to perform, thereby being promising for their utilization in laboratories with limited resources.

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.

Globalmente ocorrem cerca de 800.000 mortes de crianças menores de cinco anos associadas à diarreia, principalmente na África subsaariana, sul da Ásia e América Latina. Dentre os patógenos causadores de diarreia, Escherichia coli diarreiogênica (DEC) é o agente etiológico bacteriano mais comum, incluindo E. coli enteropatogênica (EPEC) e E. coli produtora da toxina de Shiga e seu subgrupo enterohemorrágica (STEC/EHEC). Os dados epidemiológicos indicam a importância do diagnóstico precoce e sua realização em locais com pouca infraestrutura. Desta forma o objetivo deste trabalho foi o desenvolvimento de um teste rápido, sensível e específico para o diagnóstico de EPEC e STEC/EHEC. Primeiramente, foram definidas diferentes condições do cultivo bacteriano: Dulbecco\'s modified Eagle\'s (DMEM), DMEM contendo 1% de triptona e DMEM pré-condicionado para o cultivo dos isolados de EPEC/EHEC e avaliação da produção/secreção das proteínas secretadas EspA e EspB, utilizando anticorpos monoclonais (MAb) e policlonais (PAb) anti-EspA ou anti-EspB por ELISA indireto. Para a avaliação da liberação das toxinas de Shiga para o sobrenadante do cultivo bacteriano de STEC/EHEC, foram testados diferentes condições de tratamento, o cultivo bacteriano foi tratado com Triton X-100 e o sedimento foi tratado com tampão de lise B-PER utilizando MAb e PAb anti-Stx1 ou anti-Stx2 por ELISA de captura. Subsequentemente, foi desenvolvido e avaliado o teste de aglutinação em látex para a detecção de EspB em isolados de EPEC/EHEC, e Stx1 e Stx2 em isolados de STEC/EHEC. EspB foi definida como biomarcador, o MAb anti-EspB como ferramenta para o diagnóstico de EPEC/EHEC, e a condição ideal para a produção/secreção de EspB foi o cultivo em DMEM. Para o diagnóstico de STEC/EHEC a condição ideal para liberação das toxinas Stx foi o tratamento do cultivo com Triton X-100. Tanto o ELISA, como a aglutinação em látex apresentaram sensibilidades e especificidades exigidas para testes diagnósticos de doenças negligenciadas em países em desenvolvimento e os testes de aglutinação em látex para a detecção destes patógenos foram precisos, rápidos e fáceis de executar, sendo portanto promissores para a utilização em laboratórios com mínima infraestrutura. / There are 800,000 deaths associated with diarrhea worldwide in children under five, and these are mainly in sub-Saharan Africa, Southeast Asia and Latin America. Among the causative pathogens of diarrhea, diarrheagenic Escherichia coli (DEC) is the most common bacterial etiological agent, including enteropathogenic E. coli (EPEC) and Shiga toxin-producing E. coli and its subgroup enterohemorrhagic E. coli (STEC/EHEC). Epidemiological data indicate the importance of early diagnosis and its realization in places with limited resources. Therefore, the objective of this work was to develop a rapid, sensitive and specific test for the diagnosis of EPEC and STEC/EHEC. First, different bacterial growth conditions were evaluated: Dulbecco\'s modified Eagle\'s medium (DMEM) or DMEM containing 1% tryptone, and DMEM pre-conditioned with EPEC/EHEC isolates. The production/secretion of the secreted proteins EspA and EspB was determined by indirect ELISA utilizing anti-EspA or anti-EspB monoclonal (MAb) and polyclonal (PAb) antibodies. Different treatments were tested for their effect on the release of Shiga toxins into the medium of STEC/EHEC bacterial cultures. The bacterial culture supernatant was treated with Triton X-100, and the sediment was treated with B-PER lysis buffer. The toxins release was determined by capture ELISA using anti-Stx1 or anti-Stx2 MAb and PAb. Subsequently, a latex agglutination test was developed and evaluated for the detection of EspB in EPEC/EHEC isolates and of Stx1 and Stx2 in STEC/EHEC isolates. EspB was defined as the biomarker and anti-EspB MAb as the tool for the diagnosis of EPEC/EHEC. The ideal conditions for the production/secretion of EspB were cultivation in DMEM. For the diagnosis of STEC/EHEC, the ideal conditions for the release of Stx were Triton X-100 treatment. ELISA as well as latex agglutination showed the sensitivities and specificities required for diagnostic tests of neglected diseases in developing countries. The latex agglutination test for the detection of these pathogens was precise, rapid and easy to perform, thereby being promising for their utilization in laboratories with limited resources.

The objective of this study was to evaluate the survival of non-O157:H7 STEC, Salmonella spp., and S. aureus in dry fermented sausages. Chorizo and Landjager sausages were inoculated with individual bacterial cocktails and stuffed into natural casings. Temperature, relative humidity, pH, and water activity were monitored through fermentation, drying, and storage. Bacterial counts were determined by serial dilution and plated in triplicates on selective media. Plates were incubated at 37°C for 24 hours and colony forming units per gram (CFU/g) were observed. Results of the first study validate that contaminated raw materials contribute to pathogen survival and background bacteria outcompeted the starter culture. The pH critical limit of < 5.3 was met but there was no pathogen inhibition. Results from the second study confirm that pH and water activity are not enough to eliminate pathogens when post processing interventions are not used. Critical pH (< 5.3) and water activity (< 0.85) limits were met, but pathogens still survived. In chorizo, non-O157:H7 was recovered through enrichments until the end of the study. In landjager, non-O157:H7 STEC and Salmonella were recovered through enrichments until the end of the study.The studies suggest that sausages produced without post processing interventions are a health risk to consumers.

Les Escherichia coli producteurs de Shiga-toxines (STEC) sont des bactéries pathogènes originaires du tractus digestif des ruminants qui peuvent contaminer certains aliments. Une fois ingérés, ils peuvent provoquer des pathologies graves. Le comportement des STEC n'appartenant pas au sérotype O157:H7 dans les fromages était mal connu. Par ailleurs, en dépit d'une forte prévalence des STEC dans les fromages, cet aliment est moins impliqué dans les épidémies que la viande. C'est pourquoi nous avons étudié dans un premier temps le devenir de ces STEC au cours de la fabrication de 5 schémas technologiques de fromages différents. Dans un deuxième temps, nous avons analysé le comportement et la survie de STEC ayant survécu à la fabrication d'un fromage au cours d'une digestion artificielle. Nos résultats ont montré que certaines technologies sont favorables à la croissance des STEC. Seul un affinage de plus de 60 jours a permis une réduction du taux des STEC. Les étapes de cuisson du caillé ou de coagulation longue de certaines technologies semblent inhiber la croissance des STEC. De plus, la croissance et la survie observées pour les souches E. coli O157:H7 étaient plus faibles que pour les souches E. coli non-O157:H7. L'étude de la survie des souches STEC au cours de la digestion artificielle a montré une différence de comportement entre les 2 souches STEC testées : la souche E. coli O157:H7 a survécu à la digestion tandis que celle de sérotype O26:H11 s'est fortement développée en fin de digestion.