<p>An analysis of the recorded cases of Salmonella in Sweden indicates that there was no significant increase between 1997-2008, including or excluding outbreaks. The most common serotypes of Salmonella in Sweden were found to be S. Typhimurium and S. Enteritidis, with only the incidence of S. Typhimurium significantly increasing between 1997-2008. The most common phage types identified were S. Typhimurium 40, 104, NST and NT, with both S. Typhimurium NST and NT found to be significantly increasing. The season that has the greatest incidence of salmonellosis is the summer, from June to August. The number of Salmonella outbreaks occurring each year was found to be statistically increasing, this was however almost certainly due to a change in cluster detection techniques introduced in 2008, since a re-analysis excluding this year found no significant increase between 1997-2007. The most common serotypes that caused outbreaks during the study period were found to be S. Typhimurium, S Enteritidis, S. Saintpaul, S. Stanley and S. Hadar. There was no relationship found between the serotypes, with the number of cases not being proportional to the number of outbreaks. Despite these positive results, it should be of concern that evidence was found indicating that the sources of Salmonella infection appear to be changing, with an increase in the presence of ‘non-domestic’ serotypes.</p><p> </p>

An analysis of the recorded cases of Salmonella in Sweden indicates that there was no significant increase between 1997-2008, including or excluding outbreaks. The most common serotypes of Salmonella in Sweden were found to be S. Typhimurium and S. Enteritidis, with only the incidence of S. Typhimurium significantly increasing between 1997-2008. The most common phage types identified were S. Typhimurium 40, 104, NST and NT, with both S. Typhimurium NST and NT found to be significantly increasing. The season that has the greatest incidence of salmonellosis is the summer, from June to August. The number of Salmonella outbreaks occurring each year was found to be statistically increasing, this was however almost certainly due to a change in cluster detection techniques introduced in 2008, since a re-analysis excluding this year found no significant increase between 1997-2007. The most common serotypes that caused outbreaks during the study period were found to be S. Typhimurium, S Enteritidis, S. Saintpaul, S. Stanley and S. Hadar. There was no relationship found between the serotypes, with the number of cases not being proportional to the number of outbreaks. Despite these positive results, it should be of concern that evidence was found indicating that the sources of Salmonella infection appear to be changing, with an increase in the presence of ‘non-domestic’ serotypes.

Dehidroepiandrosterona (DHEA) é um hormônio esteroide secretado pelas glândulas adrenais, gônadas e cérebro, que juntamente com sua forma sulfatada (DHEAS) constituem os esteroides mais abundantes da circulação. É um importante precursor para hormônios sexuais e exerce funções diretas sob diversos sistemas do corpo, dentre eles, o sistema imune. A relação entre sistema endócrino e imune já é bem estabelecida, tendo hormônios a capacidade de modular a resposta imune e vice-versa. Dentre os componentes da resposta imune, estão os neutrófilos, importantes células da imunidade inata que reconhecem e eliminam patógenos através de diversos mecanismos efetores como fagocitose, produção de espécies reativas de oxigênio (EROs), desgranulação, neutrophil extracellular traps (NETs). Já é descrito que o avanço da idade é acompanhado pela redução dos níveis plasmáticos de DHEA, assim como a redução da atividade efetora de neutrófilo, podendo estes dois mecanismos estarem relacionados com estado de senescência e aumento da suscetibilidade de idosos à infecções. Porém, poucos estudos existem mostrando relação entre DHEA e neutrófilos. Desta forma, o objetivo do nosso trabalho, foi avaliar a influência do hormônio DHEA nas diversas funções efetoras de neutrófilos. Para tanto, neutrófilos humanos isolados do sangue periférico foram submetidos a um pré-tratamento com DHEA e infectado com Salmonella enterica serovar Typhimurium ou estimulados com LPS. Observamos que o tratamento prévio dos neutrófilos com 0,01 ?M de DHEA é capaz aumentar a fagocitose da S. Typhimurium, assim como aumento da atividade microbicida destas células. Para investigar o mecanismo pelos quais a capacidade microbicida foi aumentada, avaliamos a produção de EROs e formação de NETs. Curiosamente o hormônio não influenciou em nenhum destes processos. Então, por fim, avaliamos o perfil de produção de citocinas in vitro produzidas pelos neutrófilos tratados com DHEA e infectados com S. Typhimurium ou estimulados com LPS (lipopolissacarídeo). Nosso estudo mostrou que os neutrófilos infectados ou estimulados reduzem a produção de quimiocinas como IL-8, MIP-1? e MIP-1?, quando comparados aos neutrófilos sem tratamento. Em adição, neutrófilos tratados com esteroide e estimulados com LPS tiveram a produção de TNF-? reduzida, a produção da IL-4 foi aumentada nos neutrófilos tratados com DHEA e infectados e a relação entre IFN-?/IL-4 foi reduzida. Nossos dados mostram que o DHEA pode estar contribuindo para o controle da infecção por S. Typhimurium, principalmente através da fagocitose e killing intracelular. Apesar dos dados de citocinas mostrarem que o DHEA está direcionando a resposta de neutrófilos para um caráter menos inflamatório, acreditamos que este possa ser um mecanismo protetor ao organismo, a fim de evitar uma resposta inflamatória exacerbada que poderia causar lesões teciduais. Concluímos então, que o DHEA é um importante esteroide com atividades imunorreguladoras que podem estar contribuindo para o controle da infecção por S. Typhimurium, porém mais estudos são necessários para o entendimento dos mecanismos utilizados por este esteroide para modular a resposta de neutrófilos, células tão importantes ao organismo humano. / Dehydroepiandrosterone (DHEA) is a steroid hormone secreted by the adrenal glands, gonads and brain, which along with its sulfated form (DHEAS) are the most abundant circulating steroid. It is an important precursor to sex hormones and exerts direct roles in several body systems, including the immune system. The relationship between the endocrine and immune system is already well established, with the hormone modulating the immune response and vice versa. Among the components of the immune response, neutrophils are important innate immunity cells that recognize and eliminate pathogens through various effector mechanisms such as phagocytosis, production of reactive oxygen species (ROS), degranulation, neutrophil extracellular traps (NETs). It is reported that advanced age is accompanied by the reduction of plasma levels of DHEA as well as the reduction of the effector activity of neutrophils, these two mechanisms may be related to the state of senescence and increase in susceptibility of the elderly to infections. However, few studies exist showing the relationship between DHEA and neutrophils activity. Thus, the aim of our study was to evaluate the influence of the hormone DHEA in the several effector functions of neutrophils. To that end, human neutrophils isolated from peripheral blood were subjected to a treatment with DHEA and infected with Salmonella enterica serovar Typhimurium or stimulated with LPS (lipopolysaccharide). We observed that the treatment of neutrophils with 0.01 ?M of DHEA can increase phagocytic rates of S. Typhimurium, as well as increase the microbicidal activity of these cells. To investigate the mechanism by which the microbicidal capacity has been increased, we evaluated the production of ROS and formation of NETs. Interestingly the hormone did not influence in any of these processes. Then, we finally evaluated in the cytokine production profile produced by neutrophils treated in vitro with DHEA and infected with S. Typhimurium or stimulated with LPS. Our study showed that pré-treated and infected neutrophils or LPS-stimulated cells reduced the production of chemokines such as IL-8, MIP-1? and MIP-1? compared to untreated neutrophils. In addition, when steroid treated neutrophils were stimulated with LPS, a reduced TNF-? production was observed and the levels of IL-4 was increased in treated neutrophils with DHEA and infected with S. Typhimurium. The ratio of IFN-? / IL-4 was reduced. Our data show that DHEA may be contributing to the control of infection by S. Typhimurium, mainly by phagocytosis and intracellular killing. Although cytokines data show that DHEA is directing the neutrophil response to a less inflammatory response, and we believe that this may be a protective mechanism of the organism in order to avoid an exacerbated inflammatory response that could cause tissue damage to the body. We conclude then, that DHEA is a steroid with important immunoregulatory activities that may be contributing to the control of infection by S. typhimurium, but more studies are needed to understand the mechanisms used by this steroid to modulate neutrophil response, cells so important to the human body.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Martina Klaučová Supervisor: prof. PharmDr. Petr Pávek, Ph.D. Consultant: PharmDr. Ivona Pávková, Ph.D. Diploma thesis title: Ames test in the drug development Background: Thesis objective is the determination of potential genotoxicity of newly developed drugs within primary testing and the introduction of the Ames microfluctuation test which can be used in common laboratory conditions. Methods: I used commercially supplied kit based on the principles of Ames test which detects reverse mutation through colour changes of the samples using bacterial strains S. typhimurium. At first I had to study literary sources and then I could design the procedures of the Ames microfluctuation test, preparation of the chemicals and storage of the strains which are optimal for all laboratories. Results: The drug samples T6445 and T6447 with 30 µM concentration tested by metabolic activation S9 on bacterial strain ST TA 98 show genotoxicity. The sample UOCHB1 with 30 µM concentration tested without activation shows possible genotoxicity on both strains ST TA 98 and ST TA 100. Other samples do not show any toxicity. I used 3 different procedures during the designation of assay. The most suitable version of the...

O reconhecimento da flagelina pelos NLRs Naip5 e NLRC4 leva à formação do complexo multiproteico denominado inflamassoma que culmina na ativação da caspase-1, com consequente clivagem da forma inativa das citocinas pró-inflamatórias IL-1b e IL-18 e morte da célula infectada. Neste trabalho pudemos observar que in vitro, a maturação de BMDCs com a estimulação com flagelina citosólica, inserida em vesículas lipídicas que permitem a transfecção da flagelina para o citosol, foi independente da ativação de NLRC4, caspase-1 e TLR5, mas somente de MyD88. Já a ativação de linfócitos T por estas BMDCs ativadas por flagelina citosólica é dependente de caspase-1 e MyD88. A neutralização da citocina IL-1a, levou à inibição da ativação de linfócitos T, indicando a contribuição desta para a montagem de resposta imune. A neutralização de IL-1a também levou a uma redução na produção de IL-12, que seria a citocina responsável pela polarização dos linfócitos para Th1. A imunização com flagelina leva ao desenvolvimento de imunidade protetora contra o desafio com S. typhimurium, igualmente dependente de caspase-1 e MyD88. Podemos dizer que a flagelina induz resposta imune tanto in vivo quanto in vitro e que, em ambos os casos, há a participação das moléculas caspase-1 e MyD88. / TLR5 activates inflammatory genes through MyD88 pathway whereas NLRC4 and NAIP5 assemble multiprotein complexes called inflammasomes, leading to caspase-1 activation and secretion of proinflammatory cytokines IL-1 and IL-18. Cytosolic flagellin (FLA-BSDot) induced upregulation of costimulatory molecules independent on TLR5, NLRC4 and Caspase-1, but dependent on MyD88. In addition, FLA-BSDot-stimulated OVA-pulsed BMDCs induced proliferation and production of IFN by OT-II splenocytes, dependent on caspase-1 and MyD88. FLA-BSDot stimulation leads to the secretion of IL-1 and IL-1. Neutralization of IL-1 inhibited BMDCs maturation in response to FLA-BSDot and led to decreased IFN production by OT-II splenocytes. Searching for the effector mechanism by which IL-1 induces Th1 polarization in response to FLA-BSDot, we observed a significant reduction in IL-12 production when IL-1 was neutralized. Also, we could see that adaptive immune responses induced by flagellin in vivo was protective against S.typhimurium lethal challenge, showing again a role for caspase-1 and MyD88. From these data we can infer that caspase-1 and MyD88 are both involved in the adaptive response induced by flagelin both in vitro and in vivo.

O reconhecimento da flagelina pelos NLRs Naip5 e NLRC4 leva à formação do complexo multiproteico denominado inflamassoma que culmina na ativação da caspase-1, com consequente clivagem da forma inativa das citocinas pró-inflamatórias IL-1b e IL-18 e morte da célula infectada. Neste trabalho pudemos observar que in vitro, a maturação de BMDCs com a estimulação com flagelina citosólica, inserida em vesículas lipídicas que permitem a transfecção da flagelina para o citosol, foi independente da ativação de NLRC4, caspase-1 e TLR5, mas somente de MyD88. Já a ativação de linfócitos T por estas BMDCs ativadas por flagelina citosólica é dependente de caspase-1 e MyD88. A neutralização da citocina IL-1a, levou à inibição da ativação de linfócitos T, indicando a contribuição desta para a montagem de resposta imune. A neutralização de IL-1a também levou a uma redução na produção de IL-12, que seria a citocina responsável pela polarização dos linfócitos para Th1. A imunização com flagelina leva ao desenvolvimento de imunidade protetora contra o desafio com S. typhimurium, igualmente dependente de caspase-1 e MyD88. Podemos dizer que a flagelina induz resposta imune tanto in vivo quanto in vitro e que, em ambos os casos, há a participação das moléculas caspase-1 e MyD88. / TLR5 activates inflammatory genes through MyD88 pathway whereas NLRC4 and NAIP5 assemble multiprotein complexes called inflammasomes, leading to caspase-1 activation and secretion of proinflammatory cytokines IL-1 and IL-18. Cytosolic flagellin (FLA-BSDot) induced upregulation of costimulatory molecules independent on TLR5, NLRC4 and Caspase-1, but dependent on MyD88. In addition, FLA-BSDot-stimulated OVA-pulsed BMDCs induced proliferation and production of IFN by OT-II splenocytes, dependent on caspase-1 and MyD88. FLA-BSDot stimulation leads to the secretion of IL-1 and IL-1. Neutralization of IL-1 inhibited BMDCs maturation in response to FLA-BSDot and led to decreased IFN production by OT-II splenocytes. Searching for the effector mechanism by which IL-1 induces Th1 polarization in response to FLA-BSDot, we observed a significant reduction in IL-12 production when IL-1 was neutralized. Also, we could see that adaptive immune responses induced by flagellin in vivo was protective against S.typhimurium lethal challenge, showing again a role for caspase-1 and MyD88. From these data we can infer that caspase-1 and MyD88 are both involved in the adaptive response induced by flagelin both in vitro and in vivo.

Les salmonelles sont l’une des premières causes d’infections bactériennes d’origine alimentaire. Depuis le début des années 1990, l’isolement de salmonelles multirésistantes aux antibiotiques a considérablement accru avec l’émergence des souches épidémiques Salmonella Typhimurium DT104 qui sont, pour la majorité, résistantes à l’ampicilline, le chloramphénicol, la streptomycine, les sulfamides et les tétracyclines. Les gènes codant ces résistances sont regroupés sur un intégron complexe de classe 1 nommé In104, localisé lui-même sur un îlot génomique de 43 kb désigné Salmonella Genomic Island 1 (SGI1). Depuis sa première identification chez S. Typhimurium DT104, SGI1 a été identifié à travers le monde chez plusieurs sérovars de Salmonella, et plus récemment chez Proteus mirabilis. Chez ces souches, la multirésistance aux antibiotiques est liée, soit à l’îlot SGI1 dans sa forme initialement décrite, soit à des variants de SGI1 correspondant à la structure initiale de SGI1 comportant des modifications au niveau de l’intégron complexe In104. L’îlot génomique Salmonella Genomic Island 1 (SGI1) représente une préoccupation importante car le phénotype de multirésistance qu’il confère aux souches bactériennes est souvent responsable d’échecs thérapeutiques pouvant entrainer des complications importantes, voire la mort. Dans ce contexte, le travail de thèse a été centré sur l’enjeu sanitaire majeur représenté par cette diffusion épidémique du clone S. Typhimurium au cours des années 1990 chez l’homme et les bovins. Les travaux entrepris dans le cadre de la thèse ont eu, en premier lieu, l’objectif d’apprécier l’évolution moléculaire de SGI1 dix années après l’émergence de ces souches en élevage bovin, puis d’évaluer la diffusion de SGI1 chez des souches naturelles appartenant à d’autres genres bactériens que Salmonella. Il a ainsi été dressé un bilan de la multirésistance aux antibiotiques chez les souches de S. Typhimurium isolées de bovins malades en France de 2002 à 2007 et une recherche de la présence de SGI1, chez d’autres espèces bactériennes que Salmonella, et par sondage à partir de leurs phénotypes de résistance, a été mise en œuvre. Les résultats obtenus ont indiqué un faible pouvoir évolutif de SGI1 qui semble en contradiction avec les capacités moléculaires majeures de recombinaison et de transfert démontrées tant in vitro qu’in vivo. Les études menées ont toutefois permis la première description d’un nouveau variant, nommé SGI1-T, qui résulte d’une recombinaison intramoléculaire. Le deuxième grand objectif de la thèse a été de contribuer à une meilleure connaissance du rôle que pourrait avoir SGI1 dans la virulence bactérienne. Une première stratégie de modélisation expérimentale (salmonellose systémique murine) a ainsi été conduite, qui visait à comparer le pouvoir virulent in vivo de souches isogéniques ne se distinguant que par la présence ou l’absence de SGI1. Une seconde approche a été également menée, qui a consisté en une évaluation du rôle de SGI1 dans la formation de biofilms, l’organisation en biofilms favorisant une meilleure colonisation bactérienne, qui peut constituer à son tour un élément d’efficacité du pouvoir virulent final. Les résultats obtenus ont confirmé le rôle positif de SGI1 dans la formation de biofilms, et plus généralement son implication dans la signalisation cellulaire du Quorum Sensing. / Salmonella is a major cause of food-borne outbreaks. Since the early 1990s, isolation of multidrug-resistant Salmonella has increased with the emergence of epidemic Salmonella Typhimurium DT104 strains which are mostly resistant to ampicilin, chloramphenicol, streptomycin, sulfonamides and tetracyclines. The genes coding these resistances are clustered on a complex class 1 integron (MDR region) located on a genomic island of 43 kb designated SGI1. Since its first identification in S. Typhimurium DT104, SGI1 has been identified worldwide in other Salmonella serotypes, and more recently in Proteus mirabilis. For these strains, multidrug resistance is conferred, either to the classical structure of SGI1, or to related variants of SGI1 corresponding to the initial structure of SGI1 with modification of the complexe integron In104. The Salmonella Genomic Island 1 (SGI1) constitutes a great concern since it confers a multidrug resistance phenotype often responsible of therapeutic failures which may cause important complications, or even death. In this context, the work has been focused on the major health issue represented by the epidemic diffusion of the Salmonella Typhimurium clone in the course of 1990s in human and cattle. As a first objective, the work allowed to appreciate the molecular evolution of SGI1 in the course of time and to assess the diffusion of SGI1 to other bacterial strains than Salmonella in natural conditions. Therefore, an overview of the multidrug resistance in Salmonella Typhimurium strains isolated from diseased cattle in France from 2002 to 2007 was carried out and a screening of natural strains from other bacterial species than Salmonella that may harbor SGI1 was undertaken. The results indicated weak molecular evolutions of SGI1, which seems in contradiction with the great capability of SGI1 to recombine and transfer, as attested in vitro as in vivo. Nevertheless, this study allowed the first description of a new SGI1 variant, named SGI1-T, which is the result of intra-molecular recombination events. Another second objective of the thesis was to contribute to a better knowledge of the role of SGI1 in bacterial virulence. A strategy of experimental modeling (murine systemic salmonellosis) was first set up to compare the levels of virulence conferred by isogenic strains differing only by the presence or the absence of SGI1. A second approach was also carried out to evaluate the role of SGI1 in biofilm formation. Indeed, the organization in biofilm facilitates bacterial colonization, which constitutes in turn an element of effectiveness of the final virulence. A positive role of SGI1 in biofilm formation was demonstrated in the framework of this study, and more generally, questions the role of SGI1 in the Quorum Sensing regulation system.

Flagelina, a subunidade estrutural dos flagelos bacterianos, pode ser empregada em estratégias vacinais como carregadora de epitopos antigênicos fusionados ou como adjuvante. Neste trabalho avaliamos a imunogenicidade e o potencial vacinal das flagelinas Salmonella enterica sorovar Typhimurium em camundongos BALB/c após administração pelas vias oral ou intraperitoneal. Os resultados indicam que animais imunizados pela via oral com linhagens flageladas de S. Typhimurium não desenvolvem respostas de anticorpos, sistêmicos ou secretados, anti-flagelina, por outro lado, ativam respostas celulares específicas para as flagelinas em função da via de administração utilizada. Observamos ainda que as diferentes flagelinas testadas apresentam efeito adjuvante quando co-administradas pela via nasal com a fímbria CFA/I de ETEC. Em suma, os resultados apresentados contribuem para um melhor conhecimento sobre as propriedades imunológicas e adjuvantes das flagelinas de S. Typhimurium e agregam informações para o uso racional dessas proteínas em formulações vacinais. / Flagellin, the structural subunit of bacterial flagella, can be used in vaccine development as a fused antigenic epitope carrier or as an adjuvant. In this work we evaluated the immunogenicity and vaccine approach of S. Typhimurium flagellin in BALB/c mice after administration by oral or intraperitoneal route. Our results indicate that mice immunized by oral route with flagellated S. Typhimurium strains do not develop any anti-flagellin antibody response, be it serum or secreted. On the other hand, specific anti-flagellin cellular response was observed depending on the chosen route of immunization with S. Typhimurium attenuated strain. Furthermore, all flagellins tested proved to be efficient adjuvants when co-administrated with CFA/I fimbriae by nasal route. Together, these results contribute to a better understanding of the immunological and adjuvant properties of S. Typhimurium flagellins and also provide information on the rational use of these proteins in vaccine development.

Flagelina, a subunidade estrutural dos flagelos bacterianos, pode ser empregada em estratégias vacinais como carregadora de epitopos antigênicos fusionados ou como adjuvante. Neste trabalho avaliamos a imunogenicidade e o potencial vacinal das flagelinas Salmonella enterica sorovar Typhimurium em camundongos BALB/c após administração pelas vias oral ou intraperitoneal. Os resultados indicam que animais imunizados pela via oral com linhagens flageladas de S. Typhimurium não desenvolvem respostas de anticorpos, sistêmicos ou secretados, anti-flagelina, por outro lado, ativam respostas celulares específicas para as flagelinas em função da via de administração utilizada. Observamos ainda que as diferentes flagelinas testadas apresentam efeito adjuvante quando co-administradas pela via nasal com a fímbria CFA/I de ETEC. Em suma, os resultados apresentados contribuem para um melhor conhecimento sobre as propriedades imunológicas e adjuvantes das flagelinas de S. Typhimurium e agregam informações para o uso racional dessas proteínas em formulações vacinais. / Flagellin, the structural subunit of bacterial flagella, can be used in vaccine development as a fused antigenic epitope carrier or as an adjuvant. In this work we evaluated the immunogenicity and vaccine approach of S. Typhimurium flagellin in BALB/c mice after administration by oral or intraperitoneal route. Our results indicate that mice immunized by oral route with flagellated S. Typhimurium strains do not develop any anti-flagellin antibody response, be it serum or secreted. On the other hand, specific anti-flagellin cellular response was observed depending on the chosen route of immunization with S. Typhimurium attenuated strain. Furthermore, all flagellins tested proved to be efficient adjuvants when co-administrated with CFA/I fimbriae by nasal route. Together, these results contribute to a better understanding of the immunological and adjuvant properties of S. Typhimurium flagellins and also provide information on the rational use of these proteins in vaccine development.

This study was conducted to determine the survival of Escherichia coli O157:H7 and Salmonella Typhimurium in marinade that was used to vacuum tumble beef inside skirts and beef tri-tip roasts. The depth of penetration of each microorganism into the individual meat products, and the survival of these microorganisms in the products as well as marinade stored over time were evaluated. Two commercial marinades were used, Reo TAMU Fajita Marinade and Legg's Cajun Style Marinade. Eighteen beef inside skirts and 18 tri-tips were used during this study. Both inside skirts and tri-tips were vacuum tumbled for a total of 1 h. Samples of products were tested immediately following tumbling (day 0), or were vacuum packaged and stored in the cooler (approximately 2 degrees C) to be tested 7 and 14 days following tumbling. Samples of the spent marinade were taken and tested initially following tumbling (day 0), and were also stored in a cooler and tested 3 and 7 days after the marinade was used. The results of the study showed that with both marinades S. Typhimurium and E. coli O157:H7 penetrated throughout the skirt meat. After having been stored for 7 days following tumbling, the log value of both S. Typhimurium and E. coli O157:H7 decreased in the meat. After 14 days of storage following tumbling, the log value of both S. Typhimurium and E. coli O157:H7 continued to decrease; however, both pathogens were still detectable. The penetration of the pathogens in the tri-tip roast varied depending on the thickness of the roast. The thicker roasts had undetectable levels of both pathogens in the geometric center; however, the thinner tri-tip roasts had detectable levels at the geometric center. The spent marinade tested on day 0, 3, and 7 showed that the microorganisms were able to survive in the marinade at refrigerated temperatures. The results of this study demonstrated that pathogens may penetrate into the interior of beef skirts and tri-tips during vacuum tumbling with contaminated marinade, and that pathogens survive during refrigerated storage of spent marinade. Industry should consider these data when evaluating potential food safety risks associated with the production of vacuum tumbling beef products.