Orientador: Tomomasa Yano / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-03T15:36:39Z (GMT). No. of bitstreams: 1 Marques_EneidaGoncalvesLemes_M.pdf: 6403857 bytes, checksum: aba606a1028881cdd6692f20d1eb780b (MD5) Previous issue date: 2003 / Resumo: O resumo poderá ser visualizado no texto completo da tese digital / Abstract: The abstract is available with the full electronic digital document / Mestrado / Microbiologia / Mestre em Genética e Biologia Molecular

Cholesterol has been shown to play important role in the pathogenesis and persistence of intracellular pathogens. Here, we modulate host cholesterol biosynthesis pathway using pharmacological agent statins, which are reversible inhibitors of HMG†CoA reductase enzyme. The aim of the study was to investigate the role of statins in inducing host protective responses against intracellular pathogens. We report reduced growth of Listeria monocytogenes (LM) and Mycobacterium tuberculosis (Mtb) in murine macrophages. We show prominent immunomodulatory activity induced by statins, mainly increased phagosomal maturation and autophagy resulting in decreased bacterial growth in macrophages. Subsequently, statin†treated mice showed decrease in bacterial loads, accompanied by reduced histopathology in the acute phase of infection during listeriosis and tuberculosis. Furthermore, we found decreased growth of Mtb in peripheral blood mononuclear cells (PBMC) and monocyte†derived macrophages (MDM) isolated from patients with familial hypercholesterolemia (FH) on statin therapy when compared to healthy subjects. Together, our results show that statins induces protection against Mtb in murine macrophages, mice and human mononuclear cells and monocyte†derived macrophages.

The effects of initial thermal processing, plant sanitation, and employee habits on the microbiological quality of blue crab (Callinectes sapidus) meat were determined in a commercial crab processing facility. Thermal processing was evaluated at 5, 7, and 8 minutes at 250ï °F for the destruction of microorganisms, including Listeria monocytogenes. F-values calculated indicated a sufficient reduction of L. monocytogenes at each processing time. Fresh picked crab meat was evaluated for microbial levels when exposed to ambient temperatures over a four hour period. It was found that time and temperature did not influence the microbial populations significantly except in the fourth hour. Plant sanitation was evaluated based on levels of adenosine triphosphate (ATP) and microbial counts. Areas found to have high levels of ATP typically had low microbial counts, thus suggesting that crab meat residual was the problem. The presence of Listeria species in the plant was determined using a commercial polyclonal antibody test. Listeria species were found under picking tables, on cooler doors, employees' aprons, and on several employees' hands. In a laboratory setting, an automated hand wash was compared with a manual hand wash for the removal of Listeria innocua, as a model for Listeria monocytogenes. It was found that a manual hand wash of 15 seconds was superior to an equal time automated wash. The microbial quality of crab meat was found to be affected by daily plant procedures, and could be changed by modifying procedures. / Master of Science

The pathogenicity of food-borne intracellular bacterium Listeria monocytogenes is greatly associated with its abilities to invade non-phagocytic cells, counteract the host innate immune system, resist bactericidal antibiotic-mediated killing, and breaking the physical barriers. In the last 30 years of research on L. monocytogenes, several virulence factors, such as Listeriolysin O (LLO), InlA, InlB, ActA, PI-PLC, and PC-PLC have already been characterized as important players that help this bacterium to achieve the key stage of infection. There are approximately 3,000 open reading frames in Listeria’s genome; however, only few virulence factors are functionally characterized. Thus, it is important to identify new virulence factors and understand how new virulence factors in Listeria help this opportunistic pathogen to counteract the host innate immune system, resist antibiotic-mediated killing, colonize vital organs, and finally successfully develop life-threatening listeriosis. In this study, inrame deletion mutagenesis was used to generate the deletion mutants of novel listerial virulence factors and a series of biochemical, in vitro and in vivo experiments were conducted to characterize the roles of these virulence factors during the infection process. In the first part of this study, an AlkD-like protein (Adlp, LmoF2365_0220) was identified and the protein is associated with oxidant tolerance and aminoglycoside antibiotic resistance. In the second part of this study, a new internalin-like protein (LmoH7858_0369) was shown to be involved in invasion of Hep-G2 cells and organ colonization in mice. The third part of this study showed that listeriolysin O (LLO) mediates cytotoxicity on brain endothelial cells, suggesting that LLO may contribute to the invasion of the central nervous system by L. monocytogenes. In summary, we identified and characterized two novel virulence factors, Adlp and LmoH7858_0369 that contributed to bacterial infection and revealed a new invasion mechanism of CNS cells that is mediated by LLO. Results from these studies provide a better understanding on the pathogenicity of L. monocytogenes and can be used as therapeutical targets or vaccine candidates

Listeria monocytogenes is one of the toughest challenges the food industry is facing today. While quaternary ammonium compounds (QAC) are typically applied at very high concentrations to kill L. monocytogenes, there are some factors that can cause its reduced efficacy in the food processing environment. Sublethal concentrations of stagnated QAC have been found both in processing plants and in waste waters released to the environment. Therefore, the purpose of this study was to investigate the ability of two L. monocytogenes strains, Bug600 (serotype 1/2a) and ScottA (serotype 4b) to persist in lethal and sublethal concentrations of first generation QAC (benzalkonium chloride, BAC) and second generation QAC (Ster-BAC), and if these strains would regrow when conditions had improved. Using BAC or Ster-BAC concentrations that mimic those found occurring in some environments, we determined L. monocytogenes survival, persistence, and recovery in high/low nutrient conditions, and also isolated two distinct morphotypes.

Attachment strength of Listeria spp. and their InlA expression was assessed. Listeria monocytogenes 19111 exhibited the strongest attachment strength with L. monocytogenes 19115, L. grayi, L. innocua, and L. monocytogenes 7644 being the weakest. InlA expression was not detected in silver stained SDS gels but was detected in Western blotting images. Internalin A was only detected in protein extracts of L. monocytogenes 19111 and 7644 with band intensities of 50.1 and 2.5 pixels, respectively. Greater InlA expression was correlated with higher attachment strength in L. monocytogenes 19111. Listeria monocytogenes 19115 did not express InlA but it had a stronger attachment than L. monocytogenes 7644 which demonstrated InlA expression. Intensity of InlA expressed in L. monocytogenes 19111 increased when temperature increased from 10 to 40 oC, and L. monocytogenes 7644 only expressed InlA at 40 oC. It was also determined that InlA was more expressed in nutrient-rich media than in nutrient-poor media.

Listeria monocytogenes is an enteric pathogen that can replicate within bile, yet this capability differs between strains. This project analyzed whether the pathogenic potential of the strain affects the ability to resist bile. We tested this hypothesis by examining the effect of bile on the morphology of a virulent strain (EGD-e) and an avirulent strain (HCC23) under aerobic and anaerobic conditions. Our data showed that exposure to bile greatly impacted the growth of HCC23. Additionally, scanning electron microscopy and transmission electron microscopy analyses indicated that bile affects the cell envelope of EGD-e and HCC23 differently. Our results suggest that differences exist in the ability of EGD-e and HCC23 to survive and replicate in the presence of bile. We propose that the virulence capability of L. monocytogenes directly correlates to its ability to resist the detergent properties of bile.

Listeria monocytogenes has emerged as a food safety concern for a number of produce commodities. While L. monocytogenes contamination can occur throughout the supply chain, contamination from the packinghouse environment represents a particular challenge and has been linked to recalls. This study aimed to investigate the prevalence, persistence, and diversity of Listeria monocytogenes (LM) and other Listeria species (LS) in produce packinghouses. A longitudinal study was performed in 11 packinghouses (commodities included micro-green, peach, apple, tomato, broccoli, cauliflower, and cucumber) in three US states. In each packinghouse, 34 to 46 sites representing zones 2-4 were selected and swabbed. Packinghouses were visited 4 times and samples were processed for Listeria by US Food and Drug Administration's Bacteriological Analytical Manual methods. Presumptive Listeria-positive isolates were confirmed by PCR. Species and allelic type (AT) were identified by sigB sequencing. Among the 1,584 samples tested, 3.2%, 2.7%, and 0.6% of the samples were positive for LM, LS, and both LM and LS, respectively. Five different species of Listeria were identified with L. monocytogenes being the most prevalent species. A high AT diversity (0.95 Simpson's Diversity Index) was observed amongst Listeria isolates. There were 15 instances of Listeria repeated isolation (site testing positive ≥2 times). Upon analysis of subtype data, only 3 sites tested positive for the same Listeria AT >2 times. Data showed in this longitudinal study that Listeria prevalence and persistence in packinghouses was low (e.g., <4% prevalence). Therefore, sanitation program development and implementation in packinghouses are critical to limit Listeria harborage and residence. / Master of Science in Life Sciences / Listeria monocytogenes is one of the deadliest foodborne pathogens, accounting for about 20% of the deaths caused by foodborne illnesses in the US. Historically, L. monocytogenes has been a big concern for Ready-to-Eat products (ice cream, deli meats, etc.), but in the last decade, there have been several listeriosis outbreaks associated with fresh produce (e.g. cantaloupes, apples, celery, packaged salad) becoming a produce safety concern. Some of these outbreaks have been traced back to the produce farm (pre-harvest) and the operations after harvesting (post-harvest). Though there is research focusing on the prevalence of Listeria in the pre-harvest environment, there is a need for studies investigating Listeria at the post-harvest level. This research project, focused on gaining a better understanding of the prevalence, persistence, and diversity of Listeria (including L. monocytogenes) in produce packinghouses. 11 packinghouses facilities were sampled four times during the packing season. The samples were obtained from different stationary (e.g. walls, drains, floors) and moving (e.g. bins, forklifts, pallets) non-food contact surfaces and equipment during operation hours. Isolates were processed to detect and isolate Listeria species (including L. monocytogenes). Listeria isolates were confirmed and fingerprinted. Listeria prevalence in these packinghouses was low (6.4%), and it varied among packinghouses. Drains, cold storages, and wet non-food contact surfaces were the sites with the highest Listeria prevalence. There were 3 cases of Listeria repeated isolation (same Listeria detected in the same site in at least 2 of the 4 visits). The diversity of Listeria in these packinghouses was high. The information gathered through this research provides a better understanding of where and what species of Listeria can be found in a produce packinghouse iv facility. This knowledge may be used to develop and implement mitigation strategies and interventions to control and/or reduce the risk of Listeria contamination in produce packinghouses.

La surveillance de la listériose en France s’est construite par étapes depuis les années 1980 sur deux piliers, la microbiologie et l’épidémiologie. Grâce à la création du Centre National de Référence des Listeria et à la mise au point de techniques de typage performantes, l’Institut Pasteur assure une surveillance microbiologique depuis 1987. Une surveillance épidémiologique initiée entre 1984 et 1992 par le Laboratoire National de la Santé, a été développée par le Réseau National de Santé Publique de 1993 à 1999, puis amplifiée par l’Institut de Veille Sanitaire à partir de 2000. Le premier objectif de cette thèse est de décrire les différentes phases de la construction de cette surveillance afin d’analyser leurs contributions respectives au cours de ces 30 dernières années. Cette construction s’est faite en 4 phases : 1. L’étape fondatrice de 1982 à 1992 a été la reconnaissance et la prise en compte du rôle des aliments dans la transmission de la maladie et dans la survenue d’épidémies. 2. La deuxième phase de 1993 à 2000 a été l’édification d’un système de surveillance opérationnel pour détecter et investiguer les épidémies en France. 3. La troisième phase de 2000 à 2005 a permis de consolider le système de surveillance et de le perfectionner en ajoutant un volet complémentaire avec des prélèvements alimentaires.4. Depuis 2005, nous sommes dans la quatrième phase avec comme objectif l’optimisation du système. Cette optimisation repose sur l’adaptation des outils de surveillance et d’alerte aux connaissances. Ainsi, après avoir montré que la durée d’incubation de la maladie varie selon la forme clinique de la maladie, nous avons proposé d’intégrer cette variation pour déterminer la période d’évaluation des expositions alimentaires à risque. L’analyse des performances du système a permis à deux reprises de proposer de nouveaux seuils de signalement plus spécifiques afin d’optimiser cette surveillance tout en réduisant son coût. Le deuxième objectif de cette thèse est de montrer la contribution des données de surveillance à une politique de santé publique. Un premier travail a consisté à mettre en perspective les variations temporelles d’incidence observées avec les différentes sources de données disponibles afin d’en analyser les déterminants. La phase de décroissance de 1987 à 1997 a été concomitante des mesures de contrôles prises par l’industrie agro-alimentaire et de la réduction de la contamination des aliments. La phase d’augmentation en 2006-2007 semble multifactorielle. L’augmentation de la prescription de traitements de réduction de l’acidité gastrique par des inhibiteurs de la pompe à protons pourrait être l’un des déterminants majeurs de cette augmentation.Dans une deuxième analyse, nous avons hiérarchisé les groupes à risque de listériose sur la base de l’estimation du taux d’incidence de listériose et de sa mortalité dans ces groupes. Cela a permis d’identifier les groupes les plus vulnérables : hémopathies, certains cancers (digestifs, cérébral et pulmonaire), maladie de Horton, cirrhose hépatique, les dialysés rénaux, les greffés, et les femmes enceintes. Une analyse épidémiologique des listérioses materno-néonatales (MN) a montré une association entre les régions avec une incidence plus faible de listérioses materno-néonatales et les régions où la séroprévalence toxoplasmique des femmes enceintes est la plus faible, ce qui suggère un effet positif des recommandations contre la toxoplasmose pour la prévention de la listériose MN. / Listeriosis surveillance was built up stage by stage in France since the 1980s on a twofold basis: microbiology and epidemiology. Thanks to the creation of the Listeria National Reference Centre (Centre National de Référence des Listeria), the Pasteur Institute has been doing microbiological surveillance since 1987. Epidemiological surveillance was initiated by the National Health Laboratory, then conducted by the National Health Network and further developed by the National Institute of Health Surveillance. This thesis aims first of all to describe the different stages in the setting up of this surveillance system in order to analyze their respective inputs during these last thirty years. The four stages are:1. From 1982 to 1992: awareness and recognition of the role of food in the transmission of listeriosis and as the source of outbreaks. 2. From 1993 to 2000: building a reliable surveillance system in order to detect and investigate outbreaks in France. 3. From 2000 to 2005: strengthening and perfecting the surveillance system by taking additional measures, such as food sampling.4. Since 2005, we have reached the fourth stage, designed to optimize the surveillance system. This optimization involves adapting surveillance and early warning tools to new knowledge and information. For instance, having established that listeriosis incubation periods vary according to the clinical form of the illness, we suggested the integration of the variation of exposure period factor when interviewing patients with the food questionnaire. On two separate occasions, analysis of the surveillance system performance results made it possible to modify the criteria for early warning so as to optimize surveillance by increasing its specificity whilst reducing costs.The second aim of this thesis is to illustrate how surveillance data can contribute to public health policies. A first study analyzed temporal trends, using all available data in order to give some explanation as to major trends. The first trend was a reduction of incidence from 1987 to 1997 that was concomitant with control measures by the food industry and a drop in food contamination. The increased trend observed in 2006-2007 appears to be due to several factors. The increased rate of sales of proton pump inhibitors medication could be the major factor in this increase. In a second study, we ranked groups at risk of acquiring listeriosis based on the incidence of listeriosis and its lethality in each group. This enabled us to identify the most vulnerable groups : hematological malignancy, some cancers (digestive, lung, and brain cancer), dialysis, cirrhosis, organ transplantation and pregnancy. Epidemiological analysis of listeriosis cases associated with pregnancy indicated an association between regions with low rate listeriosis associated with pregnancy and regions where toxoplasmosis prevalence of pregnant women is low. This suggests that recommendations for avoiding toxoplasmosis have a positive effect on preventing listeriosis during pregnancy.

Listeria monocytogenes é o microrganismo patogênico de maior relevância em carnes processadas prontas para consumo. A presença frequente de L. monocytogenes no ambiente pode levar a uma contaminação dos produtos após o processamento industrial e como esses produtos não passam por tratamento bactericida antes de serem consumidos, a saúde do consumidor pode estar em risco. Para inibir a multiplicação de L. monocytogenes nos produtos cárneos durante o armazenamento em refrigeração após o processamento, os fabricantes podem utilizar diversos aditivos antimicrobianos na formulação destes produtos. Este estudo objetivou avaliar a atividade antimicrobiana de aditivos tradicionalmente empregados em produtos cárneos processados, e de quatro novos blends preparados à base de nisina (Nisaplin&#174;) contra cepas de L. monocytogenes, fazendo-se a avaliação in vitro e in situ, em mortadelas experimentalmente contaminadas, formuladas com os compostos estudados, armazenadas à vácuo e em refrigeração (8 °C) por 70 dias. Os compostos extrato de alecrim, diacetato de sódio e Nisaplin®, quando testados in vitro, apresentaram maior eficiência na inibição das cepas de L. monocytogenes que lactato de sódio e vinagre tamponado. Quando testados in vitro, os produtos comerciais BioVia&#174; CL600 e NovaGARD&#174; LM100 e os quatro blends utilizados no preparo das mortadelas foram igualmente efetivos na inibição de L. monocytogenes. De acordo com os resultados dos testes in situ, o melhor controle de L. monocytogenes em mortadelas durante 70 dias a 8 °C ocorreu nos produtos preparados com o blend contendo extrato de alecrim, diacetato de sódio, vinagre tamponado e Nisaplin&#174;. O blend contendo extrato de alecrim, lactato de sódio e Nisaplin&#174;, foi o menos efetivo entre os blends testados. / Listeria monocytogenes is the most important microbial pathogen in ready-to-eat processed meat products. The frequent presence of L. monocytogenes in the environment can lead to product contamination after industrial processing and since these products do not have a bactericidal step before consumed, consumer health may be at risk. To inhibit the multiplication of L. monocytogenes in processed meat products during refrigerated storage, manufacturers may use various antimicrobial additives in the formulation of these products. This study aimed to evaluate the in vitro and in situ activity of additives traditionally used in processed meat products and four new blends based on nisin (Nisaplin&#174;) against L. monocytogenes, in experimentally contaminated bolognas, formulated with the studied compounds and stored under vacuum and refrigerated (8 °C) for 70 days. Rosemary extract, sodium diacetate and Nisaplin&#174;, when tested in vitro, were more effective than sodium lactate and buffered vinegar for the inhibition of the L. monocytogenesstrains. When tested in vitro, the commercial products BioVia&#174; CL600 and NovaGARD&#174; LM100 and the four blends used in bologna preparation were equally effective in inhibiting L. monocytogenes. According to the results of the in situ tests, the best control of L. monocytogenes in bolognas for 70 days at 8 °C occurred in the products prepared with the blend containing rosemary extract, sodium diacetate, buffered vinegar and Nisaplin&#174;. The blend containing rosemary extract, sodium lactate and Nisaplin&#174;, was the least effective among the tested blends.