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No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Cristina_Jansen.pdf: 1461357 bytes, checksum: 1eabd3351ea2e61e231bd4b28f9e3f34 (MD5) Previous issue date: 2015-03-23 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / A colmeia constitui-se em uma fonte de geração de produtos de alto valor nutritivo e com grandes potencialidades farmacológicas. A própolis é um dos produtos obtidos da colmeia, que é produzida pelas abelhas através da coleta de resinas e gomas de árvores, de plantas e de flores, materiais que são parcialmente digeridos e acrescentados de cera e pólen. As propriedades biológicas da própolis são atribuídas principalmente ao conteúdo de fitoquímicos. Dentre estes compostos, os que estão presentes em maior proporção são os compostos fenólicos, majoritariamente os da classe dos flavonoides. Porém, vários são os fatores que interferem na composição química da própolis, o que acaba refletindo no conteúdo e proporção dos diferentes fitoquímicos, e, portanto, em suas propriedades. O Brasil, sendo um país com grande diversidade de vegetação, favorece ampla variação na composição da própolis, contribuindo para dificultar a padronização deste produto no país. Assim, o objetivo deste estudo foi avaliar a composição de fitoquímicos em amostras de própolis oriundas do Sul do Rio Grande do Sul, afim de testar suas propriedades biológicas. Parte das amostras de própolis foram analisadas quanto aos principais parâmetros físico-químicos, e de outra parte foram preparados extratos hidroalcoólicos concentrados. Os extratos foram submetidos à determinação química de compostos fenólicos, flavonoides e carotenoides, e da análise de espectroscopia Ultravioleta-Visível. Também foram avaliados quanto à capacidade antioxidante, através dos métodos de DPPH, ABTS e FRAP; quanto à atividade antibacteriana frente a cepas de bactérias patogênicas (Staphylococcus aureus, Escherichia coli, Listeria monocytogenes); e quanto ao efeito citotóxico, o qual foi realizado através do método de redução do MTT em células da linhagem melanoma B16F10. Dentre os parâmetros físico-químicos, apenas uma amostra apresentou conteúdo de umidade acima do permitido pela legislação brasileira. A cor foi definida como amarela para as amostras oriundas de Rio Grande e de Canguçu, e castanho claro para as amostras oriundas de Pelotas e de Pedro Osório. Todas as amostras apresentaram alto teor dos fitoquímicos analisados (compostos fenólicos, flavonoides e carotenoides); porém, houve variação significativa no conteúdo destes compostos entre as diferentes amostras. Pelo espectro UV observou-se absorção máxima entre 290 e 400 nm, região característica da absorção dos flavonoides. As amostras de própolis de Pelotas e de Rio Grande apresentaram alta capacidade antioxidante determinada pelos métodos com o radical DPPH e FRAP, sendo comparáveis aos obtidos com o padrão quercetina. Constatou-se uma relação positiva entre as amostras com maior teor de fitoquímicos e a elevada ação antioxidante. Todas as amostras apresentaram atividade antibacteriana frente às cepas testadas; porém, os resultados foram superiores na inibição das bactérias Gram-positivas (S. aureus e L. monocytogenes) do que para a bactéria Gram-negativa (E. coli O157:H7), sendo que as amostras de Pelotas e Rio Grande tiveram alta ação bactericida. Todas as amostras de própolis analisadas demonstraram ação citotóxica em 48h no combate às células de câncer de melanoma de camundongo, nas concentrações de 250 e 500 μg.mL-1. Após 72h a própolis causou citotoxicidade na concentração de 25 μg.mL-1. Amostras oriundas de Pelotas e de Rio Grande apresentaram resultados promissores no combate à linhagem testada. / The beehive is a source of nutritious high-value products and with great pharmacological potential. Propolis is one of the products obtained from the beehive, produced by honey bees by collecting resin and gum trees, plants and flowers, which are partially digested and added of wax and pollen. Its biological properties are mainly attributed to phytochemicals. Among these, the highest contend are phenolic compounds, mainly the class of flavonoids. However, there are several factors that influence the chemical composition of propolis, which ends up reflecting on their properties. The Brazil is a country with great diversity of vegetation that enhances the variations in the composition of propolis, contributing to hinder its standardization in the country. Therefore, the aim of this study was to evaluate the composition of phytochemicals of propolis samples of South of Rio Grande do Sul, and test their biological properties. Part of the propolis samples were analyzed for major physical and chemical parameters and another part was used to obtain the etanolic extracts. The extracts were subjected to determination of phenolic compounds, flavonoids and carotenoids, and analysis of Ultraviolet-visible spectroscopy. Also it was determined the antioxidant capacity that was measured by the methods DPPH, ABTS and FRAP. The antibacterial activity was evaluated against strains of pathogenic bacteria (Staphylococcus aureus, Escherichia coli, Listeria monocytogenes). The cytotoxic effect was performed using the MTT reduction method melanoma cell line B16F10. For the physico-chemical parameters, only one sample showed humidity values above those permitted by brasilian legislation. The color was defined as yellow for the Rio Grande and Canguçu samples, and light brown to Pelotas and Pedro Osorio samples. The samples showed high concentration of the analyzed phytochemicals (phenolics, flavonoids and carotenoids), but there was significant variation between samples. The UV spectrum showed absorption maxima between 290 and 400 nm, characteristic of the region flavonoids. Propolis from Pelotas and Rio Grande showed the highest antioxidant capacity by method with DPPH radical and FRAP, comparable to those obtained with the standard quercetin. It was found a positive relationship between the samples with higher levels of phytochemicals and antioxidant effects. All samples showed antibacterial activity against all tested strains, but the results were better in the inhibition of Gram-positive bacteria (S. aureus and L. monocytogenes) than for Gram-negative (E. coli O157: H7), and samples of Pelotas and Rio Grande had high bactericidal action. The propolis samples analyzed showed cytotoxic action in 48 hours in fighting cells of mouse melanoma cancer at concentrations of 250 and 500 μg.mL-1. After 72h propolis caused decrease in carcinogenic lineage in concentrations 25μg.mL-1. Samples of Pelotas and Rio Grande showed promising results in combating tested lineage.

O objetivo do presente estudo foi monitorar a incidência de Staphylococcus aureus, Listeria sp., Listeria monocytogenes, Escherichia coli, coliformes totais, bactérias aeróbias mesófilas e psicrotróficas ao longo da produção de leite pasteurizado, desde a ordenha até a obtenção do produto final para estabelecer etapas e locais críticos da produção, bem como avaliar se a presença de Listeria sp. constitui um bioindicador de L. monocytogenes, e E. coli um bioindicador de outros micro-organismos patogênicos. As coletas foram feitas em 5 laticínios (A, B, C, D e E) do Estado de São Paulo, em duplicata, com intervalo de coleta variando de 3 semanas a 7 meses, de acordo com disponibilidade dos laticínios. Coletou-se um total de 236 amostras foram coletadas, sendo 36 de leite (cru e pasteurizado), 162 eram provenientes de superfícies que não tinham contato com o leite e 38 superfícies que entravam em contato com leite. Das 36 amostras de leite analisadas, 13,9% estavam contaminadas com Listeria sp. e nenhuma com L. monocytogenes; 61,1% continham E. coli e 5,6% apresentavam S. aureus. Somente o leite do laticínio C apresentou em uma das coletas micro-organismo patogênico (E. coli) no leite pasteurizado, indicando falhas no processamento ou no manejo no momento da ordenha. Das 38 amostras de superfícies com contato com o leite (38), 2,6% foram positivas para Listeria sp., 50,0% para E. coli e 5,3% para S. aureus. Das amostras de superfícies sem contato com o leite (162), 13,3% estavam contaminas com Listeria sp., 6,2% com L. monocytogenes e 25,9% com E. coli. De acordo com o limite estabelecido de aeróbios mesófilos no leite cru pela IN 62, constatou-se que 50,0% do leite cru dos laticínios A, D e E, 100% do leite cru do laticínio B e 33,3% do leite cru do laticínio C estão fora dos padrões estabelecidos pela legislação. Foi comprovado que a Listeria sp. não pode ser considerada como bioindicador de L. monocytogenes pelo teste Qui-Quadrado (p<0,05). Ao comparar as médias das amostras positivas para os microorganismos E. coli, S. aureus, Listeria sp. e L. monocytogenes dos laticínios processadores de leite tipo A com os de leite pasteurizado, somente o S. aureus no leite apresentou diferença significativa pelo teste \"T\" (p<0,05). Além dos pontos críticos de controle (PCC) checados através da Árvore Decisória (pasteurização, superfícies internas de embalagens), outros pontos merecem destaque pela elevada quantidade de patógenos (tanques de armazenamento de leite cru e pisos e paredes de câmaras frias). Os resultados obtidos ressaltam a importância da adoção de ferramentas de gestão da qualidade, como Boas Práticas de Fabricação e APPCC, para que a segurança alimentar seja garantida ao longo da cadeia de produção do leite pasteurizado nos laticínios estudados. / The aim of this study was to monitor the incidence of Staphylococcus aureus, Listeria sp., Listeria monocytogenes, Escherichia coli, total coliforms, mesophilic aerobic and psychrotrophic bacteria along the pasteurized milk production, from milking to the final product, to establish steps and critical points of production, as well as to evaluate the presence of Listeria sp. as a bioindicator of L. monocytogenes and E. coli a bioindicator of other pathogenic microorganisms. Duplicate samples were collected in 5 dairy plants (A, B, C, D, E) from the state of São Paulo, within intervals ranging from 3 weeks to 7 months, according to the dairy plants availability. A total of 236 samples were collected, being 36 of milk (raw and pasteurized), 162 from surfaces with no contact with the milk, and 38 from surfaces with contact with milk. Out of 36 milk samples analyzed, 13.9% were contaminated with Listeria sp. and none had L. monocytogenes; 61.1% were contaminated with E. coli and 5.6% with S. aureus. Only dairy plant C showed pathogenic microorganism (E. coli) in the pasteurized milk in one of the collections, indicating failures in the pasteurization or excessive bacterial load in the raw milk. Out of the 38 samples of surfaces that had contact with milk, 2.6% were positive for Listeria sp., 50.0% for E. coli and 5.3% for S. aureus. As for the samples from surfaces with no contact with milk (162), 13.3% were contaminated with Listeria sp., 6.2% with L. monocytogenes and 25.9% with E. coli. According to the Brazilian regulations for aerobic mesophiles in raw milk by Normative Instruction 62, 50.0% of samples from dairy plants A, D and E, 100% of samples from dairy plant B and 33.3% of samples from dairy plant C were above the tolerance limit adopted. The analysis of Listeria sp. could not be considered as a bioindicator of L. monocytogenes by chi-square test (p<0.05). When comparing the mean frequencies of positive samples for E. coli, S. aureus, Listeria sp. and L. monocytogenes in the processing dairy plants of type A milk (plants A and B) and the pasteurized one (plants C, D and E), only S. aureus in milk showed significant difference by \"T\" test (p<0.05). In addition to the critical control points (CCP) checked by a decision tree (pasteurization, internal surfaces of packaging), other points should be highlighted by the high number of pathogens found (bulk raw milk tanks, floors and walls of cold storage rooms). Results of this trial indicate the importance of adoption of quality management tools such as Good Manufacture Practices and HACCP, to ensure food safety along the pasteurized milk production chain in the dairy plants evaluated.

Biofilmes de Listeria monocytogenes são fontes potenciais de contaminação de alimentos processados e podem diminuir a efetividade de procedimentos de higienização e sanitização nas indústrias. No presente estudo foi avaliada a estrutura e a dispersão de biofilmes formados por duas cepas de L. monocytogenes em diferentes superfícies, como aço inoxidável, vidro e poliestireno. Foram utilizados diferentes sistemas de cultivo como microplacas de 96 poços de poliestireno e de aço inoxidável, microplacas de 24 poços contendo lâminas circulares de aço inoxidável ou de vidro e, câmaras de poliestireno contendo 8 poços com fundo de borossilicato (vidro). Os experimentos foram realizados com incubação por 1, 4 e 8 dias a 25°C. A formação de biofilme foi verificada em microplacas de 96 de poliestireno e de aço inoxidável através do método de quantificação de biomassa do biofilme por coloração com cristal violeta, e também em sistema de microplaca de 24 poços contendo lâminas circulares de aço inoxidável ou de vidro, através de enumeração em placa das células aderidas nas superfícies. As estruturas dos biofilmes foram observadas por meio de microscopia de fluorescência (para o sistema de microplaca de 24 poços contendo lâminas circulares de aço inoxidável) e através de microscopia confocal a laser (para o sistema com câmaras com fundo de vidro). Para isso, os biofilmes foram corados com o kit de viabilidade bacteriana LIVE/DEAD®, a fim de diferenciar as células viáveis (coradas com Syto 9) das células mortas (coradas com iodeto de propídeo), sendo feitas estimativas do número de células aderidas à superfície de vidro através de quantificação por fluorescência. Foram realizados testes de concentração inibitória mínima (CIM) e concentração bactericida mínima (CBM) de óleos essenciais de plantas de Cymbopogon citratus (capim-limão) e de Zingiber officinale (gengibre) e de dois sanitizantes comerciais (um à base de óleo de coco babaçu e outro à base de dióxido de cloro) frente a L. monocytogenes. Posteriormente, foi testada a eficácia desses antimicrobianos na remoção de biofilmes de L. monocytogenes formados por 4 e 8 dias a 25ºC, em superfície de aço inoxidável e de vidro, através da enumeração em placas de células aderidas e de observações microscópicas. Foi também avaliada a presença de moléculas relacionadas ao estresse oxidativo e nitrosativo em biofilmes maduros de duas cepas de L. monocytogenes formados em superfícies de aço inoxidável e de vidro, para o estudo do possível efeito do óxido nítrico (NO) exógeno e de inibidores de NO na dispersão de células do biofilme e na alteração dos níveis de expressão de genes de L. monocytogenes relacionados ao estresse oxidativo e nitrosativo (Lmo0990, Lmo0807 e Lmo1485), bem como à regulação do gene de virulência e formação de biofilme (PrfA). Foi verificada a presença de intermediários de oxigênio reativo (ROI - reactive oxygen intermediates) e de intermediários de nitrogênio reativo (RNI - reactive nitrogen intermediates) nos biofilmes de L. monocytogenes com 4 e 8 dias de incubação a 25ºC formados em superfícies de aço inoxidável e de vidro, por meio de marcadores ii fluorescentes específicos e visualizações microscópicas. A fim de confirmar indiretamente a presença de óxido nítrico (NO) em cultivos de L. monocytogenes incubados por 4 e 8 dias a 25ºC, foi realizada a dosagem de nitrito com o emprego do reagente Griess. Foram realizados testes de concentração inibitória mínima (CIM) do doador de óxido nítrico como nitroprussiato de sódio (SNP) e dos inibidores de NO como N?-Nitro-L-arginina metil éster (L-NAME) e 2-(4-carboxifenil)-4,4,5,5- tetrametilimidazolina-1-oxi-3-óxido (Carboxi-PTIO sal de potássio, C-PTIO) frente a L. monocytogenes. Foi testada a eficácia do SNP e dos inibidores de NO na remoção de biofilmes pré-formados por 4 e 8 dias de L. monocytogenes em superfície de aço inoxidável. Os resultados deste trabalho demonstraram que o biofilme de L. monocytogenes formado em vidro e em aço inoxidável apresentou uma arquitetura microscópica semelhante a um \"favo de mel\", com presença de canais de água, bem como cavidades de tamanhos variados devido à dispersão de grupos de células planctônicas ou morte celular. A utilização de óleos essenciais e/ou sanitizantes comerciais por 1h em biofilmes de L. monocytogenes formados por 4 e 8 dias, foi eficaz na redução do número de células aderidas na superfície de aço inoxidável e de vidro. No biofilme de L. monocytogenes foram detectadas moléculas relacionadas ao estresse oxidativo como peróxido de hidrogênio (H2O2) e radicais superóxidos, bem como moléculas de estresse nitrosativo como óxido nítrico (NO) e peroxinitrito. A adição de NO exógeno (por meio do doador SNP) e a adição de inibidores de NO no meio de cultivo não alteraram o crescimento planctônico de L. monocytogenes. Foi observado que a exposição a SNP e a inibidores de NO por 1h em biofilmes de L. monocytogenes pré-formados por 4 e 8 dias, não induziu a dispersão celular. A adição de NO exógeno bem como a remoção de NO do meio de cultura por moléculas inibidoras não alteraram o nível de expressão dos genes Lmo1485, Lmo0990, Lmo0807 e PrfA, em culturas de L. monocytogenes de 8 dias. A utilização de óleos essenciais de plantas e/ou sanitizantes comerciais em biofilmes pré-formados pode ser uma alternativa eficaz na eliminação de L. monocytogenes em superfícies de manipulação de alimentos, mas a melhor estratégia de controle é impedir a formação de biofilme pelo emprego de tratamentos combinados nos estágios iniciais de contaminação. Em conclusão, L. monocytogenes formou biofilme com estruturas bem definidas que podem contribuir para a sobrevivência e disseminação da bactéria no ambiente de processamento de alimentos. Apesar de L. monocytogenes não formar um biofilme espesso com multicamadas, as células aderidas apresentam geralmente maior resistência à ação dos antimicrobianos em comparação com as células planctônicas, conseguindo sobreviver e persistir na superfície, com mecanismos regulatórios bastante eficientes frente a diferentes tipos de estresse. / Biofilms of Listeria monocytogenes are potential sources of contamination of processed foods, and may interfere in the effectiveness of cleaning and sanitizing procedures in food industry. In the present study the structures and dispersion of biofilms formed by two strains of L. monocytogenes on different abiotic surfaces, such as stainless steel, glass and polystyrene were evaluated. Different cultivation systems such as polystyrene and stainless steel 96-wells microplates, 24-wells microplates containing round stainless steel or glass slides, and 8-wells polystyrene chambers with borosilicate bottom were used. The experiments were done for 1, 4 and 8 days of incubation at 25°C. Biofilm formation was observed in 96-wells microplates of polystyrene and stainless steel by the quantification of biofilm biomass by staining with crystal violet, and also in 24-wells microplates system with circular slides of stainless steel or glass, with enumeration of adhered cells on agar plates. The structure of biofilms were observed by fluorescence microscopy (for the system of 24-wells microplates containing circular slides of stainless steel) and by confocal laser scanning microscopy (for the system of glass bottom chambers). For this, biofilms were stained with the LIVE/DEAD® bacterial viability kit, in order to differentiate viable cells (stained with Syto 9) from dead cells (stained with propidium iodide). The estimative of the number of viable cells was done by measurement of fluorescence. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the essential oils plants of Cymbopogon citratus (lemon grass) and Zingiber officinale (ginger) and two commercial sanitizers (babassu coconut oil-based sanitizer and chlorine dioxide-based sanitizer) against L. monocytogenes were also determined. Subsequently, the antimicrobials were evaluated against L. monocytogenes biofilms formed by 4 and 8 days at 25°C on the stainless steel and glass surfaces by enumeration on agar plates of adhered cells and microscopic observations. The presence of molecules related to oxidative and nitrosative stresses in mature biofilms of two strains of L. monocytogenes formed on glass and stainless steel surfaces was also evaluated, and the possible role of exogenous nitric oxide (NO) and inhibitors of NO were studied for induction of biofilm dispersal cells and changed of genic expression levels of L. monocytogenes related to oxidative and nitrosative stress genes (Lmo0990, Lmo0807 and Lmo1485), as well as the regulation of virulence and biofilm formation gene (PrfA). The presence of reactive nitrogen intermediates (RNI) and reactive oxygen intermediates (ROI) was investigated in biofilms of L. monocytogenes formed on stainless steel and glass surfaces with 4 and 8 days-old at 25°C, using specific fluorescent dyes and microscopic views. In order to indirectly confirm the presence of nitric oxide (NO) in cultures of L. monocytogenes incubated for 4 and 8 days at 25°C, the dosage of nitrite was carried out with Griess reagent. The minimum inhibitory concentration (MIC) of a nitric oxide donor as sodium nitroprusside (SNP) and two NO inhibitors such as N-nitro-L-arginine methyl ester (L-NAME) and 2-(4-carboxyphenyl)-4,4,5,5- tetrametilimidazoline-1-oxy-3-oxide (Carboxy-potassium salt, C-PTIO), were iv evaluated for L. monocytogenes. It was also determined the effectiveness of SNP and of the two inhibitors of NO to eliminate biofilms of L. monocytogenes preformed for 4 and 8 days on stainless steel surfaces. The results of this work showed that L. monocytogenes biofilms formed on glass and on stainless steel surfaces, presented similar microscopic architectures in a \"honeycomb\" like structure, with water channels and hollows of different sizes, possibly due to cell dispersion or death. The exposure to essential oils and/or commercial sanitizers for 1h for L. monocytogenes biofilms formed for 4 and 8 days was effective in reducing the number of cells adhered on stainless steel and on glass surfaces. In biofilms of L. monocytogenes, molecules were detected related to oxidative stress such as hydrogen peroxide (H2O2) and superoxide radicals, as well as molecules related to nitrosative stress as nitric oxide (NO) and peroxynitrite. The addition of exogenous NO (SNP) and inhibitors of NO in the culture medium did not inhibit planktonic growth of L. monocytogenes, and exposure to SNP and to inhibitors of NO for 1 h for biofilms of L. monocytogenes preformed by 4 and 8 days did not induce cell dispersion. The addition of exogenous NO and NO removal from the culture medium by inhibitory molecules did not alter the level of expression of Lmo1485, Lmo0990, and PrfA Lmo0807 genes in cultures of 8- days-old of L. monocytogenes. The use of essential oils from plants and/or sanitizing commercial agents on pre-formed biofilms can be an effective alternative in the control of L. monocytogenes in food handling surfaces, but the best control strategy is avoid the biofilm formation by applying combined treatments in initial stages of contamination. In conclusion, L. monocytogenes formed biofilms with well defined structures that may contribute to the survival and spread of bacteria in food processing environment. Despite L. monocytogenes do not form a multilayer thick biofilms, adherent cells generally have greater resistance to antimicrobial activity compared to planktonic cells, surviving and persisting on the surface with regulatory mechanisms effective against different types of stress.

A ocorrência de surtos de doencas associadas aos vegetais minimamente processados (VMP) tem chamado a atenção para a sua segurança microbiológica. A avaliação quantitativa de riscos permite que o impacto das materias-primas e processamento seja avaliado e os resultados obtidos sejam usados para gestão e comunicação do risco. Desta forma, o presente estudo objetivou quantificar o risco de infecções por Salmonella spp. e Listeria monocytogenes a partir do consumo de VMP no Brasil. Um total de quinhentas e doze amostras de VMP foram analisadas e foi possivel enumerar e detectar Salmonella em 0,4% e 0,4% das amostras, respectivamente. L. monocytogenes foi enumerada e detectada em 0,97% e 3,1% das amostras analisadas, respectivamente. Os isolados de Salmonella spp. (n=4) e L. monocytogenes (n=69) foram confirmados por PCR e caracterizados por sorotipagem tradicional. Os isolados de L. monocytogenes foram caracterizados quanto ao ribotipo, resistencia ao cloro, taxa de multiplicação (µ), capacidade de formação de biofilmes e presença de genes de virulência. O sorovar predominante entre Salmonella spp. foi S. Typhimurium. Em relação a L. monocytogenes, observou-se prevalência do sorotipo 4b e do ribogrupo DUP-1038 e presenca de genes de virulência em 100% (inlA) e 97% (inlC e inlJ) dos isolados. A maioria dos isolados de L. monocytogenes foi resistente a exposição a 125 ppm de cloro livre, e todos foram capazes de aderir ao aco inox, atingindo concentracoes acima de 4 log UFC/cm2. Testes-desafio foram conduzidos para determinar o potencial de multiplicação (δ) de cepas de Salmonella e L. monocytogenes em nove diferentes tipos of VMPs armazenados a 7°C e 15°C por 6 dias. O armazenamento a 15°C por 6 dias resultou nos maiores aumentos nas populações de L. monocytogenes em couve picada (δ= 3,34) e rúcula ((δ= 3,22), enquanto para Salmonella, as maiores populações foram observadas em rúcula (δ= 4,05) e escarola (δ= 2,80). Testes-desafios posteriores indicaram que a multiplicação dos dois patógenos em VMP foi mais pronunciada quando os mesmos foram embalados sob atmosfera modificada em comparação a embalagem em filmes perfurados. Modelos preditivos primários e secundários descrevendo a taxa de multiplicação e tempo de lag de Salmonella spp. e L. monocytogenes em VMP em função da temperatura de armazenamento (7, 10, 15, 20, 25 e 30°C) foram gerados. Verificou-se que os modelos gerados apresentaram a precisão necessária e foram adequados para modelagem da multiplicação dos dois patógenos em VMP. Os modelos de avaliação quantitativa de risco (AQR) foram construidos para determinar a probabilidade de infecção por Salmonella spp. e L. monocytogenes devido ao consumo de VMPs. Os modelos construidos com base nos dados levantados da literatura indicaram risco de infecção por Salmonella spp. e L. monocytogenes de 8.66 x 10-3 e 1.87 x 10-8, respectivamente, sendo necessário que medidas de mitigação do risco sejam adotadas. / The occurrence of foodborne disease outbreaks linked to minimally processed vegetables (MPV) is concerning industries, consumers and governments worldwide. Quantitative risk assessments can estimate the impact of raw materials and processing practices and these estimates are used for risk management and risk communication. This study aimed at quantifying the risks of infection by Salmonella spp. and Listeria monocytogenes due to consumption of MPV in Brazil. A total of five hundred and twelve samples of MPV were analyzed and Salmonella was detected and enumerated in 0.4% and 0.4% of the samples, respectively. L. monocytogenes was enumerated and detected in 0.97% and 3.1% of the samples analyzed, respectively. Isolates of Salmonella spp. (n=4) and L. monocytogenes (n=69) were confirmed through PCR and characterized by traditional serotyping. The isolates of L. monocytogenes were characterized for their ribotype, resistance to chlorine, growth rate, (µ) and ability to form biofilms and presence of virulence factors. Among Salmonella spp., S. Thyphimurium was the most prevalent serovar. Among L. monocytogenes, prevalence of serotype 4b and ribotype DUP-1038 was observed. Virulence gene inlA was present in 100% of the isolates, and genes inlC and inlJ in 97%. The majority of L. monocytogenes isolates were resistant to up to 125 ppm of free chlorine and all isolates were able to attach to stainless steel coupons, reaching populations of up to 4 log10 CFU/cm2. Challenge tests were carried out to determine the growth potential (δ) of Salmonella and L. monocytogenes in nine types of MPV stored at 7°C and 15°C for 6 days. The storage of MPV at 15°C for 6 days resulted in the greatest increases in L. monocytogenes populations in shredded collard green (δ= 3.34) and arugula (δ= 3.22), whereas for Salmonella, the highest populations were found in arugula (δ= 4.05) and escarole (δ= 2.80). Further challenge tests indicated that multiplication of both pathogens in MPV was more pronounced when these products were packaged under modified atmosphere in comparison to packaging in perforated films. Primary and secondary predictive models describing the growth rate and lag time of Salmonella and L. monocytogenes in MPV as a function of storage temperature (7-30°C) were generated. The generated models were accurate and suitable for modeling the growth of pathogens in MPVs. Quantitative risk assessment (QRA) models were built to determine the probability of infection by Salmonella and L. monocytogenes due to consumption of MPVs. The models built using data available in the literature indicated that the risks of infection by these pathogens were 8.66 x 10-3 and 1.87 x 10-8, respectively, evidencing the need for adoption of risk mitigation measures.

Die antimikrobielle Wirkung eines Pflanzenextraktes aus fermentiertem Rooibos (Aspalathus linearis) und eines Extraktes aus Hopfen (Humulus lupulus) auf Milchsäurebildner (Lactobacillus spp., Carnobacterium spp., Leuconostoc carnosum), Verderbniserreger (Bacillus spp., Brochothrix spp., Pseudomonas fluorescens) und pathogene Mikroorganismen (Listeria monocytogenes, Salmonella Enteritidis) wird unter In-vitro-Bedingungen über einen Lagerungszeitraum von 28 Tagen bei Temperaturen von 10 °C und 25 °C untersucht. In den sich anschließenden Challengeversuchen wird evaluiert, ob sich die antimikrobiellen Wirkungen beider Pflanzenextrakte gegen Listeria monocytogenes auf die Lebensmittelmatrix Rotschmierkäse übertragen lassen. Mögliche herstellungsbedingte und sensorische Probleme aufgrund der Anwendung der Pflanzenextrakte im Lebensmittel „Weichkäse“ werden erörtert. Die Ausgangskeimzahl beträgt je Keim 102–103 KbE/ml. Der Rooibosextrakt wird in einer Konzentration von 5 mg/ml zugesetzt und der Hopfenextrakt in einer Konzentration von 30 µg/ml. Unter In-vitro-Bedingungen ist eine statistisch abgesicherte bakteriostatische Wirkung des Rooibosextraktes auf Listeria monocytogenes bei einer Lagerungstemperatur von 10 °C in den ersten 14 Tagen nachweisbar. Die Reduzierung der Verderbniserreger in den ersten 24 Stunden nach Zugabe des Rooibosextraktes bei beiden Lagerungstemperaturen bzw. der Milchsäurebildner bei 25 °C ist statistisch nicht abzusichern und bei den weiteren Probenahmen nicht mehr darstellbar. Der Rooibosextrakt hat keine nachweisbare antibakterielle Wirkung auf Salmonella Enteritidis. Alle grampositiven Testkeime lassen sich durch die Zugabe des Hopfenextaktes statistisch signifikant in ihrem Wachstum beeinflussen. Eine antibakterielle Aktivität des Hopfens zeigt sich bei Listeria monocytogenes in einer verlängerten lag-Phase und anschließendem bakteriostatischen Effekt bei 10 °C. Der Hopfenextrakt hat keinerlei Wirkung auf die gramnegativen Teststämme Pseudomonas fluorescens und Salmonella Enteritidis. Der Extrakt aus Hopfen zeigt auch in der Lebensmittelmatrix Rotschmierekäse eine signifikante Wachstumshemmung auf Listeria monocytogenes. Der Extrakt aus fermentierten Rooibos zeigt keinen antilisteriellen Effekt. Beide Pflanzenextrakte beeinflussen leicht die sensorischen Eigenschaften der Käse, die Käsereifung nicht. Erstmals liegen antibakterielle In-vitro-Studien der getesteten Pflanzenextrakte gegen lebensmittelrelevante Mikroorganismen über 28 Tage vor. Eine Anwendung von Pflanzenextrakten als antimikrobieller Lebensmittelzusatz ist erst nach entsprechender Validierung im jeweiligen Einzelprodukt möglich.

Listeria monocytogenes est une bactérie à Gram positif impliquée dans des infections graves d’origine alimentaire. La plupart des cas de listériose humaine sont causés par la consommation d'aliments réfrigérés prêts à consommer. La capacité de ces bactéries à survivre et à se multiplier dans une large gamme de conditions difficiles fait de ce pathogène une des préoccupations majeures dans les industries agro-alimentaires. Ces propriétés de L. monocytogenes sont renforcées par son aptitude à former des biofilms. Le but de ce projet était d'explorer l'adaptation de ce pathogène à la déshumidification et aux basses températures par deux approches de protéomique. La première approche, basée sur la technique d’imagerie par spectrométrie de masse (IMS) MALDI-TOF, permet de réaliser la cartographie de molécules à partir d'échantillons biologiques. Ce travail a consisté à développer cette approche, en considérant un biofilm bactérien comme un tissu, afin d’accéder à des informations sur la distribution de protéines dans des biofilms de L. monocytogenes soumis à un stress de déshumidification. En outre, une approche LC-MS/MS a été utilisée pour relier les données spectrales d’intérêt obtenues par l'IMS et l'identification des protéines. L’IMS a permis d'examiner la distribution de 47 protéines de bas poids moléculaire dans les biofilms. Cinq protéines ont été identifiées par LC-MS/MS grâce aux données m/z de l’IMS, y compris deux protéines de choc thermique. Les résultats démontrent que l'IMS peut être utilisée pour disséquer le protéome spatial d'un biofilm bactérien. La deuxième approche protéomique a consisté en une comparaison semi-quantitative relative et sans marquage (shotgun proteomic) des protéines exprimées dans différentes conditions de culture. Par cette méthode, nous avons exploré l’expression protéique en fonction du mode de croissance (biofilm vs planctonique) et de la température (10°C, 25°C et 37°C). Parmi les 920 et 931 protéines uniques identifiées, provenant respectivement de cellules sessiles et planctoniques, beaucoup sont liées à des fonctions cellulaires de base, mais certaines sont liées à la thermorégulation. Des changements ont été observés dans le protéome de L. monocytogenes en biofilm par rapport aux cellules planctoniques, ce qui indique des modes de régulation différents selon le mode de croissance. Ces comparaisons de l'expression des protéines dans plusieurs conditions (modes de croissance, températures) enrichiront les bases de données et aideront à modéliser les circuits de régulation qui conduisent à l'adaptation de L. monocytogenes aux environnements. / Listeria monocytogenes is a Gram-positive bacterium implicated in serious food-borne infections. Most cases of human listeriosis are caused by the consumption of refrigerated ready-to-eat foods. The ability of these bacteria to survive and multiply in a wide range of harsh conditions make this pathogen a major concern in agro-food industries. These properties of L. monocytogenes are enhanced by its ability to form biofilms. The aim of this project was to explore the adaptation of this pathogen to dehumidification and low temperatures by two proteomic approaches. The first approach, based on the MALDI-TOF mass spectrometry imaging (IMS), allows the mapping of molecules from biological samples. This work aimed to develop this approach, considering a bacterial biofilm as a tissue, in order to access information on the distribution of proteins in L. monocytogenes biofilms subjected to a dehumidification stress. In addition, an LC-MS/MS approach was used to link spectral data of interest obtained by IMS and protein identification. The IMS allowed to examine the distribution of 47 low molecular weight proteins within the biofilms. Five identified proteins were assigned by LC-MS/MS using IMS m/z data, including two cold-shock proteins. The results demonstrate that imaging can be used to dissect the spatial proteome of a bacterial biofilm. The second proteomic approach consisted on a relative semi-quantitative label-free (shotgun proteomic) comparison of proteins expressed under different culture conditions. With the method, we explored protein expression according to the mode of growth (biofilm vs planktonic) and temperature (10°C, 25°C and 37°C). Throughout the 920 and 931 unique proteins identified, from sessile and planktonic cells, respectively, many are connected to basic cell functions, but some are linked with thermoregulation. A shift was observed in the proteome of L. monocytogenes biofilms compared to planktonic cells indicating different patterns of regulation according to the mode of growth. These comparisons of protein expression throughout several conditions (mode of growth and temperatures) will enrich databases and help to model regulatory circuitry that drives adaptation of L. monocytogenes to environments.

Listeria monocytogenes est un pathogène intracellulaire facultatif responsable d’une infection sévère d’origine alimentaire, la listériose. L’étude du processus d’infection cellulaire de cette bactérie a permis d’élucider divers mécanismes impliqués dans les interactions hôte-pathogène et dans le fonctionnement de la cellule eucaryote. En particulier, L. monocytogenes a été l’un des modèles pionniers dans la découverte du ciblage de la chromatine et de régulateurs nucléaires par des microbes. L’étude d’un facteur de virulence de L. monocytogenes, LntA, a permis l’identification d’un de ces régulateurs : BAHD1. En recrutant des protéines impliquées dans la formation de l’hétérochromatine, telles HDAC1/2 et HP1, BAHD1 stimule la formation d’une chromatine compacte à effet répressif. Lors d’une infection de cellules épithéliales par L. monocytogenes, BAHD1 réprime la réponse immunitaire stimulée par les interférons, une fonction inhibée par LntA. BAHD1 demeurant peu étudiée, mon doctorat a eu pour premier objectif de poursuivre la caractérisation de ce régulateur épigénétique. Par ailleurs, des données préliminaires suggéraient qu’un facteur de virulence de Listeria récemment découvert, InlP, avait la potentialité d’être, comme LntA, une nucléomoduline. Mon deuxième objectif a été d’explorer cette hypothèse.Les résultats de mon premier axe montrent que BAHD1 interagit avec MIER1 et que cette interaction est cruciale pour l’association de BAHD1 aux HDAC1/2. Nous reportons également que BAHD1 modifie la chromatine en changement la méthylation et l’acétylation des histones, ainsi que la méthylation de l’ADN, au niveau d’un gène cible, ESR1. Ces résultats nous permettent de proposer que BAHD1 forme, avec MIER1, un échafaudage assemblant un nouveau complexe de remodelage de la chromatine associé aux HDAC1/2 : le complexe BAHD1. Nous avons ensuite étudié un rôle de BAHD1 dans un organe ciblé par la Listeria, le cerveau. Nos résultats indiquent qu’une déficience totale en BAHD1 altère le transcriptome global de cet organe chez la souris. Les gènes majoritairement surexprimés sont impliqués dans des fonctions du système nerveux, le métabolisme et des troubles neurologiques. Les gènes majoritairement sous-exprimés sont impliqués dans des voies de l’immunité innée, dont des gènes de réponses aux interférons. Par ailleurs, une haplo-déficience en Bahd1 provoque des troubles comportementaux. En comparaison des souris Bahd1+/+, les souris Bahd1+/- souffrent d’une anxiété accrue et d’altérations du réflex de sursaut acoustique. Ces résultats suggèrent qu’une dérégulation de BAHD1, par des stimuli de l’environnement ou par des stimuli infectieux, pourrait avoir des effets neuro-pathologiques.Le second axe de ma thèse concernait l’étude des interactions d’InlP avec des protéines nucléaires de l’hôte, identifiées par un crible double-hybride. Nous montrons d’abord qu’InlP est une internaline atypique, avec des répétitions riches en leucine caractérisées par un motif LPX2. Nous identifions, ensuite, deux protéines nucléaires ciblées par InlP : le facteur d’épissage et suppresseur de tumeur RBM5 et le corépresseur RERE. Quand InlP est produite de façon ectopique dans les cellules humaines, elle se localise dans le noyau, où elle altère la formation de corps nucléaires enrichis en RERE. Dans des cellules sur-exprimant RBM5, InlP inhibe l’effet pro-apoptotique de RBM5 et stimule la formation de corps nucléaires denses associés à RBM5. Ces résultats suggèrent qu’InlP est une nucléomoduline agissant sur la l’assemblage et le désassemblage de compartiments de stockage de protéines cibles impliquées dans la synthèse et l’épissage d’ARNs de l’hôte.Ce travail ouvrent des perspectives dans la compréhension des interactions hôte-pathogène et dans une meilleure connaissance des mécanismes patho-épigénétiques, ainsi qu’en biologie cellulaire, dans la compréhension de la dynamique des organites nucléaires sans membrane. / Listeria monocytogenes is an optional intracellular pathogen responsible for a severe foodborne infection called listeriosis. The study of the cellular infection process of this bacterium has shed light on various mechanisms involved in host-pathogen interactions and in the functioning of the eukaryotic cell. In particular, L. monocytogenes has emerged as one of the pioneering models in the discovery of microbial targeting of chromatin and nuclear regulators. The study of a virulence factor of L. monocytogenes, LntA, allowed the identification of one of these regulators : BAHD1. By recruiting proteins involved in the formation of heterochromatin, such as HDAC1/2 and HP1, BAHD1 stimulates the formation of a compact chromatin with a repressive effect. When epithelial cells are infected with L. monocytogenes, BAHD1 suppresses the immune response stimulated by interferons, a function inhibited by LntA. Since BAHD1 is still under-researched, the first objective for my thesis was to further characterize this epigenetic regulator. In addition, preliminary data suggested that a recently discovered virulence factor of Listeria, InlP, had the potential to be, like LntA, a nucleomodulin. My second objective was to explore this hypothesis.The results of my first axis show that BAHD1 interacts with MIER1 and that this interaction is crucial for the association of BAHD1 with HDAC1/2. We also report that BAHD1 modifies chromatin by changing histone methylation and acetylation, as well as DNA methylation, at a target gene, ESR1. These results allow us to propose that BAHD1 form, with MIER1, a scaffold assembling a new chromatin remodeling complex associated with HDAC1/2 : the BAHD1 complex. We then studied the role of BAHD1 in an organ targeted by Listeria, the brain. Our results indicate that a total deficiency in BAHD1 alters the overall transcriptome of this organ in mice. Most of the overexpressed genes are involved in nervous system functions, metabolism and neurological disorders. The predominantly downregulated genes are involved in innate immunity pathways, including interferon response genes. In addition, a haplodeficiency in Bahd1 causes behavioral problems. Compared to Bahd1+/+ mice, Bahd1+/- mice suffer from increased anxiety and changes in acoustic startle reflex. These results suggest that deregulation of BAHD1, through environmental or infectious stimuli, may have neuro-pathological effects.The second axis of my thesis focused on the study of InlP interactions with host nuclear proteins, identified by a double-hybrid screen. First, we show that InlP is an atypical internalin, with leucine-rich repeats characterized by an LPX2 motif. We then identify two nuclear proteins targeted by InlP: the splicing factor and tumor suppressor RBM5 and the corepressor RERE. When InlP is produced ectopically in human cells, it is localized in the nucleus, where it alters the formation of nuclear bodies enriched in RERE. In RBM5-overexpressing cells, InlP inhibits the pro-apoptotic effect of RBM5 and stimulates the formation of dense nuclear bodies associated with RBM5. These results suggest that InlP is a nucleomodulin acting on the assembly and disassembly of target protein storage compartments involved in the synthesis and splicing of host RNAs.This work opens perspectives in the understanding of host-pathogen interactions and in a better knowledge of patho-epigenetic mechanisms, as well as in cell biology and the understanding of membraneless nuclear organelles dynamics.

Numerous examples of non-linear survival curves, plotted as log surviving cells vs. time, for bacteria exposed to heat have been reported. Factors which may affect the shape of a survival curve and the heat resistance of bacteria include temperature, pH, and NaCl concentration. Many studies have examined the effect of these factors individually, but little information exists on the combined effects. The objective of this study was to mathematically model non-linear survival curves to account for these factors and their interactions simultaneously. Heat resistance of <u>Listeria monocytogenes</u>(L. <u>monocytogenes</u>) was determined in O.lM KH₂P0₄ buffer and in infant formula at three temperatures (50, 55, and 60 C), three pH levels (5, 6, and 7), and three NaCl concentrations (0, 2, 4%). Survival curves were fit using linear regression, non-linear regression with a modified logistic equation, and non-linear regression with a modified Gompertz equation. / Ph. D.

<p>Glavni cilj doktorske disertacije bio je ispitivanje op&scaron;tih odlika industrijski proizvedenih, fermentisanih suvih kobasica u Srbiji i mogućnost dodatnog unapređenja njihove mikrobiolo&scaron;ke bezbednosti. Ispitivani su mikrobiolo&scaron;ki i fizičko-hemiijski pokazatelji u industrijskom proizvodnom lancu sremske kobasice i sudžuka, tipičnih fermentisanih suvih kobasica od svinjskog i goveđeg mesa. U sremskoj kobasici praćeno je prisustvo Salmonella а Escherichia coli O157 u sudžuku. Ispitane su i promene glavnih grupa mikrobiota, pH i aw vrednosti i hemijski sastav proizvoda. Mikrobiolo&scaron;ka kontaminacija u početnim koracima proizvodnje bila je uglavnom visoka. Salmonela je ustanovljena u fazi pripreme nadeva u dva od tri proizvodna pogona, dok je E. coli O157 potvrđena u jednom uzorku usitnjenog mesa i masnog tkiva jednog proizvodnog pogona. Rezultati ispitivanja proizvodnje kobasica slični su rezultatima istraživanja u drugim zemljama. Svi uzorci gotovih sremskih kobasica bili su zadovoljavajućeg nivoa mikrobiolo&scaron;kog kvaliteta, dok većina uzoraka sudžuka nije bila zadovoljavajuća. Početno prisustvo alimentarnih patogena može se smatrati bezbednosnim rizikom.<br />Preživljavanje Salmonella Тyphimurium, Escherichia coli O157 i Listeria monocytogenes tokom proizvodnje i skladi&scaron;tenja inokulisane, sremske kobasice i sudžuka, praćeno je u drugoj fazi istraživanja. Smanjenje brojnosti sva tri patogena u skladu je sa literaturnim podacima i može se zaključiti da postupci uobičajeno primenjeni u industrijskoj proizvodnji sremske kobasice i sudžuka ne osiguravaju uvek, u dovoljnoj meri, mikrobiolo&scaron;ku bezbednost proizvoda. Ispitiana je i mogućnost pasterizacije u cilju redukcije navedenih patogena u kobasicama od svinjskog, odnosno, goveđeg mesa, uz ocenu senzorskog kvaliteta proizvoda. Rezultati su pokazali da Salmonella Typhimurium i E. coli O157 mogu da budu uklonjene pasterizacijom gotovih fermentisanih suvih kobasica а dа senzorske odlike proizvoda i dalje оstanu prihvatljive. L. monoctogenes se pokazala kao patogen koji je značajno otporniji na zagrevanje, u oba tipa kobasica, zbog čega su potrebna dalja istraživanja radi redukcije dо prihvatljivog nivoa.</p> / <p>Glavni cilj doktorske disertacije bio je ispitivanje op&scaron;tih odlika industrijski proizvedenih, fermentisanih suvih kobasica u Srbiji i mogućnost dodatnog unapređenja njihove mikrobiolo&scaron;ke bezbednosti. Ispitivani su mikrobiolo&scaron;ki i fizičko-hemiijski pokazatelji u industrijskom proizvodnom lancu sremske kobasice i sudžuka, tipičnih fermentisanih suvih kobasica od svinjskog i goveđeg mesa. U sremskoj kobasici praćeno je prisustvo Salmonella a Escherichia coli O157 u sudžuku. Ispitane su i promene glavnih grupa mikrobiota, pH i aw vrednosti i hemijski sastav proizvoda. Mikrobiolo&scaron;ka kontaminacija u početnim koracima proizvodnje bila je uglavnom visoka. Salmonela je ustanovljena u fazi pripreme nadeva u dva od tri proizvodna pogona, dok je E. coli O157 potvrđena u jednom uzorku usitnjenog mesa i masnog tkiva jednog proizvodnog pogona. Rezultati ispitivanja proizvodnje kobasica slični su rezultatima istraživanja u drugim zemljama. Svi uzorci gotovih sremskih kobasica bili su zadovoljavajućeg nivoa mikrobiolo&scaron;kog kvaliteta, dok većina uzoraka sudžuka nije bila zadovoljavajuća. Početno prisustvo alimentarnih patogena može se smatrati bezbednosnim rizikom.<br />Preživljavanje Salmonella Typhimurium, Escherichia coli O157 i Listeria monocytogenes tokom proizvodnje i skladi&scaron;tenja inokulisane, sremske kobasice i sudžuka, praćeno je u drugoj fazi istraživanja. Smanjenje brojnosti sva tri patogena u skladu je sa literaturnim podacima i može se zaključiti da postupci uobičajeno primenjeni u industrijskoj proizvodnji sremske kobasice i sudžuka ne osiguravaju uvek, u dovoljnoj meri, mikrobiolo&scaron;ku bezbednost proizvoda. Ispitiana je i mogućnost pasterizacije u cilju redukcije navedenih patogena u kobasicama od svinjskog, odnosno, goveđeg mesa, uz ocenu senzorskog kvaliteta proizvoda. Rezultati su pokazali da Salmonella Typhimurium i E. coli O157 mogu da budu uklonjene pasterizacijom gotovih fermentisanih suvih kobasica a da senzorske odlike proizvoda i dalje ostanu prihvatljive. L. monoctogenes se pokazala kao patogen koji je značajno otporniji na zagrevanje, u oba tipa kobasica, zbog čega su potrebna dalja istraživanja radi redukcije do prihvatljivog nivoa.</p> / <p>The main aim of this doctoral thesis was to investigate the general characteristics of industrially-produced Serbian dry fermented sausages and the possibility of further improving their microbiological safety. Microbiological and physicochemical indicators were studied along the industrial production chains producing Sremska and Sudzuk sausage, typical of dry, fermented sausage prepared from pork or beef, respectively. The occurrences of Salmonella in pork sausages, and of Escherichia coli O157 in beef sausages were determined. Changes in the main groups of microbiota, pH, aw values and the chemical composition of the products were evaluated. Microbiological contamination in the initial stages of production was generally high. Salmonella was confirmed from the batter-preparation phase in two of three production lines, while E. coli O157 was confirmed in one sample of meat and fatty tissue from one production line. The results of this investigation of sausage production were similar to results obtained in other countries. All samples of finished pork sausage were of acceptable microbiological quality, while the majority of beef sausage samples were not microbiologically acceptable. The initial presence of foodborne pathogens can be considered a food safety risk.<br />The survival of Salmonella Тyphimurium, Escherichia coli O157 and Listeria monocytogenes during production and storage of inoculated pork and beef sausages was determined in the second phase of the investigation. All three pathogens declined in numbers in accordance with data from the literature, and it can be concluded that normal measures for pork and beef sausage preparation in industrial production do not always ensure, to a suitable level, the microbiological safety of the products. The possibility of pasteurising finished pork and beef sausages, with the aim of reducing Salmonella Typhimurium and E. coli O157, respectively, was investigated, and at the same time, sensory evaluation of product quality was performed. The results showed that pasteurisation eliminated Salmonella Typhimurium and E. coli O157 from finished dry fermented sausages, while sensory qualities of the products remained acceptable. L. monoctogenes proved to be a significantly more heat-resistant pathogen in both types of sausage, and therefore, further research is required to determine how to reduce numbers of this pathogen to acceptable levels.</p>

Listeria monocytogenes est une bactérie pathogène impliquée dans la toxi-infection alimentaire à l’origine de la listeriose, une maladie peu fréquente mais avec un taux de mortalité de 25 % chez l’homme. Cette bactérie est capable de former un biofilm lui permettant de mieux résister aux stress environnementaux ainsi qu’aux traitements de décontamination. Une nouvelle stratégie d’analyse génomique a été développée et a permis de cibler des systèmes de sécrétion et des protéines potentiellement impliqués dans la formation de biofilm. L’inactivation de la voie SecA2 entraîne la formation d’un biofilm aérien et par conséquent fragile. Ce morphotype est capable de croître de façon sessile à 20°C sur du polystyrène alors que ce n’est pas le cas pour la souche sauvage. De nouvelles protéines sécrétées de façon SecA2 dépendante ont été identifiées par l’étude de l’exoprotéome du mutant ΔsecA2 en comparaison avec celui de la souche sauvage. Le rôle des lipoprotéines dans la formation de biofilm ainsi que leur maturation par les peptidases signal de type II, LspA et LspB, a également été abordé. La combinaison d'une analyse de l’expression des gènes codant les lipoprotéines au cours de la formation de biofilm avec l’analyse génomique basé sur le sécrétome a permis de cibler trois lipoprotéines, dont LpeA qui serait impliquée dans les phases tardives de formation de biofilm. Enfin, l’importance majeure de LspA dans la maturation des lipoprotéines, a été mise en évidence par l’étude de l’exoprotéome des doubles mutant ΔlgtΔlspA et ΔlgtΔlspB en comparaison avec celui de Δlgt. / Listeria monocytogenes is a foodborne pathogenic bacteria responsible for listeriosis, a rare but high mortality rate disease in humans (25 %). This bacterium can form biofilm allowing a better resistance to environmental stresses as well as decontamination treatments. A new strategy for genomic analysis was developed and allowed to target secretion systems and proteins potentially involved in biofilm formation. Inactivation of the SecA2 pathway leads to the formation of an aerial and fragile biofilm. This morphotype is able to grow in a sessile mode at 20 °C on polystyrene whereas this is not the case for the wild type strain. New proteins secreted in a SecA2 manner were identified by comparing the ΔsecA2 exoproteome to the one of the wild type. The role of lipoproteins in biofilm formation and their maturation by the signal peptidase II, LpsA and LspB, was also tackled. Combining expression analysis of genes encoding lipoproteins during biofilm formation with genomic analysis based on the secretome allowed targeting three lipoproteins, including LpeA, which appeared to be involved in the later stages of biofilm formation. Finally, the importance of LspA in the maturation of lipoproteins,was highlighted by comparing of the double mutant ΔlgtΔlspA and ΔlgtΔlspB exoproteomes to the one of Δlgt.