Orientador: Oswaldo Durival Rossi Junior / Banca: Antönio Nader Filho / Banca: Dirceu Rodrigues Meira / Banca: José Paes de Almeida Nogueira Pinto / Banca: Ruben Pablo Schocken-Iturrino / Resumo: O Bacillus cereus é um microrganismo ubíquo encontrado freqüentemente em produtos lácteos. As perdas econômicas e o risco que traz à saúde pública são alarmantes. Os objetivos deste trabalho foram pesquisar o Bacillus cereus em leite, verificar a sua capacidade enterotoxigênica e o risco que provoca à saúde pública e comparar diferentes técnicas utilizadas para detecção de toxinas. Para tal, foram analisadas 120 amostras de leite (30 de leite cru, 30 de leite pasteurizado, 30 de leite em pó e 30 de UAT). Para a pesquisa de enterotoxinas, foram utilizadas três técnicas. Encontraram-se contaminadas 15 (50,0%), 29 (96,7%), 22 (73,3%) e 4 (13,3%) amostras de leite cru, pasteurizado, em pó e UAT, respectivamente. Para a detecção de enterotoxinas pela técnica da alça ligada em coelhos, foram positivas, respectivamente, 1 (7,1%), 10 (35,7%) e 3 (13,6%) cepas das amostras de leite cru, pasteurizado e em pó. Para o teste de aumento de permeabilidade vascular dérmica, apresentaram-se enterotoxigênicas, respectivamente, 1 (7,1%), 1 (3,6%), 2 (9,1%) e 1 (4,0%) cepas isoladas de leite cru, pasteurizado, em pó e UAT. Para a detecção de enterotoxinas pela prova de aglutinação passiva em látex, apresentaram-se positivas 7 (63,6%), 4 (30,8%), 3 (33,3%) e 8 (80,0%) cepas isoladas, respectivamente, de leite cru, pasteurizado, em pó e UAT. Conclui-se que as amostras de leite analisadas, principalmente as amostras já processadas termicamente, deixam a desejar quanto a sua qualidade, colocando em risco a saúde dos consumidores. Conclui-se também que a técnica de aglutinação é a mais indicada para a detecção da enterotoxina produzida pelo Bacillus cereus. / Abstract: Bacillus cereus is a ubiquitous microorganism frequently found in milk products. The economic losses and the damage to public health they cause are enormous. The objectives of this work were to search for Bacillus cereus in milk, their enterotoxigenic activity to estimate the risks the use of this product may produce and to compare different techniques used to detection of enterotoxins. For that, 120 samples of milk were examined (30 of raw milk, 30 of pasteurized milk, 30 of milk powder and 30 of UHT milk). To test the enterotoxigenicity, three techniques were used. Bacillus cereus was present in 15 (50,0%), 29 (96,7%), 22 (73,0%) and 4 (13,3%) samples of raw milk, pasteurized milk, milk powder and UHT milk, respectively. For the enterotoxigenicity detection using the assay in the rabbit ileal loop, 1 (7,1%), 10 (35,7%) and 3 (13,6%) strains from raw milk, pasteurized milk and milk powder were positive. For the enterotoxigenicity detection using the assay for vascular permeability activity, 1 (7,1%), 1 (3,6%), 2 (9,1%) and 1 (4,0%) strains from raw milk, pasteurized milk, milk powder and UHT milk were positive, respectively. Using the reversed passive latex agglutination, diarrheal toxin production was shown to be 7 (63,6%), 4 (30,8%), 3 (33,3%) and 8 (80,0%) strains respectively isolated from raw milk, pasteurized milk, milk powder, and UHT milk. It was concluded that the milk samples analyzed didn't present good quality and may put in risk the health of consumers of such products. We also concluded that the reversed passive latex agglutination is the best technique to determine the enterotoxins produced from Bacillus cereus. / Doutor

Orientador: Arnaldo Yoshiteru Kuaye / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-16T12:29:08Z (GMT). No. of bitstreams: 1 Fernandes_MegdaSilva_M.pdf: 3935080 bytes, checksum: 25bc1e5988870cf168c5403b4c8d2aba (MD5) Previous issue date: 2010 / Resumo: A ricota é um tipo de queijo fresco de origem italiana, obtido pela precipitação das proteínas do soro do queijo por acidificação associada ao calor. Por suas características nutricionais, físico-químicas e bioquímicas apresenta-se propícia ao desenvolvimento microbiano. No processamento deste produto destacam-se o Bacillus cereus, pela sua capacidade de esporular e ser um contaminante potencial do leite e do ambiente e as bactérias do gênero Enterococcus, pela característica ubíqua, habilidade de sobrevivência à condições diversas de pH, temperatura e salinidade e ocorrência em casos de infecções hospitalares. Os objetivos deste trabalho foram: a) verificar as possíveis fontes de contaminação de ricota por B. cereus e Enterococcus spp. ao longo do processamento; b) identificar as espécies de enterococos, avaliar o potencial de patogenicidade e o perfil de resistência destas espécies a antibióticos de uso clínico; e, c) avaliar a conformidade das amostras de ricota aos padrões microbiológicos legais. Amostras de leite cru e pasteurizado, soro de queijo, ricotas antes e após embalagem, superfícies diversas do ambiente e do ar obtidas em três coletas de laticínio da região Sul de Minas Gerais foram submetidas à determinação de B. cereus e Enterococcus spp. As contagens de B. cereus em leite cru, pasteurizado e soro de queijo, foram de 1,4 x104, 1,2 x103 e 1,0 x103 UFC/ml, respectivamente, e, apenas uma amostra de ricota final apresentou valor maior que 102 UFC/g. Dentre as 60 amostras ambientais, destaca-se a forma de moldagem da ricota, que apresentou contaminação persistente e contagem de até 1,7x107 UFC/unidade de B. cereus. Enterococcus spp. foram encontradas em todas as amostras de ricota, com contagens entre 103 e 107 UFC/g, e em todas de leite cru, com contagens de até 1,9 x106 UFC/ml. Nas superfícies de forma e tela, vassoura, parede e ralo foram encontrados valores superiores a 105 UFC/unidade; já para tanques, bancada da área de embalagem e caixa de recolhimento do soro os números foram superiores a 102 UFC/unidade. De um total de 136 isolados, confirmados para o gênero Enterococcus, 71,3% (97/136) foram confirmados para a espécie E. faecium e 20,6% (28/136) para E. faecalis, pela técnica de PCR. Os isolados (66) de E. faecium e E. faecalis das amostras de produto final submetidas aos testes fenotípicos resultaram em 89,4% (59/66) positivos para hemólise, nenhum para gelatinase (0/66) e 98,5% (65/66) positivos para termonuclease. A maioria dos isolados de E. faecium e E. faecalis mostrou resistência a pelo menos três dos 5 antimicrobianos testados, destacando-se que 100% deles apresentaram resistência à vancomicina. De 15 amostras de ricota avaliadas após 21 dias de armazenamento sob refrigeração, 13,3% (2/15) estavam em desacordo com o padrão legal para estafilococos coagulase positiva e em nenhuma delas foi detectada a presença de Salmonella, Listeria monocytogenes e coliformes termotolerantes. A natural e inevitável contaminação da matéria-prima e do ambiente de processamento de ricota por B. cereus e Enterococcus spp., bactérias estas potencialmente patogênicas, tem na eficiência dos programas de higienização um fator indispensável para o seu controle / Abstract: The ricotta is a type of fresh cheese of Italian origin, obtained by precipitation of proteins from cheese whey by acidification associated with the heat. Because of its nutritional, physicochemical and biochemical characteristics it is conducive to microbial growth. On the processing of this product it can be emphasized the Bacillus cereus, due to its ability to sporulate and be a potential contaminant of milk and the environment, and the bacteria of the genus Enterococcus, due its ubiquitous characteristic, ability to survive the various conditions of pH, temperature and salinity and appearance in cases of hospital infections. The objectives of the present work were: (a) to verify the possible sources of ricotta contamination by B. cereus and Enterococcus spp. during processing; (b) to identify the species of enterococci, evaluate the pathogenic potential and the resistance profile of these species to antibiotics of clinical use; and (c) to assess the conformity of samples of ricotta under legal microbiological standards. Samples of raw and pasteurized milk, cheese whey, ricotta before and after packaging, various surfaces of the environment and of air obtained from three collections on a dairy industry located in southern Minas Gerais were subjected to the determination of B. cereus and Enterococcus spp. The counts of B. cereus in raw and pasteurized milk and in cheese whey were 1,4 x104, 1,2 x103 and 1,0 x103 CFU/ml, respectively, and only one sample of final ricotta had levels of B. cereus higher than 1,7x107 CFU/unity. Among the 60 environmental samples, it can be highlighted the mold where the ricotta is shaped, which showed persistent contamination and count up to 1,7x107 CFU/unity for B. cereus. All the samples of ricotta and raw milk showed the presence of Enterococcus spp. with counts between 103 and 107 CFU/g and up to 1,9 x106 CFU/ml, respectively. On the surfaces of the mold, mesh, broom, wall and drain it were found counts higher than 105 CFU/unity; for the tank, stand in the area of packaging and box for the collection of serum the counts were higher than 102 CFU/unity. Over 136 isolated of the genus Enterococcus, 71,3% (97/136) were confirmed for the species E. faecium and 20,6% (28/136) for E. faecalis by PCR technique. The isolates (66) of E. faecium and E. faecalis taken from the samples of the final product submitted to phenotypic tests resulted in 89,4% (59/66) positive for hemolysis, none for gelatinase (0 / 66) and 98,5% (65/66) positive for thermonuclease. Most of the isolates of E. faecium and E. faecalis showed resistance to at least three of the 5 antimicrobials, highlighting that 100% of them were resistant to vancomycin. From 15 samples of ricotta evaluated after 21 days of refrigerated storage, 13,3% (2/15) were in disagreement with the legal standard for coagulase-positive staphylococci and none were detected the presence of Salmonella, Listeria monocytogenes and thermotolerant coliforms. The natural and inevitable contamination of raw-materials and of the processing environment of ricotta by B. cereus and Enterococcus spp., which are potentially pathogenic bacteria, have in the efficiency of the hygiene programs a essential factor for its control / Mestrado / Mestre em Tecnologia de Alimentos

Springer and European Journal of Clinical Microbiology and Infectious Diseases, 33, 2014, 1371-79, Seasonal trend and clinical presentation of Bacillus cereus bloodstream infection: association with summer and indwelling catheter, K. Kato & Y. Matsumura & M. Yamamoto & M. Nagao & Y. Ito & S. Takakura & S. Ichiyama, figure number 2, original copyright notice is given to the publication in which the material was originally published, by adding; with kind permission from Springer Science and Business Media / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19922号 / 医博第4142号 / 新制||医||1017(附属図書館) / 33008 / 京都大学大学院医学研究科医学専攻 / (主査)教授 中川 一路, 教授 佐藤 俊哉, 教授 玉木 敬二 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Bacillus cereus est une bactérie très largement disséminée dans l'environnement. Certaines souches sont à l'origine de toxi-infections alimentaires de type émétique ou diarrhéique. Afin de coloniser l'intestin et induire un syndrome diarrhéique, B. cereus doit s'adapter aux variations d'oxygénation et de potentiel d'oxydoréduction rencontrées, se multiplier et sécréter des toxines. En comparant les capacités métaboliques et les capacités de sécrétion de l'entérotoxine Nhe de quatre souches de B. cereus, nous avons mis en évidence que la L-lactate déshydrogénase A (LdhA) était quelles que soient les conditions de croissance impliquée à la fois dans le métabolisme fermentaire et dans la toxinogénèse de B. cereus. LdhA contribue au maintien du ratio NAD+/NADH intracellulaire et son absence affecte les capacités d'expression de plusieurs gènes d'entérotoxines en anaérobiose et en aérobiose chez la souche F4430/73. Afin de déterminer si l'effet observé sur les toxines était indirect et dépendant du régulateur rédox Rex, un mutant ne synthétisant plus Rex a été construit. La caractérisation de ce mutant a mis en évidence le rôle de Rex dans le métabolisme de B. cereus, dans la réponse au stress oxydant et dans la toxinogénèse. De part sa structure et sa capacité a lié l'ADN, Rex pourrait être un facteur de transcription contrôlant l'expression des gènes codant les entérotoxines. Sa capacité de fixation sur l'ADN est dépendante du ratio NAD+/NADH. Afin de déterminer si LdhA pouvait réguler directement l'expression des gènes des toxines, ldhA a été exprimé chez E. coli et purifiée sous la forme d'une protéine fusion. Les premières expériences de retard sur gel n'ont pas permis de mettre en évidence de fixation sur les régions promotrices de toxines / Bacillus cereus is a widespread bacteria. Some strains are responsible of food-borne classified as emetic and diarrhoel syndromes. To colonize such environment and induce diarrhea, B. cereus must be able to grow in the various oxidoreduction potential and oxygenation conditions encountered in the gastrointestinal tract and to secrete toxins. By comparing the catabolic capacities of four strains of the B. cereus group grown under anoxic and oxic conditions in relation to their capacities for expressing Non hemolytoc enterotoxin (Nhe), we identified Lactate dehydrogenase A (LdhA) as both involved in the fermentative metabolism of B. cereus and toxinogenesis. We showed that disruption of ldhA affected the fermentative capacity of anaerobically grown F4430/73 cells and the expression of several enterotoxin genes under both anaerobiosis and aerobiosis. To determine whether the observed effect in the toxins expression was indirect and dependant of Rex, a rex mutant was built. The characterization of this mutant revealed the role of Rex in the metabolism of B. cereus, oxidative stress and toxins production. Rex could be a transcription factor controlling the expression of genes encoding enterotoxins. Ability of DNA binding is dependant on the ratio of NAD+/NADH. To determine whether LdhA could directly regulate the expression of toxins genes, LdhA was expressed in E. coli and purified as a fusion protein. The first gel retardation experiments have failed to demonstrate fixation of LdhA in the promoter regions of toxins

Bacillus cereus (Bc) et Bacillus thuringiensis (Bt) sont deux espèces génétiquement proches. Bc est une bactérie pathogène que peuvent causer des gastro-entérites d’origine aliméntaire. Bt est une bactérie entomopathogène, dont le cycle de vie dans la larve d’insecte est contrôlé par des systèmes de quorum sensing, comme le système Rap/Phr, que régule processus tels que la sporulation, la formation de biofilm et la conjugaison. La présence des ces genès a été identifiée dans les plasmides, et ces eleménts ont été associés à l’adaptation des spécies dans sont niche ecologique. Le but de cette étude est de comprendre le rôle des plasmides dans ces bactéries. Pour la première étude l’insecte larvae, le niche privilegie de Bt, ont été infectées par souches de Bc et Bt, avec un contenu plasmidique diffèrent. Le fitness a été evallué par le comptage de cellules végétatives et spores dans quatre temps. Les souches de Bt et Bc ont été classées dans cinq groups par rapport à sont fitness. Dans ces groups le plasmide a affecté le fitness de la bactérie positive ou négativement. Les résultats ont démontré que les souches du group du B. cereus que reçoivent a pathogène plasmid ne est pas suffisant pour une augmentation effectif de la population bactérienne, i.e., coloniser l’hôte. La deuxième étude a permis caractériser le système rap/phr porté par le plasmide cryptique pHT8_1. Les résultats démontrent que la protéine Rap8 inhibe la sporulation dans la l’insecte. L’activité de cette protéine est inhibée par le peptide de signalisation Phr8. Le système Rap/Phr8_1 a permis les bactéries exercer un strict contrôle sur la sporulation, un processus important pour assurer la survie et la dissémination des bactéries. L’ensemble des résultats de la deuxième étude montrent que les plasmides peuvent fournir avantages pour l’adaptation et evolution de B. thuringiensis dans son niche ecologique, alors que les résultats de la première étude indiqués que les souches de Bc group doivent avoir un contenu génétique approprié pour exhiber un fitness élévé en permettant une optimal multiplication and dissemination de populations bactérienne dans l’insect larvae. / Bacillus cereus (Bc) and Bacillus thuringiensis (Bt) are two closely related species. Bc is a pathogenic species responsible for gastroenteritis by food-borne. Bt is an entomopathogenic bacterium, which the lifecycle in insect larvae is controlled by quorum sensing systems, such as Rap/Phr, which regulates processes such as sporulation, biofilm formation and conjugation. The presence of these genes in plasmids has been described, furthermore, plasmids have been involved in bacterial adaptation to their ecological niche. In order to understand the role of the plasmids to these species, two complementary works were carried out. First, insect larvae, a privileged ecological niche of Bt strains, were infected with Bc and Bt strains harboring different plasmid contents. Their fitness were evaluated by vegetative cells and spore counts at four time points. Bt and Bc strains were classified into five groups according to the bacterial fitness. In these groups, the plasmid affects positively or negatively the bacterial fitness. The results demonstrated that for B. cereus group strains, getting a pathogenicity plasmid is not enough to effectively increase bacterial population, colonizing insect hosts. The second study characterized the rap/phr system encoded by the cryptic plasmid pHT8_1. The Rap8 protein inhibited the sporulation process in insect larvae. This protein was directly inhibited by the active signaling peptide Phr8. The Rap8/Phr8 system may allow the bacteria to exert a tight control of the sporulation process in the host cadaver for optimizing the multiplication, the survival and the dissemination of the bacteria. Thus, the results of the second study showed that the plasmids can provide advantages for the adaptation and the evolution of B. thuringiensis in its ecological niche, while the results of the first study indicate that B. cereus group strains must have a suitable genetic background to display a high fitness allowing optimal multiplication and dissemination of the bacterial population within insect larvae.

Mudanças nos hábitos alimentares dos consumidores têm aumentado a demanda por frutas frescas e polpas de frutas. Com isso, a busca por novos compostos com atividade antimicrobiana é de grande interesse, já que a aplicação de aditivos químicos convencionais em alimentos tem sido reavaliada devido à potencial toxicidade de alguns desses compostos. As bacteriocinas ou BLIS, produzidas por diferentes gêneros bacterianos, têm despertado grande interesse industrial para aplicação em alimentos em processos de bioconservação. No presente trabalho, foram obtidos 3 isolados bacterianos (A, B e C) a partir de polpa de abacaxi, produtores de substância antagonista frente a diferentes linhagens de Listeria monocytogenes e Bacillus sp. A natureza proteica das substâncias antagônicas produzidas pelos isolados foi confirmada pela sensibilidade à enzima ? - quimotripsina. As linhagens foram identificadas através de provas bioquímicas e moleculares como Bacillus cereus, com a colaboração da Fundação Oswaldo Cruz (Rio de Janeiro, Brasil). Para estudos mais detalhados da BLIS foi selecionado o isolado C denominado Bacillus cereus LFB-FIOCRUZ 1640. A melhor condição de incubação para produção da BLIS foi a 30ºC/24 horas, em caldo MRS. A BLIS apresentou estabilidade térmica por até 30 minutos a 80ºC e em ampla faixa de pH frente a L. monocytogenes. A purificação da BLIS foi realizada com resina Amberlite XAD-16 e de troca catiônica SP-Sepharose \"Fast Flow\", tendo o extrato apresentado um fator de purificação de 4,1 vezes e rendimento de 30%. O extrato obtido foi liofilizado e uma alíquota foi analisada por SDS-PAGE, o que permitiu a estimativa da massa molecular da BLIS de 25 kDa. A Concentração Bactericida Mínima (CBM) de 2 mg/mL foi determinada frente ao indicador L. monocytogenes e esta concentração de extrato foi aplicada em sistema modelo contendo polpa de abacaxi. A BLIS teve ação bactericida inibindo completamente L. monocytogenes por 24h/ 37ºC. Neste sentido, a aplicação da BLIS na área de alimentos e outras áreas farmacêuticas poderá ser explorada em outros trabalhos. / Changes in habits of consumers, with preference for healthy foods, have increased the demand for fruits and fruit pulps. Thus, there is great interest in searching new compounds with antimicrobial activity, since the application of conventional additives in foods is of concern due to the toxicity of some of these compounds. The bacteriocins or BLIS, produced by different bacteria genera, have increased the interest for its industrial application in food technology in processes of biopreservation. In this study, three isolates were obtained (A, B and C) from pineapple pulp, which produce antagonistic substances with activity against Listeria monocytogenes and Bacillus sp. The proteinaceous nature of the antagonistic substance produced by the isolates was confirmed by susceptibility to the enzyme ? - chymotrypsin. The strains were identified by biochemical and molecular tests as Bacillus cereus, in collaboration with Fundação Oswaldo Cruz (Rio de Janeiro, Brazil). Isolate C of Bacillus cereus was named LFB-FIOCRUZ 1640. The best condition of incubation for the BLIS production was 30°C for 24 hours in MRS broth. The BLIS was thermostable (up to 30 minutes at 80°C) and it was partially stable in the range of pH 2 to 9. The purification of the BLIS was performed with Amberllite XAD-16 and cation-exchange SP-Sepharose \"Fast Flow\" resins. The extract presented a purification factor of 4.1 times and 30% of yield. The extract was lyophilized and analyzed by SDS-PAGE. The molecular mass of the BLIS was estimated as 25 kDa. The Minimum Bactericidal Concentration (MBC) of 2 mg/mL was determined with the indicator L. monocytogenes, and this concentration of extract was applied in a model system containing pineapple pulp. The BLIS had bactericidal action which completely inhibited L. monocytogenes for 24h/37°C. The BLIS of B. cereus 1640 presents potential for application as an antimicrobial in food and other pharmaceutical areas.

Há controvérsia sobre os processos responsáveis pela atual distribuição de Florestas Tropicais Sazonalmente Secas (FTSS) na América do Sul. Este tipo de vegetação compreende uma grande proporção de todas as espécies neotropicais. Entender o que modela a sua distribuição pode fornecer novas perspectivas para a evolução deste bioma e contribuirpara os aspectos de sua conservação. O trabalho avaliou a evolução deste bioma no sul do Brasil, onde as FTSS e as Florestas Tropicais (FT) são amplamente intercaladas. Para isso, foi reconstruídaa história filogeográfica do cacto, Cereus hildmannianus, uma espécie característica e abundante das FTSS. Métodos de datação molecular, estrutura populacional e filogeografia foram realizadas para avaliar os eventos histórico-demográficospor meio de uma amostragem densa que compreendeu 24 populações e cerca de 150 amostrasde, pelo menos, uma dentre as seis regiões genômicas nuclear e cloroplastidiais selecionadas. A partir disso, foi investigado um possível cenário da dinâmica populacional de C. hildmannianus. Os resultados indicam uma separação da espécie em dois grupos principais (ST: 0,788) com eventos de expansão populacional: umem regiões costeiras e o outro no interior do sul do Brasil, concondante com a distribuição dos núcleos das FTSS. O tempo do ancestral comum mais recente de C. hildmannianus, há 2,56 milhões de anos, remete a especiação deste ao período pré-Glacial. Os resultados do padrão de distribuição de C. hildmannianus foram concordantes com as áreas de endemismo para outros táxons das FTSS. Os eventos de dispersão e de vicariância entre as FTSS e as FT podem estar associados às mudanças paleoclimáticas durante os períodos glaciais do Quaternário, promovendo eventos de retração/expansão nestas florestas. A compreensão desses padrões na história biogeográfica de populações naturais podem auxiliar futuros planos de conservação deste bioma, na América do Sul. / There is controversy about the processes responsible for the current distribution of Seasonally Dry Tropical Forests (SDTF) in South America. This vegetation type comprises a large proportion of all Neotropical species. Understanding what shapes your distribution may provide new insights into the evolution of this ecosystem and contribute to aspects of conservation. The study evaluated the evolution of this biome in southern Brazil, where SDTF and Rainforests are widely interspersed. For this, we reconstructed the phylogeographic history of the cactus, Cereus hildmannianus, a kind of characteristic and abundant SDTF. Molecular dating methods, population structure and phylogeography were performed to evaluate the historical and demographic events through a dense sampling which comprised 24 populations and about 150 samples of at least one among the six nuclear and chloroplast genomic regions selected. From this, we investigated a possible scenario of population dynamics of C. hildmannianus. The results indicate a separation of the species into two main groups (ST: 0.788) with events of population expansion: one in coastal regions and the other inside the south of Brazil, concondante with the distribution of the nuclei of SDTF. The time of the most recent common ancestor of C. hildmannianuswere 2.56 million years ago, this speciation refers to the pre-Glacial. The results of the distribution pattern C. hildmannianus were consistent with areas of endemism for other taxa of SDTF. The events of dispersal and vicariance between SDTF and Rainforests may be related to paleoclimatic changes during glacial periods of the Quaternary, promoting events shrinkage /expansion in these forests. Understanding these patterns in the biogeographic history of natural populations may aid future conservation plans this biome in South America.

Clostridium difficile est responsable de vingt-cinq pour cent des diarrhées post-antibiotiques et de la majorité des cas de colite pseudomembraneuse. C'est un bacille anaérobie à Gram positif sporulant. La bactérie est recouverte par un réseau cristallin bidimensionnel appelé couche S. Elle est formée par deux sous-unités de haut et bas poids moléculaire issues du clivage du précurseur SlpA par la protéase Cwp84. Ces deux protéines sont codées par des gènes situés dans le locus cwp et sont sécrétées par le système de sécrétion de type SecA2. Elles sont impliquées dans l'adhésion/colonisation du côlon par C. difficile. L'adhésion est une étape commune avec la formation du biofilm par les bactéries. Le biofilm est une communauté bactérienne enchâssée et protégée par une matrice extracellulaire produite par les membres de la communauté. C'est le mode de vie principal des bactéries. Nous avons caractérisé la voie de sécrétion de type SecA2 de C. difficile. La protéine SecA2 (codée également par un gène du locus cwp) est essentielle pour la survie de C. difficile. Elle a une double localisation : le cytoplasme et la membrane intracellulaire. SecA2 de B. anthracis est capable de dimériser avec les protéines SecA1 et SecA2 de C. difficile. De plus, la complémentation du mutant secA2 de B. anthracis par le gène secA2 de C. difficile est fonctionnelle. Le rôle de la couche S, de la protéase Cwp84 et de la mobilité dans le biofilm de C. difficile ont été également étudiés. La souche 630∆erm forme un biofilm fin et fragile tandis que le mutant 630∆ermcwp84::erm forme un biofilm épais et robuste. Nous avons montré que l'activité protéolytique de Cwp84 était impliquée dans la formation du biofilm. De plus, une inhibition de la traduction de l'ARN slpA avec un ARN antisens spécifique permet une augmentation de la taille du biofilm formé par la souche 630∆erm. Similairement, l'expression de l’allèle secA2 muté dominant qui bloque au moins partiellement la voie de sécrétion de type SecA2 augmente la taille du biofilm. Nos résultats suggèrent que la couche S, la protéase Cwp84 et la protéine SecA2 sont impliquées dans la formation du biofilm de C. difficile. Par ailleurs nous avons testé un panel de souches dans leur capacité à former un biofilm. Les résultats montrent que les souches non mobiles ne seraient pas capables de former un biofilm épais. Enfin, nous avons étudié la capacité de C. difficile à se développer en aérobiose au sein d’un biofilm mixte avec Bacillus cereus. Nous avons mis en évidence un recrutement et une multiplication de C. difficile dans la pellicule formée à l'interface air/liquide par B. cereus. Un rapport optimal des spores des deux espèces est requis pour le développement de C. difficile dans ces conditions. La présence de spores de C. difficile dans la pellicule suggère que les biofilms de l'environnement pourraient être des réservoirs de spores de C. difficile, et à l'origine de contaminations humaine et animale. / Clostridium difficile is responsible for twenty-five percent of post-antibiotics diarrhea and for most cases of pseudomembranous colitis. It is an anaerobic, sporulating, Gram-positive bacillus. The bacterium is covered by a two-dimensional lattice called S-layer. It is formed by two subunits of high and low molecular weight after the cleavage of the SlpA precursor by the Cwp84 protease. These two proteins are encoded by genes located in the locus cwp and are secreted by the SecA2 secretion system. They are involved in colonic adhesion/colonization by C. difficile. Adhesion is a common step with biofilm formation by bacteria. The Biofilm is a microbial community embedded in and protected by an extracellular matrix produced by the community members. The biofilm is the main bacterial way of life.We have characterized the SecA2 secretory pathway of C. difficile. The SecA2 protein (as encoded by a gene locus cwp) is essential for the survival of C. difficile. It has a dual location: cytoplasm and intracellular membrane. SecA2 of B. anthracis is able to dimerize with SecA1 and SecA2 proteins of C. difficile. Moreover, the complementation of B. anthracis secA2 mutant with secA2 gene of C. difficile is functional.The role of the S-layer, of the Cwp84 protease and of the motility in the biofilm of C. difficile have also been studied. The 630∆erm strain forms a thin and weak biofilm while the 630∆ermcwp84::erm mutant forms a thick and robust biofilm. We have shown that proteolytic activity of Cwp84 was involved in biofilm formation. Furthermore, a decrease in the translation of slpA RNA with an antisense RNA specific permits an increase in the size of the biofilm of the strain 630∆erm strain. Similarly, the expression of the dominant mutated secA2 allele, which at least partially blocks the SecA2 secretory pathway, increases the biofilm size. Our results suggest that the S-layer, the Cwp84 protease and the SecA2 protein are involved in biofilm formation of C. difficile. On the other hand, we have tested a panel of strains in their capacity to form a biofilm. The results show that non-motile strains are unable to form a thick biofilm.Finally, we have studied the ability of C. difficile to grow aerobically in a mixed biofilm with B. cereus. We highlighted a recruitment and proliferation of C. difficile in the film formed at the air/liquid interface with B. cereus. An optimal ratio of spores of both species is required for the development of C. difficile in these conditions. The presence of C. difficile spores in the film suggests that the environment biofilms could be reservoirs of spores of C. difficile, and the source of human and animal contamination.

Background: Almost all foals develop transient diarrhoea within the first weeks of life. Studies indicated different viral, bacterial, and parasitic causes, such as rotavirus, Clostridium perfringens, Escherichia coli, and Cryptosporidium are discussed. But little is known about the development of intestinal microflora in foals. The present study investigated whether the supplementation with Bacillus cereus var. toyoi would modify the developing intestinal microflora and consequently reduce diarrhoea in foals. From birth, the foals were randomly assigned to three treatment groups: placebo (10 mL isotonic NaCl, n = 8), low dosage (LD; 5 × 108 cfu B. cereus var. toyoi, n = 7) and high dosage (HD; 2 × 109 cfu B. cereus var. toyoi, n = 10). Treatment groups were supplemented orally once a day for 58 days. Faeces scoring and sampling were performed within the first 24 h after birth and on day 9, 16, 23, 30, 44, 58 of the foal’s life and also on the first day of diarrhoea. Culture-plate methods were used to analyse the bacterial microflora. Results: Eighty-eight per cent of the foals developed diarrhoea (placebo 7/8, LD 5/7, HD 10/10) during the first 58 days of life. Bacillus cereus var. toyoi supplementation had no effect on bacterial microflora. Clostridium perfringens and enterobacteria were equally prevalent in foals with diarrhoea and those who were not afflicted. Conclusions: We conclude that the supplementation of B. cereus var. toyoi had no effect on the occurrence of diarrhoea and health status in the foals.