Bacillus cereus est une bactérie aéro-anaérobie facultative à Gram positif ubiquiste pouvant s’adapter à de nombreux environnements et s’y développer. C’est un agent pathogène de l’homme capable de produire tout un éventail de protéines extracellulaires et de toxines jouant un rôle majeur dans la pathogénicité de ce micro-organisme. B. cereus croit suivant un métabolisme de type respiratoire en aérobiose et fermentaire en anaérobiose en l’absence d’accepteur final d’électrons. En aérobiose, la chaine respiratoire est une source majeure des dérivés réactifs de l'oxygène (ROS) endogènes. En anaérobiose, les ROS endogènes sont générés en réponse au stress oxydant secondaire au stress nutritionnel et au stress réducteur, lorsque les cultures sont réalisées à bas potentiel d’oxydo-réduction (POR). Les résidus méthionines (Met) sont particulièrement sensibles à l’oxydation par les ROS. L’oxydation des Met conduit à la formation de méthionine sulfoxyde (Met(O)), un dérivé oxydé stable détectable par spectrométrie de masse (MS). L'oxydation des résidus Met est réversible : leur réduction est catalysée par des méthionines sulfoxyde réductases (Msr). Pour déterminer le rôle de l’oxydation des résidus Met, nous avons réalisé une étude exhaustive par MS de la dynamique de l’exoprotéome de la souche ATCC 14579 (pBClin 15) de B. cereus en aérobiose (pO2 = 100%) et en anaérobiose (pO2 = 0%) à haut (POR initial = +140 mV) et bas potentiel redox (PORi= -350 mV). Les résultats ont montré que la dynamique des toxines était représentative de la dynamique de l’exoprotéome à la fois en termes d’abondance relative de protéines et d’oxydation des Met dans les trois conditions testées. L’analyse des résultats suggèrent que (i) l’abondance des toxines et leur taux de méthionines oxydés reflètent le niveau d’oxydation cellulaire et (ii) la sécrétion de toxines au cours de la croissance cellulaire contribue au maintien de l'homéostasie redox intracellulaire en piégeant les ROS endogènes, en particulier en phase active de croissance en aérobiose et en fin de croissance en anaérobiose. Pour étayer l’hypothèse selon laquelle, les Met des protéines extracellulaires, et des toxines en particuliers sont des composants de la machinerie cellulaire antioxydante, nous avons construit une souche mutante ne synthétisant plus MsrAB et comparer le protéome et l’exoprotéome de cette souche mutante avec celle de la souche parentale en aérobiose et anaérobiose à haut POR. Cette étude a mis en évidence l’implication de MsrAB mais également du plasmide cryptique pBClin15 dans la sécrétion des toxines et le maintien de l'homéostasie redox intracellulaire / Bacillus cereus is a Gram-positive aerobic or facultative anaerobic worldwide-distributed bacterium. In addition, B. cereus is a human pathogen able to produce a range of extracellular enzymes and toxins playing a major role in the virulence of the bacteria. In presence of oxygen, B. cereus performs respiration. Without oxygen or other electron acceptors, it performs mixed-acid fermentation. Under aerobiosis, the respiratory electron transport chain is a major source of endogenous reactive oxygen species (ROS). Under anaerobiosis, endogenous ROS are generated in response to reductive stress (mainly under high-reductive anaerobiosis) and to starvation (nutrient stress), i.e. in response to secondary oxidative stresses. Methionine residues (Met) of proteins are vulnerable to oxidation by free radicals. Oxidation of Met leads to the formation of methionine sulfoxide (Met (O)), a stable by-product detectable by mass spectrometry (MS). Met(O) can be reduced back to Met by the action of methionine sulfoxide reductase (Msr). To determine the role of oxidation of Met residues, B. cereus exoproteome time courses were monitored by MS under low oxidation-reduction potential (ORP) anaerobiosis (initial ORP = +140 mV and pO2 = 0%), high-ORP anaerobiosis (iORP = -350 mV and pO2 = 0%), and aerobiosis (pO2 = 100%). The results indicated that toxin-related proteins were the most representative of the exoproteome changes, both in terms of protein abundance and their Met(O) content in the presence and in the absence of oxygen. The analysis results suggest that (i) the abundance of toxins and their oxidized methionines rates reflect the cellular oxidation level and (ii) the secretion of toxins during growth helps to maintain redox homeostasis by keeping endogenous ROS at bay, during the exponential growth phase under aerobic conditions and at the end of growth under anaerobiosis. To support our hypothesis that Met residues of extracellular proteins, particulars of toxins are components of the cellular machinery antioxidant, we constructed a mutant strain by deleting the gene of MsrAB and compare the cellular proteome and exoproteome of this mutant strain with the wild-type strain under aerobiosis and high-ORP anaerobiosis. This study highlighted the involvement of MsrAB but also pBClin15 plasmid in the secretion of toxins and maintain of the intracellular redox homeostasis.

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.

Les personnes âgées sont souvent victimes de dénutrition pouvant conduire à de multiples dégradations de la santé. La prévention de cet état de dénutrition est une des principales préoccupations des autorités de santé. C’est dans cet objectif que le projet OPTIFEL a conçu de nouveaux produits alimentaires optimisés, afin d’améliorer l’alimentation des personnes âgées, en tenant compte de leurs besoins et souhaits, tout en préservant leur sécurité sanitaire. Bacillus cereus est une bactérie pathogène sporulée capable de se développer dans des conditions variables comme le froid, l’absence d’oxygène, des bas pH ainsi que des matrices alimentaires diverses. Elle est l’une des principales causes de Toxi-Infection Alimentaire Collective (TIAC) surtout en Institut Médico-Sociaux (IMS). Ce travail de thèse avait donc pour objectif de déterminer le comportement de B. cereus à différentes étapes de la vie d’un produit alimentaire. La conservation au froid peut permettre la multiplication de souches psychrotolérantes de B. cereus qui peuvent alors représenter un risque d’infection pour le consommateur. Les conditions anaérobies réduisent la capacité d’adaptation de B. cereus au froid et aux bas pH. La composition de l’aliment et sa préparation influence également le comportement de B. cereus, comme nous l’avons constaté avec un bouillon de carotte dans lequel la présence d’oxygène crée un effet létal pour la bactérie. Certaines souches psychrotolérantes de B. cereus sont capables de produire de la toxine émétique appelée céréulide. Nous avons montré que cette production peut se faire à partir de 8°C et atteindre des concentrations pouvant causer des intoxications à partir de 10°C. S’il est produit dans l’aliment, le cereulide ne serait pas détruit lors de la préparation de l’aliment. Par contre les cellules végétatives de B. cereus sont très thermosensibles et celles qui auraient pu se développer pourront être détruites par un réchauffage du produit avant sa consommation. Ce travail a permis de définir les paramètres importants pour limiter le risque d’infection alimentaire par B. cereus. / Elderlies are often victims of undernutrition which can lead to multiple health damages. Prevention of this state of undernutrition is one of the main concerns of health authorities. With this objective, the OPTIFEL project designed new optimized foodstuffs, to improve foods for elderlies, by taking into account their needs and wishes, while preserving their safety. Bacillus cereus is a spore-forming bacterial pathogen able to grow in various conditions as low temperature, absence of oxygen, low pH and in different types of food. It is one of the main causes of foodborne outbreaks, particularly in socio-medical institutions. The aim of this work was to determine the behavior of B. cereus at different steps of the life of the food product. Cold storage can allow multiplication of psychrotolerant B. cereus strains that can represent a risk of infection for consumers. Anaerobic conditions reduce the adaptation capacity of B. cereus at low temperature and low pH. The composition and the preparation of the food product also influence the behavior of B. cereus, as we showed with carrot broth in which oxygen created a lethal effect for the bacterium. Certain psychrotolerant B. cereus strains are able to produce an emetic toxin named cereulide. We showed that this production starts at 8 °C and can reach at 10 °C concentrations sufficient to cause intoxications. If this toxin is produced in food, it would not be destroyed during the food preparation. In contrast, vegetative cells of B. cereus are very heat-sensitive and those who would have been able to develop could be destroyed by a reheating of the food product before its consumption. This work allowed the identification of important parameters to limit the risk of foodborne disease by B. cereus.

Chez les bactéries sporulantes du genre Bacillus, des mécanismes importants tels que la sporulation et la virulence sont régulés par des systèmes de communication cellulaire qui impliquent des peptides de signalisation et des régulateurs de la famille RNPP (Rap, NprR, PlcR, PrgX). L'objectif de mon travail de thèse a été de déterminer le rôle du régulateur NprR chez les bactéries du groupe B. cereus. Ce travail se divise en trois parties complémentaires. La première partie a consisté à montrer que NprR est impliqué dans un système de communication cellulaire. Nous avons montré que NprR est un régulateur transcriptionnel de début de phase stationnaire qui est dépendant du peptide de signalisation NprX. Associé à NprX, NprR active la transcription du gène nprA qui code pour une protéase extracellulaire. Nous avons démontré que le peptide NprX est sécrété, maturé puis réimporté dans la cellule bactérienne par deux systèmes d'oligopeptide perméase (Opp et Npp). Une fois dans la cellule, la forme mature de NprX (vraisemblablement l'heptapeptide SKPDIVG) se lie à NprR et permet la transcription du gène nprA. Nous avons ensuite cherché à déterminer la fonction de ce régulateur au cours du cycle infectieux de B. thuringiensis (Bt) chez l'insecte. Nous avons montré que NprR est actif après la mort de l'insecte et permet aux bactéries de survivre, sous forme de cellules végétatives, dans les cadavres. Une analyse transcriptomique indique que NprR régule l'expression d'au moins 41 gènes qui codent notamment pour des enzymes dégradatives et un locus de gènes impliqués dans la production d'un peptide synthétisé de façon non ribosomique (la kurstakine). Nous avons démontré que les gènes codant pour les enzymes dégradatives s'expriment spécifiquement après la mort de l'hôte et que les produits de ces gènes sont essentiels pour hydrolyser différents substrats (protéines, lipides, chitine), ce qui suggère que Bt a un mode de vie nécrotrophe dans le cadavre. La kurstakine est essentielle pour la survie de Bt pendant son développement nécrotrophe et nous avons montré que cette molécule est nécessaire pour le swarming et la formation de biofilm. Par ailleurs, un mutant du gène nprR ne se développe pas et ne sporule pas efficacement dans le cadavre. L'ensemble de nos résultats indiquent que le necrotrophisme est un mode de vie hautement régulé, qui est essentiel dans le cycle infectieux de Bt car il contribue à la transmission horizontale de ce micro-organisme. Enfin, nous avons étudié la régulation de l'expression des gènes nprR et nprX. Nous avons montré que les gènes nprR-nprX sont co-transcrits à partir d'un promoteur dépendant de sigma-A (PA) situé en amont du gène nprR. La transcription à partir de ce promoteur débute lors de l'entrée en phase stationnaire et est contrôlée par deux régulateurs transcriptionnels: CodY et PlcR. Le répresseur CodY pourrait se lier à l'ADN en amont du promoteur PA et réprimer la transcription des gènes nprR-nprX pendant la phase exponentielle de croissance. Au début de la phase stationnaire, le contrôle négatif de CodY est levé et PlcR active la transcription de nprR-nprX en se liant à une boîte PlcR située en amont de PA. Nos résultats indiquent que nprX est également transcrit indépendamment de nprR à partir de deux promoteurs, PH et PE, respectivement dépendant de sigma-H et sigma-E. Les deux promoteurs permettent d'assurer la transcription de nprX en phase stationnaire tardive alors que la transcription à partir du promoteur PA est achevée. Cette étude met en évidence le role clé des régulateurs CodY, PlcR and Spo0A dans la régulation de l'expression des gènes nprR-nprX. / In sporulating Bacillus, major processes like virulence gene expression and sporulation are regulated by communication systems involving signaling peptides and regulators of the RNPP family. In this work, we investigated the role of one such regulator, NprR, in bacteria of the Bacillus cereus group. This work can be divided into three complementary parts.The first part consisted to demonstrate that NprR is involved in a quorum-sensing system. We showed that NprR is a transcriptional regulator whose activity depends on the NprX signalling peptide. In association with NprX, NprR activates the transcription of an extracellular protease gene (nprA) during the first stage of the sporulation process. We demonstrated that the NprX peptide is secreted, processed and then reimported within the bacterial cell by two oligopeptide permease systems (Opp and Npp). Once inside the cell, the mature form of NprX, presumably the SKPDIVG heptapeptide, directly binds to NprR allowing nprA transcription. The second part was to explore the function of NprR during the infectious cycle of B. thuringiensis (Bt). We showed that NprR is active after death of the insect and allows Bt to survive in the cadavers as vegetative cells. Transcriptomic analysis revealed that NprR regulates at least 41 genes encoding degradative enzymes or involved in the synthesis of a non-ribosomal peptide named kurstakin. The degradative enzymes include chitinases, proteases and lipases. The corresponding genes are specifically expressed after host death suggesting that Bt has an active necrotrophic lifestyle in the cadaver. We showed that kurstakin is essential for Bt survival during necrotrophic development. It is required for swarming mobility and biofilm formation, presumably through a pore forming activity. A nprR deficient mutant does not develop necrotrophically and does not sporulate efficiently in the cadaver. Altogether, our results show that necrotrophism is a highly regulated mechanism essential for the Bt infectious cycle, contributing to horizontal transmission. Finally, the last part of my PhD consisted to study the regulation of nprR and nprX expression. We showed that the nprR-nprX genes are cotranscribed from a sigma A-dependent promoter (PA) located upstream from nprR. The transcription from PA starts at the onset of the stationary phase and is controlled by two transcriptional regulators: CodY and PlcR. The nutritional repressor CodY binds a DNA target site upstream from PA and represses nprR-nprX transcription during the exponential growth phase. At the onset of the stationary phase, the negative control of CodY is relieved and PlcR activates nprR-nprX transcription by binding a PlcR box located upstream from PA. We showed that nprX is also transcribed independently of the nprR transcription from two promoters, PH and PE, dependent on sigma-H and sigma-E, respectively. Both promoters ensure nprX transcription during late stationary phase and sporulation while transcription from PA is complete. This study highlights the key role played by CodY, PlcR and Spo0A in nprR-nprX transcription.

Les bactéries utilisent un mode de communication appelé quorum sensing pour régulerl’expression des gènes en fonction de la densité de population et contrôler ainsi de façonmulticellulaire des processus tels que la sporulation, la compétence ou la virulence. Chez les bactériesà Gram-positif, le quorum sensing repose principalement sur la production, la sécrétion et la détectionde petits peptides de signalisation.Le projet porte sur l’étude du système quorum sensing: NprR/NprX chez Bacillus cereus, oùNprR est l’effecteur qui reconnait spécifiquement le peptide de signalisation NprX. NprR est uneprotéine bi-fonctionnelle. Seule, elle agit en tant qu’inhibiteur de la sporulation, en complexe avecNprX, elle perd sa fonction initiale au profit d’une activité facteur de transcription impliquée dans lavirulence. NprR appartient à une famille d’effecteurs de quorum sensing appelée RNPP (Rap, NprR,PlcR et PrgX) encore mal caractérisée au niveau structural. Mon projet de thèse a consisté en l’analysestructure-fonction du système NprR/NprX.Pour comprendre la régulation fonctionnelle de NprR par NprX, des études en solution (SECMALSet DLS) ont permis de mettre en évidence un switch moléculaire qui repose sur un changementd’oligomérisation. Ainsi NprX fait basculer NprR d’une conformation Apo dimérique à uneconformation compléxée tétramérique.L’étude structurale par cristallographie a aboutit à la résolution de la structure du complexeNprR/NprX. L’analyse de ce tétramère suggère la reconnaissance de 2 sites distincts sur l’ADN.L’étude structurale par SAXS, a quant à elle, permis de proposer une conformation dimérique de laforme Apo NprR, modèle conforté grâce à une étude par mutagénèse dirigée des résidus d’interface. Ils’agit d’un mode de dimérisation semblable à celui des protéines Rap (membres de la famille RNPP).La caractérisation par ITC de l’interaction NprR/NprX avec différentes formes du peptide,ainsi que l’analyse de la poche de fixation du complexe, ont permis de mieux comprendre la spécificitéd’interaction et de mettre en évidence deux résidus clés de l’effecteur : l’Asn275 essentielle à lafixation du peptide et l’Arg 126 essentielle à l’activation de la fonction facteur de transcription.Ces travaux ont contribué à une meilleure compréhension du système quorum sensingNprR/NprX grâce à l’élucidation du switch moléculaire contrôlé par NprX mais aussi à une meilleureconnaissance de la famille d’effecteurs RNPP. / Bacteria use a communication mode named quorum sensing to regulate gene expression depending on the population density and thus to control processes such as sporulation, competence or virulence in a multicellular manner. In Gram-positive bacteria, the quorum sensing relies mostly on the production, the secretion and the detection of small signaling peptides. The project focuses on the study of the quorum sensing system NprR/NprX in Bacillus cereus, where NprR is the effector, which recognizes specifically the signaling peptide NprX. NprR is a bi-functional protein. In the absence of peptide, it acts as a sporulation inhibitor while in complex with NprX, it acts as transcription factor implicated in virulence. NprR belongs to a family of quorum sensing effectors named RNPP (for the first identified members: Rap, NprR, PlcR and PrgX) still not well characterized at a structural level. My PhD project consisted to perform the structure/function analysis of the NprR/NprX system. To understand the functional regulation of NprR by NprX, I carried out different studies in solution (SEC-MALS and DLS). These results allowed me to highlight a molecular switch based on a changing of the oligomerisation state of the protein. NprX binding switches NprR from an Apo dimeric conformation to a tetrameric complex. The structural study by crystallography led to the resolution of the tetrameric NprR/NprX complex structure. The analysis of this tetramer suggests the recognition of 2 DNA binding sites. The structural study of the dimeric conformation of Apo NprR by SAXS, allowed me to propose a model similar to that of the Rap dimers (members of RNPP family). This model is supported by a directed mutagenesis study of interface residues. The characterization by ITC of the NprR/NprX interaction with different forms of the peptide, as well as the analysis of the binding pocket in the complex, led to a better understanding of the specificity of the interaction. Two key residues of the effector were highlighted: Asn275, essential to peptide binding and Arg126, essential to the activation of the transcription factor function. These results have contributed to a better understanding of the NprR/NprX quorum sensing system thanks to the elucidation of the molecular switch controlled by NprX but also in a better knowledge of the RNPP effectors family.

As bactérias patogênicas são os principais agentes que contaminam alimentos e podem prejudicar a saúde humana. Para tentar combater e controlar a contaminação de alimentos investigam-se novos compostos que apresentam atividade antimicrobiana. O ramnolipídeo (RL) é um biossurfatante (BS) produzido por Pseudomonas spp. que apresenta elevada biodegradabilidade e, baixa toxicidade além de potencial antimicrobiano. O objetivo desse trabalho foi estudar a atividade antimicrobiana do RL frente às bactérias patogênicas Gram positivas, Bacillus cereus (ATCC 33018), Listeria monocytogenes (ATCC 19112), Staphylococcus aureus (ATCC 8095) e Gram negativas, Escherichia coli (EHEC) (ATCC 43895) e Salmonella enterica (ATCC 13076) além de contribuir na elucidação do mecanismo de ação destes compostos. Os testes de susceptibilidade ao RL foram realizados a partir da determinação da concentração inibitória mínima (CIM) e concentração bactericida mínima (CBM) utilizando a técnica de micro-diluição. O efeito do pH sobre a atividade antimicrobiana foi avaliado na faixa de pH 5 a 9. Para avaliação do mecanismo de ação foram realizados ensaios de permeabilidade celular, espectroscopia de infravermelho e hidrofobicidade celular. O RL apresentou atividade antimicrobiana para as bactérias B. cereus em CIM 19,5 &#956g/mL e CBM 39,1 &#956g/mL, e para L. monocytogenes CIM 156,2 &#956g/mL e CBM 312,5 &#956g/mL. Para B. cereus apresentou efeito bactericida a partir de 30 minutos na CBM, e para L. monocytogenes em 8 horas de incubação com o RL na CBM. As bactérias Gram negativas E. coli e S. enterica mostraram-se resistentes ao RL. O pH influenciou a ação antimicrobiana do RL sendo mais efetivo em pH mais ácidos. O tratamento com RL promoveu redução da hidrofobicidade da superfície celular das bactérias sensíveis. Os espectros infravermelhos evidenciaram alterações na composição química da membrana/parede celular principalmente para bactérias Gram positivas. A permeabilidade da membrana celular aumentou de acordo com o aumento da concentração de RL. A atividade antimicrobiana do RL foi evidenciada para as bactérias Gram positivas sendo mais sensíveis B. cereus e L. monocytogenes. Os resultados obtidos neste trabalho sugerem que o RL promove alterações na permeabilidade e composição química da membrana celular bacteriana sendo um agente potencial para controle de bactérias Gram positivas de importância alimentar. / Pathogenic bacteria are main agents that contaminate food and are harmful to human health. The search for new compounds to combat and control food pathogens is of increasing interest. Rhamnolipid (RL) is a biosurfactant (BS) typically produced by Pseudomonas spp., showing high biodegradability, low toxicity and antimicrobial activity. This study aimed to evaluate the antimicrobial activity of RL against the food pathogenic Gram positive bacteria Bacillus cereus (ATCC 33018), Listeria monocytogenes (ATCC 19112), Staphylococcus aureus (ATCC 8095) and Gram negative, Escherichia coli (EHEC) (ATCC 43895) and Salmonella enterica (ATCC 13076) and also contribute to the elucidation of RL mechanism of action. Susceptibility tests were performed by determination of the minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) using the broth microdilution method. The effect of pH on antimicrobial action was also investigated ranging from 5 to 9. Mechanism of action was studied using membrane permeability, infrared spectroscopy and cell hydrophobicity assays. The MIC value for B. cereus was 19.5 &#956g/mL and MBC was 39.1 &#956g/mL. L. monocytogenes was inhibited at concentration 156.2 &#956g/mL showing MBC of 312.5 &#956g/mL. B. cereus presented bactericidal effect after 30 minutes and for L. monocytogenes after 8 hours. The Gram-negative E. coli and S. enterica were resistant to RL. The pH influence antimicrobial activity of the RL showing decreasing MIC values at acidic conditions. Cell hydrophobicity was reduced by RL for the sensitive bacteria. Infrared spectroscopy showed that RL induced changes in chemical composition of cell membrane/ wall especially for the Gram positive bacteria. Cell permeability also increases as RL concentration increases. Antimicrobial activity of RL was evidenced for Gram positive bacteria and the most sensitive were B. cereus and L. monocytogenes. The results of this study suggest that rhamnolipid biosurfactant promotes changes in the permeability and membrane chemical composition showing potential to control foodborne Gram positive bacteria.

Orientador: Anita Jocelyne Marsaioli / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-21T09:27:02Z (GMT). No. of bitstreams: 1 Araujo_FranciscaDianadaSilva_D.pdf: 5387746 bytes, checksum: ae8110c020cf470d09fb0e22f670508f (MD5) Previous issue date: 2012 / Resumo: Aspectos distintos da ecologia química de bactéria, fungo e abelha foram investigados e abordados em três capítulos. O primeiro capítulo descreve o estudo das moléculas sinalizadoras envolvidas no processo de quorum-sensing da bactéria Enterobacter sakazakii (Cronobacter spp.), que resultou na identificação de três acil-homosserina lactonas (acil-HSLs): (S)-(-)-N-heptanoil-HSL, (S)-(-)-N-dodecanoil-HSL e (S)-(-)-N-tetradecanoil-HSL. Estes semioquímicos foram degradados por enzimas de Bacillus cereus. No segundo capítulo o estudo do fungo Epicoccum nigrum possibilitou o isolamento de meleína, 4,5-dimetil-resorcinol, flavipina e de um novo metabólito, denominado epicolactona. O monitoramento destas substâncias em três mutantes de E. nigrum apontou para a produção de 5-hidroxi-meleína, ausente no fungo selvagem, indicando que a rota biossintética de policetídeos em E. nigrum foi alterada, ativando a ação de uma mono-oxigenase responsável pela oxidação da meleína. No terceiro capítulo, foram investigados feromônios produzidos por diferentes castas da abelha social Tetragonisca angustula. Bioensaios com as rainhas indicaram que lipídios cuticulares são possíveis responsáveis pela indução da cópula nos machos. Operárias fundadoras e guardas foram diferenciadas quimicamente por compostos presentes em seus extratos cefálicos e abdominais. Extratos cefálicos de machos em diferentes ciclos de vida mostraram-se semelhantes / Abstract: Different aspects of bacteria, fungus and bee chemical ecology were investigated and discussed in three chapters. First chapter describes the study of signaling molecules involved in the Enterobacter sakazakii (Cronobacter spp.) quorum sensing process that resulted in the identification of three acyl-homoserine lactones (acyl-HSLs): (S)-(-)-N-heptanoyl-HSL, (S)-(-)-N-dodecanoyl-HSL and (S)-(-)-N-tetradecanoyl-HSL. These semiochemicals were modified by Bacillus cereus enzymes. Second chapter describes the study of Epicoccum nigrum (fungus) revealing the isolation of mellein, 4,5-dimethylresorcinol, flavipin and of a novel metabolite, named epicolactone. Monitoring these compounds in three E. nigrum mutants pointed to the production of 5-hydroxymellein, absent in wild fungus strain, indicating that biosynthetic route of polyketides in E. nigrum has been modified, activating a monooxygenase responsible for mellein oxidation. Third chapter describes pheromones produced by different castes of Tetragonisca angustula social bee. Bioassays with queens indicated that cuticular lipids might be responsible for copulation induction in males. Cephalic and abdominal extracts of founded and guard workers were chemically different, while cephalic extracts of males at different life cycles were similar / Doutorado / Quimica Organica / Doutora em Ciências

Parmi les desserts à base d’ovoproduits, l’île flottante est reconnue comme particulièrement sensible d’un point de vue microbiologique car sa commercialisation souffre de la survenue intempestive d’altérations que les industriels souhaitent maîtriser. Les travaux réalisés au cours de cette thèse avaient pour objectif de mieux appréhender ces phénomènes afin de mieux les contrôler. Nous avons montré que l’altération de l’île flottante concernait principalement la crème anglaise et qu’elle s’accompagnait d’un développement bactérien conséquent, d’une fréquente diminution du pH et de modifications sensorielles liées à l’aspect et à l’odeur. Les principales bactéries détectées ont été identifiées comme appartenant au groupe Bacillus cereus et aux genres Staphylococcus et Enterococcus. Le blanc d’œuf pasteurisé, utilisé pour la fabrication des œufs en neige, s’est avéré être une source de contamination possible.Cependant, l’implication de bactéries installées sous forme de biofilms dans l’environnement de production ou véhiculées par d’autres ingrédients a aussi été fortement envisagée. La ré-inoculation, en culture pure, d’une collection bactérienne représentative dans de la crème anglaise stérile a montré que différents types de modifications sensorielles et physico-chimiques s’exprimaient d’un genre bactérien à l’autre et qu’ils étaient notamment corrélés à la capacité des bactéries à consommer les sucres et les protéines de la crème anglaise et à produire des métabolites dont des composés volatils odorants. Ces résultats à l’appui, différents tests ont pu être proposés, p / Among the chilled egg products-based desserts, the French dessert “île flottante” is recognized as particularly sensitive from a microbiological point of view, because marketing is suffering from untimely spoilage occurrence that manufacturers wish to control. The work done in this thesis aimed to better understand these phenomena in order to better control them. We have shown that the dessert spoilage mainly concerned the custard cream and it was characterized by high bacterial count, frequent pH decreasing and sensory changes of appearance and smell. The main bacteria detected were identified as belonging to the Bacillus cereus group and Staphylococcus and Enterococcus genera. The possible involvement of bacteria from the pasteurized egg white, used for the egg white foaming, in the dessert spoilage issue was established.However, the involvement of bacteria from biofilms installed in the production environment or provided by other ingredients was also strongly suspected. The spoilage potential assessment of pure culture of a representative bacterial collection in sterile custard cream has shown that different types of sensory and physicochemical changes were expressed according to bacterial genus and that these changes were particularly correlated with the ability of bacteria to consume sugars and proteins of custard and to produce various volatile compounds with specific odorous. With these results, various tests have been proposed for a better control of the white egg batches orientation according to their microbiological quality and so to guarantee their safety with

As bactérias patogênicas são os principais agentes que contaminam alimentos e podem prejudicar a saúde humana. Para tentar combater e controlar a contaminação de alimentos investigam-se novos compostos que apresentam atividade antimicrobiana. O ramnolipídeo (RL) é um biossurfatante (BS) produzido por Pseudomonas spp. que apresenta elevada biodegradabilidade e, baixa toxicidade além de potencial antimicrobiano. O objetivo desse trabalho foi estudar a atividade antimicrobiana do RL frente às bactérias patogênicas Gram positivas, Bacillus cereus (ATCC 33018), Listeria monocytogenes (ATCC 19112), Staphylococcus aureus (ATCC 8095) e Gram negativas, Escherichia coli (EHEC) (ATCC 43895) e Salmonella enterica (ATCC 13076) além de contribuir na elucidação do mecanismo de ação destes compostos. Os testes de susceptibilidade ao RL foram realizados a partir da determinação da concentração inibitória mínima (CIM) e concentração bactericida mínima (CBM) utilizando a técnica de micro-diluição. O efeito do pH sobre a atividade antimicrobiana foi avaliado na faixa de pH 5 a 9. Para avaliação do mecanismo de ação foram realizados ensaios de permeabilidade celular, espectroscopia de infravermelho e hidrofobicidade celular. O RL apresentou atividade antimicrobiana para as bactérias B. cereus em CIM 19,5 &#956g/mL e CBM 39,1 &#956g/mL, e para L. monocytogenes CIM 156,2 &#956g/mL e CBM 312,5 &#956g/mL. Para B. cereus apresentou efeito bactericida a partir de 30 minutos na CBM, e para L. monocytogenes em 8 horas de incubação com o RL na CBM. As bactérias Gram negativas E. coli e S. enterica mostraram-se resistentes ao RL. O pH influenciou a ação antimicrobiana do RL sendo mais efetivo em pH mais ácidos. O tratamento com RL promoveu redução da hidrofobicidade da superfície celular das bactérias sensíveis. Os espectros infravermelhos evidenciaram alterações na composição química da membrana/parede celular principalmente para bactérias Gram positivas. A permeabilidade da membrana celular aumentou de acordo com o aumento da concentração de RL. A atividade antimicrobiana do RL foi evidenciada para as bactérias Gram positivas sendo mais sensíveis B. cereus e L. monocytogenes. Os resultados obtidos neste trabalho sugerem que o RL promove alterações na permeabilidade e composição química da membrana celular bacteriana sendo um agente potencial para controle de bactérias Gram positivas de importância alimentar. / Pathogenic bacteria are main agents that contaminate food and are harmful to human health. The search for new compounds to combat and control food pathogens is of increasing interest. Rhamnolipid (RL) is a biosurfactant (BS) typically produced by Pseudomonas spp., showing high biodegradability, low toxicity and antimicrobial activity. This study aimed to evaluate the antimicrobial activity of RL against the food pathogenic Gram positive bacteria Bacillus cereus (ATCC 33018), Listeria monocytogenes (ATCC 19112), Staphylococcus aureus (ATCC 8095) and Gram negative, Escherichia coli (EHEC) (ATCC 43895) and Salmonella enterica (ATCC 13076) and also contribute to the elucidation of RL mechanism of action. Susceptibility tests were performed by determination of the minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) using the broth microdilution method. The effect of pH on antimicrobial action was also investigated ranging from 5 to 9. Mechanism of action was studied using membrane permeability, infrared spectroscopy and cell hydrophobicity assays. The MIC value for B. cereus was 19.5 &#956g/mL and MBC was 39.1 &#956g/mL. L. monocytogenes was inhibited at concentration 156.2 &#956g/mL showing MBC of 312.5 &#956g/mL. B. cereus presented bactericidal effect after 30 minutes and for L. monocytogenes after 8 hours. The Gram-negative E. coli and S. enterica were resistant to RL. The pH influence antimicrobial activity of the RL showing decreasing MIC values at acidic conditions. Cell hydrophobicity was reduced by RL for the sensitive bacteria. Infrared spectroscopy showed that RL induced changes in chemical composition of cell membrane/ wall especially for the Gram positive bacteria. Cell permeability also increases as RL concentration increases. Antimicrobial activity of RL was evidenced for Gram positive bacteria and the most sensitive were B. cereus and L. monocytogenes. The results of this study suggest that rhamnolipid biosurfactant promotes changes in the permeability and membrane chemical composition showing potential to control foodborne Gram positive bacteria.

<p>In&nbsp; this&nbsp; study,&nbsp; tolerance&nbsp; of&nbsp; soil&nbsp; bacteria&nbsp; to&nbsp; hexavalent&nbsp; chromium&nbsp; (Cr(VI))&nbsp; was&nbsp; investigated.&nbsp; First,&nbsp; influence&nbsp; of&nbsp; high chromium levels of anthropogenic and geogenic origin on the&nbsp; soil cultivable&nbsp; bacterial community was examined. Next, a number&nbsp; of&nbsp; bacterial&nbsp; strains&nbsp; with&nbsp; high&nbsp; Cr(VI)&nbsp; tolerance&nbsp; were&nbsp; isolated&nbsp; from&nbsp; diverse&nbsp;&nbsp; environmental&nbsp; samples&nbsp; such&nbsp; as&nbsp; soil, sediment, water and waste material.&nbsp; Strains were&nbsp; identified&nbsp; and&nbsp; tested for&nbsp; the&nbsp; level of&nbsp; Cr(VI) tolerance&nbsp; and&nbsp; the&nbsp; ability to<br />reduce toxic Cr(VI) to more innocuous Cr(III). Selected&nbsp; <em>Bacillus cereus</em>&nbsp; group strains&nbsp; were further characterized&nbsp; -&nbsp; their morphological&nbsp; and&nbsp; biochemical&nbsp; characteristics,&nbsp; 16S&nbsp; rRNA&nbsp; and&nbsp; pycA&nbsp; gene&nbsp; sequences,&nbsp; biofilm&nbsp; formation&nbsp; potential&nbsp; and resistance to other heavy metals were determined. Also, more detailed study of their tolerance level and&nbsp; Cr(VI) reduction was&nbsp; conducted.&nbsp; Strain&nbsp; with&nbsp; the highest&nbsp; resistance&nbsp; together&nbsp; with the&nbsp; control&nbsp; chromate&nbsp; sensitive&nbsp; strain&nbsp; were&nbsp; analyzed&nbsp; by STEM EDS for their cellular and endospore Cr content under different conditions. Results indicate Cr(VI) tolerant bacteria are&nbsp; present&nbsp; both&nbsp; in&nbsp; low&nbsp; and&nbsp; high&nbsp; Cr&nbsp; environments.&nbsp; Majority&nbsp; of&nbsp; isolates&nbsp; belonged&nbsp; to&nbsp; the&nbsp;<em> B.&nbsp; cereus&nbsp;</em> group&nbsp; indicating&nbsp; its overall high tolerance to&nbsp; Cr(VI). Certain strains exhibited high&nbsp; tolerance and reduction&nbsp; ability,&nbsp; indicating their possible<br />usefulness&nbsp; in practical&nbsp; bioremediation&nbsp; application.&nbsp; STEM&nbsp; EDS&nbsp; analysis&nbsp; of&nbsp; Cr(VI)-sensitive&nbsp;<em> B.&nbsp; subtilis&nbsp;</em> PY79&nbsp; strain&nbsp; and Cr(VI)-resistant&nbsp; <em>B. cereus&nbsp;</em> group strain&nbsp; NCr1a revealed&nbsp; significant differences in their response to Cr(VI)&nbsp; and in&nbsp; their&nbsp; Cr cellular and endospore content.</p> / <p>U ovom radu ispitana je tolerantnost&nbsp; zemlji&scaron;nih&nbsp; bakterija na &scaron;estovalentni hrom (Cr(VI)). Prvo, ispitan je uticaj visokog nivoa&nbsp; hroma&nbsp; antropogenog&nbsp; i&nbsp; geogenog&nbsp; porekla&nbsp; na&nbsp; kultivabilnu&nbsp; bakterijsku&nbsp; zajednicu&nbsp; zemlji&scaron;ta.&nbsp; Dalje,&nbsp; izolovani&nbsp; su bakterijski sojevi sa visokom tolerancijom na Cr(VI) iz različitih sredinskih uzoraka &nbsp; kao &scaron;to su zemlji&scaron;te, sediment, voda i otpadni materijal. Sojevi su identifikovani i određen je nivo njihove Cr(VI) tolerancije i sposobnost redukcije toksičnog Cr(VI)&nbsp; u&nbsp; manje&nbsp; toksični&nbsp; Cr(III).&nbsp; Odabrani&nbsp; sojevi&nbsp; <em>Bacillus&nbsp; cereus&nbsp;</em> grupe&nbsp; su&nbsp; dalje&nbsp; karakterisani&nbsp; &ndash;&nbsp; određene&nbsp; su&nbsp; njihove morfolo&scaron;ke i biohemijske karakteristike, 16S rDNK i&nbsp; pycA&nbsp; sekvence, potencijal formiranja biofilma i otpornost na druge te&scaron;ke&nbsp; metale.&nbsp; Takođe,&nbsp; sprovedeno&nbsp; je&nbsp; detaljnije&nbsp; ispitivanje&nbsp; njihove&nbsp; tolerancije&nbsp; i&nbsp; redukcije&nbsp; Cr(VI).&nbsp; Soj&nbsp; sa&nbsp; najvi&scaron;om otporno&scaron;ću&nbsp; je&nbsp; uporedo&nbsp; sa&nbsp; kontrolnim&nbsp; osetljivim&nbsp; sojem&nbsp; analiziran&nbsp; pomoću&nbsp; STEM&nbsp; EDS&nbsp; na&nbsp; sadržaj&nbsp; hroma&nbsp; u&nbsp; ćelijama&nbsp; I endosporama u različitim uslovima. Rezultati ukazuju da su bakterije tolerantne na Cr(VI) prisutne i u sredinama sa niskim i&nbsp; sa&nbsp; visokim&nbsp; koncentracijama&nbsp; hroma.&nbsp; Većina&nbsp; izolata&nbsp; pripadala&nbsp; je&nbsp; B.&nbsp; cereus&nbsp; grupi&nbsp; &scaron;to&nbsp; ukazuje&nbsp; na njenu&nbsp; uop&scaron;teno&nbsp; visoku otpornost na Cr(VI). Pojedini sojevi su pokazali visoku otpornost i sposobnost&nbsp; redukcije Cr(VI), &scaron;to ukazuje na mogućnost njihove praktične primene u bioremedijaciji. STEM EDS analiza osetljivog<em> B. subtilis</em> PY79 soja i Cr(VI)- rezistentnog soja <em>B.&nbsp; cereus</em>&nbsp; grupe&nbsp; NCr1a&nbsp; otkrila&nbsp; je&nbsp; značajne&nbsp; razlike&nbsp; u&nbsp; njihovom&nbsp; odgovoru na&nbsp; Cr(VI)&nbsp; i&nbsp; sadržaju&nbsp; Cr&nbsp; u njihovim&nbsp; ćelijama&nbsp; i endosporama.</p>