Cette étude s’inscrit dans le cadre d’un programme de recherche destiné à évaluer le potentiel d’utilisation en horticulture des bactéries Bacillus pumilus souche PTB180 et Bacillus subtilis souche PTB185. Elle avait pour objectifs (1) d’évaluer in vitro l’activité antagoniste contre Botrytis cinerea des souches PTB180 et PTB185, (2) d’estimer leur capacité à survivre sur la phyllosphère de la tomate et du concombre et 3) d’évaluer leur effet sur le développement de la pourriture grise (B. cinerea) sur des plants de tomate et de concombre cultivés en serre. L'activité antagoniste de PTB180 et PTB185 a été évaluée en boîtes de Pétri sur géloses, sur tissus foliaires de tomate et de concombre et sur fruits de tomate. Les deux souches ont inhibé très fortement la croissance mycélienne et la germination des spores de B. cinerea sur géloses. Sur feuilles de tomate et sur disques foliaires de concombre, PTB185 et le mélange (1:1) des deux souches ont réduit significativement (p ≤ 0,01) la croissance mycélienne de B. cinerea comparativement aux témoins. PTB180 a réprimé significativement la croissance mycélienne de B. cinerea sur les fruits de tomate. Afin d'estimer la survie de PTB180 et PTB185 sur la phyllosphère, des plants de tomate et de concombre ont été pulvérisés jusqu'à ruissellement avec une suspension (1×107 unités formatrices de colonies [UFC]/mL) de PTB180, PTB185 ou d'un mélange (1:1) des deux souches. Les populations de chaque souche ont ensuite été suivies au cours du temps sur les feuilles. Les résultats obtenus montrent que les souches survivent au moins 21 jours sur les plants de tomate et de concombre avec un taux de survie variant de 43% à 100%. De plus, pratiquement aucune variation dans les proportions de chaque souche n'a été observée au fil du temps lorsque PTB180 et PTB185 étaient appliquées en mélange. Enfin, l’application foliaire de PTB180, PTB185 et du mélange (1:1) des deux souches a permis une réduction significative de l’incidence et de la sévérité de la pourriture grise chez des plants de tomate et de concombre inoculés avec B. cinerea et cultivés en serre. Les souches PTB180 et PTB185 ont montré au cours de cette étude une forte activité antagoniste envers B. cinerea, la capacité de survivre sur la phyllosphère de plants de tomate et de concombre et de réprimer le développement de la pourriture grise chez ces derniers. Ces souches pourraient éventuellement être utilisées comme agents de lutte biologique contre la pourriture grise du concombre et de la tomate de serre.

L'augmentation et la dissémination de la résistance aux antibiotiques chez les bactéries à gram-negatif, particulièrement les Entérobactéries, les bactéries du genre Pseudomonas et Acinetobacter, représentent un problème majeur de santé publique au niveau mondial. Les infections nosocomiales causées par les bactéries multi-résistantes (BMR) ont conduit non seulement à une augmentation de la mortalité, de la morbidité, et du coût de traitement, mais aussi continuent de mettre en danger la vie des patients surtout immunodéprimés en milieu hospitalier. Bien entendu, l'utilisation abusive et non contrôlée des antibiotiques a grandement contribué à la large diffusion des déterminants de la résistance; cependant, des études récentes ont démontré que ces déterminants de la résistance pouvaient émerger à partir de sources anciennes et/ou environnementales. Ainsi, face à cette préoccupation mondiale, plusieurs études ont été rapportées avec des recommandations importantes de conduire des études épidémiologiques, moléculaires, et génomiques afin de contrôler la diffusion et l'augmentation de la résistance aux antibiotiques. De plus, durant ces 10 dernières années, nous avons assisté à l'emergence et au développement de nouvelles technologies de séquençage à haut débit coïncidant avec une augmentation exponentielle du nombre de genomes bactériens séquencés. / The increase and spread of multidrug-resistant (MDR) gram-negative bacteria especially Enterobacteriaceae, Pseudomonas, and Acinetobacter (E.P.A) species have become a major concern worldwide. The hospital-acquired infections caused by MDR bacteria have led not only to an increase in mortality, morbidity, and cost of treatment, but also continue to endanger the life of patients, especially those immunocompromised. Although the frequent misuse of antibiotic drug has greatly contributed to worldwide dissemination and resistance to antibiotics; recent studies have shown that these resistance determinants could emerge from ancient or environmental sources. Front of this worldwide concern, several studies have been reported with significant recommendations to conduct molecular epidemiology, and genomic studies, in order to control the increase and the dissemination of the antibiotic resistance. Moreover, during these last 10 years, we are witnessing the emergence and development of new technologies of high throughput sequencing and coinciding with an exponential increase of number of bacterial genomes sequenced today. Therefore, it is in this context that the project of this thesis was conducted with three essential objectives: (i) the genome sequencing of clinical MDR bacteria, the analysis and the identification of the mechanisms and the genetic determinants of antimicrobial resistance (ii) the achievement of molecular epidemiology studies from clinical MDR bacteria responsible of outbreak (iii) the development and implementation of molecular tools for monitoring and diagnosis of potential MDR bacteria.

L'augmentation et la dissémination de la résistance aux antibiotiques chez les bacilles à Gram-négatif, particulièrement les Entérobactéries, les bactéries du genre Pseudomonas et Acinetobacter, représentent un problème majeur de santé publique. Les infections nosocomiales causées par les bactéries multi-résistantes ont conduit non seulement à une augmentation de la mortalité, de la morbidité et du coût de traitement, mais aussi continuent à mettre en danger la vie des patients surtout immunodéprimés. L'utilisation abusive et non contrôlée des antibiotiques a grandement contribué à la large diffusion de la résistance aux antibiotiques. Cependant, des études récentes ont démontré que cette résistance pouvait émerger à partir de sources anciennes et/ou environnementales. Ainsi, face à cette préoccupation mondiale et suite à de nombreuses recommandations, plusieurs études épidémiologiques et moléculaires ont été rapportées afin de contrôler et surveiller la diffusion et la dissémination de la résistance aux antibiotiques. Il est cependant prioritaire de développer des nouveaux outils de surveillance de la résistance aux antibiotiques. C'est dans cette optique que ce projet de thèse s'articule avec comme objectifs :- Le développement et la mise en place de nouveaux outils et logiciels de surveillance et de diagnostic des bactéries multi-résistantes, - La réalisation des études d'épidémiologie moléculaire sur les isolats cliniques de bactéries multi-résistantes responsables d'épidémies. / The increase and spread of multidrug-resistant (MDR) gram-negative bacteria especially Enterobacteriaceae, Pseudomonas, and Acinetobacter (E.P.A) species have become a major concern worldwide. The hospital-acquired infections caused by MDR bacteria have led not only to an increase in mortality, morbidity, and cost of treatment, but also continue to endanger the life of patients, especially those immunocompromised. Although, the frequent misuse of antibiotic drug has greatly contributed to worldwide dissemination of antibiotics resistance. Recent studies have shown that these resistance determinants could emerge from ancient or environmental sources. Front of this worldwide concern, and various recommendations, several epidemiological and molecular studies have been reported in order to control the spread and the dissemination of the antibiotic resistance. However, it is a priority to develop new tools for monitoring antibiotic resistance. Therefore, it is in this context that the project of this thesis was conducted with two essential objectives: -The development and implementation of news tools and software for monitoring and diagnosis of potential MDR bacteria. -The achievement of molecular epidemiology studies from clinical MDR bacteria responsible of outbreak.

O operon groESL de C. crescentus apresenta dupla regulação. A indução deste operon por choque térmico é dependente do fator sigma de choque térmico σ32. A temperaturas fisiológicas, a expressão de groESL apresenta regulação temporal durante o ciclo celular da bactéria e o controle envolve a proteína repressora HrcA e o elemento CIRCE (controlling inverted repeat of chaperonin expression). Para estudar a atividade da proteína repressora in vitro, produzimos e purificamos de E. coli a HrcA de C. creseentus contendo uma cauda de histidinas e a ligação especifica ao elemento CIRCE foi analisada em ensaios de migração retardada em gel de poliacrilamida (EMRGP). A quantidade de DNA retardada pela ligação a HrcA aumentou significativamente na presença de GroES/GroEL, sugerindo que estas proteínas modulam a atividade de HrcA. Corroboração desta modulação foi obtida analisando fusões de transcrição da região regulatória de groESL com o gene lacZ, em células de C. crescentus produzindo diferentes quantidades de GroES/EL. HrcA contendo as substituições Pro81 AJa e Arg87Ala, aminoácidos que se localizam no domínio putativo de ligação ao DNA da proteína, mostraram ser deficientes na ligação a CIRCE, tanto in vitro como in vivo. Em adição, HrcA Ser56Ala expressa na mesma célula juntamente com a proteína selvagem produziu um fenótipo dominante-negativo, indicando que a HrcA de C. crescentus liga-se a CIRCE como um oligômero, provavelmente um dímero. As tentativas de obtenção de mutantes nulos para os genes groESL ou dnaKJ falharam, indicando que as proteínas GroES/GroEL e DnaK/DnaJ são essenciais em C. crescentus, mesmo a temperaturas normais. Foram então construídas no laboratório as linhagens mutantes condicionais SG300 e SG400 de C. crescentus, onde a expressão de groESL e de dnaKJ, respectivamente, está sob controle de um promotor induzido por xilose (PxyIX). Estas linhagens foram caracterizadas quanto á sua morfologia em condições permissivas ou restritivas, assim como quanto à capacidade de sobrevivência frente a vários tipos de estresse. As células da linhagem SG300, exauridas de GroES/GroEL, são resistentes ao choque térmico a 42°C e são capazes de adquirir alguma termotolerância. Entretanto, estas células são sensíveis aos estresses oxidativo, salino e osmótico. As células da linhagem SG400, exauridas de DnaKlJ, são sensíveis ao choque térmico, à exposição a etanol e ao congelamento, e são incapazes de adquirir termotolerância. Além disso, tanto as células exauridas de GroES/GroEL quanto as exauridas de DnaK/DnaJ apresentam problemas na sua morfologia. As células de SG300 exauridas de GroES/GroEL formam filamentos longos que possuem constrições fundas e irregulares. As células de SG400 exauridas de DnaK/DnaJ são apenas um pouco mais alongadas que as células pré-divisionais selvagens e a maioria das células não possuem septo. Estas observações indicam bloqueio da divisão celular, que deve ocorrer em diferentes estágios em cada linhagem. / In Caulobacter crescentus, the groESL operon presents a dual type of control. Heat shock induction of the operon is dependent on the heat shock sigma factor σ-32. At physiological temperatures, groESL expression is cell cycle regulated and the control involves the repressor protein HrcA and the element CIRCE (controlling inverted repeat of chaperonin ~xpression). To study the activity of HrcA in vitro, we produced and purified from E. coli a histidine-tagged version of the protein, and specific binding to the CIRCE element was analyzed in electrophoretic mobility shift assays (EMSA). The amount of retarded DNA increased significantly in the presence of GroES/GroEL, suggesting that these proteins modulate HrcA activity. Further evidence of this modulation was obtained using lacZ transcription fusions with the groESL regulatory region in C. crescentus cells producing different amounts of GroES/GroEL. The mutants proteins HrcA Pro81Ala and HrcA Arg87Ala, that contain amino acid substitutions in the putative DNA-bindíng domain of the protein, were found to be deficient in binding to CIRCE in vitro and in vivo. Furthermore, HrcA Ser56Ala expressed together with the wild type protein within the same cell, produced a dominant-negative phenotype, indicating that C. crescentus HrcA binds to CIRCE in an oligomeric form, most likely as a dimer. Attempts to obtain null mutants for groESL or dnaKJ were unsuccessful indicating the importance of GroES/GroEL and DnaK/lDnaJ to the survival of C. crescentus cells. Conditional mutants were then constructed in our laboratory in which groESL and dnaKJ expression is under the control ofaxylose inducible promoter (PxyIX) , giving rise to strains SG300 and SG400, respectively. These strains were characterized in regard to their morphology under permissive and restrictive conditions, as well as their viability under different types of environmental stresses. SG300 cells depleted of GroES/GroEL are resistant to heat shock at 42°C and can acquire some thermotolerance, but they are sensitive to oxidative, saline and osmotic stresses. SG400 cells depleted of DnaKlJ are quite sensitive to heat shock, ethanol and freezing, and are unable of acquiring thermotolerance. Cells depleted of either GroES/EL or DnaKlJ also present morphological problems. SG300 cells depleted of GroES/EL form long and pinched filaments. SG400 cells depleted of DnaKlJ are only somewhat more elongated than wild-type predivisional cells and most cells do not present septum. These observations indicate a cell division arrest, which should occur at different stages in each strain.

L’augmentation et la dissémination de la résistance aux β-lactamines chez les bacilles à Gram négatif, particulièrement les Entérobactéries, les bactéries du genre Pseudomonas et Acinetobacter, représentent un problème majeur de santé publique. Les infections nosocomiales causées par ces bactéries multi-résistantes (BMR) ont conduit à une augmentation de la mortalité, de la morbidité et du coût de traitement. L’utilisation abusive et non contrôlée de ces antibiotiques a grandement contribué à la large diffusion de cette résistance. Ainsi, face à cette préoccupation mondiale et suite à de nombreuses recommandations, plusieurs études épidémiologiques et moléculaires ont été rapportées afin de contrôler et de surveiller la diffusion et la dissémination des BMR. Contrairement à de nombreuses régions dans le monde, il existe peu d’informations concernant la caractérisation moléculaire des gènes de résistance aux β-lactamines des bacilles à Gram négatif isolés en Tunisie et surtout en Libye. C’est dans cette optique que ce projet de Thèse de Doctorat s’articule avec comme objectifs: (i) mettre en évidence la prévalence des bacilles à Gram négatifs multi-résistants isolés aux niveaux des hôpitaux tunisiens et libyens (ii) identifier le support génétique de la résistance aux β-lactamines de ces souches cliniques (iii) étudier la diversité clonale des souches multi-résistantes par typage moléculaire. / The increase and spread of β-lactam resistance in gram negative bacteria especially Enterobacteriaceae, Pseudomonas and Acinetobacter (E.P.A) species have become a major concern worldwide. The hospital-acquired infections caused by MDR bacteria have led to an increase in mortality, morbidity and cost of treatment. The frequent misuse of antibiotic drug has greatly contributed to worldwide dissemination of antibiotics resistance. Front of this worldwide concern, and various recommendations, several epidemiological and molecular studies have been reported in order to control the spread and the dissemination of these MDR. Unlike many parts of the world, there is little information concerning the molecular characterization of the β-lactam resistance genes of Gram-negative bacilli isolated in Tunisia and especially in Libya. Therefore, it is in this context that the project of this thesis was conducted with essential objectives: (i) highlight the prevalence of multi-resistant Gram negative bacilli isolated in Tunisian and Libyan hospitals (ii) identify the genetic support of resistance to β-lactams of these clinical strains (iii) study the clonal diversity of the multi-resistant strains by molecular typing (iii) study the molecular epidemiology of these BMRs in these countries in order to control the decision-making process of the treatment and the rapid identification of epidemics by implementing appropriate control measures for the spread of infections and especially developing new tools and software for the diagnosis and monitoring of potential MDR bacteria in Mediterranean countries.

Introdução: O Clostridium difficile é um bacilo Gram-positivo, anaeróbio estrito, formador de esporos, que produz toxinas que podem causar diarreia, colite pseudomembranosa, dilatação do cólon, sepse e até morte. Nos últimos anos o quadro clínico e epidemiológico das infecções por Clostridium difficile tem se modificado e as limitações das opções terapêuticas tornaram-se mais evidentes. Objetivo Primário: Comparar as metodologias de disco-difusão e ágar-diluição na detecção de sensibilidade/resistência de isolados de Clostridium difficile. Objetivos Secundários: Avaliar prospectivamente o perfil de sensibilidade/resistência de isolados clínicos hospitalares de Clostridium difficile provenientes de seis hospitais terciários da cidade de São Paulo e fornecer evidências para fundamentar o diagnóstico e o tratamento empírico das diarreias causadas por Clostridium difficile. Métodos: utilizamos os métodos de disco-difusão e ágar-diluição, de acordo com os critérios estabelecidos pelo CLSI e EUCAST. Resultados: Os coeficientes de correlação observados entre os diâmetros dos halos de inibição e Concentração Inibitória Mínima foram abaixo do esperado tornando inviável o método de disco-difusão para determinação de sensibilidade aos antimicrobianos nitazoxanida, teicoplanina e tigeciclina. Todas as 50 cepas deste estudo foram sensíveis ao metronidazol (MIC50 foi de 1 ?g/mL a MIC90 foi de 2 ug/mL). Para o método de disco-difusão, sugerimos que halos de inibição >= 33mm possam ser interpretados como sensíveis. Devido à moderada correlação, significância estatística e distribuição de halos de inibição das amostras próximos aos valores encontrados utilizando a cepa ATTC, sugere-se a utilização do método de disco-difusão para vancomicina, onde halos com diâmetro >= 22mm possam ser considerados como sensíveis pelo método. Para o moxifloxacino houve uma boa correlação entre as duas metodologias: discodifusão e de ágar-diluição (O coeficiente de Pearson foi de -0,84, e o valor de p foi menor que 0,00001), sugerindo que halos de inibição >= 18mm possam ser interpretados como sensíveis pela metodologia de disco-difusão. A nitazoxanida foi à droga que mostrou melhor atividade in vitro (MIC50 foi 0,06 ?g/mL e a MIC90 de 0,12 ug/mL). Por se mostrar uma droga com potente atividade in vitro (MIC50 e a MIC90 foi de 0,12 ug/mL), a tigeciclina poderia ser mais uma opção terapêutica em infecções por Clostridium difficile, dependendo de mais estudos para avaliar sua real eficácia clínica e segurança. Conclusão: Os resultados verificados neste estudo indicam a necessidade de mais estudos in vitro e clínicos para definir os limites de sensibilidade/resistência para a teicoplanina e a nitazoxanida, pois faltam critérios de interpretação tanto para disco-difusão quanto para ágar-diluição. Os resultados deste trabalho in vitro confirmaram a utilidade do metronidazol como uma droga eficaz no tratamento de infecção por Clostridium difficile. A nitazoxanida foi à droga que mostrou melhor atividade in vitro por método dilucional. Sugerimos a utilização do método de disco-difusão para: metronidazol, vancomicina e moxifloxacino. Os resultados desse trabalho sugerem que halos de inibição para metronidazol ( >= 33mm), moxifloxacino ( >= 18mm) e vancomicina ( >= 22mm) poderiam ser considerados como sensíveis pelo método de disco-difusão. O método de ágardiluição é um método de boa acurácia, porém trabalhoso para ser executado na rotina laboratorial / Introduction: Clostridium difficile is a Gram-positive bacillus, strictly anaerobic, spore-forming, which produces toxins that can cause diarrhea, colitis pseudomembranous, colon expansion, sepsis and even death. In recent years the clinical and epidemiological picture of infection by Clostridium difficile has been modified and limitations of therapeutic options have become more evident. Primary Objective: Comparing the methods of disk diffusion and agar dilution in the detection sensitivity/resistance isolates of Clostridium difficile. Secondary Objectives: Prospectively evaluate the profile of sensitivity/resistance of hospital clinical isolates of Clostridium difficile from six tertiary hospitals in São Paulo city and provide evidence to support the diagnosis and empirical treatment of diarrhea caused by Clostridium difficile. Methods: We use the disk diffusion method and agar dilution method, according to the established criteria by CLSI and EUCAST. Results: The observed correlation coefficients between the inhibitions diameter zone of the and Minimum Inhibitory Concentration were under expectations impeding the disk diffusion method for determining sensitivity to nitazoxanide antimicrobial, teicoplanin and tigecycline. All 50 strains of this study were sensitive to metronidazole (MIC50 was 1 Ug/ml to MIC90 was 2 ug/ml). For the method disk diffusion, we suggest that inhibition zones >= 33mm can be interpreted as sensitive. Due to the moderate correlation, statistical significance and distribution of zones of inhibition on samples of the next found values using the strain ATTC, we suggest using the disk diffusion method for vancomycin where halos diameter >= 22mm can be considered as sensitive by the method. There was a good correlation to moxifloxacin between the two methodologies: disk diffusion and agar dilution (Pearson\'s coefficient was -0.84 , and the \"p\" value was less than 0.00001), suggesting that inhibition zones >= 18mm can be interpreted as sensitive by disk diffusion method. Nitazoxanide was the drug that showed a better performance in vitro activity (MIC50 was 0.06 ?g/ml and MIC90 0.12 ug/ml). For a drug that shows potent activity in vitro (MIC50 and MIC90 was 0.12 ug/ml), the tigecycline could be a therapeutic option in infection by Clostridium difficile, depending on further studies to evaluate their real clinical efficacy and security. Conclusion: Obtained results in this study indicate the need for further studies in vitro and clinicians to define the limits of sensitivity/resistance to teicoplanin and nitazoxanide, so there is no interpretation criteria for both disk diffusion and for agar dilution. Results of this work in vitro study confirmed the utility of metronidazole as an effective drug in the treatment of infection by Clostridium difficile. Nitazoxanide was the drug that showed better performance in vitro by dilutional method. We suggest the use of disk diffusion method: metronidazole, vancomycin and moxifloxacin. This work suggest that inhibition zones for metronidazole ( >= 33mm), moxifloxacin ( >= 18mm) and vancomycin ( >= 22mm) could be considered as sensitive by disk diffusion method. The agar dilution method is a method to be accurate, but laborious to run in the laboratory routine

Introdução: O Clostridium difficile é um bacilo Gram-positivo, anaeróbio estrito, formador de esporos, que produz toxinas que podem causar diarreia, colite pseudomembranosa, dilatação do cólon, sepse e até morte. Nos últimos anos o quadro clínico e epidemiológico das infecções por Clostridium difficile tem se modificado e as limitações das opções terapêuticas tornaram-se mais evidentes. Objetivo Primário: Comparar as metodologias de disco-difusão e ágar-diluição na detecção de sensibilidade/resistência de isolados de Clostridium difficile. Objetivos Secundários: Avaliar prospectivamente o perfil de sensibilidade/resistência de isolados clínicos hospitalares de Clostridium difficile provenientes de seis hospitais terciários da cidade de São Paulo e fornecer evidências para fundamentar o diagnóstico e o tratamento empírico das diarreias causadas por Clostridium difficile. Métodos: utilizamos os métodos de disco-difusão e ágar-diluição, de acordo com os critérios estabelecidos pelo CLSI e EUCAST. Resultados: Os coeficientes de correlação observados entre os diâmetros dos halos de inibição e Concentração Inibitória Mínima foram abaixo do esperado tornando inviável o método de disco-difusão para determinação de sensibilidade aos antimicrobianos nitazoxanida, teicoplanina e tigeciclina. Todas as 50 cepas deste estudo foram sensíveis ao metronidazol (MIC50 foi de 1 ?g/mL a MIC90 foi de 2 ug/mL). Para o método de disco-difusão, sugerimos que halos de inibição >= 33mm possam ser interpretados como sensíveis. Devido à moderada correlação, significância estatística e distribuição de halos de inibição das amostras próximos aos valores encontrados utilizando a cepa ATTC, sugere-se a utilização do método de disco-difusão para vancomicina, onde halos com diâmetro >= 22mm possam ser considerados como sensíveis pelo método. Para o moxifloxacino houve uma boa correlação entre as duas metodologias: discodifusão e de ágar-diluição (O coeficiente de Pearson foi de -0,84, e o valor de p foi menor que 0,00001), sugerindo que halos de inibição >= 18mm possam ser interpretados como sensíveis pela metodologia de disco-difusão. A nitazoxanida foi à droga que mostrou melhor atividade in vitro (MIC50 foi 0,06 ?g/mL e a MIC90 de 0,12 ug/mL). Por se mostrar uma droga com potente atividade in vitro (MIC50 e a MIC90 foi de 0,12 ug/mL), a tigeciclina poderia ser mais uma opção terapêutica em infecções por Clostridium difficile, dependendo de mais estudos para avaliar sua real eficácia clínica e segurança. Conclusão: Os resultados verificados neste estudo indicam a necessidade de mais estudos in vitro e clínicos para definir os limites de sensibilidade/resistência para a teicoplanina e a nitazoxanida, pois faltam critérios de interpretação tanto para disco-difusão quanto para ágar-diluição. Os resultados deste trabalho in vitro confirmaram a utilidade do metronidazol como uma droga eficaz no tratamento de infecção por Clostridium difficile. A nitazoxanida foi à droga que mostrou melhor atividade in vitro por método dilucional. Sugerimos a utilização do método de disco-difusão para: metronidazol, vancomicina e moxifloxacino. Os resultados desse trabalho sugerem que halos de inibição para metronidazol ( >= 33mm), moxifloxacino ( >= 18mm) e vancomicina ( >= 22mm) poderiam ser considerados como sensíveis pelo método de disco-difusão. O método de ágardiluição é um método de boa acurácia, porém trabalhoso para ser executado na rotina laboratorial / Introduction: Clostridium difficile is a Gram-positive bacillus, strictly anaerobic, spore-forming, which produces toxins that can cause diarrhea, colitis pseudomembranous, colon expansion, sepsis and even death. In recent years the clinical and epidemiological picture of infection by Clostridium difficile has been modified and limitations of therapeutic options have become more evident. Primary Objective: Comparing the methods of disk diffusion and agar dilution in the detection sensitivity/resistance isolates of Clostridium difficile. Secondary Objectives: Prospectively evaluate the profile of sensitivity/resistance of hospital clinical isolates of Clostridium difficile from six tertiary hospitals in São Paulo city and provide evidence to support the diagnosis and empirical treatment of diarrhea caused by Clostridium difficile. Methods: We use the disk diffusion method and agar dilution method, according to the established criteria by CLSI and EUCAST. Results: The observed correlation coefficients between the inhibitions diameter zone of the and Minimum Inhibitory Concentration were under expectations impeding the disk diffusion method for determining sensitivity to nitazoxanide antimicrobial, teicoplanin and tigecycline. All 50 strains of this study were sensitive to metronidazole (MIC50 was 1 Ug/ml to MIC90 was 2 ug/ml). For the method disk diffusion, we suggest that inhibition zones >= 33mm can be interpreted as sensitive. Due to the moderate correlation, statistical significance and distribution of zones of inhibition on samples of the next found values using the strain ATTC, we suggest using the disk diffusion method for vancomycin where halos diameter >= 22mm can be considered as sensitive by the method. There was a good correlation to moxifloxacin between the two methodologies: disk diffusion and agar dilution (Pearson\'s coefficient was -0.84 , and the \"p\" value was less than 0.00001), suggesting that inhibition zones >= 18mm can be interpreted as sensitive by disk diffusion method. Nitazoxanide was the drug that showed a better performance in vitro activity (MIC50 was 0.06 ?g/ml and MIC90 0.12 ug/ml). For a drug that shows potent activity in vitro (MIC50 and MIC90 was 0.12 ug/ml), the tigecycline could be a therapeutic option in infection by Clostridium difficile, depending on further studies to evaluate their real clinical efficacy and security. Conclusion: Obtained results in this study indicate the need for further studies in vitro and clinicians to define the limits of sensitivity/resistance to teicoplanin and nitazoxanide, so there is no interpretation criteria for both disk diffusion and for agar dilution. Results of this work in vitro study confirmed the utility of metronidazole as an effective drug in the treatment of infection by Clostridium difficile. Nitazoxanide was the drug that showed better performance in vitro by dilutional method. We suggest the use of disk diffusion method: metronidazole, vancomycin and moxifloxacin. This work suggest that inhibition zones for metronidazole ( >= 33mm), moxifloxacin ( >= 18mm) and vancomycin ( >= 22mm) could be considered as sensitive by disk diffusion method. The agar dilution method is a method to be accurate, but laborious to run in the laboratory routine

No description available.

O operon groESL de C. crescentus apresenta dupla regulação. A indução deste operon por choque térmico é dependente do fator sigma de choque térmico σ32. A temperaturas fisiológicas, a expressão de groESL apresenta regulação temporal durante o ciclo celular da bactéria e o controle envolve a proteína repressora HrcA e o elemento CIRCE (controlling inverted repeat of chaperonin expression). Para estudar a atividade da proteína repressora in vitro, produzimos e purificamos de E. coli a HrcA de C. creseentus contendo uma cauda de histidinas e a ligação especifica ao elemento CIRCE foi analisada em ensaios de migração retardada em gel de poliacrilamida (EMRGP). A quantidade de DNA retardada pela ligação a HrcA aumentou significativamente na presença de GroES/GroEL, sugerindo que estas proteínas modulam a atividade de HrcA. Corroboração desta modulação foi obtida analisando fusões de transcrição da região regulatória de groESL com o gene lacZ, em células de C. crescentus produzindo diferentes quantidades de GroES/EL. HrcA contendo as substituições Pro81 AJa e Arg87Ala, aminoácidos que se localizam no domínio putativo de ligação ao DNA da proteína, mostraram ser deficientes na ligação a CIRCE, tanto in vitro como in vivo. Em adição, HrcA Ser56Ala expressa na mesma célula juntamente com a proteína selvagem produziu um fenótipo dominante-negativo, indicando que a HrcA de C. crescentus liga-se a CIRCE como um oligômero, provavelmente um dímero. As tentativas de obtenção de mutantes nulos para os genes groESL ou dnaKJ falharam, indicando que as proteínas GroES/GroEL e DnaK/DnaJ são essenciais em C. crescentus, mesmo a temperaturas normais. Foram então construídas no laboratório as linhagens mutantes condicionais SG300 e SG400 de C. crescentus, onde a expressão de groESL e de dnaKJ, respectivamente, está sob controle de um promotor induzido por xilose (PxyIX). Estas linhagens foram caracterizadas quanto á sua morfologia em condições permissivas ou restritivas, assim como quanto à capacidade de sobrevivência frente a vários tipos de estresse. As células da linhagem SG300, exauridas de GroES/GroEL, são resistentes ao choque térmico a 42°C e são capazes de adquirir alguma termotolerância. Entretanto, estas células são sensíveis aos estresses oxidativo, salino e osmótico. As células da linhagem SG400, exauridas de DnaKlJ, são sensíveis ao choque térmico, à exposição a etanol e ao congelamento, e são incapazes de adquirir termotolerância. Além disso, tanto as células exauridas de GroES/GroEL quanto as exauridas de DnaK/DnaJ apresentam problemas na sua morfologia. As células de SG300 exauridas de GroES/GroEL formam filamentos longos que possuem constrições fundas e irregulares. As células de SG400 exauridas de DnaK/DnaJ são apenas um pouco mais alongadas que as células pré-divisionais selvagens e a maioria das células não possuem septo. Estas observações indicam bloqueio da divisão celular, que deve ocorrer em diferentes estágios em cada linhagem. / In Caulobacter crescentus, the groESL operon presents a dual type of control. Heat shock induction of the operon is dependent on the heat shock sigma factor σ-32. At physiological temperatures, groESL expression is cell cycle regulated and the control involves the repressor protein HrcA and the element CIRCE (controlling inverted repeat of chaperonin ~xpression). To study the activity of HrcA in vitro, we produced and purified from E. coli a histidine-tagged version of the protein, and specific binding to the CIRCE element was analyzed in electrophoretic mobility shift assays (EMSA). The amount of retarded DNA increased significantly in the presence of GroES/GroEL, suggesting that these proteins modulate HrcA activity. Further evidence of this modulation was obtained using lacZ transcription fusions with the groESL regulatory region in C. crescentus cells producing different amounts of GroES/GroEL. The mutants proteins HrcA Pro81Ala and HrcA Arg87Ala, that contain amino acid substitutions in the putative DNA-bindíng domain of the protein, were found to be deficient in binding to CIRCE in vitro and in vivo. Furthermore, HrcA Ser56Ala expressed together with the wild type protein within the same cell, produced a dominant-negative phenotype, indicating that C. crescentus HrcA binds to CIRCE in an oligomeric form, most likely as a dimer. Attempts to obtain null mutants for groESL or dnaKJ were unsuccessful indicating the importance of GroES/GroEL and DnaK/lDnaJ to the survival of C. crescentus cells. Conditional mutants were then constructed in our laboratory in which groESL and dnaKJ expression is under the control ofaxylose inducible promoter (PxyIX) , giving rise to strains SG300 and SG400, respectively. These strains were characterized in regard to their morphology under permissive and restrictive conditions, as well as their viability under different types of environmental stresses. SG300 cells depleted of GroES/GroEL are resistant to heat shock at 42°C and can acquire some thermotolerance, but they are sensitive to oxidative, saline and osmotic stresses. SG400 cells depleted of DnaKlJ are quite sensitive to heat shock, ethanol and freezing, and are unable of acquiring thermotolerance. Cells depleted of either GroES/EL or DnaKlJ also present morphological problems. SG300 cells depleted of GroES/EL form long and pinched filaments. SG400 cells depleted of DnaKlJ are only somewhat more elongated than wild-type predivisional cells and most cells do not present septum. These observations indicate a cell division arrest, which should occur at different stages in each strain.

Les systèmes d’efflux tripartite de type Resistance, Nodulation and cell-Division (RND) sont essentiels dans le maintien de phénotypes de résistance multidrogues et contre les métaux lourds dans nombreuses bactéries Gram-négatives. Le transport de ces composés toxiques hors de la cellule est permis par l’assemblage d’une protéine de type antiporteur cation/proton (unité RND) insérée dans la membrane interne, connectée à une protéine insérée dans la membrane externe, pour former un canal de sorti qui traverse l’entièreté de l’enveloppe cellulaire. Le troisième composant du système, la protéine de type membrane fusion protein (MFP) qui est aussi appelée periplasmic adaptor protein (PAP), est requis pour permettre l’assemblage de tout ce complexe à trois composants. Cependant, les MFPs sont supposées jouer un rôle important et actif dans le mécanisme d’efflux du substrat. Pour mieux comprendre le rôle des MFPs au sein des systèmes d’efflux de type RND, nous avons étudié les protéines ZneB (précédemment appelée HmxB) et SilB, les composants périplasmiques des systèmes ZneCBA et SilABC responsables de la résistance aux métaux lourds chez Cupriavidus metallidurans CH34. Nous avons identifié la spécificité de liaison au substrat de ces protéines, montrant leur capacité à fixer le zinc (ZneB), ou le cuivre et l’argent (SilB). De plus, nous avons résolu la structure cristalline de ZneB à une résolution de 2.8 Å dans la forme apo- et avec un ion zinc fixé. La structure de ZneB possède une architecture générale composée de quatre domaines caractéristiques des MFPs, et la présence du site de coordination au zinc dans une région très flexible à l’interface des domaines β-barrel et membrane proximal. Les modifications structurales que la protéine subit lors de la fixation du zinc on été observée dans le cristal mais aussi en solution, ce qui suggère un rôle actif des MFPs dans le mécanisme d’efflux des métaux, vraisemblablement via la fixation et le relargage de l’ion à l’antiporteur. Les études de sélectivité de transport des antiporteurs ZneA et SilA montre que ces dernières et leurs protéines périplasmiques respectives ont des affinités similaires pour les métaux lourds. De plus, les études de transport ont apportés des arguments en faveur de l’hypothèse de capture cytoplasmique du substrat par l’antiporteur, tandis que la capacité des protéines périplasmiques à fixer les métaux lourds a apporté des arguments en faveur de l’hypothèse de capture périplasmique du substrat par l’antiporteur. Les deux modes de capture pourraient en réalité coexister ;cependant, le débat autour du compartiment cellulaire de capture du substrat par l’antiporteur est complexe et requiert de plus amples efforts afin d’être cerné. / Tripartite resistance nodulation cell division (RND)-based efflux complexes are paramount for multidrug and heavy metal resistance in numerous Gram-negative bacteria. The transport of these toxic compounds out of the cell is driven by the inner membrane proton/substrate antiporter (RND protein) connected to an outer membrane protein to form an exit duct that spans the entire cell envelope. The third component, a membrane fusion protein (MFP) also called periplasmic adaptor protein, is required for the assembly of this complex. However, MFPs are also proposed to play an important active role in substrate efflux. To better understand the role of MFPs in RND-driven efflux systems, we studied ZneB (formerly HmxB) and SilB, the MFP components of the ZneCAB and SilABC heavy metal RND-driven efflux complexes from Cupriavidus metallidurans CH34. We have identified the substrate binding specificity of the proteins, showing their ability to selectively bind zinc (ZneB), or copper and silver cations (SilB). Moreover, we have solved the crystal structure of the apo- and the metal-bound forms of ZneB to 2.8 Å resolution. The structure of ZneB displays a general architecture composed of four domains characteristic of MFPs, and it reveals the metal coordination site at the very flexible interface between the β-barrel and the membrane proximal domains. Structural modifications of the protein upon zinc binding were observed in both the crystal structure and in solution, suggesting an active role of MFPs in substrate efflux possibly through binding and release. The selectivity assays of the antiporter proteins ZneA and SilA demonstrated similar specificities in relation to their cognate MFPs toward heavy metal cations. Moreover, antiporter transport assays provide evidence for cytoplasmic substrate capture by this protein, whereas MFP substrate binding provides evidence for periplasmic substrate capture. Therefore, both modes of capture might co-exist; nevertheless, the substrate capture issue is a complex topic still needing consequent efforts to understand it. / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished