Actinobacillus pleuropneumoniae est l’agent étiologique de la pleuropneumonie porcine. La bactérie se transmet par voies aériennes et contacts directs. Plusieurs facteurs de virulence ont été identifiés, nommément les polysaccharides capsulaires, les lipopolysaccharide, les exotoxines ApxI à IV et de nombreux mécanismes d’acquisition du fer. Aucun vaccin efficace contre tous les sérotypes de la bactérie n’a encore été élaboré. Afin de mieux comprendre de quelle façon A. pleuropneumoniae régule la transcription de ses nombreux facteurs de virulence et de découvrir de nouvelles cibles potentielles pour l’élaboration de vaccins efficaces, le profil transcriptomique de la bactérie a été étudié dans des conditions simulant l’infection ainsi qu’à la suite d’une infection naturelle aiguë chez l’animal. Des biopuces de première et de seconde génération (AppChip1 et AppChip2) comportant respectivement 2025 cadres de lecture ouverts (ORF) de la version préliminaire du génome d’A. pleuropneumoniae sérotype 5b souche L20 et 2033 ORF de la version finale annotée du même génome ont été utilisées. Dans un premier temps, des expériences réalisées dans des conditions de concentration restreinte en fer ont permis d’identifier 210 gènes différentiellement exprimés, dont 92 étaient surexprimés. Plusieurs nouveaux mécanismes d’acquisition du fer ont pu être identifiés, incluant un système homologue au système YfeABCD de Yersinia pestis, impliqué dans l’acquisition du fer chélaté, ainsi que des gènes homologues aux composantes du système HmbR de Neisseria meningitidis impliqué dans l’acquisition du fer à partir de l’hémoglobine. Dans des conditions de culture permettant la formation de biofilms, les gènes tadC et tadD d’un opéron tad (« tight adherence locus ») putatif, les gènes pgaBC impliqués dans la synthèse d’un polysaccharide de la matrice du biofilm ainsi que deux gènes présentant de fortes homologies avec un gène codant pour l’adhésine auto-transporteur Hsf retrouvée chez Haemophilus influenzae ont montré une surexpression significative. Plusieurs de ces gènes ont également été retrouvés lors d’expériences réalisées avec des cellules épithéliales d’origine pulmonaire en culture, qui ont permis d’identifier 170 gènes différentiellement exprimés après la croissance planctonique au-dessus des cellules, et 131 autres suite à l’adhésion à ces cellules. Parmis les gènes surexprimés, les gènes tadB et rcpA de l’opéron tad putatif, les gènes pgaBC ainsi que le gène codant pour l’homologue d’Hsf ont été retrouvés. En présence de liquide de lavage broncho-alvéolaire (BALF), 156 gènes ont montré un profil d’expression modifié, et le gène apxIVA, identifié comme étant surexprimé, a pu être détecté pour la première fois dans des conditions de croissance in vitro. Finalement, des expériences visant à déterminer les gènes utilisés directement chez l’animal en phase aiguë de la pleuropneumonie porcine ont permis d’identifier 150 gènes qui étaient différentiellement exprimés. En plus d’identifier des gènes d’un possible opéron codant pour un fimbriae de type IV, 3 des 72 gènes surexprimés sont conservés chez tous les sérotypes d’A. pleuropneumoniae et codent pour des protéines ou lipoprotéines de surface. Nos expériences ont permis d’identifier plusieurs nouveaux facteurs de virulence potentiels chez A. pleuropneumoniae ainsi que plusieurs nouvelles cibles potentielles pour l’élaboration de vaccins efficaces contre tous les sérotypes. / Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia. Transmission of the disease occurs through direct contact or aerosols. The bacteria possess many virulence factors, namely capsular polysaccharides, lipopolysaccharides, four Apx toxins and iron acquisition mechanisms. To this day, an efficient cross-serotype vaccine has yet to be developed. In order to investigate regulation mechanisms in A. pleuropneumoniae and to identify new potential targets for the synthesis of subunit vaccines, the transcriptomic profile of the bacteria under conditions that simulate the infection and following a natural acute infection in vivo were studied. The experiences relied on first and second generation microarrays (AppChip1 and AppChip2) designed using 2025 ORFs of the draft version of the A. pleuropneumoniae serotype 5b strain L20 genome and 2033 ORFs of the final and annotated version of the same genome respectively. First, experiments were conducted under iron-restricted conditions and 210 genes were deemed differentially expressed, 92 of which were up-regulated. Some new putative iron acquisition mechanisms were identified, including genes homologous to those of the Yersinia pestis YfeABCD chelated-iron acquisition system, as well as other genes homologous to components of the HmbR iron uptake from hemoglobin system of Neisseria meningitidis. When cultured in conditions promoting biofilm production, genes tadC and tadD from a putative tad (« tight adherence locus ») operon, genes pgaABC involved in the biosynthesis of a polysaccharide of the biofilm matrix as well as two ORFs encoding a putative autotransporter adhesins similar to the Haemophilus influenzae Hsf adhesin were all significantly overexpressed. Many of these genes were also overexpressed when lung epithelial cells were infected with A. pleuropneumoniae. While 170 genes were differentially expressed after planktonic growth in the culture medium above the cells, another 131 were identified following direct adhesion to the cells. Genes tadB and rcpA of the tad locus, as well as genes pgaBC and an ORF coding for the Hsf homolog where all found among overexpressed genes. When A. pleuropneumoniae was cultured in contact with broncho-alveolar lavage fluids (BALF), 156 genes were significantly differentially expressed and gene apxIVA, which was up-regulated, was detected for the first time during in in vitro growth conditions. Finally, experiments were conducted in vivo in animals naturally infected with A. pleuropneumoniae in the late stage of the acute phase in order to identify genes that are expressed during the infection of the natural host. While 150 genes were deemed differentially expressed, genes apxIVA as well as two genes from an operon coding for a putative type IV fimbriae were up-regulated. Out of those 72 genes that were overexpressed, 3 encode proteins or lipoproteins of the outer membrane which are conserved among all serotypes of the bacteria. Overall, we were able to identify several new potential virulence factors for A. pleuropneumoniae in the course of our experiments, as well as several new potential targets for the elaboration of an efficient cross-serotype vaccine.

Les autotransporteurs monomériques, appartenant au système de sécrétion de type V, correspondent à une famille importante de facteurs de virulence bactériens. Plusieurs fonctions, souvent essentielles pour le développement d’une infection ou pour le maintien et la survie des bactéries dans l’organisme hôte, ont été décrites pour cette famille de protéines. Malgré l’importance de ces protéines, notre connaissance de leur biogenèse et de leur mécanisme d’action demeure relativement limitée. L’autotransporteur AIDA-I, retrouvé chez diverses souches d’Escherichia coli, est un autotransporter multifonctionnel typique impliqué dans l’adhésion et l’invasion cellulaire ainsi que dans la formation de biofilm et d’agrégats bactériens. Les domaines extracellulaires d’autotransporteurs monomériques sont responsables de la fonctionnalité et possèdent pratiquement tous une structure caractéristique d’hélice β. Nous avons mené une étude de mutagenèse aléatoire avec AIDA-I afin de comprendre la base de la multifonctionnalité de cette protéine. Par cette approche, nous avons démontré que les domaines passagers de certains autotransporteurs possèdent une organisation modulaire, ce qui signifie qu’ils sont construits sous la forme de modules fonctionnels. Les domaines passagers d’autotransporteurs peuvent être clivés et relâchés dans le milieu extracellulaire. Toutefois, malgré la diversité des mécanismes de clivage existants, plusieurs protéines, telles qu’AIDA-I, sont clivées par un mécanisme qui demeure inconnu. En effectuant une renaturation in vitro d’AIDA-I, couplée avec une approche de mutagenèse dirigée, nous avons démontré que cette protéine se clive par un mécanisme autocatalytique qui implique deux acides aminés possédant un groupement carboxyle. Ces résultats ont permis la description d’un nouveau mécanisme de clivage pour la famille des autotransporteurs monomériques. Une des particularités d’AIDA-I est sa glycosylation par une heptosyltransférase spécifique nommée Aah. La glycosylation est un concept plutôt récent chez les bactéries et pour l’instant, très peu de protéines ont été décrites comme glycosylées chez E. coli. Nous avons démontré que Aah est le prototype pour une nouvelle famille de glycosyltransférases bactériennes retrouvées chez diverses espèces de protéobactéries. La glycosylation d’AIDA-I est une modification cytoplasmique et post-traductionnelle. De plus, Aah ne reconnaît pas une séquence primaire, mais plutôt un motif structural. Ces observations sont uniques chez les bactéries et permettent d’élargir nos connaissances sur la glycosylation chez les procaryotes. La glycosylation par Aah est essentielle pour la conformation d’AIDA-I et par conséquent pour sa capacité de permettre l’adhésion. Puisque plusieurs homologues d’Aah sont retrouvés à proximité d’autotransporteurs monomériques putatifs, cette famille de glycosyltranférases pourrait être importante, sinon essentielle, pour la biogenèse et/ou la fonction de nombreux autotransporteurs. En conclusion, les résultats présentés dans cette thèse apportent de nouvelles informations et permettent une meilleure compréhension de la biogenèse d’une des plus importantes familles de protéines sécrétées chez les bactéries Gram négatif. / Monomeric autotransporters, a family of proteins that use the type V secretion pathway, are important mediators of virulence for many bacterial pathogens. Many functions important for host colonization and survival have been described for these proteins. Despite the recognized importance of this family of proteins, the mechanisms that are required for the biogenesis and functionality of monomeric autotransporters still remain poorly understood. The Escherichia coli adhesin involved in diffuse adherence (AIDA-I) is a classical multifunctional autotransporter protein that mediates bacterial aggregation and biofilm formation, as well as adhesion and invasion of cultured epithelial cells. Extracellular domains of autotransporters are responsible for the protein function and fold into a characteristic β-helical structure. We performed a random mutagenesis of the AIDA-I passenger domain in order to identify regions involved in the various phenotypes associated with the expression of this protein. Our study suggests that the passenger domain of AIDA-I possesses a modular organization, which means that AIDA-I is built with individual functional modules. Autotransporter passenger domains can be cleaved from the β-domain and released into the extracellular milieu. However, despite the fact that diverse cleavage mechanisms have been previously described, many autotransporters, like AIDA-I, are cleaved by an unknown mechanism. By monitoring the in vitro refolding and cleavage following by site-directed mutagenesis, we showed that AIDA-I processing is an autocatalytic event that involves two acidic residues. Our results unveil a new mechanism of auto-processing in the autotransporter family. AIDA-I is one of the few glycosylated proteins found in Escherichia coli. Glycosylation is mediated by a specific heptosyltransferase encoded by the aah gene, but little is known about the role of this modification and the mechanism involved. Our findings suggest that Aah represents the prototype of a new large family of bacterial protein O-glycosyltransferases that modify various substrates recognized through a structural motif. Furthermore, we showed that glycosylation occurs in the cytoplasm by a cotranslational mechanism. These observations are unique in bacteria and represent a significant advance in our comprehension of prokaryotic glycosylation. We also showed that glycosylation is required to ensure a normal conformation of AIDA-I and, as a consequence, is necessary for its cell-binding function. The finding that other autotransporters or large adhesin-encoding genes are linked to Aah homologue-encoding genes suggests that glycosylation may be important, if not essential, for the function of these proteins, as for AIDA-I. In conclusion, the results presented in this thesis bring new information about the autotransporter family and also give new insight into the mechanisms that are important for different aspects of the biogenesis of monomeric autotransporters.

Les thiols à faible masse moléculaire (FMM) peuvent affecter la biodisponibilité du mercure et mener à une plus grande production de méthylmercure par les microorganismes méthylants. Les résultats d’une étude réalisée au sein de la matrice extracellulaire du périphyton de la zone littorale d’un lac des Laurentides ont démontré que les concentrations en thiols à FMM retrouvées dans cette matrice sont jusqu’à 1000 fois supérieures à celles de la colonne d’eau avoisinante. Ces thiols sont significativement corrélés à la chlorophylle a du périphyton, suggérant une production par les algues. Le mercure de la matrice extracellulaire, plus spécifiquement dans la fraction colloïdale mobile, est aussi corrélé aux thiols d’origine algale. Ces résultats suggèrent qu’une accumulation de thiols s’opère dans l’espace extracellulaire du périphyton et qu’ils peuvent favoriser le transfert du mercure vers les microorganismes produisant le méthylmercure. Les résultats d’une seconde étude menée sur les méthodes de préservation d’échantillons d’eau pour la conservation des thiols à FMM ont démontré que la température optimale d’entreposage était de -80 °C et que la lyophilisation menait à une sous-estimation des concentrations mesurées. Les taux de dégradation étaient plus rapides lors de la conservation à -20 °C et variables selon la chimie de l’eau utilisée et les espèces de thiols observées. Suivant ces résultats, nous proposons un cadre de recherche pour de futures études sur la spéciation du mercure dans le périphyton et nous suggérons des précautions lors de la manipulation et de la conservation des thiols à FMM d’échantillons naturels. / Low molecular weight (LMW) thiols can affect the bioavailability of mercury and by enhancing methylmercury production by methylating microorganisms. Results of a study conducted within periphyton extracellular matrix in the littoral zone of a lake of the Laurentians have shown that LMW thiol concentrations found in this matrix are up to 1000 fold higher than those in the water column of surrounding waters. Relationships between these thiols and periphyton chlorophyll a were significant, suggesting production by algae. Mercury in the extracellular matrix, especially in the mobile colloidal fraction, was also correlated to thiols from algal sources. These results suggest thiol accumulation in the periphyton extracellular space, and that thiols likely promote mercury transfer to methylating microorganisms. A second study on preservation methods of natural water samples for LMW thiols conservation showed that a storage temperature of -80 °C was optimal and that freeze-drying led to an under-estimation of measured concentrations. Degradation rates were faster with storage at -20 °C and varied substantially depending on the chemistry of the water used as depending on thiol species observed. In regards of these results, we propose a research framework for future studies on mercury speciation processes in periphyton. We also suggest precautions on handling and storing natural samples for LMW thiol analyses.

Kulturunabhängige 16S rRNA Analyse des subgingivalen bakteriellen Biofilms bei der aggressiven Parodontitis und Vergleich mit bekannten Bakterien bzw. Phylotypen, die im Zusammenhang mit der parodontalen Flora nachgewiesen wurde. Putative Pathogene wurden bestimmt. / In this culture independent 16S rRNA study cloning and sequencing was used to analyse gingival samples from a population of 26 persons suffering from aggressive periodontitis and six healthy adult individuals.

Univerzita Karlova v Praze Přírodovědecká fakulta Studijní program: Vývojová biologie (P1520) Studijní obor: Vývojová biologie (1501V000) Oleksandr Chernyavskiy Zdokonalené metody pro snímání obrazových dat a analýzu tkání a buněk pomocí konfokální a multifotonové mikroskopie Improved Methods of Image Acquisition and Analysis of Tissues and Cells by Confocal and Multi-Photon Microscopy Abstrakt disertační práce Školitel: RNDr. Lucie Kubínová CSc Praha, 2015 Abstract The aim of this study was to develop methods and approaches for image acquisition with subsequent image analysis of data, obtained by confocal and two- photon excitation microscopy as well as their combination, enabling new possibilities of visualization and assessment of information on biological tissues and cell structures in 3D and their measurement. We focused on methods that exploited advantages of confocal and multi-photon excitation microscopy. Our further aim was to demonstrate the applicability of non-invasive approach for in vivo applications, usefulness and the relevance of these methods in several special biological applications with emphasis on improved image acquisition, analysis and evaluation of real biological specimens. The present work was not oriented on just one specific biological problem, but rather to methodological...

O gênero Methylobacterium é composto por bactérias de coloração rósea, metilotróficas facultativas (PPFM - pink-pigmented facultative methylotrophic), que podem fixar nitrogênio, nodular a planta hospedeira, produzir o fitohormônio citocinina e as enzimas pectinase e celulase, podendo dessa forma promover o crescimento vegetal devido à disponibilidade de nitrogênio e à indução de resistência sistêmica. Methylobacterium spp. têm sido descritas como endófitos ou epífitas em diferentes plantas hospedeiras, onde a sua colonização e distribuição no hospedeiro podem ser influenciadas pelo genótipo da planta ou por interações com outros microrganismos associados ao hospedeiro. Neste contexto, poucos trabalhos têm sido desenvolvidos visando um melhor entendimento da interação Methylobacterium-planta e da diversidade deste gênero bacteriano que tem sido isolado de diferentes plantas hospedeiras, exercendo diferentes funções ainda pouco conhecidas. Portanto, este trabalho tem como objetivo estudar a diversidade genética de Methylobacterium spp., por meio do seqüenciamento parcial dos genes 16S rRNA e mxaF; analisar os genes de responsáveis pela interação da Methylobacterium com a planta hospedeira e analisar os genes envolvidos na interação Methylobacterium (endófito)- Xylella fastidiosa (patógeno). Os resultados mostraram que existe uma resposta adaptativa de Methylobacterium spp. específica para cada planta hospedeira. Da mesma forma, foi observado que esta adaptação específica da bactéria à planta, também pode levar à seleção de genótipos específicos para cada planta hospedeira, embora eventos aleatórios também possam ser responsáveis pela diversidade de Methylobacterium na planta hospedeira. Na análise de expressão gênica da interação Methylobacterium-planta, foi observado que o gene relacionado ao metabolismo do metanol (mxaF) não apresentou mudança no padrão de expressão. Genes relacionados a estresse crtI (estresse sentido pela bactéria) e acdS (estresse sentido pela planta), tiveram suas expressões reduzidas na presença da planta, mostrando que a presença de exsudados das plantas não representou um estresse ao desenvolvimento bacteriano. Os genes relacionados à patogenicidade patatin e phoU não foram alterados, confirmando que Methylobacterium é um endófito, e possuem expressão induzida de tais genes quando interagindo com a planta hospedeira. Os resultados permitem concluir que nas condições avaliadas os exsudados das plantas não causam estresse à bactéria (SR1.6/6). Por meio da análise de expressão gênica in vitro de X. fastidiosa em co-cultivo com M. mesophilicum, foi observado que este fitopatógeno vascular apresentou diminuição do crescimento e da formação de biofilme. Os resultados aqui apresentados mostram que a diversidade deste grupo de endófitos é parcialmente determinada pela planta hospedeira, onde tais bactérias interagem tanto com a planta como com outros grupos, como fitopatógenos presentes neste nicho. / The genus Methylobacterium, constituted by PPFMs - pink-pigmented facultative methylotrophic, are able to fix nitrogen, nodule the host plant, produce cytokines and enzymes involved in induction of systemic resistance such as pectinase and cellulase, inducing plant growth. Methylobacterium sp. has been described as endophyte or epiphyte in different host plants, where the colonization and distribution on the host can be influenced by plant genotype or by interaction with other microorganisms associated to the host. In this context, few studies aims the better understanding of the diversity of this genus in different host, the interaction Methylobacterium-plant, and the interaction Methylobacterium-other bacteria. Therefore, this study aims to study the genetic diversity of Methylobacterium spp., by sequencing the 16S rRNA and mxaF gene; to analyze the genes responsible for the Methylobacterium-plant host interaction and to analyze the genes involved in Methylobacterium (endophyte) - Xylella fastidiosa (pathogen) interaction. Results show differential adaptive responses of Methylobacterium spp. in distinct plant species. However, the clustering according to the host plant was observed for a subset of isolates, suggesting that this diversity could be driven by stochastic events, although plant genotype may contribute to this diversity. Analyzing the Methylobacterium-plant interaction gene expression it was observed that genes related to metabolism of methanol (mxaF) was not amended. The genes related to stress such as crtI (stress sensed by the bacteria) and acdS (stress sensed by the plant) had its expression reduced with the plant showing that the plant exudates did not represent a stress to the bacteria development. The genes related to pathogenicity like patatin and phoU were not amended, confirming that Methylobacterium is an endophyte that do not induce when the bacteria interacts with the plant host. Using a genetic expression analyses of X. fastidiosa in vitro in co-cultive with M. mesophilicum, it was seen that this phytopathogen presented the growth and biofilm formation reduction. These results show that the diversity of this endophyte group is partially determinate by the plant host, where this bacterium interacts with the plant and with other groups, such as phytopathogen present in this niche.

Methylobacterium mesophilicum SR1.6/6 é uma bactéria endofítica isolada de ramo de citros previamente esterilizado superficialmente. Esta bactéria possui a capacidade de associar-se com uma ampla variedade de espécies e tecidos de plantas, principalmente nas raízes, mediado pela formação de biofilme e superfícies do hipocótilo de plântulas in vitro. Este trabalho teve como objetivo o desenvolvimento e a aplicação de um modelo para o estudo in vitro da associação entre esta bactéria e plântulas de soja. Metodologias de genômica e transcriptômica foram aplicadas para a obtenção do draft genômico desta bactéria e de um amplo perfil de sequências expressas, obtidas em dois tratamentos distintos; i) células de biofilme células bacterianas aderidas às raízes das plântulas removidas por sonicação, e ii) células planctônicas células bacterianas em suspensão (i.e. interagindo somente com exsudados radiculares). Os dados genômicos obtidos por 454-pirosequenciamento resultaram em uma cobertura de 37 vezes o tamanho do genoma e geraram 242 contigs. Entre estes, 187 contigs grandes representaram 96% do genoma (tamanho estimado de 6.8 Mb), com um conteúdo GC de 69.5%. Considerando a análise da expressão gênica, um procedimento para monitorar a aderência das células bacterianas às raízes das plântulas foi realizado por meio de microscopia eletrônica de varredura de amostras de raízes coletadas durante o experimento (i.e. 24, 48 e 72h após a inoculação). As células bacterianas obtidas em cada tratamento foram inicialmente submetidas à extração de RNA total, seguida de um processo de enriquecimento de mRNA e sequenciamento por meio da tecnologia de 454-pirosequenciamento (RNA-Seq). O amplo perfil de expressão gênica obtido foi mapeado no draft genômico, resultando em um total de 1.930 clusters gênicos. Posteriormente, estes clusters foram filtrados de acordo com sua abundância e ocorrência diferencial em cada tratamento, resultando em 280 genes diferencialmente expressos. Funções relacionadas ao metabolismo de etanol/metanol, divisão celular, resposta ao estresse oxidativo, produção de sideróforos, biossíntese de peptidoglicanos e hopanóides foram induzidas nas células bacterianas aderidas às raízes das plântulas, enquanto que genes relacionados ao metabolismo essencial das células foram observados principalmente nos tratamentos controle e planctônico. Estes dados fornecem uma base para estudos relacionados a mecanismos moduladores da interação bactériaplanta, distinguindo significativamente os tratamentos biofilme e planctônico, mostrando assim que o contato físico é essencial para o sucesso da interação em estudo. Por fim, estas análises permitiram uma ampla visualização de perfis de expressão gênica desta bactéria, utilizando o draft genômico primeiramente obtido como base para o estudo desta interação com a planta hospedeira. Estudos futuros podem ser desenvolvidos visando caracterizar os mecanismos adaptativos desta bactéria, como seu metabolismo metilotrófico e outros metabolismos específicos, os quais podem dar suporte ao comportamento endofítico deste organismo. / Methylobacterium mesophilicum strain SR1.6/6 is an endophytic bacterium, which has originally been isolated from surface-sterilized healthy citrus branch. This bacterium is able to associate with a range of plant species, rather in the roots, mediated by a biofilm structure, and in hypocotyl surfaces of in vitro seedlings. The aim of the present study was the development and application of a model to study in vitro the association between this bacterium and soybean seedlings. Genomic and transcriptomic approaches were applied resulting a draft of this bacterium genome and a broad profile of mapped mRNA sequences obtained in two different treatments; i) biofilm cells root adhered bacterial cells were removed by sonication, and ii) planktonic cells bacteria cells in suspension (i.e. interacting only with root exudates). Genomic data, obtained by 454-pyrosequencing have had an average depth of 37-fold coverage of the genome and yielded 242 contigs. Among these, 187 large contigs represented 96% of the genome sequence (estimate size of 6.8 Mb) with a GC content of 69.5%. Concerning the gene expression survey, the process to monitor the adherence of bacteria cells to the roots was performed by scanning electron microscopy of roots collected along the experiment (i.e. 24, 48 and 72 hours after inoculation). Bacterial cells obtained in each treatment were firstly submitted to RNA extraction, followed by mRNA enrichment and RNA-Seq using 454-pyrosequencing technology. The broad gene expression profile obtained was mapped into the drafted genome, resulting in a total of 1.930 gene clusters. After that, these clusters were filtered according to their abundance and differential occurrence in each treatment resulting in 280 differential expressed genes. Functions related to methanol/etanol metabolism, cell division, oxidative stress response, siderophore production, peptidoglycan and hopanoid biosynthesis were induced in bacterial cells adhered to plant roots, while genes related to essential cell metabolism were observed mostly in control and planktonic treatment. Also, these data provide insights into the mechanisms modulating plant microbe-interaction, significantly distinguishing biofilm and planktonic treatment, showing that the physical contact is a crucial step on plant-microbe interactions. In conclusion, results allowed a strongly supported analysis of gene expression, based on the genome draft of an endophytic bacterium interacting with the host plant. Further studies should focus on the adaptive mechanisms present in this bacterium, like the methylotrophic lifestyle and other specific metabolisms which might support its behavior as an endophytic bacterium.

A inibição do quorum sensing (QS) altera a comunicação bacteriana, reduzindo a expressão de fatores de virulência e a formação de biofilmes, o que pode conferir menor pressão seletiva em comparação aos antibióticos tradicionais. As frutas e hortaliças constituem uma fonte rica em compostos com propriedades potenciais de inibição do QS. Entretanto, há pouca referência sobre o potencial de pimentas do gênero Capsicum e de seus compostos isolados como inibidores do QS. Esse trabalho teve como objetivo avaliar o efeito de extratos orgânicos obtidos das variedades de pimenta-malagueta e pimentão vermelho sobre o sistema QS dependente do sinalizador AI-1 (acil homoserina lactona - AHL) em bactérias Gram-negativas. Os extratos foram obtidos por extração em fase sólida e separados em uma fração metanólica e outra amônica; sendo os compostos característicos identificados e quantificados por cromatografia líquida de alta eficiência (CLAE). A atividade antimicrobiana dos extratos foi avaliada pela determinação da concentração inibitória mínima (MIC) e pela curva de crescimento de Chromobacterium violaceum ATCC 12472, Serratia liquefaciens MG1 e Pseudomonas aeruginosa PAO1. O efeito anti-QS dos extratos foi avaliado pelos testes de difusão em ágar e quantificação da produção de violaceína em meio líquido por C. violaceum e sobre a formação de biofilme, avaliado pelo ensaio de cristal violeta e microscopia confocal, em S. liquefaciens e P. aeruginosa nas temperaturas 30 ºC e 37 ºC. Os resultados obtidos pela CLAE indicaram que o extrato metanólico de pimenta-malagueta (EMPM) continha capsaicinoides como a capsaicina e dihidrocapsaicina, luteolina e outros compostos não identificados; já o extrato amônico desta não continha os compostos capsaicinoides. Ambos os extratos de pimentão vermelho continham luteolina e compostos não identificados, mas não apresentaram capsaicinoides. Como o EMPM era representativo dos demais extratos, por conter tanto capsaicinóides quanto luteolina, o foco deste trabalho foi avaliar os efeitos do EMPM sobre fenótipos microbianos nas concentrações 5; 2,5; 1,25 e 0,625 mg/ml, além de utilizar a capsaicina como controle comparativo em concentrações equivalentes às do extrato (25, 50 e 100 µg/ml). Os resultados da atividade antimicrobiana mostraram inibição parcial do crescimento das bactérias nas concentrações sub-MIC (MIC >5 mg/ml) de 5 e 2,5 mg/ml de EMPM. A capsaicina também inibiu parcialmente o crescimento das bactérias a 100 µg/ml, com exceção de S. liquefaciens a 37 ºC, cujo crescimento foi induzido em 50 e 25 µg/ml. A produção de violaceína foi reduzida pelo EMPM a 1,25 e 0,625 mg/ml, sem afetar o crescimento de C. violaceum. Ensaios com C. violaceum CV026, estirpe biosensora capaz de produzir o pigmento na presença de AI-1 exógeno, sugerem que o possível mecanismo de atuação do extrato sobre o sistema QS em C. violaceum 12472 é sobre a síntese do sinalizador, já que não foi observada inibição da produção de violaceína em CV026 pelo extrato. Contrariamente, a capsaicina incrementou a produção do pigmento na estirpe 12472, mas ensaios com a estirpe CV026 indicaram que a capsaicina não atua como sinalizador do QS, uma vez que esta não induziu a produção de violaceína nesta estirpe. Já a formação de biofilme foi incrementada na presença do EMPM, sendo consideravelmente maior em P. aeruginosa a 30 ºC. Igualmente, observou-se indução da formação de biofilme por capsaicina em S. liquefaciens (37 ºC) e P. aeruginosa (30 ºC). Porém, a capsaicina não teve efeito sobre a formação de biofilme de S. liquefaciens quando cultivada a 30 ºC, nem P. aeruginosa a 37 ºC. Os resultados revelam que a produção de violaceína em C. violaceum ATCC 12472 é inibida pelo EMPM, mas não pela capsaicina. Já, o EMPM e a capsaicina, de forma geral, não inibem a formação de biofilme de S. liquefaciens MG1 nem P. aeruginosa PAO1. Outros estudos são necessários para elucidar os mecanismos pelos quais o EMPM e a capsaicina agem sobre os fenótipos avaliados neste trabalho. / Quorum sensing inhibition alters bacterial communication by reducing virulence factors expression and biofilm formation, exerting less selective pressure compared to antibiotics. Fruits and vegetables are rich sources of compounds with potential QS-inhibition properties. However, there are few references about the potential of peppers belonging to the genus Capsicum and its isolated compounds as QS inhibitors. This study aimed to assess the effect of organic extracts obtained from Capsicum varieties, pimenta-malagueta (red chili) and pimentão vermelho (red bell pepper), on the AI-1 dependent QS system. The extracts were obtained by solid phase extraction and split into a methanolic and an ammonic fraction. Characteristic compounds were identified and quantified by high performance liquid chromatography (HPLC). The antimicrobial activity of the extracts was assessed by determining the minimal inhibitory concentration (MIC) and the growth curve of Chromobacterium violaceum ATCC 12472, Serratia liquefaciens MG1 and Pseudomonas aeruginosa PAO1. The anti-QS effect of the extracts was evaluated by the agar diffusion assay and the quantification of violacein production was assessed in liquid medium by C. violaceum, as well as in the biofilm formation test determined by the crystal violet assay and confocal microscopy with S. liquefaciens and P. aeruginosa at 30 ºC and 37 ºC. HPLC results showed that the methanolic extract of pimenta-malagueta (EMPM) contained capsaicinoids such as capsaicin and dihidrocapsaicin, luteolin and other unidentified compounds in lower concentrations; while its ammonic extract did not have capsaicinoids. Both pimentão vermelho extracts contained luteolin and other unidentified compounds in low concentrations, but they did not contain capsaicinoids. As EMPM was representative among the extracts because it contained capsaicinoids and luteolin, the focus of this work was to assess the effect of EMPM over microbial phenotypes at concentrations of 5, 2.5, 1.25 and 0.625 mg/ml, using capsaicin as a comparative control at equivalent concentrations to those in EMPM (25, 50 and 100 µg/ml). Antimicrobial activity assays showed a partial inhibition growth of bacteria at sub-MIC concentrations (MIC >5 mg/ml) of EMPM at 5 and 2.5 mg/ml. Similarly, capsaicin partially inhibited bacterial growth at 100 µg/ml, except for S. liquefaciens at 37 ºC in which growth was induced at 50 and 25 µg/ml. Violacein production was reduced by EMPM at 1,25 and 0,625 mg/ml without affecting C. violaceum growth. Assays with C. violaceum CV026, a biosensor strain that produces violacein in the presence of exogenous AI-1, suggest that EMPM reduced violacein production in C. violaceum 12472 by interfering with the AI-1 synthesis. In contrast, capsaicin incremented violacein synthesis in strain 12472, but experiments with strain CV026 revealed that capsaicin does not function as an analog of AI-1. Biofilm formation was increased in EMPM presence, being remarkably superior in P. aeruginosa cultivated at 30 ºC, as opposed to cultivation at 37 ºC. Similarly, capsaicin induced biofilm formation in S. liquefaciens (37 ºC) and P. aeruginosa (30 ºC). However, capsaicin did not affect biofilm formation on S. liquefaciens cultured at 30 ºC, neither on P. aeruginosa at 37 ºC. These results show that violacein production in C. violaceum ATCC 12472 is inhibited by EMPM, but not by capsaicin. In general, EMPM and capsaicin did not inhibit biofilm formation in S. liquefaciens MG1 neither in P. aeruginosa PAO1. More studies are necessary to elucidate the mechanisms by which EMPM and capsaicin affect the studied phenotypes in this work.

Resistência hidráulica é altamente prevalente em cateteres venosos centrais de longa permanência. Selos de heparina são comumente utilizados para manter evitar o problema, mas mostram pouca efetividade. O objetivo do presente estudo foi avaliar a efetividade e segurança de dois selos quanto à redução de ocorrência de incidência de baixo fluxo. Setenta e cinco pacientes com doença renal crônica no estágio 5D em hemodiálise de alta eficiência no Centro Integrado de Nefrologia (Guarulhos, Brasil) foram randomizados na proporção de 1:1:1 para receber selo contendo a combinação de minociclina 3 mg/ml com o anticoagulante/quelante EDTA 30 mg/ml (M-EDTA), heparina 1.000 U/mL (Heparina) ou citrato trissódico 30% (Citrato) por 15 semanas. Sessenta e oito pacrticipantes terminaram o estudo no qual ambos participantes e investigadores foram cegos quanto à alocação do tratamento. O desfecho primátio de efetividade foi ocorrência de resistência hidráulica e o desfecho secundário de segurança foi ocorrência de reações adversas a selos. A incidência de resistência hidráulica foi significantemente maior em cateteres selados com Heparina (18/23) comparado com os selos Citrato (4/22) e M-EDTA (2/23) (p< 0.001). O estudo clínico sugere que o Citrato e M-EDTA mantem a patência de cateteres melhor que a heparina 1.000 UI/ml, sendo altamente custo-efetivos em comparação com heparina 1.000 UI/ml. Ainda, o Citrato pode ser a melhor opção de selo por não se associar com resistência antimicrobiana a longo prazo. / Hydraulic Resistance is highly prevalent among long-term central venous catheters. Heparin catheter lock solutions are commonly used to maintain catheter patency, however this problem remains high. The purpose of the CLOCK Trial was to evaluate two catheter lock solutions as to their effectiveness and safety. Seventy-five CKD 5D patients on high-efficiency hemodialysis at the Integrated Centre of Nephrology (Guarulhos, Brazil) were randomized 1:1:1 to receive a lock solution combining minocycline 3 mg/ml with the anticoagulant/chelation agent EDTA 30 mg/ml (M-EDTA) or heparin 1,000 IU/ml (Heparin) or trisodium citrate 30% (Citrate) versus Heparin for 15 weeks. A total of 68 patients completed the trial in which both investigators and patients were blinded to treatment allocation. The primary end-point was the occurrence of hydraulic resistance and secondary safety end-point was adverse drug reactions related to the lock solutions. The incidence of hydraulic resistance was significantly higher among patients on Heparin (18/23) compared to Citrate (4/22) and M-EDTA (2/23) lock solutions, (p< 0.001). The trial suggests Citrate and M-EDTA may preserve catheter patency better than Heparin and they may show higher cost-effectiveness when compared to heparin. Citrate may be a better option due the lack of association with longterm antimicrobial resistance.

Apresenta-se a investigação experimental do tratamento de esgoto sanitário, submetido apenas a gradeamento, por meio de reatores aeróbios de leito trifásico com circulação em tubos concêntricos induzida por injeção de ar. Foram construídos dois reatores com 0,25m de diâmetro externo, 0,20m de diâmetro interno e alturas de 6m (R6) e 12m (R12). O meio suporte utilizado (fase sólida) foi areia com 0,27mm de diâmetro médio. Os tempos de detenção hidráulica investigados variaram entre 1,0h e 8,0h, com concentração de sólidos de 50g/l e 100g/l. Nas etapas iniciais do trabalho houve a necessidade de vazões de ar da ordem de 6000 l/h para manter o meio em suspensão, introduzindo uma quantidade de oxigênio superior ao necessário para o tratamento. Para diminuir a vazão de ar foi utilizado um propulsor axial no tubo de subida para auxiliar a circulação do meio trifásico, o que permitiu reduzir as vazões de ar para cerca de 3500 l/h. Os melhores resultados desta fase foram obtidos com TDH = 3h e 100g/l de areia. As remoções médias de DBO bruta, e filtrada, DQO bruta e filtrada e NTK foram de 89%, 99%, 84%, 95% e 87%, respectivamente, para o R12 e de 89%, 98%, 82%, 95% e 86%, respectivamente, para o R6, sem diferenças estatisticamente significativas entre os reatores. As remoções médias de fósforo total para os reatores R12 e R6 com TDH = 3h foram 31% e 32%.Entretanto, foi produzida uma parcela de sólidos em suspensão de baixa sedimentabilidade, que prejudicava a qualidade do efluente final. As soluções investigadas para este problema foram a utilização de uma câmara de flotação no R12 e a diminuição do diâmetro do tubo de subida, implantada no R6. Ambos os reatores foram ensaiados com TDH = 3h e 100g/l de areia. O sistema de flotação foi avaliado com taxas de recirculação de 15% e 20%. O efluente final resultante ainda continha uma parcela indesejável de biomassa suspensa. A remoção média de fósforo total durante essa etapa foi de 42 %. De forma geral não houve melhorias estatisticamente significativas na operação do flotador na remoção de fósforo e nos demais parâmetros avaliados. Entretanto ensaios em laboratório mostraram que com a adição de 75 mg/l de Cloreto Férrico a remoção de fósforo total aumentou para 87 %. A redução do diâmetro do tubo interno no R6 permitiu diminuir as vazões iniciais para 1500 l/h sem problemas de suspensão do leito, sendo posteriormente aumentado para 2100 l/h por exigência do tratamento biológico. As remoções médias de DQO e DBO brutas, NT, e Fósforo Total situaram-se em 91%, 88%, 72% e 32% respectivamente. O reator apresentou bom desempenho hidrodinâmico, melhorando os problemas de sedimentabilidade do lodo e as características físicas do efluente. A pesquisa mostra que a solução de leito trifásico com circulação é tecnicamente viável para o tratamento de esgoto sanitário submetido apenas a gradeamento. A câmara de flotação integrada mostrou-se tão eficiente quanto os decantadores, com a vantagem de permitir aumento significativo na remoção de fósforo com a adição de floculantes. Os dados demonstraram também que é possível controlar as condições hidrodinâmicas para a produção de lodo com melhor sedimentabilidade e para otimizar a necessidade de injeção de ar, atuando sobre a relação entre os diâmetros interno e externo. Os aspectos econômicos envolvidos na utilização eficiente do ar constituem a principal necessidade de pesquisa visando à adoção da tecnologia para a utilização proposta. / The experimental investigation of the sanitary sewage treatment, submitted only to screening, through three-phase bed aerobic reactors with circulation in concentric tubes induced by air injection, is presented. Two reactors had been constructed, with 0.25 m external diameter, 0.20 m internal diameter, and 6m (R6) and 12m (R12) high, respectively. The support medium (solid phase) was sand with 0.27 mm medium diameter. The hydraulic detention times investigated ranged between 1.0 h and 8.0 h, with solid concentrations of 50 g/l and 100 g/l. At the initial stages of the research, the reactors needed to work with air outflow of 6000 l/h, to maintain the support medium suspended, introducing an oxygen quantity higher than the necessary to the treatment. To reduce the air outflow, an axial propeller had been used at the ascent tube to aid the circulation, allowing to reduce the air outflows to about 3500 l/h. The best results for this stage had been obtained with HDT = 3h and 100g sand/l. The averaged removal of rough and filtered BOD, rough and filtered COD, and TKN for the R12 were 89%, 99%, 84%, 95%, and 87%, respectively. For the R6 the values were 89%, 98%, 82%, 95%, and 86%, respectively, without statistically significant differences between the R6 and R12. The averaged total phosphorus removal for R12 and R6 were 31 and 32%, with HDT = 3h. However, a low sedimentability portion of suspending solids had been produced, which prejudiced the final effluent quality. The solutions investigated to solve this problem were a flotation chamber in the R12 and the reduction of the ascent tube diameter, in the R6. Both reactors had been analyzed with HDT = 3h and 100g sand/l. The flotation system had been analyzed with recirculation rate of 15% and 20%. The final effluent still contained an undesired suspended biomass portion. The medium total phosphorus removal during this stage was 42%. Generally, there was no statistically significative improvements at the flotation tank operation in the phosphorus removal and others evaluated parameters. Laboratory assays, however, showed that adding 75 mg ferric chloride, the total phosphorus removal increased to 87%. The intern tube diameter reduction at the R6 diminished the initial outflow to 1500 l/h, without bed suspension problems. The outflow was later increased to 2100 l/h, due to the biological treatment. The medium removal of CDO, BOD, NT ant total phosphorus were 91%, 88%, 72%, and 32%, respectively. The reactor presented good hydrodynamic performance, improving the sedimentability problems of the sludge and the effluent physical characteristics. The research shows that the triphasic bed with circulation solution is technically feasible for the sanitary sewerage treatment submitted only to screening. The integrated flotation chamber was as efficient as the sedimentations tanks, with the advantage of to allow significative increase in the phosphorus removal with coagulants addition. The data also shown that it is possible to control the hydrodynamic conditions to produce sludge with better sedimentability and to optimize the air injection necessity, influencing the relation between the internal and the external diameters. The economical aspects involved in the efficient use of the air constitutes the principal research necessity, aiming the adoption of the technology to the proposed use.