Os sistemas de sinalização de dois componentes são sistemas prevalentes em bactérias, permitindo a adaptação a diferentes condições ambientais. O sistema de dois componentes classicamente possui uma proteína histidina quinase, o primeiro componente, capaz de reconhecer o estímulo ambiental e fosforilar o regulador de resposta, o segundo componente. Pseudomonas aeruginosa é uma proteobactéria ubíqua, capaz de infectar hospedeiros filogeneticamente distintos. Esse patógeno oportunista apresenta um dos maiores conjuntos de sistemas de dois componentes em bactérias, que permite que ela sobreviva numa grande gama de ambientes, incluindo humanos. P. aeruginosa UCBPP-PA14 apresenta pelo menos 64 histidina quinases e 76 reguladores de resposta codificados em seu genoma. Diversos sistemas de dois componentes já foram correlacionados com a virulência, sendo o sistema GacSA o exemplo melhor caracterizado. Há poucos estudos sistemáticos sobre o envolvimendo dos reguladores de resposta na virulência de P. aeruginosa e os sinais que induzem a ativação dos reguladores de resposta precisam ser encontrados. Para identificar novos reguladores de resposta envolvidos na patogenicidade, infecções in vitro em macrófagos e in vivo em Drosophila melanogaster foram realizadas neste trabalho. Os macrófagos foram infectados com cada mutante dos reguladores de resposta ou com a linhagem selvagem, e a produção da citocina pró-inflamatória TNF-α e o clearance bacteriano foram determinados. Alternativamente, as moscas foram infectadas utilizando-se a estratégia de feeding e a sobrevivência foi verificada. Utilizando-se essas abordagens, a identificação de diversos reguladores de resposta com papel na virulência foi alcançada, além de se corfirmar o papel de reguladores de resposta já estudados. Um dos novos genes envolvidos em virulência, PA14_26570 (nomeado neste trabalho de atvR), codifica um regulador de resposta atípico com substituição no aspartato fosforilável para glutamato, o que usualmente induz um estado sempre ativo. Um mutante não polar em atvR foi construído e macrófagos infectados com a linhagem ΔatvR confirmaram um maior clearance bacteriano e maior produção de TNF-α em comparação aos macrófagos infectados com a linhagem selvagem. Para comprovar a participação de AtvR durante a patogênese, um modelo de pneumonia aguda em camundongos foi utilizado. Camundongos infectados com a linhagem ΔatvR apresentaram uma maior sobrevivência em comparação aos camundongos infectados com a linhagem selvagem. Além disso, os camundongos infectados com ΔatvR apresentaram menor carga bacteriana, aumento no recrutamento de neutrófilos ativados e aumento na produção de citocinas pró-inflamatórias (TNF-α e IFN-γ). Utilizando-se uma abordagem transcritômica (RNA-Seq), foi determindo diversos genes são regulados positivamente na linhagem superexpressando AtvR em relação à linhagem controle. Dentre esses, os clusters de respiração anaeróbia nar, nir, nor e nos estão incluídos. Esse resultado foi confirmado por qRT-PCR e análises fenotípicas, em que a linhagem ΔatvR apresentou menor crescimento e expressão da nitrato redutase durante condições de hipóxia em comparação à linhagem selvagem. Em suma, neste trabalho foi demonstrado que diversos reguladores de resposta são importantes para a virulência de P. aeruginosa em macrófagos in vitro e in vivo em Drosophila, além de caracterizar o regulador de resposta atípico AtvR, que regula a respiração anaeróbica por desnitrificação, permitindo que P. aeruginosa possa infectar e colonizar o hospedeiro com maior eficiência. / Two-component systems are widespread in bacteria, allowing the adaptation to environmental changes. A two-component system is classically composed by a sensor kinase that phosphorylates a cognate response regulator. Pseudomonas aeruginosa is a ubiquitous proteobacterium able to cause disease in several hosts. This opportunistic pathogen presents one of the largest sets of two-component systems known in bacteria, which certainly contributes to its ability to thrive in a wide range of environmental settings, including humans. P. aeruginosa UCBPP-PA14 genome codes for at least 64 sensor kinases and 76 response regulators. Some response regulators are already known to be related to virulence, with the GacSA system as the best characterized. There are no systematic studies about the involvement of P. aeruginosa response regulators in virulence. Moreover, the input signal that triggers the response regulator activation is yet to be uncovered for most systems. To find new response regulators involved in virulence, in vitro infections werecarried out using macrophages. Briefly, the macrophages were infected with each response regulator mutant or the wild-type strain, the pro-inflammatory cytokine production (TNF-α) and the bacterial clearance were evaluated. Using this approach, we identified several response regulators involved in virulence, and we also confirmed the involvement of known response regulators in this process. One of the novel virulence-related response regulators, PA14_26570 (named here as AtvR), is an atypical response regulator with a substitution in the phosphorylable aspartate to glutamate, that usually leads to an always-on state. A non-polar mutant was constructed, and macrophage infection with ΔatvR confirmed an increased bacterial clearance as well as a higher TNF-α production as compared to the wild-type strain. To ascertain the role of AtvR during the pathogenic process, an acute pneumonia model was used. Mice infected with ΔatvR showed an increased survival as compared to mice infected with the wildtype strain. In addition, ΔatvR infected mice showed reduced bacterial burden, increased neutrophil recruitment and activation, as well as increased pro-inflammatory cytokine production (TNF-α and IFN-γ). Also, using a transcriptomic approach (RNASeq), we showed that several genes were upregulated in the strain overexpressing AtvR. These genes include the anaerobic respiration clusters nar, nir, nor and nos. This result was confirmed by qRT-PCR and phenotypic analysis, in which ΔatvR showed reduced growth and nitrate reductase expression during hypoxic conditions as compared to the wild-type strain. In conclusion, we have demonstrated that several response regulators are important for P. aeruginosa virulence in vitro. In addition, we further characterized the atypical response regulator AtvR, which regulates anaerobic respiration via denitrification, allowing this bacterium to infect and colonize the host more efficiently.

Pseudomonas aeruginosa est un pathogène opportuniste responsable du développement de maladies nosocomiales. Il provoque des infections en employant différents facteurs de virulence dont le principal, associé aux infections sévères, est le système de sécrétion de type 3 (SST3). Les souches de type PA7, taxonomiquement marginales, sont dépourvues de SST3 et leur pouvoir pathogène repose sur le nouveau système de virulence ExlBA. Ce SST5b, ou TPS, est composé du transporteur ExlB qui permet la translocation d’ExlA, une toxine induisant la perméabilisation de la membrane plasmique des cellules eucaryotes.Ce travail représente la première investigation des mécanismes de régulation du système ExlBA. Ainsi, il a été mis en évidence que la déplétion en fer est un signal d’activation de l’expression des gènes exlBA. De plus, les deux principaux messagers secondaires, AMPc et di-GMPc, sont impliqués dans la régulation du TPS. En effet, la voie CyaB-AMPc/Vfr, connue pour réguler le SST3, contrôle la toxicité des souches de type PA7 grâce à une activation transcriptionnelle directe des gènes exlBA, qui peut être stimulée par la chélation du calcium extracellulaire. Parallèlement, alors qu’ExlA était supposée être sécrétée pour agir sur les cellules eucaryotes, cette étude a montré que la toxine doit être exposée à la surface de la membrane bactérienne pour provoquer la lyse de ces cellules, ceci par un mécanisme dépendant du di-GMPc. Effectivement, une forte concentration en di-GMPc empêche la sécrétion d’ExlA en induisant de façon post-traductionnelle son maintien au niveau du transporteur ExlB, ce qui favoriserait l’action de la toxine sur les membranes eucaryotes. / Pseudomonas aeruginosa is an opportunistic pathogen responsible for nosocomial diseases. It provokes infections due to several virulence factors. Among them the most aggressive is the type 3 secretion system (T3SS), associated with severe infection. PA7-like strains, that are taxonomic outliers, lack the T3SS but are still pathogenic thanks to the novel virulence system ExlBA. This T5bSS, or TPS, is composed by the transporter ExlB that allows translocation of ExlA toxin to induce permeabilisation of eukaryotic cell membrane.This study is the first investigation of regulatory mechanisms that modulate ExlBA. It provided evidence that iron depletion is an activator signal of exlBA gene expression. Furthermore, the two main second messengers, cAMP and c-di-GMP, are involved in ExlBA regulation. CyaB-cAMP/Vfr pathway, known to regulate T3SS, controls toxicity of PA7-like strains through direct transcriptional activation of exlBA. This pathway may be stimulated by an extracellular calcium chelation. At the same time, while ExlA was supposed to be secreted to kill eukaryotic cells, this work showed that the toxin must be exposed at the surface of the bacterial membrane to cause lysis of these cells, by a mechanism dependent on c-di-GMP. Indeed, a c-di-GMP high concentration prevents ExlA secretion by inducing its maintenance at the ExlB transporter, that would promote the action of the toxin on eukaryotic membranes.

De nombreux glycoclusters multivalents des calixarènes, des pillararènes ou des fullerènes ont été synthétisés au sein de notre laboratoire et ont montré d'excellentes affinités pour diverses lectines grâce à leur multivalence et au « glycoside cluster effect ». Nous avons cherché à approfondir ces résultats en ajoutant un degré de dynamisme à ces molécules. Pour cela, nous avons appliqué les concepts de la chimie combinatoire dynamique où des briques moléculaires s'auto-assemblent via des liaisons réversibles pour générer à l'équilibre thermodynamique une chimiothèque d'oligomères. Des briques moléculaires dithiophénols glycosylés sont capables de s'auto-assembler via la formation de ponts disulfures. Leurs propriétés ont été investiguées en chimie combinatoire dynamique et la distribution d'espèces résultant de l'équilibration a montré la formation exclusive des cyclotrimères et cyclotétramères, ou dyn[3]- et dyn[4]arènes. La répétition de l'expérience en présence d'une lectine modèle (ConA) a mené à l'amplification des homodyn[3]- et homodyn[4]arènes. Ces derniers ont été isolés par HPLC semi-préparative et leurs affinités pour ConA ont été mesurées en ITC dans le domaine du nanomolaire. Une extension de cette méthodologie aux lectines LecA et LecB de Pseudomonas aeruginosa est en cours / Several glycoclusters based on calixarenes, pillararenes or fullerenes have been synthesized in our laboratory. They exhibited strong affinities for several lectins through their multivalence and the “glycoside cluster effect”. The prupose of this study was to add a dynamic part to these molecules. We therefore applied the concept of dynamic combinatorial chemistry in which building blocks are able to self-assemble through reversible bonds to generate a library of oligomers. Dithiophenols bearing carbohydrate epitopes can self-assemble through the formation and exchange of disulfide bonds. Their properties in dynamic combinatorial chemistry were studied and the species distribution at the thermodynamic equilibrium revealed the selective formation of cyclotrimers and cyclotetramers named dyn[3]- and dyn[4]arenes. The equilibration in the presence of ConA, used as a model lectin, have led to the amplification of homodyn[3]- and homodyn[4]arenes. These glycodyn[n3,4]arenes have been isolated and their affinities toward ConA measured by ITC in the nanomolar range. Extension of this methodology toward the lectins LecA and LecB of Pseudomonas aeruginosa is in progress

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Thesis (Ph.D. in Microbiology) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 201-223).

Orientador: Lidiane Nunes Barbosa / Resumo: Combinações entre antimicrobianos, a exemplo de nisina (bacteriocina) e fármacos antibacterianos tradicionais, podem amenizar o problema da resistência bacteriana, pois o possível efeito sinérgico se torna estratégico, possibilitando o uso de doses menores no tratamento de doenças infecciosas com redução nos custos e na toxicidade, além de ter potencial na prevenção do surgimento das linhagens resistentes. No entanto, os mecanismos envolvidos na ação antibacteriana são importantes para pesquisas de novos fármacos. O objetivo do estudo foi investigar como a nisina, alguns fármacos antibacterianos e respectivas combinações interferem no metabolismo de Staphylococcus aureus Meticilina Resistente (MRSA) e Pseudomonas aeruginosa, através de ensaios de estresse oxidativo bacteriano, análises morfológicas por microscopia eletrônica de transmissão (MET) e análise do perfil de proteínas expressas nas bactérias quando expostas aos antimicrobianos e suas combinações em concentrações subletais. Inicialmente foram realizados ensaios visando obter os valores de concentração inibitória mínima (CIM) e concentração subletal máxima (CSM) para nisina e fármacos como tetraciclina, ciprofloxacina, vancomicina, polimixina B, oxacilina e cefalotina para ambas bactérias. Na sequencia, foram realizados ensaios para verificação de sinergismo entre nisina e antibacterianos utilizando metodologia da curva de sobrevivência, sendo escolhidas para ensaios posteriores, as combinações com demostração de sine... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Combinations of antimicrobials, such as nisin (bacteriocin) and traditional antibacterial drugs, may assuage the problem of bacterial resistance because the possible synergistic effect becomes interesting, allowing the use of smaller doses in the treatment of infectious diseases and reduction in costs and toxicity , besides having potential in the prevention of the emergence of resistant strains. However, the mechanisms involved in antibacterial action are important for research on new drugs. The aim of this study was to investigate how nisin, antibacterial drugs and their combinations interfere in the metabolism of Methicillin Resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa, verified in bacterial oxidative stress assays, morphological analyzes by transmission electron microscopy (TEM) and analysis of the protein profile expressed in bacteria when exposed to antimicrobials and combinations in sublethal concentrations. Initially, assays were performed to obtain the minimum inhibitory concentration (MIC) and maximum sublethal concentration (MSC) for nisin and drugs such as tetracycline, ciprofloxacin, vancomycin, polymyxin B, oxacillin and cephalothin for both bacteria. Subsequently, assays were performed to verify the synergism between nisin and antibacterials using time kill curve methodology and the combinations with demonstration of synergism (reduction in final bacterial count above 2 logs of CFU / mL in relation to initial inoculum) were chosen for later... (Complete abstract click electronic access below) / Doutor

Pseudomonas aeruginosa est un pathogène opportuniste responsable de maladies nosocomiales. Il provoque des infections aiguës ou chroniques en employant conjointement plusieurs facteurs de virulence. Les souches les plus agressives possèdent un système de sécrétion de type III (SST3), injectant des toxines directement dans le cytoplasme des cellules eucaryotes grâce à une nano-aiguille. Récemment, une souche clinique hyper-virulente, appelée CLJ1, a été isolée dans l'unité de soins intensifs de l'hôpital universitaire de Grenoble sur un patient souffrant d'une infection pulmonaire hémorragique. Cette souche ne possède pas les gènes codant pour le SST3 mais sécrète une pore-forming toxin, ExlA, non identifiée auparavant. ExlA est une protéine de 172 kDa, formant des pores de 1,6 nm dans la membrane plasmique de plusieurs types de cellules, à l'exception des érythrocytes. Le pore provoque la rétraction des cellules hôtes et finit par induire la mort de la cellule. Nous avons montré que CLJ1 appartenait à un nouveau clade très divergent des souches classiques de P. aeruginosa, dont les membres possèdent le gène exlA au lieu des gènes codant pour le SST3. Les souches exlA-positives que nous avons collectées dans le monde proviennent d'infections humaines et d'échantillons environnementaux. Leur cytotoxicité, sur diverses cellules humaines et sur un modèle murin d’infection pulmonaire, est corrélée avec les niveaux de sécrétion d'ExlA. En plus de la toxicité membranaire, les souches exlA-positives ont montré des activités protéolytiques élevées envers les VE et E-cadhérines, deux protéines adhésives des jonctions adhérentes requises pour l'intégrité de l'endothélium et de l'épithélium, respectivement. Nous avons démontré que la formation de pores par ExlA dans la membrane eucaryote induisait une entrée massive et rapide de calcium dans le cytosol. Cet afflux de calcium permet la maturation et l'activation d'ADAM10, une protéase eucaryote située à la membrane plasmique. L'activation d’ADAM10 induit le clivage de ses substrats naturels : les VE et E-cadhérines. ExlA fait partie de la même famille de pore forming toxin que ShlA de Serratia marcescens. Nous avons démontré que ShlA utilisait le même mécanisme qu’ExlA pour induire le clivage des cadhérines. En conclusion, les souches bactériennes produisant ExlA ou ShlA détournent un mécanisme naturel de l'hôte pour induire la perte d'intégrité tissulaire. / Pseudomonas aeruginosa is an opportunistic pathogen responsible for nosocomial diseases. It provokes acute or chronic infections due to several virulence factors acting in concert. The most aggressive strains possess a Type III Secretion System (T3SS), injecting toxins directly into the cytoplasm of eukaryotic cells thanks to a nano-needle. Recently, a hyper-virulent clinical strain, called CLJ1, was isolated from a patient suffering of hemorrhagic pulmonary infection, at the intensive care unit of Grenoble University Hospital. This strain lacks a T3SS but secretes a pore-forming toxin, ExlA, not previously identified. ExlA is a 172-kDa protein, forming 1.6-nm pores in the plasma membrane of several cell types, except erythrocytes. The pore causes the retraction of host cells and eventually induces necrotic cell death. We showed that CLJ1 belongs to a recently-discovered and highly divergent clade of P. aeruginosa, whose members possess the exlA gene instead of the genes coding for the T3SS and its effectors. The strains we collected worldwide originate from human infections and environmental samples. Their cytotoxicity on various human cells and mouse models of infection was correlated with ExlA secretion levels. In addition to membrane toxicity, exlA-positive strains displayed high proteolytic activities targeting VE and E-cadherins, two intercellular-junction adhesive proteins required for endothelium and epithelium integrity. We thus investigated the mechanisms of ExlA-induced cadherin cleavage. We demonstrated that ExlA pore formation in the eukaryotic membrane induces a massive and rapid entry of calcium into the cytosol. This calcium influx enables the maturation and activation of ADAM10, an eukaryotic protease located at the cell membrane. ADAM10 activation induces the cleavage of its natural substrates: the VE- and E-cadherins. ExlA is related to other toxins, including ShlA from Serratia marcescens, and altogether they constitute a family of pore-forming toxins with unique properties. We demonstrated that ShlA uses the same mechanism as ExlA to induce the cleavage of the cadherins. In conclusion, exlA- and shlA-positive strains hijack a natural mechanism of the host to induce the loss of tissue integrity.

La voie de signalisation impliquant l’interleukine (IL)-22 a un rôle majeur dans le maintien des fonctions de barrière des surfaces exposées du corps humain. Elle est indispensable pour la promotion de l'immunité antimicrobienne épithéliale, de l'inflammation et de la réparation tissulaire. Des situations pathologiques impliquant une altération de cette voie de signalisation ont déjà été décrites, mais rarement au niveau pulmonaire. Ce travail étudie cette voie de signalisation dans trois pathologies respiratoires. Lors de la broncho-pneumopathie chronique obstructive, un excès de sécrétion de protéases à sérine secondaire au recrutement de neutrophiles est responsable d’une protéolyse du récepteur à l’IL-22. Cette protéolyse inhibe les mécanismes de défense épithéliale dépendante de l’IL-22. Lors d’infection pulmonaire à P. aeruginosa, un facteur de virulence sécrété par cette bactérie dégrade l’IL-22 et inhibe la sécrétion épithéliale de peptides antimicrobiens. Enfin, une surexpression du récepteur à l’IL-22 a été observée dans le cancer du poumon non à petites cellules. Cette surexpression est associée à une surmortalité. / The (interleukin) IL-22/IL-22 receptor (R) pathway is critical in the maintenance of barrier function at exposed surface of the body. This pathway is also essential to promote innate mucosal immunity, inflammation and tissue homeostasis. Dysregulation of IL-22/IL-22R pathway has been described in human diseases, but has been barely studied in respiratory pathologies. This work reveals three lung diseases with altered IL-22/IL-22R pathway. During chronic obstructive pulmonary disease, the proteolytic action of neutrophil-derived enzymes cleave the IL-22R and inhibit IL-22-mediated epithelial cell response. Next, we demonstrated that P. aeruginosa used its own proteolytic system to escape from host defenses through the proteolysis of IL-22 leading to negative regulation of antimicrobial peptides. Finally, we observed that higher IL-22R expression is correlated with squamous cell lung carcinoma and is associated with increase mortality.

Les métaux biologiques jouent un rôle clé en tant que cofacteurs, contribuant à la structuration des macromolécules et catalysant les réactions biochimiques dans les cellules. Ils sont nécessaires pour une croissance bactérienne optimale mais deviennent toxiques lorsqu'ils sont présents en excès. Par conséquent, l'homéostasie de ces métaux doit être finement régulée, et tout déséquilibre dans leur concentration pourrait affecter la viabilité cellulaire. Lors de cette thèse nous avons investigué les mécanismes moléculaires impliqués dans l'homéostasie du Fe chez Pseudomonas aeruginosa, en étudiant les deux principaux sidérophores produits par cette bactérie, pyoverdine (PVD) et pyochéline (PCH). Avec notre approche, nous avons identifié des nouveaux mécanismes de régulation des voies d’acquisition du fer par PVD et PCH. Nous avons également étudié comment d'autres métaux biologiques peuvent interférer avec ces voies, et le Co a montré une forte propension à pirater et à polluer la voie PCH. / Biological metals (Fe, Zn, Co, Ni, Mn, Cu) play a key role by acting as co-factors, contributing to macromolecule structuration, and catalyzing biochemical reactions into the cells. They are required for optimal bacterial growth but also become toxic when present in excess. Consequently, the homeostasis of these metals has to be finely regulated, and any disequilibrium in their concentration into bacteria could affect cell viability. We have further investigated the molecular mechanisms implicated in Fe homeostasis in Pseudomonas aeruginosa involving the two major siderophores produced by this bacterium, pyoverdine (PVD) and pyochelin (PCH). With our approach we identified new regulation mechanisms of both PVD and PCH pathways. In parallel, we have also investigated how other biological metals than Fe can interfere with these iron uptake pathways. Our data showed a strong propensity of Co to pirate and pollute the PCH iron uptake pathway.

A maioria das bactérias não cresce como células individuais, mas em comunidades estruturadas como organismos pseudomulticelulares, ou biofilmes, estando presentes em praticamente todos os ecossistemas naturais e patogênicos. A adesão bacteriana à superfície e subsequente formação de biofilme promove mudanças metabólicas, fenotípicas e genotípicas que faz com que a sua erradicação seja extremamente difícil. Desta maneira, microrganismos que apresentam susceptibilidade a determinados antimicrobianos em testes laboratoriais convencionais, são na verdade altamente resistentes aos mesmos quando na forma de biofilmes. Bactérias na forma de biofilmes estão associadas com aproximadamente 80% das infecções médicas, ganhando destaque naquelas relacionadas a implantes médicos. Com o aumento da expectativa de vida humana, maior é a necessidade de substituição e reparo de funções biológicas e, portanto é estimado um aumento no número de pessoas hospitalizadas e que irão receber implantes biomédicos. Esses materiais, independente do seu nível de sofisticação, estão suscetíveis ao risco de colonização microbiana e infecção. O presente trabalho, conduzido de forma multidisclinar, utilizou microrganismos patogênicos e superfícies modelo, para demonstrar provas de conceito com relação a duas estratégias para o combate da formação de biofilmes de bactérias: (i) a busca por fitocompostos com atividade antiformação de biofilmes, guiado por relatos etnofarmacológicos, e o posterior recobrimento de superfície polimérica com estes compostos e, (ii) a modificação de propriedades de superfícies, através da técnica de plasma iônico, para a obtenção de superfícies antiadesivas. Assim, 45 extratos aquosos foram obtidos de 24 plantas utilizadas na medicina tradicional do bioma Caatinga. O rastreamento de atividade antibiofime e antibacteriana (nas concentrações de 0,4 e 4,0 mg/mL) evidenciou o alto potencial antibiofilme de extratos contra Staphylococcus epidermidis ATCC 35984 e indicou três plantas com atividade antimicrobiana para Pseudomonas aeruginosa ATCC 27853. Subsequentemente, o estudo foi focado na purificação dos compostos bioativos de quatro plantas: Pityrocarpa moniliformis, ativa contra S. epidermidis e, Anadenanthera colubrina, Commiphora leptophloeos e Myracrodruon urundeuva, ativas contra P. aeruginosa. O fracionamento bioguiado e a caracterização química das frações por MALDI MS MS demonstraram que os compostos ativos nos quatros casos pertencem à classe dos taninos. Estruturas complexas de proantocianidinas (composta principalmente por profisetinidina para A. colubrina e por prorobinetinidina para C. leptophloeos), e de taninos hidrolisáveis (constituído por unidades de ácido gálico em M. urundeuva) foram identificadas. Estes taninos inibiram a formação de biofilme de P. aeruginosa através da ação bacteriostática, causando danos de membrana e excesso na proliferação de vacúolos bacterianos, embora a membrana de eritrócitos tenha sido preservada. Com relação à P. moniliformis, proantocianidinas ricas em prodelfinidina (0.125 mg/mL) foram os compostos responsáveis pela completa inibição da formação de biofilme de S. epidermidis, sem afetar a viabilidade do microrganismo. Como demonstrado por diversas técnicas, os resultados indicam que tanto a superfície bacteriana (S. epidermidis) quanto a superfície dos materiais testados (vidro e poliestireno) são espontaneamente recobertos pelas proantocianidinas, tornando-as superfícies hidrofílicas. Através da técnica de “spin coating”, a superfície polimérica foi recoberta com estas proantocianidinas, convertendo-se em uma superfície fortemente hidrofílica. A habilidade de prevenir a adesão de S. epidermidis foi mantida e o material se mostrou compatível com as células epiteliais de mamíferos, indicando o grande potencial destes produtos naturais como agentes funcionais de recobrimento de superfícies. No outro enfoque deste estudo, a modificação de propriedades de superfícies via descarga de plasma dos gases N2/H2 produziu, de maneira rápida e bastante efetiva, superfícies de poliestireno capazes de impedir a adesão de bactérias altamente resistentes aos antimicrobianos. Através de espectroscopia de raio X, verificou-se que uma concentração de nitrogênio de 8,8% e a componente polar da energia de superfície superior a 15 mJ/m2, são necessários para reduzir a adesão de bactérias que apresentam superfície hidrofílica (como enterobactérias produtoras de carbapenemase e MRSA), enquanto que cepas hidrofóbicas (exemplificadas pelo S. epidermidis) mantiveram a capacidade de aderir e formar biofilmes. As interações respulsivas explicam os efeitos antiadesivos obtidos, tanto no recobrimento de superfícies por proantocianidinas quanto no tratamento por plasma iônico. / Most bacteria do not grow as individual cells, but in communities structured as pseudomulticelulares organisms, or biofilms, being present in virtually all natural ecosystems and pathogens. The bacterial adhesion to surfaces and subsequent biofilm formation promotes metabolic, phenotypic and genotypic changes, which makes their eradication extremely difficult. Thereby, microorganisms that exhibit susceptibility to antimicrobials during conventional laboratory tests are in fact highly resistant to them when in the form of biofilms. Bacteria living as biofilms are associated with approximately 80% of all medical infections, mainly those related to indewelling devices. With increasing life expectancy, greater is the need for replacement and repair biological functions and, therefore, it is estimated an increasing number of hospitalized people and who will receive biomedical implants. However, regardless of the level of material sophistication, all of them are susceptible to the risk of microbial colonization and infection. This study, conducted in a multidisclinar way, employed pathogenic microorganisms and surface models to demonstrate proofs of concept regarding two strategies to combat bacterial biofilm formation: (i) the search for phytocompounds having antibiofilm formation activity, guided by ethnopharmacological reports, and further the polymer surface coating with these compounds, and (ii) the modification of surface properties, by the ionic plasma discharge technique to obtain anti-adhesive surfaces. Thus, 45 aqueous extracts were obtained from 24 plants used in the tradicinal medicine of the Caatinga biome. The screening of antibiofim and antibacterial activities (at concentrations 0.4 and 4.0 mg/mL) showed the high antibiofilm potential of the extracts against Staphylococcus epidermidis ATCC 35984 and indicated three plants with antimicrobial activity against Pseudomonas aeruginosa ATCC 27853. Subsequently, the study was focused on the purification of bioactive compounds from four plants: Pityrocarpa moniliformis, active against S. epidermidis and, Anadenanthera colubrina, Commiphora leptophloeos and Myracrodruon urundeuva, active against P. aeruginosa. The bioguided fractionation and the chemical characterization of the fractions by MALDI MS MS showed that the active compounds in the four cases belong to the class of tannins. Complex structures of proanthocyanidins (mainly composed profisetinidin for A. colubrina and prorobinetinidin for C. leptophloeos), and hydrolysable tannins (consisting of gallic acid units in M. urundeuva) were identified. These tannins inhibited biofilm formation of P. aeruginosa through bacteriostatic action, causing membrane damage and excess on the proliferation of bacterial vacuoles while the erythrocyte membrane was preserved. With respect to P. moniliformis, proanthocyanidins riched in prodelphinidin (0.125 mg/mL) were the compounds responsible for the complete inhibition of S. epidermidis biofilm formation, without affecting the viability of the microorganism. As demonstrated by various techniques, the results indicated that both surfaces, of the bacterium (S. epidermidis) and of the tested materials (glass and polystyrene), were spontaneously covered by proanthocyanidins, becoming hydrophilic surfaces. Using spin coating technique, the surface was coated with these proanthocyanidins, making the surface highly hydrophilic. The ability to prevent adherence of S. epidermidis was maintained and the material proved to be compatible with mammalian epithelial cells, indicating the potential usefulness of these natural products as functional agents for coating surfaces. In another approach of this study, the modification of surface properties via plasma discharge using N2/H2 gases mixtures produced, by a quickly and effectively way, polystyrene surfaces able to prevent the adhesion of bacteria highly resistant to antibiotics. Through X-ray spectroscopy, it was found that a nitrogen concentration of 8.8% and the polar component of surface energy greater than 15 mJ/m2 are needed to reduced adhesion by bacteria exhibiting hydrophilic surface (such as Enterobacteriaceae carbapenemase-producing and the MRSA), while hydrophobic strains (exemplified by S. epidermidis) had the capacity to adhere and to form biofilms. The respulsive interactions could explain the anti-adhesive effects obtained in the coating of surfaces by proanthocyanidins as well as in the ionic plasma treatments.