Dans les environnements naturels, industriels ou médicaux, les microorganismes sont majoritairement présents en étant associés aux surfaces dans des communautés hautement organisées appelées biofilms. Ces édifices biologiques constituent une stratégie de survie étonnement efficace témoignant d’une grande capacité de résistance à différent stress environnementaux tels que les traitements de nettoyage et de désinfection. L’impact des biofilms d’un point de vue sanitaire est donc considérable du fait qu’ils permettent la persistance et la transmission de germes pathogènes dans l’environnement. Dans ce contexte, ce travail de thèse avait pour objectif une meilleure compréhension des phénomènes limitant l’efficacité de désinfectants au sein des biofilms en s’appuyant notamment sur des techniques innovantes d’imagerie de fluorescence non-invasive. Le but final étant d’apporter des éléments utiles à l’optimisation des traitements de désinfection. Dans une première partie, une méthode d’investigation structurale à haut-débit par microscopie confocale a été développée et utilisée pour étudier la diversité architecturale des biofilms bactériens formés par un large panel de souches. Cette étude nous a permis d’identifier des souches d’intérêt en termes de structures de biofilms formés pour la suite du travail. Nous avons notamment pu mettre en évidence la capacité de B. subtilis à former des structures importantes et avec une architecture spécifique dans un système immergé. Dans une deuxième partie, les dynamiques d’action spatiotemporelles de désinfectants ont été visualisées dans les biofilms de souches de P. aeruginosa ou B. subtilis par des approches de microscopie confocale de fluorescence en temps réel. L’utilisation de cette technique nous a permis de mettre en évidence les difficultés de pénétration du chlorure de benzalkonium au sein des structures formées par différentes souches de P. aeruginosa. La corrélation des paramètres cinétiques d’inactivation et des données obtenues par la caractérisation biochimique de la matrice suggère un rôle majeur des substances extracellulaires dans la limitation de pénétraton du désinfectant. Nous avons également pu montrer une résistance marquée du biofilm formé par une souche de B. subtilis isolée d’un dispositif médical à l’acide péracétique, à la concentration et au temps d’utilisation du biocide dans le milieu médical. De plus, les structures tridimensionnelles formées par cette souche étaient capables de protéger le pathogène Staphylococcus aureus dans un biofilm mixte vis-à-vis du même traitement soulignant l’importance des interactions multi-espèces dans la résistance des bactéries aux désinfectants et la persistance de pathogènes dans nos environnements. / In natural, industrial or medical environments, microorganisms are present mainly in being associated with surfaces in highly organized communities called biofilms. These biological structures cosntitute a surprisingly effective survival strategy showing a large ability to withstand environmental stresses such as cleaning and disinfection treatments. Therefore, biofilms have a considerable impact on public health because they allow the persistence and transmission of pathogens. In this context, this work aimed to better understand the phenomena limiting the effectiveness of disinfectants in biofilms noticeably by using innovative imaging fluorescence non-invasive techniques. The ultimate goal was to provide data which can help to optimize disinfection treatments. In the first part, a high-throughput structural method based on confocal microscopy was developed and used to study the architectural diversity of bacterial biofilms formed by a wide range of strains. This study allowed us to identify strains of interest in terms of biofilm structure for the second part of the work. In particular, we demonstrated the ability of B. subtilis to form protruding structures with a specific architecture in a submerged system. In the second part, the spatiotemporal dynamic of the action of disinfectants were visualized in the biofilms of P. aeruginosa or B. subtilis strains by a time-lapse fluorescence confocal microscopy method. Using this technique, we showed that benzalkonium chloride encountered problems of penetration in the biofilms formed by P. aeruginosa strains. The correlation of kinetic inactivation parameters and data obtained by the characterization biochemical matrix suggested a key role of extracellular substances in the penetration limitations of the disinfectant. We also observed a pronounced resistance of the biofilm formed by a strain of B. subtilis isolated from a medical device to peracetic acid at the in-use concentration and time of biocide in medical areas. In addition, three-dimensional structures formed by this strains afforded protection to the pathogen Staphylococcus aureus in mixed biofilm against the same treatment This point highlights the importance of multi-species interactions in bacterial resistance to disinfectants and in the persistence of pathogens in our environments.

L'interaction non-covalente entre un ligand et un récepteur selon un modèle clé-serrure constitue une des bases essentielles de tout système biologique. La présence de multiples clés et serrures sur les biomolécules conduit à des interactions multivalentes. Les lectines sont très fréquemment structurées en homo-multimères et sont donc des cibles de choix pour l'étude des interactions avec des structures multivalentes glycosylées. Ligands et récepteurs multivalents peuvent obéir à plusieurs mécanismes d'association conduisant à des profils thermodynamiques et cinétiques permettant de rationnaliser les améliorations spectaculaires d'affinité souvent observées. L'utilisation de ligands de faible valence et de petite taille permet une présentation contrôlée des sucres au travers d'une structure unique bien définie. Ces glycoclusters sont des plateformes adaptées à l'étude de l'influence de la topologie de la présentation des sucres sur l'interaction. La synthèse de glycoclusters a été optimisée selon une voie convergente de glycosylation puis de couplage par CuAAC permettant la synthèse de structures multi-glycosylées telles que des calix[4]arènes de différentes conformations, des peptoïdes linéaires et cycliques ou encore des porphyrines. Ces ligands ont été évalués par quatre techniques d'analyse des interactions (HIA, ELLA, SPR, ITC) principalement en présence de la lectine PA-IL de Pseudomonas aeruginosa mais également avec la Galectine-1 humaine et la lectine d'Erythrina cristagalli (légumineuse). Des glycoclusters de seconde génération ont été ensuite été préparés avec l'objectif d'optimiser les composantes enthalpiques et entropiques de l'interaction. Les résultats indiquent que de légères modifications de la présentation des sucres peuvent induire des mécanismes d'association différents. La conception de structures rigidifiées a révélé des profils thermodynamiques contre-intuitifs qui ont pu être modélisés. Par cette étude, plusieurs ligands ont montré des affinités sans précédent pour la lectine PA-IL. Le meilleur ligand multivalent de première génération a confirmé un potentiel thérapeutique prometteur in vivo. / Following Fischer's “lock-key“ concept, non-covalent interactions between a ligand and its receptor is one of the most fundamental process of any biological system. The presence of multiple keys and locks at the surface of many biomolecules leads to multivalent interactions. Lectins are appropriate partners for the study of multivalent interactions with multivalent glycoconjugates since lectins are generally organized as homomultimers. Association of ligands and receptors can occur through several mechanisms leading to distinct thermodynamic and kinetic patterns. Thermodynamic and kinetic parameters often rationalize the impressive affinity improvement observed in the context of multivalent interactions. Small and low valency multivalent ligands provide a neat organization of carbohydrates through a single well-defined structure. These glycoclusters are appropriate probes for studying the influence of the overall topology on the interaction. Glycocluster synthesis was optimized according to a convergent strategy consisting of a glycosidation reaction followed by multiple CuAAC couplings. This strategy yielded a library of glycoclusters based on conformers of calix[4]arenes, linear and cyclic peptoids and porphyrins scaffolds. Glycoclusters were evaluated thanks to a combination of four biochemical techniques (HIA, ELLA, SPR, ITC) mainly versus PA-IL, a tetrameric lectin from Pseudomonas aeruginosa. Further investigations of these ligands were performed with a plant lectin from Erythrina cristagalli and with human galectin-1. Second generation glycoclusters were prepared in order to optimize enthalpic and entropic contributions to the interaction. Results indicate that a slight modification of the glycocluster topology could induce different mechanisms. The design of glycoclusters with stiffened linkers highlights unexpected entropic patterns. Molecular modeling of these linkers provided rationalization of these entropic patterns on the basis of Boltzmann distribution. This work present glycoclusters with an unprecedented affinity for PA-IL. The best first generation glycocluster confirmed promising therapeutic potentialities in vivo.

Pseudomonas aeruginosa et Stenotrophomonas maltophilia, pathogènes opportunistes majeurs, pourraient acquérir leur résistance aux antibiotiques dans l’environnement, sous la pression exercée par les métaux lourds par co-sélection de résistance. Nous avons tout d’abord évalué la distribution et l’abondance de ces espèces dans un large panel de sols d’origine géographique différente (France et Afrique) et évalué l’influence d’activités anthropiques susceptibles d’exposer les sols en éléments métalliques sur cette distribution. Alors que la présence de P. aeruginosa est sporadique et plutôt liée à un apport exogène, S. maltophilia est présente dans tous les sols étudiés, suggérant son endémicité. L’évaluation des résistances des souches isolées de ces sols a également montré des différences entre les deux espèces. Les souches environnementales de P. aeruginosa sont pour la plupart caractérisées par un phénotype sauvage alors que celles de S. maltophilia présentent une grande diversité de phénotypes en fonction des sites, parfois similaires à ceux de souches cliniques. Cette diversité peut être attribuée à l’adaptation aux conditions environnementales très différentes rencontrées mais il est difficile d‘attribuer précisément aux métaux un rôle dans la co-sélection de ces résistances. L’étude menée sur la communauté bactérienne d’un sol contaminé a également permis de mettre en évidence une forte proportion de bactéries résistantes à différents antibiotiques représentée par des espèces qualifiées de pathogènes opportunistes ainsi que la présence du gène blaIMP, permettant la résistance à l’imipénème, utilisé en milieu clinique pour le traitement de clones multi-résistants. / Pseudomonas aeruginosa and Stenotrophomonas maltophilia, two major opportunistic pathogens, could acquire antibiotic resistance in the environment under heavy metal pressure that co-selects both resistances. We first investigated the distribution and abundance of these species in a wide range of soils of different geographical origin (France and Africa) and evaluated the influence of human activities that may expose soils to metallic elements on this distribution. While the presence of P. aeruginosa is rather sporadic and could be linked to exogenous intake, S. maltophilia is present in all studied soils, that suggests its endemicity. Evaluating resistance capacities of strains isolated from these soils also showed differences between the two species. Environmental strains of P. aeruginosa are mostly characterized by a wild type phenotype, whereas those of S. maltophilia present a wide diversity of phenotypes depending on the site, sometimes similar to those of clinical strains. This diversity could be attributed to a deep adaptation to the very different environmental conditions encountered in the original niche but it is difficult to attribute specifically to metals a role in coselection of resistance. The study conducted on the bacterial community present in a contaminated soil has also highlighted a high proportion of bacteria resistant to different antibiotics represented by species qualified as opportunistic pathogens and the presence of the gene blaIMP, enabling resistance to imipenem, used in the hospital to treat infections due to multidrug-resistant clones.

A gama-proteobactéria Pseudomonas aeruginosa é um patógeno oportunista humano frequentemente associado a pacientes com queimadura grave e aos portadores de fibrose cística. O estabelecimento de infecção depende de uma série de fatores que contribuem para a virulência deste patógeno, dentre eles a produção de sideróforos e outros sistemas de captação de ferro. Pioverdina é o principal sideróforo sintetizado por bactérias do gênero Pseudomonas e linhagens deficientes na sua produção são incapazes de estabelecer infecção em modelos animais. A regulação da biossíntese deste sideróforo envolve a agregação entre as células, indicando a dependência de contato para completa indução da sua produção. O contato com uma superfície altera o comportamento das células e diversos fenótipos são dependentes deste sinal mecânico. PrlC é uma oligopeptidase A putativamente envolvida na degradação de peptídeo-sinais e PA14_00800, uma pequena proteína com domínio de função desconhecida, codificada por um gene imediatamente à jusante de prlC. Existem poucos trabalhos na literatura sobre PrlC e seus homólogos e nenhuma informação sobre PA14_00800. Este trabalho teve como objetivo elucidar o envolvimento de PrlC e PA14_00800 na regulação da produção de pioverdina por células em contato com uma superfície. Para estabelecer uma correlação na expressão destes genes, um estudo da organização gênica foi realizado por RT-PCR, confirmando que eles fazem parte do mesmo operon e, portanto, que a expressão destes genes é regulada pelos mesmos fatores. Ensaios classicamente modulados pelo segundo mensageiro c-di-GMP, como formação de biofilme e motilidade, não apresentaram variações nas linhagens mutantes ΔprlC, ΔPA14_00800 ou Δoperon, indicando que a deleção destes genes não altera significativamente os níveis de c-di-GMP nas células. A motilidade do tipo swarming é, no entanto, severamente afetada na linhagem ΔPA14_00800 quando o meio de cultura não contém cloreto de cálcio e glicose, indicando um defeito na sinalização celular ou requerimento energértico desta linhagem nestas condições. PA14_00800 regula a fluorescência de P. aeruginosa em meio sólido e semissólido, mas não em meio líquido. Esta fluorescência depende tanto de pioverdina quanto de PQS, umamolécula de comunicação celular fluorescente, e a possibilidade de outros fatores estarem envolvidos neste fenótipo ainda está sob investigação. Análise do transcritoma por RNASeq com a linhagem ΔPA14_00800 comparada à linhagem parental foi realizada a partir de colônias destas linhagens crescidas em M9 modificado. Genes envolvidos no sistema de secreção do tipo III e do tipo VI e na biossíntese de PQS apareceram dentre os genes diferencialmente expressos, bem como genes para o catabolismo de glicose. Este trabalho foi o primeiro a investigar o papel de PA14_00800 na fisiologia de P. aeruginosa, e os conhecimentos adquiridos aqui podem ser transpostos, com cautela, para compreensão da função dos homólogos de PA14_00800 em outras bactérias. / The gamma-proteobacterium Pseudomonas aeruginosa is a human opportunistic pathogen frequently associated with patients with severe burns and those with cystic fibrosis. The establishment of infection depends on several factors that contribute to the virulence of this pathogen, among them siderophore production and other iron uptake systems. Pyoverdine is the main siderophore synthesized by the bacteria of the genus Pseudômonas and pyoverdinedeficient strains are unable to establish infection in animal models. The regulation of biosynthesis of this siderophore involves cell aggregation, indicating contact dependency for complete induction of pyoverdine production. Surface contact alters cell behavior and several phenotypes are dependent on this mechanical cue. PrlC is an oligopeptidase A putatively involved in peptide-signals degradation and PA14_00800, a small protein with a domain of unknown function, encoded by a gene immediately downstream of prlC. There are few papers in the literature on PrlC and its homologues and no information on PA14_00800. This work aimed to elucidate the role of PrlC and PA14_00800 in surface-dependent regulation of pyoverdine production. To establish a correlation in the expression of these genes, a study of the gene organization was performed by RT-PCR, confirming that they are part of an operon and therefore the expression of these genes is regulated by the same factors. Traits classically modulated by the second messenger c-di-GMP, such as biofilm formation and motility, did not show variations in the ΔprlC, ΔPA14_00800 or Δoperon, indicating that the deletion of these genes does not significantly alter the levels of c-di-GMP within the cells. Swarming motility is, however, severely affected in the strain ΔPA14_00800 when the culture medium does not contain calcium chloride and glucose, indicating a cell signaling defect or energetic requirement under these conditions. PA14_00800 regulates surface-dependent fluorescence of P. aeruginosa, in solid and semi-solid medium. This fluorescence depends on both pyoverdine and PQS, a fluorescent cell-to-cell communication molecule, and the investigation of other putative factors involved in this phenotype is still under study. Transcriptomic analysis by RNASeq with the strain ΔPA14_00800 compared to PA14 was performed from colonies ofthese strains grown in modified M9 1% agar. Genes involved in the type III and type VI secretion systems, in PQS biosynthesis and glucose catabolism were differentially expressed. This work was the first to investigate the role of PA14_00800 in the physiology of P. aeruginosa, and the knowledge obtained here can be cautiously transposed to understanding the role of PA14_00800 homologues in other bactéria.

Um grande desafio da atualidade é a crescente ocorrência, em ambientes aquáticos, de cianobactérias potencialmente tóxicas capazes de produzir cianotoxinas como a microcistina. Estratégias de controle de florações incluem a aplicação de algicidas. No entanto, esses produtos podem resultar na liberação de toxinas, que podem não ser removidas da água pelos processos convencionais de tratamento, sendo necessária uma etapa adicional como a adsorção com carvão ativado granular (CAG). A presente pesquisa avaliou com embasamento estatístico o efeito de cinco diferentes algicidas (à base de diuron, carbonato de sódio, polímero catiônico, sulfato de cobre e peróxido de hidrogênio), aplicados em três concentrações distintas, sobre a Microcystis aeruginosa, analisando-se a densidade celular, o biovolume, a concentração de potássio e de microcistina-LR extracelular/intracelular. Além disso, foram investigadas as implicações que esses produtos tiveram sobre a adsorção em dois CAGs, por meio de isotermas e modelos cinéticos. Todos os algicidas afetaram a integridade das células, resultando na liberação de microcistina. O último dia experimental (dia 7) foi o que apresentou maior concentração de microcistina extracelular, em que, em alguns casos, houve aumento de 10 vezes da concentração final de microcistina extracelular em relação à concentração inicial. O potencial de liberação de microcistina extracelular ocorreu na seguinte ordem de algicidas: polímero catiônico > sulfato de cobre > peróxido de hidrogênio > carbonato de sódio > diuron. Os algicidas também alteraram o comportamento de adsorção (de monocamada para multicamada), bem como pioraram os ajustes cinéticos. Além disso, enquanto o polímero catiônico praticamente triplicou a capacidade adsortiva dos carvões, o sulfato de cobre e o peróxido de hidrogênio pioraram o desempenho de ambos. Os algicidas possuem modos diferentes de ação e geraram consequências distintas para os indicadores avaliados, além de alterar a adsorção. Isso pode indicar a necessidade de se considerar a presença desses produtos na elaboração de projetos de estações de tratamento de água (ETAs). / A major challenge nowadays is the increasing occurrence in aquatic environments of potentially toxic cyanobacteria capable of producing cyanotoxins such as microcystin. Bloom control strategies include application of algaecides. However, these products may result in toxins release, which may not be removed from water by conventional treatment processes, requiring an additional step such as adsorption with granular activated carbon (GAC). The present study evaluated, using statistical methods, the effect of five different algaecides (diuron, sodium carbonate, cationic polymer, copper sulfate and hydrogen peroxide), applied on Microcystis aeruginosa in three different concentrations. We analyzed cell density, biovolume, potassium concentration and extracellular/intracellular microcystin-LR concentration. In addition, we investigated the implications these products had on two GACs adsorption, through isotherms and kinetic models. All algaecides affected cell integrity, resulting in microcystin release. The last experimental day (day 7) was the one that presented the highest extracellular microcystin concentration, in which, in some cases, there was a 10 times increase in the final extracellular microcystin concentration in relation to the initial concentration. The extracellular microcystin release potential occurred in the following order of algaecides: cationic polymer > copper sulfate > hydrogen peroxide > sodium carbonate > diuron. Algaecides also modified adsorption behavior (from monolayer to multilayer), as well as worsened kinetic adjustments. Furthermore, while cationic polymer almost tripled carbons adsorptive capacity, copper sulfate and hydrogen peroxide worsened the performance of both. Algaecides present different modes of action and caused distinct consequences for the evaluated indicator, besides modifying adsorption process. This may indicate the need to consider the presence of these products in water treatment plants (WTPs) designs.

Pseudomonas aeruginosa é uma proteobactéria do grupo gama, que pode se comportar como um patógeno oportunista. A linhagem PA14 apresenta duas ilhas de patogenicidade. A maior delas, PAPI-1, contém dois grupos de genes envolvidos com virulência, transcritos de maneira oposta e que estão entre duas seqüências repetidas diretas. O primeiro grupo compreende quatro genes dispostos em dois operons, que codificam para proteínas de sistemas de dois componentes (PvrS, PvrR, RcsC e RcsB). PvrS e RcsC são proteínas sensoras híbridas, que apresentam domínios de histidina-quinase e de reguladores de resposta. PvrR é um regulador de resposta com um domínio EAL com atividade de fosfodiesterase de diGMP cíclico e RcsB apresenta um domínio de ligação a DNA, além de um domínio fosfoaceptor. O outro grupo é composto de cinco genes, cupD1 a cupD5, que codificam para uma fímbria do tipo chaperone-usher e que apresenta alta similaridade com cupA, envolvido na formação de biofilme em outras linhagens de P. aeruginosa. Trabalhos anteriores mostraram que pvrS, pvrR, rcsC, rcsB e cupD2 estão relacionados com a virulência de PA14. Como estes grupos de genes parecem ter sido inseridos na ilha em um único evento de recombinação, este trabalho investigou se os sistemas de dois componentes estão relacionados com a regulação da expressão de cupD. Foi observado que a expressão de cupD é maior a 28ºC do que que a 37ºC e é influenciada positivamente pelo regulador global de expressão, MvaT, uma proteína tipo H-NS. Ensaios de β-galactosidase a partir de uma fusão de transcrição mostraram que a atividade promotora de cupD é cerca de 50% menor numa linhagem com deleção em rcsB em relação à linhagem selvagem. Nenhuma diferença consistente foi observada entre as linhagens com deleções em pvrS, pvrR, rcsC e rcsB e PA14 em relação a motilidade dos tipos swarming, swimming ou twitching ou à formação de biofilme. A linhagem de P. aeruginosa PA14 superexpressando RcsB mostrou níveis exacerbados de mRNA de cupD1, sendo a atuação de RcsB específica em cupD, já que os outros grupos de genes cup presentes em PA14 não mostraram a mesma variação na expressão, conforme analisado por RT-PCR quantitativo. Essa linhagem mostrou também um aumento na formação de biofilme, sem que a motilidade fosse alterada. Ainda visando elucidar os mecanismos de regulação de cupD, linhagens que superexpressam pvrR também foram analisadas quanto a estes fenótipos. Nesse caso, a superexpressão de pvrR diminuiu a formação de biofilme, conforme esperado, aumentou a motilidade do tipo swarming, porém não alterou a expressão de cupD. Os dados do presente trabalho demonstraram que a cupD é regulado pelos genes do sistemas de dois componentes adjacentes a ele e que o ativador de transcrição RcsB está relacionado com a formação de biofilme em tubos de vidro, provavelmente via a fímbria CupD. / Pseudomonas aeruginosa is a γ-proteobacteria that can behave as an opportunistic pathogen. The strain PA14 carries two pathogenicity islands, the largest of them, PAPI-1, contains two gene clusters between two direct repeat sequences that are transcribed in opposite directions and are involved in virulence. The first group consists of four genes arranged in two operons encoding two-component system proteins (PvrS, PvrR, RcsC and RcsB). PvrS and RcsC are hybrid sensor proteins, which contain domains of histidine kinase and response regulator domains. PvrR is a response regulator with a phosphodiesterase EAL domain and RcsB presents a C- terminal HTH DNA biding domain, in addition to a phosphoaceptor domain. The other group is composed of five genes, cupD1-5, that encodes components and assembly factors of a putative fimbrial CupD, which has high similarity with CupA, involved in the biofilm formation in other P. aeruginosa strains. Earlier work showed that pvrS, pvrR, rcsC, rcsB and cupD2 are related to the virulence of PA14. As these groups of genes appear to have been inserted on the island in a single event of recombination, this study investigated whether the two-component systems are related to the regulation of cupD expression. It was observed that cupD promoter activity is higher at 28oC than at 37oC and it is positively influenced by the global regulator, MvaT, a H-NS like protein. A lacZ transcriptional fusion showed about 50% less promoter activity of cupD from a strain with deletion in rcsB as compared to PA14. No consistent differences were found among the strains with deletions in pvrS, pvrR, rcsC and rcsB and PA14 on swarming, swimming and twitching motilities or biofilmsformation. A strain overexpressing overexpression showed heigher levels of cupD1mRNA of, and the role of RcsB as an activator is specific to cupD, as the other groups of cup genes present in PA14 did not show the same variation in the expression, as analyzed by quantitative RT-PCR. This strain also showed an increase in biofilm formation. In further assays aiming to elucidate the mechanisms of regulation of cupD, a strains overexpressing pvrR was also analyzed. Overexpression of pvrR decreased the formation of biofilm, as expected, and increased swarming motility, but did not alter the expression of cupD. The data from this study demonstrated that cupD is regulated by RcsB, and that this transcriptional activator is involved in the formation of biofilm in glass tubes, probably via CupD fimbriae.

A resistência bacteriana a antibióticos é um grave e crescente problema de saúde pública de âmbito mundial. O principal, e mais eficiente, mecanismo de resistência aos &#946-lactâmicos em bacilos Gram-negativos é a produção de &#946-lactamases, que possuem a capacidade de hidrolisar o anel &#946-lactâmicos e consequentemente inativar essa classe de antibióticos. Vale ressaltar, que atualmente os antibióticos &#946-lactâmicos são os mais utilizados clinicamente, particularmente em infecções graves. Dentre as &#946-lactamases existentes destacam-se as carbapenemases, enzimas capazes de inativar a maioria dos antibióticos &#946-lactâmicos. Uma grande preocupação é o fato dessas enzimas, em sua maioria, serem codificadas por plasmídeos, o que propicia a disseminação desses genes de resistência; portanto, é de extrema importância a realização de um rápido e efetivo monitoramento da presença de patógenos portadores desses genes de resistência, para que assim se possa prevenir a disseminação desses determinantes. Foram incluídos neste estudo 230 amostras únicas de Acinetobacter e Pseudomonas aeruginosa resistentes a imipenem detectados em pacientes internados em hospitais privados da cidade de São Paulo durante o período de fevereiro a outubro de 2013. As amostras foram avaliadas quanto à hidrólise de imipenem por espectrofotometria, quanto à presença de genes de carbapenemases por PCR e sequenciamento, e quanto à clonalidade por eletroforese em campos pulsados (PFGE) ou ERIC-PCR. Foram realizados ensaios de conjugação, transformação e sequenciamento completo de plasmídeos. Dentre as amostras de Acinetobacter spp. 80% (88) foram capazes de hidrolisar o imipenem. Dentre esses 76,1% (67) foram positivos para blaOXA-51-like, 19,3% (17) foram positivos para blaOXA-72. blaOXA-23, blaOXA-482 e blaIMP-1 foram detectados isoladamente em isolados distintos. O gene blaIMP-1 foi detectado em A. ursingii inserido em integron de classe 1 e representa a primeira descrição no Brasil. Uma nova carbapenemase OXA-482-like foi detectada em A. baumanii. Utilizando-se ERIC-PCR, observou-se uma grande diversidade de grupos clonais, com o máximo de quatro isolados por grupo. Dentre as amostras de P. aeruginosa, apenas 35,3% foram capazes de hidrolisar o imipenem. Dessas amostras, 14 possuíam o gene blaSPM-1, e isolados únicos possuíam, individualmente, os genes blaIMP, blaVIM, blaKPC-2 ou blaGES-23. O gene blaKPC-2 foi detectado inserido em contexto genético diferente dos descritos anteriormente, em plasmídeo IncU de 32 Kb, mobilizável, mas não conjugativo. Esta é a primeira descrição da sequencia completa de plasmídeo albergando o gene blaKPC-2 em P. aeruginosa no Brasil. Nas demais amostras (20) com atividade hidrolítica, não foram detectados genes de carbapenemase conhecidos, o que sugere a presença de genes de carbapenemase ainda não descritos. Em três amostras foi possível obter transformantes com plasmídeos, resistentes a carbapenêmicos. As amostras com blaSPM-1 apresentaram perfis de PFGE estreitamente relacionados. Em contraste, os perfis de PFGE das amostras com potenciais novas carbapenemases apresentaram índice de similaridade de Dice inferior ix a 80%, evidenciando grande diversidade clonal. Nossos achados evidenciam que a carbapenemase não intrínseca predominante em Acinetobacterem hospitais privados da cidade de São Paulo é OXA-72, e em hospitais privados há uma grande diversidade clonal. Em P. aeruginosa, a carbapenemase predominante é SPM-1, cuja disseminação é mediada por um único clone. Há potencialmente um número significativo de novas carbapenemases em Acinetobacter e P. aeruginosa, algumas delas mediadas por plasmídeos. / Bacterial resistance to antibiotics is a serious and growing public health problem worldwide. The main and most efficient mechanism of resistance to &#946-lactams in Gram-negative bacilli is the production of &#946-lactamases, which have the ability to hydrolyze the &#946-lactam ring and consequently inactivate this class of antibiotics. It is worth mentioning that currently &#946-lactam antibiotics are the most used clinically, particularly in severe infections. Among the existing &#946-lactamases, carbapenemases are capable of inactivating most &#946-lactam antibiotics. A major concern is that these enzymes are mostly encoded by plasmids, which facilitates the spread of these resistance genes; therefore, it is of extreme importance to carry out a rapid and effective monitoring of the presence of pathogens bearing these resistance genes, in order to prevent the dissemination of these determinants. This study included 230 unique samples of imipenem-resistant Acinetobacterand Pseudomonas aeruginosa detected in patients hospitalized in private hospitals in the city of São Paulo during the period from February to October 2013. The samples were evaluated for the imipenem hydrolysis by spectrophotometry, the presence of carbapenemase genes by PCR and sequencing, and concerning clonality by pulsed field electrophoresis (PFGE) or ERIC-PCR. Conjugation, transformation and complete sequencing of plasmids were performed. Among Acinetobacter spp. samples, 80% (88) were able to hydrolyze imipenem. Among these, 76.1% (67) were positive for blaOXA-51-like genes and 19.3% (17) were positive for blaOXA-72. The blaOXA-23, blaOXA-482 and blaIMP-1 genes were detected alone in distinct isolates. The blaIMP-1 gene was detected in A. ursingii inserted in class 1 integron and represents the first description in Brazil. A novel OXA-482-like carbapenemase was detected in A. baumanii. Using ERIC-PCR, a great diversity of clonal groups was observed, with a maximum of four isolates per group. Among P. aeruginosa samples, only 35.3% were able to hydrolyze imipenem. Of these samples, 14 had the blaSPM-1 gene, and single isolates individually possessed the blaIMP, blaVIM, blaKPC-2 or blaGES-23 genes. The blaKPC-2 gene was found inserted in a genetic context different from those described previously, in a mobilizable, but not conjugative, 32 Kb IncU plasmid. This is the first description of the complete nucleotide sequence of a plasmid harboring the blaKPC-2 gene in P. aeruginosa in Brazil. In the remaining samples (20) with hydrolytic activity, no known carbapenemase genes were detected, suggesting the presence of carbapenemase genes not yet described. In three samples it was possible to obtain transformants with plasmids, resistant to carbapenems. Samples with blaSPM-1 showed closely related PFGE profiles. In contrast, the PFGE profiles of the samples with potential new carbapenemases showed Dice similarity index lower than 80%, evidencing a great clonal diversity. Our findings show that the predominant non-intrinsic carbapenemase in Acinetobacter in the city of São Paulo is OXA-72, and in private hospitals there is great clonal diversity. In P. aeruginosa, the predominant carbapenemase is SPM-1, the spread of this enzyme is mediated by a single clone. There are potentially a significant number of new carbapenemases in Acinetobacter and P. aeruginosa, some of them plasmid mediated.

In der vorliegenden Untersuchung wurde der Postantibiotische Effekt (PAE) von Amikacin und Ceftazidim alleinig und in Kombination beider Antibiotika nach einmaliger und mehrfacher Exposition bei Pseudomonas aeruginosa bestimmt. Es wurden verschiedene Stämme mit unterschiedlicher Resistenz gegen Amikacin und Ceftazidim untersucht. Die MHK-Werte bewegten sich für Ceftazidim zwischen < 0,25 µg/ml bis 4 µg/ml und für Amikacin zwischen < 2 µg/ml bis 32 µg/ml. Der PAE lag nach der einmaligen Inkubation von Amikacin bei 0 bis 2,65 Stunden, von Ceftazidim bei 0 bis 2,78 Stunden und in der Kombination beider bei 2,4 bis 5,37 Stunden. Außerdem ergab die mehrmalige Exposition bei Amikacin nach der 1. Inkubation einen PAE von 0,75 bis 2,25 Stunden und stieg auf 2,3 bis 3,5 Stunden nach der 3. Inkubation an; bei Ceftazidim von 1,1 bis 2,18 Stunden Anstieg auf 1,18 bis 2,5 Stunden und bei der Kombination von Amikacin und Ceftazidim von 2,3 bis 3,75 Stunden war ein geringer Abfall auf 1,25 bis 3,25 Stunden zu verzeichnen. Es wurde ein Zusammenhang zwischen dem PAE und der MHK beobachtet: je höher die Resistenz, desto kürzer der PAE. Die Dosierungsintervalle wurden so gewählt, daß sie der klinisch üblichen dreifach Applikation pro Tag entsprachen. Aus unseren Untersuchungen läßt sich theoretisch eine einmalige Applikation pro Tag für Aminoglykoside und eine Kombinationstherapie Aminoglykosid plus Beta-Lactamantibiotika ableiten; vor allem auch, daß bei Mehrfachapplikation der PAE nicht kürzer wird. Für Ceftazidim erscheint eine Dauerinfusion bei fehlendem PAE sinnvoll. / In the current study we examinated the postantibiotic effect (PAE) of the antimicrobial agents amikacin and ceftazidime in vitro. We analyzed the PAE using both agents alone and in combination and after once and several expositions of different isolates of Pseudomonas. Different resistant stains of Pseudomonas were explored versus amikacin and ceftazidime. The observed minimum inhibitory concentration values (MIC) for ceftazidime ranged from < 0.25µg/ml to 4.0µg/ml and for amikacin from < 2.0µg/ml to 32µg/ml. After unique incubation the PAE of amikacin ranged from 0 to 2.65 hours while ceftazidime ranged from 0 to 2.78 hours. In combination of both we observed PAEs between 2.4 to 5.37 hours. A several exposition of amikacin showed after the first incubation PAEs between 0.75 to 2.25 hours increasing after third incubation from 2.3 to 3.5 hours whereas ceftazidime ranged from 1.1 to 2.18 hours and 1.18 to 2.5 hours. Both antimicrobial agents in combination had PAEs between 2.3 and 3.75 hours after first incubation and decreased low after third incubation between 1.25 and 3.25 hours. There is significant correlation to be seen between PAE and MIC-values: the higher resistance is, the shorter PAE will become.

Introduction: les follicules lymphoïdes (FL) sont absents du poumon normal mais ont été décrits dans les poumons de patients atteints de mucoviscidose ou de dilatations de bronches non mucoviscidosiques, suggérant un rôle pour l’infection bronchique dans la néogenèse lymphoïde (NL). Nous avons étudié la dynamique de la néogenèse lymphoïde dans l’infection bactérienne. Méthodes: les souris C57BL/6 ont reçu une instillation intratrachéale de billes d’agarose contenant du PAO1 ou du S. aureus (106 CFU/animal) permettant une infection prolongée et ont été comparées à des souris contrôles (billes stériles ou absence de billes). Les souris ont été sacrifiées à J1, J4, J7 et J14. Résultats: l’instillation unique de billes d’agarose contenant du PAO1 ou du S. aureus induit en 14 jours des FL fonctionnels situés sous l’épithélium en regard des zones d’infection. Le marquage pour CXCL12 et CXCL13 est faible chez contrôles, mais présent dans l’épithélium (CXCL13) dès J1 et présent également dans les FL (CXCL12 et CXCL13) à J14 chez les souris infectées. Le traitement des souris par un anticorps anti CXCL12 ou anti CXCL13 n’inhibe pas la formation des FL induite par l’infection à PAO1. Conclusion: nos données suggèrent un rôle pour l’infection bactérienne prolongée et l’épithélium respiratoire dans la NL des bronchopathies chroniques. Notre modèle permet d’évaluer les mécanismes de la formation et de persistance des FL dans le poumon. / Introduction: lymphoid follicles (LF) are absent in normal lungs, but are described in lungs of subjects with cystic fibrosis (CF) or non-CF bronchiectasis, suggesting a role for bacterial infection in lymphoid neogenesis. We aimed to study the dynamic of pulmonary lymphoid neogenesis (LN) during bacterial infection. Methods: C57BL/6 mice were instilled intratracheally with PAO1- or S. aureus-coated (1.106 CFU/mouse) agarose beads (which produced prolonged airway infection) and compared to controls (sterile beads or no instillation). Mice were sacrificed on day (d)1, d4, d7, and d14 after instillation. Results: chronic pulmonary infection with PAO1 or S. aureus induced organised LF in 14 days after a single challenge with PAO1- or S. aureus-coated beads. Bacteria- induced LF were exclusively localized in the subepithelium of infected airways. Staining for CXCL12 and CXCL13 was weak in airway epithelium of controls, but was positive in airway epithelium (CXCL13) at 1 day and in LF (both) of infected mice at 14 days. Treatment with anti CXCL12 or anti CXCL13 Ab did not reduce LN induced by PAO1 infection. Conclusions: chronic bacterial infection and respiratory epithelium could contribute to LN in chronic airway diseases. Our unique model allows to study mechanisms for the formation and maintenance of lung LF.

Introdução: Isolados clínicos de Pseudomonas aeruginosa multirresistentes estão associados a elevadas taxas de mortalidade. A resistência ao imipenem é uma urgência global, uma vez que é considerado o tratamento de escolha para infecções associadas a bactérias Gram negativas multirresistentes. Assim, elucidar os mecanismos de resistência é de vital importância para realizar um controle epidemiológico efetivo da disseminação deste tipo de isolado. Objetivos: Caracterizar os principais mecanismos de resistência ao imipenem em 76 isolados clínicos brasileiros de Pseudomonas aeruginosa, recuperados em 2004/2007, de 4 centros hospitalares do Estado de São Paulo. Material e métodos: Foram investigados: i) o perfil de resistência com determinação da CIM do imipenem; ii) a detecção de metalo-betalactamases (MBL) através de métodos fenotípicos e genotípicos; iii) a sensibilidade e especificidade do método de dupla difusão do disco na detecção de MBL; iv) a presença de genes codificadores de metilases 16S RNAr e sua associação com fenótipos aminoglicosídeo resistentes; v) alterações da permeabilidade por perda da porina OprD; vi) a presença ou ausência do gene oprD por PCR; vii) triagem fenotípica para expressão de bombas de efluxo através da determinação da CIM de quinolonas, cefalosporinas e carbapenêmicos na presença/ausência de inibidores específicos, realizando uma análise comparativa com o método de disco combinado; viii) os genes associados às bombas de efluxo mexA e mexE, através de PCR; ix) caracterizar a expressão das bombas de efluxo MexAB-OprM e MexEF-OprN, x) a clonalidade dos isolados por tipagem genotípica, através de ERIC-PCR, avaliando a relação genética (dendrograma) e sua associação com o predomínio de um determinado mecanismo de resistência. Resultados: Dentre os isolados de P. aeruginosa resistentes ao imipenem estudados (n=76, CIM50 e CIM90 = 32 &#181;g/mL e > 512 &#181;g/mL, respectivamente) 82% apresentaram um fenótipo de multirresistência. O principal mecanismo de resistência ao imipenem foi a produção de MBL, detectada em 74% dos isolados, e destes, 62% carregavam o gene blaSPM-1 e 12% carregavam o gene blaVIM-like. O método de dupla difusão do disco identificou a produção de MBL em 61% dos isolados. A combinação CAZ/MAA apresentou maior sensibilidade na detecção de MBL associada à SPM-1 (89%), mostrando uma especificidade de 86%. A presença do gene rmtD foi confirmada em 66% das amostras resistentes aos aminoglicosídeos, sendo que a presença concomitante do gene rmtD e do gene blaSPM-1 foi confirmada em 61% dos isolados. A deleção da porina OprD foi observada em 71% dos isolados. Dentre os isolados MBL positivos, 66% apresentaram ausência desta porina e, dentre as amostras MBL negativas, 85% não apresentaram OprD. Assim, para a resistência ao imipenem foi confirmada a contribuição de dois mecanismos, mediados pela presença de MBL e ausência de porina OprD. Em 13% (10/76) isolados, a deleção da porina OprD esteve associada à presença de seqüências de inserção (SI) em uma região anterior ao gene oprD. Por outro lado, a ausência de amplificação da região 736/1394 do gene oprD, em 11% (9/76) dos isolados, sugeriu a presença de polimorfismos. O gene mexA esteve presente em 92% dos isolados, enquanto que o gene mexE esteve presente em 82% dos isolados. A triagem de bombas de efluxo por disco combinado e análise da CIM na presença de reserpina, CCCP e PA&#946;N, utilizando levofloxacina, meropenem, aztreonam, imipenem ou levofloxacina, não teve correlação com a superexpressão dos sistemas MexAB-OprM e MexEF-OprN. Ambos os métodos careceram de especificidade e sensibilidade quando comparados ao PCR em tempo real. A superexpressão dos sistemas mexA e mexE foi confirmada em 35% (7/20) isolados MBL negativos, enquanto que 11% (6/56) isolados MBL positivos apresentaram superexpressão do gene mexA ou mexE, sendo que 7% (4/56) isolados MBL positivos superexpressaram ambos os genes. A superexpressão dos sistemas MexAB-OprM e MexEFOprN como único mecanismo de resistência ao meropenem e imipenem foi confirmada em 10% (2/20) dos isolados MBL negativos. Nos 76 isolados, a tipagem genotípica por ERIC-PCR, identificou a presença de 24 clusters (considerando 90% de similaridade na análise do dendrograma). Conclusão: A convergência de múltiplos mecanismos de resistência em P. aeruginosa parece ser um evento favorável para a seleção de clones endêmicos multirresistentes disseminados na região Sudeste do Brasil. / Introduction: Clinical isolates of Pseudomonas aeruginosa are associated with high mortality rates. Resistance to imipenem is a global concern, since it is a drug of choice for the treatment of infections produced by multidrug-resistant Gram-negative bacteria. Thus, research on resistance mechanisms is crucial to carry out an effective program for infection control and epidemiology of imipenem-resistant strains. Objective: to characterize the major mechanisms of imipenem resistance in 76 clinical isolates of P. aeruginosa recovered from clinical samples collected, from 2004 to 2007, in four hospitals in the State of São Paulo, Brazil. Material and methods: Isolates were screened for: i) resistance profile to antibacterial agents, determining the MIC of imipenem; ii) the detection of metallo-beta-lactamase (MBL) by phenotypic and genotypic methods, iii) MBL detection by using a double-disk diffusion test (D-test), determining the sensitivity and specificity of the assay; iv) the presence of genes encoding 16S rRNA methylases and their association with aminoglycoside-resistant phenotypes, v) changes in the bacterial permeability due to porin (OprD) loss; vi) the presence or absence of the oprD gene by using PCR; vii) phenotypic expression of efflux pumps by determining the MIC of quinolones, cephalosporins and carbapenems in the presence/absence of specific inhibitors, performing a comparative analysis with a combined-disk method, viii) genes encoding efflux pumps proteins (mexC and mexX) by PCR; ix) MexAB-OprM and MexEF efflux pumps expression; x) clonal relatedness, by ERIC-PCR genotyping, regarding the predominance of major resistance genotypes. Results: Among imipenem-resistant P. aeruginosa strains (n=76, MIC50 e MIC90 = 32 &#181;g/mL e > 512 &#181;g/mL, respectively) 82% showed a multidrug-resistant phenotype. The main mechanism of imipenem resistance was the MBL production detected in 74% strains, of which 62% harbored the blaSPM-1 gene, and 12% harbored the blaVIM-like gene. The D-test identified MBL production in 61% strains. In this regard, CAZ/MAA was the most sensitive combination for MBL detection associated to SPM-1 enzyme (89%), exhibiting 86% specificity. The presence of the rmtD 16S rRNA methylase gene was confirmed in 66% aminoglycoside-resistant strains. Moreover, presence of both rmtD and blaSPM-1 genes was identified in 61% strains. Loss of OprD porins was observed in 71% strains. In this regard, 66% MBL positive strains and 85% MBL negative strains showed OprD loss. Thus, MBL production and OprD loss contributed to imipenem resistance in P. aeruginosa. Most likely, in 13% (10/76) strains the porin loss was associated to insertion sequences (SI) inserted upstream of the oprD gene. On the other hand, in 11% (9/76) strains the absence of a PCR product targeting the 736/1394 region of the oprD gene, suggested the presence of polymorphisms. The mexA gene was identified in 92% strains, whereas the mexE gene was identified in 82% strains. Results obtained from efflux pump screening by using a combined-disk assay and MIC determination in the presence of reserpine, CCCP e PABN (using levofloxacin, meropenem, aztreonam or imipenem) was not correlated with results obtained from MexAB-OprM and MexEF-OprN overexpression analysis by RT-PCR. In this regard, both combined-disk and MIC assay showed lack of specificity and sensitivity in comparison to RT-PCR. Overexpression of mexA and mexE genes was confirmed in 35% (7/20) MBL-negative and 11% (6/56) MBL-positive strains, respectively, being 7% (4/56) MBL-positive strains overexpressed both genes. The overexpression of MexAB-OprM and MexEF-OprN efflux pumps, as only mechanism of resistance to meropenem and imipenem was observed in 10% (2/20) MBL-negative strains. ERICPCR typing revealed the presence of 24 clusters among 76 imipenem-resistant P. aeruginosa strains (&#8805; 90% similarity). Conclusion: The convergence of multiple mechanisms of resistance in Pseudomonas aeruginosa seems to be a favorable event for the selection of multiresistant clones endemic in the southeastern region of Brazil.