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Avaliação da presença de sinergismo antimicrobiano in vitro contra isolados de Pseudomonas aeruginosa resistentes a carbapenêmicos obtidos em hemoculturas de pacientes submetidos a transplante de células precursoras hematopoiéticas / Evaluation of antimicrobial in vitro synergy against carbapenemresistant Pseudomonas aeruginosa isolates from bloodstream infection in hematopoietic stem cell transplant recipientsJessica Fernandes Ramos 11 June 2018 (has links)
A infecção de corrente sanguínea (ICS) causada por bactérias multirresistentes tem alta mortalidade em pacientes receptores de transplante de células-tronco hematopoiéticas (TCTH). A Pseudomonas aeruginosa é um dos agentes mais frequentes e de difícil tratamento nessa população de pacientes. Objetivos: Avaliar características clínicas, microbiológicas e moleculares de 30 isolados de P. aeruginosa resistente à carbapenêmicos (PARC) em ICS de pacientes submetidos a TCTH e a presença de sinergismo antimicrobiano in vitro. Métodos: Os dados clínicos foram obtidos retrospectivamente de prontuários médicos e registrados em banco de dados. Análises bivariadas e multivariadas foram realizadas para avaliar determinantes de desfechos clínicos e uma curva de sobrevida foi construída. Determinou-se a concentração inibitória mínima (CIM) dos antimicrobianos por meio de microdiluição, foram realizados ensaios de sinergismo por método de checkerboard e time-kill, avaliação da clonalidade por eletroforese em campo pulsado e detecção de genes codificadores de mecanismos de resistência e virulência por reação em cadeia de polimerase. O sequenciamento do genoma completo (WGS) dos principais clones foi realizado por Nextera XT, utilizando a tecnologia Illumina MiSeq. Resultados: A maioria dos pacientes era do gênero feminino, com mediana de idade de 48 anos. Neutropenia foi presente em 93% dos pacientes e colonização prévia por PARC em 32%. A mortalidade em 14 dias foi 68%; a maioria dos pacientes que morreram foram transplantados alogênicos (79% vs. 17% entre receptores de transplante autólogo; p=0,012). Pacientes tratados com duas ou três drogas não apresentaram diferença estatisticamente significante na mortalidade até 14 dias após a ICS. Foram avaliados 30 isolados bacterianos. Todos apresentaram alto nível de resistência ao meropenem (MERO): CIM90 > 512 ug/mL; dois terços eram resistentes à amicacina (AMK) (CIM 2-512 ug/mL) e todos mantinham sensibilidade à colistina (COL). Muitos isolados (17/30) alcançaram efeito sinérgico in vitro pelo método time-kill com a combinação MERO mais COL, mas não com AMK. Nenhum antagonismo foi observado. Houve menor mortalidade em pacientes cujo isolado apresentou sinergismo entre COL e MERO quando comparados a pacientes portadores de isolados sem sinergismo, sem significância estatística. O gene de carbapenamase mais identificado foi blaSPM e 6 isolados apresentaram blaSPM e blaKPC. Os isolados apresentaram genes relacionados com virulência, tais como toxA, exoS e lasB; pacientes com ICS causada por P. aeruginosa que abrigava o gene lasB apresentaram maior risco de evoluir para o óbito. O WGS mostrou que os clones abrigavam SPM-1, Tn4371, mutações em porinas, em partes das bombas de efluxo, nas proteínas ligadores de penicilina (PBP) e pertenciam a ST277. Conclusão: As ICS por PARC cursaram com alta mortalidade em pacientes submetidos à TCTH. Houve uma grande proporção de resultados positivos para sinergismo entre os antimicrobianos in vitro, mas não foi possível demonstrar benefício estatisticamente significante no uso da terapia combinada com três drogas. Os clones carreavam SPM-1, Tn4371 e pertenciam a ST277 / Bloodstream infection (BSI) has high mortality in hematopoietic stem cell transplant (HSCT) recipients and Pseudomonas aeruginosa is an important and challenging organism. Objectives: To evaluate clinical, microbiological and molecular features of carbapenem-resistant P. aeruginosa (CRPA) isolates from BSI identified among HSCT patients and address in vitro synergy of antibiotic combination. Methods: Patient medical records were retrospectively reviewed and registered in a database. We used bivariate and multivariate analyzes to investigate determinants of clinical outcomes, and demonstrated overall mortality using a survival curve. We determined minimal inhibitory concentrations (MIC) for antimicrobials and in vitro synergies using checkerboard and time-kill assays, pulsed-field electrophoresis (PFGE) for clonality assessment and polymerase chain reaction (PCR) to detect carbapenamases and virulence genes were performed for all isolates. Whole genome sequence (WGS) of main clones was performed by Nextera XT, using Illumina MiSeq technology. Results: Most patients were female, median age was 48 years old. Main baseline disease was acute leukemia and 68% received allogeneic HSCT. 93% of patients had neutropenia and 32% had prior CRPA gut colonization.14-day mortality was 68%; mortality was higher among allogeneic HSCT recipients compared to autologous HSCT recipients (79% vs. 17% p = 0,012). Patients treated with two or three drugs did not present a statistically significant difference in 14-day mortality after BSI. In total, 30 bacterial isolates were analyzed; all presented a high resistance level to meropenem (MERO): MIC90 > 512ug/mL; two thirds were also resistant to amikacin (AMK) (MIC 2-512 ug/mL) and all were susceptible to colistin (COL). Many (17/30) isolates achieved in vitro synergistic effect in time-kill assay with the association of MERO and COL, but synergistic effect was not observed with AMK, by time-kill. No antagonistic effect was observed. There was a tendency towards better survival in patients whose CRPA isolate had in vitro synergy between COL and MERO without statistical significance. The most frequent carbapenamase gene identified was blaSPM, and six co-harboured both blaKPC and blaSPM. Isolates presented genes related to virulence factors such as toxA, exoS and more patients with BSI caused by P. aeruginosa harbouring gene lasB evolved to death. WGS analysis showed that clones harboured SPM-1, Tn4371 and belonged to ST277. They also presented mutations in genes related with porins and efflux pumps, as well in penicillin binding proteins (PBPs). Conclusion: CRPA BSI as associated with high mortality in HSCT recipients. A large proportion of isolates had in vitro synergy; however, we could not demonstrate statistically significant benefit in the use of combination therapy. Clones carried SPM-1, Tn4371 and belonged to ST277
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Estratégias de combate à adesão de bactérias patogênicas e formação de biofilmes : prospecção de fitocompostos e modificações de superfícies visando uso biomédico / Strategies to combat adhesion and biofilm formation of pathogenic bacteria : phytocompounds prospecting and surface modifications aiming biomedical useTrentin, Danielle da Silva January 2013 (has links)
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.
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Caracterização da superexpressão do fator sigma ECF σx em Pseudomonas aeruginosa PA14 / Characterization ofthe ECF sigma fator σx overexpression in Pseudomonas aeruginosa PA14.Ana Laura Boechat Borges 05 July 2013 (has links)
Pseudomonas aeruginosa é uma proteobactéria do grupo gama muito versátil, capaz de colonizar ambientes variados e infectar hospedeiros filogeneticamente distintos, incluindo humanos imunocomprometidos. Os fatores sigma de função extracitoplasmática (ECF) são membros de sistemas de sinalização de superfície celular (CSS), abundantes em P. aeruginosa. Vinte genes codificando fatores sigma ECF estão presentes nos genomas sequenciados de P. aeruginosa, a maioria fazendo parte de sistemas TonB relacionados à captação de ferro. Neste trabalho, seis fatores sigma pobremente caracterizados foram superexpressos na linhagem PA14 a partir de um promotor induzível por arabinose para investigar seu papel na expressão dos sistemas de dois componentes PvrSR e RcsCB, que atuam na regulação da fímbria CupD, além de sua influência no crescimento de culturas de P. aeruginosa. Não foi observado efeito positivo de nenhum dos fatores sigma testados na expressão dos sistemas de dois componentes e a superexpressão de cinco deles tampouco levou a qualquer alteração no crescimento, porém a produção de piocianina foi alterada na superexpressão de PA14_55550 e a superexpressão de PA14_26600 e PA14_46810 levou a um discreto aumento no início da formação de biofilme em PA14. Por outro lado, culturas superexpressando σx (ALB04) apresentaram um perfil alterado de lipopolissacarídeo e uma curva de crescimento bifásica, alcançando precocemente uma fase estacionária seguida de uma recuperação do crescimento até uma segunda fase estacionária. Durante a primeira fase estacionária, a maior parte das células aumenta de tamanho e morre, mas as células remanescentes retornam à morfologia selvagem e seguem para a segunda fase de crescimento exponencial. Isso não acontece devido a mutações compensatórias, uma vez que células coletadas de pontos tardios da curva e diluídas em meio novo repetem este comportamento. Apesar de trabalhos com a linhagem PAO1 associarem σx à transcrição de oprF, que codifica a principal porina não específica de Pseudomonas, nas condições dos nossos ensaios em PA14 a expressão dessa porina não foi induzida pela superexpressão de σx. Assim, os efeitos observados nessa superexpressão também não podem ser atribuídos a OprF. A transcrição de oprF em PA14 mostrou-se majoritariamente dependente da região promotora a que se atribui a ligação de σ70, ao contrário dos relatos na literatura da dependência da região de ligação a σx. Análises proteômicas foram realizadas para investigar os elementos envolvidos nesses efeitos de superexpressão de σx, o que revelou a indução de diversas enzimas envolvidas na via de biossíntese de ácidos graxos. As células superexpressando σx apresentam uma maior proporção de ácidos hexadecanoico (C16) e hexadecenoico (C16:1) e dados de anisotropia mostram uma maior fluidez da(s) membrana(s). Este trabalho é o primeiro relato de um fator sigma ECF envolvido em biossíntese de lipídeos em P. aeruginosa. / Pseudomonas aeruginosa is a very versatile gammaproteobacteria, able to colonize different environments and to infect phylogenetically distinct hosts, including immunocompromised humans. The extracytoplasmic function sigma factors (ECFs) are members of cell signaling systems (CSS), abundant in P. aeruginosa. Twenty genes coding for ECF sigma factors are present in the sequenced genomes of P. aeruginosa, most of them being part of TonB systems related to iron uptake. In this work, six poorly characterized sigma factors were overexpressed in strain PA14 from an arabinose inducible promoter to investigate their role in the expression of the two-component systems PvrSR and RcsCB, which regulates CupD fimbria, and their influence in P. aeruginosa cultures growth. None of the tested sigma factors led to two-component systems upregulation and overexpression of five of them caused no change in the growth profile, but pyocyanin production was altered in PA14_55550 overexpression and PA14_26600 and PA14_46810 overexpression led to a slight increase in biofilm initiation in PA14. By the other side, cultures overexpressing σx (ALB04) presented an altered lipopolysaccharide profile and a biphasic growth curve, reaching an early stationary phase followed by a growth resuming untill a second stationary phase. During the early stationary phase, most cells swells and dies, but the remaining cells return to wild type morphology and proceed to the second exponential phase of growth. This is not due to compensatory mutations, since cells collected from late points of the curve and diluted in fresh medium repeat this behavior. Although studies with strain PAO1 associate σx with transcription of oprF, encoding the major nonspecific porin of Pseudomonas, under our experiments conditions with PA14, this porin expression is not induced by σx overexpression. Thus, the effects observed in this overexpression cannot be attributed to OprF. Transcription of oprF in PA14 proved to be mainly controlled by the σ70-dependent promoter region instead of the σx-dependent promoter region reported in the literature. Proteomic analyses were performed to investigate the elements involved in these effects of σx overexpression, which revealed the induction of several enzymes involved in fatty acids biosynthesis. Cells overexpressing σx exhibit a greater proportion of hexadecanoic (C16) and hexadecenoic (C16: 1) acids and anisotropy data show higher fluidity of the membrane (s). This work is the first report of an ECF sigma factor involved in lipid biosynthesis in P. aeruginosa.
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Acúmulo de polifosfato e o papel do gene phoU em Pseudomonas aeruginosa. / Polyphosphate accumulation and the role of phoU in Pseudomonas aeruginosa.Luiz Gustavo de Almeida 05 December 2013 (has links)
Polifosfato inorgânico (PPi) é um polímero linear formado por diversas moléculas de ortofosfato (Pi) unidas por ligações fosfoanidridas de alta energia. Bactérias da espécie Pseudomonas aeruginosa acumulam grandes quantidades de PPi. Moléculas de Pi são captadas do meio através de dois sistemas de transporte. O principal deles é o sistema Pst, que possui alta afinidade por seu substrato. Pst é codificado por um operon de mesmo nome, formado por cinco genes. Os quatro primeiro genes codificam para as proteínas envolvidas no transporte de Pi, sendo que o último gene do operon, phoU, codifica para uma proteína cuja exata função é desconhecida. Com o objetivo de elucidar o papel deste gene e avaliar a sua relação com o acúmulo de PPi, foi construída uma mutação phoU na cepa PA14 de P. aeruginosa. O mutante não só apresentou uma maior capacidade de acumular PPi mas também se mostrou mais sensível a estresses ambientais e a antibióticos. Os resultados obtidos indicam que o gene phoU possui uma função regulatória sobre o acúmulo de PPi. O mutante phoU também apresentou níveis mais altos da molécula de alarme guanosina tetrafosfato (ppGpp). Foi proposto um modelo que explica a relação entre o gene phoU, o acúmulo de polifosfato e ppGpp. O mutante phoU foi também cultivado em cultura contínua em um quimiostato limitado em Pi por 13 dias. Diversos ensaios fenotípicos foram realizados com bactérias isoladas do quimiostato. Estes apresentaram algumas características fisiológicas distintas da cepa ancestral. / Inorganic polyphosphate (PPi) is a linear polymer composed of several molecules of orthophosphate (Pi) linked by energy-rich phosphoanhydride bonds. Bacteria of the species Pseudomonas aeruginosa accumulate large amounts of PPi. Pi molecules are captured via two trasport systems. The main one is the Pst system, which has high affinity for its substrate. Pst is encoded by an operon of the same name, consisting of five gene. The first four genes encode proteins involved in the transport of Pi and the last gene of the operon, phoU, encodes a protein whose exact function is unknown. To elucidate the role of phoU and its relation to PPi accumulation, a phoU mutant was constructed in strain PA14 of P. aeruginosa. The mutant accumulated high levels of PPi but was more sensitive to environmental stresses and antibiotics. The results indicate that phoU plays a regulatory role in the accumulation of PPi. The phoU mutant also displays high levels of the alarmone guanosine tetraphosphate (ppGpp). A model that explains the relation between phoU, ppGpp and polyphosphate accumulation is proposed. The phoU mutant was grown under steady-state conditions in a chemostat limited Pi for 13 days. Phenotypic assays performed with the bacteria isolated from the chemostat showed they acquired some distinct physiological characteristics.
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Le Pilus Conjugatif de Pseudomonas aeruginosa : Caractérisation des éléments de membrane externe / The conjugative pilus of Pseudomonas aeruginosa : Caracterisation of outer membrane componentsSpagnolo, Jennifer 26 April 2013 (has links)
La souche de P. aeruginosa PA14 est un isolat humain hautement virulent. PA14 possède deux îlots de pathogénicité. L'îlot de pathogénicité PAPI-1 de 108 kb est un élément intégratif et conjugatif (ICE), capable de s'auto-transférer à des souches de Pseudomonas par un mécanisme de conjugaison. Le mécanisme de transfert fait intervenir un pilus de Type IVb, encodé dans l'îlot PAPI-1. Mon travail de doctorat a eu pour but de caractériser à un niveau moléculaire le pilus de Type IVb (Pil-PAPI-1). J'ai d'abord, inséré un promoteur constitutif en place du promoteur endogène pour activer l'expression du locus pil2. J'ai démontré que 9 des 10 gènes sont requis pour le transfert d'ADN. J'ai ensuite initié la caractérisation de composants formant la machine de conjugaison. J'ai caractérisé le produit des gènes pilL2 et pilN2. J'ai démontré que PilL2 est une protéine de membrane externe (ME) et exposée dans le périplasme. Cette protéine est essentielle pour la fonctionnalité (transfert d'ADN) de la machinerie de conjugaison. Malgré ses caractéristiques prédites de lipoprotéine, aucune des mutations réalisées n'a pu modifier la localisation de ME de PilL2. J'ai aussi démontré que PilL2 forme un sous complexe de ME avec PilN2, la sécrétine du système. PilN2 forme des multimères stable, et présente les caractéristiques d'une liposécretine, capable d'auto-insertion et d'auto-multimérisation dans la ME. J'ai démontré que le N-ter de PilN2 mature est critique pour la formation d'un pore fonctionnel, mais n'est pas impliqué dans l'interaction avec PilL2. Ces résultats suggèrent que PilL2 et PilN2 forment un nouveau type de sous complexe de ME dans la famille des TFPb. / The P. aeruginosa PA14 strain is a highly virulent human isolate. PA14 possesses two pathogenicity islands. The 108-kb pathogenicity island PAPI-1 is an integrative and conjugative element (ICE), capable of self-transferring to any recipient Pseudomonas strain, by a conjugative mechanism. The transfer mechanism is mediated by a Type IVb pilus, encoded within the PAPI-1 Island. My PhD work aimed to characterize this Type IVb pilus (Pil-PAPI-1) at a molecular level. The pil2 locus is poorly expressed under laboratory condition. I, first, introduced a constitutive promoter to turn on expression of the pil2 locus. I demonstrated that 9 of the 10 genes are required for DNA transfer. Then, I initiated the characterization of components forming the conjugation machinery. I characterized the products of pilL2 and pilN2 genes. I demonstrated that PilL2 is an OM protein and protruding in the periplasm. This protein is essential for the functionality (DNA transfer) of the conjugative TFPb machinery. Despite its predicted lipoprotein-hallmarks, none of the mutations engineered were able to abrogate the OM-localization of PilL2. We also demonstrated that PilL2 forms an OM sub-complex with PilN2, the secretin of the system. We provide evidence that PilN2 forms stable multimers, which presents the features of a liposecretin, capable of self-insertion and self-multimerization in the OM. We demonstrated that while the N-terminus of the mature PilN2 is required for the formation of a functional pore, it is not involved in interaction with PilL2. These results suggest that PilL2 and PilN2 could form new type of OM sub-complex in the TFPb family.
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Métallome et homéostasie du fer chez Pseudomonas aerunginosa : rôle des sidérophores pyochéline et pyoverdine / Metallome and iron homeostasie of Pseudomonas aeruginosa : a role for siderophores pyocheline and pyoverdineCunrath, Olivier 28 April 2015 (has links)
Pseudomonas aeruginosa est une bactérie à Gram-négatif, pathogène et opportuniste, responsable de nombreuses et sévères infections chez l’homme. Ce microorganisme comme la plupart des organismes vivants a besoin de fer pour sa croissance ainsi que d’autres métaux biologiques comme le zinc, le cuivre, le nickel, le manganèse, le cobalt, le molybdène, le vanadium et d’autres. Afin d’acquérir le fer P. aeruginosa produit deux sidérophores majeurs, la pyoverdine (PVD), souvent considérée comme sidérophore principal, et la pyochéline (PCH). Lors de cette thèse nous avons pu démontrer les enzymes de biosynthèse de ces sidérophores adoptent une organisation spécifique aux pôles des bactéries. De plus, l’étude de la composition en métaux de P. aeruginosa dans différentes conditions de cultures a pu démontrer que la bactérie adapte sa concentration intracellulaire en métaux selon la composition du milieu extracellulaire. / Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen. It is responsible for a wide range of human diseases. Iron is an essential element for this organism, like for nearly all other organisms. To a lower extent this organisms needs other metals such as zinc, copper,nickel, manganese and other for its survival. To acquire iron, P. aeruginosa secrets two major siderophores, pyoverdine (PVD) and pyochelin (PCH). During the thesis we have shown that the enzymes involved in the biosynthesis of theses siderophores adopt a specific localization at the bacterial cell poles. Furthermore, the study of the metal composition of P. aeruginosa in different growth conditions has shown that this bacterium is able to adaptits internal metal concentration to the extracellular metal availability.
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Evolutionary ecology of social bacterial populations under antibiotic and bacteriophage pressure / Ecologie évolutive des populations bactériennes sociales sous la pression de bactériophages et d’antibiotiquesVasse, Marie 16 December 2015 (has links)
Les bactéries constituent le socle de presque tous les écosystèmes et l’étude de leurs dynamiques face aux perturbations biotiques et abiotiques est essentielle à la compréhension de leur maintien, de leur évolution et de leur diversification. Cette thèse vise à une meilleure appréhension de l’impact des bactériophages et des antibiotiques sur l’écologie évolutive des populations bactériennes et, plus particulièrement, sur l’évolution de leurs comportements sociaux. Dans une première partie, nous avons étudié comment les antibiotiques (Chapitres 1 et 2) et les phages (Chapitre 3) affectent les interactions fondées sur la production de biens publics ainsi que l’évolution de la résistance dans les populations de Pseudomonas aeruginosa, en combinant modélisation mathématique et évolution expérimentale. Nous avons montré que les phages et les antibiotiques favorisent les tricheurs face aux coopérateurs dans les environnements homogènes. Alors que l’avantage des tricheurs permet la croissance de la population et augmente la fréquence de résistance à court terme (Chapitre 1), les populations dominées par les tricheurs finissent par décliner en présence de phages, vraisemblablement suite aux pressions combinées des phages et des tricheurs (Chapitre 3). Dans une seconde partie, nous avons exploré in vitro les interactions complexes entre les phages et les antibiotiques dans le contexte des thérapies combinées. Conformément à la prédiction de la théorie de l’évolution selon laquelle plusieurs moyens de contrôle combinés sont plus efficaces que chacun séparément, nous avons montré que l’usage simultané de phages et d’antibiotiques réduit davantage la survie et la résistance des populations. Si ce résultat principal peut être modulé par différents facteurs tels que la dose d’antibiotiques (Chapitres 4 et 5), le moment d’inoculation (Chapitre 4), et le mode d’action des antibiotiques (Chapitre 5), il persiste sur le long terme (Chapitre 5). Nos résultats soulignent la complexité des interactions entre les effets négatifs des phages et des antibiotiques et l’écologie évolutive des populations bactériennes et apportent de nouveaux éléments à la fois à la compréhension de l’évolution de la socialité et à l’usage thérapeutique potentiel des phages et des antibiotiques. / Bacteria are the basis of virtually all ecosystems and examining their dynamics in the face of biotic and abiotic perturbations is essential to understanding their persistence, evolution and diversification. This thesis is directed towards a better understanding of the impact of phage and antibiotic pressure on the evolutionary ecology of bacterial populations and, in particular, on the evolution of bacterial social behaviours. First, using a combination of mathematical modelling and experimental evolution, we studied how antagonisms in the form of antibiotics (Chapters 1 and 2) and phages (Chapter 3) affect the dynamics of public goods production and strategies, and the evolution of resistance in populations of the bacterium Pseudomonas aeruginosa. We found that both phages and antibiotics favour cheats over cooperators in well-mixed environments. While the advantage to cheats leads to population growth and even increased resistance frequency in the short-term (Chapter 1), the cheat-dominated populations eventually declined in the presence of phage predators, arguably due to the combination of antagonist pressure and cheating load (Chapter 3). Second, based on the evolutionary prediction that multiple control agents will be more efficient at controlling bacterial populations and reducing the evolution of resistance, we investigated in vitro the complex interactions between phages and antibiotics in the context of combined therapies. We showed that the combination of phages and antibiotics decreased population survival and resistance evolution significantly more than either alone. While this main result may be mitigated by several factors such as antibiotic dose (Chapters 4 and 5), the timing of inoculation (Chapter 4), and antibiotic mode of action (Chapter 5), it is also obtained in longer-term assays (Chapter 5). Our results highlight the complexity of the interplay between the negative effects exerted by antibiotics and phages and the evolutionary ecology of bacterial populations, and bring new insights both to the understanding of social evolution and for the potential therapeutic use of phages and antibiotics.
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Le système CupE de la voie chaperonne - "usher" de Pseudomonas aeruginosa : assemblage, fonction et régulation. Identification du système à deux composants PprA-PprB et caractérisation de son régulonGiraud, Caroline 13 July 2011 (has links)
La bactérie à Gram négatif Pseudomonas aeruginosa est un pathogène opportuniste possédant de nombreux systèmes moléculaires contribuant à sa pathogénicité et à son développement selon un mode de vie en biofilm, qui permet une meilleure résistance aux défenses de l’hôte et aux antibiotiques. Parmi ces systèmes, on retrouve les fimbriae assemblés par la voie chaperonne – « usher » (CU). La voie CU implique une protéine formant un pore dans la membrane externe, la protéine « usher », une chaperonne périplasmique et au moins une sous unité piline qui va être assemblée en fimbriae à la surface de la bactérie.Mon travail de thèse a principalement porté sur l’étude du cluster de gènes cupE, qui code pour un système CU du clade σ-fimbriae. Ce système est différent des quatre autres systèmes CU cupA – cupD déjà caractérisés chez P. aeruginosa et appartenant au clade γ4-fimbriae. Indépendamment des gènes codant la protéine « usher » et la chaperonne, ce cluster comprend quatre autres gènes qui codent pour des sous unités pilines atypiques (une piline majeure, deux pilines mineures et une adhésine). Nous avons montré que le système CupE est fonctionnel et permet l’assemblage de fimbriae à la surface de la cellule, assemblage (oligomérisation de la sous unité majeure en fimbirae) pour lequel nous avons montré que l’adhésine est essentielle, au contraire des deux sous unités mineures. Ces fimbriae jouent un rôle important dans la formation et la structuration du biofilm, tant à des étapes précoces que lors des étapes tardives. Excepté une piline mineure, tous les éléments de la fibre sont importants pour la formation du biofilm. Ce cluster de gènes est spécifiquement exprimé en conditions de formation du biofilm et par une approche de mutagenèse aléatoire par transposition, le système à deux composants (TCS) PprA – PprB a été identifié comme un régulateur positif potentiel de ce cluster. L’implication de ce TCS dans la régulation de cupE a été vérifiée et nous avons pu démontrer par des techniques de retard sur gel que ce contrôle est direct et que PprB se lie sur des boites putatives en amont du +1 de transcription de cupE défini par 5’-RACE PCR.Ce TCS ayant été identifié au préalable comme un régulateur positif de pili de type IVB Flp, eux-mêmes acteurs du biofilm chez P. aeruginosa, nous avons caractérisé le régulon du régulateur de réponse PprB. Parmi les nouvelles cibles régulées positivement par PprB, nous avons identifié deux cibles nouvelles dont nous avons débuté la caractérisation. La première est un opéron de quatre gènes codant pour un transporteur ABC impliqué dans la résistance aux antibiotiques spécifiquement en conditions de biofilm et pour une protéine de haut poids moléculaire, substrat potentiel de ce transporteur ABC. Cette protéine, que nous avons renommé AdhA, est effectivement sécrétée par le transporteur ABC et est impliquée dans la cohésion des cellules au cours de la formation du biofilm. Il s’agit d’une nouvelle adhésine participant à la structuration du biofilm chez P. aeruginosa. La seconde cible est un gène codant pour une protéine que nous avons renommée Hvn, homologue aux halovibrines HvnA et HvnB de Vibrio fischeri. Le sécrétome d’une souche délétée du gène hvn est considérablement modifié et son absence d’effet sur la morphologie des cellules eucaryotes par rapport au sécrétome de la souche PAO1 suggère que la protéine Hvn pourrait jouer un rôle dans la virulence de P. aeruginosa.Au travers de ce travail, nous avons caractérisé le système CupE de la voie CU chez P. aeruginosa et montré qu’il assemblait des fimbriae atypiques pouvant avoir un rôle dans les différentes phases de la formation du biofilm et qu’il était sous la régulation directe et positive du TCS PprA – PprB. [...] / The Gram negative opportunistic pathogen Pseudomonas aeruginosa is equipped with molecular systems that contribute to bacterial pathogenesis and biofilm development, this latter being associated with increased resistance to host defenses and antibiotics. Among them, are the fimbriae assembled by the chaperone usher (CU) pathway. The CU pathway involves a protein called the usher that forms a pore in the outer membrane, a periplasmic chaperone and at least one fimbrial subunit assembled into fimbriae at the cell surface.My PhD study mainly focuses on the cupE gene cluster, encoding a CU system from the σ-fimbrial clade. This system is different from all the CU systems cupA – cupD already characterized in P. aeruginosa, all belonging to the γ4-fimbrial clade. Independently from the genes encoding the usher and the chaperone, this cluster comprises four other genes encoding atypical pilins (one major pilin, two minor pilins and one adhesin). We showed that this CupE system is functional and allows the assembly of fimbriae at the cell surface. Unlike the two minor pilins, the adhesin is necessary for the fimbriae assembly (oligomerisation of the major subunit into the fiber). These fimbriae play an important role in biofilm formation and structuration, at early and late steps. Except one minor pilin, all subunits are important for the CupE-dependent biofilm formation. This gene cluster is specifically expressed in biofilm conditions and a random transposon mutagenesis allowed us to identify the two component system (TCS) PprA-PprB as an activator of cupE genes. We verified the implication of this TCS in cupE regulation and, using EMSA, we showed that the PprB control on cupE is direct, with PprB binding onto putative boxes upstream the transcription start of cupE, defined by 5’-RACE PCR.As this TCS was identified before as a positive regulator for the type IVB Flp pilus, another actor in the biofilm formation, we defined the PprB regulon. Among the new targets positively controlled by PprB, we found two new targets that we started to characterize. The first one is a four gene operon encoding an ABC transporter involved in antibiotic resistance specifically in biofilm conditions and a high molecular weight protein, a potential substrate for this ABC transporter. This protein that we renamed AdhA is indeed secreted by this ABC transporter and is implicated in the cohesion between cells during the biofilm formation. It is a new adhesin participating into the biofilm structuration of P. aeruginosa. The second target is a gene encoding a protein that we renamed Hvn, and homologous to HvnA and HvnB halovibrins from Vibrio fischeri. Secretome from an hvn mutant is highly modified and the lack of effect on eukaryotic cell’s morphology in comparison to the PAO1 secretome suggests the Hvn protein can play a role in P. aeruginosa virulence.Through this work, we characterized the cupE system from the CU pathway and showed that this system can assemble atypical fimbriae having a role in the different phases of biofilm formation. This system is under the positive and direct regulation of the TCS PprA – PprB.[...]
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Régulation du transfert de l' îlot de pathogénicité PAPI-1 chez Pseudomonas aeruginosa PA14 / Regulation of the transfer of the PAPI-1 pathogenicity island in Pseudomonas aeruginosa PA14Roux, Nicolas 20 February 2015 (has links)
Les infections à Pseudomonas aeruginosa sont un problème de santé publique important et peu de solutions thérapeutiques sont disponibles face à des souches isolées multi-résistantes. Le séquençage de souches de P. aeruginosa a montré qu'en plus du génome cœur, il existe de nombreux gènes accessoires. La souche PA14 est un isolat clinique hautement virulent, de part la présence de deux îlots de pathogénicité, PAPI-1 et PAPI-2, contribuant de manière individuelle et synergique à la virulence. L'îlot PAPI-1, de 108 kb, est un élément intégratif et conjugatif (ICE), capable de s'auto-transférer à des souches de Pseudomonas par un mécanisme de conjugaison. Le mécanisme de transfert fait intervenir un pilus de type IVb encodé dans l'îlot PAPI-1.Mon travail de thèse a eu pour objectif d'identifier les régulateurs de ce locus pilPAPI-1. Ce travail a montré que ce locus est faiblement exprimé mais qu'il peut être induit par l'ajout d'acide caprique. Par une approche de mutagénèse aléatoire, j'ai démontré qu'il existe au moins deux gènes de fonctions inconnues présents dans PAPI-1 nécessaires à l'activation du locus et au transfert de l'îlot : RL103, qui coderait pour un régulateur transcriptionnel de type Ribbon-Helix-Helix (RHH) et RL102, qui coderait pour une protéine de partitionnement chromosomique. Par une approche transcriptomique, j'ai démontré que ces deux régulateurs sont aussi impliqués dans l'activation de l'expression de plus de 35% des gènes de PAPI-1. Dans leur ensemble, ces résultats ont mis en évidence que RL103 et RL102 sont deux activateurs du transfert de PAPI-1 et ont montré le premier exemple de régulateur de type RHH impliqué dans le transfert d'un ICE. / P. aeruginosa infections have become a serious threat to public health and are very difficult to treat due to the increasingly emergence of strains resistant to all known antibiotics. Sequencing of P. aeruginosa strains showed, that in addition to a conserved core genome, there are variable accessory genes. The PA14 strain is a highly virulent clinical and this is mainly due to two pathogenicity islands, PAPI-1 and PAPI-2, that contribute individually and synergistically to the virulence. The 108 kb PAPI-1 pathogenicity island is an integrative and conjugative element (ICE), capable of self-transferring to any recipient Pseudomonas strain by a conjugative mechanism. The transfer mechanism is mediated by a type IVb pilus, encoded within the PAPI-1 island. My PhD work was aimed to identifying the regulators of this pilPAPI-1 locus. This work showed that this locus is weakly expressed but may be induced by the addition of capric acid. Using a random mutagenesis approach, i have shown that there are at least two genes of unknown function (present in PAPI-1) necessary for activation of the locus pilPAPI-1 and the transfer of the island : RL103, which would encode a Ribbon Helix Helix-like transcriptional regulator and RL102, which would encode a partitioning chromosome protein. Using a transcriptomic approach with microarrays, I demonstrated that these two regulators are also involved in the activation of the expression of more than 35% of PAPI-1 genes. Taken together, these results show that Cpr and RL102 are two activators of the PAPI-1 transfer of PA14 and show the first example of a Ribbon-Helix-Helix transcriptional regulator involved in the transfer of an ICE.
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Inativação fotodinâmica de micro-organismos patogênicos explorando nanopartículas de prata e riboflavinaSÁ, Sandra Regina de 10 February 2015 (has links)
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Previous issue date: 2015-02-10 / FACEPE / A Terapia Fotodinâmica Antimicrobiana (TFDa) tem sido proposta como um método alternativo para o tratamento de infecções causadas por micro-organismos como bactérias e fungos. Tal técnica baseia-se na ação simultânea de uma fonte luminosa e um agente fotossensibilizador, que na presença de luz, geraa produção de oxigênio singleto e espécies reativas de oxigênio que ao interagirem com componentes celulares, promovem a morte celular desses micro-organismos. Esse estudo teve como objetivo avaliar a ação da TFDa explorando nanopartículas de prata (com tamanho médio de 13 nm) e moléculas de riboflavina(157 μM) em conjunto com um LED (Light Emitting Diode) com comprimento de onda no azul (λ=455 ± 20nm), sobre cepas de Streptococcus mutans, Pseudomonas aeruginosae Candida albicans. As cepas de S. mutans foram repicadas em BHI (Brain Infusion Heart) e incubadas em estufa bacteriológica a 37ºC, em atmosfera de 5% de CO2 por 48 he os inóculos de P. aeruginosa foram cultivados e estocados em TSA (Tryptic Soy Agar), em presença de O2, a 37º C, por 24 horas. Decorrido o tempo de incubação, colônias dos micro-organismos foram suspensas em Phosphate Buffered Saline (PBS) a uma densidade de1x107 Unidades Formadoras de Colônia (UFC)/mL.As culturas de Candida albicansforam cultivadas em meio SAB+Y (Agar Sabouraud Dextroseenriquecido com extrato de levedura). A incubação foi realizada à temperaturade 37ºC, durante período de 24 horas. Em seguida,foram preparadassuspensões com concentração de 5x106 células/mL,correspondente à turbidez de 0,5 na escala McFarland.O experimento foi dividido em doze grupos–o grupo controle, o grupo apenas com o tratamento com a luz (L), tratamento com a riboflavina (Rb), utilização da prata (Ag) e o sistema NPsAgRb+L. Posterior aotratamento, foi realizado diluição seriada e as placasincubadas a 37 ºC por 24 horas para bactérias e 48 hs para Candida sp. Ao final foi realizada a contagem de UFC/mLde ambos os micro-organismos. Os resultados foram submetidos à análise estatística descritiva. Na redução de UFC/mL foi estatisticamente significante em culturas de S. mutans com redução de 3 logs no número de células viáveis após 6 minutos de irradiação (p ˂0,05).A inativação fotodinâmica contra P. aeruginosa não foi observada estatística significativa após tratamento (p > 0,05). O mesmo foi observado nas culturas de C. albicans(p > 0,05), concluindo-se que a utilização de nanopartículas próximas ao fotossensibilizador foi eficiente contra bactérias Gram-positivas. / Antimicrobial Photodynamic Therapy (TFDA) has been proposed as an alternative method for treating infections caused by microrganisms such as bacteria and fungi. This technique is based on the simultaneous action of a light source and a photosensitizing agent, in the presence of light, generates the singlet oxygen and reactive oxygen species to interact with cellular components and promote cell death of these microrganisms. This study aimed to evaluate the action of TFDa exploring silver nanoparticles (with an average size of 13 nm) and riboflavin molecules (157 mM) in conjunction with an LED (Light Emitting Diode) with a wavelength in the blue (λ = 455 ± 20nm) on Streptococcus mutans, Pseudomonas aeruginosa and Candida albicans. The strains of S. mutans were subcultured in BHI (Brain Heart Infusion) and incubated in bacteriological incubator at 37 ° C in an atmosphere of 5% CO2 for 48 h and the inocula of P. aeruginosa were grown and stored in TSA (Tryptic Soy Agar) in the presence of O2 at 37ºC for 24 hours. After the incubation time, colonies of microrganisms were suspended in Phosphate Buffered Saline (PBS) at a density of 1x107 colony forming units (CFU)/mL. The Candida albicans cultures were grown in SAB + Y (Sabouraud Dextrose Agar enriched with yeast extract). Incubation was carried out at 37°C temperature for 24 hours. Then, suspensions were prepared with a concentration of 5x106 cells/mL, corresponding to 0.5 on the McFarland turbidity scale. The experiment was divided into twelve groups - a control group, the only group with treatment with light (L), treatment with riboflavin (Rb), use of silver (Ag) and NPsAgRb + L system. After the treatment was carried out serial dilutions and the plates incubated at 37°C for 24 hours for bacteria and 48 hours for Candida sp. At the end it was performed CFU counts / ml of both microrganisms. The results were submitted to descriptive statistical analysis. The reduction of CFU/mL was statistically significant in cultures of S. mutans to 3 logs reduction in the number of viable cells after 6 minutes of irradiation (p ˂ 0.05). The photodynamic inactivation against P. aeruginosa was no statistically significant after treatment (p> 0.05). The same was observed in cultures of C. albicans (p> 0.05), it was concluded that the use of nanoparticles next to the photosensitizer was effective against Gram-positive bacteria.
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