Le Pseudomonas aeruginosa est un pathogène nosocomial majeur. Le clone ST235 est le plus prévalent des clones internationaux dits à hautris que. Ce clone est très fréquemment multi résistant aux antibiotiques, ce qui complique la prise en charge des infections dont il est à l’origine.Malgré son importance clinique, la base moléculaire Du succès du clone ST235 n’est pas comprise.Dans ce travail, nous avons cherché à comprendre l’origine spacio temporelle de ce clone et les bases moléculaires de son succès. A l’aide d’outils bio informatiques existants ,nous avons trouvé que le clone ST235 a émergé en Europe en 1984 et que tous les isolates ST235 produisent l’exotoxine ExoU. Nous avons également identifié 22 gènes Contigus spécifiques de ce clone et impliqués dans l’efflux transmembranaire, dans le traitement de l’ADN et dans la transformation bactérienne. Cette combinaison unique de gènes a pu contribuer à la gravité des infections dues à ce clone et à sa capacité à acquérir des gènes de résistance aux antibiotiques. Ainsi, la diffusion mondiale de ce clone a probablement été favorisée par l’utilisation extensive des fluoroquinolones, puis il est de venu localement résistant aux amino glycosides, aux β-lactamines, et aux carbapénèmes par mutation et acquisition d’éléments de résistance. Nous avons majoritairement utilisé des outils existants,mais avons découvert que les programmes de détection des séquences d’insertions (IS, ayant un rôle important dans l’évolution des génomes bactériens) ne sont pas adaptés aux données dont nous disposions. Nous avons ainsi mis au point un outil (appelé panISa) qui détecte de façon précise et sensible les IS à partir de données brutes de séquençage de génomes bactériens. / Pseudomonas aeruginosa is a major nosocomial pathogen with ST235 being the most prevalent of the so-called ‘international’ or ‘high-risk’ clones. This clone is associated with poor clinical outcomes in part due to multi- and high-level antibiotic resistance. Despite its clinical importance, the molecular basis for the success of the ST235 clone is poorly understood. Thus this thesis aimed to understand the origin of ST235 and the molecular basis for its success, including the design of bioinformatics tools for finding insertion sequences (IS) of bacterial genomes.To fulfill these objectives, this thesis was divided into 2 parts.First, the genomes of 79 P. aeruginosa ST235 isolates collected worldwide over a 27-year period were examined. A phylogenetic network was built using Hamming distance-based method, namely the NeighborNet. Then we have found the Time to the Most Recent Common Ancestor (TMRCA) by applying a Bayesian approach. Additionally, we have identified antibiotic resistance determinants, CRISPR-Cas systems, and ST235-specific genes profiles. The results suggested that the ST235 sublineage emerged in Europe around 1984, coinciding with the introduction of fluoroquinolones as an antipseudomonal treatment. The ST235 sublineage seemingly spreads from Europe via two independent clones. ST235 isolates then appeared to acquire resistance determinants to aminoglycosides, β-lactams, and carbapenems locally. Additionally, all the ST235 genomes contained the exoU-encoded exotoxin and identified 22 ST235-specific genes clustering in blocks and implicated in transmembrane efflux, DNA processing and bacterial transformation. These unique genes may have contributed to the poor outcome associated with P. aeruginosa ST235 infections and increased the ability of this international clone to acquire mobile resistance elements.The second part was to design a new Insertion Sequence (IS) searching tool on next-generation sequencing data, named panISa. This tool identifies the IS position, direct target repeats (DR) and inverted repeats (IR) from short read data (.bam/.sam) by investigating only the reference genome (without any IS database). To validate our proposal, we used simulated reads from 5 species: Escherichia coli, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Staphylococcus aureus, and Vibrio cholerae with 30 random ISs. The experiment set is constituted by reads of various lengths (100, 150, and 300 nucleotides) and coverage of simulated reads at 20x, 40x, 60x, 80x, and 100x. We performed sensitivity and precision analyses to evaluate panISa and found that the sensitivity of IS position is not significantly different when the read length is changed, while the modifications become significant depending on species and read coverage. When focusing on the different read coverage, we found a significant difference only at 20x. For the other situations (40x-100x) we obtained a very good mean of sensitivity equal to 98% (95%CI: 97.9%-98.2%). Similarly, the mean of DR sensitivity of DR identification is high: 99.98% (95%CI: 99.957%-99.998%), but the mean of IR sensitivity is 73.99% (95%CI: 71.162%-76.826%), which should be improved. Focusing on precision instead of sensibility, the precision of IS position is significantly different when changing the species, read coverage, or read length. However, the mean of each precision value is larger than 95%, which is very good.In conclusion, P. aeruginosa ST235 (i) has become prevalent across the globe potentially due to the selective pressure of fluoroquinolones and (ii) readily became resistant to aminoglycosides, β-lactams, and carbapenems through mutation and acquisition of resistance elements among local populations. Concerning the second point, our panISa proposal is a sensitive and highly precise tool for identifying insertion sequences from short reads of bacterial data, which will be useful to study the epidemiology or bacterial evolution.

Pseudomonas aeruginosa é uma gamma-proteobacteria ubíqua, sendo a principal causa de infecção hospitalar dentre todos os patógenos relacionados com pneumonia em UTI. A defesa do hospedeiro se dá por vários mecanismos como a liberação, por fagócitos, de espécies reativas de oxigênio, como o ânion superóxido (O2?-), peróxido de hidrogênio (H2O2) e o radical hidroxila (OH?) para combater o patógeno. LsfA pertence à família das peroxirredoxinas (Prx) e ao sub-grupo de Prxs que contém somente uma cisteína catalítica (denominadas 1-Cys Prx). Prxs são enzimas capazes de remover peróxidos (incluindo peroxinitrito) em velocidades muito elevadas. Além disso, LsfA está relacionada a patogenicidade de P. aeruginosa. Dentro desse contexto, o objetivo do presente trabalho é a caracterização bioquímica e estrutural de LsfA; que pode possibilitar a identificação de inibidores dessa enzima antioxidante. Por outro lado, caracterizando cineticamente reações de oxido-redução de LsfA e caracterizando seus mecanismos de ação, podemos identificar seus substratos biológicos. Dessa maneira, utilizando diferentes técnicas, determinamos as constantes de segunda ordem de LsfA com H2O2 (na ordem de 107 M -1.s -1); para terc-butilhidroperóxido (na ordem de 106 M-1.s-1) e peroxinitrito (na ordem de 107 M-1.s-1). A redução de LsfA por ascorbato foi descrita previamente por nosso grupo (na ordem de 103 M-1.s-1); e aqui apresentamos dados preliminares sobre a redução dessa 1-Cys Prx por GSH. Além disso, fomos capazes de determinar a estrutura cristalográfica de LsfA em sua forma oxidada e superoxidada, com resolução de 2.6 e 2.0 ? respectivamente; que, como esperado, se apresentou no estado dimérico, em ambos os casos. Descrevemos aqui características sobre a estrutura do sítio ativo de LsfA, que apresenta mais eletronegativo, com a cisteína peroxidásica desprotonada, e mais hidrofóbico. Na estrutura de LsfA superoxidada, observamos a co-cristalização dessa enzima com uma molécula de polietileno glicol que pode estar mimetizando um substrato. Portanto, esses estudos levantaram importantes informações estruturais e bioquímicas de uma enzima antioxidante envolvida com a virulência de P. aeruginosa / Pseudomonas aeruginosa is a ubiquous gamma-proteobacteria that is the main cause of hospitalar infections among all pathogens related with pneumonia. Host defenses against pathogens are mainly by phagocytes, which releases reactive oxygen species, such as superoxide (O2?-), hydrogen peroxide (H2O2) and hydroxyl radical (OH?) to fight against pathogen. LsfA belongs to peroxiredoxins (Prx) family; and to the 1-Cys Prx sub-group (Prx6 sub-family) that possess only one catalytic cysteine. Prx are enzymes can remove peroxides with extremely high efficiency. LsfA was already related with P. aeruginosa virulence. So, the aim of the present work is the structural and biochemical characterization of LsfA, which may enable the discovery of inhibitors. Furthermore, the investigation of the kinetics and the mechanism of catalysis of LsfA may give insights on the chemical nature of its biological substrates. Therefore, using different techniques; the second order rate constants of LsfA with H2O2 (107 M -1.s -1), tert-butylhydroperoxide (106 M -1.s -1) and peroxynitrite (107 M -1.s -1) were determined. Our group has already determined the rate constant between ascorbate and LsfA (103 M -1.s -1) and preliminary data on the reduction of this 1-Cys Prx by glutathione is described. Furthermore, two crystallographic structures of LsfA were elucidated in distinct oxidative states (sulfenic and sulfonic acid in the CP), both in the dimeric state; at 2.6 and 2.0 ? resolution respectively. Features in the LsfA active site are also described here, such as poor exposure to the solvent. In the LsfA crystal structure where Cp is hyperoxidized to sulfinic acid, we observed the presence of an electronic density compatible with a PEG molecule that might be mimicking one of the possible substrates. Therefore, relevant structural and biochemical information were gained with our studies about an antioxidant enzyme involved with P aeruginosa virulence

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 86-98).

Physiological and compositional changes in Pseudomonas aeruginosa (ATCC 9027) were monitored during, growth on various petroleum compounds in a chemically-defined medium. Growth of P. aeruginosa was observed when furnace oil, kerosene, aviation fuel, light crude oil and hexadecane were used as carbon and energy sources. A variable and extended lag period before active growth was achieved was characteristic of petroleum-grown cells as compared to glucose-grown cells. Growth on the petroleum hydrocarbons, compared with that on glucose, resulted in changes in cell lipid composition, outer membrane proteins, cell-surface hydrophobicity, surface-tension, and pH changes in the growth medium during transition from early to late-log phase. Cell composition and physiology of cells grown in the petroleum mixtures varied due to differences in the chemical composition of the material. Production of an exopolymer (characterized as a peptidoglycolipid) was associated with petroleum-grown cells but not with glucose-grown cells. The above differences illustrate some of the dynamic and physiological and biochemical changes the microorganism undergoes to access its hydrophobic carbon and energy source.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [64]-73).

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 163-178).

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-10-20T12:53:52Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) ANÁLISE FENOTÍPICA E GENÉTICA DE FATORES VIRULÊNCIA DE ISOLADOS CLÍNICOS DE Pseudomonas aeruginosa MULTIDROGA-SENSÍVEL E MULTIDROGA-RESISTENTE.pdf: 1188065 bytes, checksum: 3d201d9ccfc4ba0e5a0ef45cce7f7056 (MD5) / Made available in DSpace on 2016-10-20T12:53:52Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) ANÁLISE FENOTÍPICA E GENÉTICA DE FATORES VIRULÊNCIA DE ISOLADOS CLÍNICOS DE Pseudomonas aeruginosa MULTIDROGA-SENSÍVEL E MULTIDROGA-RESISTENTE.pdf: 1188065 bytes, checksum: 3d201d9ccfc4ba0e5a0ef45cce7f7056 (MD5) Previous issue date: 2016-02-29 / CNPq / Pseudomonas aeruginosa é um patógeno humano oportunista responsável por causar uma enorme variedade de infecções agudas e crônicas com níveis significativos de morbidade e mortalidade. A sua plasticidade genética e metabólica possibilitou o desenvolvimento de isolados multidroga-resistentes (MDR) e a capacidade de expressar de inúmeros fatores de virulência. Este trabalho teve como objetivo correlacionar o padrão de susceptibilidade antimicrobiana, a produção de fatores de virulência através de técnicas fenotípicas (protease alcalina, hemolisina, fosfolipase C, lipase e pigmentos) e genéticas (genes aprA, lasA, lasB, plcH e toxA) e a variabilidade genética de 30 isolados clínicos de P. aeruginosa isoladas de diferentes sítios de infecção, sendo 15 isolados multidroga-sensível (MDS) e 15 MDR. Nossos resultados revelaram que 50% dos isolados foram resistentes a ceftazidima, sendo as cefalosporinas a classe antimicrobiana com mais isolados resistentes, principalmente entre isolados MDR onde todos foram resistentes. Entre os isolados MDS, todos foram sensíveis a carbapenêmicos e quinolonas. A grande diversidade apresentada no perfil de susceptibilidade às classes de antimicrobianos sugere a existência de associação de diversos mecanismos de resistência. Entre os isolados 53% foram amostras de secreção traqueal, entre estes todos os isolados sensíveis a todos os antimicrobianos testados. Em relação aos fatores de virulência os isolados MDR apresentaram menor produção de piocianina e lipase, e menor detecção dos genes toxA e lasA, enquanto os MDS, apresentaram menor produção de hemolisina e fosfolipase C. Não houve diferença entre os grupos para produção de protease alcalina e o gene aprA. Todos isolados apresentaram produção de piocianina e os genes lasB e plcH. Em relação ao perfil genético, foi encontrada uma grande diversidade, em um total de 30 isolados foi possível observar 28 perfis genéticos. A presença dos clones ocorreu entre os isolados MDR. Embora alguns estudos relatem que o acréscimo de mecanismos de resistência leva a diminuição dos fatores de virulência, sugerindo assim que, as cepas de P. aeruginosa MDR têm a virulência diminuída quando comparada com cepas MDS, neste trabalho dos resultados obtidos não constatamos esta tendência para produção de protease alcalina, hemolisina, fosfolipase C e para a detecção do gene aprA, sugerindo que esta correlação seja multifatorial. Contudo, a ocorrência destes fatores de virulência em quase todos os isolados estudados sugere um elevado nível de patogenicidade dos mesmos. Concluímos portanto, que P. aeruginosa é um patógeno capaz de acumular inúmeros fatores de virulência e frequentemente associado à multirresistência, o que dificulta o tratamento de infecções causadas por esta bactéria. / Pseudomonas aeruginosa is an opportunistic human pathogen responsible for causing a wide variety of acute and chronic infections with significant levels of morbidity and mortality. Its genetic and metabolic plasticity enabled the development of multidrug-resistant (MDR) strains and the ability to express countless virulence factors. This paper aimed to correlate the pattern of antimicrobial susceptibility, the production of virulence factors through phenotyping techniques (alkaline protease, hemolysin, phospholipase C, lipase and pigments) and genetic (gene aprA, lasB, lasB, plcH e toxA) and the genetic variability of 30 clinical isolates of P. aeruginosa isolated from different sites of infection, 15 isolates multidrug-sensitive (MDS) and 15 MDR. Our results showed reveal that 50% of the isolates were resistant to ceftazidime, cephalosporin was the antimicrobial class with more resistant isolates, especially isolates MDR that were totally resistant to them. Among the isolated MDS, all were sensitive to carbapenems and quinolones. The large diversity presented in the susceptibility profile to antimicrobial classes suggests the existence of an association of several resistance mechanisms. Among the isolated 53% came from tracheal secretions, among them all isolates susceptible to all antimicrobials tested. Regarding virulence factors MDR isolates presented lower production pyocyanin and lipase, and lower detection toxA e lasA genes, since the MDS, showed lower production of hemolysin and phospholipase C. There was no difference between groups for the production of alkaline protease and aprA gene. All isolates presented pyocyanin production and lasB and plcH genes. In relation to genetic profile, it was verified a large diversity, in a totality of 30 isolates was observed 28 genetic profiles. The presence of clones occurred among the MDR isolates. Even though some studies to report that the increase of resistance mechanisms leads to the reduction of virulence factors, suggesting that the strains of P. aeruginosa MDR have decreased virulence strains compared with MDS, this work the results obtained we have not found this tendency to alkaline protease production, hemolysin, phospholipase C and for detecting aprA gene, suggesting that this correlation is multifactorial. However, the occurrence of these virulence factor in almost all isolates studied suggests a high level of pathogenicity the same. Therefore, it can be concluded that, P. aeruginosa is a pathogen with ability to accumulate several virulence factors and often associated to multiresistant complicating the treatment of infections caused by this bacterium.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / O objetivo deste trabalho foi avaliar a glicerina resultante da transesterificaÃÃo do Ãleo de mamona como fonte de carbono e nutrientes para P. aeruginosa LAMI. O efeito da concentraÃÃo de nutrientes e de condiÃÃes ambientais foi avaliado de acordo com dois planejamentos fatoriais completos sobre o crescimento celular, produÃÃo de biossurfactante e propriedades tensoativas do surfactante produzido. A anÃlise estatÃstica dos dados foi realizada pelo software Statistica 6.0. Avaliou-se o efeito da concentraÃÃo de glicerina e de nitrato de sÃdio e do tamanho do inÃculo, de acordo com um planejamento fatorial 23. Uma anÃlise dos efeitos mostrou que o aumento da concentraÃÃo de nitrato e a reduÃÃo da concentraÃÃo de glicerina favoreceram a produÃÃo de biossurfactantes atingindo-se uma concentraÃÃo mÃxima de 1,6 g/L de ramnose. A partir deste resultado, realizou-se um planejamento fatorial completo 24 avaliando-se os fatores concentraÃÃo de nitrato e de fosfato, pH e temperatura. Os resultados mostraram que a reduÃÃo da razÃo carbono/nitrogÃnio (C/N), com um Ãtimo equivalente a 12, favoreceu a produÃÃo de ramnolipÃdeos por P. aeruginosa LAMI, bem como a reduÃÃo da concentraÃÃo de fosfato em pH 7,0 e temperatura de 37 ÂC. Nestas condiÃÃes obteve-se 2,3 g/L de ramnose, atingindo-se coeficientes de rendimento em termos de substrato (YP/S) e de biomassa (YP/X) de 0,103 g/g e 3,13 g/g,respectivamente. A produtividade volumÃtrica mÃxima foi 31,94 mg/Lh. A cinÃtica de crescimento celular e produÃÃo de biossurfactantes foi avaliada, variando-se a razÃo C/N de 21 a 86. Os perfis de produÃÃo de biomassa e de ramnolipÃdeos sugeriram uma cinÃtica mista, semiassociada ao crescimento. O biossurfactante obtido de acordo com a melhor condiÃÃo de cultivo foi capaz de formar emulsÃes com querosene, Ãleo de soja, Ãster metÃlico e Ãleo naftÃnico, com Ãndice de emulsificaÃÃo de, aproximadamente, 60 %. Uma atividade emulsificante equivalente a 3,25 unidades mostrou que o biossurfactante foi capaz de formar emulsÃes Ãleo-Ãgua. O biossurfactante foi extraÃdo do meio de cultivo livre de cÃlulas e submetido a purificaÃÃo por cromatografia. A cromatografia em camada delgada mostrou a presenÃa de dois produtos majoritÃrios. O espectro de ressonÃncia magnÃtica nuclear H1 apresentou deslocamentos quÃmicos caracterÃsticos de grupamentos quÃmicos que constituem uma molÃcula de diramnolipÃdeo tipo Rha-Rha-C10C10. Entretanto, a elucidaÃÃo completa da estrutura do ramnolipÃdeo deve ser complementada por anÃlises espectroscÃpicas de maior resoluÃÃo / The aim of this work was analysing the glycerine from castor oil transesterification as a source of carbon and nutrients to P. aeruginosa LAMI. Nutrients concentration and environmental conditions were studied using two complete factorial planning, with cellular growth, biosurfactant production and product surface active properties as response variables. The statistic analysis was done using the software Statistica 6.0. First of all, inoculum size and concentrations of glycerine and NaNO3 were analysed with a 23 factorial planning. The increase in nitrate concentration and a decrease in glycerine concentration favored biosurfactants production, reaching a maximum rhamnose concentration of 1.6 g/L. A complete 24 factorial planning was planned based on these results. Nitrate and phosphate concentrations, pH and temperature were selected factors. Results showed that a decrease in carbon/nitrogen ratio, with an optimum of 12, and phosphate concentration favored rhamnolipid production by P. aeruginosa LAMI at pH 7,0 and 37 ÂC. A rhamnose concentration of 2.3 g/l was obtained, with product yields on substrate and biomass of 0.103 and 3.13g/g,respectively. The volumetric productivity was 31.94 mg/L.h. The influence of carbon/nitrogen ration, from 21 to 86, on growth kinetics and biosurfactant production was studied. Biomass and rhamnolipids production behavior suggest a mixed kinetics, semi-associated to growth. The biosurfactant produced using the optimized conditions formed emulsions with kerosene, soybean oil, methyl esters (biodiesel) and naphtenic oil, with emulsification index of about 60%. An emulsification activity of 3.25 units was also obtained, showing that the biosurfactant may be used to forme oil-water emulsions. Finally, the biosurfactant was extracted from a free-cell fermented medium and submited to chromatographic purification. The analytical thin layer chromatography showed the presence of two mainly products. The H1 nuclear magnetic spectra showed characteristic signals of chemical groups that are typical of a dirhamnolipid Rha-Rha-C10C10 molecule. However, a complete explanation of the rhamnolipid structure must be completed by high resolution spectroscopy analysis

FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / Neste trabalho avaliamos a atividade biolÃgica de lectinas de sementes de Erythrina fusca e velutina, de algas marinhas Hypnea musciformes, Bryothamnion seaforthii e triquetrum e do diterpeno casbano, um produto natural isolado de Croton nepetaefolius, sobre Pseudomonas aeruginosa ATCC 10145. Foi comparada a aÃÃo in vitro das 5 lectinas e do diterpeno casbano, sobre colÃnias de P. aeruginosa, em placas de poliestireno. Investigada a aÃÃo das lectinas de alga marinha H.musciforme, de sementes de Erythrina velutina, e do diterpeno casbano, no processo de formaÃÃo do biofilme bacteriano de P.aeruginosa, em placas de poliestireno; e identificado entre as lectinas de E.velutina, H.musciforme e diterpeno casbano, aquele com maior potencial de aplicaÃÃo no controle do crescimento de colÃnias de P. aeruginosa. As lectinas testadas nÃo foram capazes de inibir o crescimento e a formaÃÃo de biofilme de Pseudomonas aeruginosa nas condiÃÃes experimentadas. Por outro lado, diterpeno casbano, na concentraÃÃo de 500 μg/mL em 18 horas, foi capaz de inibir o crescimento de P. aeruginosa em 40%, comparado ao controle positivo. Esta inibiÃÃo foi observada atà uma concentraÃÃo de 125 μg/mL. Entretanto, nÃo foi observada inibiÃÃo da formaÃÃo do biofilme da P. aeruginosa nas concentraÃÃes utilizadas neste estudo. / In this study the biological activity of seeds lectins from Erythrina velutina and fusca, marine algae Hypnea musciformis, Bryothamnion seaforthii and triquetrum and the diterpene casbane, a natural product isolated from Croton nepetaefolius was evaluated upon Pseudomonas aeruginosa ATCC 10145. We compared the in vitro effect of lectins and diterpene casbane on colonies of P. aeruginosa in microtiter plates. Investigated the action of lectins from marine algae H. musciforme of seeds of Erythrina velutina, and diterpeno casbano in the process of formation of P. aeruginosa biofilm on polystyrene plates, and identified among lectins: E. velutina, H. musciforme and diterpene casbane, the one with greater potential for application in controlling the growth of colonies of P. aeruginosa. The lectins tested were able to inhibit growth and biofilm formation of P. aeruginosa in the studied conditions. Moreover, diterpene casbane at a concentration of 500 mg/mL in 18 hours, was able to inhibit the growth of P. aeruginosa in 40%, compared to positive control. This inhibition was observed until a concentration of 125 mg/mL. However, the inhibition of biofilm formation of P. aeruginosa there was no observed at the concentrations used in this study.