Orientador: João Pessoa Araújo Júnior / Resumo: O Brasil é o segundo maior produtor de carne bovina do mundo e para se manter competitivo tem objetivado melhorias em higiene e segurança alimentar. Micro-organismos deteriorantes diminuem a vida de prateleira dos produtos e podem viabilizar patógenos em biofilmes predominantemente heterogêneos. Pseudomonas aeruginosa são bactérias mais comuns em carnes, e tem como característica a alta capacidade de produção de biofilme. Sendo ambientais, a contaminação da carne é facilitada por falhas de higiene e boas práticas, sendo relevante estudos sobre a sua presença em produtos cárneos. Neste trabalho, objetivou-se estudar a intensidade de produção de biofilme e sua expressão gênica por cepas de P. aeruginosa isoladas em planta de processamento bovino. As amostras foram obtidas por suabes de carcaças e superfícies em planta de processamento bovino totalizando sete coletas, divididas em dois dias, amostrando-se 22 pontos em cada coleta. Os isolados foram confirmados físico-quimicamente. A produção de biofilme por espectrofotometria classificou a intensidade em fortes, moderadas, fracas e não produtoras. Foram isoladas 32 cepas, das quais 4 demonstraram forte produção de biofilme, 3 moderadas, 4 fracas e 8 não produtoras. Foi predominante a contaminação em carcaças recém abatidas, anteriormente à refrigeração. As amostras foram confirmadas usando o alvo oprL em análise por qPCR, e a comparação da expressão de alginato, algU e algD posteriormente normalizados pelo gene 16S foi realizada... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Brazil is the second largest beef producer in the world and in order to remain competitive, it has aimed at improving hygiene and food safety. Damaging microorganisms decrease the shelf life of products and may render pathogens viable in predominantly heterogeneous biofilms. Pseudomonas aeruginosa are the most common bacteria in meats, characterized by high biofilm production capacity being environmental, meat contamination is facilitated by good practices hygiene faults being relevant studies on their presence in meat products. The objective of this study was to determine the intensity of biofilm production and its gene expression by strains of P. aeruginosa isolated from a bovine processing plant. Samples were obtained by carcass swabs and surfaces in a bovine processing plant, totaling seven samples, divided in two days, sampling 22 points in each collection. The isolates were physicochemically confirmed. The biofilm production by spectrophotometry classified the intensity as strong, moderate, weak and non-producing. Thirty-two strains were isolated, 4 of which showed strong biofilm production, 3 moderate, 4 weak and 8 non-producing. Contamination was predominant in freshly slaughtered carcass prior to refrigeration. All samples were confirmed in qPCR analysis with the oprL target, and gene expression of strong and weak samples were developmented with alginate genes, algU and algD, normalized by 16S gene. Expression analyzes of the algU and algD genes did not demonstrate s... (Complete abstract click electronic access below) / Mestre

Cyanobacteria produce a variety of secondary metabolites which possess amongst others antifungal, antibacterial, and antiviral properties. Being primary producers they are also a vital component within the food web. However, certain strains also produce toxic metabolites such as the hepatotoxins microcystin (MC) and nodularin (NOD). Their toxicity in combination with the increasing global occurrence has resulted in a drinking water guideline limit of 1 μg L-1 being issued by the World Health Organisation (WHO). However, these toxins are not only present in water, but can be accumulated by fish and shellfish. Currently, no regulations regarding cyanotoxin contaminated seafood has been established despite similar toxicity to routinely monitored marine toxins such as domoic acid (DA). To facilitate regular monitoring, a high performance liquid chromatography photo diode array (HPLC-PDA) analysis method for the detection of DA was optimised to enable the simultaneous detection of DA and nine cyanotoxins. This method was then utilised to determine cyanotoxin concentration in laboratory cyanobacteria strains. To assess the accumulation and depuration of cyanotoxins in the common blue mussel Mytilus edulis, three feeding trials were performed. During these, mussels were exposed to two cyanobacteria strains, Nodularia spumigena KAC66, Microcystis aeruginosa PCC 7813, both individually and simultaneously. A rapid dose dependent accumulation of cyanotoxins was observed with maximum concentration of 3.4 -17 μg g-1 ww accumulated by M. edulis, which was followed by a much slower depuration observed. During the final feeding trial, with N. spumigena KAC 66 and M. aeruginosa PCC7813, cyanotoxins were still detectable following 27 days of depuration. Mortality in all studies was 7% or less indicating that most mussels were unaffected by the maximum dose of 480 μg L-1 NOD (feeding study 1), 390 μg L-1 MC (feeding study 2), or 130 μg L-1 total cyanotoxins (feeding trial 3), respectively. Mortality in negative control tanks was lower throughout all three feeding trials ( < 1 - 2.6%). Consumption of a typical portion size (20 mussels) would result in ingestion of cyanotoxins at levels significantly higher than the WHO recommended tolerable daily intake (TDI) of 2.4 μg NOD and/or MCs for a 60 kg adult. This value was exceeded not only during the exposure period (maximum levels 270 - 1370 μg cyanotoxins per 20 mussels), but also at the end of the depuration period 39-600 μg cyanotoxins per 20 mussels. These results illustrated that cyanotoxin monitoring of seafood should be considered not only during, but also following bloom events. In an attempt to investigate the cyanotoxin budget of the experimental system, not only mussels, but cyanobacteria cultures, the tank water, and the mussel faeces were also analysed for their cyanotoxin content. Results showed that large quantities of MCs and NOD were unaccounted for during all exposure trials. The combined effect of cyanotoxin metabolism in M. edulis, biotic and/or abiotic degradation, protein binding, and losses during the extraction and analysis were thought to have contributed to the unaccounted cyanotoxin fraction. Mussel flesh was analysed for the presence of glutathione or cysteine conjugates, however, there was no evidence of their occurrence in the samples tested. Due to these discrepancies in the toxin budget of the system, the introduction of correction factors for the analysis of cyanotoxins in M. edulis was suggested in order to protect the general public.

Pseudomonas aeruginosa é um importante patógeno humano oportunista que causa graves infecções em pacientes imunocomprometidos e em pacientes portadores de fibrose cística (FC). O objetivo deste trabalho foi avaliar a capacidade de nanopartículas de albumina sérica bovina associadas aos antígenos totais de P. aeruginosa em proteger camundongos do desafio pela via nasal por este patógeno. Camundongos foram imunizados pela via subcutânea usando antígenos totais de P. aeruginosa, nanopartículas vazias ou nanopartículas associadas aos antígenos totais de P. aeruginosa nos dias 0, 7 e 14. A produção de anticorpos IgG total e os subtipos IgG1 e IgG2a foram avaliados por ELISA. Os camundongos imunizados foram desafiados com P. aeruginosa e os pulmões foram coletados para estudos histopatológicos e detecção da bactéria no pulmão. Nossos resultados mostram que camundongos imunizados com nanopartículas associadas aos antígenos totais de P. aeruginosa produziram altos títulos de anticorpos IgG1 anti-P. aeruginosa e títulos significativos de IgG2a. Os dados também mostram que camundongos imunizados com nanopartículas associadas aos antígenos totais de P. aeruginosa e desafiados com a bactéria apresentaram diminuição dos sinais inflamatórios, com uma redução significativa na intensidade e concentração de células inflamatórias, diminuição da hemorragia, sinais de edema e hiperemia nos pulmões se comparado aos outros grupos. A bactéria não foi detectada nos pulmões dos animais imunizados e infectados nos tempos analisados. Portanto, essa formulação é capaz de induzir uma resposta protetora nos animais infectados, sendo portanto, uma promissora plataforma para vacinas contra P. aeruginosa. / Pseudomonas aeruginosa is an important opportunistic human pathogen that causes severe infections in immunocompromised patients and also in cystic fibrosis patients. The aim of this work was to evaluate the ability of bovine serum albumin nanoparticles with entrapped antigens extracted from P. aeruginosa to protect mice from intranasal challenge with this pathogen. Mice were immunized via the subcutaneous route using P. aeruginosa antigens, empty nanoparticles or nanoparticles with entrapped P. aeruginosa antigens on days 0, 7 and 14. The total IgG antibody production and specific IgG1 and IgG2a titer were measured by ELISA. Immunized mice were challenged with live P. aeruginosa and their lungs were collected for histopathology studies and for detection of bacteria in their lungs. Our data showed that NPPa-vaccinated mice presented a high anti-Pseudomonas IgG1 and a low IgG2a antibody titles and decreased inflammatory signs, with significant reduction in intensity and concentration of inflammatory cells, lower hemorrhagic, edema and hyperemia signs in the lungs of challenge mice with live P. aeruginosa if compared to the other groups. The bacteria was not detected in the lungs from infected mice in the analyzed time. Therefore, this formulation is able to induce a functional response in an animal model of infection and thereby it is a promising platform for P. aeruginosa vaccines. / Fundação de Amparo à Pesquisa do Estado de Minas Gerais - FAPEMIG

Evalúa la eficacia de dos desinfectantes de uso hospitalario (Dezavid® y Supersafe-D®) frente a biopelículas de Pseudomonas aeruginosa y Staphylococcus aureus formadas sobre acero inoxidable. Adicionalmente se evaluó la eficacia del hipoclorito de sodio (lejía tradicional) de uso doméstico. Se formó durante 6 días una biopelícula sobre discos de acero inoxidable de 1 cm de diámetro y 1 mm de espesor, posteriormente se les trató con concentraciones de 0,05 %, 0,10 % y 1,00 % de Dezavid®; Supersafe-D® y lejía tradicional, dejando actuar el desinfectante durante el tiempo recomendado por el fabricante. Posteriormente se cuantificó las UFC/disco en comparación con un grupo de discos que no fueron sometidos a ningún desinfectante. Los desinfectantes con mayor eficacia fueron Dezavid® 1,00 %; 0,10 % y Supersafe-D® para Staphylococcus aureus ATCC y cepa clínica. En el caso de Pseudomonas aeruginosa ATCC y cepa clínica los desinfectantes con mayor eficacia fueron Dezavid® 1,00 % y Supersafe-D®. La lejía (1:20) tuvo igual eficacia que Dezavid® 1,00 %; 0,10 % y Supersafe-D® para Staphylococcus aureus ATCC y cepa clínica. En el caso de Pseudomonas aeruginosa ATCC y cepa clínica, la lejía (1:20) tuvo igual eficacia que Dezavid® 0,10 %. / Tesis

Objetivo: Avaliar a influência da infecção por Pseudomonas aeruginosa formadora de biofilme na penetração pulmonar da tobramicina através da modelagem populacional dos dados de plasma e microdialisado em animais sadios e infectados. Metodologia: A pneumonia foi desenvolvida através de inoculação de P. aeruginosa (cepa PA14) pela via intratraqueal (109 UFC/mL) a ratos Wistar. Sete dias após a inoculação os animais infectados (n = 5) receberam tobramicina 10 mg/kg i.v. bolus. Animais saudáveis (n = 6) foram utilizados como controle. As concentrações livres pulmonares foram coletadas por microdiálise (sonda CMA/20). As sondas de microdiálise foram calibradas in vitro através de diálise e retrodiálise e in vivo utilizando retrodiálise. A ligação da tobramicina às proteínas plasmáticas foi determinada por microdiálise. As concentrações do fármaco nas amostras foram determinadas por cromatografia líquida em tandem com espectrometria de massas (CLAE-EM/EM) utilizando metodologia validada. Os parâmetros farmacocinéticos foram determinados por abordagem não-compartimental (Phoenix®) e modelagem populacional (popPK) (Monolix®). Resultados e Discussão: A recuperação relativa (RR) das sondas foi independente da concentração de tobramicina e inversamente proporcional ao fluxo de perfusão. A RR determinada in vivo foi de 27,64 % ± 7,70 para animais sadios e 24,47 % ± 1,66 para animais infectados. A ligação às proteínas plasmáticas foi de 11,3 ± 1,9%. A infecção com formação de biofilme não alterou a farmacocinética plasmática da tobramicina, entretanto reduziu em cerca de 70% a penetração pulmonar do fármaco. As concentrações plasmáticas e teciduais foram simultaneamente descritas por um modelo farmacocinético populacional de dois compartimentos, tanto em animais sadios como infectados. A infecção, utilizada como covariável categórica, permitiu descrever as alterações no volume do compartimento periférico e na constante de eliminação do compartimento central devido à infecção. Conclusões: As concentrações plasmáticas da tobramicina, utilizadas para ajuste posológico, superestimam as concentrações ativas no pulmão infectado. O modelo popPK descrito permite a previsão das concentrações livres pulmonares da tobramicina em pulmão infectado, podendo auxiliar na otimização da terapia de pneumonias com P. aeruginosa formadora de biofilme. / Objective: To evaluate the influence of biofilm-forming Pseudomonas aeruginosa infection on tobramycin lung penetration by population pharmacokinetic modeling of plasma and microdialysate data in healthy and infected rats. Methodology: The infection was developed by intratracheal inoculation (109 CFU/mL) of P. aeruginosa (PA14 strain) to Wistar rats. In order to determine plasma and tissue concentrations, seven days after the inoculation the infected animals (n = 5) received tobramycin 10 mg/kg i.v. bolus dose via femoral vein. A healthy group (n = 6) was used as control. Free lung concentrations were determined in microdialysate samples obtained using CMA/20 probes. Microdialysis probes were calibrated in vitro by dialysis and retrodialysis and in vivo by retrodialysis. Tobramycin plasma protein binding was determined by microdialysis. Plasma and tissue concentrations were quantified by a developed and validated liquid chromatography in tandem with mass spectrometry (LC-MS/MS) method. Compartmental and non-compartmental analyses were carried out by Monolix™ and Phoenix™ software, respectively. Results and Discussion: Microdialysis probes relative recovery was independent of the tobramycin concentration and is inversely proportional to the perfusion flow rate investigated. The in vivo probe recovery was 27.64 % ± 7.70 (healthy rats) and 24.47 % ± 1.66 (infected rats). The plasma protein binding was 11.3 ± 1.9%. The biofilm-forming lung infection did not alter tobramycin plasma pharmacokinetics, however, reduced lung penetration in about 70%. The plasma and tissue concentrations-time profiles were simultaneously described by a two compartment popPK model in healthy and infected animals. The infection process, used as categorical covariate allowed describing the changes observed in the volume of the peripheral compartment and in constant rate of elimination from the central compartment. Conclusions: Tobramycin plasma concentrations, used for dosing adjustments, overestimate active concentrations in infected lung. The described popPK model allows predicting free tobramycin lung concentrations in infected lung and could be useful to optimize the treatment of pneumonia caused by biofilm-forming P. aeruginosa with this drug.

Pseudomonas aeruginosa (Pa) est un pathogène opportuniste responsable d’infections pulmonaires aigues graves chez les malades prédisposés. Devant l’émergence croissante de la résistance aux antibiotiques, le développement de thérapeutiques alternatives adjuvantes est indispensable et nécessite la compréhension des interactions hôte-pathogènes au cours de l’infection. La voie métabolique de dégradation du tryptophane appelée voie des kynurénines produit chez l’hôte des métabolites aux propriétés immunomodulatrices connues. Récemment, l’existence de cette voie a été mise en évidence chez Pa, bien que la nature et la quantité de métabolites produits ne soient pas parfaitement connus. La production bactérienne de kynurénines pourrait interférer avec la mise en place de la réponse immunitaire de l’hôte et sa régulation au cours des différentes phases de l’infection, altérant la balance immunitaire pulmonaire au profit du pathogène. A ce titre, la voie des kynurénines de Pa constituerait une cible thérapeutique potentielle. L’objectif de ce travail de thèse est d’étudier l’implication de la voie des kynurénines de Pa dans la virulence bactérienne et la réponse immune de l’hôte dans un modèle murin d’agression respiratoire aiguë. Pour cela, les souris sont infectées avec des souches sauvages de Pa, avec des souches mutantes ΔkynA, non productrices de kynurénines, et des souches ΔkynU, surproductrices de kynurénines. Les interactions potentielles avec la voie des kynurénines de l’hôte sont explorées en inhibant la première enzyme de la voie métabolique, l’indoleamine-2,3-dioxygenase (IDO). Enfin, le rôle du récepteur arylhydrocarbone (AhR), récepteur connu des kynurénines et impliqué dans l’immunité pulmonaire, est exploré en comparant la réponse à l’infection de souris AhR KO à celle des souris sauvages. Dans ce travail, nous décrivons tout d’abord la production des différents métabolites de la voie des kynurénines de Pa in vitro et in vivo dans le modèle d’infection respiratoire aigue, en décrivant pour la première fois la production d’acide kynurénique et de 3-hydroxy-kynurénine pour cette bactérie. Ensuite, nous montrons que les kynurénines bactériennes interfèrent avec la réponse immune de l’hôte, en majorant le recrutement cellulaire alvéolaire, tout en atténuant le niveau d’inflammation et l’activation des cellules présentatrices d’antigènes. Enfin, nous rapportons que l’IDO et l’AhR sont impliqués dans cette immunomodulation, faisant des kynurénines bactériennes des agents du dialogue hôte-pathogène au cours de l’infection respiratoire aigue. A la lumière de ces résultats, la voie des kynurénines pourrait constituer une cible thérapeutique d’intérêt dans les infections respiratoires à P. aeruginosa. / Pseudomonas aeruginosa (Pa) is a Gram-negative bacteria frequently involved in healthcare-associated pneumonia and considered as a « problem-pathogen ». To face the announced post-antibiotic era due to increasing resistance and lack of new antibiotics, new treatment strategies have to be developed. During pneumonia, lung injury results from both bacterial-mediated virulence and host response. Modulation of an overreacting host response could be an alternative therapeutic target in Pa-induced lung infection. Kynurenines are small molecules resulting from tryptophan degradation with reported immunomodulatory properties. Pa is known to produce kynurenine, but the functional enzymes, types and amounts of secreted metabolites are poorly known. Interestingly, many host cells also possess the kynurenine pathway, whose metabolites are known to control immune system homeostasis. The following experiments aim to determine whether bacterial metabolites can interfere with the host’s immune response, leading to a possible immunomodulatory interplay between bacteria and host kynurenine pathways, impacting on the pathophysiology of P. aeruginosa infection. To that goal, we use a murin model of acute lung injury. Mice were infected with WT strain of Pa, compared to mutant strains unable to produce kynurenine (ΔkynA), and mutant strains overproducing them (ΔkynU). Moreover, we studied the interactions between bacterial and host kynurenine pathways by inhibiting the first enzyme of the host pathway called indoleamine-2,3-dioxygenase (IDO). Finally, we assessed the role of the arylhydrocarbon receptor (AhR), a known receptor to kynurenine involved in lung immunity, using AhR KO mice. First, we assess types and levels of metabolites produced by Pa in an in vitro model, and the relevance of this production in vivo. We show for the first time that Pa is able to secrete kynurenine at clinically relevant levels, and other metabolites such as kynurenic acid and 3 OHkynurenine, what was unknown to date. Second, we show that bacterial metabolites were able to modulate the host innate immune response, by increasing alveolar recruitment of neutrophils, associated with decreased inflammatory cytokines levels and impairment of antigen-presenting cells activation. Finally, we report that IDO and AhR are involved in this kynurenine-mediated immunomodulation. These data suggest that pulmonary infection with a bacteria highly expressing the kynurenine pathway enzymes could lead to an imbalance in the immune response to infection, thus constituting a potential therapeutic target to improve Pa-induced pneumonia outcome.

Made available in DSpace on 2015-09-25T12:22:40Z (GMT). No. of bitstreams: 1 PDF - Erivanna Karlene dos Santos Oliveira.pdf: 2316361 bytes, checksum: a621cb80b0ca857fdd90fc5f90fbf6bf (MD5) Previous issue date: 2014-04-11 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The presence of cyanobacteria in reservoirs, lakes and rivers is a global environmental health problem as a result ofanthropogenic eutrophication. These microorganisms hinder water purifiers, to produce taste and odor difficult to remove and the cells at high density clog the sand filters, reducing its time of use. In the conventional method of water treatment, coagulation, flocculation and sedimentation are essential in the removal of cells and determine the efficiency of the following operations. In this study we analyzed the efficiency of removal of intact cells of Microcystis aeruginosaand microcystin-LR in the stages of coagulation, flocculation and sedimentation, followed by filtration through sand filters with the use of coagulants ferric chloride and aluminum sulfate, evaluating four filtration rates: 15, 30, 50 and 100 m 3 .m -2 .d -1 at pH 7.5 and a dosage of 40 mg.L -1 tests with a bench-scale (Jartest) using treated water dechlorinated and adadded with a pureculture of M. aeruginosa with approximate final densityof 10 cel.mL -1 simulating a bloom. The parametres control were turbidity, apparent color and cell concentration. It was observed that both the efficiency rates at 15 and 30 m 1 had similar removal of apparent color, turbidity and cellconcentration (79%, 80% and 92% and 80%, 80,5% and 89,10% respectively for the coagulant ferric chloride. And for aluminum sulphate was the same for both rates removals of 81,48% for apparent color, turbidity to 83,25% and to 90,50% for cell concentration, since the rate for 30 m 1 removals were 83,33%, 83,25% and 89,71%, respectively. Nocell lysiswas observedandconsequently, no further increases in concentrationsofmicrocystin-LR cyanotoxinduring treatment was observed. / A presença de cianobactérias em reservatórios, lagos e rios é um problema mundial de saúde ambiental, em consequência da eutrofização antropogênica. Esses microrganismos dificultam a potabilização da água, por produzir gosto e odor difíceis de remover e suas células em alta densidade colmatam os filtros, diminuindo sua vida útil. No método convencional de tratamento de água, coagulação, floculação e sedimentação são fundamentais na remoção de células e determinam a eficiência das operações seguintes. Neste trabalho analisou-se a eficiência na remoção de células intactas de Microcystis aeruginosa e da microcistina-LR nas etapas de coagulação, floculação e sedimentação, seguido por filtração em filtro de areia com uso dos coagulantes cloreto férrico e sulfato de alumínio, avaliando quatro taxas de filtração de 15, 30, 50 e 100 m 3 .m , em pH 7,5 e dosagem de 40 mg.L -1 com testes em escala de bancada (Jartest) utilizando-se água tratada de torneira desclorada e adicionada de uma cultura pura de M. aeruginosa com densidade final aproximada de 10 5 cel.mL -1 simulando uma floração. Os parâmetros de referência foram turbidez, cor aparente e concentração celular. Observou-se que a eficiência, tanto com as taxas de 15 e 30 m 3 .m -2 .d -1 tiveram remoções semelhantes para as variáveis cor aparente, turbidez e concentração celular com 79%, 80% e 92%; e 80%, 80,5% e 89,10%, respectivamente para o coagulante cloreto férrico. Para o sulfato de alumínio e para ambas as taxas, houve a mesma reposta com remoções de 81,48% para cor aparente, 83,25% para turbidez e 90,50% para concentração celular, já para a taxa de 30 m3 .m -2 .d -1 as remoções foram de 83,33% , 83,25% e 89,71% respectivamente. Não foi observada lise celular e consequentemente, não houve aumentos, ao longo do tratamento, das concentrações da cianotoxina microcistina -LR.

Um estudo de coorte prospectivo foi realizado com o objetivo de comparar a eficácia da polimixina B à de outros antibióticos. Foram estudados pacientes com pneumonia ou traqueobronquite associadas à ventilação mecânica causadas por Pseudomonas aeruginosa ou Acinetobacter baumannii. Critérios de inclusão para este estudo foram idade igual ou superior a dezoito anos e uso de terapia antimicrobiana apropriada por período igual ou superior a 48 horas. O desfecho primário avaliado foi mortalidade em 30 dias. Variáveis clínicas foram comparadas entre os pacientes que utilizaram polimixina B e os que utilizaram outras drogas. O modelo de regressão de Cox foi realizado. Um total de 67 episódios ocorridos em 63 pacientes foi analisado: 45(67,2%) foram tratados com polimixina B e 22 (32,8%) com comparadores. A maior parte dos comparadores (72,7%) era de beta-lactâmicos. A maioria dos episódios foi de pneumonia associada à ventilação mecânica (PAV). As infecções foram causadas por P. aeruginosa em 28 casos (41,8%), por A. baumannii em 35 casos (52,2%) e por ambos em 4 casos (6%). A mortalidade geral em 30 dias foi de 44,8% (30 de 67): 53,3% (24 de 45) no grupo da polimixina B e 27,3% (6 de 22) no grupo dos comparadores (p=0,08). A taxa de mortalidade no grupo da polimixina e comparadores foi de 65,6 e 12,0 por 1000 pacientes-dia, respectivamente (p=0,02). A análise multivariada mostrou que o uso de polimixina B foi fator independente associado à mortalidade em 30 dias (Hazard Ratio ajustada, 4,3; Intervalo de Confiança de 95%, 1,39-13,03), após ajuste para tempo de internação hospitalar antes da infecção e aumento > 100% da creatinina em relação ao valor basal durante o tratamento. O escore APACHE II no inicio da infecção foi mantido no modelo final, embora não tenha atingido significância estatística, para ajuste de possível fator confundidor residual. Não houve diferenças significativas nos desfechos secundários, incluindo tempo de ventilação mecânica após terapia adequada, superinfecção e erradicação bacteriana nas secreções respiratórias. A terapia com polimixina B foi inferior comparada a outras drogas na pneumonia e traqueobronquite associadas à ventilação mecânica causadas por P. aeruginosa e A. baumannii. / To compare the efficacy of polymyxin B with other antimicrobials in the treatment of ventilator-associated pneumonia (VAP) and tracheobronchitis (VAT) by Pseudomonas aeruginosa or Acinetobacter baumannii, a prospective cohort study was performed. Patients who received appropriate therapy for ≥48h were analyzed. The primary outcome was 30-day mortality. A total of 67 episodes were analyzed: 45 (67.2%) treated with polymyxin B and 22 (32.8%) with comparators. Thirty-day mortality was 44.8%: 53.3% (24 of 45) in the polymyxin B group and 27.3% (6 of 22) in the comparator group, P=0.08. The mortality rates in the polymyxin B and comparator group were 65.6 and 12.0 per 1000-patients-day, respectively (P=0.02) Treatment with polymyxin B was independently associated with 30-day mortality in multivariate model, with similar results in the subgroup of patients with VAP, suggesting that this antibiotic may be inferior to other drugs in the treatment of VAP and VAT by these organisms.

Este trabalho objetivou adaptar um método de controle de larvas do mexilhão dourado (Limnoperna fortunei) com a utilização de radiação ultravioleta e verificar seu efeito sobre cianobactérias e cianotoxinas presentes na água. Limnoperna fortunei (Dunker, 1857), conhecido vulgarmente como mexilhão dourado é proveniente do sudeste asiático. Foi, provavelmente, introduzido nos nossos mananciais, não intencionalmente, através da água de lastro, com os primeiros registros na América do Sul, em 1991, no Rio da Prata, nas proximidades de Buenos Aires, Argentina. No Brasil foi visto pela primeira vez na área do Delta do Jacuí, em frente ao porto de Porto Alegre, RS, no ano de 1998. Além de ameaçar à biodiversidade de ecossistemas, vem provocando a obstrução das tubulações e trocadores de calor junto às estações de tratamento de água e indústrias que utilizam água bruta para resfriamento. As estações de tratamento, além de enfrentarem problemas com o entupimento pelo mexilhão, defrontam-se também com as florações de cianobactérias. As florações, conhecidas também como blooms, são eventos de multiplicação e acumulação de microalgas ou cianobactérias nos corpos hídricos, que podem durar de algumas horas ao longo do dia a meses. As cianobactérias podem liberar cianotoxinas que estão presentes principalmente no interior das células, e são liberadas na lise celular, que ocorre principalmente por senescência natural. Os experimentos foram realizados em uma unidade piloto, onde concentrações conhecidas de larvas do mexilhão dourado foram submetidas a doses de radiação ultravioleta, na faixa de 200 a 800 mWs/cm2. As amostras de água bruta utilizadas nos testes foram avaliadas por meio de métodos analíticos adequados (APHA, 2005). Foram determinados os parâmetros de temperatura (°C), pH, turbidez (NTU), dureza (mgCaCO3/L) e sólidos suspensos (mg/L), os quais poderiam influenciar nas condições dos testes. As mesmas condições testadas para o mexilhão foram utilizadas nos experimentos com cianobactérias. As larvas de mexilhão dourado e a água bruta utilizada no experimento foram obtidos no delta do Jacui, Porto Alegre, Rio Grande do Sul, Brasil. A cianobactéria Microcystis aeruginosa, produtora de microcistina, foi cultivada em laboratório. A mortalidade instantânea das larvas aproximou-se dos 100% nas condições do teste com a dosagem de 781mWs/cm2 , com DL50 de 324 mWs/cm2. Na água residual dos experimentos de exposição à radiação UV, foram realizados ensaios ecotoxicológicos crônicos com Pimephales promelas, Ceriodaphnia dubia e Selenastrum capricornutum, a fim de detectar a presença de subprodutos que poderiam gerar toxicidade aos organismos de diferentes níveis tróficos. Os resultados desta avaliação ecotoxicológica não demonstraram toxicidade residual. Os dados obtidos demonstraram-se satisfatórios no controle daslarvas de mexilhão, entretanto não promoveram a lise das células de M. aeruginosa e a conseqüente liberação de microcistinas nas condições testadas. / L. fortunei (Dunker, 1857), commonly known as golden mussel comes from Southeast Asia. It might have been unintentionally introduced in our water sources through ballast water, with the first records in 1991, in Rio de la Plata, near Buenos Aires, Argentina, South America. In Brazil it was first seen in 1998, in Jacuí Delta, opposite Porto Alegre’s harbor. Besides threatening the biodiversity of ecosystems, this mussel has caused the obstruction of pipes and heat exchangers along the water treatment plants and industries that use raw water for cooling. Treatment plants facing problems with the clogging of mussels also have to contend with the cyanobacterial blooms. The blooms are events of multiplication and accumulation of algae or cyanobacteria in water bodies that can last from a few hours to days or months. The cyanobacteria may release cyanotoxins present mainly in cells and are released upon cell lysis, which occurs primarily by natural senescence. Thus, the aim of study is to adapt a control method of golden mussel larvae (L. fortunei) using ultraviolet radiation and verify its effect on cyanobacteria and cyanotoxins in the water. The experiments were performed in a pilot unit, where known concentrations of mussel larvae were subjected to doses of ultraviolet radiation ranging from 200 to 800 mWs/cm2, and the quality of water used, evaluated. The same conditions tested for the mussels were used in experiments with cyanobacteria. Mussel larvae and raw water used in the experiments were obtained from the Jacuí Delta, Porto Alegre, Rio Grande do Sul, Brazil. The cyanobacteria Microcystis aeruginosa, witch produces microcystin, was grown in culture in our laboratory. The instantaneous mortality of larvae was approximately 100% with 781mWs/cm2 in test conditions, with LD50 of 324 mWs/cm2. Ecotoxicological tests were performed with Pimephales promelas, Ceriodaphnia dubia, and Selenastrum capricornutum, to detect the presence of byproducts that could cause toxicity to organisms of different trophic levels in the residual water. The results of ecotoxicological evaluation showed no residual toxicity. The data showed to be satisfactory in larvae control, but did not cause lysis in cells of M. aeruginosa and the consequent release of microcystins in the water.

Pseudomonas aeruginosa is an opportunistic human pathogen that is now the leading cause of morbidity and mortality in immunocompromised individuals. Those suffering with the genetic disease cystic fibrosis (CF) commonly encounter P. aeruginosa infections. P. aeruginosa infection can present itself as an acute infection, which is characterised by highly virulent, "free-swimming" bacteria, or as a chronic infection associated with the formation of surface-adhered bacterial communities known as biofilms. The labyrinth of interconnecting signalling networks has meant that the regulatory mechanisms behind biofilm formation and virulence are largely undefined. In this dissertation, a single nucleotide polymorphism was identified within the gene, fusA1, encoding elongation factor G (EF-G). The mutation introduced minor structural changes to the protein which were likely to have functional repercussions in its involvement in protein synthesis. Phenotypic analysis revealed that the mutation conferred changes in both resistance and sensitivity to various antibiotics, as well as changes in motility, exoenzyme production, quorum sensing, metabolism, synthesis of biofilm-associated proteins and exopolysaccharide production. Most notably was the up-regulation of a major virulence determinant, the type three secretion system, typically characteristic of cells comprising an acute infection. Proteomic and transcriptomic profiling of the mutant strain provided an insight into the genetic basis behind these phenotypes, identifying the up-regulation of multidrug efflux systems and modulations to the chemotactic systems. This study also found links between several biological processes that were modulated in the mutant strain, such as crosstalk between sulfur metabolism, iron uptake and the oxidative stress response. In summary, the work presented in this dissertation highlights the susceptibility of fusA1 to spontaneous mutation and identifies a novel role for EF-G in bacterial virulence and antibiotic sensitivity, both of which have worrying implications for infection within the CF lung.