Introdução: As polimixinas são consideradas as opções terapêuticas de resgate para o tratamento das infecções de corrente sanguínea (ICS) por bacilos Gram-negativos (BGN) com resistência aos carbapenens (CR) e a combinação com outro antimicrobiano tem sido utilizada apesar da falta de evidência clínica para esta prática. Objetivo: Avaliar o uso de polimixina B intravenosa em monoterapia e em combinação com outro antimicrobiano para ICS por BGN CR. Pacientes e Métodos: Foi um estudo de coorte retrospectivo em um hospital terciário, incluindo 99 pacientes. A comparação dos tipos terapias foi através do propensity score. Resultados: A mortalidade global em 30 dias foi de 43,4%: 40,7% (24 de 59) e 47,5% (19 de 40), p=0,51, em pacientes que receberam combinação e monoterapia, respectivamente. A sepse grave/choque séptico no dia da ICS, alta pontuação do escore de bacteremia de Pitt e a presença de neoplasia como doença de base foram, independentemente, associados a maior mortalidade em 30 dias no modelo de regressão de Cox. A terapia combinada não foi significativamente associada a este resultado (hazard ratio, 0,70; intervalo de confiança de 95%, 0,36-1,36); p=0,29). Apesar de não ser significativa, houve uma tendência a um efeito benéfico da associação em pacientes com ICS por BGN CR da família Enterobacteriaceae. Não houve diferença no desenvolvimento de insuficiência renal aguda em pacientes que receberam terapia de combinação comparado com os que receberam monoterapia. Conclusão: Não houve diferença na mortalidade em 30 dias em pacientes com ICS por BGN CR tratados com polimixina B em combinação com outro antimicrobiano ou em monoterapia. A prática rotineira de combinar um segundo antibiótico em esquemas baseados em polimixinas, especialmente, se a bactéria apresenta resistência in vitro ao carbapenem, ainda necessita estudos clínicos adicionais. / Background: Polymyxins are usually the last resort therapy for carbapenem-resistant Gram-negative bacteria (CR GNB) bloodstream infections (BSIs), combination with another antimicrobial has been used despite the lack of clinical evidence supporting such practice. Objetive: We aimed to assess the use of intravenous polymyxin B in combination with another antimicrobial in comparison with polymyxin B as a single drug for CR GNB BSIs, adjusting for a propensity score for indication of combination therapy. Patient and methods: We compared combination versus monotherapy with polymyxin B for CR GNB BSIs, adjusting for a propensity score for indication of combination therapy. It was a retrospective cohort study at a tertiary-hospital including 99 patients. Results: The overall 30-day mortality was 43.4%: 40.7% (24 of 59) and 47.5% (19 of 40), P=0.51, in patients receiving combination and monotherapy, respectively. Severe sepsis/ septic shock at BSI onset higher Pitt bacteremia score and neoplasia were independently associated with higher 30-day mortality in a Cox-regression model. Combination therapy was not significantly associated with this outcome (Hazard Ratio, 0.70; 95% confidence interval, 0.36-1.36); P=0.29). Although not significant, there was a tendency to a beneficial effect of combination in patients with Enterobacteriaceae CR GNB BSIs. There was no difference in development of AKI in patients receiving combination therapy compared to those receiving monotherapy. Conclusions: There was no difference in 30-day mortality in patients with CR GNB BSIs treated with polymyxin B in combination with another antimicrobial compared with polymyxin B alone. The routine practice of combining a second antibiotic in polymyxins-based regimes, especially if the bacteria present in vitro resistance to the agent, still lacks support from clinical studies.

Introdução: As polimixinas são consideradas as opções terapêuticas de resgate para o tratamento das infecções de corrente sanguínea (ICS) por bacilos Gram-negativos (BGN) com resistência aos carbapenens (CR) e a combinação com outro antimicrobiano tem sido utilizada apesar da falta de evidência clínica para esta prática. Objetivo: Avaliar o uso de polimixina B intravenosa em monoterapia e em combinação com outro antimicrobiano para ICS por BGN CR. Pacientes e Métodos: Foi um estudo de coorte retrospectivo em um hospital terciário, incluindo 99 pacientes. A comparação dos tipos terapias foi através do propensity score. Resultados: A mortalidade global em 30 dias foi de 43,4%: 40,7% (24 de 59) e 47,5% (19 de 40), p=0,51, em pacientes que receberam combinação e monoterapia, respectivamente. A sepse grave/choque séptico no dia da ICS, alta pontuação do escore de bacteremia de Pitt e a presença de neoplasia como doença de base foram, independentemente, associados a maior mortalidade em 30 dias no modelo de regressão de Cox. A terapia combinada não foi significativamente associada a este resultado (hazard ratio, 0,70; intervalo de confiança de 95%, 0,36-1,36); p=0,29). Apesar de não ser significativa, houve uma tendência a um efeito benéfico da associação em pacientes com ICS por BGN CR da família Enterobacteriaceae. Não houve diferença no desenvolvimento de insuficiência renal aguda em pacientes que receberam terapia de combinação comparado com os que receberam monoterapia. Conclusão: Não houve diferença na mortalidade em 30 dias em pacientes com ICS por BGN CR tratados com polimixina B em combinação com outro antimicrobiano ou em monoterapia. A prática rotineira de combinar um segundo antibiótico em esquemas baseados em polimixinas, especialmente, se a bactéria apresenta resistência in vitro ao carbapenem, ainda necessita estudos clínicos adicionais. / Background: Polymyxins are usually the last resort therapy for carbapenem-resistant Gram-negative bacteria (CR GNB) bloodstream infections (BSIs), combination with another antimicrobial has been used despite the lack of clinical evidence supporting such practice. Objetive: We aimed to assess the use of intravenous polymyxin B in combination with another antimicrobial in comparison with polymyxin B as a single drug for CR GNB BSIs, adjusting for a propensity score for indication of combination therapy. Patient and methods: We compared combination versus monotherapy with polymyxin B for CR GNB BSIs, adjusting for a propensity score for indication of combination therapy. It was a retrospective cohort study at a tertiary-hospital including 99 patients. Results: The overall 30-day mortality was 43.4%: 40.7% (24 of 59) and 47.5% (19 of 40), P=0.51, in patients receiving combination and monotherapy, respectively. Severe sepsis/ septic shock at BSI onset higher Pitt bacteremia score and neoplasia were independently associated with higher 30-day mortality in a Cox-regression model. Combination therapy was not significantly associated with this outcome (Hazard Ratio, 0.70; 95% confidence interval, 0.36-1.36); P=0.29). Although not significant, there was a tendency to a beneficial effect of combination in patients with Enterobacteriaceae CR GNB BSIs. There was no difference in development of AKI in patients receiving combination therapy compared to those receiving monotherapy. Conclusions: There was no difference in 30-day mortality in patients with CR GNB BSIs treated with polymyxin B in combination with another antimicrobial compared with polymyxin B alone. The routine practice of combining a second antibiotic in polymyxins-based regimes, especially if the bacteria present in vitro resistance to the agent, still lacks support from clinical studies.

A oviposição é um estágio vulnerável do ciclo de vida de carrapatos. Rhipicephalus microplus, como todos Ixodidae e Argasidae, possui uma glândula especializada, o órgão de Gené, que produz uma cera que é depositada na superfície do ovo durante a oviposição. Além de restringir a perda excessiva de água, a cera atua como uma barreira contra o ataque de organismos invasores. Em R.microplus, como em outros carrapatos, há poucos estudos demonstrando atividade antimicrobiana em ovos. Ainda mais, não há na literatura relato de atividade antibiofilme em ovos de carrapatos e nem mesmo em qualquer outro artrópode. O objetivo do presente trabalho foi avaliar a hipótese da existência de mecanismos de defesa em ovos de R. microplus contra biofilmes bacterianos. O extrato água/metanol da cera dos ovos apresentou atividade contra o biofilme de Pseudomonas aeruginosa sem afetar a sua viabilidade. Esse extrato também demonstrou efeito antibiótico contra Staphylococcus epidermidis. Nós identificamos a molécula com ambas atividades (antibiofilme e antibiótica) como N-(3-sulfooxy-25-cholest-5-en-26-oyl)-L-isoleucina (boophiline). Na busca por possíveis mecanismos responsáveis pelo efeito antibiofilme de boophiline contra P. aeruginosa, 14 genes foram analisados por qRT-PCR. Boophiline inibe a expressão de fliC (flagelo) e cdrA (componente estrutural da matriz), cujos produtos são necessários para a formação do biofilme de P. aeruginosa. Monosfosfato de guanosina dimérico cíclico (c-di-GMP) é um importante segundo mensageiro característico de bactérias Gram-negativas. Altos níveis intracelulares de c-di-GMP promovem o estilo de vida séssil enquanto baixos níveis induzem o comportamento móvel. De acordo com essa afirmação, nós encontramos que boophiline aumenta a motilidade swarming de P. aeruginosa. Desta forma, nos questionamos se o mecanismo de ação de boophiline estaria envolvido com c-di-GMP já que o sistema quorum sensing não foi afetado pela molécula. Interessantemente, quando os níveis de c-di-GMP foram aumentados pela superexpressão de uma diguanilato ciclase, boophiline não inibiu efetivamente a formação de biofilme. Uma explicação para esse resultado é que boophiline interfere em uma via específica regulada por c-di-GMP, o que explicaria não termos obtido um decréscimo no nível total deste segundo mensageiro. Contrariamente, boophiline foi bactericida contra S. epidermidis. Mudanças morfológicas significativas foram observadas em células tratadas com a molécula, as quais foram severamente danificadas. Boophiline levou a formação anormal de septo, rompimento da membrana bacteriana e extravasamento do material intracelular. Adicionalmente avaliamos o potencial antibiofilme e anti-protozoário de filtrados de cultura obtidos de bactérias isoladas de tecidos de R. microplus. Quatorze filtrados de cultura bacteriano apresentaram notável atividade contra o biofilme de P. aeruginosa e S. epidermidis e foram citotóxicos contra Tritrichomonas foetus. Nosso trabalho é pioneiro em demonstrar a existência de proteção contra biofilmes em ovos de carrapatos bem como de bactérias associadas a carrapatos como produtoras de moléculas bioativas. Além disso, nós demonstramos que boophiline é uma nova molécula antibiofilme, sendo a primeira vez relatado na literatura que um composto age inibindo cdrA. Os dados obtidos em nosso estudo poderiam estimular novas abordagens em áreas como fisiologia e controle de artrópodes, genética e fisiologia de microrganismos e controle de biofilmes. / The oviposition is a vulnerable stage of the tick life cycle. Rhipicephalus microplus, as all Ixodidae and Argasidae, has a specialized gland, the Gene’s organ, which produce a wax that is smeared on egg surface during oviposition. In addition to restricting excessive water loss, wax acts as a barrier to attack by invading organisms. In R. microplus, as in other ticks, there are few studies showing antimicrobial activity in eggs. Moreover, there is no report of antibiofilm activity in tick eggs nor in any other arthropod. The objective of our study was to evaluate the hypothesis of the existence of defense mechanisms against bacterial biofilms in R. microplus eggs. The eggs wax water/methanol extract showed activity against Pseudomonas aeruginosa biofilm without affecting its viability. This extract also presented an antibiotic effect against Staphylococcus epidermidis. We have identified the molecule anti-biofilm and antibiotic as N-(3-sulfooxy-25-cholest-5-en-26-oyl)-L-isoleucine (boophiline). In the search for possible mechanisms responsible by antibiofilm effect of boophiline against P. aeruginosa, 14 genes were evaluated by qRT-PCR. We showed that boophiline inhibits the expression of fliC (flagellum) and cdrA (matrix structural component), whose products are necessary for biofilm formation in P. aeruginosa. Bis-(3’–5’)-cyclic dimeric guanosine monophosphate (c-di-GMP) is an important second messenger, characteristic of Gram-negative bacteria. High intracellular levels of c-di-GMP promote a sessile mode of growth, while low levels promote motile behavior. In line with this, we found that boophiline increases swarming motility, which raised the question whether it acts by altering c-di-GMP levels. Interestingly, when c-di-GMP levels were increased by overexpression of a diguanilate cyclase, boophiline no longer inhibited biofilm formation. One explanation for these results is that boophiline interferes with a specific c-di-GMP-regulated pathway, which would explain we have not obtained a decrease in the total level of this second messenger. Conversely, boophiline had a bactericidal effect against S. epidermidis. Significant morphological changes were observed in the boophiline-treated cells, which appeared to be severely damaged. Boophiline was found to cause abnormal septum formation, bacterial membrane disruption, and extravasation of intracellular material. Additionally, our work also aimed to evaluate the potential antibiofilm and anti-protozoa of culture filtrates obtained from bacteria isolated of R. microplus tissues. Fourteen bacterial culture filtrates showed remarkable activity against of P. aeruginosa and S. epidermidis biofilms, and were cytotoxic against Tritrichomonas foetus. Our work is pioneer in demonstrating the existence of protection mechanisms in tick eggs against biofilms, and ticks-associated bacteria as producers of bioactive molecules. Furthermore, we demonstrated that boophiline is a new antibiofilm molecule, and is the first reported in the literature that a molecule inhibits cdrA. The data obtained in our study could stimulate new approaches in areas such as the physiology and control of arthropods, the genetics and physiology of microorganisms, and biofilm control.

Pseudomonas aeruginosa (Pa) est une bactérie multi-résistante responsable de plus de 10% des infections nosocomiales en Europe. Pa réagit aux signaux environnementaux en activant un système de régulation complexe qui permet à la bactérie d’adopter une mode de vie planctonique (infection aigue) ou sessile (infection chronique caractérisée par la formation des biofilms). Le système à deux composants GacS/GacA joue un rôle central dans la permutation entre ces deux modes. Pendant une infection chronique, GacS HK forme des homodimeres permettant l’activation de la formation du biofilm. Nos travaux ont confirmé le rôle du domaine périplasmique de GacS HK (GacSp) dans la détection du signal et l’activation de la voie GacS/GacA. Malgré l’instabilité de GacSp, J’ai résolu la structure 3D de ce domaine par la spectroscopie RMN. GacSp adopte un repliement PDC qui caractérise un grand nombre de domaines senseurs connus pour fixer une large gamme de ligands. L’étude de la structure m’a permis de proposer un site de fixation du ligand. Les résultats présentés dans cette thèse montrent l’importance de GacSp et propose une nouvelle piste pour les études thérapeutiques visant à éradiquer le biofilm.La deuxième partie de ma thèse présente la structure cristalline du glycoside hydrolase 39 du B. cellulosilyticus WH2 présente dans le côlon (GH39wh2). GH39wh2 partage la même architecture avec les autres membres de la famille 39, cependant, elle possède un site actif plus large et plus exposé. Les analyses structurales et bioinformatiques ont permis d’attribuer GH39wh2 à un nouveau sous-groupe caractérisé par une fonction endoglycosidase inconnue. / Pseudomonas aeruginosa (Pa) is a Multidrug-Resistant bacterium responsible for more than 10% of nosocomial infections in Europe. Interestingly, Pa can switch its lifestyle between planktonic (acute infection) and sessile lifestyle (biofilm-type chronic infection) through different regulatory pathways, in which GacS/GacA Two Component System (TCS) plays a central decisive role. An active homodimer form of GacS Histidine Kinase (HK) leads to biofilm formation, while the inhibition of GacS by the hybrid HK RetS via cytoplasmic hetero-dimerization promotes acute infection. During my thesis, we unveiled for the first time the sensing role of the periplasmic detection domain of GacS HK (GacSp). I further solved the 3D structure of GacSp using NMR spectroscopy revealing its PDC-like fold, which characterizes a large number of detection domains known to bind a wide range of ligands. The structural analyses of the new GacSp structure suggest a conserved ligand-binding pocket. All together, my results present structural and functional understanding of GacSp role, which form the basis to consider this domain as a new target for anti-biofilm therapy.In the second part of my thesis, I report a 2.5Å resolution crystal structure of a family 39 glycoside hydrolase member (GH39wh2) from the human gut bacteria B. cellulosilyticus WH2. GH39wh2 shares a similar architecture as found in other related GH39 members but unveils an atypical shallow solvent-exposed groove. Complementary biochemical and bioinformatics analyses assign GH39wh2 to a new GH39 subgroup harboring a yet unknown endoglycosidase activity.

Cyanobacteria are one of the oldest forms of photosynthetic life and may have contributed significantly to the evolution of oxygen into the then anoxic environment. Cyanobacteria are also one of the best sources of natural secondary metabolites (cyanopeptides) some of which have harmful effects on the ecosystem, while others may be beneficial. It is known that these secondary metabolites are continuously produced during growth, however, it is not known whether the producing cyanobacteria actually benefit from these metabolites. The overarching aim of this study was to answer the question ‘Why do cyanobacteria produce secondary metabolites?’. With this aim in mind, preliminary work focused on understanding the growth and secondary metabolite production characteristics of Microcystis aeruginosa PCC7806. The technique of labelling secondary metabolites with 15N was successfully employed in differentiation and quantification of ex-novo and de-novo metabolites. The effect of exogenous cyanopeptides such as microcystins, aerucyclamides, anabaenopeptins, aeruginosamide, cyanopeptolin and aeruginosin on M. aeruginosa PCC7806 was evaluated using a rapid bioassay approach along with an automated cell enumeration technique. The results indicate that at least some cyanopeptides (microcystins-LR, microcystin-LF, aeruginosamide, anabaenopeptin B and aerucyclamide A) induce significant changes to cell division and metabolite production rate. In an ecological scenario, the release of such secondary metabolites by lysing cells (such as when blooms collapse), may be perceived as an alarm signal by surrounding live cells, which may in turn slow cell division and prepare for re-invasion. This may be a strategy for species survival and dominance. While the results from this study do not confirm a role for cyanopeptides, it is thought that the results are clearly indicative of the role played by cyanopeptides for the producing organism. In order to confirm a role, it is recommended that monitoring ribosomally synthesised metabolites (e.g. aerucyclamides) along with chlorophyll-a gene expression, with sophisticated techniques such as qPCR are used.

M. Tech (Department of Biotechnology, Faculty of Applied and Computer Sciences) Vaal University of Technology. / Water shortages, water pollution and climate changes are highly interrelated global issues. These have raised immense concerns about serious adverse effects on the quality, treatment and re-use of wastewater. A major role of water is for vitality of life on earth. Water is recognized as source of evolution from origin to degree of civilization, since it is an essential resource its treatment becomes a necessity for day to day for life. Nanoparticles and their application in treatment of wastewater is becoming a major area of research. It is mainly applicable to the removal of major contaminants like microorganisms. This study was carried out with an objective to investigate the antibacterial and antifungal potentials of nanoparticles. Cobalt and cobalt complexes of urea and thiourea were synthesized and characterized using UV-Vs, PL, FTIR, TEM, SEM, XRD and TGA techniques. The Co particles are in a mixture of rod, agglomerates with irregular shape around 50 – 100 nm in diameter. The Co/Thiourea particles appear to be around 10 – 30nm in size. The Co complexed with urea images showed spherical to hexagonal shape with 50 nm size in diameter. The antimicrobial activity was determined using Minimum Inhibitory and bactericidal concentration and the well diffusion method. The antibacterial and antifungal activities of ratios (1:1, 1:2, 1:3, 2:1 μg/mL) of doped cobalt nanoparticles were tested against a panel of five Gramnegative bacteria - (Escherichia coli, Pseudomonas aeruginosa, Shigella enterica, Salmonella typhi and Salmonella sonnei) human pathogenic bacteria; and two fungal strains - Aspergillus niger and Candida albicans. Zones of inhibition as a consequence of nanoparticles were compared with that of different standards like Neomycin for antibacterial activity and Amphotericin B for antifungal activity. The results showed a remarkable inhibition of the bacterial growth against the tested organisms. The most striking feature of this study is that Cobalt, Urea and Thiourea nanoparticles have antifungal activity comparable or more effective (as in case of Thiourea on A. niger) than Amphotericin B and nearly promising antibacterial activity although not comparable to Neomycin.

No description available.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Anbu is a bacterial protein with significant homology to the sub-units of the 20S proteasome and is predicted to be a novel bacterial proteasome. The goal of this project was to determine if the recombinant Anbu protein from Pseudomonas aeruginosa is a proteasome. Anbu from P. aeruginosa was successfully cloned, expressed and purified. In order to determine the catalytic activity of Anbu, the purified protein was tested with a variety of substrates and conditions. The targets analyzed included fluorescently-labeled substrates, denatured proteins, diubiquitin, and a peptide library in the hopes of obtaining a useful model substrate. Experiments were also conducted to determine what role Anbu has in the cell. Western analysis was performed on the cell lysate of wild type P. aeruginosa and insertional mutants to detect Anbu expression. The level of biofilm formation was compared between the wild type and mutants. Cultures were grown under stress conditions including the oxidative stress of diamide and the nitrosative stress of S-nitrosoglutathione. Growth rates were monitored in an attempt to detect a phenotypic difference between the wild type and the mutants lacking Anbu, HslV, and the other proteins of interest. While a substrate for Anbu has yet to be found, this protein was found to assemble into a larger structure and P. aeruginosa lacking Anbu was sensitive to the oxidative stress of diamide and the nitrosative stress of S-nitrosoglutathione.

No description available.