Submitted by Etelvina Domingos (etelvina.domingos@ufpe.br) on 2015-03-12T16:57:48Z No. of bitstreams: 2 TESE_Agrinaldo Jacinto do Nascimento Júnior.pdf: 6927683 bytes, checksum: bf123d71858e87a718a988da950251f3 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-12T16:57:48Z (GMT). No. of bitstreams: 2 TESE_Agrinaldo Jacinto do Nascimento Júnior.pdf: 6927683 bytes, checksum: bf123d71858e87a718a988da950251f3 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2013-12-19 / FACEPE CAPES INAMI / A membrana externa de bactérias Gram- negativas é constituida majoritariamente de lipopolissacárideo (LPS) e fosfolipídeo. Cada unidade de LPS é constituida por até três partes, o lipídeo A, o Core e outra nem sempre presente chamada antígeno-O. O lípideo A tem um maior carater apolar, embora possua grupos fosfatos. Já o core é a região mais carregada do LPS e possui não apenas açucares fosforilados, mas também carboxilados. Desta maneira, a superfície da membrana de LPS é negativa e a interação com os cátions necessária para neutralizar a carga da membrana. Por isso, estas membranas possuem alta capacidade em adsorver cátions e assim são candidatas a serem utilizadas como agente biorremediadores, na captura por exemplo de radionuclídeos. Numa perspectiva de saúde, o LPS atua como um antigeno para o sistema imunológico de mamíferos e a sua ação pode auxiliar a causar não apenas febre, mas até levar a morte individuos imunocomprometidos. A bactéria Gram-negativa Pseudomonas aeruginosa é um destes patógenos nosocomiais oportunistas e tem sido apontada como uma das principais responsáveis por provocar a morte de pacientes portadores de fibroce cística. A estrutura supramolecular das membranas de LPS afetam não apenas a sua permeabilidade, mas ainda a ativação do sistema imunológico do hospedeiro no momento da infecção. Para uma descrição a nível molecular da influência dos cátions, Na+ , K+, Ca2+, Mg2+ , Zn2+ e Ba2+ na membrana de LPS utilizamos uma abordagem teórica baseada na dinâmica molecular clássica atomística. O modelo da membrana foi uma bicamada constituida de 72 unidades de LPS de Pseudomonas aeruginosa do quimiotipo PAO1 ancorados em 180 moléculas de 1,2-dipalmitoil-3-fosfatidil-etanolamina (DPPE) considerando o pH = 7 e temperatura de 300K. Os resultados das análises das simulações indicaram que as membranas de LPS suportam um nível de hidratação maior do que as bicamadas de fosfolipídeos e além disso os cátions tendem a provocar a ligação cruzada entre as unidades de LPS. Ainda, o aumento do raio de hidratação dos cátions, bem como a diminuição da ligação cruzada entre as unidades de LPS tendem a promover a transição da membrana de um estado lamelar para não lamelar. Deste modo, os resultados sugerem que a escolha combinada da valência do cátion e sua capacidade relativa de coordenar os grupos fosfatos e moléculas de água podem servir como regra para modular propriedades das membranas de LPS como estrutura supramolecular, fluidez e hidratação.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2015-05-26T17:40:43Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação_de_mestrado FERNANDA correção após apresentação FINALIZADA DIGITAL.pdf: 1967282 bytes, checksum: 8e1bd13e2a7cb2f2b493bb443c3db0f9 (MD5) / Made available in DSpace on 2015-05-26T17:40:43Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação_de_mestrado FERNANDA correção após apresentação FINALIZADA DIGITAL.pdf: 1967282 bytes, checksum: 8e1bd13e2a7cb2f2b493bb443c3db0f9 (MD5) Previous issue date: 2015-02-23 / Pseudomonas aeruginosa é uma importante causa de infecções apresentando, no Brasil, altas taxas de morbidade e mortalidade. Os principais mecanismos de resistência utilizados por P. aeruginosa são a produção de β-lactamases e a expressão de bombas de efluxo. O uso de IgG e antibióticos para tratamento destas infecções tem apresentado resultados bem sucedidos, o que motiva a realização de estudos quanto a atividade in vitro da IgG humana com antibióticos frente a isolados de P. aeruginosa. O objetivo deste estudo foi detectar a presença dos genes de resistência, a relação clonal, o tempo de morte em isolados de P. aeruginosa provenientes de Infecções relacionadas à saúde (IrAS) de hospitais públicos do Recife-PE, em 2014, e verificar in vitro a taxa de fagocitose e a produção de oxido nítrico (NO) por monócitos humanos quando infectados por P. aeruginosa tratada com IgG humana em associação a Meropenem e Polimixina E. Foram avaliados 32 isolados para a detecção de genes de resistência por PCR, e para a avaliação de similaridade genética por ERIC-PCR. Destes, foram selecionados os 5 isolados apresentando maior número de genes de resistência e menor similaridade genética. Esses cinco isolados foram submetidos às associações dos sub-CIMs destes antibióticos com a IgG humana, para a determinação do tempo de morte, da taxa de fagocitose de células bacterianas e para a dosagem de NO por monócitos humanos. Foi detectado a presença do gene blaKPC em dois isolados de P. aeruginosa. Todos os isolados possuem os genes mexR, mexB, mexE e rpsL, apenas um isolado foi negativo para os genes mexA e mexF. Foram detectados 30 perfis genéticos distintos entre os isolados bacterianos. O tempo de morte dos isolados revelou que os tratamentos combinados com antibióticos, principalmente Polimixina E, são mais letais para as células bacterianas. A taxa de fagocitose por monócitos humanos revelou que quando infectados com bactéria tratada com IgG mais antibiótico, os monócitos ficam mais ativos à fagocitose e reduz drasticamente a quantidade de células bacterianas. Houve diferença significativa na produção de NO naqueles tratados com Meropenem e IgG. Com o presente estudo concluiu-se que a combinação de IgG mais antibiótico pode ser uma alternativa para o tratamento dessas infecções, pois a bactéria estará em número reduzido facilitando a fagocitose.

Blooms of cyanobacteria have increased in occurrence in the past three decades and have been reported to cause severe problems for animals and humans, leading to death in extreme instances. The majority of poisonings that have taken place have been attributed to a hepatotoxin produced by the species Microcystis aeruginosa, namely microcystin. The appearance of a cyanobacterial bloom does not give any indication as to its toxicity and therefore, it is imperative that simple, yet sensitive, bioassays are developed to overcome this problem. This study was undertaken to evaluate the effects of microcystin-LR on the liver of Xenopus laevis both in vitro and in vivo. This animal provides an opportunity to study the long-term hepatotoxic effects of the toxin compared to in vitro studies performed with mice and rats. The use of the liver slice model system as a potential bioassay to study the effects of microcystin-LR on Xenopus laevis liver was evaluated. Liver slices were cultured in RPMI- 1640 culture medium for periods ranging from 30 hours to 10 days and the liver slices were exposed to toxin concentrations ranging from 1nM to 500nM. The use of frog liver slices to study the longer-term effects of low-dose exposure to microcystin-LR was evaluated by observing the ultrastructural changes within hepatocytes using transmission electron microscopy, the release of the enzymes alanine aminotransferase and lactate dehydrogenase into the surrounding culture medium, as well as using a 3-[4,5-dimethylthiazol-2yl]-2,5- diphenyl tetrazolium bromide assay to determine the viability of the liver slices in culture. The amount of lipid peroxidation in the liver slices after exposure to microcystin-LR was assessed using the Thiobarbituric Acid Test. Results showed the frog liver slice culture system to be an inadequate method to evaluate the hepatotoxic effects of microcystin-LR. An in vivo assessment of the effects of microcystin-LR on Xenopus laevis was carried out using a total of 9 frogs (3 groups of 3 frogs). Frogs received a single intraperitoneal dose of 120mg/kg of microcystin-LR and were sacrificed at 8 and 24 hours post exposure. Microcystin-LR caused no significant change in serum lactate dehydrogenase levels, hepatosomatic index (liver weight as a percentage body weight), glutathione peroxidase activity, glycogen or lipid peroxidation. There was, however, an increase in glutathione sii transferase activity in the liver. The presence of the toxin in the liver was confirmed by immunohistochemistry. This study suggests that Xenopus laevis has, in some way, adapted to detoxifying aquatic toxins in the environment.

This study reports the findings on the effect of nitrates and phosphates on the biomass and toxin production of various strains of the unicellular non-nitrogen fixing cyanobacterium, Microcystis aeruginosa. The occurrence of blooms of Microcystis aeruginosa and microcystin in freshwater impoundments across the globe has been on the increase lately due to increased levels of eutrophication, resulting in human and animal deaths and illness, as well as drinking and recreational water foulment. A range of environmental factors have been shown to effect growth and microcystin production. Existing literature however is somewhat contradictory as to the effects of these physical and chemical factors on toxin production. Therefore Microcystis aeruginosa strains were cultured under batch and continuous conditions to determine the effect of nitrate and phosphate concentrations and ratios on biomass and toxin production. Cultures were analysed with regards to internal nutrient stores, biomass production, nutrient depletion, photosynthetic efficiency and microcystin production. Results showed that microcystin production correlated to growth rate, photosynthetic efficiency and internal nitrogen stores and that an optimal N:P ratio was associated with microcystin levels, growth rate and photosynthetic efficiency. Results therefore led to the conclusion that the nitrogen, carbon, and phosphate balance within the cell is closely associated with microcystin production. Whether or not microcystin is produced to maintain this balance or produced as a function of this balance remains to be determined.

Purified macrophage cationic proteins were used in functional assays to determine their interactions with the outer membrane and lipopolysaccharide of Pseudomonas aeruginosa. A fluorescent derivative of polymyxin B (dansyl-polymyxin) was found to bind to saturation to purified lipopolysaocharide, with similar affinity for the aminoglycoside supersensitive strain H215 and wild type strain H103 lipopolysaocharide. MCP-1 could displace more dansyl-polymyxin bound to the lipopolysaocharide of both strains, and bound with greater affinity than MCP-2. When whole cells were used, MCPs also displaced bound dansyl-polymyxin. Effects on the outer membrane of whole cells were examined by determining the initial rate of uptake of the hydrophobic fluorescent probe 1-N-phenylnaphthylamine. Uptake was enhanced in the presence of MCPs, indicating permeabilization of the outer membrane. MCP-1 caused maximal uptake of the probe at 40 µg/ml, MCP-2 at 70 µg/ml, and crude extract at only 20 µg/ml. Uptake of the probe was found to be enhanced at add pH, with maximal uptake occurring with only 7.5 µg/ml MCP-1 at pH 6.5. The data suggested that MCPs act to permeabilize the outer membranes of P. aeruginosa in a manner analagous to that defined for other polycationic agents. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Please read the abstract (Summary) in the section, 00front, of this document / Dissertation (MSc (Microbiology))--University of Pretoria, 2008. / Microbiology and Plant Pathology / unrestricted

Pseudomonas aeruginosa, a ubiquitous environmental bacterium and an opportunistic human pathogen, forms biofilms through a series of interactions between the cells and adherence to surfaces. Not only does rhamnolipid contribute to the pathogenic potential of P. aeruginosa, but it has also been reported that the bacterium utilises rhamnolipid to actively maintain the void spaces surrounding microcolonies, thus contributing to the architecture of P. aeruginosa biofilms. The P. aeruginosa rhlAB operon encodes the enzyme rhamnosyltransferase I, which produces mono-rhamnolipid, and the induction of rhlAB is dependent on the quorum sensing transcription activator RhIR complexed with the auto inducer N-butyryl-homoserine lactone. In this study, several aspects related to rhamnolipid biosynthesis and regulation in P. aeruginosa PAO1 were investigated. As a first step, a biochemical assay was developed and optimised whereby the concentration of rhamnolipid could be accurately quantified following its extraction from small sample volumes. Although the optimised rhamnolipid assay is not able to distinguish between different rhamnolipids or between different homologs of a specific rhamnolipid, it is, however, simple to perform, cost¬effective and does not rely on the use of specialised equipment. Subsequently an rhlAB-deficient mutant strain of P. aeruginosa PAOI strain was generated. For this purpose, three allelic exchange strategies, i.e. plasmid incompatibility, the use of a SacB counter-selectable marker and a combination of these approaches, were investigated by making use of newly constructed allelic exchange vector systems. The results that were obtained indicated that, of the three approaches, the latter was most efficient in generating the desired P. aeruginosa mutant strain, and 90% of the derived strains were found to be double reciprocal mutants. Reporter gene technology, using the genes encoding for stable and unstable variants of the green fluorescent protein (GFP), was finally used to investigate the transcriptional activity of the rhlA promoter in P. aeruginosa biofilms under conditions of continuous flow using glass as substratum. For this purpose, mini-CTX-GFP reporter vectors, containing stable and unstable variants of the gfp reporter gene, were constructed that allow for integration of a single copy of the transcriptional fusion in a defined, non-essential region onto the P. aeruginosa genome. Several global regulators have been reported to playa role in regulating quorum sensing and/or rhamnolipid biosynthesis in P. aeruginosa, amongst other, the sigma factors RpoS and RpoN. Therefore, rhlA promoter activity was also investigated in biofilms of P. aeruginosa strains lacking either RpoN or RpoS. Although structural differences between the biofilms formed by the P. aeruginosa wild-type PAD 1 and respective mutant strains were noted, transcription of rhlA appeared to be constitutive from 24 h onwards and did not appear to be localised to specific areas within the microcolonies or biofilms. These results, combined with those obtained by batch analysis, indicated that RpoS positively regulates rhlA transcription, whilst RpoN did not appear to influence rhlA promoter activity under the conditions used in this study. / Dissertation (MSc)--University of Pretoria, 2009. / Microbiology and Plant Pathology / unrestricted

A study was conducted to find the effect of magnesium, calcium, manganese and zinc ions on the infection of Psudomonas aeruginosa strain 1C by RNA bacteriophage 7s. When an 18 hour progeny experiment was performed, it was found that magnesium, calcium and manganese had different effects on bacteriophage production and was dependent on the bacterial growth conditions. RNA bacteriophage 7s progeny production was significantly enhanced by the addition of magnesium to cultures of Psudomonas aeruginosa 1C grown in a magnesium deficient medium. Under these environmental conditions there was a slight increase in progeny in the presence of calcium. When Psudomonas aeruginosa 1c was grown in a complete medium, the infection of cells by bacteriophage 7s was enhanced by magnesium and calcium but not manganese or zinc, as demonstrated by the One Step Growth Curve.

Opportunistic pathogens (e.g., Legionella pneumophila, Mycobacterium avium complex, Acanthamoeba polyphaga, Pseudomonas aeruginosa) residing in human-made water systems, particularly premise plumbing, are now the primary source of water-borne disease in developed countries. The prevention and control of opportunistic pathogens is a new challenge in premise plumbing due to the limited knowledge concerning the factors driving their occurrence and regrowth mechanisms, and also the complexity of premise plumbing conditions. The goal of this study is to identify key factors governing occurrence of opportunistic pathogens in drinking water distribution systems, particularly premise plumbing, via field investigations and lab-scale experiments. A molecular survey of three opportunistic pathogens (L. pneumophila, M. avium, P. aeruginosa), related groups (Legionella and mycobacteria) and two amoeba hosts (Acanthamoeba spp. and Hartmanella vermiformis) was performed in two real-word chloraminated drinking water distribution systems using quantitative polymerase chain reaction (q-PCR). A high occurrence of Legionella (" 69.0%) and mycobacteria (100%), lower occurrence of L. pneumophila (" 20%) and M. avium (" 33.3%), and rare detection of Pseudomonas aeruginosa (" 13.3%) was observed in both systems. Hartmanella vermiformis was more prevalent than Acanthamoeba. Three-minute flushing resulted in reduced gene copies of Legionella, mycobacteria, H. vermiformis and 16S rRNA genes (P<0.05) and distinct microbial community structure in postflushing water, implying strong regrowth potential of opportunistic pathogens in premise pluming. In order to examine the influence of pipe material, disinfectant type, and water age on occurrence and persistence of the target microorganisms, triplicate simulated distribution systems (SDSs) comparing iron, cement and PVC pipe materials were fed either chlorinated or chloraminated tap water, and were sampled at water ages ranging from 1d to 5.7d. q-PCR quantification of target microorganisms in both biofilm and bulk water revealed that Legionella, mycobacteria, P. aeruginosa and both amoebas naturally colonized the six SDSs, but L. pneumophila and M. avium were not detected. Disinfectant type and dose have the strongest influence on the microbiota. Disinfectant decay was noted with water age, particularly in chloraminated SDSs (due to nitrification), generally resulting in increased microbial detection frequencies and densities with water age. Influence of pipe material became apparent at water ages corresponding to low disinfectant residual. Natural colonization of Legionella spp., Mycobacterium spp., Acanthamoeba spp., H. vermiformis and M. avium was also observed in biofilms from five annular reactors, which were used to investigate effects of prior granular activated carbon (GAC) biofiltration and disinfectant type (chlorine, chloramine) on opportunistic pathogens under premise plumbing conditions. GAC pre-treatment effectively reduced total organic carbon (TOC). In most cases, total bacteria and opportunistic pathogens were higher in undisinfected annular reactors, but the levels were not proportional to the level of GAC pre-treatment/TOC. Chlorine was more effective for controlling mycobacteria and Acanthamoeba, whereas chloramine was more effective for controlling Legionella. Both chlorine and chloramine effectively reduced M. avium and H. vermiformis numbers. Pyrosequencing of 16S rRNA genes in biofilms revealed a significant effect of GAC pre-treatment and disinfectant type on the microbial community structure. Overall, the study provides insights to critical factors triggering proliferation of opportunistic pathogens in drinking water systems. Knowledge gained from this study can assist in formulating practical guidance for drinking water utilities and water consumers in terms of opportunistic pathogen prevention and control. / Ph. D.

No description available.