Made available in DSpace on 2014-06-12T18:28:52Z (GMT). No. of bitstreams: 2 arquivo1139_1.pdf: 1823767 bytes, checksum: 3c97dca6c1335d6168c89fcfde1d6c4c (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2011 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Pseudomonas aeruginosa é um patógeno oportunista frequentemente associado a infecções nosocomiais e resistência a diversos antimicrobianos. O surgimento de mecanismos de resistência aos carbapenêmicos, como a produção das enzimas carbapenemases MBL (metalo-β- lactamase) e KPC (Klebsiella pneumoniae carbapenemase), tem se destacado devido ao amplo espectro de degradação de antibióticos, levando à redução das opções terapêuticas. Este trabalho teve por objetivo caracterizar fenotipicamente e genotipicamente isolados nosocomiais de P. aeruginosa procedentes de cinco hospitais públicos do Recife coletados no período de 2006 a 2010. Características fenotípicas como morfologia da colônia, produção de pigmento, gelatinase e hemolisina, assim como o perfil de susceptibilidade a antimicrobianos foram avaliados pelo crescimento em meios específicos e pelo método de disco difusão padronizado pelo CLSI (2008), respectivamente. Os isolados resistentes aos carbapenêmicos foram submetidos à pesquisa de genes blaKPC, e os resistentes a ceftazidima (CAZ) e/ou imipenem (IPM) foram investigados quanto à produção de MBL utilizando o método de disco aproximação com o ácido 2-mercaptopropiônico (2-MPA), seguido pela pesquisa de genes blaSPM-1, blaIMP, blaVIM dentre os isolados MBL positivos no teste fenotípico. A investigação da relação clonal das amostras de P. aeruginosa foi realizada utilizando o método de sequências consenso intergênicas repetitivas de enterobactérias (ERIC PCR). Durante o período da pesquisa foram obtidos 61 isolados de P. aeruginosa, dos quais 36,96% eram mucoides; 69,81% eram produtores de piocianina; 92,86% eram gelatinase positivos e 71,70% hemolisina positivo. A sensibilidade aos antimicrobianos variou entre 44,26% e 81,97%, para o aztreonam e polimixina B, respectivamente. Observou-se também que 4,92% dos isolados eram panresistentes e 54,09% multirresistentes, dos quais 34,14% eram sensíveis apenas a polimixina B. Dentre os 26 isolados resistentes aos carbapenêmicos, 7,65% foram positivos para o gene blaKPC, enquanto que dentre os 29 isolados resistentes ao IPM e/ou CAZ, 44,83% foram positivos para pesquisa de MBL, e destes, 46,15% foram positivos para o gene blaSPM-1, não havendo detecção dos genes blaIMP e blaVIM. A tipagem molecular dos isolados revelou 21 perfis genéticos distintos. Os isolados KPC positivos pertenciam a um mesmo clone e dos isolados SPM-1 positivos, três apresentaram o mesmo perfil clonal e eram procedentes do mesmo hospital, enquanto os outros três isolados SPM-1 positivos apresentaram perfis clonais distintos. A capacidade de produção de MBL por P. aeruginosa tem sido detectada com elevada prevalência em hospitais da cidade do Recife nos últimos anos, porém a detecção de P. aeruginosa KPC positivas ainda é rara em todo o um mundo, sendo esse o primeiro relato no Brasil

Made available in DSpace on 2014-06-12T18:29:37Z (GMT). No. of bitstreams: 2 arquivo3468_1.pdf: 1099139 bytes, checksum: 574af48b24a50c6ed65c25e40fd65aa0 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2008 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Um estudo de caso-controle retrospectivo foi realizado para investigar os fatores de risco para óbito em pacientes de Unidades de Terapia Intensiva com infecção por Pseudomonas aeruginosa. Dos 131 pacientes investigados, 67 (51,1%) morreram até 30 dias após o diagnóstico de infecção por esse patógeno. A duração média de internamento hospitalar antes do diagnóstico de infecção por Pseudomonas aeruginosa foi de 28,5 ± 26,5 dias. Não foi encontrada associação entre resistência e óbito nesse estudo (multi-resistente p=0,26; panresistente p= 0,42), porém o acerto na terapêutica inicial foi inversamente proporcional ao grau de resistência. Houve uma tendência a maior mortalidade nos pacientes que receberam terapia combinada (empírica p=0,09; definitiva p= 0,08), apesar do maior acerto na terapêutica e do menor percentual de choque séptico nesse grupo de doentes. Esse achado pode ser justificado por parâmetros farmacodinâmicos que não foram estudados nesse trabalho e pelo uso de aminoglicosídeos em grande escala na terapia dupla, o que é ponto controverso nos estudos realizados sobre a eficácia dessa combinação. Análise multivariada do nosso estudo concluiu que idade [odds ratio (OR) 1.04], presença de choque séptico (OR 15.4) e hipoalbuminemia (OR 0.32) foram fatores de risco independentes para o óbito

Made available in DSpace on 2014-06-12T23:13:25Z (GMT). No. of bitstreams: 2 arquivo8723_1.pdf: 3102671 bytes, checksum: 63033e858f152a6154f2c73715fb9ff6 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2006 / O aumento do número de bactérias multi-resistentes aos antibióticos tem crescido significativamente nos últimos anos. Entre as bactérias gram-negativos, a P.aeruginosa tem demonstrado uma maior facilidade de desenvolvimento da resistência aos antibióticos. O objetivo deste estudo foi o de determinar os padrões de susceptibilidade antimicrobiana às diversas classes de antibióticos contra a Pseudomonas aeruginosa e realizar uma análise comparativa entre as unidades de terapia intensiva (UTI) e as enfermarias de dois hospitais terciários do Recife-PE. O estudo foi realizado entre setembro de 2004 e janeiro 2006. Os testes de susceptibilidade antimicrobiana foram realizados em 304 amostras diferentes de P. aeruginosa segundo os padrões do NCCLS (National Commitee for Clinical and Laborastory Standards). Os antibióticos testados foram: polimixina (88,7%) ; piperacilinatazobactam (66,2%) ; aztreonam (59,8%); amicacina (59,4%); meropenem (58,2%); imipenem (57,7%); ciprofloxacina (49,7%); gentamicina e cefepime (48,6%); ceftazidima (30,0%) e cefotaxima (6,8%). Os sitios mais frequentes de infecção foram a urina com 26,7% e a secreção traqueal com 26,1% . O estudo demonstrou ainda uma diferença estatisticamente significativa na susceptibilidade antimicrobiana comparativa entre as UTI e as enfermaria de um dos dois hospitais envolvidos , com menor susceptibilidade aos antibióticos testados nas UTI. Em conclusão, a freqüência de cepas de P.aeruginosa multi-resistente (28,0%) foi maior que a descrita na literatura nacional e mundial, principalmente quando isolamos o grupo de pacientes internados nas UTI(s). Os achados do estudo sugerem que o aparecimento de resistência antibiótica para P.aeruginosa no Recife tem sido mais frequente do que em outros centros Nacionais e Internacionais. Para reduzir a frequência destes clones multi-resistentes , monitorização epidemiológica e otimização da antibioticoterapia deverá ser considerada urgentemente

Please read the abstract in the section 00front of this document / Dissertation (MSc (Microbiology))--University of Pretoria, 2005. / Microbiology and Plant Pathology / unrestricted

No description available.

Cystic Fibrosis is one of the most common genetic recessive diseases among Caucasians and is caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene on chromosome 7. There are different classes of CFTR mutation, leading to differences in disease severity among patients. In addition to the CFTR genotype, secondary genetic factors, modifier genes, also influence CF phenotypes. Due to the dysfunction of CFTR protein and production of thickened mucus, bacterial infection in the lungs is favored and can lead to further clinical complications in CF patients. Pseudomonas aeruginosa is one of the most common bacteria detected among patients. The aim of this project was to investigate four candidate modifier genes, Factor B, Complement Factor 3, Toll-like Receptor 4 and Heme oxygenase-1, which might affect the status of Pseudomonas aeruginosa infection. A total of 22 single nucleotide polymorphisms (SNPs) were selected in these four genes and they were tested against five phenotypic traits, including age of diagnosis, FEV1% predicted andstandard deviation value, age of first Pseudomonas aeruginosa infection and Pseudomonas aeruginosa infection status. Among the selected SNPs, both case-control studies and family-based analysis were performed in order to establish any correlation between the genotypes and the phenotypes. In addition, haplotype analysis was performed to determine whether there was interaction between SNPs or whether there were unidentified SNPs in the vicinity of the selected ones that might contribute to the observed phenotypic traits. Among the 22 chosen SNPs, 13 of them were found to be significantly linked to one or more of the tested phenotypes. The three most significant associations were BF_2557 with lung function, HMOX1_9531 with lung function and BF_7202 with age of diagnosis. Several haplotypes were significantly associated with one of the five phenotypes. There was no evidence for the presence of unidentified SNPs or interaction between SNPs. Most of haplotype associations were likely due to the presence of a single SNP which was found to be significantly linked to the phenotype. Conclusively, both SNPs and haplotype analyses suggest that the four candidate genes are modifiers of disease severity in CF. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

No description available.

The oxidation of glucose by Pseudomonas aeruginosa is known to follow the sequence: glucose→ gluconic acid→2-ketogluconic acid→pyruvic acid and thence into the tricarboxylic acid cycle. The most striking aspect of this pathway is that the first two oxidative steps do not involve phosphorylated intermediates at the substrate level. In the present study radioactive phosphorus was used in an attempt to elucidate the carbohydrate metabolism of P. aeruginosa. Cell free preparations of P. aeruginosa, obtained by crushing a cell paste in the Hughes press, incubated with added cofactors, ADP and P³² resulted in the formation of labelled ADP and ATP. The presence of glucose or succinate in the reaction mixture greatly depressed the amount of ATP found. The cell free preparations were found to yield ATP as measured in the hexokinase trap, but the formation of ATP was not increased by the addition of glucose, gluconic acid, 2-ketogluconic acid or succinic acid. These results suggested that no net energy was gained by the extract by the oxidation glucose→ gluconic acid→ 2-ketogluconic acid. In manometric experiments it was found that the cell free preparation did not oxidize glucose-6-phosphate, ribose-5-phosphate, α-ketoglutarate, citrate and isocitrate. Glucose was oxidized with the uptake of two atoms of oxygen per mole of substrate. In the presence of ATP, glucose was oxidized with the uptake of only one atom of oxygen. Gluconic acid and gluconolactone were oxidized with the uptake of one atom of oxygen; ATP had no effect on these last two oxidations. From these data two reactions beyond 2-ketogluconate have been postulated. [Formulas omitted] / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

I have investigated the evolutionary response of Pseudomonas aeruginosa to salt (NaCl) stress, and the physiological mechanisms responsible for this adaptation. Populations of P. aeruginosa founded from the same ancestral genotype were selected at three different concentrations of NaCl, low, moderate and high for about 660 generations with four independent replicates for each concentration. Adaptation was measured as the fitness of the evolved populations relative to the ancestor assessed in direct, head-to-head competition experiments conducted in the same environment in which they were selected (direct response) as well as in all alternative environments (correlated response). Results suggest that selection in each salt environment led to adaptation to that environment and a modest degree of specialization that evolved because correlated responses to selection were smaller than direct responses. In order to identify the physiological mechanisms contributing to the populations' adaptation in high NaCl concentration, I chose a sample of evolved lines that showed the strongest evidence for specialization to salt and competed them against the common ancestor in KCl and sucrose. Results suggested that increased Na+ /H+ antiporter activity is probably the primary mechanism behind adaptation to high NaCl concentration, however alternative mechanisms cannot be excluded. Tolerance curves, which measure the performance of a genotype across a gradient of salt concentrations, suggested no change in the high salt group’s ability to tolerate extreme concentrations of NaCl. We conclude that high salt evolved population showed improvements to its ionic/osmotic stress resistance strategies mainly to Na+ efflux strategies but with no changes to salt niche.

Although the importance of tricarboxylic acid cycle activity in the metabolism of aerobic bacteria is well established, detailed studies on the utilization of intermediates of the cycle designed to assess the nature, importance and control of the enzymes concerned have not been performed with pseudomonads. Results of this investigation have shown that Pseudomonas aeruginosa ATCC 9027 lacks NAD or NADP linked L-malic dehydrogenase. Studies with cell fractions have shown that an NAD and NADP independent, particulate L-malic dehydrogenase catalyses the oxidation of L-malic acid to oxalacetic acid. The labelling patterns of citrate obtained from succinate-1,4-¹⁴ C and succinate-2,3-¹⁴ C have demonstrated the involvement of the particulate malic dehydrogenase and have excluded any other possibility. Phosphofructokinase could not be detected in the cell-free extract preparations and thus accounting for the non-functional Embden-Meyerhof pathway in this organism. Cells grown in succinate medium either do not have or have extremely low levels of glucose metabolizing enzymes. The glucose effect on tricarboxylic acid cycle enzymes was not observed. Further, the addition of α-keto- glutarate and glutamate to the medium did not repress these enzymes. These observations suggest that tricarboxylic acid cycle activity is of special importance for growth and metabolism in pseudomonads. The data have also indicated that during growth in a succinate medium, pentose synthesis must occur by the action of transketolase upon compounds derived from tricarboxylic acid cycle intermediates. It has been shown that both the glucose permease and the glucose metabolizing enzymes were induced simultaneously on shift from succinate to glucose medium. The glucose permease was found to be very specific since glucose uptake was not inhibited even in the presence of 100-fold excess of α-CH glucoside, 2-deoxyglucose, galactose, fructose or mannose. Particulate malic dehydrogenase was inhibited by adenine nucleotides while it was activated by GTP and GDP. The mechanism of this regulation is not clear, however, it is obviously important in the control of tricarboxylic acid cycle activity. Addition of glutamate to the medium repressed the synthesis of glutamic dehydrogenase. High levels of malic enzyme were maintained on growth in glucose and succinate media, whereas the levels were low in acetate medium. These observations demonstrated the capacity of the organism to regulate the synthesis of enzymes in response to the particular environment. Tricarboxylic acid cycle intermediates and glyoxylate have been found to exert "fine control" over the activities of isocitrate dehydrogenase and isocitrate lyase in such a way that flow of isocitrate through the tricarboxylic acid cycle and the glyoxylate cycle is precisely regulated to suit the needs of the cell. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate