35 resultados para Staphylococcus aureus alpha-toxin HaCat keratinocyte
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Dissertao apresentada para a obteno do Grau de Mestre em Gentica Molecular e Biomedicina, pela Universidade Nova de Lisboa, Faculdade de Cincias e Tecnologia
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Dissertation presented to obtain a PhD degree in Biology/ Molecular Biology by the Universidade Nova de Lisboa, Instituto de Tecnologia Qumica e Biolgica
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RESUMO: Os Staphylococcus aureus resistentes meticilina (MRSA, do ingls methicillin-resistant Staphylococcus aureus) so um dos principais agentes responsveis por infees hospitalares. Os MRSA so resistentes a praticamente todos os antibiticos -lactmicos devido a dois mecanismos principais: produo de -lactamase (bla), codificada pelo gene blaZ, e produo de uma protena de ligao penicilina (PBP2a, do ingls penicillin binding protein 2), codificada pelo gene mecA. Estes dois genes so regulados por sistemas homlogos, constitudos por um sensor-transdutor (BlaR1 e MecR1) e um repressor (BlaI e MecI), de tal modo que ambos os sistemas so capazes de co-regular os genes mecA e blaZ, embora com eficincias de induo muito diferentes. De facto, a induo mediada pelo sistema mecI-mecR1 to lenta que se acredita que este sistema no est funcional na maioria das estirpes MRSA. No entanto, dados recentes do nosso laboratrio, demonstram a ausncia de relao entre a presena do gene mecI e o nvel de resistncia meticilina em estirpes MRSA epidmicas, e tambm que, o fentipo de resistncia da grande maioria das estirpes no perturbado pela sobre-expresso em trans do repressor mecI. Curiosamente, as duas estirpes em que a expresso da resistncia foi afetada pela sobre-expresso do mecI so negativas para o locus da -lactamase, o que sugere que este locus pode interferir diretamente com a represso do gene mecA mediada pelo MecI. Nesta tese de mestrado esta hiptese foi explorada usando estratgias de biologia molecular e ensaios fenotpicos da resistncia aos -lactmicos. Os resultados obtidos demonstram que a presena do plasmdeo nativo da -lactamase no s anula a represso mediada pelo MecI, como tambm aumenta o nvel de resistncia das estirpes parentais. Vrias hipteses foram ento formuladas para explicar estas observaes. Dados preliminares, em conjunto com evidncias experimentais publicadas, sugerem que o BlaI forma hetero-dmeros com o MecI que, aps a induo, so inativados eficientemente pelo BlaR1. Em concluso, estes resultados apresentam novas perspetivas para o mecanismo de regulao do mecA e para uma nova importante funo do opero da -lactamase para o fentipo das estirpes MRSA.-------------------ABSTRACT: Methicillin-resistant Staphylococcus aureus (MRSA) is an important nosocomial pathogen and is also emerging in the community. MRSA is cross-resistant to virtually all -lactam antibiotics and has acquired two main resistance mechanisms: production of -lactamase (bla), coded by blaZ, and production of penicillin binding protein 2a (PBP2a), coded by mecA. Both genes are regulated by homologous sensor-transducers (BlaR1 and MecR1) and repressors (BlaI and MecI), and coregulation of mecA and blaZ by both systems has been demonstrated, although with remarkable different efficiencies. In fact, induction of mecA by mecI-mecR1 is so slow that it is believed it is not functional in most MRSA strains. However, recent data from our laboratory has unexpectedly demonstrated that not only there is no correlation between the presence of mecI gene and the resistance level in epidemic MRSA strains, but also that for most strains there were no significant changes on the resistance phenotype upon the mecI overexpression in trans. Interestingly, the two strains in which mecI overexpression affected the resistance expression were negative for the bla locus, suggesting that this locus may interfere directly with the MecI-mediated repression of mecA and account for those puzzling observations. In this master thesis we have explored this hypothesis using molecular biology strategies and phenotypic analysis of -lactam resistance. The data obtained demonstrate that the presence of a wild-type plasmid containing the bla locus not only disrupts the MecImediated repression, but also significantly enhances the expression of resistance. Several preliminary hypotheses were formulated to explain these observations and preliminary data, together with published evidence, support the working model that BlaI forms functional hetero-dimers with MecI, which upon induction are readily inactivated by BlaR1. These results provide new insights into the regulatory mechanism(s) of mecA and open new perspectives for the role of -lactamase operon in the MRSA phenotype.
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RESUMO: O efluxo de compostos antimicrobianos um mecanismo importante na multirresistncia em bactrias. Bombas de efluxo codificadas em plasmdeos, como a QacA e a Smr, esto implicadas na susceptibilidade reduzida a biocidas, geralmente utilizados na preveno e controlo de infeces nosocomiais, incluindo as causadas por estirpes de Staphylococcus aureus resistentes meticilina (MRSA). Neste trabalho pretendeu-se avaliar a relevncia de QacA e Smr no perfil de susceptibilidade dos isolados clnicos MRSA SM39 e SM52, que transportam os plasmdeos pSM39 e pSM52 com os determinantes qacA e smr, respectivamente. A actividade de efluxo das estirpes SM39 e SM39 curada (sem pSM39) e das estirpes SM52 e RN4220:pSM52 (estirpe susceptvel RN4220 transformada com pSM52) foi caracterizada por: (1) determinao da concentrao mnima inibitria (CMI) de biocidas, corantes e antibiticos, na ausncia e presena dos inibidores de efluxo tioridazina, clorpromazina, verapamil e reserpina; e (2) fluorometria em tempo-real. A determinao de CMIs demonstrou que a actividade de efluxo mediada por QacA e Smr est envolvida na susceptibilidade reduzida aos biocidas e corantes testados, que incluram o brometo de hexadeciltrimetilamnio, a cetrimida, o cloreto de benzalcnio, a berberina, o cloreto de dequalnio, a pentamidina e o brometo de etdeo. Os ensaios fluoromtricos confirmaram a elevada actividade de efluxo presente nas estirpes com os genes qacA ou smr. A determinao de CMIs para antibiticos -lactmicos em conjunto com o teste da nitrocefina revelou a presena simultnea do gene qacA e de uma -lactamase no plasmdeo pSM39. Este trabalho evidencia a importncia das bombas de efluxo QacA e Smr na resistncia a biocidas em estirpes MRSA e na sobrevivncia destas estirpes em ambiente hospitalar e na comunidade, para alm de destacar a questo da potencial co-resistncia entre biocidas e antibiticos.--------------- ABSTRACT: Drug efflux has become an important cause of multidrug resistance (MDR) in bacteria. Plasmid-encoded MDR efflux pumps, such as QacA and Smr, are implicated in reduced susceptibility to biocides, generally used in the prevention and control of nosocomial infections, including the ones caused by methicillin-resistant Staphylococcus aureus (MRSA). In this work, we aimed to evaluate the relevance of QacA and Smr to the susceptibility profile of the clinical MRSA isolates SM39 and SM52, which harbor the plasmids pSM39 and pSM52 that carry the determinants qacA and smr, respectively. Efflux activity of strain SM39 and its plasmid-free counterpart, SM39 cured, SM52 and RN4220:pSM52 (susceptible strain RN4220 transformed with pSM52) was characterized by: (1) determination of minimum inhibitory concentration (MIC) of biocides, dyes and antibiotics, in the absence and presence of the efflux inhibitors thioridazine, chlorpromazine, verapamil and reserpine; and (2) real-time fluorometry. MIC determination showed that QacA and Smr mediated efflux was involved in the reduced susceptibility profile to the biocides and dyes tested, which included hexadecyltrymethylammonium bromide, cetrimide, benzalkonium chloride, berberine, dequalinium chloride, pentamidine and ethidium bromide. Fluorometric assays confirmed the higher efflux activity present in strains harboring qacA or smr genes. Moreover, MIC determination for -lactam antibiotics together with the nitrocefin test confirmed the presence of a -lactamase in the plasmid carried by SM39 strain, pSM39. This work highlights the relevance of QacA and Smr to the biocide resistance in MRSA strains, and consequently to their survival and maintenance in the hospital environment and in the community. Furthermore, the presence of a -lactamase and qacA determinants in the the same plasmid reinforces the question of the potencial biocide/antibiotic co-resistance in MRSA strains.
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Dissertao para obteno do Grau de Doutor em Biologia
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Dissertation presented to obtain the Ph.D degree in Biology
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Dissertao para a obteno do grau de doutor em Biologia pelo Instituto de Tecnologia Qumica e Biolgica. Universidade Nova de Lisboa
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Dissertation presented to obtain the Ph.D degree in Biology
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Dissertao para obteno do Grau de Mestre em Microbiologia mdica
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A thesis submitted for the Degree of Master in Medical microbiology
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Staphylococcus aureus is one of the most important contemporary human pathogens. The evolutionary success of this species is closely related to its remarkably capacity to acquire antibiotic resistance traits. In this perspective, it is important to extend our knowledge concerning the mechanisms of antibiotic resistance in S. aureus and to identify new antimicrobials targets.(...)
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Methicillin-resistant Staphylococcus aureus (MRSA), a human pathogen confined to hospitals (HAMRSA) for over 30 years have been emerging worldwide in the last two decades as a leading cause of severe infections in healthy individuals in the community (CA-MRSA). Despite its clinical significance, in the beginning of our studies no information existed on the prevalence, and population structure of CA-MRSA in Portugal. Moreover, it remained to be clarified how CA-MRSA emerged in our country. In particular, it was not known if CA-MRSA emerged locally by acquisition of the staphylococcal cassette chromosome mec (SCCmec) by established methicillin-susceptible S. aureus (MSSA) in the community, if they were imported from abroad or have escaped from the hospital.(...)
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Part of the work described in this chapter, was the subject of the following publication: D. Vieira, T. a. Figueiredo, A. Verma, R. G. Sobral, A. M. Ludovice, H. de Lencastre, and J. Trincao, Purification, crystallization and preliminary X-ray diffraction analysis of GatD, a glutamine amidotransferase-like protein from Staphylococcus aureus peptidoglycan, Acta Crystallogr. Sect. F Struct. Biol. Commun., vol. 70, no. 5, pp. 14, Apr. 2014.
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The cell wall of Staphylococcus aureus is a highly complex network mainly composed of highly cross-linked peptidoglycan (PG) and teichoic acids (TAs), both important for the maintenance of the integrity and viability of bacteria. The penicillin binding proteins (PBPs), which catalyse the final stage of PG biosynthesis, are targets of -lactam antibiotics and have been a key focus of antibacterial research. S. aureus has four native PBPs, PBP1-4 carried by both methicillin-sensitive (MSSA) and resistant (MRSA) strains. PBP4 is required for the synthesis of the highly cross-linked PG and, as shown in recent studies, is essential for the expression of -lactam resistance in community-acquired strains (CA-MRSA). This protein has a septal localization that seems to be spatially and temporally regulated by an unknown intermediate of the wall teichoic acids (WTA) biosynthesis pathway. Therefore, if WTA synthesis is compromised, PBP4 becomes dispersed throughout the entire cell membrane. The aim of this project was to identify the WTA precursor responsible for the septal recruitment of PBP4. In order to do so, inducible mutants of tarB and tarL genes in the background of NCTCPBP4-YFP were constructed allowing for the study of PBP4 localization in the presence and absence of these specific tar genes.With this work we were able to show that the absence of TarB or TarL leads to the delocalization of PBP4, indicating that TarL or a protein/WTA precursor whose localization/synthesis is dependent on TarL is responsible for the recruitment of PBP4.
