Formação de biofilmes por Enterococcus faecium sensível e multirresistente aos antimicrobianos: características à proteômica

• Autor(a) principal: Silva, Beatriz Nascimento Monteiro da
• Data de Publicação: 2012
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Biblioteca Digital de Teses e Dissertações da UERJ
• Texto Completo: http://www.bdtd.uerj.br/handle/1/14430
• topic: Biofilm formation; Enterococcus faecium; Vancomicyn; Proteomic analysis; Biofilmes; Enterococcus faecium; Vancomicina; Proteômica; Biofilme; Proteômica; Vancomicina; CNPQ::CIENCIAS BIOLOGICAS::MICROBIOLOGIA::MICROBIOLOGIA APLICADA::MICROBIOLOGIA MEDICA

<i>Enterococcus faecium</i> has emerged as an important pathogen related to the scenario of the hospitalar infections, particularly strains bearing characteristics of multidrug resistance. Despite being identified as the etiological agents in various infections, their virulence traits remain unclear. However, it is known that the ability to form biofilms may be implicated as one of the attributes capable of promoting the pathogenic role of these microorganisms. Therefore, this study aimed to investigate the ability of biofilm formation by two strains of <i>E. faecium</i>: SS-1274 (derived from the type strain) and CL-6729 (belonging to a clonal complex globally dispersed). Quantitative analysis of biofilm formation were carried out by using the semiquantitative crystal violet assay after 24h and 72 h of incubation in poliestirene microtiter plates. Growth curves were obtained for biomass quantification by crystal violet assay after a range of incubation period from 2h to 74 h. The influence of subinhibitory concentrations (subMICs) of ampicillin, gentamicin and vancomycin in the biofilm formation of <i>E. faecium</i> is further characterized in assays for quantification of biomass. The presence of DNA and protein in extracellular polymeric substance (EPS) was evaluated by detachment assays and by fluorimetry. The architecture, spatial distribution and reaction against the fluorochromes Syto9 (DNA stain) and Sypro Ruby Protein (protein stain) were observed by confocal laser scanning microscopy (CLSM). Proteomic analysis was evaluated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) and by ESI-Q-TOF mass spectrometry. Our results showed that the multidrug resistance strain (CL-6729) formed greater biomass than the reference strain (SS-1274) at the different periods. The growth curve analyses suggest different stages during the process of maturation of these structures. Despite the lower amount of biomass of SS-1274, the profile of the growth curve, during the period measured, was similar to CL-6729. The analysis of extracellular polymeric substance by confocal microscopy and quantitative assays revealed that protein appears to be an important constituent for the biofilms constituition of both strains. However biofilms formed in the presence of antimicrobials, especially those formed in the presence of ¼ of CMI and during 24h seem to suffer changes in the constitution. The results for mass spectrometry suggest that biofilm cells of <i>Enterococcus faecium</i> can also be metabolically active because the identification of a considerable number of proteins related to the metabolism and cell division, similarly to planktonic cells. However, further investigations are needed to quantify the differences related to its expression.