Proteomica aplicada ao estudo da resistencia a antibioticos em estirpes de Escherichia coli isoladas de animais selvagens
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2009
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A resistência a antibióticos tem vindo a sofrer um aumento considerável nos
últimos anos, constituindo-se como um problema crescente de saúde pública. Este facto surge associado a mecanismos genéticos, como a transferência horizontal de genes, que levam à detecção de estirpes bacterianas comensais e patogénicas em diferentes organismos de origem doméstica e selvagem. Este trabalho consistiu na análise
proteómica de estirpes de Escherichia coli comensais multiresistentes isoladas de
animais selvagens. Após a caracterização a nível monodimensional, por SDS-PAGE, de
estirpes resistentes a antibióticos beta-lactâmicos, foi depois possível obter perfis
proteicos bidimensionais (IEF x SDS-PAGE). Para a obtenção destes perfis, foi
fundamental a aplicação e optimização de técnicas de extracção, solubilização,
equilibração e limpeza de amostras. Como resultado, foi possível obter géis
bidimensionais bem diferenciados que levaram à sequenciação de proteínas por
espectrometria de massa (MALDI-TOF) que, correlacionando esses dados com as bases
de dados bioinformáticas permitiu identificar e caracterizar as proteínas presentes. Da sequenciação dos polipéptidos resultou a identificação de proteínas relacionadas com serótipos de carácter patogénico e virulento de E. coli, O6 e O157:H7, como a flavoproteína wrbA com o accession Q8X4B4, de peso molecular 20690,38086 e ponto isoeléctrico 5,93 tendo estes resultados sido obtidos com um intervalo de confiança de 100%.
Através deste trabalho, foi possível identificar e caracterizar proteínas obtidas de uma estirpe de E. coli (5A BLLE) isolada de uma gaivota. No proteoma obtido
verificou-se a presença de proteínas relacionadas com mecanismos de resistência a
antibióticos (7,3%), entre outras, permitindo assim levantar novas perspectivas de investigação, nomeadamente pela identificação dos genes específicos e das vias metabólicas responsáveis pela multiresistência bacteriana.
Antibiotic resistance as suffered a major increase in the last few years, existing as a growing public health problem. This fact is associated to genetic mechanisms like horizontal gene transfer which lead to the detection of commensal and pathogenic bacterial strains in different organisms of commercial and wild origin. This study consisted on a proteomic analysis of multiresistant Escherichia coli commensal strains isolated from wild animals. After a characterization of strains resistant to beta-lactam antibiotics at the monodimensional level by SD-PAGE, it was possible obtain bidimensional proteic profiles (IEF x SDS-PAGE). In obtaining these profiles was crucial the application and optimization of techniques concerning protein extraction, solubilization, equilibration and samples clean up. As a result, were obtained well diferentiated bidimensional gels that led to the protein sequencing by mass spectrometry (MALDI-TOF) which in correlation with bioinformatic data bases allowed identifying and characterizing the proteins present. From the polypeptide sequencing resulted the identification of proteins related to E. coli serotypes of pathogenic and virulent character O6 and O157:H7, like flavoprotein wrbA with accession Q8X4B4, molecular weight 20690,38086 and isoelectric point 5,93. These results were obtained with a confidence interval of 100%. Through this study it was possible to identify and characterize proteins obtained from an E. coli strain (5A BLLE) isolated from a yellow-legged seagull. In the obtained proteome were found proteins related to antibiotic resistance mechanisms (7,3%) among others, allowing to raise new investigation perspectives naimly for the identification of the specific genes and metabolic pathways responsible for bacterial multiresistance.
Antibiotic resistance as suffered a major increase in the last few years, existing as a growing public health problem. This fact is associated to genetic mechanisms like horizontal gene transfer which lead to the detection of commensal and pathogenic bacterial strains in different organisms of commercial and wild origin. This study consisted on a proteomic analysis of multiresistant Escherichia coli commensal strains isolated from wild animals. After a characterization of strains resistant to beta-lactam antibiotics at the monodimensional level by SD-PAGE, it was possible obtain bidimensional proteic profiles (IEF x SDS-PAGE). In obtaining these profiles was crucial the application and optimization of techniques concerning protein extraction, solubilization, equilibration and samples clean up. As a result, were obtained well diferentiated bidimensional gels that led to the protein sequencing by mass spectrometry (MALDI-TOF) which in correlation with bioinformatic data bases allowed identifying and characterizing the proteins present. From the polypeptide sequencing resulted the identification of proteins related to E. coli serotypes of pathogenic and virulent character O6 and O157:H7, like flavoprotein wrbA with accession Q8X4B4, molecular weight 20690,38086 and isoelectric point 5,93. These results were obtained with a confidence interval of 100%. Through this study it was possible to identify and characterize proteins obtained from an E. coli strain (5A BLLE) isolated from a yellow-legged seagull. In the obtained proteome were found proteins related to antibiotic resistance mechanisms (7,3%) among others, allowing to raise new investigation perspectives naimly for the identification of the specific genes and metabolic pathways responsible for bacterial multiresistance.
Descrição
Dissertação de Mestrado em Genética Molecular, Comparativa e Tecnológica
Palavras-chave
Escherichia coli , Proteómica , Eletroforese , Resistência microbiana a medicamentos