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Optimization of a bioassay to evaluate Escherichia coli stress responses
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Na última década, a espectroscopia por transformada de Fourier (FTIR) tem vindo a ser aplicada às mais diversificadas áreas, sendo uma técnica cada vez mais utilizada em microbiologia, por permitir obter resultados num curto espaço de tempo e por ter um baixo custo por análise. No entanto, a reprodutibilidade desta técnica é influenciada por um grande número de fatores, entre os quais, o estado fisiológico das células, o método de manipulação da amostra e as condições de crescimento. Para tentar solucionar este problema, o presente trabalho pretende otimizar um bioensaio, baseado em espectroscopia de infravermelho, para avaliar a resposta metabólica dos microrganismos quando expostos a condições de stress, utilizando a bactéria Escherichia coli como modelo, uma vez que as suas vias metabólicas são muito semelhantes com as de outros microrganismos.
O bioensaio foi otimizado em termos da concentração de nutriente do meio de cultura, (tendo sido testadas as concentrações 1.25, 2.5 e 5x), fase de crescimento da bactéria E. coli (onde foram utilizadas células na fase exponencial, no início da fase estacionária e na fase estacionária) e o tempo de exposição das células aos agentes de stress (1, 8 e 24 h). Os agentes de stress utilizados foram: álcool, lixívia, cloreto de sódio, ácido clorídrico e hidróxido de sódio. Estes agentes foram escolhidos por teres diferentes mecanismos de ação nas células dos microrganismos. É de extrema importância o desenvolvimento e otimização de bioensaios que permitam avaliar a resposta metabólica das células dos microrganismos quando expostos a condições adversas, para aumentar o conhecimento sobre o metabolismo microbiano e, por exemplo, para o desenvolvimento de novos antibióticos. Após a otimização do bioensaio, foi decidido testar o ensaio expondo as células de E. coli a seis antibióticos diferentes, cujos mecanismos de ação são conhecidos. Desta maneira, é possível observar se a otimização do bioensaio cumpre o objetivo de diferenciar amostras expostas a diferentes substâncias. Observou-se que as condições que permitem obter uma maior reprodutibilidade e sensibilidade do bioensaio são diferentes para cada tipo de agente de stress, mas regra geral, expor as células aos agentes de stress durante 24 horas, usar o meio 1.25x concentrado e as células na fase de crescimento estacionária permite distinguir os vários mecanismos de ação no metabolismo das células de E. coli.
A metodologia descrita permitiu identificar o impacto de cada parâmetro na resolução metabólica da impressão digital biomolécula adquirida pela espectroscopia FTIR e, portanto, o bioensaio otimizado maximiza a capacidade metabólica de impressão digital do método, nomeadamente ao caracterizar diferentes respostas metabólico ao stress.
In the last decade, Fourier Transform Infrared Spectroscopy (FTIR) has been applied in several areas, being an increasingly used technique in microbiology, especially for whole cell metabolomics fingerprint. However, the reproducibility of this technique is influenced by a large number of factors such as the physiological state of cells, sample manipulation and growth conditions. In order to solve this problem, the present work intends to optimize a bioassay for evaluating the response of microorganisms to stress conditions, using Escherichia coli as a model, as its metabolic pathways share pronounced similarities to mainstream pathways across other microorganisms. The bioassay was optimized in terms of nutrient medium concentration, E.coli growth phase and exposed time to the stress agents. The stress agents used were: ethanol, bleach, sodium chloride, hydrochloric acid and sodium hydroxide. These agents were chosen for having different mechanisms of action in microorganism’s cells. A bioassay which allows to evaluate the metabolic response of the microorganism cell when exposed to stress conditions is important to increase knowledge about microbial metabolism and for example for the development of new drugs. After the bioassay optimization, it was decided to test the assay exposing the E. coli cells to six antibiotics, whose mechanism of action is known. This allows to observe if the optimization of the bioassay meets the objective of differentiating samples exposed to different substances. It was observed that the conditions that allow higher reproducibility and sensitivity of the bioassay are different for each type of stress agent, but as a general rule exposing the cells to stress agents for 24 hrs, use the nutrient medium 1.25x concentrate and the cells in the stationary growth phase were the global optimal conditions to distinguish the various mechanisms of action on the metabolism of E. coli cells. The approach described allowed to identify the impact of each parameter on the metabolic resolution of the biomolecular fingerprint acquired by FTIR spectroscopy, and therefore, the adapted bioassay maximizes the metabolic fingerprinting ability of the method, namely to characterize different metabolic stress responses.
In the last decade, Fourier Transform Infrared Spectroscopy (FTIR) has been applied in several areas, being an increasingly used technique in microbiology, especially for whole cell metabolomics fingerprint. However, the reproducibility of this technique is influenced by a large number of factors such as the physiological state of cells, sample manipulation and growth conditions. In order to solve this problem, the present work intends to optimize a bioassay for evaluating the response of microorganisms to stress conditions, using Escherichia coli as a model, as its metabolic pathways share pronounced similarities to mainstream pathways across other microorganisms. The bioassay was optimized in terms of nutrient medium concentration, E.coli growth phase and exposed time to the stress agents. The stress agents used were: ethanol, bleach, sodium chloride, hydrochloric acid and sodium hydroxide. These agents were chosen for having different mechanisms of action in microorganism’s cells. A bioassay which allows to evaluate the metabolic response of the microorganism cell when exposed to stress conditions is important to increase knowledge about microbial metabolism and for example for the development of new drugs. After the bioassay optimization, it was decided to test the assay exposing the E. coli cells to six antibiotics, whose mechanism of action is known. This allows to observe if the optimization of the bioassay meets the objective of differentiating samples exposed to different substances. It was observed that the conditions that allow higher reproducibility and sensitivity of the bioassay are different for each type of stress agent, but as a general rule exposing the cells to stress agents for 24 hrs, use the nutrient medium 1.25x concentrate and the cells in the stationary growth phase were the global optimal conditions to distinguish the various mechanisms of action on the metabolism of E. coli cells. The approach described allowed to identify the impact of each parameter on the metabolic resolution of the biomolecular fingerprint acquired by FTIR spectroscopy, and therefore, the adapted bioassay maximizes the metabolic fingerprinting ability of the method, namely to characterize different metabolic stress responses.
Descrição
Tese de mestrado em Microbiologia Aplicada, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2017
Palavras-chave
Resposta ao stress Escherichia coli Espectroscopia de infravermelho por transformada de Fourier Bioensaio Impressão metabólica Teses de mestrado - 2017