A carregar...
Publicação
Determinação da actividade de efluxo de Hoechst 33258 em Escherichia coli
| Nome: | Descrição: | Tamanho: | Formato: | |
|---|---|---|---|---|
| 1.83 MB | Adobe PDF |
Autores
Orientador(es)
Resumo(s)
Para a detecção da actividade de efluxo em bactérias são normalmente usadas moléculas fluorescentes, que são substratos de bombas de efluxo e cuja concentração intracelular pode ser facilmente detectada por fluorescência. O brometo de etídio é o fluoróforo e substrato de efluxo de eleição para a caracterização da actividade de efluxo
em bactérias e serviu de base para o desenvolvimento e optimização do método fluorométrico semiautomático na Unidade de Microbiologia Médica, IHMT/NOVA, para utilização no fluorimetro e termociclador em tempo-real Rotor-Gene 3000.
Neste trabalho, adaptamos o método fluorométrico semiautomático em placa de 96 poços para o uso com o fluoróforo Hoechst 33258, um corante menos tóxico que o brometo de etídio, de modo a avaliar a sua eventual aplicabilidade para a caracterização da actividade de efluxo em bactérias, usando duas estirpes de referência de E. coli que
diferem entre si na actividade do seu principal sistema de efluxo, o AcrAB-TolC: a estirpe AG100, com o sistema de efluxo AcrAB-TolC funcional e a estirpe AG100A, com o sistema AcrAB-TolC inactivo. Concomitantemente foi avaliado o efeito sinérgico de compostos inibidores de efluxo na resistência intrínseca aos antibióticos, biocidas e
corantes nestas duas estirpes.
Os resultados obtidos demostram que (i) o Hoechst 33258 é um substrato da bomba de efluxo AcrAB-TolC; (ii) que a tioridazina e a clorpromazina potenciam a acumulação de Hoechst 33258 a níveis similares ao das células cuja a actividade de efluxo foi inactivada; (iii) é possível diferenciar estirpes de E. coli com diferentes níveis de
acumulação de Hoechst 33258, nomeadamente, a estirpe AG100 com o sistema de efluxo AcrAB activo e funcional, da estirpe isogénica AG100A, que apresenta o sistema de efluxo AcrAB-TolC inactivado; (iv) o Hoechst 33258 pode ser usado para avaliar a actividade de efluxo em E. coli; (v) o Hoechst 33258 pode ser considerado uma alternativa ao brometo de etídio e (vi) o método fluorométrico semiautomático em placa de 96 poços usando fluorimetro Synergy HT pode ser usado para detectar do transporte de Hoechst 33258 em E. coli.
A aplicação do método a novas moléculas fluorescentes permitirá aprofundar o conhecimento sobre a actividade dos sistemas de efluxo bem como identificar novos substratos e novos inibidores numa diversidade de bactérias.
For the detection of efflux activity in bacteria it is commonly used fluorescent molecules, which are efflux pump substrates and whose intracellular concentration can be easily detected by fluorescence. Ethidium bromide is the fluorophore and the substrate of choice for the characterization of efflux activity and was the basis for the development and optimization of the semiautomated fluorometric method at the Unit of Medical Microbiology, IHMT/NOVA, for use within the fluorimeter and real-time thermocycler Rotor-Gene 3000. In this work, we adapted the semiautomated fluorometric method in a 96-well plate format for the testing of the fluorophore Hoechst 33258, a less toxic compound than ethidium bromide, to study its possible applicability for the characterization of efflux activity in bacteria. We used two reference strains of E. coli that differ from each other in the activity of its main efflux system, the AcrAB-TolC efflux system: the AG100 strain, with a functional AcrAB-TolC efflux system and the AG100A strain with an inactive AcrAB-TolC efflux system. Simultaneously, it was evaluated the synergistic effect of efflux inhibitors in the resistance to antibiotics, biocides and dyes in these two strains. The results obtained demonstrate that (i) the Hoechst 33258 is a substrate of the AcrAB-TolC efflux pump; (ii) thioridazine and chlorpromazine can enhance the accumulation of Hoechst 33258 at levels similar to the cells whose efflux activity was inactivated; (iii) it is possible to differentiate E. coli strains with different levels of accumulation of Hoechst 33258, in particular, the strain AG100 with the AcrAB-TolC efflux system active and functional and the AG100A isogenic strain, with the AcrABTolC efflux system efflux inactivated; (iv) the Hoechst 33258 can be used to evaluate the efflux activity in E. coli; (v) the Hoechst 33258 can be considered an alternative to ethidium bromide; and (vi) the semiautomated fluorometric method in a 96-well plate format using the Synergy HT can be used to detect the transport of Hoechst 33258 in E. coli. The application of the method to new fluorescent molecules will allow to increase the knowledge about the activity of efflux systems as well as identify new substrates and inhibitors in a variety of bacterial species.
For the detection of efflux activity in bacteria it is commonly used fluorescent molecules, which are efflux pump substrates and whose intracellular concentration can be easily detected by fluorescence. Ethidium bromide is the fluorophore and the substrate of choice for the characterization of efflux activity and was the basis for the development and optimization of the semiautomated fluorometric method at the Unit of Medical Microbiology, IHMT/NOVA, for use within the fluorimeter and real-time thermocycler Rotor-Gene 3000. In this work, we adapted the semiautomated fluorometric method in a 96-well plate format for the testing of the fluorophore Hoechst 33258, a less toxic compound than ethidium bromide, to study its possible applicability for the characterization of efflux activity in bacteria. We used two reference strains of E. coli that differ from each other in the activity of its main efflux system, the AcrAB-TolC efflux system: the AG100 strain, with a functional AcrAB-TolC efflux system and the AG100A strain with an inactive AcrAB-TolC efflux system. Simultaneously, it was evaluated the synergistic effect of efflux inhibitors in the resistance to antibiotics, biocides and dyes in these two strains. The results obtained demonstrate that (i) the Hoechst 33258 is a substrate of the AcrAB-TolC efflux pump; (ii) thioridazine and chlorpromazine can enhance the accumulation of Hoechst 33258 at levels similar to the cells whose efflux activity was inactivated; (iii) it is possible to differentiate E. coli strains with different levels of accumulation of Hoechst 33258, in particular, the strain AG100 with the AcrAB-TolC efflux system active and functional and the AG100A isogenic strain, with the AcrABTolC efflux system efflux inactivated; (iv) the Hoechst 33258 can be used to evaluate the efflux activity in E. coli; (v) the Hoechst 33258 can be considered an alternative to ethidium bromide; and (vi) the semiautomated fluorometric method in a 96-well plate format using the Synergy HT can be used to detect the transport of Hoechst 33258 in E. coli. The application of the method to new fluorescent molecules will allow to increase the knowledge about the activity of efflux systems as well as identify new substrates and inhibitors in a variety of bacterial species.
Descrição
Palavras-chave
Microbiologia médica Biologia molecular Efluxo de Hoechst Escherichia coli Bactérias Bombas de efluxo
Contexto Educativo
Citação
Editora
Instituto de Higiene e Medicina Tropical