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Transporte do brometo de etídio através da parede celular de Mycrobacterium smegmatis: desenvolvimento e aplicação de metodologias de quantificação do transporte e correlação com a resistência aos antibióticos
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O efluxo activo e a permeabilidade celular reduzida são consideradas as principais causas da resistência intrínseca micobacteriana a diversos antimicrobianos. Neste estudo comparámos a capacidade de extrusão do brometo de etídio (EtBr), um substrato universal de bombas de efluxo, pela estirpe selvagem M. smegmatis mc2155 com
estirpes mutantes deletadas para LfrA e MspA, respectivamente a principal bomba de efluxo e porina de M. smegmatis, na presença e ausência dos inibidores de bombas de efluxo (IBEs) verapamil, tioridazina, clorpromazina e carbonil cianeto mclorofenilhidrazona
(CCCP) e correlacionámos estes resultados com a capacidade destes
IBEs em reduzir a susceptibilidade de M. smegmatis a diversos antibióticos.
Os resultados obtidos mostram que, na ausência da principal porina de M. smegmatis, MspA, a acumulação de EtBr diminui e as células tornam-se igualmente mais resistentes a diversos antibióticos. Quando a principal bomba de efluxo de M. smegmatis, LfrA, é deletada a estirpe mutante apresenta um incremento na acumulação de EtBr e um aumento na susceptibilidade ao EtBr, isoniazida, rifampicina, etambutol e ciprofloxacina. Na presença dos IBEs testados, com excepção do CCCP, observa-se uma redução da concentração mínima inibitória para a estreptomicina, rifampicina, amicacina, ciprofloxacina, claritromicina e eritromicina.
Estes resultados colocam em evidência que a porina MspA é um canal importante para a entrada de EtBr e antibióticos nas células e que o efluxo activo através da bomba de efluxo LfrA está envolvido na resistência de baixo nível a diversos antibióticos e EtBr em M. smegmatis.
As metodologias desenvolvidas neste trabalho e os resultados obtidos
proporcionaram um contributo para o melhor conhecimento dos dois principais mecanismos responsáveis pela resistência intrínseca micobacteriana a diversos antimicrobianos, considerada como um importante factor facilitador da aquisição e estabilização de um património genético que conduz ao desenvolvimento de resistência
de alto nível em micobactérias.
Active efflux systems and reduced cell wall permeability are considered to be the main causes of mycobacterial intrinsic resistance to many antimicrobials. In this study we have compared M. smegmatis wild-type strain mc2155 with mutants for LfrA and MspA, the main efflux pump and porin of M. smegmatis, respectively, for their ability to extrude ethidium bromide (EtBr), a known efflux pump substrate, in the presence or absence of the efflux pump inhibitors (EPIs) verapamil, thioridazine, chlorpromazine and carbonyl cyanide m-chlorophenylhydrazone (CCCP), and correlated these results with the ability of the EPI to reduce the susceptibility of M. smegmatis to several antimicrobials. The results obtained show that, in the absence of the major porin of M. smegmatis, MspA, the accumulation of EtBr decreased and the cells became equally more resistant to several antibiotics. When the major M. smegmatis efflux pump, LfrA, is deleted the mutant strain show increased accumulation of EtBr and increased susceptibility to EtBr, isoniazid, rifampicin, ethambutol and ciprofloxacin. Reduction of the minimum inhibitory concentration for streptomycin, rifampicin, amikacin, ciprofloxacin, clarithromycin and erythromycin is observed in the presence of the EPI tested, with the exception of CCCP. These results put in evidence that MspA porin is an important channel for entrance of EtBr and antibiotics into the cells and that active efflux via the LfrA efflux pump is involved in low-level resistance to several antibiotics and EtBr in M. smegmatis. The methodologies developed in this work and the results obtained provided a contribution to the knowledge of the two major mechanisms responsible for mycobacteria intrinsic resistance to several antibiotics, considered an important facilitator factor to the acquisition and stabilization of a genetic background for the development of high-level resistance in mycobacteria.
Active efflux systems and reduced cell wall permeability are considered to be the main causes of mycobacterial intrinsic resistance to many antimicrobials. In this study we have compared M. smegmatis wild-type strain mc2155 with mutants for LfrA and MspA, the main efflux pump and porin of M. smegmatis, respectively, for their ability to extrude ethidium bromide (EtBr), a known efflux pump substrate, in the presence or absence of the efflux pump inhibitors (EPIs) verapamil, thioridazine, chlorpromazine and carbonyl cyanide m-chlorophenylhydrazone (CCCP), and correlated these results with the ability of the EPI to reduce the susceptibility of M. smegmatis to several antimicrobials. The results obtained show that, in the absence of the major porin of M. smegmatis, MspA, the accumulation of EtBr decreased and the cells became equally more resistant to several antibiotics. When the major M. smegmatis efflux pump, LfrA, is deleted the mutant strain show increased accumulation of EtBr and increased susceptibility to EtBr, isoniazid, rifampicin, ethambutol and ciprofloxacin. Reduction of the minimum inhibitory concentration for streptomycin, rifampicin, amikacin, ciprofloxacin, clarithromycin and erythromycin is observed in the presence of the EPI tested, with the exception of CCCP. These results put in evidence that MspA porin is an important channel for entrance of EtBr and antibiotics into the cells and that active efflux via the LfrA efflux pump is involved in low-level resistance to several antibiotics and EtBr in M. smegmatis. The methodologies developed in this work and the results obtained provided a contribution to the knowledge of the two major mechanisms responsible for mycobacteria intrinsic resistance to several antibiotics, considered an important facilitator factor to the acquisition and stabilization of a genetic background for the development of high-level resistance in mycobacteria.
Descrição
Palavras-chave
Micobatérias Microbiologia médica Mycobacterium smegmatis Terapêutica Brometo de etílio Bombas de efluxo
Contexto Educativo
Citação
Editora
Instituto de Higiene e Medicina Tropical