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Impacto da sobre-expressão de bombas de efluxo de estirpes E. coli MDR clínicas na resistência aos compostos antimicrobianos
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A multirresistência aos antibióticos é actualmente uma questão de enorme relevância clínica, que coloca sérias limitações em termos de opções terapêuticas.
Este problema tem ganho particular visibilidade em Enterobacteríaceas, assistindose a um aumento de infecções por estirpes de Escherichia coli multirresistentes.
Com este trabalho pretendem-se estudar os mecanismos fisiológicos de
resistência aos antibióticos em E. coli, com particular ênfase na detecção e quantificação da componente de efluxo nesta resistência.
Foram estudadas 8 estirpes clínicas de E. coli multirresistentes, isoladas no serviço de Patologia Clínica do Hospital dos SAMS, de pacientes internados neste hospital entre o último semestre de 2007 e o primeiro semestre de 2008. A presença de actividade de bombas de efluxo nestas estirpes foi avaliada por:
(1) Determinação de concentrações mínimas inibitórias para a ciprofloxacina, trimetoprim e gentamicina na presença/ausência de inibidores de bombas de efluxo (Carbonyl cyanide m-chlorophenylhydrazone, phenylalanyl arginyl-β-
naphthylamide, verapamil, tioridazina e clorpromazina);
(2) Detecção da possível sobre-expressão de bombas de efluxo por aumento da extrusão de brometo de etídeo, um substrato de bombas de efluxo, por métodos qualitativos em placa e quantitativos por fluorimetria em tempo real, utilizando um termociclador em tempo real (Rotor-Gene 3000TM da Corbett).
(3) Quantificação relativa dos níveis de expressão de mRNA (qRT-PCR) dos genes acrA, acrB, acrE, acrF, marA, yhiV e emrB, codificantes e reguladores de bombas de efluxo relacionadas com a resistência aos antibióticos em E. coli. Das 8 estirpes de E. coli testadas, 4 demonstraram possuir um efluxo aumentado de brometo de etídeo, que é fortemente inibido pelos inibidores de bombas de efluxo testados, tendo-se verificado concomitante reversão/redução da resistência aos
antibióticos nestas estirpes quando na presença de inibidores de bombas de efluxo. A análise por qRT-PCR das 2 estirpes com maior actividade de efluxo, foi possível verificar a sobre-expressão dos genes codificantes para as bombas de efluxo estudados, destacando-se a sobre-expressão (50x aumentado) do principal sistema de efluxo em E.
coli, o sistema acrAB, numa das estirpes altamente resistente aos antibióticos.
Igualmente foi possível caracterizar uma estirpe clínica de E.coli multirresistente com actividade de efluxo aumentada devido à sobre expressão do sistema AcrEF.
Verificou-se que a sobre-expressão de bombas de efluxo é uma realidade nas estirpes clínicas de E. coli, contribuindo significativamente para a multirresistência aos antibióticos. A utilização de técnicas simples e rápidas com recurso a substratos fluorescentes, poderá contribuir para detectar precocemente a resistência aos
antibióticos por sobre-expressão de bombas de efluxo. No futuro, o uso de inibidores de bombas de efluxo como compostos adjuvantes da terapêutica poderá vir a constituir uma opção clínica a considerar de forma a recuperar a actividade de antibióticos em estirpes resistentes.
The multidrug resistance to antibiotics is in nowadays an important issue with clinical significance, and that brings restrictions to therapeutic options. This problem has gained visibility mostly in Enterobactereaceae, with an increase in Escherichia coli (E. coli) multidrug resistant infections. The aim of this work is to study physiologic mechanisms of antibiotic resistance in E. coli, with particular emphasis in the detection and quantification of efflux component in bacterial resistance. We have studied 8 multidrug resistant E. coli strains, isolated in the Clinical Pathology Laboratory of SAMS Hospital, Lisbon from hospitalized patients between the last semester of 2007 and the first semester of 2008. The presence of efflux pumps activity in these strains was evaluated with different methods: (1) Determination of the Minimum inhibitory concentration (MICs) for ciprofloxacin, gentamicin, trimethoprim in the presence/absence of efflux pump inhibitors (Carbonyl cyanide m-chlorophenylhydrazone, phenylalanyl arginyl-β- naphthylamide, verapamil, thioridazine e chlorpromazine); (2) Detection of the efflux pumps over expression due to an increase in expulsion of ethidium bromide, that is an efflux pump substrate, with qualitative (solid medium plates) and quantitative methods (real time fluorimetry, with the use of the Rotor- Gene 3000TM Corbett termocycler). (3) Relative quantification of mRNAs (qRT-PCR) of acrA, acrB, acrE, acrF, marA, yhiV e emrB efflux pumps encoded genes in E. coli.From the 8 strains of E. coli studied, 4 showed increased ethidium bromide efflux, and we were able to observe reversion/reduction of their antibiotic resistance when in presence of efflux pump inhibitors. The qRT-PCR analysis of the two strains that showed the most active efflux activity revealed the over expression of their efflux-pump genes, with particular importance on the most important efflux system of E. coli, the AcrAB efflux system (50x increased). It was also demonstrated and characterized a clinical E. coli MDR strain with its MDR phenotype associated with the overexpression and increased activity of the AcrEF system. The methods used in this work allowed the detection of the increase efflux activity in multidrug resistant E. coli clinical strains and this increased activity was correlated with the over expression of their efflux-pump systems, with direct relation to their multidrug resistance. Therefore, over expression of efflux pump is a fact in E. coli clinical strains and this has direct implications in their multidrug resistance. The application of simple and fast techniques might contribute to early detect antibiotic resistance due to efflux pumps over expression in clinical strains. In the future, the application of efflux pump inhibitors as helper compounds in antibiotherapy might be a clinical option to consider.
The multidrug resistance to antibiotics is in nowadays an important issue with clinical significance, and that brings restrictions to therapeutic options. This problem has gained visibility mostly in Enterobactereaceae, with an increase in Escherichia coli (E. coli) multidrug resistant infections. The aim of this work is to study physiologic mechanisms of antibiotic resistance in E. coli, with particular emphasis in the detection and quantification of efflux component in bacterial resistance. We have studied 8 multidrug resistant E. coli strains, isolated in the Clinical Pathology Laboratory of SAMS Hospital, Lisbon from hospitalized patients between the last semester of 2007 and the first semester of 2008. The presence of efflux pumps activity in these strains was evaluated with different methods: (1) Determination of the Minimum inhibitory concentration (MICs) for ciprofloxacin, gentamicin, trimethoprim in the presence/absence of efflux pump inhibitors (Carbonyl cyanide m-chlorophenylhydrazone, phenylalanyl arginyl-β- naphthylamide, verapamil, thioridazine e chlorpromazine); (2) Detection of the efflux pumps over expression due to an increase in expulsion of ethidium bromide, that is an efflux pump substrate, with qualitative (solid medium plates) and quantitative methods (real time fluorimetry, with the use of the Rotor- Gene 3000TM Corbett termocycler). (3) Relative quantification of mRNAs (qRT-PCR) of acrA, acrB, acrE, acrF, marA, yhiV e emrB efflux pumps encoded genes in E. coli.From the 8 strains of E. coli studied, 4 showed increased ethidium bromide efflux, and we were able to observe reversion/reduction of their antibiotic resistance when in presence of efflux pump inhibitors. The qRT-PCR analysis of the two strains that showed the most active efflux activity revealed the over expression of their efflux-pump genes, with particular importance on the most important efflux system of E. coli, the AcrAB efflux system (50x increased). It was also demonstrated and characterized a clinical E. coli MDR strain with its MDR phenotype associated with the overexpression and increased activity of the AcrEF system. The methods used in this work allowed the detection of the increase efflux activity in multidrug resistant E. coli clinical strains and this increased activity was correlated with the over expression of their efflux-pump systems, with direct relation to their multidrug resistance. Therefore, over expression of efflux pump is a fact in E. coli clinical strains and this has direct implications in their multidrug resistance. The application of simple and fast techniques might contribute to early detect antibiotic resistance due to efflux pumps over expression in clinical strains. In the future, the application of efflux pump inhibitors as helper compounds in antibiotherapy might be a clinical option to consider.
Descrição
Palavras-chave
Ciências biomédicas Biologia molecular Micobactérias Enterobactérias Escherichia coli Terapêutica Resistência
Contexto Educativo
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Instituto de Higiene e Medicina Tropical