Relação entre o mecanismo de efluxo e a resistência aos antimicobacterianos em isolados clínicos de Mycobacterium tuberculosis

Abstract

O tratamento com antimicrobianos é a principal estratégia de controle da tuberculose (TB). Entretanto, o aumento do número de casos de TB com cepas de Mycobacterium tuberculosis resistente aos antimicrobianos cria um cenário que dificulta a cura do paciente e o controle da doença. Embora várias mutações em loci específicos tenham sido identificadas como base de resistência, outros mecanismos, como o sistema de efluxo, podem contribuir para a resistência e o estabelecimento dessas mutações. O objetivo deste estudo foi avaliar a contribuição do mecanismo de efluxo em isolados clínicos de M. tuberculosis resistentes aos principais antimicrobianos do esquema básico (isoniazida, INH; e rifampicina, RIF) e de multirresistência (ofloxacina, OFX; e amicacina, AMK). Três clássicos inibidores de bombas de efluxo - EPI - (verapamil, VP; tioridazina, TZ; e clorpromazina, CPZ) foram selecionados para detectar o efluxo. Primeiramente, foram analisadas três cepas MDR, duas pré-XDR e a cepa sensível de referência H37Rv. Foi utilizada a metodologia de checkerboard combinada com o tetrazolium microplate-based assay para analisar a interação entre os EPIs com os fármacos. Foi observado que os EPIs diminuem efetivamente a resistência aos antibióticos utilizados, com reduções de 4 a 64 vezes. Para avaliar a atividade do efluxo em tempo real foi utilizado o método fluorimétrico com o brometo de etídio (BrEt), em presença dos EPIs. Foi demonstrado que todas as cepas apresentaram efluxo intrínseco, porém a acumulação e o efluxo do BrEt foi mais evidente na cepa pre-XDR com resistência adicional a AMK. A quantificação transcricional do RNAm dos genes de bombas de efluxo (mmpl7, mmr, Rv1258, p55, efpA, Rv2459) e do regulador transcricional whib7, quando as cepas foram expostas às concentrações subinibitórias dos antibióticos, foi analisada por RT-qPCR. Houve um aumento do nível transcricional de todos os genes em uma cepa MDR e na cepa Pre-XDR com resistência adicional a OFX, quando expostas a pelo menos um dos fármacos envolvidos na resistência.

Treatment with antimicrobials is the main TB control strategy. However, the increase in the number of TB cases with Mycobacterium tuberculosis strains resistant to antimicrobial creates a scenario that hinders patient's healing and disease control. Although several genetic mutations in specific loci involved in drug resistance have been identified as base of resistance, other mechanisms such as efflux system may contribute to resistance and to the establishment of these mutations. The main goal of this study was to assess the overall contribution of efflux mechanism in M. tuberculosis clinical isolates resistant to the main first line drugs (isoniazid, INH; and rifampicin, RIF) and second line (ofloxacin, OFX; and amikacin, AMK). Three classical inhibitors of efflux pumps -EPI- (verapamil, VP; thioridazine, TZ; and chlorpromazine, CPZ) were selected to detect the efflux. Firstly, we analyzed three MDR strains, two Pre-XDR and an H37Rv reference susceptible strain. We used the checkerboard method combined with the tetrazolium microplate-based assay to analyze the interaction between EPIs and drugs. It was observed that EPIs effectively reduce the MIC of antibiotics, with four-fold to 64-fold reduction. Efflux activity was evaluated in real-time by fluorimetric method with ethidium bromide (EtBr), in the presence of EPIs. All strains showed intrinsic efflux, however the accumulation and efflux of EtBr was evident most in the pre-XDR strain with additional resistance to AMK. The quantification of mRNA transcriptional level of efflux pump genes (mmpl7, mmr, Rv1258, p55, efpA, Rv2459) and the transcriptional regulator, whib7, when strains were exposed to antibiotics subinibitory concentrations was examined by RT-qPCR. There was an increase in the transcriptional level of all genes in a MDR strain and pre-XDR strain with additional OFX resistance, when exposed to at least one of the drugs involved in resistance, INH, RIF or OFX. However, there was no correlation between the reduction of antibiotic resistance levels with the EPI and the expression of genes encoding the pumps. Finally, in order to demonstrate the time to detection (TTD) of the bacterial growth in the antimicrobial environment in presence and absence of EPI, were applied BACTECTM MGITTM system 960 and Epicenter V5.53A equipped with software TB eXiST. Were used the MDR strains, a pre-XDR strain with additional resistance to AMK, and a monorresistant to OFX strain. In general, strains have showed a slower grew in the presence of VP, TZ and/or CPZ when combined with INH or RIF. This suggests that efflux is essential for the strain to grow faster in the presence of certain antibiotics. In addition, the efflux can act synergistically with the presence of mutations in order to reduce the biological cost of the bacteria and promoting growth at high drug concentrations. In conclusion, the described results demonstrated that the efflux system plays an important role in resistance to antibiotics used in the treatment of TB, and that the use of efflux inhibitors may potentiate the antimicrobial activity.