Abstract
Drug resistant tuberculosis continues to increase and new approaches for its treatment are necessary. The identification of M. tuberculosis clinical isolates presenting efflux as part of their resistant phenotype has a major impact in tuberculosis treatment. In this work, we used a checkerboard procedure combined with the tetrazolium microplate-based assay (TEMA) to study single combinations between antituberculosis drugs and efflux inhibitors (EIs) against multidrug resistant M. tuberculosis clinical isolates using the fully susceptible strain H37Rv as reference. Efflux activity was studied on a real-time basis by a fluorometric method that uses ethidium bromide as efflux substrate. Quantification of efflux pump genes mRNA transcriptional levels were performed by RT-qPCR. The fractional inhibitory concentrations (FIC) indicated synergistic activity for the interactions between isoniazid, rifampicin, amikacin, ofloxacin, and ethidium bromide plus the EIs verapamil, thioridazine and chlorpromazine. The FICs ranged from 0.25, indicating a four-fold reduction on the MICs, to 0.015, 64-fold reduction. The detection of active efflux by real-time fluorometry showed that all strains presented intrinsic efflux activity that contributes to the overall resistance which can be inhibited in the presence of the EIs. The quantification of the mRNA levels of the most important efflux pump genes on these strains shows that they are intrinsically predisposed to expel toxic compounds as the exposure to subinhibitory concentrations of antibiotics were not necessary to increase the pump mRNA levels when compared with the non-exposed counterpart. The results obtained in this study confirm that the intrinsic efflux activity contributes to the overall resistance in multidrug resistant clinical isolates of M. tuberculosis and that the inhibition of efflux pumps by the EIs can enhance the clinical effect of antibiotics that are their substrates.
Highlights
Tuberculosis (TB) remains a public health issue worldwide (World Health Organization, 2014)
In the present study we have explored the contribution of the efflux mechanisms to the overall resistance to isoniazid, rifampicin, amikacin and ofloxacin in five MDR M. tuberculosis clinical isolates from Brazil by (i) the analysis of the synergistic effect of the efflux inhibitors (EIs) verapamil, thioridazine and chlorpromazine on the Minimum Inhibitory Concentrations (MIC) of the antibiotics and ethidium bromide by a tetrazolium microplate-based assay (TEMA) on checkerboard format; (ii) the analysis of real-time efflux activity, using ethidium bromide as efflux substrate, by a semi-automated fluorometric method in presence and absence of each EI; and (iii) the analysis of mRNA transcriptional levels of selected efflux pump genes in these strains
The EIs verapamil, thioridazine and chlorpromazine were tested at 1⁄2 MIC with and without 0.4% glucose and ethidium bromide at the equilibrium concentration determined for each strain and the assays performed like described above
Summary
Tuberculosis (TB) remains a public health issue worldwide (World Health Organization, 2014). Prolonged exposure to subinhibitory concentrations of antituberculosis drugs facilitate the progressive acquisition of chromosomal mutations and provide the natural ground for the development of bacteria with high-level resistance phenotypes due to the acquisition of mutations in the antibiotic target This chain of events is relevant in long-term therapies such as that used in tuberculosis treatment, where a sustained pressure of sub-inhibitory concentrations of antibiotics can result in an increased efflux activity and allow the selection of spontaneous high-level drug resistant mutants (Machado et al, 2012; Schmalstieg et al, 2012). In the present study we have explored the contribution of the efflux mechanisms to the overall resistance to isoniazid, rifampicin, amikacin and ofloxacin in five MDR (two of which pre-XDR) M. tuberculosis clinical isolates from Brazil by (i) the analysis of the synergistic effect of the EIs verapamil, thioridazine and chlorpromazine on the MICs of the antibiotics and ethidium bromide by a tetrazolium microplate-based assay (TEMA) on checkerboard format; (ii) the analysis of real-time efflux activity, using ethidium bromide as efflux substrate, by a semi-automated fluorometric method in presence and absence of each EI; and (iii) the analysis of mRNA transcriptional levels of selected efflux pump genes in these strains
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