RESUMO
The aim of the present study was to (i) evaluate the in vitro action of rifampicin (RIF), ethambutol or isoniazid with efflux pumps inhibitors (EPIs) in Mycobacterium tuberculosis (Mtb) H37Rv and (ii) evaluate the morphological and efflux pumps (EPs) transcriptional changes by the action of rifampicin + verapamil combination (RIF + VP). The minimal inhibitory concentration and synergic effect of drug combinations were determined by Resazurin Microtiter Plate Assay and Resazurin Drugs Combination Microtiter Assay, respectively. VP showed greater capacity of ethidium bromide accumulation and RIF + VP had the lower fractional inhibitory concentration index. The RIF + VP exerted a similar reduction of viable cell counts to RIF by time-kill curve, but decreases in the expression of EPs genes were observed by Real time PCR at 72 h of RIF + VP exposure. Accumulative morphological changes (wrinkled and rounding) caused by each drug were observed by scanning electron microscopy after RIF + VP exposure. The downexpression of EPs related genes exposed to RIF + VP, suggest an effective inhibitory activity of VP in Mtb H37Rv. The role of EPs and the use of EPIs open up a powerful approach and the RIF + VP combination should be studied in Mtb more thoroughly.
Assuntos
Antibióticos Antituberculose/farmacologia , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Rifampina/farmacologia , Verapamil/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Relação Dose-Resposta a Droga , Quimioterapia Combinada , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Viabilidade Microbiana , Microscopia Eletrônica de Varredura , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/ultraestrutura , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacosRESUMO
Tuberculosis (TB) is a health public problem and a long combination therapy is necessary to treat patients. In recent years, some drugs, not routinely used in treatment of TB, have appeared as promising new anti-TB therapies in patients with resistance to classical drugs. The aim of this study was: (i) to evaluate a modified checkerboard assay, Resazurin drugs combination microtiter assay (REDCA) to detect drugs interaction in Mycobacterium tuberculosis; (ii) to evaluate the interaction between isoniazid (INH) or ethambutol (EMB) with levofloxacin (LEVO) in susceptible and resistant M. tuberculosis Brazilian clinical isolates. M. tuberculosis H37RV ATCC 27294 and 19 clinical isolates (10 resistant and 9 susceptible) were tested. The fractional inhibitory concentration index (FICI) ≤ 0.5 was considered synergistic. Synergism in M. tuberculosis H37RV and resistant M. tuberculosis Brazilian isolates was observed with EMB vs. LEVO. No synergism was observed with INH vs. LEVO by both assays. No statistical difference was observed by the two assays studied. REDCA showed to be a simple assay for detecting synergism between drugs in M. tuberculosis. The results with EMB vs. LEVO are promising and it can be a new option in future investigations of drugs interactions against M. tuberculosis with the view to reduce EMB adverse effects.
