RESUMEN
Tuberculosis (TB) is an important public health problem worldwide and the emergence of multidrug-resistant (MDR) TB and extensively drug-resistant (XDR) TB worsened the global context. The resistance in Mycobacterium tuberculosis, the causative agent of TB, can partially derive from efflux pumps (EPs) activity in plasma membrane. Due to the recent discovery of piperine (PIP), an organic alkaloid compound, increasing the bioavailability of various drugs, the current assay evaluated the combined activity of PIP and anti-TB drugs in susceptible and resistant M. tuberculosis clinical isolates. The minimum inhibitory concentrations for isoniazid, rifampicin, ethambutol, streptomycin and PIP were determined by resazurin microtiter assay and the combined effects of anti-TB drugs with PIP determined by resazurin drug combination microtiter assay and time-kill curve. The efflux pump inhibitor activity of PIP was determined by bromide accumulation assay and cytotoxicity carried out in VERO cells and J774. A1 macrophages. PIP showed to have EPI activity and RIF + PIP and SM + PIP combinations showed synergistic effect, but low effect in enhancing the killing in M. tuberculosis H37Rv and in the clinical isolates studied, which had different resistance profiles. Future studies are needed to further clarify the importance of PIP as an adjunctive drug in the therapy against TB.
Asunto(s)
Alcaloides/farmacología , Antituberculosos/farmacología , Proteínas Bacterianas/efectos de los fármacos , Benzodioxoles/farmacología , Proteínas de Transporte de Membrana/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Piperidinas/farmacología , Alcamidas Poliinsaturadas/farmacología , Alcaloides/metabolismo , Animales , Antituberculosos/metabolismo , Proteínas Bacterianas/metabolismo , Benzodioxoles/metabolismo , Chlorocebus aethiops , Farmacorresistencia Bacteriana/efectos de los fármacos , Sinergismo Farmacológico , Quimioterapia Combinada , Proteínas de Transporte de Membrana/metabolismo , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/metabolismo , Piperidinas/metabolismo , Alcamidas Poliinsaturadas/metabolismo , Células VeroRESUMEN
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.
Asunto(s)
Antibióticos Antituberculosos/farmacología , Proteínas Bacterianas/efectos de los fármacos , Proteínas de Transporte de Membrana/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Rifampin/farmacología , Verapamilo/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Viabilidad Microbiana , Microscopía Electrónica de Rastreo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/ultraestructura , Factores de Tiempo , Transcripción Genética/efectos de los fármacosRESUMEN
BACKGROUND: At the triple border Brazil/Paraguay/Argentina there is easy mobility from one city to another for economic and tourism activities. This constant and fast population mobility is mainly to visit Iguazu Falls, in the Iguazu River, on the border of the Brazilian state of Paraná and the Argentina. As the incidence of tuberculosis is high in this setting, our study aimed to establish a first baseline of circulating genotypic lineages of Mycobacterium tuberculosis. METHODOLOGY/PRINCIPAL FINDINGS: This study included 120 patients from 10 cities in southwestern Paraná, Brazil with pulmonary symptoms, from July 2009 to July 2011. Information about sex, age, clinical features and address was collected by reviewing the national tuberculosis notification database. Of these, 96 (80%) isolates were identified as M. tuberculosis and 22 (22.9%) were drug resistant (20, 20.8% INH mono-resistant and 2, 2.1% multidrug-resistant). All isolates were subjected to genotyping by Spoligotyping and MIRU-VNTR typing. The distribution of the isolates analyzed by spoligotyping revealed 30 distinct patterns. The four mainly detected clades were Latin American and Mediterranean (LAM), ill-defined T, Haarlem (H) and S. The MIRU-VNTR showed 85 distinct patterns. Spoligotyping combined to MIRU-VNTR allowed 90 distinct patterns. CONCLUSIONS/SIGNIFICANCE: Our study demonstrated that there is significant molecular diversity in circulating M. tuberculosis, with predominance of the LAM and T clades in cities of southwestern Paraná, Brazil, bordering Argentina and Paraguay.
Asunto(s)
Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/genética , Tuberculosis/microbiología , Adolescente , Adulto , Anciano , Técnicas de Tipificación Bacteriana/métodos , ADN Bacteriano/genética , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , América del Sur , Adulto JovenRESUMEN
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.