Isoniazid and verapamil modulatory activity and efflux pump gene expression in Mycobacterium tuberculosis.

INTRODUCTION: Resistance to first-line anti-tuberculosis drugs is a major concern in the treatment of the disease. New strategies, such as the use of efflux pump inhibitors (EPIs), are being investigated to improve the outcome of the treatment. Verapamil (VP), one such inhibitor, was shown to inhibit several efflux pump (EP) Mycobacterium tuberculosis proteins and demonstrate synergic activity with anti-TB drugs.OBJECTIVE: To evaluate the combinatory effect of isoniazid (INH) and VP in M. tuberculosis.METHODS: Minimal inhibitory concentrations and combinatory effects of INH+VP were determined using respectively resazurin microtitre assay plate (REMA) and resazurin drugs combination microtitre assay (REDCA). From the results, we selected three bacilli with different susceptibility profiles and assessed their expression of 10 EP genes through quantitative reverse transcription polymerase chain reaction after exposure to INH, VP and INH + VP for 48 h.RESULTS: A significant reduction of INH MIC was observed in INH-susceptible isolates upon combination with VP. In brief, gene expression assays revealed expression patterns that could be correlated with each resistance profile, presence or absence of gene mutations and combinatory effect with VP.CONCLUSION: Combining VP with INH showed important results in drug-susceptible strains, and clinical trials on combined VP + anti-TB drugs should be discussed.

Pyrazinamide susceptibility testing in Mycobacterium tuberculosis using the fast resazurin microtiter assay plate.

SETTING: Department of Clinical Analysis and Biomedicine, State University of Maringa, Maringa, PR, Brazil. OBJECTIVE: To evaluate the performance of the resazurin microtiter assay (REMA) plate at pH 5.5 in detecting Mycobacterium tuberculosis susceptibility to pyrazinamide (PZA). DESIGN: The minimal inhibitory concentration (MIC) of PZA in M. tuberculosis H37Rv and M. bovis AN5 reference strains and in 34 clinical M. tuberculosis isolates (26 PZA-susceptible and eight PZA-resistant) was determined using REMA at pH 5.5 and compared to REMA at pH 6.0. RESULTS: REMA at pH 5.5 was helpful in discriminating PZA-susceptible from resistant M. tuberculosis isolates when â©¿50 µg/ml PZA was considered as the cut-off for PZA susceptibility. Furthermore, it provided results in 8 days. However, two PZA-resistant isolates failed to grow at pH 5.5. CONCLUSION: As the REMA method is rapid, inexpensive, easy to perform and read, it would be of great usefulness in low-income countries for detecting PZA-resistant M. tuberculosis. REMA at pH 5.6-5.9 should be evaluated on an extended panel of clinical M. tuberculosis isolates with a greater range of MIC values in different laboratories for a better understanding of its utility in differentiating PZA-resistant from PZA-susceptible isolates.

Potential of Casiopeínas® Copper Complexes and Antituberculosis Drug Combination against Mycobacterium tuberculosis.

New compounds with antituberculosis activity and their combination with classic drugs have been evaluated to determine possible interactions and antagonism. The aim of this study was to evaluate the in vitro activity of Casiopeínas® copper-based compounds (CasIIIia, CasIIIEa, and CasIIgly) alone and combined with isoniazid (INH), rifampicin, or ethambutol (EMB) against resistant and susceptible Mycobacterium tuberculosis. Seventeen clinical M. tuberculosis isolates (5 multi-drug resistant and 2 resistant to INH and/or EMB) were subjected to determination of the minimal inhibitory concentration (MIC) by the resazurin microtiter assay and combination assessment by the resazurin drug combination microtiter assay. The Casiopeínas® alone showed a remarkable effect against resistant isolates with MIC values from 0.78 to 12.50 µg/ml. Furthermore, a synergistic effect mainly with EMB is shown for both resistant and susceptible clinical isolates. Casiopeínas® are promising candidates for future investigation into the development of antituberculosis drugs, being one of the first examples of essential metal-based drugs used in this field.