Bioluminescense method for high-throughput screening of compounds against Mycobacterium tuberculosis

Resumo

There has been an increase of Mycobacteriumtuberculosis strains that are resistant to the current anti-TBagents, mainly through acquired resistance by therapeuticfailure, This fact has underscored the need of a quickdevelopment of antimycobacterial drugs that are moreeffective than those currently in use. Moreover, newmethodologies to determine the bactericidal activity of thesecompounds have been proposed. This study describes theuse of bioluminescent strains of Mycobacteriumtuberculosis H37Rv – ATCC 27974 and Mycobacteriumtuberculosis Erdman ATCC 35801 both containing theplasmid pSMT1 constructed with luxA and luxB genes fromVibrio harveyi in a screening to evaluate theantimycobacterial activities of anti-TB agents. Thestandardization of the technique was performed usingisoniazid and rifampicin, as a drug standard and the resultsof Minimal Inhibitory Concentration (MIC) were 0.03 μg/mLand 0.03 μg/mL, respectively. These values were totallycompatible with those obtained with Microplate Alamar BlueAssay (MABA). The standardization of the bioluminescence measurement of intracellular antimycobacterial activity wasperformed using the J774 murine macrophage-like cell lineinfected with Mycobacterium tuberculosis Erdmancontaining the plasmid pSMT1 and rifampicin as a drugstandard and the result of MIC was 0.16 μg/mL similar withthose obtained with the technique of colony forming unit(CFU). A total of 32 compounds were evaluated, 19 crudeplants extracts and 13 synthetic compounds and the resultsof Minimal Inhibitory Concentration were compared withthose obtained with the MABA. 02 crude plants extractsand 02 synthetic compounds were evaluated and the resultsof MIC and percent of inhibition were compatible with thoseobtained with the CFU technique. The overall agreementsbetween the MICs obtained by MABA and the resultsobtained with the luciferase reporter strain of Mycobacteriumtuberculosis and with bioluminescence measurement ofintracellular antimycobacterial activity and the CFUtechnique encourage the use of this recombinantmycobacteria in high-throughput screening of compoundsagainst Mycobacterium tuberculosis.