Simple and Rapid Method To Determine Antimycobacterial Potency of Compounds by Using Autoluminescent Mycobacterium tuberculosis

Amajor obstacle in the process of discovery of drugs againstMycobacterium tuberculosis is its extremely slow growth rate and long generation time (20 to 24 h). Consequently, determination of MICs andminimum bactericidal concentrations (MBCs) of potential drug candidates using current methods requires 7 days (resazurin-basedMIC assay [REMA]) and 1month (CFU enu-meration), respectively. We employed a synthetic luciferase operon optimized for expression in high-GC-content bacteria and adapted it for use in mycobacteria. Using luminescence-based readouts, we were able to determine theMICs and bactericidal activities of approved tuberculosis (TB) drugs, which correlated well with currently used methods. Although luminescence-based readouts have been used previously to determine theMICs and bactericidal activities of approved TB drugs, in this study we adapted this assay to carry out a pilot screen using a library of 1,114 compounds belonging to diverse chemical scaffolds. We found that MICs derived from a 3-day luminescence assay matched well with REMA-basedMIC values. To determine the bacte-ricidal potencies of compounds, a 1:10 dilution of the cultures from theMIC plate was carried out on day 7, and the bactericidal concentrations determined based on time to positivity in 2 weeks were found to be comparable withMBC values determined by the conventional CFU approach. Thus, the luminescent mycobacterium-based approach not only is very simple and inexpensive but also allowed us to generate the information in half the time required by conventional methods. Tuberculosis (TB) continues to be a major threat to publichealth worldwide, accounting for 1.3 million deaths annually, and is also a leading cause of death among people coinfected wit