title: Simple and rapid method to determine antimycobacterial potency of compounds by using autoluminescent Mycobacterium tuberculosis.
abstract: A major obstacle in the process of discovery of drugs against Mycobacterium tuberculosis is its extremely slow growth rate and long generation time (∼20 to 24 h). Consequently, determination of MICs and minimum bactericidal concentrations (MBCs) of potential drug candidates using current methods requires 7 days (resazurin-based MIC assay [REMA]) and 1 month (CFU enumeration), 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 the MICs 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 the MICs 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-based MIC values. To determine the bactericidal potencies of compounds, a 1:10 dilution of the cultures from the MIC 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 with MBC 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.
authors: AstraZeneca India Pvt. Ltd., Bangalore, India.
authors: Sreevalli
authors: S
authors: Sharma
authors: Global Health Institute, École polytechnique fédérale de Lausanne (EPFL) Station 19, Lausanne, Switzerland.
authors: Ekaterina
authors: E
authors: Gelman
authors: AstraZeneca India Pvt. Ltd., Bangalore, India.
authors: Chandan
authors: C
authors: Narayan
authors: AstraZeneca India Pvt. Ltd., Bangalore, India.
authors: Deepa
authors: D
authors: Bhattacharjee
authors: AstraZeneca India Pvt. Ltd., Bangalore, India.
authors: Vijayashree
authors: V
authors: Achar
authors: AstraZeneca India Pvt. Ltd., Bangalore, India.
authors: Vaishali
authors: V
authors: Humnabadkar
authors: AstraZeneca India Pvt. Ltd., Bangalore, India.
authors: V
authors: V
authors: Balasubramanian
authors: AstraZeneca India Pvt. Ltd., Bangalore, India.
authors: Vasanthi
authors: V
authors: Ramachandran
authors: Global Health Institute, École polytechnique fédérale de Lausanne (EPFL) Station 19, Lausanne, Switzerland neeraj.dhar@epfl.ch neeladin@gmail.com.
authors: Neeraj
authors: N
authors: Dhar
authors: AstraZeneca India Pvt. Ltd., Bangalore, India neeraj.dhar@epfl.ch neeladin@gmail.com.
authors: Neela
authors: N
authors: Dinesh
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D008163:Luminescent Measurements; D008826:Microbial Sensitivity Tests; D009169:Mycobacterium tuberculosis / Q000187:drug effects*; D015203:Reproducibility of Results
chemical_list: D000995:Antitubercular Agents