RAPID SCREENING OF NATURAL-PRODUCTS FOR ANTIMYCOBACTERIAL ACTIVITY BYUSING LUCIFERASE-EXPRESSING STRAINS OF MYCOBACTERIUM-BOVIS BCG AND MYCOBACTERIUM-INTRACELLULARE

The object of this study was to investigate the ability of a rapid luc iferase assay to detect antimycobacterial activity in plant extracts, Recombinant strains of Mycobacterium bovis BCG (rBCG) and Mycobacteriu m intracellulare expressing firefly luciferase were used as the test o rganisms, Assays were conducted in a 96-well mini-tube format under bi osafety level 2 conditions. Control and test wells were sampled immedi ately after inoculation and after 3 (recombinant M, intracellulare) an d 5 (rBCG) days of incubation to measure luminescence with a microplat e luminometer, and the relative change in luminescence was calculated as a percentage of control values, As an alternative test method, Alam ar blue was added after 12 days of incubation, and changes in color we re read visually, a total of 480 extracts were tested, Sixteen extract s were active against rBCG, and of those, seven were also active again st recombinant M, intracellulare, With activity defined as a relative change in luminescence of less than or equal to 1% (i,e,, greater than or equal to 99% inhibition) and a persistence of blue color after add ition of Alamar blue, there was 99.0% agreement between the two method s, Our results suggest that the luciferase assay is rapid and accurate and has the potential to greatly accelerate the evaluation of antimyc obacterial activity in plant extracts in vitro, With this method, it i s possible to screen a large number of samples in a short period of ti me.