Microplate Alamar blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium

In response to the need for rapid, inexpensive, high-throughput assays for antimycobacterial drug screening, a microplate-based assay which uses Alamar blue reagent for determination of growth was evaluated. MICs of 30 antimicrobial agents against Mycobacterium tuberculosis H(37)Rv, M. tuberculosis H37Ra, and Mycobacterium avium were determined in the microplate Alamar blue assay (MABA) with both visual and fluorometric readings and compared to MICs determined in the BACTEC 460 system, For all three mycobacterial strains, there was less than or equal to 1 dilution difference between MABA and BACTEC median MICs in four replicate experiments for 25 to to of the 30 antimicrobics. Significant differences between MABA and BACTEC MICs were observed with 0, 3, and 5 of 30 antimicrobial agents against H(37)Rv, H37Ra, and M. avium, respectively. Overall, MICs determined either visually or fluorometrically in MABA were highly correlated with those determined in the BACTEC 460 system, and visual MABA and fluorometric MABA MICs were highly correlated, MICs of rifampin, rifabutin, minocycline, and clarithromycin were consistently lower for H,,Ra compared to H,,RV in ail assays but were similar for most other drugs, M. tuberculosis H37Ra may be a suitable surrogate for the more virulent II,,RV strain in primary screening of compounds for antituberculosis activity. MABA is sensitive, rapid, inexpensive, and nonradiometric and offers the potential for screening, with or without analytical instrumentation, large numbers of antimicrobial compounds against slow-growing mycobacteria.