Resazurin reduction assays for screening of anti-tubercular compounds against dormant and actively growing Mycobacterium tuberculosis, Mycobacterium bovis BCG and Mycobacterium smegmatis.
OBJECTIVE
To develop simple, rapid, low-cost and robust assays for screening drugs against dormant and actively growing mycobacteria.


METHODS
Actively growing aerobic and hypoxia-adapted dormant cultures of Mycobacterium tuberculosis, Mycobacterium bovis BCG and Mycobacterium smegmatis were tested for susceptibility to standard antimicrobial drugs by resazurin reduction assay. The visual and fluorimetric MICs were compared with those obtained by the standard cfu assay.


RESULTS
Drug MICs for M. tuberculosis and M. bovis BCG were determined by the aerobic resazurin microplate assay (REMA) and correlated well with those obtained by the cfu assay. Metronidazole and nitrofurans showed comparable bactericidal activity in the hypoxic resazurin reduction assay (HyRRA). The HyRRA assay was noted to be superior to the cfu assay in that it distinguished between metabolically active dormant bacteria and non-viable organisms, unlike the cfu assay that could not differentiate between these two populations. The HyRRA assay performed with good concordance in both fluorimetric and visual formats to distinguish between bactericidal and bacteriostatic effects of a drug.


CONCLUSIONS
The REMA and HyRRA assays will be useful for anti-tubercular anti-dormancy compound screening and drug susceptibility testing in a safe, reliable, easy and cost-effective manner particularly in low resource countries. The application of the assays in M. smegmatis or M. bovis BCG offers the distinct advantage of rapidly and safely screening anti-tubercular compounds in a high-throughput format.
Taneja
Neetu Kumra
NK
Department of Biotechnology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India.
Tyagi
Jaya Sivaswami
JS
D000332:Aerobiosis; D000693:Anaerobiosis; D000995:Antitubercular Agents / Q000494:pharmacology*; D015169:Colony Count, Microbial; D004353:Drug Evaluation, Preclinical; D005470:Fluorometry; D007202:Indicators and Reagents; D008826:Microbial Sensitivity Tests; D009163:Mycobacterium bovis / Q000187:drug effects*; D020102:Mycobacterium smegmatis / Q000187:drug effects*; D009169:Mycobacterium tuberculosis / Q000187:drug effects*; D010078:Oxazines / Q000737:chemistry*; D010084:Oxidation-Reduction; D014966:Xanthenes / Q000737:chemistry*
D000995:Antitubercular Agents; D007202:Indicators and Reagents; D010078:Oxazines; D014966:Xanthenes; C005843:resazurin