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JAC Advance Access originally published online on June 22, 2007
Journal of Antimicrobial Chemotherapy 2007 60(2):288-293; doi:10.1093/jac/dkm207
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Resazurin reduction assays for screening of anti-tubercular compounds against dormant and actively growing Mycobacterium tuberculosis, Mycobacterium bovis BCG and Mycobacterium smegmatis

Neetu Kumra Taneja and Jaya Sivaswami Tyagi*

Department of Biotechnology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India

Received 2 March 2007; returned 11 April 2007; revised 17 April 2007; accepted 9 May 2007

* Corresponding author. Tel: +91-011-26588491; Fax: +91-011-26588663; E-mail: jstyagi{at}aiims.ac.in

Objectives: 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.

Keywords: M. tuberculosis , M. bovis BCG , M. smegmatis , dormancy , REMA , HyRRA


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