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Antimicrobial Agents and Chemotherapy, April 2007, p. 1380-1385, Vol. 51, No. 4
0066-4804/07/$08.00+0     doi:10.1128/AAC.00055-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Low-Oxygen-Recovery Assay for High-Throughput Screening of Compounds against Nonreplicating Mycobacterium tuberculosis{triangledown}

Sang Hyun Cho,1 Saradee Warit,1 Baojie Wan,1 Chang Hwa Hwang,1 Guido F. Pauli,1,2 and Scott G. Franzblau1*

Institute for Tuberculosis Research,1 Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 606122

Received 12 January 2006/ Returned for modification 23 February 2006/ Accepted 19 December 2006

Screening for new antimicrobial agents is routinely conducted only against actively replicating bacteria. However, it is now widely accepted that a physiological state of nonreplicating persistence (NRP) is responsible for antimicrobial tolerance in many bacterial infections. In tuberculosis, the key to shortening the 6-month regimen lies in targeting this NRP subpopulation. Therefore, a high-throughput, luminescence-based low-oxygen-recovery assay (LORA) was developed to screen antimicrobial agents against NRP Mycobacterium tuberculosis. M. tuberculosis H37Rv containing a plasmid with an acetamidase promoter driving a bacterial luciferase gene was adapted to low oxygen conditions by extended culture in a fermentor with a 0.5 headspace ratio. The MICs of 31 established antimicrobial agents were determined in microplate cultures maintained under anaerobic conditions for 10 days and, for comparative purposes, under aerobic conditions for 7 days. Cultures exposed to drugs under anaerobic conditions followed by 28 h of "recovery" under ambient oxygen produced a luminescent signal that was, for most compounds, proportional to the number of CFU determined prior to the recovery phase. No agents targeting the cell wall were active against NRP M. tuberculosis, whereas drugs hitting other cellular targets had a range of activities. The calculated Z' factor was in the range of 0.58 to 0.84, indicating the suitability of the use of LORA for high-throughput assays. This LORA is sufficiently robust for use for primary high-throughput screening of compounds against NRP M. tuberculosis.

* Corresponding author. Mailing address: Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, MC 964, Rm. 412, Chicago, IL 60612. Phone: (312) 355-1715. Fax: (312) 355-2693. E-mail: sgf{at}uic.edu

{triangledown} Published ahead of print on 8 January 2007.

Antimicrobial Agents and Chemotherapy, April 2007, p. 1380-1385, Vol. 51, No. 4
0066-4804/07/$08.00+0     doi:10.1128/AAC.00055-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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