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Antimicrobial Agents and Chemotherapy, August 2002, p. 2533-2539, Vol. 46, No. 8
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.8.2533-2539.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Simple Fibroblast-Based Assay for Screening of New Antimicrobial Drugs against Mycobacterium tuberculosis

Takemasa Takii,1,2* Yoshifumi Yamamoto,1 Taku Chiba,1 Chiyoji Abe,3 John T. Belisle,2 Patrick J. Brennan,2 and Kikuo Onozaki1

Department of Molecular Health Sciences, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya,1 Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, Kiyose, Tokyo, Japan,3 Department of Microbiology, Colorado State University, Fort Collins, Colorado2

Received 30 August 2001/ Returned for modification 3 December 2001/ Accepted 15 May 2002

In this study, we propose a simple and reproducible host-cell-based assay for the screening of antimycobacterial drugs that is suitable for drug discovery. The method evaluates both antimycobacterial activity of the drugs and their cytotoxicity to host cells. The basis of this simple fibroblast-based assay (SFA) is that cells of human lung fibroblast cell line MRC-5, which are highly sensitive to mycobacterial cytotoxicity, are killed by virulent Mycobacterium tuberculosis strain H37Rv bacilli in response to the viability of bacilli. Clinically used antimycobacterial drugs inhibited the mycobacterial cytotoxicity to MRC-5 cells in a dose-dependent manner. MICs of isoniazid, streptomycin, rifampin, and ethambutol determined by this SFA (0.428, 1.816, 0.013, and 3.465 µg/ml, respectively) were within 1 log of MICs determined by the broth dilution test (BDT) using Middlebrook 7H9 medium. The MIC of pyrazinamide, which exhibits bactericidal activity only at a high dose by BDT (1,231 µg/ml at pH 6.6 and 492 µg/ml at pH 5.8), was 3.847 µg/ml in the modified method of SFA. On the other hand, sodium azide, a toxic agent for both mammalian cells and bacteria, exhibited cytotoxicity to fibroblasts at a dose lower than that required to inhibit mycobacterial growth. Thus, this fibroblast-based method enabled us to evaluate both antibacterial activity of drugs and their cytotoxicity to human cells within a short period of time.

* Corresponding author. Mailing address: Department of Molecular Health Sciences, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-Dori, Mizuho-Ku, Nagoya 467-8603, Japan. Phone: 81-52-836-3421. Fax: 81-52-836-3419. E-mail: ttakii{at}phar.nagoya-cu.ac.jp.

Antimicrobial Agents and Chemotherapy, August 2002, p. 2533-2539, Vol. 46, No. 8
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.8.2533-2539.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Takii, T., Hamasaki, S., Hirano, K., Abe, C., Onozaki, K. (2005). Simple Fibroblast-Based Assay To Test the Pyrazinamide Susceptibility of Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 49: 804-807 [Abstract] [Full Text]  


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