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Journal of Clinical Microbiology, May 1998, p. 1392-1398, Vol. 36, No. 5
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Simplified Quantitative Assay System for Measuring Activities of Drugs against Intracellular Legionella pneumophila

Futoshi Higa,* Nobuchika Kusano, Masao Tateyama, Takashi Shinzato, Noriko Arakaki, Kazuyoshi Kawakami, and Atsushi Saito

The First Department of Internal Medicine, Faculty of Medicine, University of the Ryukyus, 903-01 Okinawa, Japan

Received 2 September 1997/Returned for modification 15 October 1997/Accepted 20 January 1998

We developed a new simple assay for the quantitation of the activities of drugs against intracellular Legionella pneumophila. The cells of a murine macrophage-like cell line (J774.1 cells) allowed the intracellular growth and replication of the bacteria, which ultimately resulted in cell death. The infected J774.1 cell monolayers in 96-well microplates were first treated with antibiotics and were further cultured for 72 h. The number of viable J774.1 cells in each well was quantified by a colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and an enzyme-linked immunosorbent assay reader. The number of growing bacteria in each well was also determined by counting the numbers of CFU on buffered charcoal yeast extract-alpha agar plates. Viable J774.1 cell counts, determined by the colorimetric assay, were inversely proportional to the number of intracellular replicating bacteria. The minimum extracellular concentrations (MIECs) of 24 antibiotics causing inhibition of intracellular growth of L. pneumophila were determined by the colorimetric assay system. The MIECs of beta-lactams and aminoglycosides were markedly higher than the MICs in buffered yeast extract-alpha broth. The MIECs of macrolides, fluoroquinolones, rifampin, and minocycline were similar to the respective MICs. According to their intracellular activities, clarithromycin and sparfloxacin were the most potent among the macrolides or fluoroquinolones tested in this study. Our results indicated that the MTT assay system allows comparative and quantitative evaluations of the intracellular activities of antibiotics and efficient processing of a large number of samples.

* Corresponding author. Mailing address: The First Department of Internal Medicine, Faculty of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-01, Japan. Phone: 81-98-895-3331, ext. 2438. Fax: 81-98-895-3086. E-mail: fhiga{at}med.u-ryukyu.ac.jp.

Journal of Clinical Microbiology, May 1998, p. 1392-1398, Vol. 36, No. 5
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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