SIMPLIFIED QUANTITATIVE ASSAY SYSTEM FOR MEASURING ACTIVITIES OF DRUGS AGAINST INTRACELLULAR LEGIONELLA-PNEUMOPHILA

We developed a new simple assay for the quantitation of the activities of drugs against intracelllular Legionella pneumophila. The cells of a murine macrophage-like cell line (J774.1 cells) allowed the intracel lular growth and replication of the bacteria, which ultimately resulte d in cell death, The infected J774.1 cell monolayers in 96-well microp lates were first treated with antibiotics and were further cultured fo r 72 h, The number of viable J774.1 cells in each well was quantified by a colorimetric assay with (4,5-dimethylthiazol-2-yl)-2,5-diphenylte trazolium bromide (MTT) and an enzyme-linked immunosorbent assay reade r. The number of growing bacteria in each well was also determined by counting the numbers of CFU on buffered charcoal yeast extract-alpha a gar plates, Viable J774.1 cell counts, determined by the colorimetric assay, were inversely proportional to the number of intracellular repl icating bacteria. The minimum extracellular concentrations (MIECs) of 24 antibiotics causing inhibition of intracellular growth of L. pneumo phila were determined by the colorimetric assay system. The MIECs of b eta-lactams and aminoglycosides were markedly higher than the MICs in buffered yeast extract-alpha broth, The MIECs of macrolides, fluoroqui nolones, rifampin, and minocycline were similar to the respective MICs . According to their intracellular activities, clarithromycin and spar floxacin 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 o f samples.