When an actively growing culture of the H37Ra strain of Mycobacterium tuberculosis was exposed to isoniazid at a concentration of 0.5 ,g/ml, the cells began to lose their ability to synthesize mycolic acids immediately. After 60 min, the cells had completely lost this ability. The synthesis of the three mycolate components-amycolate, methoxymycolate, and f-mycolate-was inhibited. The viability of the isoniazid-treated cells was unaffected up to about 60 min of exposure, after which time there was a gradual decline in the viability to about 18% after 180 min. Correspondingly, growth of the drug-treated cells slowed down and stopped after 24 hr. The inhibition of the synthesis of mycolic acids was reversible if the drug was removed before the loss of viability set in. Incubation of the viable cells in the absence of the drug for 24 hr restored the mycolate synthesis. These results strongly suggest that the inhibition of the synthesis of the mycolic acids is closely associated with the primary mechanism of action of isoniazid on the tubercle bacilli. The sequence of events which leads to the loss of viability of cells exposed to isoniazid is described.Isoniazid is the mainstay of antituberculosis therapy. Since its discovery as a tuberculostatic agent (12), much work has been done to elucidate its mechanism of action (32). The precise nature of the primary action of isoniazid on the tubercle bacilli is unknown today.One of the more significant early observations was that tubercle bacilli lose their acid-fastness when exposed to isoniazid (14), suggesting that the composition of a certain lipid might be somewhat altered. The altered component could be the mycolic acids which have been implicated in the acidfastness of this organism (11). Studies of several workers suggested an intimate relationship between the effect of the drug and the synthesis of specific lipid components unique to the cell envelope of the tubercle bacilli (4, 10, 25, 26). Recently, Winder and associates found evidence that isoniazid interfered with the formation of the cell envelope, specifically the synthesis of mycolic acids (30,31).We have investigated the effect of isoniazid on the in vivo synthesis of mycolic acids in M. tuberculosis H37Ra using '4C-acetate as the metabolite. We found that isonizaid at a concentration of 0.5 pg/ml immediately began to reduce the cells' ability to synthesize mycolic acids. The gradual decline in viability of the cells began only after complete inhibition of the synthesis of mycolic acids had occurred. In addition, the effect of the drug on the synthesis of mycolates was reversible if the drug was removed before the cells lost their viability. These findings are used to describe the possible mechanism of the action of isoniazid. MATERLALS AND METHODS

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