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The metabolic response of freshly harvested, nude-mouse-derived Mycobacterium leprae to biophysical parameters was studied to facilitate an understanding of axenic culture requirements. Quantitation of intracellular ATP and the rate of [U-14C]palmitic acid incorporation into phenolic glycolipid I (PGL-I) were used as metabolic indicators after axenic incubation in modified Dubos medium under various biophysical conditions. PGL-I synthesis was optimal at 33 degrees C, whereas ATP was optimally maintained at less than or equal to 33 degrees C. Both metabolic indices showed sharp reductions at 37 degrees C. After 5 days of incubation, PGL-I synthesis and ATP maintenance showed pH optima of 5.1 to 5.6, with the higher value appearing optimal for ATP maintenance after extended incubation. Metabolic activity was negatively affected by strong reducing agents, and ATP maintenance was optimal when the gaseous environment was maintained at 2.5 to 10% oxygen. The results may partially explain the failure to cultivate the leprosy bacillus in vitro.

The ability of Mycobacterium leprae to oxidize palmitic acid during incubation in an axenic medium was studied. By using a Buddemeyer-type detection system, partially purified nude-mouse-derived M. leprae was found to produce 14CO2 from 14C-labeled palmitic acid in a linear fashion for at least 1 week. Procedures known to remove residual host tissue did not diminish the rate of 14CO2 evolution, indicating that bacterial metabolism was being measured. Palmitate oxidation was temperature sensitive, with an apparent optimum of 33 degrees C, but pH insensitive. Bacilli exposed to a variety of antileprosy drugs for 1 or 2 weeks displayed significantly reduced rates of 14CO2 evolution upon subsequent addition of 14C-labeled palmitic acid. This activity could be readily detected with 10(6) bacilli, thus indicating its potential for use in clinical susceptibility testing.