Inhibition of metabolism and growth of mycobacterium leprae by Gamma irradiation

ABSTRACT

Mycobacterium leprae is uncultivable on artificial medium, but viability can be maintained without multiplication for a limited time in vitro. In this study, we evaluated gamma-irradiation (gamma-irr) as a means to kill this slowly growing organism. Freshly harvested, viable, athymic, nu/nu mouse-derived M. leprae were exposed to varying doses of gamma-irr from a 60Co source. Two indicators of bacterial viability were determined metabolism, measured by oxidation of 14C-palmitic acid to 14CO2 in the BACTEC 460 system, and multiplication, measured by titration in the mouse foot pad. gamma-Irr of both M. leprae and M. lufu, a cultivable control mycobacterium, resulted in a dose-dependent inhibition of viability. gamma-Irr of up to 10(3) rad had little effect on the metabolic activity of either organism. For M. leprae, 10(4)-10(5) rad caused an intermediate inhibitory effect; whereas 10(6) rad yielded almost total inhibition. In the mouse foot pad assay, up to 10(4) rad had little effect on M. leprae growth; however, 10(5) rad resulted in at least a 2-log reduction in the number of bacilli recovered and no M. leprae growth was measurable after exposure to 10(6) rad. With M. lufu, 10(5) rad inhibited metabolic activity by 99% and caused > or = 2-log reduction in the number of colony forming units (CFU). No CFU of M. lufu were recovered after exposure to 10(6) rad. Scanning electron microscopy revealed the presence of some aberrant protrusions on the cell surface of lethally irradiated M. leprae; whereas boiling and autoclaving caused obvious morphological denaturation. These data suggest that gamma-irr is an effective way to kill M. leprae without causing extensive damage to the cell architecture. Killing M. leprae by gamma-irr may be preferable when comparing cellular responses to live versus dead bacilli in vitro and in vivo.