Adaptation of mycobacteria on solid, egg-based media to anaerobic conditions and characterization of their diagnostic phenotypes

Abstract

Recent evidence indicates that human pulmonary tuberculous granulomas are hypoxic, and a related dormancy model has been established for Mycobacterium tuberculosis using liquid media in tightly sealed tubes. In the present study, we examined the growth capacity of various mycobacterial species on solid, egg-based media under oxygen-limited conditions. During primary anaerobic cultivation on an Ogawa egg-based medium, all of the inoculated mycobacterial strains persisted for 10 weeks without detectable colony formation on the surface of the medium. Because these anaerobic cultures were restored and possessed the capacity to grow within 3 weeks following transfer to microaerobic conditions (5% or 10% oxygen tension), it was evident that the organisms persisted in a non-replicative dormant state as seen with anaerobic cultures in liquid media. The colonies grown under microaerobic conditions were also capable of growth under anaerobic conditions. Anaerobic growth could be observed only when the microaerobically grown colonies were subcultured under anaerobic conditions, suggesting that preliminary adaptation to the environment under reduced oxygen tensions is an essential process to enable growth under anaerobic conditions. A similar mode of anaerobic growth on slants of an egg-based medium was also observed with clinical isolates of M. tuberculosis. All of the clones that were adapted to grow in the anaerobic environments retained their species-specific colonial morphologic features, while, among the biochemical characteristics tested, heat-stable catalase activity and niacin accumulation in the medium disappeared during adaptation to anaerobic growth. Based on these results, we conclude that egg-based media actively support the anaerobiosis of mycobacteria exhibiting either a non-replicative dormant state or anaerobic growth, both of which are associated with latency and reactivation of tuberculosis. It was also evident that the use of egg-based media would be of great advantage in the diagnostic characterization of the reactivated clones of mycobacteria.