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.

Cutaneous tuberculosis—ambiguous transmission, bacterial diversity with biofilm formation in humoral abnormality: case report illustration

“…Cutaneous tuberculosis is more difficult to diagnose and therapy than its pulmonary form because there are other competing biofilm-forming bacteria as presented in the Buruli ulcer case ( 27 ) and there is limited access to oxygen ( 28 ). Only some microorganisms in the specific niche will survive competition for oxygen, nutrients (low-nutrient environments), and space, especially in the context of host factors.…”

“…It is worth noting that only three species were isolated from both aerobic and anaerobic cultures ( Table 2 ), and paucibacillary growth in skin ulcers ( Table 1 ) may be caused by limited oxygen access. One study showed that the anaerobic growth of MTB could be observed only when microaerobic-grown colonies were subcultured under anaerobic conditions ( 28 ). Therefore, our observation of anaerobic growth predominance ( Table 2 ) suggests that biofilm is the adaptation to the reduced oxygen tensions and MTB can grow under micro- or anaerobic conditions.…”

Evolution ofM. bovisBCG Vaccine: Is Niacin Production Still a Valid Biomarker?

“…The copy number of VNTR 3690 varies among Indian clinical isolates of M. tuberculosis (one to twelve copies), M. tuberculosis H37Rv TMC102 (four copies), M. tuberculosis H37Ra (two to four copies), and M. bovis BCG (one copy) [ 99 ]. A detailed comparison among virulent M. tuberculosis , M. bovis, and M. bovis BCG based on published literature [ 14 , 15 , 17 – 23 ] and on our own work is summarized in Table 1 .…”