Intraspecies diversity of dormant forms of Mycobacterium smegmatis

The non-spore-forming gram-positive bacterium Mycobacterium smegmatis mc² 155, related to M. tuberculosis, was revealed to be capable of forming different types of dormant forms (DFs) during the life cycle of its cultures. The relationship between the intraspecies diversity of DFs and the cultivation conditions of the mycobacterium was established. The DFs possessed the following common properties: (i) maintenance of viability for a long period of time (5 months), (ii) resistance to deleterious factors such as heat treatment, and (iii) morphological and ultrastructural peculiarities that distinguish DFs from vegetative cells. The diversity of M. smegmatis DFs manifested itself in differences in terms of structural organization, conditions required for growth renewal, and capacity to produce antibiotic-resistant variants upon germination on selective media. Well-differentiated cystlike dormant cells (CDCs) were formed in the cultures grown in synthetic SR1 medium with fivefold-decreased nitrogen content. The structural organization of CDCs differed from that of other DF types mainly in the presence of club-shaped cells, thickened lamellar cell walls, coarse cytoplasm texture, and large electron-transparent triacylglyceride inclusion bodies. It was possible to use mycobacterial CDCs as a source of PCR-competent DNA. CDC populations were heterogeneous in cell buoyant density, and the individual fractions, which we isolated, were found to differ in thermal stability and the ability to revert to growth under standard conditions. Coccoid DFs, which retained their colony-forming capacity for a long time but were less heat-resistant than the CDCs, were formed by mycobacteria grown in standard Sauton's medium with initial pH value decreased to 6.2. Poorly differentiated DFs resulted from growing mycobacterial cultures in Sauton's medium with a fivefold-decreased phosphorus content. Upon germination of various DF types, the variants resistant to kanamycin (200 μg/ml) and tetracycline (20 μg/ml) were obtained. CDC suspensions incubated for 5 months demonstrated the highest percentage (1.5%) of antibiotic-resistant clones. The data obtained on the DF diversity of M. smegmatis, a fast-growing relative of M. tuberculosis, contribute to our understanding of the flexibility of the survival strategy of this bacterium in nature and in the host organism.