Non-exponential growth of Mycobacterium leprae Thai-53 strain cultured in vitro.

To determine why cultivation of Mycobacterium leprae in vitro has not been successful, we attempted to culture this bacterium in media supplemented with a variety of biological materials. We observed a slight increase in the number of cells in medium supplemented with human blood plasma and an extract of nude mouse tissue after more than 3 months of cultivation at 30°C. To ascertain whether this increase was real growth, we analyzed the growth by droplet digital PCR. The analysis had the following two results: the slow increase in the copy number of cell-associated DNA and the release of a large amount of DNA into the culture medium from bacterial cells during cultivation. These results were supported by the electron microscope examination of M. leprae in infected mouse tissues, which showed that most of the replicated bacteria degenerated and that only a small number of cells survived. Based on these results, we hypothesize that during M. leprae growth, many of the replicated cells degenerate and that only a few remaining cells participate in the next growth stage. This means that, unlike other cultivable bacteria, the growth of M. leprae is not exponential and that the increase in the number of cells is therefore extremely slow. Thus, to judge the success of M. leprae cultivation, growth should be observed over a long period of time, and the increase in the number of viable cells must be carefully measured.