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. 2016 Mar;5(1):66-73.
doi: 10.1016/j.ijmyco.2015.12.001. Epub 2015 Dec 28.

Populations of Latent Mycobacterium Tuberculosis Lack a Cell Wall: Isolation, Visualization, and Whole-Genome Characterization

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Free PMC article

Populations of Latent Mycobacterium Tuberculosis Lack a Cell Wall: Isolation, Visualization, and Whole-Genome Characterization

Ali Akbar Velayati et al. Int J Mycobacteriol. .
Free PMC article

Abstract

Objective/background: Mycobacterium tuberculosis (MTB) causes active tuberculosis (TB) in only a small percentage of infected people. In most cases, the infection is clinically latent, where bacilli can persist in human hosts for years without causing disease. Surprisingly, the biology of such persister cells is largely unknown. This study describes the isolation, identification, and whole-genome sequencing (WGS) of latent TB bacilli after 782days (26months) of latency (the ability of MTB bacilli to lie persistent).

Methods: The in vitro double-stress model of latency (oxygen and nutrition) was designed for MTB culture. After 26months of latency, MTB cells that persisted were isolated and investigated under light and atomic force microscopy. Spoligotyping and WGS were performed to verify the identity of the strain.

Results: We established a culture medium in which MTB bacilli arrest their growth, reduce their size (0.3-0.1μm), lose their acid fastness (85-90%) and change their shape. Spoligopatterns of latent cells were identical to original H37Rv, with differences observed at spacers two and 14. WGS revealed only a few genetic changes relative to the already published H37Rv reference genome. Among these was a large 2064-bp insertion (RvD6), which was originally detected in both H37Ra and CDC1551, but not H37Rv.

Conclusion: Here, we show cell-wall free cells of MTB bacilli in their latent state, and the biological adaptation of these cells was more phenotypic in nature than genomic. These cell-wall free cells represent a good model for understanding the nature of TB latency.

Keywords: Mycobacterium tuberculosis; Tuberculosis latency; Whole-genome sequencing.

Conflict of interest statement

Conflicts of interest

The authors declare no conflicts of interest.

Figures

Fig. 1
Fig. 1
Presence of rod and round shape cells in unfiltered sediments of MTB culture tubes after 782 days of oxygen and nutrient deprivation.
Fig. 2
Fig. 2
Most of cells from filtered sediments of MTB cultures appeared to be round or oval shape.
Fig. 3
Fig. 3
The average size of “cell wall free” cells of latent MTB bacilli was ranging from 0.1 to 0.3 μm.
Fig. 4
Fig. 4
Reads from two NRITLD60 libraries (“NRITLD60 frags” and “NRITLD60 jumps”) aligned to a ~2 kb region of H37Rv (base position 1,986,400 to 1,988,400) are shown in IGV [47]. Beginning at roughly 1,987,100 and until base position 1,987,711, there is a dropout in aligned coverage in both the fragment and jump reads thus illustrating the 616 base deletion reported by Pilon (1,987,095–1,987,711).

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