title: Populations of latent Mycobacterium tuberculosis lack a cell wall: Isolation, visualization, and whole-genome characterization.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Mycobacteriology Research Centre, National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran.
authors.forename: Ali Akbar
authors.initials: AA
authors.lastname: Velayati
authors.affiliation: Genome Sequencing & Analysis Program, The Broad Institute of MIT & Harvard, Cambridge, MA, USA; Delft Bioinformatics Lab, Delft University of Technology, Delft, The Netherlands.
authors.forename: Thomas
authors.initials: T
authors.lastname: Abeel
authors.affiliation: Genome Sequencing & Analysis Program, The Broad Institute of MIT & Harvard, Cambridge, MA, USA.
authors.forename: Terrance
authors.initials: T
authors.lastname: Shea
authors.affiliation: The Republican Research and Practical Centre for Epidemiology and Microbiology, Minsk, Belarus.
authors.forename: Gennady
authors.initials: G
authors.lastname: Konstantinovich Zhavnerko
authors.affiliation: Genome Sequencing & Analysis Program, The Broad Institute of MIT & Harvard, Cambridge, MA, USA.
authors.forename: Bruce
authors.initials: B
authors.lastname: Birren
authors.affiliation: Department of Global Health and Social Medicine, Harvard Medical School, Boston, MA, USA.
authors.forename: Gail H
authors.initials: GH
authors.lastname: Cassell
authors.affiliation: Genome Sequencing & Analysis Program, The Broad Institute of MIT & Harvard, Cambridge, MA, USA.
authors.forename: Ashlee M
authors.initials: AM
authors.lastname: Earl
authors.affiliation: Department of Microbiology, The Public Health Agency of Sweden, Solna, Sweden.
authors.forename: Sven
authors.initials: S
authors.lastname: Hoffner
authors.affiliation: Mycobacteriology Research Centre, National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran. Electronic address: pfarnia@hotmail.com.
authors.forename: Parissa
authors.initials: P
authors.lastname: Farnia
mesh_terms: D002473:Cell Wall / Q000648:ultrastructure*; D016680:Genome, Bacterial; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D055985:Latent Tuberculosis / Q000382:microbiology*; D009169:Mycobacterium tuberculosis / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification* / Q000648:ultrastructure
keywords: Mycobacterium tuberculosis; Tuberculosis latency; Whole-genome sequencing