title: Cytological and transcript analyses reveal fat and lazy persister-like bacilli in tuberculous sputum.
abstract: BACKGROUND
  Tuberculous sputum provides a sample of bacilli that must be eliminated by chemotherapy and that may go on to transmit infection. A preliminary observation that Mycobacterium tuberculosis cells contain triacylglycerol lipid bodies in sputum, but not when growing in vitro, led us to investigate the extent of this phenomenon and its physiological basis.
  
  
  RESULTS
  Microscopy-positive sputum samples from the UK and The Gambia were investigated for their content of lipid body-positive mycobacteria by combined Nile red and auramine staining. All samples contained a lipid body-positive population varying from 3% to 86% of the acid-fast bacilli present. The recent finding that triacylglycerol synthase is expressed by mycobacteria when they enter in vitro nonreplicating persistence led us to investigate whether this state was also associated with lipid body formation. We found that, when placed in laboratory conditions inducing nonreplicating persistence, two M. tuberculosis strains had lipid body levels comparable to those found in sputum. We investigated these physiological findings further by comparing the M. tuberculosis transcriptome of growing and nonreplicating persistence cultures with that obtained directly from sputum samples. Although sputum has traditionally been thought to contain actively growing tubercle bacilli, our transcript analyses refute the hypothesis that these cells predominate. Rather, they reinforce the results of the lipid body analyses by revealing transcriptional signatures that can be clearly attributed to slowly replicating or nonreplicating mycobacteria. Finally, the lipid body count was highly correlated (R(2) = 0.64, p < 0.03) with time to positivity in diagnostic liquid cultures, thereby establishing a direct link between this cytological feature and the size of a potential nonreplicating population.
  
  
  CONCLUSIONS
  As nonreplicating tubercle bacilli are tolerant to the cidal action of antibiotics and resistant to multiple stresses, identification of this persister-like population of tubercle bacilli in sputum presents exciting and tractable new opportunities to investigate both responses to chemotherapy and the transmission of tuberculosis.
authors.affiliation: Department of Infection, Immunity and Inflammation, University of Leicester Medical School, Leicester, United Kingdom.
authors.forename: Natalie J
authors.initials: NJ
authors.lastname: Garton
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Waddell
authors.forename: Anna L
authors.initials: AL
authors.lastname: Sherratt
authors.forename: Su-Min
authors.initials: SM
authors.lastname: Lee
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Smith
authors.forename: Claire
authors.initials: C
authors.lastname: Senner
authors.forename: Jason
authors.initials: J
authors.lastname: Hinds
authors.forename: Kumar
authors.initials: K
authors.lastname: Rajakumar
authors.forename: Richard A
authors.initials: RA
authors.lastname: Adegbola
authors.forename: Gurdyal S
authors.initials: GS
authors.lastname: Besra
authors.forename: Philip D
authors.initials: PD
authors.lastname: Butcher
authors.forename: Michael R
authors.initials: MR
authors.lastname: Barer
mesh_terms: D005714:Gambia / Q000453:epidemiology; D020869:Gene Expression Profiling; D006801:Humans; D008055:Lipids / Q000032:analysis*; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000166:cytology / Q000235:genetics / Q000502:physiology*; D020411:Oligonucleotide Array Sequence Analysis; D013183:Sputum / Q000382:microbiology*; D014280:Triglycerides / Q000032:analysis*; D014397:Tuberculosis, Pulmonary / Q000453:epidemiology / Q000382:microbiology*
chemical_list: D008055:Lipids; D014280:Triglycerides