Cytological and transcript analyses reveal fat and lazy persister-like bacilli in tuberculous sputum.
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.
Garton
Natalie J
NJ
Department of Infection, Immunity and Inflammation, University of Leicester Medical School, Leicester, United Kingdom.
Waddell
Simon J
SJ
Sherratt
Anna L
AL
Lee
Su-Min
SM
Smith
Rebecca J
RJ
Senner
Claire
C
Hinds
Jason
J
Rajakumar
Kumar
K
Adegbola
Richard A
RA
Besra
Gurdyal S
GS
Butcher
Philip D
PD
Barer
Michael R
MR
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*
D008055:Lipids; D014280:Triglycerides