Research Article: Cytological and Transcript Analyses Reveal Fat and Lazy Persister-Like Bacilli in Tuberculous Sputum

Date Published: April 1, 2008

Publisher: Public Library of Science

Author(s): Natalie J Garton, Simon J Waddell, Anna L Sherratt, Su-Min Lee, Rebecca J Smith, Claire Senner, Jason Hinds, Kumar Rajakumar, Richard A Adegbola, Gurdyal S Besra, Philip D Butcher, Michael R Barer, Olivier Neyrolles

Abstract: BackgroundTuberculous 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.Methods and FindingsMicroscopy-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 (R2 = 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.ConclusionAs 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.

Partial Text: Mycobacterium tuberculosis infects one in three worldwide and kills more people each year than any other bacterial pathogen. Routine treatment of tuberculosis requires combination antibiotic therapy for a minimum of six months, and places a substantial burden on health care systems, particularly in resource-poor countries. Over eight million new cases every year testify to this obligate pathogen’s ongoing success in transmission [1], yet we know little about what the organism needs to achieve this essential step.

We applied a combination of ex vivo and in vitro analyses to study the phenotypes of M. tuberculosis cells in smear-positive sputum samples. We found a subpopulation of lipid body–positive acid-fast cells in all samples for which >100 bacilli were analysable. Our further in vitro studies revealed nonreplicating persistence, as defined by Wayne and colleagues [46], to be a condition in which M. tuberculosis cells are induced to form lipid bodies at frequencies comparable to those observed among tubercle bacilli in sputum. Consistent with this finding, transcriptome analysis of M. tuberculosis in sputum revealed signals compatible with slow or non-growth and absence of aerobic respiration. Moreover, the time to positivity in diagnostic liquid culture was shown to be directly related to sputum lipid body content, adding further weight to the view that lipid body–positive cells are not replicating. While other explanations remain possible, we conclude that the lipid body–positive cells in sputum have a persister-like phenotype, with important implications for the treatment and transmission of tuberculosis. Further studies should elucidate the impact of chemotherapy on the frequency of lipid body–positive populations of M. tuberculosis in patient sputum, and the relationship between this candidate biomarker and both infectivity and the clinical response to treatment.



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