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

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.

Dormant Mycobacterium tuberculosis Fails To Block Phagosome Maturation and Shows Unexpected Capacity To Stimulate Specific Human T Lymphocytes

“…Moreover, a resuscitation-promoting factor-dependent population of M. tuberculosis was recently observed in clinical TB samples, providing further evidence of the presence of D-Mtb in sputa (20). Because sputa of infecting individuals are likely to reflect the population of bacilli present in open TB lung lesions (19), these data suggest that a large population of D-Mtb can be found in active TB lesions and that dormant bacteria are not necessarily only associated to latency. The analysis of the M. tuberculosis population in sputa also supports the hypothesis that D-Mtb may be transmitted to susceptible individuals through the infecting droplets released from patients with active TB.…”

“…Interestingly, morphologic and transcriptome analysis of M. tuberculosis in sputum of infected individuals revealed the presence of high percentages of D-Mtb in active TB (19). Moreover, a resuscitation-promoting factor-dependent population of M. tuberculosis was recently observed in clinical TB samples, providing further evidence of the presence of D-Mtb in sputa (20).…”

The epidemiology, pathogenesis, transmission, diagnosis, and management of multidrug-resistant, extensively drug-resistant, and incurable tuberculosis

“…[175][176][177][178] The antibiotics are no longer present because clearance occurs before a single M tuberculosis has replicated, and certainly by the second and third replications. This timing makes the probability of generation of mutants, or even amplifying pre-existing ones, less likely, particularly for nonreplicating persistors and semi dormant bacilli, aptly described as "fat and lazy" by Garton and colleagues 179 because of their lipid content and slow doubling times, which can take weeks. 180 The pharmacokinetic mismatch hypothesis for acquired drug resistance was directly tested for isoniazid and rifampicin in the hollow fibre model of tuberculosis, on the basis of M tuberculosis doubling times of 24 h and 240 h. 181,182 This preclinical model was chosen because of the ease in which acquired drug resistance arises in the model.…”

Protein kinase C-delta (PKCδ), a marker of inflammation and tuberculosis disease progression in humans, is important for optimal macrophage killing effector functions and survival in mice

“…10 This feature of accumulating fewer lipids during an inflammatory condition by PKCd À / À mice upon Mycobacterium tuberculosis (Mtb) infection might impair its ability to undergo slow-replicating, persistence phase as reported in human sputum. 11 Lipids are a prerequisite for the persistence of Mtb in macrophages that transforms them into foamy macrophages that contribute to tissue pathology and facilitate dissemination. 12 Most studies on the role of PKCd were performed using inhibitors in vitro that lacked in specificity to a particular PKC isoform.…”