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.
Methods and Findings
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 (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.
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.
Studying sputum from humans with pulmonary tuberculosis, Michael Barer and colleagues detect mycobacteria containing lipid bodies. Analyses linking this cytological feature to a slow-growing phenotype sheds light on persistence.
Every year, nearly nine million people develop tuberculosis—a contagious infection usually of the lungs—and about two million people die from the disease. Tuberculosis is caused by Mycobacterium tuberculosis, bacteria that are spread in airborne droplets when people with the disease cough or sneeze. The symptoms of tuberculosis include a persistent cough, weight loss, and night sweats. Diagnostic tests include chest X-rays, the tuberculin skin test, and sputum analysis. For the last of these tests, a sample of sputum (mucus and other matter brought up from the lungs by coughing) is collected and then taken to a laboratory where bacteriologists look for M. tuberculosis using special stains—tuberculosis-positive sputum contains “acid-fast bacilli”—and also try to grow bacteria from the sample. Tuberculosis can be cured by taking several powerful antibiotics for several months. It is very important that this treatment is completed to ensure that all the M. tuberculosis bacteria in the body are killed and to prevent the emergence of drug-resistant bacteria.
Why Was This Study Done?
Strenuous efforts are being made to reduce the global burden of tuberculosis but with limited success so far for many reasons. One barrier to success is the efficiency with which M. tuberculosis spreads from one person to another. Very little is known about this part of the bacteria's life cycle. If scientists could understand more about the transmission of M. tuberculosis between people, they might identify new therapeutic and preventative targets. In the study, therefore, the researchers examine the acid-fast bacilli in tuberculosis-positive sputum samples to get a snapshot of M. tuberculosis at the point of its transmission to a new person and ask how the characteristics of these bacilli compare with those of M. tuberculosis growing in the laboratory.
What Did the Researchers Do and Find?
The researchers collected sputum samples from patients with tuberculosis in the UK and The Gambia before they received any treatment, and looked for the presence of acid-fast bacilli containing “lipid bodies.” These small structures contain a fat called triacylglycerol. M. tuberculosis accumulates triacylglycerol when it is exposed to several stresses present during infection (for example, reduced oxygen or hypoxia) and the researchers suggest that the presence of this fat may help the bacteria survive during transmission and establish a new infection. They found that all the samples contained some lipid body–positive acid-fast bacilli. Next, the researchers showed that M. tuberculosis grown in the laboratory under hypoxic conditions, which induce the bacteria to enter an antibiotic-tolerant condition called a “nonreplicating persistent” (NRP) state, also accumulated lipid bodies. This result suggests that the lipid body–positive acid-fast bacilli in sputum might be in an NRP state. To test this idea, the researchers compared the pattern of mRNAs (the templates from which proteins are produced; the pattern of mRNAs is called the transcriptome and gives an idea of which proteins a cell is making under given conditions) made by growing cultures of M. tuberculosis, by M. tuberculosis maintained in the NRP state, and by the acid-fast bacilli in several sputum samples. The transcriptome of the sputum sample revealed production of many proteins made in the NRP state. Finally, the researchers showed that the time needed to grow M. tuberculosis from sputum samples increased as the proportion of lipid body–positive acid-fast bacilli in the sputum increased, just as one would suspect if the presence of lipid bodies signifies nongrowing cells.
What Do These Findings Mean?
It has been generally assumed that the acid-fast bacilli in sputum collected from patients with tuberculosis are rapidly replicating M. tuberculosis released from infected areas of the lungs. By identifying a population of bacteria that contain lipid bodies and that are in an NRP-like state in all the samples of sputum examined from two geographical sites, this study strongly challenges this assumption. The characteristics of this population of bacteria, the researchers suggest, might help them survive the adverse conditions that M. tuberculosis encounters during transmission between people and might partly explain why complete clearance of M. tuberculosis requires extended treatment with antibiotics. To establish the clinical significance of these findings, future studies will need to examine whether antibiotic treatment affects the frequency of lipid body–positive M. tuberculosis bacteria in patients' sputum and whether there is any relationship between this measurement and infectiousness, or clinical response to treatment.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050075.
The MedlinePlus encyclopedia contains pages on tuberculosis and on sputum culture (in English and Spanish)
The US National Institute of Allergy and Infectious Diseases provides information on all aspects of tuberculosis
The US Centers for Disease Control and Prevention Division of Tuberculosis Elimination provides several fact sheets and other information resources about tuberculosis
The World Health Organization provides information on efforts to reduce the global burden of tuberculosis