Resuscitation-promoting Factors Reveal an Occult Population of Tubercle Bacilli in Sputum

Abstract

Rationale: Resuscitation-promoting factors (Rpfs) are a family of secreted proteins produced by Mycobacterium tuberculosis (Mtb) that stimulate mycobacterial growth. Although mouse infection studies show that they support bacterial survival and disease reactivation, it is currently unknown whether Rpfs influence human infection. We hypothesized that tuberculous sputum might include a population of Rpf-dependent Mtb cells. Objectives: To determine whether Rpf-dependent Mtb cells are present in human sputum and explore the impact of chemotherapy on this population. Methods: In tuberculous sputum samples we compared the number of cells detected by conventional agar colony-forming assay with that determined by limiting dilution, most-probable number assay in the presence or absence of Rpf preparations. Measurements and Main Results: In 20 of 25 prechemotherapy samples from separate patients, 80-99.99% of the cells demonstrated by cultivation could be detected only with Rpf stimulation. Mtb cells with this phenotype were not generated on specimen storage or by inoculating sputum samples with a selection of clinical isolates; moreover, Rpf dependency was lost after primary isolation. During chemotherapy, the proportion of Rpf-dependent cells was found to increase relative to the surviving colony-forming population. Conclusions: Smear-positive sputum samples are dominated by a population of Mtb cells that can be grown only in the presence of Rpfs. These intriguing proteins are therefore relevant to human infection. The Rpf-dependent population is invisible to conventional culture and is progressively enhanced in relative terms during chemotherapy, indicating a form of phenotypic resistance that may be significant for both chemotherapy and transmission.

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.…”

Rifamycin action on RNA polymerase in antibiotic-tolerant Mycobacterium tuberculosis results in differentially detectable populations

“…We wondered if the additional casein or malachite green in 7H11 medium would change cfu counts, but the numbers of cfus were similar on agar plates made with 7H10 or 7H11 medium (7H11 includes a casein digest and contains about fourfold more malachite green than 7H10). Addition of fresh CF from log-phase Mtb, shown in patient sputum to enhance DD Mtb recovery (13,14), did not change MPN estimates of DD Mtb when supplemented in the MPN plates to a final concentration of 50% (Fig. S2A).…”

“…By this method, VBNC populations would be characterized as dead. In 2010, a study of 25 untreated TB patients in the United Kingdom found that 80-99% of the viable bacteria in the sputum of 80% of the subjects could not be detected as cfus (13). Instead, the initial bacterial number was estimated by the most probable number (MPN) method (13) based on a replicate series of limiting dilutions (LDs), herein termed an MPN-LD assay.…”

Early diagnosis and effective treatment regimens are the keys to tackle antimicrobial resistance in tuberculosis (TB): A report from Euroscicon's international TB Summit 2016

“…They can then be reverted to their actively replicating physiology by exposure to resuscitation promoting factors (Rpfs) and are also known as Rpf-dependent cells. 48 Rpfs are secreted or cell-wall bound enzymes produced by actively dividing mycobacteria. They are structurally and functionally similar to lysozymes and are essential for resuscitating dormant cells.…”

“…48 These cells are highly abundant in sputum and extra pulmonary TB samples with their relative proportions varying from 0 to 99.99% of the total M. tuberculosis population. Jaishree Garhyan (Jawaharlal Nehru University, India) reported bone marrow to be a niche for persisters as the mesenchymal stem cells survive for an extended period of time and reside in immune suppressed environments.…”