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.

Figure 1.

Resuscitation-promoting factor E (RpfE) and culture supernatant stimulate growth of Mycobacterium tuberculosis (Mtb) cells undetectable by conventional methods from sputa. The most probable number (MPN) count obtained in 7H9 medium (gray columns) or the same medium supplemented either with Rpf-containing culture supernatant (Rpf+SN; open columns) or recombinant RpfE (hatched columns) is compared with the cell count determined by colony-forming unit (CFU) assay (solid columns). Error bars indicate 95% confidence limits for MPN counts and standard deviation values for CFU.

Figure 2.

Effect of resuscitation-promoting factor (Rpf)-free supernatants on resuscitation of Mycobacterium tuberculosis (Mtb) cells from sputum. Rpf-free culture supernatant was isolated from the quintuple Rpf deletion Mtb mutant (Rpf–SN). The resuscitation index (RI) was calculated as the ratio of the various most probable number (MPN) counts obtained to the colony-forming unit (CFU) counts. Asterisks indicate samples in which the RI exceeds 1 (in each case the lower 95% confidence interval for the RI was >0.48). The various MPN counts were obtained in 7H9 liquid medium with the addition of Rpf-containing culture supernatant (Rpf+SN) (open columns) or Rpf-free culture supernatant (Rpf–SN) (solid columns). Error bars indicate 95% confidence limits for MPN counts.

Figure 3.

Inhibitory activity of sputum samples on the growth of Mycobacterium tuberculosis (Mtb) H37Rv cells in liquid medium. Mid-log-phase Mtb cells were spiked into two different culture-negative sputum samples from patients with tuberculosis and were incubated for 24 hours at 4°C. Control cells were kept in phosphate-buffered saline for 24 hours at 4°C. Solid columns, colony-forming units (CFU); gray columns, most probable number (MPN) in 7H9 medium; open columns, MPN in the presence of Rpf-containing culture supernatant (Rpf+SN).

Figure 4.

Storage of a freshly expectorated tuberculous sputum sample does not produce additional resuscitation-promoting factor (Rpf)-dependent Mycobacterium tuberculosis (Mtb) cells. Sputum from patient 25 was assessed for colony-forming units (CFU), most probable number (MPN), and MPN_Rpf+SN counts immediately after collection (within 3 h) and at the times shown after storage at 4°C. Solid columns, CFU; gray columns, MPN in 7H9 medium; open columns, MPN in the presence of Rpf-containing culture supernatant (Rpf+SN).

Figure 5.

Chemotherapy induces a decline in resuscitation-promoting factor (Rpf)-dependent cells that is correlated with but slower than the accompanying fall in colony-forming unit (CFU) counts. Changes in CFU and most probable number (MPN) counts were calculated as the log₁₀ ratio of CFU or Rpf-supplemented MPN count before the onset of treatment to the corresponding count during treatment. Treatment sputum samples were collected 7–11 days after the start of chemotherapy.

Figure 6.

Effect of rifampin treatment in vitro on viability of Mycobacterium tuberculosis (Mtb) cells in sputum. Decontaminated sputum samples were incubated in 7H9 medium containing PANTA antibiotic supplement for 14 hours, and then treated with rifampin (1 μg/ml) in 7H9 medium containing PANTA for 7 days at 37°C with shaking. Solid columns, colony-forming units (CFU) before rifampin treatment; gray columns, CFU after 7 days of treatment (note: these were all negative, i.e., CFU < 10/ml); open columns, MPN in Rpf+SN before treatment; hatched columns, MPN in Rpf+SN after treatment. Error bars indicate 95% confidence limits for MPN counts and standard deviation values for CFU.