Detection and Quantification of Differentially Culturable Tubercle Bacteria in Sputum from Patients with Tuberculosis

Abstract

Rationale: Recent studies suggest that baseline tuberculous sputum comprises a mixture of routinely culturable and differentially culturable tubercle bacteria (DCTB). The latter seems to be drug tolerant and dependent on resuscitation-promoting factors (Rpfs).

Objectives: To further explore this, we assessed sputum from patients with tuberculosis for DCTB and studied the impact of exogenous culture filtrate (CF) supplementation ex vivo.

Methods: Sputum samples from adults with tuberculosis and HIV-1 and adults with no HIV-1 were used for most probable number (MPN) assays supplemented with CF and Rpf-deficient CF, to detect CF-dependent and Rpf-independent DCTB, respectively.

Measurements and main results: In 110 individuals, 19.1% harbored CF-dependent DCTB and no Rpf-independent DCTB. Furthermore, 11.8% yielded Rpf-independent DCTB with no CF-dependent DCTB. In addition, 53.6% displayed both CF-dependent and Rpf-independent DCTB, 1.8% carried CF-independent DCTB, and 13.6% had no DCTB. Sputum from individuals without HIV-1 yielded higher CF-supplemented MPN counts compared with counterparts with HIV-1. Furthermore, individuals with HIV-1 with CD4 counts greater than 200 cells/mm³ displayed higher CF-supplemented MPN counts compared with participants with HIV-1 with CD4 counts less than 200 cells/mm³. CF supplementation allowed for detection of mycobacteria in 34 patients with no culturable bacteria on solid media. Additionally, the use of CF enhanced detection of sputum smear-negative individuals.

Conclusions: These observations demonstrate a novel Rpf-independent DCTB population in sputum and reveal that reduced host immunity is associated with lower prevalence of CF-responsive bacteria. Quantification of DCTB in standard TB diagnosis would be beneficial because these organisms provide a putative biomarker to monitor treatment response and risk of disease recurrence.

Keywords: HIV; culturability; limiting dilution assay; resuscitation-promoting factors; tuberculosis.

Figure 1.

Participant disposition flow chart. (A) Most probable number (MPN) assays were set up with culture filtrate (CF) and resuscitation-promoting factor (Rpf)⁻ CF supplementation of growth media. CF was isolated from wild type Mycobacterium tuberculosis and Rpf⁻ CF from a quintuple rpf gene-knockout mutant, allowing for the detection of Rpf-dependent or Rpf-independent differentially culturable tubercle bacteria (DCTB), respectively. To control for the CF effect, MPN assays with no CF supplementation were also performed, which allowed for detection of CF-independent DCTB. (B) A total of 156 patients were analyzed in this study. These included individuals that had strong clinical indication for TB disease either through a positive smear or positive GeneXpert result. Of these, 46 patients were excluded because of lack of culture-confirmed M. tuberculosis infection detected by MGIT, MPN, and/or CFU. Patients were also deemed ineligible if they were on antibiotic treatment or infected with drug-resistant strains. Of the 110 eligible patients, 21 and 13 had CF-dependent and Rpf-independent DCTB, respectively, whereas 59 patients had mixed DCTB populations. Sputum from 15 patients did not harbor DCTB, and two patients carried CF-independent DCTB. (C) A glossary of terms used in the figure. ^*Mixtures of CF-dependent DCTB and Rpf-independent DCTB populations. ^†In some cases, the data for laboratory diagnosis (GeneXpert, MGIT, HAIN, and smear status) were not available. HAIN = Hain Lifesciences Line Probe Assay Genotype MTBDR plus; MGIT = mycobacteria growth indicator tube.

Figure 2.

Distribution of differentially culturable tubercle bacteria (DCTB) in a cross-sectional group of patients with tuberculosis. Shown on the y-axis are individual patients with their relative proportions of DCTB (given as the quantum of resuscitatable bacteria, reported as the resuscitation index [RI = MPN/CFU]) on the x-axis. Culture filtrate (CF)-dependent DCTB, calculated as the log(CF⁺ MPN/CFU), is reflected in red. If the calculation of CF-dependent or Rpf-independent DCTB yielded a negative value, this was adjusted to 0 because only the growth stimulatory effects of CF/Rpf⁻ CF were considered. Rpf-independent DCTB, calculated as the log(Rpf⁻ MPN/CFU), is reflected in blue. CF-independent DCTB, calculated as the log(MPN No CF/CFU), is shown in green. In cases where the CFU was zero, a value of 1 was used to reflect the absence of culturable bacteria, which indicates that the entire population detected in the MPN assay constituted DCTB. The combined colored bars reflect patients with both CF-dependent and independent DCTB populations and absence of bars indicated no detectable DCTB. Inset depicts log CFU counts in samples with no detectable DCTB population. MPN = most probable number; Rpf = resuscitation-promoting factors.

Figure 3.

Measures of bacterial load stratified by HIV-1 infection status or CD4 T-cell counts. (A) Scatterplot depicting bacterial load distributions in HIV infected/uninfected individuals. (B) Scatterplot depicting bacterial load distributions in individuals with high versus low CD4 T-cell counts. Error bars represent medians and interquartile ranges. To determine statistical significance, the Mann–Whitney U test was used with a 95% confidence interval. CF⁺ MPN (red), Rpf⁻ MPN (blue), MPN no CF (green). Two participants had missing CD4 counts. In all categories the CF⁺ MPN/Rpf⁻ MPN yielded higher bacterial counts when compared with the MPN no CF (P < 0.0001). *Significant with a 95% confidence interval. CF = culture filtrate; MPN = most probable number; ND = no bacterial growth detected; Rpf = resuscitation-promoting factors.