Assessing the propensity of TB clinical isolates to form viable but non-replicating subpopulations

Abstract

Current tuberculosis (TB) treatment is typically effective against drug-susceptible Mycobacterium tuberculosis, but can fail due to acquired drug resistance or phenotypic resistance. M. tuberculosis persisters, a subpopulation of viable but non-replicating (VBNR) antibiotic-tolerant bacteria, are thought to contribute to poor TB treatment outcomes. In this exploratory study, we investigated treatment-naïve drug-susceptible clinical isolates collected from people with TB, who subsequently had unsuccessful treatment outcomes. These were compared to isolates from cured individuals in terms of their ability to form VBNR subpopulations. Clinical isolates from individuals with unfavorable treatment outcomes form larger subpopulations of VBNR M. tuberculosis (2.67-13.71%) than clinical isolates from cured cases (0- 1.63%) following infection of THP-1 macrophages. All isolates were drug susceptible based on phenotypic and genotypic analysis. Whole genome sequencing identified 23 non-synonymous genomic variants shared by treatment failure clinical isolates, that were not present in isolates from cured cases. This exploratory study highlights the ability of treatment-naïve clinical isolates to form heterogeneous populations containing VBNR M. tuberculosis. We also demonstrate that clinical isolates from individuals with unsuccessful treatment outcomes form higher percentages of VBNR M. tuberculosis. The findings of this exploratory study suggest that an increased propensity to form VBNR subpopulations may impact TB treatment outcome.

Keywords: Heterogenous; Persistence; Persister; TB; Treatment failure; Tuberculosis; VBNR.

Fig. 1

Calculating the VBNR subpopulation using flow cytometry data. The figure shows histogram data for Turbo FP635 fluorescence intensity measured at 0 h (red) and 120 h (orange) post THP-1 infection and for 120 h in vitro culture (dotted line). Histograms (a–d) is representative of a technical replicate for the isolate S153dx from the cured group. Histograms e-h is representative of a technical replicate for isolate S43dx from the failed/recurrent group. M. tuberculosis recovered from macrophages at 0 h post infection shows maximum red fluorescence intensity (a, e). Measured Turbo FP635 signal larger than the mean fluorescence intensity was deemed high red, as measured by the bar. This gate was applied to l20h Turbo FP635 intensities measured for M. tuberculosis recovered at 120 h post infection (c, g) and in vitro cultured M. tuberculosis (b, f). The macrophage-enriched VBNR subpopulation percentage was calculated by subtracting the percentage in vitro cultured M. tuberculosis from the percentage intracellular M. tuberculosis at 120 h. A histogram overlay for the cured sample (d) shows a complete overlap for Turbo FP635 intensity measured at 120 h post macrophage infection and in vitro cultured M. tuberculosis. This is indicative of a miniscule or absent VBNR subpopulation. The histrogram overlay for the representative failed isolate (h) shows that the fluorescence intensity signal measured at 120 h post infection does not fully overlap with that of the in vitro cultured bacteria, suggesting a high red fluorescent subpopulation of bacteria, suggestive of a VBNR population.

Fig. 2

Fluorescent intensities 120 H post THP-1 infection. Population-wide replication dynamics of baseline isolates obtained from cured treatment group (A–F). Population-wide replication dynamics of baseline isolates obtained from failed/recurrent treatment group (G–L). Intracellular bacteria lysed from macrophages 0 h (red), in vitro bacteria 120 h (dotted black line), intracellular bacteria lysed from macrophages 120 h (orange). Data is representative of two biological duplicates consisting of two to three technical replicates. A high level of red fluorescence was detected at 0 h (A–L), post induction, which decreased in isolates from cured individuals (A–F) in both in vitro cultures and in macrophage infections at 120 h, suggestive of replication of M. tuberculosis under these conditions. In contrast, red fluorescence intensity remained high in M. tuberculosis from failed/recurrent isolates which were recovered from macrophages at 120 h, suggesting slow or non-replication of these bacteria.

Fig. 3

VBNR frequency in isolates obtained from the cured and failed/recurrent patient groups following macrophage infections. Isolates from failed/recurrent patients exhibit a higher proportion of VBNR populations compared to the isolates from the cured patient group and M. tuberculosis H37Rv. Mean values of two independent biological replicate experiments consisting of two to three technical replicates plotted with a significant p-value of 0.0022 between M. tuberculosis isolates from the cured vs. failed/recurrent group.