Clinical Strains of Mycobacterium tuberculosis Representing Different Genotype Families Exhibit Distinct Propensities to Adopt the Differentially Culturable State

Abstract

Growing evidence points to the presence of differentially culturable tubercle bacteria (DCTB) in clinical specimens from individuals with active tuberculosis (TB) disease. These bacteria are unable to grow on solid media but can resuscitate in liquid media. Given the epidemiological success of certain clinical genotype families of Mycobacterium tuberculosis, we hypothesize that different strains may have distinct mechanisms of adaptation and tolerance. We used an in vitro carbon starvation model to determine the propensity of strains from lineages 2 and 4 that included the Beijing and LAM families respectively, to generate DCTB. Beijing strains were associated with a greater propensity to produce DCTB compared to LAM strains. Furthermore, LAM strains required culture filtrate (CF) for resuscitation whilst starved Beijing strains were not dependent on CF. Moreover, Beijing strains showed improved resuscitation with cognate CF, suggesting the presence of unique growth stimulatory molecules in this family. Analysis of starved Beijing and LAM strains showed longer cells, which with resuscitation were restored to a shorter length. Cell wall staining with fluorescent D-amino acids identified strain-specific incorporation patterns, indicating that cell surface remodeling during resuscitation was distinct between clinical strains. Collectively, our data demonstrate that M. tuberculosis clinical strains from different genotype lineages have differential propensities to generate DCTB, which may have implications for TB treatment success.

Keywords: Beijing strains; LAM strains; Mycobacterium tuberculosis; differentially culturable tubercle bacteria; tuberculosis.

Figure 1

Participant disposition flow chart and assessment of DCTB in baseline sputum samples. (A) Participant disposition flow chart for individuals recruited in the BMG and MGIT Plus studies for the DCTB data analysis. Samples excluded for various reasons are indicted with the letters a-k. The Beijing family belongs to lineage 2 (East-Asian) whilst lineage 4 (Euro-American) includes the T, S, LAM and X families. (B) Schematic diagram adapted from Gordhan et al. [4] shows the methodology used for DCTB assessment in sputum specimens in real time obtained at baseline. Sputum samples were decontaminated and assessed by CFU and the Most Probable Number (MPN) limiting dilution assays containing culture filtrate (CF) to detect DCTB. To control for the effect of CF in growth stimulation, fresh Middlebrook 7H9 media was used (No CF MPN) which allowed for quantification of conventionally culturable bacteria. DCTB ratios were obtained by dividing the MPN values (with or without CF) by CFU counts. (C) Proportion of DCTB detected in the different M. tuberculosis genotype families. DCTB had to be present in only one of the conditions that is with or without CF, within each family. Genotype families with less than 10 samples were excluded from the analysis. The Beijing family showed the greatest amount of DCTB (p-value ≤ 0.05) using a chi-squared t-test, compared to the other families. (D) Assessment of the effect of CF on the resuscitation of DCTB in the various M. tuberculosis families. The amount of DCTB was calculated as log (MPN/CFU) for both the CF-independent and CF-dependent DCTB for each strain type. Using the Mann-Whitney test (GraphPad), the Beijing family showed significant resuscitation with CF (p-value < 0.05) and more DCTB (p-value = 0.0518) were detected within this family compared to the other genotype families. * = p-value < 0.05, ** = p-value < 0.01, *** = p-value < 0.001 and ns = not significant.

Figure 2

Generation of DCTB in H37Rv using an in vitro carbon starvation model. (A) Schematic diagram showing the conditions for carbon starvation of H37Rv and downstream assessment for the detection of DCTB. The DCTB population in H37RV grown in 7H9 media and starved in PBS was detected by the MPN and CFU assays, followed by the assessment of the resuscitated cells (with media and CF), starved and axenic cells by microscopy. (B) Microscopy of stained starved cells before and after resuscitation with and without CF compared to an axenic culture of H37Rv grown in 7H9 media. (C) Assessment of culturability of cells under the different culture conditions. Up to one hundred TADA-stained cells from each of the culture conditions were assessed for the proportion of stained and unstained cells under a fluorescent microscope. (D) Comparison of cell length differences for the unstarved, starved and resuscitated cells. The scale bar represents 2 µM. A one-way ANOVA test showed that the starved H37Rv cells were significantly shorter (**** = p-value ≤ 0.0001) compared to the resuscitated cells which were significantly longer than the cells grown in 7H9 (**** = p-value ≤ 0.0001).

Figure 3

Assessment of the propensity of the clinical Beijing and LAM isolates to produce DCTB. (A) Each of the 5 Beijing and LAM strains and the H37RV control laboratory strain were starved in PBS for two weeks, after which DCTB from each culture were resuscitated with media and CF from H37Rv. The resuscitation index was calculated using the MPN/CFU ratio. The experiment was performed in triplicate. T-test analysis showed that both Beijing and LAM strains resuscitated significantly more CF-dependent DCTB compared to resuscitation with media (p-value ≤ 0.01). (B) Average DCTB resuscitation index for the 5 Beijing and 5 LAM strains. * = p-value < 0.05, ** = p-value < 0.01 and *** = p-value < 0.001 All three genotype families demonstrate greater resuscitation of the starved strains with CF compared to media (p-value ≤ 0.001–≤0.01) with no difference in resuscitation between the Beijing and LAM families (p-value ≤ 0.01).

Figure 4

The effects of CFs derived from clinical isolates in the resuscitation of DCTB. (A) A schematic representation of the procedure followed to supplement MGIT tubes with CF from the Beijing and LAM clinical strains. (B) MGIT time to positivity (TTP) of starved H37Rv supplemented with Beijing-CF and LAM-CF compared to CF from H37RV and media. A one-way ANOVA test showed that CF from both Beijing and LAM strains significantly decreased TTP compared to media and H37Rv-CF (* = p-value ≤ 0.05 and **** = p-value ≤ 0.0001). (C) Resuscitation Index of the five starved Beijing strains compared to starved H37Rv, resuscitated in media, H37Rv-CF and Beijing- CF (from Beijing 2). A two-way ANOVA statistical test showed that Beijing-CF allowed greater detection of DCTB in starved Beijing 1 and Beijing 4 cells when compared to the media control (* = p-value ≤ 0.05, *** = p-value ≤ 0.001 and ns = not significant). (D) Resuscitation Index of five independent starved LAM strains compared to starved H37Rv, resuscitated in media, H37Rv-CF and LAM-CF (from LAM 5). A two-way ANOVA statistical test showed no significant resuscitation of DCTB with LAM-CF compared to starved cultures resuscitated with media and H37Rv-CF. Two of the LAM strains showed significant resuscitation with LAM CF compared to media (* = p-value ≤ 0.05 and ns = not significant). Resuscitation index was plotted as the MPN/CFU ratio. The experiments for each strain were performed in triplicate.

Figure 5

Cytological profiling of axenic, starved and resuscitated cells by fluorescent microscopy. Starved cells were resuscitated in 7H9 media and CF, and stained with the TADA probe for 24 h at 37 °C. The experiment was performed in triplicate. One hundred cells from each condition were analyzed for staining patterns using a SR-SIM fluorescent microscope. (A) Microcopy showing the presence of six different staining patterns for H37RV resuscitated cells. Scale bars represent 2 µM. (B) The proportion of cells resuscitated with media and CF, showing the distribution of the six identified cell staining patterns. (C) Comparison of TADA staining distribution plots of exponentially grown axenic culture of H37RV and resuscitated bacteria. The pattern of probe incorporation along the poles and cell wall was assessed for 30 cells under each condition. Similar staining patterns were assessed for 30 axenically grown and resuscitated Beijing cells (D) and LAM cells (E). The staining pattern for the 30 cells for the three strains were further analyzed under nutrient- rich culture conditions (F), resuscitated with media (G) and resuscitated with CF (H). In each case, the solid line represents H37Rv, the dashed line represents the Beijing strain, and the dotted line is the LAM strain.