Expression of antimicrobial drug tolerance by attached communities of Mycobacterium tuberculosis

Abstract

There is an urgent need to improve methods used to screen antituberculosis drugs. An in vitro assay was developed to test drug treatment strategies that specifically target drug-tolerant Mycobacterium tuberculosis. The H37Rv strain of M. tuberculosis survived antimicrobial treatment as attached microbial communities when maintained in tissue culture media (RPMI-1640) with or without lysed human peripheral blood leukocytes. When cultured planktonically in the presence of Tween-80, bacilli failed to form microbial communities or reach logarithmic phase growth yet remained highly susceptible to antimicrobial drugs. In the absence of Tween, bacilli tolerated drug therapy by forming complex microbial communities attached to untreated well surfaces or to the extracellular matrix derived from lysed human leukocytes. Treatment of microbial communities with DNase I or Tween effectively dispersed bacilli and restored drug susceptibility. These data demonstrate that in vitro expression of drug tolerance by M. tuberculosis is linked to the establishment of attached microbial communities and that dispersion of bacilli targeting the extracellular matrix including DNA restores drug susceptibility. Modifications of this in vitro assay may prove beneficial in a high-throughput platform to screen new antituberculosis drugs especially those that target drug-tolerant bacilli.

Keywords: Mycobacterium tuberculosis; biofilm; drug resistant; drug tolerant; microbial communities.

Figure 1. M. tuberculosis cultured in nutrient rich mammalian cell culture media fails to proliferate in vitro

The H37Rv strain of M. tuberculosis was cultured in mammalian cell culture media (RPMI-1640 + 2% bovine plasma) with and without Tween-80 and in the presence of lysed human leukocytes show no net increase in viable bacilli numbers out to seven days. Data is expressed as (A) mean log₁₀ colony forming units (CFUs) ± standard error of the mean (SEM) or (B) mean percent survival ± SEM of CFUs cultured on 7H11 agar. n=3.

Figure 2. M. tuberculosis cultured in nutrient rich mammalian cell culture media express differences in tolerance to isoniazid (INH) in vitro

The H37Rv strain of M. tuberculosis was cultured in mammalian cell culture media (RPMI-1640 + 2% bovine plasma) containing (A) Tween-80 (0.05%), (B) in the absence of Tween-80 (0.05%) and (C) in the presence of lysed human leukocytes was treated for seven days with INH (10μg/ml) or equal volumes of the carrier water. Data is expressed as the mean percent survival of viable colony forming units (CFUs) ±standard error of the mean (SEM) on 7H11 agar. n=6 separate experiments. ** = P≤ 0.01, *** = P≤ 0.001 compared to carrier controls.

Figure 3. M. tuberculosis cultured in nutrient rich mammalian cell culture media express differences in tolerance to rifampin (RIF) in vitro

The H37Rv strain of M. tuberculosis was cultured for seven days in mammalian cell culture media (RPMI-1640 + 2% bovine plasma) containing (A) Tween-80 (0.05%), (B) in the absence of Tween-80 (0.05%) and (C) in the presence of lysed human leukocytes. Cultures were treated with RIF (50μg/ml) or equal volumes of the carrier dimethyl sulfoxide (DMSO, < 0.01%). Data is expressed as the mean percent survival of viable colony forming units (CFUs) ± standard error of the mean (SEM) on 7H11 agar. n=3 separate experiments. * = P≤ 0.05 ** = P≤ 0.01, *** = P≤ 0.001 compared to carrier controls.

Figure 4. M. tuberculosis cultured in nutrient rich mammalian cell culture media express differences in tolerance to combination antimicrobial drug treatment in vitro

The H37Rv strain of M. tuberculosis was cultured for seven days in mammalian cell culture media (RPMI-1640 + 2% bovine plasma) containing (A) Tween-80 (0.05%), (B) in the absence of Tween-80 (0.05%) and (C) in the presence of lysed human leukocytes. Cultures were treated for seven days with of isoniazid (INH, 10μg/ml), rifampin (50μg/ml) and pyrazinamide (PZA, 3mg/ml) combined (RHZ) or equal volumes of the carrier dimethyl sulfoxide (DMSO, < 0.01%). Data is expressed as the mean percent survival of viable colony forming units (CFUs) ± standard error of the mean (SEM) on 7H11 agar. n=2 separate experiments. * = P≤ 0.05 ** = P≤ 0.01, *** = P≤ 0.001 compared to carrier controls.

Figure 5. The expression of in vitro antimicrobial drug tolerance by M. tuberculosis is associated with attached communities of bacilli with different staining characteristics

The H37Rv strain of M. tuberculosis was cultured for fourteen days in mammalian cell culture media (RPMI-1640 + 2% bovine plasma) containing (A) Tween-80 (0.05%), (B) in the absence of Tween-80 (0.05%) and (C) in the presence of lysed human leukocytes, paraformaldehyde fixed and stained with rhodamine (red) and the DNA stain TO-PRO-3 and viewed by confocal microscopy. (A) In the presence of Tween-80, bacilli stain poorly with rhodamine and are attached as individual or small clusters of bacilli (Z stack height, 6.4μm). (B) In the absence of Tween-80 and lysed leukocytes, the majority of bacilli stain with rhodamine and are firmly attached to well surfaces to form complex colonies of bacilli (Z stack height, 20.6μm). (C) In the presence of lysed human leukocytes, rhodamine positive bacilli form complex raised colonies (Z stack height, 29.8μm) attached to host-derived extracellular DNA. Grid = 45μm square.

Figure 6. Viable and non-viable bacilli are spatially stratified when cultured as attached communities of M. tuberculosis

The H37Rv strain of M. tuberculosis was cultured in mammalian cell culture media (RPMI-1640 + 2% bovine plasma) containing (A) Tween-80 (0.05%), (B) in the absence of Tween-80 (0.05%). The H37Rv strain of M. tuberculosis was cultured for seven days in mammalian cell culture media (RPMI-1640 + 2% bovine plasma) containing (A) Tween-80 (0.05%), (B) in the absence of Tween-80 (0.05%) and stained with the live-dead (Syto 9 and propidium iodide, Bac-Light, Life Technologies) prior to fixation with paraformaldehyde and viewed by confocal microscopy.

Figure 7. Treatment of drug tolerant M. tuberculosis with DNase I partially restores in vitro susceptibility to isoniazid

The H37Rv strain of M. tuberculosis was cultured in mammalian cell culture media (RPMI-1640 + 2% bovine plasma) containing (A) Tween-80 (0.05%), (B) in the absence of Tween-80 (0.05%) and (C) in the presence of lysed human leukocytes. Cultures were treated for seven days with INH (10μg/ml) or equal volumes of the carrier water with or without DNase I (0.5 mg/ml) added on day seven and ten. Data is expressed as the mean percent survival of viable colony forming units (CFUs) ± standard error of the mean (SEM) on 7H11 agar. n=6 separate experiments. * = P≤ 0.05 ** = P≤ 0.01, *** = P≤ 0.001 compared to carrier controls.