Mycobacterium tuberculosis Is Selectively Killed by Rifampin and Rifapentine in Hypoxia at Neutral pH

ABSTRACT

The activities of rifampin, rifapentine, bedaquiline, PA-824, clofazimine, nitazoxanide, isoniazid, amikacin, moxifloxacin, niclosamide, thioridazine, and pyrazinamide were tested against nonreplicating (dormant) Mycobacterium tuberculosis H37Rv under conditions of hypoxia at pHs 5.8 and 7.3, mimicking environments of cellular granulomas and caseous granulomas, respectively. At pH 5.8, several drugs killed dormant bacilli, with the best being rifampin and rifapentine. At pH 7.3, only rifampin and rifapentine efficiently killed dormant bacilli, while all other drugs showed little activity.

TEXT

The etiologic agent of tuberculosis (TB) is Mycobacterium tuberculosis. Two billion people are latently infected with this organism, and in 10% of them, it reactivates to active TB in their lifetime (1). Currently, treatments require 6 months of combination therapy with isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol for active TB and 9 months of INH or 3 months of rifapentine (RFP) and INH for latent TB (2, 3). Active and latent TB infections comprise mixtures of cellular and caseous granulomas, with tubercle bacilli ranging from actively replicating (AR) to dormant nonreplicating (NR) stages (4). In cellular granulomas, replicating bacilli are killed by current therapy, while in low vascularized caseous granulomas, low oxygen pressure restricts the growth from the AR to the NR stage in their hypoxic centers, enabling M. tuberculosis to move into a dormant drug-refractory state.

The pH of the phagolysosomes of activated macrophages present in cellular granulomas is acidic (5), while in necrotic centers of hypoxic cholesterol/triacylglycerol-rich caseous granulomas, where dormant M. tuberculosis lives, the pH is 7.2 to 7.5 (6–8). No information on drug activity against NR M. tuberculosis grown in hypoxic neutral conditions is known. In this study, we investigated the growth patterns of NR M. tuberculosis at different pHs and measured the activities of 12 drugs against NR cells under hypoxic conditions at pHs 5.8 and 7.3, mimicking the environments of cellular granulomas and caseous granulomas, respectively.

Briefly, M. tuberculosis strain H37Rv was grown at 37°C for 40 days in 20- by 125-mm screw-cap tubes containing Dubos-Tween-albumin broth (DTAB) as previously described (9–11), but in the present study, the DTAB was adjusted to pHs 5.8, 6.6, 7.0, 7.4, and 7.6. For preparation of NR cells, log-phase cultures were diluted in DTAB and incubated in tubes with the caps tightly screwed and tight rubber caps put under the screw caps. Growth in the tubes was monitored by measuring pH, optical density at 600 nm, and CFU/ml on Middlebrook 7H10 (7H10) agar plates incubated at 37°C for 3 weeks. To determine drug activity against NR cells, 12- and 19-day-old hypoxic cultures (H12 and H19, respectively) were incubated with drugs for 7, 14, and 21 days. Drugs were added by syringe to H12 and H19 cultures at the following maximum concentrations of the drug in serum (C_max) (10, 11): 8 μg/ml (RIF), 10 μg/ml (RFP), 1 μg/ml (bedaquiline [BQ]), 2 μg/ml (PA-824 [PA]), 1 μg/ml (clofazimine [CL]), 10 μg/ml (nitazoxanide [NZ]), 2 μg/ml (INH), 8 μg/ml (amikacin [AK]), 4 μg/ml (moxifloxacin [MX]), 0.3 μg/ml (niclosamide [NC]), and 0.5 μg/ml (thioridazine [TH]). Pyrazinamide was used at 100 μg/ml. After drug exposure, 1 ml of NR culture was washed and resuspended in 1 ml of DTAB, and 0.2 ml of the dilution was inoculated into 7H10 plates for determination of numbers of CFU/ml.

The kinetics of growth (CFU and optical density) and pHs of NR M. tuberculosis cultures at different initial pHs are shown in Fig. 1. All CFU numbers rapidly increased up to day 8 and then increased more slowly up to about day 16, followed by a decline to day 40 (Fig. 1A). At pHs 5.8 and 6.6, the numbers of CFU/ml were higher than those at pHs 7.0 and 7.4, and at pH 7.6, the numbers of CFU/ml were the lowest. The optical densities at all pHs increased up to about day 16, followed by a stabilization up to day 40 (Fig. 1B). The pHs of pH 5.8 and 6.6 culture media slightly increased, while the pHs of pH 7.0 and 7.4 culture media slightly decreased (Fig. 1C). The pH of pH 7.6 culture medium decreased by 0.24 from day 0 to 40. On the basis of these observations, we decided to test drug activity against NR cells using hypoxic 12-day-old (H12) and 19-day-old (H19) cells at pHs 5.8 and 7.3, obtained from pH 5.8 and 7.4 cultures, respectively.

FIG 1.

Growth and pH values of nonreplicating (NR) M. tuberculosis H37Rv cultures in Dubos-Tween-albumin broth adjusted at pHs 5.8, 6.6, 7.0, 7.4, and 7.6, respectively. Results are shown as mean (standard deviation) log₁₀ CFU/ml (A), optical density at 600 nm (OD₆₀₀) (B), and pH values (C) from two experiments.

The activities of 12 drugs against H12 and H19 NR cells on days 7, 14, and 21 are shown in Fig. 2. On day 21, H12 cells at pH 5.8 (Fig. 2A) were efficiently killed by RIF and RFP (≥6.2- and 5.3-log₁₀-CFU reductions, respectively). The log₁₀-CFU reductions for the other drugs were 3.6 (CL), 3.0 (BQ), 2.2 (PA), 2.1 (NZ), 1.3 (NC), 1.0 (PZA), 0.9 (MX), 0.6 (TH), and 0.2 (AK and INH) (Fig. 2A and C). The CFU reductions of H19 cells at pH 5.8 were higher than those of H12 cells for most drugs (Fig. 2B and D).

FIG 2.

Survival of nonreplicating (NR) cultures of M. tuberculosis after 0, 7, 14, and 21 days of exposure to drugs, as estimated by CFU counts. Twelve-day-old and 19-day-old hypoxic (H12 and H19, respectively) cultures at pH 5.8 (A to D) and pH 7.3 (E to H) were incubated with drugs. Ctrl, control; RIF, rifampin; RFP, rifapentine; BQ, bedaquiline; PA, PA-824; CL, clofazimine; NZ, nitazoxanide; INH, isoniazid; AK, amikacin; MX, moxifloxacin; NC, niclosamide; TH, thioridazine; PZA, pyrazinamide. The drug concentrations used were 8, 10, 1, 2, 1, 10, 2, 8, 4, 0.3, 0.5, and 100 μg/ml, respectively. Dashed lines indicate the limit of detection (5 CFU/ml). Means and standard deviations of values obtained from two experiments are shown.

H12 cells at pH 7.3 were efficiently killed by RIF and RFP (day 21, ≥5.2-log₁₀-CFU reduction) but not by CL, BQ, NC, PZA, TH, or INH (7 to 21 days, ≤0.4-log₁₀-CFU reduction) (Fig. 2E and G); MX, AK, PA, and NZ were slightly active (7 to 21 days, 0.7 to 1.1 log₁₀-CFU reduction). H19 cells at pH 7.3 were efficiently killed by RIF and RFP (day 21, ≥5.2-log₁₀-CFU reduction) but not by BQ, NC, PZA, MX, TH, AK, or INH (7 to 21 days, ≤0.4-log₁₀-CFU reduction) (Fig. 2F and H); PA, NZ, and CL were slightly active (7 to 21 days, 0.7 to 0.9-log₁₀-CFU reduction).

Overall, lipophilic RIF and RFP (logP 3.85 and 4.83, respectively) (see https://www.drugbank.ca/) were very active against dormant H12 and H19 cells, irrespective of acidic (pH 5.8) or neutral (pH 7.3) conditions. As we previously observed (11), at pH 5.8, lipophilic drugs (CL, BQ, PA, NZ, NC, and TH, logP ≥ 2.14) were usually more active against NR cells than hydrophilic drugs (PZA, MX, AK, and INH, logP ≤ 0.01). In contrast, under conditions mimicking caseous granulomas (hypoxia at pH 7.3), only the rifamycins RIF and RFP rapidly killed NR M. tuberculosis, while all other lipophilic and hydrophilic drugs tested had no or little effect. RIF is known to accumulate and maintain therapeutic levels in caseum, where dormant M. tuberculosis resides (12, 13), and a 3-month treatment of latent TB with RFP and INH was found to be noninferior to 9 months of INH treatment alone (3, 14). Overall, our Wayne model experiment in hypoxia at pH 7.3 mimicking caseum may be important for testing the activity of new drugs and/or combinations against NR M. tuberculosis and guiding the selection of future therapies inhibiting the reactivation of latent TB to active TB.