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.KEYWORDS Mycobacterium tuberculosis, dormancy, hypoxia, killing, pH, rifampin, rifapentine, tuberculosis T he 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

Niclosamide: Beyond an antihelminthic drug

“…skin or intestinal tuberculosis infections [57] . Subsequently, a number of research groups have reported the ability of niclosamide and related salicylanilide derivatives to inhibit the growth of M. tuberculosis and reported the effect of pH on growth inhibition [58] , [59] , [60] .…”

The Combination Rifampin-Nitazoxanide, but Not Rifampin-Isoniazid-Pyrazinamide-Ethambutol, Kills Dormant Mycobacterium tuberculosis in Hypoxia at Neutral pH

“…Briefly, for preparation of NR, hypoxic (H) cells, M. tuberculosis H37Rv log-phase cultures were diluted in Dubos-Tween-albumin broth (DTAB) adjusted to pH 7.3 (10,11) and incubated at 37°C for 12 days (H12) in 20-by 125-mm stirred tubes (120 rpm) containing 16 ml of culture (0.5 headspace ratio) (9) with the caps tightly screwed and tight rubber caps put under the screw caps. Control tubes with 1.5 g/ml of methylene blue as an indicator of oxygen depletion were also incubated.…”

“…Noticeable decolorization of methylene blue was observed around day 12. Drugs were added by syringe to H12 cultures at their maximum concentration of drug in serum (C max ) (g/ml) (10,12,13) as follows: R, 8; I, 2; E, 4; PA-824 (PA), 2; nitazoxanide (NZ), 10; sutezolid (SZ), 1. Pyrazinamide was used at 100 g/ml.…”

Drug-Resistant Tuberculosis 2020: Where We Stand

“…NRP-2 cells developed a thickened outer layer that helped in restricting RIF entry [85]. Our group used the Wayne model at different pHs: pH 6.6, the pH or culture media [86], pH 5.8, to mimic the environment of cellular granulomas [87], pH 7.3, to mimic the environment of caseous granulomas [88]. We found that at pH 5.8, several drugs killed NR bacilli, with the best being the rifamycins RIF and rifapentine (RFP), while at pH 7.3, only RIF and RFP killed dormant bacilli out of 12 drugs tested [88].…”

“…Our group used the Wayne model at different pHs: pH 6.6, the pH or culture media [86], pH 5.8, to mimic the environment of cellular granulomas [87], pH 7.3, to mimic the environment of caseous granulomas [88]. We found that at pH 5.8, several drugs killed NR bacilli, with the best being the rifamycins RIF and rifapentine (RFP), while at pH 7.3, only RIF and RFP killed dormant bacilli out of 12 drugs tested [88]. Since the rifamycins were the only agents sterilizing caseum obtained from rabbits [35,36], our model could mimic caseum to measure drug activity against NR Mtb in this environment.…”