Reduced drug uptake in phenotypically resistant nutrient-starved nonreplicating Mycobacterium tuberculosis

Abstract

During active tuberculosis a spectrum of physiologically different Mycobacterium tuberculosis bacilli reside in human tissues. Subpopulations of the pathogen survive antibiotic treatment for a prolonged time in a dormant state of phenotypic drug resistance, a phenomenon independent of genetic mutations. Here, we used an established culture model of nutrient deprivation to shift down M. tuberculosis from growth to nonreplicating survival, which is characterized by a drastic loss of drug susceptibility. Liquid chromatography coupled with mass spectrometry techniques were employed to quantify drug penetration in replicating and nutrient-starved nonreplicating bacilli. We found that intracellular concentrations of fluoroquinolones, rifamycins, and linezolid were lower in nonreplicating M. tuberculosis. Studies with pump inhibitors suggest that the observed differences were independent of efflux processes. We conclude that decreased drug permeability contributes to phenotypic drug resistance of dormant M. tuberculosis.

Fig 1

Intracellular amounts of antituberculosis agents in exponentially growing and nutrient-starved nonreplicating M. tuberculosis (Mtb) following a 30-min incubation period at 10 μM. The drug content is expressed as the amount of drug per CFU. (Inset) Data for thioridazine. The experiments were done in biological triplicates. Standard deviations are shown as error bars. MXF, moxifloxacin; OFX, ofloxacin; LVX, levofloxacin; RIF, rifampin; RIB, rifabutin; RIP, rifapentine; EMB, ethambutol; MEF, mefloquine; TRZ, thioridazine; LNZ, linezolid. *, P < 0.05; ** P < 0.01; *** P < 0.005; ns, not significant.

Fig 2

Effects of efflux pump inhibitors verapamil (VER) (75 μM) and reserpine (RES) (20 μM) on ofloxacin (OFX) (A) and rifampin (RIF) (B) accumulation in replicating and nonreplicating M. tuberculosis. Relative drug accumulation is expressed as percentages of average uninhibited accumulation in the replicating bacteria. Data from one representative experiment out of three are shown as means and standard deviations (n = 3).

Fig 3

Intracellular ofloxacin (OFX) contents of replicating and nonreplicating M. tuberculosis measured at regular time intervals over a 30-min incubation period. Drug content is expressed as the amount of drug per CFU. Data from one representative experiment out of two are shown as means and standard deviations of two measurements, from which curves were calculated by nonlinear fit.