Eradication of bacterial persisters with antibiotic-generated hydroxyl radicals

Abstract

During Mycobacterium tuberculosis infection, a population of bacteria likely becomes refractory to antibiotic killing in the absence of genotypic resistance, making treatment challenging. We describe an in vitro model capable of yielding a phenotypically antibiotic-tolerant subpopulation of cells, often called persisters, within populations of Mycobacterium smegmatis and M. tuberculosis. We find that persisters are distinct from the larger antibiotic-susceptible population, as a small drop in dissolved oxygen (DO) saturation (20%) allows for their survival in the face of bactericidal antibiotics. In contrast, if high levels of DO are maintained, all cells succumb, sterilizing the culture. With increasing evidence that bactericidal antibiotics induce cell death through the production of reactive oxygen species (ROS), we hypothesized that the drop in DO decreases the concentration of ROS, thereby facilitating persister survival, and maintenance of high DO yields sufficient ROS to kill persisters. Consistent with this hypothesis, the hydroxyl-radical scavenger thiourea, when added to M. smegmatis cultures maintained at high DO levels, rescues the persister population. Conversely, the antibiotic clofazimine, which increases ROS via an NADH-dependent redox cycling pathway, successfully eradicates the persister population. Recent work suggests that environmentally induced antibiotic tolerance of bulk populations may result from enhanced antioxidant capabilities. We now show that the small persister subpopulation within a larger antibiotic-susceptible population also shows differential susceptibility to antibiotic-induced hydroxyl radicals. Furthermore, we show that stimulating ROS production can eradicate persisters, thus providing a potential strategy to managing persistent infections.

Fig. 1.

A system for identifying persister cells. (A) Kill kinetics for M. smegmatis cultures treated with CIP and INH or RIF and INH. (B) Similar killing followed by a stabilization of bacterial numbers is observed in M. tuberculosis cultures treated with CIP and INH or RIF and INH. (C) The M. smegmatis persister population generated with CIP and INH (dashed line, squares) is cross-tolerant to the addition of RIF added at a bactericidal concentration of 30 μg/mL (solid line, circles). (D) The M. tuberculosis persister population generated with CIP and INH (dashed line, squares) is cross-tolerant to the addition of RIF, added at day 7 to a bactericidal concentration of 0.1 μg /mL (solid line, circles).

Fig. 2.

M. smegmatis and M. tuberculosis persister populations are not observed in maximally aerobic conditions. (A) M. smegmatis cells treated with CIP and INH are steadily killed in open, aerated flasks (dashed line) and those in closed, septum-sealed flasks stabilize at 0.01% of the population (solid line). (B) Similar behavior is observed for M. smegmatis exposed to RIF and INH. (C) M. tuberculosis cells treated with OFX and INH exhibit biphasic killing with continued death of cells in aerated flasks (dashed line) and those in septum sealed flasks stabilize (solid line). (D) Similar behavior is observed for M. tuberculosis exposed to RIF and INH.

Fig. 3.

Increased availability to oxygen allows killing of persister cells. (A) M. smegmatis stationary phase cultures treated in open, aerobic conditions (dashed line) lose more than five logs of viability over three weeks, while cultures maintained in closed, septum-sealed flasks (solid line) during antibiotic treatment lose only two logs of viability. (B) Dissolved-oxygen saturation remains greater than 90% in open, aerated flasks (dashed line) while closed, septum-sealed flasks (solid line) become moderately depleted in oxygen, with DO saturation dropping to 75%. (C) An established M. smegmatis persister population in a closed, septum-sealed flask is transferred to open, aerated conditions at day 3. After transfer the persister population immediately begins losing viability.

Fig. 4.

Preadaptation to reduced DO levels does not enhance persister cell formation or survival. (A) The DO saturation of control septum-sealed flasks (dashed line) or nitrogen-injected septum-sealed flasks (solid line) containing M. smegmatis cells treated with CIP and INH. Data points represent the average of eight replicates. Error bars represent SDs. (B) M. smegmatis cells in control septum-sealed flasks (dashed line, circles) or nitrogen-injected septum-sealed flasks (solid line, squares) are treated with CIP and INH. Similar kill kinetics and persister population size are observed.

Fig. 5.

Modulating the free-radical concentration affects the number of persisters identified. (A) M. smegmatis cells treated with CIP and INH in open, aerated conditions in the presence (dashed line) or absence (solid line) of the free-radical quencher thiourea at a concentration of 150 mM. The presence of thiourea restores a biphasic kill kinetic under open aerobic conditions and a persister population is again identified. (B) The M. smegmatis persister population identified after antibiotic treatment with CIP and INH is killed with the addition of CFZ at day 3.