Eradication of bacterial persisters with
antibiotic-generated hydroxyl radicals
Sarah Schmidt Granta,b,c,1, Benjamin B. Kaufmanna,c,d,1, Nikhilesh S. Chandd,e, Nathan Haseleya,d,f,
and Deborah T. Hunga,b,c,d,2
aBroad Institute of MIT and Harvard, Cambridge, MA 02142;bDivision of Pulmonary and Critical Care Medicine, Department of Medicine, Brigham and
Women’s Hospital, Boston, MA 02114;cDepartment of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General
Hospital, Boston, MA 02114;dDepartment of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115;eDepartment of Molecular and
Cellular Biology, Harvard University, Cambridge, MA 02138; andfHarvard–MIT Division of Health Sciences and Technology, Cambridge, MA 02139
Edited by* Eric S. Lander, Broad Institute of MIT and Harvard, Cambridge, MA, and approved June 11, 2012 (received for review March 2, 2012)
During Mycobacterium tuberculosis infection, a population of bac-
teria 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 antibi-
otic-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-suscepti-
ble population, as a small drop in dissolved oxygen (DO) satura-
tion (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 bac-
tericidal 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 per-
sister 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 eradi-
cates the persister population. Recent work suggests that environ-
mentally induced antibiotic tolerance of bulk populations may
result from enhanced antioxidant capabilities. We now show that
the small persister subpopulation within a larger antibiotic-suscep-
tible 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.
losis remains a leading infectious cause of death globally (1). In
2009, 1.7 million people died from tuberculosis (TB), with the
majority of deaths occurring in the developing world (2). Suc-
cessful treatment of active, symptomatic TB requires a minimum
of 6 mo of combination therapy (typically four drugs) and is
frequently complicated by drug toxicities (3). Treating clinically
asymptomatic, latent infections, during which bacteria evade the
host immune system and persist in infected individuals for dec-
ades, requires an even longer treatment course, typically 9 mo.
Many patients infected with M. tuberculosis, both in the United
States and globally, are thus unable to comply with the complex
currently recommended therapeutic regimens (4). The rise of
drug-resistant infection around the world further complicates
successful TB treatment. It is estimated that 3.3% of new cases
of TB are resistant to multiple standard TB drugs (2).
During both active and latent infection, it has been proposed
that a subpopulation of bacteria enters a reversible nonreplicating
state, refractory to traditional antibiotics (5). This phenomenon
is characterized by the reduced efficacy of antibiotics against
nonreplicating or slowly replicating bacteria in the absence of
genotypic resistance (5–7). In vivo it has been observed when
mice are infected with M. tuberculosis, and subsequently treated
with antibiotics. In this infection model, antibiotics reduce bac-
nfection with the bacterial pathogen Mycobacterium tubercu-
terial cell numbers but do not sterilize the mouse (8). A plateau
is typically reached during which numbers of viable bacteria
stabilize. In addition to the mouse infection model, the inability
to sterilize has been observed in the zebra fish (Mycobacterium
marinum), guinea pig (M. tuberculosis), and macrophage
(M. tuberculosis) infection models (9–11). In vitro, the survival
of a similar small subpopulation can also be observed when
a culture is exposed to high doses of antibiotics (12, 13). The
surviving cells, often called persisters (14), retain genetic sus-
ceptibility to the antibiotic. The ability of M. tuberculosis to enter
this physiological state, where intact cells lie dormant and survive
despite exposure to bactericidal concentrations of antibiotics,
may contribute to the need for long and complex treatment
regimens to eradicate TB infection.
Many other human pathogens, including Escherichia coli,
Staphylococcus aureus, and Pseudomonas aeruginosa, are simi-
larly able to enter a drug-refractory state (14–17). Work in E. coli
mutants has suggested that, in a bacterial population, persisters
exist before antibiotic exposure (18). In contrast, there is also
evidence to suggest that antibiotic tolerance can be induced in
response to cellular stresses, including antibiotic treatment. For
example, a small fraction of a population of E. coli, when faced
with increasing levels of antibiotic, secretes the signaling mole-
cule indole to induce transcriptional changes in neighboring
cells, most notably in the upregulation of efflux pumps and oxi-
dative stress protective mechanisms, resulting in antibiotic tol-
erance (19). Additionally, induction of the SOS response in
E. coli has been shown to induce both β-lactam and fluo-
roquinolone antibiotic tolerance (20–22). Recent transcriptome
profiling of persisters also revealed that several stress response
regulons, including the SOS response, as well as several toxin–
antitoxin (TA) genes, are upregulated in persister populations in
E. coli (15) and M. tuberculosis (12). The mechanism by which
TA loci may induce persistence in E. coli was recently described.
Many toxin genes, activated by degradation of the cognate an-
titoxin, encode mRNases that rapidly degrade mRNA, stopping
translation and inducing antibiotic tolerance (23).
Recent work has identified reactive oxygen species as an im-
portant antibiotic-induced cellular stress. These studies demon-
strate that bactericidal antibiotics with a variety of different
mechanisms of action increase ROS production within cells via
the Fenton reaction (24–27). Numerous ROS, and in particular
hydroxyl radicals, are toxic to cells and can result in cell death.
Author contributions: S.S.G., B.B.K., N.S.C., N.H., and D.T.H. designed research; S.S.G.,
B.B.K., N.S.C., and N.H. performed research; S.S.G. and B.B.K. analyzed data; and S.S.G.,
B.B.K., and D.T.H. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1S.S.G. and B.B.K. contributed equally to this work.
2To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| July 24, 2012
| vol. 109
| no. 30
Tolerance to antibiotics may therefore depend on the ability of
the cell to defend itself against ROS, as suggested by several
recent studies (28–30). For example, the coordinated stringent
response to nutrient limitation in P. aeruginosa and E. coli was
shown to increase antioxidant enzyme expression and decrease
production of prooxidant molecules, resulting in antibiotic tol-
erance (28). Bacteria also produce nitric oxide (NO) as well as
hydrogen sulfide (H2S), both of which result in antibiotic toler-
ance via suppression of the Fenton reaction as well as increased
antioxidant enzyme expression in both Gram-positive and Gram-
negative bacteria (29, 30).
One challenge in studying antibiotic-tolerant persister cell
physiology has been the difficulty in reproducibly generating and
isolating this small subpopulation from the much larger antibi-
otic-susceptible population. In this work, we describe a model
capable of reliably producing a subpopulation of persisters in
both M. tuberculosis and the nonpathogenic model organism
Mycobacterium smegmatis. We used this model to examine fac-
tors important for the formation and survival of the persister
population. We demonstrate that persister cells constitute a dis-
tinct subpopulation within the larger culture population. We
found that the survival of persisters requires a small (20%) drop
in dissolved-oxygen (DO) saturation. If the DO concentration is
maintained at high levels, this population is killed over time. We
demonstrate that the drop in oxygen saturation does not affect
the survival of the dominant antibiotic-susceptible population,
nor does the drop in DO signal persister cell formation. We
hypothesize that high DO conditions may increase levels of ROS
generated during antibiotic treatment, leading to killing of per-
sisters. Supporting this theory we found that the hydroxyl-radical
scavenger thiourea protects the persister subpopulation even
when DO is maintained at high levels. Conversely, we used the
antibiotic clofazimine (CFZ) to induce ROS generation and
found that CFZ can successfully eradicate the persister pop-
ulation. Taken together these data suggest that the persister
subpopulation has differential susceptibility to antibiotic-induced
hydroxyl radicals compared with the larger, antibiotic-susceptible
population. There is growing evidence for the critical role of
ROS and oxidative damage in antibiotic tolerance in other
populations that are refractory to antibiotic killing, and in this
work we demonstrate the important role of ROS in a stochastic
persister population model.
We initially sought to detect a subpopulation of persisters in
a larger population of susceptible, logarithmically growing
mycobacteria. Cultures of M. smegmatis were grown to stationary
phase and then diluted in fresh media roughly 15-fold to an
optical density (OD600) of 0.2. Cells were next exposed to pairs of
bactericidal antibiotics with different modes of action to prevent
the emergence of genetically resistant clones. For example, the
DNA gyrase inhibitor ciprofloxacin (CIP) at a concentration well
above the minimum inhibitory concentration (MIC) was paired
with a bacteriostatic concentration of the cell-wall biosynthesis
inhibitor isoniazid (INH). M. smegmatis bacilli treated in this
manner show approximately four logs of killing over the first 24 h
but exhibit no further reduction in cell number during the fol-
lowing seven days (Fig. 1A and Fig. S1A). We did not observe
significant differences in the mean number of persisters identified
when CIP was used alone versus in combination with low-dose
INH (Fig. S1B). Similarly, treated M. tuberculosis bacilli show
three logs of killing over the first four days before stabilization in
cell number is achieved (Fig. 1B). Along with surviving in the
antibiotic in which they were initially challenged, M. smegmatis
and M. tuberculosis persisters also demonstrated tolerance to
subsequent challenges with bactericidal levels of rifampicin
(RIF) or streptomycin (STM), antibiotics with completely un-
related mechanisms of action (Fig. 1 C and D and Fig. S1C).
During these initial experiments to characterize the persister
subpopulation, we observed substantial experiment-to-experi-
ment variability in the size of the final persister population. In
particular we noted that perturbation of the culture during
sampling reduced the size of the persister population compared
with undisturbed cultures (Fig. S2). This observation led us to
hypothesize that the sampling process, which required removing
a foil cap, thus introducing ambient air into the flask headspace,
may increase the DO saturation of the culture, thereby affecting
the number of persisters identified. To test this hypothesis, we
characterized the M. smegmatis persister population in two
controlled conditions in which oxygen saturations would not be
affected by sampling. In the first system, a highly aerobic con-
dition that we will refer to as the “open system,” an oxygen-
permeable membrane was used to cover a shaking baffled
Erlenmeyer flask, increasing the oxygen transfer rate into the
media and resulting in an equilibrium state of high DO. [Note:
At 37 °C, the maximum dissolved oxygen in water is ∼6.7 mg/L
(31)]. In the second system, the sealed or “closed system” con-
dition, a rubber septum was used to seal a flat-bottom Erlen-
meyer flask (headspace/media ratio 5:1), thus preventing
ongoing oxygen diffusion from the atmosphere. Consequently,
oxygen levels within the flask drop as cell metabolism consumes
oxygen within the media and headspace. In both models, a nee-
dle and syringe were used to sample the cultures for bacterial
enumeration to minimize perturbation of the systems. In the
closed system model, we found that for RIF/INH, CIP/INH, and
STM/INH, the number of viable M. smegmatis cells stabilized
two days after the initial kill in a biphasic manner, as we had
previously observed. In contrast, bacilli in the continuously aer-
ated system continued to die (Fig. 2 A and B and Fig. S3).
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 per-
sister population generated with CIP and INH (dashed line, squares) is cross-
tolerant to the addition of RIF, added at day 7 to a bactericidal concentra-
tion of 0.1 μg /mL (solid line, circles).
A system for identifying persister cells. (A) Kill kinetics for M.
| www.pnas.org/cgi/doi/10.1073/pnas.1203735109Grant et al.
To understand whether M. tuberculosis cultures were similarly
sensitive to the DO system, we treated cells in tightly capped
square media bottles with either a headspace ratio of 4:1,
designed to approximate septum-sealed conditions, or a com-
paratively aerated 60:1 ratio. Similar to what we had observed
with M. smegmatis, in M. tuberculosis the reduced oxygen con-
dition exhibited slower killing and a stable persister population,
in contrast to the more aerated condition where the number of
survivors steadily declined (Fig. 2 C and D). To determine
whether the functional relationship between DO and antibiotic
susceptibility of persisters is specific to mycobacteria we tested
the Gram-negative pathogen P. aeruginosa as well as E. coli using
our model system (SI Materials and Methods). We found, similar
to what was observed in mycobacteria, that fewer cells survive
antibiotic treatment in the maximally aerated conditions com-
pared with the septum-sealed conditions (Fig. S4). These results
demonstrate that in vitro models used to identify persister pop-
ulations are extremely sensitive to changes in oxygen tension. In
addition, these observations suggest that high levels of DO may
either prevent the formation or interfere with the maintenance
of the persister population.
In stationary phase a subpopulation of bacteria also exhibit
phenotypic antibiotic tolerance, thought to be due to nutrient
exhaustion and changes in the pH of the media (5, 32). Given
our finding that persisters identified within a freshly diluted
culture are sensitive to antibiotic killing in the presence of suf-
ficiently high DO levels, we next sought to determine if the an-
tibiotic-tolerant population in a fully stationary phase, undiluted
culture also becomes susceptible to antibiotic treatment when
placed in highly aerated conditions. A culture of M. smegmatis
was grown to stationary phase, transferred into either closed or
open conditions, and then treated with bactericidal concen-
trations of CIP and INH. Over three weeks of treatment, sta-
tionary phase cultures in the closed system exhibited only
a modest two-log drop in census, similar to what has been
reported previously for stationary phase cultures (33). In con-
trast, the treatment of stationary phase cultures in the open
system resulted in a reduction in census of more than five orders
of magnitude to the lower limit of detection. Based on the ob-
servation that in a stationary phase culture the antibiotic-tolerant
subpopulation loses antibiotic tolerance when oxygen levels are
sufficiently increased, we conclude that tolerance is not due solely
to nutrient limitation or changes in pH (Fig. 3A).
Hypoxia has previously been shown to play a role in inducing
antibiotic tolerance in M. tuberculosis (34). In the most-studied
hypoxia model, commonly referred to as the Wayne model, an-
tibiotic tolerance in the bulk population appears as cells adapt to
microaerophilic (DO saturation ∼ 1%) and anaerobic (DO sat-
uration ≤ 0.06%) conditions. To determine the degree of oxygen
depletion in the experiments described above (Fig. 2) we mea-
sured the dissolved-oxygen saturation in both the open and closed
models. To our surprise we found only a 20% difference in DO
saturation between the two conditions. In the closed system, the
oxygen saturation fell over three days before stabilizing at 75%,
but in the open system, oxygen saturations equilibrated around
95% (Fig. 3B). Thus, in contrast to the previously described
Wayne model, our data demonstrate that very small decreases in
DO are sufficient to promote bacterial survival, albeit of a small
persister subpopulation, in the presence of bactericidal antibiotic
concentrations. We next tested whether decreasing the inoculum,
which should decrease the rate of oxygen consumption and
therefore lead to higher DO saturation, would decrease the size
of the persister subpopulation. Indeed, we found that with a 10-
fold lower starting inoculum, we did not identify a stable persister
population above the level of detection (Fig. S5), perhaps in part
explaining the “inoculum effect” (35). To further confirm the
effect that increasing oxygen levels has on the ability of the
antibiotics to kill the persister population, we transferred M.
smegmatis persisters generated in the closed system to the open
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.
M. smegmatis and M. tuberculosis persister populations are not
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 satu-
ration 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.
Increased availability to oxygen
Grant et al. PNAS
| July 24, 2012
| vol. 109
| no. 30
system. This transfer into aerated conditions led to the sub-
sequent death of persisters (Fig. 3C), thus demonstrating that
persister cells are in fact vulnerable or can become vulnerable to
antibiotics under conditions of higher DO saturation.
The observation that a 20% drop in DO saturation, the result
of metabolic oxygen consumption, results in a stable persister
population suggests that the reduction in oxygen saturation ei-
ther stimulates persister formation or facilitates the survival of
a preexisting bacterial population alive at the time of oxygen
depletion. To differentiate between these scenarios, we tested
whether preadapting a population of cells to lower DO levels
might result in a higher percentage of persisters or even induce
antibiotic tolerance in the entire population. We used gas dis-
placement with nitrogen gas to preadapt cultures to 75% DO
saturation for one hour before treating with high concentrations
of CIP and INH (Fig. 4A). Following antibiotic treatment the
oxygen-displaced flasks fell from 75% DO saturation to 60% DO
saturation over the next four days, while the control flasks
dropped from 100 to 80% oxygen saturation (Fig. 4A). Despite
these differences in DO, the kill kinetics and mean size of the
persister populations for the oxygen-displaced flasks were sta-
tistically indistinguishable from the control flasks seven days af-
ter treatment (Fig. 4B, t test P value ∼ 0.67). These data suggest
that reduced oxygen levels are not driving increased persister cell
formation before antibiotic exposure but instead are facilitating
the survival of persisters. Furthermore, because the reduction of
the DO level by nitrogen displacement did not change the initial
first phase of killing compared with nondisplaced cultures, the
20% drop in DO saturation appears to have no effect on the
survival of the dominant antibiotic-susceptible population. We
therefore conclude that within the bulk culture there is a bacte-
rial subpopulation (which forms the second-phase plateau)
whose sensitivity to subtle changes in oxygen when challenged
with antibiotics differs from the majority of the population.
This observation, combined with recent work demonstrating
both the role of ROS in antibiotic killing and cellular antioxidant
responses in antibiotic tolerance, led us to speculate that the
sensitivity of the persister population to changes in oxygen levels
may be linked to changes in the number of toxic free radicals
generated with antibiotic treatment. Exposure to bactericidal
antibiotics has been found to result in the intracellular genera-
tion of toxic free radicals, resulting in bacterial cell death. Oxy-
gen diffuses freely across cell membranes (36), resulting in an
intracellular concentration in equilibrium with that of the sur-
rounding media, with previous measurements demonstrating
that reactive oxygen species are generated in direct proportion to
oxygen concentration (36, 37). The intracellular free-radical
concentration following antibiotic exposure is therefore pre-
dicted to be higher in the open model compared with the closed
model. To confirm this hypothesis we used the fluorescent re-
porter dye 3′-(p-hydroxy-phenyl) fluorescein (HPF) to measure
relative hydroxyl-radical levels in norfloxacin-treated E. coli cells
in maximally aerated media compared with media with a DO
level of 75%. As expected, we found higher levels of hydroxyl
radicals in maximally aerated media (P value below 0.0002, Fig.
S6, SI Materials and Methods).
To test the role of free radicals in the killing of both the pre-
dominant antibiotic-susceptible population and in the smaller
persister subpopulation, we blocked the effects of hydroxyl rad-
icals after CIP treatment using the free-radical quencher thio-
urea (24) in the aerated, open system. We found that in high DO
conditions, thiourea successfully restored the biphasic kill kinetic
and an antibiotic-tolerant population was again identified (Fig.
5A). We also observed a reduced kill kinetic for the larger an-
tibiotic-susceptible population, consistent with earlier work
demonstrating the role of ROS in antibiotic killing in E. coli (24).
From these data we conclude that under high DO conditions, the
persister population is killed following antibiotic treatment as
a consequence of an increased number of toxic hydroxyl radicals
generated with antibiotic treatment.
We conversely hypothesized that increasing hydroxyl radicals
within cells could successfully eradicate the persister population
in the closed system. We used the antibiotic clofazimine, which
results in increased intracellular ROS production due to redox
cycling (38). CFZ, recently identified as a competing substrate
for the enzyme NDH-2 within the electron transport chain, is
reduced by NDH-2 and subsequently oxidized by O2, resulting in
increased ROS (38). When we challenged persisters with CFZ,
the number of surviving persisters decreased by three orders of
magnitude, contrasting with the tolerance observed for rifampin
and streptomycin (Fig. 5B). In the presence of thiourea the ad-
dition of CFZ did not change the number of persisters in a sta-
tistically significant manner, suggesting that CFZ is killing the
persister population through increased intracellular ROS gen-
eration (Fig. S7, t test P value ∼0.42). These data, combined with
the nitrogen displacement data demonstrating that subtle
changes in oxygen saturation do not affect the survival of the
dominant antibiotic-susceptible population, suggest a model in
which persisters respond differently to antibiotic-induced hy-
droxyl radicals compared with the larger antibiotic-susceptible
population, except under maximal DO conditions.
formation or survival. (A) The DO saturation of control septum-sealed flasks
(dashed line) or nitrogen-injected septum-sealed flasks (solid line) contain-
ing 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.
Preadaptation to reduced DO levels does not enhance persister cell
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 con-
ditions 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.
Modulating the free-radical concentration affects the number of
| www.pnas.org/cgi/doi/10.1073/pnas.1203735109Grant et al.
The important role that oxygen plays in antibiotic killing of
bacteria has also long been recognized, bolstered by observations
that in abundance it can improve antibiotic efficacy, and when
absent can result in complete antibiotic tolerance (24, 34). We
now find that much smaller changes in oxygen tension than
previously described affect the antibiotic killing of bacterial
persisters. Although these small reductions in oxygen tension do
not alter the kill kinetic of the larger antibiotic-susceptible
population, these changes dramatically affect the size and sur-
vival ability of the smaller persister subpopulation. In an envi-
ronment where dissolved oxygen saturations are high, this
subpopulation rapidly dies and can no longer be identified. We
observe the same relationship between oxygen and antibiotic
sensitivity in Gram-negative bacteria (P. aeruginosa and E. coli),
which demonstrates that this phenomenon is not unique to
mycobacteria. These results suggest that the persister sub-
population differs from the larger population in its ability to
withstand hydroxyl radicals generated by antibiotic challenge.
Previous work in E. coli has demonstrated that antibiotic killing
in susceptible populations is mediated by hydroxyl radicals (24),
in part through oxidation of the guanine nucleotide pool (39).
With the addition of thiourea we find that the kill kinetic of the
bulk population is reduced, suggesting that hydroxyl radicals may
similarly mediate antibiotic killing in mycobacteria and that
persisters may survive antibiotic challenge due to a unique ca-
pacity to withstand the generation of toxic free radicals. This
ability of persisters to survive when the DO level is reduced is in
contrast to the larger, susceptible population. When DO levels
are near saturation, however, the protective mechanisms in
persisters are insufficient and they succumb to antibiotic chal-
lenge. When decreased free-radical concentrations are achieved
either through a reduction in oxygen saturation or the addition
of thiourea, a free-radical quencher, the drug-tolerant persister
subpopulation is able to survive in the presence of otherwise
bactericidal antibiotic concentrations. In contrast, strategies to
increase cellular ROS, such as the addition of the antibiotic
CFZ, results in killing of the drug-tolerant population. Antibi-
otic-generated hydroxyl-radical–mediated killing may therefore
represent an Achilles’ heel of persistence.
The impact small changes in oxygen tension have on the
maintenance and survival of persisters may explain incon-
sistencies within the M. tuberculosis persister literature. Recently,
two studies were published with conflicting results concerning
the enrichment of drug-tolerant persisters in late exponential
and stationary phase cultures (12, 13), with one identifying an
enrichment of persisters in these cultures while the other did not.
Similarly, there are discrepancies regarding the cross-tolerance
of M. tuberculosis persisters to heterologous drugs. Based on the
data presented here, the origin of these divergent observations
may lie in differing culture densities, which impacts the rate of
oxygen depletion and oxygen saturation at the time of treatment,
thereby changing antibiotic efficacy.
Within its human host, M. tuberculosis sometimes survives
within low-oxygen physical niches, which may further protect M.
tuberculosis from hydroxyl-radical-mediated antibiotic killing.
The most extreme example is the granuloma, a collection of M.
tuberculosis-infected macrophages and immune cells, which was
recently demonstrated to possess a hypoxic core with an oxygen
tension of 3 mm Hg (40). Because phenotypic drug tolerance is
observed in vitro under hypoxic conditions, it has been suggested
that standard TB therapy is likely ineffective in the granuloma.
This work suggests that much more subtle changes in oxygen
tension may dramatically reduce antibiotic efficacy with the goal
of sterilization. During in vivo infections, M. tuberculosis likely
encounters a gradient of oxygen tensions in tissue that range
from 100 mm Hg in the alveolus to 60 mm Hg in normal lung
tissue to 3 mm Hg in the center of a granuloma (40, 41). The
data presented here suggest that effective sterilization of M. tu-
berculosis may be limited by the gradual decline in oxygen ten-
sion even before hypoxic conditions are obtained.
There is a growing body of evidence demonstrating the im-
portant role that ROS play in bactericidal antibiotic–mediated
killing (24–26). This understanding of how bactericidal antibiotics
result in cell death raises the hypothesis that drug tolerance may
be mediated by increased abilities within a cell to detoxify ROS.
Recent work has suggested that antibiotic tolerance of the bulk
population, induced by changes in the environment, specifically
starvation, may be the result of enhanced antioxidant strategies
(28). Our work focuses on the small persister subpopulation
within a larger culture, and we demonstrate that this sub-
population is differentiated from the larger antibiotic-susceptible
population by different sensitivities to hydroxyl-radical-mediated
damage from antibiotic exposure. Our findings are supported by
recent work demonstrating that E. coli persisters produce fewer
hydroxyl radicals following antibiotic challenge, and a possible
mechanism is suggested by work demonstrating that E. coli
secretes the signaling molecule indole that activates oxidative
stress responses and increased drug tolerance (19, 42, 43). Al-
though the lack of oxygen in anaerobic or microaerophilic con-
ditions has long been known to affect the efficacy of antibiotics,
we demonstrate that just a 20% change in oxygen saturation
significantly affects antibiotic efficacy by allowing the survival of
a subpopulation of cells. At the same time, we show that this
subpopulation remains vulnerable to the common antibiotic-in-
duced hydroxyl-radical–mediated death pathway if sufficiently
high free-radical concentrations can be maintained, induced for
example by the antibiotic clofazimine. Previous efforts toward
eradicating persister populations have focused on identifying
small molecules that specifically kill this population, or which
potentiate antibiotic efficacy in this population (44, 45). Next-
generation approaches to antibiotic discovery may lie in identi-
fying small molecules that potentiate hydroxyl-radical formation
or subvert cellular mechanisms that protect against hydroxyl-
radical damage (38, 46). This work now argues that such
approaches may have the important added benefit of elimi-
nating persister populations, thus sterilizing an infection (46).
Materials and Methods
Bacterial Strains, Plasmids, and Culture Conditions. M. smegmatis was grown
at 37 °C in Middlebrook 7H9 broth supplemented with 10% ADS (albumin-
dextrose-saline, vol/vol), 0.2% glycerol, and 0.05% Tween-80. The M. smeg-
matis mc2155 strain expressing GFP contains an episomal pUV15tetORm de-
rivative in which the repressor tetR was removed, allowing constitutive GFP
expression (47). M. tuberculosis H37Rv was grown at 37 °C in Middlebrook 7H9
broth supplemented with 10% OADC (oleic acid-albumin-dextrose complex,
vol/vol), 0.2% glycerol, and 0.05% Tween-80, or on Middlebrook 7H10 plates
supplemented with 10% OADC enrichment. All drugs and antibiotics for this
study were from Sigma-Aldrich.
Antibiotic Killing Experiments in M. smegmatis. Freezer stocks of mc2155 were
diluted 1:2,000 in 7H9 medium and cultured for three days unless otherwise
specified. Cultures were then diluted ∼15-fold in prewarmed media to an
OD600of 0.2 and antibiotics added. Experiments were conducted in 125-mL
flat-bottom Erlenmeyer flasks covered with either a rubber septum (Sigma-
Aldrich) or 125-mL baffled Erlenmeyer flasks covered with an oxygen-per-
meable membrane (AeraSeal; Excel Scientific). At various time points 1 mL of
cells were collected using a 1-mL syringe connected to a 16-gauge needle.
The viability of cells is measured using a most probable number (MPN) assay
(48) when possible or plating for colony-forming units otherwise. In some
experiments, to increase sensitivity at low cell densities, cells were washed in
fresh media. For the MPN assay, cells were aliquoted into Costar 96-well
black clear-bottom plates and then serially diluted 10-fold. After 10 d, plates
were read in M5 SpectroMax plate reader using GFP fluorescence as the
readout. The MPN was calculated using 4–8 replicates (49). Unless otherwise
specified, antibiotics were used at the following concentrations: cipro-
floxacin 5 μg/mL, isoniazid 40 μg/mL, rifampin 30 μg/mL, streptomycin 2.5 μg/
Grant et al. PNAS
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mL, and clofazimine 40 μg/mL. For heterologous drug challenge, the second
antibiotic was diluted in 0.5 mL fresh media and injected on day 3 through
the septum. For the purposes of calculating the geometric mean and SD,
samples with measurements below the threshold of detection (30 cells/mL)
were assumed to contain 1 cell/mL All graphed data points in figures rep-
resent the average of three biologic replicates and the error bars represent
the SDs, except for the initial time point in MPN experiments, which reflects
a single measurement. Data in figures were fitted to single or double ex-
ponential decays plus an additive constant in cases where cell populations
plateaued. In some cases, fits were constrained to a constant before
decaying exponentially to mimic the delay in action of some drug combi-
nations or the delayed physical addition of a drug. When multiple conditions
came from a common starter culture, line fits were constrained to begin
with the same initial number of cells. Confidence intervals of fit parameters
were typically quite large, limiting our ability to compare kinetic parameters
Antibiotic Killing Experiments in M. tuberculosis. Freezer stocks of H37Rv were
diluted 1:50 in fresh 7H9 media and cultured until late log phase, OD600
between 0.6 and 1.0. Cultures were then diluted to an OD600 of 0.2 and
either 12 or 0.75 mL (for maximally aerobic conditions) aliquoted into square
media bottles (Nalgene, 30 mL). Three duplicate media bottles were set up
for each time point measured. Antibiotics were added at the desired con-
centration. At indicated time points 300 μL of cells were removed from each
media bottle being assayed, and because unsealing of the cap can affect
oxygenation, cultures were then discarded. Collected cells were washed
once in fresh media, serially diluted, and plated on 7H10 plates. Colonies
were counted at day 20 and 25. For heterologous drug challenge, square
media bottles were covered with rubber septa (Sigma) and rifampin at
a concentration of 0.1 μg /mL in 0.5 mL of fresh media was injected
through the septum. Unless otherwise specified, antibiotics were used at
the following concentrations: ciprofloxacin 8 μg /mL (10 × MIC), ofloxacin
1 μg /mL (10 × MIC), rifampin 0.1 μg /mL (10 × MIC), and INH 0.1 μg /mL
(1 × MIC).
Dissolved-Oxygen Measurements. Dissolved oxygen levels within flasks were
determined using the Fluorometrix Cellphase Dissolved Oxygen Sensors (DO-
1). The oxygen-sensing patches were attached to the bottoms of 125-mL
Erlenmeyer flasks before autoclaving and setting up the culture. The oxygen-
sensing patches were calibrated to media at 37 °C. All measurements were
obtained at 37 °C.
Nitrogen Displacement. Experiments were conducted in 125-mL flat-bottom
Erlenmeyer flasks covered with rubber septa (Sigma). After adding cells to
each flask, a 16-gauge needle was inserted into each septum to prevent
injected into the flask via a second needle. Both needles were then removed.
ACKNOWLEDGMENTS. We thank T. Kawate and J. Gomez for helpful
discussions. B.B.K. thanks the Massachusetts General Hospital Executive
Committee on Research and New York Community Trust Heiser Postdoctoral
Fellowships for support. S.S.G. is supported by National Institutes of Health
Grant K08 AI085033. N.H. is supported by National Human Genome Research
Institute Grant T32 HG002295.
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| www.pnas.org/cgi/doi/10.1073/pnas.1203735109Grant et al.