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Eradication of Bacterial Persisters with Antibiotic-Generated Hydroxyl Radicals

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Eradication of Bacterial Persisters with Antibiotic-Generated Hydroxyl Radicals Eradication of bacterial persisters with antibiotic-generated hydroxyl radicals Sarah Schmidt Grant a,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; and fHarvard–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- terial cell numbers but do not sterilize the mouse (8). A plateau teria likely becomes refractory to antibiotic killing in the absence of is typically reached during which numbers of viable bacteria genotypic resistance, making treatment challenging. We describe stabilize. In addition to the mouse infection model, the inability an in vitro model capable of yielding a phenotypically antibi- to sterilize has been observed in the zebra fish (Mycobacterium otic-tolerant subpopulation of cells, often called persisters, within marinum), guinea pig (M. tuberculosis), and macrophage populations of Mycobacterium smegmatis and M. tuberculosis.We (M. tuberculosis) infection models (9–11). In vitro, the survival find that persisters are distinct from the larger antibiotic-suscepti- of a similar small subpopulation can also be observed when ble population, as a small drop in dissolved oxygen (DO) satura- a culture is exposed to high doses of antibiotics (12, 13). The tion (20%) allows for their survival in the face of bactericidal surviving cells, often called persisters (14), retain genetic sus- antibiotics. In contrast, if high levels of DO are maintained, all cells ceptibility to the antibiotic. The ability of M. tuberculosis to enter succumb, sterilizing the culture. With increasing evidence that bac- this physiological state, where intact cells lie dormant and survive tericidal antibiotics induce cell death through the production of despite exposure to bactericidal concentrations of antibiotics, reactive oxygen species (ROS), we hypothesized that the drop in may contribute to the need for long and complex treatment DO decreases the concentration of ROS, thereby facilitating per- regimens to eradicate TB infection. sister survival, and maintenance of high DO yields sufficient ROS Many other human pathogens, including Escherichia coli, to kill persisters. Consistent with this hypothesis, the hydroxyl- Staphylococcus aureus, and Pseudomonas aeruginosa, are simi- radical scavenger thiourea, when added to M. smegmatis cultures larly able to enter a drug-refractory state (14–17). Work in E. coli maintained at high DO levels, rescues the persister population. mutants has suggested that, in a bacterial population, persisters Conversely, the antibiotic clofazimine, which increases ROS via exist before antibiotic exposure (18). In contrast, there is also an NADH-dependent redox cycling pathway, successfully eradi- evidence to suggest that antibiotic tolerance can be induced in cates the persister population. Recent work suggests that environ- response to cellular stresses, including antibiotic treatment. For mentally induced antibiotic tolerance of bulk populations may example, a small fraction of a population of E. coli, when faced result from enhanced antioxidant capabilities. We now show that with increasing levels of antibiotic, secretes the signaling mole- the small persister subpopulation within a larger antibiotic-suscep- cule indole to induce transcriptional changes in neighboring tible population also shows differential susceptibility to antibiotic- cells, most notably in the upregulation of efflux pumps and oxi- induced hydroxyl radicals. Furthermore, we show that stimulating dative stress protective mechanisms, resulting in antibiotic tol- ROS production can eradicate persisters, thus providing a potential erance (19). Additionally, induction of the SOS response in strategy to managing persistent infections. E. coli has been shown to induce both β-lactam and fluo- roquinolone antibiotic tolerance (20–22). Recent transcriptome nfection with the bacterial pathogen Mycobacterium tubercu- profiling of persisters also revealed that several stress response Ilosis remains a leading infectious cause of death globally (1). In regulons, including the SOS response, as well as several toxin– 2009, 1.7 million people died from tuberculosis (TB), with the antitoxin (TA) genes, are upregulated in persister populations in majority of deaths occurring in the developing world (2). Suc- E. coli (15) and M. tuberculosis (12). The mechanism by which cessful treatment of active, symptomatic TB requires a minimum TA loci may induce persistence in E. coli was recently described. of 6 mo of combination therapy (typically four drugs) and is Many toxin genes, activated by degradation of the cognate an- frequently complicated by drug toxicities (3). Treating clinically titoxin, encode mRNases that rapidly degrade mRNA, stopping translation and inducing antibiotic tolerance (23). asymptomatic, latent infections, during which bacteria evade the fi MICROBIOLOGY host immune system and persist in infected individuals for dec- Recent work has identi ed reactive oxygen species as an im- ades, requires an even longer treatment course, typically 9 mo. portant antibiotic-induced cellular stress. These studies demon- strate that bactericidal antibiotics with a variety of different Many patients infected with M. tuberculosis, both in the United mechanisms of action increase ROS production within cells via States and globally, are thus unable to comply with the complex the Fenton reaction (24–27). Numerous ROS, and in particular currently recommended therapeutic regimens (4). The rise of hydroxyl radicals, are toxic to cells and can result in cell death. 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). Author contributions: S.S.G., B.B.K., N.S.C., N.H., and D.T.H. designed research; S.S.G., During both active and latent infection, it has been proposed B.B.K., N.S.C., and N.H. performed research; S.S.G. and B.B.K. analyzed data; and S.S.G., that a subpopulation of bacteria enters a reversible nonreplicating B.B.K., and D.T.H. wrote the paper. state, refractory to traditional antibiotics (5). This phenomenon The authors declare no conflict of interest. is characterized by the reduced efficacy of antibiotics against *This Direct Submission article had a prearranged editor. nonreplicating or slowly replicating bacteria in the absence of 1S.S.G. and B.B.K. contributed equally to this work. – genotypic resistance (5 7). In vivo it has been observed when 2To whom correspondence should be addressed. E-mail: [email protected]. mice are infected with M. tuberculosis, and subsequently treated This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. with antibiotics. In this infection model, antibiotics reduce bac- 1073/pnas.1203735109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1203735109 PNAS | July 24, 2012 | vol. 109 | no. 30 | 12147–12152 Downloaded by guest on September 25, 2021 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 Fig. 1. A system for identifying persister cells. (A) Kill kinetics for M. the survival of the dominant antibiotic-susceptible population, smegmatis cultures treated with CIP and INH or RIF and INH. (B) Similar nor does the drop in DO signal persister cell formation. We killing followed by a stabilization of bacterial numbers is observed
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