persister cells and the paradox of chronic infections

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Persister Cells and theParadox of Chronic InfectionsDormant persister cells are tolerant to antibiotics and are largelyresponsible for recalcitrance of chronic infections

Kim Lewis

Chronic infections are often causedby pathogens that are susceptible toantibiotics, but the disease may bedifficult or even impossible to erad-icate with antimicrobial therapy.

This paradox has been known for a very longtime, and a solution appears to be in sight.

Many recalcitrant chronic infections are asso-ciated with biofilms—communities of microor-ganisms that tend to adhere to surfaces and areenclosed in a layer of exopolymers. Biofilmsform on indwelling medical devices or withintissues and are responsible for infections of cath-eters, orthopedic prostheses, heart valves, mid-dle ear (otitis), unhealing wounds, and lungs ofpatients with cystic fibrosis (CF). These infec-tions may require surgery to remove a heartvalve or prosthesis, and ultimately cause thedeath of CF patients. How are biofilms able toresist killing by antibiotics?

Early attempts to explain biofilm resistance tokilling failed to pinpoint a specific mechanism,resulting in the popular idea that recalcitrance is

multicomponent. The usual suspects include re-tarded penetration of drugs through the exo-polymer matrix, slow growth of cells, and antibi-otic resistance mechanisms that are specificallyexpressed in the biofilm. Yet it appears that themain culprit may have been overlooked.

A decade ago, our group was studying killingof biofilms of Pseudomonas aeruginosa, theprincipal pathogen in CF infections, by antibiot-ics and noticed that death of cells was distinctlybiphasic (Fig. 1A). Most cells in the biofilm werereadily killed at low concentrations of antibiot-ics, but a small subpopulation appeared invinci-ble. It seemed clear that these persister cells,rather than the bulk, were responsible for theinfection’s recalcitrance.

Originally described by Joseph Bigger in 1944in the study of a planktonic population of Staph-ylococcus, persister cells remained a mere curi-osity to the small number of experts who knewof their existence. Rediscovering persisters inbiofilms, and the intriguing possibility that thesecells are the main culprit of recalcitrant chronic

infections, renewed our interest in under-standing the nature of these unusual cells.

Why Are Persisters Tolerant

to Antibiotics?

If some cells in a clonal population of apathogen were resistant to killing by antibi-otics, our first guess would be that this pop-ulation includes resistant mutants. Wherepersisters are involved, this guess would beincorrect—regrowing surviving cells pro-duces a culture with the same small sub-population of survivors, from 10-5to 10-2 ofthe original population. This simple experi-

Summary

• Both bacteria and fungi produce small numbersof dormant persister cells whose function issurvival.

• Persisters are not mutants, but phenotypic vari-ants of the wild type, and are tolerant to killingby antibiotics.

• Toxin/antitoxin modules function as persistergenes.

• Antimicrobial therapy selects for high persis-tence mutants.

Kim Lewis is a Pro-fessor in the De-partment of Biol-ogy, and Director ofthe AntimicrobialDiscovery Center,Northeastern Uni-versity, Boston.

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ment shows that phenotypic variants are pro-duced within a population of genetically identi-cal cells by an apparently stochastic process.Random fluctuations in the expression of persis-ter genes (Fig. 1B) will send some cells down aparticular development pathway. There is anincreasing appreciation of this type of pheno-typic variation, known as bistability, which hasbeen described for chemotaxis, competence, andsporulation.

Persisters neither die nor grow in the presenceof antibiotic, which suggests that they are dor-mant. Natalie Balaban and her coauthors atRockefeller University tracked individual cellsof an E. coli hipA7 (“high persister”) mutantand found that persisters did not grow and,unlike regular cells from the same population,were not lysed by ampicillin. Guessing that per-sisters are dormant and probably have low levelsof protein synthesis allowed us to separate themfrom the bulk of the population by using a strainof E. coli that has a degradable GFP gene clonedunder the control of a ribosomal promoter. In agrowing population, regular cells of this strainare green, but any cell that entered into dor-

mancy will become dim, since GFP de-grades and is not replenished (Fig. 2).Indeed, such a culture splits into twosubpopulations—a bright one, sensitiveto antibiotics, and a small, dim one thatis not killed by the same agents.

How would dormancy help a micro-organism survive exposure to antibiot-ics? One key is that bactericidal antibi-otics kill not by merely inhibitingtargets, but by corrupting them, leadingto production of toxic products. Fluo-roquinolones such as ciprofloxacin turntopoisomerases into DNA endonucle-ases; aminoglycosides interrupt transla-tion, causing production of misfoldedtoxic peptides; and cell wall synthesisinhibitors lead to the activation of au-tolysins and cell lysis. Jim Collins andcolleagues at Boston University in Bos-ton, Mass., recently reported that bac-tericidal antibiotics additionally lead toproduction of toxic reactive oxygenspecies.

If a cell is dormant and the targetsinactive, antibiotics can no longer kill.Of course, this protection comes at aprice—persisters forfeit propagation, at

least until they awaken. However, if a popula-tion of cells encounters antibiotics, the persisterswill be the only ones waking up.

We use the term antibiotic tolerance in orderto differentiate the dormancy protection frommechanistically distinct and more familiarforms of antibiotic resistance. All resistancemechanisms essentially prevent binding of theantibiotic to the target. Resistant cells can thengrow in the presence of elevated levels of antibi-otic, which manifests itself as an increase in theminimal inhibitory concentration (MIC). Thepresence of dormant persisters has no effect onthe MIC, since these cells are not growing andlack resistance mechanisms. Dormancy resolvesthe paradox of untreatable infections, explain-ing how a pathogen that has no resistance mech-anisms can resist being killed by antibiotics.

In Search of the Mechanism of

Persister Formation

To identify genes coding for a complex functionand then decipher the underlying mechanism,microbiologists have successfully used screening

F I G U R E 1

(A) Dose-dependent killing with a bactericidal antibiotic reveals a small subpopulation oftolerant cells, persisters. (B) Candidate persister genes of E. coli. Persisters are formedthrough parallel redundant pathways.

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Lewis: From Russia with Love, Particularly for MicrobiologyKim Lewis calls himself a “garden-variety American,” born in NewYork, but raised in Moscow after hisfamily moved there “for idealisticreasons” when he was two. He at-tributes his happy childhood there tobeing “shielded by the grown-upsfrom the unpleasant reality of the po-lice state,” he says. “I left Russia be-cause I had to live free, which I donow.”

Lewis, 57, professor in the North-eastern University Department of bi-ology and director of its Antimicro-bial Discovery Center, says that hisinterest in biology goes back as far ashe can remember. “The first book Ichecked out of the library at age fivewas about animals,” he says. Hismother, whose work entailed trans-lating Russian literature into English,later gave him the collected works ofGerman zoologist Alfred EdmundBrehm (1829–1884), including theLife of Animals, which made a bigimpression. “One of my earlydreams was to discover new animals,and then I realized with regret thatthis was not going to happen,” hesays. “Little did I know that manyyears later microbiology would giveme the opportunity not only to findnew organisms, but that the majorityof microbes are ‘uncultured,’ andwaiting to be explored.”

Before the Soviet Union brokeapart, educators there encouragedstudents to specialize early. Thus,Lewis enrolled in a Moscow biologyschool while in the 8th grade. “Talk-ing to other kids who were similarlyobsessed with biology was one of themost pleasurable experiences I canrecall,” he says. By junior highschool, he was reading works by theanimal physiologist Konrad Lorenz,whose research on imprinting is con-sidered a fundamental contribution.“The experiments seemed so clev-er—like presenting seagull chickswith a long stick with a red ball at theend emulating the seagull’s beak, towhich the chicks responded enthusi-

astically—that I was gravely con-cerned that I would not be able tothink of my own experiments,”Lewis says.

He need not have worried. In the10th grade, he read a textbook byProfessor Vladimir Skulachev ofMoscow University on the emergingfield of bioenergetics. After visitingtheprofessor,hevolunteeredtoworkin his lab. Skulachev “is one of themost imaginative scientists I havemet, and that, of course, had a biginfluence on my future choice ofprojects,” Lewis says.

“Perhaps the strongest impressionwas made by reading a rather thintextbook on biochemistry that be-longed to my stepfather, who wasfinishing medical school at the time,”he continues. “In the chapter on pro-tein synthesis, the textbook matter-of-factly explained how informationis translated from DNA into proteinsinto functions. The essential mecha-nism of heredity was actually under-stood at the molecular level! I knewthat whatever I chose to study, Iwould want to understand things atthe level of molecules.”

Lewis received his B.S. degree inbiochemistry from Moscow Univer-sity in 1976, and his Ph.D., also inbiochemistry from Moscow Univer-sity, four years later. He returned tothe United States in 1987, losing hisacademic research position at Mos-cow University when he applied toemigrate.

Lewis served as a research associ-ate at the University of Wisconsin,Madison, from 1987 to 1988, andthen assistant professor of biology atthe Massachusetts Institute of Tech-nology to 1994. He spent the nextthree years as an associate professorof medical and research technologyat the University of Maryland Schoolof Medicine in Baltimore, and thenreturned to Boston, to become a re-search associate professor in theTufts University biotechnology cen-

ter. He joined Northeastern Univer-sity there in 2001.

Lewis is now a full-fledged micro-biologist, studying antimicrobialdrug tolerance while also searchingfor novel antimicrobial drugs. “Go-ing into microbiology was an easychoice—it uniquely allows you to re-main a biologist without necessarilylimiting your choices to a narrowfield,” he says. “Microorganisms in-teract as pathogens or symbiontswith every living creature on theplanet, and in this regard your hori-zons are as broad as you wish.”

Persister cells are a special interest.They are dormant variants of bacte-ria that are highly tolerant to killingby all known antibiotics. Persistercells are largely responsible for re-lapsing chronic infections caused bybiofilms, Lewis says. “Biofilmsseemed like a perfect . . . paradox.Bacterial cells that are susceptible toantibiotics become recalcitrant whenforming a biofilm, without actuallyacquiring any apparent resistancemechanism. Analyzing this paradoxlead to the discovery of dormant per-sister cells in the biofilm.”

His wife, Tanya, a molecular biol-ogist, works on transgenic animalmodels of human diseases at the No-vartis Research Institute in Cam-bridge, Mass. He has two daughters,Sasha, an attorney in New York, andMaria, who works in land manage-ment in Moscow. His favorite vaca-tion is“kayakingwithmywife some-where up north.”

Inhis spare time,Lewis enjoysmu-sic, reading, and art. “Growing up inRussia,oneacquired the typical setof19th-century interests—reading, art,andclassicalmusic,”he says. “This isnot due to any refinement on mypart; those were simply the onlychoices available. Now I also enjoyjazz and rock.”

Marlene Cimons

Marlene Cimons lives and writes inBethesda, Md.



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of transposon insertion libraries in search ofmutants lacking the phenotype of interest. Map-ping the insertions produces a catalog of theprincipal genes. This straightforward approachhas lead to the identification of genes coding forsuch properties as sporulation, virulence, andflagellum formation.

In E. coli, we can do even better than a transpo-son insertion screen. Recently, a fairly complete setof knockout strains was created by Hirotada Moriand his colleagues at the University of Nagoya (theKeyo collection). This collection is being gener-ously shared with the scientific community. Wescreened the Keyo library for persister genes andfound not a single mutant unable to make persis-ters. A number of strains showed diminished per-sister levels (as judged by the number of cells

surviving treatment with high levels ofantibiotic). Strains with diminished per-sister levels were primarily deleted ingenes coding for global regulators (dnaK,dnaJ, hupAB, ihfAB, dksA).

From these findings, we concludedthat persisters are not formed through asingle control pathway ending in anexecution mechanism. Rather, there ap-pear to be independent, parallel mech-anisms of persister formation (Fig. 1B).As if a temporary phenotype and thesmall fraction of cells exhibiting it werenot enough of an obstacle!

When the usual molecular genetic ap-proaches fail, there is always transcrip-tome analysis. Persisters isolated by cellsorting and collected by simply lysing apopulation of growing E. coli with ampi-cillin produced a transcriptome showingupregulation of genes whose productscould potentially lead to dormancy—thetoxin-antitoxin (TA) modules. Discov-ered originally as a plasmid maintenancemechanism, the toxins come in a varietyof classes and shut down cellular func-tions. If a daughter cell loses a plasmid,the more labile antitoxin diminishes, re-leasing the toxin that either kills the cellor causes stasis.

Whole-genome sequencing showedthat TA modules are present in bacte-rial chromosomes as well, but theirfunction was unknown. It seemed that“toxins” are excellent candidates forpersister genes. According to Ken Ger-

des at the University of Newcastle in Newcastle,U.K. and his collaborators, some of the E. colitoxins—RelE and MazF—act as mRNA endo-nucleases and inhibit protein synthesis. This in-hibition can be reversed by expression of theirantitoxins.

We found that ectopic expression of RelEcauses multidrug tolerance, emulating nativepersisters. Alex Neyfakh and Nora Vazquez-Laslop at the University of Illinois in Chicago,Ill., also found multidrug tolerance in cells over-expressing MazF.

Another interesting E. coli toxin is coded by thehipBA TA locus. Harris Moyed selected for high-persister strains in the early 1980s and identified ahipA7 gain-of-function allele which increased per-sister production 100-fold in an exponentially

F I G U R E 2

A strain of E. coli that carries a degradable GFP under the control of a ribosomal promotercan be used to physically sort persisters. In a growing population, regular cells expressGFP and are green, but a cell that entered dormancy has diminished protein synthesisand becomes dim due to GFP degradation. Cell sorting then allows to separate the brightand the dim subpopulations. Antibiotic kills mostly the bright, but not the dim sortedcells.



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growing culture. Eugene Koonin andKira Makarova at the National Institutesof Health in Bethesda, Md., noticed thatHipA belongs to the phosphatidylinositolfamily of protein kinases, and in collabo-ration with Dick Brennan and MariaSchumacher, we found that HipA is aserine protein kinase which phosphory-lates and inhibits elongation factor EF-Tu(Fig. 3). Overexpression of HipA pro-duces drug tolerance.

It appears that different toxins cancause dormancy by inhibiting proteinsynthesis through independent mecha-nisms. However, knocking out the toxinsdoes not produce a discernible pheno-type, probably because there is a highdegree of redundancy. There are now 15identified TA modules in E. coli, and 80in M. tuberculosis, and these numberscontinue to increase.

Although persisters can form stochas-tically, there is also a deterministic com-ponent. For example, increasing cell den-sity in a growing culture results in a sharpincrease in persister levels, reachingaround 1% in E. coli in stationary state.The component responsible for this risein unknown. We reasoned that a givenTA module under conditions when it ishighly expressed will act as a determinis-tic component of persister formation. Insearch of such an inducible TA module,we turned to the SOS response.

Stress Response, a Key

Component

of Persister Formation

The canonical SOS response is triggered by DNAdamage and induces expression of DNA repairenzymes and protective proteins. Damage is recog-nized by the RecA protein, which in turn causesself-cleavage of the LexA repressor occupying anoperator sequence upstream from SOS genes (Fig.4). The Lex-box operator is present upstream ofseveral TA modules. Knocking out one of thesemodules, TisAB, causes a sharp decrease in persis-ter formation in a culture treated with ciprofloxa-cin, an antibiotic that leads to DNA damage. Thissuggests that most persister formation is TisB de-pendent under conditions of SOS response. Over-expression of the TisB toxin causes high levels of

tolerance not only to fluoroquinolones, butalso to unrelated antibiotics—�-lactams and ami-noglycosides. Thus, an unexpected side-effect ofciprofloxacin is production of multidrug-tolerantcells resistant to killing by all antibiotics, prob-ably contributing to recalcitrance of infections.Indeed, we did not suspect that treatment withone antibiotic can promote production of cellstolerant to all antibiotic therapy.

TisB seems an unlikely candidate for a persisterprotein since it is a typical antimicrobial peptide.TisB is 29 amino acids long, hydrophobic, posi-tively charged, and acts by disrupting the mem-brane potential, leading to a drop in ATP and,when artificially overexpressed, to cell death, ac-cording to Cecilia Unoson of Uppsala Universityin Uppsala, Sweden, and her collaborators. Ap-

F I G U R E 3

The HipA toxin of E. coli forms a complex with the antitoxin HipB, and both act asrepressors, binding to the promoter region of the hipBA locus (A). When released fromHipB, the HipA kinase phosphorylates elongation factor Ef-Tu, inhibiting protein synthesisand causing dormancy.



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parently, mild overexpression of the protein dur-ing the SOS response decreases the proton motiveforce enough to cause dormancy but not death.ATP depletion seems like a perfect switch for acellular systems shutdown.

The experiments with TisB pinpoint withconsiderable confidence a mechanism of per-sister formation, ending a 60-year quest. Itseems likely that TisB is only the first in whatwill undoubtedly be a large number of mech-anisms for persister formation. Apart fromsetting a precedent, the process of elucidatingthe role of TisB may also serve as a key fordiscovering these additional mechanisms—finding conditions when a particular candi-date is expressed.

Dormancy and the Curious Case

of Antimicrobial Peptides

Antimicrobial peptides are widely dis-tributed among bacteria, fungi, plants,and animals. In humans, antimicrobialpeptides are an important componentof the innate immune response.

Among plants, antimicrobial pep-tides produced by Medicago legumeswere identified as the long-sought fac-tors causing terminal differentiation oftheir symbionts, nitrogen-fixing rhizo-bia, according to Peter Mergaert atCentre National de la Recherche Scien-tifique in Gif-sur-Yvette, France, andhis collaborators. At high concentra-tions, the Medicago peptides causemembrane disruption and death of bac-teria. At low physiological concentra-tions, bacterial cells stop dividing andenter into an apparent irreversible dor-mancy. The supply of nutrients by theplant is apparently sufficient to allowthese cells to continue to fix nitrogen.

Finding TisB-producing persistersand Medicago peptides inducing dor-mancy in rhizobia raises an intriguingquestion regarding conventional anti-microbial peptides known as “killertoxins.” Might exported antimicrobialpeptides produced by bacteria, such aslantibiotics, play an (additional) role ofa community sedative? Bacteria areprotected from their own antimicrobialpeptides primarily through efflux; whenthese peptides are released in the envi-ronment, they may play a dual role—

killing susceptible neighboring species while in-ducing dormancy in the producing colony. Liketypical antibiotics, antimicrobial peptides areusually induced upon entering the stationarystate, when cells would benefit from increasedtolerance to threats.

Clinical Significance of Persister Cells

It appears that there are two faces of the patho-gen threat—resistance in acute infections, andpersister tolerance in chronic infections (Fig. 5).There are many parallels between these twomechanistically distinct protective mecha-nisms—intrinsic resistance/intrinsic ability to

F I G U R E 4

The common antibiotic ciprofloxacin causes DNA damage by converting its targets, DNAgyrase and topoisomerase, into endonucleases. This activates the canonical SOSresponse, leading to increased expression of DNA repair enzymes. At the same time, theLexA repressor that regulates expression of all SOS genes also controls transcription ofthe TisAB toxin/antitoxin module. The TisB toxin is an antimicrobial peptide, which bindsto the membrane, causing a decrease in proton motive force and ATP. This produces asystems shutdown, blocking antibiotic targets, which ensures multidrug tolerance.



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produce tolerant persisters; resistant/tolerant (hip) mutants; and induction ofboth types by stress responses.

We have discussed the role of bio-films in chronic infections, and it seemsthat the primary role of a biofilm is toprovide a protective habitat for persis-ters by shielding them from the immunesystem. Persisters have broader signifi-cance than in the context of biofilminfections, and these cells are likely toplay a critical role in recalcitrance totherapy whenever the immune responseis limited. Such cases would include dis-seminated infections in immunocom-promised patients undergoing cancerchemotherapy or infected with HIV.

Persisters are also likely to play animportant role in immunocompetentindividuals in cases where the pathogen is lo-cated at sites poorly accessible by components ofthe immune system. These include the centralnervous system, where pathogens such as Me-ningococcus and Neisseria cause debilitatingmeningitis and brain abscesses, and the gastro-intestinal tract, where hard-to-eradicate Helico-bacter pylori cause gastroduodenal ulcers andgastric carcinoma.

Tuberculosis is perhaps the most prominentcase of a chronic infection by a pathogen evad-ing the immune system. The acute infection mayresolve spontaneously or as a result of antimi-crobial therapy, but the pathogen often remainsin a “latent” form. It is estimated that 1 in 3people carry latent M. tuberculosis, and 10% ofcarriers develop an acute infection at some stagein their lives. Virtually nothing is known aboutthis latent form that serves as the main reservoirof tuberculosis. One simple possibility is that thelatent form of the pathogen is persisters.

Persisters in Chronic Infections

Persisters are a plausible candidate for explain-ing the recalcitrance of some chronic infections.But plausibility is not causality, and exploringthe involvement of persisters in disease is nottrivial. Introducing isolated persisters into ananimal and examining antibiotic tolerance willnot work, since persisters will revive and turninto regular cells. A helpful clue comes fromexperiments aimed at selecting for high-persistermutants in vitro. A culture is treated with a high

concentration of a bactericidal antibiotic, sur-

viving persisters are reinoculated, and the selec-tion is repeated. After several cycles, the cultureis enriched for high-persister mutants. This ap-proach is useful for identifying candidate persis-ter genes. But this is also the procedure used inantimicrobial therapy of chronic infections—periodic application of high doses of bacteri-cidal antibiotics. If this leads to the selection ofhip mutants, it would mean that persisters pro-vide a selective advantage to the pathogen, andare indeed causally connected to recalcitrance ofthe disease. In this sense the experiment hasalready been done, and we simply need to lookat the result.

The most dramatic case of chronic infectioncomes from cystic fibrosis, when patients areperiodically treated with high doses of antibiot-ics over the course of decades. We took advan-tage of the longitudinal isolates of P. aeruginosacollected from a single CF patient over thecourse of many years by Jane Burns and col-leagues at Seattle Children’s Hospital, and mea-sured the level of persisters by adding high dosesof ofloxacin and determining the number ofsurviving cells. This experiment showed thatpersister levels increased dramatically, by 100-fold, in late isolates of this clonal lineage. Simi-lar increases in persister levels were observed inlate isolates from most CF patients examined.Importantly, in approximately half of the cases,the pathogen showed no increase in MIC toantibiotics used to treat this infection. This ex-periment suggests that the major culprit respon-

F I G U R E 5

Resistant pathogens play a major role in acute infections, and a variety of acquiredmechanisms lead to multidrug resistance. In the parallel world of chronic infections,tolerant persister cells survive the attack of antibiotics and repopulate the infection,causing a relapse. Lengthy antibiotic treatment favors selection of high-persister mu-tants, further complicating therapy.



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sible for the incurable infection is the presence ofpersister cells.

A similar study examined the role of persistersin a recalcitrant infection of Candida albicans.This fungal pathogen also produces persisters,though they are probably analogous rather thanhomologous to their bacterial counterparts. Incancer patients undergoing chemotherapy, theimmune system is compromised, and the oppor-tunistic pathogen C. albicans forms a character-istic white biofilm known as oral thrush, ororopharyngeal candidiasis. Patients are treateddaily with topical chlorhexidine, and after 3weeks the infection may resolve, but it oftenremains recalcitrant to treatment. C. albicansstrains were collected from patients and testedfor survival to high doses of amphotericin B.Invariably, all isolates from nonresolving infec-tion appeared to be high-persister mutants. Thetwo cases examined so far come from com-pletely unrelated microorganisms and suggest ageneral role for hip strains and persister cells inrecalcitrant chronic infections.

Prospects for Persister Eradication

The incidence of chronic infections in the devel-oped world is comparable to acute infections,but we do not have a single agent capable ofeffectively eliminating dormant cells. In part,this is due to the FDA requirement to test newdrugs against exponentially growing pathogens.Most antibiotics would fail if tested against sta-tionary cells, and all will fail if tested againstpersisters. While resistance is a serious problem,we do have a very large arsenal of antibiotics totreat pathogens in acute infections. Knowingthat persisters are largely responsible for recal-citrance of chronic infections is an importantfirst step to developing therapeutic agents, butthe road will not be easy—developing an anti-biotic even in an “easy” case of a narrow-spec-trum compound acting against exponentiallygrowing cells is extremely difficult. Only threenovel compounds have been developed over thepast 30 years–linezolid (oxazolidinone), dapto-

mycin (peptidolipid), and synercid (Quinupris-tin/dalfopristin). All three act only against gram-positive species.

Multidrug-resistant, gram-negative patho-gens such as P. aeruginosa, Burkholderia cepa-cia, Acinetobacter baumannii, and Klebsiellapneumoniae are becoming major problems,with no new drugs in sight. An overreliance onthe major source of antibiotics—cultivable bac-teria—reduced the pace of discovery to a trickleby the early 1960s. Gram-negative pathogenshave a formidable penetration barrier made oftwo membranes and transenvelope MDRpumps that export amphipathic compoundsacross this barrier. Synthetic drugs are almostinvariably extruded by the MDRs. Add thesedifficulties to the need to kill dormant cellswhich form by redundant mechanisms, and theprospect of eradicating a chronic infection ap-pears bleak.

A possible solution may lie in the mode ofaction of a minor group of antibiotics that arevery different from conventional target-specificcompounds. These are prodrugs, used now pri-marily as anti-TB therapeutics. Compoundssuch as isoniazid (INH) or ethionamide are ac-tivated by bacteria-specific enzymes into reac-tive compounds. Metronidazole is the onlyknown broad-spectrum prodrug, being acti-vated by nitrate reductase, which is present inmany bacteria. The beauty of prodrugs is thatthey can in principle kill dormant cells, since areactive molecule can disrupt such cellular com-ponents as the membrane and DNA, and theircovalent binding to targets produces an irrevers-ible sink, countering efflux. Indeed, metronida-zole is highly effective against gram-negativespecies, but its application is limited to anaero-bic conditions where nitrate reductase is ex-pressed. Known prodrugs do not sterilize aninfection, but their mode of action carries thepromise of developing compounds with a betterfit to their activating enzyme, which may pro-duce sufficient amounts of reactive compoundsthat will kill persisters.

ACKNOWLEDGMENTS

Kim Lewis is supported by grants from the National Institutes of Health, the Army Research Office, and the Bill and MelindaGates Foundation.



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SUGGESTED READING

Balaban, N. Q., J. Merrin, R. Chait, L. Kowalik, and S. Leibler. 2004. Bacterial persistence as a phenotypic switch. Science305:1622–1665.Correia, F. F., A. D’Onofrio, T. Rejtar, L. Li, B. L. Karger, K. Makarova, E. V. Koonin, and K. Lewis. 2006. Kinase activityof overexpressed HipA is required for growth arrest and multidrug tolerance in Escherichia coli. J. Bacteriol. 188:8360–8367.Dorr, T., M. Vulic, and K. Lewis. 2010. Ciprofloxacin causes persister formation by inducing the TisB toxin in Escherichiacoli. PLOS Biol 8:e1000317.Dubnau, D., and R. Losick. 2006. Bistability in bacteria. Mol. Microbiol. 61:564–572.Hansen, S., K. Lewis, and M. Vulic. 2008. The role of global regulators and nucleotide metabolism in antibiotic tolerance inEscherichia coli. Antimicrob. Agents Chemother. 52:2718–2726.Keren, I., N. Kaldalu, A. Spoering, Y. Wang, and K. Lewis. 2004. Persister cells and tolerance to antimicrobials. FEMSMicrobiol Lett 230:13–18.LaFleur, M. D., Q. Qi, and K. Lewis. 2010. Patients with long-term oral carriage harbor high-persister mutants of Candidaalbicans. Antimicrob. Agents Chemother. 54:39–44.Lewis, K. 2007. Persister cells, dormancy and infectious disease. Nature Rev. Microbiol. 5:48–56.Schumacher, M. A., K. M. Piro, W. Xu, S. Hansen, K. Lewis, and R. G. Brennan. 2009. Molecular mechanisms ofHipA-mediated multidrug tolerance and its neutralization by HipB. Science 323:396–401.


persister cells and the paradox of chronic infections

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