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Drug Resistance Updates 29 (2016) 76–89

Contents lists available at ScienceDirect

Drug Resistance Updates

jo ur nal homep age: www.elsev ier .com/ locate /drup

olecular mechanisms and clinical implications of bacterialersistence

oran Elie Michiels, Bram Van den Bergh, Natalie Verstraeten, Jan Michiels ∗

entre of Microbial and Plant Genetics, KU Leuven, Leuven, Belgium

r t i c l e i n f o

eywords:ntibiotic toleranceersistersesistancehronic infectionsathogens

a b s t r a c t

Any bacterial population harbors a small number of phenotypic variants that survive exposure to highconcentrations of antibiotic. Importantly, these so-called ‘persister cells’ compromise successful antibi-otic therapy of bacterial infections and are thought to contribute to the development of antibioticresistance. Intriguingly, drug-tolerant persisters have also been identified as a factor underlying failureof chemotherapy in tumor cell populations. Recent studies have begun to unravel the complex molecularmechanisms underlying persister formation and revolve around stress responses and toxin–antitoxinmodules. Additionally, in vitro evolution experiments are revealing insights into the evolutionary and

adaptive aspects of this phenotype. Furthermore, ever-improving experimental techniques are stimu-lating efforts to investigate persisters in their natural, infection-associated, in vivo environment. Thisreview summarizes recent insights into the molecular mechanisms of persister formation, explains howpersisters complicate antibiotic treatment of infections, and outlines emerging strategies to combat thesetolerant cells.

© 2016 Elsevier Ltd. All rights reserved.

. Introduction

We are currently facing an antibiotic crisis that is threateningur medical care, as effective antibiotics are becoming increas-ngly scarce, or even nonexistent for some pathogens (World Healthrganization, 2014). The emergence of drug resistance is predom-

nantly cited as the main cause, but other strategies by whichathogens overcome antibiotic killing also play a role and may haveeen underestimated so far. Indeed, shortly after the introductionf the first antibiotic, now more than seven decades ago, it wasbserved that antibiotics fail in completely sterilizing antibiotic-ensitive bacterial populations, even when applied at very highoncentrations (Hobby et al., 1942). Instead, a small number ofhenotypic variants within these populations, called ‘persisters’,ithstand antibiotic killing by transiently adopting an alternativehenotypic state (Bigger, 1944; Lewis, 2010). This phenomenonas largely ignored for the remainder of the 20th century, but inter-

st revived when persisters were found to play a major role in the

ntibiotic recalcitrance of biofilms, which are strongly linked withhronic infections (Hall-Stoodley et al., 2004; Spoering and Lewis,001; Verstraeten et al., 2016).

∗ Corresponding author at: KU Leuven – Centre of Microbial and Plant Genetics,asteelpark Arenberg 20, Box 2460, B-3001 Leuven, Belgium.

E-mail address: jan.michiels@kuleuven.be (J. Michiels).

ttp://dx.doi.org/10.1016/j.drup.2016.10.002368-7646/© 2016 Elsevier Ltd. All rights reserved.

In this review article we will discuss recent insights andcommon themes of the molecular mechanisms of persistence.Additionally, we will summarize the current knowledge on per-sistence in an in vivo context and the clinical implications ofpersister cells, discuss how the dynamics of antibiotic treatmentaffect persistence, and give an overview of promising emergingstrategies to target persisters clinically. Eradication of persistersmight indeed improve the outlook of the current antibiotic crisis,not in the least because persistence may also lead to acceleratedevolution of resistance. Improved knowledge on persistence mayeven cross-pollinate other research fields, as a similar phenomenoncomplicates the use of chemotherapy in cancer.

2. Bacterial persistence: phenotypic heterogeneity enablingantibiotic survival

Persistence is the occurrence of rare, transiently antibiotic-tolerant phenotypic variants in isogenic bacterial populations, andthus represents an example of phenotypic heterogeneity (Dharand McKinney, 2007). Regular cells can develop into a persister,and persister cells can revert to the sensitive phenotype, both

processes being directed by specific switching rates (Fig. 1a). Con-sequently, persister levels in a population are governed by aninterplay between these switching rates and the growth rates ofboth cell types (Patra and Klumpp, 2013; Van den Bergh et al.,

J.E. Michiels et al. / Drug Resistance Updates 29 (2016) 76–89 77

Fig. 1. Basic characteristics of persistence. (a) In an isogenic bacterial population, normal and persister cells interconvert according to certain switching rates, indicated as ra

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nd rb. Both switching rates can be influenced positively or negatively by the growtn antibiotic-resistant population and an antibiotic-sensitive population with a subpeveal the presence of a subpopulation of tolerant persisters.

016b). Switching can occur spontaneously, but is at the same timetrongly influenced by the growth phase and environmental fac-ors (Balaban et al., 2004; Keren et al., 2004a) (Fig. 1a). Persisterells have been observed in every tested species so far, includingmportant human pathogens as Pseudomonas aeruginosa, Staphy-ococcus aureus, and Mycobacterium tuberculosis (De Groote et al.,009; Keren et al., 2011, 2004a). Moreover, persistence is not lim-

ted to bacteria, as it is observed in eukaryotic microorganismss Candida albicans (LaFleur et al., 2006) and Saccharomyces cere-isiae (Bojsen et al., 2016), and even in tumor cell populationssee Box 1).

The experimental hallmark of persistence is a biphasic killingurve, obtained when a lethal dose of a bactericidal antibiotic isdded to a bacterial population and the number of surviving cellss followed over time (Fig. 1b). During the first phase, the bulk ofhe population is killed and the number of surviving cells declinesapidly. The second phase is characterized by a slower killing rate,aused by the presence of a highly antibiotic-tolerant persisterubpopulation (Fig. 1b). Since surviving persisters are geneticallyndistinguishable from the bulk of the population, their regrowthfter antibiotic removal yields a new, antibiotic-sensitive popu-ation containing the same number of persisters as the originalopulation. Obviously, persistence is fundamentally different fromesistance: contrary to resistant mutants, persisters are unable toivide in the presence of antibiotics, and their tolerant phenotype

s lost upon resumption of division and is thus nonheritable. Whileany different resistance mechanisms exist, ultimately they all

esult in the prevention of drug-target binding. In contrast, theolerance of persisters is primarily attributed to an altered physiol-gy, lending protection against antibiotic toxicity (Kint et al., 2012;ewis, 2010).

.1. The distinctive physiology of persisters

Already in his seminal paper in 1944, Bigger proposed the pres-nce of dormant, nondividing variants in an otherwise activelyrowing population as explanation for the tolerant phenotypef the S. aureus persister cells he observed. Indeed, he showedxperimentally that growth inhibition by cold treatment, nutri-nt removal, or bacteriostatic concentrations of boric acid increaseshe number of persisters (Bigger, 1944). Most antibiotics interfereith active cellular processes, such as macromolecular synthesis,

nd their efficacy is therefore dependent on the physiological activ-

ty of the cell (Eng et al., 1991; Tuomanen et al., 1986). Antibioticarget inactivity in dormant cells is thus an attractive explanationor persistence, and a great deal of recent work has indeed beenonsistent with this concept. Single-cell observations of persisters

se and a variety of environmental factors. (b) Comparison of the time–kill curves oftion of persisters. In the antibiotic-sensitive population, biphasic time–kill kinetics

demonstrated that they are slowly- or nongrowing, indicative ofa dormant state (Balaban et al., 2004; Maisonneuve et al., 2013).Transcriptome analysis of isolated persisters revealed reducedmetabolic activity, showing downregulation of genes involved inenergy production and nonessential cellular functions such asflagellar synthesis (Keren et al., 2011, 2004b; Shah et al., 2006).Moreover, induction of growth inhibition by interfering with majorcellular processes consistently causes increased persistence. Forexample, overexpression of translation-inhibiting toxins causescell stasis and persistence, which is reversed upon relief of trans-lation inhibition by expression of the cognate antitoxin (Korchand Hill, 2006; Pedersen et al., 2002; Vázquez-Laslop et al., 2006).Similarly, almost 100% of Escherichia coli cells become persisterupon exposure to the transcription inhibitor rifampicin, the trans-lation inhibitor tetracycline, or carbonyl cyanide m-chlorophenylhydrazine, which blocks ATP synthesis (Kwan et al., 2013).

Consistent with global dormancy underlying tolerance, persis-ters are often tolerant to multiple antibiotics (Lewis, 2010; Wiuffet al., 2005; Wiuff and Andersson, 2007) and other stresses suchas prophage induction (Pearl et al., 2008) or serum complement-mediated lysis (Putrins et al., 2015). However, no correlation existsbetween persister levels for different antibiotics among environ-mental E. coli strains (Hofsteenge et al., 2013; Stewart and Rozen,2011) or Acinetobacter baumannii clinical isolates (Barth et al.,2013), casting doubt on the idea of a single homogeneous, dormantsubpopulation of persisters. Indeed, persisters have been argued torepresent instead a heterogeneous collection of cells, displaying dif-ferent physiologies that confer varying degrees of tolerance againstdifferent antibiotics (Allison et al., 2011a; Amato and Brynildsen,2015; Levin et al., 2014).

Recently, several studies have also questioned the dormantnature of persister cells. Two reports on Mycobacterium found thatpersisters are actively dividing cells. In a first study, drug tolerancein macrophage-residing Mycobacterium marinum and M. tubercu-losis was associated with an actively replicating population anddepended on the macrophage-induced expression of efflux pumps(Adams et al., 2011). In another study, Mycobacterium smegmatisisoniazid tolerance was correlated with fluctuating levels of thedrug-activating catalase-peroxidase enzyme KatG, but it was notdependent on the growth rate (Wakamoto et al., 2013). However,as isoniazid is a prodrug requiring enzymatic activation, the lattermechanism might reflect a specific case of drug escape rather than atrue example of persistence, since the growing persister population

was never exposed to any active drug (Maisonneuve and Gerdes,2014; Wood et al., 2013). Growth arrest alone also cannot explainpersistence in P. aeruginosa biofilms. A starvation-induced block incell division leads to drug tolerance only when it is accompanied

78 J.E. Michiels et al. / Drug Resistanc

Box 1: An intriguing parallel: drug-tolerant persisters incancerCancer is an evolutionary phenomenon of clonal cell popu-lations, and it has been proposed that we can learn muchby drawing parallels with the fields of microbial experimen-tal evolution and infectious diseases (Glickman and Sawyers,2012; Sprouffske et al., 2012). Similar to bacterial populationsunder antibiotic stress, cancer cells rapidly adapt to anti-cancertherapy by acquiring mutations conferring drug resistance(Lambert et al., 2011). However, mutational drug resistance can-not explain ‘retreatment response’: when cancer relapses afterremission, it is often still responsive to the same chemothera-peutic agent that was given before (Cara and Tannock, 2001).This suggests that cancer cell populations contain phenotypicvariants that are reversibly drug-tolerant and can give rise tocancer relapse after chemotherapy, just as bacterial persistersunderlie recurrence of infection after antibiotic treatment (Kimand Tannock, 2005; Menchón and Condat, 2011).During high-dose drug therapy of different tumor-derived can-cer cell lines, Sharma et al. (2010) consistently detected thepresence of a drug-tolerant cancer cell subpopulation. These‘drug-tolerant persisters’ (DTPs) were mostly nonproliferativeand transiently expressed stem cell markers. Their forma-tion depended on chromatin-modifying enzymes and signalingthrough the insulin-like growth factor 1 receptor (IGF1R). In afollow-up study, DTPs were additionally found to require alde-hyde dehydrogenase (ALDH) activity, a previously establishedstem cell marker, which protects against toxicity from reactiveoxygen species (Raha et al., 2014). Inhibition of ALDH activityby disulfiram causes accumulation of lethal levels of reac-tive oxygen species and eliminates DTPs. This finding couldpresent a novel therapeutic strategy to limit cancer recurrence,as a treatment regimen combining erlotinib and desulfiram sig-nificantly delayed tumor relapse in a xenograft mouse model.Many other studies have confirmed the existence of DTPs thatcan cause post-therapeutic tumor recurrence, and identifiedpossible therapeutic approaches to eliminate them (Chen et al.,2012; Knoechel et al., 2014; Vinogradova et al., 2016). Some-times, relapse occurs after many years of sustained remission.In one case, dormant pre-leukemic stem cells caused acutelymphoblastic leukemia relapse 22 years after the initial diag-nosis and presumed cure (Ford et al., 2015).In the examples described above, DTPs often display prop-erties of cancer stem cells (CSCs). CSCs or tumor-initiatingcells are indeed proposed to be an important cause for post-therapeutic tumor recurrence as they were found to be moretolerant toward cytotoxic agents and radiation therapy (Wichaet al., 2006). The precise relation between CSCs and DTPs isunknown and probably complex (Sharma et al., 2010). Themolecular mechanisms underlying therapy tolerance of CSCsare multifactorial, but remarkable similarities with bacterialpersistence mechanisms have been unraveled. Both activemechanisms such as drug efflux and passive protection by aquiescent cellular state seem to play a role (Eyler and Rich,2008). Indeed, just as antibiotics mainly work against actively-dividing bacteria, cytotoxic cancer therapeutics induce celldeath most strongly in highly-proliferating tumor cells.

bfldbisrnt2

encoded in a toxin–antitoxin (TA) locus. As exemplified below,

y an active stress response (Nguyen et al., 2011). In another study,uorescence-activated cell sorting with fluorescent markers for cellivision and metabolic activity showed an increased likelihood toecome a persister for cells from the nondividing and metabolically

nactive subpopulation. However, more than 99% of this inactiveubpopulation is killed, and many persisters are still found amongapidly growing cells (Orman and Brynildsen, 2013). Whether or

ot dormancy is sufficient to explain antibiotic survival of persis-er cells remains an active matter of scientific debate (Wood et al.,013).

e Updates 29 (2016) 76–89

Interestingly though, both views are perhaps not as contradic-tory as initially thought, since recent work indicates that activeand passive mechanisms may in fact contribute in a complemen-tary fashion to the tolerant phenotype of persisters. Firstly, highactivity of certain cellular processes might confer a metabolic costupon the cell, leading to energy depletion, growth retardation, andconsequently, persistence. For example, virulence factor expres-sion in Salmonella was shown to be bistable, and lead to slowgrowth and persistence in the highly-expressing subpopulation(Arnoldini et al., 2014). In E. coli, activation of motility significantlycontributes to the formation of persisters tolerant to gentamicin(Shan et al., 2015). Similarly, active cells that maintain high respi-ratory activity during stationary phase partially digest endogenousRNA and proteins, causing slow resumption of growth and highpersistence upon antibiotic challenge in fresh medium (Ormanand Brynildsen, 2015). Secondly, a recent study identified activeefflux mechanisms in persisters that were otherwise not growingor dividing, suggesting that persisters can take advantage of twoseemingly contradicting strategies to survive antibiotic exposure:‘passive defense’ through dormancy and ‘active defense’ by efflux-mediated reduction of intracellular accumulation of antibiotics (Puet al., 2016).

2.2. Stochastic and deterministic factors govern spontaneous andinduced persistence

2.2.1. Stochasticity: noise underlies the formation andresuscitation of persisters

In a landmark study by Nathalie Balaban and colleagues,microfluidic observation of single E. coli cells showed that non-growing persisters form spontaneously before antibiotic exposureand in the apparent absence of stress (Balaban et al., 2004). Thus,it was concluded that persisters are not induced as a responseto the antibiotic, but constitute drug-independent, pre-existingvariants. This pre-existing phenotypic heterogeneity is protectiveagainst unexpected episodes of lethal antibiotic exposure, and isan example of a bet-hedging strategy, conferring a competitiveadvantage in periodically changing environments (Kussell et al.,2005; Stepanyan et al., 2015). The most prominent mechanismthat generates phenotypic heterogeneity is stochastic noise inmolecular processes (Ackermann, 2015). All cellular processes areinherently noisy (Elowitz et al., 2002), leading to fluctuations inprotein levels among individual cells of a population. However,noise alone is insufficient to generate large phenotypic differ-ences unless it is amplified by regulatory processes. Noise can beamplified by positive or double-negative regulatory feedback loops.When this feedback is combined with a nonlinear response, coex-istence of phenotypically distinct variants is the result (Eldar andElowitz, 2010; Smits et al., 2006). Nonlinear responses are com-mon among transcription factors, e.g. when cooperative bindingof DNA sequences or posttranslational modifications are involved,and often manifest as threshold-based phenomena (Veening et al.,2008).

In the case of persistence, experimental studies have con-firmed that noisy expression of persister genes underlies persisterformation, although the precise regulatory networks generatingphenotypic bistability are mostly unknown. HipA, a well-describedpersister protein, induces the formation of dormant persisters assoon as its cellular concentration exceeds a certain threshold level(Rotem et al., 2010). In a typical population, HipA levels fluctuateabove and below the threshold, resulting in co-occurrence of grow-ing and dormant cells. HipA is a prototypical example of a toxin

these loci play a pivotal role in bacterial persistence. Corroborat-ing the results of Rotem et al. (2010), microfluidic observations ofexponentially growing cells of E. coli confirmed that stochastic TA

J.E. Michiels et al. / Drug Resistanc

Fig. 2. Persistence results from an interplay between stochastic and deterministicfactors. In a bacterial population, the expression level of a persister protein fluctuatesbetween individual cells, resulting in a smooth distribution around the average ofthe population. Cells where the persister protein spontaneously exceeds a certainthreshold concentration will switch to the persister state and become tolerant toantibiotics. When environmental factors increase the average expression level oftt

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analysis on isolated persisters (Keren et al., 2011, 2004b; Shah

he persister protein, additional persisters are induced because more cells attainhe threshold level required for switching to the persister state.

xpression causes cessation of growth and that this correlates withigh tolerance against �-lactam antibiotics (Maisonneuve et al.,013).

Surviving persisters can only form a new population if theyesuscitate from the persister state to resume growth. Resuscitations also thought to occur stochastically, but due to experimental hur-les, no precise mechanisms have been verified yet. Nevertheless,athematical modeling suggests that the unique characteristics of

ranscriptional autoregulation in TA operons, in particular condi-ional cooperativity, play an important role (Cataudella et al., 2013,012). In the case of HipA-mediated persistence, autoinhibitiony an intermolecular phosphorylation event has been proposeds an initial step to emerge from dormancy and resume growthSchumacher et al., 2012). The recently described Salmonella TacToxin catalyzes the acetylation of charged tRNA molecules, whichnhibits translation and causes persistence (Cheverton et al., 2016).nterestingly, these researchers uncovered an additional protein,th, which detoxifies the corrupted tRNAs, relieves inhibition ofranslation, and allows growth resumption of persisters. However,ow the activities of TacT and Pth are regulated and how this wouldause the switch to flip, remains to be elucidated.

.2.2. Determinism: environmental factors modulate persistenceSpontaneous formation of persisters in unstressed populations

oes not adequately capture all characteristics of the persistencehenomenon. Indeed, persister levels have been shown to beependent on several environmental factors, implicating a deter-inistic aspect in the mechanisms underlying persister formation.

ut how are these stochastic and deterministic elements recon-iled? Both concepts can be integrated by the notion that theopulation average expression level of persister proteins can beirected by environmental factors. As such, the number of individ-al cells that reach a certain threshold value and adopt the persistertate will change accordingly (Fig. 2) (Lewis, 2010; Maisonneuvend Gerdes, 2014).

Persister formation is critically dependent on the growth phase:heir numbers are very low in lag and early-exponential phase,ut increase dramatically from mid-exponential toward stationaryhase (Keren et al., 2004a). This is probably mainly due to decreas-

ng nutrient availability, as nutrient transitions and starvation haveeen identified as important triggers of persister formation (Fung

t al., 2010; Keren et al., 2004a; Maisonneuve et al., 2011; Nguyent al., 2011). Specifically, diauxic carbon source transitions haveeen highlighted as important factors enhancing persistence, both

e Updates 29 (2016) 76–89 79

in planktonic populations and biofilms (Amato et al., 2013; Amatoand Brynildsen, 2015, 2014).

In addition to starvation, a plethora of other environmentalstimuli affect persister levels. Pre-exposure of bacteria to sublethalstresses from various origins causes sharply increased persister lev-els, as was shown for heat stress (Mordukhova and Pan, 2014;Murakami et al., 2005), hyperosmotic stress (Murakami et al.,2005), acid stress (Hong et al., 2012), and oxidative stress (Honget al., 2012; Wu et al., 2012). Also, pre-exposure to subinhibitorylevels of antibiotics increases persistence (Dörr et al., 2009; Johnsonand Levin, 2013; Kwan et al., 2013). Importantly, increased persis-tence was observed in Klebsiella pneumoniae when antibiotics wereadded gradually instead of at once, mimicking the gradual increasein antibiotic serum concentration after clinical administration (Renet al., 2015).

Persister levels are also governed by chemical signaling, reveal-ing a social dimension to persistence. Initial investigations did notidentify increased persistence when spent medium, rich in quorumsensing molecules, was added to cultures of E. coli or P. aeruginosa(Lewis, 2010). Later, quorum sensing mutants were found toexhibit impaired persistence, and quorum sensing molecules werereported to increase persistence in P. aeruginosa, Streptococcus spp.,and A. baumannii (Bhargava et al., 2014; Leung and Lévesque, 2012;Möker et al., 2010), though not in S. aureus or E. coli (Möker et al.,2010). In the latter, the stationary phase signaling molecule indolewas later shown to be involved in persister formation (Vega et al.,2012). In a mixed-species environment, indole produced by E. colieven increased tolerance in Salmonella Typhimurium, a speciesthat does not natively produce indole (Vega et al., 2013). Remark-ably, others observed an inverse relation between indole signalingand persistence (Hu et al., 2014), indicating that further work isnecessary to clarify how it contributes to persister formation. Nev-ertheless, other lines of research also suggest the presence of asocial dimension in persistence: persistence can result from thecostly expression of a cooperative trait (Arnoldini et al., 2014), facil-itates the utilization of beta-lactamase as a public good (Medaneyet al., 2015), and can be modeled as a social phenomenon (Gardneret al., 2007).

While most reports have focused on environmental factors lead-ing to elevated persistence, some conditions have in fact beenshown to decrease persister levels, indicating that environmentalmodulation of persistence is bidirectional. For example, alkalineconditions (Lebeaux and Chauhan, 2014), hypo-ionic environments(Jiafeng et al., 2015), and the addition of amino acids (Duan et al.,2016) or glycolytic nutrients (Allison et al., 2011b) have beenshown to drastically reduce levels of persisters in the presence ofaminoglycoside treatment. In general, switching from persisters tonormal cells appears to be strongly increased under environmen-tal conditions favorable for active growth, such as upon transfer tofresh medium (Gefen et al., 2008; Van den Bergh et al., 2016b).

2.3. Toxin–antitoxin modules and stress responses underliemolecular mechanisms of persistence

As described above, many environmental conditions influencepersistence. Additionally, many involved genes have been iden-tified through selection for antibiotic survival in mutagenizedpopulations (Moyed and Bertrand, 1983; Slattery et al., 2013;Torrey et al., 2016), screening of deletion mutant libraries (Hansenet al., 2008; Ma et al., 2010), transposon insertion libraries (DeGroote et al., 2009; Hu and Coates, 2005; Li and Zhang, 2007)and overexpression libraries (Spoering et al., 2006), transcriptome

et al., 2006), and Tn-Seq or similar approaches (Girgis et al., 2012;Shan et al., 2015). Yet, despite all this knowledge, complete molec-ular pathways spanning all steps from the initial trigger to the

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ownstream effector have been elucidated in only a select num-er of cases in E. coli, and predominantly revolve around stressesponses and TA modules.

.3.1. Stress responsesTranscriptome analysis of samples enriched in persisters

evealed the upregulation of many stress response genes. Lewisnd colleagues found increased expression of genes involved inhe SOS response, phage-shock response, and heat- and cold-shockesponse (Keren et al., 2004b). Indole-induced persistence was cou-led with increased expression of genes involved in oxidative stressnd phage-shock pathways, and deletions of these genes substan-ially reduces indole-induced persistence (Vega et al., 2012). In. coli, mutants lacking the oxidative stress response, stringentesponse, or SOS response show important persistence defects forultiple antibiotics (Wu et al., 2015). In addition, as discussed

bove, many different stressors are known to elicit increased per-istence. Together, these results suggest that stress responses play

prominent role in persister formation pathways.

.3.2. Toxin–antitoxin modulesTA loci comprise two genes, encoding a stable toxin and an

nstable antitoxin, inhibiting the toxin. TA modules were ini-ially discovered in the context of bacterial plasmids, where theyncrease plasmid maintenance by killing or inhibiting growth oflasmid-free cells. Later, it was found that they are also wide-pread among bacterial chromosomes, but their function has longemained elusive (Van Melderen, 2010). To date, six different typesf TA loci have been described based on their genetic organizationnd mechanism of action (Page and Peti, 2016). Recently, TA mod-les, mainly from type I and type II, emerged as central effectors

n persistence. Type I TA loci contain a protein toxin and a non-oding RNA antitoxin that base-pairs to the mRNA of the toxino inhibit its translation. Toxins of type I TA systems are small,ydrophobic peptides that insert into membranes, leading to lossf membrane potential and growth cessation. The most prevalentnd best studied TA modules are the type II systems. Type II anti-oxins are proteins that bind to and inhibit their cognate toxinrotein. Some type II toxins inhibit replication by interfering withNA gyrase, but most function as translation inhibitors. The major-

ty of translation-inhibiting type II toxins have endoribonucleasectivity, but others inhibit translation by inactivating ribosomelongation factors, inactivating glutamyl-tRNA synthetase (GltX),r acetylating charged tRNAs (Cheverton et al., 2016; Page and Peti,016).

Several lines of evidence point to a role for TA loci in per-istence. The first recognized persister gene, hipA, later turnedut to be a part of the hipBA type II TA operon (Black et al.,991; Moyed and Bertrand, 1983). Furthermore, transcriptometudies revealed strong upregulation of multiple type II toxinsn persisters, mainly mRNases such as relE, mazF, dinJ, and mqsRKeren et al., 2004b; Shah et al., 2006). Moreover, persistences induced by ectopic overexpression of type II toxins and res-ued by the expression of their cognate antitoxins, as was shownor example with hipBA, relBE, and mazEF (Korch and Hill, 2006;edersen et al., 2002). Similarly, overexpression of type I tox-ns tisB and hokB causes increased persistence (Dörr et al., 2010;erstraeten et al., 2015b). In most cases however, deletion of sin-le TA modules is insufficient to yield a detectable persistenceefect, probably owing to their functional redundancy and com-lementary contribution to persistence. In contrast, successive

eletion of several type II mRNase toxins in E. coli causes a pro-ressive reduction in the level of persisters (Maisonneuve et al.,011), strongly supporting their complementary contribution toersistence.

e Updates 29 (2016) 76–89

2.3.3. Persister formation pathways: stress responses andtoxin–antitoxin modules

In 2010, Lewis and colleagues were the first to delineate thecomplete molecular picture of a persistence pathway (Fig. 3). DNAdamage resulting from ciprofloxacin treatment induces the SOSresponse, activates RecA, and relieves LexA-mediated inhibition oftranscription of the tisB/istR-1 type I TA module (Dörr et al., 2010,2009). TisB is a small, hydrophobic peptide that inserts in the mem-brane and disrupts the proton motive force (pmf) and productionof ATP (Unoson and Wagner, 2008), resulting in shutdown of bac-tericidal antibiotic targets and consequently multidrug tolerance(Dörr et al., 2010).

Other identified pathways consistently include the stringentresponse as the central stress response mediating persistence. Byobserving transcriptional fusions of two representative type II TAloci in single cells, Maisonneuve et al. (2013) showed that inductionof persistence in growing E. coli cultures is dependent on stochas-tic toxin expression, and that toxin expression is coupled withhigh levels of (p)ppGpp. They further uncovered the connectionbetween (p)ppGpp and TA induction, which involves accumulationof inorganic polyphosphate, activation of the Lon protease, degra-dation of antitoxins, and inhibition of translation and growth byincreased levels of free mRNase toxins (Fig. 3). The type II toxinHipA has no RNase activity, and while it was known that persis-tence of the high-persistent hipA7 mutant depends on the stringentresponse (Korch et al., 2003), it was at this point still unclear how itcould be integrated into this model. Two independent recent stud-ies found that HipA operates upstream of the mRNase toxins inthis pathway. Indeed, HipA inactivates glutamyl-tRNA synthetase(GltX) by phosphorylation of a conserved serine residue (Germainet al., 2013; Kaspy et al., 2013) (Fig. 3). This results in defectiveaminoacylation and the generation of ‘hungry’ codons at the ribo-somal A site, which is a strong trigger for activation of the stringentresponse. Increased levels of (p)ppGpp subsequently mediate per-sistence through activation of the mRNase toxins as describedabove.

The stringent response also mediates another persistence path-way in E. coli, independent of Lon and type II TA-modules. Instead,the essential conserved GTPase Obg induces persistence by activat-ing transcription of the type I toxin hokB, in a (p)ppGpp-dependentfashion (Verstraeten et al., 2015b) (Fig. 3). Interestingly, (p)ppGpphas been described to bind Obg (Persky et al., 2009), but the exactmechanism of hokB transcriptional activation remains unknown.HokB is a toxic membrane protein, and for plasmid-associatedhok/sok modules, rapid postsegregational killing of plasmid-freecells has been described (Gerdes et al., 1986). However, simi-lar to the TisB-mediated persistence pathway described above,intermediate levels of HokB induce membrane depolarizationand multidrug tolerance through dormancy (Verstraeten et al.,2015b).

Apart from increasing levels of free type II mRNase antitoxinsand inducing transcription of the type I HokB toxin, (p)ppGpp itselfhas been found to be part of a metabolic TA module comprising(p)ppGpp as a metabolite toxin and SpoT as an enzymatic anti-toxin (Amato et al., 2013). Moreover, the biochemical (p)ppGppnetwork exhibits double-negative feedback regulation, suggestingthat it could generate a bistable persistence phenotype. Activationof the (p)ppGpp-SpoT metabolic TA module after a diauxic car-bon source shift triggers distinct pathways for the formation ofofloxacin and ampicillin persisters, respectively (Amato et al., 2013;Amato and Brynildsen, 2015). Both pathways require inhibition oftranscription by DksA and trans-translation, while involvement ofnucleoid-associated proteins and inhibition of negative DNA super-

coiling are specific for ofloxacin persisters, and dependency on ClpAand broad inhibition of peptidoglycan synthesis are specific forampicillin persisters (Fig. 3). However, a role for TA modules in

J.E. Michiels et al. / Drug Resistance Updates 29 (2016) 76–89 81

Fig. 3. Molecular pathways underlying persistence in E. coli. In a subset of cells in a bacterial population, stress responses are activated spontaneously, or by an environmentaltrigger, and activate an additional molecular cascade leading to the induction of the terminal effector, mostly a toxin from a type I or type II TA module. In a pathway identifiedby Dörr et al. (2010), ciprofloxacin treatment induces the SOS response, which relieves LexA-mediated repression of transcription of the type I tisB toxin. TisB subsequentlyforms membrane pores, causing loss of pmf and ATP, leading to multidrug tolerance. As found by Maisonneuve et al. (2013), spontaneous or starvation-induced high levels of(p)ppGpp cause accumulation of polyphosphate by inhibition of PPX. Polyphosphate activates Lon, which directs proteolysis of the antitoxin of type II TA systems, liberatingfree toxins that induce dormancy by inhibition of translation. Verstraeten et al. (2015a) identified Obg as a central regulator mediating another stringent response-dependentpersistence pathway, culminating in the induction of HokB, a type I toxin that causes persistence by disruption of the pmf. HipA phosphorylates and inactivates glutamyl-tRNA synthetase, leading to uncharged tRNAs that induce the stringent response by RelA-mediated (p)ppGpp synthesis. Increased (p)ppGpp subsequently causes persistenceas described above. Amato et al. (2013) and Amato and Brynildsen (2015) revealed how a diauxic carbon shift triggers distinct pathways for the formation of persisterstolerant to ofloxacin and ampicillin. Both pathways require the stringent response, DksA, and trans-translation. Nucleoid-associated proteins and the Clp protease systemare additionally required for ofloxacin- and ampicillin-tolerant persisters, respectively. Although terminal effectors were not identified, transcription inhibition, inhibition ofD tified

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. Clinical implications of persistence

The frequent treatment failure in patients suffering from Staphy-ococcus infections was the major motivation for Bigger’s study onhe bactericidal action of penicillin which lead to the first descrip-ion of bacterial persistence (Bigger, 1944). Today, persistence isndeed thought to play an important role in antibiotic therapy fail-re in the clinic (Fauvart et al., 2011). The finding of Kim Lewisnd colleagues that persisters are a major culprit for the antibioticecalcitrance of bacteria residing in biofilms, led them to pro-ose a simple and attractive model for the relapsing nature ofiofilm-related infections (Lewis, 2001; Spoering and Lewis, 2001)Fig. 4). The immune system can efficiently eradicate populations of

ongrowing bacteria, for example during treatment with bacterio-tatic antibiotics. Consequently, the small number of persisters thatemain after antibiotic therapy is not of clinical concern, as longs they are accessible for immune components. However, when

as the physiological basis for tolerance. Abbreviations: PPX, polyphosphatase; PPK,

bacteria adopt a biofilm-associated lifestyle, they become embed-ded in a self-produced protective matrix, called extracellular poly-meric substance. In a biofilm, the immune response is hampered,meaning that persisters can survive and re-establish an infectionwhen the antibiotic pressure drops (Fig. 4). Biofilm formation isindeed strongly linked with the chronic and recurrent nature ofmany infections (Costerton, 1999).

The model for persister-mediated relapse of biofilm-associatedinfections can be extended to any other situation involving animpaired immune response. For example, an immunodeficientstate is common among cancer patients, transplant patients receiv-ing immunosuppresive therapy, and HIV/AIDS patients. Moreover,bacteria have evolved a variety of strategies to escape the action ofthe host immune response, even in immunocompetent individuals.Some pathogens pertinaciously reside in body sites where they areshielded from immune components, and they are often impossible

to eradicate despite prolonged and aggressive antibiotic treat-ments. For example, Salmonella Typhi can persist in the gallbladder(Gonzalez-Escobedo et al., 2011), Helicobacter pylori chronicallyresides in the stomach (Salama et al., 2013), and P. aeruginosa, S.

82 J.E. Michiels et al. / Drug Resistance Updates 29 (2016) 76–89

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ig. 4. Model for persister-mediated relapse of bacterial infections. Antibiotic treatersisters. However, persisters residing in biofilms or host cells are protected fromesponse. Upon cessation of antibiotic therapy, persisters are able to resume growt

ureus, and other pathogens persistently thrive in the thickenedespiratory mucus of cystic fibrosis patients (Filkins and O’Toole,015). Bacteria can also evade the immune response by invad-

ng host cells (Fig. 4). Indeed, many pathogens involved in chronicnd recurrent infections, such as M. tuberculosis, uropathogenic. coli, and Salmonella Typhimurium are facultatively intracellu-ar (Hunstad and Justice, 2010; Kaiser et al., 2014; Sampson et al.,016). Importantly, the clinical implications of persisters are proba-ly not limited to this direct effect on therapy failure. As we discuss

n Box 2, it is expected that persisters also have a major contributiono the evolution of drug resistance.

.1. Studying persistence in vivo

The majority of persistence research has been carried out initro, and consequently knowledge on in vivo persistence mecha-isms is currently rather limited. Fortunately, several studies coulduccessfully extend in vitro findings to in vivo models or clini-al isolates, indicating the relevance and clinical applicability ofn vitro persistence research. For example, in vitro strategies toradicate E. coli and S. aureus persisters are successful in a mouseodel of urinary tract infection and deep-seated thigh infection,

espectively (Allison et al., 2011b; Conlon et al., 2013). Mutationsn hipA, an E. coli persister gene found by an in vitro mutagene-is screen, are common among clinical isolates of uropathogenic. coli (Schumacher et al., 2015). In Salmonella, mutations in thehpAB TA locus confer high persistence in vitro (Slattery et al., 2013),nd shpAB was later shown to contribute to in vivo persistence in

mouse model of typhoid fever (Helaine et al., 2014). However,n vitro and in vivo mechanisms of persistence might also differ.

. tuberculosis high-persistence mutants identified by an in vivocreen of a transposon mutant library using a murine infectionodel do not exhibit the same phenotype when grown in vitro.

mposing stresses that are thought to occur in vivo or growing

ipes out all non-persister bacteria, and the immune defense gets rid of planktonicimmune response, and survive the action of both the antibiotic and the immunecause infection relapse.

the bacteria in macrophage cell culture also cannot reproduce thehigh-persistence phenotype observed in vivo (Dhar and McKinney,2010).

Because persisters only constitute a small fraction of bacte-rial populations and continuously interconvert with the normalcell type, they are difficult to study in animal models. The ele-gant application of single-cell techniques relying on fluorescentproteins, time-lapse microscopy, and flow cytometry has recentlyallowed the direct study of in vivo persisters in animal infectionmodels for different pathogens. Intravenous infection of zebrafishlarvae with ‘green fluorescent protein’ (gfp)-expressing M. mar-inum allowed the observation of the bacterial burden in infectedanimals (Adams et al., 2011). By quantitatively tracking the flu-orescent signal in the infected larvae, M. marinum was shownto exhibit biphasic killing kinetics during in vivo antibiotic treat-ment. Residual bacteria were shown to be tolerant persisters: theirantibiotic susceptibility is unchanged from the parental strain. Thisin vivo imaging approach also revealed that the tolerant persis-ters are mainly located in macrophages. Remarkably, the persisterswere actively replicating, and their tolerance was attributed tothe high-level expression of drug efflux pumps. Manina et al.(2015) used time-lapse microscopy and a fusion of the 16S ribo-somal RNA gene with an unstable GFP variant to quantitate growthdynamics and transcriptional activity of single M. tuberculosis cells.It was found that bacteria explanted from the lungs of chron-ically infected mice harbor a subpopulation of nongrowing butmetabolically active bacteria, possibly responsible for relapse afteran initial successful course of antibiotics (Manina and McKinney,2013).

Helaine et al. (2014) used fluorescence dilution to investi-

gate the formation of nonreplicating S. Typhimurium persistersin a mouse typhoid model. Bacteria recovered from the mesen-teric lymph nodes of antibiotic-treated mice had not undergoneany replication and could resume growth in vitro in the absence

J.E. Michiels et al. / Drug Resistance Updates 29 (2016) 76–89 83

Box 2: Persistence and the evolution of drug resistanceIn addition to their direct contribution to clinical therapy failure,persisters may act as a ‘catalyst’ for the emergence of geneticresistance. Indeed, persisters form a continuous reservoir ofviable cells in the presence of antibiotics, and mathemati-cal modeling suggests increased development of resistancethrough persistence (Levin and Rozen, 2006). Although growthand division usually does not occur in the persister subpopula-tion, nondividing, stressed cells still accumulate mutations bymechanisms independent of DNA replication, a phenomenoncalled stationary-phase mutation, stress-induced mutation,or adaptive mutation (Bjedov et al., 2003; Loewe et al.,2003). Many of the stress responses involved in persistencehave been found to accelerate these mutational processes ingrowth-restricted environments (Cohen et al., 2013). Indeed,both the stringent response and the SOS response, knowndrivers of persister formation, have been linked with stress-induced mutation and the evolution of antibiotic resistance.For example, when P. aeruginosa lacks a functional stringentresponse, it does not form resistant colonies on ofloxacin-supplemented agar plates (Nguyen et al., 2011). Stress-inducedmutation critically depends on the SOS response, among otherstress responses (Al Mamun et al., 2012; Foster, 2007). More-over, stress responses have been linked with horizontal genetransfer. For example, an increased SOS response stronglyaccelerates the horizontal dissemination of antibiotic resis-tance genes in E. coli and V. cholerae (Beaber et al., 2004).Interestingly, the biofilm environment, which serves as a pro-tective niche for in vivo persisters, has been found to increaseboth mutation rates (Driffield et al., 2008; Ryder et al., 2012)and horizontal gene transfer (Savage et al., 2013).Although persistence and resistance are clearly fundamentallydifferent strategies, in some cases, persister cells and resis-tant mutants seem to rely on related tactics to circumventthe consequences of antibiotic treatment. For example, effluxpumps are important mediators of antibiotic resistance (Li andNikaido, 2009), and have additionally been shown to be impor-tant for intracellular persistence in mycobacteria (Adams et al.,2011) and paraquat-induced and spontaneous persistence inE. coli (Pu et al., 2016; Wu et al., 2012). In P. aeruginosa, fos-fomycin resistance mechanisms were found to be implicatedin persistence against ofloxacin treatment (De Groote et al.,2011). Mutations in genes of the nuo and opp operons of E. coliwere selected during periodic aminoglycoside treatment andcaused high persistence against different antibiotics (Van denBergh et al., 2016b). Additionally, mutations in these operonswere reported to occur in aminoglycoside-resistant mutants(Lázár et al., 2013; Suzuki et al., 2014). These overlaps betweenpersistence and resistance mechanisms could suggest that theevolutionary path toward genetic resistance is more readilyaccessible when high-persistence mutations in the same orrelated pathways or genes have already occurred.Despite the compelling theoretical arguments, the linkbetween persistence and the evolution of resistance isstill largely unexplored experimentally. In one study focus-ing on antibiotic tolerance but not specifically persistence,the antibiotic-tolerant Streptoccus pneumoniae vncS mutantexhibited an increased ability to acquire penicillin and strep-tomycin resistance by transformation with genomic DNAof a clinically resistant isolate (Novak et al., 1999). It wasalso reported that multidrug-resistant strains are often highlypenicillin-tolerant (Liu et al., 1985), and high persistence andresistance were recently found to be positively correlatedamong different Pseudomonas species (Vogwill et al., 2016).Obviously, understanding bacterial persistence is not onlyrequired to fight chronic and recrudescent infections, but willalso help to devise strategies that limit the emergence andspread of antibiotic-resistant pathogens.As discussed in Box 1, tolerant phenotypic variants, calleddrug-tolerant persisters (DTPs), are also found in cancer cellpopulations, and a parallel argumentation applies for DTPs as

a reservoir for the emergence of therapy resistance. Interest-ingly, two recent studies have independently confirmed thatin addition to the selection of pre-existing resistant clones,acquired resistance can also result from de novo mutations inDTPs (Hata et al., 2016; Ramirez et al., 2016). Thus, unravelingand targeting the molecular mechanisms underlying forma-tion of DTPs may represent a novel and promising approach to

prevent therapy resistance in cancer treatment (Oxnard, 2016).

of antibiotics, showing that they are persisters. In this model,Salmonella replicates in macrophages, and it was shown thatthe acidic and nutrient-poor conditions of the intracellular envi-ronment cause a 100- to 1000-fold increase in the number ofpersisters, through a mechanism dependending on TA modulesand the Lon protease. In another study, a large subset of toler-ant Salmonella could not be eradicated from the cecum-draininglymph nodes by ciprofloxacin treatment of infected mice (Kaiseret al., 2014). These tolerant pathogens were capable of re-initiatinginfection when injected in the peritoneal cavity of new mice,and can therefore account for relapse of infection. Corroborat-ing the findings of Helaine et al. (2014), tolerant bacteria residedmainly intracellularly, in dendritic cells, and were slow-growing asinferred from fluctuations in the proportion of differentially taggedstrains and plasmid dilution experiments. The correlation betweenin vivo growth rate and tolerance was investigated further usingan engineered fluorescent protein that exhibits a time-dependentspectral shift (Claudi et al., 2014). Remarkably, although nondivid-ing bacteria survive best, their impact is small because they arerare. Instead, the majority of survivors originates from the largersubpopulation with an intermediate growth rate and tolerancelevel.

It is worth noting that many of the factors that have been iden-tified as triggers of in vitro persister formation are encounteredby bacteria in vivo (Balaban, 2011). Firstly, microbial pathogensare confronted with nutrient limitation during infection, as thehost actively restricts access to essential nutrients, most notablyiron (Kwaik and Bumann, 2013). Secondly, the pathogenicity ofsome bacteria, such as P. aeruginosa and S. aureus, depends on quo-rum sensing, as it regulates the expression of important virulencefactors (Rutherford and Bassler, 2012). Thirdly, reactive oxygenspecies are important antimicrobial molecules used by cells of theinnate immune response to fight of infections (Yang et al., 2013).Finally, in vivo persistence could even be promoted by the antibi-otics that are used to treat the infection (Ren et al., 2015).

3.2. A link between persistence and virulence?

The stringent response and toxin–antitoxin modules are emerg-ing as the central regulator and effectors of persistence in differentbacterial species (Maisonneuve and Gerdes, 2014). At the sametime, the virulent characteristics of most bacterial pathogenshave been found to depend critically on the stringent response(Dalebroux et al., 2010), and toxin–antitoxin modules are knownto influence biofilm formation and virulence in a number ofpathogenic bacteria (Wang and Wood, 2011; Wen et al., 2014).Many other identified mediators of persistence play a dual roleby also regulating aspects of bacterial virulence, including quo-rum sensing (Rutherford and Bassler, 2012), indole signaling(Bommarius et al., 2013), and the SOS response (Gotoh et al.,2010).

Remarkably, it seems that an even more direct link between

persistence and virulence exists. In Salmonella, bistable expres-sion of the type III secretion system, an important virulencefactor, causes the coexistence of two phenotypically distinctsubpopulations: a slow-growing, virulent subpopulation and a

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ast-growing, avirulent subpopulation (Sturm et al., 2011). Inter-stingly, the decreased growth rate of the virulent subpopulationlso causes it to be highly antibiotic-tolerant (Arnoldini et al.,014). Without antibiotic treatment, genetic cheaters of this sys-em, namely avirulent mutants that never express the type IIIecretion system, can outgrow the wild type that displays phe-otypic bistability. However, the lack of the type III secretionystem makes the avirulent cheaters unable to invade host tis-ues. Interestingly, it was shown that only wild type clonesnd not avirulent mutants are able to reseed the infection after

course of antibiotic therapy (Diard et al., 2014). Moreover,issue invasion was a necessary step for infection relapse, con-rming the observation that in vivo persisters mainly reside inost cells (Helaine et al., 2014; Kaiser et al., 2014). Thus, antibi-tic treatment influences within-host evolution of pathogens byelecting for both virulence and the associated persister pheno-ype.

The overlap in mechanisms mediating persistence and virulenceuggests that therapies targeting these mechanisms could have awofold effect, inhibiting both the pathogen’s ability to replicatend disseminate within a host and its tendency to form antibiotic-olerant persisters that cause relapse of infection. In this respect,t is interesting to note that a combination of tobramycin withnti-virulence quorum sensing inhibitors showed increased effi-acy against P. aeruginosa in a chronic, foreign body-associatediofilm infection in mice (Christensen et al., 2012). Since quorumensing is involved in P. aeruginosa persistence (Möker et al., 2010),t is likely that a reduction in the number of persisters is respon-ible for the increased therapeutic response in this chronic biofilmnfection model.

.3. Contribution of persistence to clinical therapy failure

As to the underlying causes of the antibiotic crisis, genetic antibi-tic resistance has always dominated the scene, and antimicrobialusceptibility testing by assessing the MIC is mainstream practiceo guide antimicrobial therapy (Jenkins and Schuetz, 2012). How-ver, many pathogens are documented to be highly susceptible inaboratory testing yet unresponsive in clinical therapy, implicatingntibiotic tolerance or persistence. The clinical implications of per-istence are not as well established as is the case for resistance, buthere are many indications that it can considerably contribute toherapy failure, and is currently largely overlooked (Van den Berght al., 2016a). More than half a century ago, it was already describedhat prolonged antibiotic therapy of M. tuberculosis infections in

ice could not eradicate all pathogens, and the survivors wereighly susceptible upon in vitro testing (Mccune and Tompsett,956). In S. pneumoniae, loss-of-function of the VncS histidineinase suppresses autolytic activity and causes high tolerance toancomycin, with unchanged MIC (Novak et al., 1999). In a rabbiteningitis model, the parent strain was readily killed upon intra-

enous vancomycin treatment, while the density of the tolerantncS mutant remained constant. Strikingly, vncS loss-of-functionutations were also found in clinical isolates, indicating the rele-

ance of this tolerance mechanism in clinical settings (Novak et al.,999). Similarly, high-persistence mutations in hipA are commonmong clinical isolates of uropathogenic E. coli strains (Schumachert al., 2015).

HIV/AIDS patients are often confronted with multiple roundsf relapsing Salmonella bloodstream infections. Isolates from thenitial infection and subsequent episodes are genetically identical,ointing to relapse of the primary strain due to tolerant persis-

ers rather than reinfection (Gordon et al., 2002; Okoro et al.,012). Urinary tract infections similarly recur after antibiotic ther-py in 20–30% of affected women (Ejrnaes et al., 2006; Foxman,003). Confirming a causal role for persistence, a comparison of

e Updates 29 (2016) 76–89

uropathogenic E. coli isolated from same-strain recurrent versusacute infections showed equal antibiotic susceptibilities (MICs)but higher persister levels in the strains from recurrent infec-tions (Goneau et al., 2014). In chronic lung infections of cysticfibrosis patients, P. aeruginosa persister levels increase over timewhile MICs remain largely unchanged, indicating that persistenceunderlies the recalcitrance of cystic fibrosis lung infections(Mulcahy et al., 2010).

3.4. Periodic antibiotic treatment selects for increased persistence

A large body of experimental work has established that bacteriacan rapidly evolve antibiotic resistance during antibiotic treatment,both in vitro and in vivo. In the case of persistence, much less isknown about its dynamics and evolvability in view of antibac-terial therapy. Mathematical models suggest that bet-hedgingphenotypes such as persistence are an evolutionary adaptationto environments characterized by fluctuating episodes of stress,and that the level of persisters reflects the frequency of stressperiods (Kussell et al., 2005). Based on these theoretical pre-dictions, one could hypothesize that pathogenic bacteria evolvehigh persister levels rather than genetic resistance when antibi-otics are applied in a high-dose periodic fashion, which is thecase in certain infections, such as those of cystic fibrosis patients,and with certain antibiotics, such as aminoglycosides (Stankowiczet al., 2015). As described above, selection for high persistencehas already been observed during chronic infections in vivo, butsince detailed information about the antibiotic treatment his-tory of these clinical isolates is lacking, no clear conclusions onthe evolutionary aspects of persistence can be drawn from thesestudies.

However, recent work has shed light on the evolutionarydynamics of persistence by evolving bacterial populations in vitrounder precisely controlled antibiotic treatment schedules. Usually,in vitro evolution experiments under antibiotic stress allow thebacteria to grow in the continuous presence of elevating concen-trations of antibiotics, successfully revealing adaptive paths towardresistance (Jansen et al., 2013) (Fig. 5). In an alternative approach,growth and stress are uncoupled and applied to the populationin a cyclic fashion by periodically applying high concentrationsof antibiotics and subsequently transferring surviving persisters tofresh, antibiotic-free medium (Fig. 5). Fridman et al. (2014) inter-mittently exposed E. coli populations to high concentrations ofampicillin and observed the rapid evolution of antibiotic tolerancewithout any change in MIC. Ampicillin mainly affects dividing bac-teria, and increased tolerance was conferred by a delay in regrowthupon transfer to antibiotic-containing medium. Remarkably, theincrease in lag time was tuned to the duration of antibiotic expo-sure. These findings were confirmed and extended by Van denBergh et al. (2016b), who found that once-daily dosing of aminogly-cosides rapidly selects for mutants forming up to 100% of persisters.Interestingly, the number of days without stress between consec-utive antibiotic treatments correlates with the evolved level ofpersisters, confirming long-standing theoretical predictions of bet-hedging and persistence (Kussell et al., 2005). Worryingly, similarevolutionary dynamics were observed in the ESKAPE pathogens, agroup of highly important nosocomial pathogens including Entero-coccus faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa,and Enterobacter spp, indicating that this might represent a noveland currently overlooked strategy of pathogens to circumventantibiotic eradication in clinical infections (Michiels et al., 2016).

In S. aureus, periodic treatment with daptomycin also led to hightolerance without a change in MIC (Mechler et al., 2015). In contrastto the results of Fridman et al. (2014), increased tolerance in theseexamples is not caused by an extended lag phase, as nongrowing

J.E. Michiels et al. / Drug Resistance Updates 29 (2016) 76–89 85

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tationary phase cultures were challenged directly with antibioticsble to kill nondividing cells.

.5. Combating bacterial persister cells

Although it has become clear that persistence plays an impor-ant role in clinical antibiotic therapy failure, it is currently notaken into account when making treatment decisions for bacterialnfections. Clearly, efforts should be made to include an assess-

ent of persistence to guide antibiotic therapy (Brauner et al.,016; Van den Bergh et al., 2016a). However, since persister cellsre often multidrug-tolerant, there is also an urgent need for newompounds specifically designed to target persisters. To date, mul-iple strategies have been described but in most cases their in vivopplicability remains to be investigated.

One approach that has been taken is the screening of small-olecule libraries, which identified compounds that selectively kill

onreplicating mycobacteria (Bryk et al., 2008) or promote per-ister resuscitation and killing in combination with conventionalntibiotics (Kim et al., 2011). Others have argued to consider ratio-al strategies against persisters, by using adjuvant molecules totimulate antibiotic-mediated killing (Allison et al., 2011a). Forxample, persisters tolerant to aminoglycosides can be sensitizedy stimulating metabolic activity and consequently proton motiveorce-dependent antibiotic uptake. Allison et al. (2011b) reportedhat the combination of aminoglycosides with specific metabo-ites enhances efficacy against E. coli and S. aureus biofilms, andmproves the treatment of chronic infections in a mouse model for. coli urinary tract infection. Similar results were obtained withobramycin in P. aeruginosa biofilms formed on abiotic surfacesBarraud et al., 2013) but could not be replicated in biofilms grownn cystic fibrosis-derived airway cells (Price et al., 2015). Otherombinations of antibiotics with metabolites have been reported toncrease anti-persister activity, such as gentamicin with L-arginineLebeaux and Chauhan, 2014), daptomycin with glucose (Prax et al.,016), and fluoroquinolones with L-serine (Duan et al., 2016).

Proteins involved in persister formation are other potentialargets for rationally-designed anti-persister therapies. However,ew such examples exist, probably because the persisters of aingle population are likely heterogeneous and formed by dis-inct mechanisms (Allison et al., 2011a; Amato and Brynildsen,015; Levin et al., 2014). In one recent example, HipA inhibitorsere identified using an in silico screening approach and shown

o reduce persistence (Li et al., 2016). Another successful strat-

gy targets essential cellular features that are important for thentegrity and viability of bacteria, whether they are in a persistertate or not. For example, cationic, membrane-penetrating peptidesr a combination of these peptides with bacteriophage-derived

experiments with antibiotics. As theory predicts, and recent evolution experimentstence mutants rather than antibiotic resistance, contrary to environments with aatch the increasing MICs.

peptidoglycan-degrading endolysins have been shown to be highlyeffective against bacterial persisters (Briers et al., 2014; Chen et al.,2011; Defraine et al., 2016). Acyldepsipeptides represent a novelclass of antibiotics effective against Gram-positive bacteria thatwork by stimulating uncoordinated, ATP-independent proteolysisby activation of the ClpP protease (Brötz-Oesterhelt et al., 2005;Kirstein et al., 2009). Conlon et al. (2013) reasoned that thesemolecules might also be able to trigger protein degradation indormant persisters. Indeed, ADEP4, a semisynthetic acyldepsipep-tide, exerts strong bactericidal activity against S. aureus populationsthat are tolerant to conventional antibiotics. ClpP null mutantsresistant against ADEP4 arise rapidly, but display increased sen-sitivity toward other antibiotics such as rifampicin. As such, acombination therapy of rifampicin and ADEP4 can successfully ster-ilize a mouse model of chronic, deep-seated thigh infection that isuntreatable with conventional antibiotics.

Remarkably, recent data indicates that the mainstay DNA-crosslinking anti-cancer drugs mitomycin C and cisplatin holdpromise for the treatment of bacterial infections, as they are ableto eradicate bacterial persisters. A low, clinically achievable doseof mitomycin C eradicates Borrelia burgdorferi persisters in vitro(Sharma et al., 2015). Similarly, Kwan et al. (2015) reported mit-omycin C-mediated killing of persisters of E. coli, P. aeruginosa,and S. aureus in different in vitro and in vivo models, and killingof both normal and persister cells was attributed to DNA cross-linking. Later, cisplatin was also shown to exhibit broad-spectrumantibacterial activity against growing, nongrowing, and persistercells (Chowdhury et al., 2016). An additional advantage of mito-mycin C and cisplatin is that they have already been FDA-approvedas chemotherapeutic cancer drugs. Repurposing of existing drugsconsiderably diminishes the time and costs associated with themarket introduction of a new drug. However, these general DNAcross-linking agents affect host DNA as well and are associated withserious side effects, rendering their clinical applicability question-able.

4. Concluding remarks

In recent years, tremendous progress has been made in ourunderstanding of bacterial persistence. However, many ques-tions still remain unresolved despite intensive scientific efforts.Persistence has turned out to be a highly complex phenomenon,governed by many different and heterogeneous molecular mecha-nisms and requiring both active and passive responses for defense

against antibiotic lethality. Although the stringent response andTA modules appear to be broadly conserved among the diversemolecular pathways of E. coli persistence, it is unclear if this holdstrue for other species. For example, the stringent response and TA

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odules have apparently no role in the formation of persister cellsn S. aureus (Conlon et al., 2016).

It is intriguing to note the many similarities that exist betweenersisters in the fields of microbiology and cancer. Due to the pres-nce of drug-tolerant persisters that are phenotypically distinctut genetically identical to the rest of the population, clinical ther-py of both bacterial infections and cancer often fails despite drugusceptibility. In both cases, surviving persisters are expected toontribute to the development of drug resistance, and this hypoth-sis was recently confirmed for drug-tolerant persisters in cancer.emarkably, mitomycin C and cisplatin, compounds with a longistory as anti-cancer drugs, were recently found to be highly activegainst bacterial persisters.

Crucially, because many lines of evidence suggest that persis-ers complicate antibiotic eradication of infections, they shouldrgently be given more attention to help control the current antibi-tic crisis. Therefore, more insights into the in vivo mechanisms ofersister formation are required, and proposed persister-targetingherapies should advance to clinical testing to determine their real-ife applicability.

cknowledgements

J.E.M. received a doctoral fellowship from the Agency for Inno-ation by Science and Technology (IWT). This work was supportedy grants from the FWO (G047112N, G0B2515N), the KU Leuvenesearch Council (PF/10/010) and the Interuniversity Attractionoles Program initiated by the Belgian Science Policy Office (IAP7/28).

eferences

ckermann, M., 2015. A functional perspective on phenotypic heterogeneity inmicroorganisms. Nat. Rev. Microbiol. 13, 497–508.

dams, K., Takaki, K., Connolly, L., 2011. Drug tolerance in replicating mycobacteriamediated by a macrophage-induced efflux mechanism. Cell 145, 39–53.

l Mamun, A.A.M., Lombardo, M., Shee, C., Lisewski, A.M., Gonzalez, C., Lin, D.,Nehring, R.B., Saint-ruf, C., Gibson, J.L., Frisch, R.L., Lichtarge, O., Hastings, P.J.,Rosenberg, S.M., 2012. Identity and function of a large gene networkunderlying mutagenic repair of DNA breaks. Science 338, 1344–1348.

llison, K.R., Brynildsen, M.P., Collins, J.J., 2011a. Heterogeneous bacterialpersisters and engineering approaches to eliminate them. Curr. Opin.Microbiol. 14, 593–598.

llison, K.R., Brynildsen, M.P., Collins, J.J., 2011b. Metabolite-enabled eradication ofbacterial persisters by aminoglycosides. Nature 473, 216–220.

mato, S.M., Brynildsen, M.P., 2015. Persister heterogeneity arising from a singlemetabolic stress. Curr. Biol. 25, 2090–2098.

mato, S.M., Brynildsen, M.P., 2014. Nutrient transitions are a source of persistersin Escherichia coli biofilms. PLOS ONE 9, e93110.

mato, S.M., Orman, M.A., Brynildsen, M.P., 2013. Metabolic control of persisterformation in Escherichia coli. Mol. Cell 50, 475–487.

rnoldini, M., Vizcarra, I.A., Pena-Miller, R., Stocker, N., Diard, M., Vogel, V.,Beardmore, R.E., Hardt, W.-D., Ackermann, M., 2014. Bistable expression ofvirulence genes in Salmonella leads to the formation of an antibiotic-tolerantsubpopulation. PLoS Biol. 12, e1001928.

alaban, N.Q., 2011. Persistence: mechanisms for triggering and enhancingphenotypic variability. Curr. Opin. Genet. Dev. 21, 768–775.

alaban, N.Q., Merrin, J., Chait, R., Kowalik, L., Leibler, S., 2004. Bacterial persistenceas a phenotypic switch. Science 305, 1622–1625.

arraud, N., Buson, A., Jarolimek, W., Rice, S.A., 2013. Mannitol enhances antibioticsensitivity of persister bacteria in Pseudomonas aeruginosa biofilms. PLOS ONE8, e84220.

arth, V.C., Rodrigues, B.Á., Bonatto, G.D., Gallo, S.W., Pagnussatti, V.E., Ferreira,C.A.S., de Oliveira, S.D., 2013. Heterogeneous persister cells formation inAcinetobacter baumannii. PLOS ONE 8, e84361.

eaber, J.W., Hochhut, B., Waldor, M.K., 2004. SOS response promotes horizontaldissemination of antibiotic resistance genes. Nature 427, 72–74.

hargava, N., Sharma, P., Capalash, N., 2014. Pyocyanin stimulates quorumsensing-mediated tolerance to oxidative stress and increases persister cellspopulation in Acinetobacter baumannii. Infect. Immun. 8, 3417–3425.

igger, J., 1944. Treatment of staphylococcal infections with penicillin byintermittent sterilisation. Lancet 244, 497–500.

jedov, I., Tenaillon, O., Gérard, B., Souza, V., Denamur, E., Radman, M., Taddei, F.,Matic, I., 2003. Stress-induced mutagenesis in bacteria. Science 300,1404–1409.

e Updates 29 (2016) 76–89

Black, D.S., Kelly, A.J., Mardis, M.J., Moyed, H.S., 1991. Structure and organization ofhip, an operon that affects lethality due to inhibition of peptidoglycan or DNAsynthesis. J. Bacteriol. 173, 5732–5739.

Bojsen, R., Regenberg, B., Gresham, D., Folkesson, A., 2016. A common mechanisminvolving the TORC1 pathway can lead to amphotericin B-persistence inbiofilm and planktonic Saccharomyces cerevisiae populations. Sci. Rep. 6, 21874.

Bommarius, B., Anyanful, A., Izrayelit, Y., Bhatt, S., Cartwright, E., Wang, W.,Swimm, A.I., Benian, G.M., Schroeder, F.C., Kalman, D., 2013. A family of indolesregulate virulence and Shiga toxin production in pathogenic E. coli. PLOS ONE8, e54456.

Brauner, A., Fridman, O., Gefen, O., Balaban, N.Q., 2016. Distinguishing betweenresistance, tolerance and persistence to antibiotic treatment. Nat. Rev.Microbiol. 14, 320–330.

Briers, Y., Walmagh, M., Grymonprez, B., Biebl, M., Pirnay, J.-P., Defraine, V.,Michiels, J., Cenens, W., Aertsen, A., Miller, S., Lavigne, R., 2014. Art-175 is ahighly efficient antibacterial against multidrug-resistant strains and persistersof Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 58,3774–3784.

Brötz-Oesterhelt, H., Beyer, D., Kroll, H.-P., Endermann, R., Ladel, C., Schroeder, W.,Hinzen, B., Raddatz, S., Paulsen, H., Henninger, K., Bandow, J.E., Sahl, H.-G.,Labischinski, H., 2005. Dysregulation of bacterial proteolytic machinery by anew class of antibiotics. Nat. Med. 11, 1082–1087.

Bryk, R., Gold, B., Venugopal, A., Singh, J., Samy, R., Pupek, K., Cao, H., Popescu, C.,Gurney, M., Hotha, S., Cherian, J., Rhee, K., Ly, L., Converse, P.J., Ehrt, S., Vandal,O., Jiang, X., Schneider, J., Lin, G., Nathan, C., 2008. Selective killing ofnonreplicating mycobacteria. Cell Host Microbe 3, 137–145.

Cara, S., Tannock, I.F., 2001. Retreatment of patients with the same chemotherapy:implications for clinical mechanisms of drug resistance. Ann. Oncol. 12, 23–27.

Cataudella, I., Sneppen, K., Gerdes, K., Mitarai, N., 2013. Conditional cooperativityof toxin–antitoxin regulation can mediate bistability between growth anddormancy. PLoS Comput. Biol. 9, e1003174.

Cataudella, I., Trusina, A., Sneppen, K., Gerdes, K., Mitarai, N., 2012. Conditionalcooperativity in toxin–antitoxin regulation prevents random toxin activationand promotes fast translational recovery. Nucleic Acids Res. 40, 6424–6434.

Chen, J., Li, Y., Yu, T.-S., McKay, R.M., Burns, D.K., Kernie, S.G., Parada, L.F., 2012. Arestricted cell population propagates glioblastoma growth afterchemotherapy. Nature 488, 522–526.

Chen, X., Zhang, M., Zhou, C., Kallenbach, N.R., Ren, D., 2011. Control of bacterialpersister cells by Trp/Arg-containing antimicrobial peptides. Appl. Environ.Microbiol. 77, 4878–4885.

Cheverton, A.M., Gollan, B., Przydacz, M., Wong, C.T., Mylona, A., Hare, S.A., Helaine,S., 2016. A Salmonella toxin promotes persister formation through acetylationof tRNA. Mol. Cell, http://dx.doi.org/10.1016/j.molcel.2016.05.002.

Chowdhury, N., Wood, T.L., Martínez-Vázquez, M., García-Contreras, R., Wood, T.K.,2016. DNA-crosslinker cisplatin eradicates bacterial persister cells. Biotechnol.Bioeng., http://dx.doi.org/10.1002/bit.25963.

Christensen, L.D., Van Gennip, M., Jakobsen, T.H., Alhede, M., Hougen, H.P., Høiby,N., Bjarnsholt, T., Givskov, M., 2012. Synergistic antibacterial efficacy of earlycombination treatment with tobramycin and quorum-sensing inhibitorsagainst Pseudomonas aeruginosa in an intraperitoneal foreign-body infectionmouse model. J. Antimicrob. Chemother. 67, 1198–1206.

Claudi, B., Spröte, P., Chirkova, A., Personnic, N., Zankl, J., Schürmann, N., Schmidt,A., Bumann, D., 2014. Phenotypic variation of Salmonella in host tissues delayseradication by antimicrobial chemotherapy. Cell 158, 722–733.

Cohen, N.R., Lobritz, M.A., Collins, J.J., 2013. Microbial persistence and the road todrug resistance. Cell Host Microbe 13, 632–642.

Conlon, B.P., Nakayasu, E.S., Fleck, L.E., LaFleur, M.D., Isabella, V.M., Coleman, K.,Leonard, S.N., Smith, R.D., Adkins, J.N., 2013. Activated ClpP kills persisters anderadicates a chronic biofilm infection. Nature 503, 365–370.

Conlon, B.P., Rowe, S.E., Gandt, A.B., Nuxoll, A.S., Donegan, N.P., Zalis, E.A., Clair, G.,Adkins, J.N., Cheung, A.L., Lewis, K., 2016. Persister formation in Staphylococcusaureus is associated with ATP depletion. Nat. Microbiol. 1, 16051.

Costerton, J.W., 1999. Bacterial biofilms: a common cause of persistent infections.Science 284, 1318–1322.

Dalebroux, Z.D., Svensson, S.L., Gaynor, E.C., Swanson, M.S., 2010. ppGpp conjuresbacterial virulence. Microbiol. Mol. Biol. Rev. 74, 171–199.

De Groote, V.N., Fauvart, M., Kint, C.I., Verstraeten, N., Jans, A., Cornelis, P., Michiels,J., 2011. Pseudomonas aeruginosa fosfomycin resistance mechanisms affectnon-inherited fluoroquinolone tolerance. J. Med. Microbiol. 60, 329–336.

De Groote, V.N., Verstraeten, N., Fauvart, M., Kint, C.I., Verbeeck, A.M., Beullens, S.,Cornelis, P., Michiels, J., 2009. Novel persistence genes in Pseudomonasaeruginosa identified by high-throughput screening. FEMS Microbiol. Lett. 297,73–79.

Defraine, V., Scheurmans, J., Grymonprez, B., Govers, S.K., Aertsen, A., Fauvart, M.,Michiels, J., Lavigne, R., Briers, Y., 2016. Efficacy of Artilysin® Art-175 againstresistant and persistent Acinetobacter baumannii. Antimicrob. AgentsChemother. 60, 3480–3488.

Dhar, N., McKinney, J.D., 2010. Mycobacterium tuberculosis persistence mutantsidentified by screening in isoniazid-treated mice. Proc. Natl. Acad. Sci. U. S. A.107, 12275–12280.

Dhar, N., McKinney, J.D., 2007. Microbial phenotypic heterogeneity and antibiotic

tolerance. Curr. Opin. Microbiol. 10, 30–38.

Diard, M., Sellin, M.E., Dolowschiak, T., Arnoldini, M., Ackermann, M., Hardt, W.-D.,2014. Antibiotic treatment selects for cooperative virulence of SalmonellaTyphimurium. Curr. Biol. 24, 2000–2005.

istanc

D

D

D

D

E

E

E

E

E

F

F

F

F

F

F

F

G

G

G

G

G

G

G

G

G

G

H

H

H

J.E. Michiels et al. / Drug Res

örr, T., Lewis, K., Vulic, M., 2009. SOS response induces persistence tofluoroquinolones in Escherichia coli. PLoS Genet. 5, e1000760.

örr, T., Vulic, M., Lewis, K., 2010. Ciprofloxacin causes persister formation byinducing the TisB toxin in Escherichia coli. PLoS Biol. 8, e1000317.

riffield, K., Miller, K., Bostock, J.M., O’Neill, A.J., Chopra, I., 2008. Increasedmutability of Pseudomonas aeruginosa in biofilms. J. Antimicrob. Chemother.61, 1053–1056.

uan, X., Huang, X., Wang, X., Yan, S., Guo, S., Abdalla, A.E., Huang, C., Xie, J., 2016.l-Serine potentiates fluoroquinolone activity against Escherichia coli byenhancing endogenous reactive oxygen species production. J. Antimicrob.Chemother., dkw114.

jrnaes, K., Sandvang, D., Lundgren, B., Holm, S., Monsen, T., Lundholm, R., Ferry, S.,Frimodt-moller, N., 2006. Pulsed-field gel electrophoresis typing ofEscherichia coli strains from samples collected before and after pivmecillinamor placebo treatment of uncomplicated community-acquired urinary tractinfection in women. J. Clin. Microbiol. 44, 1776–1781.

ldar, A., Elowitz, M.B., 2010. Functional roles for noise in genetic circuits. Nature467, 167–173.

lowitz, M.B., Levine, A.J., Siggia, E.D., Swain, P.S., 2002. Stochastic gene expressionin a single cell. Science 297, 1183–1186.

ng, R.H., Padberg, F.T., Smith, S.M., 1991. Bactericidal effects of antibiotics onslowly growing and nongrowing bacteria. Antimicrob. Agents Chemother. 35,1824–1828.

yler, C.E., Rich, J.N., 2008. Survival of the fittest: cancer stem cells in therapeuticresistance and angiogenesis. J. Clin. Oncol. 26, 2839–2845.

auvart, M., De Groote, V.N., Michiels, J., 2011. Role of persister cells in chronicinfections: clinical relevance and perspectives on anti-persister therapies. J.Med. Microbiol. 60, 699–709.

ilkins, L.M., O’Toole, G.A., 2015. Cystic fibrosis lung infections: polymicrobial,complex, and hard to treat. PLoS Pathog. 11, e1005258.

ord, A., Mansur, M.B., Furness, C.L., van Delft, F.W., Okamura, J., Suzuki, T.,Kobayashi, H., Kaneko, Y., Greaves, M., 2015. Protracted dormancy ofpre-leukaemic stem cells. Leukemia 29, 2202–2207.

oster, P.L., 2007. Stress-induced mutagenesis in bacteria. Crit. Rev. Biochem. Mol.Biol. 42, 373–397.

oxman, B., 2003. Epidemiology of urinary tract infections: incidence, morbidity,and economic costs. Dis. Mon. 49, 53–70.

ridman, O., Goldberg, A., Ronin, I., Shoresh, N., Balaban, N.Q., 2014. Optimizationof lag time underlies antibiotic tolerance in evolved bacterial populations.Nature 99, 418–421.

ung, D.K.C., Chan, E.W.C., Chin, M.L., Chan, R.C.Y., 2010. Delineation of a bacterialstarvation stress response network which can mediate antibiotic tolerancedevelopment. Antimicrob. Agents Chemother. 54, 1082–1093.

ardner, A., West, S.A., Griffin, A.S., 2007. Is bacterial persistence a social trait?PLoS ONE 2, e752.

efen, O., Gabay, C., Mumcuoglu, M., Engel, G., Balaban, N.Q., 2008. Single-cellprotein induction dynamics reveals a period of vulnerability to antibiotics inpersister bacteria. Proc. Natl. Acad. Sci. U. S. A. 105, 6145–6149.

erdes, K., Rasmussen, P.B., Molin, S., 1986. Unique type of plasmid maintenancefunction: postsegregational killing of plasmid-free cells. Proc. Natl. Acad. Sci. U.S. A. 83, 3116–3120.

ermain, E., Castro-Roa, D., Zenkin, N., Gerdes, K., 2013. Molecular mechanism ofbacterial persistence by HipA. Mol. Cell 52, 1–7.

irgis, H.S., Harris, K., Tavazoie, S., 2012. Large mutational target size for rapidemergence of bacterial persistence. Proc. Natl. Acad. Sci. U. S. A. 109,12740–12745.

lickman, M.S., Sawyers, C.L., 2012. Converting cancer therapies into cures: lessonsfrom infectious diseases. Cell 148, 1089–1098.

oneau, L.W., Yeoh, N.S., Macdonald, K.W., Cadieux, P.A., Burton, J.P., Razvi, H.,Reid, G., 2014. Selective target inactivation rather than global metabolicdormancy causes antibiotic tolerance in uropathogens. Antimicrob. AgentsChemother. 58, 2089–2097.

onzalez-Escobedo, G., Marshall, J.M., Gunn, J.S., 2011. Chronic and acute infectionof the gall bladder by Salmonella Typhi: understanding the carrier state. Nat.Rev. Microbiol. 9, 9–14.

ordon, M.A., Banda, H.T., Gondwe, M., Gordon, S.B., Boeree, M.J., Walsh, A.L.,Corkill, J.E., Hart, C.A., Gilks, C.F., Molyneux, M.E., 2002. Non-typhoidalsalmonella bacteraemia among HIV-infected Malawian adults: high mortalityand frequent recrudescence. AIDS 16, 1633–1641.

otoh, H., Kasaraneni, N., Devineni, N., Dallo, S.F., Weitao, T., 2010. SOSinvolvement in stress-inducible biofilm formation. Biofouling 26, 603–611.

all-Stoodley, L., Costerton, J.W., Stoodley, P., 2004. Bacterial biofilms: from thenatural environment to infectious diseases. Nat. Rev. Microbiol. 2, 95–108.

ansen, S., Lewis, K., Vulic, M., 2008. Role of global regulators and nucleotidemetabolism in antibiotic tolerance in Escherichia coli. Antimicrob. AgentsChemother. 52, 2718–2726.

ata, A.N., Niederst, M.J., Archibald, H.L., Gomez-caraballo, M., Siddiqui, F.M.,Mulvey, H.E., Maruvka, Y.E., Ji, F., Bhang, H.C., Krishnamurthy Radhakrishna, V.,Siravegna, G., Hu, H., Raoof, S., Lockerman, E., Kalsy, A., Lee, D., Keating, C.L.,

Ruddy, D.A., Damon, L.J., Crystal, A.S., Costa, C., Piotrowska, Z., Bardelli, A.,Iafrate, A.J., Sadreyev, R.I., Stegmeier, F., Getz, G., Sequist, L.V., Faber, A.C.,Engelman, J.A., 2016. Tumor cells can follow distinct evolutionary paths tobecome resistant to epidermal growth factor receptor inhibition. Nat. Med. 22,262–269.

e Updates 29 (2016) 76–89 87

Helaine, S., Cheverton, A.M., Watson, K.G., Faure, L.M., Matthews, S.A., Holden,D.W., 2014. Internalization of Salmonella by macrophages induces formation ofnonreplicating persisters. Science 343, 204–208.

Hobby, G., Meyer, K., Chaffee, E., 1942. Observations on the mechanism of action ofpenicillin. Exp. Biol. Med. 50, 281–285.

Hofsteenge, N., van Nimwegen, E., Silander, O.K., 2013. Quantitative analysis ofpersister fractions suggests different mechanisms of formation amongenvironmental isolates of E. coli. BMC Microbiol. 13, 25.

Hong, S.H., Wang, X., O’Connor, H.F., Benedik, M.J., Wood, T.K., 2012. Bacterialpersistence increases as environmental fitness decreases. Microb. Biotechnol.5, 509–522.

Hu, Y., Coates, A.R.M., 2005. Transposon mutagenesis identifies genes whichcontrol antimicrobial drug tolerance in stationary-phase Escherichia coli. FEMSMicrobiol. Lett. 243, 117–124.

Hu, Y., Kwan, B.W., Osbourne, D.O., Benedik, M.J., Wood, T.K., 2014. Toxin YafQincreases persister cell formation by reducing indole signaling. Environ.Microbiol. 17, 1275–1285.

Hunstad, D.A., Justice, S.S., 2010. Intracellular lifestyles and immune evasionstrategies of uropathogenic Escherichia coli. Annu. Rev. Microbiol. 64, 203–221.

Jansen, G., Barbosa, C., Schulenburg, H., 2013. Experimental evolution as anefficient tool to dissect adaptive paths to antibiotic resistance. Drug Resist.Updat. 16, 93–107.

Jenkins, S.G., Schuetz, A.N., 2012. Current concepts in laboratory testing to guideantimicrobial therapy. Mayo Clin. Proc. 87, 290–308.

Jiafeng, L., Fu, X., Chang, Z., 2015. Hypoionic shock treatment enablesaminoglycosides antibiotics to eradicate bacterial persisters. Sci. Rep. 5, 14247.

Johnson, P.J.T., Levin, B.R., 2013. Pharmacodynamics, population dynamics, and theevolution of persistence in Staphylococcus aureus. PLoS Genet. 9, e1003123.

Kaiser, P., Regoes, R.R., Dolowschiak, T., Wotzka, S.Y., Lengefeld, J., Slack, E., Grant,A.J., Ackermann, M., Hardt, W.-D., 2014. Cecum lymph node dendritic cellsharbor slow-growing bacteria phenotypically tolerant to antibiotic treatment.PLoS Biol. 12, e1001793.

Kaspy, I., Rotem, E., Weiss, N., Ronin, I., Balaban, N.Q., Glaser, G., 2013.HipA-mediated antibiotic persistence via phosphorylation of theglutamyl-tRNA-synthetase. Nat. Commun. 4, 3001.

Keren, I., Kaldalu, N., Spoering, A.L., Wang, Y., Lewis, K., 2004a. Persister cells andtolerance to antimicrobials. FEMS Microbiol. Lett. 230, 13–18.

Keren, I., Minami, S., Rubin, E., Lewis, K., 2011. Characterization and transcriptomeanalysis of Mycobacterium tuberculosis persisters. MBio 2, e00100–e111.

Keren, I., Shah, D., Spoering, A.L., Kaldalu, N., Lewis, K., 2004b. Specialized persistercells and the mechanism of multidrug tolerance in Escherichia coli. J. Bacteriol.186, 8172–8180.

Kim, J.J., Tannock, I.F., 2005. Repopulation of cancer cells during therapy: animportant cause of treatment failure. Nat. Rev. Cancer 5, 516–525.

Kim, J.-S., Heo, P., Yang, T.-J., Lee, K.-S., Cho, D.-H., Kim, B.T., Suh, J.-H., Lim, H.-J.,Shin, D., Kim, S.-K., Kweon, D.-H., 2011. Selective killing of bacterial persistersby a single chemical compound without affecting normal antibiotic-sensitivecells. Antimicrob. Agents Chemother. 55, 5380–5383.

Kint, C.I., Verstraeten, N., Fauvart, M., Michiels, J., 2012. New-found fundamentalsof bacterial persistence. Trends Microbiol. 20, 1–9.

Kirstein, J., Hoffmann, A., Lilie, H., Schmidt, R., Helga, R.W., Heike, B.O., Mogk, A.,Turgay, K., Rübsamen-Waigmann, H., Brötz-Oesterhelt, H., Mogk, A., Kürsad, T.,2009. The antibiotic ADEP reprogrammes ClpP, switching it from a regulated toan uncontrolled protease. EMBO Mol. Med. 1, 37–49.

Knoechel, B., Roderick, J.E., Williamson, K.E., Zhu, J., Lohr, J.G., Cotton, M.J., Gillespie,S.M., Fernandez, D., Ku, M., Wang, H., Piccioni, F., Silver, S.J., Jain, M., Pearson,D., Kluk, M.J., Ott, C.J., Shultz, L.D., Brehm, M.A., Greiner, D.L., Gutierrez, A.,Stegmaier, K., Kung, A.L., Root, D.E., Bradner, J.E., Aster, J.C., Kelliher, M.A.,Bernstein, B.E., 2014. An epigenetic mechanism of resistance to targetedtherapy in T cell acute lymphoblastic leukemia. Nat. Genet. 46, 364–370.

Korch, S.B., Henderson, T.A., Hill, T.M., 2003. Characterization of the hipA7 allele ofEscherichia coli and evidence that high persistence is governed by (p)ppGppsynthesis. Mol. Microbiol. 50, 1199–1213.

Korch, S.B., Hill, T.M., 2006. Ectopic overexpression of wild-type and mutant hipAgenes in Escherichia coli: effects on macromolecular synthesis and persisterformation. J. Bacteriol. 188, 3826–3836.

Kussell, E., Kishony, R., Balaban, N.Q., Leibler, S., 2005. Bacterial persistence: amodel of survival in changing environments. Genetics 169,1807–1814.

Kwaik, Y.A., Bumann, D., 2013. Microbial quest for food in vivo: “nutritionalvirulence” as an emerging paradigm. Cell. Microbiol. 15, 882–890.

Kwan, B.W., Chowdhury, N., Wood, T.K., 2015. Combatting bacterial infections bykilling persister cells with mitomycin C. Environ. Microbiol. 17, 4406–4414.

Kwan, B.W., Valenta, J.A., Benedik, M.J., Wood, T.K., 2013. Arrested proteinsynthesis increases persister-like cell formation. Antimicrob. AgentsChemother. 57, 1468–1473.

LaFleur, M.D., Kumamoto, C.A., Lewis, K., 2006. Candida albicans biofilms produceantifungal-tolerant persister cells. Antimicrob. Agents Chemother. 50,3839–3846.

Lambert, G., Estévez-salmeron, L., Oh, S., Liao, D., Emerson, B.M., Tlsty, T.D., Austin,R.H., 2011. An analogy between the evolution of drug resistance in bacterialcommunities and malignant tissues. Nat. Rev. Cancer 11, 375–382.

Lázár, V., Pal Singh, G., Spohn, R., Nagy, I., Horváth, B., Hrtyan, M., Busa-Fekete, R.,Bogos, B., Méhi, O., Csörg"o, B., Pósfai, G., Fekete, G., Szappanos, B., Kégl, B., Papp,B., Pál, C., 2013. Bacterial evolution of antibiotic hypersensitivity. Mol. Syst.Biol. 9, 700.

8 istanc

L

L

L

L

LL

L

L

L

L

L

M

M

M

M

M

M

M

M

M

M

M

M

M

M

M

M

N

N

O

8 J.E. Michiels et al. / Drug Res

ebeaux, D., Chauhan, A., 2014. pH-mediated potentiation of aminoglycosides killsbacterial persisters and eradicates in vivo biofilms. J. Infect. Dis. 210,1357–1366.

eung, V., Lévesque, C.M., 2012. A stress-inducible quorum-sensing peptidemediates the formation of persister cells with noninherited multidrugtolerance. J. Bacteriol. 194, 2265–2274.

evin, B.R., Concepción-Acevedo, J., Udekwu, K.I., 2014. Persistence: a copaceticand parsimonious hypothesis for the existence of non-inherited resistance toantibiotics. Curr. Opin. Microbiol. 21, 18–21.

evin, B.R., Rozen, D.E., 2006. Non-inherited antibiotic resistance. Nat. Rev.Microbiol. 4, 556–562.

ewis, K., 2010. Persister cells. Annu. Rev. Microbiol. 64, 357–372.ewis, K., 2001. Riddle of Biofilm Resistance. Antimicrob. Agents Chemother. 45,

999–1007.i, T., Yin, N., Liu, H., Pei, J., Lai, L., 2016. Novel inhibitors of toxin HipA reduce

multidrug tolerant persisters. ACS Med. Chem. Lett. 7, 449–453.i, X.-Z., Nikaido, H., 2009. Efflux-mediated drug resistance in bacteria: an update.

Drugs 69, 1555–1623.i, Y., Zhang, Y., 2007. PhoU is a persistence switch involved in persister formation

and tolerance to multiple antibiotics and stresses in Escherichia coli.Antimicrob. Agents Chemother. 51, 2092–2099.

iu, H.H., Tomasz, A., 1985. Penicillin tolerance in multiply drug-resistant neutralisolates of Streptococcus pneumoniae. J. Infect. Dis. 152, 365–372.

oewe, L., Textor, V., Scherer, S., 2003. High deleterious genomic mutation rate instationary phase of Escherichia coli. Science 302, 1558–1560.

a, C., Sim, S., Shi, W., Du, L., Xing, D., Zhang, Y., 2010. Energy production genessucB and ubiF are involved in persister survival and tolerance to multipleantibiotics and stresses in Escherichia coli. FEMS Microbiol. Lett. 303, 33–40.

aisonneuve, E., Castro-Camargo, M., Gerdes, K., 2013. (p)ppGpp controls bacterialpersistence by stochastic induction of toxin–antitoxin activity. Cell 154,1140–1150.

aisonneuve, E., Gerdes, K., 2014. Molecular mechanisms underlying bacterialpersisters. Cell 157, 539–548.

aisonneuve, E., Shakespeare, L.J., Girke, M., Gerdes, K., 2011. Bacterial persistenceby RNA endonucleases. Proc. Natl. Acad. Sci. U. S. A. 108, 13206–13211.

anina, G., Dhar, N., McKinney, J.D., 2015. Stress and host immunity amplifyMycobacterium tuberculosis phenotypic heterogeneity and induce nongrowingmetabolically active forms. Cell Host Microbe 17, 32–46.

anina, G., McKinney, J., 2013. A single-cell perspective on non-growing butmetabolically active (NGMA) bacteria. Curr. Top. Microbiol. Immunol. 374,135–161.

ccune, R.M., Tompsett, R., 1956. Fate of Mycobacterium tuberculosis in mousetissues as determined by the microbial enumeration technique. I. Thepersistence of drug-susceptible tubercle bacilli in the tissues despiteprolonged antimicrobial therapy. J. Exp. Med. 104, 737–762.

echler, L., Herbig, A., Paprotka, K., Fraunholz, M., Nieselt, K., Bertram, R., 2015. Anovel point mutation promotes growth phase-dependent daptomycintolerance in Staphylococcus aureus. Antimicrob. Agents Chemother. 59,5366–5376.

edaney, F., Dimitriu, T., Ellis, R.J., Raymond, B., 2015. Live to cheat another day:bacterial dormancy facilitates the social exploitation of �-lactamases. ISME J.10, 778–787.

enchón, S.A., Condat, C.A., 2011. Quiescent cells: a natural way to resistchemotherapy. Physica A 390, 3354–3361.

ichiels, J.E., Van den Bergh, B., Verstraeten, N., Fauvart, M., Michiels, J., 2016.In vitro emergence of high persistence upon periodic aminoglycoside challengein the ESKAPE pathogens. Antimicrob. Agents Chemother. AAC,00757-16.

öker, N., Dean, C.R., Tao, J., 2010. Pseudomonas aeruginosa increases formation ofmultidrug-tolerant persister cells in response to quorum-sensing signalingmolecules. J. Bacteriol. 192, 1946–1955.

ordukhova, E.A., Pan, J.-G., 2014. Stabilization ofhomoserine-O-cuccinyltransferase (MetA) decreases the frequency ofpersisters in Escherichia coli under stressful conditions. PLOS ONE 9,e110504.

oyed, H.S., Bertrand, K.P., 1983. hipA, a newly recognized gene of Escherichia coliK-12 that affects frequency of persistence after inhibition of murein synthesis.J. Bacteriol. 155, 768–775.

ulcahy, L.R., Burns, J.L., Lory, S., Lewis, K., 2010. Emergence of Pseudomonasaeruginosa strains producing high levels of persister cells in patients withcystic fibrosis. J. Bacteriol. 192, 6191–6199.

urakami, K., Ono, T., Viducic, D., Kayama, S., Mori, M., Hirota, K., Nemoto, K.,Miyake, Y., 2005. Role for rpoS gene of Pseudomonas aeruginosa in antibiotictolerance. FEMS Microbiol. Lett. 242, 161–167.

guyen, D., Joshi-Datar, A., Lepine, F., Bauerle, E., Olakanmi, O., Beer, K., McKay, G.,Siehnel, R., Schafhauser, J., Wang, Y., Britigan, B.E., Singh, P.K., 2011. Activestarvation responses mediate antibiotic tolerance in biofilms andnutrient-limited bacteria. Science 334, 982–986.

ovak, R., Henriques, B., Charpentier, E., Normark, S., Tuomanen, E., 1999.Emergence of vancomycin tolerance in Streptococcus pneumoniae. Nature 399,590–593.

koro, C.K., Kingsley, R.A., Quail, M.A., Kankwatira, A.M., Feasey, N.A., Parkhill, J.,Dougan, G., Gordon, M.A., 2012. High-resolution single nucleotidepolymorphism analysis distinguishes recrudescence and reinfection inrecurrent invasive nontyphoidal Salmonella Typhimurium disease. Clin. Infect.Dis. 54, 955–963.

e Updates 29 (2016) 76–89

Orman, M.A., Brynildsen, M.P., 2015. Inhibition of stationary phase respirationimpairs persister formation in E. coli. Nat. Commun. 6, 7983.

Orman, M.A., Brynildsen, M.P., 2013. Dormancy is not necessary or sufficient forbacterial persistence. Antimicrob. Agents Chemother. 57, 3230–3239.

Oxnard, G.R., 2016. The cellular origins of drug resistance in cancer. Nat. Med. 22,232–234.

Page, R., Peti, W., 2016. Toxin–antitoxin systems in bacterial growth arrest andpersistence. Nat. Chem. Biol. 12, 208–214.

Patra, P., Klumpp, S., 2013. Population dynamics of bacterial persistence. PLOS ONE8, e62814.

Pearl, S., Gabay, C., Kishony, R., Oppenheim, A., Balaban, N.Q., 2008. Nongeneticindividuality in the host-phage interaction. PLoS Biol. 6, e120.

Pedersen, K., Christensen, S.K., Gerdes, K., 2002. Rapid induction and reversal of abacteriostatic condition by controlled expression of toxins and antitoxins. Mol.Microbiol. 45, 501–510.

Persky, N.S., Ferullo, D.J., Cooper, D.L., Moore, H.R., Lovett, S.T., 2009. TheObgE/CgtA GTPase influences the stringent response to amino acid starvationin Escherichia coli. Mol. Microbiol. 73, 253–266.

Prax, M., Mechler, L., Weidenmaier, C., Bertram, R., 2016. Glucose augments killingefficiency of daptomycin challenged Staphylococcus aureus persisters. PLOSONE 11, e0150907.

Price, K.E., Orazi, G., Ruoff, K.L., Hebert, W.P., O’Toole, G.A., Mastoridis, P., 2015.Mannitol does not enhance tobramycin killing of Pseudomonas aeruginosa in acystic fibrosis model system of biofilm formation. PLOS ONE 10, e0141192.

Pu, Y., Zhao, Z., Li, Y., Zou, J., Ma, Q., Zhao, Y., Ke, Y., Zhu, Y., Chen, H., Baker, M.A.B.,Ge, H., Sun, Y., Xie, X.S., Bai, F., 2016. Enhanced efflux activity facilitates drugtolerance in dormant bacterial cells. Mol. Cell 62, 284–294.

Putrins, M., Kogermann, K., Lukk, E., Lippus, M., Varik, V., Tenson, T., 2015.Phenotypic heterogeneity enables uropathogenic Escherichia coli to evadekilling by antibiotics and serum complement. Infect. Immun. 83, 1056–1067.

Raha, D., Wilson, T.R., Peng, J., Peterson, D., Yue, P., Evangelista, M., Wilson, C.,Merchant, M., Settleman, J., 2014. The cancer stem cell marker aldehydedehydrogenase is required to maintain a drug-tolerant tumor cellsubpopulation. Cancer Res. 74, 3579–3590.

Ramirez, M., Rajaram, S., Steininger, R.J., Osipchuk, D., Roth, M.A., Morinishi, L.S.,Evans, L., Ji, W., Hsu, C.-H., Thurley, K., Wei, S., Zhou, A., Koduru, P.R., Posner,B.A., Wu, L.F., Altschuler, S.J., 2016. Diverse drug-resistance mechanisms canemerge from drug-tolerant cancer persister cells. Nat. Commun. 7, 10690.

Ren, H., He, X., Zou, X., Wang, G., Li, S., Wu, Y., 2015. Gradual increase in antibioticconcentration affects persistence of Klebsiella pneumoniae. J. Antimicrob.Chemother. 70, 3267–3272.

Rotem, E., Loinger, A., Ronin, I., Levin-Reisman, I., Gabay, C., Shoresh, N., Biham, O.,Balaban, N.Q., 2010. Regulation of phenotypic variability by a threshold-basedmechanism underlies bacterial persistence. Proc. Natl. Acad. Sci. U. S. A. 107,12541–12546.

Rutherford, S.T., Bassler, B.L., 2012. Bacterial quorum sensing: its role in virulenceand possibilities for its control. Cold Spring Harb. Perspect. Med. 2,a012427.

Ryder, V.J., Chopra, I., O’Neill, A.J., 2012. Increased mutability of staphylococci inbiofilms as a consequence of oxidative stress. PLoS ONE 7, e47695.

Salama, N.R., Hartung, M.L., Müller, A., 2013. Life in the human stomach:persistence strategies of the bacterial pathogen Helicobacter pylori. Nat. Rev.Microbiol. 11, 385–399.

Sampson, S.L., Helaine, S., Holden, D.W., Sampson, S.L., 2016. Elucidatingpopulation-wide mycobacterial replication dynamics at the single-cell level.Microbiology, http://dx.doi.org/10.1099/mic.0.000288.

Savage, V.J., Chopra, I., O’Neill, A.J., 2013. Staphylococcus aureus biofilms promotehorizontal transfer of antibiotic resistance. Antimicrob. Agents Chemother. 57,1968–1970.

Schumacher, M.A., Balani, P., Min, J., Chinnam, N.B., Hansen, S., Vulic, M., Lewis, K.,Brennan, R.G., 2015. HipBA–promoter structures reveal the basis of heritablemultidrug tolerance. Nature 524, 59–64.

Schumacher, M.A., Min, J., Link, T.M., Guan, Z., Xu, W., Ahn, Y.-H., Soderblom, E.J.,Kurie, J.M., Evdokimov, A., Moseley, M.A., Lewis, K., Brennan, R.G., 2012. Role ofunusual P loop ejection and autophosphorylation in HipA-mediatedpersistence and multidrug tolerance. Cell Rep. 2, 518–525.

Shah, D., Zhang, Z., Khodursky, A., Kaldalu, N., Kurg, K., Lewis, K., 2006. Persisters: adistinct physiological state of E. coli. BMC Microbiol. 6, 53.

Shan, Y., Lazinski, D., Rowe, S., Camilli, A., Lewis, K., 2015. Genetic basis of persistertolerance to aminoglycosides in Escherichia coli. MBio 6, e00078-15.

Sharma, B., Brown, A.V., Matluck, N.E., Hu, L.T., Lewis, K., 2015. Borrelia burgdorferi,the causative agent of Lyme disease, forms drug-tolerant persister cells.Antimicrob. Agents Chemother. 59, 4616–4624.

Sharma, S.V., Lee, D.Y., Li, B., Quinlan, M.P., Takahashi, F., Maheswaran, S.,McDermott, U., Azizian, N., Zou, L., Fischbach, M.A., Wong, K.-K., Brandstetter,K., Wittner, B., Ramaswamy, S., Classon, M., Settleman, J., 2010. Achromatin-mediated reversible drug-tolerant state in cancer cellsubpopulations. Cell 141, 69–80.

Slattery, A., Victorsen, A.H., Brown, A., Hillman, K., Phillips, G.J., 2013. Isolation ofhighly persistent mutants of Salmonella enterica serovar Typhimurium revealsa new toxin–antitoxin module. J. Bacteriol. 195, 647–657.

Smits, W.K., Kuipers, O.P., Veening, J.-W., 2006. Phenotypic variation in bacteria:the role of feedback regulation. Nat. Rev. Microbiol. 4, 259–271.

Spoering, A.L., Lewis, K., 2001. Biofilms and planktonic cells of Pseudomonasaeruginosa have similar resistance to killing by antimicrobials. J. Bacteriol. 183,6746–6751.

istanc

S

S

S

S

S

S

S

T

T

U

V

V

V

V

V

V

V

V

tolerance to different antibiotics. Front. Microbiol. 6, 1003.Wu, Y., Vulic, M., Keren, I., Lewis, K., 2012. Role of oxidative stress in persister

J.E. Michiels et al. / Drug Res

poering, A.L., Vulic, M., Lewis, K., Vulic, M., Lewis, K., 2006. GlpD and PlsBparticipate in persister cell formation in Escherichia coli. J. Bacteriol. 188,5136–5144.

prouffske, K., Merlo, L.M.F., Gerrish, P.J., Maley, C.C., Sniegowski, P.D., 2012.Cancer in light of experimental evolution. Curr. Biol. 22, R762–R771.

tankowicz, M.S., Ibrahim, J., Brown, D.L., 2015. Once-daily aminoglycoside dosing:an update on current literature. Am. J. Heal. Pharm. 72, 1357–1364.

tepanyan, K., Wenseleers, T., Duénez-Guzmán, E.A., Muratori, F., Van den Bergh,B., Verstraeten, N., De Meester, L., Verstrepen, K.J., Fauvart, M., Michiels, J.,2015. Fitness trade-offs explain low levels of persister cells in theopportunistic pathogen Pseudomonas aeruginosa. Mol. Ecol. 24, 1572–1583.

tewart, B., Rozen, D.E., 2011. Genetic variation for antibiotic persistence inEscherichia coli. Evolution 66, 933–939.

turm, A., Heinemann, M., Arnoldini, M., Benecke, A., Ackermann, M., Benz, M.,Dormann, J., Hardt, W.-D., 2011. The cost of virulence: retarded growth ofSalmonella Typhimurium cells expressing type III secretion system 1. PLoSPathog. 7, e1002143.

uzuki, S., Horinouchi, T., Furusawa, C., 2014. Prediction of antibiotic resistance bygene expression profiles. Nat. Commun. 5, 5792.

orrey, H.L., Keren, I., Via, L.E., Lee, J.S., Lewis, K., 2016. High persister mutants inMycobacterium tuberculosis. PLoS ONE 11, e0155127.

uomanen, E., Cozens, R., Tosch, W., Zak, O., Tomasz, A., 1986. The rate of killing ofEscherichia coli by beta-lactam antibiotics is strictly proportional to the rate ofbacterial growth. J. Gen. Microbiol. 132, 1297–1304.

noson, C., Wagner, E.G.H., 2008. A small SOS-induced toxin is targeted against theinner membrane in Escherichia coli. Mol. Microbiol. 70, 258–270.

an den Bergh, B., Michiels, J.E., Fauvart, M., Michiels, J., 2016a. Should we developscreens for multidrug tolerance? Expert Rev. Anti. Infect. Ther., 7210.

an den Bergh, B., Michiels, J.E., Wenseleers, T., Windels, E.M., Vanden Boer, P.,Kestemont, D., De Meester, L., Verstrepen, K.J., Verstraeten, N., Fauvart, M.,Michiels, J., 2016b. Frequency of antibiotic application drives rapidevolutionary adaptation of Escherichia coli persistence. Nat. Microbiol. 1, 16020.

an Melderen, L., 2010. Toxin–antitoxin systems: why so many, what for? Curr.Opin. Microbiol. 13, 781–785.

ázquez-Laslop, N., Lee, H., Neyfakh, A.A., 2006. Increased persistence inEscherichia coli caused by controlled expression of toxins or other unrelatedproteins. J. Bacteriol. 188, 3494–3497.

eening, J.-W., Smits, W.K., Kuipers, O.P., 2008. Bistability, epigenetics, andbet-hedging in bacteria. Annu. Rev. Microbiol. 62, 193–210.

ega, N.M., Allison, K.R., Khalil, A.S., Collins, J.J., 2012. Signaling-mediated bacterialpersister formation. Nat. Chem. Biol. 8, 431–433.

ega, N.M., Allison, K.R., Samuels, A.N., Klempner, M.S., Collins, J.J., 2013.

Salmonella typhimurium intercepts Escherichia coli signaling to enhanceantibiotic tolerance. Proc. Natl. Acad. Sci. U. S. A. 110, 14420–14425.

erstraeten, N., Knapen, W., Fauvart, M., Michiels, J., 2016. A historical perspectiveon bacterial persistence. In: Methods in Molecular Biology. Springer Science,New York, pp. 3–13.

e Updates 29 (2016) 76–89 89

Verstraeten, N., Knapen, W., Fauvart, M., Michiels, J., 2015a. Membranedepolarization-triggered responsive diversification leads to antibiotictolerance. Microb. Cell 2, 299–301.

Verstraeten, N., Knapen, W.J., Kint, C.I., Liebens, V., Van den Bergh, B., Dewachter,L., Michiels, J.E., Fu, Q., David, C.C., Fierro, C.A., Marchal, K., Beirlant, J., Versées,W., Hofkens, J., Jansen, M., Fauvart, M., Michiels, J., 2015b. Obg and membranedepolarization are part of a microbial bet-hedging strategy that leads toantibiotic tolerance. Mol. Cell 59, 9–21.

Vinogradova, M., Gehling, V.S., Gustafson, A., Arora, S., Tindell, C.A., Wilson, C.,Williamson, K.E., Guler, G.D., Gangurde, P., Manieri, W., Busby, J., Flynn, E.M.,Lan, F., Kim, H., Odate, S., Cochran, A.G., Liu, Y., Wongchenko, M., Yang, Y.,Cheung, T.K., Maile, T.M., Lau, T., Costa, M., Hegde, G.V., Jackson, E., Pitti, R.,Arnott, D., Bailey, C., Bellon, S., Cummings, R.T., Albrecht, B.K., Harmange, J.-C.,Kiefer, J.R., Trojer, P., Classon, M., 2016. An inhibitor of KDM5 demethylasesreduces survival of drug-tolerant cancer cells. Nat. Chem. Biol. 12, 531–538.

Vogwill, T., Comfort, A.C., Furió, V., MaClean, R.C., 2016. Persistence and resistanceas complementary bacterial adaptations to antibiotics. J. Evol. Biol. 29,1223–1233.

Wakamoto, Y., Dhar, N., Chait, R., Schneider, K., Signorino-Gelo, F., Leibler, S.,McKinney, J.D., 2013. Dynamic persistence of antibiotic-stressed mycobacteria.Science 339, 91–95.

Wang, X., Wood, T.K., 2011. Toxin–antitoxin systems influence biofilm andpersister cell formation and the general stress response. Appl. Environ.Microbiol. 77, 5577–5583.

Wen, Y., Behiels, E., Devreese, B., 2014. Toxin–antitoxin systems: their role inpersistence, biofilm formation, and pathogenicity. Pathog. Dis. 70, 240–249.

Wicha, M.S., Liu, S., Dontu, G., 2006. Cancer stem cells: an old idea — a paradigmshift. Cancer Res. 66, 1883–1891.

Wiuff, C., Andersson, D.I., 2007. Antibiotic treatment in vitro of phenotypicallytolerant bacterial populations. J. Antimicrob. Chemother. 59, 254–263.

Wiuff, C., Zappala, R., Regoes, R.R., Garner, K., Baquero, F., Levin, B.R., 2005.Phenotypic tolerance: antibiotic enrichment of noninherited resistance inbacterial populations. Antimicrob. Agents Chemother. 49, 1438–1494.

Wood, T.K., Knabel, S.J., Kwan, B.W., 2013. Bacterial persister cell formation anddormancy. Appl. Environ. Microbiol. 79, 7116–7121.

World Health Organization, 2014. Antimicrobial Resistance: Global Report onSurveillance. WHO, Geneva, Switzerland.

Wu, N., He, L., Cui, P., Wang, W., Yuan, Y., Liu, S., Xu, T., Zhang, S., Wu, J., Zhang, W.,Zhang, Y., 2015. Ranking of persister genes in the same Escherichia coli geneticbackground demonstrates varying importance of individual persister genes in

tolerance. Antimicrob. Agents Chemother. 56, 4922–4926.Yang, Y., Bazhin, A.V., Werner, J., Karakhanova, S., 2013. Reactive oxygen species in

the immune system. Int. Rev. Immunol. 32, 249–270.