Distinguishing between resistance, tolerance and persistence to antibiotic treatment

ABSTRACT Antibiotic tolerance is associated with the failure of antibiotic treatment and the relapse of many bacterial infections. However, unlike resistance, which is commonly measured

using the minimum inhibitory concentration (MIC) metric, tolerance is poorly characterized, owing to the lack of a similar quantitative indicator. This may lead to the misclassification of

tolerant strains as resistant, or vice versa, and result in ineffective treatments. In this Opinion article, we describe recent studies of tolerance, resistance and persistence, outlining

how a clear and distinct definition for each phenotype can be developed from these findings. We propose a framework for classifying the drug response of bacterial strains according to these

definitions that is based on the measurement of the MIC together with a recently defined quantitative indicator of tolerance, the minimum duration for killing (MDK). Finally, we discuss

genes that are associated with increased tolerance — the 'tolerome' — as targets for treating tolerant bacterial strains. Access through your institution Buy or subscribe This is a

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EVOLUTIONARY TRAJECTORY OF TOLERANCE USING ANTIBIOTICS WITH DIFFERENT METABOLIC DEPENDENCIES Article Open access 09 May 2022 THE PHYSIOLOGY AND GENETICS OF BACTERIAL RESPONSES TO ANTIBIOTIC

COMBINATIONS Article 03 March 2022 MOLECULAR MECHANISMS OF ANTIBIOTIC RESISTANCE REVISITED Article 21 November 2022 REFERENCES * McKeegan, K. S., Borges-Walmsley, M. I. & Walmsley, A.

R. Microbial and viral drug resistance mechanisms. _Trends Microbiol._ 10, S8–S14 (2002). Article  CAS  PubMed  Google Scholar  * Scholar, E. M. & Pratt, W. B. (eds) _The Antimicrobial

Drugs_ (Oxford Univ. Press, 2000). Google Scholar  * D'Costa, V. M., McGrann, K. M., Hughes, D. W. & Wright, G. D. Sampling the antibiotic resistome. _Science_ 311, 374–377 (2006).

Article  CAS  PubMed  Google Scholar  * Bigger, J. W. Treatment of staphylococcal infections with penicillin by intermittent sterilisation. _Lancet_ 244, 497–500 (1944). Article  Google

Scholar  * Hobby, G. L., Meyer, K. & Chaffee, E. Observations on the mechanism of action of penicillin. _Proc. Soc. Exp. Biol. Med._ 50, 281–285 (1942). Article  CAS  Google Scholar  *

Horne, D. & Tomasz, A. Tolerant response of _Streptococcus sanguis_ to β-lactams and other cell-wall inhibitors. _Antimicrob. Agents Chemother._ 11, 888–896 (1977). Article  CAS  PubMed

  PubMed Central  Google Scholar  * Balaban, N. Q., Gerdes, K., Lewis, K. & McKinney, J. D. A problem of persistence: still more questions than answers? _Nat. Rev. Microbiol._ 11,

587–591 (2013). Article  CAS  PubMed  Google Scholar  * Kester, J. C. & Fortune, S. M. Persisters and beyond: mechanisms of phenotypic drug resistance and drug tolerance in bacteria.

_Crit. Rev. Biochem. Mol. Biol._ 49, 91–101 (2014). Article  CAS  PubMed  Google Scholar  * Handwerger, S. & Tomasz, A. Antibiotic tolerance among clinical isolates of bacteria. _Annu.

Rev. Pharmacol. Toxicol._ 25, 349–380 (1985). Article  CAS  PubMed  Google Scholar  * Tuomanen, E., Cozens, R., Tosch, W., Zak, O. & Tomasz, A. The rate of killing of _Escherichia coli_

by β-lactam antibiotics is strictly proportional to the rate of bacterial growth. _J. Gen. Microbiol._ 132, 1297–1304 (1986). CAS  PubMed  Google Scholar  * McDermott, W. Microbial

persistence. _Yale J. Biol. Med._ 30, 257–291 (1958). CAS  PubMed  Google Scholar  * Lederberg, J. & Zinder, N. Concentration of biochemical mutants of bacteria with penicillin. _J. Am.

Chem. Soc._ 70, 4267–4268 (1948). Article  CAS  PubMed  Google Scholar  * Gefen, O. & Balaban, N. Q. The importance of being persistent: heterogeneity of bacterial populations under

antibiotic stress. _FEMS Microbiol. Rev._ 33, 704–717 (2009). Article  CAS  PubMed  Google Scholar  * Balaban, N. Q., Merrin, J., Chait, R., Kowalik, L. & Leibler, S. Bacterial

persistence as a phenotypic switch. _Science_ 305, 1622–1625 (2004). Article  CAS  PubMed  Google Scholar  * Wakamoto, Y. et al. Dynamic persistence of antibiotic-stressed mycobacteria.

_Science_ 339, 91–95 (2013). Article  CAS  PubMed  Google Scholar  * Depardieu, F., Podglajen, I., Leclercq, R., Collatz, E. & Courvalin, P. Modes and modulations of antibiotic

resistance gene expression. _Clin. Microbiol. Rev._ 20, 79–114 (2007). Article  CAS  PubMed  PubMed Central  Google Scholar  * Blair, J. M., Webber, M. A., Baylay, A. J., Ogbolu, D. O. &

Piddock, L. J. Molecular mechanisms of antibiotic resistance. _Nat. Rev. Microbiol._ 13, 42–51 (2015). Article  CAS  PubMed  Google Scholar  * Chait, R., Craney, A. & Kishony, R.

Antibiotic interactions that select against resistance. _Nature_ 446, 668–671 (2007). Article  CAS  PubMed  Google Scholar  * Wiegand, I., Hilpert, K. & Hancock, R. E. Agar and broth

dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. _Nat. Protoc._ 3, 163–175 (2008). Article  CAS  PubMed  Google Scholar  * Mattie, H.

Antibiotic efficacy _in vivo_ predicted by _in vitro_ activity. _Int. J. Antimicrob. Agents_ 14, 91–98 (2000). Article  CAS  PubMed  Google Scholar  * Paterson, D. L. et al. Outcome of

cephalosporin treatment for serious infections due to apparently susceptible organisms producing extended-spectrum β-lactamases: implications for the clinical microbiology laboratory. _J.

Clin. Microbiol._ 39, 2206–2212 (2001). Article  CAS  PubMed  PubMed Central  Google Scholar  * Ishida, K., Guze, P. A., Kalmanson, G. M., Albrandt, K. & Guze, L. B. Variables in

demonstrating methicillin tolerance in _Staphylococcus aureus_ strains. _Antimicrob. Agents Chemother._ 21, 688–690 (1982). Article  CAS  PubMed  PubMed Central  Google Scholar  * Wolfson,

J., Hooper, D., McHugh, G., Bozza, M. & Swartz, M. Mutants of _Escherichia coli_ K-12 exhibiting reduced killing by both quinolone and β-lactam antimicrobial agents. _Antimicrob. Agents

Chemother._ 34, 1938–1943 (1990). Article  CAS  PubMed  PubMed Central  Google Scholar  * Mueller, M., de la Pena, A. & Derendorf, H. Issues in pharmacokinetics and pharmacodynamics of

anti-infective agents: kill curves versus MIC. _Antimicrob. Agents Chemother._ 48, 369–377 (2004). Article  CAS  PubMed  PubMed Central  Google Scholar  * Barry, L. A. et al. _Methods for

determining bactericidal activity of antimicrobial agents; approved guideline_. (National Committee for Clinical Laboratory Standards, 1999). Google Scholar  * Keren, I., Kaldalu, N.,

Spoering, A., Wang, Y. P. & Lewis, K. Persister cells and tolerance to antimicrobials. _Fems Microbiol. Lett._ 230, 13–18 (2004). Article  CAS  PubMed  Google Scholar  * Pasticci, M. B.

et al. Bactericidal activity of oxacillin and glycopeptides against _Staphylococcus aureus_ in patients with endocarditis: looking for a relationship between tolerance and outcome. _Ann.

Clin. Microbiol. Antimicrob._ 10, 26 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * Fridman, O., Goldberg, A., Ronin, I., Shoresh, N. & Balaban, N. Q. Optimization of

lag time underlies antibiotic tolerance in evolved bacterial populations. _Nature_ 513, 418–421 (2014). Article  CAS  PubMed  Google Scholar  * Regoes, R. R. et al. Pharmacodynamic

functions: a multiparameter approach to the design of antibiotic treatment regimens. _Antimicrob. Agents Chemother._ 48, 3670–3676 (2004). Article  CAS  PubMed  PubMed Central  Google

Scholar  * Gefen, O., Gabay, C., Mumcuoglu, M., Engel, G. & Balaban, N. Q. Single-cell protein induction dynamics reveals a period of vulnerability to antibiotics in persister bacteria.

_Proc. Natl Acad. Sci. USA_ 105, 6145–6149 (2008). Article  CAS  PubMed  PubMed Central  Google Scholar  * Helaine, S. et al. Dynamics of intracellular bacterial replication at the single

cell level. _Proc. Natl Acad. Sci. USA_ 107, 3746–3751 (2010). Article  CAS  PubMed  PubMed Central  Google Scholar  * Amato, S. M., Orman, M. A. & Brynildsen, M. P. Metabolic control of

persister formation in _Escherichia coli_. _Mol. Cell_ 50, 475–487 (2013). Article  CAS  PubMed  Google Scholar  * Maisonneuve, E., Castro-Camargo, M. & Gerdes, K. (p)ppGpp controls

bacterial persistence by stochastic induction of toxin–antitoxin activity. _Cell_ 154, 1140–1150 (2013). Article  CAS  PubMed  Google Scholar  * Chao, L. & Levin, B. R. Structured

habitats and the evolution of anticompetitor toxins in bacteria. _Proc. Natl Acad. Sci. USA_ 78, 6324–6328 (1981). Article  CAS  PubMed  PubMed Central  Google Scholar  * Rodionov, D. G.

& Ishiguro, E. E. Effects of inhibitors of protein synthesis on lysis of _Escherichia coli_ induced by β-lactam antibiotics. _Antimicrob. Agents Chemother._ 40, 899–903 (1996). Article 

CAS  PubMed  PubMed Central  Google Scholar  * Orman, M. A. & Brynildsen, M. P. Dormancy is not necessary or sufficient for bacterial persistence. _Antimicrob. Agents Chemother._ 57,

3230–3239 (2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Johansen, H. K., Jensen, T. G., Dessau, R. B., Lundgren, B. & Frimodt-Moller, N. Antagonism between penicillin

and erythromycin against _Streptococcus pneumoniae in vitro_ and _in vivo_. _J. Antimicrob. Chemother._ 46, 973–980 (2000). Article  CAS  PubMed  Google Scholar  * Thonus, I. P., Fontijne,

P. & Michel, M. F. Ampicillin susceptibility and ampicillin-induced killing rate of _Escherichia coli_. _Antimicrob. Agents Chemother._ 22, 386–390 (1982). Article  CAS  PubMed  PubMed

Central  Google Scholar  * Mascio, C. T., Alder, J. D. & Silverman, J. A. Bactericidal action of daptomycin against stationary-phase and nondividing _Staphylococcus aureus_ cells.

_Antimicrob. Agents Chemother._ 51, 4255–4260 (2007). Article  CAS  PubMed  PubMed Central  Google Scholar  * de Steenwinkel, J. E. et al. Time–kill kinetics of anti-tuberculosis drugs, and

emergence of resistance, in relation to metabolic activity of _Mycobacterium tuberculosis_. _J. Antimicrob. Chemother._ 65, 2582–2589 (2010). Article  CAS  PubMed  Google Scholar  * Evans,

D. J., Allison, D. G., Brown, M. R. & Gilbert, P. Susceptibility of _Pseudomonas aeruginosa_ and _Escherichia coli_ biofilms towards ciproflaxin: effect of specific growth rate. _J.

Antimicrob. Chemother._ 27, 177–184 (1991). Article  CAS  PubMed  Google Scholar  * Manina, G., Dhar, N. & McKinney, J. D. Stress and host immunity amplify _Mycobacterium tuberculosis_

phenotypic heterogeneity and induce nongrowing metabolically active forms. _Cell Host Microbe_ 17, 32–46 (2015). Article  CAS  PubMed  Google Scholar  * Kitano, K. & Tomasz, A.

_Escherichia coli_ mutants tolerant to β-lactam antibiotics. _J. Bacteriol._ 140, 955–963 (1979). Article  CAS  PubMed  PubMed Central  Google Scholar  * Bernier, S. P. et al. Starvation,

together with the SOS response, mediates high biofilm-specific tolerance to the fluoroquinolone ofloxacin. _PloS Genet._ 9, e1003144 (2013). Article  CAS  PubMed  PubMed Central  Google

Scholar  * Sandberg, A. et al. Intra- and extracellular activities of dicloxacillin against _Staphylococcus aureus in vivo_ and _in vitro_. _Antimicrob. Agents Chemother._ 54, 2391–2400

(2010). Article  CAS  PubMed  PubMed Central  Google Scholar  * Dorr, T., Davis, B. M. & Waldor, M. K. Endopeptidase-mediated β-lactam tolerance. _PloS Pathog._ 11, e1004850 (2015).

Article  PubMed  PubMed Central  CAS  Google Scholar  * Dorr, T., Vulic, M. & Lewis, K. Ciprofloxacin causes persister formation by inducing the TisB toxin in _Escherichia coli_. _PloS

Biol._ 8, e1000317 (2010). Article  PubMed  PubMed Central  CAS  Google Scholar  * Wiuff, C. & Andersson, D. I. Antibiotic treatment _in vitro_ of phenotypically tolerant bacterial

populations. _J. Antimicrob. Chemother._ 59, 254–263 (2007). Article  CAS  PubMed  Google Scholar  * Johnson, P. J. T. & Levin, B. R. Pharmacodynamics, population dynamics, and the

evolution of persistence in _Staphylococcus aureus_. _PloS Genet._ 9, e1003123 (2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Gefen, O., Fridman, O., Ronin, I. &

Balaban, N. Q. Direct observation of single stationary-phase bacteria reveals a surprisingly long period of constant protein production activity. _Proc. Natl Acad. Sci. USA_ 111, 556–561

(2014). Article  CAS  PubMed  Google Scholar  * Lewis, K. Persister cells, dormancy and infectious disease. _Nat. Rev. Microbiol._ 5, 48–56 (2007). Article  CAS  PubMed  Google Scholar  *

Nguyen, D. et al. Active starvation responses mediate antibiotic tolerance in biofilms and nutrient-limited bacteria. _Science_ 334, 982–986 (2011). Article  CAS  PubMed  PubMed Central 

Google Scholar  * Levin-Reisman, I. et al. Automated imaging with ScanLag reveals previously undetectable bacterial growth phenotypes. _Nat. Methods_ 7, 737–739 (2010). Article  CAS  PubMed

  Google Scholar  * Luidalepp, H., Joers, A., Kaldalu, N. & Tenson, T. Age of inoculum strongly influences persister frequency and can mask effects of mutations implicated in altered

persistence. _J. Bacteriol._ 193, 3598–3605 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * Madar, D. et al. Promoter activity dynamics in the lag phase of _Escherichia

coli_. _BMC Syst. Biol._ 7, 136 (2013). Article  PubMed  PubMed Central  CAS  Google Scholar  * Joers, A., Kaldalu, N. & Tenson, T. The frequency of persisters in _Escherichia coli_

reflects the kinetics of awakening from dormancy. _J. Bacteriol._ 192, 3379–3384 (2010). Article  CAS  PubMed  PubMed Central  Google Scholar  * Putrinš, M., Kogermann, K., Lukk, E. &

Lippus, M. Phenotypic heterogeneity enables uropathogenic _Escherichia coli_ to evade killing by antibiotics and serum complement. _Infect. Immun._ 83, 1056–1067 (2015). Article  PubMed 

PubMed Central  CAS  Google Scholar  * Pearl, S., Gabay, C., Kishony, R., Oppenheim, A. & Balaban, N. Q. Nongenetic individuality in the host–phage interaction. _PloS Biol._ 6, 957–964

(2008). Article  CAS  Google Scholar  * Baranyi, J. Stochastic modelling of bacterial lag phase. _Int. J. Food Microbiol._ 73, 203–206 (2002). Article  PubMed  Google Scholar  * Akerlund,

T., Nordstrom, K. & Bernander, R. Analysis of cell size and DNA content in exponentially growing and stationary-phase batch cultures of _Escherichia coli_. _J. Bacteriol._ 177, 6791–6797

(1995). Article  CAS  PubMed  PubMed Central  Google Scholar  * Hartman, B. J. & Tomasz, A. Expression of methicillin resistance in heterogeneous strains of _Staphylococcus aureus_.

_Antimicrob. Agents Chemother._ 29, 85–92 (1986). Article  CAS  PubMed  PubMed Central  Google Scholar  * Levin, B. R. & Rozen, D. E. Non-inherited antibiotic resistance. _Nat. Rev.

Microbiol._ 4, 556–562 (2006). Article  CAS  PubMed  Google Scholar  * Nataro, J. P., Blaser, M. J. & Cunningham-Rundles, S. (eds) in _Persistent Bacterial Infections_. 3–10 (ASM Press,

2000). Book  Google Scholar  * Rotem, E. et al. Regulation of phenotypic variability by a threshold-based mechanism underlies bacterial persistence. _Proc. Natl Acad. Sci. USA_ 107,

12541–12546 (2010). Article  CAS  PubMed  PubMed Central  Google Scholar  * Korch, S. B. & Hill, T. M. Ectopic overexpression of wild-type and mutant _hipA_ genes in _Escherichia coli_:

effects on macromolecular synthesis and persister formation. _J. Bacteriol._ 188, 3826–3836 (2006). Article  CAS  PubMed  PubMed Central  Google Scholar  * Moyed, H. S. & Bertrand, K. P.

_hipA_, a newly recognized gene of _Escherichia coli_ K-12 that affects frequency of persistence after inhibition of murein synthesis. _J. Bacteriol._ 155, 768–775 (1983). Article  CAS 

PubMed  PubMed Central  Google Scholar  * Levin-Reisman, I. & Balaban, N. Q. in _Bacterial Persistence: Methods and Protocols_ (eds Michiels, J. & Fauvart, M.) 75–81 (Humana Press,

2015). Google Scholar  * El Meouche, I., Siu, Y. & Dunlop, M. J. Stochastic expression of a multiple antibiotic resistance activator confers transient resistance in single cells. _Sci.

Rep._ 6, 19538 (2016). Article  CAS  PubMed  PubMed Central  Google Scholar  * Adams, K. N. et al. Drug tolerance in replicating mycobacteria mediated by a macrophage-induced efflux

mechanism. _Cell_ 145, 39–53 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * Kayser, F. H., Benner, E. J. & Hoeprich, P. D. Acquired and native resistance of

_Staphylococcus aureus_ to cephalexin and other β-lactam antibiotics. _Appl. Microbiol._ 20, 1–5 (1970). Article  CAS  PubMed  PubMed Central  Google Scholar  * El-Halfawy, O. M. &

Valvano, M. A. Antimicrobial heteroresistance: an emerging field in need of clarity. _Clin. Microbiol. Rev._ 28, 191–207 (2015). Article  CAS  PubMed  PubMed Central  Google Scholar  *

Adams, K. N., Szumowski, J. D. & Ramakrishnan, L. Verapamil, and its metabolite norverapamil, inhibit macrophage-induced, bacterial efflux pump-mediated tolerance to multiple

anti-tubercular drugs. _J. Infect. Dis._ 210, 456–466 (2014). Article  CAS  PubMed  PubMed Central  Google Scholar  * Mattie, H., Sekh, B. A., van Ogtrop, M. L. & van Strijen, E.

Comparison of the antibacterial effects of cefepime and ceftazidime against _Escherichia coli in vitro_ and _in vivo_. _Antimicrob. Agents Chemother._ 36, 2439–2443 (1992). Article  CAS 

PubMed  PubMed Central  Google Scholar  * Coates, A. R. & Hu, Y. Targeting non-multiplying organisms as a way to develop novel antimicrobials. _Trends Pharmacol. Sci._ 29, 143–150

(2008). Article  CAS  PubMed  Google Scholar  * Feng, J. et al. Identification of novel activity against _Borrelia burgdorferi_ persisters using an FDA approved drug library. _Emerg.

Microbes Infect._ 3, e49 (2014). Article  CAS  PubMed  PubMed Central  Google Scholar  * Kim, J. S. et al. Selective killing of bacterial persisters by a single chemical compound without

affecting normal antibiotic-sensitive cells. _Antimicrob. Agents Chemother._ 55, 5380–5383 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * Fleck, L. E. et al. A screen for

and validation of prodrug antimicrobials. _Antimicrob. Agents Chemother._ 58, 1410–1419 (2014). Article  PubMed  PubMed Central  CAS  Google Scholar  * Conlon, B. P. et al. Activated ClpP

kills persisters and eradicates a chronic biofilm infection. _Nature_ 503, 365–370 (2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Roostalu, J., Jõers, A., Luidalepp, H.,

Kaldalu, N. & Tenson, T. Cell division in _Escherichia coli_ cultures monitored at single cell resolution. _BMC Microbiol._ 8, 68 (2008). Article  PubMed  PubMed Central  Google Scholar

  * Claudi, B. et al. Phenotypic variation of _Salmonella_ in host tissues delays eradication by antimicrobial chemotherapy. _Cell_ 158, 722–733 (2014). Article  CAS  PubMed  Google Scholar

  * Mattie, H., Zhang, L. C., van Strijen, E., Sekh, B. R. & Douwes-Idema, A. E. Pharmacokinetic and pharmacodynamic models of the antistaphylococcal effects of meropenem and cloxacillin

_in vitro_ and in experimental infection. _Antimicrob. Agents Chemother._ 41, 2083–2088 (1997). Article  CAS  PubMed  PubMed Central  Google Scholar  * Arnoldini, M. et al. Bistable

expression of virulence genes in _Salmonella_ leads to the formation of an antibiotic-tolerant subpopulation. _PLoS Biol._ 12, e1001928 (2014). Article  PubMed  PubMed Central  CAS  Google

Scholar  * Nickel, J. C., Ruseska, I., Wright, J. B. & Costerton, J. W. Tobramycin resistance of _Pseudomonas aeruginosa_ cells growing as a biofilm on urinary catheter material.

_Antimicrob. Agents Chemother._ 27, 619–624 (1985). Article  CAS  PubMed  PubMed Central  Google Scholar  * Hayes, C. S. & Low, D. A. Signals of growth regulation in bacteria. _Curr.

Opin. Microbiol._ 12, 667–673 (2009). Article  CAS  PubMed  PubMed Central  Google Scholar  * Bhuyan, B. K., Fraser, T. J. & Day, K. J. Cell proliferation kinetics and drug sensitivity

of exponential and stationary populations of cultured L1210 cells. _Cancer Res._ 37, 1057–1063 (1977). CAS  PubMed  Google Scholar  * Sharma, S. V. et al. A chromatin-mediated reversible

drug-tolerant state in cancer cell subpopulations. _Cell_ 141, 69–80 (2010). Article  CAS  PubMed  PubMed Central  Google Scholar  * Jayaraman, R. Bacterial persistence: some new insights

into an old phenomenon. _J. Biosci._ 33, 795–805 (2008). Article  CAS  PubMed  Google Scholar  * Cohen, N. R., Lobritz, M. A. & Collins, J. J. Microbial persistence and the road to drug

resistance. _Cell Host Microbe_ 13, 632–642 (2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Potrykus, K. & Cashel, M. (p)ppGpp: still magical? _Annu. Rev. Microbiol._ 62,

35–51 (2008). Article  CAS  PubMed  Google Scholar  * Kaspy, I. et al. HipA-mediated antibiotic persistence via phosphorylation of the glutamyl-tRNA-synthetase. _Nat. Commun._ 4, 3001

(2013). Article  PubMed  CAS  Google Scholar  * Germain, E., Castro-Roa, D., Zenkin, N. & Gerdes, K. Molecular mechanism of bacterial persistence by HipA. _Mol. Cell_ 52, 248–254 (2013).

Article  CAS  PubMed  Google Scholar  * Hahn, J., Tanner, A. W., Carabetta, V. J., Cristea, I. M. & Dubnau, D. ComGA–RelA interaction and persistence in the _Bacillus subtilis_ K-state.

_Mol. Microbiol._ 97, 454–471 (2015). Article  CAS  PubMed  PubMed Central  Google Scholar  * Gerdes, K. & Maisonneuve, E. Bacterial persistence and toxin–antitoxin loci. _Annu. Rev.

Microbiol._ 66, 103–123 (2012). Article  CAS  PubMed  Google Scholar  * Girgis, H. S., Harris, K. & Tavazoie, S. Large mutational target size for rapid emergence of bacterial

persistence. _Proc. Natl Acad. Sci. USA_ 109, 12740–12745 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Hansen, S., Lewis, K. & Vulic, M. Role of global regulators and

nucleotide metabolism in antibiotic tolerance in _Escherichia coli_. _Antimicrob. Agents Chemother._ 52, 2718–2726 (2008). Article  CAS  PubMed  PubMed Central  Google Scholar  * Spoering,

A. L., Vulic, M. & Lewis, K. GlpD and PlsB participate in persister cell formation in _Escherichia coli_. _J. Bacteriol._ 188, 5136–5144 (2006). Article  CAS  PubMed  PubMed Central 

Google Scholar  * Vazquez-Laslop, N., Lee, H. & Neyfakh, A. A. Increased persistence in _Escherichia coli_ caused by controlled expression of toxins or other unrelated proteins. _J.

Bacteriol._ 188, 3494–3497 (2006). Article  CAS  PubMed  PubMed Central  Google Scholar  * Shan, Y., Lazinski, D., Rowe, S. E., Camili, A. & Lewis, K. Genetic basis of persister

tolerance to aminoglycosides in _Escherichia coli_. _mBio_ 6, e00078-15 (2015). Article  PubMed  PubMed Central  CAS  Google Scholar  * Balaban, N. Q. Persistence: mechanisms for triggering

and enhancing phenotypic variability. _Curr. Opin. Genet. Dev._ 21, 768–775 (2011). Article  CAS  PubMed  Google Scholar  * Casadesus, J. & Low, D. A. Programmed heterogeneity:

epigenetic mechanisms in bacteria. _J. Biol. Chem._ 288, 13929–13935 (2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Huh, D. & Paulsson, J. Non-genetic heterogeneity from

stochastic partitioning at cell division. _Nat. Genet._ 43, 95–100 (2011). Article  CAS  PubMed  Google Scholar  * Tsimring, L. S. Noise in biology. _Rep. Progress Phys._ 77, 026601 (2014).

Article  CAS  Google Scholar  * Gelens, L., Hill, L., Vandervelde, A., Danckaert, J. & Loris, R. A general model for toxin–antitoxin module dynamics can explain persister cell formation

in _E. coli_. _PLoS Comput. Biol._ 9, e1003190 (2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Maisonneuve, E., Shakespeare, L. J., Jorgensen, M. G. & Gerdes, K.

Bacterial persistence by RNA endonucleases. _Proc. Natl Acad. Sci. USA_ 108, 13206–13211 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * Helaine, S. et al. Internalization of

_Salmonella_ by macrophages induces formation of nonreplicating persisters. _Science_ 343, 204–208 (2014). Article  CAS  PubMed  PubMed Central  Google Scholar  * Aldridge, B. B. et al.

Asymmetry and aging of mycobacterial cells lead to variable growth and antibiotic susceptibility. _Science_ 335, 100–104 (2012). Article  CAS  PubMed  Google Scholar  * Kieser, K. J. &

Rubin, E. J. How sisters grow apart: mycobacterial growth and division. _Nat. Rev. Microbiol._ 12, 550–562 (2014). Article  CAS  PubMed  PubMed Central  Google Scholar  * Vaubourgeix, J. et

al. Stressed mycobacteria use the chaperone ClpB to sequester irreversibly oxidized proteins asymmetrically within and between cells. _Cell Host Microbe_ 17, 178–190 (2015). Article  CAS 

PubMed  PubMed Central  Google Scholar  * Wu, Y. X., Vulic, M., Keren, I. & Lewis, K. Role of oxidative stress in persister tolerance. _Antimicrob. Agents Chemother._ 56, 4922–4926

(2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Song, Y., Rubio, A., Jayaswal, R. K., Silverman, J. A. & Wilkinson, B. J. Additional routes to _Staphylococcus aureus_

daptomycin resistance as revealed by comparative genome sequencing, transcriptional profiling, and phenotypic studies. _PLoS ONE_ 8, e58469 (2013). Article  CAS  PubMed  PubMed Central 

Google Scholar  * Brehm-Stecher, B. F. & Johnson, E. A. Single-cell microbiology: tools, technologies, and applications. _Microbiol. Mol. Biol. Rev._ 68, 538–559 (2004). Article  CAS 

PubMed  PubMed Central  Google Scholar  * Ping, W. et al. Robust growth of _Escherichia coli_. _Curr. Biol._ 20, 1099–1103 (2010). Article  CAS  Google Scholar  * Iino, R., Matsumoto, Y.,

Nishino, K., Yamaguchi, A. & Noji, H. Design of a large-scale femtoliter droplet array for single-cell analysis of drug-tolerant and drug-resistant bacteria. _Front. Microbiol._ 4, 300

(2013). Article  PubMed  PubMed Central  Google Scholar  * Shah, D. et al. Persisters: a distinct physiological state of _E. coli_. _BMC Microbiol._ 6, 53 (2006). Article  PubMed  PubMed

Central  CAS  Google Scholar  * Orman, M. A. & Brynildsen, M. P. Establishment of a method to rapidly assay bacterial persister metabolism. _Antimicrob. Agents Chemother._ 57, 4398–4409

(2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Jarzembowski, T., Wisniewska, K., Jozwik, A. & Witkowski, J. Heterogeneity of methicillin-resistant _Staphylococcus

aureus_ strains (MRSA) characterized by flow cytometry. _Curr. Microbiol._ 59, 78–80 (2009). Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS The authors thank N.

Shoresh for illuminating discussions regarding this manuscript, and the members of the Balaban laboratory, I. Kaspy and G. Glaser for comments and suggestions. This work is supported by the

Minerva Center for Stochastic Decision Making in Microorganisms, a European Research Council (ERC) Starting Grant (260871) and the Israel Science Foundation (492/15). AUTHOR INFORMATION

AUTHORS AND AFFILIATIONS * Asher Brauner, Ofer Fridman, Orit Gefen and Nathalie Q. Balaban are at the Racah Institute of Physics and the Harvey M. Kruger Family Center for Nanoscience and

Nanotechnology, Edmond J. Safra Campus, The Hebrew University of Jerusalem, Jerusalem 91904, Israel., Asher Brauner, Ofer Fridman, Orit Gefen & Nathalie Q. Balaban Authors * Asher

Brauner View author publications You can also search for this author inPubMed Google Scholar * Ofer Fridman View author publications You can also search for this author inPubMed Google

Scholar * Orit Gefen View author publications You can also search for this author inPubMed Google Scholar * Nathalie Q. Balaban View author publications You can also search for this author

inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Nathalie Q. Balaban. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. POWERPOINT

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al._ Distinguishing between resistance, tolerance and persistence to antibiotic treatment. _Nat Rev Microbiol_ 14, 320–330 (2016). https://doi.org/10.1038/nrmicro.2016.34 Download citation *

Published: 15 April 2016 * Issue Date: May 2016 * DOI: https://doi.org/10.1038/nrmicro.2016.34 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content:

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