SpringerLink
Log in

A General Method for Measuring Persister Levels in Escherichia coli Cultures

  • Protocol
Bacterial Persistence

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1333))

Abstract

Genetically homogeneous bacterial cultures contain persisters, cells that are not killed by bactericidal antibiotics. These cells are suggested to be involved in the establishment of chronic infections. Persister levels depend on growth conditions. Here, we discuss the parameters that have to be considered when measuring persister levels and provide a sample protocol to do it.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (Canada)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (Canada)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.00
Price excludes VAT (Canada)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free ship** worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (Canada)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free ship** worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Bigger JW (1944) Treatment of staphylococcal infections with penicillin by intermittent sterilization. Lancet 244(6320):497–500

    Article  Google Scholar 

  2. Lewis K (2010) Persister cells. Annu Rev Microbiol 64:357–372

    Article  CAS  PubMed  Google Scholar 

  3. Keren I, Kaldalu N, Spoering A, Wang Y, Lewis K (2004) Persister cells and tolerance to antimicrobials. FEMS Microbiol Lett 230(1):13–18

    Article  CAS  PubMed  Google Scholar 

  4. Balaban NQ, Merrin J, Chait R, Kowalik L, Leibler S (2004) Bacterial persistence as a phenotypic switch. Science 305(5690):1622–1625

    Article  CAS  PubMed  Google Scholar 

  5. Joers A, Kaldalu N, Tenson T (2010) The frequency of persisters in Escherichia coli reflects the kinetics of awakening from dormancy. J Bacteriol 192(13):3379–3384

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  6. Orman MA, Brynildsen MP (2013) Dormancy is not necessary or sufficient for bacterial persistence. Antimicrob Agents Chemother 57(7):3230–3239

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  7. Epstein SS (2009) Microbial awakenings. Nature 457(7233):1083

    Article  CAS  PubMed  Google Scholar 

  8. Kussell E, Kishony R, Balaban NQ, Leibler S (2005) Bacterial persistence: a model of survival in changing environments. Genetics 169(4):1807–1814

    Article  PubMed Central  PubMed  Google Scholar 

  9. Ratcliff WC, Denison RF (2011) Bacterial persistence and bet hedging in Sinorhizobium meliloti. Commun Integr Biol 4(1):98–100

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  10. Fredriksson A, Nystrom T (2006) Conditional and replicative senescence in Escherichia coli. Curr Opin Microbiol 9(6):612–618

    Article  CAS  PubMed  Google Scholar 

  11. Adams KN, Takaki K, Connolly LE, Wiedenhoft H, Winglee K, Humbert O, Edelstein PH, Cosma CL, Ramakrishnan L (2011) Drug tolerance in replicating mycobacteria mediated by a macrophage-induced efflux mechanism. Cell 145(1):39–53

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  12. Wakamoto Y, Dhar N, Chait R, Schneider K, Signorino-Gelo F, Leibler S, McKinney JD (2013) Dynamic persistence of antibiotic-stressed mycobacteria. Science 339(6115):91–95

    Article  CAS  PubMed  Google Scholar 

  13. Ezraty B, Vergnes A, Banzhaf M, Duverger Y, Huguenot A, Brochado AR, Su SY, Espinosa L, Loiseau L, Py B, Typas A, Barras F (2013) Fe-S cluster biosynthesis controls uptake of aminoglycosides in a ROS-less death pathway. Science 340(6140):1583–1587

    Article  CAS  PubMed  Google Scholar 

  14. Javid B, Sorrentino F, Toosky M, Zheng W, Pinkham JT, Jain N, Pan M, Deighan P, Rubin EJ (2014) Mycobacterial mistranslation is necessary and sufficient for rifampicin phenotypic resistance. Proc Natl Acad Sci U S A 111(3):1132–1137

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. Martinez JL, Blazquez J, Baquero F (1994) Non-canonical mechanisms of antibiotic resistance. Eur J Clin Microbiol Infect Dis 13(12):1015–1022

    Article  CAS  PubMed  Google Scholar 

  16. Keren I, Shah D, Spoering A, Kaldalu N, Lewis K (2004) Specialized persister cells and the mechanism of multidrug tolerance in Escherichia coli. J Bacteriol 186(24):8172–8180

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  17. Spoering AL, Lewis K (2001) Biofilms and planktonic cells of Pseudomonas aeruginosa have similar resistance to killing by antimicrobials. J Bacteriol 183(23):6746–6751

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  18. Allison KR, Brynildsen MP, Collins JJ (2011) Metabolite-enabled eradication of bacterial persisters by aminoglycosides. Nature 473(7346):216–220

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  19. Orman MA, Brynildsen MP (2013) Establishment of a method to rapidly assay bacterial persister metabolism. Antimicrob Agents Chemother 57(9):4398–4409

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  20. Maisonneuve E, Castro-Camargo M, Gerdes K (2013) (p)ppGpp controls bacterial persistence by stochastic induction of toxin-antitoxin activity. Cell 154(5):1140–1150

    Article  CAS  PubMed  Google Scholar 

  21. Nguyen D, Joshi-Datar A, Lepine F, Bauerle E, Olakanmi O, Beer K, McKay G, Siehnel R, Schafhauser J, Wang Y, Britigan BE, Singh PK (2011) Active starvation responses mediate antibiotic tolerance in biofilms and nutrient-limited bacteria. Science 334(6058):982–986

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  22. Dorr T, Lewis K, Vulic M (2009) SOS response induces persistence to fluoroquinolones in Escherichia coli. PLoS Genet 5(12):e1000760

    Article  PubMed Central  PubMed  Google Scholar 

  23. Dorr T, Vulic M, Lewis K (2010) Ciprofloxacin causes persister formation by inducing the TisB toxin in Escherichia coli. PLoS Biol 8(2):e1000317

    Article  PubMed Central  PubMed  Google Scholar 

  24. Goneau LW, Yeoh NS, Macdonald KW, Cadieux PA, Burton JP, Razvi H, Reid G (2014) Selective target inactivation rather than global metabolic dormancy causes antibiotic tolerance in uropathogens. Antimicrob Agents Chemother 58(4):2089–2097

    Article  PubMed Central  PubMed  Google Scholar 

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

    Article  PubMed Central  PubMed  Google Scholar 

  26. Helaine S, Thompson JA, Watson KG, Liu M, Boyle C, Holden DW (2010) Dynamics of intracellular bacterial replication at the single cell level. Proc Natl Acad Sci U S A 107(8):3746–3751

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  27. Roostalu J, Joers A, Luidalepp H, Kaldalu N, Tenson T (2008) Cell division in Escherichia coli cultures monitored at single cell resolution. BMC Microbiol 8:68

    Article  PubMed Central  PubMed  Google Scholar 

  28. Gefen O, Gabay C, Mumcuoglu M, Engel G, Balaban NQ (2008) Single-cell protein induction dynamics reveals a period of vulnerability to antibiotics in persister bacteria. Proc Natl Acad Sci U S A 105(16):6145–6149

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  29. Luidalepp H, Joers A, Kaldalu N, Tenson T (2011) Age of inoculum strongly influences persister frequency and can mask effects of mutations implicated in altered persistence. J Bacteriol 193(14):3598–3605

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  30. Levin-Reisman I, Gefen O, Fridman O, Ronin I, Shwa D, Sheftel H, Balaban NQ (2010) Automated imaging with ScanLag reveals previously undetectable bacterial growth phenotypes. Nat Methods 7(9):737–739

    Article  CAS  PubMed  Google Scholar 

  31. Gill WP, Harik NS, Whiddon MR, Liao RP, Mittler JE, Sherman DR (2009) A replication clock for mycobacterium tuberculosis. Nat Med 15(2):211–214

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  32. Keren I, Minami S, Rubin E, Lewis K (2011) Characterization and transcriptome analysis of Mycobacterium tuberculosis persisters. MBio 2(3):e00100–e00111

    Article  PubMed Central  PubMed  Google Scholar 

  33. Canas-Duarte SJ, Restrepo S, Pedraza JM (2014) Novel protocol for persister cells isolation. PLoS One 9(2):e88660

    Article  PubMed Central  PubMed  Google Scholar 

  34. Cuny C, Dukan L, Fraysse L, Ballesteros M, Dukan S (2005) Investigation of the first events leading to loss of culturability during Escherichia coli starvation: future nonculturable bacteria form a subpopulation. J Bacteriol 187(7):2244–2248

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  35. Makinoshima H, Nishimura A, Ishihama A (2002) Fractionation of Escherichia coli cell populations at different stages during growth transition to stationary phase. Mol Microbiol 43(2):269–279

    Article  CAS  PubMed  Google Scholar 

  36. Oliver JD (2005) The viable but nonculturable state in bacteria. J Microbiol 43 Spec No:93–100

    Google Scholar 

  37. Buerger S, Spoering A, Gavrish E, Leslin C, Ling L, Epstein SS (2012) Microbial scout hypothesis, stochastic exit from dormancy, and the nature of slow growers. Appl Environ Microbiol 78(9):3221–3228

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  38. Ma C, Sim S, Shi W, Du L, **ng D, Zhang Y (2010) Energy production genes sucB and ubiF are involved in persister survival and tolerance to multiple antibiotics and stresses in Escherichia coli. FEMS Microbiol Lett 303(1):33–40

    Article  CAS  PubMed  Google Scholar 

  39. Moyed HS, Bertrand KP (1983) hipA, a newly recognized gene of Escherichia coli K-12 that affects frequency of persistence after inhibition of murein synthesis. J Bacteriol 155(2):768–775

    PubMed Central  CAS  PubMed  Google Scholar 

  40. Wolfson JS, Hooper DC, McHugh GL, Bozza MA, Swartz MN (1990) Mutants of Escherichia coli K-12 exhibiting reduced killing by both quinolone and beta-lactam antimicrobial agents. Antimicrob Agents Chemother 34(10):1938–1943

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  41. Hofsteenge N, van Nimwegen E, Silander OK (2013) Quantitative analysis of persister fractions suggests different mechanisms of formation among environmental isolates of E. coli. BMC Microbiol 13:25

    Article  PubMed Central  PubMed  Google Scholar 

  42. Wiuff C, Andersson DI (2007) Antibiotic treatment in vitro of phenotypically tolerant bacterial populations. J Antimicrob Chemother 59(2):254–263

    Article  CAS  PubMed  Google Scholar 

  43. Vazquez-Laslop N, Lee H, Neyfakh AA (2006) Increased persistence in Escherichia coli caused by controlled expression of toxins or other unrelated proteins. J Bacteriol 188(10):3494–3497

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  44. Lioy VS, Machon C, Tabone M, Gonzalez-Pastor JE, Daugelavicius R, Ayora S, Alonso JC (2012) The zeta toxin induces a set of protective responses and dormancy. PLoS One 7(1):e30282

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  45. Tabone M, Lioy VS, Ayora S, Machon C, Alonso JC (2014) Role of toxin zeta and starvation responses in the sensitivity to antimicrobials. PLoS One 9(1):e86615

    Article  PubMed Central  PubMed  Google Scholar 

  46. Tripathi A, Dewan PC, Barua B, Varadarajan R (2012) Additional role for the ccd operon of F-plasmid as a transmissible persistence factor. Proc Natl Acad Sci U S A 109(31):12497–12502

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  47. Tripathi A, Dewan PC, Siddique SA, Varadarajan R (2014) MazF-induced growth inhibition and persister generation in Escherichia coli. J Biol Chem 289(7):4191–4205

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  48. Madar D, Dekel E, Bren A, Zimmer A, Porat Z, Alon U (2013) Promoter activity dynamics in the lag phase of Escherichia coli. BMC Syst Biol 7(1):136

    Article  PubMed Central  PubMed  Google Scholar 

  49. Andrews JM (2001) Determination of minimum inhibitory concentrations. J Antimicrob Chemother 48(Suppl 1):5–16

    Article  CAS  PubMed  Google Scholar 

  50. Luidalepp H, Hallier M, Felden B, Tenson T (2005) tmRNA decreases the bactericidal activity of aminoglycosides and the susceptibility to inhibitors of cell wall synthesis. RNA Biol 2(2):70–74

    Article  CAS  PubMed  Google Scholar 

  51. Udekwu KI, Parrish N, Ankomah P, Baquero F, Levin BR (2009) Functional relationship between bacterial cell density and the efficacy of antibiotics. J Antimicrob Chemother 63(4):745–757

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  52. Kram KE, Finkel SE (2014) Culture volume and vessel affect long-term survival, mutation frequency, and oxidative stress of Escherichia coli. Appl Environ Microbiol 80(5):1732–1738

    Article  PubMed Central  PubMed  Google Scholar 

  53. Keren I, Wu Y, Inocencio J, Mulcahy LR, Lewis K (2013) Killing by bactericidal antibiotics does not depend on reactive oxygen species. Science 339(6124):1213–1216

    Article  CAS  PubMed  Google Scholar 

  54. MacKenzie FM, Gould IM (1993) The post-antibiotic effect. J Antimicrob Chemother 32(4):519–537

    Article  CAS  PubMed  Google Scholar 

  55. Reasoner DJ, Geldreich EE (1985) A new medium for the enumeration and subculture of bacteria from potable water. Appl Environ Microbiol 49(1):1–7

    PubMed Central  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the European Regional Development Fund through the Center of Excellence in Chemical Biology and Estonian Science Foundation grant 8822.

Author information

Authors and Affiliations

  1. Institute of Technology, University of Tartu, Nooruse 1, Tartu, 50411, Estonia

    Niilo Kaldalu, Arvi Jõers, Henri Ingelman & Tanel Tenson

Authors
  1. Niilo Kaldalu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Arvi Jõers
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Henri Ingelman
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tanel Tenson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tanel Tenson .

Editor information

Editors and Affiliations

  1. Department of Microbial and Molecular Systems, KU Leuven - University of Leuven, Heverlee, Belgium

    Jan Michiels

  2. Department of Microbial and Molecular Systems, KU Leuven - University of Leuven, Heverlee, Belgium

    Maarten Fauvart

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Kaldalu, N., Jõers, A., Ingelman, H., Tenson, T. (2016). A General Method for Measuring Persister Levels in Escherichia coli Cultures. In: Michiels, J., Fauvart, M. (eds) Bacterial Persistence. Methods in Molecular Biology, vol 1333. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2854-5_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2854-5_3

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2853-8

  • Online ISBN: 978-1-4939-2854-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation