Persister Resuscitation

  • Arvi Jõers
  • Marta Putrinš
  • Niilo Kaldalu
  • Hannes Luidalepp
  • Tanel TensonEmail author


By definition, persister cells must resume growth after the bactericidal treatment. Growth resumption can also lead to the reoccurrence of the infections and is, therefore, the reason why persisters are considered clinically important. Furthermore, treatments that enforce the dormant bacteria to resuscitate during the antibiotic treatment might become a key for eradicating persistent infections. Unfortunately, resuscitation of dormant bacteria is still poorly studied and very little is known about resuscitation of persisters during infection.

In this chapter, we have summarized the knowledge of factors that affect growth resumption of bacterial cells in general, and more specifically, after antibiotic treatment. We also touch the potentially relevant field of Viable but nonculturable (VBNC) cells. To illustrate how different can be persister resuscitation in vivo compared to the in vitro conditions we draw an example from urinary tract infection. Understanding the mechanisms of bacterial growth resumption inside the host is one very promising direction where the field could move in order to find new therapeutic options against persistent infections.



This work was supported by Estonian Research Council (grant PRG335), and by the European Regional Development Fund (through the Centre of Excellence in Molecular Cell Engineering). We thank David Schryer for correcting the English language.


  1. Allison, K. R., Brynildsen, M. P., & Collins, J. J. (2011). Metabolite-enabled eradication of bacterial persisters by aminoglycosides. Nature, 473(7346), 216–220. Scholar
  2. Athamna, A., Athamna, M., Medlej, B., Bast, D. J., & Rubinstein, E. (2004). In vitro post-antibiotic effect of fluoroquinolones, macrolides, -lactams, tetracyclines, vancomycin, clindamycin, linezolid, chloramphenicol, quinupristin/dalfopristin and rifampicin on Bacillus anthracis. Journal of Antimicrobial Chemotherapy, 53(4), 609–615. Scholar
  3. Ayrapetyan, M., Williams, T. C., Baxter, R., & Oliver, J. D. (2015). Viable but nonculturable and persister cells coexist stochastically and are induced by human serum. Infection and Immunity, 83(11), 4194–4203. Scholar
  4. Ayrapetyan, M., Williams, T., & Oliver, J. D. (2018). Relationship between the viable but nonculturable state and antibiotic persister cells. Journal of Bacteriology, 200(20).
  5. Azevedo, N. F., Bragança, S. M., Simões, L. C., Cerqueira, L., Almeida, C., Keevil, C. W., & Vieira, M. J. (2012). Proposal for a method to estimate nutrient shock effects in bacteria. BMC Research Notes, 5, 422.
  6. Balaban, N. Q., Merrin, J., Chait, R., Kowalik, L., & Leibler, S. (2004). Bacterial persistence as a phenotypic switch. Science (New York), 305(5690), 1622–1625. Scholar
  7. Balaban, N. Q., Helaine, S., Lewis, K., Ackermann, M., Aldridge, B., Andersson, D. I., Brynildsen, M. P., Bumann, D., Camilli, A., Collins, J. J., Dehio, C., Fortune, S., Ghigo, J. M., Hardt, W. D., Harms, A., Heinemann, M., Hung, D. T., Jenal, U., Levin, B. R., Michiels, J., Storz, G., Tan, M. W., Tenson, T., Van Melderen, L., Zinkernagel, A. (2019). A definitions and guidelines for research on antibiotic persistence. Nature Review Microbiology, 17(7), 441–448. (Erratum in: Nature Review Microbiology. 2019 Apr 29) Scholar
  8. Blango, M. G., & Mulvey, M. A. (2010). Persistence of uropathogenic Escherichia coli in the face of multiple antibiotics. Antimicrobial Agents and Chemotherapy, 54(5), 1855–1863. Scholar
  9. Bogosian, G., Aardema, N. D., Bourneuf, E. V., Morris, P. J., & O’Neil, J. P. (2000). Recovery of hydrogen peroxide-sensitive culturable cells of Vibrio vulnificus gives the appearance of resuscitation from a viable but nonculturable state. Journal of Bacteriology, 182(18), 5070–5075.
  10. Browne, H. P., Forster, S. C., Anonye, B. O., Kumar, N., Neville, B. A., Stares, M. D., Goulding, D., & Lawley, T. D. (2016). Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation. Nature, 533(7604), 543–546. Scholar
  11. Buerger, S., Spoering, A., Gavrish, E., Leslin, C., Ling, L., & Epstein, S. S. (2012). Microbial scout hypothesis, stochastic exit from dormancy, and the nature of slow growers. Applied and Environmental Microbiology, 78(9), 3221–3228. Scholar
  12. Bunker, S. T., Bates, T. C., & Oliver, J. D. (2004). Effects of temperature on detection of plasmid or chromosomally encoded Gfp- and lux-Labeled pseudomonas fluorescens in soil. Environmental Biosafety Research, 3(2), 83–90.CrossRefGoogle Scholar
  13. Celesk, R. A., & Robillard, N. J. (1989). Factors influencing the accumulation of ciprofloxacin in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy, 33(11), 1921–1926.CrossRefGoogle Scholar
  14. Colwell, R. R., Brayton, P., Herrington, D., Tall, B., Huq, A., & Levine, M. M. (1996). Viable but non-culturable Vibrio cholerae O1 revert to a cultivable state in the human intestine. World Journal of Microbiology and Biotechnology, 12(1), 28–31. Scholar
  15. Defraine, V., Fauvart, M., & Michiels, J. (2018). Fighting bacterial persistence: Current and emerging anti-persister strategies and therapeutics. Drug Resistance Updates, 38, 12–26. CrossRefGoogle Scholar
  16. Dhar, N., & McKinney, J. D. (2007). Microbial phenotypic heterogeneity and antibiotic tolerance. Current Opinion in Microbiology, 10(1), 30–38. Scholar
  17. Epstein, S. S. (2009). Microbial awakenings. Nature, 457(7233), 1083. Scholar
  18. Epstein, S. S. (2013). The phenomenon of microbial uncultivability. Current Opinion in Microbiology, 16(5), 636–642. Scholar
  19. Erman, A., Hergouth, V. K., Blango, M. G., Kos, M. K., Mulvey, M. A., & Veranič, P. (2017). Repeated treatments with chitosan in combination with antibiotics completely eradicate uropathogenic Escherichia coli from infected mouse urinary bladders. Journal of Infectious Diseases, 216(3), jix023. Scholar
  20. Fisher, R. A., Gollan, B., & Helaine, S. (2017). Persistent bacterial infections and persister cells. Nature Reviews Microbiology. CrossRefGoogle Scholar
  21. Foxman, B. (2010). The epidemiology of urinary tract infection. Nature Reviews. Urology, 7(12), 653–660 (Nature publishing group, a division of Macmillan publishers limited. All Rights Reserved). CrossRefGoogle Scholar
  22. Fridman, O., Goldberg, A., Ronin, I., Shoresh, N., & Balaban, N. Q. (2014). Optimization of lag time underlies antibiotic tolerance in evolved bacterial populations. Nature, 513(7518), 418–421. Scholar
  23. Ghosh, S., & Setlow, P. (2009). Isolation and characterization of superdormant spores of Bacillus species. Journal of Bacteriology, 191(6), 1787–1797. Scholar
  24. Gilbert, N. M., O’Brien, V. P., & Lewis, A. L. (2017). Transient microbiota exposures activate dormant Escherichia coli infection in the bladder and drive severe outcomes of recurrent disease. PLOS Pathogens, 13(3), e1006238. Scholar
  25. Goormaghtigh, F., Fraikin, N., Putrinš, M., Hallaert, T., Hauryliuk, V., Garcia-Pino, A., Sjödin, A., et al. (2018). Reassessing the role of type II toxin-antitoxin systems in formation of Escherichia coli type II persister cells. MBio, 9(3), e00640–e00618. Scholar
  26. Jõers, A., & Tenson, T. (2016). Growth resumption from stationary phase reveals memory in Escherichia coli cultures. Scientific Reports, 6(1), 24055. Scholar
  27. Joers, A., Kaldalu, N., & Tenson, T. (2010). The frequency of persisters in Escherichia coli reflects the kinetics of awakening from dormancy. Journal of Bacteriology, 192(13), 3379–3384. Scholar
  28. Jones, D. M., Sutcliffe, E. M., & Curry, A. (1991). Recovery of viable but non-culturable Campylobacter jejuni. Journal of General Microbiology, 137(10), 2477–2482. Scholar
  29. Jong, d., Imke, G., Haccou, P., & Kuipers, O. P. (2011). Bet hedging or not? A guide to proper classification of microbial survival strategies. BioEssays: News and Reviews in Molecular, Cellular and Developmental Biology, 33(3), 215–223. Scholar
  30. Kaldalu, N., Jõers, A., Ingelman, H., & Tenson, T. (2016). A general method for measuring persister levels in Escherichia coli cultures. Methods in Molecular Biology, 1333. Google Scholar
  31. Kim, J.-S., Heo, P., Yang, T.-J., Lee, K.-S., Cho, D.-H., Kim, B. T., Suh, J.-H., et al. (2011). Selective killing of bacterial persisters by a single chemical compound without affecting normal antibiotic-sensitive cells. Antimicrobial Agents and Chemotherapy, 55(11), 5380–5383. Scholar
  32. Kim, J.-S., Chowdhury, N., Yamasaki, R., & Wood, T. K. (2018). Viable but non-culturable and persistence describe the same bacterial stress state. Environmental Microbiology, 20(6), 2038–2048. Scholar
  33. Lagier, J.-C., Armougom, F., Million, M., Hugon, P., Pagnier, I., Robert, C., Bittar, F., et al. (2012). Microbial culturomics: Paradigm shift in the human gut microbiome study. Clinical Microbiology and Infection, 18(12), 1185–1193. Scholar
  34. Levin, B. R., Concepción-Acevedo, J., & Udekwu, K. I. (2014). Persistence: A copacetic and parsimonious hypothesis for the existence of non-inherited resistance to antibiotics. Current Opinion in Microbiology, 21, 18–21. Scholar
  35. Levin-Reisman, I., Gefen, O., Fridman, O., Ronin, I., Shwa, D., Sheftel, H., & Balaban, N. Q. (2010). Automated imaging with ScanLag reveals previously undetectable bacterial growth phenotypes. Nature Methods, 7(9), 737–739. Scholar
  36. Luidalepp, H., Jõers, A., Kaldalu, N., & Tenson, T. (2011). Age of inoculum strongly influences persister frequency and can mask effects of mutations implicated in altered persistence. Journal of Bacteriology, 193(14), 3598–3605. Scholar
  37. MacKenzie, F. M., & Gould, I. M. (1993). The post-antibiotic effect. The Journal of Antimicrobial Chemotherapy, 32(4), 519–537.CrossRefGoogle Scholar
  38. Marques, C. N. H., Morozov, A., Planzos, P., & Zelaya, H. M. (2014). The fatty acid signaling molecule Cis-2-decenoic acid increases metabolic activity and reverts persister cells to an antimicrobial-susceptible state. Applied and Environmental Microbiology, 80(22), 6976–6991. Scholar
  39. Matilla, M. A. (2018). Shedding light into the mechanisms of formation and resuscitation of persistent bacterial cells. Environmental Microbiology, 20(9), 3129–3131. Scholar
  40. Mizunaga, S., Kamiyama, T., Fukuda, Y., Takahata, M., & Mitsuyama, J. (2005). Influence of inoculum size of Staphylococcus aureus and Pseudomonas aeruginosa on in vitro activities and in vivo efficacy of fluoroquinolones and carbapenems. Journal of Antimicrobial Chemotherapy, 56(1), 91–96. Scholar
  41. Mukamolova, G. V., Kaprelyants, A. S., Young, D. I., Young, M., & Kell, D. B. (1998). A bacterial cytokine. Proceedings of the National Academy of Sciences of the United States of America, 95(15), 8916–8921. Scholar
  42. Mukamolova, G. V., Murzin, A. G., Salina, E. G., Demina, G. R., Kell, D. B., Kaprelyants, A. S., & Young, M. (2006). Muralytic activity of Micrococcus luteus Rpf and its relationship to physiological activity in promoting bacterial growth and resuscitation. Molecular Microbiology, 59(1), 84–98. Scholar
  43. Mulvey, M. A., Schilling, J. D., & Hultgren, S. J. (2001). Establishment of a persistent Escherichia coli reservoir during the acute phase of a bladder infection. Infection and Immunity, 69(7), 4572–4579. Scholar
  44. Mysorekar, I. U., & Hultgren, S. J. (2006). Mechanisms of uropathogenic Escherichia coli persistence and eradication from the urinary tract. Proceedings of the National Academy of Sciences of the United States of America, 103(38), 14170–14175. Scholar
  45. Nikitushkin, V. D., Demina, G. R., Shleeva, M. O., Guryanova, S. V., Ruggiero, A., Berisio, R., & Kaprelyants, A. S. (2015). A product of {RpfB} and {RipA} joint enzymatic action promotes the resuscitation of dormant mycobacteria. FEBS Journal, 282(13), 2500–2511. Scholar
  46. Oliver, J. D. (2005). The viable but nonculturable state in bacteria. Journal of Microbiology (Seoul, Korea), 43, 93–100.Google Scholar
  47. Oliver, J. D., Hite, F., McDougald, D., Andon, N. L., & Simpson, L. M. (1995). Entry into, and resuscitation from, the viable but nonculturable state by Vibrio vulnificus in an estuarine environment. Applied and Environmental Microbiology, 61(7), 2624–2630.PubMedPubMedCentralGoogle Scholar
  48. Park, J. T., & Uehara, T. (2008). How bacteria consume their own exoskeletons (turnover and recycling of cell wall peptidoglycan). Microbiology and Molecular Biology Reviews, 72(2), 211–227. Scholar
  49. Pin, C., & Baranyi, J. (2008). Single-cell and population lag times as a function of cell age. Applied and Environmental Microbiology, 74(8), 2534–2536. Scholar
  50. Pinto, D., Almeida, V., Almeida Santos, M., & Chambel, L. (2011). Resuscitation of Escherichia coli VBNC cells depends on a variety of environmental or chemical stimuli. Journal of Applied Microbiology, 110(6), 1601–1611. Scholar
  51. Porter, J., Edwards, C., & Pickup, R. W. (1995). Rapid assessment of physiological status in Escherichia coli using fluorescent probes. The Journal of Applied Bacteriology, 79(4), 399–408.CrossRefGoogle Scholar
  52. Pu, Y., Li, Y., Jin, X., Tian, T., Qi, M., Zhao, Z., Lin, S.-Y., et al. (2018). ATP-dependent dynamic protein aggregation regulates bacterial dormancy depth critical for antibiotic tolerance. Molecular Cell. CrossRefGoogle Scholar
  53. Rappé, M. S., & Giovannoni, S. J. (2003). The uncultured microbial majority. Annual Review of Microbiology, 57, 369–394. Scholar
  54. Ravagnani, A., Finan, C. L., & Young, M. (2005). A novel firmicute protein family related to the actinobacterial resuscitation-promoting factors by non-orthologous domain displacement. TL - 6. BMC Genomics, 6, 39. Scholar
  55. Reasoner, D. J., & Geldreich, E. E. (1985). A new medium for the enumeration and subculture of bacteria from potable water. Applied and Environmental Microbiology, 49(1), 1–7.PubMedPubMedCentralGoogle Scholar
  56. Rosen, D. A., Hooton, T. M., Stamm, W. E., Humphrey, P. A., & Hultgren, S. J. (2007). Detection of intracellular bacterial communities in human urinary tract infection. PLoS Medicine, 4(12), e329. Scholar
  57. Shah, I. M., Laaberki, M.-H. H., Popham, D. L., & Dworkin, J. (2008). A eukaryotic-like {Ser/Thr} kinase signals bacteria to exit dormancy in response to peptidoglycan fragments. Cell, 135(3), 486–496. Scholar
  58. Skjot-Rasmussen, L., Hammerum, A. M., Jakobsen, L., Lester, C. H., Larsen, P., & Frimodt-Moller, N. (2011). Persisting clones of Escherichia coli isolates from recurrent urinary tract infection in men and women. Journal of Medical Microbiology, 60(4), 550–554. Scholar
  59. Srimani, J. K., Huang, S., Lopatkin, A. J., & You, L. (2017). Drug detoxification dynamics explain the postantibiotic effect. Molecular Systems Biology, 13(10), 948–948. Scholar
  60. Stewart, E. J. (2012). Growing unculturable bacteria. Journal of Bacteriology, 194(16), 4151–4160. Scholar
  61. Vázquez-Laslop, N., Lee, H., & Neyfakh, A. A. (2006). Increased persistence in Escherichia coli caused by controlled expression of toxins or other unrelated proteins. Journal of Bacteriology, 188(10), 3494–3497. Scholar
  62. Zhanel, G. G., & Craig, W. A. (1994). Pharmacokinetic contributions to postantibiotic effects. Clinical Pharmacokinetics, 27(5), 377–392. Scholar
  63. Zimmermann, R., Iturriaga, R., & Becker-Birck, J. (1978). Simultaneous determination of the total number of aquatic bacteria and the number thereof involved in respiration. Applied and Environmental Microbiology, 36(6), 926–935.PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • Arvi Jõers
    • 1
  • Marta Putrinš
    • 1
  • Niilo Kaldalu
    • 1
  • Hannes Luidalepp
    • 2
  • Tanel Tenson
    • 1
    Email author
  1. 1.Insitute of TechnologyUniversity of TartuTartuEstonia
  2. 2.Quretec OÜTartuEstonia

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