The Viable but Nonculturable State in Bacteria

  • Oliver James D. (Department of Biology, University of North Carolina at Charlotte)
  • Published : 2005.02.28

Abstract

It had long been assumed that a bacterial cell was dead when it was no longer able to grow on routine culture media. We now know that this assumption is simplistic, and that there are many situations where a cell loses culturability but remains viable and potentially able to regrow. This mini-review defines what the 'viable but nonculturable' (VBNC) state is, and illustrates the methods that can be used to show that a bacterial cell is in this physiological state. The diverse environmental factors which induce this state, and the variety of bacteria which have been shown to enter into the VBNC state, are listed. In recent years, a great amount of research has revealed what occurs in cells as they enter and exist in this state, and these studies are also detailed. The ability of cells to resuscitate from the VBNC state and return to an actively metabolizing and culturable form is described, as well as the ability of these cells to retain virulence. Finally, the question of why cells become nonculturable is addressed. It is hoped that this mini-review will encourage researchers to consider this survival state in their studies as an alternative to the conclusion that a lack of culturability indicates the cells they are examining are dead.

Keywords

References

  1. Adams, B.L., TC. Bates, and J.D. Oliver. 2003. Survival of Helicobacter pylori in a natural freshwater environment. Appl. Environ. Microbiol. 69, 7462-7466.
  2. Asaka, M., AR. Sepulveda, T Sugiyama, and D.Y. Graham. 2001. Gastric Cancer, p. 481-498. In' H. Mobley, GL. Mendez, and s.r Hazell (eds.), Helicobacter pylori: physiology and genetics. American Society for Microbiology, Washington, D.C.
  3. Banin, E, T Israely, A Kushmaro, Y Loga, E Orr, and E. Rosenber. 2000. Penetration of the coral-bleacing bacterium Vibrio shiloi into Oculina patagonica. Appl. Environ. Microbiol. 66, 3031-3036.
  4. Barer, M.R. 1997. Viable butnon-culturable and dormant bacteria: time to resolve an oxymoron and a misnomer? J. Med. Microbioi. 46, 629-631.
  5. Barer, M.R. arid CR. Harwood. 1999. Bacterial viability and culturability. Adv. Micro. Physiol. 41, 93c137.
  6. Barer, M.R., A.S. Kaprelyants, D.H. Weichart, C.R. Harwood, and D.B. Kell. 1998..Microbial stress and culturability: conceptual and operational domains. Microbiology 144, 2009-2010.
  7. Barrett, T 1998. Ph.D. thesis, Univ. Aberdeen.
  8. Bates T;B. Adams, and J.D. Oliver, 2003. Survival of VBNC Helicobacter pylori cocci following antibiotic treatment. Intern. Conf. on Helicobacter pylori. Aarhus, Denmark.
  9. Begosian, G and EY. Bourneuf. 2001. A matter of bacterial life and death. EMBO Reports 2, 770-774.
  10. Begosian, G, N.D. Aardema, EY. Boumeuf, PJ.L. Morris, and J.P. ONeil. 2000. Recovery of hydrogen peroxide-sensitive culturable cells of Vibrio vulnificus gives the appearance of resuscitation from a viable but nonculturable state. J. Bacteriol. 182, 5070-5075.
  11. Bej, AK., N. Vickery, F.E Brasher, C. Jeffreys, A Jones, D.D. A. DePaola, and D.W. Cook. 1997. Use of PCR to determine genomic diversity and distribution of siderophore-mediated iron acquisition genes in clinical and environmental isolates of Vibrio vulnificus. Abstr. QI77, p. 485. Abstr. Annu. Meet. Amer. Soc. Microbial
  12. Beumer, R.R., J. de Vries, and EM. Rombouts. 1992. Campylobacter jejuni non-culturable coccoid cells. Intern. J. Food Microbiol. 15, 153-163.
  13. Bloomfield, S.F, GS.A.B. Stewart, e.E.R. Dodd, I.R. Booth, and E.GM. Power. 1998. The viable but nonculturable phenomenon explained? Microbiology 144, 1-3.
  14. Breeuwer, P. and T. Abee. 2000. Assessment of viability of microorganismsemploying fluorescence techniques. Intern. J. Food Microbiol. 55, 193-200.
  15. Bunker, S.T., T.C. Bates, and J.D. Oliver. 2004. Effects of temperature on detection of plasmid and chromosomally encoded gfpand lux-labeled Pseudomonas j1uorescens in soil. Environ. Biosafety Res. 3, 1-8.
  16. Catrenich, e.E. and K.M. Makin. 1991. Characterization of the morphologic conversion of Helicobacter pylori from bacillary to coccoid forms. Scand. J. Gastroenterol. 181, 5,8-64.
  17. Colwell, R.R., P.R. Brayton, DJ. Grimes, D.R. Roszak, S.A Huq, and L.M. Palmer. 1985. Viable, but non-culturable Vibrio cholerae and related pathogens in the environment: implication for release of genetically engineered microorganisms. Bio/Technology 3,817-820.
  18. Colwell, RR, P.R. Brayton, D. Herrington, B.D. Tall, A. Huq, and M.M. Levine. 1996. Viable but nonculturable Vibrio cholerae 01 revert to a culturable state in human intestine. World J. Microb. Biotechnol. 12, 28-31.
  19. Creach, V, A-C. Baudoux, G Bertru, and B. Le Rouzic. 2003. Direct estimate of active bacteria: CTC use and limitations. J. Microbiol. Meth. 52, 19-28.
  20. Day, AP. and J.D. Oliver. 2004. Changes in membrane fatty acid composition during entry of Vibrio vulnificus into the viable but nonculturable state. J. Microbiol. 42, 69-73.
  21. Federighi, M., J.L. Tholozan, J.M. Cappelier, J.P. Tissier, and J.L. Jouve. 1998. Evidence of non-coccoid viable but non-culturable Campylobacter jejuni cells in microcosm water by direct viable count, CTC-DAPI double staining, and scanning electron microscopy. Food Microbiol. 15, 539-550.
  22. Gunasekera, T.S., A Serensen, P.V Attfield, SJ. Sorensen, and D.A Veal. 2002. Inducible gene expression by nonculturable bacteria in milk after pasteurization. Appl. Environ. Microbial. 68, 1988-1993.
  23. Hegarty, J.P., M.T. Dowd, and KH. Baker. 1999. Occurrence of Helicobacter pylori in surface water in the United States. J. Appl. Microbial. 87, 697-70l.
  24. Heim, S.,M.D.M. Lleo, B. Bonato, e.A Guzman, and.P Canepari. 2002. The viable but nonculturable state and starvation are different stress responses of Enterococcus faecalis, as determined by proteome analysis. J. Bacteriol. 184, 6739-6745.
  25. Htilsmann, A., T.M. Rosche, I.-S. Kong, H.M. Hassan, D.M Beam, and J.D. Oliver. 2003. RpoS-dependent stress response and exoenzyme production in Vibrio vulnificus. Appl. Environ. Microbiol. 69, 6114-6120.
  26. Hulten, K, S.w. Han, H. Enroth, P.D. Klein, AR Opekun, R.H. Gilman, D.G Evans, L. Engstrand, D.Y Graham, and FA EIZaatari. 1996. Helicobacter pylori in the drinking water in Peru. Gastroenterology 110, 1031-1035.
  27. Jones, D.M., E.M. Sutcliffe, and A. Curry. 1991. Recovery of viable but non-culturable Campylobacter jejuni. J. Gen. Microbiol. 137, 2477-2482.
  28. Kell, D.B., AS. Kapreylants, D.H. Weichart, e.L. Harwood, and M.R Barer. 1998. Viability and activity in readily culturable bacteria: a review and discussion of the practical issues. Ant. van Leeuvenhoek 73,169-187.
  29. Kogure, K, U. Simidu, and N. Taga. 1979. A tentative direct microscopic method for counting living marine bacteria. Can. J. Microbial. 25,415-420.
  30. Kong, 1:-5., A Huelsmann, T'C, Bates, H. Hassan, and J.D. Oliver. 2004. The role of reactive oxygen species in the viable but nonculturable state in Vibrio vulnificus. FEMS Microbial. Ecol. 50, 133-142.
  31. Lambert; J.R., S.K Lin, and J. Aranda-Michel. 1995. Helicobacter pylori. Scand. J. Gastroenterol. 30(Suppl. 208), 33-46.
  32. u-e, M.M., S. Pierobon, M.e. Tafi, e. Signoreto, and P. Canepari, 2000. mRNA detection by reverse transcription-PCR for monitoring viability overtime in an Enterococcus faecalis viable but nonculturable population maintained in a laboratory microcosm. Appl. Environ. Microbiol. 66,4564-4567.
  33. Lleo, M.M., B. Bonato, M.e. Tafi, e. Signoretto, M. Boaretti, and P. Canepari. 200 I. Resuscitation rate in different enterococcal species in the viable but non-culturable state. J. Appl. Microbioi. 91, 1095-1102.
  34. Makino, S.-I., T. Kii, H. Asakura, T. Shirahata, T. Ikeda, K Takeshi, and K Itoh. 2000. Does enterohaeinorrhagic Escherichia coli 0157 enter the VNC state in salmon roe? Appl. Environ. Microbial. 66, 5536-5539.
  35. McDougald, D., J.I. Prosser, L.A Glover, and J.D. Oliver. 1995. Effect of temperature and plasmid carriage on nonculturability in organisms targeted for release. FEMS Microbiol. Ecol. 17, 229-238.
  36. McFeters, GA, FP. Yu, B.H. Pyle, and P.S. Stewart. 1995. Physiological assessment of bacteria using fluorochromes. J. Microbiol. Meth. 21,1-13.
  37. McGovern, VP. and J.D. Oliver. 1995. Induction of cold responsive proteins in Vibrio vulnificus. J. Bacteriol. 177,4131-4133.
  38. Mizunoe, Y, S.N. Wai, A Takade, and S.-i. Yoshida. 1999. Restorationof culturability of starvation-stressed and low-temperature-stressed Escherichia coli 0157 cells by using $H_2O_2$degrading compounds. Arch. Microbiol. 172, 63-67.
  39. Mizunoe, Y, S.N. Wai, T. Ishikawa, A. Takade, and S.-i. Yoshida. 2000. Resuscitation of viable but nonculturable cells of Vibrio parahaemolyticus induced at low temperature under starvation. FEMS Microbiol. Lett. 186,115-120.
  40. Moreno, "y., M.A. Ferrus, J.L. Alonso, A Jimenez, and J. Hernandez. 2003. Use of fluorescent in situ hybridization to evidence the presence of Helicobacter pylori in water. Water Res. 37, 2251 02256.
  41. Morton, D. and J.D. Oliver. 1994. Induction of carbon starvation proteins in Vibrio vulnificus. Appl. Environ. Microbiol. 60, 3653-3659.
  42. Mukamolova, G.Y., N.D. Yanopolskaya, D.B. Kell, and AS. KapreIyants. 1998a. On resuscitation from the donnant state of Micrococcus luteus. Ant. Van Leeuwen. 73, 237-243.
  43. Mukamolova, GV, A.S. Kaprelyants, D.l. Young, M. Young, and D.B. Kell. 1998b. A bacterial cytokine. Proc. Natl. Acad. Sci. USA 95, 8916-8921.
  44. Mukamolova, GV, S.S. Kanner, D.B. Kell, and A.S. Kaprelyants. 1999. Stimulation of the multiplication of Micrococcus luteus by an autocrine growth factor. Arch Microbial. 172, 9-14.
  45. Oliver, J.D. 1993. Formation of viable but nonculturable cells, p. 239-272. In S. Kjelleberg (ed.), Starvation in Bacteria. Plenum Press, NY.
  46. Oliver, J.D. 2000a. Problems in detecting dormant (VBNC) cells and the role of DNA elements in this response, p. 1-15. In J.K. Jansson, J.D. van Elsas, and MJ. Bailey (eds.), Tracking Genetically-Engineered Microorganisms. Landes Biosciences, Georgetown, TX.
  47. Oliver, J.D. 2000b. The public health significance of viable but nonculturable bacteria, p. 277-299. In R.R. Colwell and DJ. Grimes (eds.), Nonculturable Microorganisms in the Environment. American Society for Microbiology Press, Washington, D.C.
  48. Oliver, J.D. 2000c. The viable but nonculturab1e state and cellular resuscitation, p. 723-730. In CR. Bell, M. Brylinsky, and P. Johnson-Green (eds.), Microbial Biosystems: New Frontiers. Atlantic Canada Soc. Microb. Ecol., Halifax, Canada.
  49. Oliver, J.D. 2005. Viable but nonculturable bacteria in food environments. In P.M. Fratarnico and A.K. Bhunia (eds.), Food Borne Pathogens: Microbiology and Molecular Biology. Horizon Scientific Press, Norfolk, U.K. (in press).
  50. Oliver, J.D. and R Bockian. 1995. In vivo resuscitation, and virulence towards mice, of viable but nonculturable cells of Vibrio vulnificus. Appl. Environ. Microbiol. 61, 2620-2623.
  51. Oliver, J.D., L. Nilsson, and S. Kjelleberg. 1991. The formation of nonculturable cells of Vibrio vulnificus and its relationship to the starvation state. Appl. Environ. Microbiol. 57, 2640-2644.
  52. Oliver, J.D., M. Dagher, and K. Linden. 2005. Induction of Escherichia coli and Salmonella typhimurium into the viable but nonculturable state following chlorination of wastewater. J. Water and Health (in press).
  53. Oliver, J.D., F. Hite, D. McDougald, N.L. Andon, and L.M. Simpson. 1995. Entry into, and resuscitation from, the viable but nonculturable state by Vibrio vulnificus in an estuarine environment. Appl. Environ. Microbiol. 61,2624-2630.
  54. Peterson, W. 1. 1991. Helicobacter pylori and peptic ulcer disease. N. Engl. J. Med. 324, 1043-1048.
  55. Porter, J., e. Edwards, and RW. Pickup. 1995. Rapid assessment of physiological status in Escherichia coli using fluorescent probes. J. Appl. Bacteriol. 4, 399-408.
  56. Pruzzo, c. R Tarsi, M.M. uee, e. Signoretto, M. Zampini, R.R Colwell, and P. Canepari. 2002. Rapid assessment of physiological status in Escherichia coli using fluorescent probes. Curr. Microbiol. 45, 105-11O.
  57. Rahman, 1., M. Shahamat, P.A. Kirchman, E. Russek-Cohen, and R.R. Colwell. 1994. Methionine uptake and cytopathogenicity of viable but nonculturable Shigella dysenteriae Type 1. Appl. Environ. Microbiol. 60, 3573-3578.
  58. Rahman, 1., M. Shahamat, MAR. Chowdhury, and RR Colwell. 1996. Potential virulence of viable but nonculturable Shigella dysenteriae Type 1. Appl. Environ. Microbiol. 62, 115-120.
  59. Rodriguez, GG, D. Phipps, K. Ishiguro, and H.F. Ridgway. 1992. Use of a fluorescent redox probe for direct visualization of actively respiring bacteria. Appl. Environ. Microbiol. 58, 18011808.
  60. Rosenberg, E. and Y. Ben-Haim. 2002. Microbial diseases of corals and global warming. Environ. Microbiol. 4, 318-326.
  61. Saux, M.F.-L., D. Hervio-Heath, S. Loaec, RR Colwell, and M. Pommepuy. 2002. Detection of cytotoxin-hemolysin mRNA in nonculturable populations of environmental and clinical Vibrio vulnificus strains in artificial seawater. Appl. Environ. Microbial. 68, 5641-5646.
  62. Signoretto, C; M.M. Lleo, and P. Canepari. 2002. Modification of the peptidoglycan of Escherichia coli in the viable but nonculturable state. Curr. Microbiol. 44, 125-131.
  63. Signoretto, c, M.M. uee, M.e. Tafi, and P. Canepari. 2000. Cell wall chemical composition of Enterococcus faecalis in the viable but nonculturable state. Appl. Environ. Microbiol. 66, 19531959.
  64. Steinert, M., L. R. Emody, Amann et al. 1997. Resuscitation of viable but nonculturable Legionella pneumophila Philadelphia JR32 by Acanthamoeba castellanii. Appl. Environ. Microbiol. 63, 2047-2053.
  65. Tholozan, J.L., J.M. Cappelier, J.P. Tissier, G Delattre, and M. Federighi. 1999. Physiological characterization of viable-but-nonculturable Camplyobacterjejuni cells. Appl. Environ. Microbial. 65, 1ll0-1116.
  66. Warner, J.M. and J.D. Oliver. 1998. Randomly amplified polymorphic DNA analysis of starved and viable but noncu1turab1e Vibrio vulnificus cells. Appl. Environ. Microbial. 64, 30253028.
  67. Whitesides, M.D. and J.D. Oliver. 1997. Resuscitation of Vibrio vulnificus from' the viable but nonculturab1e state. Appl. Environ. Microbiol. 64, 3025-3028.
  68. Wolf, P. and J.D: Oliver. 1992. Temperature effects on the viable but nonculturable state of Vibrio vulnificus. FEMS Microbiol. Ecol. 101, 33-39.
  69. Xu, H.-S., N. Roberts, F.L. Singleton, RW. Attwell, DJ. Grimes, and RR. Colwell. 1982. Survival and viability of nonculturable Escherichia coli and Vibrio cholerae in the estuarine and marine environment. Microb. Ecol. 8, 313-323.
  70. Yaron, S. and K. Matthews. 2002. A reverse transcriptase-polymerase chain reaction assay for detection of viable Escherichia coli O157:H7: investigation of specific target genes. J. Appl. Microbiol. 92, 633-640.
  71. Zimmerman, R, R Iturriaga, and J. Becker-Birck. 1978. Simultaneous determination of the total number of aquatic bacteria and the number thereof involved in respiration. Appl. Environ. Microbiol. 36, 926-935.