Nonculturability: adaptation or debilitation?
Diane McDougald,
Scott A Rice,
Dieter Weichart,
Staffan Kjelleberg,
Diane McDougald
School of Microbiology and Immunology, The University of New South Wales, Sydney, N.S.W. 2052, Australia
Search for more papers by this authorScott A Rice
School of Microbiology and Immunology, The University of New South Wales, Sydney, N.S.W. 2052, Australia
Search for more papers by this authorDieter Weichart
Institute of Biological Sciences, University of Wales, Aberystwyth, Dyfed SY23 3DA, UK
Search for more papers by this authorCorresponding Author
Staffan Kjelleberg
School of Microbiology and Immunology, The University of New South Wales, Sydney, N.S.W. 2052, Australia
*Corresponding author. Tel.: +61 (2) 9385 2102; Fax: +61 (2) 9385 1591; E-mail: [email protected]Search for more papers by this authorDiane McDougald,
Scott A Rice,
Dieter Weichart,
Staffan Kjelleberg,
Diane McDougald
School of Microbiology and Immunology, The University of New South Wales, Sydney, N.S.W. 2052, Australia
Search for more papers by this authorScott A Rice
School of Microbiology and Immunology, The University of New South Wales, Sydney, N.S.W. 2052, Australia
Search for more papers by this authorDieter Weichart
Institute of Biological Sciences, University of Wales, Aberystwyth, Dyfed SY23 3DA, UK
Search for more papers by this authorCorresponding Author
Staffan Kjelleberg
School of Microbiology and Immunology, The University of New South Wales, Sydney, N.S.W. 2052, Australia
*Corresponding author. Tel.: +61 (2) 9385 2102; Fax: +61 (2) 9385 1591; E-mail: [email protected]Search for more papers by this authorAbstract
Investigation of bacterial survival in natural environments has indicated that some organisms lose culturability on appropriate media under certain conditions and yet still exhibit signs of metabolic activity and thus viability. This reproducible loss of culturability by many bacterial species led to the description of bacterial cells in this state as ‘viable but nonculturable’ (VBNC). It is suggested that the VBNC state is part of the life cycle of non-differentiating bacteria induced by environmental stress. The purpose of this review is to summarize some of the reports which support and refute the hypothesis that the VBNC response is a programmed response. Methods currently used in the determination of viability will be discussed with respect to their advantages and disadvantages. Reports which indicate resuscitation in vitro and in vivo, as well as those that show retention of infectivity and pathogenicity in the case of pathogenic organisms are presented as well.
References
- 1 Kjelleberg, S., Albertson, N., Flärdh, K., Holmquist, L., Jouper-Jaan, Å., Marouga, R., Östling, J., Svenblad, B. and Weichart, D. (1993) How do non-differentiating bacteria adapt to starvation Antonie van Leeuwenhoek 63, 333–341.
- 2 Oliver, J.D. (1993) Formation of viable but nonculturable cells. In: Starvation in Bacteria (Kjelleberg, S., Ed.), pp. 239–272. Plenum, New York.
- 3 Oliver, J.D. (1991) Formation of nonculturable Vibrio vulnificus cells and its relationship to the starvation state. Appl. Environ. Microbiol. 57, 2640–2644.
- 4 Paludan-Müller, C., Weichart, D., McDougald, D. and Kjelleberg, S. (1996) Analysis of starvation conditions that allow for prolonged culturability of Vibrio vulnificus at low temperature. Microbiology 142, 1675–1684.
- 5 Weichart, D., Oliver, J.D. and Kjelleberg, S. (1992) Low temperature induced non-culturability and killing of Vibrio vulnificus. FEMS Microbiol. Lett. 100, 205–210.
- 6 Nyström, T., Olsson, R.M. and Kjelleberg, S. (1992) Survival, stress resistance, and alteration in protein expression in the marine Vibrio sp. strain S14 during starvation for different individual nutrients. Appl. Environ. Microbiol. 58, 55–65.
- 7 Rahman, I., Shahamat, M., Kirchman, P.A., Russek-Cohen, E. and Colwell, R.R. (1994) Methionine uptake and cytopathogenicity of viable but nonculturable Shigella dysenteriae type 1. Appl. Environ. Microbiol. 60, 3573–3578.
- 8 Nwoguh, C.E., Harwood, C.R. and Barer, M.R. (1995) Detection of induced β-galactosidase activity in individual non-culturable cells of pathogenic bacteria by quantitative cytological assay. Mol. Biol. 17, 545–554.
- 9 Kogure, K., Simidu, U. and Taga, N. (1979) A tentative direct microscopic method for counting living marine bacteria. Can. J. Microbiol. 25, 415–420.
- 10 Rodriguez, G.G., Phipps, D., Ishiguro, K. and Ridgeway, H.F. (1992) Use of a fluorescent redox probe for direct visualization of actively respiring bacteria. Appl. Environ. Microbiol. 58, 1801–1808.
- 11 Thom, S.M., Horobin, R.W., Seidler, E. and Barer, M.R. (1993) Factors affecting the selection and use of tetrazolium salts as cytochemical indicators of microbial viability and activity. J. Appl. Bacteriol. 74, 433–443.
- 12 Ullrich, S., Karrasch, B., Hoppe, H.-G., Jeskulke, K. and Mehrens, M. (1996) Toxic effects on bacterial metabolism of the redox dye 5-cyano-2,3-ditolyl tetrazolium chloride. Appl. Environ. Microbiol. 62, 4587–4593.
- 13 Korgaonkar, K.S. and Ranade, S.S. (1966) Evaluation of acridine orange fluorescence test in viability studies of Escherichia coli. Can. J. Microbiol. 12, 185–190.
- 14 Porter, K.G. and Feig, Y.S. (1980) The use of DAPI for identifying and counting aquatic microflora. Limnol. Oceanogr. 25, 943–948.
- 15 Davey, H.M. and Kell, D.B. (1996) Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single-cell analyses. Microbiol. Rev. 60, 641–696.
- 16 Linder, K. and Oliver, J.D. (1989) Membrane fatty acid and virulence changes in the viable but nonculturable state of Vibrio vulnificus. Appl. Environ. Microbiol. 55, 2837–2842.
- 17 Morgan, J.A.W., Cranwell, P.A. and Pickup, R.W. (1991) Survival of Aeromonas salmonicida in lake water. Appl. Environ. Microbiol. 57, 1777–1782.
- 18 Mukamolova, G.V., Kormer, S.S., Yanopolskaya, N.D. and Kaprelyants, A.S. (1995) Properties of dormant cells in stationary-phase cultures of Micrococcus luteus during prolonged incubation. Mikrobiologiya 64, 284–288.
- 19 Mukamolova, G.V., Kaprelyants, A.S. and Kell, D.B. (1995) Secretion of an antibacterial factor during resuscitation of dormant cells in Micrococcus luteus cultures held in an extended stationary phase. Antonie van Leeuwenhoek 67, 289–295.
- 20 Kondo, K., Takade, A. and Amako, K. (1994) Morphology of the viable but nonculturable Vibrio cholerae as determined by the freeze fixation technique. FEMS Microbiol. Lett. 123, 179–184.
- 21 Rollins, D.M. and Colwell, R.R. (1986) Viable but nonculturable stage of Campylobacter jejuni and its role in survival in the natural aquatic environment. Appl. Environ. Microbiol. 52, 531.
- 22 Effendi, I. and Austin, B. (1995) Dormant/unculturable cells of fish pathogen Aeromonas salmonicida. Microbiol. Ecol. 30, 183–192.
- 23 Weichart, D. and Kjelleberg, S. (1996) Stress resistance and recovery potential of culturable and viable but nonculturable cells of Vibrio vulnificus. Microbiology 142, 845–853.
- 24 Rahman, I., Shahamat, M., Chowdhury, M.A.R. and Colwell, R.R. (1996) Potential virulence of viable but nonculturable Shigella dysenteriae type 1. Appl. Environ. Microbiol. 62, 115–120.
- 25 Yamamoto, H., Hashimoto, Y. and Ezaki, T. (1996) Study of nonculturable Legionella pneumophila cells during multiple-nutrient starvation. FEMS Microbiol. Ecol. 20, 149–154.
- 26 Brauns, L.A., Hudson, M.C. and Oliver, J.D. (1991) Use of the polymerase chain reaction in detection of culturable and nonculturable Vibrio vulnificus cells. Appl. Environ. Microbiol. 57, 2651–2655.
- 27 Weichart, D., McDougald, D., Jacobs, D. and Kjelleberg, S. (1997) In situ analysis of nucleic acids in cold-induced non-culturable Vibrio vulnificus. Appl. Environ. Microbiol. (in press).
- 28 Colwell, R.R., Brayton, P.R., Grimes, D.J., Roszak, D.B., Huq, S.A. and Palmer, L.M. (1985) Viable but non-culturable Vibrio cholerae and related pathogens in the environment: Implications for release of genetically engineered microorganisms. Bio/Technology 3, 817–820.
- 29 Grimes, D.J. and Colwell, R.R. (1986) Viability and virulence of Escherichia coli suspended by membrane chamber in semitropical ocean water. FEMS Microbiol. Lett. 34, 161–165.
- 30 Colwell, R.R., Brayton, P., Herrington, D., Tall, B., Huq, A. and Levine, M.M. (1996) Viable but non-culturable Vibrio cholerae 01 revert to a culturable state in the human intestine. World J. Microbiol. Biotechnol. 12, 28–31.
- 31 Jones, D.M., Sutcliffe, E.M. and Curry, A. (1991) Recovery of viable but non-culturable Campylobacter jejuni. J. Gen. Microbiol. 137, 2477–2482.
- 32 Saha, S.K., Saha, S. and Sanyal, S.C. (1991) Recovery of injured Campylobacter jejuni cells after animal passage. Appl. Environ. Microbiol. 57, 3388–3399.
- 33 Stern, N.J., Jones, D.M., Wesley, I.V. and Rollins, D.M. (1994) Colonization of chicks by non-culturable Campylobacter spp. Lett. Appl. Microbiol. 18, 333–336.
- 34 Medema, G.J., Schets, F.M., van de Giessen, A.W. and Havelaar, A.H. (1992) Lack of colonization of 1 day old chicks by viable, non-culturable Campylobacter jejuni. J. Appl. Bacteriol. 72, 512–516.
- 35 Beumer, R.R., de Vries, J. and Rombouts, F.M. (1992) Campylobacter jejuni non-culturable coccoid cells. Int. J. Food Microbiol. 15, 153–163.
- 36 Hussong, D., Colwell, R.R., O'Brien, M., Weiss, E., Pearson, A.D., Weiner, R.M. and Burge, W.D. (1987) Viable Legionella pneumophila not detectable by culture on agar media. Bio/Technology 5, 947–950.
- 37 Oliver, J.D. and Bockian, R. (1995) In vivo resuscitation, and virulence towards mice, of viable but nonculturable cells of Vibrio vulnificus. Appl. Environ. Microbiol. 61, 2620–2623.
- 38 Roszak, D.B., Grimes, D.J. and Colwell, R.R. (1984) Viable but nonrecoverable stage of Salmonella enteritidis in aquatic systems. Can. J. Microbiol. 30, 334–338.
- 39 Evdokimova, N.V., Dorofeev, A.G. and Panikov, N.S. (1994) Dynamics of survival and transition to dormant state of nitrogen-starving bacteria Pseudomonas fluorescens. Mikrobiologiya 63, 99–104.
- 40 Nilsson, L., Oliver, J.D. and Kjelleberg, S. (1991) Resuscitation of Vibrio vulnificus from the viable by nonculturable state. J. Bacteriol. 173, 5054–5059.
- 41 Ravel, J., Knight, I.T., Monahan, C.E., Hill, R.T. and Colwell, R.R. (1995) Temperature induced recovery of Vibrio cholerae from the viable but nonculturable state: growth or resuscitation Microbiology 141, 377–383.
- 42 Oliver, J.D., Hite, F., McDougald, D., Andon, N.L. and Simpson, L.M. (1995) Entry into, and resuscitation from, the viable but nonculturable state by Vibrio vulnificus in an estuarine environment. Appl. Environ. Microbiol. 61, 2624–2630.
- 43 Roth, W.G., Leckie, M.P. and Dietzler, D.N. (1988) Restoration of colony-forming activity in osmotically stressed Escherichia coli by betaine. Appl. Environ. Microbiol. 54, 3142–3146.
- 44 Wai, S.N., Moriya, T., Kondo, K., Misumi, H. and Amako, K. (1996) Resuscitation of Vibrio cholerae 01 strain TSI-4 from a viable but nonculturable state by heat shock. FEMS Microbiol. Lett. 136, 187–191.
- 45 Kaprelyants, A.S., Mukamolova, G.V., Davey, H.M. and Kell, D.B. (1996) Quantitative analysis of the physiological heterogeneity within starved cultures of Micrococcus luteus by flow cytometry and cell sorting. Appl. Environ. Microbiol. 62, 1311–1316.
- 46 Kaprelyants, A.S., Mukamolova, G.V. and Kell, D.B. (1994) Estimation of dormant Micrococcus luteus cells by penicillin lysis and by resuscitation in cell-free spent culture medium at high dilution. FEMS Microbiol. Lett. 115, 347–352.
- 47 Votyakova, T.V., Kaprelyants, A.S. and Kell, D.B. (1994) Influence of viable cells on the resuscitation of dormant cells in Micrococcus luteus cultures held in an extended stationary phase. The population effect. Appl. Environ. Microbiol. 60, 3284–3291.
- 48 Kaprelyants, A.S., Mukamolova, G.V., Davey, H.M. and Kell, D.B. (1996) The resuscitation of dormant cells of Micrococcus luteus. Society for General Microbiology, 135th Meeting, University of Essex, UK.
- 49 Turpin, P.E., Maycroft, K.A., Rowlands, C.L. and Wellington, E.M.H. (1993) Viable but non-culturable salmonellas in soil. J. Appl. Bacteriol. 74, 421–427.
- 50 Pommepuy, M., Butin, M., Derrien, A., Gourmelon, M., Colwell, R.R. and Cormier, M. (1996) Retention of enteropathogenicity by viable but nonculturable Escherichia coli exposed to seawater and sunlight. Appl. Environ. Microbiol. 62, 4621–4626.
