1887

Abstract

The estuarine, human-pathogenic bacterium responds to low temperature by the formation of viable but nonculturable (VBNC) cells, while starvation at moderate temperatures allows for maintenance of culturability of this organism. Recovery of cold-incubated populations of was restricted to the culturable fraction in slide cultures and most probable number assays. These populations, however, gave between 1.1- and 8-fold higher c.f.u. counts on soft agar plates than on ordinary agar plates, indicating that a small and variable fraction of the cell population was injured rather than nonculturable. Thus, the population of cold-incubated cells is composed of culturable, injured and nonculturable cells, with the numbers of the culturable and injured cells rapidly decreasing during cold incubation. Recovery of nonculturable cells of the organism, however, could not be obtained by any combination of temperature and nutrient shifts in any of the assays. VBNC cells of the organism were assessed with regard to their persistence and stress resistance in comparison to growing and starved cells. The sonication resistance of VBNC cells was initially similar to that of growing cells, but increased during prolonged cold incubation. The final resistance of cold-incubated VBNC cells was equal to the markedly increased resistance of starving cells, which also displayed increased resistance against exposure to ethanol and mechanical stress. Our results indicate that in spite of the apparent absence of recovery under a wide range of laboratory conditions, VBNC cells of undergo changes at low temperature which potentially allow them to persist for extended periods.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-142-4-845
1996-04-01
2024-12-05
Loading full text...

Full text loading...

/deliver/fulltext/micro/142/4/mic-142-4-845.html?itemId=/content/journal/micro/10.1099/00221287-142-4-845&mimeType=html&fmt=ahah

References

  1. Allen-Austin D., f Austin B., Colwell R.R. Survival of A eromonas salmonicida in river water. FEMS Microbiol Eett 1984; 21:143–146
    [Google Scholar]
  2. Azam F., Fenchel T., Field J.G., Gray J.S., Meyer-Reil L.A., Thingstad F. The ecological role of water-column microbes in the sea. Mar Ecol Prog Ser 1983; 10:257–263
    [Google Scholar]
  3. Barer M.R., Gribbon L.T., Harwood C.R., Nwoguh C.E. The viable but non-culturable hypothesis and medical microbiology. Rev Med Microbiol 1993; 4:183–191
    [Google Scholar]
  4. Binnerup S.J., Jensen D.F., Thordal-Christensen H., Soerensen J. Detection of viable, but nonculturable Pseudomonas fluorescens DF57 in soil using a microcolony epi-fluorescence technique. FEMS Microbiol Ecol 1993; 12:97–105
    [Google Scholar]
  5. Buchanan C.E., Sowell M.O. Synthesis of penicillin-binding protein 6 by stationary-phase Escherichia coli. J Bacteriol 1982; 151:491–494
    [Google Scholar]
  6. Colwell R.R., Brayton P.R., Grimes D.J., Roszak D.B., Huq S.A., Palmer L.M. Viable but nonculturable Vibrio cholerae and related pathogens in the environment: implications for release of genetically engineered microorganisms. Bio/Technology 1985; 3:817–820
    [Google Scholar]
  7. Diaper J.P., Edwards C. The use of fluorogenic esters to detect viable bacteria by flow cytometry. J Appl Bacteriol 1994; 77:221–228
    [Google Scholar]
  8. Dufour P., Colon M. The tetrazolium reduction method for assessing the viability of individual bacterial cells in aquatic environments: improvements, performance and applications. Hydrobiologia 1992; 232:211–218
    [Google Scholar]
  9. Firth J.R., Diaper J.P., Edwards C. Survival and viability of Vibrio vulnificus in seawater monitored by flow cytometry. Eett Appl Microbiol 1994; 18:268–271
    [Google Scholar]
  10. Fry J.C., Zia T. A method for estimating viability of aquatic bacteria by slide culture. J Appl Bacteriol 1982; 53:189–198
    [Google Scholar]
  11. Givskov M., Eberl L., Moller S., Kongsbak Poulsen L., Molin S. Responses to nutrient starvation in Pseudomonas putida KT2442: analysis of general cross-protection, cell shape, and macromolecular content. J Bacteriol 1994; 176:7–14
    [Google Scholar]
  12. Grimes D.J., Colwell R.R. Viability and virulence of Escherichia coli suspended by membrane chamber in semitropical ocean water. FEMS Microbiol Eett 1986; 34:161–165
    [Google Scholar]
  13. Hartke A., Bouche S., Gansel X., Boutibonnes P., Auffray Y. Starvation-induced stress resistance in Eactococcus lactis subsp. lactis IL1403. Appl Environ Microbiol 1994; 60:3474–3478
    [Google Scholar]
  14. Heinmets F., Taylor W.W., Lehman J.J. The use of metabolites in the restoration of the viability of heat and chemically inactivated Escherichia coli. J Bacteriol 1954; 67:5–12
    [Google Scholar]
  15. Heldal M., Bratbak G. Production and decay of viruses in aquatic environments. Mar Ecol Prog Ser 1991; 72:205–212
    [Google Scholar]
  16. Hennes K.P., Simon M. Significance of bacteriophages for controlling bacterioplankton growth in a mesotrophic lake. Appl Environ Microbiol 1995; 61:333–340
    [Google Scholar]
  17. Hoben H.J., Somasegaran P. Comparison of the pour, spread, and drop plate methods for enumeration of Rhizobium spp in inoculants made from presterilized peat. Appl Environ Microbiol 1982; 44:1246–1247
    [Google Scholar]
  18. Husevdg B. Starvation survival of the fish pathogen Aeromonas salmonicida in seawater. FEMS Microbiol Ecol 1995; 16:25–32
    [Google Scholar]
  19. Hussong D., Colwell R.R., O'Brien M., Weiss E., Pearson A.D., Weiner R.M., Burge W.D. Viable Eegionella pneumophila not detectable by culture on agar media. Bio/Technology 1987; 5:947–950
    [Google Scholar]
  20. Jenkins D.E., Schultz J.E., Matin A. Starvation-induced cross protection against heat or H202 challenge in Escherichia coli. J Bacteriol 1988; 170:3910–3914
    [Google Scholar]
  21. Jenkins D.E., Chaisson S.A., Matin A. Starvation-induced cross protection against osmotic challenge in Escherichia coli. J Bacteriol 1990; 172:2779–2781
    [Google Scholar]
  22. Jones D.M., Sutcliffe E.M., Curry A. Recovery of viable but non-culturable Campylobacter jejuni. J Gen Microbiol 1991; 137:2477–2482
    [Google Scholar]
  23. Jouper-Jaan A., Goodman A.E., Kjelleberg S. Bacteria starved for prolonged periods develop increased protection against lethal temperatures. FEMS Microbiol Ecol 1992; 101:229–236
    [Google Scholar]
  24. Kaprelyants A.S., Kell D.B. Rapid assessment of bacterial viability and vitality by rhodamine 123 and flow cytometry. J Appl Bacteriol 1992; 72:410–422
    [Google Scholar]
  25. Kaprelyants A.S., Gottschal J.C., Kell D.B. Dormancy in non-sporulating bacteria. FEMS Microbiol Rev 1993; 104:271–286
    [Google Scholar]
  26. Kaprelyants A.S., Mukamolova G.V., Kell D.B. Estimation of dormant Micrococcus luteus cells by penicillin lysis and by resuscitation in cell-free spent culture medium at high dilution. FEMS Microbiol Lett 1994; 115:347–352
    [Google Scholar]
  27. Kaspar C.W., Tamplin M.L. Effects of temperature and salinity on the survival of Vibrio vulnificus in seawater and shellfish. Appl Environ Microbiol 1993; 59:2425–2429
    [Google Scholar]
  28. Kogure K., Simidu U., Taga N. A tentative direct microscopic method for counting living marine bacteria. Can J Microbiol 1979; 25:415–420
    [Google Scholar]
  29. Kondo K., Takade A., Amako K. Morphology of the viable but nonculturable Vibrio cholerae as determined by the freeze fixation technique. FEMS Microbiol Eett 1994; 123:170–184
    [Google Scholar]
  30. Leduc M., Frzhel C., Siegel E., Van Heijenoort J. Multilayered distribution of peptidoglycan in the periplasmic space of Escherichia coli. J Gen Microbiol 1989; 135:1243–1254
    [Google Scholar]
  31. Lee K., Ruby E.G. Symbiotic role of the viable but nonculturable state of Vibrio fischeri in Hawaiian coastal seawater. Appl Environ Microbiol 1995; 61:278–283
    [Google Scholar]
  32. Lewis P.J., Nwoguh C.E., Barer M.R., Harwood C.R., Errington J. Use of digitized video microscopy with a fluorogenic enzyme substrate to demonstrate cell- and compartment-specific gene expression in Salmonella enteridis and bacillus subtilis. Mol Microbiol 1994; 13:655–662
    [Google Scholar]
  33. Van Der Linden M.P.G., De Haan L., Hoyer M.A., Keck W. Possible role of Escherichia coli penicillin-binding protein 6 in stabilization of stationary-phase peptidoglycan. J Bacterial 1992; 174:7572–7578
    [Google Scholar]
  34. Linder K., Oliver J.D. Membrane fatty acid and virulence changes in the viable but nonculturable state of Vibrio vulnificus. Appl Environ Microbiol 1989; 55:2837–2842
    [Google Scholar]
  35. McCann M.P., Kidwell J.P., Matin A. The putative sigma factor KatF has a central role in development of starvation-mediated general resistance in Escherichia coli. J Bacteriol 1991; 173:4188–4194
    [Google Scholar]
  36. Morgan J.A.W., Cranwell P.A., Pickup R.W. Survival of Aeromonas salmonicida in Lake Water. Appl Environ Microbiol 1991; 57:1777–1782
    [Google Scholar]
  37. Morita R.Y. Bioavailability of energy and the starvation state. In Starvation in Bacteria 1993 Edited by Kjelleberg S. New York & London: Plenum Press; pp 1–23
    [Google Scholar]
  38. Neidhardt F., Bloch P.L., Smith D.F. Culture medium for Enterobacteriaceae. J Bacteriol 1974; 119:736–747
    [Google Scholar]
  39. Nilsson L., Oliver J.D., Kjelleberg S. Resuscitation of Vibrio vulnificus from the viable but nonculturable state. J Bacteriol 1991; 173:5054–5059
    [Google Scholar]
  40. Nystrbm T., Mirden P., Kjelleberg S. Relative changes in incorporation rates of leucine and methionine during starvation survival of two bacteria isolated from marine waters. FEMS Microbiol Ecol 1986; 38:285–292
    [Google Scholar]
  41. Nystrom T., Olsson R.M., Kjelleberg S. Survival, stress resistance, and alterations in protein expression in the marine Vibrio sp. strain S14 during starvation for different individual nutrients. Appl Environ Microbiol 1992; 58:55–65
    [Google Scholar]
  42. Oliver J.D. Formation of viable but nonculturable cells. In Starvation in Bacteria 1993 Edited by Kjelleberg S. New York & London: Plenum Press; pp 239–272
    [Google Scholar]
  43. Oliver J.D., Bockian R. In vivo resuscitation, and virulence towards mice, of viable but nonculturable cells of Vibrio vulnificus. Appl Environ Microbiol 1995; 61:2620–2623
    [Google Scholar]
  44. Oliver J.D., Nilsson L., Kjelleberg S. Formation of nonculturable cells of Vibrio vulnificus and its relationship to the starvation state. Appl Environ Microbiol 1991; 57:2640–2644
    [Google Scholar]
  45. Oliver J.D., Hite F., McDougald D., Andon N.L., Simpson L.M. Entry into, and resuscitation from, the viable but nonculturable state by Vibrio vulnificus in an estuarine environment. Appl Environ Microbiol 1995; 61:2624–2630
    [Google Scholar]
  46. Ostling J., Goodman A., Kjelleberg S. Behaviour of IncP-1 plasmids and a miniMu transposon in a marine Vibrio sp. isolation of starvation inducible lac operon fusions. FEMS Microbiol Ecol 1991; 86:83–94
    [Google Scholar]
  47. Postgate J.R., Hunter J.R. Acceleration of bacterial death by growth substrates. Nature 1963; 198:273
    [Google Scholar]
  48. Proctor L.M., Okubo A., Fuhrman J.A. Calibrating estimates of phage-induced mortality in marine bacteria: ultra-structural studies of marine bacteriophage development from one-step growth experiments. Microb Ecol 1993; 25:161–182
    [Google Scholar]
  49. Quesnel L.B. A genealogical study of clonal development of Escherichia coli. J Appl Bacteriol 1963; 26:127–151
    [Google Scholar]
  50. Ravel J., Knight I.T., Monahan C.E., Hill R.T., Colwell R.R. Temperature-induced recovery of Vibrio cholerae from the viable but nonculturable state: growth or resuscitation. Microbiology 1995; 141:377–383
    [Google Scholar]
  51. Rodriguez G.G., Phipps D., Ridgway H.F. Use of a fluorescent redox probe for direct visualization of actively respiring bacteria. Appl Environ Microbiol 1992; 58:1801–1808
    [Google Scholar]
  52. Rose A.S., Ellis A.E., Munro A.L.S. Evidence against dormancy in the bacterial fish pathogen Aeromonas salmonicida subsp salmonicida. FEMS Microbiol Eett 1990; 68:105–108
    [Google Scholar]
  53. Roszak D.B., Colwell R.R. Survival strategies of bacteria in the natural environment. Microbiol Rev 1987; 51:365–379
    [Google Scholar]
  54. Roszak D.B., Grimes D.J., Colwell R.R. Viable but nonrecoverable stage of Salmonella enteridis in aquatic systems. Can J Microbiol 1984; 30:334–338
    [Google Scholar]
  55. Saha S.K., Saha S., Sanyal S.C. Recovery of injured Campylobacter jejuni cells after animal passage. Appl Environ Microbiol 1991; 57:3388–3389
    [Google Scholar]
  56. Simpson L.M., White V.K., Zane S.F., Oliver J.D. Correlation between virulence and colony morphology in Vibrio vulnificus. Infect Immun 1987; 55:269–272
    [Google Scholar]
  57. Terleckyj B., Willet N.P., Shockman G.D. Growth of several cariogenic strains of oral streptococci in a chemically defined medium. Infect Immun 1975; 11:649–655
    [Google Scholar]
  58. Torrella F., Morita R.Y. Microcultural study of bacterial size changes and microcolony and ultramicrocolony formation by heterotrophic bacteria in seawater. Appl Environ Microbiol 1981; 41:518–527
    [Google Scholar]
  59. Votyakova T.V., Kaprelyants A.S., Kell D.B. 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 1994; 60:3284–3291
    [Google Scholar]
  60. Weichart D., Oliver J.D., Kjelleberg S. Low temperature induced non-culturability and killing of Vibrio vulnificus. FEMS Microbiol Eett 1992; 100:205–210
    [Google Scholar]
  61. Williamson F.A., Palframan K.R. An improved method for collecting and staining microorganisms for enumeration by fluorescence light microscopy. J Microsc 1989; 154:267–272
    [Google Scholar]
  62. Zimmermann R., Iturriaga R., Becker-Birck J. Simultaneous determination of the total number of aquatic bacteria and the number thereof involved in respiration. Appl Environ Microbiol 1978; 36:926–935
    [Google Scholar]
/content/journal/micro/10.1099/00221287-142-4-845
Loading
/content/journal/micro/10.1099/00221287-142-4-845
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error