Epidemiological Significance of Viable but Nonculturable Microorganisms

  • Anwarul Huq
  • Irma N. G. Rivera
  • Rita R. Colwell


Although the term “viable but nonculturable” (VBNC) has been used in the literature during the past two decades to describe a survival strategy of microorganisms, notably human pathogens in the aquatic environment, there is an abundance of philosophical musings on this subject in the microbiological literature. For example, Radsimosky in 1930 (119) noted a significant difference in the number of autotrophic and organotrophic bacteria in water samples. Direct microscopic enumeration, used in water bacteriology at the time, yielded counts 200 to 5,000 times higher than culture counts on bacteriological plates (16, 17). The difference between results of bacterial enumeration by direct observation and subsequent measurement of oxygen demand led Butkevitch and Butkevitch (18) to conclude that a significant portion of a given bacterial population, which did not appear as colonies on plates, must be present in a resting stage. Knaysi (82) and Jennison (70) were able to demonstrate metabolic activity of organisms observed by direct microscopy to be present in a sample but failing to grow, hence not appearing as colonies on plates. ZoBell (156) reconfirmed earlier findings that plate culture counts of seawater, although the widely used method at the time (and to this day), yield only a small percentage of the bacteria actually present in a given sample.


Vibrio Cholerae Indirect Fluorescent Antibody VBNC State Epidemiological Significance Coccoid Form 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Alfimov, N. M. 1954. Comparative evaluation of methods for determination of the bacterial count in the sea water. Microbiology 23:693.PubMedGoogle Scholar
  2. 2.
    Allen-Austin, D., B. Austin, and R. R. Colwell. 1984. Survival of Aeromonas salmonicida in river water. FEMS Microbiol. Lett. 21:143–146.CrossRefGoogle Scholar
  3. 3.
    Amann, R., W. Ludarg, and K. Schleifer. 1994. Identification of uncultured bacteria: a challenging test for molecular taxonomists. ASM News 60:360–365.Google Scholar
  4. 4.
    Bakker, E. P., H. Rottenberg, and S. R. Caplan. 1976. An estimation of the light-induced electrochemical potential difference of protons across the membrane of Halobacterium halobium. Biochim. Biophys. Acta 440:557–572.PubMedCrossRefGoogle Scholar
  5. 5.
    Barcina, I., J. M. Gonzalez, J. Iniberri, and L. Egea. 1989. Effect of visible light on progressive dormancy of E. coli cells during the survival process in natural fresh water. Appl. Environ. Microbiol. 55:246–251.PubMedGoogle Scholar
  6. 6.
    Bej, A. K., M. H. Mahbubani, J. L. D. Cesare, and R. M. Atlas. 1991. PCR-gene probe detection of microorganisms using filter concentrated samples. Appl. Environ. Microbiol. 57:3529–3534.PubMedGoogle Scholar
  7. 7.
    Berlin, D. L., D. S. Herson, D. T. Hicks, and D. G. Hoover. 1999. Response of pathogenic Vibrio species to high hydrostatic pressure. Appl. Environ. Microbiol. 65:2776–2780.PubMedGoogle Scholar
  8. 8.
    Beumer, R. R., J. De Vries, and F. M. Rombouts. 1992. Campylobacter jejuni non-culturable coccoid cells. Int. J. Food Microbiol. 15:153–163.PubMedCrossRefGoogle Scholar
  9. 9.
    Blake, P. A., M. H. Merson, R. E. Weaver, D. G. Hollis and P. C. Hueblein. 1979. Disease caused by a marine vibrio: clinical characteristics and epidemiology. N. Engl. J. Med. 300:1–5.PubMedCrossRefGoogle Scholar
  10. 10.
    Bocuzzi, V. M., W. L. Straube, J. Ravel, R. R. Colwell, and R. T. Hill. 1998. Preparation of DNA extracted from environmental water samples for PCR amplification. J. Microbiol. Methods 31:193–199.CrossRefGoogle Scholar
  11. 11.
    Bode, G., F. Mauch, and P. Melfertheiner. 1993. The coccoid forms of Helicobacter pylori. Criteria for their viability. Epidemiol. Infect. 111:483–490.PubMedCrossRefGoogle Scholar
  12. 12.
    Bowden, W. B. 1977. Comparison of two direct-count techniques for enumerating aquatic bacteria. Appl. Environ. Microbiol. 33:1229–1232.PubMedGoogle Scholar
  13. 13.
    Bozue, J. A., and W. Johnson. 1996. Interaction of Legionella pneumophila with Acanthamoeba castellani: uptake by coiling phagocytosis and inhibition of phagosome-lysosome fusion. Infect. Immun. 64:668–673.PubMedGoogle Scholar
  14. 14.
    Brayton, P., M. L. Tamplin, A. Huq, and R. R. Colwell. 1987. Enumeration of Vibrio cholerae O1 in Bangladesh waters by fluorescent-antibody direct viable count. Appl. Environ. Microbiol. 53: 2862–2865.PubMedGoogle Scholar
  15. 15.
    Buck, G. E. 1990. Campylobacter pylori as gastroduodenal disease. Clin. Microbiol. Rev. 3:1–12.PubMedGoogle Scholar
  16. 16.
    Butkevitch, V. S. 1932. Zur Methodik der bacteriologischen Meresuntersuchungem und einige Augaben über die Verteilung dur Bakteriea im Wasser und inden Boden des Barends Meeres. Trans. Oceanogr. Inst. Moscow 2:5–39.Google Scholar
  17. 17.
    Butkevitch, V. S. 1938. On the bacterial population of Caspian and Azov Seas. Microbiology (Moscow) 7:1005–1021.Google Scholar
  18. 18.
    Butkevitch, N. V., and V. S. Butkevitch. 1936. Multiplication of sea bacteria depending on the composition of the medium and temperature. Microbiology (Moscow) 5:3223.Google Scholar
  19. 19.
    Byrd, J. J., and R. R. Colwell. 1990. Maintenance of plasmids pBR322 and pUC8 in noncultivable Escherichia coli in the marine environment. Appl. Environ. Microbiol. 56:2104–2107.PubMedGoogle Scholar
  20. 20.
    Byrd, J. J., H. S. Xu, and R. R. Colwell. 1991. Viable but non-culturable bacteria in drinking water. Appl. Environ. Microbiol. 57:875–878.PubMedGoogle Scholar
  21. 21.
    Cappelier, J. M., and M. Federighi. 1998. Demonstration of viable but nonculturable state for Campylobacter jejuni. Rev. Med. Vet. 149:319–326Google Scholar
  22. 22.
    Cholera Working Group, International Center for Diarrhoeal Diseases Research, Bangladesh. 1993. Large epidemic of cholera-like disease in Bangladesh caused by Vibrio cholerae 0139 synonym Bengal. Lancet 342:387–390.CrossRefGoogle Scholar
  23. 23.
    Chongsanguan, M., W. Chaicumga, P. Moolarsart, P. Kandhasingha, T. Shinada, H. Kurazono, and Y. Takeda. 1993. Vibrio cholerae 0139 Bengal in Bangkok. Lancet 342:430–431.CrossRefGoogle Scholar
  24. 24.
    Chowdhury, M. A. R., H. Yamanaka, S. Miyoshi, K. M. S. Aziz, and S. Shimoda. 1989. Ecology of Vibrio mimicus in aquatic environments. Appl. Environ. Microbiol. 55:2073–2078.PubMedGoogle Scholar
  25. 25.
    Chowdhury, M. A. R., H. Yamanaka, S. Miyoshi, and S. Shimoda. 1990. Ecology and seasonal distribution of Vibrio parahaemolyticus in aquatic environments of a temperate region. FEMS Microbiol. Ecol. 74:1–9.CrossRefGoogle Scholar
  26. 26.
    Chowdhury, M. A. R., S. Miyoshi, H. Yamanaka, and S. Shimoda. 1992. Ecology and distribution of toxigenic V. cholerae in aquatic environments of a temperate region. Microbios 72:203–213.PubMedGoogle Scholar
  27. 27.
    Chowdhury, M. A. R., R. T. Hill, and R. R. Colwell. 1994. A gene for the enterotoxin zonula occludens toxin is present in Vibrio mimicus and Vibrio cholerae 0139. FEMS Microbiol. Lett. 119: 377–380.PubMedCrossRefGoogle Scholar
  28. 28.
    Chowdhury, M. A. R., R. T. Hill, A. Huq, and R. R. Colwell. 1995. Physiology and molecular genetics of viable but nonculturable microorganisms, p. 105–122. In M. Levin, C. Grim, and J. S. Angle (ed.), Biotechnology and Risk Assessment. Univ. of Maryland Biotechnology Inst., Baltimore, Md.Google Scholar
  29. 29.
    Chowdhury, M. A. R., B. Xu, R. Montilla, J. A. K. Hasan, A. Huq, and R. R. Colwell. 1995. A simplified immunofluorescence technique for detection of viable cells of Vibrio cholerae O1 and O139. J. Microbiol. Methods 24:165–170.CrossRefGoogle Scholar
  30. 30.
    Chun, J., A. Huq, and R. R. Colwell. 1999. Analysis of 16S-23S rRNA intergenic spacer regions of Vibrio cholerae and Vibrio mimicus. Appl. Environ. Microbiol. 65:2202–2208.PubMedGoogle Scholar
  31. 31.
    Cole, S. P., D. Cirillo, M. F. Kagnoff, D. G. Guiney, and L. Eckmann. 1997. Coccoid and spiral Helicobacter pylori differ in their abilities to adhere to gastric epithelial cells and induce interleukin-8 secretion. Infect. Immun. 65:843–846.PubMedGoogle Scholar
  32. 32.
    Collins, V. G., and C. Kipling. 1957. The enumeration of waterborne bacteria by a new direct count method. J. Appl. Bacteriol. 20:257–264.CrossRefGoogle Scholar
  33. 33.
    Colwell, R. R., J. Kaper, and S. W. Joseph. 1977. Vibrio cholerae, Vibrio parahaemolyticus and other vibrios: occurrence and distribution in Chesapeake Bay. Science 198:394–396.PubMedGoogle Scholar
  34. 34.
    Colwell, R., P. Brayton, D. Grimes, D. Roszak, S. Huq, and L. Palmer. 1985. Viable, but nonculturable Vibrio cholerae and related pathogens in the environment: implications for release of genetically engineered microorganisms. Bio/Technology 3:817–820.CrossRefGoogle Scholar
  35. 35.
    Colwell, R. R., M. L. Tamplin, P. R. Brayton, A. L. Gauzens, B. D. Tall, D. Harrington, M. M. Levine, S. Hall, A. Huq, and D. A. Sack. 1990. Environmental aspects of V. cholerae in transmission of cholera, p. 327–343. In R. B. Sack and Y. Zinnaka (ed.), Advances in Research on Cholera and Related Diarrhoeas, 7th ed. K. T. K. Scientific Publishers, Tokyo, Japan.Google Scholar
  36. 36.
    Colwell, R. R., J. A. K. Hasan, A. Huq, L. Loomis, R. J. Siebling, M. Torres, S. Galvez, S. Islam, and D. Bernstein. 1992. Development and evaluation of a rapid, simple sensitive monoclonal antibody-based co-agglutination test for direct detection of V cholerae O1. FEMS Microbiol. Lett. 97:215–220.CrossRefGoogle Scholar
  37. 37.
    Colwell, R. R., A. Huq, K. A. Cunningham, and G. Losonsky. 1992. Prospective study of divingassociated illnesses, p. 63–70. Proc. Int. Symp. on Hazards of Diving in Polluted Waters. Maryland Sea Grant College Publication No. UM-SG-TS-92-02. University of Maryland, College Park, Md.Google Scholar
  38. 38.
    Colwell, R. R., and A. Huq. 1994. Vibrios in the environment: viable but nonculturable Vibrio cholerae, p. 117–133. In I. K. Wachsmuth, O. Olsvik, and P. A. Blake (ed.), Vibrio cholerae and Cholera: Molecular to Global Perspectives. ASM Press, Washington, D.C.Google Scholar
  39. 39.
    Colwell, R. R., and A. Huq. 1999. Global microbial ecology: biogeography and diversity of Vibrios as a model. J. Appl. Microbiol. Symp. Suppl. 85:134S–137S.Google Scholar
  40. 40.
    Colwell, R. R., and W. M. Spira. 1992. The ecology of Vibrio cholerae, p. 107–127. In D. Barua and W. B. Greenough III (ed.), Cholera Plenum Medical Book Company, New York, N.Y.Google Scholar
  41. 41.
    Colwell, R. R., P. Brayton, D. Herrington, B. Tall, A. Huq, and M. M. Levine. 1996. Viable but non-culturable Vibrio cholerae O1 revert to a cultivable state in the human intestine. World J. Microbioi Biotechnol. 12:28–31.CrossRefGoogle Scholar
  42. 42.
    Davis, B. R., G. R. Fanning, J. M. Madden, A. G. Steigerwalt, H. B. Bradford, Jr., H. L. Smith, Jr., and D. J. Breuner. 1981. Characterization of biochemically atypical Vibrio cholerae strains and designation of a new pathogenic species, Vibrio mimicus. J. Clin. Microbiol. 14:631–639.Google Scholar
  43. 43.
    Dawe, L. L., and W. R. Penrose. 1978. Bactericidal property of seawater: death or deliberation? Appl. Environ. Microbiol. 35:829–833.PubMedGoogle Scholar
  44. 44.
    Duncan, S., L. A. Glover, K. Killham, and J. I. Prosser. 1994. Luminescence-based detection of activity of starved and viable but non-culturable bacteria. Appl. Environ. Microbiol. 60:1308–1316.PubMedGoogle Scholar
  45. 45.
    Finlay, B., and S. Falkow. 1997. Common themes in microbial pathogenicity revisited. Microbiol. Mol. Biol. Rev. 61:136–169.PubMedGoogle Scholar
  46. 46.
    Fry, J. C. 1990. Direct methods and biomass estimation. Methods Microbiol. 22:41–85.CrossRefGoogle Scholar
  47. 47.
    Fuhrman, J. A., D. E. Comeau, A. Hagstrom, and A. M. Cham. 1988. Extraction from natural planktonic microorganisms of DNA suitable for molecular biological studies. Appl. Environ. Microbiol. 54:1426–1429.PubMedGoogle Scholar
  48. 48.
    Gonzalez, J. M., J. Iriberri, L. Egea, and I. Barcina. 1992. Characterization of culturable protistan grazing and death of enteric bacteria in aquatic ecosystem. Appl. Environ. Microbiol. 58:998–1004.PubMedGoogle Scholar
  49. 49.
    Goodwin, C. S., and J. A. Armstrong. 1990. Microbiological aspects of Helicobacter pylori (Campylobacter pylori). Eur. J. Clin. Microbiol. Infect. Dis. 9:1–13.PubMedCrossRefGoogle Scholar
  50. 50.
    Grimes, D. J., and R. R. Colwell. 1986. Viability and virulence of Escherichia coli suspended by membrane chamber in semi-tropical ocean water. FEMS Microbiol. Lett. 34:161–165.CrossRefGoogle Scholar
  51. 51.
    Hasan, J. A. K., A. Huq, M. L. Tamplin, R. Siebeling, and R. R. Colwell. 1994. A novel kit for rapid detection of V. cholerae O1. J. Clin. Microbiol. 32:249–252.PubMedGoogle Scholar
  52. 52.
    Hasan, J. A. K., M. A. R. Chowdhury, M. Shahabuddin, A. Huq, L. Loomis, and R. R. Colwell. 1994. Polymerase chain reaction for the detection of cholera. Toxin genes in viable but nonculturable V cholerae O1. World J. Microbiol. Biotechnol. 10:568–571.CrossRefGoogle Scholar
  53. 53.
    Hasan, J. A. K., D. Bernstein, A. Huq, L. Loomis, M. L. Tamplin, and Rita R. Colwell. 1994. Cholera DFA: an improved direct fluorescent monoclonal antibody staining kit for rapid detection and enumeration of Vibrio cholerae O1. FEMS Microbiol. Lett. 120:143–148.PubMedCrossRefGoogle Scholar
  54. 54.
    Heidelberg, J. F., K. R. O’Neill, D. Jacobs, and R. R. Colwell. 1993. Enumeration of Vibrio vulnificus on membrane filters with a fluorescently labeled oligonucleotide probe specific for kingdom-level 16S rRNA sequences. Appl. Environ. Microbiol. 59:3474–3476.PubMedGoogle Scholar
  55. 55.
    Heidelberg, J. F., M. Shahamat, M. A. Levin, I. Rahman, and R. R. Colwell. 1997. Effect of aerosolization on culturability and viability of gram-negative bacteria. Appl. Environ. Microbiol. 63: 3585–3588.PubMedGoogle Scholar
  56. 56.
    Hoff, K. A. 1989. Survival of Vibrio anguillarum and Vibrio salmonicida at different salinity. Appl. Environ. Microbiol. 55:1775–1786.PubMedGoogle Scholar
  57. 57.
    Höller, C., D. Witthuhn, and B. Janzen-Blunck. 1998. Effect of low temperatures on growth, structure, and metabolism of Campylobacter coli SP10. Appl. Environ. Microbiol. 64:581–587.PubMedGoogle Scholar
  58. 58.
    Hughes, J. M., J. M. Boyce, R. J. Levine, M. U. Khan, K. M. A. Aziz, M. I. Huq, and G. T. Curlin. 1982. Epidemiology of El Tor cholera in rural Bangladesh: importance of surface water in transmission. Bull W.H.O. 60:395–404.PubMedGoogle Scholar
  59. 59.
    Huq, A., E. B. Small, P. A. West, M. I. Huq, R. Rahman, and R. R. Colwell. 1983. Ecological relationships between Vibrio cholerae and planktonic crustacean copepods. Appl. Environ. Microbiol. 45:275–283.PubMedGoogle Scholar
  60. 60.
    Huq, A., E. Small, P. West, and R. R. Colwell. 1984. The role of planktonic copepods in the survival and multiplication of Vibrio cholerae in the environment, p. 521–534. In R. R. Colwell (ed.), Vibrios in the Environment. John Wiley & Sons, New York, N.Y.Google Scholar
  61. 61.
    Huq, A., R. R. Colwell, R. Rahman, A. Ali, M. A. R. Chowdhury, S. Parveen, D. A. Sack, and E. Russek-Cohen. 1990. Detection of V. cholerae O1 in the aquatic environment by fluorescent monoclonal antibody and culture method. Appl. Environ. Microbiol. 56:2370–2373.PubMedGoogle Scholar
  62. 62.
    Huq, A., J. A. K. Hasan, G. Losonsky, V. Diomin, and R. R. Colwell. 1994. Colonization of professional divers by toxigenic Vibrio cholerae O1 and V cholerae non-O1 at dive sites in the United States, Ukraine and Russia. FEMS Microbiol. Lett. 120:137–142.PubMedCrossRefGoogle Scholar
  63. 63.
    Huq, A., and R. R. Colwell. 1995. A microbiological paradox: viable but nonculturable bacteria with special reference to Vibrio cholerae. J. Food Protect. 59:96–101.Google Scholar
  64. 64.
    Hussong, D., R. R. Colwell, M. O’Brien, E. Weiss, A. D. Pearson, R. M. Weiner, and W. D. Bürge. 1987. Viable Legionella pneumophila not detectable by culture on agar media. Bio/Technology 5:947–952.CrossRefGoogle Scholar
  65. 65.
    Islam, M. S., B. S. Drasar, and D. J. Bradley. 1990. Long-term persistence of toxigenic V cholerae O1 in the mucilagenous sheath of a blue-green alga, Anabaena variabilis. J. Trop. Med. Hyg. 93: 133–139.PubMedGoogle Scholar
  66. 66.
    Islam, M. S., M. K. Hasan, M. A. Miah, G. C. Sur, A. Felsenstein, M. Venkatesan, R. B. Sack, and M. J. Albert. 1993. Use of the polymerase chain reaction and fluorescent antibody methods for detecting viable but nonculturable Shigella dysenteriae type 1 in laboratory microcosms. Appl. Environ. Microbiol. 59:536–540.PubMedGoogle Scholar
  67. 67.
    James, B. W., W. S. Mauchline, P. J. Dennis, C. W. Keevil, and R. Wait. 1999. Poly-3-hydroxibutyrate in Legionella pneumophila, an energy source for survival in low-nutrient environments. Appl. Environ. Microbiol. 65:822–827.PubMedGoogle Scholar
  68. 68.
    Janda, J. M., E. J. Botton, and M. Reltano. 1983. Aeromonas species in clinical microbiology: significance, epidemiology, and specification. Diagn. Microbiol. Infect. Dis. 1:221–228.PubMedCrossRefGoogle Scholar
  69. 69.
    Jannasch, H. W. 1969. Estimations of bacterial growth rates in natural waters. J. Bacteriol. 99: 156–160.PubMedGoogle Scholar
  70. 70.
    Jennison, M. W. 1937. Relations between plate counts and direct microscopic counts of E. coli during logarithmic growth period. J. Bacteriol. 33:461–469.PubMedGoogle Scholar
  71. 71.
    Jentsch, T. J., A. M. Garcia, and H. F. Lodish. 1989. Primary structure of a noted 4-acetamido-4′isothiocyanostilbene-2-2′disulphonic acid (SITS)-binding membrane protein lights expressed in Torpedo California electroplax. Biochem. J. 261:155.PubMedGoogle Scholar
  72. 72.
    Jepras, R. I., J. Carter, S. C. Pearson, F. E. Paul, and M. J. Wilkinson. 1995. Development of a robust flow cytometric assay for determining numbers of viable bacteria. Appl. Environ. Microbiol. 61:2696–2701.PubMedGoogle Scholar
  73. 73.
    Jiang, X., and T. Chai. 1996. Survival of Vibrio parahaemolyticus at low temperatures under starvation conditions and subsequent resuscitation of viable, nonculturable cells. Appl. Environ. Microbiol. 62:1300–1305.PubMedGoogle Scholar
  74. 74.
    Johnston, J. M., S. F. Becker, and L. M. McFarland. 1986. Gastroenteritis in patients with stool isolates of Vibrio vulnificus. Am. J. Med. 80:336–338.PubMedCrossRefGoogle Scholar
  75. 75.
    Jones, D. M., E. M. Sutcliffe, and A. Curry. 1991. Recovery of viable non-culturable Campylobacter jejuni. J. Gen. Microbiol. 137:2477–2482.PubMedGoogle Scholar
  76. 76.
    Kaneko, T., and R. R. Colwell. 1973. Ecology of Vibrio parahaemolyticus in Chesapeake Bay. J. Bacteriol. 113:24–32.PubMedGoogle Scholar
  77. 77.
    Karsinkin, G. S., and S. J. Kusnetsov. 1931. Neue Methoden in der Limnologie. Arb. Limnol. Sta. Kossino. 13:47–48. (In Russian, with German summary.)Google Scholar
  78. 78.
    Kepner, R. L., Jr., and J. R. Pratt. 1994. Use of fluorochromes for direct enumeration of total bacteria in environmental samples: past and present. Microbiol. Rev. 58:603–615.PubMedGoogle Scholar
  79. 79.
    Khan, M. U., G. T. Curlin, and M. I. Huq. 1979. Epidemiology of Shigella dysenteriae type 1 infections in Dacca urban area. J. Trop. Geogr. Med. 31:213–223.Google Scholar
  80. 80.
    Kim, Y. M., B. H. Lee, S. H. Lee, and T. S. Lee. 1990. Distribution of Vibrio vulnificus in seawater of Kwangan Beach, Pusan, Korea. Bull. Korean Fish. Soc. 22:385–390.Google Scholar
  81. 81.
    Klein, P. D., D. Y. Graham, A. Gaillour, A. R. Opekum, and E. O. Smith. 1991. Water source as risk factor for H. pylori infection in Peruvian children. Lancet 337:1503–1505.PubMedCrossRefGoogle Scholar
  82. 82.
    Knaysi, G. 1935. A microscopic method of distinguishing dead from living cells. J. Bacteriol. 30: 193–206.PubMedGoogle Scholar
  83. 83.
    Kogure, K., U. Simidu, and N. Taga. 1979. A tentative direct microscopic method for counting living marine bacteria. Can. J. Microbiol. 25: 415–420.PubMedCrossRefGoogle Scholar
  84. 84.
    Kondo, K., A. Takade, and K. Amako. 1994. Morphology of the viable but non-culturable Vibrio cholerae as determined by the freeze fixation technique. FEMS Microbiol. Lett. 123:179–184.PubMedCrossRefGoogle Scholar
  85. 85.
    Kurath, G., and Y. Morita. 1983. Starvation survival and physiological studies of a marine Pseudomonas spp. Appl. Environ. Microbiol. 45:1206–1211.PubMedGoogle Scholar
  86. 86.
    Lambert, J. R., S. K. Lin, and J. Aranda-Michel. 1995. Helicobacter pylori. Scand. J. Gastroenterol. 30(Suppl. 208):33–46.CrossRefGoogle Scholar
  87. 87.
    Lebaron, P., N. Parthuisot, and P. Catala. 1998. Comparison of blue nucleic acid dyes for flow cytometric enumeration of bacteria in aquatic systems. Appl. Environ. Microbiol. 64:1725–1730.PubMedGoogle Scholar
  88. 88.
    Lee, K., and E. G. Ruby. 1995. Symbiotic role of the viable but non-culturable state of V. fischeri in Hawaiian coastal waters. Appl. Environ. Microbiol. 61:278–283.PubMedGoogle Scholar
  89. 89.
    Lennette, E. H., A. Balows, W. J. Hausier, Jr., and H. J. Shadomy (ed.). 1985. Manual of Clinical Microbiology, 4th ed. American Society of Microbiology, Washington, D.C.Google Scholar
  90. 90.
    Linder, K., and J. D. Oliver. 1989. Membrane fatty acid and virulence changes in the viable but nonculturable state of Vibrio vulnificus. Appl. Environ. Microbiol. 55:2837–2842.PubMedGoogle Scholar
  91. 91.
    Losonsky, G. A., J. A. K. Hasan, A. Huq, S. Kaintuch, and R. R. Colwell. 1994. Serum antibody responses of divers to waterborne pathogens. J. Clin. Diagnos. Lab. Immun. 1:182–185.Google Scholar
  92. 92.
    Magarinos, B., J. L. Romalde, J. L. Barja, and A. E. Toranzo. 1994. Evidence of a dormant but infective state of the fish pathogen Pasteurella piscicida in seawater and sediment. Appl. Environ. Microbiol. 60:180–186.PubMedGoogle Scholar
  93. 93.
    Mai, U. E. H., M. Shahamat, and R. R. Colwell. 1990. Survival of Helicobacter pylori in the aquatic environment, p. 90–96. In H. Menge, M. Gregor, G. N. J. Tytgat, B. J. Marshal, and C. A. M. McNulty (ed.), Proceedings of the 2nd International Symposium on Helicobacter pylori, August 25–26, Bad Nauheim, Berlin. Springer-Verlag, Berlin, Germany.Google Scholar
  94. 94.
    Martins, M. T., P. S. Sanchez, M. I. Z. Sato, P. R. Brayton, and R. R. Colwell. 1993. Detection of Vibrio cholerae O1 in the aquatic environment in Brazil employing direct immunofluorescence microscopy. World J. Microbiol. Biotechnol. 9:390–392.CrossRefGoogle Scholar
  95. 95.
    Martins, M. T., I. G. Rivera, D. L. Clark, and B. H. Olson. 1992. Detection of virulence factors in culturable Escherichia coli isolates from water samples by DNA probes and recovery of toxinbearing strains in minimal o-nitrophenol-beta-D-galactopyranoside-4-methylumbelliferyl-beta-D-glucoronide media. Appl. Environ. Microbiol. 58:3095–3100.PubMedGoogle Scholar
  96. 96.
    Mason, J. D., L. A. R. Allman, J. M. Stark, and D. Lloyd. 1995. The ability of membrane potential dyes and calcofluor white to distinguish between viable and nonviable bacteria. J. Appl. Bacteriol. 78:309–315.PubMedCrossRefGoogle Scholar
  97. 97.
    Maurelli, A. T., A. E. Hromockyj, and M. L. Bernardini. 1992. Environmental regulation of Shigella virulence. Curr. Top. Microbiol. Immunol. 180:95–116.PubMedCrossRefGoogle Scholar
  98. 98.
    McFeters, G. A., and D. G. Stuart. 1972. Survival of coliform bacteria in natural waters: field and laboratory studies with membrane filter chambers. Appl. Environ. Microbiol. 24:805–811.Google Scholar
  99. 99.
    Medema, G. J., F. M. Schets, A. W. van de Giessen, and A. H. Haveljar. 1992. Lack of colonization of 1 day chicks by viable non-culturable Campylobacter jejuni. J. Appl. Bacteriol. 72: 512–516.PubMedCrossRefGoogle Scholar
  100. 100.
    Mekalanos, J. J. 1992. Environmental signals controlling expression of virulence determinants in bacteria. J. Bacteriol. 174:1–7.PubMedGoogle Scholar
  101. 101.
    Miller, J. F., J. J. Mekalanos, and S. Falkow. 1989. Coordinate regulation and sensory transduc-tion in the control of bacterial virulence. Science 243:916–922.PubMedCrossRefGoogle Scholar
  102. 102.
    Moran, A. P., and M. E. Upton. 1987. Factors affecting production of coccoid forms by Campylobacter jejuni on solid media during incubation. J. Appl. Bacteriol. 62:527–537.PubMedCrossRefGoogle Scholar
  103. 103.
    Morgan, J. A. W., C. Winstanley, R. W. Pickup, J. A. Jones, and J. R. Saunders. 1989. Direct phenotypic and genotypic detection of a recombinant pseudomonal population released into lake water. Appl. Environ. Microbiol. 55:2537–2544.PubMedGoogle Scholar
  104. 104.
    Morgan, J. A. W., G. Rhodes, and R. W. Pickup. 1993. Survival of nonculturable Aeromonas salmonicida in lake water. Appl. Environ. Microbiol. 59:874–880.PubMedGoogle Scholar
  105. 105.
    Narikawa, S., S. Kawai, H. Aoshima, O. Kawamata, R. Kawaguchi, K. Hikiji, M. Kato, S. Iino, and Y. Mizushima. 1997. Comparison of the nucleic acids of helical and coccoid forms of Helicobacter pylori. Clin. Diagn. Lab. Immunol. 4:285–290.PubMedGoogle Scholar
  106. 106.
    Nilius, M., A. Ströhle, G. Bode, and P. Malfertheiner. 1993. Coccoid like forms (CLF) of Helicobacter pylori. Enzyme activity and antigenicity. Int. J. Med. Microbiol. Virol. Parasitol. Infect. Dis. 280:259–272.Google Scholar
  107. 107.
    O’Neill, K. R., S. H. Jones, and D. J. Grimes. 1992. Seasonal incidence of Vibrio vulnificus in the Great Bay Estuary of New Hampshire and Maine. Appl. Environ. Microbiol. 58:3257–3262.PubMedGoogle Scholar
  108. 108.
    Oliver, J. D., and D. Wanucha. 1989. Survival of V. vulnificus at reduced temperatures and elevated nutrients. J. Food Safety 10:79–86.CrossRefGoogle Scholar
  109. 109.
    Oliver, J. D. 1993. Formation of viable but non-culturable cells, p. 239–272. In S. Kjelleberg (ed.), Starvation in Bacteria. Plenum Press, New York, N.Y.Google Scholar
  110. 110.
    Pace, J. L., T. Chai, H. A. Rossi, and X. Jiang. 1997. Effect of bile on Vibrio parahaemolyticus. Appl. Environ. Microbiol. 63:2372–2377.PubMedGoogle Scholar
  111. 111.
    Paszko-Kolva, C., M. Shahamat, H. Yamamoto, T. Sawyer, J. Vives-Rego, and R. R. Colwell. 1991. Survival of Legionella pneumophila in the aquatic environment. Microbiol. Ecol. 22:75–83.CrossRefGoogle Scholar
  112. 112.
    Paszko-Kolva, C., M. Shahamat, and R. R. Colwell. 1992. Long-term survival of Legionella pneumophila serogroup 1 under low nutrient conditions and associated morphological changes. FEMS Microbiol. Ecol. 102:45–55.CrossRefGoogle Scholar
  113. 113.
    Peterson, W. L. 1991. Helicobacter pylori and peptic ulcer disease. N. Engl. J. Med. 324:1043–1048.PubMedCrossRefGoogle Scholar
  114. 114.
    Phadnis, S. H., M. H. Parlow, M. Levy, D. Iiver, C. M. Caulkins, J. B. Connors, and B. E. Dunn. 1996. Surface localization of Helicobacter pylori urease and a heat shock protein homolog requires bacterial autolysis. Infect. Immun. 64:905–912.PubMedGoogle Scholar
  115. 115.
    Pommepuy, M., M. Butin, A. Derrien, M. Gourmelon, R. R. Colwell, and M. Cormier. 1996. Retention of enteropathogenicity by viable but nonculturable Escherichia coli exposed to seawater and sunlight. Appl. Environ. Microbiol. 62:4621–4626.PubMedGoogle Scholar
  116. 116.
    Pommepuy M., L. Fiksdal, M. Gourmelon, H. Melikechi, M. L. Caprais, M. Cormier, and R. R. Colwell. 1996. Effect of seawater on Escherichia coli β-galactosidase activity. J. Appl. Bacteriol. 81:174–180.PubMedCrossRefGoogle Scholar
  117. 117.
    Porter, K. G., and Y. S. Fieg. 1980. The use of DAPI for identifying and counting aquatic microflora. Limnol Oceanogr. 25:943–948.CrossRefGoogle Scholar
  118. 118.
    Postgate, J. R. 1969. Viable counts and viability, p. 611–628. In J. R. Norris and D. W. Ribbons (ed.), Methods in Microbiology. Academic Press, Inc., London, United Kingdom.Google Scholar
  119. 119.
    Radsimosky, R. 1930. Vorlantige Augaben über die Dichtigheit der berkteriellen Besiedlung einiger Gewasser. Trav. Sta. Biol. Dmiepre. 5:385–402.Google Scholar
  120. 120.
    Rahman, I., 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.PubMedGoogle Scholar
  121. 121.
    Rahman, I., M. Shahamat, M. A. R. Chowdhury, and R. R. Colwell. 1996. Potential virulence of viable nonculturable Shigella dysenteriae type I. Appl. Environ. Microbiol. 62:115–120.PubMedGoogle Scholar
  122. 122.
    Ramamurthy, T., R. Garg, S. K. Sharma, G. B. Nair, T. Shimada, T. Takeda, T. Karasawa, H. Kuraziano, A. Pal, and Y. Takeda. 1993. Emergence of novel strains of V cholerae with epidemic potential in Southern and Eastern India. Lancet 341:703–705.PubMedCrossRefGoogle Scholar
  123. 123.
    Ravel, J., R. T. Hill, and R. R. Colwell. 1994. Isolation of a Vibrio cholerae transposon-mutant with an altered viable but nonculturable response. FEMS Microbiol. Lett. 120:57–62.PubMedCrossRefGoogle Scholar
  124. 124.
    Ravel, J., I. T. Knight, C. E. Monahan, R. T. Hill, and R. R. Colwell. 1995. Temperatureinduced recovery of Vibrio cholerae from the viable but nonculturable state: growth or resuscitation?. Microbiology 141:377–383.PubMedCrossRefGoogle Scholar
  125. 125.
    Rivera, I. G., M. A. R. Chowdhury, A. Huq, D. Jacobs, M. T. Martins, and R. R. Colwell. 1995. Enterobacterial repetitive intergenic consensus sequences and the PCR to generate fingerprints of genomic DNA from Vibrio cholerae O1, 0139, and non-O1. Appl. Environ. Microbiol. 61:2898–2904.PubMedGoogle Scholar
  126. 126.
    Rodriguez, G. G., 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. 58:1801–1808.PubMedGoogle Scholar
  127. 127.
    Rollins, D. M., and R. R. Colwell. 1986. Viable but non-culturable stage of Campylobacter jejuni and its role in survival in the natural aquatic environment. Appl. Environ. Microbiol. 52:531–538.PubMedGoogle Scholar
  128. 128.
    Rosenberg, M. L., K. K. Hazlet, J. Schaefer, J. C. Wells, and R. C. Pruneda. 1976. Shigellosis from swimming. JAMA 236:1849–1852.PubMedCrossRefGoogle Scholar
  129. 129.
    Roszak, D. B., D. J. Grimes, and R. R. Colwell. 1984. Viable but non-recoverable stage of Salmonella enteritidis in aquatic systems. Can. J. Microbiol. 30:334–338.PubMedCrossRefGoogle Scholar
  130. 130.
    Roszak, D. B., and R. R. Colwell. 1987. Survival strategies of bacteria in the natural environment. Microbiol. Rev. 51:365–379.PubMedGoogle Scholar
  131. 131.
    Rottenberg, H. 1979. The measurement of membrane potential and ApH in cells, organelles, and vesicles. Methods Enzymol. 55:547–569.PubMedCrossRefGoogle Scholar
  132. 132.
    Sack, D. A., C. O. Tackt, M. B. Cohen, R. B. Sack, G. A. Losonsky, J. Shimko, J. P. Nataro, R. Edelman, M. M. Levine, R. A. Giannella, G. Schiff, and D. Lang. 1998. Validation of a volunteer model of cholera with frozen bacteria as the challenge. Infect. Immun. 66:1968–1972.PubMedGoogle Scholar
  133. 133.
    Saha, S. K., S. Saha, and S. C. Sanyal. 1991. Recovery of injured Campylobacter jejuni cells after animal passage. Appl. Environ. Microbiol. 57:3388–3389.PubMedGoogle Scholar
  134. 134.
    Sato, M. I. Z., P. S. Sanchez, I. G. Rivera, and M. T. Martins. 1995. Survival of culturable Vibrio cholerae O1 and non-O1 in seawater, freshwater and wastewater and effect of the water environmental on enterotoxin production. Rev. Microbiol. (Brazil) 26:83–89.Google Scholar
  135. 135.
    Shahamat, M., U. Mai, C. Paszko-Kolva, M. Kessel, and R. Colwell. 1993. Use of autoradiography to assess viability of Helicobacter pylori in water. Appl. Environ. Microbiol. 59:1231–1235.PubMedGoogle Scholar
  136. 136.
    Shiba, T., R. T. Hill, W. L. Straube, and R. R. Colwell. 1995. Decrease in culturability of Vibrio cholerae caused by glucose. Appl. Environ. Microbiol. 61:2583–2588.PubMedGoogle Scholar
  137. 137.
    Shirai, H., M. Nishibuchi, T. Ramamurthy, S. K. Bhattacharya, S. C. Pal, and Y. Takeda. 1991. Polymerase chain reaction for detection of cholera enterotoxin operon of V cholerae. J. Clin. Microbiol. 29:2517–2521.PubMedGoogle Scholar
  138. 138.
    Sleightholme, V., and D. Roberts. 1994. Viable but non-culturable V. cholerae O1: a short review. Public Health Lab. Serv. Microbiol. Digest 11:77–80.Google Scholar
  139. 139.
    Sommerville, C. C., I. T. Knight, W. L. Straube, and R. R. Colwell. 1989. Simple rapid method for direct isolation of nucleic acid from aquatic environment. Appl. Environ. Microbiol. 55:548–559.Google Scholar
  140. 140.
    Sorscher, E. J., C. M. Fuller, and R. J. Bridges. 1992. Identification of a membrane protein from T84 cells using antibodies made against a DIDS-binding peptide. Am. J. Physiol. 262:C136.PubMedGoogle Scholar
  141. 141.
    Steinert, M., L. Emödy, R. Amann, and J. Hacker. 1997. Resuscitation of viable but noncultur-able Legionella pneumophila Philadelphia JR32 by Acanthamoeba castellanii. Appl. Environ. Microbiol. 63:2047–2053.PubMedGoogle Scholar
  142. 142.
    Stevenson, L. H. 1978. A case for bacterial dormancy in aquatic systems. Microb. Ecol. 4:127–133.CrossRefGoogle Scholar
  143. 143.
    Strugger, S. 1948. Fluorescence microscope examination of bacteria in soil. Can. J. Res. Sect. 26: 188–193.CrossRefGoogle Scholar
  144. 144.
    Tholozan, J. L., J. M. Cappelier, J. P. Tissier, G. Gelattre, and M. Federighi. 1999. Physiological characterization of viable-but-nonculturable Campylobacter jejuni cells. Appl. Environ. Microbiol. 65:1110–1116.PubMedGoogle Scholar
  145. 145.
    Turpin, P. E., K. A. Maycroft, C. L. Rowlands, and E. M. H. Wellington. 1993. Viable but non-culturable salmonellas in soil. J. Appl. Bacteriol. 74:421–427.PubMedCrossRefGoogle Scholar
  146. 146.
    Valentine, R. C., and J. R. G. Bradfield. 1954. The urea method for bacterial viability counts with electron microscope and its relation to other viability counting methods. J. Gen. Microbiol. 11:349–357.PubMedGoogle Scholar
  147. 147.
    Virta, M., M. Karp, S. Rönnemaa, and E. M. Lilius. 1997. Kinetic measurements of the membranolytic activity of serum complement using bioluminescent bacteria. J. Immunol. Methods 201: 215–221.PubMedCrossRefGoogle Scholar
  148. 148.
    Warner, J. M., and J. D. Oliver. 1998. Randomly amplified polymorphic DNA analysis of starved and viable but nonculturable Vibrio vulnificus cells. Appl. Environ. Microbiol. 64:3025–3028.PubMedGoogle Scholar
  149. 149.
    Weichart, D., J. D. Oliver, and S. Kjelleberg. 1992. Low temperature induced non-culturability and killing of Vibrio vulnificus. FEMS Microbiol. Lett. 100:205–210.Google Scholar
  150. 150.
    Weichart, D., D. McDougald, D. Jacobs, and S. Kjelleberg. 1997. In situ analysis of nucleic acids in cold-induced nonculturable Vibrio vulnificus. Appl. Environ. Microbiol. 63:2754–2758.PubMedGoogle Scholar
  151. 151.
    Whitesides, M. D., and J. D. Oliver. 1997. Resuscitation of Vibrio vulnificus from the viable but nonculturable state. Appl. Environ. Microbiol. 63:1002–1005.PubMedGoogle Scholar
  152. 152.
    Wolf, P. W., and J. D. Oliver. 1992. Temperature effects on the viable but nonculturable state of V vulnificus. FEMS Microbiol. Ecol. 101:33–39.Google Scholar
  153. 153.
    Xu, H. S., N. Roberts, F. L. Singleton, R. W. Attwell, D. J. Grimes, and R. R. Colwell. 1982. Survival and viability of nonculturable Escherichia coli and Vibrio cholerae in the estuarine and marine environment. Microb. Ecol. 8:313–323.CrossRefGoogle Scholar
  154. 154.
    Xu, H. S., N. C. Roberts, L. B. Adams, P. A. West, R. J. Seibeling, A. Huq, M. I. Huq, R. Rahman, and R. R. Colwell. 1984. An indirect fluorescent antibody staining procedure for detection of Vibrio cholerae serovar O1 cells in aquatic environmental samples. J. Microb. Methods 2: 221–231.CrossRefGoogle Scholar
  155. 155.
    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.Google Scholar
  156. 156.
    ZoBell, C. E. 1946. Marine Microbiology: a Monograph on Hydrobacteriology, p. 41–58. Carnica Botanica Co., Waltham. Mass.Google Scholar

Copyright information

© ASM Press, Washington, D.C. 2000

Authors and Affiliations

  • Anwarul Huq
    • 1
    • 2
  • Irma N. G. Rivera
    • 1
    • 3
  • Rita R. Colwell
    • 1
    • 2
  1. 1.Center of Marine Biotechnology, Columbus CenterUniversity of Maryland Biotechnology InstituteBaltimoreUSA
  2. 2.Department of Cell and Molecular BiologyUniversity of MarylandCollege ParkUSA
  3. 3.Department of Microbiology, Biomedical Science InstituteSão Paulo UniversitySão PauloBrazil

Personalised recommendations