Characterization of Viable But Nonculturable Condition of Escherichia coli Induced with Copper

구리에 의해 유도된 VBNC 대장균의 특성

  • Ku, Hyung-Keun (Health Metrology Center, Division of Metrology for Quality Life, Korea Research Institute of Standards and Science) ;
  • Park, Sang-Ryoul (Health Metrology Center, Division of Metrology for Quality Life, Korea Research Institute of Standards and Science) ;
  • Kim, Sook-Kyung (Health Metrology Center, Division of Metrology for Quality Life, Korea Research Institute of Standards and Science)
  • 구형근 (한국표준과학연구원, 삶의질 표준본부, 보건측정센터) ;
  • 박상열 (한국표준과학연구원, 삶의질 표준본부, 보건측정센터) ;
  • 김숙경 (한국표준과학연구원, 삶의질 표준본부, 보건측정센터)
  • Published : 2008.09.28


VBNC (Viable but nonculturable) state is an adaptive response of cells in adverse environments, which lead cell not grow on routine nutrient agar. In this study, we induced VBNC in Escherichia coli using copper and verify the characterization of it. After treatment of copper, we didn't detect any cells via plate cultivation, namely, colony forming unit (CFU) was zero. However, we identified the existence of VBNC by staining live cells with Live/Dead BacLight bacterial viability kit and counting them through flow cytometry. Then we isolated genomic DNA and RNA from VBNC-induced cells and analyzed the stability of them. Degradation of RNA is more severe than that of DNA and RNA is degraded as specific fragments. In addition, we showed the morphology of VBNC cell by Bio-Transmission Electron Microscope (Bio-TEM). VBNC cell showed impaired periplasmic space and inner and outer membrane were separated and the amount of cytosol were significantly decreased.


VBNC;Escherichia coli;copper;Bio-TEM


  1. Grey, B. and T. R. Steck. 2001. Concentrations of copper thought to be toxic to Escherichia coli can induce the viable but nonculturable condition. Appl. Environ. Microbiol. 67: 5325-5327
  2. Gupte, A. R., C. L. De Rezende, and S. W. Joseph. 2003. Induction and resuscitation of viable but nonculturable Salmonella enterica serovar typhimurium DT104. Appl. Environ. Microbiol. 69: 6669-6675
  3. Heim, S., M. M. Lleo, B. Bonato, C. 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
  4. Jones, D. M., E. M. Sutcliffe, and A. Curry. 1991. Recovery of viable but non-culturable Campylobacter jejuni. J. Gen. Microbiol. 137: 2477-2482
  5. Ordax, M., E. Marco-Noales, M. M. Lopez, and E. G. Biosca. 2006. Survival strategy of Erwinia amylovora against copper: induction of the viable-but-nonculturable state. Appl. Environ. Microbiol. 72: 3482-3488
  6. Wai, S. N., Y. Mizunoe, A. Takade, and S. Yoshida. 2000. A comparison of solid and liquid media for resuscitation of starvation-and low-temperature-induced nonculturable cells of Aeromonas hydrophila. Arch. Microbiol. 173: 307-310
  7. 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 cholera in the estuarine and marine environment. Microb. Ecol. 8: 313-323
  8. Muela, A., C. Seco, E. Camafeita, I. Arana, M. Orruno, J. A. Lopez, and I. Barcina. 2008. Changes in Escherichia coli outer membrane subproteome under environmental conditions inducing the viable but nonculturable state. FEMS Microbiol. Ecol. 64: 28-36
  9. Vora, G. J., C. E. Meador, M. M. Bird, C. A. Bopp, J. D. Andreadis, and D. A. Stenger. 2005. Microarray-based detection of genetic heterogeneity, antimicrobial resistance, and the viable but nonculturable state in human pathogenic Vibrio spp. Proc. Natl. Acad. Sci. USA. 102: 19109-19114
  10. Day, A. P. 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
  11. Smith B. and J. D. Oliver. 2006. In situ and in vitro gene expression by Vibrio vulnificus during entry into, persistence within, and resuscitation from the viable but nonculturable state. Appl. Environ. Microbiol. 72: 1445-1451
  12. Lleo, M. M., S. Pierobon, M. C. Tafi, C. Signoretto, and P. Canepari. 2000. mRNA detection by reverse transcription-PCR for monitoring viability over time in an Enterococcus faecalis viable but nonculturable population maintained in a laboratory microcosm. Appl. Environ. Microbiol. 66: 4564-4567
  13. Whitesides, M. D. and J. D. Oliver. 1997. Resuscitation of Vibrio vulnificus from the Viable but Nonculturable State. Appl. Environ. Microbiol. 63: 1002-1005
  14. Arana, I., M. Orruno, D. Perez-Pascual, C. Seco, A. Muela, and I. Barcina. 2007. Inability of Escherichia coli to resuscitate from the viable but nonculturable state. FEMS Microbiol. Ecol. 62: 1-11
  15. Keep, N. H., J. M. Ward, G. Robertson, M. Cohen-Gonsaud, and B. Henderson. 2006. Bacterial resuscitation factors: revival of viable but non-culturable bacteria. Cell. Mol. Life Sci, 63: 2555-2559
  16. Oliver, J. D., L. Nilsson, and S. Kjelleberg. 1991. Formation of nonculturable Vibrio vulnificus cells and its relationship to the starvation state. Appl. Environ. Microbiol. 57: 2640-2644
  17. 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
  18. Beumer, R. R., J. de Vries, and F. M. Rombouts, 1992. Campylobacter jejuni non-culturable coccoid cells. Int. J. Food Microbiol. 15: 153-163
  19. Alexander, E., D. Pham, and T. R. Steck. 1999. The viable-but-nonculturable condition is induced by copper in Agrobacterium tumefaciens and Rhizobium leguminosarum. Appl. Environ. Microbiol. 65: 3754-3756
  20. Vattakaven, T., P. Bond, G. Bradley, and C. B. Munn. 2006. Differential effects of temperature and starvation on induction of the viable-but-nonculturable state in the coral pathogens Vibrio shiloi and Vibrio tasmaniensis. Appl. Environ. Microbiol. 72: 6508-6513
  21. Porter, J., C. Edwards, and R. W. Pickup. 1995. Rapid assessment of physiological status in Escherichia coli using fluorescent probes. J. Appl. Bacteriol. 79: 399-408
  22. Pujol, M., E. Badosa, and E. Montesinos. 2007. Epiphytic fitness of a biological control agent of fire blight in apple and pear orchards under Mediterranean weather conditions. FEMS Microbiol. Ecol. 59: 186-193