한국미생물·생명공학회지 (Microbiology and Biotechnology Letters)

  • 제36권3호
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  • Pages.209-214
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  • 2008
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  • 1598-642X(pISSN)
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  • 2234-7305(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
권호 표지

구리에 의해 유도된 VBNC 대장균의 특성

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

  • 구형근 (한국표준과학연구원, 삶의질 표준본부, 보건측정센터) ;
  • 박상열 (한국표준과학연구원, 삶의질 표준본부, 보건측정센터) ;
  • 김숙경 (한국표준과학연구원, 삶의질 표준본부, 보건측정센터)
  • 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)
  • 발행 : 2008.09.28
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초록

VBNC(Viable but nonculturable)란 생존에 불리한 환경하에서 살아 있으나 일반 영양배지에서 자라지 못하는 미생물의 상태를 나타낸다. 본 연구는 구리를 이용해 Escherichia coli에서 VBNC를 유도하고 이의 특성을 살펴보았다. 구리를 처리한 후 전통적인 평판 배양법에 의한 집락 형성계수(colony forming unit, CFU)를 측정한 결과 배양되지는 않으나, Live/Dead BacLight bacterial viability kit 염색 후 유세포계수기로 측정한 결과 살아있는 미생물로 계수되어 VBNC 상태가 확인하였다. VBNC 유도된 미생물로부터 genomic DNA와 RNA를 분리하고 이들의 안정성을 관찰하였는데 DNA에 비해 RNA의 붕괴가 많이 진행되었음을 확인할 수 있었고 RNA의 붕괴는 특정크기로 붕괴되는 것으로 관찰되었다. 또한 생물전용 투과전자현미경(Bio-Transmission Electron Microscope, Bio-TEM)을 통해 VBNC 세포의 형태를 관찰하였는데 VBNC 상태에서는 정상상태에 비해 periplasmic space가 온전하지 못하고 세포내막과 세포 외막이 분리되었으며 세포질의 양이 현저히 감소됨이 관찰되었다.

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.

키워드

참고문헌

  1. 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
  2. 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 https://doi.org/10.1111/j.1574-6941.2007.00362.x
  3. Beumer, R. R., J. de Vries, and F. M. Rombouts, 1992. Campylobacter jejuni non-culturable coccoid cells. Int. J. Food Microbiol. 15: 153-163 https://doi.org/10.1016/0168-1605(92)90144-R
  4. 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
  5. 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 https://doi.org/10.1128/AEM.67.11.5325-5327.2001
  6. 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 https://doi.org/10.1128/AEM.69.11.6669-6675.2003
  7. 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 https://doi.org/10.1128/JB.184.23.6739-6745.2002
  8. Jones, D. M., E. M. Sutcliffe, and A. Curry. 1991. Recovery of viable but non-culturable Campylobacter jejuni. J. Gen. Microbiol. 137: 2477-2482 https://doi.org/10.1099/00221287-137-10-2477
  9. 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 https://doi.org/10.1007/s00018-006-6188-2
  10. 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 https://doi.org/10.1128/AEM.66.10.4564-4567.2000
  11. 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 https://doi.org/10.1111/j.1574-6941.2008.00453.x
  12. 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
  13. 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 https://doi.org/10.1128/AEM.72.5.3482-3488.2006
  14. 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 https://doi.org/10.1111/j.1365-2672.1995.tb03154.x
  15. 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 https://doi.org/10.1111/j.1574-6941.2006.00227.x
  16. 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 https://doi.org/10.1007/s00284-001-0062-0
  17. 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 https://doi.org/10.1128/AEM.72.2.1445-1451.2006
  18. 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 https://doi.org/10.1128/AEM.00798-06
  19. 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
  20. 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 https://doi.org/10.1007/s002030000142
  21. Whitesides, M. D. and J. D. Oliver. 1997. Resuscitation of Vibrio vulnificus from the Viable but Nonculturable State. Appl. Environ. Microbiol. 63: 1002-1005
  22. 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 https://doi.org/10.1007/BF02010671