• 미생물학회지
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• 제52권3호
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• pp.313-318
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• 2016

Viable but nonculturable (VBNC) 상태에 들어간 세균은 일반적인 증균 배지에서는 집락을 형성하지 않지만, 죽은 것이 아니라 낮은 대사활성상태로 유지되고 있다. 본 연구에서는 $10^{\circ}C$의 저온 빈영양 해수에서 Edwardsiella tarda를 VBNC 상태로 유도한 후, 해수 온도를 10에서 $25^{\circ}C$로 상승시킬 때 첨가된 유기물의 종류에 따라 VBNC 상태인 균의 소생 가능성을 알아보고자 하였다. E. tarda가 접종된 빈영양 해수 microcosm을 $10^{\circ}C$에 유지하였을 때 VBNC 유도 기간은 42-84일까지 다양하였다. 유도 기간 동안 acridine orange direct counting법으로 계수한 총 균수는 초기 접종 농도인 약 $10^8cells/ml$로 일정하였으며, direct viable counting법으로 계수한 생존 균수는 약 $10^4cells/ml$로 감소되었다. VBNC E. tarda에 효모추출물, 넙치근육추출물 그리고 혈청을 첨가하여 $25^{\circ}C$에서 소생을 유도한 결과 전체 시료 개수의 37%, 23%, 37%에서 각각 소생이 확인되었으며 소생된 E. tarda의 특성은 VBNC 유도 전 원래의 세균과 일치하였다. 소생된 E. tarda를 넙치(Paralichthys olivaceus)에 복강 주사 하였을 때 접종 후 5일 이내에 시험어가 모두 사망함으로써 VBNC 상태의 E. tarda가 독력을 유지하고 있었음을 시사하였다. 그러므로 E. tarda는 우리나라 남해 연안 겨울의 저온 빈영양 해수에서 VBNC 상태로 유도되었다가 여름과 가을 시기에 수온 상승과 더불어 소생되어 양식 넙치에 지속적인 발병 요인이 되고 있는 것으로 생각된다.

• Journal of Microbiology and Biotechnology
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• 제17권9호
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• pp.1558-1562
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• 2007

To elucidate the influence of pipe materials on the VBNC (viable but nonculturable) state and bacterial numbers in drinking water, biofilm and effluent from stainless steel, galvanized iron, and polyvinyl chloride pipe wafers were analyzed. Although no HPC (heterotrophic plate count) was detected in the chlorinated influent of the model system, a DVC (direct viable count) still existed in the range between 3- and 4-log cells/ml. Significantly high numbers of HPC and DVC were found both in biofilm and in the effluent of the model system. The pipe material, exposure time, and the season were all relevant to the concentrations of VBNC and HPC bacteria detected. These findings indicate the importance of determining the number of VBNC cells and the type of pipe materials to estimate the HPC concentration in water distribution systems and thus the need of determining a DVC in evaluating disinfection efficiency.

• The Plant Pathology Journal
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• 제29권4호
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• pp.374-385
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• 2013

Environmental stresses induce several plant pathogenic bacteria into a viable but nonculturable (VBNC) state, but the basis for VBNC is largely uncharacterized. We investigated the physiology and morphology of the copper-induced VBNC state in the plant pathogen Ralstonia solanacearum in liquid microcosm. Supplementation of $200{\mu}M$ copper sulfate to the liquid microcosm completely suppressed bacterial colony formation on culture media; however, LIVE/DEAD BacLight bacterial viability staining showed that the bacterial cells maintained viability, and that the viable cells contain higher level of DNA. Based on electron microscopic observations, the bacterial cells in the VBNC state were unchanged in size, but heavily aggregated and surrounded by an unknown extracellular material. Cellular ribosome contents, however, were less, resulting in a reduction of the total RNA in VBNC cells. Proteome comparison and reverse transcription PCR analysis showed that the Dps protein production was up-regulated at the transcriptional level and that 2 catalases/peroxidases were present at lower level in VBNC cells. Cell aggregation and elevated levels of Dps protein are typical oxidative stress responses. $H_2O_2$ levels also increased in VBNC cells, which could result if catalase/peroxidase levels are reduced. Some of phenotypic changes in VBNC cells of R. solanacearum could be an oxidative stress response due to $H_2O_2$ accumulation. This report is the first of the distinct phenotypic changes in cells of R. solanacearum in the VBNC state.

• 한국미생물·생명공학회지
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• 제36권3호
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• pp.209-214
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• 2008

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.

• Journal of Microbiology and Biotechnology
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• 제27권3호
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• pp.417-428
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• 2017

Diseases caused by foodborne or waterborne pathogens are emerging. Many pathogens can enter into the viable but nonculturable (VBNC) state, which is a survival strategy when exposed to harsh environmental stresses. Pathogens in the VBNC state have the ability to evade conventional microbiological detection methods, posing a significant and potential health risk. Therefore, controlling VBNC bacteria in food processing and the environment is of great importance. As the typical one of the gram-negatives, Escherichia coli (E. coli) is a widespread foodborne and waterborne pathogenic bacterium and is able to enter into a VBNC state in extreme conditions (similar to the other gram-negative bacteria), including inducing factors and resuscitation stimulus. VBNC E. coli has the ability to recover both culturability and pathogenicity, which may bring potential health risk. This review describes the concrete factors (nonthermal treatment, chemical agents, and environmental factors) that induce E. coli into the VBNC state, the condition or stimulus required for resuscitation of VBNC E. coli, and the methods for detecting VBNC E. coli. Furthermore, the mechanism of genes and proteins involved in the VBNC E. coli is also discussed in this review.

• Journal of Microbiology
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• 제43권spc1호
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• pp.93-100
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• 2005

It had long been assumed that a bacterial cell was dead when it was no longer able to grow on routine culture media. We now know that this assumption is simplistic, and that there are many situations where a cell loses culturability but remains viable and potentially able to regrow. This mini-review defines what the 'viable but nonculturable' (VBNC) state is, and illustrates the methods that can be used to show that a bacterial cell is in this physiological state. The diverse environmental factors which induce this state, and the variety of bacteria which have been shown to enter into the VBNC state, are listed. In recent years, a great amount of research has revealed what occurs in cells as they enter and exist in this state, and these studies are also detailed. The ability of cells to resuscitate from the VBNC state and return to an actively metabolizing and culturable form is described, as well as the ability of these cells to retain virulence. Finally, the question of why cells become nonculturable is addressed. It is hoped that this mini-review will encourage researchers to consider this survival state in their studies as an alternative to the conclusion that a lack of culturability indicates the cells they are examining are dead.

• Journal of Microbiology
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• 제42권2호
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• pp.69-73
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• 2004

Vibrio vulnificus, a Gram-negative bacterium found in estuarine waters, is responsible for over 95％ of all seafood-related deaths in the United States. As a result of a temperature downshift to 5$^{\circ}C$, this organism enters the viable but nonculturable (VBNC) state. Changes in the membrane fatty acid (FA) composition of V. vulnificus may be a contributing factor to the ability of this organism to enter into and survive in the VBNC state. This hypothesis was tested by incubating the organism at 5$^{\circ}C$ in arti-ficial sea water and analyzing the cells' FAs during the initial hours of temperature and nutrient down-shift. Prior to downshift, the predominant FAs were 16:0, 16:1 and 18:0. During the first four hours of downshift, statistically significant changes occurred in 15:0, 16:1, 16:0, 17:0, and 18:0. These results indicate that changes in FA composition occur prior to entry of V. vulnificus into the VBNC state, suggesting that the ability to maintain membrane fluidity may be a factor in this physiological response. Cells in which fatty acid synthesis was inhibited did not survive, indicating that active fatty acid metab-olism is essential for entry of cells into the VBNC state.

• 한국어병학회지
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• 제31권2호
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• pp.93-99
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• 2018

Edwardsiella tarda predominantly causes edwardsiellosis in fish at high temperature, but is rarely isolated from water when water temperature is low. However, E. tarda is viable but nonculturable (VBNC) in low water temperature, but it can be revived when water temperature rises and cause disease to fish. Therefore, in order to prevent disease, it is very important to identify pathogens that are in the VBNC state in environmental water. In this study, E. tarda cells in the VBNC state were detected by the ethidium monoazide (EMA)-PCR method using the low-temperature oligotrophic sea water microcosm obtained by inoculation of E. tarda at a concentration of $10^8CFU/ml$. In order to distinguish between live and dead bacteria in E. tarda, each sample was treated with EMA at different concentrations, photoactivated with a 500 W halogen lamp, and PCR was performed with E. tarda specific primer. At the concentration of $10^7CFU/ml$ bacterium, DNA amplification was observed only in the live cells when treated with $60{\mu}g/ml$ of EMA, and smaller amounts of live cells could be distinguished from dead cells by adjusting the EMA concentration. In addition, the VBNC cells of E. tarda in the oligotrophic low temperature seawater microcosm were estimated to be in the range of $10^4{\sim}10^5CFU/ml$ by EMA-PCR. Therefore, it is possible to detect VBNC cells that will act as potential pathogens in environmental water using EMA-PCR method, and quantitative confirmation using concentration change is also possible.

• Journal of Microbiology
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• 제42권2호
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• pp.74-79
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• 2004

Ingestion of shellfish-associated Vibrio parahaemolyticus is the primary cause of potentially severe gas-troenteritis in many countries. However, only Kanagawa phenomenon (hemolysin) positive (KP$\^$+/) strains of V. parahaemolyticus are isolated from patients, whereas >99％ of strains isolated from the environment do not produce this hemolysin (i.e. are KP$\^$-/). The reasons for these differences are not known. Following a temperature downshift, Vibrio parahaemolyticus enters the viable but noncultur-able (VBNC) state wherein cells maintain viability but cannot be cultured on routine microbiological media. We speculated that KP$\^$+/ and KP$\^$-/ strains may respond differently to the temperature and salinity conditions of seawater by entering into this state which might account for the low numbers of cul-turable KP$\^$+/ strains isolated from estuarine waters. The response of eleven KP$\^$+/ and KP$\^$-/ strains of V. parahaemolyticus following exposure to a nutrient and temperature downshift in different salinities, similar to conditions encountered in their environment, was examined. The strains included those from which the KP$\^$+/ genes had been selectively removed or added. Our results indicated that the ability to produce hemolysin did not affect entrance into the VBNC state. Further, VBNC cells of both biotypes could be restored to the culturable state following an overnight temperature upshift.

• 한국미생물·생명공학회지
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• 제28권2호
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• pp.117-123
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• 2000

P. phosphoreum의 생존과 생체발광도는 온도에 의해 많은 영향을 받는다. 냉동 저장한 세포의 경우 glycerol의 보호 작용으로 세포농도와 생균수는 측정기간 동안 일정하게 유지된 반면 생체발광도는 glycerol 첨가 직후 급속히 감소하였으며, 저장 이후에도 감소된 생체발광도가 활성화되지 못하였다. 최적 생육온도인 $20^{\circ}C$의 경우 저장 초기 세포가 성장함에 따라 세포수의 증가를 보였으나 일정 시간 이후 세포 분해 현상으로 인하여 생균수 및 세포 집락수의 감소를 나타내었으며, 생체발광도는 저장 3일 이후 소멸되었다. 이와는 대조적으로 $4^{\circ}C$에 저장한 세포의 생체발광도는 저장 10일 동안 지속되어 가장 높은 생체발광 유지도를 나타내었으나 장기간 저온 저장으로 인하여 세포가 VBNC 상태에 돌입됨에 따라 총균수와 생균수는 일정한 반면 저장 10일 이후 세포 집락수의 급격한 감소를 나타내었으며, 저장 20일 이후 간균에서 구균으로 세포 형태상의 변화를 나타내었다. 이에 따라 세포 저장 시 접종원의 농도를 달리하여 VBNC 상태와 생체발광도의 관련성을 조사한 결과 VBNC 세포가 증가할수록 생체발광도의 감소를 나타내었다. 따라서 VBNC 세포를 감소시키기 위하여 세포를 고정화하여 저장한 결과 별도의 활성제 없이 실온에서 다시 활성화되어 고정화하지 않은 세포에 비해 2.3배 높은 생체발광유지도를 나타내었으며, 저온저장에 따른 platebility 소실과 세포 응축현상이 나타나지 않았다. 이러한 결과는 세포의 고정화 방법을 이용하여 $4^{\circ}C$에서도 세포의 생존 및 생체발광 유지도를 향상시킬 수 있으며, 동결 건조법의 단점을 보완해 줄 것으로 생각된다.