• Microbiology and Biotechnology Letters
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• v.36 no.3
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• pp.209-214
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• 2008

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.

• Korean Journal of Microbiology
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• v.52 no.3
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• pp.313-318
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• 2016

Bacteria in the viable but nonculturable (VBNC) state fail to produce colonies on routine bacteriological media, but are still alive in the state of very low metabolic activity. The aim of the present study was to induce the VBNC state of the Edwardsiella tarda using sea water microcosm under starvation conditions at $10^{\circ}C$ and to investigate resuscitation of the VBNC cells in temperatures changed from 10 to $25^{\circ}C$, with and without additives. E. tarda entered into the VBNC state within about 42-84 days of incubation in the microcosm. Throughout this period, the total cell counts as determined using acridine orange direct counting remained near the original inoculum level of ${\sim}10^8cells/ml$. The live cell counts measured with direct viable counting, on the other hands, declined to ${\sim}10^4cells/ml$. When the VBNC cells were incubated with addition of yeast extract, fish muscle extract or serum at $25^{\circ}C$, the ratios of resuscitated samples were 37%, 23%, and 37%, respectively. The characteristics of resuscitated E. tarda were consistent with those of the original E. tarda. When the resuscitated E. tarda were intraperitoneally injected into olive flounders, all fishes died within 5 days, indicating that the VBNC E. tarda might retain its pathogenic potential. Therefore, E. tarda under starvation conditions in the winter enter into the VBNC state and the VBNC E. tarda cells resuscitated at summer and autumn seawater temperature are considered to be pathogen continuously to olive flounder on the southern coast of Korea.

• The Plant Pathology Journal
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• v.29 no.4
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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.

• Journal of Microbiology and Biotechnology
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• v.27 no.3
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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 fish pathology
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• v.31 no.2
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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 and Biotechnology
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• v.17 no.9
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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.

• Journal of the Korean Society of Food Science and Nutrition
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• v.45 no.7
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• pp.1041-1048
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• 2016

Biofilm cells and viable but non-culturable (VBNC) state may play a role in the survival of Campylobacter jejuni under unfavorable environmental conditions. The objective of this study was to investigate the behavior of C. jejuni biofilm cells and VBNC cells on smoked duck. The transfer of C. jejuni biofilm cells to smoked duck and its ability to resuscitate from biofilm and VBNC cells on smoked duck was investigated. Transfer experiments were conducted from C. jejuni biofilm cells to smoked duck after 5 min, 1 h, 3 h, and 24 h contact at room temperature, and the efficiency of transfer (EOT) was calculated. In addition, smoked duck was inoculated with C. jejuni biofilm and VBNC cells and then stored at 10, 24, 36, and $42^{\circ}C$ to examine the cells' ability to resuscitate on smoked ducks. The 5 min contact time between C. jejuni biofilm cells and smoked duck showed a higher EOT (0.92) than the 24 h contact time (EOT=0.08), and the EOT decreased as contact time increased. Furthermore, C. jejuni biofilm cells on smoked duck were not recovered at 10, 24, and $36^{\circ}C$, and C. jejuni VBNC cells were not resuscitated at $42^{\circ}C$. Although the resuscitation of C. jejuni biofilm and VBNC cells was not observed on smoked duck, microbial criteria of C. jejuni is needed in poultry and processed poultry products due to risk of its survival and low infectious dose.

• Journal of Microbiology
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• v.42 no.2
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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.

• Journal of Microbiology
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• v.42 no.2
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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.

• Korean Journal of Microbiology
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• v.42 no.2
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• pp.116-124
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• 2006

In this study was performed to analyze quantitatively the number of viable but non-culturable bacteria in the Pine and Quercus forest soil by improved direct viable count (DVC) and plate count (PC) methods. The number of living bacteria of Pine and Quercus forest soil by PC method were less then 1% of DVC method. This result showed that viable but non-culturable (VBNC) bacteria existed in the forest soil with high percentage. Diversity and structure of VBNC bacterial populations in forest soil were analyzed by direct extracting of DNA and 16S rDNA-ARDRA from Pine and Quercus forest soil. Each of them obtained 111 clones and 108 clones from Pine and Quercus forest soil. Thirty different RFLP types were detected from Pine forest soil and twenty-six different RFLP types were detected from Quercus forest soil by HeaIII. From ARDRA groups, dominant clones were selected for determining their phylogenetic characteristics based on 16S rDNA sequence. Based on the 16S rDNA sequences, dominant clones from ARDRA groups of Pine forest soil were classified into 7 major phylogenetic groups ${\alpha}$-proteobacteria (12 clones), ${\gamma}$-proteobacteria (3 clones), ${\delta}$-proteobacteria (1 clone), Flexibacter/Cytophaga (1 clone), Actinobacteria (4 clones), Acidobacteria (4 clones), Planctomycetes (5 clones). Also, dominant clones from ARDRA groups of Quercus forest soil were classified into 6 major phylogenetic groups : ${\alpha}$-proteobacte,ia (4clones), ${\gamma}$-proteobacteria (2 clones), Actinobacteria (10 clones), Acidobacteria (8 clones), Planctomycetes (1 clone), and Verrucomicobia (1 clone). Result of phylogeneric analysis of microbial community from Pine and Quercus forest soils were mostly confirmed at uncultured or unidentified bacteria, VBNC bacteria of over 99% existent in forest soil were confirmed variable composition of unknown micro-organism.