• Journal of fish pathology
• /
• v.37 no.1
• /
• pp.49-60
• /
• 2024

A viable but non-culturable (VBNC) state is a survival strategy adopted by bacteria when faced with unfavorable environmental conditions, rendering them unable to grow on nutrient agar while maintaining low metabolic activity. This study explored the impact of temperature and nutrient availability on inducing VBNC state in Edwardsiella piscicida, the most important bacterial fish pathogen, and assessed its pathogenicity at VBNC state. E. piscicida was suspended in filtered sterile seawater and exposed to three different temperatures (4, 10, and 25℃) to induce the VBNC state. Subsequently, the induced VBNC cells were subjected to resuscitation by either raising the temperature to 28℃ or inoculating them in brain heart infusion broth supplemented with 1% NaCl. A propidium monoazide (PMA)-qPCR method was also developed to selectively quantify live (VBNC or culturable) E. piscicida cells. The results showed that the bacteria entered the VBNC state after approximately 1 month at 4℃ and 25℃, and 2 months at 10℃. The VBNC E. piscicida cells were successfully revived within 3 days in a nutrient-rich environment at 28℃, highlighting the significance of temperature and nutrition in inducing and resuscitating the VBNC state. In pathogenicity tests, resuscitated E. piscicida cells exhibited high pathogenicity in olive flounder comparable to cultured bacteria, while VBNC cells showed no signs of infection, suggesting they are unlikely to resuscitate in fish. In conclusion, this study contributes to our understanding of fish pathogen ecology by investigating the characteristics of the VBNC state under varying temperature and nutrition conditions.

• Journal of Dairy Science and Biotechnology
• /
• v.41 no.4
• /
• pp.157-162
• /
• 2023

In the field of microbiology, numerous types of bacteria live dormant to survive stresses such as pasteurization and antibiotics. Some bacteria become 'persisters' by inactivating their ribosomes, allowing them to 'sleep' through stress and revive when the stress has been removed. Under stress, some cells morph into hollow, lifeless structures known as 'cell shells.' In microbiology, these cells have been confused with viable cells in the 'viable but non-culturable cells' phenomenon. Therefore, this review addressed the concept that when revival occurs, the always-viable persister cells revive, instead of the dead cell husks.

• Korean Journal of Microbiology
• /
• v.38 no.3
• /
• pp.180-185
• /
• 2002

The direct viable count (DVC) and plate count (PC) methods was used to measure the number of bacteria in potable groundwater samples collected from bottled water from the market, mineral water, and edible groundwater near the urban areas and the stock farming congested areas. As a result, the number of living bacteria by DVC was comprised 30~80% of the total direct count (TDC), whereas the number of living bacteria by PC was around l~30% of DVC. Such results show that viable but non-culturable (VBNC) bacteria exist in the potable groundwater with high percentages. On the other hand, upon measuring the value from the conventional nutrient broth (NB), $10^-2$ fold diluted nutrient broth (DNB), and R2A broth, the values from the DNB and R2A showed 2~50 times higher than the conventional NB medium. These results indicate that oligotrophic bacterial groups which can multiply in the low nutrient broth abundantly exist in the oligotrophic environment like potable groundwater.

• Proceedings of the Korean Society of Life Science Conference
• /
• 2007.10a
• /
• pp.61.1-61.1
• /
• 2007

See Full Text

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

• Korean Journal of Microbiology
• /
• v.39 no.3
• /
• pp.181-186
• /
• 2003

This study was performed to analyze quantitatively the number of living bacteria in forest soil samples collected from Mt. Keryong using improved direct viable count (DVC) and plate count (PC) methods. The number of living bacteria by DVC comprised 18～44% of the total direct count (TDC), whereas the number of living bacteria by PC was less than 1% of TDC. These results showed that viable but non-culturable (VBNC) bacteria existed in the soil with high percentages. Besides, DVC was proved to make it possible to make a quantitative detection of the VBNC bacteria. On the other hand, upon measuring the value from the conventional nutrient broth (NB) and $10^{-2}$ folded diluted nutrient broth (DNB), the values from the DNB showed 5 to 10 times higher than those from the conventional NB medium. These results indicate that oligotrophic bacterial groups, which could multiply in the low nutrient broth, abundantly exist in the soil ecosystem. It would also be possible to apply this kind of method to other substrate to make a quantitative detection of soil bacterial groups.

• Journal of Microbiology and Biotechnology
• /
• v.27 no.5
• /
• pp.995-1004
• /
• 2017

Culture-dependent methods, such as heterotrophic plate counting (HPC), are usually applied to evaluate the bacteriological quality of hemodialysis water. However, these methods cannot detect the uncultured or viable but non-culturable (VBNC) bacteria, both of which may be quantitatively predominant throughout the hemodialysis water treatment system. Therefore, propidium monoazide (PMA)-qPCR associated with HPC was used together to profile the distribution of the total viable bacteria in such a system. Moreover, high-throughput sequencing of 16S rRNA gene amplicons was utilized to analyze the microbial community structure and diversity. The HPC results indicated that the total bacterial counts conformed to the standards, yet the bacteria amounts were abruptly enhanced after carbon filter treatment. Nevertheless, the bacterial counts detected by PMA-qPCR, with the highest levels of $2.14{\times}10^7copies/100ml$ in softener water, were much higher than the corresponding HPC results, which demonstrated the occurrence of numerous uncultured or VBNC bacteria among the entire system before reverse osmosis (RO). In addition, the microbial community structure was very different and the diversity was enhanced after the carbon filter. Although the diversity was minimized after RO treatment, pathogens such as Escherichia could still be detected in the RO effluent. In general, both the amounts of bacteria and the complexity of microbial community in the hemodialysis water treatment system revealed by molecular approaches were much higher than by traditional method. These results suggested the higher health risk potential for hemodialysis patients from the up-to-standard water. The treatment process could also be optimized, based on the results of this study.

• Korean Journal of Microbiology
• /
• v.43 no.3
• /
• pp.201-209
• /
• 2007

The CFDA (6-carboxyfluorescein diacetate) direct viable count method and plate count (PC) method using conventional nutrient broth (NB) medium and $10^{-2}$ diluted NB (DNB) medium were applied to samples collected from Mt. Yongdoo In Andong, in an effect to determine the number of living bacteria pine mushroom forest soil. The number of living bacteria determined via plate count in NB medium comprised $5{\sim}8%$ of the CFDA direct viable count, and the bacteria in the DNB medium comprised $40{\sim}47%$. This result indicated that viable but nonculturable (VBNC) bacteria existed in the pine mushroom forest soil at a high percentage. The phylogenetic characteristics of the VBNC bacterial populations in the samples of pine mushroom (Tricholoma matsutake) forest soil were analyzed via the direct extraction of DNA and 16S rDNA-ARDRA. The 115 clones from pine mushroom forest soil were clustered into 31 different RFLP phylotypes by ARDRA. Based on the 16S rDNA sequences, the 31 ARDRA clusters were classified into 6 phylogenetic groups: ${\alpha}-,\;{\beta}-,\;{\gamma}-Proteobacteria$, Acidobacteria, Actinobacteria and Firmicutes. Among these bacterial populations, approximately 85% were classified as members of phylum Acidobacteria. The Acidobacteria phylum was shown to exist abundantly in the pine mushroom forest soil.

• Korean Journal of Microbiology
• /
• v.53 no.1
• /
• pp.29-38
• /
• 2017

In this study, we investigated the phylogenetic diversity of the bacterial community and isolation of extremely halophilic bacteria using culturomics in a gray solar saltern. The number of bacterial living cells, enumerated in a gray solar saltern by direct fluorescence microscopy was three to four orders of magnitude greater than those enumerated by plate counts, suggesting the distribution of 'viable but non-culturable bacteria'. The biodiversity of bacterial communities in a gray solar saltern was investigated by pyrosequencing, 1,778 OTUs of bacteria were comprised of 18 phyla 46 classes 85 orders 140 families 243 genera with 6.16 diversity index. Archaea communities were composed of 3 phyla 6 classes 7 orders 7 families 38 genera with 4.95 diversity index from 643 OTUs. Totally 137 isolates were isolated by 59 different cultural methods based on culturomics considering culture media and conditions suitable for the growth of extremely halophilic bacteria. Phylogenetic analyses of extremely halophilic isolates based on 16S rRNA gene sequences, extremely halophilic isolates were composed of 4 phyla and 11 genera. Haloterrigena and Haloferax can be successfully isolated from culturomics. These culturomics were effective methods for collection of diversity of extremely halophilic bacteria.

• 한국생물공학회:학술대회논문집
• /
• 2001.11a
• /
• pp.918-922
• /
• 2001

In a previous study, we have isolated a number of lactobacilli from Korean women, and one of them (KLB46) was identified as Lactobacillus crispatus by 16S rRNA gene sequencing. For the ecological treatment of bacterial vaginosis (BV) cell suspension of L. crispatus KLB46 was instillated into BV patients. L. crispatus KLB46 was found to persist for several days in cell suspension with no nutrients. In this study, in order to assess the influence of starvation on physiological activity, we compared the viability and culturability of KLB46 following suspension in various buffer solutions. A pair of in situ fluorescent dye was used to assess viability (i.e. membrane integrity) and the culturability was examined by plate count assay. A rapid epifluorescence staining method using the LIVE/DEAD Bacterial Viability Kit $(BacLight^{TM})$ was applied to estimate both viable and total counts of bacteria in cell suspension. $BacLight^{TM}$ is composed of two nucleic acid-binding stains ($SYTO\;9^{TM}$ and propidium iodide). $SYTO\;9^{TM}$ penetrates all bacterial membranes and stains the cells green while propidium iodide only penetrates cells with damaged membranes, therefore the combination of the two stains produces red fluorescing cells. Optimal staining conditions for $BacLight^{TM}$ were found to be with 0.0835M $SYTO\;9^{TM}$ and 0.05M propidium iodide for 15 min incubation at room temperature in dark. When cells were microscopically examined during 140 hours of starvation, the culturability decreased markedly while the viability remained relatively constant, which suggests that large fraction of KLB46 cells became viable but non-culturable (VBNC) upon starvation.