훈제오리에서 캠필로박터균 생물막 및 Viable But Non-Culturable(VBNC) 상태에서의 행동특성

조혜진(Hye Jin Jo),전혜리(Hye Ri Jeon),윤기선(Ki Sun Yoon) 한국식품영양과학회 2016 한국식품영양과학회지 Vol.45 No.7

본 연구에서 가금류의 주요 병원성 식중독 균을 인위적으로 오염시킨 훈제오리육을 진공포장 조건에서 10, 15, 24°C에 저장하면서 유통기한 동안 관찰한 미생물의 증식 및 생존 결과 Campylobacter jejuni는 저장기간 이내에 사멸하는 경향을 보였으며, Salmonella Typhimurium과 Listeria monocytogenes는 균주의 성장 속도에는 차이가 있었으나 증식하는 경향을 보였다. 훈제오리의 유통온도는 10°C이며 유통기한이 약 30일인 것을 고려했을 때, 초기 오염 수준이 Campylobacter 균주에 의한 식중독을 유발하게 되는 균수 500 CFU/g 수준 이하에서는 유통기한 내에 문제가 없을 것으로 생각한다. 그러나 낮은 온도에서 저항성이 증가하며 살아있으나 배양은 불가능한 상태인 VBNC 상태의 C. jejuni의 특성에 따라 적절한 조건에서 회복되어 병원성을 일으킬 가능성이 있으므로 C. jejuni에 대한 지속적인 관리가 필요하다. 또한, S. Typhimurium과 L. monocytogenes의 경우 일반적인 유통/보관 온도인 10°C에서도 증식이 가능하며, 특히 가공품 및 RTE 식품은 적절한 가열처리 없이 소비할 경우 식중독 발생 가능성이 높다는 점에서 제품 제조 단계에서부터 위생적인 관리가 필요하다. 본 연구에서는 C. jejuni biofilm cells을 인위적으로 오염시킨 훈제오리육을 진공포장 하여 일반 유통/보관 온도인 10°C와 실온, 그리고 일반적으로 C. jejuni가 증식 가능한 온도인 36°C에서 저장하였으나, C. jejuni biofilm cell은 훈제오리에서는 모든 온도에서 재증식이 불가능한 것으로 관찰되었다. 또한, 10°C의 저온에서 유도한 VBNC 상태의 C. jejuni를 훈제오리에 인위적으로 오염시키고 혐기적 조건에서 42°C에 1일간 저장하며 VBNC 상태의 C. jejuni의 재증식 가능성을 분석하였으나, 최적 증식 온도인 42°C에서도 재증식은 관찰되지 않았다. 이처럼 본 연구에서는 biofilm을 형성한 C. jejuni도 VBNC 상태의 C. jejuni는 살아 있으나 훈제오리에서의 증식은 관찰되지 않았다. 따라서 훈제오리에서의 C. jejuni의 위험성은 매우 적은 것으로 생각한다. 그러나 C. jejuni의 경우 매우 적은 양으로도 식중독을 일으킬 수 있고 C. jejuni biofilm 및 VBNC의 특성에 따라 잠재적인 위험성을 포함하는 동시에 유통/보관 온도인 냉장 온도에서 더 잘 살아남는다는 점에서 식중독 발생의 주요 원인으로 작용할 수 있는 교차오염과 전이를 예방하는 것이 중요하므로 이에 대한 관리가 강조되어야 할 것으로 생각한다. 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°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°C, and C. jejuni VBNC cells were not resuscitated at 42°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.

Edwardsiella tarda의 비배양성 생존상태(VBNC) 유도 및 소생 특성

강남이,김은희,Kang, Nam I,Kim, Eunheui 한국미생물학회 2016 미생물학회지 Vol.52 No.3

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. 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.

난배양성(viable but non-culturable; VBNC) Edwardsiella piscicida의 특성 연구

김아현,이윤항,노형진,허영웅,김남은,김도형 한국어병학회 2024 한국어병학회지 Vol.37 No.1

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 avail-ability 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°C) to induce the VBNC state. Subsequently, the induced VBNC cells were subjected to resuscitation by either raising the temperature to 28°C 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°C and 25°C, and 2 months at 10°C. The VBNC E. piscicida cells were successfully revived within 3 days in a nutrient-rich environment at 28°C, 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.

Altered Gene Expression and Intracellular Changes of the Viable But Nonculturable State in Ralstonia solanacearum by Copper Treatment

Um, Hae Young,Kong, Hyun Gi,Lee, Hyoung Ju,Choi, Hye Kyung,Park, Eun Jin,Kim, Sun Tae,Murugiyan, Senthilkumar,Chung, Eunsook,Kang, Kyu Young,Lee, Seon-Woo The Korean Society of Plant Pathology 2013 Plant Pathology Journal Vol.29 No.4

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.

Altered Gene Expression and Intracellular Changes of the Viable But Nonculturable State in Ralstonia solanacearum by Copper Treatment

엄해영,이선우,공현기,이형주,최혜경,박은진,김선태,정은숙,강규영,Senthilkumar Murugiyan 한국식물병리학회 2013 Plant Pathology Journal Vol.29 No.4

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 copperinduced VBNC state in the plant pathogen Ralstonia solanacearum in liquid microcosm. Supplementation of 200 μM 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. H2O2 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 H2O2 accumulation. This report is the first of the distinct phenotypic changes in cells of R. solanacearum in the VBNC state.

Ethidium monoazide (EMA) - PCR 법을 이용한 비배양성 생존 상태(VBNC)의 Edwardsiella tarda 검출

강남이,김은희,Kang, Nam I,Kim, Eunheui 한국어병학회 2018 한국어병학회지 Vol.31 No.2

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.

16S rDNA-ARDRA법을 이용한 소나무림과 상수리나무림 토양 내 VBNC 세균군집의 계통학적 특성 비교

한송이,김윤지,황경숙,Han Song-Ih,Kim Youn-Ji,Whang Kyung-Sook 한국미생물학회 2006 미생물학회지 Vol.42 No.2

직접 생균수 측정법(DVC)과 평판계수법(PC)을 이용하여 소나무림과 상수리나무림 토양에 분포하는 세균군집의 정량적 평가를 실시한 결과, DVC법에 의해 계수된 생균수에 대해 평판법에 의해 계수된 생균수 1% 미만으로 나타났다. 이상의 결과로부터 산림토양 내에 평판배양법으로는 배양이 곤란한 난배양성(viable but non culturable; VBNC) 세균이 99% 이상 존재해 있는 것으로 판단되었다. 이들 VBNC 세균의 군집구조 해석을 위하여 토양으로부터 직접 DNA를 추출하고 16S rDNA-ARDRA 분석을 통하여 계통학적 특성을 검토하였다. 소나무림과 삼수리나무림 토양으로부터 각각 111 clones, 108 clones을 획득하고 HaeIII 절편양상에 따라 30 groups과 26 groups의 ARDRA group으로 분류하였다. 각 ARDRA group으로부터 대표 clone을 선발하여 16S rDNA 염기서 열을 결정한 결과, 소나무림 토양의 경우 ${\alpha}$-proteobacteria (12 clones), ${\gamma}$-proteobacteria (3 clones), ${\delta}$-proteobncteria (1clone), Flexibacter/Cytophaga (1 clone), Actinobacteria (4 clones), Acidobacteria (4 clones), 그리고 Planctomycetes (5 clone)의 7개의 계통군이 확인되었으며, 상수리나무림 토양에서는 ${\alpha}$-proteobacteria (4 clones), ${\gamma}$-proteobacteria (2 clones), Actinobacteria (10 clones), Acidobacteria (8 clones), Planctomycetes (1 clone), 그리고 Verrucomicrobia (1clone)로 6개의 다양한 계통군이 확인되었다. 이상, 소나무림과 상수리나무림 토양 내에 존재하는 99% 이상의 VBNC 세균군집의 대부분은 미배양성 혹은 미동정균으로 계통학적으로 다양한 미지의 미생물로 구성되어 있음이 확인되었다. 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.

플랑크톤 등 해양생물체 및 연안 해,하수에 대한 콜레라균 오염도 및 서식가능성 연구

이복권,강연호,박미선,유재연,김성한,김준영 국립보건연구원 1999 국립보건원보 Vol.36 No.-

플랑크톤 등 해양생물체 및 연안 해,하수에 대한 콜레라균 오염도 및 서식가능성 연구

이복권,강연호,김준영,김성한 국립보건연구원 2000 국립보건원보 Vol.37 No.-

Changes in Membrane Fatty Acid Composition during Entry of Vibrio vulnificus into the Viable But Nonculturable State

Ashley P. Day,James D. Oliver 한국미생물학회 2004 The journal of microbiology Vol.42 No.2

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 5oC, 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 5oC in artificial sea water and analyzing the cells’ FAs during the initial hours of temperature and nutrient downshift. 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 metabolism is essential for entry of cells into the VBNC state.