PCR-RFLP에 의한 Vibrio core group을 포함한 Vibrio 종의 구분

Jin-Sook Park(박진숙) 한국생명과학회 2012 생명과학회지 Vol.22 No.2

Vibrio속의 core 균주(Vibrio alginolyticus, Vibrio parahaemolyticus)를 포함하여 총 6 종의 Vibrio 균주(V. fluvialis, V. proteolyticus, V. vulnificus, V. mimicus)와 Grimontia (Vibrio) hollisae의 16S rDNA를 PCR 증폭하여 AluⅠ, CfoⅠ, DdeⅠ, HaeⅢ, MspⅠ, RsaⅠ의 6 종의 제한효소를 처리 후 RFLP 분석을 수행하였다. 2 종의 core 균주와 V. proteolyticus는 4 종의 제한효소(CfoⅠ, DdeⅠ, MspⅠ, RsaⅠ)에서 동일한 제한효소 패턴을 나타내었다. 제한효소의 패턴의 조합에 의해 6 종의 Vibrio 종은 6 개의 RFLP type으로 구분되었다. 특히 AluⅠ의 경우, 실험된 6 종의 Vibrio속에 대하여 각기 다른 6 개의 종 특이적 RFLP type을 나타내었다. 제한효소 패턴에 근거하여 작성한 덴드로그램에서 Vibrio core group 균주인 V. alginolyticus 와 V. parahaemolyticus는 90% 이상의 매우 높은 유사도를 나타내었다. 반면 Grimontia hollisae는 실험된 모든 제한효소 패턴에서 Vibrio속 세균과는 분명히 구분되는 RFLP type을 나타내었다. 따라서 PCR-RFLP는 제한효소를 적절히 선택한다면 Vibrio 속 세균의 신속한 구분에 여전히 유용하다. The 16S rDNA - RFLP types for six Vibrio species (V. fluvialis, V. proteolyticus, V. vulnificus, V. mimicus) including two core group members, V. alginolyticus and V. parahaemolyticus, and Grimontia (Vibrio) hollisae were determined using PCR-RFLP analysis. Six tetrameric restriction enzymes (AluⅠ, CfoⅠ, DdeⅠ, HaeⅢ, MspⅠ, and RsaⅠ) were selected for RFLP analysis. V. alginolyticus, V. parahaemolyticus, and V. proteolyticus showed the same RFLP pattern following digestion with four of the six used restriction enzymes: CfoⅠ, DdeⅠ, MspⅠ, and RsaⅠ. Various restriction enzyme combinations generated digests recognizable as distinct RFLP types for each of the assayed Vibrio species. In particular, AluⅠsingle digestion produced species specific band patterns that enabled the differentiation between these Vibrio species. Dendrogram based on restriction patterns showed that two Vibrio core group members, V. alginolyticus and V. parahaemolyticus were closely related having a similarity over 90%. Although the observed RFLP pattern for Grimontia hollisae shared several common bands with other Vibrio spp., G. hollisae results were still clearly distinct from Vibrio spp. RFLP types for all restriction enzymes tested. If restriction enzymes are aptly selected, PCR-RFLP analysis is still a rapid and effective tool for differentiating Vibrio species.

용혈독소를 생산하는 기수성 비브리오균의 생리${\cdot}$생태적 특성과 수산식품의 위생 대책 1. 용혈독소를 생산하는 새로운 병원성 Vibrio sp.의 분리와 동정

김영만,최길배,장동석,KIM Young-Man,CHOI Gil-Bae,CHANG Dong-Suck 한국수산과학회 1997 한국수산과학회지 Vol.30 No.3

금강 하구의 물에서 분리된 sucrose 분해성 Vibrio 속 중에 용혈독소를 생산하는 균주의 독성을 시험한 결과와 기존에 알려진 12종의 병원성 Vibrio 녹과 비교한 결과는 다음과 같다. 1. 분리된 장소의 환경조건은 염도가 $4.7\%_{\circ}$, pH가 7.6, 수온이 $24^{\circ}C$ 및 conductivity가 $7800{\mu}MHOS$이었다. 2. 생리, 생화학적 특성과 염분 요구도를 비교한 결과 sucrose를 분해하는 병원성 Vibrio인 V. alginolyticus, V. cholerae, V. cincinnatiensis, V. fluvialis, V. furnissii 및 V. metschnikouii와는 확실히 구별되었다. 3. 생육가능한 염도는 $0.5\~7.5\%_{\circ}$이었으며 생육가능한 pH는 $4.5\~9.5$이었다. 4. TCBS 평판한천배지에서 균의 집락은 전형적인 황색집락이었으며, 전자현미경에 나타난 균의 형태은 콤마상 간균이었다. 5. 새앙쥐에 대한 치사독성이 확실하였으며, 사람과 면양 적혈구에 대한 용혈성이 확인되었고 rat 피부혈관 투과성 항진작용이 양성이었다. 이상의 결과로 이 균은 병원성이 확인되었고 기존에 알려지지 않은 Vibrio 속으로 확인되어 이 균을 Vibrio sp. D5로 명명하였다. To determine the physiological, biochemical characteristics and toxicity of hemolysin produced by a novel sucrose positive Vibrio (Vibrio sp. D5) isolated from estuary of Kum river, it was compared with already known sucrose positive Vibrio. Salinity, pH, temperature and conductivity of place where Vibrio sp. D5 was isolated were $4.7\%_{\circ},\;7.6,\;24^{\circ}C$ and $7800{\mu}MHOS$, respectively. Physiological and biochemical characteristics distingiushed Vibrio sp. D5 from other sucrose positive Vibrio: V. alginoipicus, V. cholerae, V. cincinnatiensis, V. fluvialis, V. furnissii and V. metschnikovii. The range of salinity and pH for growth of Vibrio sp. D5 were $0.5\%\~7.5\%$ and $4.5\~9.5$, respectively. Vibrio sp. D5 exhibited typical yellow colony on TCBS agar plate and curved rod type upon transmission electron microscopy (TEM). Vibrio sp. D5 had lethal toxicity against mouse in case of intraperitoneal injection with its culture and showed hemolysin activity on human blood agar and sheep blood agar. Ubrio sp. D5 also demonstrated vascular permeability activity toward rat. From the above results, Vibrio sp. D5 was ascertained to be a novel pathogenic Vibrio.

A Profile of Naturally Occurring Plasmids from Selected Strains of Vibrios

Kim, Young-Hee The Korean Environmental Sciences Society 1997 Environmental sciences Vol.1 No.2

The naturally occurring plasmids of Vibrio species have been isolated in part to investigate their genetic traits. Among six different Vibrio species tested, Vibrio anguillarum, Vibrio fluvialis, Vibrio vulnficus, Vibrio mimicus and Vibrio furnissi did not show any presence of plasmid. One environmental isolate of Vibrio parahaemolyticus harboring plasmid was observed. The isolated plasmid was 8.7 kb by analysis with restriction endonuclease digestion. No common feature was shown relationships between the presence of plasmid and resistance against commonly used antibiotic compounds from the tested Vibrios.

용혈독소를 생산하는 기수성 비브리오균의 생리·생태적 특성과 수산식품의 대책 : 용혈독소를 생산하는 새로운 병원성 Vibrio sp.의 분리와 동정

장동석,김영만,최길배 동의대학교 생활과학연구소 1998 生活科學論集 Vol.2 No.-

금강 하구의 물에서 분리된 sucrose 분해성 Vibrio 속중에 용혈독소를 생산하는 균주의 독성을 시험한 결과와 기존에 알려진 12종의 병원성 Vibrio 속과 비교한 결과는 다음과 같다. 1. 분리된 장소의 환경조건은 염도가 4.7‰, pH가 7.6 수온이 24℃ 및 conductivity가 7800μMHOS이었다. 2. 생리, 생화학적 특성과 염분 요구도를 비교한 결과 sucrose를 분해하는 병원성 Vibrio인 V. alginolyticus, V. cholerae, V. cincinnatiensis, V. fluvialis, V. furnissii V. metschnikovii와는 확실히 구별되었다. 3. 생육가능한 염도는 0.5~7.5‰이었으며 생육가능한 pH는 4.5~9.5이었다. 4. TCBS 평판한천배지에서 균의 집락은 전형적인 황색집락이었으며, 전자현미경에 나타난 균의 형태는 콤마상 간균이었다. 5. 새앙쥐에 대한 치사독성이 확실하였으며, 사람과 면양 적혈구에 대한 용혈성이 확인되었고 rat 피부혈관 투과성 항진작용이 양성이었다. 이상의 결과로 이 균은 병원성이 확인되었고 기존에 알려지지 않은 Vibrio 속으로 확인되어 이 균을 Vibrio sp. D5로 명명하였다. To determine the physiological, biochemical characteristics and toxicity of hemolysin produced by a novel sucrose positive Vibrio (Vibrio sp. D5) isolated from estuary of Kum river, it was compared with already known sucrose positive Vibrio. Salinity, pH, temperature and conductivity of place where Vibrio sp. D5 was isolated were 4.7‰, 7.6, 24℃ and 7800μMHOS, respectively. Physiological and biochemical characteristics distinguished Vibrio sp. D5 from other sucrose positive Vibrio: V. alginolyticus, V. cholerae, V. cincinnatiensis, V. fluvialis, V. furnissii and V. metschnikovii. The range of salinity and pH for growth of Vibrio sp. D5 were 0.5%~7.5% and 4.5~9.5, respectively. Vibrio sp. D5 exhibited typical yellow colony on TCBS agar plate and curved rod type upon transmission electron microscopy (TEM). Vibrio sp. D5 had lethal toxicity against mouse in case of intraperitoneal injection with its culture and showed hemolysin activity on human blood agar and sheep blood agar. Vibrio sp. D5 also demonstrated vascular permeability activity toward rat. From the above results, Vibrio sp. D5 was ascertained to be a novel pathogenic Vibrio.

기수에서 분리된 용혈독소를 생산하는 미분류 Vibrio sp.의 세균학적 특징 -1. V. mimicus와 유사한 Vibrio sp. D9의 세균학적 특성

김영만,오희경,KIM Young-Man,OH Hee-Kyung 한국수산과학회 2000 한국수산과학회지 Vol.33 No.6

A hemolysin producing strain was isolated from Kum rivet estuary located in west part of Korea. In the process of identification the isolated strain was similar to V. mimicus but did not show characteristics of known Vibrio species; therefore, the strain was designated as Vibrio sp. D9 ( V. kumkang) tentatively and further identification study was carried out by comparing its bacteriological characteristics, Morphologically Vibrio sp. D9 was a typical straight roe with a polar flagellium. Among known Vibrio species no identical strains were found when using automatic bacteria identification system ($MicioLog^(TM)$system, release 4.0, Biolog Inc., USA) which evaluated the ability of metabolizing 95 kinds of carbon and nitrogen sources. Vibrio sp. D9 showed 18 and 13 different responses as compared to V. mimicus and V. cholerae, respectively. Clear hemolysis zones were observed with the strain against human and sheep blood agar plate, Hemolytic toxicity was confirmed by strong vascular permeability and fatal toxicity against mouse was also observed. Thus the strain was a pathogenic vibrio. Growth conditions for Vibrio sp. D9 were salinity of $0{\~}5.0{\%}$, pH of $6.4{\~}9.8$, temperature of $15{\~}41^{\circ}C$, respectively.

16S-3S rRNA Intergenic Spacer Region을 이용한 Vibrio fluvialis의 검출

강현실 ( Kang Hyeon Sil ),허문수 ( Heo Mun Su ),이제희 ( Lee Je Hui ) 한국환경생물학회 2003 환경생물 : 환경생물학회지 Vol.21 No.1

본 연구는 위장관염을 일으키는 Vibrio fluvialis의 16S 23S rRNA intergenic spacer region을 분석하였다. ISR을 PCR 증폭 후 plasmid vector에 클로닝하여 염기서열을 분석하였다. 그 결과, ISR의 염기서열은 tRNA gene 조성과 크기에 따라 총 6개의 type으로 분류되었다. 각 type은 tRNA gene 조성과 수에 따라 ISR-A, ISR-E, ISR-E1, ISR-ⅠA, ISR-EKV, ISR-EKAV로 명명하였으며, ISR-A는 tRNA^(Ala); ISR-ⅠA, tRNA^(Ⅱe)-tRNA^(Ala); ISR-EKV, tRNA^(Glu)-tRNA^(Lvs)-tRNA^(Val); ISR-EKAV, tRNA^(Glu)-tRNA^(Lys)-tRNA^(Ala)-tRNA(Val); ISR-E와 E1은 tRNA^(Glu)를 갖고 있었다. 이 중 ISR-EKV type은 minor type으로 존재하고 있으며, 여러 Vibrio종에서 V.Fluvialis를 검출하기 위한 species-speific primer 제작에 이용하였다. 제작된 primer의 특이성은 여러 Vibrio의 genomic DNA를 분리하여 PCR 반응으로 확인하였다. 그 결과, 제작된 primer는 V. fluvialis에 종 특이성이 있으며 여러 Vibrio종으로부터 빠른 검출이 가능함을 확인하였다. We have examined the 16S rRNA intergenic spacer regions(ISR) of Vibrio Fluvialis. ISRs were PCR amplified, cloned into a plasmid vector and then sequenced. As results of ISR nucleotide sequence analysis, total of 6 clones were isolated depending on the size. The clones were different in both the number and the composition of the tRNA genes, and were designated ISR-A, ISR-E, ISR-E1, ISR-IA, ISR-EKV, ISR-EKAV. ISR-A contains tRNA^(Ala) ; ISR-IA, tRNA^(Ile)-tRNA^(Ala) ; ISR-EKV, tRNA^(Glu)-tRNA^(Lys)-tRNA^(Val) ; ISE-EKAV, tRNA^(Glu)-tRNA^(Lys)-tRNA^(Ala)-tRNA^(Val) ; ISR-E and E1, tRNA^(Glu) clusters. ISR-EKV was shown to be a minor type out of the six ISR tyoes and showed a very limited homology between ISR-EKV from V. Fluvialis and ISRs from other Vibrio species. Therefore ISR-EKV sequence was used to design species-specific primers to detect V. fluvialis from other Vibrio species by PCR reaction . The specificity of the primers was examined using genomic DNA of other Vibrios as Templates for PCR reaction. The result showed that PCR can be a useful method to detect V. fluvialis among Vibrio species in a single PCR reaction.

Patterns and drivers of Vibrio isolates phylogenetic diversity in the Beibu Gulf, China

Chen Xing,Du Hong,Chen Si,Li Xiaoli,Zhao Huaxian,Xu Qiangsheng,Tang Jinli,Jiang Gonglingxia,Zou Shuqi,Dong Ke,Adams Jonathan M.,Li Nan,Jiang Chengjian 한국미생물학회 2020 The journal of microbiology Vol.58 No.12

Members of the genus Vibrio are ubiquitous in aquatic environments and can be found either in a culturable or a viable but nonculturable (VBNC) state. Despite widespread concerns as to how to define the occurrence and dynamics of Vibrio populations by culture-independent approaches, further physiological research and relevant biotechnological developments will require the isolation and cultivation of the microbes from various environments. The present work provides data and perspectives on our understanding of culturable Vibrio community structure and diversity in the Beibu Gulf. Finally, we isolated 1,037 strains of Vibrio from 45 samples and identified 18 different species. Vibrio alginolyticus, V. cyclitrophicus, V. tasmaniensis, V. brasiliensis, and V. splendidus were the dominant species that had regional distribution characteristics. The correlation between the quantitative distribution and community structure of culturable Vibrio and environmental factors varied with the Vibrio species and geographical locations. Among them, salinity, nitrogen, and phosphorus were the main factors affecting the diversity of culturable Vibrio. These results help to fill a knowledge gap on Vibrio diversity and provide data for predicting and controlling pathogenic Vibrio outbreaks in the Beibu Gulf.

Repetitive Sequence-based Genomic Fingerprinting을 이용한 Vibrio 속 균의 분자분류 및 동정

김규원 ( Gyu Won Kim ),정헌종 ( Hun Jong Chung ),박철민 ( Chul Min Park ),김기정 ( Kijeong Kim ),김원용 ( Won Yong Kim ),정상인 ( Sang In Chung ) 대한내과학회 2006 대한내과학회지 Vol.71 No.2

Background: The aims of this study were to compare the suitability of repetitive-PCR genomic fingerprinting procedures to investigate genetic relatedness of the genus Vibrio and its applicability for the molecular identification of Vibrio vulnificus. Methods: Forty-eight Vibrio strains were included for this study. REP-, ERIC-, BOX- and SERE-PCR were compared with 13 members of the genus Vibrio. Results: REP-, BOX- and SERE-PCR showed V. vulnificus strains could not be separated well from other Vibrio species. However, approximately 320 bp of highly discriminatory specific fragments was recovered from V. vulnificus strains by ERIC-PCR. Conclusions: ERIC-PCR could be used as rapid classification and identification methods of V. vulnificus from other members of the genus Vibrio.(Korean J Med 71:189-197, 2006)

Vibrio 속 세균 동정에 대한 자동화동정 시스템의 비교

박진숙,권주리 한국미생물학회 2002 미생물학회지 Vol.38 No.2

해수로부터 Vibrio속 세균 21균주를 분리하여 이들의 표현형적 특성을 조사하고 자동화동정 시스템인 Vitek과 MIDI를 이용하여 동정하고 결과를 비교하였다. Vitek과 MIDI를 이용한 Vibrio속 세균의 동정시 일치하는 결과는 단 1건(TL33, V. alginolyticus)이었으며,Vitek의 경우 21균주중 16균주(76%), MIDI의 경우 21균주 중 6균주 (29%)가 동정되었다. Vibrio속 세균의 동정에는 MIDI에 비하여 Vitek시스템이 유용한 것으로 사료된다. Twenty one Vibrio strains were isolated from costal water and their phenotypic properties were determined. We identified bacterial isolates by using automated identification systems, Vitek and MIDI, and compared their identification results with each other. The comparison of them provided identical species information for only one isolates, TL33( V. alginolyticus). From Vitek and MIDI,16 (76%), and 6 (29%) out of 21 isolates were identified to the species level, respectively. Vitek was more useful than MIDI for identification of Vibrio species.

생굴 섭취로 인한 고병원성 Vibrio균 식중독 위해평가

하지명,이지연,오혜민,신일식,김영목,박권삼,윤요한 한국식품위생안전성학회 2020 한국식품위생안전성학회지 Vol.35 No.1

This study investigated the probability of foodborne illness caused by raw oyster consumption contaminated with high risk Vibrio species such as V. vulnificus and V. cholerae. Eighty-eight raw oyster samples were collected from the south coast, west coast and Seoul areas, and examined for the prevalence of high risk Vibrio species. The growth patterns of V. vulnificus and V. cholerae in raw oysters were evaluated, and consumption frequency and amounts for raw oyster were investigated from a Korean National Health and Nutrition Examination Survey. With the collected data, a risk assessment simulation was conducted to estimate the probability of foodborne illness caused by intake of raw oysters, using @RISK. Of 88 raw oysters, there were no V. vulnificus- or V. cholerae-positive samples. Thus, initial contamination levels of Vibrio species in raw oysters were estimated by the statistical methods developed by Vose and Sanaa, and the estimated value for the both Vibrio spp. was -3.6 Log CFU/g. In raw oyster, cell counts of V. vulnificus and V. cholerae remained unchanged. The incidence of raw oyster consumers was 0.35%, and the appropriate probabilistic distribution for the consumption amounts was the exponential distribution. A risk assessment simulation model was developed with the collected data, and the probability of the foodborne illness caused by the consumption of raw oyster was 9.08×10 -15 for V. cholerae. Consumption frequency was the first factor, influencing the probability of foodborne illness.