• Journal of Microbiology and Biotechnology
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• v.14 no.2
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• pp.284-289
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• 2004

A PCR assay for the rapid detection of Vibrio vulnificus strains was developed using a virulence gene for elastase found in various Vibrio species. The DNA sequences in the elastase gene facilitated the identification of a species-specific probe for pathogenic V. vulnificus strains from both clinical and environmental sources. Using an elastase gene-based PCR reaction, a species-specific 507-bp PCR product was visualized by agarose gel electrophoresis. Three different DNA extraction methods were then compared to improve the simplicity and rapidity of detection. A PCR assay using the conventional DNA extraction or boiling method was able to detect as few as 25 V. vulnificus cells, making the detection limits at least 1-log-scale lower than that for the EDT A-treated DNA extraction method. In particular, the boiling method, which does not require purification of the chromosomal DNA, was very effective in terms of simple and rapid detection. Meanwhile, the detection limit in a mixed bacterial culture that included other bacteria, such as Escherichia coli or Bacillus subtilis, was two V. vulnificus cells, which was 1-log-scale lower than that for the control. Accordingly, when coupled with a new DNA extraction method, the elastase gene-based PCR can provide a rapid, specific, and sensitive method for identifying V. vulnificus in clinical and environmental samples.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.19 no.2
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• pp.118-126
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• 1986

Vibrio vulnificus is a recently recognized halophilic organism that nay cause serious human infections. Patients infected with V. vulnificus often have a history of exposure to the sea, suggesting that the organism may be common inhabitant of marine environment. The purpose of this experiment is to investigate the distribution and bacteriological characteristics of V. vulnificus. The strain used in this experiment was isolated from sea water and sea products such as common octopus (Octopus variabilis), ark shell (Anadara broughtonii), blue crab (Ericheir japonica), and sea squirt (Synthia roretzi) collected in Pusan area from July to October in 1985. V. vulnificus was frequently isolated in August when temperature of sea water was around $26^{\circ}C$ and rarely isolated in October when temperature of sea water was around $18.5^{\circ}C$. The distinctive biochemical characteristics of V. vulnificus were ONPG hydrolysis positive and fermented lactose and not grown in peptone water contained $8\%$ NaCl. The optical density at 660 nm of the growth of V. vulnificus was reached maximum level after 8 hours of culture at $35^{\circ}C$ in brain heart infusion broth but that of V. vulnificus was little increased at $15^{\circ}C$ for 14 hours. Optimum temperature and pH for the growth of V. vulnificus were around $35^{\circ}C$ and 8.0. The specific growth rate and the generation time of V. vulnificus isolated from the samples were $1.21\;hr^{-1}$, 34 min at $35^{\circ}C$ and $0.61\;hr^{-1}$, 69 min at $25^{\circ}C$, respectively. V. vulnificus did not grow on eosin-methylene-blue agar, salmonella-shigella agar, deoxycholate agar but grew well on Endo agar, xylose-lysine-deoxycholate agar and hektoen enteric agar. On Endo agar, the colonies of V. vulnificus were red and achieved a diameter of 2 to 4 mm as a feature enabling differentiation of V. vulnificus from other Vibrio spp. V. vulnificus grow well on TCBS agar forming green colonies. V. vulnificus refrigerated at $4^{\circ}C$ exhibited a linear decline of its viablity as 1 log cycle in every 16 hours storage, while V. vulnificus freezed at $-18^{\circ}C$ almost became extinct.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.52 no.1
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• pp.27-34
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• 2019

The seasonal and spatial variation of pathogenic Vibrio species, such as V. parahaemolyticus, V. vulnificus, V. alginolyticus, and V. cholerae were investigated in seawater and in bivalves off the Gyeongnam coast of Korea, which is an important area for shellfish production, during the period 2013-2016. V. parahaemolyticus, V. vulnificus, V. alginolyticus, and V. cholerae were detected in 12.1%, 5.2%, 15.4%, and 0.9% of seawater samples, respectively. V. parahaemolyticus, V. vulnificus, V. alginolyticus, and V. cholera were detected in 21.9%, 7.1%, 12.2%, and 0.0% of shellfish samples, respectively. The Vibrio spp. in seawater and bivalve samples were detected at high levels during the summer to early autumn; however, the levels were low during the winter. Therefore, their occurrence was seasonally dependent and correlated with high water temperature, which is also the biggest factor contributing to foodborne outbreaks associated with Vibrio. Relatively high detection rates of the strains were also found in the sea area that was continually exposed to inland wastewater. Our findings show that continuous monitoring is needed to reveal the patterns of occurrence of these pathogens from marine samples collected off the Korean coast, to reduce seafood-borne outbreaks caused by Vibrio.

• Journal of Life Science
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• v.11 no.1
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• pp.27-29
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• 2001

Vibrio vulnificus endotoxin was extracted, analyzed the chemical composition, tested its biological activity, and compared to those of Escherichia coli and Salmonella typhimurium. The major fatty acid of three endotoxins were different each other; V. vulnificus endotoxins were different each other; V. vulnificus endotoxin was myristric acid (C14:0), E. coli was lauric acid (C12:0), S. typhimurium was capric acid (C10:0). The biological activities of V. vulnificus endotoxin were similar to those of E. coli and S. typhimurium in terms of the gelation activity of the Limulus amebocyte lysate and the lethal toxicity. But the result of enzyme (AST, ALT, and LDH) analysis showed that the enzyme activity of V. vulnificus endotoxin was similar to that of E. coli, but smaller than that of S. typimurium.

• Journal of Microbiology and Biotechnology
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• v.11 no.2
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• pp.302-309
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• 2001

Colony blot hybridization using a VVHP DNA probe derived from the sequence of the hemolysin gene, vvhA, was specific in identifying all V. vulnificus strains, thereby, eliminating the need for any additional phenotypic identification. The colony blot hybridization procedure revealed a sensitivity and broad applicability sufficient for the direct enumeration of V. vulnificus in various natural samples, without the use of enrichment or culturing on selective medis. V. vulnificus was detected in all natural samples collected during August and May at concentrations ranging from $2.1{\times}10^1\;to\;4.0{\times}10^3$ organisms per ml. However, during November and February, when the mean temperatures of the seawater were $12^{\circ}C$ and $5^{\circ}C$, respectively, V. vulnificus was not detected in any natural samples.

• Journal of Microbiology and Biotechnology
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• v.18 no.11
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• pp.1841-1847
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• 2008

Vibrio vulnificus is a causative agent of serious diseases in humans, resulting from the contact of wound with seawater or consumption of raw seafood. Several studies aimed at detecting V. vulnificus have targeted vvh as a representative virulence toxin gene belonging to the bacterium. In this study, we targeted the rpoS gene, a general stress regulator, to detect V. vulnificus. PCR specificity was identified by amplification of 8 V. vulnificus templates and by the loss of a PCR product with 36 non-V. vulnificus strains. The PCR assay had the 273-bp fragment and the sensitivity of 10 pg DNA from V. vulnificus. SYBR Green I-based real-time PCR assay targeting the rpoS gene showed a melting temperature of approximately $84^{\circ}C$ for the V. vulnificus strains. The minimum level of detection by real-time PCR was 2 pg of purified genomic DNA, or $10^3$ V. vulnificus cells from pure cultured broth and $10^3$ cells in 1 g of oyster tissue homogenates. These data indicate that real-time PCR is a sensitive, species-specific, and rapid method for detecting this bacterium, using the rpoS gene in pure cultures and in infected oyster tissues.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.54 no.6
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• pp.918-926
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• 2021

We isolated 28 Vibrio vulnificus strains from seawater and fisheries and investigated the positive rate of eight virulence genes. Additionally, we evaluated the susceptibility of these strains to 25 antimicrobials. The positive rates of fur, vvhA, tcp, rtxA, vcgC, viuB, vvp, and acfA were 100, 92.9, 92.9, 67.9, 64.3, 25.0, 14.3, and 7.1%, respectively. A disk diffusion susceptibility test revealed that, all the investigated strains had the highest resistance to amoxicillin and oxacillin, followed by that to streptomycin (96.4%), cefoxitin (92.9%), clindamycin (82.1%), amikacin (67.9%), vancomycin (46.4%), nalidixic acid (7.1%), penicillin G (7.1%), and ampicillin (3.6%). Moreover, they were susceptible to 10 other antimicrobials, including cefotaxime, chloramphenicol, erythromycin, gentamicin, and rifampicin. Notably, amoxicillin, oxacillin, and streptomycin had average minimum inhibitory concentrations of 132.6, 603.4, and 23.1 ㎍/mL against V. vulnificus, respectively. These observations provide new insights regarding the necessity for sanitation of commercial fisheries and can potentially, help reduce the risk posed by fisheries contaminated with bacteria resistant to antimicrobials.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.22 no.6
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• pp.385-390
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• 1990

To establish an appropriate measure for the prevention of food poisoning from raw fish flesh (sashimi), distribution and population density of Vibrio vulnificus were investigated as environmental factors were changed. The bacteria were detected only in August during the study period from February to November, 1989. The number of bacteria was $2.0\~6.0/100ml$. In August, salinity and pH were low, but water temperature and COD were high as compared to the rest of months.

• The Journal of the Korean Society for Microbiology
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• v.35 no.3
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• pp.225-237
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• 2000

Vibrio vulnificus, a halophilic vibrio is an estuarine gram-negative bacteria that is associated with severe and frequently fatal wound infections and life-threatening septicemia. Bacteriocins are defined as antibacterial substance produced by various species of bacteria which are usually active against closely related organisms. Bacteriocins have found widespread application in epidemiological studies as specific markers of bacteria. It was proposed by Ha et al. (1990. J. Korean. Soc. Microbiol. 25: 586.) to give the bacteriocins produced by V. vulnificus the name "vulnificins". In the present study, a total of 72 strains of V. vulnificus isolated from patients and oysters were subjected to screen potential producers and indicators of vulnificin, applying ultraviolet induction method. Sensitivity of several strains of Serratia marcesans, Pseudomonas aeruginosa, Shigella flexneri, Salmonella typhi and Yersinia enterocolitica to vulnificins were also examined out. All the tested strains of V. vulnificus produced vulnificins active against indicator strains with various different inhibitory patterns. The spectrum of vulnificin activity and sensitive spectrum of indicator strains were considerably broad. Interestingly, almost all strains of S. marcescens, P. aeruginosa, Salmonella sp., Shigella sp. and Y. enterocolitica tested were sensitive to 1-7 vulnificin(s). Taken together, the present study demonstrated that all of the isolates of V. vulnificus produced vulnificins and that 8 good vulnificin producers and 10 good indicators were detected. These strains can be employed efficiently for establishing vulnificin typing scheme of V. vulnificus and for the detection of bacteriocinogeny and sensitivity in V. vulnificus. Biological role of vulnificin remains to be further elucidated.

• The Journal of the Korean Society for Microbiology
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• v.22 no.2
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• pp.109-116
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• 1987

As a process to establish an effective preventive measure of V. vulnificus septicemia, bactericidal effect of distilled water against V. vulnificus was studied. When about $2.0{\times}10^7\;CFU/ml$ of V. vulnificus was inoculated in distilled water, a dramatic decrease in the number of viable bacteria by 5 to $6LOG_{10}$ was observed in 5 minutes. Bactericidal kinetic curves could be divided into the first rapid killing phase until 1 minute and the later slow killing phase after then, showing the heterogeneity of the bacterial population inoculated. When V. vulnificus was inoculated in saline solutions having various salinities, significant decrease in the number of viable bacteria was noted only at salinities under 0.2%. The higher was the concentration of NaCl, the greater was the degree of protection against osmotic shock. When glucose, NaCl, $MgCl_2$, and $CaCl_2$ were diluted with deionized water to give same osmolarities and V. vulnificus was inoculated in each of them to compare the bactericidal curves plotted during the first 5 minutes after inoculation, the protection efficiencies were in the order of $MgCl_2>CaCl_2{\gg}NaCl{\gg}glucose$. Above results indicate that treatment(or thorough washing) of contaminated sea animals or other products with distilled water can be used as a preventive measure of V. vulnificus septicemia, and divalent cations can protect V. vulnifcus to osmotic shock with high efficiency.