• Journal of Microbiology
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• v.44 no.2
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• pp.226-232
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• 2006

Swarming has proven to be a good in vitro model for bacterial surface adherence and colonization, and the swarming differentiation of a bacterium has been shown to be coupled with changes in the expression of virulence factors associated with its invasiveness, particularly in the early stages of infection. In this study, we attempted to determine whether the expression of vvhA, which encodes for hemolysin/cytolysin (VvhA), is either upregulated or downregulated during the swarming differentiation of V. vulnificus. The insertional inactivation of vvhA itself exerted no detectable effect on the expression of V. vulnificus swarming motility. However, in our lacZ-fused vvhA transcriptional reporter assay, vvhA expression decreased in swarming V. vulnificus as compared to non-swarming or planktonic V. vulnificus. The reduced expression of vvhA in swarming V. vulnificus increased as a result of the deletional inactivation of luxS, a gene associated with quorum sensing. These results show that vvhA expression in swarming V. vulnificus is downregulated via the activity of the LuxS quorum-sensing system, suggesting that VvhA performs no essential role in the invasiveness of V. vulnificus via the adherence to and colonization on the body surfaces required in the early stages of the infection. However, VvhA may playa significant role in the pathophysiological deterioration occurring after swarming V. vulnificus is differentiated into planktonic V. vulnificus.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.27 no.2
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• pp.107-113
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• 1994

Among the currently recognized pathogenic vibrios, V. vulnificus and V. cholerae non O1 are the most serious bacteria from the point of view of sea food hygiene in Korea. In this paper, the authors compared the hemolytic activities of the crude hemolysin produced by V. vulnificus and V. cholerae non O1 isolated from shellfish collected in Chungmoo, Korea. The authors also attempted to improve the purification method of V. vulnificus hemolysin(VVH) and tried to make antiserum with the purified hemolysin. VVH was produced in abundance in heart infusion broth containing $2\%$ NaCl in a shaking cultivation process(140rpm) at $37^{\circ}C$ for 15 hours. While hemolysin production patterns of V. cholerae non O1 were quite different by the strain during the culture times compared with the V. vulnificus. Hemolytic activity of the VVH on sheep erythrocytes was stronger than those of rabbit, but hemolytic activities of the hemolysin produced by V. cholerae non O1 on rabbit erythrocytes were as much as twice as strong as on those of sheep and horse. VVH was purified by two steps of hydrophobic column chromatography on Phenyl-Sepharose HP with Fast Protein Liquid Chromatography(FPLC). Purification fold and yield of VVH was much improved by changing the elution buffer's pH from 6.0 to 9.8 and adding $1\%$ CHAPS(a zwitter ionic detergent) and $50\%$ ethylene glycol to the 10mM glycine buffer during the repeated hydrophobic column chromatography. Homogeneity of the purified hemolysin was shown by polyacrylamide gel electrophoresis. According to the five times repeated purification results, the specific activity was increased 27500 times and the yield was improved by $23.4\%$ on average. About $250{\mu}g$ of purified hemolysin was harvested from the 2400ml of culture supernatant of V. vulnificus. Molecular weight of VVH was estimated to be 50KDa by the SDS-PAGE and the neutralization scores of the obtained antiserum acting against VVH were $2000{\sim}8500$.

• Korean Journal of Microbiology
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• v.41 no.3
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• pp.168-176
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• 2005

Vibrio vulnificus, one of the marine bacterial pathogens causing septicemia, was detected using molecular methods, namely, PCR and/or Southern hybridization, and real-time PCR. Extracted and purified total DNAs by using commercial kits were used as templates for PCR. Multiplex-PCR was conducted by employing three sets of primers for the genes, hemolysin (vvhA), phosphomannomutase (pmm), and metalloprotease (vvpE), for V vulnificus virulence. The presence of DMSO ($5\%$) and BSA ($0.1\%$) in PCR reaction mixture improved a detection efficiency by higher PCR band intensities. TaqMan real-time PCR was carried out by using gene segment of vvhA as a target. Detection limit of PCR/Southern hybridization without enrichments was to be around $10^2\;cells\;g^{-1}$ of sample. However, those three methods using the enrichment at $35^{\circ}C$ in APW showed high sensitivity ($2\~10\;cells\;g^{-1}$ of sediments). Highly sensitive detection of V vulnificus by real-time PCR was achieved within $5\~6$ hr, whereas the detection by PCR/Southern hybridization required about 36 hr. Thus, it was evident that real-time PCR is the most rapid and efficient method for detecting V vulnificus in tidal flat sediments.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.53 no.5
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• pp.688-693
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• 2020

Vibrio vulnificus is a Gram-negative marine bacterium known to cause septicemia. This study was conducted to investigate the distribution of V. vulnificus along the coast of Gadeok Island in Korea and to determine the molecular characteristics of isolated strains sampled between March and November 2019 from seawater. The strains were mostly detected between July and September, when the average water temperature and average salinity were 22.2-26.2℃ and 14.2-29.9 psu, respectively. V. vulnificus was not detected in seawater below 15℃. In September, the highest population of V. vulnificus was observed at 2,100 MPN (most probable number)/100 mL, attributable to decreased salinity from heavy rains. In addition, the detection rate of V. vulnificus was higher at the sampling station near the Nakdong River. Virulence-related genes were also identified among the isolates, such as vvhA (97.1%), viuB (44.1%), and vcgC (57.4%). In particular, viuB and vcgC were only observed in V. vulnificus isolated from June to September, when the detection rate was high and water temperature was above 20℃, suggesting the role of seasonal characteristics.

• Journal of Food Hygiene and Safety
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• v.35 no.1
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• pp.6-12
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• 2020

Prevalence of toxin genes and profiles of antibiotic resistance in Vibrio vulnificus were investigated for prevention of Vibrio sepsis and selection of effective antibiotics. A total of 23 V. vulnificus strains were isolated from Vibrio sepsis patients, fish, and water samples collected from fish tanks in restaurants in Jeonnam province during 2015-2017 period. Prevalence of toxin genes including, RtxA, viuB and vvhA were assessed and susceptibilities to 15 different antibiotics were determined. As a result of the toxin gene profile, the RtxA toxin gene was detected in 19 (82.6%) out of 23 strains, and vvhA and viuB toxin genes were positive in all strains. These results showed that V. vulnificus tested in this study possessed at least one more toxin gene, and the toxin gene detection rate was higher than in previous reports. Therefore, there is always a risk of Vibrio sepsis through eating fish or having contact with aquarium water at seafood restaurants. Especially, it was deemed necessary to provide preventive education about Vibrio sepsis for workers in such restaurants. The results of antibiotic susceptibility tests presented 94.4% resistance to cepoxitin antibiotics but all strains showed susceptibility to 14 kinds of antibiotics including chloramphenicol and tetracycline. The currents antibiotic therapy using chloramphenicol and teteracycline against Vibrio sepsis was judged to be useful.

• Korean Journal of Microbiology
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• v.38 no.4
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• pp.281-286
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• 2002

Detection of Vibrio vulnificus in fish farm and searching for the bactericidal methods on this bacteria were studied. To detect this microorganism in sea water, mud, fish and mussels, selective isolation methods and detection of vvhA gene were used from January to October,2000. V. vulnificus was detected from May when the water temperature was over $17^{\circ}C$. From June to September, higher than $19^{\circ}C$, this bacteria could be isolated from most of the samples. Freezing and refrigerating did not inhibit the growth of V. vulnificus. Citric acid did not show the bactericidal effect, but more than 500 mg/l of EDTA did. With the aid of UV and photocatalyst, $TiO_{2}$ showed bactericidal effect after 15 minute treatment. Photocatalytic system consisted of glass bead coated with $TiO_{2}$ and UV illumination showed bactericidal effect on V. vulnificus at the turnover rate of 0.2/min.

• Korean Journal of Microbiology
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• v.43 no.4
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• pp.256-263
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• 2007

We performed the biochemical characteristics, molecular epidemiologocal analysis, and drug susceptibility test on V. vulnificus isolated from environmental sources in Incheon. For this study, 233 strains were isolated from seawater, sediment, shellfish. V. vulnificus isolates were divided into 15 biochemical groups, which were positive for ONPG and Amygdalin test. Among the 209 strains, 206 (98.6%) strains and 110 (52.6%) strains revealed positive for vvhA and viuB gene, and the viuB gene detection rates of V. vulnificus from seawater, shellfish and sediment were 48%, 48.5% and 61.6%, respectively. From disc diffusion test on 175 isolates, most of strains were sensitive to Imipenem (100.0%), Sulfamethoxazole/trimethoprim (98.9%), Tetracycline, Ciprofloxacin (98.3%), Ampicillin/sulbactam (97.1%), Ohloramphenicol (96.6%), Cefepime (94.9%) and Ceftriaxone (94.8%), multi-drug resistance rates was 31.5% of seawater, 34.4% of sediment and 29.2% of shellfish. PFGE was performed on 233 V. vulnificus isolates with the objective of investigating the extent of genetic diversity of these isolates in our region. We could find that at least 126 different PFGE patterns were generated according by 90% of similarity and 13 clusters by 58% of similarity. The major cluster was type I (44.6%) during the most of the year, and type J was frequent pattern in June and October. There were 9 distinct PFGE types in July, 8 types in August, 7 types in June, 6 types in September, 5 types in October 3 types in May and 1 type in March.

• 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.

• Fisheries and Aquatic Sciences
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• v.26 no.9
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• pp.558-568
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• 2023

Vibrio vulnificus is an aquatic bacterium causing septicemia and wound infection in humans. To understand this pathogen at the genomic level, it was performed whole genome sequencing of a cefoxitin-resistant strain, V. vulnificus 1908-10 possessing virulence-related genes (vvhA, viuB, and vcgC) isolated from Gadeok island coastal seawater in South Korea. The genome of V. vulnificus 1908-10 consisted of two circular contigs and no plasmid. The total genome size was estimated to be 5,018,425 bp with a guanine-cytosine (GC) content of 46.9%. We found 119 tRNA and 34 rRNA genes respectively in the genome, along with 4,352 predicted protein sequences. Virulence factor (VF) analysis further revealed that V. vulnificus 1908-10 possess various virulence genes in classes of adherence, antiphagocytosis, chemotaxis and motility, iron uptake, quorum sensing, secretion system, and toxin. In the comparison of the presence/absence of virulence genes, V. vulnificus 1908-10 had fur, hlyU, luxS, ompU, pilA, pilF, rtxA, rtxC, and vvhA. Of the 30 V. vulnificus comparative strains, 80% of the C-genotype strains have all of these genes, whereas 40% of the E-genotype strains have all of them. In particular, pilA were identified in 80% of the C-type strains and 40% of the E-type strains, showing more difference than other genes. Therefore, V. vulnificus 1908-10 had similar VF characteristics to those of type C strains. Multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin of V. vulnificus 1908-10 contained 8 A-type repeats (GXXGXXXXXG), 25 B.1-type repeats (TXVGXGXX), 18 B2-type repeats (GGXGXDXXX), and 7 C-type repeats (GGXGXDXXX). The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) showed that the RtxA protein of V. vulnificus 1908-10 had the effector domain in the order of cross-liking domain (ACD)-C58_PaToxP-like domain- α/β hydrolase-C58_PaToxP-like domain.

• Journal of Food Hygiene and Safety
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• v.38 no.5
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• pp.381-389
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• 2023

Vibrio vulnificus, the most fatal waterborne and foodborne pathogens of 50% fatality rate in the world, is common in seawater and occurs particularly in warmer months. In this study, we investigated the toxin genes using reverse transcription-polymerase chain reaction (RT-PCR), antibiotic resistance status using Vitek, and genetic characteristics using pulsed-field gel electrophoresis (PFGE) of different V. vulnificus strains isolated from the Jeju Island seawater, distribution fishery products, and fish tanks. We examined a total of 487 samples and isolated a total of 46 strains (including overlapping strains) of V. vulnificus, 44 strains from seawater and 1 strain each from fishery products and fish tank. We detected toxin gene vvhA in all 46 strains and rtxA, viu in 8 strains (17.4%) and 9 strains (19.6%) strains, respectively. Antibiotic resistance tests indicated 100% resistance to cefoxitin antibiotics. The PFGE analysis of the 46 strains identified a total of 6 types showed 100% homology and the degree of similarity was 81.3-98.0%; however, there were no similarity between the regions and samples. These results indicate that V. vulnificus isolated from the seawater, fishery products, and fish tanks should be continuously monitored as cases of food poisoning caused by V. vulnificus with toxin genes have been reported in Jeju Island.