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Distribution of Pathogenic Vibrio Species in the Coastal Seawater of South Korea (2017–2018)

Objectives Pathogenic Vibrio species are widely distributed in warm estuarine and coastal environments, and can infect humans through the consumption of raw or mishandled contaminated seafood and seawater. For this reason, the distribution of these bacteria in South Korea was investigated. Methods Seawater samples were collected from 145 coastal area points in the aquatic environment in which Vibrio species live. Environmental data (i.e., water temperature, salinity, turbidity, and atmospheric temperature) was collected which may help predict the distribution of the species (data not shown). Seawater samples were filtered, and incubated overnight in alkaline peptone water, at 37°C. Using species-specific polymerase chain reaction methods, screening tests were performed for the hlyA, ctxA, vvhA, and tlh genes. Clones of pathogenic Vibrio species were isolated using 3 selective plating media. Results In 2017, total seawater isolation rates for Vibrio vulnificus, Vibrio cholerae (non-pathogenic, non-O1, non-O139 serogroups), and Vibrio parahaemolyticus were 15.82%, 13.18%, 65.80%, respectively. However, in 2018 isolation rates for each were 21.81%, 19.40%, and 70.05%, respectively. Conclusion The isolation rates of pathogenic Vibrio species positively correlated with the temperature of seawater and atmosphere, but negatively correlated with salinity and turbidity. From 2017 to 2018, the most frequent seawater-isolated Vibrio species were V. parahaemolyticus (68.10 %), V. vulnificus (16.54%), and non-toxigenic V. cholerae (19.58%). Comprehensive monitoring, prevention, and control efforts are needed to protect the public from pathogenic Vibrio species.


Introduction
More than 100 Vibrio bacterial species which are ubiquitous in seawater, marine, and estuarine environments, have been identified worldwide. Among all Vibrio species, the most clinically significant include V. cholerae, V. parahaemolyticus, and V. vulnificus [1,2]. Cholera is an infectious disease caused by V. cholerae; a symptom of which is severe diarrhea and this may lead to dehydration, and death if untreated. Moreover, cholera outbreaks are seasonal, and in specific biogeographical zones with warmer temperatures, and ecology [1]. Cholera is an ancient disease and V. cholerae reportedly has killed millions of people from warm tropical to cold temperate regions of the world. Evidently, Vibrio infections occur during summer and early fall, corresponding to the period of warmer seawater temperatures [3]. Almost all cases of food-borne Vibrio infections are associated with a recent history of handling or consuming seafood (primarily raw oysters) [1][2][3][4][5].
By the end of the 20th century, it was reported that V.
cholerae was completely eliminated from most parts of the *Corresponding author: Sang Moon Soh developed world. However, it remained endemic to many tropical regions [4]. In recent years, this species has been detected in temperate regions and in many developed countries worldwide [6][7][8][9][10][11]. Its extreme diversity has facilitated its prevalence in North America, and among the prominent halophilic Vibrio species, V. parahaemolyticus is the leading cause of seafood-associated disease globally [7,9]. In the 1950s, seafood-borne Vibrio species were reported, wherein a strain which caused the pandemic outbreak and its variants, were identified in far East Japan [3,12]. In South Korea, the main cause of seafood-borne disease was determined to be infection by V. parahaemolyticus, leading to 18 outbreaks Korea [17,20]. Since South Korea has specific peninsular plateau geographical characteristics, and its people traditionally favor seafood consumption, KCDC has been investigating the seawater distribution of Vibrio species, and analyzing the genetic characteristics of isolated strains and their relationship with environmental factors, to use these data for the prevention of human infectious disease [21,22]. Based on these results, the KCDC aims to prevent the emergence of human infection due to the abnormal growth of pathogenic Vibrio species resulting from climate change. These surveillance results may be helpful to track the reasons for epidemic outbreaks of Vibrio species.  Figure 1).

Sample collection and amplification
More than 500 mL of seawater was collected within 1 meter of the seawater surface. Collected seawater samples were filtered using a 0.45-μm nitrocellulose membrane filter paper (Pall laboratories, NY, USA). After filtration, membranes were incubated in 5 mL of alkaline peptone water (Oxoid, Basingstoke, UK) for 8-16 hours, at 37°C.

DNA extraction
For DNA extraction, 1 mL of seawater-enriched broth sample was used. Seawater samples were pelleted by centrifugation at 12,000 x g for 2 minutes, at room temperature. After discarding the supernatant, 200 μL of distilled water was added to each sample. Samples were then heated to 100°C for 10 minutes, and subsequently cooled to −20°C for 10 minutes. DNA was collected by centrifugation at 12,000 x g for 2 minutes, at room temperature. The extracted DNA samples were stored at −20°C until required.

Screening of Vibrio species
Specifically-designed primers were used to amplify a fragment from each of the species-specific genes: V. cholerae

Vibrio species culture and colony isolation
Seawater-enriched broth samples were spread on selective medium plates, namely CHROMagar Vibrio (CHROMagar, Paris, France), thiosulfate citrate bile salt agar (Difco, MI, USA), and Tryptic Soy Agar (Difco, Michigan, USA), and incubated for > 16 hours at 37°C. After incubation, colonies that were suspected as Vibrio species were picked from each plate, and were confirmed using the VITEK II (BioMérieux, Paris, France) system and real-time PCR.

Genetic determination of V. vulnificus
Colonies that were suspected to be of V. vulnificus were extracted from each plate and confirmed by PCR methods for environmental and clinical types. Tri-primer PCR methods      (Tables 4   and 5).    Data are presented as n (%). PCR = polymerase chain reaction.  expanded to monitor coastal areas in Korea.

As previously described, V. vulnificus infections in South
Korea are the leading cause of septicemia and high mortality,

Conflicts of Interest
All authors declare that there are no conflicts of interest.