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ORIGINAL ARTICLE  
Year : 2017  |  Volume : 10  |  Issue : 2  |  Page : 417-422
Vibrio harveyi, V. parahaemolyticus, and V. vulnificus detection in Thai shellfishes by the triplex PCR method


Department of Applied Science, Faculty of Science and Technology, Suan Sunandha Rajabhat University, Bangkok, Thailand

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Date of Web Publication22-Jun-2017
 

   Abstract 

Context: Shellfish sold in Southeast Asian markets are highly contaminated. Uncooked seafood samples were collected from markets in Bangkok, Thailand, which were contaminated with Vibrio species (27%) and in which antibiotic resistance was relatively high. Aims: To simultaneously detect V. harveyi, V. parahaemolyticus, and V. vulnificus in shellfish samples including life mussel (Perna viridis), blood clam (Tegillarca granosa), and baby clam (Paphia undulata) in the local markets in Bangkok. The sensitivity, accuracy, and specificity of triplex PCR method were also evaluated. Materials and Methods: The 150 frozen shellfishes were purchased from five local markets in Bangkok. Each sample was homogenized, enriched, and prepared for colony counting and isolation. Three Vibrio species were identified according to biochemical tests and then later confirmed by triplex PCR. The accuracy and specificity of triplex PCR were evaluated and compared with conventional tests. The sensitivity of triplex PCR was explained as total count (CFU/mL) of Vibrio species. Results: Using the biochemical test and triplex PCR method in the three Vibrio species of marine shellfishes, 14 and 15 isolates of V. harveyi, 8 isolates of V. parahaemolyticus, and undetectable and 1 isolate for V. vulnificus were identified, respectively. The accuracy of triplex PCR was higher than the conventional method. Triplex PCR was shown to be sensitive as total bacterial count for three Vibrio detection in marine shellfishes was 1.2 × 107-2 × 106 CFU/mL. Conclusions: Triplex PCR assay was higher in sensitivity, accuracy, and specificity, which proved to be convenient, simple, and effective method for Vibrio detection and identification in shellfishes.

Keywords: Food-borne pathogens, shellfish, triplex PCR, Vibrio species

How to cite this article:
Thongkao K, Sudjaroen Y. Vibrio harveyi, V. parahaemolyticus, and V. vulnificus detection in Thai shellfishes by the triplex PCR method. Ann Trop Med Public Health 2017;10:417-22

How to cite this URL:
Thongkao K, Sudjaroen Y. Vibrio harveyi, V. parahaemolyticus, and V. vulnificus detection in Thai shellfishes by the triplex PCR method. Ann Trop Med Public Health [serial online] 2017 [cited 2019 Jun 19];10:417-22. Available from: http://www.atmph.org/text.asp?2017/10/2/417/208735

   Introduction Top


Recently, seafood is popular worldwide, because it is considered to be healthy, and this has resulted in an increase in both production and domestic consumption of seafood in Southeast Asia. However, Southeast Asian countries export seafood to developed countries, and its transportation varies and seldom reported. Raw food items contaminated with food‐borne pathogens and antibiotic‐resistant bacteria have been studied in Southeast Asia.[1],[2],[3] Nakaguchi reported prevalence of three genetic markers of Vibrio parahaemolyticus in seafood like fish, shrimp, squid, crab, and molluscan shellfish that were purchased from provinces in Thailand and three Southeast Asian countries, and data suggest that the molluscan shellfish sold in Southeast Asian markets are highly contaminated.[4] As Vuddhakul et al. reported, only molluscan shellfish harbored virulent strains in southern Thailand as compared to various other types of seafood.[5] Uncooked seafood samples were collected from open markets and supermarkets in Bangkok, Thailand, which were contaminated with 27% of Vibrio species and in which resistance to antibiotics was found to be relatively high.[6]

Vibrio species are the large members of isolated infectious bacteria in humans and marine animals, which include V. alginolyticus,  V.cholerae Scientific Name Search , V. fluvialis, V. harveyi, V. mimicus, V. parahaemolyticus, and V. vulinificus. Vibrio infections may cause low immunity in opportunistic host or infected host. The virulent factors affect host, such as extracellular proteins, siderophore, Type III secretion, and endotoxin. The transmission of Vibrio infections is primarily through the consumption of raw or undercooked shellfish or exposure of wounds to warm seawater, which may cause diseases in both aquatic animals and humans. Pathogenic Vibrio infections cause three of major clinical illnesses: gastroenteritis/diarrhea, wound infections, and septicemia. For example V. parahaemolyticus and V. vulnificus infections are good evidence of human infections caused by infected marine animals.[7] V. harveyi is found in the aquatic environment and defined as nonpathogenic for humans; however, they are pathogenic for marine animals and they, albeit rarely, have association with infections in humans.[8] In terms of human disease, V. harveyi was found in wound infections, specifically in a leg wound resulting from a shark bite in SC, USA.[9] A pediatric oncology patient was presented with central line sepsis caused by V. harveyi after swimming in the sea.[10] V. harveyi may be related to flrA gene involved in the regulation of V. cholerae flagella synthesis, nanH gene encoded for V. cholerae neuraminidase, tdh gene involved in V. parahaemolyticus virulence, and luxA gene is included in the lux operon involved in bioluminescent expression and quorum sensing.[11]

The detection and identification of Vibrios by the morphology of colonies on selective media TCBS (Thiosulfate citrate bile salt sucrose agar) are used to isolate Vibrio species. Vibrio species are often identified due to pH changing made from sucrose and non-sucrose consumption. Changing of pH will be converts the color of the dyes in medium, which can beyellow, green, or blue color and then Vibrios can be classify.[12] The conventional biochemical tests, including oxidase, triple sugar iron (TSI), sulfur reduction, indole production, motility (MIL), methyl red (MR) and Voges–Proskauer (VP), and salt tolerance tests are also necessary for Vibrio identifications. Media confirmation on bacterial isolates can be achieved by using commercial identification systems such as the API 20E test kit.[13] However, conventional method has many limitations such as they are laborious and time consuming, and several Vibrio species display similar biochemical characteristics, and hence the interpretation within such related species becomes difficult. Thongkao et al. developed triplex PCR which proved to be an accurate method, which was also tested on three Vibrio species. The detection limits of V. harveyi, V. parahaemolyticus, and V. vulnificus in pure cultures and spiked shrimp samples were ranged from 1.05 to 4.8 × 103 CFU/mL and 1.9 to 7 × 104 CFU/g, respectively. This multiplex PCR assay was rapid due to the reduction of time and labor spending for sample processes with high accuracy. This method may suitable for food‐borne pathogen detection in shrimp and also for horizontal gene transfer study among Vibrios in aquatic animals.[14],[15]

Diarrhea is a serious public health issue in developing countries. The prevention requires strict hygiene related to drinking water and food in these countries. Vibrio infections are caused by consumption of raw or partially cooked seafood contaminated especially with virulent strains. All Vibrios are gram‐negative and halophilic bacteria, which inhabit marine and estuarine environments and appear at water temperature above 15°C. Additionally, this bacterium is one of the most important food‐borne pathogens in tropical and subtropical areas.[1],[2],[4],[5],[6],[12] In Thailand, seafood and related products are transport from nearby towns, such as Samut Sakhon Samut Songkhram, Rayong, and Chon Bui or Southern of Thailand to Bangkok, which are the centers of seafood product markets and also the contaminations of food‐borne pathogens are relatively often. Thus, a rapid and accurate method, triplex PCR, described from previous study [14],[15] may be appropriate to detect Vibrios in seafood samples for public prevention. This study was conducted to simultaneously detect V. harveyi, V. parahaemolyticus, and V. vulnificus in shellfish samples including life mussel (Perna viridis), blood clam (Tegillarca granosa), and baby clam (Paphia undulata) from local markets at high residential area in Bangkok. The sensitivity, accuracy, and specificity of the triplex PCR method were also evaluated and compared with the conventional method.


   Materials and Methods Top


Sample collection, sample preparation, and bacterial isolation

The 150 of chilled samples were included life mussel (Perna viridis), blood clam (Tegillarca granosa), and baby clam (Paphia undulata), which were purchased from five local markets in Bangkok, Thailand. One gram of flesh from each sample was dissected out, minced, and homogenized with a glass homogenizer in 9 mL of 2% NaCl Alkaline peptone water and then incubated at 37°C. One milliliter of homogenized samples were enriched on 0, 3, and 6 h and diluted by 10-fold serial dilution (1:100, 1:1000 and 1:10,000) in phosphate buffer solution (PBS). Each dilution (100 µL) was spread on thiosulfate-citrate-bile salts-sucrose (TCBS) agar plate and then incubated at 37°C overnight for colony count The bacterial colonies were counted at the dilution yielding 30–300 colony forming units (CFUs), and CFU mL‐1 of bacterial suspension was calculated. The colonies with different morphologies from each homogenate sample were collected with sterile tooth pick, dissolved in phosphate buffer saline, and immediately extracted DNA (NaOH/Tris‐HCl) before storage at −20°C prior to identify. The three Vibrio species were identified according to biochemical tests, and then later confirmed by triplex PCR.

Conventional bacterial identification

The screenings of different colonies of each homogenate sample were characterized according by morphology of Vibrio sp. on TCBS agar and conventional biochemical tests. The bacteria isolates were confirmed by conventional biochemical tests as described by Barrow and Feltham and Kaysner et al.[16],[17]. The set biochemical tests including oxidase, TSI, lysine decarboxylase, ornithine decarboxylase, arginine decarboxylase, indole, MR‐VP, and salt tolerance tests were carried on the classification isolates to identify different species.

DNA extraction and triplex PCR amplification

For DNA preparation from homogenate samples, homogenized samples (1 mL) were enriched on 0, 3, and 6 h and diluted by ten-fold serial dilution (1:100, 1:1000 and 1:1,000) in PBS. Each prepared samples was transferred to microcentrifuge tube (size = 1.5 mL) and centrifuged at 200 g for 5 min for remove cell debris. The supernatant was transferred to new centrifuge tubes and centrifuged at 18,000 g for 5 min. After removal of the supernatant, the pellet was re-suspended in 100 µL of 25 mmol NaOH, and then the mixture was heated at 95°C for 5 min. After neutralization with 8 µL of 1 mmol L−1 Tris-HCl buffer (pH 7.5), the suspension was centrifuged at 18,000 g for 5 min. 2 µL of each supernatant was then used as a template DNA for the triplex PCR method.

Primers of triplex PCR method were specific to vhhP2 gene of V. harveyi, tlh gene of V. parahaemolyticus, and rpoS gene of V. vulnificus [Table 1]. The three Vibrio species identified by biochemical tests were later confirmed by triplex PCR. The total volume of reaction mixtures contained 5 µL of 10x PCR buffer, 5 µL of 50 mM MgCl2, 4 µL of 2.5 mM deoxyribonucleotide phosphate, 5 µL of 500 m MKCl, 1 µL of each primer, 0.25 µL of 0.5 units Taq DNA polymerase, and 1 µL of template DNA and deionized water added up to 50 µL. The cycling conditions were as follows: predenaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 59°C for 30 s, and extension at 72°C for 30 s, with a final extension at 72°C for 10 min at the end of 35 cycles.[14],[15]
Table 1: The oligonucleotide primers, target genes, Tm values, and amplicon sizes of triplex PCR for in this study

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Sensitivity, accuracy, and specificity evaluation of triplex PCR

The sensitivity of triplex PCR was explained as total count (CFU/mL) of three Vibrio species in marine shellfish at 0, 3, and 6 h. The accuracy and specificity of triplex PCR were evaluated and compared with conventional biochemical tests, and formulae were used to calculate as described by Paydar et al.[18]


   Results Top


The biochemical identification of Vibrio species is shown in [Table 2]. The positive detections for one, two and three Vibrio species were represented as one, two, and three bands [Figure 1], which were corresponded to V. harveyi (one specie), V. harveyi and V. vulnificus (two species), and V. harveyi, V. vulnificus and V. parahaemolyticus (three species), respectively. In total, 150 samples for the detection of the three Vibrio species in marine shellfishes including blood clam, baby clam, and green mussel from local market were used for the biochemical test and 14 isolates of V. harveyi, 8 isolates of V. parahaemolyticus, and an undetectable number of V. vulnificus were identified, respectively. However, according to triplex PCR, there were 15 isolates of V. harveyi, 8 isolates of V. parahaemolyticus, and 1 isolate of V. vulnificus detected. The comparison of positive results between conventional and triplex PCR methods and the calculation of accuracy and specificity are represented in [Table 3]. The accuracy and specificity of biochemical test and triplex PCR were 91.67% and 100% and 96% and 100%, respectively. The sensitivity of triplex PCR method was described as positive in the range of total Vibrio count in shellfishes at 0, 3, and 6 h [Table 4]: In blood clam the range was 7.2 × 102–1.2 × 103, 1 × 103–3.42 × 106, and 3.76 × 103–6.7 × 106 CFU/mL, respectively; in baby clam the range was 1.2 × 103–8.8 × 103, 8.42 × 104–1.47 × 107, and 7.86 × 106– 1.51 × 107CFU/mL, respectively, and in life mussel the range was 4.0 × 102–3.2 × 103, 1.54 × 105–5.05 × 107, and 7.2 × 102–1.1 × 103 CFU/mL, respectively. The range of total bacterial count for three Vibrio detections in marine shellfishes was 1.2 × 107–2 × 106 CFU/mL.
Table 2: The biochemical characteristics for Vibrio specie classification by conventional biochemical tests

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Figure 1: Identification and detection of three Vibrio species by Triplex PCR at 0 hr (10 CFU/ml‐1), 3 hr (10-103 CFU/ml‐1) and 6 hr (10-103 CFU/ml‐1); (a) baby clam, (b) green mussel, (c) blood clam

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Table 3: The numbers of Vibrio isolation and identification with accuracy and specificity of conventional and triplex PCR methods

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Table 4: The ranges of total colony count of Vibrio isolates in marine shellfishes at 0, 3, and 6 h

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   Discussion Top


This Triplex PCR assay was rapid method due to the reduction of time and labor spending for sample processes with high accuracy. In addition, reagent spending for triplex PCR was also cost effective. This method can be applied in epidemiological studies especially food‐borne pathogen detection compared with conventional methods, because it can be used on many samples and provides report in short time.[14] Triplex PCR was used at 0 h after DNA was extracted and had higher sensitivity than the conventional biochemical test. This method might be affected by DNA from dead cells or viable but nonculturable cells (VBNC), which state that bacteria cells do not form colonies on high-nutrient solid media but are considered alive because metabolic activity can still be detected.[19],[20] Moreover, triplex PCR can be used to detect the contamination of Vibrio species in seafood samples that the detection limit could be increased effective of primer by after-pre- enrichment of the bacteria in alkaline peptone water (APW) at 3 or 6 h. However, total counts of Vibrio species incubated at 6 h were lower rather than 3 h incubation in some marine samples. The low amount of Vibrio species at low concentration may be because the growth of Vibrio species is inhibited by other non-Vibrio species. In addition, in the conventional method, the amount of reagent might be higher than triplex PCR, due to which the method has lower cost effectiveness, lower sensitivity, and accuracy, and the interpretation is difficult in case of similarity in morphology colony characteristics in biochemical tests.

The supply of seafood from natural resources is limited, culture-based sources make up for its shortage in many countries. Large quantities of seafood are produced by the culture method not only for domestic consumption, but also for export. To ensure its safety and quality, it is necessary to determine the bacterial contamination in seafood, which are produced and sold. We report 15, 8, and 1 isolates of V. harveyi, V. parahaemolyticus, and V. vulnificus, respectively, from three favored shellfishes consumed by using the PCR‐based technique. Compared with conventional methods, triplex PCR showed higher accuracy and specificity (100%), especially in V. parahaemolyticus and V. vulnificus, which were common pathogens in seafood. V. harveyi was non-pathogenic bacteria for human, however, there are markers of poor hygienic condition of marine farm where marine animal living, thus, other pathogenic Vibrio can be also occurred. V. harveyi is also the one of the autochthonous marine microbial communities, and in specific ecological niches such as fish farms where antibiotics are frequently used, they might become a reservoir of antibiotic‐resistant bacteria forms and virulence genes or their homolog; it could also be present in strains from environmental sources, and the acquisition of such genes might take place in the aquatic environment.[11]

Our results reported two pathogenic Vibrio isolations (V. parahaemolyticus and V. vulnificus) from shellfishes, and distribution of them in shellfishes was corresponded to previous studies.[5],[6],[21],[22] Raw or uncooked shellfishes may be risk by increasing number of Vibrio species and in Thai food culture, shellfish type was cook levels, which related to favor of food recipes or cooking, such as, oyster is almost uncooked, bloody calm is inappropriately cooked by short parboiling, whereas mussel and baby clam are fully cooked by stir-fry. Blood clam was often prepared by scalding for short‐time before eating, whereas life mussel and baby clam were cooked by many methods or recipes, such as stir‐fry, steaming, and boiling, which were safer than blood clam cooking, and in high temperature and the cooking time was long. Thus, awareness of increasing scalding time or changing the cooking style of blood clam should be spread, as it may reduce the amount of bacteria, which can prevent seafood‐borne infection or diarrhea.

This presented triplex PCR assay was high in sensitivity, accuracy, and specificity. This method can be applied in epidemiological studies especially food-borne pathogen detection, compared with conventional methods, because it is convenient, simple, and effective method for the detection and identification of bacterial contamination in shellfishes. Vibrio contamination in Thai shellfishes was reported in this study, and awareness of shellfish cooking may prevent Vibrio infection or diarrhea.

Acknowledgment

The authors express their sincere appreciation to Research and Development Institute, Suan Sunandha Rajabhat University, Bangkok, Thailand for the grant support of this work. We would like to sincerely thank to the Research Division, Faculty of Science and Technology, Suan Sunandha Rajabhat University for partial research facility support.

Financial support and sponsorship

Nil

Conflicts of interest

There are no conflicts of interest.[24]

 
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Correspondence Address:
Kanittada Thongkao
Faculty of Science and Technology, Suan Sunandha Rajabhat University 1 U-Thong-Nok Rd., Dusit, Bangkok
Thailand
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ATMPH.ATMPH_770_16

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