Advertisement

BMC Microbiology

, 18:65 | Cite as

Influence of food matrix type on extracellular products of Vibrio parahaemolyticus

  • Rundong Wang
  • Lijun Sun
  • Yaling Wang
  • Yijia Deng
  • Zhijia Fang
  • Ying Liu
  • Qi Deng
  • Dongfang Sun
  • Ravi Gooneratne
Open Access
Research article
Part of the following topical collections:
  1. Applied microbiology

Abstract

Background

Two strains of Vibrio parahaemolyticus (ATCC 17802 and 33847) in shrimp, oyster, freshwater fish, pork, chicken and egg fried rice were evaluated for production of hemolysin and exoenzymes of potential importance to the pathogenicity of this bacterium.

Results

The two strains of V. parahaemolyticus produced hemolysin, gelatinase, caseinase, phospholipase, urease, DNase and amylase in selected food matrices. Significantly higher (p < 0.05) hemolytic activity was produced by V. parahaemolyticus in egg fried rice > shrimp > freshwater fish > chicken > oyster > pork. But the exoenzyme activities were not consistent with the hemolytic activity profile, being significantly higher (p < 0.05) in shrimp > freshwater fish > chicken > oyster > pork > egg fried rice. Filtrates of V. parahaemolyticus from shrimp, freshwater fish and chicken given intraperitoneally to adult mice induced marked liver and kidney damage and were highly lethal compared with the filtrates of V. parahaemolyticus from oyster > egg fried rice > pork.

Conclusion

From in vitro and in vivo tests, it appears that the food matrix type has a significant impact on the activity of extracellular products and the pathogenicity of V. parahaemolyticus. From a food safety aspect, it is important to determine which food matrices can stimulate V. parahaemolyticus to produce additional extracellular factors. This is the first report of non-seafood including freshwater fish and chicken contaminated with V. parahaemolyticus to have been shown to be toxic to mice in vivo.

Keywords

Vibrio parahaemolyticus Food matrices Extracellular products Pathogenicity 

Background

Vibrio parahaemolyticus is a gram-negative, facultative, anaerobic, halophilic bacterium that inhabits marine or estuarine environments [1, 2]. The natural host for this bacterium is variable because it lives in water and is concentrated in shellfish which can serve as reservoirs [3, 4, 5]. V. parahaemolyticus can contaminate raw or undercooked shrimp, fish, oyster and cause abdominal pain, acute gastroenteritis, diarrhea, and infection by the O3: K6 pandemic strain resulted in a massive number of human deaths [6, 7, 8] in several countries including China, Japan and the United States [9, 10, 11].

Following contamination of food with V. parahaemolyticus, both the bacterial cells and extracellular products contribute to the pathogenicity and among them, the extracellular products play a dominant role [12, 13, 14]. Of all extracellular products, hemolysin (thermostable direct hemolysin, thermostable-related hemolysin) is regarded as the most important virulence factor, and controls a variety of biological activities including hemolytic activity, cytotoxicity, and enterotoxicity [15, 16, 17], besides other factors such as exoenzymes [18, 19]. Among these, gelatinase and caseinase belong to a family of proteolytic enzymes that can cause tissue damage and hydrolyze various protein substrates including hemoglobin and other small amounts of biologically active peptides [20, 21]. Phospholipases involved in nutrient acquisition through the degradation of membrane lipids may also cause harm to the host [22]. DNase can act as endonuclease and contribute to DNA hydrolysis, amylase can hydrolyze carbohydrate to provide energy for the growth of V. parahaemolyticus [23] and urease may act as hemolysin [24].

Seafood has long been considered to be the only carrier of V. parahaemolyticus. Therefore, from a food safety aspect, more attention has been paid to seafood products. However, there is new evidence that V. parahaemolyticus can also contaminate non-seafood matrices (a prevalence of ~ 32.5%) such as poultry, pork, freshwater fish, eggs and their products including egg fried rice, by cross contamination of seafood to non-seafood and via cooking utensils [25, 26, 27], which suggest that V. parahaemolyticus can also cause food infection via many non-seafood types. Our previous studies [28] found that the virulence factors of V. parahaemolyticus can trigger high or low pathogenicity in different foods. But, little is known of the composition of extracellular products in different food matrices.

To better assess the risk of V. parahaemolyticus in different food matrices, a clearunderstanding of the extracellular products is essential. In this study, we examined the importance of extracellular products, hemolysins, gelatinase, caseinase, phospholipase, urease, DNase and amylase to the pathogenicity of V. parahaemolyticus in selected seafood and non-seafood products and tested their combined pathogenicity in a mouse model.

Methods

Bacterial strains and growth conditions

V. parahaemolyticus strains ATCC 17802 and ATCC 33847 were stored in 25% glycerol at − 20 °C. Each strain was grown in brain heart infusion (BHI) broth (BLBT, Beijing, China) containing 3% NaCl, at 37 °C for 24 h. The inoculum was thrice passaged in BHI-3% NaCl. The final concentration of inoculants were adjusted to ~ 104 CFU/ml and used to inoculate the food matrices.

Food matrices preparation and inoculation

Shrimp (Litopenaeus vannamei), oyster (Crassostrea), freshwater fish (Tilapia), pork and chicken were purchased from a local supermarket in Zhanjiang, China, and the meat was used in the study. Egg fried rice (rice:egg = 1:1) was cooked at 80 °C for 20 min, in the laboratory.

Test portions, 100 g each (n = 3) of shrimp meat, oyster, freshwater fish meat, pork, chicken, and egg fried rice, added salt at 3% in sterile Erlenmeyer flasks were sterilized by autoclave (YXQ-L-50A, Shanghai Boxun, Shanghai, China) at 100 °C for 20 min to kill native bacteria. Then each sample was inoculated with either 1 mL of the final V. parahaemolyticus ATCC 17802 or ATCC 33847 (described above, cell number ~ 103 CFU/g). Inoculated samples were mixed thoroughly in a vortex mixer (XW-80A, Qilinbei, Haimen, China) for 10 min and incubated at 37 °C until the bacterial counts were approximately 109 CFU/g.

After incubation, the inoculated food samples were separately washed with 100 mL 0.01 M phosphate-buffered saline (PBS, pH 7.2), and the solution centrifuged (Thermo Lynx 6000, Thermo Scientific, Waltham, MA) at 12000 rpm for 20 min at 4 °C. The supernatants were filtered (0.22 μm, Millipore, Billerica, MA) and stored at − 20 °C until use. The control food matrix samples were subjected to the same procedure except that these samples were not inoculated with V. parahaemolyticus.

Hemolytic activity

The relative hemolytic activity test measured the total hemolysins in the samples and were detected as described by Takamatsu et al. [29] modified by Jiang et al. [30]. Rabbit hemocytes were obtained by centrifugation of blood (3500 rpm, 4 °C, 5 min) three times (washed with PBS each time) and diluted to 5% with PBS. Subsequently, a sample (400 μL) of each food matrix was mixed with 100 μL of 5% rabbit red blood cells in 1.5-mL sterile tubes and incubated at 37 °C for 1.5 h. Unlysed erythrocytes were allowed to pelletize overnight at 4 °C, then 200-μL portions of the supernatant were transferred to 96-well flat-bottomed microplates (Nunc, Thermo Scientific, Waltham, MA) and the absorbance measured at 570 nm with a microplate reader (Varioskan Flash, Thermo Scientific, Waltham, MA). For controls, the same procedure was employed except the samples were changed to food matrix filtrates without the V. parahaemolyticus inocula. The results are reported as: Arelative hemolytic activity = Asample − Acontrol.

Production of extracellular enzymes

In separate plates, 0.5% (w/v) gelatin [31], 0.2% (w/v) casein [32], 3% (v/v) egg yolk emulsion, 2.5% (w/v) urea [31], 0.01% (w/v) toluidine blue or 0.2% (w/v) soluble starch [14] were added to tryptone soya agar (TSA) to determine gelatinase, caseinase, phospholipase, urease, DNase and amylase enzyme activities.

The exoenzyme activities of sample and control filtrates were determined by the Oxford Cup Method [33]. Briefly, 180 μL of the filtrate in triplicate were added into the Oxford cups in TSA plates containing different substrates. All the plates were incubated at 37 °C for 12 h. The positive reaction of a clear halo was detected with gelatinase and caseinase following addition of 70% trichloroacetic acid. The positive reaction to phospholipase, urease, and DNase were characterized by the presence of opaque halo, yellow halo, and pink halo respectively. To detect amylase, 5 mmol/L KI-I2 solution was added to the TSA plates after the 12-h incubation and a clear halo indicated a positive reaction. All positive zones around the cup were measured.

Mice pathogenicity test

Lethality study

One hundred and eight female KM mice (20 ± 2 g, 6 weeks old) were obtained from Animal Center of Guang Dong Province. During the experimental period, mice were reared under standard laboratory conditions (12 h light-dark cycle, temperature of 20 ± 1 °C, humidity 60 ± 5%) in 18 stainless steel cages with free access to distilled water and sterilized food. The mice were acclimatized to this environment for 5 days randomly assigned to 18 groups (n = 6). Twelve experimental groups were injected with millipore-filtered food matrix filtrates contaminated with either V. parahaemolyticus ATCC 17802 or ATCC 33847 strain, and six control groups with control food matrix filtrates, intraperitoneally (i.p.) at 0.2 mL / 10 g body weight (bw). The mortality rate of mice was recorded for 48 h.

Biochemical indices

For biochemical studies, another 108, 6-week-old female KM mice (obtained from Animal Center of Guang Dong Province) were injected i.p. with V. parahaemolyticus food matrix filtrates as per the above described protocol. Mice were euthanized by exsanguination while under ether vapor narcosis (in a funnel) at 12 h. Blood was sampled by percutaneous cardiac puncture and centrifuged at 3500 rpm for 10 min to obtain serum for detection of three liver-specific enzymes (aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP)) and the kidney-specific enzyme blood urea nitrogen (BUN), using detection kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), to assess tissue damage.

Ethics approval and consent to participate

All mouse experiments were conducted according to the guidelines provided by the.

Animal Care and Welfare Committee of Guangdong Ocean University (License Number: SYXK 2014–0053).

Statistical analysis

All data were analyzed using the software SPSS 19.0 (SPSS Inc., Chicago, IL, USA). Differences between the means were tested by one-way ANOVA, with the level of significance set at p < 0.05.

Results

Hemolytic activity

The relative absorbance of different food matrices filtrates reflected the hemolytic activity of V. parahaemolyticus in food samples. The V. parahaemolyticus ATCC 33847 showed a higher hemolytic activity than ATCC 17802 in all selected food samples. Irrespective of the V. parahaemolyticus strain, the hemolytic activity was significantly higher in egg fried rice > shrimp > freshwater fish > chicken > oyster > pork (Fig. 1).
Fig. 1

Hemolytic activity of V. parahaemolyticus in different food matrices. Error bars represent standard deviations of mean values from triplicate experiments (Control groups excluded). Means with different lowercase letters were significantly different (p < 0.05) among different food matrices

Production of extracellular enzymes

ATCC 17802 and ATCC 33847 strains were tested for six exoenzymes previously reported to be responsible for V. parahaemolyticus virulence. The two pathogenic strains of V. parahaemolyticus produced a wide variety of extracellular enzymes including gelatinase, caseinase, phospholipase, urease, DNase and amylase in the selected food matrices (Table 1). Extracellular factor activity was generally higher with the ATCC 33847 strain than with ATCC 17802. Overall, both V. parahaemolyticus strains produced significantly high activities (p < 0.05) of gelatinase, caseinase, phospholipase, urease, DNase and amylase in shrimp > freshwater fish > chicken > oyster > pork > egg fried rice.
Table 1

Extracellular enzyme composition and activity of Vibrio parahaemolyticus in different food matricesf

Measurement index

Enzyme

Strains

Food matrix

Shrimp

Oyster

Freshwater fish

Pork

Chicken

Egg fried rice

Positive circle diameter (mm)

Gelatinase

ATCC 17802

21.98 ± 2.10a

15.92 ± 1.39d

20.72 ± 1.98b

15.00 ± 1.67e

17.52 ± 1.44c

16.56 ± 1.12c

ATCC 33847

29.42 ± 1.71a

17.20 ± 1.39c

21.12 ± 1.45b

14.88 ± 1.56e

18.29 ± 0.99c

16.16 ± 0.91d

Caseinase

ATCC 17802

25.62 ± 2.25a

18.16 ± 1.41c

20.32 ± 1.32b

17.10 ± 1.08d

24.40 ± 3.06a

13.94 ± 2.64e

ATCC 33847

26.20 ± 1.19a

18.08 ± 0.99c

22.80 ± 0.92b

12.60 ± 1.52e

19.40 ± 1.38c

14.40 ± 2.58d

Phospholipase

ATCC 17802

14.90 ± 1.24b

9.20 ± 1.31e

12.50 ± 0.79c

14.04 ± 1.10b

14.12 ± 0.27b

16.80 ± 1.04a

ATCC 33847

16.78 ± 0.49b

10.00 ± 1.39d

17.00 ± 0.52b

13.80 ± 1.53c

13.30 ± 1.05c

18.20 ± 0.89a

Urease

ATCC 17802

21.22 ± 1.23a

16.02 ± 1.98c

18.46 ± 0.58b

15.33 ± 0.74d

16.92 ± 0.32c

11.58 ± 1.02e

ATCC 33847

24.30 ± 0.69a

15.88 ± 1.34c

19.60 ± 1.08b

15.10 ± 1.19d

17.10 ± 0.52c

10.94 ± 1.25e

DNase

ATCC 17802

26.28 ± 1.76a

12.30 ± 0.96d

19.76 ± 1.03b

13.54 ± 2.10d

16.40 ± 0.99c

12.42 ± 1.45d

ATCC 33847

27.90 ± 0.81a

13.90 ± 0.68d

18.82 ± 1.20b

12.42 ± 1.16e

17.04 ± 0.36c

14.00 ± 1.05d

Amylase

ATCC 17802

18.34 ± 1.11a

16.64 ± 1.21c

17.08 ± 0.29b

15.40 ± 1.08c

17.52 ± 0.40b

15.30 ± 1.64c

ATCC 33847

20.80 ± 1.62a

17.12 ± 0.53d

17.99 ± 0.65c

13.70 ± 1.58e

18.98 ± 0.25b

18.20 ± 0.78c

Note: f Mean ± standard deviation of three replicates. Means in the same line with different superscript letters are significantly different (p < 0.05). Results were negative for the filtrates of all food matrices not inoculated with V. parahaemolyticus

Mice pathogenicity tests

Lethality

The mortality of mice injected with the food matrix filtrates was higher with the shrimp matrix than other food matrices (Table 2) probably because of the higher extracellular enzyme activity). The mortality rate was highest in shrimp > freshwater fish > chicken > oyster > egg fried rice > pork. Strain ATCC 33847 appeared more virulent in that it caused more deaths. There were no deaths in the control groups.
Table 2

Mortality in mice injected intraperitoneally with different food matrix filtrates (n = 6)

Measurement index

Strains

Food matrix filtrates

Shrimp

Oyster

Freshwater fish

Pork

Chicken

Egg-fried rice

Controla

Death rate

ATCC17802

2/6

1/6

2/6

0/6

1/6

0/6

0/36

ATCC33847

3/6

1/6

2/6

0/6

2/6

1/6

Note: Each mouse was injected intraperitoneally with 0.2 mL / 10 g bw of food matrix filtrate and the death rate recorded at 48 h. a Control mice were injected with filtrates of shrimp, oyster, freshwater fish, pork, chicken and egg fried rice that were not inoculated with V. parahaemolyticus

Liver and kidney damage in mice

The serum biochemical parameters indicative of liver and kidney function measured at 12 h in mice injected with different food matrix filtrates are shown in Fig. 2. In pork samples, no significant changes (p > 0.05) were detected in most of the parameters compared with the controls. AST and ALT activity indicative of liver damage were significantly higher (p < 0.05) in mice given shrimp, freshwater fish, chicken and egg fried rice filtrates compared with the respective controls. ALP was significantly higher (p < 0.05) only in mice given shrimp. BUN activity indicative of kidney damage was significantly elevated (p < 0.05) in all test mice injected with oyster > freshwater fish > shrimp > chicken > pork > egg fried rice, compared with the respective controls. The food matrix filtrates of ATCC 33847 affected liver and kidney function more than ATCC 17802.
Fig. 2

Liver and kidney function indices detection. The mice were sacrificed after giving different food matrix filtrates intraperitoneally for 12 h. The test groups given filtrates of V. parahaemolyticus ATCC 17802 (or ATCC33847) samples, control groups given filtrates of foods that were not inoculated with V. parahaemolyticus. Means with asterisks (*) are significantly different (p < 0.05) from the respective controls.

Discussion

A correlation between virulence and the production of extracellular products by food contaminant bacteria [34, 35, 36] including by V. alginolyticus [37] and V. vulnificus [38] but little is known about the specific extracellular products of V. parahaemolyticus and its pathogenicity in different food matrices. To our knowledge, this study is the first to examine the extracellular products – hemolysin and six exo-enzymatic activities in two pathogenic V. parahaemolyticus strains in selected seafood and non-seafood and assess relative risk.

Hemolysin is an important virulence factor responsible for the pathogenicity of V. parahaemolyticus because it can lyse cells, especially red blood cells, and cause systemic infections [39]. In the hemolytic activity test, the two pathogenic V. parahaemolyticus strains produced hemolysin not only in seafood but also in non-seafood. The significantly higher (p < 0.05) hemolytic activity observed in egg fried rice than in shrimp >freshwater fish > chicken> oyster > pork (Fig. 1). We hypothesized that the nutrition factors in egg fried rice can also promote V. parahaemolyticus to produce more hemolysin, which is in agreement with Taniguchi et al. [40] and Shinoda et al. [41] who identified a lecithin-dependent hemolysin that can also cause hemolysis. So, we believe that the high lecithin concentration in eggs may induce V. parahaemolyticus to produce more hemolysin in egg fried rice. This is the first evidence of V. parahaemolyticus producing more hemolysin in lecithin-enriched food, which means that some non-seafood may in fact be equally pathogenic than the traditionally affected seafood and therefore worthy of more attention. V. parahaemolyticus, like many other bacteria, require a source of iron and its hemolytic activity and virulence are greatly enhanced on exposure to elevated iron concentrations [42, 43]. Hence, we believe that it is also important to pay more attention to monitoring of foods with a higher iron content. Although the mortality rates of mice injected with different food matrix filtrates (containing V. parahaemolyticus extracellular products) were highest in shrimp > freshwater fish > chicken > oyster > egg fried rice > pork, it was not possible to prove this statistically because of the limited number of mice used in the study.

Vibrio strains are known to produce a series of exoenzymes that contribute to expression of pathogenicity. In this study, no differences were observed in the composition of exoenzymes between the two pathogenic V. parahaemolyticus strains in different food matrices, which means that there is a high food safety risk no matter what food matrix type is contaminated by V. parahaemolyticus [44].

Results from our study suggest that V. parahaemolyticus produce significantly higher activity (p < 0.05) of gelatinase, caseinase, urease, DNase and amylaes in shrimp matrix than freshwater fish (Table 2) and are in agreement with the results of Liu et al. [31] and Zhang and Austin [32] who reported that higher phospholipase, gelatinase and caseinase activities were detected in Vibrio species isolated from marine shrimp, fish, and shark. The virulence of pathogenic Vibrios is related to their ability to produce exoenzymes [45]. As shown in Table 1, the exoenzyme activities in chicken were greater than in the oyster matrix, which suggested that V. parahaemolyticus produced more exoenzymes in the chicken and hence that some non-seafoods also pose a high risk to humans. The lower exoenzyme activities observed in pork and egg fried rice (Table 1) are interesting because Iuchi and Tanaka [46] showed that production of exoenzymes in V. parahaemolyticus was repressed by various carbohydrates present in the medium. We believe that the high concentration of carbohydrates in egg fried rice may have suppressed V. parahaemolyticus’s ability to secrete exoenzymes. Analyzing the activities of different exoenzymes in different food matrices provides a way to comprehensively study the pathogenic mechanism of V. parahaemolyticus. However, further studies are required to determine which factor(s) have the most influence on the production of exoenzymes in pork and chicken.

Although cytotoxicity assays [15, 47] are often used to study the pathogenicity of vibrio extracellular products, in vitro tests do not adequately represent the true toxicity in vivo [48]. In our study, the mouse model was used to determine the toxicity of V. parahaemolyticus extracellular products. It was observed that shrimp filtrate was highly lethal to adult mice (Table 2) and caused more damage to liver and kidney (Fig. 2) than other food matrix filtrates, followed by freshwater fish and chicken filtrates. It was interesting to observe that egg fried rice, which showed the highest hemolytic activity, did not cause significant pathogenicity to mice. This is in contrast to the traditional view that hemolysin is the major virulence factors of V. parahaemolyticus and that the high hemolytic activity is responsible for most of the tissue damage [49]. We believe that the pathogenicity of V. parahaemolyticus extracellular products is dependent not only on hemolysin, but also on the mixture of other secreted enzymes. Xu et al. [13] and Bhattacharjee et al. [50] demonstrated that pathogenic V. parahaemolyticus, although lacking hemolysin, can still cause cytotoxicity and death in mice. Other studies have also suggested that hemolysin is not necessarily the only virulence factor of pathogenic V. parahaemolyticus [39, 51]. Liver and kidney damage, as shown by elevated clinical chemistry indices such as ALT, ALP, AST and BUN activity (Fig. 2), were observed in mice given shrimp filtrate and to a lesser extent in mice given pork or egg-fried-rice filtrates. According to Maeda and Yamamoto [52], the high levels of exoenzyme activity alone could cause extensive damage to host tissue. In addition, damage to spleen and stomach were observed in mice given shrimp filtrate (unpublished observation). Our mouse results are in agreement with the findings of Moreno and Landgraf [38] and provide further proof that exoenzymes play a vital role in the pathogenicity of V. parahaemolyticus. Hence it is important to consider the extracellular enzymes activities also in risk assessment. Besides, the type III secretion (T3SS) system of V. parahaemolyticus also play a role in lethality in the murine infection model [14] although the mechanism of action of the T3SS system that influences the virulence is not well understood.

Baffone and others [34, 53] demonstrated that most of the extracellular products identified in V. alginolyticus and V. vulnificus are not directly associated with pathogenicity but require the bacterial cells also to be present to cause pathogenicity, unlike V. parahaemolyticus where the extracellular products alone can be pathogenic. In our studies also, the extracellular products of V. parahaemolyticus alone were pathogenic to mice. It is suggested that the pathogenesis mechanism of V. parahaemolyticus is different to other Vibrio types. Besides the invasion damage caused by the bacteria, the virulence factors of V. parahaemolyticus are highly toxic to tissues. If the food matrices are contaminated by V. parahaemolyticus, transient heating could remove most of the bacteria, but some thermo-tolerant products including thermostable direct hemolysin, thermostable related hemolysin, and other thermo-tolerant enzymes that can survive at 85 °C for 10 min [54] would possess biological activity to induce tissue damage. Hence, food should be heated to at least 85 °C for 10 min to destroy the activity of pathogenic thermo-tolerant products of V. parahaemolyticus. Besides, the food producers could incorporate probiotics (eg: Lactobacillus pentosus, Streptomyces) [55, 56] to inhibit the growth of V. parahaemolyticus and reduce the production of pathogenic extracellular products. If humans are infected with V. parahaemolyticus, bacteriophage therapy [57, 58] could be used to control and inhibit the virulence of Vibrio species. Such methods can be regarded as better strategies in view of the ever-increasing anti-microbial resistance in both humans and animals.

Conclusions

The present study suggests that the food matrix type has a marked effect on the pathogenicity of extracellular products of V. parahaemolyticus. Higher hemolytic activity observed in egg fried rice is an important new finding from a food safety aspect. Significantly higher activity of exoenzymes observed in shrimp and freshwater fish was strongly linked to high pathogenicity. This is the first report to show that besides the extracellular products in shrimp produced by V. parahaemolyticus, some non-seafood such as chicken infected with V. parahaemolyticus may also be toxic to mice in vivo. Although, for non-seafood matrices such as chicken it is unlikely that high levels of V. parahaemolyticus could be reached by cross-contamination from seafood matrices or via cooking utensils, the high pathogenicity still exists and need to be paid attention. It appears that exoenzymes, in addition to hemolysin, are involved in the pathogenesis of V. parahaemolyticus in food matrices.

Notes

Acknowledgements

The authors wish to thanks Prof. Dr. Lijun Sun, College of Food Science and Technology, Guangdong Ocean University for his contribution on the design and supervised the entire study.

Funding

The design of the study, experimentation, and interpretation of the data was funded by the National Science Fund (NO. 31371746), and the higher educational program for cultivating major scientific research projects of Guangdong Ocean University (Nos GDOU 2013050205, 2014050203) and the scientific research program of administration of quality and technology supervision of guangdong province (NO. 2015ZZ02) for their financial support.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors’ contributions

RW participated in the project conception, carried out all the experimental work, analyzed and interpreted the data and wrote the manuscript. LS and YW were corresponding author, designed and supervised the entire project. YD, ZF, YL, QD, DS and RG contributed to the design and interpretation of experimental results, as well as editing and revising the manuscript. All authors have read and approved the final manuscript.

Ethics approval and consent to participate

The animal work presented in this study was approved by the Animal Care and Welfare Committee of Guangdong Ocean University (SYXK 2014–0053).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. 1.
    Hlady WG, Klontz KC. The epidemiology of Vibrio infections in Florida, 1981– 1993. J Infect Dis. 1996;173:1176–83.CrossRefPubMedGoogle Scholar
  2. 2.
    Daniels NA, MacKinnon L, Bishop R, Altekruse S, Ray B, Hammond RM, Thompson S, Wilson S, Bean NH, Griffin PM, Slutsker L. Vibrio parahaemolyticus infections in the United States, 1973–1998. J Infect Dis. 2000;81:1661–6.CrossRefGoogle Scholar
  3. 3.
    Su YC, Liu C. Vibrio parahaemolyticus: a concern of seafood safety. Food Microbiol. 2007;24:549–58.CrossRefPubMedGoogle Scholar
  4. 4.
    Abd-Elghany SM, Sallam KI. Occurrence and molecular identification of Vibrio parahaemolyticus in retail shellfish in Mansoura. Egypt Food Control. 2013;33:399–405.CrossRefGoogle Scholar
  5. 5.
    Letchumanan V, Pusparajah P, Tan L T, Yin W F, Lee L H, Chan K G. Occurrence and antibiotic resistance of Vibrio parahaemolyticus from shellfish in Selangor, Malaysia. Front Microbiol 2105; 6:1417.Google Scholar
  6. 6.
    McLaughlin JB, DePaola A, Bopp CA, MartinekK A, Napolilli NP, Allison CG, Murray SL, Thompson EC, Bird MM, Middaugh JP. Outbreak of Vibrio parahaemolyticus gastroenteritis associated with Alaskan oysters. New Engl J Med. 2005;353:1463–70.CrossRefPubMedGoogle Scholar
  7. 7.
    Letchumanan V, Chan K, Lee L. Vibrio parahaemolyticus: a review on the pathogenesis, prevalence and advance molecular identification techniques. Front Microbiol. 2014;5:705.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Albuquerque CR, Araújo RL, Souza OV, Vieira RH. Antibiotic-resistant Vibrios in farmed shrimp. Biomed Res Int. 2015;2015:505914.Google Scholar
  9. 9.
    Romilio TE, Katherine G, Nicolas P. Insight into the origin and evolution of the Vibrio parahaemolyticus pandemic strain. Front Microbiol. 2017;8:1397.CrossRefGoogle Scholar
  10. 10.
    Shinoda S. Sixty years from the discovery of Vibrio parahaemolyticus and some recollections. Biocontrol Sci. 2011;16:129–37.CrossRefPubMedGoogle Scholar
  11. 11.
    Xu M, Yamamoto K, Honda T, Xu M. Construction and characterization of an isogenic mutant of Vibrio parahaemolyticus having a deletion in the thermostable direct hemolysin-related hemolysin gene (trh). Publications Office of the European Union. 2010;176:4757–60.Google Scholar
  12. 12.
    Broberg CA, Calder TJ, Orth K. Vibrio parahaemolyticus cell biology and pathogenicity determinants. Microbes Infect. 2011;13:992–1001.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Vongxay K, Wang S, Zhang X, Wu B, Hu H, Pan Z, Chen S, Fang C. Pathogenetic characterization of Vibrio parahaemolyticus isolates from clinical and seafood sources. Int J Food Microbiol. 2008;126:71–5.CrossRefPubMedGoogle Scholar
  14. 14.
    Hiyoshi H, Kodama T, Iida T, Honda T. Contribution of Vibrio parahaemolyticus virulence factors to cytotoxicity, enterotoxicity and lethality in mice. Infect Immun. 2010;78(4):1772–80.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Honda T, Iida T. The pathogenicity of Vibrio parahaemolyticus and the role of the thermostable direct haemolysin and related haemolysins. Rev Med Microbiol. 1993;4:106–13.CrossRefGoogle Scholar
  16. 16.
    Kaper JB, Campen RK, Seidler RJ, Baldini MM, Falkow S. Cloning of the thermostable direct or Kanagawa phenomenon associated hemolysin of Vibrio parahaemolyticus. Infect Immun. 1984;45:290–2.PubMedPubMedCentralGoogle Scholar
  17. 17.
    Nishibuchi M, Hill WE, Zon G, Payne WL, Kaper JB. Synthetic oligodeoxyribonucleotide probes to detect Kanagawa phenomenon-positive Vibrio parahaemolyticus. J Clin Microbiol. 1986;23:1091–5.PubMedPubMedCentralGoogle Scholar
  18. 18.
    Alam M, Miyoshi SI, Yamamoto S, Tomochika KI, Shinoda S. Expression of virulence related properties by an intestinal adhesiveness of Vibrio mimicus strains isolated from aquatic environments. Appl Environ Microbiol. 1996;62:3871–4.PubMedPubMedCentralGoogle Scholar
  19. 19.
    Osawa R, Okitsu T, Morozumi H, Yamai S. Occurrence of urease-positive Vibrio parahaemolyticus in Kanagawa, Japan, with specific reference to presence of thermostable direct hemolysin (TDH) and the TDH-related hemolysin genes. Appl Environ Microbiol. 1996;62:725–7.PubMedPubMedCentralGoogle Scholar
  20. 20.
    Harrington DJ. Bacterial collagenases and collagen-degrading enzymes and their role in human disease. Infect Immun. 1996;64:1885–91.PubMedPubMedCentralGoogle Scholar
  21. 21.
    Hase CC, Finkelstein RA. Bacterial extracellular zinc-containing metalloproteases. Microbiol Rev. 1993;57:823–37.PubMedPubMedCentralGoogle Scholar
  22. 22.
    Fiore AE, Michalski JM, Russel RG, Sears CL, Kaper JB. Cloning, characterization, and chromosomal mapping of a phospholipase (lecithinase) produced by Vibrio cholerae. Infect Immun. 1997;65:3112–7.PubMedPubMedCentralGoogle Scholar
  23. 23.
    Rodrigues DP, Ribeiro RV, Alves RM, Hofer E. Evaluation of virulence factors in environmental isolates of Vibrio species. Mem Inst Oswaldo Cruz. 1993;88(4):589–92.CrossRefPubMedGoogle Scholar
  24. 24.
    Cai YL, Ni Y. Purification, characterization, and pathogenicity of urease produced by Vibrio parahaemolyticus. J ClinLab Anal. 1996;10:70–3.Google Scholar
  25. 25.
    Wu YN, Wen J, Ma Y, Ma XC, Chen Y. Epidemiology of foodborne disease outbreaks caused by Vibrio Vibrio parahaemolyticus, China, 2003–2008. Food Control. 2014;46:197–202.Google Scholar
  26. 26.
    Chao GX, Jiao XA, Zhou XH, Yang ZQ, Huang JL, Zhou LP, Qian XQ. Distribution, prevalence, molecular typing, and virulence of Vibrio parahaemolyticus isolated from different sources in coastal province Jiangsu, China. Food Control. 2009;20:907–12.CrossRefGoogle Scholar
  27. 27.
    Tunung R, Margaret S, Jeyaletchumi P, Chai LC, Ghazali FM, Nakaguchi Y, Nishibuchi M, Son R. Prevalence and quantification of Vibrio parahaemolyticus in raw salad vegetables at retail level. J Microbiol Biotechnol. 2010;20:391–6.PubMedGoogle Scholar
  28. 28.
    Wang RD, Sun LJ, Wang YL, Deng YJ, Liu Y, Xu DF, Liu HM, Ye RY, Gooneratne R. Pathogenicity of Vibrio parahaemolyticus in different food matrices. J Food Protect. 2016;79:288–93.CrossRefGoogle Scholar
  29. 29.
    Takamatsu D, Osaki M, Sekizaki T. Thermosensitive suicide vectors for gene replacement in Streptococcus suis. Plasmid. 2001;46:140–8.CrossRefPubMedGoogle Scholar
  30. 30.
    Jiang HW, Wang KG, Zhang YQ, Huang Q, Hu XX, Yang RY. A quantitative determination of Vibrio parahaemolyticus hemolytic activity. Military Med Sci. 2013;37(4) [In Chinese]Google Scholar
  31. 31.
    Liu PC, Lee KK, Chen SN. Isolates of Vibrio harveyi from diseased kuruma prawns Penaeus japonicus. Curr Microbiol. 1996;22:413–6.Google Scholar
  32. 32.
    Zhang XH, Austin B. Pathogenicity of Vibrio harveyi to salmonids. J Fish Dis. 2000;23:93–102.CrossRefGoogle Scholar
  33. 33.
    Liu XF, Li Y, Li JR, Cai LY, Li XX, Chen JR, Lyu SX. Isolation and characterisation of Bacillus spp. antagonistic to Vibrio parahaemolyticus for use as probiotics in aquaculture. World J Microb Biot. 2015;31:791–805.Google Scholar
  34. 34.
    Baffone W, Citterio B, Vittoria E, Casaroli A, Pianetti A, Campana R, Bruscolini F. Determination of several potential virulence factors in Vibrio spp isolated from sea water. Food Microbiol. 2001;18:479–88.CrossRefGoogle Scholar
  35. 35.
    Edberg SC, Gallo P, Kontnick C. Analysis of the virulence characteristics of bacteria isolated from bottled, water cooler and tap water. Microb Ecol Health Dis. 2009;9:67–77.CrossRefGoogle Scholar
  36. 36.
    Shin-Ichi M. Extracellular proteolytic enzymes produced by human pathogenic Vibrio species. Frontiers Microbiol. 2013;4 339–339Google Scholar
  37. 37.
    Liu XF, Zhang H, Liu X, Gong Y, Chen Y, Cao Y, Hu C. Pathogenic analysis of Vibrio alginolyticus infection in a mouse model. Folia Microbiol. 2014;59:167–71.CrossRefGoogle Scholar
  38. 38.
    Moreno ML, Landgraf M. Virulence factors and pathogenicity of Vibrio vulnificus strains isolated from seafood. J Appl Microbiol. 1998;84:747–51.CrossRefPubMedGoogle Scholar
  39. 39.
    Park K, Ono T, Rokuda M, Jang M, Iida T, Honda T. Cytotoxicity and enterotoxicity of the thermostable direct hemolysin-deletion mutants of Vibrio parahaemolyticus. Microbiol Immunol. 2004;48:313–8.CrossRefPubMedGoogle Scholar
  40. 40.
    Taniguchi H, Hirano H, Kubomura S, Higashi K, Mizuguchi Y. Comparison of the nucleotide sequences of the genes for the thermostable direct hemolysin and the thermolabile hemolysin from Vibrio parahaemolyticus. Microb Pathogenesi. 1986;1:425–32.CrossRefGoogle Scholar
  41. 41.
    Shinoda S, Matsuoka H, Tsuchie T, Miyoshi S, Yamamoto S, Taniguchi H, Mizuquchi Y. Purification and characterization of a lecithin-dependent haemolysin from Escherichia coli transformed by a Vibrio parahaemolyticus gene. J Gen Microbiol. 1991;137:2705–11.CrossRefPubMedGoogle Scholar
  42. 42.
    Kustusch RJ, Kuehl CJ, Crosa JH. Power plays: iron transport and energy transduction in pathogenic vibrios. Biol Met. 2011;24:559–66.Google Scholar
  43. 43.
    Wright AC, Simpson LM, Oliver JD. Role of iron in the pathogenesis of Vibrio vulnificus infections. Infect Immun. 1981;34:503–7.PubMedPubMedCentralGoogle Scholar
  44. 44.
    Chen J, Zhang RH, Qi XJ, Zhou B, Wang JK, Chen Y, Zhang HX. Epidemiology of foodborne disease outbreaks caused by Vibrio parahaemolyticus during 2010–2014 in Zhejiang Province, China. Food Control. 2017;77:110–15.Google Scholar
  45. 45.
    Nottage AS, Birkbeck TH. Purification of a proteinase produced by the bivalve pathogen Vibrio alginolyticus NCMB 1339. J Fish Dise. 1987;10:211–20.CrossRefGoogle Scholar
  46. 46.
    Iuchi S, Tanaka S. Catabolite-like repression of extracellular enzyme production in Vibrio parahaemolyticus. Microbiol Immunol. 1980;24:803–14.CrossRefPubMedGoogle Scholar
  47. 47.
    Oliver JD, Wear JE, Thomas MB, Warner M, Linder K. Production of extracellular enzymes and cytotoxicity by Vibrio vulnificus. Diagn Microbiol Infect Disenso. 1986;5:99–111.CrossRefGoogle Scholar
  48. 48.
    Yeung PS, Wiedmann M, Boor KJ. Evaluation of a tissue culture-based approach for differentiating between virulent and avirulent Vibrio parahaemolyticus strains based on cytotoxicity. J Food Protect. 2007;70:348–54.CrossRefGoogle Scholar
  49. 49.
    Nair GB, Ramamurthy T, Bhattacharya SK, Dutta B, Takeda Y, Sack DA. Global dissemination of Vibrio parahaemolyticus serotype o3:k6 and its serovariants. Clin Microbiol Rev. 2007;20:39–48.CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Bhattacharjee RN, Park KS, Okada K, Kumagai Y, Uematsu S, Takeuchi O, Akira S, Iida T, Honda T. Microarray analysis identifies apoptosis regulatory gene expression in HCT 116 cells infected with thermostable direct hemolysin-deletion mutant of Vibrio parahaemolyticus. Biochem. Bioph Res Co. 2005;335:328–34.CrossRefGoogle Scholar
  51. 51.
    Lynch T, Livingstone S, Buenaventura E, Lutter E, Fedwick J, Buret AG, Graham D, Devinney R. Vibrio parahaemolyticus disruption of epithelial cell tight junctions occurs independently of toxin production. Infect Immun. 2005;73:1275–83.CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Maeda H, Yamamoto T. Pathogenic mechanisms induced by microbial proteases in microbial infections. Biol Chem Hoppe Seyler. 1996;377:217–26.PubMedGoogle Scholar
  53. 53.
    Baffone W, Pianetti A, Bruscolini F, Barbieri E, Citterio B. Occurrence and expression of virulence-related properties of Vibrio species isolated from widely consumed seafood products. Int J Food Microbiol. 2000;54:9–18.CrossRefPubMedGoogle Scholar
  54. 54.
    Sakurai J, Matsuzaki A, Miwatani T. Purification and characterization of thermostable direct hemolysin of Vibrio parahaemolyticus. Infect Immun. 1973;8:775–80.PubMedPubMedCentralGoogle Scholar
  55. 55.
    Sha YJ, Wang BJ, Liu M, Jiang KY, Wang L. Interaction between Lactobacillus pentosus HC-2 and Vibrio parahaemolyticus E1 in Litopenaeus vannamei in vivo and in vitro. Aquaculture. 2016;465:117–23.CrossRefGoogle Scholar
  56. 56.
    Tan LTH, Lee LH, Goh BH, Chan KG, Wright V. Streptomyces bacteria as potential probiotics in aquaculture. Front Microbiol. 2016;1:1–8.Google Scholar
  57. 57.
    Letchumanan V, Chan KG, Pusparajah P, Saokaew S, Duangjai A, Goh BH, Ab Mutalib NS, Lee LH. Insights into bacteriophage application in controlling Vibrio species. Front Microbiol. 2016;7:1114.PubMedPubMedCentralGoogle Scholar
  58. 58.
    Jassim SAA, Limoges RG. Natural solution to antibiotic resistance: bacteriophages “the living drugs”. World J Microbiol Biotechnol. 2014;30:2153–70.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© The Author(s). 2018

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors and Affiliations

  1. 1.College of Food Science and Technology, Guangdong Provincial Key Laboratory of Aquatic Product Processing and Safety, Key Laboratory of Advanced Processing of Aquatic Products of Guangdong Higher Education InstitutionGuangdong Ocean UniversityZhanjiangChina
  2. 2.Centre for Food Research and Innovation, Department of Wine, Food and Molecular BiosciencesLincoln UniversityCanterburyNew Zealand

Personalised recommendations