Establishment Of Predictive Model And Risk Assessment For Foodborne Vibrio Spp. In Shrimp

Vibrio vulnificus and Vibrio parahaemolyticus are gram-negative halophilic bacteria that naturally occur in warm estuarine water and marine environment worldwide. They are recognized as the important cause of human foodborne disease associated with the consumption of contaminated seafood. The most common clinical presentation of foodborne V. vulnificus infection is primary septicemia, with an average mortality rate exceeding 50%. V. parahaemolyticus accounted for the largest number of outbreaks and cases in China, and the most common clinical presentation of V. parahaemolyticus infection is gastroenteritis. We also studied the growth kinetics model of V. vulnificus and V. parahaemolyticus in broth and shrimp. The survival rates of V. vulnificus and V. parahaemolyticus inoculated into shrimp samples were measured at low temperature, their survival curves were plotted respectively. The model of V. parahaemolyticus was inactivated by the Ultra High Pressure (UHP) was studied. In the present study, we determined not only the prevalence and levels of V. vulnificus in retail shrimps collected in warmer months, but also the virulence characteristics and antibiotic sensitivity of V. vulnificus isolated from the shrimps. Using data of V. vulnificus predictive model and surveillance, we carried out the quantitative risk assessment of V. vulnificus in shrimp in warmer season. This paper suggests measures to control V. vulnificus and V. parahaemolyticus.At first, the optimum pH, temperature and salinity for V. vulnificus VvFJ04 growth in tryptic soy broth (TSB) were determined by single-factor analysis respectively. The response surface model was applied to study optimization of the growth parameters for VvFJ04, Y=0.42+0.00451x₁+0.03x₂+0.005671x₃+0.026x₁x₂ +0.004875x₁x₃-0.002875x₂x₃+0.001078x₁²-0.051x₂²-0.013x₃². The results showed that the optimum conditions for germination as follows: pH was 8.29, temperature was 32°C and salinity was 2.38%. The experimental results indicated that the proposed model can be used for describing and predicting the growth feature of VvFJ04 at different environmental factors.The optimum temperature, salinity, and pH for V. parahaemolyticus ATCC 17802 growth were determined by single-factor analysis respectively. The effects of different temperatures (28.59°C–45.41°C), concentrations of sodium chloride (NaCl, 1.32%–4.68%) and pH values (5.32–8.68) on the growth rates of V. parahaemolyticus were studied. The primary growth curves were fitted using the Baranyi equation. A response surface model (RSM) was prepared, predictions of V. parahaemolyticus growth rates could be obtained from growth curves. Mathematical evaluation demonstrated that the standard error of prediction (%SEP), the root-mean-squares error (RMSE), accuracy factor (Af) and bias factor (Bf) were within acceptable range.The future study was to understand the growth kinetics characteristics of V. vulnificus strains VvFJ04, VvHB09, VvSH09 and V. parahaemolyticus ATCC 17802 in 2% NaCl TSB and Pacific white shrimp. The primary growth models of V. vulnificus and V. parahaemolyticus were fitted well by the modified Gompertz equation, modified Logistic equation, Baranyi equation at from 15°C to 40°C. The determination coefficient R² values were greater than 0.91. The lag time, growth rate, and maximum population density of each primary model were compared. At 15°C, V. parahaemolyticus and V. vulnificus VvFJ04 grew very slowly in 2% NaCl TSB broth, VvHB09 and VvSH09 did not grow in TSB. Three V. vulnificus did not grow in shrimp at 15°C, only V. parahaemolyticus grew slowly in shrimp at 15°C. The growth rates of V. vulnificus and V. parahaemolyticus were maximum at 35°C and 40°C. The Gompertz equation, Ratkowsky equation and Davey equation were used to predict the secondary models of the growth rate and lag time for V. vulnificus and V. parahaemolyticus.In order to study the variation of inactive V. vulnificus and V. parahaemolyticus in shrimp under 5°C and–18°C. Then Linear, Weibull and Logistic models were applied to predict curves of inactive V. vulnificus and V. parahaemolyticus, respectively. At 5°C, VvHB09 showed more resistance to cool pressure. At–18°C, V. parahaemolyticus strain ATCC 17802 and VvSH09 showed more resistance to cold pressure. Moreover, Linear model was proved to be more accurate and useful in fitting V. vulnificus and V. parahaemolyticus inactivation curves based on mathematical evaluation than nonlinear models, Weibull and Logistic model.In order to study the effect of inactive V. parahaemolyticus ATCC 17802 by the Ultra High Pressure, recurring to Design Expert and the Box-Behnken experiment method to design and study the effect of the UHP cooperated with different temperature and time on the deadly ratio of V. parahaemolyticus, the response surface model of V. parahaemolyticus sterilized by the UHP was built, Y=4.34+0.69x₁+2.48x₂ +0.97x₃+0.6x₁x₂+0.22x₁x₃+0.39x₂x₃–0.27x₁²+0.18x₂²+0.29x₃². The optimum process parameters for inactivation of six logarithms V. parahaemolyticus were obtained as: temperature was 25.2°C, pressure was 230.3 MPa, and pressure holding time was 13.6 min. The effect of UHP on the microstructure of V. parahaemolyticus ATCC17802 was investigated by Transmission Electron Microscope (TEM). The results showed that the cell wall of V. parahaemolyticus was fractured, breached and leaked out cytoplasm. To quantify the prevalence and the levels of V. vulnificus in shrimps, the shrimp samples were collected from ten cities in China. The most probable number (MPN) method was used combining with the polymerase chain reaction (PCR) which was used to classify the isolates by detecting V. vulnificus hemolysin gene (vvhA). One hundred and forty out of 239 samples were positive for V. vulnificus, with the geometric mean level at 153.3 MPN/g. The prevalence and levels of V. vulnificus in shrimps varied in different geographic areas (P<0.05), with the level values for the samples in the South cities (179.6 MPN/g) remarkably higher than that in the North cities (7.6 MPN/g). In addition, we found that 79 of 128 V. vulnificus positive samples contained C-type (Clinical) strains and 8 contained E-type (Environmental) strains. Seven shrimp samples contained untypeable V. vulnificus and 34 samples had both C-type and E-type strains. The antimicrobial susceptibility of the isolates to 12 antibiotics was tested as well. Most of the 169 isolates remained susceptible to the majority of antimicrobials tested, whereas some strains tend to be less sensitive to amikacin, ampicillin, gentamicin and tetracycline. In conclusion, V. vulnificus is commonly found in retail marine shrimp in Chinese seafood market, and most of the shrimp samples contained C-type strains, which may pose a potential threat to human health.A microbiological risk assessment is to assess the potential hazard caused by pathogens in food which may affect public health, in order to guide risk managers to establish related measures. A quantitative microbial risk assessment of V. vulnificus in shrimp was undertaken. The assessment was conducted in accordance with the widely accepted procedures for risk assessment, which involoves hazard identification, hazard characterization, exposure assessment, and risk characterization. Using the data on the prevalence and levels of V. vulnificus in shrimp, the frequency and quantity of shrimp consumption and predictive model, exposure assessment model was established. The outcome of the exposure assessment model was combined with a beta-poisson dose-response model to estimate the probability of disease. Simulation results showed that the probability of V. vulnificus infection was 1.977×10^-5. It provided measure to reduce the risk of V. vulnificus.The results of this paper provide technical support to reduce the risk of two bacteria at seafood harvest and consumption period. The basic data of this research build the risk management to prevent and control foodborne illness causing by V. vulnificus and V. parahaemolyticus.