Vibrio Parahaemolyticus In Shellfish:Survey On Its Abundance, Quantitative Microbiological Risk Assessment And Characteristics Of Isolates

Vibrio parahaemolyticus is a worldwide important pathogen associated with seafood consumption, and the typical disease it caused is gastroenteritis. V. parahaemolyticus is the first cause of bacterial foodborne disease according to data from China. Molluscan shellfish accumulate microorganisms from the environment during filter feeding, bringing up risk of infection. This study surveyed six provinces in China to determine the temporal and spatial distribution of V. parahaemolyticus in raw shellfish. Based on quantitative data of V. parahaemolyticus, we built a mathematical model to describe the growth and survival of V. parahaemolyticus in raw shellfish during the whole processing and distribution chain from farm to table. Combined with the dose response relationship, risk of illness per serving and per year were calculated. We also analyzed the biochemical, genetic, antibiotic resistant and molecular subtyping characteristics of V. parahaemolyticus isolates.1. The abundance of V. parahaemolyticus in shellfishThe prevalence and abundance of total V. parahaemolyticus were 70.6% and 44.6 MPN/g, respectively. V. parahaemolyticus densities followed a geographical trend, with Guangxi and Sichuan shellfish possessing total V. parahaemolyticus levels 100-fold higher than those of Liaoning and Shandong regions. V. parahaemolyticus densities followed a seasonal trend, with the peak appeared in summer and autumn. No significant differences between oysters and clams were observed in the total V. parahaemolyticus levels. Aqua farms born the highest levels of total V. parahaemolyticus, followed by retail markets and catering markets. The catering markets showed the lowest levels of total V. parahaemolyticus, whereas there were 25.1% samples exceeded the limit of 1,000 MPN/g which was set in GB 29921.The prevalence and abundance of pathogenic V. parahaemolyticus were 26.7% and 0.5 MPN/g, respectively. Pathogenic V. parahaemolyticus densities followed a geographical trend, with Liaoning shellfish possessing the highest pathogenic V. parahaemolyticus levels and Guangxi the lowest. Pathogenic V. parahaemolyticus densities followed a seasonal trend, with the peak of summer and the bottom of winter. There were significant differences and clams had pathogenic V. parahaemolyticus levels higher than oysters. Catering markets born the highest numbers of pathogenic V. parahaemolyticus, followed by retail markets then aqua farms.2. Quantitative risk assessment of V. parahaemolyticus in raw shellfishBased on the surveyed contamination data of V. parahaemolyticus, cold chain assessment and room temperature assessment were carried out. The risk of V. parahaemolyticus in harvested shellfish was assessed after cold chain circulating. The results of cold chain assessment showed that the simulated levels of V. parahaemolyticus at home were higher than the levels of aqua farms. Risk of illness per serving of Guangxi was the lowest as 2.36×10-5, while risk of the other 3 provinces were around 10-4. Combined with demographic data, risk of illness per year was assessed and Guangxi had the lowest number of cases as 323, with Shandong the highest as 4942. Risk of illness per serving was 10-6 for four provinces, and risk of illness per year was less than 100 cases, when the levels of V. parahaemolyticus meeting the limit of GB 29921. The protection levels of GB 29921 were more than 87% for all four provinces.The levels of V. parahaemolyticus at home similuted in the room temperature assessment were higher than similuted in the cold chain assessment. Risk of illness per serving and per year similuted in the room temperature assessment were also higher than cold chain assessment. The protection levels of GB 29921 were more than 98% for all four provinces in the room temperature assessment.The sensitivity analysis results in Guangxi of cold chain assessment showed that 8 factors were risk factors. Risk factors were lined according to the correlation coefficients as this order:temperature> grams per serving> cooldown time> transport time> the initial levels of pathogenic V. parahaemolyticus at aqua farms (Cp)> probability of eating raw> prevalence of pathogenic V. parahaemolyticus> prevalence of total V. parahaemolyticus. The initial levels of pathogenic V. parahaemolyticus at aqua farms ranked fifth in risk factors in Guangxi with correlation coefficient of 0.21, and the correlation coefficients for both prevalence of pathogenic V. parahaemolyticus and total V. parahaemolyticus were only 0.02 and 0.01, respectively.3. Analysis on characteristics of V. parahaemolyticus isolatesBiochemical tests of V. parahaemolyticus were carried out using API 20E and VITEK 2 GN systems. The results showed that the clinical strains and environmental strains had significant differences in four biochemical tests of dSOR、SUCT、AMY and 1LATk. Only 3 biochemical tests among 8 common ones were consistent in two systems, which were ONPG/BGAL, H2S and SAC. Besides, there were 3 biochemical tests out of the remaining 5 non-consistent tests which had significant differences among the two systems, which were GLU/OFF, CIT and LDC.Gene results showed that OMP gene was 100% positive.89.25% clinical strains were positive for tdh, and all were negative for trh.1 out of 88 environmental strains was positive for tdh, and one positive for trh. The results of three genes in T3SS2 clusters showed that 98.92% clinical isolates were positive and 1 environmental strain was positive.96.77% clinical strains were positive for PGS, and 67.05% environmental strains were positive. The ERIC results showed that 88.17% clinical isolates were positive versus 10.23% environmental strains were positive.9 strains of environmental strains were positive for SXT.The antibiotic characteristics to 8 antibiotics of all isolates were consistent and ampicillin resistance or intermediary was the most prevalent. The geometric mean of ampicillin MIC value of all isolates is greater than the interpretation value of resistance. There were 4 strains which showed multiple antibiotic resistance and/or intermediate resistance.Compared to PFGE, ERIC-PCR and REP-PCR have the advantages of more faster and more convenient. They are more suitable for analysis on a large number of strains and were applied in subtyping many different bacteria. The results of this study showed that ERIC-PCR and REP-PCR can be used for molecular typing of V. parahaemolyticus. ERIC-PCR had larger numbers of amplified bands, and less group numbers. REP-PCR had smaller numbers of amplified bands, and better typing ability. A higher similarity coefficients were found among strains isolated from different sample species, different sampling section, different provinces by the two typing methods. It may indicate that the presence of dominant strains of V. parahaemolyticus in the environment.