| Background Vibrio parahaemolyticus has gradually become an important food-borne pathogens in coastal countries, the data show that since 1998, the scale of food poisoning caused by Vibrio parahaemolyticus showed a clear upward trend over food poisoning caused by the salmonella, leapt to the top. In our country, Vibrio parahaemolyticus is the primary food-borne pathogens that cause bacterial food poisoning in coastal areas, and is the leading cause of national foodborne disease caused by microbe. Food poisoning caused by Vibrio parahaemolyticus always occurred in summer and autumn, often resulted in collective disease which is seriously harmful to the health of consumers.Objective To Learn commercial aquatic pollution levels with Vibrio parahaemolyticus and Vibrio parahaemolyticus foodborne infection in sentinel hospitals in Beijing; To build Vibrio parahaemolyticus in aquatic food microbiological risk assessment laboratory databases, through the study of serotype, virulence genotypes, resistance patterns and molecular typing of Vibrio parahaemolyticus in different aquatic products (sea products, freshwater products); To provide traceability of the pathogen that caused foodborne disease outbreaks in Beijing, by research on the correlation between aquatic products contaminated and clinical cases infected by Vibrio parahaemolyticus based on molecular typing and epidemiological science.Method1. Select three large market in Beijing:Xinfadi agricultural products wholesale market, Yuegezhuang agricultural wholesale center and Jinshen seafood wholesale market. Collect different kinds of freshwater products and sea products, use MPN method for quantitative test to access Vibrio parahaemolyticus pollution rate and pollution in different kinds of aquatic products.2. Select Chinese Medicine University Dongfang Hospital, Beijing Fengtai Hospital, China Aerospace Science and Industry Group 731 Hospital as sentinel hospitals, choose the outpatients with vomiting or diarrhea and other gastrointestinal symptoms as monitoring samples. Acquire infection rate, by collecting stool samples to detect Vibrio parahaemolyticus.3. Use PCR method to detect virulence genes tlh, tdh, trh of Vibrio parahaemolyticus isolates separated from aquatic products and clinical cases.4. Use Slide agglutination method for serotyped derived from fish isolated strains and clinical isolates of Vibrio parahaemolyticus isolates separated from aquatic products and clinical cases.5. Refer CLSI recommended broth dilution method for susceptibility testing of Vibrio parahaemolyticus isolates separated from aquatic products and clinical cases.6. Use PFGE method for molecular typing study of Vibrio parahaemolyticus isolates separated from aquatic products and clinical cases, Use Bionumerics analysis software for DNA fingerprinting cluster analysis to quantify the level of gene phylogenetic relationship between different strains. Result1.164 aquatic product samples were collected from July to September. Vibrio parahaemolyticus was isolated from 80 aquatic product samples. Total incidence of Vibrio parahaemolyticus was 48.78%. The incidence of Vibrioi parahaemolyticus in sea products was 53.04%, and the incidence of Vibrioi parahaemolyticus in freshwater products was 38.78%. The average bacteria concentration in sea products was 36.54MPN/g, and the average bacteria concentration in freshwater products was 66.63MPN/g. Among 80 positive samples, there are 14 samples’ bacteria concentration> 210MPN/g, showing serious pollution. In different kinds of aquatic products, the contamination rates of shellfish and shrimp are higher than that of fish and crabs. There are no significant differences of pollution rates in different months.2. In three sentinel hospitals 21 Vibrio parahaemolyticus strains were detected out of clinical cases from late July to late August. There were 33 patients due to vomiting or diarrhea and other gastrointestinal symptoms for treatment in August, the test results showed that 17 patients infected with Vibrio parahaemolyticus, the infection rate was 51.52%.3. PCR detection of virulence genes results showed that 101 Vibrio parahaemolyticus isolates, tlh gene were all positive, trh genes were all negative. Among 21 clinical strains,20 strains’tdh virulence gene were positive, tdh virulence genes carring rate of clinical isolates was 95.24%. Among 80 aquatic products strains, only one tdh virulence gene was positive, tdh virulence gene carrying rate of aquatic products isolates was 1.25%.4. Among 21 clinical strains, O3:K6 serotype had 13 strains, O4:K8 serotype had 6 strains, with the proportion of 61.90%,28.57% respectively.80 aquatic products strains were divided into 9 O serogroups, among which 02 (35.00%),01 (13.75%),05 (12.5%) were the main serotypes. Completely typing strains accounted for 37.50% of the total 80 aquatic products strains (30/80), in which O2:K28 is the largest, accounting for 43.33% of completely typing strains (13/30). Three new serotypes were found, O1:K33, O5:K33 and O11:K34.5. The result of antimicrobial susceptibility test showed that 21 clinical strains appear resistance only to ampicillin, the resistance rate is 28.57%(6/21), to the remaining 11 antibiotics are all sensitive. Among 80 aquatic products strains,50 strains are resistant to at least one antibiotic, the total resistance rate is 62.50%. There are 10 dual resistant strains, dual resistance rate is 12.50%. In the 12 kinds of antibiotics for detection, all strains were 100% sensitive to ceftazidime, cefotaxime, cefepime, chloramphenicol and meropenem. Ampicillin has the most resistant strains, the resistant rate is 45.00%(36/80), followed by gentamicin which resistance rate is 25.00%(20/80). Resistance rates of tetracycline, ciprofloxacin, nalidixic acid and trimethoprim/sulfamethoxazole are all 1.25%(1/80). Polymyxin B has 5.00%(4/80) mid-sensitive strains, but not resistant strains.6. PFGE molecular typing results of Vibrio parahaemolyticus showed that 101 strains digested by restriction endonucleases sfi I showing good typing ability, the typing rate was 96.04%(97/101),92 kinds belt types were produced, the number of bands in each strain was among 12-22. The cluster analysis showed that similarity of Vibrio parahaemolyticus strains from different sources was 48.3%-100%. There were three clones group, intergroup similarity> 88.7%, including 6 aquatic isolates and 11 clinical isolates. There are close genetic relationships between isolates from different sources, up to 100% genetic similarity.Conclusion1. Beijing commercial aquatic products have high Vibrio parahaemolyticus pollution levels. Shellfish and shrimp have higher contamination rates than other kinds of aquatic products, which showed serious food security risk. We should focus on strengthening monitoring Vibrio parahaemolyticus contamination in shellfish and shrimp. The possibility of cross contamination between fresh products and sea products is large.2. The infection rate of Vibrio parahaemolyticus is high in Beijing foodborne infections, the infection rates between different sexes have no difference.3. The difference between virulence genotypes of Vibrio parahaemolyticus from aquatic products isolates and clinical isolates is large. Contaminated Aquatic products may not be the main cause of Beijing residents infected with Vibrio parahaemolyticus. It remains to be studied that whether Vibrio parahaemolyticus in food change virulence after entering the body, in addition to widely recognized virulence genes tdh and trh, Vibrio parahaemolyticus there may be other unknown causative factor, the pathogenic mechanism needs further study. In addition to the widely recognized virulence genes tdh and trh, Vibrio parahaemolyticus may has other unknown pathogenic factors, and the pathogenic mechanism needs further study.4.03:K.6 and 04:K8 serotypes of Vibrio parahaemolyticus are dominant strains led to Vibrio parahaemolyticus infection in Beijing. The serotype of Vibrio parahaemolyticus strains from aquatic products is mainly 02 group, we found three new serotype, in which two are unreported that prompt serotypes Changes may occur. O2:KUT and 02:K3 serotypes of aquatic products isolates have high potential risk to cause foodborne illness, which should attract attention and be strengthened monitoring.5. Vibrio parahaemolyticus resistance phenomenon in Beijing is not serious. The use of antibiotics in clinic should consider regional factor to avoid resistant strains increasing. Vibrio parahaemolyticus drug resistance is affected by origin, the situation is complex and should be strengthened monitoring. Multidrug-resistant strains have not been found in Beijing area, but in shellfish and shrimp appeared dual-resistant strains, suggest that restrictions on the use of antibiotics in aquaculture should be strengthened.6. The PFGE band patterns of Vibrio parahaemolyticus from clinical cases are centralized, 03:K6 serotypes of clinical isolates have similar band pattern show high close genetic relationship, suggest that the patients may have the same source of infection. The PFGE band patterns of Vibrio parahaemolyticus from aquatic products is dispersed. The strains with high similarity mostly came from the same time and the same gathering place, suggest that there may be cross-contamination. The strains with high similarity came from different times and places of, suggest the aquatic products had the same pollution source and purchase channels, the health sector should strengthen the supervision to source. Clinical isolates and aquatic products isolates reached 100% genetic similarity, but the time was not relevant, suggest that patients may eat food contaminated by the same sources. Clinical isolates and aquatic products isolates have high genetic similarity, the time was closely related, suggest that patients may eat the exact aquatic products contaminated by Vibrio parahaemolyticus. However, because of the lack of epidemiological data, this study was unable to track the infection source which needs to be improved. |
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