Study On Proportion Of Pathogenic And Nonpathogenic Vibrio Parahaemolyticus In Mixed Culture

Foodborne gastroenteritis associated with the consumption of raw or undercooked seafood,e.g.,oysters,is caused by pathogenic Vibrio parahaemolyticus.In the coastal areas of China,the pathogenic strain is the leading cause of bacterial food poisoning.Pathogenic V.parahaemolyticus was initially identified as the causative agent in a food poisoning case in Japan in 1950,and with widespread consumption of seafood,this bacterium has accounted for the majority of cases of food poisoning in Japan,North America,and Southeast Asia.Although V.parahaemolyticus is thought to be an important cause of seafood-associated disease outbreaks throughout the world,its pathogenic strains have seldom been isolated from food and environmental samples,and the majority of pathogenic isolates are recovered from clinical samples.Currently,based on a series of molecular biology techniques,several DNA-based methods have been developed for the detection of pathogenic V.parahaemolyticus in environmental and food samples.Many studies have found that the ratios of pathogenic strains to total V.parahaemolyticus obtained with DNA-based methods are much higher than those with conventional culture methods.In conventional culture methods,the pathogenic strains need to be isolated from the background flora,and thus time-consuming enrichment is always necessary.On the other hand,the DNA-based methods are less dependent on enrichment.Thus,we hypothesized that the culturing process has a negative effect on the rate of isolation of pathogenic V.parahaemolyticus in environmental and in food samples.1.Optimized the hybridization conditions of colony in situ hybridization for pathogenic Vibrio parahaemolyticus.Selected three oligoprobes based on the tdh,trh and tox R gene of Vibrio parahaemolyticus,optimized the pretreatment method and hybridization procedure and color detection of colony in situ hybridization forpathogenic Vibrio parahaemolyticus.The results showed that the high specificity,low background and clear color result can be obtained by using the method of 2h 80? hybridization,30 min 37?pre-hybridization and 37? 8h hybridization,which has the advantages of high efficiency,accurate and can distinguish pathogenic strains when compared to the traditional detection method.2.Pathogenic strains of Vibrio parahaemolyticus are rarely isolated from seafood samples.In this study,pathogenic(ATCC 33847: tdh+,trh-,tlh+;ATCC 17802: tdh-,trh+,tlh+)and non-pathogenic(G2: tdh-,trh-,tlh+;G8: tdh-,trh-,tlh+)V.parahaemolyticus strains were mixed at 9:1 and 1:1 ratios and cultured at 37°C in tryptic soy broth(TSB)or cooked shrimp for 10 h.The rate of isolation of the pathogenic strains after 10 h was determined by colony hybridization and PCR,and the results showed that the isolation rate of the pathogenic strains decreased after co-culturing in TSB and cooked shrimp.Out of the 24 co-cultured groups,the rates of isolation of the pathogenic strain in 20 were decreased and among them,the rates in 18 decreased by more than half,and those in the rates of 17 groups decreased by more than 40%.The isolation rate of the pathogenic strains was more prominent in the shrimp samples,and when the strain ATCC 17802 was mixed with strain G8 in a 9:1 ratio in the shrimp samples,no pathogenic strain was detected after 10 h.The interactions between non-pathogenic and pathogenic V.parahaemolyticus are possible reasons for the rare isolation of V.parahaemolyticus from seafood samples.3.Detected the proportion of pathogenic strains in total Vibrio parahaemolyticus before and after co-culture by in situ hybridization and colony PCR technique.Compared the total number of single strains and mixed strains after 8h culture in TSB.Analysed the proportion of pathogenic strains in co-culture procedur,compare the difference of co-cultured in two different temperature.We hypothesized that the pathogic V.parahaemolyticus decrease in co-culture due to poor resistance in lack of nutrition and temperature decline in the environment,while more research is in need to determine the real mechanism.