Resistance,Genetic Diversity And Rapid Detection Methods Of Vibrio Parahaemolyticus Recovered From Commonly Consumed Aquatic Products In Shanghai

Vibrio parahaemolyticus is a Gram-negative bacterium that is often found in coastal waters.The bacterium can cause human gastroenteritis,diarrhoeal illness,and even death.Continuous monitoring of V.parahaemolyticus contamination in aquatic products is crucial for assuring food safety.In this study,we isolated and characterized 561 Vibrio parahaemolyticus strains recovered from 25 species of commonly consumed shellfish,crustaceans,and fish collected in July and August of 2017 in Shanghai,China.Among these species,Callista chinensis,Corbicula aurea and Lutraria arcuata have not been previously detected for V.parahaemolyticus.The results revealed a very low occurrence of pathogenic V.parahaemolyticus carrying toxin genes trh?0.2%?and tdh?0.0%?.However,high percentages of resistance to antimicrobial agents ampicillin?93.0%?,rifampin?82.9%?,streptomycin?75.4%?and kanamycin?50.1%?was found.Approximately 77.5%of the isolates had multidrug-resistant?MDR?phenotypes with37 resistance patterns.High incidence of tolerance to heavy metals Hg²⁺?74.7%?and Zn²⁺?56.2%?was observed in the isolates.Different resistance profiles were found among different species and kinds of aquatic products.Enterobacterial repetitive intergenic consensus-polymerase chain reaction amplification?ERIC-PCR?-based fingerprinting of the MDR isolates revealed 428 ERIC-genotypes,which demonstrated remarkable genetic variation among the isolates.In this study,based on the loop-mediated isothermal amplification technique?LAMP?,a set of 6 primers were designed targeting the major toxin genes tdh and trh of V.parahaemolyticus,respectively.With the capillary as the reaction rector,a specific,sensitive,low-cost,visual and micro assay was developed to detect the toxin genes.The optimum conditions of the capillary LAMP?cLAMP?were as follows:6 mmol/L or 8mmol/L magnesium ion,1.44 mmol/L or 1.28 mmol/L dNTPs,0.48 U Bst DNA polymerase,the ratio of internal to external primers of 8:1,reaction temperature of 65?,and reaction time of 60 min.The detection limits for the tdh and trh genes were 3 fg/?L and 34 fg/?L for genomic DNA,9.85×10³ CFU/mL and 8.25×10⁵ CFU/mL for bacterial culture,respectively.No cross-reactivity was observed for the other common bacterial pathogens Vibrio cholera,Vibrio vulnificus,Vibrio alginolyticus,Aeromonas hydrophila,Escherichia coli,Staphylococcus aureus.Taken together,the results in this study revealed divergent resistance phenotypes and ERIC-genotypes of the V.parahaemolyticus isolates recovered from commonly consumed aquatic products.The data support the urgent need for improving aquatic animal health management,and provide technical support for the development of rapid,visual and micro-assay kit for the detection of pathogenetic V.parahaemolyticus.