Isolation And Identification Of Vibrio Parahaemolyticus From Penaeus Vannamei And Study On The Synergistic Antibacterial Effect Of Nanomaterials With Antibiotics

Vibrio parahaemolyticus is one of the main pathogens of Penaeus vannamei,and once the infection breaks out,it will cause huge economic losses to the shrimp culture industry.Antibiotics are widely used in aquaculture as chemicals as killing or inhibiting agents for bacteria.The residue of antibiotics in the environment not only pollutes the oceans,but also induces the emergence of antibiotics resistant bacteria and even multi-antibiotics resistant bacteria in the shrimp culture environment.The accumulation of antibiotics resistant and multi-antibiotics-resistant Vibrio parahaemolyticus in the environment will bring new difficulties to the prevention and control of pathogenic bacteria in shrimp culture.In recent years,studies have found that some nanomaterials can restore the antibacterial activity of traditional antibiotics to a certain extent,and the combination of nanomaterials and antibiotics can be used to restore the sensitivity of antibiotics resistant Vibrio parahaemolyticus to antibiotics,providing new ideas for the prevention and control of Vibrio parahaemolyticus in aquaculture.In this study,Vibrio parahaemolyticus isolated from typical marine culture areas of Penaeus vannamei was used as research objects to explore the antibacterial effects and mechanisms of nanomaterials and antibiotics.Firstly,Vibrio parahaemolyticus was isolated and identified from Penaeus vannamei in three regions,and the whole genome sequencing was carried out.Then,the appropriate antibacterial combination of antibiotics and nanomaterials was screened and the mechanism was studied.Finally,the combined drug was applied to the intestinal culturable bacteria and antibiotic resistant microbiota of Penaeus vannamei,and the effect of the combination drug on the microbial communities was discussed.The specific research contents of this paper are as follows:(1)Isolation,identification and drug resistance analysis of Vibrio parahaemolyticus from three sampling points.The results showed that the Vibrio parahaemolyticus isolated from three areas were resistant to antibiotics,especially the strains isolated from Dongying resistant to four antibiotics.Whole genome sequencing was performed on the strains isolated.The strains isolated from three different regions belong to three different types according to multilocus sequence typing.From the pathogenic system analysis,it was found that the virulence factor genes carried by the isolates from the three regions were similar,and the number of antibiotics resistant genes carried was small and slightly different.(2)Screening of nanomaterial/oxytetracycline(OTC)combination with OTCresistant strains and preliminary exploration of synergistic antibacterial mechanism.The combination of nanomaterials and OTC with high antibacterial activity against OTC-resistant strain DY-C1-1 isolated from Dongying was screened.It was found that the combination of silver nanoparticles(Ag NPs)and OTC was the best.The mechanism of action was explored and it was found that Ag NPs / OTC enhanced cell membrane damage and intracellular reactive oxygen species(ROS)content.(3)Effect of Ag NPs/OTC combination on intestinal culturable bacteria and antibiotic resistant bacteria in Dongying cultured Penaeus vannamei.It was found that Ag NPs/OTC had little effect on the diversity and variability of intestinal flora.But it can inhibit the growth of Aerococcus,Vagococcus and norank＿f＿Intrasporangiaceae.And Vibrio has strong tolerance to Ag NPs/OTC.Using antibiotics to culture antibiotics resistant bacteria,it was found that community diversity decreased significantly and diversity changed significantly.After Ag NPs/OTC treatment,the diversity and diversity of antibiotics resistant bacteria changed little,but the overall abundance of antibiotics resistant bacteria decreased,and the community species abundance decreased.The study was carried out in three aspects: basic characters of shrimp-derived Vibrio parahaemolyticus in typical Penaeus vannamei culture areas in China,evaluation of the antimicrobial effect of combined nanomaterials/antibiotics and the effect of combined drugs on intestinal culturable and antibiotics resistant flora,the results of which will provide a theoretical basis and scientific basis for the control of antibiotics resistant Vibrio parahaemolyticus with certain practical significance.