Isolation And Identification Of Lytic Phage Of Vibrio Parahaemolyticus And Its Targeted Antibacterial Effect In Salmon

Vibrio parahaemolyticus(Vp)is one of the main pathogens of seafood.Eating seafood contaminated by Vibrio parahaemolytic,s can cause gastroenteritis,fever and other symptoms,which seriously affect human health.In order to explore an effective method for controlling Vibrio parahaemolyticus in seafood at room temperature,this study used Vibrio parahae,molyticus as the host strain,5 strains of lytic phage isolated from Yangzhou city sewag,and study its biological characteristics,phage microbicides production and its application in the field of biological control,lay a foundation for developing new deputy hemolytic vibrio fungicide.The results are as follows:(1)Using 42 strains of Vp as host,30 strains of Vp phage were isolated from the sewage of Yangzhou City,and 5 strains of phage with good lyticity were screened for further study.The morphology of the phage was observed by transmission electron microscope,including Siphoviridae,Tectiviridae and Myoviridae.The phage has good stability at 60℃ and pH 4～10.The results of cleavage spectroscopy showed that VppYZU81 had the broadest cleavage spectrum and could lyse 25 host strains;the mixture of 5 phage strains(VppMIX)had a lysis rate 43 host strains.Studies of one-step growth curves and MOI indicate differences in phage lysis.(2)The whole genome sequencing of 5 phages showed that all 5 strains were new phages.The the total length of phage VppYZU64,VppYZU68,VppYZU81,VppYZU92 and VppYZU110 genomes were 76,153 bp,58,495 bp,42,897 bp,80,670 bp and 144,768 bp,respectively;The average length of the coding genes were 663.96 bp,698.81 bp,915 bp,654.18 bp,and 931.20 bp,and there are 100,27,40,114,35 open reading frames(ORFs).Functional genes mainly include genes that regulate nucleic acid metabolism and expression,structural protein genes,assembly protein genes,and cleavage-related genes.(3)The bacteriophage evaluated the bacteriostatic effect of the bacteriophage and citric acid bactericide at 25 0C and 37 C in the medium and the salmon model.In the medium at 25 ℃,when 500 μg/mL citric acid(pH 5.70)was combined with 106 PFU/mL VppYZU64 to inhibit the host bacteria,the total number of colonies in the control group for 8 h increased from the initial 3.11 lgCFU/mL to the lowest pathogenic dose(5.00 lgCFU/mL),the total number of colonies increased to 105 CFU/mL at 14 h,16 h,and 22 h in citric acid treatment group,phage treatment group and combined treatment group,and phage and citric acid had combined antibacterial effect.When HC1 with pH 5.70 combined with VppYZU64 inhibited the host bacteria,the total number of colonies increased to 105 CFIU/mL at 11 h and 16 h,respectively,in the hydrochloric acid treatment group and the combined treatment group.The antibacterial effect of hydrochloric acid is lower than that of citric acid,and the antibacterial effect is not obvious when combined with phage.500 μg/mL citric acid was co-operated with the phage mixture VppMIx at 25 ℃ for the host mixture VpMIx,and the total number of colonies was increased to 5.00 lg CFU/mL for 31 h in the combined treatment group.The antibacterial effect of VppMIx combined with citric acid was better than that of single phage combined with citric acid,and the inhibition time was extended by 4 h.(4)The salmonella tablets were subjected to sensory evaluation to determine the appropriate concentration of 600 μg/mL,and the concentration of citric acid was combined with VppMIx(106 PFU/mL)for infection of VpMIx(104 lgCFU/mL).Salmon fillet.The number of colonies of salmon slices stored at 25 C for 6 h in citric acid treatment group(BA),phage treatment group(BP)and combined treatment group(BPA)decreased by 0.59 lgCFU/g,2.02 lgCFU/g,3.84 lgCFU/g(P<0.05);the total number of colonies in each experimental group increased to 5.00 lgCFU/g at 11 h,16 h,and 23 h,respectively.After storage at 37 C for 3 h,the total number of colonies in the BA,BP,and BPA experimental groups decreased by 0.83 lgCFU/g,3.31 lgCFU/g,and 3.42 lgCFU/g(P<0.05).The experimental groups were at 4,7,and 10 h,respectively up to 105 CFU/g.When VppYZU64 combined with 600 μg/mL citric acid inhibited biofilm,the biofilm inhibition rate was up to 93.80%in the combined treatment group.The results of scanning electron microscopy showed that the combined treatment group had the best biofilm removal effect.(5)The frequency of Bacteriophage insensitive mutants(BIMs)of single phage was 10-3～10-5,and the BIMs frequency of VppMIX has decreased to 10-6.The phage-insensitive mutant strain was unstable.After 20 passages of 35 insensitive strains of VppYZU64,25 strains recovered their sensitivity to phage.The isolated insensitive mutant VPYZU64-1 had a smaller colony edge than the parental strain VPYZU64,and the cell surface of VPYZU64-1 cells was shrunk by scanning electron microscopy.