Isolation And Characterization Of Vibrio Parahaemolyticus Phage And Its Application Of Receptor Binding Domain

Vibrio parahaemolyticus is a foodborne pathogen that is widespread in estuarine,marine and coastal environments and can not only cause severe acute hepatopancreatic necrosis disease(AHPND)in aquatic organisms,but also cause human infection with acute gastroenteritis.At present,in order to quickly and effectively control the proliferation of pathogenic V.parahaemolyticus,the prevention and treatment of antibiotics were widely used in aquaculture,transportation and sales,which leaded to the development and spread of multi-antibiotic-resistant bacteria in pathogenic microbial communities.Bacteriophages are viruses that can specifically recognize,adsorb and lyse bacteria.Phage therapy is safe,pollution-free and has low side effects,and is considered to be a scientific and efficient biological control method.At the same time,phage and its specific binding modules can be used as important molecular elements for efficient and rapid detection of pathogenic bacteria.In this study,a lytic phage v B＿Vpa P＿GHSM17 of V.parahaemolyticus was isolated from the sewage from the Guangzhou Huangsha seafood market.By electron microscope observation,phage GHSM17 was a short-tailed phage with an icosahedral structure,with head length,diameter and tail length of 54±2,53±2,and 19.5±1 nm,respectively,and could form plaque on bacterial lawns.The spotting method was used to determine the host spectrum of the phage.GHSM17 could lyse 9 strains(9/17)of V.parahaemolyticus,but could not lyse other species of bacteria.The one-step growth curve of phage GHSM17 showed that it had an incubation period of 20 min and a rapid growth stage of 100 min,and the lysis amount was about 316 pfu/infected cell.Furthermore,GHSM17 exhibited a relatively wide temperature and p H stability range,indicating that the phage retains its activity well under room temperature conditions.After 70 min of UV irradiation with 100 W,the phages were all inactivated.Preliminary bactericidal experiments showed that GHSM17 inhibited the growth of V.parahaemolyticus within 8 h,making it a potential biofungicide candidate.Whole-genome sequencing of GHSM17 genome:The whole genome of GHSM17 was43.2 kb in length and encoded 45 ORFs.The 23 ORFs were similar to genes encoding known functional proteins,including DNA metabolic modules,lysis modules,packaging modules,structural modules and additional functional modules.Comparative genomics results and ANIm indicated that GHSM17 belonged to a new member of the genus Maculvirus in the family Autographiviridae.Phylogenetic tree analysis based on terminal enzyme large subunit and RNA polymerase further verified the taxonomic status of GHSM17.The three putative filament protein genes gp9/14/15 of phage GHSM17 were amplified by PCR and coupled with the green fluorescent protein gene to construct three vectors p ET-28a-GFP-gp9/14/15,and finally expressed and purified recombinant tail proteins GFP-gp9,GFP-gp14 and GFP-gp15.All three recombinant tail proteins exhibited excellent binding activity to V.parahaemolyticus,among which GFP-gp15 exhibited the strongest binding activity and species specificity,and was the best candidate for subsequent binding modules.GFP-gp15 was used to detect V.parahaemolyticus,the detection limit was 2×10~2CFU/m L,and the bacterial recovery rate in spiked oyster samples was over 40%.Multidrug-resistant V.parahaemolyticus endangers the global economy and human health.Bacteriophages have extremely broad research and development prospects in the control and rapid detection of multidrug-resistant bacteria.In this study,a vibrio lytic phage was isolated,and the physiological and biochemical characteristics and genomic analysis of the phage were carried out to further explore its potential as a biofungicide candidate and the application prospect of developing a rapid detection method.