| Vibrio parahaemolyticus,as a halophilic gram-negative bacterial cell,can not only cause severe vibriosis in marine animal for disruption of economic development in aquaculture,but also cause infections even death in human by diarrhea,vomiting, abdominal cramps,nausea,fever and other symptoms.The main pathogenic factors of pathogenic Vibrio parahaemolyticus were TDH,TRH,TLH,T3SS1,T3SS2,Pir Avp,and Pir Bvp.As traditional method of preventing and controlling Vibrio parahaemolyticus, antibiotics was widely used in aquaculture farms,transportation chains and sales chains.However,fishermen blindly abused,leading to the outbreak of multidrug-resistant pathogenic bacteria.Therefore,it became very urgent to expand and develop of new probiotics.Bacteriophage was the most abundant virus population in the biosphere.It can release hundreds of progeny phages by infecting and lysing the host.Compared with antibiotics,the phage can only specifically identify a genus or a species of pathogenic bacteria without destroying normal microbial community.The research and application of phages with broad-spectrum and efficient lysing ability to solve the problem of multi-drug resistance will be a scientific and efficient biological control method with broad prospects.In this study,three virulent phages,named v B＿VpS＿BA3（phage BA3）v B＿Vp P＿BA6 （phage BA6）and v B＿VpS＿CA8（phage CA8）,were isolated and purified from the sewage from the Huangsha aquatic products trading market in Guangzhou using foodborne Vibrio parahaemolyticus O1-1 and O3-11 as hosts,respectively.Regarding the host range of the61 Vibrio parahaemolyticus isolates,BA3,BA6 and CA8 had an infectivity of 8.2%,19.7%and 36.1%.By combining their morphological characteristics with the formal guidelines of the International Committee on Taxonomy of Viruses,phage BA6 was composed of an icosahedral head and a short tail,belonging to Podoviridae.Phage BA3 and CA8 exhibited typical characteristics of phages belonging to the family,Siphoviridae,in the order, Caudovirales.Bioinformatics analysis showed that phage BA3,BA6 and CA8 all carried linear ds DNA genome,the complete genome sequence of BA6 was 50,520 bp（115-bp repeat sequence）with 64 ORFs and GC content was 41.77%.The BA3 genome size was58,648 bp with 85 ORFs and GC content was 46.30%,while the CA8 genome size wad58,480 bp with 84 ORFs and GC content was 46.42%.The ORFs were associated with phage structure,packing,host lysis,DNA metabolism,and additional functions.Furthermore,nucleotide BLAST,ANIm,comparative genomic features revealed that BA3and CA8 represented different isolates but novel members of the family,Siphoviridae.Lastly,nucleotide pairwise sequence alignment based on BLASTN revealed that phages BA3 and CA8 were 97.32%identical to each other while ANI was 96.48%,thus,they can be considered different isolates of the same phage species.Here,biological characteristics of BA3 and CA8 was performed.Vibrio parahaemolyticus strain,O1-1/O3-11,when infected at an multiple of infection of 0.1,generated the maximum bacteriophage titer,indicating that 0.1 was the optimal multiple of infection for the phage BA3/CA8 titer.The assays revealed latent periods,defined as the time interval between the adsorption and the beginning of the first burst,of～20 min（phage BA3）and～30 min（phage CA8）.It was determined to be～100 pfu/cell for phage BA3 and～180pfu/cell for phage CA8 under the laboratory growth conditions.Test on controling Vibrio parahaemolyticus by phage showed that,phage BA3/CA8 can significantly inhibit food-borne Vibrio parahaemolyticus O1-1/O3-11 within 24 hours under the condition of multiple infection of 0.1.Bacterial killing assays showed that bacterial concentration was reduced by 3.65 and 3.63 log cfu/m L when O3-11 and CA8 were co-cultured for 16 and 24hours,respectively.All the data indicated that there was genetic diversity in the functional genes of both phages,which will be applied on the controlling of pathogenic bacteria in food and the treatment of animal bacterial diseases in the field of aquaculture in the future.Phage therapy with high research value and application prospects can promote economic development and safeguard human health. |
