Study On The Biological Characteristics Of Salmonella Phage S5 And Its Preliminary Application

Salmonella is an unencapsulated,non-spore-forming Gram-negative bacterium with rounded ends and belongs to the Enterobacteriaceae family.So far,more than2,600 serotypes have been identified worldwide.Salmonella has no host specificity and can cause a variety of animal and human diseases such as cow,sheep,pigs,chickens,and ducks,which is an important zoonotic pathogens.At the same time,Salmonella,as a common food-borne pathogen,can cause acute gastroenteritis,food poisoning,sepsis and other diseases in humans and animals.Salmonella not only hinders the development of livestock and poultry breeding industry,but also poses a serious threat to human health and public health and safety.Antibiotics are one of the main means to prevent and treat Salmonella infection.However,the long-term,widespread and irrational use of antibiotics makes the drug resistance of Salmonella increasing day by day and the drug resistance mechanism is complex,resulting in a great decrease in the therapeutic effect of antibacterial drugs,and clinical therapy difficulty.Under the pressure of comprehensive restriction use of antibacterial drugs in farms nowadays,phage therapy provides new ideas and methods as an alternative to antibiotics.In this study,a lytic Salmonella phage was isolated and purified from the sewage of a chicken farm in Chengdu,Sichuan Province.Through the isolation and identification of the phage and its biological characteristics research,as well as the preliminary study of the prevention and therapy test of the Salmonella phage,it provides a good research material for the development of new antibacterial agents for Salmonella induced infections.1.Biological characteristics of Salmonella phage S5 and bioinformatics analysis of phage isolatesIn this study,20 Salmonella strains preserved in the laboratory were used as host bacteria,and a Salmonella phage was isolated from sewage samples in a chicken farm by the double-layer agar plate method and named S5.Preliminary identification results showed that the phage formed round,radiolucent plaques with neat and clear edges on double-layer agar plates.After negative staining with 2%phosphotungstic acid for 5min,their morphological structure was observed by transmission electron microscopy.Transmission electron microscopy results showed that the head of phage S5 was ortho-icosahedral symmetrical with a diameter of 50 nm,it had a long tail with a body length of 200 nm.According to the classification rules of the International Committee on Taxonomy of Viruses,this phage is a member of the order Tail Phage,family Long-tailed Phages.The results showed that the phage could successfully lyse2 strains of Salmonella Kastrup and 13 strains of Salmonella typhimurium preserved in the laboratory,and the lysis rate was 75%(15/20).The titer of phage lysate was8×10⁹PFU/mL as determined by double-layer agar plate method and plate counting method by measuring the biological characteristics such as thermal stability and pH stability,optimal multiplicity of infection(MOI),and one-step growth curve of phage S5,the results showed that the phage titer remained basically unchanged at the temperature from 30°C to 60°C for 30 min or 1 h,there had little effect on phage activity at pH 4.0?pH11.0,and pH 9.0?11.0 was the optimal pH.The optimal multiplicity of infection for this phage was 0.0001.The results of the measured one-step growth curve showed that the titer remained basically unchanged within 5min(incubation period)after infecting the host bacteria,and from 5 min to 100 min after the phage infecting the host bacteria,the phage proliferated in large numbers,with an outbreak period of about 95 min and an average lysis amount of 50 PFU/cell.The above results showed that the phage S5 isolated and identified in this study had a wide temperature stability and acid-base tolerance range,belonged to a long-tailed bacteriophage family with strongly lytic activity.The results of bioinformatics analysis showed that phage S5 is a double-stranded DNA virus with linear ds DNA of nucleic acid type.The genome is 72519 bp in length,has a GC content of 39.45%,containing nine t RNAs and 109 predicted ORFs,and no genes carrying any deleterious products such as bacterial virulence factors were found.Phage S5 is a virulent phage with high application safety.It provides a good research material for further research and development of phage preparations and lays a foundation for the development of new antibacterial agents for infections caused by Salmonella.2.Control effect of Salmonella phage S5 on Salmonella infection in chicksIn this study,the median lethal dose(LD₅₀)of Salmonella was determined by challenge test,and 2.5×10⁹PFU/mL phage was intraperitoneally injected at five time points including 6 hours before,3 hours before,at the same time,3 hours after,and 6 hours after Salmonella infection,respectively,verifying that the phage had a preventive and therapeutic effect on salmonellosis and exploring the optimal therapeutic dose.The results showed that the LD₅₀of Salmonella was 1×10⁸CFU/mL,and inoculation of phage lysate 6 hours before,3 hours before,at the same time,3hours after,and 6 hours after Salmonella infection could effectively prevent and control salmonellosis and reduce mortality.Inoculation of phage lysate at these two time points,the same time and 3 hours after Salmonella infection,can achieve better control effect in experimental chicks infected with Salmonella,with a protection rate as high as 90%,and the protection rate of prevention and control effect is greater than that of the antibiotic treatment control group.The results showed that the protective effect of phage on chicks increased with the higher concentration of phage.When the Salmonella challenge dose was 1×10⁸CFU/mL,the phage therapeutic dose was2.5×10¹⁰PFU/mL could achieve the best therapeutic effect.The results showed that phage S5 had a good preventive and therapeutic effect on salmonellosis,which laid a foundation for the development of phage preparations.