| In recent years,due to the unreasonable use and even abuse of antibiotics,the problem of Salmonella resistance has become more serious.Bacteriophage is a virus that can specifically adsorb and infect bacteria and has a natural bactericidal ability.In order to find a new antibacterial treatment,6 multi-drug resistant Salmonella strains(SA209,SH01,SH16,SH19,SH20,SH171)were used as host bacteria,mixed with treated sewage from a large-scale farm in Anyang City.After centrifugation,phage containing solution was obtained,the phage was isolated and identified by the spot method and the double-layer plate method.The obtained phage isolate was purified and concentrated,and the morphology and structure of the obtained phage isolate were observed by transmission electron microscope,and then the nucleic acid type of the phage was determined.The optimum multiplicity of infection,one-step growth curve,temperature stability,acid-base stability,phage lysis profile were investigated,and the phage organic solvent sensitivity,salt ion concentration on the phage activity,and phage in vitro antibacterial effect were further determined.The results will provide a scientific basis for bacteriophages as a new antibacterial agent for the prevention and treatment of Salmonella infection,which has important theoretical significance and application value.The main findings are as follows:1.Phage isolation and identificationUsing Salmonella SA209 and SHO1 as host bacteria,2 strains of Salmonella phage were isolated and named PSA-1 phage and PSH-1 phage,respectively.2.Phage morphology identificationThe heads of the bacteriophage PSA-1 and PSH-1 are both typical icosahedral structures;the total length of the two phages is 75.9 nm and 272.5 nm,respectively,and the head length is about 61.1 nm and 69.3 nm,respectively;the phage PSA-1 does not have the obvious tail structure and belongs to short-tailed bacteriophage;bacteriophage PSH-1 has a tail of about 203.2 nm,which belongs to long-tailed bacteriophage.3.The optimal multiplicity of infection of phageThe phage was infected with host bacteria by setting different infection complex numbers(0.001,0.01,0.1,1,10,100),and then the phage titer was determined.The results showed that the optimal multiplicity of infection of bacteriophage PSA-1 was 0.001,and the optimal multiplicity of infection of bacteriophage PSH-1 was 0.01.4.One-step growth curve of phageThe incubation period of bacteriophage PSA-1 infecting the host bacteria is 10 min,the lysis period is 20 min,and then it enters the stable phase,with an average lysed amount of 108 PFU/cell.The incubation period of bacteriophage PSH-1 infecting the host bacterium is 20min,and the lysis period is after 80 minutes,the phage PSH-1 enters the stable phase,with an average cracking capacity of 495 PFU/cell.5.Temperature and pH stabilityPSA-1 and PSH-1 were treated at different temperatures(40? 50?,60?,70?,80?)and different pH(2,3,4,5,6,7,8,9,10,11,12,13).The phage stability was determined by the determination of the phage titer.Both PSA-1 and PSH-1 maintained stable activity at pH 3-11 and 40?-50?.6.Organic solvent sensitivity testSome of the nucleocapsids of phages are coated with a capsule composed mainly of proteins and lipids,which will be destroyed when exposed to organic solvents,resulting in reduced potency or inactivity of the phage.PSA-1 and PSH-1 were treated with chloroform and isopropanol for a period of time,and the titer of the two phages was not affected,indicating that the nucleocapsids of PSA-1 and PSH-1 had no capsule structure.7.Identification of phage nucleic acid typeThe genetic material of PSA-1 and PSH-1 was extracted with viral DNA/RNA extraction kit.After the treatment with DNase I and RNase A,the nucleic acids of the two phages were confirmed to be DNA by gel electrophoresis.8.In vitro antibacterial test of phagePSA-1 and PSH-1 could inhibit the growth of host Salmonella in milk at different temperatures by adding phage and inoculating host bacteria in commercial milk. |
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