Research On Isolation And Identification Of Porcine ExPEC Phage And Its Biological Characters

Escherichia coli is an essential cause of neonatal diarrhea,weaner diarrhea,sepsis,mastitis and other diseases in newborn piglets.Extraintestinal Pathogenic Escherichia coli（ExPEC）is an important member of E.coli.Not only can it normally colonize the intestinal tract,but also colonize the extraintestinal tissue through its unique virulence factors.Therefore,the extraintestinal multi-tissue organ can get infected,causing symptoms such as pneumonia,endocarditis,nephritis,meningitis and so on.In recent years,the clinical detection rate of ExPEC has shown an increasing trend year by year,and the economic losses caused to the pig industry have become increasingly significant.Currently,antibiotic drugs are commonly used in the clinical prevention and treatment of E.coli disease caused by ExPEC.However,with the overuse of antibiotics,the problem of ExPEC drug resistance has become more prominent,and the number of porcine ExPEC with multi-drug resistance is increasing,which seriously affects the prevention and treatment effect of porcine ExPEC infection.It is imperative to develop a safe and efficient alternative of antibiotics for the prevention and treatment of porcine ExPEC infections.As a type of viruses that invade bacteria,phages are ideal for the treatment of bacterial infections as alternative antibiotics because of their short research and development cycle,low cost,safety and efficiency.In this study,the biological characteristics of the selected phages are studied by isolating and selecting the porcine ExPEC phages.It aims to provide theoretical basis for the application and development of porcine ExPEC phages,and provide new ideas for the effective prevention and control of porcine ExPEC infection.In this study,three clinical isolates of porcine ExPEC dominant serotypes(serotypes are O₉₃,O₃₈and O₁₂₇,respectively,numbered in order AHquan18-1,JSxu18-3 and HByan18-2)are employed as host bacteria for the following experiments:（1）Collect samples of manure sewage from pig farms,separate lytic bacteriophages by double-layer agar plate method,and proliferate and purify the separate phages.By measuring their lysis profile,the phages are initially selected and the morphological characteristics of the selected phages are observed using transmission electron microscopy.（2）Physiological and biochemical characteristics such as phage thermal stability,p H stability,multiplicity of infection（MOI）,and one-step growth curve are measured.（3）Extract the whole genome of phages,identify their nucleic acid types,use high-throughput sequencing technology to sequence the whole genome of phages,and analyze them in bioinformatics such as gene function prediction,functional annotation and phylogenetic tree construction.The results showed that:（1）Five strains of phages are isolated and named PC2,PC3,PH3,PH4 and PW4 respectively.The lytic rates of five phages to 72 porcine ExPEC strains are 2.78%（2/72）,8.33%（6/72）,8.33%（6/72）,6.94%（5/72）and5.56%（4/72）respectively.In addition,PC3 can also lyse a genetically engineered strain of E.coli DH5α.Three bacteriophages（PC2,PC3 and PH4）are screened for follow-up tests.Transmission electron microscopy（TEM）observed that the heads of all three phages show a positive icosahedral structure with obvious tails,belonging to the Caudovirales.PC2 is siphoviridae,PC3 and PH4 belonged to the Myoviridae.（2）Three strains of phages show good stability in the environment of temperature40℃～50℃and pH5～11.The optimal MOI for PC2 is 0.00001,the incubation period is about 20 min,and the average lysis volume is 11 PFU/m L.The optimal MOI of PC3 is 0.0001,the incubation period is about 20 min,and the average lysis volume is515 PFU/m L.The optimal PH4 MOI is 0.001,the incubation period is about 20 min,and the average lysis volume is 226 PFU/m L.（3）All the nucleic acids of the three phage strains are double-stranded DNA.The total length of PC2 genome is 45201 bp,GC content is 54.57%,54 coding gene sequences are predicted,and 25 coding genes with known function are identified.There is only 1 base difference between the PC3and PH4 genome sequences.The total length of PC3 and PH4 genomes is 155895 bp,among which the GC content is 48.79%,192 coding gene sequences are predicted,and 43 coding genes with known function are identified.PC2,PC3 and PH4 genomes do not carry virulence genes and antibiotic resistance genes.The results show as follows:（1）In this study,three porcine ExPEC phages（PC2,PC3 and PH4）are successfully isolated and selected.PC2 is siphoviridae,and PC3and PH4 are Myoviridae.（2）The three strains of phages have good heat resistance and acid and alkali environmental resistance,and at the same time have a short incubation period and strong pyrolysis performance.（3）None of the three phage genomes encoded virulence genes and antibiotic resistance-related genes,which has good application prospects.（4）PC3 phage host spectrum can cover PH4,and the best MOI and p H stability are better than PH4,and there is no difference between them in genome analysis.Therefore,PC2 and PC3 phages can be used for later clinical application researches.