The Bacteriophage Endolysin P9ly And Study On Its Antimicrobial Activity

With the massive use of antibiotics,the problem of bacterial resistance is becoming increasingly serious.Prevention and treatment of drug-resistant bacterial infections are becoming increasingly important,the development of new antimicrobial agents that can replace traditional antibiotics is urgent.The lytic enzyme encoded by the genome of phage can hydrolyze the peptidoglycan of bacterial cell wall and lyse the bacterial body,which can not only kill drug-resistant bacteria rapidly and efficiently,but also is less likely to develop drug resistance.Therefore,lysozyme is a very promising antibiotic replacement and supplementation agent.In this paper,Shigella phage PDS9,which has been isolated from fecal samples of healthy broiler chickens in Fufeng breeder farm in Nanning,Guangxi Zhuang Autonomous Region,was used to study its biological properties,observe the morphology using transmission electron microscopy,extract the genome for sequencing and analysis.The gene sequence of the genome encoding the lytic enzyme was subjected to biological tests such as PCR amplification and molecular cloning,followed by purification of its induced expression to obtain the target protein and study its enzymatic properties.The lytic activity of lytic enzyme P9ly against common laboratory strains was studied by Oxford cup method,scanning electron microscopy,plate coating method and turbidimetric analysis.Finally,the bactericidal effect of P9ly in combination with antibiotics was studied by plate coating method and turbidity method to lay the theoretical foundation for the further application of the subsequent lytic enzyme.The results showed that phage PDS9 belongs to the family of long-tailed phage with a broad host spectrum,it can lyse not only the multi-drug resistant host bacterium BDS9,but also Shigella fowleri and Shigella sonnei.The optimal infection complex of phage PDS9 is 0.1,the incubation period is about 20 min,the outbreak period lasts 90 min,the potency remains basically stable between 4 and 40?and the p H value remains high in the range of 5 to 11.Phage PDS9 is a double-stranded DNA virus with a full-length genome of 169,768 bp,GC content of 39.46%,263ORFs,of which 199 are known functional coding sequences.The lysozyme gene P9ly is located on ORF190 with a size of 489 bp,which is highly similar to Lyz-like superfamily and belongs to T4-like lysozyme.The recombinant protein of lysozyme gene P9ly has a molecular weight of 21.51KDa,the optimum action temperature is 37?,the optimum p H is between7 and 8,the metal ion K⁺has a promoting effect on P9ly activity was promoted by metal ion K⁺,Mg²⁺,Mn²⁺,Ca²⁺had obvious inhibitory effect,the enzyme activity was the best when the salt ion concentration was 300m M.P9ly(112?g/m L)can form a significant inhibition circle with the host bacteria BDS9,E.coli and S.aureus;P9ly(75?g/m L)showed a significant lytic effect on the host bacteria BDS9,E.coli and S.aureus as observed by scanning electron microscopy;P9ly(75?g/m L)could reduce the bacterial count by at least one order of magnitude when it incubated with the host bacteria BDS9,E.coli,E.coli O157,Salmonella,Staphylococcus aureus,Staphylococcus argenteus,Shigella fowleri,Shigella sonnei and Salmonella enterica for 60 min at 37?;P9ly(50?g/m L)showed a significant degradation effect on the cell wall of host bacteria BDS9,E.coli,E.coli O157,Salmonella,Staphylococcus aureus,Shigella fowleri,Shigella sonnei,Klebsiella pneumoniae,Staphylococcus argenteus,Salmonella enterica,and Pseudomonas aeruginosa.It can be seen that lysozyme P9ly has a broad antibacterial spectrum and better antibacterial activity.P9ly(75?g/m L)and ceftriaxone sodium(5?g/m L)were mixed in equal volume and incubated with Shigella BDS9,Staphylococcus aureus,E.coli,Shigella fowleri,Shigella sonnei,Shigella dysenteriae,Staphylococcus argenteus,Salmonella enterica and Salmonella for 60 min at 37?,and their antibacterial effect was better than both of them alone.The combination of the above two reduced the concentration of ceftriaxone sodium by half and showed the same antibacterial activity.In summary,this thesis investigates the biological properties of the Shigella phage PDS9 and its lytic enzyme P9ly.The lytic enzyme P9ly has a broader antibacterial spectrum and good antibacterial activity,and its combination with ceftriaxone sodium can exhibit higher antibacterial activity,and at the same time can reduce the antibacterial concentration of ceftriaxone sodium,which is important for the prevention and control of bacterial infectious diseases.