Genomic Analysis Of Bacteriophage P46FS4 Infecting Salmonella And The Study Of Its Endolysin

Named by american bacteriologist D.E.Salmon,Salmonella is a gram-negative bacterium and belonging to the enterobacteriaceae,which is a common foodborne zoonoses.In nature,it is a widespread and extre mely harmful foodborne pathogen.Because of the abuse of antibiotics,the situation of Salmonella resistance to antibiotics has become increasingly serious.More and more multi-drug resistant strains has made Salmonella drug resistance a widespread problem at home and abroad.People began to limited the use of antibiotics and develop alternative antibiotic drugs.The use of phage and endolysin in the treatment of pathogenic bacteria can not only reduce the current environmental pressure caused by the abuse of antibiotics,but also not easily produce residue.In this study,Salmonella bacteriophage was measured its biological characteristics and genomic analysis.The expression and modification of P46FS4 endolysin were performed to achieve better antibacterial effect in vitro.The contents of this study include the following:1.In this study,Salmonella was used as the host strain.A bacteriophage was isolated from sewage and named P46FS4.Observed by transmission electron microscopy(TEM),the hesd of the bacteriophage was an icosahedral capsid.The diameter of the head was 90.5 nm and the length of the tail was 123.1 nm.It belonged to the Caudovirales and Myoviridae family.The biological characteristics of Salmonella phage P46FS4 showed that its optimal multiplicity of infection(MOI)was 0.001,and the titer was up to 10~8PFU/ml.The host rang showed that the phage was able to lyse 4 strains of E.coli(4/16)and 56 strains of Salmonella(56/62).The bacteriophage P46FS4 can still maintain 10~7 PFU/m L under 50℃,and the titer drops sharply when it above 60℃.Under the condition of 1%chloroform treatment,the titer of the bacteriophage P46FS4 was able to maintain 10~6PFU/m L within12 h,and was 0 after 18 h.The one-step growth curve of phage P46FS4 showed that its incubation period was about 50 minutes and lysis period was 120 minutes with a lysis amount of 64 PFU.2.The P46FS4 genome is 158,706 bp with a GC content of 50.44%,and the sequence number of Gen Bank was MT078988.Using bioinformatics software for gene prediction and functional annotation,a total of 213 CDS and 3 t RNA genes were predicted.88 CDS were functionally known,while other 122 CDS sequences corresponded to unkown putative genes,and 3 CDS were unpredictable.Through genome-wide sequence alignment analysis,it was found that P46FS4 has the closest relationship to Salmonella SKML-39,and there are co-linearity and homology between the ir genomes.3.This study successfully expressed a soluble endolysin protein,which can effectively inhibit bacteria in vitro.In addition,in order to improve the antibacterial effect of endolysin in vitro,different amounts of hydrophobic amino acids were added to the C-terminus of endolysin.The results show that different amount of hydrophobic amino acids have different antibacterial effects in vitro.Compared with other proteins,the addition of 3hydrophobic amino acids at the C-terminus of the endolysin can achieve a significant antibacterial effect without the host bacteria being treated.Adding 12 hydrophobic amino acids to the C-terminus,it has a more significant antibacterial effect than other proteins after treating the host bacteria with EDTA or chloroform.In summary,the Salmonella bacteriophage P46FS4 isolated in this study is belonging to the Caudovirales and Myoviridae family with a wide host rang.By expressing phage endolysin and modifying its C-terminal hydrophobic amino acid,the endolysin is changed to achieve better antibacterial effect.This study lays a foundation for the clinical application of the prevention and treatment of bacterial diseases.