Biological Characteristics Of A Novel Strain Of MRSA Phage SauPS-28 And Primary Expression Of Its Endolysin

Objective: In order to evaluate the new therapeutic methods of drug-resistant bacteria,the biological characteristics of phage and the expression of lysin were studied,which provided basic reference for phage therapy.Methods: A novel methicillin-resistant Staphylococcus aureus phage,named SauPS-28,was isolated from environmental samples using methicillin-resistant Staphylococcus aureus as bait.The morphology and structural feature of SauPS-28 were observed by transmission electron microscopy(TEM);the optimal multiplicity of infection(MOI),growth curve,acid-base and temperature stability were measured by plaque counting method with DLA(double agar plate).Whole genome was analyzed and annotated by homologous alignment after sequencing.Phylogenetic tree was mapped by MEGA-X software basing on the genome-wide comparison of ten other phages with similar homology.Structural proteins of phage were analyzed by sodium dodecylsulphate polyacrylamide gel electrophoresis(SDS-PAGE).Main bactericidal functional protein of phage,lysin,was cloned and expressed,and the expression conditions were optimized.Results:(1)SauPS-28 forming a uniformly sized,clear and translucent circular plaque.(2)SauPS-28 had a positive polyhedral structure at the top and a slender and long tail,which is consistent with the characteristics of Siphoviridae(long-tailed phages).(3)Seven drug-resistant bacteria could be lysed by SauPS-28,indicating that the host range of this phage is relatively wide.(4)SauPS-28 produced the highest titer of daughter phages at an multiplicity of infection of 0.01(5)The growth curve results showed that the latent period of SauPS-28 was about 30 min,and the rise period was about 40 min,which leveled off after 70 min.(6)SauPS-28 maintains good biological activity in the range of p H 6 to 9 and temperature 4℃ to 37℃.(7)Whole SauPS-28 genome digested by Eco R Ⅰ resulted in multiple bands,indicating that the SauPS-28 genome is double-stranded DNA.(8)SDS-PAGE showed that SauPS-28 has at least 12 structural proteins.(9)Total length of SauPS-28 genome was 43286 bp,and 68 ORFs were predicted,and the 36 th ORF was the lysin.(10)Lysin Lys28 was successfully expressed with the highest expression at an induction time of 5h,final IPTG concentration of 1 mmol/L and temperature of 30℃.Conclusion: In this study,a novel MRSA phage,SauPS-28,was isolated,its biological characteristics were investigated.Its whole genome was sequenced and analyzed,and its derived lysin protein,Lys28,was initially expressed.phage genome provides basic data for further exploration of the interaction mechanism between phage and host bacteria;phage structural protein analysis helps to understand the structural framework and related functions of phage.