Preparation And Separation/purification Of Klebsiella Phage

The crisis of antibiotic resistance has been brought the global attention and will lead to the era of no antibiotics available.Bacteriophages are expected to be a potential treatment for multidrug-resistant bacterial infections due to their great antibacterial effect.The purpose of this study is to explore the safety of Klebsiella phage,and to provide a feasible method for the preparation and purification of large scale bacteriophages.In this paper,the safety of Klebsiella phage was investigated with bioinformatics method,and the preparation of phage by two-step fermentation to produce 1,3-propanediol was investigated.First of all,a phage stored in our lab was analysis based on the genomics bioinformatics,mainly focus on the building of evolutionary tree which based on homology of the tail fiber protein gp45.Besides,phages downloaded were listed with their function modules.The stability of phages was evaluated by statistics of the number and sites of restriction enzymes in genomes.The safety of phage was confirmed by comparing all phage ORFs?open reading frame?with genes in resistant gene database and the virulence genes database.In addition,the lysogenicity and virulence of Kp34 phages were analyzed,and it was found that among the 9 phages,in which two strains were isolated from hospital clinical samples,and only 3 strains were found to be template phage,and Kp34 phage had a good application prospect.Secondly,after the simple screening of fermentation medium,glycerol was chosen as substrate,Klebsiella pneumonia S2 was used for production of 1,3-propanediol using glycerol.From the point of energy saving,downstream processing,the safe dispose of the fermentation of Klebsiella pneumonia and cost of phage preparation,we tried a two-step fermentation conjugation phage preparation and product of 1,3-propanediol.In which,fermentation time,MOI and infection medium were optimized.When the fermentation time was 15 h,the titer was 2.6×10¹⁰PFU/mL,and the concentration of 1,3-propylene glycol was 39.73 g/L.The initial host bacteria concentration had a great influence on the final obtained phage titer.The titer obtained from batch feed fermentation broth at 9 h was up to 9.25×10¹00 PFU/mL,which effectively increased the phage titer.In addition,the optimal MOI was found to be influenced by the age of the host bacteria.Finally,focus on problems existing in the separation and purification of large-scale phage,we explored two-step salting-out extraction to realize the separation and purification of phage from host cells,cell debris,proteins and endotoxin.In the first SOE consisted in sodium citrate-ethyl acetate system,phages partitioned to the bottom phase,assisted by centrifugal to remove most of the bacteria and debris.Then,in the second SOE,n-propanol was poued into the bottom phase in first SOE,in which,phage preferred to the interphase.With the two-step SOEs,77%phages were recovered,96%proteins,96%cells and 83%endotoxins were removed.The purification fold of phage for protein,cell and endotoxin were 19.31,18.04 and5.19,respectively.Importantly,the concentration fold of phage was 500 compared with that of crude phage.The research indicated that SOE may be an effective technique for purification of phages in the future.This paper focuses on the application of phages,and from the perspective of industrial application,we tried to solve the problems of safety of genome,preparation,separation and purification,which will pave the way for the wider application of phages.