The Biological Characteristics Of Klebsiella Pneumoniae Phage VB?KpnP?ZK1 And Its Depolymerase

Klebsiella pneumoniae is a common opportunistic zoonotic pathogen that can cause various bacterial infections and even death in humans and livestock.With the long-term irregular use of antibiotics in clinical and aquaculture,the problem of bacterial resistance has become increasingly serious.In particular,the drug resistance of K.pneumoniae has attracted the attention of the WHO and there is an urgent need to develop new antibacterial drugs.Especially the hypervirulent K.pneumoniae(hv Kp)strain dominated by serotype K1 has higher mortality,which seriously threatens human health and public health safety.As a natural killer of bacteria,bacteriophages can infect and kill bacteria.Some phages can encode depolymerases,which can efficiently and specifically hydrolyze capsular polysaccharides produced by bacteria.The capsular polysaccharide is an important virulence factor of serotype K1 K.pneumoniae.Once the capsular polysaccharide is destroyed,the body can easily remove the bacteria.Therefore,phages and their depolymerases show potential in preventing and controlling the infection of capsular polysaccharide-producing strains.In this study,the phage v B?Kpn P?ZK1(abbreviated as ZK1)was isolated using serotype K1 hv KP as the host.ZK1 can form plaques with a translucent halo,and was characterized and was found to belong to the family Podoviridae.ZK1 has good physical and chemical stability.The one-step growth curve showed that a eclipse period of ZK1 was only 1 min,a latent phase was only 2 min and a burst volume of about 161 PFU/cell,showing high efficiency.The mouse bacteremia model caused by Wkp26 infection was successfully established,and the in vivo therapeutic effect of phage ZK1 was evaluated.The results showed that 8×10~6PFU/mouse phage ZK1could effectively protect mouse and improve the survival rate of mouse,reduce the bacterial load in blood and main tissues,reduce the degree of pathological damage,and show the antibacterial effect and application potential in vivo.DNA extraction and genome sequencing analysis of the phage showed that ZK1had the highest homology of 93%with other phages,and 57 open reading frames were predicted.It was preliminarily determined that the tail fibers protein Depo16should have depolymerase activity through gene location,gene sequence and amino acid sequence alignment.Depo16 was expressed and showed significant depolymerization activity on K.pneumoniae K1 capsules specifically.And the results of capsular staining and scanning electron microscopy further proved that could remove the thick K1 capsular polysaccharide layer that surrounds the surface of K.pneumoniae.Depo16 showed stable depolymerization activity when exposed to temperatures of 4 to 50?and p H 4-9.Although Depo16 itself has no bactericidal activity,Depo16 and serum exhibit a synergistic effect when killing K.pneumoniae serotype K1 clinical isolates.Through this study,a phage that can specifically infect and kill serotype K1 K.pneumoniae and a high-efficiency depolymerase that can hydrolyze K1 capsular polysaccharide have been obtained.Both of them show potential in fighting serotype K1 K.pneumoniae,laying a solid experimental foundation for further therapeutic research.