Study On The Activity Of Klebsiella Pneumoniae Phage And Its Antibacterial Enzymes

Klebsiella pneumoniae(Kp),an important zoonotic opportunistic pathogen,is becoming increasingly drug resistant under conditions of long-term antibiotic use.Phages and their derivatives are expected to be used as promising antibiotic substitutes for the prevention and control of Kp.In this study,Kp phages were isolated from sewage samples and phages,endolysins and depolymerases with good antibacterial activity in vitro and in vivo were obtained;The metabolic and immune response of phage vB_KoxP_ZX8 in mice was analysed to address the issue of phage metabolic kinetics that needs to be addressed prior to industrial application of phages;The mechanism of host Kp111-2 resistance to phage vB_KpnP_ZX1 was analysed to address the issue of phage resistance prior to industrial application of phages.1.Screening and genomic analysis of Kp phagesFour Kp phages were isolated from sewage samples and subjected to biological characterisation,genomic analysis and in vitro antimicrobial activity to obtain phages with antimicrobial applications.The results showed that the four phages had a capsule-specific host range and broad temperature and pH adaptations.None of them contained virulence factors or drug resistance genes,and only vB_KpnP_ZX1 encoded three lysogenicity-related genes(integrase,repressor and anti-repressor).All four phages encoded a large number of novel proteins and endolysin,and only vB_KpnP_ZX1 encoded a depolymerase.Compared to the other three phages,vB_KoxP_ZX8 had the best in vitro antibacterial activity,clarifying bacterial cultures by lysis within 30 min at MOI=1 and maintained for 6 h.2.Metabolic kinetics and antibacterial study of phage vB_KoxP_ZX8In vivo safety analysis,metabolism analysis,antibody level analysis,antibacterial activity analysis and phage resistance analysis of phage vB_KoxP_ZX8 in mice were performed to address the issue of phage metabolic kinetics.The results showed that the metabolism of vB_KoxP_ZX8 by single intraperitoneal injections of 5×107 pfu and 5×109 pfu in mice was 24 h and 48 h,respectively,with the spleen,liver and thymus being the major metabolizing organs.vB_KoxP_ZX8 did not cause any adverse reactions within one week after injection and only the high dose of phage caused fluctuations in inflammatory factors within 3 days.Within one month after injection of vB_KoxP_ZX8,low doses of phage induced low levels of specific IgM and IgG antibodies(both with a maximum potency of 1:16);high doses of phage induced low levels of specific IgM and IgG antibodies(with a maximum potency of 1:64 and 1:32,respectively)and neutralising antibodies with a maximum potency of 1:256.The binding activity of phage-specific IgG antibodies to capside and tail fiber protein was characterised by ELISA.An in vivo antimicrobial assay showed that a single intraperitoneal injection of 5×107 pfu of vB_KoxP_ZX8 1 h after KpADl challenge increased survival from 0 to 100%in bacteraemic mice,did not induce the production of phage-resistant strains and significantly improved histopathology caused by bacterial infection.3.Analysis of phage receptor and receptor binding protein of vB_KpnP_ZX1The phage receptor and the receptor binding protein of the new phage vB_KpnP_ZX1 were analysed in order to solve the problem of phage resistance.Since vB_KpnP_ZX1 encodes an integrase,the possibility of vB_KpnP_ZX1 integration into host Kp111-2 was first excluded by phage gene overexpression assays,while two genes inhibiting phage proliferation were identified:anti-repressor and transcriptional regulator 1.Tail fiber proteins are primary receptor-binding proteins that initiate phage infestation by binding to cell surface capsular polysaccharides,as demonstrated by competitive adsorption assays.The capsular polysaccharide was identified as the major phage receptor by screening phage-resistant strains.Kp111-2 reduces bacterial surface capsular polysaccharides to block phage adsorption by single gene mutations in wbaP,wbaZ or wzc and is associated with reduced biofilm formation and decreased virulence.Furthermore,screened adaptive phage mutants of lysed phageresistant strains reinfect the bacteria by recognising new polysaccharide sites through endoglucanase mutations.Notably,bacteria can still rapidly evolve phage resistance in the face of adaptive phage mutant strains.4.Characterisation of the capsular polysaccharide degradation activity and antibacterial activity of depolymerase Dep_ZX1Sequence analysis,expression purification,environmental stability analysis,capsular degradation activity,biofilm degradation activity and in vitro and in vivo antibacterial activity of depolymerase Dep_ZX1 were carried out to analyse the key enzymes in phage infection of bacteria.The degradation activity of Dep_ZX1 against K57-type capsule polysaccharide was characterised by antibacterial zone test and TEM image.Dep_ZX1 at 10μg/mL significantly prevented the biofilm formation,degraded the biofilm formed and reduced the minimum bactericidal concentration of Kp111-2 in the biofilm when combined with the antibiotics gentamicin,streptomycin and kanamycin.Stability tests showed that Dep_ZX1 could be used at 25-50℃ and pH 4.0-10.0.Dep_ZX1 was able to increase the phagocytic capacity and serum bactericidal capacity of mouse phagocytes,and 50 μg of Dep_ZX1 was able to increase the survival rate of bacteremic mice from 0 to 100%by intraperitoneal injection 1 h before and after Kp111-2 challenge.5.Characterisation of the peptidoglycan degradation activity and antibacterial activity of endolysin Lys_ZX4Sequence analysis,expression purification,environmental stability analysis,peptidoglycan degradation activity and in vitro antibacterial activity of the endolysin Lys_ZX4 were performed to analyse the key active enzyme in phage lysis of bacteria.Neither Lys_ZX4 nor 0.5 mM EDTA alone showed antibacterial activity due to the outer membrane barrier of Gram-negative bacteria,Lys_ZX4-NCA with the antimicrobial peptide KWKLFKI showed antibacterial activity,and Lys_ZX4 or Lys_ZX4-NCA combined with 0.5 mM EDTA showed significantly higher antibacterial activity.The in vitro antibacterial test showed that 100 μg/mL of endolysin Lys_ZX4 or Lys_ZX4-NCA combined with 0.5 mM EDTA for 4 h killed 99.9%of KpO4,and the antibacterial effect was higher against log phase bacteria than against mature phase bacteria.Lys_ZX4-NCA showed broad-spectrum antibacterial activity against Gramnegative bacteria,including Klebsiella pneumoniae,Escherichia coli,Proteus mirabilis and Citrobacter.Stability tests showed that Lys_ZX4-NCA was suitable for temperatures of 2565℃ and pH of 6.0-9.0,and its antibacterial activity was inhibited by 1 mM Mg2+,10 mM Ca2+ and 20%serum.Ly s_ZX4-NCA was more suitable for the treatment of specific infections,and treatment with EDTA on the skin of KpO4-infected mice for 4 h reduced the number of bacteria by 3 logarithms.In conclusion,the novel phage vB_KoxP_ZX8 screened in this study was able to specifically degrade Kl-type Kp,with low levels of specific antibodies,high antibacterial activity and no induction of phage resistance in mice,and is expected to be used to control Kl-type Kp-induced infections;The novel depolymerase Dep_ZX1 was able to specifically degrade K57-type Kp capsular polysaccharide and degrade biofilm,improve phagocytosis,serum bactericidal activity and survival of bacteremic mice,and is expected to be used to control K57-type Kp infections or ductal infections caused by biofilms;The constructed recombinant endolysin Lys_ZX4-NCA has broad-spectrum antibacterial activity against Gram-negative bacteria and is expected to be used for local infections caused by Gramnegative bacteria;Kp111-2 reduces bacterial surface capsular polysaccharide blocking phage adsorption by single mutations in wbaP,wbaZ or wzc,and Kp phage vB_KpnP_ZX1 recognises new polysaccharide sites for re-infection of bacteria by mutations in endoglucanase is a novel finding in the face of phage-resistant strains.