Efficacy Of Bacteriophage In The Treatment Of Multidrug-resistant Klebsiella Pneumoniae-induced Pneumonia

Background:Klebsiella pneumoniae（Kpn）,a Gram-negative bacterium belonging to the Enterobacteriaceae family,is widespread in a variety of environments and is the main cause of hospital and community acquired pneumonia.At the same time,the effectiveness of most available antibiotics against Klebsiella pneumoniae decreased significantly in the face of antibiotic pressure and evolving Kpn itself.Multidrug-resistant（MDR）Klebsiella pneumoniae,such as broad-spectrumβ-lactam and carbapenem resistance,has been identified as a serious public health threat and has been named a"superbug"by the World Health Organization.As a new method against MDR bacteria,phage therapy has shown positive clinical effects,and more and more evidence shows that phage is effective as a synergistic or alternative therapeutic strategy for antibiotics.Phages are highly strain-specific viruses that kill the host bacteria with precision and metabolize automatically after removing them from the body.Although there are some problems with using phages,including bacterial resistance to phages and the rapid elimination of phages by the immune system as potential invaders.However,phage cocktails are thought to be an effective way to overcome these problems.Objective:To explore the efficacy of phage cocktail and single phage in the treatment of MDR Klebsiella pneumoniae infection,and to provide a new perspective for the clinical treatment of multidrug-resistant Klebsiella pneumoniae infection.Methods:Using ST11 strain C10 and ST383 strain C6 as host bacteria,two bacteriophages were isolated from hospital sewage.Morphological and biological characteristics of the two bacteriophages were measured by electron microscope,host spectrum,optimal multiplicity of infection（MOI）,one-step growth curve,in vitro host cleavage potency,thermal stability,pH stability,and adsorption receptor identification.Phage genome sequencing and gene function annotation were performed.Phage cytotoxicity was detected using A549 cell model.In animal experiments,C57BL/6J mice aged 7 weeks were infected and administered by nasal drops.The efficacy of phage single preparation and cocktail was confirmed by the survival curve of mice,the bacterial load and cytokine levels in the lungs and histopathology of lung,liver and kidney.Results:The phages targeting MDR Klebsiella pneumoniae were isolated from hospital sewage and named pKp11 and pKp383.Transmission electron microscopy（TEM）showed that phage pKp11 belonged to the Podoviridae family,and phage pKp383 belonged to the Siphoviridae family.Two phages can lyse 12 sequence types（STs）of Klebsiella pneumoniae that carry multiple resistance genes.The optimal MOI of pKp11 and pKp383 were 0.01 and 0.001,the incubation period was 10 min,and the outbreak time was 100 and 110 min,respectively.When MOI was above 10^-5,both bacteriophages could effectively inhibit the host strain.In addition,pKp11 and pKp383 remain stable at 4～50°C and pH 6～10.Lipopolysaccharide（LPS）of Klebsiella pneumoniae is a specific adsorption receptor for pKp11 and pKp383.Phage pKp11 and pKp383 showed no toxicity or drug resistance related genes.Even high concentration（10⁹PFU/m L）of phage on A549 cells showed no cytotoxicity.Animal experiments showed that mice treated with phage had significantly higher survival rates than untreated mice infected with Klebsiella pneumoniae isolates.In the single treatment group,bacterial load,inflammatory cytokines and pathological injury were reduced within 48h,while in the phage cocktail group,inflammation was rapidly inhibited and lung tissue injury was cured within 24h.In addition,no histopathological changes in the liver or kidneys were observed in any of the phage treatment groups.Conclusion:Phage pKp11 and pKp383 can lysate some strains of ST11 and ST383carrying multidrug-resistant genes,which have the characteristics of wide host range,high lysate activity and strong environmental adaptability.The two bacteriophages did not carry virulence and drug resistance genes and showed no cytotoxicity in vitro.Bacteriophage therapy especially bacteriophage cocktails can effectively treat pneumonia caused by the prevalent Klebsiella pneumoniae isolates ST11 and ST383in mice.