Isolation,characterization And Application Of A Phage Infecting Carbapenem-resistant Klebsiella Pneumoniae

Background:Carbapenem-resistant Klebsiella pneumoniae?CRKP?strains are resistant to almost all first-line antibiotics and commonly cause hard-to-treat and fatal infections.And,Hypervirulent?hypermucoviscosity?Klebsiella pneumoniae can cause severe infections,such as liver abscesses and meningitis,in healthy people.The emergence and increase in carbapenem-resistant hypermucoviscous Klebsiella pneumoniae?CR-HMKP?may lead to more serious and complex public health problems.A bacteriophage?phage?is a type of virus that can kill bacteria precisely and efficiently.In the 1920s,phages with highly effective bactericidal activity were recognized as potential antibacterials and used to treat bacterial infections.We have studied a novel phage's basic physiological features,performed an analysis of its genome sequence and evaluated the potential applications of kpssk3 in a mouse model.Materials and methods1.A lytic phage infecting CRKP was isolated from raw sewage from Southwest Hospital using the host strain NY03 by double-layered agar method.2.To determine the morphological characteristics of kpssk3,the phage was concentrated by caesium chloride?CsCl?gradient centrifugation,stained with 2%phosphotungstic acid and observed using a transmission electron microscope at a voltage of 100 kV.3.A one-step growth experiment was performed to reveal the latency time and burst size of the phage.4.The host range of phage kpssk3 was determined by a spot test and a double-layer agar plate test using 77 different bacterial strains.To further test the lytic activity of kpssk3 and its ability to infect different bacteria,a double-layer agar plate test was performed to determine the efficiency of plating.5.To investigate the stability of kpssk3 under various conditions,we determined its titer at different temperatures?4?,25?,37?,45?,50?,55?,60?,65?and 70??and pH values?2-11?.The double-layer agar method was used to determine the phage titer after temperature and pH treatment.6.A standard phenol-chloroform extraction protocol was used with modifications for phage DNA isolation.DNA was sequenced using the Illumina MiSeq platform.Open reading frames?ORFs?were predicted using GeneMarkS.The functions of the proteins encoded by each ORF were predicted using BLASTp and further confirmed using the Pfam database.ORFs were also compared against Virulence Factors of Pathogenic Bacteria and Antibiotic Resistance Genes Database to verify the safety of the phage.rRNA and tRNA genes were analyzed using RNAmmer and tRNAscan-SE,respectively.To investigate the evolutionary history of kpssk3 and to determine its taxonomic position,a phylogenetic tree was constructed using Molecular Evolutionary Genetic Analysis based on the amino acid sequences of the major capsid protein.7.The CRKP-induced bacteremia mouse model was established by ip injection of 2×LD₁₀₀ dosage of early log-phase NY03,which was followed 3 hours later by treatment with different antibacterial agents,including phage,polymyxin B,tigecycline and ceftazidime/avibactam plus aztreonam.8.Fecal samples were collected from the phage-treated mice at 3 time points before?0 d?and after?3 and 10 d?phage therapy to study the change of gut microbiome by high-throughput 16S rDNA sequence analysis.9.The cytotoxicity of different concentration of kpssk3 to HeLa cells was tested using CCK-8 assay.At animal level,2 different doses of kpssk3 was injected into mice intraperitoneally twice daily.After 5 days,the mice were euthanized,at which point the spleen,lung,kidney and liver were harvested for histopathological analysis.Results:1.A CRKP phage was successfully isolated from sewage from Southwest Hospital,and named kpssk3.2.The morphological characteristics of kpssk3 observed by transmission electron microscopy,including a symmetrical,polyhedral head and a short tail,indicate that it belongs to the family Podoviridae.3.Phage kpssk3 has a relatively short latent period of approximately 10 min,and the burst size is approximately 200 phage particles per cell.4.Phage kpssk3 lysed 92.59%?25/27?of the clinical CRKP isolates,and did not form plaques on double-layer agar plates that contained lawns of other strains.5.The phage titer remained basically invariable from 4 to 45?at pH values from pH 4.0to 11.0.6.Phage kpssk3 had a linear dsDNA genome,40,539 bp in length,containing 42 open reading frames.No rRNA,tRNA,antibiotic resistance,virulence or integrase genes were found in the phage genome.It is a T7-like virus in the subfamily Autographivirinae.7.Intraperitoneal application of a single dose of phage kpssk3 3 hours postinfection protected 100%BALB/c mice against bacteremia induced by NY03.Three groups of antibiotics were used to rescue the infected mice,with the 7-day survival rates being 20,20and 90%,respectively.8.16S rDNA sequence analysis revealed no notable distortion of the gut microbiota except the decrease in Chao1 and ACE indexes.9.Phage kpssk3 was not shown to be cytotoxic to mammalian cells in vitro and in vivo.Conclusions:In summary,kpssk3 is a newly isolated lytic phage.Its strict host specifcity,possible ability to degrade capsular exopolysaccharides,favorable stability,and ideal genetic characteristics make kpssk3 a promising candidate for phage therapy.Phage kpssk3significantly improved the survival rate of mice with CRKP-induced bacteremia.Additionally,both in vivo and in vitro studies showed no significant cytotoxicity by kpssk3,and there was no evidence of significant changes in intestinal flora during treatment.kpssk3 provides a new and effective method for the treatment of CRKP bacteremia and has great clinical application potential.