A novel bacteriophage for targeting Klebsiella pneumoniae and its preparation method

Abstract

This invention discloses a novel bacteriophage for targeting Klebsiella pneumoniae and its preparation method, relating to the fields of microbiology and medical technology; comprising the following steps: S1: providing an environmental sample containing a lytic bacteriophage; S2: processing and filtering the environmental sample to obtain an initial filtrate containing the bacteriophage; S3: co-culturing the initial filtrate with the host bacterium Klebsiella pneumoniae CRKP-2503074069 to obtain a culture mixture; S4: filtering the culture mixture to obtain a bacteriophage lysate. The bacteriophage of this invention contains a glycoside hydrolase family 28 domain, a capsular polysaccharide depolymerase; this enables it not only to lyse bacteria but also to enzymatically lyse the thick capsule of Klebsiella pneumoniae, explaining the formation of its characteristic target-like plaques and endowing it with the additional ability to disrupt biofilm structures.

Description

Technical Field

[0001] This invention relates to the fields of microbiology and medical technology, and in particular to a novel bacteriophage for targeting Klebsiella pneumoniae and its preparation method. Background Technology

[0002] Klebsiella pneumoniae is a Gram-negative, encapsulated microorganism belonging to the Enterobacteriaceae family. It is a significant pathogen causing hospital-associated infections, particularly pneumonia, bloodstream infections, and sepsis in immunocompromised patients. As a member of the ESKAPEE pathogens, Klebsiella pneumoniae possesses a strong ability to acquire and transmit antimicrobial resistance determinants, rendering many traditional antibiotics ineffective.

[0003] Carbapenem-resistant Klebsiella pneumoniae (CRKP) has been listed by the WHO as a priority pathogen requiring the development of novel treatments. Although polymyxins were once used as a "last line of defense" antibiotic, their clinical application is increasingly limited by nephrotoxicity, neurotoxicity, and plasmid-mediated resistance. Furthermore, the thick capsular polysaccharide (CPS) of Klebsiella pneumoniae not only helps it evade immune clearance but also acts as a physical barrier restricting antibiotic penetration. Highly virulent variants (hvKp) further exacerbate this challenge due to enhanced CPS production and the presence of additional virulence factors.

[0004] To address the increasingly serious problem of antimicrobial resistance, bacteriophages are once again being considered a promising alternative. Lysogenic phages can infect and kill bacterial hosts with high specificity, self-amplify at the infection site, and co-evolve with drug-resistant mutants. Notably, many phages infecting encapsulated bacteria encode capsular depolymerases—a class of enzymes associated with viral particles that degrade cytosolic plasma structure (CPS) and facilitate host recognition. These enzymes not only enhance phage infectivity but also make bacteria more easily cleared by the host immune system. Although there are increasing reports on Klebsiella pneumoniae phages, the diversity of capsular serotypes and the rapid emergence of phage-resistant variants continue to necessitate the discovery and characterization of new phages. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by proposing a novel bacteriophage for targeting Klebsiella pneumoniae and its preparation method.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing a novel bacteriophage against Klebsiella pneumoniae, comprising the following steps: S1: Provide an environmental sample containing lysing bacteriophages; S2: Process and filter the environmental sample to obtain an initial filtrate containing bacteriophages; S3: Co-culture the initial filtrate with the host bacterium Klebsiella pneumoniae CRKP-2503074069 to obtain a culture mixture; S4: Filter the culture mixture to obtain phage lysate; S5: The phage lysate is inoculated onto the surface of a solid culture medium containing the host bacteria, and after culturing, a single transparent phage plaque is picked out; S6: The selected plaques are purified in multiple rounds until a uniform plaque morphology is obtained, resulting in purified lytic phage KPTJT11.

[0007] Preferably, step S6 is followed by an amplification and concentration step: S7: The purified phage was mixed with host bacteria in the logarithmic growth phase for infection with a multiplicity of infection of 1, and the lysate was collected by centrifugation after culture. S8: The lysate is precipitated and concentrated using polyethylene glycol and sodium chloride to obtain a high-titer phage formulation.

[0008] Preferably, the environmental sample is a sewage, vegetable, or poultry manure sample.

[0009] A lytic phage prepared by the method described above, wherein the phage is Klebsiella pneumoniae phage KPTJT11, which exhibits a short-tailed phage morphology and has an isometric head under a transmission electron microscope.

[0010] Preferably, the phage contains a gene encoding a tail spike protein having a GH28 domain, wherein the GH28 domain is located in the C-terminal region of the tail spike protein.

[0011] A phage composition comprising the above-described lytic phage and a pharmaceutically acceptable carrier.

[0012] Preferred: the use of the phage composition in the preparation of formulations for inhibiting or eliminating biofilms formed by Klebsiella pneumoniae.

[0013] Preferred: The multiplicity of infection between the bacteriophage and bacteria is 0.01 to 1 when used.

[0014] Preferred: the use of the phage composition in the preparation of a medicament for the prevention or treatment of infections caused by Klebsiella pneumoniae.

[0015] The beneficial effects of this invention are as follows: 1. The bacteriophage of the present invention contains a glycoside hydrolase family 28 domain, which is a capsular polysaccharide depolymerase; this enables it not only to lyse bacteria, but also to enzymatically decompose the thick capsule of Klebsiella pneumoniae, which explains the formation of its characteristic target-like plaques and endows it with the additional ability to disrupt biofilm structures.

[0016] 2. In a mouse model of intraperitoneal infection, a single injection of this invention significantly improved the survival rate of infected animals and significantly reduced the bacterial load in major organs such as the liver, spleen, and lungs; the bacteriophage alone was non-toxic to mice, demonstrating its in vivo efficacy and preliminary safety. Attached Figure Description

[0017] Figure 1 This is a diagram showing the isolation and morphological characterization of bacteriophage KPTJT11 in this invention; Figure 2 This is a schematic diagram showing the life cycle parameters and environmental stability of bacteriophage KPTJT11 in this invention; Figure 3 This is a genomic feature map of bacteriophage KPTJT11 in this invention; Figure 4 This is a comparative genomics and depolymerase correlation analysis diagram of bacteriophage KPTJT11 in this invention; Figure 5 This is a schematic diagram illustrating the anti-biofilm activity of bacteriophage KPTJT11 against in vitro Klebsiella pneumoniae biofilms in this invention. Figure 6 This image shows the therapeutic effect of bacteriophage KPTJT11 in a mouse model infected with Klebsiella pneumoniae, as described in this invention. Detailed Implementation

[0018] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0019] Example 1: 1 Materials and Methods 1.1 Isolation, purification and amplification of bacteriophages In phage isolation, waste samples from the host environment are typically screened to identify phages capable of infecting and lysing host bacteria (such as clinical Klebsiella pneumoniae strains). In this study, vegetable and chicken manure samples were collected and screened from the Yuzhong and Tianjiatuo vegetable and poultry markets in Chongqing, China. 50g of manure samples were immersed in 30ml of commercially available TM buffer (20mM Tris-HCl, pH 7.2; 10mM NaCl; 20mM MgSO4) and incubated overnight at 4°C with shaking. The samples were then centrifuged at 10000g for 10min at 4°C. The supernatant was filtered through a 0.22μm filter. 100μL of the filtrate was added to 10ml of LB medium along with an equal volume of clinical Klebsiella pneumoniae strain CRKP-2503074069 and incubated at 37°C with shaking for 6h. The mixture was then centrifuged and filtered again through a 0.22μm filter. The filtrate was spotted onto the surface of clinical Klebsiella pneumoniae CRKP-2503074069 bacteria, air-dried, and incubated at 37°C for 24 hours. Clear spots were picked up with a Pasteur pipette and immersed in 500 μL LLB medium to ensure sufficient liquid flow through the filter membrane during filtration.

[0020] During purification, individual plaques are typically picked from mixed plaques on a culture plate and subjected to multiple rounds of serial dilutions and plating to ensure plaque homogeneity. For the phages used in this study, the isolated phages underwent six consecutive rounds of single-plaque isolation and 10-fold serial dilutions until homogeneous plaques appeared. Each time a plaque was picked, it was placed in 500 μL of LB broth, vigorously vortexed, and then filtered to remove media debris. 100 μL of the resulting supernatant was serially diluted 10-fold, and then 100 μL of each dilution was mixed with 100 μL of Klebsiella pneumoniae with a LOD600 of 0.2 in a 5 mL tube and incubated at 37°C for 5 min to allow the phages to adsorb onto the bacteria. Subsequently, approximately 3 mL of semi-solid medium at 45°C was added to the mixture, the tube was sealed, gently shaken, and then slowly poured onto a solid LB agar plate and incubated at 37°C.

[0021] To obtain higher phage titers, large-volume phage solutions were prepared. For KPTJT11, 400 ml of bacterial culture with an OD600 of 0.2 was prepared and aliquoted into 50 ml centrifuge tubes. 1 ml of phage filtrate was added to each tube, and the mixture was incubated at 37°C with shaking for 6 h. The mixture was then centrifuged at 10,000 rpm and filtered through a 0.22 μm bacterial filter. The filtrate was treated with DNase I and RNase for 30 min to remove bacterial nucleic acids. To neutralize the effect of DNase I, 0.25 mol EDTA solution was added to the solution. The filtrate was then further purified and concentrated by adding PEG8000 (10% w / v) and NaCl (5% w / v). After incubation overnight at 4°C, the solution was centrifuged at 18,000 rpm for 20 min. The supernatant was discarded, and the precipitate was resuspended in TM buffer. The filtrate was then further filtered, and the phage concentration was determined by plaque assay on semi-solid LB medium.

[0022] 1.2 Observation using transmission electron microscopy Phage particles were precipitated with polyethylene glycol (PEG8000) and resuspended in phage buffer (20 mM Tris-HCl, pH 7.2; 10 mM NaCl; 20 mM MgSO4). The suspension was dialyzed against a 0.25 µm VSWP membrane filter (Merck Millipore, Darmstadt, Germany) for 20 min. An ultrathin carbon membrane (approximately 3 × 3 mm) was floated from a mica sheet into 100 µL of purified phage suspension (2 × 10⁻⁶ mm). 10 The surface was treated with PFU / mL. After adsorption for 20 min, the grid was transferred to a 1% (v / v) EG-grade glutaraldehyde droplet for fixation for 20 min. The sample was negatively stained with 2% uranium acetate and observed using a Tecnai 10 TEM (FEI, Thermo Fisher Scientific) at an accelerating voltage of 80 kV. Images were acquired using a MegaView G2 CCD camera (Emsis, Münster, Germany). TEM sample preparation followed the method described by Hoyles et al.

[0023] 1.3 Sequencing, Data Filtering and Quality Control Phage samples were sent to Chengdu PhagetimeBiotech.Co.Ltd and underwent rigorous quality control before subsequent analysis. Qualified samples were used for nucleic acid extraction. When constructing sequencing libraries, the insert size was approximately 200-500 bp. Library quality assessment ensured suitability for high-throughput sequencing. Qualified libraries were sequenced using the DNBSEQ-T7 platform with a paired-end 150 bp strategy. The raw data generated by the platform was used for subsequent bioinformatics analysis.

[0024] FastP was used to filter and quality control the raw sequencing reads. Reads containing a high proportion of ambiguous nucleotides, adapter sequences, and low-quality reads were removed. The resulting clean reads were used for genome assembly and downstream analysis. Denovo genome assembly was performed using metaSPAdes, and multiple k-mer values ​​were tested to obtain optimal assembly results. Subsequently, BWA was used to align the clean reads back to the assembled genome to calculate sequencing depth and assess assembly coverage and integrity.

[0025] To predict coding sequences and tRNA genes, Prokka was used for genome annotation. The predicted protein sequences were further aligned with the NR database using BLASTp to obtain functional annotations and homologous sequence information. EggNOG-mapper was used to functionally annotate the predicted genes, and information from multiple databases, including COG, GO, KEGG, CAZy, BiGG, and PFAM, was integrated to assign inferred biological functions.

[0026] To identify potential virulence factors, gene sequences were compared with the VFDB database using BLAST. ResFinder was used to predict antibiotic resistance genes to check for any known resistance determinants integrated into the phage genome. Phage lifestyle was predicted using the deep learning-based tool DeepPhage. Based on the DeepPhage scoring system, the phage was classified as virulent (lytic). A lifestyle score ≥ 0.5 indicated a lytic phage, while a score < 0.5 indicated a temperate phage.

[0027] 1.4 Comparative genomics analysis Comparative genomics analysis is essential for comparing genome sequences of different species, identifying divergent or convergent features, assessing evolutionary changes, and providing functional annotation and taxonomic interpretation. The assembled genome of bacteriophage KPTJT11 was analyzed to characterize its genomic features and structural proteins. This genome was compared with reference phage genomes obtained from public databases, and linear genome comparisons were visualized using Easyfig v2.2.5. Whole-genome phylogenetic relationships were assessed using VICTOR and further validated using VIPtree software. Predicted coding sequences (CDSs) encoding structural proteins and host recognition proteins (including sting proteins) were extracted from the genome annotation. The amino acid sequence of sting proteins was analyzed using the Pfam database to identify conserved domains and potential depolymerase catalytic motifs. Phylogenetic relationships of sting proteins were analyzed using the NGPhylogeny web server and compared with relevant phage sequences in the Pfam database. The structure of sting proteins was predicted using AlphaFold, and the predicted structures were compared with known proteins in the Protein Data Bank (PDB) to validate the structures.

[0028] 1.5 Characteristics of the Bacteriophage Life Cycle The working multiple of infection (MOI) of bacteriophages was determined using 96-well plates. Six wells were set up per row, with 200 ml of logarithmically growing bacteria in each well. The MOI ranged from 1 to 0.01, and a blank control was included. Different dilutions of purified bacteriophage were added to each well to achieve the desired MOI, and the plates were incubated for 6 hours. Results were analyzed using GraphPad Prism.

[0029] The one-step growth curve and lysis rate of phages were determined using the previously established MOI. Bacteria were cultured to the logarithmic growth phase (OD6000.3) and infected with phages at a low MOI of 1. After allowing phage adsorption for 5 min, the cells were centrifuged and washed to remove unadsorbed phages, then resuspended in fresh culture medium. Bacterial-phage complex samples were taken every 10 min for 2 h. Samples at each time point were diluted 10-fold and plated in triplicate. Plaque-forming units were counted using viable plate counting, growth curves were plotted, lysis rates were calculated, and results were calculated and analyzed using GraphPad Prism software.

[0030] Fragmentation rate (PFU / cell) = PFUPlateu / PFUInitial − PFUInitial / PFUInitial 1.6 Influence of Environmental Parameters on KPTJT11 The effects of environmental factors on bacteriophages were assessed using the plaque assay. In the temperature tolerance experiment, bacteriophages were placed at 4℃, 20℃, 30℃, 40℃, 50℃, 60℃, 70℃, 80℃ and 90℃, respectively, for 10 min at each temperature. Then, they were serially diluted 10-fold and inoculated three times on LB plates using the plaque assay.

[0031] In the pH stability experiment, 500 ml of bacteriophage was added to equal volumes of solutions with different pH values, including pH ranges of 2-3, 4-6, 7, 8-9, and 10-12, and incubated overnight at 4°C. Subsequently, 100 μL of each solution was serially diluted 10-fold and triple-inoculated onto LB agar plates using the plaque method. Plaque-forming units were counted using the viable cell plate count method, and PFU / mL was calculated.

[0032] The tolerance of the phage to ultraviolet irradiation was also tested. 1 ml of concentrated phage was placed under a UV lamp on a standard workbench. Every 10 minutes, 100 μl of phage was serially diluted 10-fold and inoculated triple-times onto LB agar plates using the plaque method. Plaque-forming units were counted using the viable cell plate count method, and PFU / mL was calculated.

[0033] The viral envelope is sensitive to chloroform. To determine whether the bacteriophage is enveloped, it was treated with different concentrations of chloroform for 1 hour, and mixed thoroughly on a vortex mixer. Plaque-forming units were counted using the viable plate count method, and PFU / mL was calculated.

[0034] 1.7 The role of bacteriophages in biofilms The bacteria were cultured in 96-well plates for 72 hours, and their anti-biofilm activity was assessed using phages with different MOIs. The culture plates were incubated at 37°C for 24 hours.

[0035] 1.8 Mouse infection model 1.81 Determination of bacterial infection dose in a mouse model of Klebsiella pneumoniae infection This study used male BALB / c mice (6-8 weeks old, weighing 20-25g). The necessary approvals for the animal experiments were obtained from the National Research Center's Animal Ethics Committee and Biosafety Committee (IACU-CQMU-2026-02013). Relevant guidelines were strictly followed throughout the experiment. An experimental pneumonia model was established using clinical Klebsiella pneumoniae strains via intraperitoneal injection (IP). The inoculation dose was prepared according to the method of Anad et al.: 30ml of overnight BALB culture was centrifuged, and the precipitate was washed three times with PBS. The concentration was adjusted to four different levels (OD6000 0.6, 0.7, 0.8, 0.9), with each concentration serving as an independent experimental group. Four mice in each group were injected with the corresponding concentration. After 72 hours of observation, OD6000 0.9 was determined to be the optimal dose.

[0036] 1.82 Therapeutic effect of KPTJT11 on survival rate of mouse model of Klebsiella pneumoniae infection A new batch of mice was selected and acclimatized for 5 days before the experiment. Simultaneously, phage was concentrated using PEG8000, and its concentration was determined by the spot method. The MOI determination method was as described previously. Mice were divided into 4 groups of 5 mice each. Group 1 was the PBS solution group, Group 2 was the phage group, Group 3 was the bacteria + phage treatment group, and Group 4 was the bacteria-only group. Each group was injected with 200 μL of the corresponding solution. Group 3 received phage treatment 1 hour after infection. Mouse weight was measured and survival percentage recorded every 12 hours, with continuous observation for 72 hours.

[0037] 1.83 Effects of KPTJT11 on Bacterial Load in Organs at Different Time Points in a Mouse Model of Klebsiella pneumoniae As previously mentioned, three groups of mice were established: Group 1 (PBS), Group 2 (model group), and Group 3 (KPTJT11 treatment group). Mice were acclimatized for 5 days. Each group received the prescribed treatment via intraperitoneal injection. Phage treatment was administered 1 hour post-infection. The total observation period was 72 hours, with 3 mice from each group sacrificed every 24 hours. Peritoneal organs, including the spleen, liver, and lungs, were carefully removed. 1 g of each organ was weighed, homogenized, and then added to 1 ml of PBS to prepare a 1 ml / g solution. The resulting solutions were serially diluted 10-fold, and 100 μL of each dilution was triple-inoculated onto MacConkey agar plates. Organ colony forming units (CFU / g) were determined using the viable cell count method.

[0038] 2 Results like Figure 1-6 ;in: Figure 1 (A) Morphology of KPTJT11 plaques after incubation at 37°C on Klebsiella pneumoniae CRKP-2503074069 (lawn). The plaques are transparent, round (approximately 3 mm) and develop a halo around the periphery after prolonged incubation, exhibiting a characteristic bullseye-like morphology. (B) Magnified image showing the bullseye-like plaque structure, indicating the presence of capsular polysaccharide depolymerase activity. (C) Transmission electron microscopy image of KPTJT11, showing an isometric head (approximately 52 nm in diameter) and a short tail, consistent with Podovirus morphology. Figure 2(A) Determination of the optimal multiple of infection (MOI) of KPTJT11, determining the optimal MOI = 1. (B) One-step growth curve of KPTJT11 at the optimal MOI, showing a latency of approximately 5 min and an average lysis yield of approximately 40 plaque-forming units per infected cell. (C) Thermal stability of KPTJT11 after treatment at different temperatures, showing its stability within the range of 4-60℃. (D) pH stability of KPTJT11 after incubation over a wide pH range (pH 4-12). (E) Effect of ultraviolet (UV) irradiation on the activity of KPTJT11, showing that the phage titer decreases with prolonged irradiation time. (F) Sensitivity of KPTJT11 to chloroform treatment, showing that the survival rate decreases with increasing chloroform concentration, and complete inactivation occurs at 95%. Figure 3 The genome structure of KPTJT11 is shown to be a circular single-stranded DNA genome of approximately 40 kb with a GC content of 51.9%. The figure displays 47 predicted coding sequences (CDSs) involved in DNA replication, DNA packaging, and viral particle assembly. GCskew analysis suggests the putative origin and endpoint of replication. No antibiotic resistance genes or tRNA genes were detected. Figure 4 (A) A linear genome comparison (Easyfig) of KPTJT11 with closely related Klebsiella pneumoniae short-tailed phages (vB_KpnP_IME205, vB_KpnPIL_33, and K5) shows strong genomic collinearity and >70% nucleotide sequence identity, while also revealing differential regions encoding host recognition and structural proteins. (B) Phylogenetic analysis of the tail spike protein based on Pfam-annotated sequences demonstrates the evolutionary relationship between the KPTJT11 tail spike protein and related phage homologs. (C) The domain composition of the KPTJT11 tail spike protein shows that it possesses a conserved N-terminal Phage_T7_tail domain and a C-terminal Glyco_hydro_28 catalytic domain associated with depolymerase activity. (D) A comparison of the three-dimensional structure of the KPTJT11 tail spike protein predicted by AlphaFold with related structures in Protein Data Bank (PDB) shows structural similarity. Figure 5 (A) KPTJT11 inhibited early biofilm formation at different multiples of infection (MOIs), showing a significant and dose-dependent inhibition of biofilm formation compared to the untreated control; (B) The clearance effect of KPTJT11 on pre-formed biofilms was significantly decreased at MOIs of 1 and 0.1, while no significant effect was observed at MOI of 0.01. Figure 6(A) Survival curves of BALB / c mice after intraperitoneal infection with Klebsiella pneumoniae CRKP-2503074069 and subsequent phage therapy, showing a significantly higher survival rate in the phage-treated group compared to the infected control group. (BD) Bacterial load (log) in the liver (B), lung (C), and spleen (D) at 24, 48, and 72 hours post-infection. 10 CFU / g tissue). Compared with untreated infected mice, phage treatment significantly reduced bacterial load in all organs at 48h and 72h, but no significant effect was observed at 24h; 2.1 Isolation and Characterization of KPTJT11 This lytic bacteriophage was isolated from vegetable and poultry feces samples in wastewater from Chongqing, China. After incubation at 37°C for 6-12 hours on the host bacterium *Klebsiella pneumoniae* CRKP-2503074069 (lawn), the phage formed transparent circular plaques approximately 3 mm in diameter. After 24 hours, the plaques remained 3 mm in diameter, but a turbid edge approximately 1 cm in diameter formed around them. After 48 hours, the turbid edge increased to approximately 13 mm, while the central transparent plaque remained at 3 mm. Figure 1 A). This centrally transparent, peripherally turbid region is described as a "bullet-shaped" morphology, usually indicating the presence of depolymerase activity, and is also characteristic of most bacteriophages infecting encapsulated bacteria. Figure 1 B). TEM observation showed that KPTJT exhibited a short-tailed phage morphology with an isometric head of 52 nm in diameter, typical of a short-tailed phage. Figure 1 C).

[0039] 2.2 Lifecycle Parameters The optimal multiplicity of infection (MOI) for KPTJT11 was determined to be 1 ( Figure 2 A). A one-step growth curve experiment was conducted under this MOI to evaluate its replication kinetics. The latency period was approximately 5 minutes, followed by a rapid exponential growth phase, reaching a plateau at 35 minutes. Figure 2 B). Based on the ratio of terminal to initial phage titers, the apparent lysis rate is approximately 3 × 10⁻⁶. 4 PFU / cell. However, this value was significantly overestimated due to secondary infection events during the experiment. The actual lysis rate should be estimated to be within the typical range for short-tailed phages.

[0040] KPTJT11 exhibits broad thermal stability, maintaining >90% infectivity in the range of 4°C to 50°C, with a slight decrease at 60°C, and complete inactivation at ≥70°C. Figure 2 C). This phage is stable over a wide pH range (4-12), but rapidly inactivated under strongly acidic conditions (pH≤3). Figure 2D). Ultraviolet irradiation caused the phage titer to decrease logarithmically over time, decreasing by approximately 6 log(D) after 60 minutes. Figure 2 E). Chloroform sensitivity tests showed that KPTJT11 maintained full activity under ≤25% chloroform conditions, partially retained activity under 50-75% chloroform conditions, and was completely inactivated under 95% chloroform conditions. Figure 2 F), which is consistent with the structure of its non-enveloped viral particles.

[0041] Genomic characteristics of 2.3KPTJT11 The complete genome of bacteriophage KPTJT11 was assembled into a circular double-stranded DNA molecule, 41,236 bp in length, with a GC content of 51.9%. GCskew analysis showed a distinct inflection point near the predicted origin of replication. A total of 44 open reading frames (ORFs) were predicted, with an average length of 780 bp, accounting for 91.2% of the entire genome. No tRNA genes or genes encoding integrase were found. Based on functional annotation, the predicted ORFs can be divided into three main functional modules: (i) DNA replication, transcription, and genome processing modules, including DNA polymerases, helicases, ligases, and nuclease-related proteins; (ii) viral particle structure, morphogenesis, and assembly modules, including capsid, packaging, and structural linker proteins; and (iii) host interaction and cleavage modules, including holin, endolysin, and cell wall degrading enzymes. Functional annotations further categorize ORFs into three main modules: (i) DNA replication and nucleotide metabolism (e.g., DNA polymerase, helicase, primase); (ii) viral particle structure and assembly (e.g., major capsid proteins, portal proteins, tail fibers, tail spikes); and (iii) host lysis (holmins, endolysin). Notably, no genes associated with antibiotic resistance, virulence factors, or lysogenicity were detected, supporting the biosafety profile of KPTJT11. Figure 3 ).

[0042] The genome encodes 447 predicted coding sequences (CDSs), including proteins associated with DNA replication, DNA packaging, and viral particle assembly. Figure 3 ).

[0043] 2.4 Taxonomic Classification and Host Prediction Genome-wide phylogenetic analysis using VIPtree and VICTOR classified KPTJT11 into the family Podoviridae, subfamily Autographivirinae, and genus Przondovirus. This phage diverged from reference phages such as KP32, IME205, K11, KN4-1, Pharr, and K5, forming an independent clade, suggesting that KPTJT11 is a novel species within this genus. Inter-genome similarity calculations using VIRIDIC showed 87.6% nucleotide identity with vB_KpnP_IME205 and 85.3% with K5, both below the 95% species boundary threshold defined by ICTV, confirming its classification as a novel species.

[0044] Host prediction using the CHERRY tool showed that *Klebsiella pneumoniae* was the most likely host, with a confidence score of 0.79. DeePhage lifestyle prediction yielded a lysis score of 0.99, further confirming that KPTJT11 is a strictly virulent phage. Spot-speck host range assays showed that KPTJT11 lysed only one of the five clinical *Klebsiella pneumoniae* strains tested, indicating a narrow host specificity.

[0045] 2.5 Comparative genomics and identification of depolymerase domains Genome alignment was performed using Easyfig v2.2.5. The results showed that KPTJT11 exhibited high collinearity and >70% nucleotide identity with three closely related Klebsiella pneumoniae short-tailed phages: vB_KpnP_IME205, vB_KpnPIL_33, and K5. The differentially expressed regions were mainly located in genes encoding structural proteins and host recognition proteins, including the putative tail spike protein (TSP; ORF008). Figure 4 A). Pfam domain analysis was performed on the tail spur protein (815 amino acids) of KPTJT11. Its N-terminal region (residues 1-180) contains a conserved Phage_T7_tail domain (PF03906), a typical feature of short-tailed phage tail fibers. Its C-terminal region (residues 257-580) contains a Glyco_hydro_28 domain (PF00295), belonging to glycoside hydrolase family 28 (GH28), which is associated with pectin cleavage or capsular polysaccharide depolymerase activity. Figure 4 C). Phylogenetic analysis of the tail spike protein amino acid sequence showed that KPTJT11 clustered with KN4-1 and other phages encoding GH28, and was clearly separated from phages carrying other depolymerase families. Figure 4 B).

[0046] To further support its depolymerase function, the three-dimensional structure of KPTJT11TSP was predicted using AlphaFold2 and compared with the resolved structures in the Protein Data Bank (PDB). The prediction model showed that its C-terminal domain has typical right-handed parallel β-helix folds, which is highly similar to the GH28 depolymerase of Klebsiella pneumoniae phage KN4 (PDB: 7VPG). Figure 4 D). These results strongly suggest that KPTJT11 encodes a functional capsular depolymerase, which may be the reason for the halo phenotype in plaque experiments.

[0047] 2.6 Anti-biofilm activity Klebsiella pneumoniae commonly forms biofilms on the trachea, lungs, and other mammalian tissues. Therefore, experiments were conducted to evaluate the ability of this bacteriophage to clear and prevent biofilm formation in vitro under different MOIs. Figure 5 As shown in Figure A, compared with the untreated control, this phage significantly inhibited biofilm formation, with P values ​​of <0.0001, 0.0031, 0.0048, 0.0330, and 0.8566, respectively. Except for the lowest MOI group, all other groups inhibited early biofilm development and effectively disrupted biofilms in a dose-dependent manner. Subsequently, the phage's ability to clear pre-formed biofilms was tested. The results showed that, compared with the untreated control, this phage could significantly clear pre-formed biofilms (…). Figure 5 B). The differences between the MOI1 and 0.1 groups were highly significant (P<0.0001), while there was no significant difference in the MOI0.01 group.

[0048] 2.7 Therapeutic effect of KPTJT11 in a mouse model of intraperitoneal infection The minimum lethal dose (MLD) of Klebsiella pneumoniae CRKP-2503074069 was determined to be 5 × 10⁻⁶ mice. 8 CFU caused a 75% mortality rate within 72 hours. When used for phage therapy, mice were administered 1×10⁻⁶ CFU intraperitoneally 1 hour after infection. 8 At 72 h, the survival rate of the phage-treated group was 80%, significantly higher than the 20% in the untreated infection group (P<0.01, log-rank test). Figure 6 A). The survival rate of mice in both the PBS group and the phage-only group was 100% throughout the experiment, confirming that KPTJT11 is non-toxic.

[0049] To assess bacterial clearance, bacterial load was measured in the liver, lungs, and spleen at 24, 48, and 72 hours post-infection. Compared with the untreated control group, phage treatment significantly reduced CFU counts in all three organs at 48 and 72 hours (P < 0.001 for each time point and each organ; two-way ANOVA combined with multiple comparison tests). No significant decrease was observed at 24 hours (P > 0.05). Figure 6 (BD). These data indicate that KPTJT11 can effectively control Klebsiella pneumoniae infection in vivo by reducing systemic bacterial dissemination.

[0050] 3 Discussion 3.1 Biological characteristics and environmental stability of KPTJT11 In this study, a novel lytic phage, KPTJT11, targeting clinically multidrug-resistant Klebsiella pneumoniae strains was isolated and characterized. KPTJT11 exhibits a typical short-tailed phage morphology, with an icosahedral head (approximately 52 nm) and a short, non-contractile tail. Figure 1 C), consistent with members of the genus Przondovirus in the family Przondophalidae. This phage forms clear plaques (approximately 3 mm) surrounded by a turbid halo that enlarges over time. Figure 1 (A, B) This phenotype strongly suggests that it possesses capsular polysaccharide (CPS) depolymerase activity. Similar halo-shaped plaques have been reported in several Klebsiella pneumoniae short-tailed phages, such as ΦK64-1, KP32, and KP34, in which virus particle-associated depolymerases can degrade the bacterial capsule immediately adjacent to the lysis region.

[0051] One-step growth analysis showed that its latency was short, about 5 minutes, and the lysis rate was about 40 PFU / cell. Figure 2 B). Although the apparent cleavage amount calculated from the initial titer was significantly higher (approximately 3 × 10⁻⁶), 4 The lysis rate was approximately 40 PFU / cell, but this should be attributed to secondary infection events—a recognized limitation of one-step growth experiments when dealing with highly lytic phages under suboptimal MOI conditions. The corrected lysis rate of approximately 40 PFU / cell is consistent with the reported range (20–100 PFU / cell) for other Klebsiella pneumoniae short-tailed phages, supporting the high replication efficiency of KPTJT11.

[0052] KPTJT11 exhibits good stability over a wide temperature (4-60℃) and pH (4-12) range, but is rapidly inactivated only under high temperature (≥70℃) or strong acid (pH≤3) conditions. Figure 2(C, D). This thermal stability and pH tolerance are comparable to other short-tailed phages such as Escherichia coli phage T7 and Klebsiella pneumoniae phage KP32, and it is suitable for routine storage, transportation, and laboratory procedures. However, its significant sensitivity to strong acid conditions suggests a key obstacle to oral administration, which can be overcome through formulation strategies such as enteric coating or combined administration with antacids. It becomes inactive over time under UV irradiation (…). Figure 2 E) Consistent with the known susceptibility of double-stranded DNA phages to UV-induced damage to thymine dimers and capsid proteins, suggesting the need for protective measures in environmental applications. Tolerant at low concentrations of chloroform but completely inactivated at 95% chloroform. Figure 2 F), confirming that KPTJT11 is a non-enveloped phage, which is consistent with the structural characteristics of the class Caudoviricetes.

[0053] 3.2 Genomic characteristics and tail spur protein encoding depolymerase The complete genome of KPTJT11 is a circular double-stranded DNA molecule, 41,236 bp in length, with a GC content of 51.9%, and is highly similar in structure to the genomes of other Przondovirus members. No integrase, repressor, or antirepressor protein genes were found, and coupled with a lysis score of 0.99 given by DeePhage, KPTJT11 can be clearly classified as a strictly lytic phage. More importantly, no genes encoding antibiotic resistance determinants, virulence factors, or known toxins were detected, which meets a key biosafety prerequisite for a therapeutic phage candidate.

[0054] A notable feature of KPTJT11 is that its tail spike protein (TSP; ORF008) contains a C-terminal glycosidase family 28 (GH28) domain (PfamPF00295). Figure 4 C). GH28 enzymes are well-known for hydrolyzing α-1,4-glycosidic bonds in pectin and galacturonic acid polysaccharides in plants, fungi, and bacteria. However, their presence in bacteriophages is relatively rare. To date, depolymerases encoding GH28 have only been sporadically reported in a few Klebsiella pneumoniae phages (such as KN3, KN4, and K30), and are generally associated with CPS degradation. The structure of KPTJT11TSP predicted by AlphaFold2 shows that its GH28 domain has a typical right-handed parallel β-helix fold and highly overlaps with the resolved structure of the Klebsiella pneumoniae phage KN4 depolymerase (PDB:7VPG). Figure 4D). This strong structural conservation strongly supports annotating the KPTJT11TSP as a CPS depolymerase and explains its halo-shaped plaque phenotype at the molecular level. Phylogenetic analysis of the TSP sequence further shows that KPTJT11 clusters with phages carrying GH28, rather than with phages encoding other depolymerase families (such as Pectate_lyase, GH13). Figure 4 B), further reinforcing its uniqueness in taxonomy and function.

[0055] The discovery of the GH28 depolymerase in KPTJT11 not only expands the diversity of known Klebsiella pneumoniae phage capsule-targeting enzymes but also provides a potential enzymatic tool for therapeutic and biotechnological applications. Previous studies have shown that recombinant depolymerases can enhance the sensitivity of encapsulated bacteria to serum bactericidal and phagocytic effects and disrupt biofilms without relying on live phage particles. This study aims to clone and characterize the GH28 domain of KPTJT11 as a cell-free anticapsulation agent.

[0056] 3.3 Anti-biofilm activity and in vivo therapeutic effect Klebsiella pneumoniae readily forms stable biofilms on both living and non-living surfaces, leading to persistent infection and medical device-related complications. KPTJT11 exhibits a significant and dose-dependent effect in inhibiting early biofilm formation and effectively disrupts pre-formed biofilms at an MOI ≥ 0.1. Figure 5 (A, B). This anti-biofilm activity may be attributed to a dual mechanism: (i) direct lysis and killing of planktonic and embedded bacteria in the biofilm; and (ii) enzymatic degradation of the CPS matrix by TSP-associated depolymerases, thereby disrupting biofilm structural integrity and facilitating phage penetration. Similar biofilm-disrupting capabilities have also been reported in other Klebsiella pneumoniae phages encoding depolymerases, such as ΦK64-1 and KPO1K2.

[0057] The therapeutic potential of KPTJT11 was validated in a mouse model of intraperitoneal infection. A single intraperitoneal injection of KPTJT11 (1×10⁻⁶) was administered 1 hour post-infection. 8 PFU significantly improved survival rate (80%, compared to 20% in the untreated control group); Figure 6 A), and significantly reduced the bacterial load in the liver, spleen, and lungs at 48 h and 72 h post-infection. Figure 6(BD). These results are consistent with recent preclinical studies that also demonstrated the effectiveness of Klebsiella pneumoniae phage in mouse models of sepsis and pneumonia. Notably, no toxicity was observed in mice administered the phage alone, further supporting the biosafety inferred from genomic analysis. The lack of significant bacterial clearance at 24 hours suggests that gradual accumulation of phage progeny may be necessary, or that synergy with the host immune system is required, consistent with previous assumptions regarding other phage-based therapeutic systems.

[0058] 3.4 Narrow host range: limitations can also be opportunities Host range analysis showed that KPTJT11 lysed only one of the five clinical Klebsiella pneumoniae strains tested. While this narrow specificity is typical of many capsular receptor-dependent short-tailed phages, as they recognize strain-specific K antigen structures, it also constitutes a major obstacle to clinical translation. Single phage formulations are inherently limited by the extensive serotype diversity of Klebsiella pneumoniae (>80 K types), therefore clinical application may require: (i) patient-specific phage matching; (ii) co-lacing with phages with complementary host ranges to form cocktail formulations; or (iii) engineering phage receptor-binding proteins to broaden host tropism.

[0059] First, adding KPTJT11 to a phage cocktail covering common K types (such as K1, K2, K5, K57, and K64) allows for broader coverage while leveraging its excellent anti-biofilm and in vivo therapeutic effects. Second, the modular structure of TSP—a conserved N-terminal domain and a variable C-terminal catalytic domain—makes it suitable for receptor retargeting via domain substitution or site-directed mutagenesis. Third, the purified GH28 depolymerase itself can also be developed as a standalone anti-capsule agent because its immunogenicity may be lower than that of live phage particles, and it exhibits greater stability.

[0060] 3.5 Limitations and Future Directions This study has several limitations. First, the exact capsular serotype of the host strain CRKP-2503074069 has not been determined, therefore the receptor specificity of KPTJT11 cannot be precisely defined. Future studies will use host whole-genome sequencing and wzi gene sequencing or capsular-specific PCR for K genotyping. Second, the therapeutic effect of KPTJT11 was only evaluated under conditions similar to prophylactic administration (administered 1 hour after infection). Whether it remains effective at later time points or after established infection needs further investigation. Third, the immunogenicity and pharmacokinetics of KPTJT11 in vivo have not been evaluated; these parameters are crucial for predicting phage duration and potential immune clearance with repeated administration. Fourth, although the strong inference of TSP's depolymerase activity based on plaque morphology and structural homology still requires direct biochemical verification through heterologous expression and zymography. Finally, the bacterial regrowth observed at 6 hours in liquid culture suggests the possible rapid emergence of phage-resistant mutants. Therefore, it is necessary to systematically study the tolerance mechanism and design strategies to alleviate tolerance (such as phage-antibiotic synergy and combined cocktail formulations).

[0061] In summary, KPTJT11 is a novel, well-characterized, and biosafety-compliant lytic phage exhibiting significant anti-biofilm activity and in vivo therapeutic potential against clinically MDR Klebsiella pneumoniae strains. The presence of the GH28 family depolymerase domain in its tail spike protein distinguishes KPTJT11 from many previously reported Klebsiella pneumoniae phages and opens up more possibilities for its translational development. Despite its narrow host range, KPTJT11 remains an important addition to the phage arsenal against Klebsiella pneumoniae, and can be developed as a component of phage cocktails or as a source of recombinant depolymerases. This study further strengthens the potential of phage strategies as a viable alternative to antimicrobial resistance and traditional antibiotics.

[0062] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a novel bacteriophage targeting Klebsiella pneumoniae, characterized in that, Includes the following steps: S1: Provide an environmental sample containing lysing bacteriophages; S2: Process and filter the environmental sample to obtain an initial filtrate containing bacteriophages; S3: Co-culture the initial filtrate with the host bacterium Klebsiella pneumoniae to obtain a culture mixture; S4: Filter the culture mixture to obtain phage lysate; S5: The phage lysate is inoculated onto the surface of a solid culture medium containing the host bacteria, and after culturing, a single transparent phage plaque is picked out; S6: Perform multiple rounds of purification on the selected plaques until a uniform plaque morphology is obtained, resulting in purified lytic phages.

2. The method for preparing a novel bacteriophage against Klebsiella pneumoniae according to claim 1, characterized in that, The step S6 is followed by amplification and concentration steps: S7: The purified phage was mixed with host bacteria in the logarithmic growth phase for infection with a multiplicity of infection of 1, and the lysate was collected by centrifugation after culture. S8: The lysate is precipitated and concentrated using polyethylene glycol and sodium chloride to obtain a high-titer phage formulation.

3. The method for preparing a novel bacteriophage against Klebsiella pneumoniae according to claim 1, characterized in that, The environmental samples are sewage, vegetable or poultry manure samples.

4. A lytic phage prepared by the method according to any one of claims 1-3, characterized in that, The bacteriophage is a Klebsiella pneumoniae bacteriophage, which appears as a short-tailed bacteriophage with an isometric head under a transmission electron microscope.

5. A method for preparing a novel bacteriophage against Klebsiella pneumoniae according to claim 4, characterized in that, The phage contains a gene encoding a tail spike protein with a GH28 domain located in the C-terminal region of the tail spike protein.

6. A bacteriophage composition, characterized in that, It comprises the lytic phage of any one of claims 4-5 and a pharmaceutically acceptable vector.

7. Use of the phage composition according to claim 6 in the preparation of formulations for inhibiting or eliminating biofilms formed by Klebsiella pneumoniae.

8. The application according to claim 7, characterized in that, When used, the multiplicity of infection between bacteriophage and bacteria is 0.01 to 1.

9. The use of the phage composition according to claim 6 in the preparation of a medicament for the prevention or treatment of infections caused by Klebsiella pneumoniae.

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