Klebsiella pneumoniae bacteriophage, bacteriophage composition thereof and application thereof

Abstract

The present application relates to a Klebsiella pneumoniae bacteriophage, a bacteriophage composition thereof and application thereof, and belongs to the field of microorganisms. The present application provides a lytic bacteriophage Kp_WF01 infecting Klebsiella pneumoniae, and biological property research, morphological observation and whole-genome phylogenetic analysis are performed on the bacteriophage, and it is determined that the bacteriophage Kp_WF01 is a new species of Caudoviricetes Class Autographivirales Order Autotranscriptaviridae Family Przondovirus Genus. The in-vivo treatment effect and safety of the bacteriophage Kp_WF01 are evaluated by using a mouse model infected with CRKP, and the results show that the bacteriophage Kp_WF01 can rescue the CRKP-infected mouse, can be used as an effective therapeutic preparation against CRKP infection, and the treatment effect of the bacteriophage Kp_WF01 is better than that of imipenem.

Description

Technical Field

[0001] This invention relates to a Klebsiella pneumoniae bacteriophage, a bacteriophage composition thereof, and its application, belonging to the field of microbiology. Background Technology

[0002] Klebsiella pneumoniae ( Klebsiella pneumoniae Klebsiella pneumoniae (KP) is a Gram-negative, encapsulated, opportunistic pathogen that can cause serious illnesses such as pneumonia, liver abscess, urinary tract infections, and bacteremia. Some strains are highly pathogenic because they produce a mucoid capsule, protecting themselves from the host's immune system and antibiotics. Hypervirulent Klebsiella pneumoniae... K. pneumoniae Klebsiella pneumoniae (HVKP) produces capsular polysaccharides, becoming a significant cause of severe and fatal infections in healthy community populations, posing a major challenge to phage therapy (Tang et al., 2023). The overuse of antibiotics in clinical practice has led to the emergence of multidrug-resistant (MDR) Klebsiella pneumoniae, among which carbapenem-resistant Klebsiella pneumoniae (CRKP) is a life-threatening pathogen. Since carbapenems are considered a last resort in clinical treatment, carbapenem resistance is becoming increasingly serious. Therefore, there is an urgent need to develop alternative antimicrobial drugs, and phage therapy is a promising candidate for solving this problem (Baqer et al., 2022; Choi et al., 2024).

[0003] Bacteriophages are widely distributed and diverse in nature, making them one of the most abundant organisms on Earth. They are widely present in the natural environment, with wastewater and sewage directly connected to hospital environments being the richest sources (Kakasis et al., 2019). Bacteriophages are viruses that specifically infect bacteria. They form a highly efficient and evolved parasitic relationship with the host by recognizing and binding to specific receptors on the surface of bacterial cells. Unlike broad-spectrum antibiotics, bacteriophages are highly selective, acting only on their targeted specific strains and being ineffective against other strains. This high specificity greatly improves the safety of their use, minimizing potential damage to beneficial bacteria in the body or environment. Based on these characteristics, phage therapy, using bacteriophages as antimicrobial agents, has fewer side effects than antibiotics and shows promising application prospects in combating multidrug-resistant bacterial infections (Peng et al., 2023).

[0004] Although research on phage therapy has a history of nearly a century, the emergence of antibiotic resistance has made phages a hot research topic again (Gordillo & Barr, 2019). The overuse of antibiotics has led to the rapid spread of resistance, while the development of new antibiotics is extremely slow, further highlighting the enormous potential of phage therapy as an alternative (Nilsson, 2019). Currently, although phages are used to treat multidrug-resistant Klebsiella pneumoniae infections (Yang et al., 2024), phage-resistant strains also frequently emerge. Therefore, to apply more phages to clinical treatment, it is necessary to continuously isolate new host-specific phages to address the newly evolved bacterial pathogens arising from the endless struggle between phages and bacteria (Oduor et al., 2020). Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a Klebsiella pneumoniae phage, its phage composition, and its applications. The phage provided by this invention is a lytic phage Kp_WF01 that infects carbapenem-resistant Klebsiella pneumoniae. Through biological characteristic studies, morphological and genetic evolutionary relationship analysis of this phage, it has been found that phage Kp_WF01 has broad application prospects as a new potential antibacterial agent.

[0006] The technical solution of the present invention is as follows: A Klebsiella pneumoniae bacteriophage ( Klebsiella pneumoniae phage Kp_WF01 was deposited on June 17, 2024, at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO: M 20241241.

[0007] The aforementioned Klebsiella pneumoniae bacteriophage Kp_WF01 has a hexagonal head, approximately 60 nm in diameter, and is tailless, thus morphologically classified as a tailless bacteriophage. Based on biological characteristics and evolutionary relationships, this bacteriophage is... Caudoviricetes Outline Autographivirales Head Autotranscriptaviridae division Przondovirus A new species of the genus.

[0008] A phage composition comprising the above-mentioned Klebsiella pneumoniae phage Kp_WF01.

[0009] According to a preferred embodiment of the present invention, the phage composition further includes Pseudomonas aeruginosa phage Pa_WF01 and / or Acinetobacter baumannii phage Ab_WF01.

[0010] The use of the above-mentioned Klebsiella pneumoniae phage Kp_WF01 or the above-mentioned phage composition in the preparation of products that inhibit or kill Klebsiella pneumoniae.

[0011] According to a preferred embodiment of the present invention, the product includes medicines, feed, bactericides, disinfectants, and cleaning agents.

[0012] According to a preferred embodiment of the present invention, the Klebsiella pneumoniae is a carbapenem-resistant Klebsiella pneumoniae.

[0013] In this invention, the Klebsiella pneumoniae includes human-derived Klebsiella pneumoniae and other Klebsiella pneumoniae; other Klebsiella pneumoniae may be, for example, Klebsiella pneumoniae in water or Klebsiella pneumoniae attached to other species.

[0014] A product that inhibits or kills Klebsiella pneumoniae includes the above-mentioned Klebsiella pneumoniae bacteriophage Kp_WF01 or the above-mentioned bacteriophage composition.

[0015] According to a preferred embodiment of the present invention, the product includes medicines, feed, bactericides, disinfectants, and cleaning agents.

[0016] More preferably, the drug is an in vitro therapeutic drug or an in vivo therapeutic drug.

[0017] According to a preferred embodiment of the present invention, the Klebsiella pneumoniae is a carbapenem-resistant Klebsiella pneumoniae.

[0018] In the specific implementation process, corresponding auxiliary materials may also be added to the product.

[0019] Beneficial effects: 1. A lytic bacteriophage Kp_WF01 infecting Klebsiella pneumoniae was isolated and identified from sewage surrounding the Second Affiliated Hospital of Shandong Medical University. Biological characteristics, morphological observation, and evolutionary analysis of this bacteriophage were conducted, confirming that bacteriophage Kp_WF01 is... Caudoviricetes Outline Autographivirales Head Autotranscriptaviridae division Przondovirus A new species of the genus.

[0020] 2. This invention evaluated the in vivo therapeutic efficacy and safety of phage Kp_WF01 using a CRKP-infected mouse model. The results showed that phage Kp_WF01 can rescue CRKP-infected mice and can serve as an effective therapeutic agent against CRKP infection. Furthermore, the therapeutic effect of phage Kp_WF01 is better than that of imipenem. As a novel potential antibacterial agent, it has broad application prospects and provides a theoretical basis and technical support for the clinical application of phage therapy for CRKP infection. Attached Figure Description

[0021] Figure 1The image shows the morphology of bacteriophage Kp_WF01; where A is a plaque of bacteriophage Kp_WF01 on the culture medium; B is a morphological image of bacteriophage Kp_WF01 under a transmission electron microscope, and the scale bar represents 100 nm. Figure 2 Figure 1 shows the results of the biological characteristics study of bacteriophage Kp_WF01; where A is a bar chart of the optimal infection multiplicity of bacteriophage Kp_WF01; B is a bar chart of the temperature stability of bacteriophage Kp_WF01; C is a bar chart of the pH stability of bacteriophage Kp_WF01; and D is the growth curve of CRKP and its infected bacteriophage Pa_WF01. Figure 3 Genome map of bacteriophage Kp_WF01; Figure 4 A phylogenetic tree of phage Kp_WF01 based on the whole genome sequence; Figure 5 Evaluation of the therapeutic effect of phage Kp_WF01 on CRKP-infected mice; where A is a line graph of mouse survival rate; B is a bar graph of serum bacterial killing test; C is a scatter plot of bacterial load in various organs of mice; D is a scatter plot of phage titer in various organs of mice. Figure 6 H&E staining images of mouse liver, spleen, and lung tissue sections; Figure 7 The image shows the in vitro antibacterial effect of the bacteriophage Kp_WF01 composition. Detailed Implementation

[0022] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings, tables, and specific embodiments. Examples of the embodiments are shown in the accompanying drawings. It should be understood that the specific embodiments described in the following description of the invention are merely illustrative examples of specific implementations of the invention and are intended to explain the invention, but do not constitute a limitation thereof.

[0023] The endpoints of the ranges and any values ​​disclosed herein are not limited to the exact ranges or values, which should be understood to include those close to them.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified. All data were analyzed using GraphPadPrism 8.0 software. The significance of differences between two groups was determined by unpaired t-tests, and the significance of differences among multiple groups was determined by analysis of variance. Values ​​were considered significant when p < 0.01.

[0025] In the following examples, the carbapenem-resistant Klebsiella pneumoniae used Klebsiella pneumoniae CRKP), carbapenem-resistant Pseudomonas aeruginosa Pseudomonas aeruginosa CRPA), methicillin-resistant Staphylococcus aureus (MRSA) Staphylococcus aureus MRSA), carbapenem-resistant Acinetobacter baumannii Acinetobacter baumannii CRAB), Multidrug-resistant Enterococcus faecalis Enterococcus faecalis MDR-Ef) and drug-resistant Escherichia coli (Carbapenem-resistant) Escherichia coli The above-mentioned strains (CREC) were provided by the Clinical Laboratory of the Affiliated Hospital of Shandong Second Medical University, Shandong Province. They were isolated from the blood, feces, sputum, etc. of hospitalized patients. The public can obtain them from the Affiliated Hospital of Shandong Second Medical University, or with the consent of the Affiliated Hospital of Shandong Second Medical University (i.e., the applicant). The above-mentioned biological materials are only used to repeat the relevant experiments of this invention and cannot be used for other purposes.

[0026] The *Pseudomonas aeruginosa* bacteriophage Pa_WF01 used was deposited on April 11, 2024, at the China Center for Type Culture Collection (CCTCC), located at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO: M 2024661. It has been disclosed in patent document CN118374460A (application number 202410805183.8), and this application does not involve the deposit of this microorganism.

[0027] The Acinetobacter baumannii phage Ab_WF01 used was deposited on December 11, 2023, at the China Center for Type Culture Collection (CCTCC), located at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO: M 20232519. It has been disclosed in patent document CN117431221A (application number 202311764467.9), and this application does not involve the deposit of this microorganism.

[0028] Example 1 Isolation and purification of bacteriophage Kp_WF01 Wastewater samples were collected from the wastewater treatment plant of the Affiliated Hospital of Shandong Second Medical University. The wastewater samples were centrifuged at 5000g for 10 minutes to remove impurities, and then filtered through a 0.22μm microporous membrane to remove bacteria, yielding the wastewater filtrate. CRKP was incubated at 37℃ and 220rpm on a shaker until OD reached... 600 =0.6 (1×108 (CFU / mL). Mix 10 mL of wastewater filtrate with 10 mL of liquid LB medium, then add 200 μL of CRKP bacterial suspension to the mixture and incubate overnight at 37°C and 220 rpm on a shaker. The next day, remove the culture medium, centrifuge at 5000 g for 10 minutes, and collect the supernatant. Filter the supernatant through a 0.22 μm microporous membrane to remove bacteria and obtain the initial phage isolation solution. Use a dot assay with CRKP as the indicator strain to detect the presence of phage plaques. Purify the phage at least three times using the double-layer agar plate method until a uniform single plaque is observed. The purified phage is named Kp_WF01. The purification results are as follows: Figure 1 As shown in A, uniformly sized, round, transparent patches with a diameter of 4-5 mm were observed on a double-layer agar plate, with halos around the patches.

[0029] Example 2 Biological characteristics of bacteriophage Kp_WF01 (1) Morphological observation of bacteriophage Kp_WF01 The morphology of the bacteriophage was observed using a transmission electron microscope. Bacteriophage Kp_WF01 was attached to a carbon-coated copper grid and negatively stained with 2% phosphotungstic acid for 5 min. The morphology of the bacteriophage was then observed using a transmission electron microscope (Hitachi HT7700, Tokyo, Japan) at 80 kV.

[0030] Electron micrographs of bacteriophage Kp_WF01 are shown below. Figure 1 As shown in Figure B, the bacteriophage has only a hexagonal head with a diameter of approximately 60 nm and no tail. Based on these morphological characteristics and the standards of the International Committee on Taxonomy of Viruses (ICTV), bacteriophage Kp_WF01 should be morphologically a tailless bacteriophage.

[0031] (2) Identification of the host range of bacteriophages Carbapenem-resistant Klebsiella pneumoniae (CRKP), carbapenem-resistant Pseudomonas aeruginosa (CRPA), methicillin-resistant Staphylococcus aureus (MRSA), carbapenem-resistant Acinetobacter baumannii (CRAB), multidrug-resistant Enterococcus faecalis (MDR-Ef), and drug-resistant Escherichia coli (CREC) were plated on Columbia blood agar plates and incubated overnight at 37°C. The next day, single colonies were picked from the plates and transferred to 5 mL of LB broth, then cultured at 37°C with shaking at 220 rpm. The strains were identified by amplifying the 16S rRNA gene using universal bacterial primers F27 and R1492, and the gene sequence was aligned using the NCBI Basic Local Alignment Search (BLAST) tool. Correctly identified strains were stored at -80°C after the addition of glycerol.

[0032] The host range of phage Kp_WF01 was determined by spot assay to detect the above-mentioned clinically resistant isolates (including CRKP, CRPA, MRSA, CRAB, MDR-Ef, and CREC). The procedure was as follows: 100 μL of the above-mentioned resistant bacterial suspension in the exponential phase was evenly spread on an LB agar plate. 5 μL of phage solution was dropped onto the plate with the resistant bacteria, and the plate was incubated overnight at 37°C. The next day, the presence or absence of phage plaques indicating bacterial lysis was observed. The results are shown in Table 1. The results indicate that phage Kp_WF01 only has a lytic effect on CRKP.

[0033] Table 1. Host range and plaque formation rate of bacteriophage Kp_WF01

[0034] Note: Lysis activity: "+" indicates that a lysing zone can be produced, "-" indicates that a lysing zone cannot be produced; Plaque formation rate: determined by calculating the ratio of plaque forming units (PFUs) of each phage-sensitive strain to the PFUs of the indicator strain, "++" indicates a plaque formation rate > 1, "+" indicates a plaque formation rate > 0.1, "-" indicates a plaque formation rate = 0.

[0035] (3) Optimal multiple of infection detection for phage Kp_WF01 To determine the optimal multiple of infection (MOI) of the phage, a series of 10-fold dilutions of phage Kp_WF01 were performed using SM buffer. To assess the minimum concentration at which phage Kp_WF01 could effectively inhibit host cell growth, CRKP cells were infected with Kp_WF01 at different MOIs, and the phage titer at each dilution was determined using a double-layer agar plate.

[0036] The results are as follows Figure 2 As shown in Figure A, the phage titer reaches its highest level, approximately 2 × 10⁻⁶, when the MOI is 0.001. 8 PFU / mL. Therefore, in subsequent experiments, we used MOI=0.001 as the standard dose.

[0037] (4) One-step growth curve To investigate the burst potential of bacteriophages, the infection kinetics of bacteriophage Kp_WF01 were determined using a one-step growth curve method. Bacteriophage Kp_WF01 (MOI=0.001) was compared with CRKP (1×10⁻⁶). 8The mixture (CFU / mL) was incubated at 37°C for 15 minutes. The incubated sample was centrifuged at 12000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was washed twice with preheated sterile LB liquid medium. The centrifuged particles were resuspended in 20 mL of LB liquid medium and then incubated at 37°C and 220 rpm on a shaker. Samples were collected every 10 minutes, and the phage titer was determined using the double-layer agar plate method until 60 minutes. The burst strength was calculated using the method described in the literature (Kropinski, 2018).

[0038] Based on the ratio of bacteriophages released during the incubation period to the bacteria that initially infected the cell, the burst potential of bacteriophages is approximately 13 PFU per infected cell.

[0039] (5) Thermal stability and pH stability ① Under aseptic conditions, add 1 mL of phage to each of the seven 1.5 mL EP tubes and incubate them in water baths at 4℃, 25℃, 37℃, 50℃, 60℃, 70℃ and 80℃ for 1 hour. Then, cool the phage samples on ice for 10 minutes and determine the phage titer using the double agar plate method.

[0040] The results are as follows Figure 2 As shown in B, phage Kp_WF01 exhibited the highest activity at 25°C, and the stability of the phage subsequently decreased with increasing temperature, completely losing its activity when the temperature reached 60°C.

[0041] ② Add 1M hydrochloric acid solution or sodium hydroxide solution to a test tube containing 15mL LB liquid medium to adjust the pH of the medium to 0 to 14, and filter it with a 0.22μm filter to remove bacteria. Then add 500μL of bacteriophage to each test tube and incubate it in a water bath at 37℃ for 1 hour. Use the double agar plate method to detect the titer of bacteriophage at different pH levels.

[0042] The results are as follows Figure 2 As shown in C, phage Kp_WF01 exhibits optimal activity at pH 7-10 and remains active in the pH range of 4-12, but further increases or decreases in pH will cause phage Kp_WF01 to lose its activity.

[0043] (6) Detection of host cell lysis activity The host bacterium (CRKP) was cultured in LB liquid medium and infected with phage Kp_WF01 with an MOI of 0.001. The medium was incubated at 37°C with shaking at 220 rpm, and samples were taken every hour. The OD values ​​were analyzed spectrophotometrically. 600 nm The bacterial turbidity was measured for 10 hours. The experiment was repeated three times, with the bacterial suspension without phage Kp_WF01 used as a control.

[0044] The results are as follows Figure 2 As shown in D, in the control group, the absorbance of bacterial cultures not infected with bacteriophages increased rapidly, and the OD value increased after 4 hours. 600 nm The concentration reached 0.8, then plateaued. Compared with the control group, the growth of bacteria infected with phage Kp_WF01 was significantly inhibited, indicating that phage Kp_WF01 has a good inhibitory effect on CRKP.

[0045] Example 3 Bacteriophage whole genome sequencing and bioinformatics analysis Genomic DNA was extracted from bacteriophage Kp_WF01 using the TIANamp Viral Genomic DNA / RNA Extraction Kit (Tiangen, Beijing, China) according to the manufacturer's instructions. The whole genome of bacteriophage Kp_WF01 was sequenced using the DNBSEQ-T7 sequencing platform from Chengdu Xinshiji Biotechnology Co., Ltd., and its genome sequence is shown in SEQ ID NO.1. MetaSPAdes software was used for de novo genome assembly (Nurk et al., 2017). The coding genes and tRNAs of the genome were annotated using Prokka, and then Blastp was used to align the protein sequences with the NR library to obtain sequence information with high similarity for each gene in the NR library (Seemann et al., 2014). EggNOG-mapper was used for gene functional annotation (Cantalapiedra et al., 2021). Blastp was used to perform homology alignment of gene sequences with the VFDB database to predict virulence genes (Liu et al., 2022). Resfinder was used for analysis to predict the presence of drug resistance genes on the genome (Bortolaia et al., 2022). The circular map of the phage genome was constructed using the CGview server (Grant JR and Stothard P, 2008). The innermost circle shows the GC offset, the middle circle indicates the GC content, and the outermost circle represents the predicted coding protein sequence (CDS) of phage Kp_WF01. Arrows indicate the direction of transcription. Different CDS represent different functions: morphogenetic proteins, host lysis proteins, DNA replication and metabolic proteins, and hypothetical proteins and miscellaneous RNAs.

[0046] The genome map of bacteriophage Kp_WF01 is as follows: Figure 3As shown, the complete genome sequence of phage Kp_WF01 is 40772 bp in size, with a GC content of 53.28%. The phage Kp_WF01 genome contains 46 open reading frames (ORFs). Using Blastx to search the NCBI database, approximately 89.13% (41 out of the 46 ORFs) of the predicted ORFs were identified as known putative functional proteins. Furthermore, the remaining 5 ORFs did not match any proteins in the database and were classified as putative proteins. No antibiotic resistance or virulence genes were detected in the phage Kp_WF01 genome, indicating that this phage has little ability to mediate horizontal transfer of antibiotic resistance genes. Functional gene annotations for phage Kp_WF01 were predicted into five groups: proteins involved in DNA replication / modification, metabolism, cleavage, DNA packaging, and phage structure-related proteins. In addition, a comparative analysis of the similarity between phage Kp_WF01 and other phages was performed using BLAST in the NCBI database. The results showed that Klebsiella pneumoniae phage vB_Kpn419800-EKp24 ( Przondovirus The genus member (GenBank ID: PX694421.1) showed the highest similarity to phage Kp_WF01, exhibiting 93.31% identity and 98% query coverage.

[0047] Example 4 Phage phylogenetic analysis Phylogenetic analysis was performed using a phylogenetic tree constructed based on the whole genome sequence of Klebsiella pneumoniae phage to infer the evolutionary relationship between phage Kp_WF01 and other phages. Multiple alignments were performed using the Clustal W algorithm. The phylogenetic tree was constructed using the neighbor-joining method in MEGA X software, with the bootstrap set to 1000 (Kumar S et al., 2018). GenBank accessions were listed before the Klebsiella pneumoniae phage name. Phage classification was color-coded: blue. Caudoviricetes Outline Przondovirus Genus; orange, Caudoviricetes Unclassified; purple, Caudoviricetes Outline Autotranscriptaviridae Unclassified; green, Caudoviricetes Outline Drexlerviridae division Webervirus Genus; black, unclassified. Phage Kp_WF01 is indicated in red.

[0048] Figure 4 The bacteriophage Kp_WF01 and the Klebsiella pneumoniae bacteriophage HMGUkp3 clustered together; both are tailless bacteriophages. Caudoviricetes Outline AutographiviralesThis result was confirmed by TEM. Furthermore, Kp_WF01 and three other unclassified Klebsiella pneumoniae phages (phi1_146020, phi1_146019, phi1_146027) originated from the same clade. According to the phage species classification criteria, the two phages showed ≥95% identity and were classified as the same species (Turner D, Kropinski AM, Adriaenssens EM. A Roadmap for Genome-Based Phage Taxonomy. Viruses. 2021; 13(3):506. https: / / doi.org / 10.3390 / v13030506). Phage Kp_WF01 showed less than 95% genome similarity to known closely related phages and clustered with other unclassified phages on the phylogenetic tree. The combined results of Examples 3 and 4 indicate that phage Kp_WF01 is... Caudoviricetes Outline Autographivirales Head Autotranscriptaviridae division Przondovirus A new species of the genus.

[0049] The bacteriophage Kp_WF01 is a Klebsiella pneumoniae bacteriophage ( Klebsiella pneumoniae Phage Kp_WF01 was deposited on June 17, 2024, at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, with accession number CCTCC NO: M 20241241.

[0050] Example 5 Phage therapy in mouse infection models The mice used in the experiment were SPF-grade 6-8 week old C57BL / 6j mice (randomly selected males / females), purchased from Qingdao Mouse Doll Experimental Animal Production Co., Ltd.

[0051] (1) Phage therapy in mouse infection models ① To evaluate the safety and efficacy of phage therapy in vivo, the experiment was divided into 5 groups, with 10 mice in each group. Three of these groups served as the infection group, receiving an intraperitoneal injection of 100 μL of CRKP (1 × 10⁻⁶). 8The control group received 100 μL of PBS buffer (pH 7.0) intraperitoneally. One hour after injection, the infection group received 100 μL of phage Kp_WF01 (MOI = 0.001) as the Kp_WF01 treatment group (CRKP+Kp_WF01), 100 μL of imipenem (IPM, 5 mg / mL) as the IPM treatment group (CRKP+IPM), and 100 μL of SM buffer as the no-treatment group (CRKP+SM buffer). One hour after injection, the control group received 100 μL of SM buffer as the buffer control group (PBS+SM buffer) and 100 μL of phage Kp_WF01 (MOI = 0.001) as the phage control group (PBS+Kp_WF01). All mice were immunized with 100 μL of cyclophosphamide (200 mg / kg) intraperitoneally every 48 hours for 7 consecutive days, and the mortality rate of each group was observed daily.

[0052] The results are as follows Figure 5 As shown in Figure A, all mice infected with CRKP died 3 days after infection without treatment. At 5 days post-infection, the survival rate in the Kp_WF01 treatment group (approximately 70%) was higher than that in the antibiotic IPM treatment group (40%). Furthermore, all mice in the control group remained alive after 7 days. Mice in the Kp_WF01 treatment group showed better treatment outcomes compared to the IPM treatment group, indicating that phage therapy can rescue CRKP-infected mice.

[0053] ② To further evaluate the therapeutic potential of the phage, mice were divided into two groups of 10 mice each. The infection group received an intraperitoneal injection of 100 μL of CRKP (1 × 10⁻⁶). 8 CFU / mL), the normal group was intraperitoneally injected with 100 μL PBS buffer (pH 7.0), and blood was collected from healthy mice or mice infected with CRKP 2 hours after injection to obtain serum. CRKP (3 × 10⁻⁶ CFU / mL) was added to the serum. 7 CFU was resuspended in 10 μL of fresh LB liquid medium, and then 1 mL of active or inactivated serum was added at a 1:100 volume ratio (heated in a 56°C water bath for 30 minutes). The mixture was then incubated with phage Kp_WF01 (MOI = 0.001) at 37°C with low-speed shaking at 60 rpm for 1 hour, followed by bacterial count. CRKP cultured in LB liquid medium served as a control to verify normal bacterial growth; CRKP resuspended in 10 μL of fresh LB liquid medium served as a positive control (Mock); and a mixture of CRKP and phage Kp_WF01 incubated (without serum) served as a negative control (Control).

[0054] The results are as follows Figure 5As shown in Figure B, the bacterial count in the Control group was significantly lower than that in the Mock group, indicating that phage Kp_WF01 can effectively target Klebsiella pneumoniae. Compared with normal mouse serum (both active and inactivated serum) combined with phage, the bacterial count in CRKP-infected mouse serum (both active and inactivated serum) combined with phage was lower, with the lowest bacterial count observed in active serum from infected mice combined with phage. These results indicate that phage Kp_WF01 can enhance serum complement activity in Klebsiella pneumoniae (CRKP)-infected mice and synergistically interact with the immune response in infected mice.

[0055] ③ To further evaluate its therapeutic effect, mice were grouped according to Experiment ① in this embodiment, with 6 mice in each group. They were sacrificed on the 3rd day after treatment, and the bacterial clearance and phage titer in the mice's organs were detected. Lung, liver, and spleen tissues were aseptically harvested, weighed, and homogenized with 1 mL of PBS buffer (pH 7.0). The prepared organ homogenates were serially diluted with PBS buffer (pH 7.0), and the diluted solutions were spread on LB agar plates. Bacterial load and phage titer were measured. Finally, bacterial load and phage titer were calculated based on colony count and plaque-forming units.

[0056] like Figure 5 As shown in C, bacterial load in the lung, liver, and spleen tissues of mice was measured on day 3 post-treatment. Compared with the untreated group, the bacterial load in the above organs of mice in both the Kp_WF01 treatment group and the IPM treatment group was significantly reduced, and the bacterial load in the Kp_WF01 treatment group was significantly lower than that in the IPM treatment group, indicating that the treatment effect of bacteriophage Kp_WF01 is superior to that of antibiotic IPM. Figure 5 As shown in D, phage titers in the lung, liver, and spleen tissues of mice were measured on day 3 post-treatment. The phage titers in the three organs of mice in the Kp_WF01 treatment group were significantly higher than those in the untreated group and the IPM treatment group (P<0.05). This result is consistent with... Figure 5 The C-binding analysis further confirms that phage Kp_WF01 can serve as an effective therapeutic agent against CRKP infection, and indicates that phage Kp_WF01 has a good alternative to IPM.

[0057] (2) Pathological observation of mouse tissues and organs To analyze the pathological and histological changes in the liver, lungs, and spleen of mice, the livers, lungs, and spleens collected for the above experiments were fixed in 4% paraformaldehyde for 24 hours, then dehydrated by graded anhydrous ethanol, embedded in paraffin, and 3 μm thick tissue sections were cut and stained with H&E. The pathological changes in each organ of the mice were observed under an optical microscope (200X).

[0058] The results are as follows Figure 6As shown, no obvious pathological changes were observed in any organ of mice in the phage control group and the buffer control group, and the tissue and cell structure was basically normal. In all groups, a small amount of hepatocyte steatosis (yellow arrow) and occasional minor hemorrhage (blue arrow) were observed in the liver cells. A small amount of brownish-yellow pigment deposition (yellow arrow) was observed in the spleen, with no significant differences between the experimental groups. In the untreated group and the IPM-treated group, a small amount of cell necrosis (orange arrow) and occasional congestion (green arrow) were observed in the spleen tissue, and a large number of granulocyte infiltrations were observed in the alveolar walls of the lung tissue (green arrow). However, in the organs of the phage Kp_WF01-treated group, these pathological results were mostly significantly improved, indicating that phage Kp_WF01 has the potential to treat CRKP infection.

[0059] Example 6 Antibacterial efficacy detection of bacteriophage Kp_WF01 and its composition The host bacterium CRKP was evenly spread on an LB agar plate. Then, 5 μL of a combination of two bacteriophages, Kp_WF01 and Pseudomonas aeruginosa bacteriophage Pa_WF01 (titer ratio 1:1), and 5 μL of a combination of three bacteriophages, Kp_WF01, Pseudomonas aeruginosa bacteriophage Pa_WF01 and Acinetobacter baumannii bacteriophage Ab_WF01 (titer ratio 1:1:1), were dropped onto the LB agar plate coated with CRKP using the spot method. The plates were incubated overnight at 37°C. The next day, the presence of phage plaques was observed on the LB agar plate.

[0060] The results are as follows Figure 7 As shown, the circular transparent plaques represent phage plaques on CRKP. The left side shows a combination of two phages, Kp_WF01 and Pseudomonas aeruginosa phage Pa_WF01, while the right side shows a combination of three phages, Kp_WF01, Pseudomonas aeruginosa phage Pa_WF01, and Acinetobacter baumannii phage Ab_WF01. The small white dots in the transparent plaques represent resistant strains. The results indicate that the combination of Kp_WF01 and Pseudomonas aeruginosa phage Pa_WF01 can lyse CRKP, while the lytic activity of the combination of Kp_WF01, Pseudomonas aeruginosa phage Pa_WF01, and Acinetobacter baumannii phage Ab_WF01 is higher than that of the combination of only two phages.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and do not constitute a limitation on the content of the present invention. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including combining various technical features in any other suitable manner. These simple modifications and combinations should also be regarded as the content disclosed in the present invention and all fall within the protection scope of the present invention.

Claims

1. A Klebsiella pneumoniae bacteriophage ( Klebsiella pneumoniae phage Kp_WF01, characterized in that, It was deposited on June 17, 2024 at the China Center for Type Culture Collection, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO: M 20241241.

2. A bacteriophage composition, characterized in that, It contains the Klebsiella pneumoniae phage Kp_WF01 as described in claim 1.

3. The phage composition according to claim 2, characterized in that, The phage composition further includes Pseudomonas aeruginosa phage Pa_WF01 and / or Acinetobacter baumannii phage Ab_WF01.

4. The use of the Klebsiella pneumoniae phage Kp_WF01 of claim 1 or the phage composition of claim 2 or 3 in the preparation of products that inhibit or kill Klebsiella pneumoniae.

5. The application as described in claim 4, characterized in that, The products include medicines, feed, bactericides, disinfectants, and cleaning agents.

6. The application as described in claim 4, characterized in that, The Klebsiella pneumoniae mentioned is a carbapenem-resistant Klebsiella pneumoniae.

7. A product that inhibits or kills Klebsiella pneumoniae, characterized in that, Includes the Klebsiella pneumoniae phage Kp_WF01 as described in claim 1 or the phage composition as described in claim 2 or 3.

8. The product as described in claim 7, characterized in that, The products include medicines, feed, bactericides, disinfectants, and cleaning agents.

9. The product as described in claim 8, characterized in that, The drug is either an in vitro therapeutic drug or an in vivo therapeutic drug.

10. The product as described in claim 7, characterized in that, The Klebsiella pneumoniae mentioned is a carbapenem-resistant Klebsiella pneumoniae.

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