Klebsiella pneumoniae bacteriophage vBKpnMpha0233 and application thereof

Abstract

The invention discloses a klebsiella pneumoniae bacteriophage vBKpnMpha0233 and application thereof, and belongs to the technical field of microorganisms. The Klebsiella pneumoniae phage vBKpnMpha0233 is preserved in the Guangdong Microbiological Culture Collection Center on November 13, 2025, the preservation number is GDMCC (China General Microbiological Culture Collection Center) NO: 67292-B1, and the preservation address is Institute of Microbiology, Academy of Sciences, Guangdong Province. The Klebsiella pneumoniae adsorption rate of the bacteriophage is as high as 92%, the incubation period is short, the filial generation outbreak amount of single host bacteria is high, efficient amplification and rapid bacteriostasis can be achieved under the extremely low MOI (0.00001), and the bacteriophage has good pH and temperature tolerance and adapts to the complex environment; genome analysis shows that the strain is a brand new strain, does not contain resistance genes, is low in virulence risk, has good biological safety and is suitable for various bacterium control scenes such as agriculture, environmental protection and clinic.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Klebsiella pneumoniae bacteriophage vB_KpnM_pha0233 and its applications. Background Technology

[0002] Klebsiella pneumoniae (Kp) belongs to the Enterobacteriaceae family and was originally called Friedlander's bacterium. It is a Gram-negative, non-motile, capsuled bacterium found in soil, surface water, and other environmental environments, and commonly parasitizes the skin, respiratory tract, and intestines of humans and animals. Kp is an opportunistic pathogen, most likely to infect immunocompromised individuals in hospital settings. As one of the few Gram-negative bacilli capable of causing primary pneumonia, it is also a major cause of hospital-acquired pneumonia (HAP), which can cause, but is not limited to, sepsis, meningitis, and urinary tract infections (UTIs), leading to severe organ damage and life-threatening diseases.

[0003] Currently, antibiotics are mainly used clinically to treat Klebsiella pneumoniae (Kp) infections, including quinolones, β-lactams (penicillins, cephalosporins, carbapenems), aminoglycosides, and polymyxins. However, Kp exhibits natural resistance to penicillin, and its population members often develop acquired resistance to multiple antibiotics. With the widespread clinical use of antibiotics, the resistance rate of Kp to the aforementioned common clinical antibiotics is gradually increasing, showing a trend of multidrug resistance. The overlap of high toxicity and high resistance is becoming increasingly common, and its prevalence is showing a trend towards diversification and globalization.

[0004] To date, even carbapenems, considered the "last line of defense," have been breached against Klebsiella pneumoniae, and the proportion of carbapenem-resistant Klebsiella pneumoniae has been increasing year by year. Meanwhile, polymyxins, as a basic polypeptide antibiotic, are highly effective against most Gram-negative bacilli, but their use is limited due to their neurotoxicity and nephrotoxicity. Given the current severe drug resistance situation and the inability of new drug development to meet clinical needs, polymyxins have regained importance and are considered a last resort for treating carbapenem-resistant Gram-negative bacilli; however, some Klebsiella pneumoniae strains have shown resistance to polymyxins. Therefore, finding alternative drugs and strategies for treating drug-resistant Klebsiella pneumoniae infections has become indispensable. With the significant increase in multidrug-resistant Klebsiella pneumoniae, bacteriophages have been identified as potential treatments for clinical infections.

[0005] Bacteriophages (phages) are viruses that specifically infect bacterial hosts. Initially isolated to target pathogens, phage therapy is defined as the direct administration of virulent bacteriophages to patients to lyse the bacterial pathogens causing clinically relevant infections. It is an investigational anti-infective therapy targeting refractory, multidrug-resistant, and / or biofilm-mediated infections. As natural antibacterial agents, bacteriophages are abundant in nature, readily available, and have low isolation and purification costs and inherent toxicity, making them ideally suited as part of a multidimensional strategy to combat antibiotic resistance. Clinically, to overcome resistance and enhance treatment efficacy, phage therapy is often used as an alternative, adjunctive, or complementary treatment to traditional antibiotic therapy, sharing similarities with antibiotic therapy but also possessing unique therapeutic advantages. Summary of the Invention

[0006] To address the aforementioned technical limitations, this invention provides a Klebsiella pneumoniae bacteriophage vB_KpnM_pha0233 and its applications.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] This invention first provides a bacteriophage strain, namely Klebsiella pneumoniae phage vB_KpnM_pha0233, which was deposited on November 13, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 67292-B1, and the deposit address is the Institute of Microbiology, Guangdong Academy of Sciences.

[0010] This invention screened a Klebsiella pneumoniae phage vB_KpnM_pha0233 from the natural environment, which has high lytic activity, environmental stability and safety. This phage can specifically recognize and lyse Klebsiella pneumoniae to clear the infection and overcome the problem of antibiotic resistance in Klebsiella pneumoniae.

[0011] The present invention also provides the use of the aforementioned Klebsiella pneumoniae bacteriophage vB_KpnM_pha0233 in the preparation of a medicament for treating diseases caused by Klebsiella pneumoniae.

[0012] The present invention also provides the application of the aforementioned Klebsiella pneumoniae bacteriophage vB_KpnM_pha0233 in the preparation of Klebsiella pneumoniae antibacterial agents.

[0013] Furthermore, the antibacterial agent is used in food preservation, environmental disinfection, livestock breeding, poultry farming, aquaculture, water treatment, or the biomedical industry.

[0014] The present invention also provides a phage preparation comprising the aforementioned Klebsiella pneumoniae phage vB_KpnM_pha0233.

[0015] Furthermore, the phage preparation also includes a buffer solution as a stabilizer, wherein the buffer solution is SM buffer, DPBS buffer, physiological saline or glycerol solution, preferably SM buffer.

[0016] SM buffer: Take 1.972 g of magnesium sulfate heptahydrate and 5.58 g of sodium chloride, add 50 mL of 1M Tris-HCl (pH 7.5) and 5 mL of 2% gelatin, add ddH2O to dissolve completely, and bring the volume to 1 L. Sterilize at 121°C and 0.15 MPa for 20 min by moist heat. An additional 0.1-1% BSA can be added.

[0017] DPBS buffer: Dissolve 0.2g KCl, 0.2g KH2PO4, 8g NaCl, and 2.8865g Na2HPO4·12H2O in 800 mL of distilled water. Adjust the pH to 7.0-7.3 with HCl solution, then bring the volume to 1000 mL and sterilize at 121℃ and 0.15 MPa for 20 min by moist heat.

[0018] Glycerin solution: Dissolve glycerin thoroughly with ddH2O to achieve a final concentration of 5-50%, and then filter to remove bacteria using a 0.22μm filter membrane.

[0019] The applications of the phages described in the present invention mainly cover the fields of human health, animal breeding, and environmental biological prevention and control. The multiplicity of infection (MOI) range in which the phages can effectively lyse Klebsiella pneumoniae in a liquid environment is not less than 0.00000001 and not higher than 2000. A more optimal range is 0.0000001 < MOI < 1000, and an even more optimal range is 0.0000005 < MOI < 2. In human medicine, the most promising application direction of the phages is for various infections caused by multi-drug resistant Klebsiella pneumoniae (such as pneumonia, bacteremia, etc.), providing a potential alternative treatment option for patients who are insensitive or intolerant to conventional antibiotics. In animal breeding, their applications can focus on the aquaculture industry, for preventing and treating fish skin ulcers, septicemia, and crustacean diseases caused by Klebsiella pneumoniae under specific conditions (such as high-density farming, stress response), thus helping to reduce the use of antibiotics and ensure breeding safety. In environmental biological prevention and control, the phages can be specifically used to eliminate the host bacteria enriched in environments such as medical facilities and water bodies, and have application potential in hospital infection control and environmental microorganism management. In addition, the phages also show certain application prospects in preventing and treating pet- and livestock-related bacterial diseases, controlling plant bacterial diseases, as well as in food preservation, water and soil cleaning, and other fields.

[0020] Advantages of the present invention:

[0021] 1. The adsorption rate of phage vB_KpnM_pha0233 against Klebsiella pneumoniae kpn045 is as high as 92%. And only 12 minutes are required to complete the first round of proliferation and release progeny phages after adsorption, significantly shortening the latent period of the phages and solving the technical defects of the long latent period and slow bacteria control start of conventional phages. The proliferation burst period of the phages is concentrated in 10 - 35 minutes, and the progeny phage burst amount per single host bacterium reaches 136 PFU / cell; the progeny phages can quickly enter a new round of "adsorption - desorption - proliferation" cycle, enabling the number of free phages in the system to form a fluctuating effect of efficient amplification, achieving rapid suppression of the host bacteria. The optimal MOI is as low as 0.00001, significantly reducing the initial dosage of the phages, saving production and application costs significantly; at the same time, the rapid proliferation characteristic can form a bacteria control pressure in a short time, avoiding the risk of ecological interference caused by high-dose phages, and adapting to scenarios with high requirements for the timeliness of bacteria control such as food preservation, clinical first aid, and emergency treatment of industrial wastewater.

[0022] 2. Bacteriophage vB_KpnM_pha0233 maintains stable activity within a broad pH range of 4-11. Compared to most existing bacteriophages that can only survive in neutral to weakly acidic / alkaline environments (pH=5-9), the pH tolerance range of this invention is significantly broadened. It can tolerate complex acid-base scenarios such as the acidic environment of food processing, the weakly alkaline water of agricultural irrigation, and the weakly acidic exudate of clinical wounds, eliminating the need for additional buffers to adjust the system pH and significantly reducing application costs. This bacteriophage maintains high activity after incubation at 4-50℃ for 90 min, tolerating the low-temperature environment of cold chain transportation, room temperature storage conditions, and the physiological temperature of human / livestock skin. Activity decreases significantly after incubation at 65℃ for 30 min, and is completely inactivated after incubation at 65℃ for 60 min or 80℃ for 30 min or more. This characteristic ensures the activity stability of the bacteriophage during routine applications and allows for rapid inactivation after use through high-temperature treatment, reducing the risk of interference with beneficial microorganisms in the ecological environment and improving application safety. Combining its wide pH and temperature tolerance characteristics, this bacteriophage can be widely applied to various scenarios such as preservation of acidic fruits and vegetables, microbial control in alkaline soils, local drug administration for clinical wound infections, and antibacterial treatment in industrial circulating water. It solves the technical pain point that existing bacteriophages are difficult to scale up due to insufficient environmental tolerance, and has outstanding industrialization potential.

[0023] 3. Bacteriophage vB_KpnM_pha0233 effectively inhibited the proliferation of host bacteria within 0–8 h under different MOI conditions, resulting in decreased OD of the bacterial culture. 600 The value remained at a low level; the outbreak time of resistant bacteria was negatively correlated with MOI, with higher MOI indicating earlier emergence of resistant bacteria. However, when using a low MOI (≤0.0001), the outbreak time of resistant bacteria was delayed to 12 hours, significantly extending the effective control window and overcoming the technical defect that high MOI phages easily induce resistant bacteria rapidly. This makes it suitable for scenarios requiring long-term bacterial control, such as agriculture and environmental protection. In vitro, this phage significantly inhibited and cleared host bacterial biofilms, with a significant dose-dependent effect (p<0.0001): there was no significant difference in the inhibitory effect of low and high doses of phage on biofilms, but the low-dose phage (MOI=0.00001) showed a significantly better biofilm clearance effect than the high-dose phage (MOI=1) (P<0.01). This characteristic significantly reduced the phage dosage required for biofilm clearance, saving production costs while reducing interference from high-dose phages with beneficial environmental bacteria, thus meeting the needs of clinical biofilm-related infections and industrial pipeline biofilm clearance. Combining the dual characteristics of low MOI, long-lasting resistance to drug resistance, and low-dose, highly effective biofilm clearing, this phage can be widely used in clinical treatment of chronic wound infections (biofilm-dominated infections), antibacterial treatment of industrial circulating water pipelines, and microbial control in livestock and poultry farming environments. It is both practical and economical, with significant industrialization potential.

[0024] 4. Genome-wide and phylogenetic analysis of bacteriophage vB_KpnM_pha0233 revealed that this strain is a novel bacteriophage and contains no genes related to antibiotic resistance. Genome sequence alignment and phylogenetic analysis showed significant differences in genome structure, composition, and evolutionary branching compared to previously reported bacteriophages, making it a novel bacteriophage strain never before disclosed. This characteristic provides the invention with an independent intellectual property barrier, preventing technological homogenization and offering an exclusive advantage for subsequent industrial applications. A comprehensive screening of the bacteriophage genome revealed no antibiotic resistance-related gene sequences, eliminating the risk of lateral transfer of resistance genes to host bacteria or environmental microorganisms during infection and proliferation. This fundamentally avoids the technical challenges of "superbug" induction and complies with biosafety application standards in agriculture, industry, and other fields. Annotation and analysis by two authoritative platforms, CARD (Antibiotic Resistance Gene Database) and PhageLeads (Phage Virulence Gene Database), revealed no virulence-related genes. Only one homologous sequence fragment of a virulence gene was annotated in the NCBI Virus database. Compared to naturally occurring phages that showed virulence genes detected on multiple platforms, this phage exhibits significantly lower virulence risk and possesses a sound foundation for safe application. Given its absence of resistance genes and low virulence risk, this phage can be preferentially applied in scenarios with relatively mild biosafety requirements, such as agricultural planting, microbial control, industrial circulating water sterilization, and livestock and poultry breeding environment sterilization. After subsequent functional verification of the virulence gene, its application can be further expanded to fields with higher safety requirements, such as food preservation. Attached Figure Description

[0025] Figure 1 This is the morphology of bacteriophage vB_KpnM_pha0233 under a transmission electron microscope.

[0026] Figure 2 This shows the plaque morphology of bacteriophage vB_KpnM_pha0233 on a double-layer plate.

[0027] Figure 3 It is the particle size of bacteriophage vB_KpnM_pha0233.

[0028] Figure 4 This is the whole genome annotation diagram of bacteriophage vB_KpnM_pha0233.

[0029] Figure 5This is a phylogenetic analysis of phage vB_KpnM_pha0233; A: A heatmap of AAI (mean amino acid similarity) values ​​and genome BLAST distance phylogenetic (GBDP) analysis of 41 Klebsiella pneumoniae phage genomes; AAI values ​​range from 85% (blue) to 100% (red), depicting the degree of genetic similarity among phages; the phylogenetic trees on the left and top represent GBDP analysis, showing the phylogenetic relationships among the 41 phages; the symbols and colors at the bottom correspond to taxonomic differences at the family, genus, or species level; the vertical axis is labeled... The labels represent families, genera, or species; the matching shapes and colors indicate consistency at a specific taxonomic level. B: Collinearity comparison of the vB_KpnM_pha0233 genome with OR532806.1 and NC_047900.1. Arrows indicate the position and orientation of the CDS in each genome (right arrows indicate positive strands, left arrows indicate negative strands). Lines between genomes indicate sequence similarity (as shown in the lower left corner). CDS are color-coded according to functional categories, as shown in the legend in the lower right corner. C41: Genomic similarity between pairs of phage strains.

[0030] Figure 6 It is the optimal infection multiplicity for bacteriophage vB_KpnM_pha0233.

[0031] Figure 7 This is the adsorption curve of bacteriophage vB_KpnM_pha0233.

[0032] Figure 8 This is a one-step growth curve of bacteriophage vB_KpnM_pha0233.

[0033] Figure 9 This is the result of the thermal stability of bacteriophage vB_KpnM_pha0233.

[0034] Figure 10 These are the pH stability results for bacteriophage vB_KpnM_pha0233.

[0035] Figure 11 This is the in vitro antibacterial curve of bacteriophage vB_KpnM_pha0233.

[0036] Figure 12 It refers to the inhibitory effect of bacteriophage vB_KpnM_pha0233 on biofilm formation.

[0037] Figure 13 It refers to the biofilm removal capability of bacteriophage vB_KpnM_pha0233. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0039] Unless otherwise specified, all raw materials and reagents used in the examples are commercially available products.

[0040] The host bacterium used in this invention is Klebsiella pneumoniae kpn045, which was obtained from the following literature and renamed kpn045 in this invention. It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 13, 2025, along with the Klebsiella pneumoniae phage vB_KpnM_pha0233 of this invention, with the accession number GDMCC NO: 67292-B1.

[0041] Lai, Y., et al. (2025). "Comparison of the microbiome of sputum from HIV / AIDS patients using PacBio 16S rRNA sequencing and ddPCR." BMC Infectious Diseases 25(1).

[0042] The main reagents and culture media involved in the examples are as follows:

[0043] (1) SM buffer: Weigh 0.9776 g anhydrous MgSO4 and 5.85 g NaCl into a clean beaker, add 50 mL 1 mol / L Tris-HCl (pH 7.5) and 5.0 mL 2% gelatin, add 800 mL ddH2O and stir thoroughly to dissolve, then use ddH2O to make up to 1 L, dispense into 500 mL blue cap bottles, sterilize at 121℃ and 0.15 MPa for 20 min, cool and store at room temperature for later use.

[0044] (2) LB liquid culture medium: Weigh 10 g Tryptone, 5 g Yeast extract, and 5 g NaCl into a clean beaker. Add 900 mL ddH2O to the beaker and stir thoroughly to completely dissolve the powder. Adjust the pH to 7.0 and bring the volume to 1 L using ddH2O. Then dispense 100 mL into each 250 mL Erlenmeyer flask, seal with 8 layers of gauze and wrap with newspaper, sterilize at 121℃ and 0.15 MPa for 20 min, cool, and store at room temperature for later use.

[0045] (3) 1.5% LB solid medium: Prepare LB liquid medium according to the method in (2) and dispense it into 250 mL Erlenmeyer flasks. Then add 1.5 g agar powder to every 100 mL of liquid medium, seal with rubber stoppers and wrap with newspaper, sterilize at 121℃ and 0.15 MPa for 20 min, cool and store at room temperature for later use.

[0046] (4) 0.6% LB semi-solid culture medium: Prepare LB liquid culture medium according to the method in (2) and dispense it into 250 mL Erlenmeyer flasks. Then add 0.6 g agar powder to every 100 mL of liquid culture medium, seal with rubber stoppers and wrap with newspaper, sterilize at 121℃ and 0.15 MPa for 20 min, cool and store at room temperature for later use.

[0047] Example 1: Phage Isolation and Purification

[0048] Host bacterium (Klebsiella pneumoniae kpn045) resuscitation: Klebsiella pneumoniae kpn045 cryovials were taken out of the -80℃ freezer and thawed on ice. Then, in a biosafety cabinet, an appropriate amount of bacterial suspension was taken with a disposable inoculation loop and streaked on a 1.5% LB solid medium plate for resuscitation. The plate was sealed with sealing film and incubated upside down in a 37℃ incubator for 24 h.

[0049] Preparation of host bacteria (Klebsiella pneumoniae Kpn045 bacterial suspension): The next day, a single colony was picked from the plate and inoculated into a 30 mL virus sampling tube containing 8 mL of LB liquid medium. The sampling tube was sealed with sealing film and cultured at 37℃ and 220 rpm for 12 h to obtain fresh Kpn045 bacterial suspension.

[0050] Environmental sample processing: Take 50 mL of activated sludge sample from Fuzhou University Town Wastewater Treatment Plant into a 50 mL centrifuge tube, centrifuge at 4℃ and 20000 g for 10 min, and collect the supernatant through a 0.22 μm filter membrane into a sterile sampling tube.

[0051] Co-culturing of bacterial suspension with environmental samples: KPN045 was revived and cultured in liquid form in 96-well plates. Single colonies were picked and inoculated into 96-well plates containing 400 μL of LB liquid medium. The plates were sealed with a sealing film and incubated at 37°C and 700 rpm in a microplate shaker for 12-24 h. The 96-well plates containing bacterial suspension were centrifuged at 2344 g for 1 min at 4°C, the supernatant was discarded, and the bacterial pellet was retained. 500 μL of 2×LB liquid medium was added to each well of the plate containing the bacterial pellet for resuspending. Then, 500 μL of the treated environmental sample filtrate was added to each well of the bacterial suspension, and the mixture was thoroughly mixed and incubated at 37°C and 700 rpm in a microplate shaker for 5-7 days.

[0052] Isolation and Verification of Bacteriophages in Co-culture Medium

[0053] Centrifuge the 96-well plate containing co-culture medium at 4°C and 2344 g for 10 min to precipitate the bacterial cells. Collect the supernatant in a 1.5 mL sterile EP tube and store at 4°C.

[0054] Add 3 mL of kpn045 bacterial suspension to an LB agar plate, spreading it evenly. Remove excess suspension using a 1 mL pipette. Open the plate and allow it to air dry. Once dry, add 3 μL of phage solution to the corresponding position on the plate. After the phage solution dries, seal the plate with sealing film and incubate upside down at 37°C for 24 h. Observe the formation of plaques. The presence of plaques indicates the presence of phage capable of lysing kpn045 in the sample supernatant. Store the sample supernatant showing a positive result at 4°C; this is the phage stock solution.

[0055] Phage purification using double-layer agar plate method

[0056] Take the phage stock solution validated by the drop method from the 4°C freezer. First, dispense 900 μL of sterile SM buffer into a 96-well plate. Add 100 μL of phage stock solution to the first row and mix by pipetting. Then, take 100 μL of phage solution from the first row of diluents and add it to the second row and mix by pipetting. Repeat the above operation at a ratio of 1:10 to dilute the phage stock solution to the appropriate multiple.

[0057] Add 100 μL of diluted phage solution to a 1.5 mL sterile EP tube containing 400 μL of host bacterial culture, mix well by pipetting, and incubate at 37°C for 10 min. After incubation, add the mixture to a virus sampling tube containing 7 mL of liquid LB semi-solid medium, mix well by pipetting, and quickly pour onto LB solid medium plates. After the medium solidifies and the surface water marks dry, seal with sealing film and incubate upside down at 37°C overnight.

[0058] The following day, plaque formation was observed, and individual plaques were picked and transferred to 1.5 mL EP tubes containing 500 μL of sterile SM buffer. The tubes were incubated overnight at 4°C to leach the phages. Subsequently, 500 μL of bacterial culture was added to the EP tube containing the leachate, and the tubes were incubated overnight at 37°C for small-scale phage amplification. After amplification, the culture was centrifuged at 20,000 g for 10 min at 4°C, and the supernatant was transferred to a new 1.5 mL EP tube to obtain the phage solution. This completed one round of phage purification. The above steps were repeated 2-4 times, resulting in 3-5 rounds of purification to obtain pure phage.

[0059] Phage amplification and titer determination

[0060] Add 500 μL of purified phage solution to a 15 mL centrifuge tube containing 5 mL of bacterial culture, mix well by pipetting, seal with a sealing film, and incubate at 37℃ and 220 rpm for 5-7 days to achieve large-scale phage amplification. Continue incubation until the culture medium becomes clear or a large number of dead bacterial cells precipitate at the bottom of the centrifuge tube. Then, centrifuge the culture medium at 4℃ and 20000 g for 10 min and transfer the supernatant to a new 15 mL centrifuge tube. Name the phage pha0233.

[0061] Following the phage purification procedure, the supernatant of the phage amplification culture was diluted and the titer was determined by the double-layer plate method. Each experimental group was measured three times, and the number of phage plaques on the plates was recorded. The average value was used to calculate the phage titer.

[0062] Example 2 Identification of bacteriophages

[0063] 1. Morphological observation of bacteriophages

[0064] (1) Observation of phage morphology using transmission electron microscopy (TEM): Take 20 μL of phage with a titer of 10 9 -10 11 High-titer phage solution (PFU / mL) was dropped onto a copper mesh surface and allowed to stand for 10 min to allow phage adsorption. Excess phage solution was then blotted away with filter paper. Subsequently, 20 μL of 2% phosphotungstic acid was added to the copper mesh surface for 90 s negative staining. Excess staining solution was blotted away with filter paper, and the copper mesh was dried overnight before observing phage morphology under a 100 kV accelerated voltage transmission electron microscope.

[0065] like Figure 1 As shown, under a transmission electron microscope, the head of bacteriophage pha0233 is approximately 97 × 102 nm in size, the tail is approximately 121 nm before contraction and approximately 56 nm after contraction, exhibiting the typical morphology of a Myoviridae bacteriophage, and no lipid envelope is observed on its surface. According to the classification criteria of the International Committee on Taxonomy of Viruses (ICTV), this bacteriophage is determined to belong to the order Caudovirales, family Myoviridae, and is named vB_KpnM_pha0233.

[0066] (2) First, dispense 900 μL of sterile SM buffer into a 96-well plate. Add 100 μL of the phage stock solution to the first row and mix by pipetting. Then, add 100 μL of the diluent from the first row to the second row and mix by pipetting. Repeat the above operation to dilute the phage stock solution to the appropriate factor. Add 100 μL of the diluted phage solution to a 1.5 mL sterile EP tube containing 400 μL of host bacteria solution, mix by pipetting, and incubate at 37°C for 5 min. After incubation, add the mixture to a virus sampling tube containing 7 mL of liquid 0.6% LB semi-solid medium, mix by pipetting, and quickly pour it onto an LB solid medium plate. After the medium solidifies and the surface water marks dry, seal the plate and incubate it upside down at 37°C overnight. Turn on the desk lamp and copy plate, place the double-layer plate on the copy plate, and observe the formation of phage plaques.

[0067] like Figure 2 As shown, after 12 h of culture in a double-layer agar plate, phage pha0233 formed phage plaques with a diameter of 1.0±0.5 mm. The plaques were transparent with clear edges and surrounded by a depolymerase halo.

[0068] (3) Dilute the phage fluid with particle-free ultrapure water at a certain ratio, inject it into a 1 mL syringe, and determine the phage particle size using a NanoSight Pro nanoparticle tracking analyzer. Figure 3 As shown, the average size of phage pha0233 particles is approximately 104 nm.

[0069] 2. Phage genomics and phylogenetic analysis

[0070] (1) Whole genome annotation and analysis

[0071] The full-length genome of phage pha0233 is 158,187 bp, with a GC content of 46.40%. Using annotation tools such as PhaBox, Phastest, Prokka, Phagescope, NCBI Virus, and Genemarks, a total of 233 open reading frames (ORFs) were predicted, including 209 coding sequences (CDS). (See...) Figure 4The proteins encoded by these CDS are divided into the following nine modules according to their functions: (1) 27 phage structure-related proteins, including head closing proteins, tail fibrin proteins, and base plate proteins; (2) 21 DNA packaging and replication-related proteins, including portal proteins, terminal enzyme large subunits, DNA helicases, and DNA ligases; (3) 9 DNA repair and recombination-related proteins, including homing endonucleases, DNA repair proteins, and recombinant endonuclease protein subunits; (4) 10 regulatory-related proteins, including greening regulators, late promoter transcription regulators, and transcription repressors; (5) 25 transport and metabolism-related proteins, including guanylate kinase, ribose-phospho-pyrophosphate kinase, and thymidylate synthase; (6) 5 cleavage-related proteins, including Rz-like transmembrane proteins, endosomalins, and tail lysozymes; (7) 4 membrane proteins; (8) 1 virulence-related protein; and (9) 108 putative proteins, accounting for 51.67% of the total predicted CDS. Eight tRNAs were annotated in the predicted CDS, indicating that this phage can achieve precise adaptation to the host translation system through actively encoding tRNAs. This can expand the host spectrum of the phage to some extent and serve as a vector for horizontal gene transfer, promoting genome evolution. Meanwhile, no integrase was annotated in the phage genome, indicating that this strain is a virulent phage. Furthermore, neither CARD nor PhageLeads analysis tools detected any potential antibiotic resistance genes or virulence genes in phage pha0233. However, one virulence-related gene was annotated using NCBI Virus amino acid sequence alignment.

[0072] (2) Phylogenetic analysis

[0073] like Figure 5 The whole genome sequence of bacteriophage pha0233 was uploaded to the NCBI Viruses nt database for BLASTN. The alignment results were sorted from highest to lowest Query Coverage, and the top 40 bacteriophage sequences were selected and submitted to Virus Classification and Tree Building Online Resource (VICTOR) and CompareM for viral phylogenetic, taxonomic, and average amino acid identity (AAI) analysis. The results were then combined using R language to create a visualization heatmap and phylogenetic tree. Simultaneously, the Virus Intergenomic Distance Calculator (VIRIDIC) was used to calculate the similarity between the paired bacteriophage genomes, applying default thresholds for species (>95%) and genus (>70%).

[0074] Phylogenetic and taxonomic analyses of VICTOR show that pha0233 is most closely related to OR532806.1 and NC_047900.1. While it belongs to the same genus but a different species as the other 40 bacteriophages, pha0233 is classified as a new species at the species level. CompareM calculations show that the AAI values ​​of pha0233, OR532806.1, and NC_047900.1 are 92.44% and 92.91%, respectively, which does not definitively confirm whether pha0233 is a new species. Further double-sequence linear alignments were performed on the whole genomes of pha0233 with OR532806.1 and NC_047900.1, respectively. The results showed that although the coverage rates of pha0233 and these two strains were 89% and 92%, respectively, the alignment results revealed that the similarity of most gene regions covered by pha0233 and these strains was below 95%, indicating significant differences. Furthermore, pha0233 contained unaligned blank regions, suggesting that these genes are unique to pha0233. Combined with the tRNA and DNA recombination-related proteins annotated with the pha0233 gene, this indicates that pha0233 not only acquired some new genes during evolution but also underwent homologous recombination events, thus forming a separate branch during evolution. VIRIDIC's pairwise genomic similarity results for 41 bacteriophages also showed that the similarity values ​​between pha0233 and the other 40 bacteriophages were all below the threshold for new species (95%), corroborating the VICTOR classification results and proving that pha0233 is a novel bacteriophage.

[0075] Based on the morphological and genomic characteristics of phage pha0233, it was identified as Klebsiella pneumoniae phage vB_KpnM_pha0233, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 13, 2025, with accession number GDMCC NO: 67292-B1, and the deposit address is Institute of Microbiology, Guangdong Academy of Sciences.

[0076] Example 3: Optimal Multiple of Infection for Bacteriophages

[0077] The multiplicity of infection (MOI) refers to the ratio of the number of bacteriophages to the number of host bacteria at the time of infection, that is, the number of bacteriophages infected by each bacterium.

[0078] Phage titers were determined using the double-layer plate method, and the host bacterial concentration was adjusted to 1×10⁻⁶ using LB liquid medium. 9CFU / mL, then the concentration of the host bacteria remained unchanged, and LB liquid medium was added according to the target MOI to adjust the titer of the phage fluid and perform a 1:10 serial dilution.

[0079] Phage suspensions and host bacterial suspensions were mixed thoroughly according to the multiplicity of infection ratios of 1, 0.1, 0.01, 0.001, 0.0001, and 0.00001. The mixtures were incubated at 37 °C for 4 h, followed by centrifugation at 20,000 g at 4 °C for 10 min. The supernatant was collected into sterile 1.5 mL EP tubes, and the phage titer in the supernatant was determined using the double-layer plate method. Three replicates were set up for each experimental group.

[0080] Optimal multiplicity of infection results as follows Figure 6 As shown, the phage titer of pha0233 gradually increases as the MOI decreases. The phage titers of MOI=0.00001 and MOI=0.000001 are basically equal. MOI=0.00001 is selected as the optimal MOI for phage pha0233, that is, when the phage and host bacteria are mixed at a ratio of 1:100000, the phage proliferation efficiency is the highest.

[0081] Example 4 Adsorption curve of bacteriophage

[0082] Methods: The titer of phage fluid was determined by the double-layer plate method, and the titer of the phage fluid was diluted to 1×10⁻⁶. 3 PFU / mL was used as the phage stock solution for later use, and the host bacteria were simultaneously resuscitated and cultured in liquid. 400 μL of bacterial culture was aliquoted into 1.5 mL EP tubes and cultured for 20 min. 100 μL of the diluted phage stock solution was added every 2 min. At 0 min, 100 μL of SM buffer was added as a blank control. The mixture was then centrifuged at 20000 g for 10 min at 4 °C. 100 μL of the supernatant was incubated with 400 μL of host bacteria culture at 37 °C for 10 min, and the titer was determined. Each experimental group was divided into three replicates.

[0083] like Figure 7 As shown, the adsorption of phage pha0233 reached a maximum of 92% at 6 min, and the progeny phage was released at 12 min. The number of free phages in the supernatant fluctuated between 16 and 20 min, possibly due to the adsorption-desorption of the progeny phage.

[0084] Example 5: One-step growth curve of bacteriophage

[0085] Phage titer was determined using the double-layer plate method. 1 mL of phage solution was thoroughly mixed with 10 mL of host bacteria solution and incubated at 37°C for 7 min to allow phage adsorption. The mixture was then centrifuged at 20,000 g for 1 min at 4°C, and the unadsorbed phage in the supernatant was discarded. The precipitate was washed with LB liquid medium preheated to 37°C, centrifuged again, and resuspended in 10 mL of LB liquid medium preheated to 37°C. The precipitate was then incubated in a shaker at 37°C for 3 h. Samples were taken at intervals, 200 μL each time, for a total of 11 samplings. Each collected sample was centrifuged separately at 20,000 g at 4°C for 10 min, and 100 μL of the supernatant was collected into a 1.5 mL EP tube containing 400 μL of host bacteria. The phage titer in the supernatant was then determined using the double-layer plate method. A one-step phage growth curve was plotted with infection time on the x-axis and phage titer on the y-axis.

[0086] like Figure 8 As shown, the one-step growth curve of phage pha0233 shows that it has an extremely short latency period. After adsorbing the host bacteria for 10 min, it rapidly lyses the host bacteria and releases progeny phages. Its lysis cycle is about 110 min, and the burst period is about 10-35 min. During this period, the phage titer increases rapidly. The fluctuation in phage titer between 35 min and 110 min may be due to the adsorption-desorption of progeny phages. It reaches a plateau at 110 min, and the phage titer tends to stabilize. The burst amount of phage pha0233 is calculated to be about 136 PFU / cell.

[0087] Example 6: Thermal stability of bacteriophages

[0088] The titer of phage fluid was determined using the double-layer plate method, and the concentration of phage fluid was adjusted to 1×10⁻⁶. 8 After PFU / mL, 170 μL of phage suspension was added to a sterile PCR tube and incubated at 4℃, 20℃, 37℃, 50℃, 65℃, and 80℃ for 30 min, 60 min, and 90 min, respectively. Then, 100 μL was taken and the titer of phage in each experimental group was determined using the double-layer plate method. Each experimental group was set up in triplicate.

[0089] like Figure 9 As shown, phage pha0233 exhibits high activity within 90 min of incubation at 4-50℃, but its activity decreases significantly after 30 min of incubation at 65℃, and it is completely inactivated after 60 min of incubation at 65℃ and 30 min or more of incubation at 80℃.

[0090] Example 7 pH stability of bacteriophages

[0091] The titer of phage fluid was determined using the double-layer plate method, and the concentration of phage fluid was adjusted to 1×10⁻⁶. 8 After PFU / mL, 100 μL of phage suspension was added to a 1.5 mL centrifuge tube containing 900 μL of SM buffer at different pH values ​​(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12). The mixture was incubated at 4℃ for 60 min. Subsequently, the titer of phage in each experimental group was determined using the double-layer plate method, with three replicates for each experimental group.

[0092] like Figure 10 As shown, phage pha0233 maintains stable activity over a very wide pH range (4-11), with relatively reduced activity at pH=3, extremely low activity at pH=2, and complete inactivation only under extremely alkaline conditions (pH=12).

[0093] Example 8 In vitro antibacterial curve

[0094] Phage titers were determined using the double-layer plate method, and host bacteria were revived and cultured in liquid form. The bacterial concentration was then adjusted to 1×10⁻⁶ using LB liquid medium. 9 CFU / mL. Add 1 mL of the above bacterial culture to a 250 mL Erlenmeyer flask containing 100 mL of LB liquid medium. Add phage fluid to the flasks according to MOIs of 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, and 0.000001 respectively. Mix well, and zero the flask using LB liquid medium as a blank control. Measure the OD using a UV spectrophotometer. 600nm The values ​​were then recorded, and the conical flasks were incubated at 37°C and 220 rpm for 12 h, with the OD value of the bacterial culture in the conical flasks measured every 1 h. 600nm Values ​​were set up in triplicate for each experimental group, based on the measured OD values. 600nm Plot the in vitro antibacterial curve of the bacteriophage.

[0095] like Figure 11 The in vitro antibacterial results of bacteriophage pha0233 showed that different MOIs effectively inhibited the growth of host bacteria within 0-8 h, keeping the OD value of the bacterial culture at a low level. Subsequently, a large-scale proliferation of resistant bacteria occurred, leading to an increase in the OD600 value. Furthermore, the higher the MOI, the earlier the resistant bacteria appeared. When the MOI was relatively low (MOI≤0.0001), resistant bacteria appeared only after 12 h. In summary, the inhibitory effect of bacteriophages on the growth of host bacteria is time-dependent.

[0096] Example 9: Inhibitory ability of bacteriophages on biofilm formation

[0097] First, Klebsiella pneumoniae was resuscitated and cultured in liquid culture, and the bacterial concentration was adjusted to 1×10⁻⁶ using culture medium.9 CFU / mL, 1 mL of bacterial culture was added to a 24-well plate. For the negative control group, 100 μL of LB liquid medium was added. For the positive control group (antibiotic group), 100 μL of ceftazidime at concentrations of 400 μg / mL (1 / 4 MIC), 800 μg / mL (1 / 2 MIC), and 1600 μg / mL (MIC) were added. For the phage experimental group, 100 μL of ceftazidime at concentrations of 10 CFU / mL were added. 5 PFU / mL (low dose, MOI=0.00001), 10 10 High-dose (MOI=1) phages were used, and 1.1 mL of LB liquid medium was added to 24-well plates for the blank control group. All groups were incubated at 37℃ for 24 h. After incubation, the bacterial culture was discarded, and the cells were washed three times with 1.5 mL of sterile PBS buffer (pH=7.4) and air-dried. The biofilm was then fixed with 1.5 mL of methanol for 15 min, the methanol was discarded, and the cells were air-dried. After staining with 1.1 mL of 0.1% crystal violet for 15 min, the cells were washed three times with 1.5 mL of PBS buffer and air-dried. Finally, the crystal violet was dissolved in 1.1 mL of 95% ethanol for 15 min, and the OD was measured using a microplate reader. 590nm Values ​​were calculated. Three replicates were set up for each group. The average absorbance after subtracting the blank control group (LB liquid medium) from the measurement results was used for significance analysis.

[0098] like Figure 12 Different concentrations of ceftazidime and bacteriophages effectively inhibited the formation of KPN045 biofilm (P<0.0001). There was no significant difference in the inhibitory effect on biofilm between low-dose (MOI=0.0001) and high-dose (MOI=1) bacteriophages, and the inhibitory effects of both were not significantly different from those of ceftazidime at a concentration of 1 / 2 MIC. At the same time, there was no difference between high-dose bacteriophages and ceftazidime at a concentration of MIC. Relatively speaking, the inhibitory effect of low-dose bacteriophages was not as good as that of ceftazidime at a concentration of MIC.

[0099] Example 10: The ability of bacteriophages to scavenge biofilms

[0100] First, Klebsiella pneumoniae was resuscitated and cultured in liquid culture, and the bacterial concentration was adjusted to 1×10⁻⁶ using culture medium. 9 CFU / mL, add 1 mL of bacterial culture to a 24-well plate (for the blank control group, add 1 mL of LB liquid medium to a 24-well plate), and incubate at 37°C for 24 h to form a biofilm. After incubation, discard the bacterial culture in each well, add 1 mL of sterile PBS buffer (pH=7.4) to each well to wash 3 times, and then air dry. Subsequently, add 900 μL of LB liquid medium to each well.

[0101] Add 100 μL of the corresponding reagent to each well according to the following groups:

[0102] Negative control group: 100 μL LB liquid culture medium.

[0103] Positive control group, i.e. antibiotic group: 100 μL of ceftazidime solution with concentrations of 12.5 μg / mL (1 / 4 MIC), 25 μg / mL (1 / 2 MIC), and 50 μg / mL (MIC).

[0104] Phage experimental group: 100 μL concentration of 10 5 PFU / mL (low dose, MOI=0.00001), 10 10 PFU / mL (high dose) phage solution.

[0105] Blank control group: 100 μL LB liquid culture medium

[0106] After adding the samples, the 24-well plates were incubated at 37°C for 24 h. After incubation, the bacterial culture in each well was discarded, and the plates were washed three times with 1 mL of sterile PBS buffer (pH=7.4) and then air-dried. 1 mL of methanol was added to each well to fix the biofilm, and the staining time was 15 min. The methanol was then discarded, and the plates were air-dried. Subsequently, 1 mL of 0.1% crystal violet was added to each well for staining, and the plates were washed three times with 1 mL of PBS buffer and then air-dried. Finally, 1 mL of 95% ethanol was added to each well to dissolve the crystal violet, and the OD was measured using a microplate reader after 15 min. 590nm Values ​​were calculated. Each experimental group was set up in triplicate. The average absorbance after subtracting the blank control group (LB liquid medium) from the measurement results was used for significance analysis.

[0107] like Figure 13 Both ceftazidime and bacteriophage at different concentrations could effectively remove the biofilm formed by kpn045 (P<0.0001). The removal effect of high-dose (MOI=1) bacteriophage was better than that of low-dose bacteriophage (P<0.01), and there was no significant difference in removal effect between ceftazidime at a concentration of 1 / 2 MIC and ceftazidime at a concentration of 1 / 4 MIC. However, the removal capacity of both was not as good as that of ceftazidime at a concentration of 1 / 4 MIC.

Claims

1. A bacteriophage strain, characterized in that, The bacteriophage mentioned is Klebsiella pneumoniae phage vB_KpnM_pha0233, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 13, 2025, with accession number GDMCC NO: 67292-B1, and the deposit address is the Institute of Microbiology, Guangdong Academy of Sciences.

2. Use of the Klebsiella pneumoniae bacteriophage vB_KpnM_pha0233 as described in claim 1 in the preparation of a medicament for treating diseases caused by Klebsiella pneumoniae.

3. The application of Klebsiella pneumoniae bacteriophage vB_KpnM_pha0233 as described in claim 1 in the preparation of Klebsiella pneumoniae antibacterial agents.

4. The application according to claim 3, characterized in that, The antibacterial agent is used in food preservation, environmental disinfection, livestock breeding, poultry farming, aquaculture, water treatment, or the biomedical industry.

5. A phage preparation, characterized in that, It contains the Klebsiella pneumoniae phage vB_KpnM_pha0233 as described in claim 1.

6. The phage preparation according to claim 5, characterized in that, It also contains a buffer solution as a stabilizer, which is DPBS buffer, physiological saline, SM buffer or glycerol solution.

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