Klebsiella pneumoniae bacteriophage vBKpnMpha0172 and application thereof

Abstract

The invention discloses a klebsiella pneumoniae bacteriophage vBKpnMpha0172 and application thereof, and belongs to the technical field of microorganisms. The Klebsiella pneumoniae phage vBKpnMpha0172 is preserved in the Guangdong Microbiological Culture Collection Center on November 13, 2025, the preservation number is GDMCC NO: 67291-B1, and the preservation address is Institute of Microbiology, Academy of Sciences, Guangdong Province. The bacteriophage has excellent temperature and pH environment tolerance, the optimal infection complex number is as low as 0.00001, the initial use dosage of the bacteriophage is greatly reduced, high adsorption rate and rapid outbreak proliferation characteristics are combined, efficient lysis pressure on host bacteria can be formed in a short time, and the application range of the bacteriophage is widened. The method has a good application prospect in the industries of food preservation, environment disinfection, livestock breeding, poultry breeding, aquaculture, water treatment, biomedical treatment and the like.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Klebsiella pneumoniae bacteriophage vB_KpnM_pha0172 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_pha0172 and its applications.

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

[0008] This invention first provides a bacteriophage strain, namely Klebsiella pneumoniae phage vB_KpnM_pha0172, which was deposited on November 13, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 67291-B1, and the deposit address is the Institute of Microbiology, Guangdong Academy of Sciences.

[0009] This invention screened a Klebsiella pneumoniae phage vB_KpnM_pha0172 with high lytic activity, environmental stability and safety from the natural environment. This phage can specifically recognize and lyse Klebsiella pneumoniae to clear the infection and overcome the problem of antibiotic resistance in Klebsiella pneumoniae.

[0010] The present invention also provides the use of the aforementioned Klebsiella pneumoniae bacteriophage vB_KpnM_pha0172 in the preparation of a medicament for treating diseases caused by Klebsiella pneumoniae.

[0011] The present invention also provides the application of the aforementioned Klebsiella pneumoniae bacteriophage vB_KpnM_pha0172 in the preparation of Klebsiella pneumoniae antibacterial agents.

[0012] Furthermore, the antibacterial agent is used in food preservation, environmental disinfection, livestock breeding, poultry farming, aquaculture, water treatment, or the biomedical industry.

[0013] The present invention also provides a phage preparation comprising the aforementioned Klebsiella pneumoniae phage vB_KpnM_pha0172.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] Glycerin solution: Dissolve glycerin thoroughly with ddH2O to achieve a final concentration of 5-50%, and then filter it through a 0.22μm filter membrane for sterilization.

[0018] The applications of the phages described in the present invention mainly cover the fields of human health, animal husbandry, 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 this phage 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 husbandry, its application 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 farming safety. In environmental biological prevention and control, this phage can be specifically used to remove the host bacteria enriched in environments such as medical facilities and water bodies, and has application potential in hospital infection control and environmental microbial management. In addition, this phage also shows certain application prospects in preventing and treating pet- and livestock-related bacterial diseases, controlling plant bacterial diseases, and in food preservation, water and soil cleaning, and other fields.

[0019] Advantages of the present invention:

[0020] 1. The optimal multiplicity of infection (MOI) determined for phage vB_KpnM_pha0172 against Klebsiella pneumoniae kpn042 is 0.00001 (10 -5 ). This extremely low MOI parameter breaks through the conventional understanding in the prior art that phages are mostly applied with medium to high MOIs (0.1 - 10), and shows technical effects far beyond expectations at this optimal parameter: the adsorption rate of the phage to the target host bacteria is as high as 96%, enabling host recognition and infection to be completed in a short time, greatly shortening the latency period, and laying a foundation for rapid proliferation; only about 1 h after infection, the burst size of progeny phages can reach 1×10 5 PFU / mL, achieving rapid amplification of the phages; the extremely low MOI significantly reduces the initial dosage of the phages used, significantly saving production costs; combined with the characteristics of high adsorption rate and rapid burst proliferation, it can form a high-efficiency lysis pressure on the host bacteria in a short time, solving the technical defects such as high costs, proliferation saturation, and non-target microorganism interference caused by high MOIs in the prior art.

[0021] 2. Bacteriophage vB_KpnM_pha0172 exhibits excellent temperature and pH tolerance, demonstrating significant potential for practical applications. Specifically, it maintains high activity after incubation at 4–50°C for 90 min, tolerating fluctuations in ambient temperature. At 50°C, only a slight decrease in activity occurs without significant functional loss. However, it is completely inactivated after incubation at 65–80°C for 30 min or more, indicating controllable heat inactivation characteristics, facilitating disinfection and safe handling in practical applications. Furthermore, this phage maintains stable activity across a broad pH range of 3–11, only becoming completely inactivated under extreme acidic or alkaline conditions (pH=2, pH=12), overcoming the limitation of most phages that can only survive in neutral to weakly acidic or alkaline environments. The aforementioned temperature and pH tolerance characteristics enable this bacteriophage to be adapted to various complex environments, such as food preservation (e.g., antibacterial activity in acidic fruits and vegetables and weakly alkaline meat products), agricultural planting (e.g., microbial control in acidic soil and alkaline irrigation water), medical assistance (e.g., infection control in local weakly acidic or alkaline wounds), and wastewater treatment (e.g., purification of industrial wastewater with fluctuating pH levels). This solves the technical bottleneck of existing bacteriophages being difficult to apply on a large scale due to their poor environmental tolerance, and has significant industrialization value.

[0022] 3. The antibacterial effect of bacteriophage vB_KpnM_pha0172 exhibits a significant MOI-dependent effect, and precise bacterial control can be achieved in different scenarios by adjusting the multiplicity of infection: When a relatively high MOI (≥0.0001) is used, the bacteriophage can rapidly inhibit the proliferation of host bacteria within 0-5 h, reducing the OD of the bacterial culture. 600 The value is maintained at a low level, meeting the needs of short-term, high-efficiency bacterial control in food preservation and emergency treatment of wound infections. When using a relatively low MOI (≤0.00001), although the antibacterial effect is weaker than that of the high MOI group within 0-5 hours, long-term stable antibacterial effect can be achieved with prolonged action time, and no large-scale proliferation of resistant bacteria occurs within 12 hours. This overcomes the shortcomings of early outbreak of resistant bacteria and short duration of bacterial control under high MOI, and is suitable for long-term bacterial control scenarios such as agricultural root irrigation and sewage treatment. The characteristic of no outbreak of resistant bacteria within 12 hours under low MOI (≤0.00001) conditions breaks through the technical bottleneck of existing bacteriophages that "high doses easily induce resistance and low doses are ineffective in bacterial control", which greatly reduces the risk of bacterial resistance in long-term application and enhances the clinical and industrial application value of bacteriophage preparations. In addition, this bacteriophage can significantly inhibit and clear host bacterial biofilms in vitro (p<0.0001), and has unique advantages for clinically refractory biofilm-related infections (such as medical device-related infections and chronic wound infections) and biofilm blockage in industrial pipelines, filling the technological gap of low clearance rate of mature biofilms by conventional antibiotics.

[0023] 4. Genome-wide and phylogenetic analysis of bacteriophage vB_KpnM_pha0172 indicates that this bacteriophage is a novel strain and does not contain any genes related to antibiotic resistance or virulence. Compared to some bacteriophages or antibiotic preparations carrying resistance genes, this bacteriophage does not pose a risk of lateral transfer of antibiotic resistance genes to the host bacteria or environmental microorganisms during infection and proliferation. This effectively avoids the technical challenges of "superbug" development and complies with biosafety standards in clinical anti-infection and agricultural microbial control. The bacteriophage genome does not encode virulence factors (such as hemolysin, enterotoxins, and adhesion factors), meaning that infection of the host bacteria will not lead to increased virulence, nor will the expression of virulence genes harm beneficial organisms outside the target environment (such as human gut probiotics and agricultural beneficial microorganisms). This overcomes the technical limitation of some natural bacteriophages carrying virulence genes that restrict their application. This safety feature allows bacteriophage vB_KpnM_pha0172 to be directly applied in scenarios with stringent biosafety requirements, such as clinical wound infection treatment, infant food preservation, and probiotic protection in livestock and poultry farming. It eliminates the need for additional gene editing to remove harmful genes, significantly reducing formulation development costs and approval cycles, and possesses outstanding industrialization value. Attached Figure Description

[0024] Figure 1 This is the morphology of bacteriophage vB_KpnM_pha0172 under a transmission electron microscope.

[0025] Figure 2 This shows the morphology of phage vB_KpnM_pha0172 plaques on a double-layer plate.

[0026] Figure 3 It is the particle size of bacteriophage vB_KpnM_pha0172.

[0027] Figure 4 This is the whole genome annotation diagram of bacteriophage vB_KpnM_pha0172.

[0028] Figure 5This is a phylogenetic analysis of phage vB_KpnM_pha0172; 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 70% (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 classification at the family, genus, or species level. Differences; the vertical axis labels represent families, genera, or species, and the matching shapes and colors indicate consistency at a specific taxonomic level; B: Collinearity comparison between the vB_KpnM_pha0172 genome and NC_049454.1; the arrows indicate the position and orientation of the CDS in each genome (right arrows indicate positive strands, left arrows indicate negative strands); the lines between genomes indicate sequence similarity (as shown in the lower left corner), and the CDS are color-coded according to functional categories, as shown in the legend in the lower right corner; C41: Genome similarity between pairs of phage strains.

[0029] Figure 6 It is the optimal infection multiplicity for bacteriophage vB_KpnM_pha0172.

[0030] Figure 7 This is the adsorption curve of bacteriophage vB_KpnM_pha0172.

[0031] Figure 8 This is a one-step growth curve of bacteriophage vB_KpnM_pha0172.

[0032] Figure 9 The results show the thermal stability of bacteriophage vB_KpnM_pha0172.

[0033] Figure 10 These are the pH stability results for bacteriophage vB_KpnM_pha0172.

[0034] Figure 11 This is the in vitro antibacterial curve of bacteriophage vB_KpnM_pha0172.

[0035] Figure 12 It is the inhibitory effect of bacteriophage vB_KpnM_pha0172 on biofilm formation.

[0036] Figure 13 This refers to the biofilm removal capability of bacteriophage vB_KpnM_pha0172. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0038] Unless otherwise specified, all raw materials and reagents used in the examples are commercially available products.

[0039] The host bacterium used in this invention is Klebsiella pneumoniae kpn042, which was obtained from the following literature and renamed kpn042 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_pha0172 of this invention, with the accession number GDMCC NO: 67291-B1.

[0040] Jarvis WR, Highsmith AK. Bacterial growth and endotoxin production inlipid emulsion. J Clin Microbiol. 1984 Jan;19(1):17-20. doi: 10.1128 / jcm.19.1.17-20.1984. PMID: 6361059; PMCID: PMC270969.

[0041] The host bacterium was obtained from the above literature and has been renumbered and named kpn042 in this invention.

[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 kpn042) resuscitation: Klebsiella pneumoniae kpn042 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 Kpn042 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 Kpn042 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: KPN042 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 mixed by pipetting 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 kpn042 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 kpn042 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. The phage is named pha0172.

[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, the head of bacteriophage pha0172 is approximately 88 × 10⁵ nm in size, the tail is approximately 10⁹ nm before contraction and approximately 71 nm after contraction, exhibiting a 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 belongs to the order Caudovirales, family Myoviridae, and is named vB_KpnM_pha0172.

[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 pha0172 formed phage plaques with a diameter of 1.0±0.5 mm. The plaques were transparent with clear edges and no depolymerase halo around them.

[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 pha0172 particles is approximately 158 nm.

[0069] 2. Phage genomics and phylogenetic analysis

[0070] (1) Whole genome annotation and analysis

[0071] The full-length genome of bacteriophage pha0172 is 47616 bp, with a GC content of 52.77%. Using annotation tools such as PhaBox, Phastest, Prokka, Phagescope, NCBI Virus, and Genemarks, a total of 78 open reading frames (ORFs) were predicted, including 56 coding sequences (CDS). (See...) Figure 4 These CDS-encoded proteins are divided into the following seven modules according to their functions: (1) 18 phage structure-related proteins, including head closure proteins, tail fibrils, and base plate proteins; (2) 8 DNA packaging and replication-related proteins, including portal proteins, terminal enzyme large subunits, and terminal enzyme small subunits; (3) 8 regulatory-related proteins, including greening regulators, excision enzymes, and transcriptional repressors; (4) 7 transport and metabolism-related proteins, including signal recognition granule proteins and NAD+. +(5) Four types of lysis-related proteins, including Rz-like transmembrane proteins, endosin, and perforin; (6) Two types of membrane proteins; (7) Eight putative proteins. One tmRNA-SsrA and its binding protein SmpB were annotated in the predicted CDS. This result means that the phage has stronger host adaptability and more precise self-translational regulation ability. It is a "functional advantage gene" formed by its long-term co-evolution with the host. It has important theoretical value for understanding phage-bacterial interaction and developing phage therapy (such as modifying tmRNA to enhance lysis efficiency). Meanwhile, integrase and excision enzymes were also annotated in the phage genome, indicating that this phage is a temperate phage with both lysogen and lysis life cycles. However, no lysogeny was observed in pha0172 during the study. Its high lysis activity indicates that it was always in a lysis state. Furthermore, no potential antibiotic resistance genes or virulence genes were found in phage pha0172 by CARD and PhageLeads analysis tools.

[0072] (2) Phylogenetic analysis

[0073] like Figure 5 The whole genome sequence of bacteriophage pha0172 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] CompareM calculations showed that pha0172 and NC_049454.1 had an AAI value of 99.98%, indicating a very high overall proteomic similarity between them. However, the AAI values ​​with other phages were all less than 92%. VICTOR phylogenetic and classification analyses showed that although pha0172 and NC_049454.1 were clustered together, suggesting a possible common ancestor, species classification showed that pha0172, NC_049454.1, and other phages were classified as different species within the same genus, indicating that pha0172 was classified as a new species at the species level. Further double-sequence linear alignment of the whole genomes of pha0172 and NC_049454.1 revealed a high degree of consistency in the core genome sequence (high collinearity). However, pha0172 exhibited large regions in certain functional modules (Transport and metabolism, Membrane proteins and structure) that were absent in NC_049454.1, appearing as unique regions of pha0172 on the alignment map. This suggests that pha0172 may have acquired a unique gene cassette. New genes are the basis for new functions and are one of the important manifestations of "novelty," indicating that although the structural framework genes are largely the same, pha0172 has acquired new genes during evolution, making it a novel bacteriophage. VIRIDIC's pairwise genomic similarity results for 41 bacteriophages also showed that the similarity values ​​between pha0172 and the other 40 bacteriophages were all below the threshold for new species (95%), corroborating the VICTOR classification results and confirming that pha0172 is a novel bacteriophage.

[0075] Based on the morphological and genomic characteristics of phage pha0172, it was identified as Klebsiella pneumoniae phage vB_KpnM_pha0172, and was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 13, 2025, with accession number GDMCC NO: 67291-B1, at the 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 pha0172 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 pha0172, 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] The titer of phage fluid was determined using 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 number of free phages in the supernatant of phage pha0172 reached its lowest point after 8 minutes of incubation, meaning that its adsorption rate reached its highest point at 8 minutes, at 96%, and remained in the incubation period for 20 minutes without releasing progeny phages.

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

[0085] Phage titer was determined using the double-layer plate method. At the optimal MOI of 0.00001, 1 mL of phage solution was thoroughly mixed with 10 mL of host bacterial solution and incubated at 37°C for 7 min to allow phage adsorption. The mixture was then centrifuged at 4°C, 20000 g for 1 min, 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 20000 g, 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. Subsequently, the phage titer in the supernatant was determined using the double-layer plate method. A one-step growth curve of the phage was plotted with infection time as the x-axis and phage titer as the y-axis.

[0086] like Figure 8 As shown, the one-step growth curve of phage pha0172 reveals a short latency period. It rapidly lyses the host bacteria and releases progeny phages 20 minutes after adsorption, with a lysis cycle of approximately 55 minutes. The burst phase lasts approximately 20-50 minutes, during which the phage titer increases rapidly, reaching a plateau at 65 minutes, where the titer tends to stabilize. The calculated burst yield of phage pha0172 is extremely high, approximately 1 × 10⁻⁶. 5 PFU / mL.

[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 pha0172 exhibits high activity within 4-50℃ for 90 min, with a slight decrease in activity at 50℃, and complete inactivation at 65-80℃ for 30 min or more.

[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. 8After 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 pha0172 maintains stable activity over a very wide pH range (3-11) and is inactivated only under extreme acidic and alkaline conditions (pH=2, 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 In vitro antibacterial results of bacteriophage pha0172 showed that when its MOI was relatively high (MOI≥0.0001), it could effectively inhibit the growth of host bacteria within 0-5 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 OD value. 600nm The higher the MOI (Mean Inhibition Index), the earlier resistant bacteria appear. When the MOI is relatively low (MOI ≤ 0.00001), the antibacterial effect is not as significant as the above within 0-5 h, but it can still effectively inhibit bacterial growth over time, and no large-scale proliferation of resistant bacteria occurs within 12 h. In summary, it can be concluded that 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 12.5 μg / mL (1 / 4 MIC), 25 μg / mL (1 / 2 MIC), and 50 μg / mL (MIC) were added. For the phage experimental group, 100 μL of ceftazidime at concentrations of 10 μg / mL (MIC) and 10 μg / mL (MIC) were added. 5 PFU / mL (low-dose MOI=0.00001), 10 10 Phages at PFU / mL (high dose, MOI=1) were added to 24-well plates, along with 1.1 mL of LB liquid medium for the blank control group. All groups were incubated at 37℃ for 24 h. 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. The cells were then stained with 1.1 mL of 0.1% crystal violet for 15 min, 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 Klebsiella pneumoniae kpn042 biofilm (P<0.0001). There was no significant difference in the inhibitory effect on biofilm between low dose (MOI=0.00001) and high dose (MOI=1) of bacteriophages. The inhibitory effect of both was not significantly different from that of ceftazidime at a concentration of 1 / 2 MIC, but was less than 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 clear biofilms formed by Klebsiella pneumoniae kpn042 (P<0.0001). The high-dose (MOI=1) bacteriophage showed better clearance than the low-dose bacteriophage (P<0.001), and its clearance effect was not significantly different from that of ceftazidime at a concentration of 1 / 2 MIC, but it was not as good as that of ceftazidime at a concentration of MIC. The clearance effect of the low-dose (MOI=0.00001) bacteriophage on the formed biofilm was between that of ceftazidime at a concentration of 1 / 2 MIC and 1 / 4 MIC.

Claims

1. A bacteriophage strain, characterized in that, The bacteriophage mentioned is Klebsiella pneumoniae phage vB_KpnM_pha0172, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 13, 2025, with accession number GDMCC NO: 67291-B1, and the deposit address is Institute of Microbiology, Guangdong Academy of Sciences.

2. Use of the Klebsiella pneumoniae bacteriophage vB_KpnM_pha0172 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_pha0172 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 bacteriophage preparation, characterized in that, It contains the Klebsiella pneumoniae phage vB_KpnM_pha0172 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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