Phage for splitting K1 capsular klebsiella pneumoniae and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a bacteriophage for splitting K1 capsular klebsiella pneumoniae and application of the bacteriophage. The technical problem to be solved by the invention is to provide a new choice for treating high-toxicity klebsiella pneumoniae infection. According to the technical scheme, the phage Yonganavius KP03 for splitting K1 capsular klebsiella pneumoniae is provided, and the preservation number of the phage is CCTCC (China Center For Type Culture Collection) NO: M20241155. The bacteriophage is specific in antibacterial spectrum and short in incubation period, can be applied to preparation of drugs for preventing and treating high-toxicity klebsiella pneumoniae, and has good treatment and killing effects on animals infected with multi-drug-resistant klebsiella pneumoniae and environments polluted by the multi-drug-resistant klebsiella pneumoniae.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a bacteriophage capable of lysing K1 capsule type Klebsiella pneumoniae and application thereof. Background Art

[0002] Klebsiella pneumoniae is an important zoonotic pathogen. In addition to causing severe bovine mastitis, it is also one of the common conditional pathogens in human medicine. It can cause various diseases including pneumonia, urinary tract infection, liver abscess, bacteremia, conjunctivitis and meningitis, causing considerable economic losses to the breeding and medical industries. With the unlimited use of antibiotics in clinical and breeding industries, the drug resistance of Klebsiella pneumoniae has become increasingly severe, especially the emergence of carbapenem-resistant hypervirulent strains (carbapenem-resistant hypervirulent Klebsiella pneumoniae), which has made the antibiotic treatment of Klebsiella pneumoniae almost "unavailable". The increasing resistance of pathogenic bacteria to antibiotics poses a huge threat to human health, public health safety and the healthy development of the breeding industry. Klebsiella pneumoniae is one of the most drug-resistant opportunistic pathogens in zoonotic organisms. Some Klebsiella pneumoniae, including K1 and K2 capsular types, are extremely virulent and can cause a variety of infectious diseases in animals and humans. Infections caused by these highly drug-resistant and highly virulent Klebsiella pneumoniae strains are extremely difficult to prevent and control, and there is currently a lack of effective drugs and treatment strategies.

[0003] Bacteriophages are a type of virus that specifically infects bacteria. They can infect microorganisms such as bacteria, fungi, actinomycetes or spirochetes and cause the lysis of host bacteria. They are natural bactericidal substances and have been widely used in the treatment of otolaryngology, cavity medicine, ophthalmology, dermatology, pediatrics and lung diseases. As the global medical field faces an increasingly severe threat of bacterial drug resistance, bacteriophages have great potential in the treatment or prevention of diseases due to their efficient and specific bactericidal effects. Previously, highly virulent and drug-resistant Klebsiella pneumoniae have been reported, but there are few studies on highly virulent Klebsiella pneumoniae phages. It is of great significance to develop new phages that have specific and potent preventive and therapeutic effects on highly virulent Klebsiella pneumoniae. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a new option for treating highly virulent Klebsiella pneumoniae infection.

[0005] The technical solution of the present invention is Klebsiella pneumoniae phage Yongunavirus KP03, which is preserved in China Center for Type Culture Collection (CCTCC), with a preservation address of Wuhan University, Wuhan, China, zip code 430072; the preservation number is CCTCCNO: M20241155.

[0006] The present invention also provides the use of the bacteriophage Yongunavirus KP03 in preparing a preparation for preventing and treating K1 capsular Klebsiella pneumoniae.

[0007] The present invention also provides a method for preparing a preparation for preventing and treating K1 capsular Klebsiella pneumoniae, comprising the following steps:

[0008] S1. Cultivate K1 capsular Klebsiella pneumoniae to the early logarithmic growth stage, add bacteriophage YongunavirusKP03, and culture until the solution is clear, add 1% chloroform to rupture the bacteria, add NaCl to a final concentration of 1 mol / L, stir until completely dissolved, place in an ice bath for 6 h, centrifuge, collect the supernatant, and obtain a lysate;

[0009] S2. Add PEG8000 to the lysate, gently invert to dissolve it (no vigorous inversion), place in an ice bath, centrifuge, and collect the precipitate;

[0010] The precipitate was dissolved in S3 and SM buffer, and chloroform was added for extraction. The upper aqueous phase was collected and filtered to obtain the preparation.

[0011] Specifically, in step S1, the ratio of the number of live bacteriophages added to the number of live Klebsiella pneumoniae host bacteria is 1:10.

[0012] In step S1, after culturing until the solution is clear, DNaseI and RNaseA are added to a final concentration of 1 μg / mL, and the mixture is shaken and cultured for 30 minutes.

[0013] Furthermore, in step S1, the centrifugation conditions are 4°C and 10,000×g for 15 min.

[0014] In step S2, the amount of PEG8000 added is: 10 g PEG8000 is added to every 100 mL of lysate. The purpose of adding PEG8000 is to precipitate the phages, thereby concentrating the phages and further improving the phage titer.

[0015] Particularly, in step S2, the centrifugation conditions are 4°C and 12000 rpm for 15 min.

[0016] Specifically, in step S3, a filter membrane with a pore size of 0.22 μm is used for filtration.

[0017] The present invention also provides a preparation for preventing and treating K1 capsular Klebsiella pneumoniae obtained by the preparation method.

[0018] Furthermore, the preparation is a spray or an injection.

[0019] Beneficial effects of the present invention: The present invention discloses a novel phage that can lyse K1 capsule-type highly virulent Klebsiella pneumoniae. The phage not only has a good disinfecting effect on the host multidrug-resistant highly virulent Klebsiella pneumoniae isolated from cattle farm sewage, but also has a strong disinfecting effect on the other three multidrug-resistant Klebsiella pneumoniae isolated from the hospital. The antibacterial spectrum is relatively specific, and can be used to prepare drugs for the prevention and treatment of highly virulent Klebsiella pneumoniae. The phage has a short incubation period, can quickly kill the host bacteria, has low toxic and side effects, is highly safe, has a relatively wide temperature and acid-base tolerance range, and has good hydrophilicity. It is easy to be made into a spray and an injection, and has a good therapeutic and killing effect on animals infected with multidrug-resistant Klebsiella pneumoniae and environments contaminated by multidrug-resistant Klebsiella pneumoniae.

[0020] The bacteriophage Yongunavirus KP03 for lysing K1 capsular Klebsiella pneumoniae of the present invention was deposited in the China Center for Type Culture Collection (CCTCC) on June 6, 2024, with the deposit address: Wuhan University, Wuhan, China, Postal Code 430072; the deposit number is CCTCC NO: M20241155. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 , plaque image of bacteriophage Yongunavirus KP03.

[0022] Figure 2 , transmission electron micrograph of bacteriophage Yongunavirus KP03.

[0023] Figure 3 , one-step growth curve of bacteriophage Yongunavirus KP03.

[0024] Figure 4 , temperature stability diagram of bacteriophage Yongunavirus KP03.

[0025] Figure 5 , acid-base tolerance diagram of bacteriophage Yongunavirus KP03.

[0026] Figure 6 , the minimum lethal dose of Klebsiella pneumoniae GXNNKP03 in mice.

[0027] Figure 7 , The effect of different doses of bacteriophage Yongunavirus KP03 on mouse survival rate. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present application will be described in detail below in combination with the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present application are within the scope of protection of the present application.

[0029] LB nutrient agar, LB broth, MacConkey MAC agar, and SM buffer were purchased from Beijing Solebow Technology Co., Ltd.; agar powder was purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.; 1 mol / L Tris, NaCl, MgSO4, EDTA (disodium ethylenediaminetetraacetic acid), PBS, DNase I, RNaseA (TaKaRa), solid calcium chloride, concentrated HCl, etc. were commercially available.

[0030] Example 1 Isolation and Preparation of Klebsiella pneumoniae Phage KP03

[0031] Under sterile conditions, 20 μL of mastitis milk sample was dripped onto MAC agar medium, placed in a 37°C constant temperature incubator for 24 h, and the growth of the colonies was observed. Typical colonies that were large, raised, mucous, and pink were selected for separation and purification to obtain Klebsiella pneumoniae GXNNKP03 (deposited in China Center for Type Culture Collection (CCTCC), deposit address: Wuhan University, Wuhan, China, zip code 430072). Klebsiella pneumoniae GXNNKP03 was streaked and inoculated onto 1.5% LB agar medium, and after culturing overnight, a single clone was picked and inoculated into 5 mL LB liquid medium, and then cultured at 37°C for 5 to 6 h as a host bacterial culture for standby use.

[0032] The phage was isolated by sewage enrichment method: centrifugation at 4°C and 8000rpm for 10min, the purpose is to remove large particles of waste residue from natural environmental sewage from farms, sewage treatment plants, ponds, etc., take the supernatant, add CaC12 to a final concentration of 1mmo1 / L, act for about 15min, and then filter with a 0.22μm pore filter. Take 100mL of filtrate and add it to 2×100mL LB liquid culture medium, and add Klebsiella pneumoniae liquid in logarithmic phase growth at a ratio of 1%. After overnight culture at 37°C and 180rpm, take an appropriate volume and centrifuge at 10000rpm for 10min, take the supernatant, and filter through a 0.22μm pore filter to remove residual bacteria, and collect the supernatant as the phage stock solution. Add 200 μL of host bacterial solution in logarithmic growth phase to 5-6 mL of 0.5% LB agar medium, mix well and pour onto 1.5% LB agar plate. After solidification, add 10 μL of phage stock solution. Add the same amount of sterile water to the control group. After the plate is dry, place it at 37°C for 12 hours to observe whether plaques appear. If plaques are formed, it proves that phages exist.

[0033] The phage was purified by double-layer agar plate method. The phage stock solution was diluted 10 times with PBS buffer, and 100 mL of the bacterial dilution was mixed with 200 mL of logarithmic host bacteria for 10 minutes. It was added to 5-6 mL of 0.5% LB agar medium and mixed thoroughly. It was spread on a 1.5% LB agar plate. After the upper layer solidified, it was moved to a 37°C constant temperature incubator for overnight culture. A single plaque was picked and purified until plaques of uniform size appeared. This was a purified phage, named Klebsiella pneumoniae phage Yongunavirus KP03. The results are shown in Figure 2. Figure 1 Store at 4℃ until use.

[0034] Use double-layer plates to detect phage titer: dilute the purified bacteria in 10-fold gradients, take 0.1 mL of the corresponding bacterial dilutions and mix them with 0.2 mL of the host bacterial solution, spread on double-layer agar plates, and culture at 37°C for about 8 hours. Count the plaques on each plate. Select plates with about 100 to 300 plaques, and calculate the initial phage concentration based on the dilution multiple to obtain the phage titer. Phage titer (PFU / mL) = dilution multiple × number of plaques × 10, the phage Yongunavirus KP03 titer is 1.33 × 10 9 PFU / mL.

[0035] The isolated phage was named KP03 and deposited in the China Center for Type Culture Collection (CCTCC) at Wuhan University, Wuhan, China.

[0036] Example 2 Amplification and concentration of bacteriophage

[0037] The host bacteria were cultured at 37℃ and 160rpm with shaking until the initial logarithmic growth. Phages were added at the optimal infection multiplicity, and the solution was cultured at 37℃ and 160rpm with shaking until the solution was clear. DNaseI and RNaseA were added to a final concentration of 1μg / mL, and then placed in a shaker for shaking culture for 30min, and then 1% chloroform was added to break the bacteria. NaCl was added to a final concentration of 1mol / L, and the mixture was stirred and mixed evenly. After complete dissolution, the mixture was placed in an ice bath for 6h. The lysate was centrifuged at 4℃ and 10000×g for 15min, and the supernatant was collected. 10g of PEG8000 was added to every 100mL of the collected supernatant, and it was gently inverted to dissolve it. It was forbidden to be violent. It was placed in an ice bath at 4℃ overnight to precipitate the phage particles. After that, it was centrifuged at 4℃ and 12000rpm for 15min, and the supernatant was discarded, leaving the precipitate. The centrifuge tube containing the precipitate was opened and inverted to allow the remaining liquid to drain. Add 2 mL of SM buffer to the centrifuge tube to dissolve the precipitate, extract with an equal volume of chloroform 2 to 3 times, take the upper aqueous phase, filter it through a 0.22 μm pore size filter membrane into a new tube to obtain the phage concentrate.

[0038] Example 3 Transmission electron microscopic observation of Klebsiella pneumoniae phage KP03

[0039] Pipette a drop of phage stock solution (about 20 μL) onto the copper mesh, let it stand for 15 minutes, remove the excess liquid, let it stand for 15 minutes, remove the excess liquid, add a drop of 2% phosphotungstic acid solution, stain for 5 minutes, then immediately remove the excess staining liquid on the side, and observe the morphology under a transmission electron microscope after drying. The observation results are as follows: Figure 2 As shown in the figure, electron microscopy observation shows that the head of the phage is a regular icosahedral structure with a head diameter of about 55nm, belonging to the order Caudata and the family Siphoviridae. According to the "Taxonomy of Viruses - The Eighth Report of the International Committee on Taxonomy of Viruses" published by the International Committee on Taxonomy of Viruses (ICTV) in 2015, the phage belongs to the family Siphoviridae.

[0040] Example 4 Determination of the Optimal Multiplicity of Infection of Klebsiella Pneumoniae Phage KP03

[0041] The multiplicity of infection (MOI) is the ratio of the number of phages to the number of host bacteria at the beginning of infection. Specifically, it measures the ratio of the number of phages adsorbed on bacteria to the number of bacteria in culture. There is an optimal multiplicity of infection for the combination of phages and host bacteria. The determination method is as follows: Cultivate the host bacteria to the logarithmic phase, and the number of colonies is about 10 8CFU / mL, add phage at a MOI of 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, incubate at 37°C for 10 minutes, add 10mL LB liquid culture medium, shake culture at 37°C and 160rpm for 6 hours, centrifuge at 10000rpm for 10 minutes, take the supernatant and filter it with a 0.22μm pore size filter membrane, then dilute it with LB liquid culture medium in a 10-fold gradient, and determine the titer by double-layer agar plate method. Each independent experiment was repeated 3 times. The results are shown in Table 1. The optimal infection multiplicity of Klebsiella pneumoniae phage KP03 is 0.1. It shows that the titer of the offspring phage obtained by adding phage and host bacteria at a ratio of 1:10 is the highest.

[0042] Table 1 Optimal multiplicity of infection of Klebsiella pneumoniae phage KP03

[0043] Multiplicity of infection MOI (PFU / CFU) Phage titer (PFU / mL) 0.00001 <![CDATA[1.2×10 9 ]]> 0.0001 <![CDATA[2.1×10 9 ]]> 0.001 <![CDATA[2.5×10 9 ]]> 0.01 <![CDATA[4.6×10 9 ]]> 0.1 <![CDATA[5.2×10 9 ]]> 1 <![CDATA[2.5×10 9 ]]> 10 <![CDATA[2.1×10 9 ]]> 100 <![CDATA[1.0×10 9 ]]>

[0044] Example 5 Determination of one-step growth curve of Klebsiella pneumoniae phage KP03

[0045] The method for determining the one-step growth curve is as follows: take 1 mL of bacterial solution with a concentration of 1×10 8 CFU / mL, and mixed with phage according to the optimal infection multiplicity, incubated at 37℃, adsorbed for 5min, centrifuged at 4℃, 5000rpm for 5min, discarded the supernatant, and then added 1.5mL LB liquid culture medium preheated to 37℃ to resuspend, and then centrifuged at 4℃, 5000rpm for 5min, discarded the supernatant, and repeated this step twice to ensure that the free phage without adsorption is removed. Add to a centrifuge tube containing 10mL LB liquid culture medium preheated at 37℃ and resuspend, place the above centrifuge tube in a shaker at 37℃, 180rpm, and sample 200mL every 10min, centrifuged at 4℃, 10000rpm for 5min, marked with serial numbers, and sampled continuously for 100min. The titer of the phage was determined every 10min using the double-layer agar plate method, and a one-step growth curve was drawn with the horizontal axis being the infection time and the vertical axis being the phage titer. Each independent test was repeated 3 times.

[0046] The results are as follows Figure 3 As shown, the titer of phage KP03 did not change significantly in the first 10 minutes of infecting the host bacteria, then the phage titer increased rapidly and stabilized after 40 minutes, that is, the incubation period of the phage was about 10 minutes, the outbreak period was about 40 minutes, and the outbreak amount was 152 PFU / Ce11.

[0047] Example 6 Temperature tolerance experiment of Klebsiella pneumoniae bacteriophage KP03

[0048] Take 10 sterile EP tubes, take 1 mL of phage lysate and place them in 1.5 mL EP tubes, and place them in a water bath at 4°C, 25°C, 37°C, 50°C, 60°C, 70°C, and 80°C for 60 min. Take 100 mL of each tube and use the double-layer plate method to determine the phage titer. Plot the temperature as the horizontal axis and the corresponding phage titer as the vertical axis, and derive the optimal growth temperature range of the phage based on the changes in bacterial titer. Each independent experiment was repeated 3 times. The results are as follows: Figure 4 As shown, the suitable temperature of phage KP03 is 4-60°C, and the suitable temperature range of this phage is relatively wide.

[0049] Example 7 pH tolerance experiment of Klebsiella pneumoniae phage KP03

[0050] Take 9mL LB broth, adjust its pH value to 3, 4, 5, 6, 7, 8, 9, 10, 11 respectively, add 1mL phage lysis solution, and leave at room temperature for 2h. Use the double-layer plate method to determine the phage titer, and measure it in parallel 3 times. Plot the graph with pH as the horizontal axis and the corresponding phage titer as the vertical axis, and obtain the optimal pH range for phage growth based on the change in phage titer. Each independent experiment was repeated 3 times. The results are as follows. Figure 5 As shown, the suitable pH range of bacteriophage KP03 is 3-11, and the suitable pH range of this bacteriophage is relatively wide.

[0051] Example 8 Host spectrum analysis of Klebsiella pneumoniae phage KP03

[0052] serial number Strain name Strain origin Animal origin Serotype Lysis 1 GXNKP01 Nanning, Guangxi Niu Yuan K1 + 2 GXQKP02 Qinzhou, Guangxi Niu Yuan K1 + 3 GXNNKP03 Nanning, Guangxi Niu Yuan K1 + 4 GXYLKP05 Yulin, Guangxi Niu Yuan K1 + 5 GXNNKP06 Nanning, Guangxi Niu Yuan K1 + 6 GXHCKP07 Hechi, Guangxi Niu Yuan K1 + 7 GXHCKP08 Hechi, Guangxi Niu Yuan K1 + 8 GXNNKP09 Nanning, Guangxi Niu Yuan K1 + 9 GXNNKP10 Nanning, Guangxi Niu Yuan K1 + 10 GXYLKP11 Yulin, Guangxi Niu Yuan K1 + 11 GXNNKP12 Nanning, Guangxi Niu Yuan K2 - 12 GXQZKP13 Qinzhou, Guangxi Niu Yuan K2 - 13 GXNNKP14 Nanning, Guangxi Niu Yuan K2 - 14 GXNNKP15 Nanning, Guangxi Niu Yuan K2 - 15 GXNNKP16 Nanning, Guangxi Niu Yuan K2 - 16 GXNNKP17 Nanning, Guangxi Niu Yuan K2 - 17 GXBBKP18 Bobai, Guangxi Niu Yuan K54 - 18 GXBBKP19 Bobai, Guangxi Niu Yuan K54 - 19 GXBBKP20 Bobai, Guangxi Niu Yuan K54 - 20 GXBSKP21 Baise, Guangxi Niu Yuan K57 - 21 GXBSKP22 Baise, Guangxi Niu Yuan K57 - 22 GXBSKP23 Baise, Guangxi Niu Yuan K57 -

[0053] Note: In Table 1, “+” means that the phage can lyse the strain, and “_” means no lysis.

[0054] Example 9 Determination of the minimum lethal dose of Klebsiella pneumoniae GXNNKP03 in mice

[0055] Adjust the spare phage titer to 10 9 PFU / mL is for standby use, and phage KP03 host spectrum analysis. 22 strains of highly toxic Klebsiella pneumoniae from different sources and categories preserved in this laboratory were collected (separation method is shown in Example 1), and host spectrum analysis was carried out with phage. The specific operation is as follows: 0.2mL of 22 strains of Klebsiella pneumoniae overnight culture are taken respectively, 8mL of 0.7%LB semi-solid culture medium at about 45°C is added, and evenly spread on the solid 1.5%LB solid culture medium prepared in advance, and then each flat plate is evenly divided into four regions, wherein three regions take 10 μL of standby phage and drip on the surface, and another region drips and is physiological saline for comparison, and after the droplet is dried, it is inverted at 37°C and cultivated for 10h, and the observation result is recorded as "+" if plaque is produced, otherwise it is "-".

[0056] The results are shown in Table 2: Klebsiella pneumoniae KP03 can lyse the highly virulent Klebsiella pneumoniae of the K1 capsule type preserved in this laboratory, but cannot lyse the other K2, K54, and K57 types of Klebsiella pneumoniae, indicating that KP03 has specific lysis ability for K1 type Klebsiella pneumoniae.

[0057] Table 2 Host spectrum of Klebsiella pneumoniae phage KP03 of the present invention

[0058] Thirty-six mice were randomly divided into six groups (six mice in each group) and treated with different doses of bacterial solution (1.2×10 5 , 1.2×10 4 , 1.2×10 3 , 1.2×10 2 , 1.2×10 1 CFU / mouse) was injected intraperitoneally into experimental mice (6-8 week-old SPF grade BALB / c female mice), and the control group was injected intraperitoneally with an equal amount of sterile PBS. The survival status of mice in each group was recorded every 12 hours. The results of mouse survival are shown in Figure 6 As shown. Based on the death of mice in 7 days, the minimum lethal dose of highly pathogenic Klebsiella pneumoniae GXNNKP03 was obtained. Results The survival rate of mice injected intraperitoneally with 12 CFU of GXNNKP03 pathogen was 85% within 7 days. At the same time, the infected mice showed severe clinical symptoms, refused to eat and drink, had frizzy hair, and had stiff limbs. Intraperitoneal injection of 1.2×10 2 The survival rate of mice infected with CFU GXNNKP03 pathogen was 80% within 7 days; 1.2×10 3 The survival rate of mice infected with CFU GXNNKP03 pathogen was 20% within 7 days; 1.2×10 4 The survival rate of mice infected with CFU GXNNKP03 pathogens within 7 days was 0%; 1.2×10 5 The survival rate of mice infected with the pathogen CFUGXNNKP03 was 0 within 7 days; the conclusion is that the minimum lethal dose of highly virulent Klebsiella pneumoniae GXNNKP03 in mice is 1.2×10 4 CFU.

[0059] Example 10 In vivo safety test of bacteriophage KP03

[0060] Twelve 6- to 8-week-old SPF-grade BALB / c female mice were randomly divided into two groups, with 6 mice in each group. Each mouse in the experimental group was intraperitoneally injected with 1 mL of 10 9PFU / mL of phage KP03 preparation, and each mouse in the control group was intraperitoneally injected with an equal volume of sterile PBS. The two groups of mice were fed for one week and observed for clinical changes. Afterwards, the mice were killed, dissected and the internal organs, digestive tract and mucosa were observed. The results showed that after one week of continuous observation of the two groups of mice, the mice were in good mental state and had no abnormalities in daily behavior; the mice were killed by cervical dislocation, and no abnormalities were found in the tissues and organs of the dissection. This suggests that phage KP03 is safe and has no toxic side effects on mice.

[0061] Example 11 Effects of different doses of bacteriophage on the survival rate of mice

[0062] The specific experimental plan is as follows: 30 SPF BALB / c female mice aged 6 to 8 weeks were randomly divided into 5 groups, with 6 mice in each group. The phage treatment group was intraperitoneally injected with 2×MLD Klebsiella pneumoniae GXNNKP03 (1.2×10 4 CFU / mouse) 1 hour later, 1.2×10 3 PFU / mouse, 1.2×10 4 PFU / mouse, 1.2×10 5 The mice were treated with phages at a dose of PFU / mouse; the negative control group was treated with sterile PBS instead of PBS during both the challenge and treatment; the positive control group was treated with the same amount of sterile PBS after intraperitoneal injection of bacteria. The mortality of mice in each group was recorded every 12 hours.

[0063] Results after treatment with bacteriophage KP03 Figure 7 As shown, the attack bacteria 2×MLD (1.2×10 4 After the phage treatment group was infected with highly pathogenic Klebsiella pneumoniae (CFU / mouse), all untreated mice died within 48 hours. Compared with the control group, the survival rate of mice in the phage treatment group was significantly improved, with only 1.52×10 4 The phage dose of 1.52×10 5 The PFU dose of phage had the highest protection rate for mice, with a 5-day survival rate of 95%, indicating that the phage had a good therapeutic effect and greatly improved the survival rate of mice.

Claims

1. A bacteriophage Yongunavirus KP03 that lyses K1-capsulated Klebsiella pneumoniae, characterized in that: Its accession number is CCTCCNO: M20241155.

2. Use of the bacteriophage Yongunavirus KP03 according to claim 1 in the preparation of a preparation for preventing and treating K1 capsular Klebsiella pneumoniae.

3. A method for preparing a preparation for preventing and treating K1 capsular Klebsiella pneumoniae, characterized in that: The steps include: S1. Cultivate K1 capsular Klebsiella pneumoniae to the early logarithmic growth stage, add the bacteriophage Yongunavirus KP03 described in claim 1, and culture until the solution is clear, add 1% chloroform to rupture the bacteria, add NaCl to a final concentration of 1 mol / L, stir until completely dissolved, place in an ice bath for 6 h, centrifuge, collect the supernatant, and obtain a lysate; S2. Add PEG8000 to the lysate, gently invert to dissolve it (no vigorous inversion), place in an ice bath, centrifuge, and collect the precipitate; The precipitate was dissolved in S3 and SM buffer, and chloroform was added for extraction. The upper aqueous phase was collected and filtered to obtain the preparation.

4. The preparation method according to claim 3, characterized in that: In step S1, when the bacteriophage YongunavirusKP03 is added, the ratio of the number of live bacteriophage to the number of Klebsiella pneumoniae host bacteria is 1:

10.

5. The preparation method according to claim 3, characterized in that: In step S1, after culturing until the solution is clear, DNaseI and RNase A are added to a final concentration of 1 μg / mL and cultured with shaking for 30 minutes.

6. The preparation method according to claim 3, characterized in that: In step S1, the centrifugation conditions are 4°C and 10,000×g for 15 min.

7. The preparation method according to claim 3, characterized in that: Step S2, the amount of PEG8000 added is: 10 g PEG8000 is added to every 100 mL of lysate.

8. The preparation method according to claim 3, characterized in that: In step S2, the centrifugation conditions are 4°C and 12000 rpm for 15 min.

9. The preparation method according to claim 3, characterized in that: In step S3, a filter membrane with a pore size of 0.22 μm is used for filtration.

10. The preparation for preventing and treating K1-capsular Klebsiella pneumoniae obtained by the preparation method according to any one of claims 3 to 9; further, the preparation is a spray or an injection.

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