Phage for specifically splitting high-toxicity capsular klebsiella pneumoniae and application thereof
By screening out the bacteriophage vB_KpnP_Henu1_3 that specifically cleaves Klebsiella pneumoniae, the treatment problem of multidrug-resistant Klebsiella pneumoniae was solved, and the rapid and safe Klebsiella pneumoniae lysis and disinfection effect was achieved.
Patent Information
- Application Number
- CN202510521103.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-24
- Publication Date
- 2025-07-11
AI Technical Summary
The existing antibiotics have poor therapeutic effects on multidrug-resistant Klebsiella pneumoniae, and new antibacterial strategies are urgently needed. Bacteriotherapy has significant advantages.
The bacteriophage vB_KpnP_Henu1_3, which specifically cleaves Klebsiella pneumoniae, was screened for the preparation of drugs and disinfectants with polyhedral head and long tail structure, high titer, stable temperature and pH.
The bacteriophage vB_KpnP_Henu1_3 can quickly and effectively lyse Klebsiella pneumoniae, has good temperature and acid-base tolerance, high safety, and is widely used in medical devices and environmental disinfection.
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Figure CN120290496A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of microorganisms, and specifically to a phage capable of specifically lysing highly virulent capsular Klebsiella pneumoniae and its application. Background Art
[0002] Klebsiella pneumoniae (Kp) is an opportunistic pathogen that can cause a series of infectious diseases in humans and is also a common pathogen in hospitals. Klebsiella pneumoniae exists in the upper respiratory tract and intestines of the human body. When the body's immunity decreases, it can cause respiratory tract, urogenital tract infections, trauma, sepsis, and diarrhea. Usually, carbapenem antibiotics are commonly used to treat Klebsiella pneumoniae infections. However, with the continuous update and extensive use of antibiotics, the bacterial resistance to antibacterial drugs has been continuously increasing. Klebsiella pneumoniae has the characteristics of multidrug resistance, heterogeneous resistance, and even pan-resistance. Colistin, the last line of defense for treating multidrug-resistant Gram-negative bacterial infections, has also lost its effect. To prevent drug-resistant bacteria from becoming a fatal reality, new strategies are urgently needed to reduce the risk of severe infections by means of vaccines, new drugs, improved medical services, improved accessibility of existing antimicrobial drugs, and the development of relevant guidance on how to use antimicrobial drugs most effectively; Phages are the most abundant organisms in the biosphere. They are a type of virus that can specifically and selectively kill bacteria and have no adverse effects on eukaryotic cells. Compared with antibiotic treatment of bacterial diseases, phages have significant advantages in preventing and treating diseases. Phages have a high degree of specificity. Due to the differences in their adsorption sites and the structures of bacterial surface receptors, one phage only targets a single or the same subtype of pathogenic bacteria without destroying the normal flora in the animal body. Phages only proliferate at the site of bacterial infection. A small amount of phages can proliferate on a large scale at the infection site and quickly kill pathogenic bacteria. As the host bacteria are cleared, the phages gradually excrete from the body due to the loss of their targets. Phages are widely distributed and numerous, and the screening of new phages usually only takes a few days or weeks. Phages are safe for human and animal cells and do not produce toxicity. Therefore, phage therapy has the potential to become an effective alternative to antibiotics and is one of the strategies for controlling drug-resistant bacteria. For this reason, we have proposed a phage capable of specifically lysing highly virulent capsular Klebsiella pneumoniae and its application. Summary of the Invention
[0003] The technical problem to be solved by the present invention is to overcome the existing defects and provide a phage capable of specifically lysing highly virulent capsular Klebsiella pneumoniae and its application, which can effectively solve the problems in the background art.
[0004] To achieve the above object, the present invention provides the following technical solution: A phage that specifically lyses highly virulent capsular Klebsiella pneumoniae and its application, including a phage named Klebsiella phage vB_KpnP_Henu1_3, which belongs to the family Siphoviridae and has a polyhedral head; the titer is ≥1010, the growth latent period is 20 min, the rapid growth period is 20 - 60 min, the temperature stability range is 4 - 55 °C, and the pH stability range is 4 - 12.
[0005] Furthermore, the application of the phage in degrading the biofilm of Klebsiella pneumoniae serotype K1 with high virulence.
[0006] Furthermore, the phage is used to prepare a drug or biological agent for preventing or treating Klebsiella pneumoniae infectious diseases, and the drug or biological agent uses phage vB_KpnP_Henu1_3 as the sole active ingredient.
[0007] Furthermore, the Klebsiella pneumoniae includes Klebsiella pneumoniae Kp1049.
[0008] Furthermore, the application of the above-mentioned Klebsiella pneumoniae in preparing a drug or biological agent for preventing or treating Klebsiella pneumoniae infectious diseases; such as lysate, phage drug preparation, phage cocktail composition, combination of phage and antibiotic, etc.
[0009] Furthermore, the application of Klebsiella pneumoniae phage in preparing a phage disinfectant or inhibitor: for sterilization and disinfection of medical device environment, hospital environment, hospital ward, animal breeding environment and other environments.
[0010] Compared with the prior art, the beneficial effects of the present invention are: The present invention uses Klebsiella pneumoniae Kp1049 as the host, samples from untreated hospital sewage, and screens out the phage vB_KpnP_Henu1_3, which can efficiently lyse Klebsiella pneumoniae Kp1049, providing a phage source for the treatment of Klebsiella pneumoniae in hospitals. The phage vB_KpnP_Henu1_3 can not only enrich the germplasm resource library of Klebsiella pneumoniae phages but also provide an alternative basic material for the biological control of Klebsiella experimental bacteria. The phage vB_KpnP_Henu1_3 has good temperature and acid-base tolerance and can maintain a stable titer level during long-term storage at room temperature. This phage can show an obvious lysis effect on Klebsiella pneumoniae Kp1049 within 1 hour, having good therapeutic effects and application prospects. After whole-genome sequencing, the phage vB_KpnP_Henu1_3 was found to have no virulence and drug resistance-related genes, ensuring the safety of this phage preparation during use. The phage vB_KpnP_Henu1_3 has a high safety factor and a wide application range in the preparation of phage disinfectants and phage preparations for medical device surfaces, hospital environments, and other environments, and has good application prospects. Brief Description of the Drawings
[0011] Figure 1 It is the plaque map of the Klebsiella pneumoniae phage vB_KpnP_Henu1_3 in a phage for specifically lysing highly virulent capsular Klebsiella pneumoniae and its application of the present invention; Figure 2 It is the transmission electron microscope image of the Klebsiella pneumoniae phage vB_KpnP_Henu1_3 in a phage for specifically lysing highly virulent capsular Klebsiella pneumoniae and its application of the present invention; Figure 3 It is the optimal multiplicity of infection map of the Klebsiella pneumoniae phage vB_KpnP_Henu1_3 in a phage for specifically lysing highly virulent capsular Klebsiella pneumoniae and its application of the present invention; Figure 4 It is the one-step growth curve map of the Klebsiella pneumoniae phage vB_KpnP_Henu1_3 in a phage for specifically lysing highly virulent capsular Klebsiella pneumoniae and its application of the present invention; Figure 5 It is the temperature stability map of the Klebsiella pneumoniae phage vB_KpnP_Henu1_3 in a phage for specifically lysing highly virulent capsular Klebsiella pneumoniae and its application of the present invention; Figure 6 It is the pH stability map of the Klebsiella pneumoniae phage vB_KpnP_Henu1_3 in a phage for specifically lysing highly virulent capsular Klebsiella pneumoniae and its application of the present invention; Figure 7 Genomic map of Klebsiella pneumoniae phage vB_KpnP_Henu1_3 in a phage that specifically lyses highly virulent capsular Klebsiella pneumoniae and its application Figure 8 Antibacterial activity map of Klebsiella pneumoniae phage vB_KpnP_Henu1_3 in a phage that specifically lyses highly virulent capsular Klebsiella pneumoniae and its application Figure 9 Anti-biofilm activity map of Klebsiella pneumoniae phage vB_KpnP_Henu1_3 in a phage that specifically lyses highly virulent capsular Klebsiella pneumoniae and its application Figure 10 Antibacterial activity map of Klebsiella pneumoniae phage vB_KpnP_Henu1_3 in Galleria mellonella in a phage that specifically lyses highly virulent capsular Klebsiella pneumoniae and its application Figure 11 Antibacterial activity map of Klebsiella pneumoniae phage vB_KpnP_Henu1_3 in mice in a phage that specifically lyses highly virulent capsular Klebsiella pneumoniae and its application Detailed implementation manners
[0012] To make the technical means, creative features, achieved purposes and functions of the present invention easy to understand, the present invention will be further described below in conjunction with specific implementation manners.
[0013] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention. Example 1
[0014] Isolation and purification of the Klebsiella pneumoniae phage of the present invention Using Klebsiella pneumoniae Kp1049 as the host bacterium, pick a single colony and inoculate it into LB liquid medium, and culture it at 37°C until the logarithmic phase; take 4000 rpm centrifugation of the unsterilized sewage from the hospital sewer for 5 min, filter the supernatant with a 0.22 μm needle filter, mix the filtered sewage with the host bacterium in the logarithmic phase, then quickly mix the mixture with water agar cooled to a suitable temperature, and pour it onto a pre-prepared LB solid petri dish. After solidification, culture it overnight at 37°C; if clear and distinct plaques are formed, it proves that the phage has been successfully isolated; otherwise, it has not.
[0015] If plaques appear on the petri dish, pick a single clear plaque and place it in physiological saline. After shaking for 2 minutes, centrifuge at 4000 rpm for 1 minute. Take 10 μL of the supernatant and mix it with the host bacteria in the logarithmic phase. Then, use the double-layer petri dish method to purify it continuously 3-4 times until plaques with uniform size and shape are formed, and the purified phage is obtained; pick a single plaque and inoculate it into 100 mL of the host bacteria solution in the logarithmic growth phase, expand the culture for 6 hours, centrifuge at 12000 rpm for 15 minutes, and filter it through a 0.22 μm filter membrane to obtain a phage solution with a high titer; the results are as follows Figure 1 , the phage formed clear plaques on the double-layer petri dish, with a halo around it and clear and regular edges, and it was a Klebsiella pneumoniae phage. Example 2
[0016] Biological characteristics of the Klebsiella pneumoniae phage of the present invention: (1) Observation of the morphological characteristics of phage vB_KpnP_Henu1_3 Observe the morphology of the phage through a transmission electron microscope. Attach the phage to a copper grid coated with carbon and stain it with 2% phosphotungstic acid negative staining solution for 5 minutes. Observe the morphology of the phage using a transmission electron microscope (Hitachi HT7700, Tokyo, Japan) at 80 kV.
[0017] The morphology of the phage is as follows Figure 1 shown. The phage head is about 50 nm in diameter, hexagonal, and has a long tail; according to these morphological characteristics and the regulations of the International Committee on Taxonomy of Viruses (ICTV), the phage should be a long-tailed phage morphologically.
[0018] (2) Determination of the optimal multiplicity of infection MOI of phage vB_KpnP_Henu1_3 To determine the optimal multiplicity of infection (MOI) of phage vB_KpnP_Henu1_3, perform a series of 10-fold serial dilutions of phage vB_KpnP_Henu1_3 using SM buffer; to evaluate the lowest concentration at which phage vB_KpnP_Henu1_3 can effectively inhibit the growth of host cells, infect Klebsiella pneumoniae Kp1049 with phage vB_KpnP_Henu1_3 at different MOIs, and use the double-layer agar plate method to detect the titer of phage vB_KpnP_Henu1_3 after infection at different MOIs.
[0019] The results are as follows Figure 3 shown. When the MOI is 0.01, the phage titer reaches the highest level, about 4×108 PFU / mL; therefore, we used MOI = 0.01 as the optimal infection dose in subsequent experiments.
[0020] One-step growth curve of bacteriophage vB_KpnP_Henu1_3 To explore the latent period and burst size of the infection of bacteriophage vB_KpnP_Henu1_3, the one-step growth curve method was used to determine the infection kinetics of bacteriophage vB_KpnP_Henu1_3; bacteriophage vB_KpnP_Henu1_3 was mixed with Kp1049 at an MOI of 0.01 and incubated at 37 °C for 15 minutes; the incubated sample was centrifuged at 12000 rpm for 10 minutes to discard the supernatant, and the precipitate was washed twice with sterile LB liquid medium preheated at 37 °C. The centrifuged pellet was resuspended in 20 mL of LB liquid medium and then placed on a shaker at 37 °C and shaken at 220 rpm. Samples were collected every 10 minutes and the titer of bacteriophage vB_KpnP_Henu1_3 was detected by the double-layer agar plate method until 120 minutes; the latent period and burst size were calculated using the method described in the literature (Kropinski, 2018).
[0021] The one-step growth curve of bacteriophage vB_KpnP_Henu1_3 is as shown in the appendix Figure 4 As shown, the results show that the latent period of bacteriophage vB_KpnP_Henu1_3 is about 10 minutes. Calculated based on the ratio of the phages released during the latent period to the initially infected bacteria, the burst size of bacteriophage vB_KpnP_Henu1_3 is about 297 PFU per infected cell.
[0022] Effect of temperature on the stability of bacteriophage vB_KpnP_Henu1_3 Under sterile conditions, 5 1.5 mL Eppendorf tubes were taken and 1 mL of bacteriophage vB_KpnP_Henu1_3 solution was added to each tube. After incubation in a water bath at 4 °C, 25 °C, 37 °C, 42 °C, and 55 °C for 12 hours, the titer of bacteriophage vB_KpnP_Henu1_3 at different temperatures was determined by the double-layer agar plate method.
[0023] The results of the thermal stability test of bacteriophage vB_KpnP_Henu1_3 are as shown in the appendix Figure 5 As shown, bacteriophage vB_KpnP_Henu1_3 has high viability at 4 - 55 °C.
[0024] Effect of pH on the stability of bacteriophage vB_KpnP_Henu1_3 1 M hydrochloric acid solution or sodium hydroxide solution was added to LB liquid medium to adjust the pH to 2 - 12. After filtering and sterilizing with a 0.22 μm filter, 2 μL of bacteriophage vB_KpnP_Henu1_3 solution was added to 200 μL of LB liquid medium at each different pH. The mixture was incubated in a water bath at 37 °C for 12 hours, and the titer of bacteriophage vB_KpnP_Henu1_3 at different pH values was detected by the double-layer agar plate method.
[0025] The pH stability test results of phage vB_KpnP_Henu1_3 are shown in the appendix Figure 6 As shown, phage vB_KpnP_Henu1_3 showed the best activity at pH 7 and still maintained high activity in the range of pH 4 - 11; in addition, further increase or decrease in pH resulted in partial loss of the activity of phage vB_KpnP_Henu1_3. Example 3
[0026] Host range analysis of the Klebsiella pneumoniae phage of the present invention: The clinically collected Klebsiella pneumoniae was inoculated into LB liquid medium and cultured overnight at 37°C with shaking at 220 rpm; 500 μL of the cultured bacterial solution was taken and added to the semi-solid medium. After mixing by inverting up and down, it was poured into an LB solid culture dish to prepare a double-layer agar plate; after the plate solidified, 2 μL of the serially diluted phage solution was dropped on the double-layer agar plate prepared above, and then placed in a 37°C bacterial incubator overnight. The formation of plaques on the plate was observed the next day.
[0027] Among the 30 clinically collected Klebsiella pneumoniae strains, phage vB_KpnP_Henu1_3 could form clear plaques on the double-layer plates of 5 Klebsiella pneumoniae strains numbered Kp1049, Kp0311, Kp0822, Kp407, and Kp0822; capsular serotype typing showed that all 7 host strains of phage vB_KpnP_Henu1_3 belonged to serotype K1. Phage vB_KpnP_Henu1_3 is a phage that specifically lyses Klebsiella pneumoniae of serotype K1. Example 4
[0028] Genome sequencing and analysis of the Klebsiella pneumoniae phage of the present invention: Add sodium chloride with a final concentration of 0.5 M and polyethylene glycol 8000 with a final mass-volume concentration of 20% (w / v) to 100 mL of Klebsiella phage proliferation solution to obtain a mixture. Mix the mixture evenly, incubate overnight at 4°C, and centrifuge to obtain a precipitate. After resuspending with 2 mL of PBS, add an equal volume of chloroform, shake for 30 seconds, and centrifuge at 4000 rpm for 10 minutes. Transfer the supernatant to a new centrifuge tube, add RNase and DNase I, mix well, incubate at 37°C for 1 h, and then add EDTA. After mixing well, add proteinase K and SDS in sequence, and incubate at 55°C for 1 hour to fully lyse the phage capsid protein. Add an equal volume of DNA extraction phenol reagent, shake for 30 s, and centrifuge at 4000 rpm for 10 minutes; transfer the supernatant to a new centrifuge tube, add an equal volume of chloroform, shake and mix well, and centrifuge at 4000 rpm for 10 min; take the supernatant to a new centrifuge tube, and add 1 / 10 volume of sodium acetate solution (pH = 5.2) and 2 volumes of pre-cooled absolute ethanol in sequence; mix gently, incubate at -20°C for 1 hour, centrifuge at 12000×g at 4°C for 10 minutes, and discard the supernatant. Add an appropriate amount of 70% ethanol solution and mix well, centrifuge at 8000×g at 4°C for 8 minutes, discard the supernatant, and repeat this washing step once. Dry the DNA naturally at room temperature, add an appropriate amount of TE buffer, and store at -20°C.
[0029] Transport the whole genome DNA sample of the extracted phage vB_KpnP_Henu1_3 to Nanjing Personal Biotechnology Co., Ltd. for sequencing. The sequencing method is the second-generation sequencing technology based on the Illumina Miseq high-throughput sequencing platform; use Illumina sequencing technology to complete the genome amplification and deep sequencing of the phage, construct an Illumina PE library, use ABySS to assemble the obtained reads, use SPAdes to splice the genome, and perform gene function annotation; the results are as follows Figure 7 , the full genome length of phage vB_KpnP_Henu1_3 is 49808 bp.
[0030] The GC content is 64%, and the genome does not contain antibiotic resistance genes and virulence factors; as can be seen from the map, phage vB_KpnP_Henu1_3 has a total of 58 protein-coding genes, including 27 hypothetical proteins, 4 unknown proteins, and 26 proteins with known functions. Example 5
[0031] The in vitro antibacterial activity of the Klebsiella pneumoniae phage of the present invention: Seven sterilized Erlenmeyer flasks were inoculated with Klebsiella pneumoniae strain Kp1049 in the logarithmic growth phase into LB medium at a ratio of 1:100. The culture was shaken at 220 rpm at 37°C. When the OD600 value of the culture broth was approximately 0.2, it was infected with phage vB_KpnP_Henu1_3 at MOIs of 1000, 100, 10, 1, 0.1, and 0.01, respectively. The uninfected culture broth was used as a positive control. 2 mL of samples were taken every 0.5 hours, and the OD600 value was measured using an ultraviolet spectrophotometer. Finally, a curve showing the effect of the phage on the growth of Klebsiella pneumoniae was plotted with time on the x-axis and the OD600 value on the y-axis.
[0032] The results of the effect of phage vB_KpnP_Henu1_3 on bacterial growth are shown in the appendix Figure 8 As shown, phage vB_KpnP_Henu1_3 can significantly inhibit the growth of bacteria within 4 hours and has good antibacterial activity. Example 6
[0033] The anti-biofilm activity of the Klebsiella pneumoniae phage of the present invention: To study the inhibitory effect of phage vB_KpnP_Henu1_3 on biofilm formation, Klebsiella pneumoniae Kp1049 cultured overnight was diluted to 107 CFU / mL with LB medium and mixed with phage vB_KpnP_Henu1_3 at different MOIs (100, 10, 1, 0.1, 0.01, 0.001, 0.0001); 200 μL of the mixed solution was added to a 96-well sterile microplate as the experimental group, 200 μL of LB medium as the negative control group, and 200 μL of the bacterial suspension as the positive control group. Each group had 3 replicates; after incubation at 37°C for 24 hours, it was then washed three times with sterile PBS buffer. Fixed with methanol for 15 minutes, 200 μL of 0.1% ammonium oxalate crystal violet solution was added to each well. After 20 minutes, it was washed three times with ultrapure water, dried, and dissolved with 200 μL of 33% acetic acid for 10 minutes. The optical density was measured using a microplate reader at 595 nm.
[0034] The inhibitory effect of phage vB_KpnP_Henu1_3 on bacterial biofilms is shown in the appendix Figure 9 As shown, phage vB_KpnP_Henu1_3 can significantly inhibit the formation of bacterial biofilms and has good anti-biofilm activity. Example 7
[0035] The therapeutic effect of the Klebsiella pneumoniae phage of the present invention on Galleria mellonella infection: Observation of the survival rate of Galleria mellonella larvae Sixty Galleria mellonella larvae were randomly divided into six groups of 10 larvae each, with five of the groups being the infection groups. 10 μL of Klebsiella pneumoniae Kp1049 bacterial solution (1×109 CFU / mL) was injected into the left caudal leg of the Galleria mellonella larvae. One hour after infection, 10 μL of phage vB_KpnP_Henu1_3 solution (MOI = 0.01, 0.1, 1, 10, 100) was respectively injected into the right caudal leg of the five groups of Galleria mellonella larvae for treatment. The control group was injected with 10 μL of PBS buffer. The survival rate of the Galleria mellonella larvae in each group was observed daily for 7 consecutive days.
[0036] Bacterial load in Galleria mellonella larvae Twenty Galleria mellonella larvae were randomly divided into four groups of 5 larvae each, with three of the groups being the infection groups. 10 μL of Klebsiella pneumoniae Kp1049 bacterial solution (1×109 CFU / mL) was injected into the left caudal leg of the Galleria mellonella larvae. One hour after infection, 10 μL of phage vB_KpnP_Henu1_3 solution (MOI = 1, 10, 100) was respectively injected into the right caudal leg of the three groups for treatment. The control group was injected with 10 μL of PBS buffer. After 48 hours, the tissues were homogenized and bacterial counts were performed. Example 8
[0037] Therapeutic effect of the Klebsiella pneumoniae phage of the present invention on mouse infection: Experimental mice: SPF-grade 6 - 8-week-old C57BL / 6J mice (randomly male / female) Observation of mouse survival rate Thirty mice were randomly divided into six groups of 5 mice each. Four of the groups were the infection groups. 100 μL of Klebsiella pneumoniae Kp1049 bacterial solution (1×1010 CFU / mL) was intraperitoneally injected into the mice. One hour after infection, 100 μL of phage vB_KpnP_Henu1_3 solution (MOI = 0.01, 0.1, 1, 10, 100) was respectively injected into the four groups of mice for treatment. The control group was injected with 100 μL of PBS buffer. The survival rate of the mice in each group was observed daily for 7 consecutive days.
[0038] Bacterial load in various organs of mice Twelve mice were randomly divided into three groups of 4 mice each. Two of the groups were the infection groups. 100 μL of Klebsiella pneumoniae Kp1049 bacterial solution (1×109 CFU / mL) was intraperitoneally injected into the mice. One hour after infection, 100 μL of phage vB_KpnP_Henu1_3 solution (MOI = 10, 100) was respectively injected into the two groups of mice for treatment. The control group was injected with 100 μL of PBS buffer. After 48 hours, the organs of the mice (heart, liver, spleen, lung, kidney) were taken, and the tissues were homogenized and bacterial counts were performed.
[0039] Although embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.
Claims
1. A phage that specifically lyses highly virulent capsular Klebsiella pneumoniae and its application, including the phage, characterized in that: The phage is named Klebsiella phage vB_KpnP_Henu1_3. The phage vB_KpnP_Henu1_3 belongs to the family Siphoviridae and has an icosahedral head; the titer is ≥1010, the growth latent period is 20 min, the rapid growth period is 20 - 60 min, the temperature stability range is 4 - 55 °C, and the pH stability range is 4 - 12.
2. The phage for specifically lysing highly virulent capsular Klebsiella pneumoniae according to claim 1 and its application, characterized in that: Application of the phage in degrading the biofilm of Klebsiella pneumoniae with high-virulence capsular serotype K1.
3. A phage for specifically lysing highly virulent capsular Klebsiella pneumoniae according to claim 2 and its application, characterized in that: The phage is used for preparing a drug or biological agent for preventing or treating Klebsiella pneumoniae infectious diseases, and the drug or biological agent uses phage vB_KpnP_Henu1_3 as the sole active ingredient.
4. A phage that specifically lyses highly virulent capsular Klebsiella pneumoniae according to claim 1 and its application, characterized in that: The Klebsiella pneumoniae includes Klebsiella pneumoniae Kp1049.