CN116790512A - Novel bacteriophage for cracking K2 capsular type high-virulence klebsiella pneumoniae and application thereof - Google Patents

Abstract

The application belongs to the technical field of biology, and particularly relates to a novel phage for cracking K2 capsular type high-virulence klebsiella pneumoniae and application thereof, wherein the klebsiella pneumoniae phage vB_KpnP_B1 is preserved in China Center for Type Culture Collection (CCTCC) in 2023, and the preservation address is as follows: in the above technical scheme, the preparation comprises Klebsiella pneumoniae phage vB_KpnP_B1 as described above; the preparation is a spray or injection, the phage not only has a better disinfection effect on Kp_B of K2 capsular type high-virulence Klebsiella pneumoniae isolated from pig farm sewage, but also has a strong disinfection effect on Klebsiella pneumoniae isolated from hospitals, and the phage has a relatively specific antibacterial spectrum, so that the phage can be applied to preparation of medicines for preventing and treating the high-virulence Klebsiella pneumoniae.

Description

Novel bacteriophage for cracking K2 capsular type high-virulence klebsiella pneumoniae and application thereof

Technical Field

The application relates to the technical field of biology, in particular to a novel bacteriophage for cracking K2 capsular type high-virulence klebsiella pneumoniae and application thereof.

Background

Klebsiella pneumoniae (Klebsiella pneumoniae, KP) is a gram-negative bacterium belonging to the genus Klebsiella (Klebsiella) phage (bacteriophage) of the family Enterobacteriaceae (Enterobacteriaceae), klebsiella bacteriophage vB _kpnp_b1, date of preservation: 2023-03-07, accession number: china center for type culture Collection. Klebsiella pneumoniae (Klebsiella pneumoniae) is an important zoonotic pathogen, which can cause serious nosocomial and community infections, including pneumonia, urinary tract infection, liver abscess, bacteremia, conjunctivitis, meningitis and the like, is one of the common conditional pathogenic bacteria in human medicine and clinic, and causes no small economic loss to the breeding industry.

With unlimited use of antibiotics in clinic and aquaculture, the drug resistance of klebsiella pneumoniae is also more severe, and particularly, the emergence of a strain (carbapenem-resistant hypervirulent Klebsiella pneumoniae) with high toxicity for producing carbapenemase is more involved in the almost "drug-free" environment of antibiotic treatment of klebsiella pneumoniae. The drug resistance of pathogenic bacteria to antibiotics is increasingly enhanced, and great threats are brought 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 of human and livestock, and some Klebsiella pneumoniae including K1 and K2 capsules is extremely virulent and can cause various infectious diseases of animals and humans. The infection caused by Klebsiella pneumoniae strains with strong drug resistance and strong toxicity is extremely difficult to prevent and control, and effective drugs and treatment strategies are lacking at present.

Phage (bacteriophage) is a natural bactericidal substance which is a kind of virus that specifically infects bacteria, can infect microorganisms such as bacteria, fungi, actinomycetes or spirochetes and cause lysis of host bacteria, and has been widely used in the treatment of otolaryngology, stomatology, ophthalmology, dermatology, pediatrics, pulmonary diseases and the like. When the worldwide medical field faces an increasingly severe threat of bacterial resistance, phages have great potential for treating or preventing diseases due to their efficient, specific bactericidal action. At present, high-virulence and drug-resistant klebsiella pneumoniae has been reported, but the research on the high-virulence klebsiella pneumoniae phage is less, and the development of novel phage with specificity and strong prevention and control effects on the high-virulence klebsiella pneumoniae has very important significance.

Disclosure of Invention

Aiming at the technical problems, the application provides a novel bacteriophage for cracking K2 capsular type high-virulence klebsiella pneumoniae and application thereof, and aims to provide a novel treatment scheme for infection of the high-virulence klebsiella pneumoniae.

In view of this, the present application provides a new K2 capsular type high virulence klebsiella pneumoniae phage vb_kpnp_b1, which is preserved in the chinese typical culture collection (CCTCC) at 2023, month 03, 07, with a preservation address: eight paths of 299 Chinese representative culture collection of university of Wuhan in Wuhan, hubei province have a collection number of CCTCC M2023261.

In the above technical scheme, further, the preparation comprising the klebsiella pneumoniae phage vb_kpnp_b1; wherein the preparation is a spray or an injection.

In the technical scheme, further, the klebsiella pneumoniae phage vB_KpnP_B1 is applied to preparation of a preparation for preventing and treating high-virulence klebsiella pneumoniae infection.

In the above technical scheme, further, the klebsiella pneumoniae phage vb_kpnp_b1 is used for killing klebsiella pneumoniae in a culture environment or a medical environment. The Klebsiella pneumoniae bacteriophage vB_KpnP_B1 is adopted for multiple drug-resistant high-virulence Klebsiella pneumoniae in medical environments (such as ground, instruments, walls and the like) and culture environments (such as food tanks, fences, feeds, drinking water, feces and the like).

The beneficial effects of the application are as follows:

1. the novel bacteriophage for cracking K2 capsular type high-virulence klebsiella pneumoniae and application thereof are characterized in that the klebsiella pneumoniae bacteriophage vB_KpnP_B1 is a newly discovered bacteriophage, the bacteriophage not only has a good disinfection effect on the host multi-drug resistant high-virulence klebsiella pneumoniae separated from pig farm sewage, but also has a strong disinfection effect on the other 3 multi-drug resistant klebsiella pneumoniae separated from hospitals, has a relatively specific antibacterial spectrum, and can be applied to preparing medicines for preventing and treating the high-virulence klebsiella pneumoniae.

2. The novel bacteriophage for cracking K2 capsular type high-virulence klebsiella pneumoniae and the application thereof have the advantages that the bacteriophage vB_KpnP_B1 has short incubation period, can quickly kill host bacteria in a culture medium, has small toxic and side effects, high safety, wide temperature and acid-base tolerance range and good hydrophilicity, is easy to prepare into spray and injection, and has good treatment and killing effects on animals infected with multi-drug resistant klebsiella pneumoniae and the environment polluted by the multi-drug resistant klebsiella pneumoniae.

Drawings

FIG. 1 is a Klebsiella pneumoniae phage vB_KpnP_B1 plaque of the application;

FIG. 2 is a scanning electron microscope image of the klebsiella pneumoniae phage vB_KpnP_B1 of the present application;

FIG. 3 is a one-step growth curve of the Klebsiella pneumoniae phage vB_KpnP_B1 of the application;

FIG. 4 is the effect of temperature on the activity of the klebsiella pneumoniae phage vB_KpnP_B1 of the present application;

FIG. 5 is the effect of pH on the activity of Klebsiella pneumoniae phage vB_KpnP_B1 of the application;

FIG. 6 is a minimal lethal dose of Kp-B, a high virulence Klebsiella pneumoniae of the application;

FIG. 7 shows the therapeutic effect of Klebsiella pneumoniae bacteriophage vB_KpnP_B1 mice of the present application;

Detailed Description

The technical solutions of the embodiments of the present application will be clearly described below with reference to the drawings in the embodiments of the present application, and it is apparent that the described embodiments are some embodiments of the present application, but not all embodiments. All other embodiments, which are obtained by a person skilled in the art based on the embodiments of the present application, fall within the scope of protection of the present application.

In the description of the present application, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments in accordance with the present application. For ease of description, the dimensions of the various features shown in the drawings are not drawn to actual scale. Techniques, methods, and apparatus known to one of ordinary skill in the relevant art may not be discussed in detail, but should be considered part of the specification where appropriate. In all examples shown and discussed herein, any specific values should be construed as merely illustrative, and not a limitation. Thus, other examples of the exemplary embodiments may have different values. It should be noted that: like reference numerals and letters denote like items in the following figures, and thus once an item is defined in one figure, no further discussion thereof is necessary in subsequent figures.

It should be noted that the terms "first," "second," and the like in the description and in the claims are used for distinguishing between similar objects and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used may be interchanged, as appropriate, such that embodiments of the present application may be implemented in sequences other than those illustrated or described herein, and that the objects identified by "first," "second," etc. are generally of a type, and are not limited to the number of objects, such as the first object may be one or more. In addition, "and/or" in the specification and claims means at least one of the connected objects, and the character "/", generally means a relationship in which the associated objects are "or".

It should be noted that, in the description of the present application, the terms like "front, rear, upper, lower, left, right", "horizontal, vertical, horizontal", and "top, bottom", etc. generally refer to the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, and are merely for convenience of describing the present application and simplifying the description, and these orientation terms do not indicate and imply that the apparatus or elements referred to must have a specific orientation or be constructed and operated in a specific orientation, and thus should not be construed as limiting the scope of the present application; the orientation word "inner and outer" refers to inner and outer relative to the contour of the respective component itself.

It should be noted that, in the present application, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element. Furthermore, it should be noted that the scope of the methods and apparatus in the embodiments of the present application is not limited to performing the functions in the order shown or discussed, but may also include performing the functions in a substantially simultaneous manner or in an opposite order depending on the functions involved, e.g., the described methods may be performed in an order different from that described, and various steps may be added, omitted, or combined. Additionally, features described with reference to certain examples may be combined in other examples.

Example 1:

the strains, reagents and media related to the examples:

the host bacteria used in the experiment are a clinical strain KP-B of high virulence klebsiella pneumoniae, which is separated from a pig in a pig farm, and is preserved in China center for type culture collection (CCTCC M2023261) together with the klebsiella pneumoniae phage vB_KpnP_B1.

LB nutrient agar, LB broth, SM buffer purchased from Beijing Soy Bao technology Co., ltd; agar powder is purchased from Beijing Ding Guo and prosperous biotechnology limited company; 1mol/L Tris, naCl, mgSO4, EDTA (disodium ethylenediamine tetraacetate), PBSDNAse I, RNase A (TaKaRa), solid calcium chloride, concentrated HCl, etc. are commercially available.

Example 2:

isolation and preparation of klebsiella pneumoniae phage vb_kpnp_b1:

the host bacteria are streaked and inoculated on 1.5% LB agar medium, after being cultured overnight, the monoclonal is selected and inoculated on 5mL LB liquid medium, and the culture is carried out for 5-6 hours at 37 ℃ in a shaking way and then is used as a host bacteria culture for standby.

The phage separation adopts a sewage enrichment method: centrifuging at 4deg.C and 8000rpm for 10min to remove large granule residues, collecting supernatant, adding CaCl2 to a final concentration of 1mmol/L, standing for about 15min, and filtering with 0.22 μm pore filter membrane. 100mL of the filtrate was added to 2X 100mL of LB medium, 1% of Klebsiella pneumoniae liquid grown in log phase was added, and after overnight culture at 37℃and 180rpm, an appropriate volume was taken and centrifuged at 10000rpm for 10min, the supernatant was taken and filtered through a filter membrane with a pore size of 0.22 μm to remove residual bacteria, and the supernatant was collected as phage stock solution. 200 mu L of logarithmic growth phase host bacteria liquid is added into 5-6 ml of 0.5% LB agar medium, after being evenly mixed, the mixture is poured into a 1.5% LB agar plate, 10 mu L of phage stock solution is dripped after solidification, the control group is dripped with equal amount of sterile water, after the plate is dried, the plate is placed at 37 ℃ for 12 hours of culture, and whether plaque appears or not is observed. If plaque formation is present, the presence of phage is demonstrated.

Phage were purified by double-layer agar plate method, phage stock was diluted 10-fold with PBS buffer, 100. Mu.L of phage dilution 200. Mu.L of log-phase host bacteria were mixed, allowed to act for 10min, added to 5-6 ml of 0.5% LB agar medium and mixed well, plated on 1.5% LB agar plates, allowed to solidify on top, and transferred to a constant temperature incubator at 37℃for overnight culture. The single plaques were picked and purified until plaques of uniform size appeared, obtaining purified single phages. Preserving at 4 ℃ for standby.

Phage titers were detected using double-layer plates: the purified phage is subjected to 10-time gradient dilution, 0.1mL of phage dilution liquid with a plurality of gradients corresponding to 0.2mL of phage dilution liquid are fully and evenly mixed with host bacteria liquid, double-layer agar plates are paved, the culture is carried out at the constant temperature of 37 ℃ for about 8 hours, and plaque counting is carried out on each plate. Selecting a plate with plaque about 100-300, and calculating according to dilution times to obtain initial concentration of phage to obtain phage titer. Phage titer (PFU/ml) =dilution x number of plaques x 10, phage titer 1.52 x 10 ¹⁰ PFU/ml。

The phage obtained by separation is named as vB_KpnP_B1 and is preserved in China Center for Type Culture Collection (CCTCC) of university of Wuhan in China, wherein the preservation date is 2023, 03 and 07, and the preservation number is CCTCC M2023261.

Example 3

Amplification and concentration of phages:

the host bacteria were cultured at 37℃and 160rpm with shaking until the logarithmic growth phase. Phage were added at the optimal multiplicity of infection, shaking culture was performed at 37℃and 160rpm until the solution was clear, DNase I and RNase A were added to a final concentration of 1. Mu.g/mL, followed by shaking culture in a shaker for 30min, and then 1% chloroform was added to disrupt the bacteria. NaC L is added to a final concentration of 1mol/L, and stirred and mixed uniformly until the NaC is completely dissolved, and then the NaC is subjected to ice bath for 6 hours. The lysate was centrifuged at 10000 Xg for 15min at 4℃and the supernatant was collected. The collected supernatant was added to PEG8000 in an amount of 10g per 100mL, gently inverted to dissolve, vigorously forbidden, and ice-bathed overnight in a refrigerator at 4℃to precipitate phage particles. After that, the mixture was centrifuged at 12000rpm at 4℃for 15 minutes, and the supernatant was discarded to leave a precipitate. The centrifuge tube containing the sediment was uncapped and inverted so that the remaining liquid was drained. Adding 2mL of SM buffer solution into a centrifuge tube, repeatedly washing and dissolving phage particles, extracting for 2-3 times by using equal volume of chloroform to obtain an upper water phase, and filtering by using a filter membrane with the aperture of 0.22 mu m into a new tube to obtain phage concentrate.

Example 4:

transmission electron microscope observation of klebsiella pneumoniae phagemid vb_kpnp_b1:

sucking a drop of about 20 mu L of the solution of the example 2 on a copper net, letting the solution stand and precipitate for 15min, sucking the redundant liquid, adding a drop of 2% phosphotungstic acid solution, dyeing for 5min, immediately removing redundant dyeing liquid on the side, drying and observing the shape under a transmission electron microscope; the observation result is shown as figure 2, and the electron microscope observation shows that the head of the phage is in a regular icosahedral structure, the diameter of the head is about 55nm, and the phage belongs to the order of the phage with tail, and belongs to the family of brachyury. According to the eighth report of the International Commission on viral Classification-International Commission on viral Classification (ICTV) published 2015, the phage belongs to the family Brevicariidae (Brevicaridae).

Example 5:

determination of optimal multiplicity of infection (multiplicity of infection is the ratio of the number of phages at the initial stage of infection to the number of host bacteria) for klebsiella pneumoniae phage vb_kpnp_b1:

the multiplicity of infection (Multiplicity of infection, MOI) is the ratio of the number of phages adsorbed to the bacteria to the number of bacteria in culture. Phage bind to host bacteria with optimal multiplicity of infection. The measurement method is as follows: culturing the host bacteria to logarithmic phase, and the colony number is about 10 ⁸ CFU/mL, phage were added at MOI of 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, incubated at 37℃for 10min, 10mL LB liquid medium was added, after shaking culture at 37℃and 160rpm for 6h, centrifuged at 10000rpm for 10min, the supernatant was filtered with a filter membrane having a pore size of 0.22 μm, and then diluted with LB liquid medium at a 10-fold gradient, and titer was measured by a double-layer agar plate method. Each independent experiment was repeated 3 times. The results are shown in Table 1, the optimal multiplicity of infection of Klebsiella pneumoniae phage vB_KpnP_B1 is 0.001.

TABLE 1 optimal multiplicity of infection of Klebsiella pneumoniae phage vB_KpnP_B1

Example 6:

determination of phage one-step growth curve of klebsiella pneumoniae phages vb_kpnp_b1:

the method for determining the one-step growth curve is as follows: 1mL of bacterial solution was taken at a concentration of 1X 10 ⁸ CFU/mL, and mixing with phage according to the optimal multiplicity of infection, standing at 37 ℃, incubating, adsorbing for 5min, centrifuging at 4 ℃, 5000rpm for 5min, discarding supernatant, then adding 1.5mL of LB liquid medium preheated to 37 ℃, then centrifuging at 4 ℃, 5000rpm for 5min, discarding supernatant, repeating this step twice, ensuring that free phage not adsorbed are removed cleanly, adding into a centrifuge tube containing 10mL of LB liquid medium preheated at 37 ℃ for resuspension, placing the centrifuge tube in a shaking table at 37 ℃, shaking culture at 180rpm, sampling 200 μl every 10min, centrifuging at 4 ℃,10000rpm for 5min, and continuously sampling for 100min with standard serial number. The phage titer was measured every 10min by a double-layer agar plate method, and a one-step growth curve was drawn with the abscissa as the infection time and the ordinate as the phage titer. Each independent experiment was repeated 3 times.

As a result, as shown in FIG. 3, the titer of phage vB_KpnP_B1 was not significantly changed 10min before the infection of the host bacteria, after which the phage titer was rapidly increased and was stabilized after 40min, i.e., the incubation period of the phage was about 10min, the outbreak period was about 40min, and the outbreak amount was 152PFU/Cell.

Example 7:

temperature tolerance experiment of klebsiella pneumoniae phage vb_kpnp_b1:

taking 10 sterile EP tubes, respectively taking 1mL of phage lysate, placing in 1.5mL of EP tube, respectively carrying out water bath at 30 ℃,40 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃ for 20min, 40min and 60min, respectively taking 100 mu L of phage titer by adopting a double-layer flat plate method. And (3) plotting the temperature on the abscissa and the corresponding phage titer on the ordinate, and obtaining the optimal growth temperature range of the phage according to the phage titer change. Each independent experiment was repeated 3 times. As a result, as shown in FIG. 4, the phage vB-KpnP-B1 had a suitable temperature of 30-60℃and the phage had a wide suitable temperature range.

Example 8:

PH tolerance test of Klebsiella pneumoniae phage vB_KpnP_B1:

9mL of LB broth was taken, the pH was adjusted to pH 3, 4, 5, 6, 7, 8, 9, 10, 11, 1mL of phage lysate was added, and the mixture was left at room temperature for 2 hours. Phage titer was determined using a double-layer plate method, and was performed 3 times in parallel. And (3) plotting the pH on the abscissa and the corresponding phage titer on the ordinate, and obtaining the optimal growth pH range of the phage according to the phage titer change. Each independent experiment was repeated 3 times. As a result, as shown in FIG. 5, the phage vB-KpnP-B1 had a suitable pH range of 4 to 10, and the phage had a wide suitable pH range.

Example 9:

analysis of the host profile of klebsiella pneumoniae phage vb_kpnp_b1:

the phage titer for use in example 1 was adjusted to 10 ⁹ PFU/ml was used and phage vB_KpnP_B1 host profiling. 40 strains of Klebsiella pneumoniae of different origin and class from this laboratory were collected and subjected to host profiling using the phage of example 1, as follows: taking 0.2ml of 40 Klebsiella pneumoniae overnight cultures, adding 0.7% LB semisolid culture medium 8m l at about 45 ℃, uniformly spreading on a prepared solid 1.5% LB solid culture medium, and then equally dividing each flat plate into four areas, wherein 10 μl of phage prepared in example 1 is dripped on the surface of each area, physiological saline is dripped in the other area to serve as a control, the liquid drops are dried and then are inverted at 37 ℃ for culturing for 10 hours, and the observed result is marked as "+" if plaque is generated, otherwise, the observed result is "-".

The results are shown in Table 2: the klebsiella pneumoniae vB_KpnP_B1 can lyse K2 capsular type high virulence klebsiella pneumoniae, and can not lyse K1, K47, K64 and K19 type klebsiella pneumoniae, which shows that vB_KpnP_B1 has specific lysis capacity for the K2 type klebsiella pneumoniae.

Table 2 shows the cleavage spectrum of the Klebsiella pneumoniae phage vB_KpnP_B1 of the application

(remark: ND not measured; unknown to the Unk)

Example 10:

determination of the minimum mortem mass of mice caused by klebsiella pneumoniae KP-B:

36 mice were randomly divided into 6 groups (6 mice per group) and bacterial solutions (2.4X10 s) were used at different doses ⁵ 、2.4×10 ⁴ 、2.4×10 ³ 、2.4×10 ² 、2.4×10 ¹ CFU/mice) were intraperitoneally injected with experimental mice (SPF grade BALB/c female mice 6-8 weeks old), control groups were intraperitoneally injected with an equal amount of sterile PBS, and survival of each group was recorded every 12 hours. The minimal lethal dose of highly pathogenic Kp-B from mice was obtained based on 7 days of death.

As shown in FIG. 6, the survival rate of 24CFU Kp-B pathogenic bacteria mice injected intraperitoneally was 50% in 7 days, and the infected mice showed serious clinical symptoms, refused to eat drinking water, were fried up by hair, and were stiff in limbs. Intraperitoneal injection 2.4X10 ² The survival rate of CFU Kp-B pathogenic bacteria mice is 40% within 7 days; intraperitoneal injection 2.4X10 ³ The survival rate of the CFU Kp-B pathogenic bacteria mice is 20% within 7 days; intraperitoneal injection 2.4X10 ⁴ Survival rate of CFU Kp-B pathogenic bacteria mice in 36h is 0; intraperitoneal injection 2.4X10 ⁵ Survival rate of CFU Kp-B pathogenic bacteria mice 24 is 0; conclusion that the minimum total lethal dose of mice caused by Kp-B of Klebsiella pneumoniae with high virulence is 2.4X10 ⁴ CFU。

Sterile detection assay of phage vB_KpnP_B1: and (3) coating 500 μl of the prepared phage vB_KpnP_B1 preparation on a blood plate, culturing at 37 ℃ for 72 hours, and observing whether pathogenic microorganisms grow on the blood plate to judge whether pathogenic microorganism pollution exists in the phage preparation. Phage vB_KpnP_B1 preparation was cultured on blood plates for 72h without pathogenic microorganism growth, and the next experiment was performed after sterility check.

Example 11:

in vivo safety assay of phage vB_KpnP_B1:

12 female BALB/c mice with the age of 6-8 weeks and SPF grade are randomly divided into two groups of 6 mice. The intraperitoneal injection of 1mL of each mouse in the experimental group is 10 ¹⁰ PFU/mL phage vB_KpnP_B1 formulation, control group each mice were intraperitoneally injected with an equal volume of sterile PBS. Two groups of mice were fed for one week and observed for clinical changes. Thereafter, the mice were sacrificed, dissected and observed for visceral, digestive, and mucosal conditions. The results show that the mice have good mental state and no abnormality in daily behaviors after being observed for two groups of mice for one week; mice were sacrificed by cervical removal, and no abnormality was observed in each tissue and organ by anatomic examination. The phage vB_KpnP_B1 is suggested to be good in safety and free of toxic and side effects on mice.

Example 12:

effect of different doses of phage on survival of mice:

the specific experimental scheme is as follows: 30 female BALB/c mice with the age of 6-8 weeks and SPF grade are randomly divided into 5 groups, and 6 female mice are selected from each group. Phage treatment group was 2×MLD Klebsiella pneumoniae KP-B (2.4X10) by intraperitoneal injection of mice ⁴ CFU/mouse) for 1 hour, 1.52×10 injections were given intraperitoneally, respectively ³ PFU/mouse, 1.52X10 ⁴ PFU/mouse, 1.52X10 ⁵ Phage treatment at PFU/mouse dose; the negative control group is replaced by sterile PBS during both sterilization and treatment; the positive control group was treated with equal amount of sterile PBS after intraperitoneal injection for challenge. Mice death was recorded every 12 hours for each group.

The results after vB_KpnP_B1 treatment are shown in FIG. 7, with a 2×MLD (2.4X10) ⁴ CFU/mouse) after highly pathogenic Kp-B, untreated mice all died within 36 hours. Compared with the control group, the phage treatment group significantly improves the survival rate of mice by only 1.52×10 ⁴ PFU doses of phage increased survival to 60% in 7 days in mice to 1.52X10) ⁵ The protection rate of the phage with PFU dose to mice is highest, and the survival rate of the mice in 7 days is up to 100%, which shows that the phage has good treatment effectThe therapeutic effect improves the survival rate of mice to a great extent.

The foregoing descriptions of specific exemplary embodiments of the present application are presented for purposes of illustration and description. It is not intended to limit the application to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the application and its practical application to thereby enable one skilled in the art to make and utilize the application in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the application be defined by the claims and their equivalents.

Claims (4)

1. Novel phage for lysing K2 capsular type high virulence Klebsiella pneumoniae and application thereof, comprising Klebsiella pneumoniae phage vB_KpnP_B1, and is characterized in that: the Klebsiella pneumoniae phage vB_KpnP_B1 is preserved in China Center for Type Culture Collection (CCTCC) in 2023, and the preservation address is as follows: eight paths of 299 Chinese representative culture collection of university of Wuhan in Wuhan, hubei province have a collection number of CCTCC M2023261.

2. The novel bacteriophage for lysing K2 capsular type high virulence klebsiella pneumoniae and application thereof according to claim 1, wherein the bacteriophage is characterized in that: the Klebsiella pneumoniae phage vB_KpnP_B1 is a preparation.

3. The novel bacteriophage for lysing K2 capsular type high virulence klebsiella pneumoniae and application thereof according to claim 1, wherein the bacteriophage is characterized in that: the application of the klebsiella pneumoniae phage vB_KpnP_B1 in preparation of a preparation for preventing and treating multiple drug resistant klebsiella pneumoniae infection.

4. The novel bacteriophage for lysing K2 capsular type high virulence klebsiella pneumoniae and application thereof according to claim 1, wherein the bacteriophage is characterized in that: the Klebsiella pneumoniae phage vB_KpnP_B1 can be used for killing Klebsiella pneumoniae in a culture environment or a medical environment.