CN118374398B

CN118374398B - A Klebsiella pneumoniae phage capable of lysing multiple K-capsule Klebsiella pneumoniae and having long-lasting antibacterial effect and its application

Info

Publication number: CN118374398B
Application number: CN202410510102.1A
Authority: CN
Inventors: 王喜亮; 田甲; 黄金梅; 张圣海; 李孟霖; 张晓东
Original assignee: Wuhan Grenon Biotechnology Co ltd
Current assignee: Wuhan Grenon Biotechnology Co ltd
Priority date: 2024-04-26
Filing date: 2024-04-26
Publication date: 2025-03-18
Anticipated expiration: 2044-04-26
Also published as: CN118374398A

Abstract

The present invention provides a Klebsiella pneumoniae phage that can lyse a variety of K-capsule Klebsiella pneumoniae and has a long-lasting antibacterial effect, and its application, which belongs to the field of biotechnology. The phage was deposited in the China Center for Type Culture Collection on March 4, 2024, and the deposit address is: Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M 2024373. The phage GRNKpP10 has a lytic effect on Klebsiella pneumoniae. In addition, the phage GRNKpP10 has lytic properties on a variety of K-capsule Klebsiella pneumoniae, and has a long-lasting antibacterial effect on a variety of K-type antigenic Klebsiella pneumoniae; the phage GRNKpP10 has the advantages of high value-added titer, safety and efficiency, and good tolerance to temperature and pH. The present invention can effectively prevent diseases caused by pathogenic Klebsiella pneumoniae in various environments.

Description

Klebsiella pneumoniae bacteriophage capable of cracking various K-type capsular Klebsiella pneumoniae and lasting in bacteriostasis and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a Klebsiella pneumoniae bacteriophage capable of cracking various K-type capsular Klebsiella pneumoniae and lasting in bacteriostasis and application thereof.

Background

Klebsiella pneumoniae (Klebsiella pneumoniae; kp) is the most important gram-negative bacterium (commonly known as Klebsiella pneumoniae) in the genus Enterobacter, and the resulting disease accounts for more than 95% of Klebsiella infections. Klebsiella pneumoniae is a pathogen for zoonotic diseases, which can cause a series of infections of animals and human beings, including respiratory tract, urinary tract, soft tissue and blood infections, and is a global new disease, and is also a cause of suppurative liver abscess complicated with metastatic meningitis and endophthalmitis.

Klebsiella pneumoniae possesses a variety of virulence factors including siderophores, pili, capsular polysaccharides (capsular polysaccharides, CPS), lipopolysaccharides (LPS), and the like. CPS is considered to be the most important virulence factor in Kp, and Klebsiella pneumoniae can be classified into 82K serotypes depending on the type of capsular polysaccharide (K antigen). Wherein the K1, K2 and K5 serotypes are closely related to severe infectious diseases of the human and animal body. The K1 strain usually causes friedelane's pneumonia, and particularly suppurative liver abscess complications. The K2 and K5 serotypes often lead to community-acquired pneumonia. Nasal sclerosis is associated with the K3 serotype. The clinical isolates of the Klebsiella pneumoniae subspecies from atrophic rhinitis are mostly from the K4 serotype and very few from the K5 serotype. Strains K54 and K57 are associated with the onset of the group invasive liver abscess syndrome. The K1, K2, K5, K20, K54 and K57 types are virulent strains compared to other serotypes, and are common in cases of pneumonia. Especially with the strongest toxicity of K1 and K2 types, the trend of the current trend is more and more serious in recent years, and the phenomenon is the main pathogenic bacteria of clinical bacterial liver abscess syndrome, which is found in Asia at the earliest and is proved in all parts of the world at present. The greater virulence of the K1, K2, K54 and K57 types may be due to their greater toxic effects in vivo. Bacterial resistance has been an important issue in the world public health field. In addition, rapid transmission of carbapenems and multidrug resistance by klebsiella pneumoniae are also of great concern.

At present, the prevention and control of the klebsiella pneumoniae are mainly treated by antibiotics, but the current klebsiella pneumoniae drug resistance is more serious, and the curative effect has certain limitation. Therefore, the invention provides the Klebsiella pneumoniae phage capable of cracking various K-type capsular Klebsiella pneumoniae and having lasting bacteriostasis and the application thereof.

Disclosure of Invention

In order to prevent diseases caused by Klebsiella pneumoniae or to reduce the prevalence caused by the problems, it is an object of the present invention to provide a phage against Klebsiella pneumoniae.

The second object of the invention is to provide an application of klebsiella pneumoniae phage GRNKpP10 in preparing a medicament for treating or preventing diseases caused by klebsiella pneumoniae infection.

In order to achieve the aim, the invention is realized by the following technical scheme that the Klebsiella pneumoniae bacteriophage which can lyse a plurality of Klebsiella pneumoniae capsules and has lasting bacteriostasis is classified and named as Klebsiella pneumoniae bacteriophage GRNKpP10, the applicant separates a Klebsiella pneumoniae bacteriophage GRNKpP from water in a sea area around the region of Guangdong province, and the bacteriophage is sent to China center for preservation of typical culture, the preservation address is China university of Wuhan and the preservation number is CCTCC NO: M2024373 in 3 months 4 days of 2024.

Further, a complex of klebsiella pneumoniae phage GRNKpP is also included, including but not limited to, the complex of klebsiella pneumoniae phage GRNKpP with other phages, other probiotics.

The Klebsiella pneumoniae phage GRNKpP or phage complex is applied to the industries of environmental disinfection, livestock breeding, poultry breeding, aquaculture, biomedical treatment and the like to prepare a Klebsiella pneumoniae bacteriostatic agent.

Further, the method is also applied to the preparation of medicines for treating or preventing diseases caused by klebsiella pneumoniae infection.

Further, the method is also applied to preparing feeds or feed additives for antagonizing klebsiella pneumoniae.

Further, the klebsiella pneumoniae is a K1 capsular type, a K11 capsular type, a K22 capsular type, a K24 capsular type, a K31 capsular type, a K37 capsular type and a K64 capsular type klebsiella pneumoniae.

The invention has the beneficial effects that:

1. The invention firstly screens out a Ktype Klebsiella pneumoniae which is cracked by a needle and has lasting bacteriostasis, the bacteriophage GRNKpP has high cracking rate on the Klebsiella pneumoniae, the bacteriophage GRNKpP has good effect on inhibiting different Klebsiella pneumoniae capsules, and has great reference significance on prevention, control and treatment of the Klebsiella pneumoniae in future.

2. The klebsiella pneumoniae phage GRNKpP provided by the invention has high fermentation rate, the titer can reach 8.8x ¹⁰ PFU/mL when the temperature is 28 ℃ and the optimal multiplicity of infection MOI=0.001, and the fermentation titer can reach 7.5x ¹⁰ PFU/mL when the test fermentation is carried out to 6h in the industrial fermentation process.

3. The klebsiella pneumoniae phage GRNKpP10 provided by the invention has good environmental tolerance, and the titer of the klebsiella pneumoniae phage GRNKpP is not obviously changed by orders of magnitude when the klebsiella pneumoniae phage GRNKpP is processed for 24 hours within the pH range of 4-11.

4. The klebsiella pneumoniae phage GRNKpP provided by the invention has no obvious order of magnitude difference in phage titer after 16W at 4 ℃, has no obvious order of magnitude difference in phage titer after 12W at 25 ℃, has no obvious order of magnitude difference in phage titer after 2W at 37 ℃, has good stability at 4-37 ℃ and can be effectively stored.

5. The klebsiella pneumoniae bacteriophage GRNKpP provided by the invention can be used for cracking various K-type klebsiella pneumoniae (K1-type, K11-type, K22-type, K24-type, K31-type, K37-type and K64-type klebsiella pneumoniae), and the klebsiella pneumoniae bacteriophage GRNKpP10 has antibacterial durability and can effectively inhibit the growth of the klebsiella pneumoniae for 24 hours.

6. The klebsiella pneumoniae phage GRNKpP provided by the invention can effectively treat the problem of ascitic fluid of the larceny bass caused by klebsiella pneumoniae, and the survival rate of the larceny bass is effectively improved by a prevention and treatment group, the survival rate of a blank group is 100%, the survival rate of the larceny bass of a virus attack group is 20%, the survival rate of the treatment group is 85%, the survival rate of a prevention group is 90%, and the survival rate of enrofloxacin group is 85%. Provides important reference value for preventing and treating klebsiella pneumoniae in future, and can be used as a biological antibacterial agent for preventing and treating klebsiella pneumoniae diseases of largehead jewfish and other animals.

Drawings

FIG. 1 is a plaque of Klebsiella pneumoniae phage GRNKpP;

FIG. 2 shows titers of Klebsiella pneumoniae phage GRNKpP under different infectious complex conditions;

FIG. 3 is a graph showing the stability of Klebsiella pneumoniae phage GRNKpP at different pH values;

FIG. 4 is a graph showing the stability of Klebsiella pneumoniae phage GRNKpP10 at various temperatures;

FIG. 5 is a schematic diagram of fermentation dynamics of Klebsiella pneumoniae phage GRNKpP;

FIG. 6 is a schematic diagram of the persistence of bacteriostasis of Klebsiella pneumoniae phage GRNKpP 10.

Detailed Description

The invention is further described in connection with the following detailed description, in order to make the technical means, the creation characteristics, the achievement of the purpose and the effect of the invention easy to understand.

Referring to fig. 1 to 6, the present invention provides the following technical solutions:

example 1:

Separation and purification of klebsiella pneumoniae phage GRNKpP10

30 Parts of sewage is collected in a certain area of Guangdong province, 20mL of each part is centrifuged at 6000rpm for 10min, the supernatant is sterilized by a 0.22 mu m filter, 10mL of filtrate, 10mL of double TSB culture medium and 1mL of Klebsiella pneumoniae (the concentration is about 5X 10 ⁸ CFU/mL) are uniformly mixed, and the mixture is subjected to shaking culture at 200rpm in a shaking table at 37 ℃ for overnight fermentation. The fermentation broth was centrifuged at 6000rpm for 10min, the supernatant was sterilized with a 0.22 μm filter, 100. Mu.L of the filtrate was mixed with 300. Mu.L of Klebsiella pneumoniae (concentration: about 1X 10 ⁸ CFU/mL), TSB semi-solid (containing 0.65% agar) was added, poured onto TSA bottom plate after gently inverting the mixture evenly, and after solidification, the petri dish was placed in a 37℃incubator for overnight culture. The clear and bright plaque spots are picked up, the bacteriophage filtrate is obtained through the sterilization of a microporous filter membrane with the thickness of 0.22 mu m after the oscillation desorption in 1mL of SM solution, the bacteriophage filtrate is inoculated into 5mL of TSB liquid culture medium, 500 mu L of corresponding host Klebsiella pneumoniae bacterial liquid is added and mixed uniformly, 200rpm oscillation culture is carried out at 37 ℃ for overnight, 6000rpm centrifugation is carried out for 10min, the supernatant is taken out, the bacteriophage filtrate is obtained through the sterilization of the microporous filter membrane with the thickness of 0.22 mu m, and the plaque morphology is observed by adopting a double-layer flat plate method. And repeating the operation for 3-5 times to obtain the plaque with consistent shape and size.

A Klebsiella pneumoniae phage GRNKpP10 is separated from a collected water sample, and is transparent, round and 1.0mm in diameter with plaque formed by the Klebsiella pneumoniae phage and a host bacterium, and has an obvious lysozyme ring as shown in figure 1. The phage is sent to China Center for Type Culture Collection (CCTCC) for preservation in 2024, and is classified and named as Klebsiella pneumoniae phage (Klebsiella pneumoniae phage) GRNKpP, wherein the address is China, university of Wuhan, and the preservation number is CCTCC NO: M2024373.

Example 2:

Titer determination of Klebsiella pneumoniae phage GRNKpP10 under different infection complex conditions

Host bacteria Klebsiella pneumoniae KP048 of Klebsiella pneumoniae phage GRNKpP is inoculated into an EP tube with 3mL TSB, and shake culture is carried out for 5-8h at 37 ℃ to obtain KP048 bacterial liquid. Bacterial solutions were inoculated at 1.5% inoculum size into 100mL of TSB medium, phage GRNKpP were inoculated until KP048 grew to log initiation, and cultures were performed at 200rpm shaking in a shaker for 12h at different multiplicity of infection (multiplicity of infection, MOI; MOI = phage number/bacteria number, see specifically fig. 2,3 replicates per group.temperature settings 28 ℃ and 35 ℃.

The results are shown in FIG. 2, and the fermentation titer is up to 8.8X10 ¹⁰ PFU/mL at 28℃MOI=0.001.

Example 3:

PH stability test of Klebsiella pneumoniae phage GRNKpP10

0.1ML of Klebsiella pneumoniae phage GRNKpP fermentation broth is added into 9.9mL of TSB liquid culture medium with pH value of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0 and 12.0 respectively (the titer after dilution is 5 x 10 ⁷ pfu/mL), the mixture is placed into a 25 ℃ incubator for incubation for 24 hours, sampling is carried out, pH values are adjusted to original culture medium ph=7.4 by hydrochloric acid and sodium hydroxide solution respectively, and then double dilution is carried out, phage titer is measured by a double-layer plate method, each point is cultivated in duplicate to obtain average value, and experiment is repeated for 3 times.

As shown in FIG. 3, the titer of Klebsiella pneumoniae phage GRNKpP treated for 24 hours at pH ranging from 4 to 11 was not significantly changed.

Example 4:

temperature stability test of Klebsiella pneumoniae phage GRNKpP10

The klebsiella pneumoniae phage GRNKpP fermentation broth was filtered by centrifugation, and 1mL was dispensed into a 1.5mL centrifuge tube (initial titer 5×10 ⁷ pfu/mL). Centrifuge tubes containing phage were placed at constant temperature of 4, 25, 37, 45, 55, 65, 75 ℃ for 0h, 1h, 24h, 48h, 92h, 1w, 2w, 4w, 8w, 12w, 16w, respectively. After the action time is over, the sample tube is taken out and immediately placed in an ice bath for cooling, and phage titer is measured by adopting a double-layer flat plate method after proper dilution. Each point was averaged in duplicate and the experiment was repeated 3 times.

As shown in FIG. 4, klebsiella pneumoniae phage GRNKpP has a long survival time at 45 ℃ and below, no significant magnitude difference in phage titer after 16W at 4 ℃, no significant magnitude difference in phage titer after 12W at 25 ℃, no significant magnitude difference in phage titer after 2W at 37 ℃, and good stability at 4-37 ℃ and can be effectively stored.

Example 5:

Host range of phage GRNKpP10

Klebsiella pneumoniae selected in the experiment is divided into 12K-type capsules, namely K1, K2, K3, K5, K11, K22, K24, K26, K31, K37, K38 and K64, each Klebsiella pneumoniae 5 strain is inoculated into an EP tube of 3mL TSB respectively, shaking is carried out at a constant temperature of 37 ℃ for 8 hours, klebsiella pneumoniae bacterial liquid is obtained, a sufficient TSA bottom plate is prepared, 0.5mL Klebsiella pneumoniae bacterial liquid and 6mL TSB semi-solid medium (containing 0.65% agar) with a temperature of about 50 ℃ are uniformly mixed and slowly paved on the TSA bottom plate, after the flat plate medium is solidified to normal temperature, 5 mu L of bacteriophage GRNKpP with a titer of 5X 10 ⁸ U/mL is respectively dripped on a flat plate, after the flat plate is dried, the flat plate is reversely placed into a 37 ℃ constant temperature biochemical incubator for culturing for about 8 hours, and the result is observed. Each set of experiments was repeated 3 times.

As shown in Table 1, klebsiella pneumoniae phage GRNKpP is capable of lysing all K1, K11, K22, K24, K31, K37 and K64 type Klebsiella pneumoniae, and is incapable of lysing K2, K3, K5, K26 and K36 type Klebsiella pneumoniae.

Experimental results prove that the phage GRNKpP has a very outstanding effect on the response of the cracking property of one or more of K1 type, K11 type, K22 type, K24 type, K31 type, K37 type and K64 type Klebsiella pneumoniae, and has great value on the targeted prevention, control and treatment of the Klebsiella pneumoniae in the future.

TABLE 1 lytic Effect of phage GRNKpP10 on different capsular Klebsiella pneumoniae

"Indicates no lysis," + "indicates lysis and plaque is clear, and" ++ "indicates lysis and plaque is clear.

Example 6:

fermentation preparation of klebsiella pneumoniae phage GRNKpP10

Single colony of Klebsiella pneumoniae KP048 is picked on a plate, inoculated into 3mL of TSB culture medium and cultured for 8 hours at 200rpm at 37 ℃ to obtain host bacterial liquid. The bacterial suspension was inoculated in an inoculum size of 2% into 120mL of TSB medium, and cultured at 37℃at 150rpm until the early logarithmic phase, and the OD600 of the bacterial solution was 0.1.

Industrial fermentation is carried out in a 10L fermenter, a fermentation system is 6L, a culture medium is TSB, the fermentation temperature is 28 ℃, the initial pH value is 7.4, klebsiella pneumoniae is inoculated according to an inoculum size of 1% by adopting a flame method, bacteriophage GRNKpP is added into the fermentation system under the optimal infection condition MOI=0.001, sterile air is introduced in the fermentation process, 3 permillage of defoamer is added, and the fermentation preparation time is 12h. From the beginning of fermentation, 20ml of fermentation broth was taken from the sampling port at 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, centrifuged at 6000rpm for 10min, and the supernatant was sterilized with a microporous filter membrane of 0.22 μm to obtain a phage-containing filtrate, and the titer thereof was measured, as described in example 2.

After fermentation is finished, taking out all mixed liquid of phage and host bacteria from a sampling port, inoculating the mixed liquid into a sterile container, centrifuging at 6000rpm for 10min, taking supernatant, filtering the supernatant into a sterile filter device by a vacuum pump, obtaining phage fermentation liquid, and storing the phage fermentation liquid at 4 ℃.

As shown in FIG. 6, the titer of Klebsiella pneumoniae phage GRNKpP reaches the maximum of 7.5X10 ¹⁰ PFU/mL in the preparation of 6h by fermentation, and the titer is stabilized at 10 ¹⁰ PFU/mL in the period of 6h to 12h, and as shown by the result, the industrial phage production by adopting the fermentation method is feasible, and effective reference data is provided for phage product preparation.

Example 7:

Experiment of Klebsiella pneumoniae bacteriophage GRNKpP on prevention and treatment of Klebsiella pneumoniae infection in ascitic disease of Lateolabrax

The largehead jewfish (Micropterus salmoides) is a relatively famous and precious fish with economic benefit and fresh water culture, and klebsiella pneumoniae is a conditional pathogenic bacterium of the largehead jewfish, can cause the largehead jewfish to suffer from ascites, has slow response and has obvious enteritis and ascites symptoms as anatomical symptoms.

The experimental largehead jewfish is provided by a largehead jewfish farm in Hubei Jingzhou, the selected largehead jewfish has normal and healthy appearance, the heads of the selected largehead jewfish are about 40 to 500g, the largehead jewfish is uniformly placed in a large-scale aquarium to be suitable for feeding for 7d before the experiment, the pH value of a water body is 7.3+/-0.1, the temperature is 25 ℃, the dissolved oxygen is 6.0+/-0.1 mg/L, and the experiment is prepared when the death rate of the largehead jewfish is stabilized within 0% -5% in one week. The Klebsiella pneumoniae used in this example was KP002, and the phage selected was Klebsiella pneumoniae phage GRNKpP of the present invention.

The experimental design is as follows, the largehead jewfish is divided into 5 groups at random, 20 jewfish in each group, and the experiment lasts for 7d:

1) Control group, each largehead jewfish is injected with an equal volume of physiological saline (belly injection);

2) The toxicity attack group is that each largehead jewfish is injected with 100 mu L of bacterial liquid with the concentration of 5 multiplied by 10 ⁹ CFU/mL (the final concentration is 5 multiplied by 10 ⁸ CFU/tail).

3) The treatment group comprises 100 mu L of bacterial liquid with the concentration of 5X 10 ⁹ CFU/mL for each micropterus salmoides, and injecting the phage GRNKpP X10 ⁷ PFU/carrying out treatment only after 2 hours;

4) The prevention group is that phage GRNKpP X10 ⁷ PFU/unit is injected 1 day before the experiment is started, and then each micropterus salmoides is injected with 100 mu L of bacterial liquid of 5X 10 ⁹ CFU/mL;

5) In the enrofloxacin treatment group, 100 mu L of bacterial liquid with the final concentration of 5 multiplied by 10 ⁹ CFU/mL is injected into each largehead jewfish. After 24 hours, enrofloxacin is added in the indicated amounts for treatment.

The experiment was continued for 7 days, and the symptoms and death number of micropterus salmoides were recorded. The survival rate of the largemouth bass was calculated for each group in the following manner.

Total A is the total number of the largemouth bass in each group;

Death A, namely, the total death number of the largemouth bass in each group is tested, and only;

And C, testing the survival rate of each group of micropterus salmoides,%.

The experimental results are shown in Table 2, the survival rate of the blank group is 100%, the survival rate of the offending group of micropterus salmoides is 0%, the survival rate of the treatment group is 85%, the survival rate of the prevention group is 90%, and the survival rate of the enrofloxacin group is 90%. Experimental results show that the klebsiella pneumoniae phage GRNKpP can effectively treat the problem of ascitic fluid of the larceny bass caused by klebsiella pneumoniae, the survival rate of the larceny bass is effectively improved by a prevention and treatment group, and the example provides an important reference value for the prevention and treatment of the klebsiella pneumoniae in the future and can be used as a biological antibacterial agent for the prevention and treatment of the klebsiella pneumoniae diseases of the larceny bass and other animals.

TABLE 2 control effect of phage GRNKpP10 on Klebsiella pneumoniae in Lateolabrax

Experimental group	Blank group	Toxin eliminating group	Treatment group	Preventive group	Enrofloxacin group
						Survival rate	100%	20%	85%	90%	85%

Example 8:

antibacterial persistence of klebsiella pneumoniae phage GRNKpP10

The Klebsiella pneumoniae KP002 single colony is picked on a flat plate, inoculated into 3mL of TSB culture medium, and cultured for 8 hours at 150rpm at 37 ℃ to obtain host bacterial liquid for later use. The experimental set-up was as follows, with this example examining the persistence of bacteriostasis of klebsiella pneumoniae phage GRNKpP.

(1) Negative control group, phage GRNKpP alone, at a concentration of 1X 10 ⁶ pfu/mL.

(2) Positive control group, KP002 alone, inoculation amount 1%.

(3) KP002+ GRNKpP10 group was added with phage at a concentration of 1X 106pfu/mL, and KP002 was added at an inoculum size of 1%.

The experimental time was 12h, and OD ₆₀₀ was measured at 1h, 2h, 4h, 6h, 8h, 12h, 24h, respectively.

Experimental results show that the OD ₆₀₀ of the KP002+ GRNKpP10 group is maintained below 0.1 in 1-24 hours, the OD ₆₀₀ of the positive group OD ₆₀₀ reaches the maximum value in 8 hours and is 2.2, so that GRNKpP has antibacterial durability, and an effective biological measure is provided for the prevention and treatment of the klebsiella pneumoniae in the future.

While the fundamental and principal features of the invention and advantages of the invention have been shown and described, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but may be embodied in other specific forms without departing from the spirit or essential characteristics thereof.

Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.

Claims

1. A Klebsiella pneumoniae phage GRNKpP10 that can lyse multiple K-capsule Klebsiella pneumoniae and has a long-lasting antibacterial effect, characterized in that: the phage was sent to the China Center for Type Culture Collection for preservation on March 4, 2024, and the preservation address is: Wuhan University, Wuhan, China; the preservation number is CCTCC NO: M2024373.

2. An application of the Klebsiella pneumoniae phage GRNKpP10 as claimed in claim 1, characterized in that it is applied to prepare an antibacterial agent for Klebsiella pneumoniae in environmental disinfection, animal husbandry, poultry farming, aquaculture or biomedical industry.

3. The use of the Klebsiella pneumoniae phage GRNKpP10 according to claim 1, characterized in that it is used to prepare a drug for treating or preventing a disease caused by Klebsiella pneumoniae infection.

4. The use of the Klebsiella pneumoniae phage GRNKpP10 according to claim 1, characterized in that it is used to prepare feed or feed additives that antagonize Klebsiella pneumoniae.

5. The use according to any one of claims 2 to 4, characterized in that the Klebsiella pneumoniae is K1 capsule type, K11 capsule type, K22 capsule type, K24 capsule type, K31 capsule type, K37 capsule type and K64 capsule type Klebsiella pneumoniae.