Klebsiella pneumoniae bacteriophage and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a klebsiella pneumoniae bacteriophage and application thereof.The klebsiella pneumoniae bacteriophage is named as vBKpnSP14 and preserved in the China General Microbiological Culture Collection Center on September 17, 2025, and the preservation number is CGMCC NO.46679. The klebsiella pneumoniae bacteriophage vBKpnSP14 has the high cracking rate on klebsiella pneumoniae, and the klebsiella pneumoniae bacteriophage vBKpnSP14 has the advantages that the klebsiella pneumoniae bacteriophage vBKpnSP14 has the high cracking rate on klebsiella pneumoniae; the bacteriophage has an effect of cracking klebsiella pneumoniae and edwardsiella tarda, has a cross-species cracking characteristic, and can solve the problem that in the prior art, a bacteriophage capable of cracking klebsiella pneumoniae and edwardsiella tarda at the same time lacks.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a strain of Klebsiella pneumoniae bacteriophage and its application. Background Technology

[0002] Klebsiella pneumoniae and Edwardsiella tarda are important zoonotic pathogens in aquaculture. Klebsiella pneumoniae is a Gram-negative bacterium, typically short rod-shaped, with smooth, moist, mucous-like colonies. It can cause septicemia, skin ulcers, and other diseases in various farmed aquatic animals, including grass carp, largemouth bass, common carp, shrimp, crab, and yellow-throated box turtles, resulting in significant economic losses to the aquaculture industry. Human infection with Klebsiella pneumoniae can cause pneumonia, enteritis, and even liver abscesses and septicemia.

[0003] Edwardsiella tarda is a Gram-negative, rod-shaped, non-spore-forming bacterium with flagella, capable of motility, and widely distributed in both freshwater and marine environments. Edwardsiella tarda primarily invades the host through the digestive tract or wounds on the body surface, causing systemic infection. Symptoms include skin ulceration, internal organ bleeding, and liver and kidney necrosis in farmed species such as largemouth bass, eel, yellow catfish, tilapia, and turbot, severely impacting the economic benefits of aquaculture. Human infection with Edwardsiella tarda can lead to diarrhea, cellulitis, and septicemia.

[0004] Both pathogens can enter the food chain through contaminated aquatic products such as fish and shrimp, seriously endangering human health. Currently, the main method of control for Klebsiella pneumoniae and Edwardsiella tarda in aquaculture is the use of antibiotics. However, the long-term use of antibiotics has led to a year-on-year increase in the detection rate of multidrug resistance in both bacteria, making prevention and control more difficult. Furthermore, antibiotics cause environmental pollution and can accumulate in the food chain, harming human health. With the promulgation of antibiotic reduction and ban orders both domestically and internationally, there is an urgent need to explore more alternative antibiotic treatment strategies. Bacteriophages are bacterial viruses, natural enemies of bacteria, with strong self-replication capabilities and high bactericidal effects, without producing toxic side effects on the body, and are considered an alternative to antibiotic treatment. In the field of aquaculture, bacteriophages have been successfully used to prevent and control bacterial infections in various aquatic animals. Although existing technologies exist for Klebsiella pneumoniae bacteriophages, such as vB_KpnP_ZK1, Kp_phage_507, and GRNKpP10, these Klebsiella pneumoniae bacteriophages only have lytic activity against Klebsiella pneumoniae and do not have cross-genera lytic ability. Summary of the Invention

[0005] The purpose of this invention is to provide a Klebsiella pneumoniae bacteriophage vB_KpnS_P14 with cross-genus and species lytic ability, addressing the problems of low drug targeting, long treatment duration, and poor treatment efficacy in aquatic animals infected with Klebsiella pneumoniae and Edwardsiella tarda, whether alone or in combination. This bacteriophage exhibits rapid and significant antibacterial activity and can be used to prepare drugs for the prevention and / or treatment of the aforementioned bacterial infections, environmental cleaners or disinfectants, food or feed additives, and water additives, achieving effective control of Klebsiella pneumoniae and Edwardsiella tarda infections.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A Klebsiella pneumoniae bacteriophage named vB_KpnS_P14 was deposited on September 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.46679. The bacteriophage has a major diameter of 61 nm, a transverse diameter of 57 nm, and a tail length of 210 nm.

[0007] This invention proposes a Klebsiella pneumoniae phage vB_KpnS_P14 with cross-genus lysis capability. This phage can simultaneously lyse Klebsiella pneumoniae and Edwardsiella tarda, exhibiting strong lysis activity. It provides a good phage source for the industrial production of phage preparations for the prevention and control of mixed infections of Klebsiella pneumoniae and Edwardsiella tarda.

[0008] Preferably, the Klebsiella pneumoniae phage belongs to the Longtail Phage family.

[0009] Preferably, the Klebsiella pneumoniae phage activity is stable at a pH of 4-12, and the Klebsiella pneumoniae phage activity is stable at a temperature of 45℃-60℃.

[0010] Preferably, the incubation period of the Klebsiella phage is 0 min to 10 min, the lysis period is 10 min to 90 min, and it enters the plateau phase after 90 min.

[0011] The use of the aforementioned Klebsiella pneumoniae phage in the preparation of an antibacterial agent, wherein the antibacterial agent lyses Klebsiella pneumoniae and / or Edwardsiella tarda.

[0012] Preferably, the Klebsiella pneumoniae bacteriophage is used in food preservation, environmental disinfection, livestock breeding, poultry farming, aquaculture, or the biomedical industry to prepare antibacterial agents for Klebsiella pneumoniae and / or Edwardsiella tarda.

[0013] Preferably, the antibacterial agent is composed of the Klebsiella pneumoniae phage, a protectant, and a synergist.

[0014] Preferably, the protective agent is gelatin and glycerin, and the synergist is EDTA.

[0015] The use of the Klebsiella pneumoniae phage in the preparation of feed additives or drugs for the prevention and / or treatment of Klebsiella pneumoniae and / or Edwardsiella tarda infections.

[0016] Preferably, the feed additive consists of the Klebsiella pneumoniae bacteriophage, chitosan oligosaccharide, trehalose, and skim milk powder.

[0017] The application of the bacteriophage in the preparation of reagents with cross-genus lysis capability, wherein the bacteriophage is the sole active ingredient in the reagent.

[0018] The present invention also provides a food or feed additive whose active ingredient includes the Klebsiella pneumoniae phage vB_KpnS_P14 of the present invention.

[0019] Preferably, the bacteriophage is inoculated or added at a multiplicity of infection of 0.00001 to 10.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention proposes a Klebsiella pneumoniae phage vB_KpnS_P14 with cross-genus lysis capability. This phage can simultaneously lyse Klebsiella pneumoniae and Edwardsiella tarda, exhibiting strong lysis activity. It provides a good phage source for the industrial production of phage preparations for the prevention and control of mixed infections of Klebsiella pneumoniae and Edwardsiella tarda.

[0021] The Klebsiella pneumoniae phage vB_KpnS_P14 provided by this invention was isolated in nature, does not contain virulence genes, has no drug resistance, has no chemical residues, has no adverse impact on the ecological environment, and has high safety.

[0022] The Klebsiella pneumoniae phage vB_KpnS_P14 provided by this invention has an extremely low optimal MOI of 0.0001. A small amount of phage can achieve the maximum progeny phage yield, meaning that adding a trace amount of phage can still effectively kill bacteria.

[0023] The Klebsiella pneumoniae phage vB_KpnS_P14 provided by this invention maintains good activity within a pH range of 5.0-12.0, and its titer remains at 8.5 × 10⁻⁶ after 20 minutes of treatment at 65°C. 6 It has a pfu / mL or higher and exhibits good temperature stability.

[0024] The Klebsiella pneumoniae bacteriophage vB_KpnS_P14 provided by this invention significantly inhibits the proliferation of both Klebsiella pneumoniae and Edwardsiella tarda after 2 hours of addition. The bacteriophage exhibits highly efficient inhibitory effects on the proliferation of both Klebsiella pneumoniae and Edwardsiella tarda. Attached Figure Description

[0025] Figure 1 Plaques formed by bacteriophage vB_KpnS_P14.

[0026] Figure 2 Electron micrograph of bacteriophage vB_KpnS_P14.

[0027] Figure 3 The optimal infection multiplicity for bacteriophage vB_KpnS_P14.

[0028] Figure 4 The acid-base stability of bacteriophage vB_KpnS_P14.

[0029] Figure 5 Temperature stability of bacteriophage vB_KpnS_P14.

[0030] Figure 6 This is a one-step growth curve of bacteriophage vB_KpnS_P14.

[0031] Figure 7 A circular diagram of the genome of bacteriophage vB_KpnS_P14.

[0032] Figure 8 A graph validating the lysis ability of bacteriophage vB_KpnS_P14; Figure 8 In the image, A represents the phage pattern formed by vB_KpnS_P14 on a GDDP10 culture plate of Edwardsiella tarda; B represents the phage pattern formed by vB_KpnS_P14 on a P14 culture plate of Klebsiella pneumoniae.

[0033] Figure 9 The growth inhibition curve of bacteriophage vB_KpnS_P14 against Klebsiella pneumoniae.

[0034] Figure 10 The growth inhibition curve of bacteriophage vB_KpnS_P14 against Edwardsiella tarda is shown.

[0035] Figure 11 The growth inhibition curve of bacteriophage vB_KpnS_P14 against Klebsiella pneumoniae on snakehead meat is shown. Detailed Implementation

[0036] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0037] Example 1: Isolation, purification and preservation of Klebsiella pneumoniae phage vB_KpnS_P14.

[0038] (1) Isolation and identification of host bacteria: Diseased fish samples and aquaculture water were collected from the largemouth bass farm in Luqiao Town, Weishan County, Shandong Province, and the Weishan Lake Modern Fishery Park. The diseased largemouth bass were placed in a clean bench and wiped with alcohol for disinfection. The abdomen of the fish was cut open, and a sterile inoculation loop was inserted into the spleen tissue. Then, the plates were streaked on BHI solid medium and incubated at 37°C for 12 h. Ten single colonies were randomly selected from the diseased fish at the two locations and inoculated into 8 mL of LB liquid medium. The culture was carried out overnight at 37°C. The bacterial suspensions from the two locations were sent to Qingke Biotechnology for 16S rDNA sequencing identification. Molecular identification showed that the dominant pathogens at the two locations were Klebsiella pneumoniae and Edwardsiella tarda, respectively. The single-clone bacterial suspensions were stored at -80°C and named P14 and GDDP10, respectively.

[0039] (2) Isolation of bacteriophage vB_KpnS_P14: Bacteriophage was isolated using the double-layer plate drop method. Aquaculture water sample from a diseased farm was centrifuged at 8000 r / min for 5 min. The supernatant was filtered through a 0.22 μm filter to obtain the filtrate. 0.1 mL of the filtrate and 0.1 mL of the Klebsiella pneumoniae suspension from (1) were added to 5 mL of LB broth and cultured at 37℃ and 180 rpm for 4 hours to obtain the culture medium. The culture medium was then filtered through a 0.22 μm filter membrane for sterilization and temporarily stored at 4℃. 0.5 mL of the filtrate and 0.1 mL of the Klebsiella pneumoniae suspension were mixed thoroughly, and then 5 mL of 50℃ LB semi-solid medium was added. The mixture was poured into a sterile plate, solidified, and then incubated upside down in a 37℃ incubator overnight. The presence of phage plaques on the plate was observed.

[0040] (3) Purification and preservation of phage vB_KpnS_P14: A single phage plaque from (2) was extracted into 1 mL of sterile SM buffer, shaken for 1 min, and allowed to stand for 30 min to obtain an extract. 0.1 mL of the extract and 0.1 mL of the Klebsiella pneumoniae suspension from (1) were mixed evenly, and 5 mL of LB semi-solid medium was added to prepare a double-layer plate. The extraction of a single phage plaque was repeated 5-6 times to form phage plaques of uniform shape and size on the semi-solid plate. The phage was named vB_KpnS_P14. The phage solution was mixed with 50% glycerol at a ratio of 1:1 and stored at -80℃.

[0041] like Figure 1 As shown, the purified phages formed round, clear plaques with a diameter of 2-3 mm and a distinct halo. Plaques with halos had a diameter of 7-8 mm. Following international nomenclature rules, they were named vB_KpnS_P14.

[0042] Example 2: Electron microscopic observation of bacteriophage vB_KpnS_P14.

[0043] Take 20 μL of liquid containing crude phage particles and drop it onto a copper grid. Allow it to settle naturally for 15 min. Then, use filter paper to absorb the excess liquid from the side. Add one drop of 2% phosphotungstic acid to the copper grid to stain the phage for 10 min. Then, use filter paper to absorb the staining solution from the side. After the sample dries, observe the phage morphology using an electron microscope.

[0044] Depend on Figure 2 It is known that this bacteriophage has a long diameter of 61 nm, a transverse diameter of 57 nm, a tail length of 210 nm, and a head with a polyhedral structure. According to the Ninth Report on the Classification of Viruses by the International Organization for Taxonomy of Viruses, this bacteriophage can be classified as belonging to the family Longtailed Phagesidae.

[0045] Example 3: Determination of the optimal multiplicity of infection.

[0046] Adjust the concentration of the host bacterium Klebsiella pneumoniae to 10. 8 CFU / mL, the phage fluid was diluted and added to 5 mL of LB broth at multiples of infection ratios of 10:1, 1:1, 1:10, 1:100, 1:1000, 1:1000, 1:10000, and 1:100000. The mixture was then incubated at 37°C with shaking for 4 h, followed by filtration through a 0.22 μm filter membrane. Phage titer was determined using the double-layer plate method.

[0047] The results are as follows Figure 3 As shown, the titer is highest when the multiplicity of infection is 1:10000, indicating that the optimal multiplicity of infection for vB_KpnS_P14 is 1:10000.

[0048] Example 4: Determination of pH stability of bacteriophages.

[0049] The pH of LB liquid medium was adjusted to 1–14 using 1 mol / L HCl and 1 mol / L NaOH, respectively. 900 μL of LB liquid medium at each pH was then mixed with 100 μL of bacteriophage to achieve a final concentration of 1 × 10⁻⁶. 10 PFU / mL, mixed thoroughly, incubated in a water bath at 37℃ for 1 h, and the phage titer was determined using the double-layer plate method under different pH conditions. Results are as follows: Figure 4 As shown.

[0050] Depend on Figure 4 It can be seen that bacteriophages maintain high activity in the pH range of 5.0 to 12.0, and the highest titer, reaching 10, is achieved at pH 6.0. 9 The activity gradually decreases with increasing or decreasing pH, but maintains high potency at pH 4.0 and 12.0. 6 The pfu / mL level is above 1. These results indicate that bacteriophage vB_KpnS_P14 exhibits good acid-base tolerance. The optimal pH range for the growth of bacteriophage vB_KpnS_P14 is 5.0–12.0.

[0051] Example 5: Determination of temperature stability of bacteriophages.

[0052] With a valence of 2×10 10 Phage stock solution of PFU / mL was aliquoted into sterile centrifuge tubes at 1 mL per tube and incubated in water baths at 45℃, 55℃, 65℃, and 70℃ for 20 min, 40 min, and 60 min, respectively, with three replicates for each temperature. The potency of each sample was determined using the bilayer plate method after sampling at each time point. Results are as follows: Figure 5 As shown.

[0053] Depend on Figure 5 It is known that bacteriophages maintain stable activity and titer within a temperature range of 45℃ to 55℃. 9 The titer remained above pfu / mL after treatment at 65℃ for different times. 8 pfu / mL. Essentially inactivated at 70°C.

[0054] Example 6: Plotting the one-step growth curve of bacteriophage.

[0055] Take 500 μL of logarithmic growth phase 10 8 CFU / mL Klebsiella pneumoniae P14 suspension and an equal volume of 10 5Phage solution of PFU / mL was mixed at the optimal MOI of 0.0001, incubated in a water bath for 5 min at 37°C, centrifuged at 6000 rpm for 5 min, and the supernatant was discarded. The precipitate was then washed twice with LB broth. The precipitate was resuspended in 100 mL of preheated LB broth at 37°C and immediately placed in a shaker at 180 rpm for incubation. Samples were taken at 0 min, 5 min, and 10 min. After 10 min, samples were taken every 10 min, and the phage titer was immediately measured using the double-layer plate method. A phage growth curve was plotted with time on the x-axis and the logarithm of the phage titer on the y-axis to obtain the phage latency and average lysis rate. Average lysis rate = phage titer at the end of the outbreak / host bacterial concentration at the beginning of infection.

[0056] Depend on Figure 6 It can be seen that the titer of the bacteriophage does not change significantly within 10 minutes after infecting the host bacteria, indicating that its incubation period is about 10 minutes. The titer of the bacteriophage increases significantly within 10 minutes to 90 minutes, indicating that the lysis period of the bacteriophage is about 80 minutes. After 90 minutes, the titer of the bacteriophage tends to stabilize and enters the plateau phase.

[0057] Example 7: Phage genome analysis.

[0058] 1×10⁻⁶ was obtained using the double-layer plate amplification method. 9 Phage solution was prepared at PFU / mL, then centrifuged at 8000 rpm for 15 minutes to obtain the supernatant. After filtration and sterilization, the genomic DNA of phage vB_KpnS_P14 was extracted using a viral genome extraction kit from Tiangen Biotech (Beijing) Co., Ltd. The qualified DNA was sent to a sequencing company for sequencing.

[0059] Sequencing revealed that the full-length genome of bacteriophage vB_KpnS_P14 is 50453 bp, with a GC content of 50.96%. The genome diagram is shown below. Figure 7 The results showed that the phage genome was annotated to 80 open reading frames. Sequence alignment of protein-coding genes was performed using Diamond software, annotating a total of 80 proteins, including perforin and endosomal proteins. This provides a basis for the expression of broad-spectrum lysins and perforin, thereby expanding the application of vB_KpnS_P14 strains and improving therapeutic efficacy. Furthermore, the phage genome does not contain antibiotic resistance genes or virulence factors, making it safe for use in aquaculture environments.

[0060] Example 8: Pyrolysis rate experiment.

[0061] Take 1 mL of 1×10 2 CFU / mL phage sample and 1 mL of 1×10 6CFU / mL Klebsiella pneumoniae culture was incubated at 37°C with shaking for 15 minutes. After mixing, it was diluted with physiological saline to 10⁻¹–10⁻³, in three gradients. 100 μL of each gradient was spread onto LB agar plates and incubated at 37°C for 12 hours. Each gradient was repeated three times. Simultaneously, 1 mL of physiological saline and 1 mL of 1×10⁻⁶ CFU / mL Klebsiella pneumoniae culture were added. 6 Using CFU / mL host bacterial culture as a control, repeat the above steps. Count the colonies on plates with 30–300 colonies. Repeat the experiment three times and take the average value. Phage lysis rate = (1 - number of colonies in the treatment group / number of colonies in the control group) × 100%.

[0062] The vB_Kpns_P14 bacteriophage has a lysis rate of 99% and a good lysis effect on the host, making it suitable for the prevention and control of Klebsiella pneumoniae in aquaculture.

[0063] Example 9: Determination of host profile of bacteriophage vB_KpnS_P14.

[0064] The tested strains included 12 strains of Klebsiella pneumoniae and 4 strains of Edwardsiella tarda. The double-layer plate method was used to detect whether bacteriophage vB_KpnS_P14 had lytic activity against different strains, and the host spectrum of bacteriophage vB_KpnS_P14 was determined.

[0065] The 12 Klebsiella pneumoniae strains were identified as Klebsiella pneumoniae P14, P15, P16, KL1, KL2, KL3, KL4, KL5, SDLM1, HNQ5, HNQ6, and HNQ7; the 4 Edwardsiella tarda strains were identified as Edwardsiella tarda GDDP10, GDDP11, SDLX1, and SDL01. All strains used in the experiment were purchased from Shandong Xinde Technology Co., Ltd.

[0066] Judgment criteria: +3, large and clear plaques, complete bacterial lysis; +2, clear plaques, but with a faint hazy background; +1, incomplete lysis, high turbidity in the spotting area; -, no plaques.

[0067] The results are shown in Table 1. The bacteriophage vB_KpnS_P14 of this invention showed a lysis coverage of 75% against Klebsiella pneumoniae strains, exhibiting a broad host spectrum. Furthermore, vB_KpnS_P14 also demonstrated lysis ability against the three tested Edwardsiella tarda strains.

[0068] Table 1: Lysis spectrum of Klebsiella pneumoniae phage vB_KpnS_P14.

[0069] Example 10: Detection of in vitro antibacterial activity of bacteriophage vB_KpnS_P14.

[0070] Klebsiella pneumoniae P14 and Edwardsiella tarda GDDP10 were inoculated separately into LB medium and cultured at 37°C until the logarithmic growth phase. The bacterial concentration was adjusted to 10⁻⁶. 6 CFU / mL, initial phage concentration 10 6 PFU / mL. After serially diluting the phage solution 10-fold, 100 μL of different concentrations of phage were inoculated into 5 mL of LB medium at MOIs of 1, 0.1, 0.01, and 0.001, respectively, with 100 μL of *Klebsiella pneumoniae* and 100 μL of *Edwardsiella tarda*. The medium was incubated at 37°C with shaking at 180 rpm for 12 hours. Each group had three replicates, with the control group consisting of bacterial suspension without phage. The OD of the bacteria was measured every hour. 600 Values ​​were used to plot the growth inhibition curves of Klebsiella pneumoniae P10 and Edwardsiella tarda GDDP10, as shown below. Figure 9 , Figure 10 As shown.

[0071] The results showed that bacteriophage vB_Kpns_P14 inhibited the growth of both Klebsiella pneumoniae and Edwardsiella tarda. For Klebsiella pneumoniae, the OD of the control group from 0h to 11h was significantly lower. 600 The value continued to rise, and after 11 hours, OD 600 The value reached 0.77. However, after adding phage, the OD value decreased at different MOI values ​​from 0 to 5 hours. 600 The value remains basically at the initial value, with OD remaining at a constant level for 5 to 8 hours. 600 The value increased significantly, with OD levels rising significantly over 8 to 12 hours. 600 The value rises slowly, but OD 600 The value remained around 0.5, significantly lower than the OD value of the control group. 600 The value indicates that bacteriophage vB_Kpns_P14 has a significant inhibitory effect on Klebsiella pneumoniae.

[0072] For Edwardsiella tarda, the OD of the control group was... 600 The value continued to rise, and after 11 hours, OD 600 The value stabilized at around 0.8. However, after adding bacteriophage, at MOI=0.001, the OD value increased from 0 to 1 hour. 600 The value increases, with OD rising between 1 and 11 hours. 600 The value decreases slowly; the remaining MOI values ​​are within 0 to 8 hours of OD. 600 The value decreases slowly, with OD values ​​decreasing over 8 to 11 hours. 600 The value remained below 0.2. The OD value of the experimental group... 600 OD values ​​compared to the control group 600 The value was significantly lower than before, indicating that the addition of bacteriophage vB_Kpns_P14 can significantly inhibit the growth of Edwardsiella tarda and has a strong lytic effect on Edwardsiella tarda.

[0073] Example 11: Inhibition experiment of bacteriophage vB_Kpns_P14 on Klebsiella pneumoniae in snakehead meat.

[0074] (1) Preparation of sterile meat pieces. After killing the snakehead, remove the skin and bones in a clean bench and cut into 1cm×1cm cubes. Then soak the fish meat in sodium hypochlorite for 5 minutes, take it out, wash it repeatedly with sterile ddH2O 3 times, place it in a sterile petri dish, and sterilize it by irradiating both sides under a UV lamp for 15 minutes.

[0075] (2) Artificial contamination of fish meat. The host bacterium *Klebsiella pneumoniae* was cultured to 1×10⁻⁶. 8 The concentration was determined by centrifuging at 6000 rpm for 5 minutes, discarding the supernatant, washing the bacterial pellet twice with sterile PBS buffer, and diluting with PBS buffer to prepare a 10 CFU / mL solution. 5 CFU / mL bacterial suspension. Take 10 μL of 10... 5 A CFU / mL dose of host bacteria was added to one surface of the fish meat, and the mixture was gently shaken to spread it evenly across the surface of the fish meat, resulting in 10³ CFU / cm² of artificially contaminated bacteria.

[0076] (3) Preparation and processing of bacteriophages. 10¹ bacteriophages were obtained using liquid propagation at the optimal multiplicity of infection. 0 The phage stock solution was diluted to 10 PFU / mL with PBS buffer. 6 PFU / mL.

[0077] Phage antibacterial group: 10 μL of 10 6 PFU / mL, 10 7 PFU / mL, 10 8 PFU / mL phage solution was added dropwise to one side of a sterile meat block and air-dried in a laminar flow hood. This step was repeated twice. 10 μL of 10... 5 CFU / mL Klebsiella pneumoniae solution was evenly dropped onto the side containing the bacteriophage and then placed in a clean bench to air dry.

[0078] Control Group A: 10 μL of sterile PBS buffer was added to one side of the sterile meat piece and air-dried in a laminar flow hood. This step was repeated twice. 10 μL of PBS buffer was then added to one side of the sterile meat piece. 5 CFU / mL Klebsiella pneumoniae culture was evenly dropped onto the side containing sterile PBS buffer and placed in a clean bench to air dry.

[0079] Control group B: 10 μL of sterile PBS buffer was added to one side of the sterile meat block and placed in a laminar flow hood to air dry. This step was repeated 4 times.

[0080] (3) Detection of bacterial load on meat surface. Three parallel experiments were conducted in each group. The petri dishes containing the samples were sealed with sealing film and incubated at 4℃ and 28℃, respectively. Samples were taken at 0h, 1h, 3h, 6h, and 9h. The meat pieces were placed in a 6-well plate containing 6mL of sterile PBS buffer, shaken for 10min, and then 100μL of the liquid was taken and serially diluted with sterile PBS buffer. The host bacterial count was then determined using the spread plate method. A bacterial growth inhibition curve was plotted with time on the x-axis and the logarithm of Klebsiella pneumoniae bacterial load on the y-axis. The results are as follows: Figure 11 As shown.

[0081] Depend on Figure 11 It can be seen that the bacterial load in control group A increased slowly over time. No colonies were produced in control group B, proving that the meat pieces were not contaminated by other bacteria throughout the entire process. After adding bacteriophages with different MOIs to the surface of the meat pieces beforehand, the Klebsiella pneumoniae bacterial load decreased in all cases, indicating that bacteriophage vB_Kpns_P14 has an inhibitory effect on Klebsiella pneumoniae on the surface of snakehead meat. Furthermore, the Klebsiella pneumoniae bacterial load was lowest at MOI=1000, indicating that the antibacterial effect was most significant at MOI=1000. At MOI=100 and MOI=1000, the proliferation of Klebsiella pneumoniae on the surface of snakehead meat was significantly inhibited within 1 hour.

[0082] Klebsiella pneumoniae is widely present in aquaculture environments and is frequently isolated from the bodies of diseased snakehead and largemouth bass. Snakehead meat is often consumed raw without high-temperature cooking, making it susceptible to Klebsiella pneumoniae contamination during processing. Based on the above results, bacteriophage vB_Kpns_P14 shows great promise for eliminating Klebsiella pneumoniae contamination in snakehead meat.

[0083] Example 12: Validation of the cross-genus lysis ability of bacteriophage vB_Kpns_P14.

[0084] 200 μL of Klebsiella pneumoniae P14 and 200 μL of Edwardsiella tarda GDDP10 were thoroughly mixed with LB semi-solid medium cooled to approximately 55°C, and then poured onto prepared LB solid medium to create double-layer plates. 5 μL of LB 10... 8 PFU / mL phage solution was added dropwise to the surface of a semi-solid culture medium, air-dried in a clean bench, and then incubated overnight at 37°C to observe for the presence of phage plaques.

[0085] The results are as follows Figure 8 As shown, bacteriophage vB_Kpns_P14 exhibits lytic activity against both Klebsiella pneumoniae P14 and Edwardsiella tarda GDDP10. The bacteriophage plaques are clear and exhibit a distinct halo.

[0086] Example 13: A reagent with cross-genus cleavage capability.

[0087] This embodiment provides a reagent with cross-genera lysis capability. After enriching and culturing the phage vB_Kpns_P14 obtained in Example 1 on plates, the phage was washed with SM buffer to obtain 1×10⁻⁶ phages. 10 Phage fluid at PFU / mL. Dilute 10–100,000 times with sterile water or SM, aliquot, and store at 4°C or room temperature to obtain reagents with cross-genus lysis capabilities. The SM buffer formulation is 50 mM Tris-HCl, 100 mM NaCl, 8 mM MgSO4, pH 7.5.

[0088] The reagent was used to test its ability to lyse across genera, and the results showed that it had a lytic effect on both Klebsiella pneumoniae and Edwardsiella tarda.

[0089] Example 14: A biological bactericide.

[0090] This embodiment provides a biological bactericide. The bacteriophage vB_Kpns_P14 screened in Example 1 is mixed with a protectant and a synergist in any proportion and dispensed into sterile spray bottles. The protectant is gelatin and glycerin, and the synergist is 0.1 mM EDTA. It can be used on the surface of aquatic animal meat as a preservative to inhibit the proliferation of Klebsiella pneumoniae and Edwardsiella tarda.

[0091] The antibacterial activity of this biocide was tested, and the results showed that the spray formulation had an inhibitory effect on both Klebsiella pneumoniae and Edwardsiella tarda.

[0092] Example 15: An aquatic feed additive.

[0093] This embodiment provides an aquatic feed additive, which is made by mixing the bacteriophage vB_Kpns_P14 obtained in Example 1 with chitosan oligosaccharide, trehalose, and skim milk powder in any proportion. After pre-freezing at -80℃ and vacuum freeze-drying, it is mixed with pregelatinized starch in any proportion, granulated to a particle size of 2 mm, and then sealed and packaged.

[0094] The cross-genera lysis ability of this feed additive was tested, and the results showed that the reagent had inhibitory and lysis effects on both Klebsiella pneumoniae and Edwardsiella tarda.

[0095] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0096] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0097] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A strain of Klebsiella pneumoniae phage, characterized in that, The Klebsiella pneumoniae bacteriophage described was vB_KpnS_P14, which was deposited on September 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.46679.

2. The Klebsiella pneumoniae phage according to claim 1, characterized in that, The Klebsiella pneumoniae bacteriophage mentioned belongs to the family Longtail Phageidae.

3. The Klebsiella pneumoniae phage according to claim 1, characterized in that, The Klebsiella pneumoniae phage activity is stable at pH 4-12, and the Klebsiella pneumoniae phage activity is stable at temperature 45℃-60℃.

4. The Klebsiella pneumoniae phage according to claim 1, characterized in that, The incubation period of the Klebsiella phage is 0 min to 10 min, the lysis period is 10 min to 90 min, and it enters the plateau phase after 90 min.

5. The application of the Klebsiella pneumoniae bacteriophage according to claim 1 in the preparation of an antibacterial agent, characterized in that, The antibacterial agent is used to lyse Klebsiella pneumoniae and / or Edwardsiella tarda.

6. The application according to claim 5, characterized in that, The antibacterial agent is composed of the Klebsiella pneumoniae phage, a protectant, and a synergist.

7. The application according to claim 6, characterized in that, The protective agent is gelatin and glycerin, and the synergist is EDTA.

8. The use of the Klebsiella pneumoniae phage of claim 1 in the preparation of feed additives or drugs for the prevention and / or treatment of Klebsiella pneumoniae and / or Edwardsiella tarda infections.

9. The application according to claim 8, characterized in that, The feed additive consists of the Klebsiella pneumoniae bacteriophage, chitosan oligosaccharide, trehalose, and skim milk powder.

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