Phage cocktail and application thereof

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a phage cocktail and application thereof. The bacteriophage cocktail comprises five bacteriophages PGPA20, PGPA60, PAB26, Lkpp-25 and Lkpp-36 which are separated from the natural world, and the bacteriophage cocktail has relatively strong bactericidal activity on pseudomonas aeruginosa, acinetobacter baumannii and K1 / K2 type klebsiella pneumoniae, and has specificity on the pseudomonas aeruginosa, the acinetobacter baumannii and the K1 / K2 type klebsiella pneumoniae; the bacteriophage can be compounded with other substances for use, and a safe and non-toxic bacteriophage killing product is provided for in-vivo and in-vitro infection treatment of pseudomonas aeruginosa, acinetobacter baumannii and K1 / K2 type klebsiella pneumoniae and disinfection and purification of pseudomonas aeruginosa, acinetobacter baumannii and K1 / K2 type klebsiella pneumoniae in the breeding industry and other environments.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a bacteriophage cocktail and its applications. Background Technology

[0002] Hospital-acquired infections are a significant burden on global public health, particularly due to multidrug-resistant Gram-negative bacteria such as *Pseudomonas aeruginosa*, *Klebsiella pneumoniae*, and *Acinetobacter baumannii*. *Pseudomonas aeruginosa* is an opportunistic pathogen that can cause acute or chronic infections in immunocompromised individuals with chronic obstructive pulmonary disease (COPD), cystic fibrosis, sepsis, and ventilator-associated pneumonia (VAP). *Klebsiella pneumoniae* is a common clinical bacterial pathogen that often poses a serious threat to clinical treatment. Serotypes K1, K2, K5, K20, K54, and K57 are considered associated with highly virulent *Klebsiella pneumoniae*, with serotypes K1 and K2 accounting for approximately 70% of highly virulent *Klebsiella pneumoniae* isolates. Infections caused by these serotypes, such as pneumonia, sepsis, meningitis, and purulent liver abscesses, are associated with high mortality rates. *Acinetobacter baumannii* is a Gram-negative bacterial pathogen that can cause VAP and bloodstream infections in patients in intensive care units. Carbapenem-resistant Acinetobacter baumannii is resistant to most existing antibiotics and is associated with high morbidity and mortality. As members of the broad-spectrum antibiotic-resistant pathogens, the high resistance of Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae makes treating these infections even more challenging. Therefore, developing novel clinical treatment strategies, such as a phage cocktail therapy that synergistically targets Pseudomonas aeruginosa, Acinetobacter baumannii, and K1 / K2 Klebsiella pneumoniae, may be a highly promising strategic direction for addressing this serious public health challenge.

[0003] Bacteriophages are naturally occurring obligate parasitic viruses that infect bacteria. They attach to specific surface receptors on host bacteria and replicate within the host to induce host-specific lysis. As an alternative to antibiotics, bacteriophages offer significant advantages. First, bacteriophages are among the most abundant organisms in nature, making them easy to isolate and obtain. Second, bacteriophages possess lytic activity and host specificity, avoiding indiscriminate damage to the normal human microbiota. Finally, bacteriophages can penetrate biofilms. Therefore, phage therapy has become a promising solution to the growing crisis of drug-resistant infections. Phage therapy can be used as a single phage or as a mixture of phages. Compared to a single phage, phage cocktails offer a broader antibacterial spectrum and stronger lytic activity, inhibiting biofilm formation more rapidly. Therefore, the development of phage cocktails is of great significance for treating drug-resistant Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae. Summary of the Invention

[0004] The purpose of this section is to outline some aspects of the embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0005] To address the aforementioned technical problems, according to one aspect of the present invention, the present invention provides the following technical solution:

[0006] A phage cocktail comprising five phages isolated from nature: PGPA20, PGPA60, PAB26, Lkpp-25, and Lkpp-36. Phages PGPA20 and PGPA60 were deposited at the China Center for Type Culture Collection (CCTCC) on October 28, 2025, with accession numbers CCTCC NO: M 20252375 and CCTCC NO: M20252376, respectively. Phages PAB26, Lkpp-25, and Lkpp-36 were deposited at the CTCC on November 3, 2025, with accession numbers CCTCC NO: M 20252414, CCTCC NO: M 20252413, and CCTCC NO: M20252415, respectively.

[0007] Preferably, the volume ratio of phages PGPA20, PGPA60, PAB26, Lkpp-25 and Lkpp-36 in the phage cocktail is 1:1:1:1:1.

[0008] The use of a phage cocktail as an active ingredient in the preparation of a pharmaceutical composition for the prevention or treatment of infectious diseases caused by Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae.

[0009] Preferably, the phage cocktail is used to kill Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae.

[0010] Preferably, the pharmaceutical composition is a liquid formulation, a lyophilized formulation, or an oral solid formulation.

[0011] The use of a phage cocktail as an active ingredient in the preparation of a spray for killing Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae in animal husbandry and medical environments.

[0012] Compared with existing technologies, the beneficial effects of this invention are: the phage cocktail has strong bactericidal activity against Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae K1 / K2, and is specific to these bacteria. It can be used in combination with other substances to provide a safe and non-toxic phage disinfection product for the treatment of Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae infections in vivo and in vitro, as well as for the disinfection and purification of these bacteria in aquaculture and other environments. Attached Figure Description

[0013] To more clearly illustrate the technical solutions of the embodiments of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and detailed embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0014] Figure 1 The image shows phage plaques of phages PGPA20, PGPA60, PAB26, Lkpp-25, and Lkpp-36 in a phage cocktail of the present invention.

[0015] Figure 2 Morphological observation of phages PGPA20, PGPA60, PAB26, Lkpp-25 and Lkpp-36 of a phage cocktail of the present invention;

[0016] Figure 3 The following are temperature stability line graphs of phages PGPA20, PGPA60, PAB26, Lkpp-25, and Lkpp-36 in a phage cocktail according to the present invention (A is the temperature stability line graph of phage PGPA20; B is the temperature stability line graph of phage PGPA60; C is the temperature stability line graph of phage PAB26; D is the temperature stability line graph of phage Lkpp-25; E is the temperature stability line graph of phage Lkpp-36).

[0017] Figure 4 The present invention provides a bar chart of pH stability for a phage cocktail consisting of phages PGPA20, PGPA60, PAB26, Lkpp-25, and Lkpp-36 (A is the pH stability bar chart for phage PGPA20; B is the pH stability bar chart for phage PGPA60; C is the pH stability bar chart for phage PAB26; D is the pH stability bar chart for phage Lkpp-25; and E is the pH stability bar chart for phage Lkpp-36).

[0018] Figure 5 The diagram shows the antibacterial curves of phages PGPA20, PGPA60, PAB26, Lkpp-25, and Lkpp-36 in a phage cocktail according to the present invention (A is the antibacterial curve of PGPA20 and PGPA60 alone at MOI=10, 1, and 0.1; B is the antibacterial curve of PAB26 at MOI=10, 1, and 0.1; C is the antibacterial curve of Lkpp-25 and Lkpp-36 alone at MOI=10, 1, and 0.1; and D is the antibacterial curve of the phage cocktail composed of PGPA20, PGPA60, PAB26, Lkpp-25, and Lkpp-36). Detailed Implementation

[0019] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0020] Secondly, the present invention is described in detail with reference to the schematic diagrams. When detailing the embodiments of the present invention, for ease of explanation, the cross-sectional views illustrating the device structure may be partially enlarged, not according to the usual scale. Furthermore, the schematic diagrams are merely examples and should not limit the scope of protection of the present invention. In addition, actual fabrication should include three-dimensional spatial dimensions of length, width, and depth.

[0021] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0022] A phage cocktail containing five phage strains, all isolated from sewage, all capable of forming clear plaques on LB agar medium. The inclusion includes bacteriophages PGPA20, PGPA60, PAB26, Lkpp-25, and Lkpp-36. Phages PGPA20 and PGPA60 were deposited at the China Center for Type Culture Collection (CCTCC) on October 28, 2025, with accession numbers CCTCC NO: M 20252375 and CCTCC NO: M 20252376, respectively, and are classified as *Pseudomonas aeruginosaphage* PGPA20 and *Pseudomonas aeruginosa phage* PGPA60, respectively. Phages PAB26, Lkpp-25, and Lkpp-36 were deposited at the CCTCC on November 3, 2025, with accession numbers CCTCC NO: M20252414, CCTCC NO: M 20252413, and CCTCC NO: M, respectively. 20252415, classified and named as *Acinetobacter baumannii* phage PAB26, *Klebsiella pneumoniae* phage Lkpp-25, and *Klebsiella pneumoniae* phage Lkpp-36, respectively. Address: Wuhan University, Wuhan, China. The volume ratio of phages PGPA20, PGPA60, PAB26, Lkpp-25, and Lkpp-36 is 1:1:1:1:1.

[0023] Example 1: Isolation and preparation of bacteriophages

[0024] The wastewater samples used in this invention were collected from Changchun Park in Changchun City. The host bacteria were *Pseudomonas aeruginosa* GPA20 and GPA60, *Acinetobacter baumannii* AB26, and *Klebsiella pneumoniae* kpp-25 and kpp-36, respectively. The wastewater was collected and filtered through gauze. The filtrate was then used as a substitute for ddH2O in the preparation of LB medium, resulting in three 200mL bottles of LB medium. Then, 1mL of overnight cultured *Pseudomonas aeruginosa* GPA20 and GPA60, 1mL of overnight cultured *Acinetobacter baumannii* AB26, and 1mL of overnight cultured *Klebsiella pneumoniae* Lkpp-25 and Lkpp-36 were added to each of the three LB medium bottles, and the cultures were incubated at 37℃ for 10-12 hours. The next day, 2mL of the overnight culture was collected, centrifuged at 12,000 rpm for 5 minutes, and the supernatant was filtered through a 0.22μm filter and stored. The resulting filtrate was then used for plaque testing to check for the presence of bacteriophages capable of lysing the host bacteria.

[0025] The plaque test was performed as follows: Single colonies of *Pseudomonas aeruginosa* GPA20 and GPA60, *Acinetobacter baumannii* AB26, and *Klebsiella pneumoniae* kpp-25 and kpp-36 were picked and placed in 5 mL of LB broth and incubated at 37°C with shaking for 12 h. Then, 100 μL of the overnight culture was transferred to an LB agar plate. After drying, 10 μL of the filtrate was added to the plate. After drying, the plate was inverted and incubated at 37°C for 12 h, and then the formation of plaques was observed. If clear plaques formed, it indicates that the filtrate contained bacteriophages capable of killing the host bacteria.

[0026] The obtained filtrate was serially diluted, and then 100 μL of the diluted filtrate and 100 μL of the overnight cultured host bacteria were thoroughly mixed. After incubating at room temperature for a period of time, the mixture was added to the preheated LB semi-solid medium. After mixing, the mixture was quickly poured onto LB solid medium to make a double-layer plate. After solidification, the plate was inverted and incubated in a 37°C incubator for 16-20 h to observe the growth of the phage plaques.

[0027] Example 2: Phage amplification and purification

[0028] On a double-layer plate where plaques have formed, pick a clear and transparent single plaque and place it in 5 mL of LB liquid medium. Simultaneously add 100 μL of host bacterial suspension containing the phage. Then, incubate the 5 mL LB liquid medium at 37°C in a shaker until the liquid gradually becomes clear. Centrifuge at 12,000 rpm for 10 min, collect the supernatant, and perform serial dilutions again. Repeat this process 2-3 times to purify single plaques.

[0029] Add 3 mL of host bacterial culture and 300 μL of phage culture to 300 mL of LB liquid medium, respectively. Incubate at 37 °C with shaking for 4–5 h, then centrifuge at 12,000 rpm for 10 min and collect the supernatant, which is the phage lysis buffer. Add 3 μL of DNase A and DNase I to the phage lysis buffer and incubate at room temperature for 30 min. Then add NaCl to a final concentration of 1 mol / L and mix thoroughly. After incubating on ice for 1–2 h, centrifuge at 10,000 rpm at 4 °C for 15 min and collect the supernatant. Add 30 g of PEG8000 to the supernatant and allow it to dissolve completely, then incubate on ice for another 12 h. Centrifuge at 12,000 rpm at 4 °C for 20 min. Finally, wash the precipitate thoroughly with 2 mL of SM solution and extract with an equal volume of chloroform to obtain purified phage.

[0030] The purified phage solution was serially diluted and plated in double-layer plates. After incubation at 37°C for 12 hours, plaque counting was performed on the double-layer plates. The plaques from the purified phage are shown below. Figure 1 As shown.

[0031] The purified bacteriophages PGPA20, PGPA60, PAB26, Lkpp-25, and Lkpp-36 were deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession numbers CCTCC NO: M20252375, M20252376, M20252414, M20252413, and M20252415, respectively.

[0032] Example 3: Transmission electron microscopy observation of bacteriophages PGPA20, PGPA60, PAB26, Lkpp-25 and Lkpp-36

[0033] The purified phage from Example 2 was observed under an electron microscope. The specific procedure was as follows: 10 μL of the purified phage was dropped onto a copper grid, and after precipitation for 15 minutes, excess liquid was absorbed with filter paper. Subsequently, it was stained with 2% phosphotungstic acid for 1-2 minutes, and after drying, it was observed using a transmission electron microscope (Hitachi H-7650). The observation results are as follows: Figure 2 As shown, the heads of PGPA20, PGPA60, PAB26, Lkpp-25, and Lkpp-36 all exhibit icosahedral shapes.

[0034] Example 4: Temperature stability determination of bacteriophages PGPA20, PGPA60, PAB26, Lkpp-25 and Lkpp-36

[0035] 1.5 mL of phages PGPA20, PGPA60, PAB26, Lkpp-25, and Lkpp-36 were respectively placed in sterile centrifuge tubes and then incubated at 4℃, 37℃, 50℃, 60℃, and 70℃, respectively. 200 μL samples were collected every 20 min until 80 min. The phage titers in different samples were determined by serially diluting the samples and plating them into double-layer plates. The results are as follows: Figure 3 As shown, within 80 minutes, the titers of bacteriophages PGPA20, PGPA60, PAB26, Lkpp-25, and Lkpp-36 remained relatively stable at or below 50°C. The titer of PGPA20 gradually decreased with increasing incubation time at 60°C, while the titers of PGPA60, PAB26, Lkpp-25, and Lkpp-36 remained relatively stable at 60°C. From 70°C onwards, the titers of all five bacteriophages gradually decreased with increasing incubation time, and even disappeared. This indicates that all five bacteriophages possess strong temperature tolerance.

[0036] Example 5: pH stability determination of bacteriophages PGPA20, PGPA60, PAB26, Lkpp-25 and Lkpp-36

[0037] 450 μL of SM solution with a pH of 2-13 was placed in a sterile centrifuge tube. Then, 50 μL of phages PGPA20, PGPA60, PAB26, Lkpp-25, and Lkpp-36 were added and vortexed to mix. The mixture was then incubated at 37°C for 1 h. Finally, the phage titers in different samples were determined by serial dilution and plating onto double-layer plates. The results are as follows: Figure 4 As shown, the bacteriophages PGPA20, PGPA60, PAB26, Lkpp-25, and Lkpp-36 maintained relatively stable bactericidal activity in environments with pH values ​​of 4, 5, 6, 7, 8, 9, 10, and 11. However, their activity decreased or even disappeared in acidic environments with pH values ​​below 4 or alkaline environments with pH values ​​above 11. This indicates that all five bacteriophages possess strong pH tolerance.

[0038] Example 6: Determination of the host spectrum of bacteriophages PGPA20, PGPA60, PAB26, Lkpp-25 and Lkpp-36

[0039] The titers of phages PGPA20, PGPA60, PAB26, Lkpp-25, and Lkpp-36 obtained in Example 2 were all adjusted to 10. 9 PFU / mL was prepared for use. Sixty-two strains of *Pseudomonas aeruginosa*, 52 strains of *Acinetobacter baumannii*, and 60 strains of *Klebsiella pneumoniae* were selected for testing. The host spectrum of *Pseudomonas aeruginosa* bacteriophages PGPA20 and PGPA60, *Acinetobacter baumannii* bacteriophage PAB26, and *Klebsiella pneumoniae* bacteriophages Lkpp-25 and Lkpp-36 were analyzed using plaque assays. The specific procedures are as follows:

[0040] Take 100 μL of the overnight culture of the test strain and spread it evenly on LB solid medium. Then add 10 μL of bacteriophages PGPA20, PGPA60, PAB26, Lkpp-25 and Lkpp-36 to the surface respectively, and incubate overnight at 37°C. Observe the results the next day. If there are empty plaques, mark it as "+" and otherwise as "-". Phage PGPA20 of *Pseudomonas aeruginosa* produced plaques on plates of 49 *P. aeruginosa* strains with a lysis rate of 79.03% (49 / 62) (Table 1); phage PGPA60 produced plaques on plates of 28 *P. aeruginosa* strains with a lysis rate of 45.16% (28 / 62) (Table 2); phage PAB26 produced plaques on plates of 22 *Acinetobacter baumannii* strains with a lysis rate of 42.31% (22 / 52) (Table 3); phage Lkpp-25 produced plaques on plates of 41 *Klebsiella pneumoniae* strains with a lysis rate of 68.33% (41 / 60) (Table 4); and phage Lkpp-36 produced plaques on plates of 22 *Klebsiella pneumoniae* strains with a lysis rate of 41.67% (25 / 60) (Table 5). Based on the principle of host spectrum complementarity, the combined use of Pseudomonas aeruginosa phages PGPA20 and PGPA60 achieved a lysis rate of 95.16% (59 / 62). Klebsiella pneumoniae phage Lkpp-25 can produce plaques on plates containing all K1 and some K2, K5, and K7 serotypes of Klebsiella pneumoniae; while phage Lkpp-36 exhibits higher specificity, producing plaques on plates containing all K2 and some K5 serotypes of Klebsiella pneumoniae. Combining Klebsiella pneumoniae phages Lkpp-25 and Lkpp-36 can simultaneously combat both K1 and K2 serotypes of Klebsiella pneumoniae. Therefore, preparing these five phages into a phage cocktail can significantly increase the bactericidal spectrum, maximally inhibiting or even eliminating Pseudomonas aeruginosa, Acinetobacter baumannii, and K1 / K2 Klebsiella pneumoniae.

[0041] Table 1 Host spectrum of phage PGPA20

[0042] serial number Bacterial name Hollow spot condition serial number Bacterial name Hollow spot condition 1 SJ-1 + 32 X-11 + 2 SJ-2 + 33 X-12 - 3 SJ-5 - 34 X-15 + 4 SJ-6 - 35 X-20 - 5 SJ-10 + 36 X-33 - 6 SJ-18 - 37 BPA01 + 7 SJ-22 + 38 BPA06 - 8 SJ-23 + 39 BPA14 + 9 SJ-24 + 40 BPA27 + 10 SJ-33 - 41 BPA31 + 11 SJ-37 - 42 BPA34 + 12 SJ-44 + 43 BPA37 + 13 SJ-46 + 44 BPA45 + 14 SJ-53 + 45 BPA49 + 15 SJ-54 + 46 Y-3 - 16 SJ-59 + 47 Y-5 + 17 SJ-60 + 48 Y-7 + 18 SJ-61 + 49 Y-12 - 19 SJ-65 - 50 Y-16 + 20 SJ-66 - 51 Y-17 + 21 SJ-75 + 52 Y-21 + 22 SJ-76 + 53 Y-22 + 23 PAO1 + 54 Y-23 + 24 S5 + 55 Y-24 + 25 S6 + 56 G-5 + 26 X-2 + 57 G-6 + 27 X-6 + 58 G-9 + 28 X-7 + 59 BPA64 + 29 X-8 + 60 BPA65 + 30 X-9 + 61 BPA61 + 31 X-10 + 62 BPA58 +

[0043] Table 2 Host spectrum of phage PGPA60

[0044] serial number Bacterial name Hollow spot condition serial number Bacterial name Hollow spot condition 1 SJ-1 + 32 X-11 + 2 SJ-2 + 33 X-12 + 3 SJ-5 + 34 X-15 - 4 SJ-6 + 35 X-20 - 5 SJ-10 - 36 X-33 + 6 SJ-18 + 37 BPA01 + 7 SJ-22 - 38 BPA06 - 8 SJ-23 + 39 BPA14 - 9 SJ-24 - 40 BPA27 - 10 SJ-33 + 41 BPA31 + 11 SJ-37 + 42 BPA34 + 12 SJ-44 - 43 BPA37 - 13 SJ-46 + 44 BPA45 - 14 SJ-53 - 45 BPA49 - 15 SJ-54 + 46 Y-3 - 16 SJ-59 - 47 Y-5 - 17 SJ-60 + 48 Y-7 - 18 SJ-61 - 49 Y-12 + 19 SJ-65 + 50 Y-16 - 20 SJ-66 + 51 Y-17 - 21 SJ-75 + 52 Y-21 - 22 SJ-76 - 53 Y-22 - 23 PAO1 + 54 Y-23 - 24 S5 + 55 Y-24 - 25 S6 + 56 G-5 - 26 X-2 - 57 G-6 - 27 X-6 + 58 G-9 - 28 X-7 - 59 BPA64 - 29 X-8 + 60 BPA65 - 30 X-9 + 61 BPA61 + 31 X-10 - 62 BPA58 -

[0045] Table 3 Host spectrum of phage PAB26

[0046] serial number Bacterial name Hollow spot condition serial number Bacterial name Hollow spot condition 1 AB-4 - 27 AB-Y54 + 2 AB-5 + 28 AB-Y58 - 3 AB-6 + 29 AB-Y69 + 4 AB-7 - 30 ATCC17978 + 5 AB-8 - 31 AB-Y64 - 6 AB-13 - 32 Ab-100 - 7 AB-14 - 33 Ab-101 - 8 AB-18 + 34 Ab-105 + 9 AB-19 - 35 Ab-127 + 10 AB-20 - 36 Ab-22(g) + 11 AB-21 + 37 AB-H1 + 12 AB-22 - 38 AB-H2 + 13 AB-23 - 39 AB-H3 + 14 AB-24 - 40 AB-H4 - 15 AB-25 - 41 AB-H5 - 16 AB26 + 42 AB-H6 + 17 AB-27 + 43 AB-H7 + 18 AB-28 - 44 AB-H8 + 19 AB-29 - 45 AB-H9 - 20 AB-30 - 46 AB-H10 - 21 AB-31 - 47 AB-H11 - 22 AB-32 + 48 AB-H12 - 23 AB-33 - 49 AB-H13 - 24 AB-34 - 50 AB-H14 - 25 AB-35 + 51 AB-38 + 26 AB-37 - 52 AB-Y28 +

[0047] Table 4 Host spectrum of bacteriophage Lkpp-25

[0048] serial number serotype Bacterial name Hollow spot condition serial number serotype Bacterial name Hollow spot condition 1 K1 kp10 + 31 K2 kpp27 + 2 K1 kpp14 + 32 K2 kpp41 + 3 K1 kpp15 + 33 K2 wkp5 + 4 K1 kpp22 + 34 K2 wkp17 + 5 K1 kpp33 + 35 K2 wkp77 + 6 K1 kpp40 + 36 K2 zkp57 - 7 K1 Lkp-10 + 37 K20 kpp8 - 8 K1 Lkp-11 + 38 K5 kp12 + 9 K1 Lkp-17 + 39 K5 kpp2 - 10 K1 Lkp-18 + 40 K5 kpp4 + 11 K1 Lkp-19 + 41 K5 kpp13 + 12 K1 Lkp-26 + 42 K5 kpp26 + 13 K1 Lkp-32 + 43 K5 kpp34 - 14 K1 Lkp-34 + 44 K5 kpp36 + 15 K1 Lkp-36 + 45 K5 kpp37 + 16 K1 Lkp-44 + 46 K5 kpp38 - 17 K2 kp1 + 47 K5 kpp43 + 18 K2 kp3 - 48 K5 kpp45 + 19 K2 kp13 + 49 K5 Lkp-8 - 20 K2 kp27 + 50 K5 wkp7 + 21 K2 kp30 + 51 K5 wkp22 + 22 K2 kp33 - 52 K5 zkp74 + 23 K2 kp56 - 53 K5 zkp85 + 24 K2 kp59 - 54 K54 kpp44 + 25 K2 kp60 - 55 K57 kpp10 - 26 K2 kp69 - 56 K57 kpp12 + 27 K2 kp79 - 57 K57 kpp17 - 28 K2 kp90 - 58 K57 kpp28 - 29 K2 kp100 - 59 K57 Lkp-1 - 30 K2 kpp6 + 60 K57 Lkp-2 +

[0049] Table 5 Host spectrum of bacteriophage Lkpp-36

[0050] serial number serotype Bacterial name Hollow spot condition serial number serotype Bacterial name Hollow spot condition 1 K1 kp10 - 31 K2 kpp27 + 2 K1 kpp14 - 32 K2 kpp41 + 3 K1 kpp15 - 33 K2 wkp5 + 4 K1 kpp22 - 34 K2 wkp17 + 5 K1 kpp33 - 35 K2 wkp77 + 6 K1 kpp40 - 36 K2 zkp57 + 7 K1 Lkp-10 - 37 K20 kpp8 - 8 K1 Lkp-11 - 38 K5 kp12 - 9 K1 Lkp-17 - 39 K5 kpp2 - 10 K1 Lkp-18 - 40 K5 kpp4 - 11 K1 Lkp-19 - 41 K5 kpp13 - 12 K1 Lkp-26 - 42 K5 kpp26 - 13 K1 Lkp-32 - 43 K5 kpp34 - 14 K1 Lkp-34 - 44 K5 kpp36 + 15 K1 Lkp-36 - 45 K5 kpp37 - 16 K1 Lkp-44 - 46 K5 kpp38 - 17 K2 kp1 + 47 K5 kpp43 - 18 K2 kp3 + 48 K5 kpp45 - 19 K2 kp13 + 49 K5 Lkp-8 - 20 K2 kp27 + 50 K5 wkp7 + 21 K2 kp30 + 51 K5 wkp22 + 22 K2 kp33 + 52 K5 zkp74 + 23 K2 kp56 + 53 K5 zkp85 + 24 K2 kp59 + 54 K54 kpp44 - 25 K2 kp60 + 55 K57 kpp10 - 26 K2 kp69 + 56 K57 kpp12 - 27 K2 kp79 + 57 K57 kpp17 - 28 K2 kp90 + 58 K57 kpp28 - 29 K2 kp100 + 59 K57 Lkp-1 - 30 K2 kpp6 + 60 K57 Lkp-2 -

[0051] Example 7: Determination of the antibacterial curves of bacteriophages PGPA20, PGPA60, PAB26, Lkpp-25, and Lkpp-36 and bacteriophage cocktails.

[0052] Pseudomonas aeruginosa BPA31, Acinetobacter baumannii AB26, and Klebsiella pneumoniae wkp17 were cultured to the logarithmic growth phase OD. 600 =0.6 for later use. First, add 100 μL of clean LB liquid medium to a 96-well plate, then add 50 μL of Pseudomonas aeruginosa BPA31, Acinetobacter baumannii AB26, and Klebsiella pneumoniae wkp17 in logarithmic growth phase, respectively. Then, according to MOIs of 10, 1, and 0.1, add 50 μL of phages PGPA20, PGPA60, PAB26, Lkpp-25, and Lkpp-36, respectively, along with a phage cocktail, and mix thoroughly. Incubate at 37°C for 12 h, and measure the OD every 1 h using a microplate reader. 600 The value of . For example Figure 5 As shown in A, Figure 5 When A is MOI=10, 1, and 0.1, the inhibition curves of PGPA20 and PGPA60 alone, compared with Pseudomonas aeruginosa BPA31 alone, show that the growth of Pseudomonas aeruginosa BPA31 was inhibited after the addition of phage PGPA20 and PGPA60, and the antibacterial effect of phage PGPA20 on BPA31 was more significant. BPA31 only began to show an upward trend after about 9 hours of co-culture. Figure 5 As shown in B, Figure 5 B represents the inhibition curves of PAB26 when MOI = 10, 1, and 0.1. Phage PAB26 can inhibit the growth of Acinetobacter baumannii AB26, and the antibacterial effect of phage PAB26 on AB26 is better with the increase of MOI. Figure 5 The results show that, Figure 5 When C was set to MOI = 10, 1, and 0.1, the inhibition curves of Lkpp-25 and Lkpp-36 alone showed that Lkpp-25 and Lkpp-36, when infected with Klebsiella pneumoniae wkp17 at MOI = 10, 1, and 0.1 ratios respectively, all exhibited inhibitory effects, and the overall antibacterial effects of the corresponding groups were similar. Subsequently, these five bacteriophages were used to construct a phage cocktail, and the antibacterial effects of the phage cocktail against Pseudomonas aeruginosa BPA31, Acinetobacter baumannii AB26, and Klebsiella pneumoniae wkp17 were determined. The results are as follows: Figure 5 As shown in D, Figure 5 D represents the inhibition curve of a phage cocktail composed of PGPA20, PGPA60, PAB26, Lkpp-25, and Lkpp-36. The phage cocktail significantly inhibited the growth of *Pseudomonas aeruginosa* and *Acinetobacter baumannii*. Although its inhibitory effect on *Klebsiella pneumoniae* was slightly weaker, it still inhibited the growth of *Klebsiella pneumoniae* overall.

[0053] Although the present invention has been described above with reference to embodiments, various modifications can be made and components can be replaced with equivalents without departing from the scope of the invention. In particular, as long as there is no structural conflict, the features in the disclosed embodiments can be combined with each other in any manner. The lack of an exhaustive description of these combinations in this specification is merely for the sake of brevity and resource conservation. Therefore, the present invention is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. A cocktail of bacteriophages, characterized in that, The phage cocktail comprises five strains of phages PGPA20, PGPA60, PAB26, Lkpp-25 and Lkpp-36 isolated from nature, wherein the phages PGPA20 and PGPA60 have been deposited with the China Center for Type Culture Collection on October 28, 2025, and the deposit numbers are CCTCC NO: M 20252375 and CCTCC NO: M 20252376, respectively; the phages PAB26, Lkpp-25 and Lkpp-36 have been deposited with the China Center for Type Culture Collection on November 3, 2025, and the deposit numbers are CCTCC NO: M 20252414, CCTCC NO: M 20252413 and CCTCC NO: M 20252415, respectively.

2. The phage cocktail of claim 1, characterized in that, The volume ratio of the phages PGPA20, PGPA60, PAB26, Lkpp-25 and Lkpp-36 in the phage cocktail is 1:1:1:1:

1.

3. Use of the phage cocktail according to any one of claims 1-2 as an active ingredient in the preparation of a pharmaceutical composition for preventing or treating infectious diseases caused by Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae.

4. Use according to claim 3, characterized in that, The phage cocktail is used for killing Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae.

5. Use according to claim 3, characterized in that, The pharmaceutical composition is a liquid preparation, a lyophilized preparation or an oral solid preparation.

6. Use of the phage cocktail according to claim 2 as an active ingredient in the preparation of a spray for killing Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae in animal breeding environments and medical environments.

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