Phage capable of simultaneously inhibiting K1 and K2 type klebsiella pneumoniae, depolymerases and application of phage and depolymerases

Abstract

The invention provides a bacteriophage capable of simultaneously and accurately inhibiting K1 and K2 type klebsiella pneumoniae, depolymerases and application, and relates to the technical field of biology, and the amino acid sequence of the bacteriophage depolymerases Depo81 is as shown in SEQ ID NO.1; the phage depolymerizing enzyme Depo81 is prepared from a klebsiella pneumoniae phage vBkpnPZB27 strain, and the preservation number of the vBkpnPZB27 strain is CCTCC (China Center for Type Culture Collection) NO: M20252087; the bacteriophage vBkpnPZB27 strain has the biological characteristic advantages of high value-added titer, safety, high efficiency and better tolerance to temperature and pH value. The bacteriophage depolymerases Depo81 provided by the invention are novel in sequence, have better acid-base and high-temperature tolerance, can synchronously remove K1 and K2 serotype klebsiella pneumoniae capsular polysaccharides, are expected to become novel antibacterial drug adjuvants for preventing and treating klebsiella pneumoniae infection, can be prepared into efficient and safe disinfection or cleaning preparations, and have broad application prospects. The method has a remarkable application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to bacteriophages, depolymerases, and applications that simultaneously inhibit K1 and K2 types of Klebsiella pneumoniae. Background Technology

[0002] Klebsiella pneumoniae ( Klebsiella pneumoniae Klebsiella pneumoniae, a Gram-negative opportunistic pathogen, is one of the main pathogens causing hospital-acquired and community-acquired infections. The capsular polysaccharides of K1 and K2 types possess a structure that strongly resists clearance by host phagocytes and inhibits complement system activation. Clinical data show that the prevalence of highly virulent K1 and K2 strains of Klebsiella pneumoniae is significantly higher in the Asia-Pacific region than in other parts of the world. K1 strains typically cause Friedlanderella pneumonia, with purulent liver abscesses being a prominent complication, while K2 strains typically cause community-acquired pneumonia. In recent years, K1 and K2 types of Klebsiella pneumoniae have exhibited multidrug resistance. These strains often carry resistance genes such as β-lactamases and carbapenemases via plasmids, leading to ineffective treatment with first-line antibiotics such as cephalosporins and carbapenems. Statistics show that the mortality rate from carbapenem-resistant strains is rising, and the development of traditional antibiotics lags far behind the spread of resistance genes, necessitating alternative treatment strategies.

[0003] Depolymerases are a class of enzymes that catalyze the breaking down of polymer molecular chains into smaller fragments or monomers. They are widely found in prokaryotes, eukaryotes, and microorganisms. Their core function is to participate in metabolism, biological defense, and environmental nutrient cycling by breaking glycosidic, phosphodiester, or peptide bonds in polymers. Bacteriophages, natural bacterial predators, can encode capsular polysaccharide depolymerases. By enzymatically breaking down the glycosidic bonds in bacterial capsules, they disrupt the physical barrier, allowing phages to adsorb onto and invade bacterial surfaces. Simultaneously, they enhance the host's immune cells' ability to clear bacteria. Due to their high host specificity, precise targeting, and lack of interference with normal flora, depolymerases have become important candidate tools for combating drug-resistant bacteria. Summary of the Invention

[0004] To prevent diseases or aquaculture problems caused by Klebsiella pneumoniae and to provide important candidate antibiotic alternatives / adjuvants against highly virulent drug-resistant Klebsiella pneumoniae, this invention provides bacteriophage vB_kpnP_ZB27, depolymerase Depo81, and their applications, which can simultaneously and precisely inhibit K1 and K2 types of Klebsiella pneumoniae. Depolymerase Depo81 has the advantages of high acid and alkali resistance and high thermal stability, making it convenient to store and use, with broad requirements for the use environment and high application value.

[0005] This invention proposes a bacteriophage strain that can simultaneously inhibit K1 and K2 types of Klebsiella pneumoniae. Klebsiella Phage vB_kpnP_ZB27, characterized by bacteriophage Klebsiella The accession number of the phage vB_kpnP_ZB27 strain is CCTCC M 20252087, the accession date is September 22, 2025, and the depositary institution is the China Center for Type Culture Collection.

[0006] The bacteriophages proposed in this invention Klebsiella Application of phage vB_kpnP_ZB27 in the preparation of antibacterial agents or feed additives that inhibit Klebsiella pneumoniae.

[0007] Preferably, the Klebsiella pneumoniae is K1 capsular type or K2 capsular type Klebsiella pneumoniae.

[0008] This invention proposes a phage depolymerization enzyme, Depo81, wherein the depolymerization enzyme Depo81 is the phage described in claim 1. Klebsiella The phage vB_kpnP_ZB27-derived protein can simultaneously inhibit K1 and K2 types of Klebsiella pneumoniae.

[0009] Preferably, the amino acid sequence of the phage depolymerase Depo81 is shown in SEQ ID NO.1.

[0010] A nucleic acid molecule encoding the aforementioned phage depolymerase Depo81, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0011] The application of the phage depolymerase Depo81 proposed in this invention in clearing the capsules of K1 or K2 capsular Klebsiella pneumoniae.

[0012] The present invention relates to the application of the phage depolymerase Depo81 in the preparation of antibacterial agents, bactericidal adjuvants, or feed additives that inhibit Klebsiella pneumoniae.

[0013] Preferably, the Klebsiella pneumoniae is K1 capsular type or K2 capsular type Klebsiella pneumoniae.

[0014] The present invention proposes the application of the above-mentioned phage depolymerase Depo81 and its sequence in serotyping, rapid identification of bacterial capsule types, auxiliary clinical diagnosis, and epidemiological investigation.

[0015] Compared with the prior art, the beneficial effects of the present invention are: (1) This invention has obtained a bacteriophage that can simultaneously and precisely inhibit K1 and K2 types of Klebsiella pneumoniae. Klebsiella Phage vB_kpnP_ZB27 and a depolymerase Depo81 that can simultaneously and precisely inhibit Klebsiella pneumoniae of types K1 and K2, this bacteriophage KlebsiellaPhage kpnP_ZB27 and depolymerase Depo81 can be used in environmental disinfection, animal husbandry, poultry farming, aquaculture or biomedical industry to prepare antibacterial agents or bactericidal adjuvants for Klebsiella pneumoniae, and can also be used to assist in clinical diagnosis and epidemiological investigation. (2) The depolymerase Depo81 of the present invention can specifically degrade and remove the capsular polysaccharides of K1 and K2 Klebsiella pneumoniae, which helps to prevent or treat diseases caused by K1 and K2 Klebsiella pneumoniae infection and avoid the development of drug resistance; and the depolymerase Depo81 of the present invention has the advantages of acid and alkali resistance and high thermal stability, which makes it convenient to store and use, has a wide range of requirements for the use environment, and has high application value. Attached Figure Description

[0016] Figure 1 This is the plaque morphology of the bacteriophage vB_kpnP_ZB27 proposed in this invention.

[0017] Figure 2 The optimal MOI detection result for the bacteriophage vB_kpnP_ZB27 proposed in this invention.

[0018] Figure 3 This is a schematic diagram of the one-step growth curve of the bacteriophage vB_kpnP_ZB27 proposed in this invention.

[0019] Figure 4 The results show the temperature stability of the bacteriophage vB_kpnP_ZB27 proposed in this invention.

[0020] Figure 5 The results show the acid-base stability of the bacteriophage vB_kpnP_ZB27 proposed in this invention.

[0021] Figure 6 This is a diagram showing the expression of the depolymerase Depo81 protein proposed in this invention.

[0022] Figure 7 The results show the stability of the depolymerase Depo81 proposed in this invention; where A represents pH stability and B represents temperature stability.

[0023] Figure 8 The diagram shows the effect of the depolymerase Depo81 proposed in this invention on inhibiting the capsular polysaccharide of Klebsiella pneumoniae.

[0024] Figure 9 This is a staining diagram showing the inhibition of Klebsiella pneumoniae capsular polysaccharide by the depolymerase Depo81 proposed in this invention. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0026] bacteriophage in this invention Klebsiella Phage vB_kpnP_ZB27 is deposited at the China Center for Type Culture Collection, Wuhan University, with accession number CCTCC M 20252087 and deposit date of September 22, 2025.

[0027] bacteriophages in Table 1 Klebsiella The strains in the phage vB_kpnP_ZB27 host spectrum are from our laboratory collection.

[0028] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available. Example 1 Isolation and purification of bacteriophage strain vB_kpnP_ZB27

[0029] Water samples were collected from a wastewater treatment plant in Anhui Province. The water sample was centrifuged at 10000g for 10 min, and the supernatant was sterilized by 0.45μm filtration. MgSO4 was added to a final concentration of 50mM, stirred thoroughly, and allowed to stand for 20 min. The mixture was then vacuum filtered, the supernatant was discarded, and the filter membrane was sonicated for 5 min to obtain the phage filtrate. 500 mL of the phage mixture, 500 mL of double-strength TSB medium (containing 2mM CaCl2 to a final concentration), and 50 mL of logarithmic-phase bacterial culture were mixed thoroughly and incubated at 37℃ with shaking for 8 h. The culture was centrifuged at 4000g for 20 min, and the phage lysate was filtered using a 0.45μm filter. 100 mL of Klebsiella pneumoniae culture in the logarithmic phase and 100 mL of phage lysate were added to 5 mL of 0.7% TSB-Ca soft agar, mixed thoroughly, and spread evenly on the surface of dry 1.5% TSB agar. After static incubation at 37℃ for 8 hours, clearly visible phage plaques were obtained. Clear and bright phage plaques were selected, desorbed by shaking in 1 mL of SM solution, and then sterilized by passing through a 0.22 μm filter membrane to obtain phage filtrate. This filtrate was inoculated into 5 mL of TSB liquid medium, and 500 μL of the corresponding host Klebsiella pneumoniae culture was added and mixed well. The mixture was incubated overnight at 37℃ with shaking at 200 rpm. After centrifugation at 6000 rpm for 10 min, the supernatant was passed through a 0.22 μm filter membrane to obtain phage filtrate. The morphology of the phage plaques was observed using the double-layer plate method. Repeating this process 3-5 times yielded phage plaques with consistent shape and size.

[0030] A strain of Klebsiella pneumoniae bacteriophage vB_kpnP_ZB27 was isolated from the collected water sample. Figure 1As shown, a distinct lysis zone surrounds the plaque, indicating that the phage strain vB_kpnP_ZB27 encodes a depolymerase. This phage was deposited at the China Center for Type Culture Collection on September 22, 2025, and is classified and named as: Klebsiella pneumoniae phage. Klebsiella Phage vB_kpnP_ZB27, Address: Wuhan University, Wuhan, China, Accession Number: CCTCC M 20252087. Example 2 Determination of the host spectrum of bacteriophage vB_kpnP_ZB27

[0031] Spread 1.5% TSB agar evenly onto a dry, sterile petri dish and allow it to dry. Add 100 mL of bacterial culture in the logarithmic growth phase to 5 mL of 0.4% TSA medium, mix well, spread evenly onto the dried plate, and allow it to air dry until the soft agar solidifies. Add 2 mL of phage culture to the soft agar using the spotting method, allow it to air dry, and then incubate at 37°C for 6 h. The lysis effect is divided into clear (+) and no plaques (-) in the spotting area, and the results are shown in Table 1. Host spectrum determination of vB_kpnP_ZB27 was performed using 45 bacterial strains. The results showed that phage vB_kpnP_ZB27 only had a targeted lysis effect on K1 and K2 capsular Klebsiella pneumoniae.

[0032] Table 1. Mechanism of action of bacteriophage and depolymerase Depo81 bacterial strains K type Phage / depolymerase activity bacterial strains K type Phage / depolymerase activity Kp176 K1 + Kp199 K54 - Kp177 K2 + Kp200 K5 - Kp178 K1 + Kp201 K31 - Kp179 K64 - Kp202 K1 + Kp180 K1 + Kp203 K57 - Kp181 K20 - Kp204 K80 - Kp182 NT - Kp205 K1 + Kp183 K30 - Kp206 NT - Kp184 K5 - Kp207 K2 + Kp185 K1 + Kp208 K27 - Kp186 K1 + Kp209 K27 - Kp187 K20 - Kp210 NT - Kp188 K2 + Kp211 K24 - Kp189 K64 - Kp212 NT - Kp190 K35 - Kp213 K38 - Kp191 K35 - Kp214 K57 - Kp192 K35 - Kp215 K64 - Kp193 K54 - Kp216 K2 + Kp194 K54 - Kp217 K1 + Kp195 K57 - Kp218 K26 - Kp196 K50 - Kp219 K5 - Kp197 K35 - Kp220 NT - Kp198 K57 In the table, "+" indicates that the phage and depolymerase Depo81 have depolymerization activity against the bacterial capsule, and "-" indicates that the phage and depolymerase Depo81 do not have depolymerization activity against the bacterial capsule. Example 3 Determination of the optimal multiple of infection of bacteriophage vB_kpnP_ZB27

[0033] The bacterial cells were cultured to the pre-log phase to achieve a bacterial concentration of 10. 8 CFU / mL, phage fluid and host bacterial culture were added at ratios of 1:1, 1:10, 1:100, 1:100, 1:1000, 1:10000, and 1:100000. The mixture was incubated at 37°C and 200 rpm for 4 h, centrifuged at 12000 rpm for 10-15 min, filtered through a 0.45 µm filter, and the titer was determined using the double-layer agar plate method.

[0034] like Figure 2 The optimal MOI for the bacteriophage vB_kpnP_ZB27 is 1:10000. Example 4 Determination of one-step growth curve of bacteriophage vB_kpnP_ZB27

[0035] The bacterial cells were cultured to the pre-log phase to achieve a bacterial concentration of 10. 8 CFU / mL was added to the host bacteria and phage culture medium at an optimal multiplicity of infection (MOI) of 1:10000, and the mixture was incubated at 37°C with shaking. Samples were taken at 0 min, every 5 min for the first 20 min, and every 10 min thereafter. The samples were centrifuged at 12000 rpm for 30 s, filtered through a 0.45 mm filter, and the phage titer was determined at each time point. A one-step growth curve was plotted with infection time on the x-axis and phage titer on the y-axis.

[0036] like Figure 3 As shown, the adsorption period of bacteriophage vB_kpnP_ZB27 is 0-10 min, the lysis period is 10-70 min, and the stationary phase is after 70 min. The one-step growth curve of this bacteriophage shows its strong proliferation ability: short latency, efficient lysis and release, ultimately achieving a high extracellular titer. Example 5 Stability of bacteriophage vB_kpnP_ZB27

[0037] Phage stability includes its tolerance to temperature and pH. In temperature tests, phage vB_kpnP_ZB27 was cultured for 1 hour at 20°C, 30°C, 40°C, 50°C, 60°C, and 70°C, respectively. To assess pH tolerance, 100 μL of phage suspension was mixed with 900 μL of LB medium at different pH values ​​(2-13) and incubated at 37°C for 1 hour. Phage titers were determined using the double-layer agar method.

[0038] like Figure 4 As shown, it exhibits good activity at temperatures ranging from 20℃ to 60℃, with a phage activity >10. 6 PFU / mL; such as Figure 5 As shown, bacteriophage vB_kpnP_ZB27 retains high activity within a pH range of 3-12, with bacteriophage activity >10. 5 PFU / mL. These results indicate that the bacteriophage vB_kpnP_ZB27 provided by this invention exhibits good tolerance to high temperatures and strong acids and alkalis. Example 6 Phage genome sequencing and analysis

[0039] Add DNase I and RNase A to 500 mL of the phage concentrate from Example 1, and incubate at 37°C for 60 min (inverting every 30 min). Add 10% SDS, proteinase K, and EDTA, and incubate at 65°C for 60 min (inverting every 30 min). Add an equal volume of Tris-saturated phenol, vortex for 30 s, centrifuge at 12000 g for 5 min, and transfer the supernatant to a new centrifuge tube. Add an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1), vortex for 30 s, centrifuge at 12000 g for 5 min, and transfer the supernatant to a new centrifuge tube. Extract with an equal volume of chloroform, vortex for 30 s, centrifuge at 12000 g for 5 min, and transfer the supernatant to a new centrifuge tube. Repeat this operation twice. Add an equal volume of isopropanol, mix well, incubate at -20°C for 30 min, centrifuge at 12000 g for 20 min at 4°C, and collect the precipitate. Wash the precipitate with 200 mL of 70% ethanol, centrifuge at 12000 g for 5 min, discard the supernatant, and retain the precipitate. Repeat twice. Dry the DNA precipitate at room temperature, add 20 mL of sterile water preheated to 65 °C, and store at -20 °C for later use.

[0040] Illumina sequencing results show that bacteriophage vB_kpnP_ZB27 is a linear, dsDNA bacteriophage. The complete genome sequence of bacteriophage vB_kpnP_ZB27 is 50486 bp in length, with a GC content of 50.61% and contains 82 coding sequences (CDS). Example 7 Cloning, expression, and purification of the depolymerase Depo81 gene from bacteriophage strain vB_kpnP_ZB27

[0041] The 2538 bp depolymerase gene predicted from Klebsiella pneumoniae phage strain vB_kpnP_ZB27 was amplified by PCR using the upstream primer sequence: 5'-CGGGATCCATGGCACTATACAGAGAAGGCAA-3' and the downstream primer sequence: 5'-CCCTCGAGTTACAGGGATATTTCCCACATAACT-3'. The PCR product was purified by restriction endonuclease digestion with BamHI and XhoI and ligated into the pET28a plasmid. Clones matching the expected size were identified and screened by agarose gel electrophoresis. The PCR amplification reaction system was 50 μL, including 25 μL of high-fidelity enzyme PrimeSTAR® HS, 17 μL of ddH2O, 2 μL of upstream primer F and 2 μL of downstream primer, and 4 μL of phage genome. The PCR amplification reaction program was 95℃ for 5 min, followed by 30 cycles of the following process: 95℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min; 72℃ for 10 min. After the recombinant plasmid was confirmed to be correct by DNA sequencing, it was transformed into E. coli DE3 to obtain DE3 / pET28a-Depo81.

[0042] DE3 / pET28a-Depo81 cells were inoculated into 200 mL of liquid LB medium containing 50 μg / mL kanamycin and cultured at 37 °C until the logarithmic growth phase. Isopropyl-β-D-thiogalactoside was added to a final concentration of 0.1 mM, and the cells were induced at 16 °C for 12–16 h. The bacterial culture was centrifuged at 10,000 g for 10 min at 4 °C, the supernatant was discarded, and the cells were resuspended in cell lysis buffer. The cells were then sonicated (120 W, 3 s on, 5 s off, 10 min total). The lysate was centrifuged at 12,000 g for 30 min at 4 °C, and the supernatant was collected.

[0043] The crude protein was purified using the Mag-Beads HIS fusion protein purification magnetic bead purification instructions to obtain purified phage depolymerase Depo81. Depo81 has a molecular weight of 90.4 kDa. The results are as follows... Figure 6 As shown. The concentration of phage depolymerase Depo81 was determined and stored at -80°C.

[0044] The target protein is phage depolymerase Depo81, the amino acid sequence of which is shown in SEQ ID NO.1; the nucleotide sequence encoding phage depolymerase Depo81 is shown in SEQ ID NO.2. Example 8 Validation of the activity of phage depolymerase Depo81

[0045] The activity of the phage depolymerase Depo81 was detected using a dot assay. The specific steps are as follows: 45 plants were selected Klebsiella Pneumoniae The strain underwent host range testing. Klebsiella Pneumoniae After the bacteria have reached the logarithmic growth phase, mix them thoroughly with 0.4% LB agar at approximately 50°C and spread them evenly on a 1.5% agar solid plate. After drying, add 5 μL of serially diluted depolymerase protein solution to each of the two-layer plates and incubate at 37°C, observing the appearance of transparent halos.

[0046] The enzymatic degradation profile of depolymerase Depo81 is shown in Table 1. The formation of plaques by phage depolymerase Depo81 against Klebsiella pneumoniae strains indicates that Depo81 has depolymerization activity against the bacterial capsule. Conversely, the absence of plaques indicates that Depo81 has no depolymerization activity against the bacterial capsule. The results show that depolymerase Depo81 exhibits depolymerization activity against all tested K1 and K2 serotypes of Klebsiella pneumoniae. Example 9 Temperature and pH stability of depolymerase Depo81

[0047] Temperature tolerance: The temperature stability of the depolymerase was assessed using the double-layer agar plate observation method. The depolymerase was aliquoted into 1.5 mL centrifuge tubes and treated at 20, 30, 40, 50, 60, and 70 °C for one hour. Subsequently, 3 μL of depolymerase at concentrations of 1, 10, and 100 μg / mL were added to a solution containing approximately 10 μg / mL of Klebsiella pneumoniae. 8 The samples were placed on a double-layer plate (CFU / mL), dried, and incubated at 37°C for 8 hours. The clear zone was then observed. The results are as follows: Figure 7 As shown in Part B.

[0048] Acid-base tolerance: Adjust PBS to the appropriate pH (2, 3, 4, 6, 7, 8, 9, 10, 11, 12), filter sterilize using a filter head, add 100 μL of diluted enzyme solution to 900 μL of PBS at different pH values ​​and treat for one hour. Then, drop 3 μL of depolymerase at concentrations of 1, 10, and 100 μg / mL into a solution containing approximately 100 μg / mL of Klebsiella pneumoniae. 8 The samples were placed on a double-layer plate (CFU / mL), dried, and incubated at 37°C for 8 hours. The clear zone was then observed. The results are as follows: Figure 7 As shown in Part A.

[0049] like Figure 7 As shown, the presence of a translucent halo indicates that the phage depolymerase Depo81 exhibits good activity at pH 2-12 and at temperatures of 20℃-50℃, with activity decreasing at 60℃ and ceasing at 70℃. These results demonstrate that the depolymerase Depo81 provided by this invention has good tolerance to high temperatures and strong acids and alkalis. Example 10 Phage depolymerase Depo81 inhibits Klebsiella pneumoniae capsular polysaccharide

[0050] Take 3 ml of strains Kp 202 (K1 type) and Kp 177 (K2 type) cultured to the logarithmic growth phase, centrifuge at 5000 rpm for 5 min, collect the precipitate into a 1.5 mL tube, wash three times with 1 mL sterile PBS and resuspend. Add 50 μg of Depo81 to the treatment group and an equal volume of sterile PBS to the control group. Incubate at 37℃ with shaking for 1 h, then centrifuge again to observe the precipitate. Results are as follows: Figure 8 As shown, the colony precipitates in the untreated group were light and loose with a large area; while the colony precipitates treated with depolymerase were firm and dense with a small area, indicating that the phage depolymerase Depo81 can degrade the capsular polysaccharide of this strain.

[0051] Further, a suitable amount of colony precipitate was picked up with a sterile inoculation loop and transferred to a 1.5 mL tube, then resuspended in a suitable amount of sterile water. The treatment group was given 50 μg of Depo81, and the control group was given an equal amount of sterile water. After incubation at 37°C for 1 hour, a small amount of bacterial suspension was smeared and air-dried. Capsule staining solution A (5 g / L carbocarbonate fuchsin, preheated in a 37°C water bath for 30 minutes beforehand) was used for staining for 3 minutes, followed by rinsing with water. Then, solution B (a mixture of 3 g / L ferric chloride, 15 g / L tannic acid, and 20 g / L potassium aluminum sulfate in a 5:2:2 ratio) was used for staining for 3 minutes, followed by rinsing with water. Finally, solution C (methylene blue ethanol saturated solution) was used for staining for 30 seconds, followed by rinsing with water, air-drying, and observation under an oil immersion microscope. Results are as follows: Figure 9 As shown, the bacterial capsules in the untreated group were clearly stained; while the outer ring of the bacteria treated with depolymerase showed almost no capsule secretion, indicating that the phage depolymerase Depo81 can degrade the capsular polysaccharide of this strain.

[0052] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Although embodiments of this application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of this application. The scope of this application is defined by the appended claims and their equivalents, all of which should be included within the protection scope of this application.

Claims

1. A bacteriophage that can simultaneously inhibit K1 and K2 types of Klebsiella pneumoniae Klebsiella phage vB_kpnP_ZB27, characterized in that, bacteriophage Klebsiella The accession number of the phage vB_kpnP_ZB27 strain is CCTCC M20252087, the deposit date is September 22, 2025, and the depositary institution is the China Center for Type Culture Collection.

2. The phage as described in claim 1 Klebsiella Application of phage vB_kpnP_ZB27 in the preparation of antibacterial agents or feed additives that inhibit Klebsiella pneumoniae.

3. The bacteriophage according to claim 2 Klebsiella The application of phage vB_kpnP_ZB27 in the preparation of antibacterial agents or feed additives for inhibiting Klebsiella pneumoniae is characterized by, The Klebsiella pneumoniae mentioned is either K1 capsular type or K2 capsular type Klebsiella pneumoniae.

4. A phage depolymerase, Depo81, characterized in that, Depolymerase Depo81 is the phage described in claim 1. Klebsiella The phage vB_kpnP_ZB27-derived protein can simultaneously inhibit K1 and K2 types of Klebsiella pneumoniae.

5. The phage depolymerase Depo81 according to claim 4, characterized in that, The amino acid sequence of the phage depolymerase Depo81 is shown in SEQ ID NO.

1.

6. A nucleic acid molecule encoding the phage depolymerase Depo81 as described in claim 4 or 5, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

2.

7. The use of the phage depolymerase Depo81 as described in claim 4 or 5 in clearing the capsules of K1 or K2 capsular Klebsiella pneumoniae.

8. The use of the phage depolymerase Depo81 as described in claim 4 or 5 in the preparation of a bacteriostatic agent, bactericidal adjuvant, or feed additive for inhibiting Klebsiella pneumoniae.

9. The application of the phage depolymerase Depo81 according to claim 8 in the preparation of a bacteriostatic agent, bactericidal adjuvant, or feed additive for inhibiting Klebsiella pneumoniae, characterized in that, The Klebsiella pneumoniae mentioned is either K1 capsular type or K2 capsular type Klebsiella pneumoniae.

10. The application of the phage depolymerase Depo81 and its sequence as described in claim 4 or 5 in serotyping, rapid identification of bacterial capsule types, and auxiliary clinical diagnosis and epidemiological investigation.

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