Acinetobacter baumannii bacteriophage, depolymerase, preparation method and application

Abstract

This invention relates to the field of phage technology, specifically disclosing an Acinetobacter baumannii phage, a depolymerase, its preparation method, and its applications. The Acinetobacter baumannii phage P919 provided by this invention exhibits strong specific lytic ability against Acinetobacter baumannii type KL3, and its encoded depolymerase possesses excellent temperature stability below 70°C, effectively degrading the capsular polysaccharide of Acinetobacter baumannii type KL3.

Description

Technical Field

[0001] This invention relates to the field of phage technology, specifically to an Acinetobacter baumannii phage, a depolymerase, its preparation method, and its application. Background Technology

[0002] Acinetobacter baumannii is widely distributed in the natural environment and is an opportunistic pathogen that can infect humans, various aquatic animals, livestock, poultry, and wild animals, posing a serious threat to public health. This pathogen is a major cause of hospital-acquired infections in humans, and can cause pneumonia, bacteremia, endocarditis, skin and soft tissue infections, urinary tract infections, and meningitis, among other things.

[0003] The main challenge in treating Acinetobacter baumannii infection lies in its multidrug resistance and numerous capsule types. In recent years, this pathogen has developed widespread resistance to most first-line antibiotics used in clinical practice. 70% of these are carbapenem-resistant Acinetobacter baumannii (CRAB), and carbapenems are considered the last line of defense against Gram-negative bacteria. The mortality rate of hospital-acquired pneumonia and bloodstream infections caused by CRAB has been reported to be close to 70%. Currently used antimicrobial agents for CRAB (such as polymyxins and tigecycline) are only used as a last resort due to their pharmacokinetic properties and toxicity. One of the key virulence factors of Acinetobacter baumannii is capsular polysaccharides (CPS), which play a crucial role in evading the host immune system, resisting desiccation, and enhancing drug resistance. Therefore, CPS is also a key target for the treatment of Acinetobacter baumannii infection. Currently, at least 237 k-site reference sequences have been identified and more than 65 k-types have been assigned. Among them, the KL3, KL2, KL9, KL13, and KL49 capsule types are the most common.

[0004] Carbapenem-resistant Acinetobacter baumannii lacks effective treatment options due to severe antibiotic resistance. There is a need to develop a product that specifically degrades the capsule to stimulate the immune system to kill Acinetobacter baumannii type KL3. Summary of the Invention

[0005] To develop a product that specifically degrades the capsule to promote the immune system's eradication of Acinetobacter baumannii type KL3, this invention provides an Acinetobacter baumannii phage, a depolymerase, its preparation method, and its applications. The Acinetobacter baumannii phage P919 provided by this invention exhibits strong specific lytic ability against Acinetobacter baumannii type KL3, and its encoded depolymerase possesses excellent temperature stability below 70°C, effectively degrading the capsular polysaccharides of Acinetobacter baumannii type KL3.

[0006] This invention provides a bacteriophage of Acinetobacter baumannii ( AcinetobacterbaumanniiAcinetobacter baumannii phage P919 was deposited at the China Center for Type Culture Collection on October 11, 2025, with accession number CCTCC NO: M 20252192.

[0007] The Acinetobacter baumannii phage P919 provided by this invention has a strong specific lysis ability against Acinetobacter baumannii KL3. Its encoded depolymerase has excellent temperature stability below 70°C, which can effectively degrade the capsular polysaccharide of Acinetobacter baumannii KL3 and promote the phagocytosis of Acinetobacter baumannii KL3 by macrophages.

[0008] The present invention also provides a phage depolymerase gene, isolated from Acinetobacter baumannii phage P919, the sequence of which is shown in SEQ ID NO.1.

[0009] The present invention also provides a phage depolymerase P919DPO, encoded by the phage depolymerase gene, the amino acid sequence of which is shown in SEQ ID NO.2.

[0010] This invention also provides a method for preparing phage depolymerase P919DPO, comprising the following steps:

[0011] The phage depolymerase P919DPO gene fragment shown in SEQ ID NO.1 was cloned, and the P919DPO gene fragment was ligated into the pET28a plasmid vector to obtain a recombinant plasmid. The recombinant plasmid was transferred into host cells, and recombinant host cells containing the recombinant plasmid were screened. The recombinant host cells were cultured to obtain a culture medium. The culture medium was separated into solid and liquid phases, and the liquid was collected to obtain the phage depolymerase P919DPO.

[0012] Furthermore, the host cell is Escherichia coli BL21(DE3).

[0013] Furthermore, the cultivation conditions are: 37℃~38℃, 180rpm~200rpm.

[0014] Furthermore, the phage depolymerase P919DPO gene fragment shown in SEQ ID NO.1 was amplified using phage P919 genomic DNA as a template and SEQ ID NO.3 to SEQ ID NO.4 as primers.

[0015] The present invention also provides the application of Acinetobacter baumannii phage P919 or phage depolymerase P919DPO in the preparation of Acinetobacter baumannii KL3 disinfection products, wherein the Acinetobacter baumannii phage P919 or phage depolymerase P919DPO is used to specifically degrade the capsular polysaccharide of Acinetobacter baumannii KL3.

[0016] Furthermore, the Acinetobacter baumannii phage P919 or the phage depolymerase P919DPO promotes phagocytosis by macrophages by degrading the capsular polysaccharide of Acinetobacter baumannii type KL3.

[0017] Furthermore, the disinfection product is a phage depolymerase P919DPO solution with a concentration of 1.5 μg / mL to 150 μg / mL, and the solvent is PBS.

[0018] This invention also provides the application of bacteriophage strain P919 or bacteriophage depolymerase P919DPO in the preparation of drugs that degrade the capsular polysaccharide of Acinetobacter baumannii type KL3.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] This Acinetobacter baumannii phage, P919, exhibits strong specific lytic activity against Acinetobacter baumannii type KL3. It can be used alone or in combination with other substances, providing a safe and non-toxic phage disinfection product for environmental disinfection and purification. The depolymerase possesses excellent temperature stability below 70℃ and can effectively degrade the capsular polysaccharide of Acinetobacter baumannii type KL3; it can also effectively enhance the body's immune clearance function, demonstrating great potential as a novel antibacterial agent.

[0021] Information on the Preservation of Biological Materials

[0022] P919, referred to in this invention as Acinetobacter baumannii phage P919, was deposited on October 11, 2025, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20252192. The address of the depository is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, 430072, China. Acinetobacterbaumannii phage P919. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. 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.

[0024] Figure 1 This is a P919 phage plaque.

[0025] Figure 2 This is a transmission electron microscope image of P919 negative staining.

[0026] Figure 3 This is a circled diagram of the bacteriophage genome.

[0027] Figure 4 The 3D structure of the trimer of the depolymerase P919DPO is shown.

[0028] Figure 5 This is a gel image after purification of the depolymerase P919DPO.

[0029] Figure 6 To detect the activity of depolymerase P919DPO on capsular polysaccharides;

[0030] In the figure, a shows the morphology of the phage depolymerase P919DPO after treatment for 12 h at concentrations of 150 μg / mL, 15 μg / mL, 1.5 μg / mL, 0.15 μg / mL and 0.015 μg / mL, and PBS is the negative control.

[0031] b shows the morphology of the phage depolymerase P919DPO after treatment for 24 h at concentrations of 150 μg / mL, 15 μg / mL, 1.5 μg / mL, 0.15 μg / mL and 0.015 μg / mL. PBS is the negative control.

[0032] c represents the morphology of the phage depolymerase P919DPO after treatment for 36 h at concentrations of 150 μg / mL, 15 μg / mL, 1.5 μg / mL, 0.15 μg / mL, and 0.015 μg / mL. PBS serves as the negative control.

[0033] Figure 7 Temperature stability experiment of phage depolymerase P919DPO;

[0034] In the figure, a represents the degradation of capsular polysaccharides by phage depolymerase after treatment at 25℃, 70℃, and 80℃ for 1 hour;

[0035] b represents the degradation of capsular polysaccharides by phage depolymerase after treatment at 25℃, 70℃, and 80℃ for 2 hours;

[0036] c represents the degradation of capsular polysaccharides by phage depolymerases after treatment at 25℃, 70℃, and 80℃ for 3 hours.

[0037] Figure 8 The effect of phage depolymerase P919DPO on the phagocytic activity of THP-1 cells;

[0038] In the figure, A represents the effect of phage depolymerase P919DPO on the phagocytosis of non-myxotropic strain ZWAb014 by THP-1 cells;

[0039] B represents the effect of phage depolymerase P919DPO on the phagocytosis of non-myxotropic strain ZWAb024 by THP-1 cells. Detailed Implementation

[0040] 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. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative 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, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0041] Example 1: A bacteriophage strain P919, bacteriophage depolymerase and its application.

[0042] I. Experimental Materials and Methods

[0043] 1. Main reagents and instruments:

[0044] Transmission electron microscopy (TEM) was performed using a Hitachi H-7650 microscope. PBS was purchased from Beijing Solarbio Science & Technology Co., Ltd., and its composition was 8 mM Na₂HPO₄, 137 mM NaCl, and 2 mM NaH₂PO₄, with a pH of 7.4. The rapid extraction kit for λ phage genomic DNA was purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd.

[0045] 2. Plasmids and bacterial samples:

[0046] The pET28a plasmid was derived from Beijing Qingke Biotechnology Co., Ltd. The *Escherichia coli* BL21(DE3) strain was also derived from Beijing Qingke Biotechnology Co., Ltd.

[0047] 3. Materials:

[0048] The wastewater samples used in this invention were obtained from Zhangjiagang City Hospital. The THP-1 cell line was preserved in our laboratory.

[0049] 4. Phage isolation and identification

[0050] The bacteriophage P919 was isolated from medical wastewater at Zhangjiagang City Hospital using co-culture and double-layer agar methods, with KL3 capsular Acinetobacter baumannii ZWAb014 as the host bacterium, and is preserved at the Jiangsu Provincial Center for Disease Control and Prevention. The specific steps include the following:

[0051] Preparation of Acinetobacter baumannii strain ZWAb014: A single colony of Acinetobacter baumannii ZWAb014 was picked from an LB agar plate and inoculated into 5 mL of LB broth medium. The culture was then incubated at 37°C and 180 rpm until the logarithmic growth phase, i.e., OD600≈0.6, and set aside for later use.

[0052] A 10 mL wastewater sample collected from Zhangjiagang City Hospital was centrifuged at 5000g for 5 minutes. The supernatant was then filtered through a 0.22 μm filter membrane, and the wastewater sample filtrate was collected. 100 μL of the wastewater sample filtrate was added to 100 μL of Acinetobacter baumannii ZWAb014 bacterial suspension and mixed thoroughly before being poured into LB semi-solid medium. After the medium solidified, the plates were inverted and incubated overnight at 37°C for 12 hours. After 12 hours, a single plaque was picked and placed in 1 mL of 1×PBS, vortexed, and diluted 10⁻⁶ times. 5 The dilution was increased by 100 μL. Then, 100 μL of the dilution and 100 μL of Acinetobacter baumannii ZWAb014 bacterial suspension were used to obtain single phage plaques using the double-layer agar plate method to purify the phage. This operation was repeated 5 times. The purification was considered successful if the morphology and size of the phage plaques on the double-layer agar plates were consistent. A single phage plaque was picked from the successfully purified double-layer agar plate and placed in 1 mL of 1×PBS. After shaking and mixing, 100 μL of phage suspension and 100 μL of Acinetobacter baumannii strain ZWAb014 bacterial suspension were used to perform the double-layer agar plate method. The plates were incubated at 37°C for 8 hours. Then, 5 mL of LB broth was added to the plates, and the plates were incubated overnight at 4°C and shaken at 80 rpm for 2 hours. After shaking, the liquid was collected and filtered through a 0.22 μm filter membrane. The purified phage was stored in LB broth at 4°C to obtain the phage P919 enrichment solution.

[0053] Acinetobacter baumannii ZWAb014 was inoculated into LB liquid medium and cultured at 37°C with shaking at 180 rpm for 12 hours to obtain Acinetobacter baumannii ZWAb014 culture. 100 μL of Acinetobacter baumannii ZWAb014 culture and 100 μL of phage P919 enrichment solution were added to LB semi-solid medium, thoroughly mixed, and then poured into LB solid plates. After solidification, the plates were inverted and incubated at 37°C for 24 hours. The plaque formation of phage strain P919 was observed and recorded.

[0054] The results are as follows Figure 1 As shown, Acinetobacter baumannii phage P919 forms plaques on the bacterial moss, and the presence of halos around the plaques indicates that Acinetobacter baumannii phage P919 encodes a depolymerase.

[0055] The purified Acinetobacter baumannii phage P919 was observed under an electron microscope. The specific procedure was as follows: 10 μL of phage strain P919 was added to a copper grid and allowed to precipitate for 5 minutes. Excess liquid was absorbed with filter paper, and the sample was stained with 2% (v / v) phosphotungstic acid for 90 seconds. After drying, it was observed under a transmission electron microscope. Phosphotungstic acid is abbreviated as PTA.

[0056] Transmission electron microscopy results as follows Figure 2As shown, Acinetobacter baumannii phage P919 has a polyhedral head with a diameter of approximately 63±1 nm, a tail sheath diameter of approximately 70±1 nm, and a neck diameter of approximately 12±1 nm between the head and tail sheath. Its tail filaments are faintly visible in transmission electron microscopy images. Morphologically, P919 belongs to myotail phage.

[0057] 5. Lysis of Acinetobacter baumannii phage P919 on Acinetobacter baumannii with different capsule types.

[0058] The 36 Acinetobacter baumannii strains were identified as follows: ZWAb001–ZWAb006, ZWAb013–ZWAb020, ZWAb023–ZWAb026, ZWAb028, ZWAb089–ZWAb098, ZWAb113–ZWAb115, ZWAb117–ZWAb118, and ZWAb121–ZWAb123. ZWAb001–ZWAb006 and ZWAb013–ZWAb020 were all sourced from the Jiangsu Provincial Center for Disease Control and Prevention. ZWAb023–ZWAb026 and ZWAb028 were all sourced from the Second Affiliated Hospital of Soochow University. ZWAb089–ZWAb098 were all sourced from the Second People's Hospital of Nanjing. ZWAb113~ZWAb115, ZWAb117~ZWAb118 and ZWAb121~ZWAb123 were all provided by Zhangjiagang First People's Hospital.

[0059] All 36 strains of Acinetobacter baumannii mentioned above are documented in the applicant's published articles, the details of which are as follows:

[0060] Zheng Sinica,2024,39(5):767-781.DOI:10.1016 / J.VIRS.2024.08.002.

[0061] The applicant needs to declare that all Acinetobacter baumannii strains listed in Table 1 of this invention will be made available to the public free of charge for twenty years.

[0062] The above 36 Acinetobacter baumannii strains were cultured overnight at 37°C to obtain bacterial cultures. 100 μL of each bacterial culture was mixed with 100 μL of phage P919 enrichment solution and transferred to LB semi-solid plates. After thorough mixing, the mixture was poured into LB solid plates, inverted, and incubated overnight at 37°C. The lysis of Acinetobacter baumannii strains by phage strain P919 was observed. Overnight incubation refers to a culture time of 12 hours.

[0063] Table 1. Lysis of Acinetobacter baumannii with different capsule types by phage strain P919

[0064]

[0065]

[0066]

[0067] Note: Phage action spectrum, + indicates lysis, - indicates non-lysis.

[0068] The information on the 36 Acinetobacter baumannii strains and the lysis profile of bacteriophage strain P919 are shown in Table 1. The results showed that the bacteria lysed by bacteriophage strain P919 all belonged to the KL3 serotype; other serotypes could not be lysed. Therefore, bacteriophage strain P919 possesses the function of specifically lysing KL3 type Acinetobacter baumannii.

[0069] 6. Phage genome analysis and depolymerase identification

[0070] Following the instructions for use of the λ phage genomic DNA rapid extraction kit, whole-genome sequencing was performed at Beijing Novogene Technology Co., Ltd. The phage genome was annotated using Pharrokka, and depolymerase prediction was performed using DePolymerase Predictor software. Proteins with scores greater than 95 were selected for recombinant expression validation. Furthermore, phage depolymerases typically function and are active as trimers with continuous β-sheets. Therefore, alphafold3 was used to predict their structure. The presence or absence of virulence genes was detected using a pathogenicity factor database, and antibiotic resistance genes were detected through comparative analysis using a comprehensive antibiotic resistance database.

[0071] The genome sequencing analysis results of bacteriophage P919 are as follows: Figure 3 As shown, genome sequencing analysis of bacteriophage P919 revealed the absence of virulence factors, antibiotic resistance genes, and lysogenic genes. Based on the latest classification standards of the International Committee on Taxonomy of Viruses, bacteriophage P919 belongs to the genus Oblonenskvirus. The protein P919DPO (P919_CDS0076) encoded by the bacteriophage was identified as a depolymerase. Its three-dimensional structure is shown below. Figure 4 As shown.

[0072] The virulence factor database is abbreviated as VFDB. The comprehensive drug resistance database is abbreviated as CARD.

[0073] 7. Prokaryotic expression of depolymerase P919DPO

[0074] Using phage P919 genomic DNA as a template, the phage RBP gene was amplified by polymerase chain reaction (PCR) using 2×PhantaMax Master Mix DNA polymerase. The primer pair was: upstream primer F shown in SEQ ID NO.3 and downstream primer R shown in SEQ ID NO.4. The PCR amplification program was 95°C for 5 minutes, followed by 35 cycles of amplification, including 95°C for 15 seconds, 50°C for 15 seconds, and 72°C for 1 minute. The PCR amplification product was directionally inserted into the expression vector pET-28a using the ClonExpress II One Step Cloning Kit. Bam HI and Hin The recombinant vector was obtained from the dIII restriction enzyme site. The recombinant vector was then transferred to *E. coli* DH5α via chemical transformation for proliferation. After Sanger sequencing confirmed the absence of any mutations in the recombinant vector, plasmids were extracted from *E. coli* DH5α and transformed into *E. coli* BL21(DE3) for protein expression.

[0075] SEQ ID NO.3:

[0076] cagcaaatgggtcgcggatccATGACTAATCCAACACTTATTACAACCC;

[0077] SEQ ID NO.4:

[0078] ctcgagtgcggccgcaagcttTTAAATATTGTAGTACGGAATCTTACGAGG.

[0079] Escherichia coli BL21(DE3) containing the expression vector was inoculated into LB broth supplemented with kanamycin at a final concentration of 50 μg / ml and cultured at 37°C and 180 rpm until OD200. 600When the value reached 0.6, 1 mM isopropyl β-d-thiogalactoside was added to a final concentration, and the mixture was then incubated at 16°C and 120 rpm for 12 hours to induce expression. After induction, the bacterial pellet was collected by centrifugation and washed three times with 1X PBS. The bacterial pellet was resuspended in binding buffer and sonicated on ice to obtain cell lysate. The cell lysate was centrifuged at 10000g for 20 minutes at 4°C to remove insoluble impurities. Finally, protein purification was performed using HisGrip Elite according to the manufacturer's instructions. The concentration of purified protein was determined using a BCA protein assay kit. Protein separation was performed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the purification effect was assessed by Fast Blue Protein Staining Solution staining.

[0080] The PCR amplification reaction system constructed from the P919DPO prokaryotic expression vector of depolymerase was 50 μL, including 25 μL of high-fidelity enzyme 2×Phanta Flash Master Mix, 17 μL of ddH2O, 2 μL of upstream primer F and 2 μL of downstream primer R, and 4 μL of phage P919 genome.

[0081] The PCR amplification reaction program was 95℃ for 5 minutes, followed by 30 cycles of the following process: 95℃ for 15 seconds, 55℃ for 15 seconds, 72℃ for 2 minutes; 72℃ for 10 minutes.

[0082] The conditions for ultrasonic disruption are: 3 seconds of operation, 10 seconds of rest, 60% power, a total disruption time of 15 minutes, and an ice bath throughout the process.

[0083] The gene sequence encoding the phage depolymerase P919DPO is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by this gene is shown in SEQ ID NO.2.

[0084] SEQ ID NO.1:

[0085]

[0086] amino acid sequence:

[0087] SEQ ID NO.2:

[0088] .

[0089] 8. Activity of depolymerase P919DPO in degrading capsular polysaccharides

[0090] The dot assay was used to detect the activity of phage depolymerase P919DPO against KL3 capsular polysaccharide. The specific steps are as follows:

[0091] Acinetobacter baumannii bacteria were grown overnight at 37°C for 12 hours to obtain a culture. 200 μL of the culture was added to LB semi-solid medium, thoroughly mixed, and then poured into LB solid plates. After air drying, 10 μL of phage depolymerase P919DPO at concentrations of 150 μg / mL, 15 μg / mL, 1.5 μg / mL, 0.15 μg / mL, and 0.015 μg / mL were added to bilayer plates and incubated at 37°C for 36 hours. The morphology of the depolymerase was recorded at 12, 24, and 36 hours. PBS buffer was used as a negative control. The phage depolymerase P919DPO at concentrations of 150 μg / mL, 15 μg / mL, 1.5 μg / mL, 0.15 μg / mL, and 0.015 μg / mL was obtained by dissolving and diluting with PBS at pH 7.4.

[0092] The results are as follows Figure 6 As shown, the depolymerase P919DPO forms a translucent halo on the bacterial moss of KL3 capsular Acinetobacter baumannii ZWAb014. Furthermore, the halo gradually enlarges over time. This indicates that the depolymerase P919DPO possesses degradative activity against KL3 capsular polysaccharides.

[0093] 9. Study on the action spectrum of depolymerase P919DPO

[0094] The dot matrix assay was used to detect the action spectrum of phage depolymerase P919DPO. The specific steps are as follows:

[0095] Thirty-six different KL-type Acinetobacter baumannii bacteria were grown overnight at 37°C for 12 hours. 200 μL of the culture was transferred to LB semi-solid medium, thoroughly mixed, and then poured into LB solid plates. After air-drying, 10 μL of phage depolymerase P919DPO at a concentration of 150 μg / mL was added to a double-layer plate, and the plates were incubated at 37°C for 24 hours. The effect of the depolymerase was recorded.

[0096]

[0097] Note: "+" indicates that the phage depolymerase P919DPO has depolymerization activity against the bacterial capsule, and "-" indicates that the phage depolymerase P919DPO does not have depolymerization activity against the bacterial capsule.

[0098] The results are shown in Table 2. The depolymerase exhibited a broader spectrum of action than bacteriophages, and was also effective against ZWAb024 and ZWAb025, which are not lysed by bacteriophages.

[0099] 10. Temperature stability of depolymerase

[0100] A dot-matrix assay was used to detect the temperature stability of capsular polysaccharide degradation by phage depolymerase P919DPO. The specific steps are as follows:

[0101] Phage depolymerase P919DPO was treated at 25℃, 70℃, and 80℃ for 1 hour, 2 hours, and 3 hours, respectively. Acinetobacter baumannii bacteria were grown overnight at 37℃. 100 μL of the culture was transferred to LB semi-solid plates, thoroughly mixed, and then poured into LB solid plates. After air drying, the concentrations of phage depolymerase P919DPO treated at different temperatures and times were adjusted to 150 μg / mL, 15 μg / mL, and 1.5 μg / mL. Then, 10 μL of each phage depolymerase P919DPO was dropped onto a double-layer plate and incubated at 37℃ for 24 hours. The morphology of the depolymerase was recorded.

[0102] like Figure 7 As shown, phage depolymerases exhibit good temperature stability below 70°C, but rapidly lose activity above 80°C.

[0103] 11. Depolymerase promotes macrophage phagocytosis.

[0104] ZWAb014 is a non-myxotropic strain, indicating that its capsule expression level is normal. ZWAb024 is a myxotropic strain, indicating that its capsule expression level is increased, and it has higher resistance in the external environment and in the body immune environment.

[0105] Normally growing THP-1 cells were placed at 5 x 10 cells per well. 5 Cells were seeded at a density of 100 ng / mL PMA into 24-well cell culture plates and induced to differentiate into mononuclear macrophages for 24 h. Then, the culture was replaced with PMA-free medium and cultured for another 48 h. Single colonies of the test strains ZWAb014 and ZWAb024 were picked and inoculated into LB broth and cultured at the logarithmic growth phase (OD2). 600 =0.7, centrifuged at 5000g for 10 minutes to collect the bacterial pellet, washed three times with 1xPBS, and adjusted the bacterial count to 5 x 10^6 cells / mL using cell culture medium 1640. 7 CFU / ml. 1 ml of bacterial suspension was added to each macrophage well to MOI = 100. Then, 15 μg, 1.5 μg, 0.15 μg, 15 ng, 1.5 ng, and 0.15 ng of depolymerase were added sequentially to each well. The cells were centrifuged at 300 g for 5 minutes and then incubated at 37°C and 5% CO2 for 1 hour. To remove extracellular bacteria, the cells were washed 6 times with 1X PBS, then treated with 300 μg / ml gentamicin for 30 minutes, followed by 6 washes with 1X PBS. Macrophages were then lysed with 1 ml of sterile ddH2O for 1 minute at room temperature. The cell lysate was serially diluted using 96-well plates, and bacterial CFU were counted on LB agar plates. The experiment was repeated three times independently.

[0106] The results are as follows Figure 8 As shown, the phage depolymerase P919DPO promoted the phagocytosis of non-myxotropic strain ZWAb014 and myxotropic strain ZWAb024 by THP-1 cells. Although the phage could not lyse ZWAb024, the depolymerase P919DPO has a broader spectrum of action and can degrade the capsule of ZWAb024, thus promoting phagocytosis by THP-1 cells.

[0107] Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.

[0108] 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 bacteriophage of Acinetobacter baumannii ( Acinetobacterbaumannii Phage) P919, characterized in that, The Acinetobacter baumannii phage P919 was deposited at the China Center for Type Culture Collection on October 11, 2025, with accession number CCTCC NO: M 20252192.

2. The application of Acinetobacter baumannii phage P919 or phage depolymerase P919DPO in the preparation of KL3 type Acinetobacter baumannii disinfectant products, characterized in that, As described in claim 1, the Acinetobacter baumannii phage P919 is isolated from the Acinetobacter baumannii phage P919, and its sequence is shown in SEQ ID NO.1; the Acinetobacter baumannii phage P919 or the phage depolymerase P919DPO is used to specifically degrade the capsular polysaccharide of Acinetobacter baumannii type KL3.

3. The application according to claim 2, characterized in that, The Acinetobacter baumannii phage P919 or the phage depolymerase P919DPO promotes macrophage phagocytosis of Acinetobacter baumannii type KL3 by degrading its capsular polysaccharide.

4. The application according to claim 3, characterized in that, When the disinfectant product is prepared from the phage depolymerase P919DPO, the disinfectant product is a phage depolymerase P919DPO solution with a concentration of 1.5 μg / mL to 150 μg / mL, and the solvent of the solution is PBS.

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