Novel acinetobacter baumannii phage vBAbaMpha0264 and application thereof

Abstract

The invention discloses a novel acinetobacter baumannii phage vBAbaMpha0264 and application thereof, and belongs to the technical field of microorganisms. The acinetobacter baumannii phage vBAbaMpha0264 is preserved in the Guangdong Microbiological Culture Collection Center on August 28, 2025, the preservation number of the acinetobacter baumannii phage vBAbaMpha0264 is GDMCC (China General Microbiological Culture Collection Center) NO: 66893-B1, and the preservation address of the acinetobacter baumannii phage vBAbaMpha0264 is Institute of Microbiology, Academy of Sciences, Guangdong. The bacteriophage vBAbaMpha0264 has high cracking activity, environmental stability and safety, shows a breakthrough host range, and can be used for effectively cracking a plurality of acinetobacter bacteria including hospital acinetobacter and acinetobacter sysei. Meanwhile, the bacteriophage has excellent chloroform tolerance, and the stability of the bacteriophage is obviously superior to that of the existing bacteriophage. Due to the characteristics, the phage vBAbaMpha0264 overcomes the technical prejudice of'narrow spectrum and instability 'of the traditional phage, and a brand new technical scheme is provided for developing a universal antibacterial preparation covering more pathogenic bacteria.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microorganisms, and particularly relates to a novel Acinetobacter baumannii phage vB_AbaM_pha0264 and application thereof. BACKGROUND

[0002] Currently, due to the abuse of antibiotics, the problem of bacterial drug resistance is becoming increasingly serious, and drug-resistant pathogenic bacteria frequently appear in clinical infections, significantly reducing the treatment effect, and some infections are facing the dilemma of lack of effective treatment. Traditional antibiotic development has a long cycle and high cost, and is prone to cause dysbiosis and secondary drug resistance, making it difficult to cope with the rapid evolution of drug resistance. Developing new, safe and efficient antibacterial strategies has become an urgent need. Under this background, phage therapy has attracted widespread attention due to its strong targeting and high safety. As a specific bacteriophage, phage can specifically recognize and invade host bacteria, ultimately lyse bacteria and release progeny phage, achieving continuous clearance of pathogenic bacteria. This process is highly specific and only acts on target bacteria without affecting normal microbial flora. Because its bactericidal mechanism is independent of antibiotics, it still has high killing ability for drug-resistant bacteria and is not prone to cross-resistance. At the same time, phage can self-amplify with the proliferation of host bacteria at the infection site, and is particularly suitable for the treatment of chronic, recurrent and biofilm-related infections. However, the clinical conversion of phage therapy depends on high-quality phage resources with high efficiency, stability and clear host spectrum. Therefore, the isolation of new phages with strong lytic activity is a key foundation for promoting the development of this technology.

[0003] Existing technologies show that most Acinetobacter baumannii-specific phages have narrow host range, making it difficult to achieve cross-species lysis, which limits their practical application value. This characteristic has been widely recognized as an inherent limitation of phage application (KONCZ M, STIRLING T, HADJ MEHDI H, et al. Genomic surveillance as a scalable framework for precision phage therapy against antibiotic-resistant pathogens [J]. Cell, 2024, 187(21): 5901-18e28.). For example, phage vB_AbaS_qsb1 (see Chinese patent application CN119162120A) can only lyse some Acinetobacter baumannii strains (aba001 and aba010), and the host spectrum is extremely limited. Experimental verification shows that it cannot lyse common Acinetobacter bacteria such as Acinetobacter nosocomialis and Acinetobacter seimii, showing typical "narrow spectrum" characteristics. SUMMARY

[0004] In view of the above technical limitations, the present application aims to provide a novel Acinetobacter baumannii phage with broad-spectrum lytic ability and its application.

[0005] The phage is Acinetobacter baumannii phage vB_AbaM_pha0264, which has been preserved in Guangdong Microbial Culture Collection Center on August 28, 2025, with a preservation number of GDMCC NO: 66893-B1 and a preservation address of Guangdong Provincial Academy of Microbiology.

[0006] The present application screens a strain of Acinetobacter baumannii phage vB_AbaM_pha0264 with high lytic activity, environmental stability and safety from the natural environment, which can specifically recognize and lyse Acinetobacter baumannii to eliminate infection and overcome the problem of antibiotic resistance of Acinetobacter baumannii.

[0007] The present application also provides the application of Acinetobacter baumannii phage vB_AbaM_pha0264 in inhibiting Acinetobacter baumannii in food production environment and facilities.

[0008] The present application also provides the application of Acinetobacter baumannii phage vB_AbaM_pha0264 in preparing an Acinetobacter baumannii bacteriostatic agent, which is used for eliminating Acinetobacter baumannii in food and production facilities, environment and preservation and transportation appliances.

[0009] The present application also provides a phage preparation, which comprises Acinetobacter baumannii phage vB_AbaM_pha0264.

[0010] The present application also provides the application of the phage preparation in preparing a medicine for preventing or treating diseases caused by Acinetobacter baumannii infection.

[0011] The present application also provides a phage composition, which comprises Acinetobacter baumannii phage vB_AbaM_pha0264.

[0012] The present application also provides a water disinfectant comprising Acinetobacter baumannii phage vB_AbaM_pha0264 or the above-mentioned phage composition.

[0013] Acinetobacter baumannii phage vB_AbaM_pha0264 can be used for treating and preventing infectious diseases caused by Acinetobacter baumannii in humans, animals or plants.

[0014] The phage preparation or phage composition described above, in addition to the phage vB_AbaM_pha0264, further comprises a buffer as a stabilizer, which can be one of DPBS buffer, physiological saline, SM buffer, glycerol solution, etc.

[0015] DPBS buffer: 0.2g of KCl, 0.2g of KH2PO4, 8g of NaCl, 2.8865g of Na2HPO4·12H2O, add 800mL of distilled water to dissolve thoroughly, adjust pH to 7.0-7.3 with HCl solution, and then make up to 1000mL, wet heat sterilization at 121℃, 0.15MPa for 20min.

[0016] SM buffer: take 1.972g of magnesium sulfate heptahydrate and 5.58g of sodium chloride, add 50mL of 1M Tris-HCl (pH 7.5) and 5mL of 2% gelatin, add ddH2O to dissolve thoroughly, and make up to 1L, wet heat sterilization at 121℃, 0.15MPa for 20min. Additional 0.1-1% BSA can be added on this basis.

[0017] Glycerol solution: use ddH2O to dissolve glycerol to a final concentration of 5-50%, and filter sterilize with a 0.22μm filter.

[0018] Beneficial effects: the phage vB_AbaM_pha0264 obtained by the present application not only completely covers the host spectrum of the phage vB_AbaS_qsb1, but also can efficiently lyse multiple strains of Acinetobacter baumannii (aba012, aba017, aba021 and aba026) that cannot be lysed by the phage vB_AbaS_qsb1, and further exhibits a breakthrough host range, and can effectively lyse multiple Acinetobacter bacteria, including Acinetobacter ano034 and Acinetobacter ase037, that cannot be infected by the phage vB_AbaS_qsb1. At the same time, the phage has excellent chloroform tolerance, and the stability is significantly better than that of existing phages. The above characteristics make the phage vB_AbaM_pha0264 overcome the technical prejudice of traditional phages "narrow spectrum and instability", and provide a new technical scheme for developing a universal antibacterial preparation covering a wider range of pathogenic bacteria.

[0019] The present application provides the bacteriophage vB_AbaM_pha0264 of Acinetobacter baumannii, which can specifically recognize and lyse Acinetobacter baumannii to eliminate infection, and exhibits good bacteriostatic effect in vivo and in vitro. The bacteriophage vB_AbaM_pha0264 in vivo (in vivo) bacteriostatic dose MOI value should be not less than 0.001, and not more than 1000; more preferably, the range is 0.01<MOI<200; and more preferably, the range is 0.02<MOI<150. The bacteriophage vB_AbaM_pha0264 in vitro (in vitro) bacteriostatic dose MOI value should be not less than 0.00001, and not more than 2000; more preferably, the range is 0.0001<MOI<1000; and more preferably, the range is 0.001<MOI<200.

[0020] The application field thereof covers human, animals and plants, and specifically includes: treating and preventing human infectious diseases caused by Acinetobacter baumannii; preventing and treating corresponding infections of pets, livestock and aquatic animals (such as fish and shrimp); and preventing and controlling related bacterial diseases of plants. In addition, the bacteriophage can also be used to eliminate Acinetobacter baumannii in the environment (such as object surface and water body), and is suitable for the fields of environmental disinfection, water purification and food preservation, and exhibits diverse application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Figure 1 is the virus particle morphology (×40K) of the bacteriophage vB_AbaM_pha0264, and the scale is 200nm (A), and the plaque morphology of the bacteriophage vB_AbaM_pha0264 (B).

[0022] Figure 2 Figure 2 is the whole genome annotation map of the bacteriophage vB_AbaM_pha0264.

[0023] Figure 3 Figure 3 is the genome collinearity analysis of the bacteriophage vB_AbaM_pha0264 and its similar bacteriophages.

[0024] Figure 4 Figure 4 is the terminal enzyme large subunit phylogenetic tree of the bacteriophage vB_AbaM_pha0264 and related groups.

[0025] Figure 5 Figure 5 is the optimal infection multiple of the bacteriophage vB_AbaM_pha0264.

[0026] Figure 6 Figure 6 is the one-step growth curve of the bacteriophage vB_AbaM_pha0264.

[0027] Figure 7 Figure 7 is the temperature stability of the bacteriophage vB_AbaM_pha0264.

[0028] Figure 8 Acid-base stability of phage vB_AbaM_pha0264

[0029] Figure 9 Chloroform stability of phage vB_AbaM_pha0264

[0030] Figure 10 Bacteriostatic experiment of phage vB_AbaM_pha0264 in vitro

[0031] Figure 11 Biofilm formation inhibition ability of phage vB_AbaM_pha0264

[0032] Figure 12 Biofilm removal ability of phage vB_AbaM_pha0264

[0033] Figure 13 Preventive intervention of phage vB_AbaM_pha0264 on Acinetobacter baumannii infection

[0034] Figure 14 Immediate treatment of phage vB_AbaM_pha0264 on Acinetobacter baumannii infection

[0035] Figure 15 Rescue treatment of phage vB_AbaM_pha0264 on Acinetobacter baumannii infection

[0036] Figure 16 Host range of phage vB_AbaM_pha0264 and vB_AbaS_qsb1; wherein, strains aba001-aba032 are Acinetobacter baumannii (aba), an033, ano034, ano039 are Acinetobacter nosocomialis, api035 is Acinetobacter pittii (api), and ase036-ase038 are Acinetobacter seifertii.

[0037] Figure 17 Lysis ability (spotting method) of phage vB_AbaM_pha0264 and vB_AbaS_qsb1 on different Acinetobacter bacteria; the clear transparent circle at the edge of the figure is a plaque, that is, the phage has lysis ability on the bacteria; if there is no plaque, the phage has no lysis ability on the bacteria. DETAILED DESCRIPTION

[0038] The application will be further described in detail below in combination with the drawings and examples.

[0039] The raw materials and reagents used in the examples are commercially available products unless otherwise specified.

[0040] The host bacteria used in the present application is Acinetobacter baumannii aba001, which can be obtained from the research published by the inventors' team, see the literature: Wang, J., et al. (2025). "A novel genus of virulent phage targeting Acinetobacter baumannii: Efficacy and safety in a murine model of pulmonary infection." PLOS Pathogens 21(6).

[0041] Note: The Acinetobacter baumannii number in this document is ioag01, which is the same strain as the Acinetobacter baumannii aba001 described in the present application. After the reorganization of the strain preservation library of the Institute of Applied Genomics, Fujian University, the Acinetobacter baumannii with number ioag01 was renumbered as aba001.

[0042] The strains aba001-aba032, an033, ano034, api035, ase036-ase038, ano039 used in Example 11 can be obtained from the following literature:

[0043] Wang, J., et al. (2025). "A novel genus of virulent phage targeting Acinetobacter baumannii: Efficacy and safety in a murine model of pulmonary infection." PLOS Pathogens 21(6)

[0044] Lin, J., et al. (2021). "Metagenomic Sequencing Revealed the Potential Pathogenic Threats of Banknotes." ACS Omega 6(5):3499-3507.

[0045] Lin, J., et al. (2021). "Metagenomic sequencing revealed the potential of banknotes as a repository of microbial genes." BMC Genomics22(1): 173.

[0046] Example 1 Isolation and purification of phage

[0047] (1) Preparation of host bacteria liquid in logarithmic growth phase: a single, morphologically typical colony was picked from a fresh plate and inoculated into 10 mL of LB liquid medium, which was incubated at 37°C in a constant-temperature shaker at 200 rpm / min for 12 hours to obtain host bacteria liquid in logarithmic growth phase.

[0048] (2) An environmental sewage (Xiamen Hongai Hospital sewage) sample was centrifuged at 20,000 x g for 10 min at 4°C to remove the precipitate and collect the supernatant. The supernatant was filtered through a 0.22 μm sterile filter membrane to remove residual bacteria and obtain the filtrate. 5 mL of the filtrate was added to 5 mL of 2x LB liquid medium, mixed, and then inoculated with 5 mL of host bacteria liquid in logarithmic growth phase, which was incubated at 37°C at 220 rpm / min overnight. After the incubation, the phage activity was detected by double-layer agar plate method, and clear and isolated plaques were observed. A single plaque with uniform morphology was picked and transferred to a sterile centrifuge tube containing 500 μL of SM buffer, and the phage was released by blowing to prepare a single plaque extract. 500 μL of the single plaque extract was added to 500 μL of 2x LB medium and 500 μL of logarithmic phase host bacteria liquid, which was incubated at 37°C at 220 rpm overnight. The culture was centrifuged at 20,000 x g for 10 min at 4°C to collect the supernatant, which was the phage purified liquid obtained by the first round of purification. The above-mentioned single plaque picking, extraction, amplification and centrifugation steps were repeated for at least 5 rounds of single plaque purification. Finally, a high-purity phage solution (purified phage suspension) was obtained, and the phage was named vB_AbaM_pha0264.

[0049] Example 2 Identification of phage

[0050] (1) Morphological observation of phage

[0051] 20 μL of the phage suspension with a titer of 1 x 10 10 PFU / mL was added to the surface of a copper mesh, and after standing for 10 min, the excess suspension was absorbed with filter paper. Then 20 μL of 2% phosphotungstic acid solution was added for negative staining, and after 90 s, the excess staining solution was absorbed with filter paper. After the copper mesh was dried, it was placed under an electron microscope at an acceleration voltage of 100 kV to observe the morphology of the phage.

[0052] Under the transmission electron microscope, the phage vB_AbaM_pha0264 had a morphological structure with a head of about 70 x 70 nm, a positive icosahedral structure. The tail was about 120 nm before contraction and about 70 nm after contraction, showing a typical morphology of the myoviridae family of phages, and no lipid envelope was observed on the surface. Figure 1A)。Phage vB_AbaM_pha0264 formed plaques with a diameter of 1.0 ± 0.5 mm after 12 h incubation in double-layer agar plates, and the plaque body was transparent with clear edges and no surrounding lysozyme halo was observed Figure 1 B)。

[0053] (2) Phage genome characteristics

[0054] The genome of phage vB_AbaM_pha0264 was 102930 bp in length with a GC content of 37.37%. A total of 327 open reading frames (ORFs) were predicted, including 161 coding sequences (CDS), by using a combination of annotation tools such as Phastest, Prokka, Phagescope, NCBI, and Genemarks. Among them, there were 16 phage structural proteins, including tail fiber protein, tail sheath protein, and tail tape measure protein; 10 DNA replication-related proteins, including DNA helicase, DNA ligase, and DNA polymerase; 19 other functional proteins, including thymidylate synthase, dihydrofolate reductase, and ribonucleotide reductase; 1 DNA packaging-related protein, i.e., large subunit of terminase; no lysozyme or other phage lysis-related proteins; and the remaining 91 were hypothetical proteins, accounting for the majority of the total predicted proteins. No integrase was found in phage vB_AbaM_pha0264, and no potential antibiotic resistance genes and virulence genes were found. Figure 7 ).

[0055] The three phages with the highest similarity to phage vB_AbaM_pha0264 were Acinetobacter phage vB_AbaM_CP14 isolated from Dalian, Acinetobacter phage phi1_092033 isolated from Chengdu, and Acinetobacter phage Liucustia isolated from Russia. The nucleotide sequence identity of these three phages to phage vB_AbaM_pha0264 was more than 95%, and the query coverage was also more than 85%. Although there was a high degree of sequence similarity between them, linear alignment found that there were differences in their genomic composition. This indicates that phage vB_AbaM_pha0264 has experienced gene recombination events in its evolutionary process, and phage vB_AbaM_pha0264 is a novel phage with unique evolutionary characteristics. Figure 3 ).

[0056] A phylogenetic tree was constructed using the large subunits of the terminal enzymes. The results showed that bacteriophage vB_AbaM_pha0264 belongs to the same branch as Acinetobacter bacteriophages vB_AbaM_B09_Aci01-1 and vB_AbaM_CP14, with a bootstrap value of 97%. Therefore, bacteriophage vB_AbaM_pha0264 is considered to belong to the Myocaudidae family within the order Caudata. Figure 4 ).

[0057] Based on the morphological and genomic characteristics of bacteriophage vB_AbaM_pha0264, it was identified as Acinetobacter baumanniiphage vB_AbaM_pha0264, and was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 28, 2025, with accession number GDMCC NO: 66893-B1, at the Institute of Microbiology, Guangdong Academy of Sciences.

[0058] Example 3: Optimal Multiple of Infection for Bacteriophages

[0059] Acinetobacter baumannii aba001 bacterial suspension in the logarithmic growth phase was mixed with phage suspension to achieve multiplicity of infection (MOI) values ​​of 0.001, 0.01, 0.1, 1, and 10. The mixture was incubated at 37°C and 220 rpm for 3 hours. After centrifugation at 20,000 g for 1 minute at 4°C, the supernatant was collected. After appropriate dilution, 100 μL of the supernatant was mixed with 300 μL of Acinetobacter baumannii aba001 bacterial suspension in the logarithmic growth phase and incubated at 37°C for 10 minutes. The number of plaques was then counted using the double-layer plate method. The MOI with the highest phage complexity efficiency was determined as the optimal MOI. Phage complexity efficiency was calculated using the formula: Phage complexity efficiency = (Number of endpoint phages / Number of initial phages) × 100%.

[0060] The results are as follows Figure 5 As shown, the optimal multiplicity of infection for bacteriophage vB_AbaM_pha0264 is 0.001, meaning that the bacteriophage proliferation efficiency is optimal when the bacteriophage and host bacteria are mixed at a ratio of 1:1000.

[0061] Example 4: One-step growth curve of bacteriophage

[0062] The bacteria liquid of Acinetobacter baumannii aba001 in logarithmic growth phase and the bacteriophage suspension were mixed at the optimal multiplicity of infection, and after the adsorption of the bacteriophage, 20000g centrifugation was performed at 4°C for 1min, and the supernatant was discarded. After resuspension of the precipitate using 10mL of LB liquid medium, it was placed at 37°C under 220r / min culture. During the period, 1mL was taken each time, placed at 4°C under 20000g centrifugation for 10min, and 100μL of supernatant was collected. 300μL of the bacteria liquid of Acinetobacter baumannii aba001 in logarithmic growth phase was added, and 37°C incubation was performed for 10min. Then the number of plaques was counted by using double-layer plate method. The one-step growth curve was drawn with the infection time as the abscissa and the number of bacteriophages as the ordinate, and the maximum burst size of the bacteriophage was calculated. The maximum burst size of the bacteriophage was calculated according to the formula: maximum burst size of the bacteriophage = number of bacteriophages in the plateau phase / initial number of bacteria.

[0063] The results are shown in Figure 6 The one-step growth curve of the bacteriophage vB_AbaM_pha0264 showed that it had a very short latent period, rapidly lysed the host bacteria and released the progeny bacteriophages within 10min after adsorbing the host bacteria. Its lysis cycle was about 120min, and the burst period was about 20-90min, during which the titer of the bacteriophage increased rapidly, and reached the plateau phase at 120min, and the titer of the bacteriophage tended to be stable. The calculated burst size of the bacteriophage vB_AbaM_pha0264 was about 15PFU / cell.

[0064] Example 5 Temperature sensitivity of the bacteriophage

[0065] The bacteriophage suspension was placed at different temperatures for 1h respectively. After gradient dilution, 100μL was added to 300μL of the bacteria liquid of Acinetobacter baumannii aba001 in logarithmic growth phase, and after 37°C incubation for 10min, the number of plaques was counted by using double-layer plate method to evaluate the change of the titer of the bacteriophage.

[0066] The results are shown in Figure 7 The bacteriophage vB_AbaM_pha0264 remained stable within the range of 4-42°C, and the titer of the bacteriophage did not change significantly (p>0.05) within 60min. When the temperature rose to 50°C, the titer of the bacteriophage decreased by about 30% after 60min; at 60°C, the survival rate decreased to 10% after 60min; and when the temperature was ≥70°C, the bacteriophage was completely inactivated within 60min.

[0067] Example 6 Acid-base sensitivity of the bacteriophage

[0068] The phage suspension was added to SM buffer with different pH values and incubated at 25°C for 1 h. After gradient dilution, 100 μL was added to 300 μL of Acinetobacter baumannii aba001 bacteria in the logarithmic growth phase, and incubated at 37°C for 10 min. The number of plaques was counted using the double-layer plate method to evaluate the change in phage titer.

[0069] As shown in Figure 8 , the phage vB_AbaM_pha0264 remained highly stable in the pH 4-8 range, and the activity did not change significantly within 60 min (p>0.05). In strong acid (pH 3) and strong alkali (pH 9-11) conditions, the phage titer decreased after 60 min, but more than 50% of the phage was still retained. In the extreme acid and alkali environment (pH≤2 or pH≥12), the phage was completely inactivated within 60 min.

[0070] Example 7 Chloroform sensitivity of the phage

[0071] Different volumes of chloroform were added to the phage suspension, mixed thoroughly, and incubated at 25°C for 30 min. The aqueous phase was taken. After gradient dilution, 100 μL was added to 300 μL of Acinetobacter baumannii aba001 bacteria in the logarithmic growth phase, and incubated at 37°C for 10 min. The number of plaques was counted using the double-layer plate method to evaluate the change in phage titer.

[0072] As shown in Figure 9 , when the concentration of chloroform reached 25%, the titer of the phage vB_AbaM_pha0264 did not change significantly within 60 min (p>0.05), showing excellent chloroform tolerance. It can be used as a surface disinfectant additive: for places that require strong, broad-spectrum, and stable disinfection, such as hospitals and laboratories, especially for handling equipment contaminated with organic matter; for environmental remediation: applied to contaminated environments containing organic solvents (such as chemical plant wastewater) to target and eliminate Acinetobacter baumannii.

[0073] Example 8 In vitro bacteriostatic test of the phage

[0074] The logarithmic growth phase of Acinetobacter baumannii aba001 bacteria was taken, and the OD 600nm of the bacterial solution was measured. After adjusting the concentration of the bacterial solution, it was added to a shaking flask containing 100 mL of LB liquid medium so that the number of bacteria was 7×10 9 CFU. Then, the phage suspension was immediately added so that the multiplicity of infection of each group was 0.01, and the volume was adjusted with SM buffer. Then, it was incubated at 37°C on a 220 rpm shaker. Every hour, the OD 600nm was measured. The in vitro bacteriostatic curve of the phage was plotted with time as the horizontal coordinate and OD 600nm as the vertical coordinate.

[0075] Results are shown in Figure 10 Figure 6 and Figure 7. Within 12h, the bacteriophage vB_AbaM_pha0264 showed strong antibacterial ability against bacteria, and could significantly inhibit bacterial growth. After 12h, the inhibitory effect of the bacteriophage vB_AbaM_pha0264 on bacteria weakened, and resistant strains began to appear and entered a plateau within 12h.

[0076] Example 9 Biofilm test

[0077] The bacterial liquid of the logarithmic growth phase of Acinetobacter baumannii aba001 was centrifuged at 5000g for 5min to collect the bacterial cells, resuspended with LB liquid medium, and the OD 600nm was measured and the bacterial liquid concentration was adjusted to 1x10 8 PFU / mL. 100μL of the bacterial liquid was added to a 96-well cell culture plate, and incubated at 37°C for 24h. The planktonic bacteria were removed, the biofilm was washed three times with DPBS buffer and air-dried. After air-drying, the biofilm was fixed with 4% paraformaldehyde, and stained with 1% crystal violet for 15min. The excess dye was removed by washing three times with 100μL of DPBS buffer. 33% acetic acid was added to the wells to completely release the crystal violet bound to the biofilm, and the OD 595nm was measured to quantify the biofilm formation.

[0078] (1) To evaluate the inhibitory effect of the bacteriophage on the biofilm, an equal amount of bacteriophage was added to the bacterial suspension.

[0079] (2) To evaluate the bacteriophage's ability to remove the biofilm, after removing the planktonic bacteria and washing, 100μL of the bacteriophage suspension was added, and after incubation for 6h, the fixation and staining steps were performed.

[0080] Results are shown in Figure 11 , 12 respectively: the bacteriophage vB_AbaM_pha0264 could significantly inhibit biofilm formation (P<0.0001) and could also significantly remove the formed biofilm (P<0.0001).

[0081] Therefore, the bacteriophage vB_AbaM_pha0264 can be used to treat chronic / refractory bacterial infections: to treat chronic wound infections caused by Acinetobacter baumannii, such as diabetic foot ulcers, burn wounds, etc.

[0082] The bacteriophage vB_AbaM_pha0264 can be used for the prevention and control of medical device and implant-related infections: to remove Acinetobacter baumannii biofilm on the surface of devices such as catheters (urinary catheters, central venous catheters), artificial joints, etc.

[0083] Example 10 G.mellonella survival curve and health index score

[0084] In vivo efficacy verification was carried out by using Galleria mellonella larvae infection model. Healthy larvae with a weight of 250-300 mg were selected and randomly divided into groups, 10 larvae in each group, and the experiment was repeated three times independently. In the prophylactic treatment group, each larva was pre-injected with bacteriophage suspension, and the model control group was injected with the same volume of normal saline; 12 hours later, each group was inoculated with A. baumannii except the blank control group. In the immediate treatment group, each larva was first inoculated with A. baumannii to establish an infection model, and 4 hours later, bacteriophage injection was given. In the dying treatment group, after 12 hours of A. baumannii infection, the dead individuals were removed, and the surviving larvae were injected with bacteriophage. All treatment groups and control groups of larvae were cultured at 37°C in the dark, and the survival status was recorded every 12 hours for 72 hours. At the same time, the health status of the larvae was scored to comprehensively evaluate the in vivo protective effect of the bacteriophage. The low, medium and high doses of treatment were MOI = 0.1, 1 and 10, respectively.

[0085] Logarithmic growth phase A. baumannii aba001 bacterial solution was collected, washed with physiological saline and counted. If the injected sample is a bacteriophage suspension, the washing step is omitted. Galleria mellonella larvae with a weight of about 250 mg, a body color of beige and no gray spots were selected as experimental models, randomly divided into groups, and each larva was injected with 10 μL of sample. The blank control group was injected with the same amount of normal saline. The larvae were placed in a 37°C incubator for culture. If a second injection is required, it is injected on the opposite side. The survival status of the larvae was observed and recorded at regular intervals during the observation period.

[0086] As shown in Figure 13 , for the prophylactic intervention of A. baumannii infection, the low-dose bacteriophage vB_AbaM_pha0264 intervention can improve the survival rate of 56.67% of G. mellonella larvae. The medium dose can improve 60.00%, and the high dose can improve 73.33%.

[0087] As shown in Figure 14 , for the immediate treatment of A. baumannii infection, the low-dose bacteriophage vB_AbaM_pha0264 treatment can improve the survival rate of 13.33% of G. mellonella larvae. The medium dose can improve 26.67%, and the high dose can improve 60.00%.

[0088] As shown in Figure 15 , for the dying treatment of A. baumannii infection, the low-dose bacteriophage vB_AbaM_pha0264 treatment can improve the survival rate of 13.33% of G. mellonella larvae. The medium dose can improve 36.67%, and the high dose can improve 40.00%.

[0089] Example 11 Host range of the phage

[0090] The host range of the phage vB_AbaM_pha0264 was analyzed by the spot method, and the specific method was as follows: first, 1 mL of bacterial liquid to be tested was coated on a solid culture medium plate, and the excess bacterial liquid was discarded and dried for 15 min. Then 5 μL of the phage suspension with a titer of 1 x 10 9 PFU / mL of the phage suspension was spotted on the plate. After the phage suspension was dried, a sealing film was used to seal the plate, and the plate was incubated at 37 °C for 4 h. Whether the phage plaque was formed at the position of the phage droplet was observed.

[0091] Phage-resistant bacteria screening: Acinetobacter baumannii bacterial liquid was coated on an LB plate. After the surface of the plate was dried, 5 μL of the phage vB_AbaS_qsb1 (see Chinese patent application CN119162120A) suspension with a titer of 5 x 10 8 PFU / mL was spotted on the surface of the plate. After the surface of the plate was dried, the plate was incubated at 37 °C overnight. Single colonies in the phage plaques were picked and streaked on an LB plate, and were subcultured for 3 times in succession to purify the stable phage-resistant bacteria. The single colonies were taken in an LB liquid medium and cultured at 37 °C with shaking at 220 rpm / min to prepare a bacterial suspension. The bacterial liquid was coated on an LB plate, and after the surface of the plate was dried, 5 μL of the phage vB_AbaS_qsb1 suspension with a titer of 5 x 10 9 PFU / mL was spotted on the surface of the plate. After the surface of the plate was dried, the plate was incubated at 37 °C overnight. Whether the phage plaques were formed was observed. If the phage plaques were formed, the strain was removed. If the phage plaques were not formed, 100 μL of the phage vB_AbaS_qsb1 suspension with a titer of 5 x 10 9 PFU / mL was mixed with 500 μL of the phage vB_AbaS_qsb1 suspension, and was incubated at 37 °C for 10 min. Then preheated LB semi-solid medium was added to the mixture and mixed to prepare a double-layer plate, which was incubated at 37 °C overnight. Whether the phage plaques were formed was observed. If the phage plaques were not formed, it was determined that the strain had stable phage resistance. Escherichia coli was used as a negative control, and the host bacteria were used as a positive control to prove the reliability of the screening results.

[0092] The results are shown in Table 1 and Table 2, respectively: Figure 16 The phage vB_AbaM_pha0264 can lyse Acinetobacter bacteria including Acinetobacter baumannii, Acinetobacter nosocomialis (ano), and Acinetobacter seifertii (ase), and has a cross-species lysis ability. In addition, the phage vB_AbaM_pha0264 can also lyse all the phage vB_AbaS_qsb1 resistant strains screened, and has a high clinical application value and environmental disinfection potential.

[0093] AsFigure 17 As shown, the host range of the phage vB_AbaM_pha0264 was significantly expanded, not only completely covering the host spectrum of the phage vB_AbaS_qsb1, but also efficiently lysing multiple strains of Acinetobacter baumannii (such as aba012, aba017, aba021 and aba026) that could not be lysed by vB_AbaS_qsb1. In addition, it also effectively lysed multiple strains of Acinetobacter bacteria, including hospital Acinetobacter (ano034) and Seti Acinetobacter (ase037). This indicates that vB_AbaM_pha0264 has a wider host range and can overcome the "narrow spectrum" technical prejudice of traditional phages.

Claims

1. A bacteriophage strain, characterized in that, The bacteriophage mentioned is Acinetobacter baumannii bacteriophage ( Acinetobacter baumannii Phage vB_AbaM_pha0264 was deposited on August 28, 2025, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC NO: 66893-B1), located at the Institute of Microbiology, Guangdong Academy of Sciences.

2. The application of the bacteriophage as described in claim 1 in inhibiting Acinetobacter baumannii, Acinetobacter nosocomialis, or Acinetobacter serumi in food production environments and facilities.

3. The use of the bacteriophage as described in claim 1 in the preparation of antibacterial agents for Acinetobacter baumannii, Acinetobacter hospitalis, or Acinetobacter sergei.

4. The application according to claim 3, wherein the antibacterial agent is used to eliminate Acinetobacter baumannii in food and production facilities, environment, and storage and transportation equipment.

5. A bacteriophage preparation, characterized in that, It includes Acinetobacter baumannii phage as described in claim 1. Acinetobacter baumannii phage)vB_AbaM_pha0264.

6. The use of the phage preparation as described in claim 5 in the preparation of a medicament for the prevention or treatment of diseases caused by Acinetobacter baumannii, Acinetobacter nosocomialis, or Acinetobacter serosa infection.

7. A bacteriophage composition, characterized in that, It includes the Acinetobacter baumannii phage vB_AbaM_pha0264 as described in claim 1.

8. The phage formulation according to claim 5 or the phage composition according to claim 7, characterized in that, It also contains a buffer solution as a stabilizer, which is DPBS buffer, physiological saline, SM buffer or glycerol solution.

9. A water disinfectant, characterized in that, It includes the bacteriophage as described in claim 1 or the bacteriophage composition as described in claim 7.

10. The use of the bacteriophage as described in claim 1 in the treatment and prevention of infectious diseases in humans, animals or plants caused by Acinetobacter baumannii, Acinetobacter novolognae or Acinetobacter sergei.

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