Acinetobacter baumannii phage and application thereof

Abstract

The invention relates to the technical field of biology, and discloses an acinetobacter baumannii bacteriophage and application thereof, the acinetobacter baumannii bacteriophage is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC M20252580. The bacteriophage belongs to the brachycaudal bacteriophage family, the genome is double-stranded linear DNA, the full length is 41020bp, the bacteriophage does not contain integrase, virulence factors and drug-resistant genes, and the bacteriophage has high biological safety. The bacteriophage is high in titer, can specifically recognize and efficiently split acinetobacter baumannii including multi-drug-resistant strains, and has the capacity of permeating and removing bacterial biofilms at the same time. The invention also discloses application of the bacteriophage in preparation of antibacterial compositions, sterilization and disinfection preparations and food preservation and fresh-keeping. The bacteriophage provided by the invention is high in sterilization efficiency, strong in specificity, safe and non-toxic, and an effective biological solution is provided for prevention and control of acinetobacter baumannii drug resistance and biological membrane pollution.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a strain of Acinetobacter baumannii bacteriophage and its applications. Background Technology

[0002] Currently, *Acinetobacter baumannii*, a Gram-negative opportunistic pathogen, exhibits high environmental adaptability. This bacterium is widely distributed in healthcare wastewater systems, soil environments, and various food processing chains. In individuals with compromised immune systems, it easily induces catheter-related bloodstream infections and ventilator-associated pneumonia. In clinical practice and environmental monitoring, isolated strains often demonstrate multidrug resistance or even pan-drug resistance. Globally, the detection rate of carbapenem-resistant strains is on the rise, increasing the mortality risk of related infections. With the discharge of waste containing this bacterium, the pathogen and its resistance agents are rapidly spreading into natural ecosystems, posing a serious challenge to public health security.

[0003] Current methods for controlling contamination by the aforementioned pathogens primarily rely on antibiotic intervention and chemical disinfectant treatment. In clinical treatment, conventional approaches often utilize antibiotics to block bacterial cell wall synthesis or interfere with protein metabolism pathways. In environmental sanitation, the application of chlorine-containing agents, oxidants, and other chemicals aims to destroy bacterial structure through contact oxidation. For the treatment of medical device or food substrate surfaces, the focus is on utilizing the broad-spectrum bactericidal properties of chemical agents to kill free-floating bacteria, thereby interrupting transmission routes and reducing bacterial load.

[0004] However, existing technologies have several shortcomings in practical applications. Traditional chemical killing mechanisms are ineffective at penetrating mature bacterial biofilms. The physical barrier formed by the extracellular polysaccharide matrix hinders the penetration of bactericidal factors, making it difficult to eliminate persistent bacteria deep within the membrane and easily leading to re-contamination. In current biocontrol efforts, many naturally isolated bacteriophage strains exhibit low lytic activity, and their proliferation titers cannot meet the requirements for rapid sterilization. The genetic background of some bacteriophages is ambiguous, posing a risk of carrying virulence factors or drug resistance genes, and presenting a safety hazard of horizontal gene transfer. Furthermore, faced with complex biofilm structures, bacteriophages lacking specific polysaccharide depolymerization capabilities struggle to disrupt the matrix framework, limiting their ability to achieve thorough purification in complex environments.

[0005] Therefore, the present invention provides a strain of Acinetobacter baumannii bacteriophage and its application to address the shortcomings of the prior art. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a strain of Acinetobacter baumannii phage and its applications, solving the problems of insufficient lysis efficiency, limited penetration into mature biofilms, and poor adaptability in complex matrices of existing phages.

[0007] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides an Acinetobacter baumannii phage ( Acinetobacter baumannii phage The bacteriophage, named Acinetobacter baumannii phage AB4P4, was deposited at the China Center for Type Culture Collection (CCTCC) on November 17, 2025, with accession number CCTCC M 20252580.

[0008] The biological characteristics of the Acinetobacter baumannii phage are as follows: titer can reach 4.010. 11 PFU / mL; electron microscopy morphology shows that its head is a spherical or near-spherical icosahedral structure with a diameter of about 32 nm, and the tail is a compact short tail structure with a length of about 11 nm. There are no obvious tail fibers. Taxonomically, it belongs to the order Caudate Phages and the family Brachypophagidae.

[0009] At the genomic level, the genetic material of the Acinetobacter baumannii phage is double-stranded linear DNA, with a full-length genome of 41,020 bp and a G+C content of 39.37%. Genomic analysis shows that it contains a total of 196 open reading frames (ORFs), of which 50 ORFs have clearly defined functional annotations, and all are located on the positive strand. The GenBank accession number of the phage is PX270320.

[0010] Preferably, the phage comprises a nucleotide sequence encoding a DNA polymerase (as shown in SEQ ID NO.1) and a nucleotide sequence encoding a major capsid protein (as shown in SEQ ID NO.2).

[0011] By adopting the above technical solution, the bacteriophage AB4P4 provided by the present invention has the following effects: High lytic activity: This bacteriophage belongs to the family Short-tailed Phagesaceae and exhibits short latency and high burst proliferation characteristics, reaching up to 4.010. 11 The PFU / mL titer indicates that it has high replication efficiency within the host and can rapidly lyse the host bacteria.

[0012] High biosafety: Based on whole-genome sequencing analysis, no tRNA genes, integrase genes, known virulence factors, drug resistance genes, or pathogenicity-related genes were detected in the AB4P4 genome. This genetically eliminates the risk of it acting as a lysogenic phage to mediate horizontal transfer of drug resistance genes, ensuring its safety in practical applications.

[0013] Clear genetic background: The well-defined genome structure and sequence information of key enzymes (such as DNA polymerase and capsid proteins) provide precise data support for subsequent molecular modification, detection kit development, and functional mechanism research.

[0014] In a second aspect, the present invention provides an application of the Acinetobacter baumannii phage, including using the Acinetobacter baumannii phage AB4P4 to lyse Acinetobacter baumannii or to prepare a composition for lysing Acinetobacter baumannii.

[0015] Preferably, the lysed Acinetobacter baumannii includes multidrug-resistant Acinetobacter baumannii that is resistant to aminoglycosides, penicillins, cephalosporins, carbapenems, tetracyclines, fluoroquinolones, or sulfonamides.

[0016] By adopting the above technical solution, the present invention achieves effective control of multidrug-resistant bacteria, and its mechanism of action and effects are as follows: Specific recognition and adsorption: Bacteriophage AB4P4 specifically recognizes receptors (such as outer membrane proteins or lipopolysaccharides) on the surface of Acinetobacter baumannii through its tail structure. Even drug-resistant strains that have undergone antibiotic target mutations can still be recognized and infected by this bacteriophage as long as their surface receptors have not changed.

[0017] Independent replication mechanism: Bacteriophages use their own DNA polymerase (SEQ ID NO.1) and host resources to replicate efficiently. This process does not depend on bacterial cell wall synthesis or protein synthesis pathways, and is therefore not affected by bacterial resistance mechanisms to traditional antibiotics (such as β-lactams and aminoglycosides).

[0018] Enzyme-mediated cell lysis: In the late stage of the phage proliferation cycle, by expressing lysis system proteins (such as perforin and endosomalase), the integrity of the bacterial cell membrane is disrupted and the peptidoglycan layer is degraded, leading to an imbalance of intracellular osmotic pressure and bacterial disintegration and death, thereby achieving the elimination of drug-resistant bacteria.

[0019] Thirdly, the present invention provides a bactericidal and disinfectant preparation, the active ingredient of which includes the aforementioned Acinetobacter baumannii phage AB4P4, and the phage content in the preparation is at least 10. 7 PFU / mL.

[0020] Preferably, the bactericidal disinfectant is used to remove biofilms formed by Acinetobacter baumannii.

[0021] By adopting the above technical solution, this bactericidal disinfectant has advantages in removing biofilms: Biofilm permeability: Acinetobacter baumannii readily forms biofilms mainly composed of extracellular polysaccharides, DNA, and proteins, hindering the penetration of chemical disinfectants. The nanoscale size of bacteriophage AB4P4 particles allows them to enter the biofilm through its water channels.

[0022] Matrix degradation mechanism: The phage genome encodes enzymes with polysaccharide depolymerization activity (such as hypothesized chitinase or caudate depolymerase), which can specifically degrade the extracellular polysaccharide matrix (EPS) of biofilms and disrupt the physical structure of biofilms.

[0023] Deep sterilization mechanism: While degrading the substrate, the bacteriophage infects dormant or persistent bacteria deep in the biofilm. By lysing the bacteria, it releases progeny bacteriophages, which continue to infect surrounding bacteria, creating a cascade amplification effect. This disintegrates the biofilm system from the inside out, solving the problem that traditional chemical disinfectants are unable to remove bacteria from biofilms.

[0024] Fourthly, the present invention provides an application of Acinetobacter baumannii phage in a food matrix for reducing or inhibiting Acinetobacter baumannii contamination, wherein the food matrix includes meat, dairy products or other foods susceptible to Acinetobacter baumannii contamination.

[0025] By adopting the above technical solution, the following effects are achieved: Precise targeting: Bacteriophage AB4P4 specifically lyses Acinetobacter baumannii without destroying the original beneficial bacteria (such as lactic acid bacteria) or sensory quality in food, and does not change the flavor and texture of food.

[0026] No residual toxicity: This bacteriophage is mainly composed of protein and nucleic acid. After completing the sterilization task, it is ingested with food and degraded into amino acids and nucleotides by enzymes in the digestive tract. It is non-toxic and harmless to the human body, providing a green and safe means of food preservation.

[0027] This invention provides a strain of Acinetobacter baumannii bacteriophage and its applications. It has the following beneficial effects: 1. The Acinetobacter baumannii phage AB4P4 provided by this invention belongs to the family Brachyphageidae. Its genome encodes an autonomous DNA polymerase and specific lysis system proteins, and its replication mechanism is independent of the host. It exhibits high fermentation titer, demonstrating high proliferation efficiency and bactericidal efficacy. This characteristic enables AB4P4 to overcome existing bacterial resistance mechanisms, effectively lysing Acinetobacter baumannii resistant to multiple antibiotics such as carbapenems and aminoglycosides, thereby reducing the bacterial load at the infection site or in the environment.

[0028] 2. This invention addresses the problem of Acinetobacter baumannii easily forming biofilms, leading to the ineffectiveness of conventional disinfectants. Bacteriophages, with their nanoscale structural size and specific lysis mechanism, can effectively penetrate and cross biofilm barriers. By specifically infecting and lysing bacteria deep within the biofilm while simultaneously disrupting the extracellular matrix that maintains the biofilm structure, AB4P4 can dismantle mature biofilm systems from the inside, eliminating persistent bacteria that are difficult for conventional chemical reagents to reach. This provides a powerful means for deep cleaning of medical environments and food substrates.

[0029] 3. Based on whole-genome sequencing analysis, this invention shows that the genome of bacteriophage AB4P4 is double-stranded linear DNA, containing no integrase genes or tRNA genes, and no known virulence factors, drug resistance genes, or pathogenic genes were detected. This genomic characteristic confirms at the molecular level that AB4P4 is a strictly virulent bacteriophage, eliminating the risk of lysogeny or the mediating of horizontal transfer of drug resistance genes during its application, thus ensuring its safety and reliability as a bactericidal agent in clinical, environmental, and food applications. Attached Figure Description

[0030] Figure 1 This is a diagram showing the plaque formation results of phage AB4P4 of the present invention in the double-layer plate method; Figure 2 This is a transmission electron microscope image of the bacteriophage AB4P4 of the present invention. Figure 3 This is a genome annotation diagram of phage AB4P4 of the present invention; Figure 4 This is a phylogenetic tree analysis result of the genome of bacteriophage AB4P4 of the present invention; Figure 5 The graph shows the optimal multiple of infection (MOI) determination results for phage AB4P4 of the present invention; Figure 6 This is a graph showing the one-step growth curve determination results of the bacteriophage AB4P4 of the present invention. Figure 7 This is a graph showing the results of the thermal stability test of the bacteriophage AB4P4 of the present invention; Figure 8 This is a graph showing the pH tolerance test results of bacteriophage AB4P4 of the present invention; Figure 9 This is a diagram illustrating the evaluation of biofilm inhibition using the viable cell counting method of the present invention. Figure 10 This is a diagram illustrating the evaluation of biofilm inhibition using the crystal violet staining method of the present invention; Figure 11 This diagram illustrates the evaluation of biofilm clearance using the viable bacteria counting method of the present invention. Figure 12This is a diagram illustrating the evaluation of biofilm clearance using the crystal violet staining method of the present invention. Figure 13 This is a comparison diagram of phage treatment and SM buffer treatment in chicken breast samples at 4℃ according to the present invention; Figure 14 This is a comparison diagram of phage treatment and SM buffer treatment in milk samples at 4°C according to the present invention; Figure 15 This is a comparison diagram of phage treatment and SM buffer treatment in chicken breast samples at 25°C according to the present invention; Figure 16 This is a comparison diagram of phage treatment and SM buffer treatment in milk samples at 25°C according to the present invention. Detailed Implementation

[0031] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all 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.

[0032] Preparation example: Resuscitation, propagation, and identification of Acinetobacter baumannii strains: Use a sterile inoculating loop to pick up the sample stored in the glycerol cryopreservation solution. Acinetobacter baumannii AB4 strain was streaked on LB solid medium and incubated overnight at 37°C. Single colonies were picked and purified for three generations to obtain morphologically homogeneous pure cultures. The purified single colonies were inoculated into LB liquid medium and incubated at 37°C with shaking at 180 rpm for about 12 hours to obtain bacterial suspensions in the logarithmic to stationary phases for subsequent experiments.

[0033] The strain was identified before the experiment. First, a preliminary determination was made based on colony morphology and routine physiological and biochemical characteristics; then, bacterial genomic DNA was extracted, and 16S rRNA gene sequencing was used to further confirm the species of the strain.

[0034] Example: Isolation and purification of Acinetobacter baumannii bacteriophage: Wastewater samples were collected from wastewater treatment plants and surrounding sewage wells in Chongqing and immediately returned to the laboratory. Samples were filtered through four layers of sterile gauze to remove large particulate impurities, and then centrifuged at 4°C and 10,000 rpm for 20 min. The resulting supernatant was filtered through a 0.22 μm microporous membrane to remove bacteria and suspended particles. The filtrate was stored at 4°C and used as a phage source. 30 mL of the treated filtrate was added to 30 L of 1 M CaCl2, 30 L of 1 M MgCl2, 15 mL of sterile LB medium, and 2 mL of logarithmic growth medium. Acinetobacter baumannii AB4 bacterial culture (obtained from the preparation example, approximately 110) 9 (CFU / mL). After mixing, incubate at 37℃ and 180 rpm with shaking for 8 h. Centrifuge the culture at 10000 rpm for 10 min, collect the supernatant and filter through a 0.22 μm filter membrane. The enrichment process is repeated three times to increase the phage concentration, finally obtaining the phage enrichment solution.

[0035] Separation and purification were performed using the double-layer agar plate method. 100 μL of enrichment solution was mixed with 100 μL of logarithmic growth phase AB4 bacterial culture (approximately 110 μL). 9 Mix (CFU / mL) and add 5 mL of pre-melted LB semi-solid agar (0.5%, w / v). Invert the plate onto the surface of a culture dish with a bottom layer of LB solid agar (1.0%, w / v). Incubate the plate at 37°C for 12–16 h and observe plaque formation. Pick a single plaque with clear edges and transfer it to a centrifuge tube containing 1 mL of SM buffer (2 g / L MgSO4·7H2O, 5.8 g / L NaCl, 50 mL / L 1 M Tris-HCl, pH 7.5). Mix well and filter through a 0.22 μm filter. Repeat the purification process for 5 rounds to obtain plaques with uniform morphology and regular edges. Store the purified phage suspension at 4°C for a short period.

[0036] To obtain high-purity phage, 100 mL of phage stock solution was taken, and DNase I and RNase A (final concentration 1 g / mL) were added. The solution was incubated at 37°C for 30 min to remove residual nucleic acid. NaCl was then added to a final concentration of 1 M, and the solution was incubated at 4°C for 1 h. PEG8000 was then added to a final concentration of 10% (w / v), and the solution was incubated at 4°C for 12 h. After precipitation, the solution was centrifuged at 10,000 rpm for 20 min at 4°C, the supernatant was discarded, and the precipitate was resuspended in 1 mL of SM buffer. An equal volume of chloroform was added to the resuspended solution, and the mixture was gently mixed to separate the layers. The solution was centrifuged at 5,000 rpm for 10 min at 4°C, and the aqueous phase was collected as the high-purity phage solution. For short-term storage, 4°C was maintained; for long-term storage, 50% (v / v) glycerol was added and the solution was incubated at -80°C. To ensure enrichment stability, the above procedures were repeated three times in independent batches, and the results were consistent. After screening, a high-titer Acinetobacter baumannii phage with a broad lytic spectrum was obtained and named AB4P4.

[0037] Test Examples 1-6: Test Example 1: Morphological observation, titer and lysis spectrum determination of bacteriophage AB4P4 First, phage titer was determined: The phage AB4P4 enrichment solution obtained in Example 1 was serially diluted 10-fold using SM buffer (10... -1 Up to 10 -10 Take 100 μL of each dilution and mix it with 100 μL of B4 bacterial culture in the logarithmic growth phase (approximately 10 μL of total volume). 8 The mixture (CFU / mL) was incubated at 37°C and 180 rpm for 30 min in a constant-temperature shaker to promote phage adsorption. Then, 5 mL of pre-dissolved LB semi-solid medium was added, mixed thoroughly, and immediately poured into plates pre-layered with LB agar. After incubation at 37°C for 12-16 h, the number of clearly identifiable plaques was observed and counted, and the phage titer was calculated based on the dilution factor. Phage titer calculation: Phage titer (PFU / mL) = Average number of plaques on 3 parallel double-layer plates (selecting 30 plaques = 300) / Dilution gradient 10 (results retained to three significant figures).

[0038] The results are as follows Figure 1 As shown, phage AB4P4 forms clear plaques. The titer of liquid-propagated Acinetobacter baumannii phage AB4P4 was 410 as determined by the double-layer plate method. 11 PFU / mL.

[0039] Next, phage electron microscopy was performed: Take 10L of high-titer phage storage solution (>10 9 PFU / mL was added dropwise to the surface of a copper grid coated with a 200-mesh carbon membrane and allowed to stand at room temperature for 10 min to promote adsorption. Then, 2% (w / v) sodium phosphotungstenate solution was added dropwise for negative staining. After standing for approximately 30 s, excess stain was blotted away with fibrous filter paper and the phage was allowed to air dry. The phage head and tail structures were observed and recorded under a transmission electron microscope.

[0040] The results are as follows Figure 2 As shown, the head of Acinetobacter baumannii phage AB4P4 has a spherical or near-spherical icosahedral structure with a compact short tail structure and no obvious tail fibers. It is classified as a phage with tails, belonging to the order Caudata and family Baudata. The head diameter is 32 nm and the tail length is 11 nm.

[0041] Finally, phage lysis profiles were determined. The test strains included 64 strains of Acinetobacter baumannii (…). Acinetobacter baumannii ), 12 strains of Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), 12 strains of Escherichia coli ( Escherichia coli ), 12 strains of Klebsiella pneumoniae ( Klebsiella pneumoniae ), 6 strains of Staphylococcus aureus ( Staphylococcus aureus ) and 3 strains of Enterococcus faecalis ( Enterococcus faecalis All strains were derived from samples and environmental samples and stored at -80℃ (containing 20% ​​v / v glycerol). Each strain was cultured in LB medium to the logarithmic growth phase (OD). 600 0.5, approximately 10 8 CFU / mL). Mix 200 L of bacterial culture with 5 mL of LB semi-solid medium containing 0.5% (w / v) agar, gently mix, and pour into the bottom layer of an LB solid plate containing 1.0% (w / v) agar. Let stand for 5 min until solidified. Then, take 5 L of phage storage solution (10 9 A sample of PFU / mL was spotted onto the surface of an agar plate, and after air drying, the plate was inverted and incubated at 37°C for 12–16 h. An equal volume of SM buffer was used as a negative control. The formation of clear plaques was observed to determine lysis activity. The results showed that the Acinetobacter baumannii phage AB4P4 could lyse 13 strains of Acinetobacter baumannii, with a lysis rate of 20.31% (13 / 64).

[0042] Test Example 2: Genome sequencing and analysis of bacteriophage AB4P4 FastP was used for quality control of the raw sequencing data, including splicing adapter sequences and removing low-quality reads and high N-content reads to obtain high-quality clean reads. MetaSPAdes software was used to perform de novo assembly of the clean reads, with different k-mer lengths tested to obtain the optimal assembly results. Then, BWA software was used to align the clean reads to the assembled genome sequence for statistical coverage. RAS and Prokka were used to annotate coding genes and tRNAs in the genome. BlastoP was then used to align protein sequences with the NR database to obtain sequence information with high similarity for each gene in the NR database. EggNOG-mapper26 was used for functional annotation of genes, including annotation information from COG, GO, KEGG, CAZy, BiGG, and PFAM databases. Phage lifecycles were predicted using PhageAI. Blaster was used to search VFDB and ResFinder to identify potential virulence and resistance genes. tRNA Scan-SE29 was used to scan for possible tRNA genes in the phage genome. A phylogenetic tree was constructed using MEGA7 software based on maximum likelihood, using default model parameters. VIRIDIC was used to calculate the pairwise genome distance / similarity of bacteriophage genomes.

[0043] Genome feature analysis showed that the genome of Acinetobacter baumannii phage AB4P4 is double-stranded linear DNA, with a genome size of 41020 bp and a G+C content of 39.37%.

[0044] like Figure 3 As shown in the figure, the outermost blue arrows represent coding genes (CDS), and the direction of the arrows indicates the transcription direction; the middle black wavy circle represents GC content; and the innermost purple and green waves represent GC skew. Analysis of the AB4P4 phage genome predicted 196 open reading frames (ORFs), and annotation confirmed that 50 of these ORFs have a clear function, all located on the sense strand. Combined with... Figure 3 The English annotations in the text indicate that the functions of these 50 encoded proteins are categorized as follows: Structural and assembly proteins: including capsid protein (ORF60), tail protein, scaffolding protein, head-to-tail connector protein, and internal virion protein A / B, etc.

[0045] DNA replication and modification proteins include DNA polymerase I (ORF98), DNA helicase (ORF96), DNA primase, and DNA ligase.

[0046] Host cleavage proteins include holin / anti-holin, internal virion protein with endolysin domain, and putative chitinase.

[0047] Transcription and packaging proteins: including DNA-directed RNA polymerase, DNA-binding proteins, and large / small terminal enzyme subunits.

[0048] Furthermore, no tRNA genes, integrase genes, virulence factors, drug resistance genes, or pathogenicity-related genes were detected in the AB4P4 genome, suggesting that it has good application safety.

[0049] Phylogenetic analysis, using NCBI BLASTn alignment, showed that AB4P4 phage shared 95.64% nucleotide similarity and 90% sequence coverage with Acinetobacter baumannii phage SWHAb1 (NC_047896.1). Considering its genome length (41,020 bp), structure (circular double-stranded DNA), and functional annotation, although P4 is highly similar to SWHAb1, its sequence is below the phage species threshold (>98-99%), suggesting that P4 may belong to a novel strain within the genus Friunavirus of the family Autographiviridae.

[0050] like Figure 4 As shown, phylogenetic tree analysis further supports this conclusion: P4 clusters with other members of the Friunavirus genus in the same lineage and has not formed an independent evolutionary branch, indicating that it belongs to the genus but has independent evolutionary characteristics.

[0051] Analysis of key proteins revealed that the tail filament protein of the bacteriophage determines its host range and cleavage profile. The amino acid sequence of one tail filament protein in this bacteriophage showed significant differences from those of a well-known bacteriophage with high similarity, with a maximum similarity of 73.7% (tail filament protein amino acid sequence shown in SEQ ID NO. 1). The holin protein of the bacteriophage determines its host cleavage ability; its amino acid sequence also showed significant differences from those of a well-known bacteriophage with high similarity, with a maximum similarity of 95.7% (holin amino acid sequence shown in SEQ ID NO. 2).

[0052] Test Example 3: Determination of the growth characteristics of bacteriophage AB4P4 Determine the optimal multiple of infection (MOI) for bacteriophages: The phage storage solution was mixed with Acinetobacter baumannii B4 bacterial culture in the logarithmic growth phase (OD). 600 0.5, approximately 10 8 Phage titers (CFU / mL) were mixed at different MOI gradients (0.001, 0.01, 0.1, 1, 10, 100, 1000), with each tube containing 2 mL of LB medium. The mixtures were incubated at 37°C with shaking at 180 rpm for 6 h. Subsequently, phage titers (PFU / mL) were determined using the double-layer agar plate method. Each MOI was set up in triplicate, and the experiment was independently repeated 5 times. The average titer for each group was calculated. The MOI corresponding to the highest titer was defined as the optimal multiplicity of infection under those conditions.

[0053] The results are as follows Figure 5 As shown, the optimal MOI for this Acinetobacter baumannii phage AB4P4 is 0.01, and the maximum titer is 4.010. 11 PFU / mL.

[0054] Determine the one-step growth curve of bacteriophages: Take 5 mL of Acinetobacter baumannii B4 bacterial culture in the logarithmic growth phase (OD) 600 0.5, approximately 10 8 The phage stock solution (CFU / mL) was mixed with the phage stock solution at the optimal multiple of infection (MOI), and thoroughly vortexed before incubation at 37°C for 15 min to promote adsorption. After adsorption, the mixture was centrifuged at 12,000 g for 2 min at 4°C, the supernatant was discarded, and the precipitate was washed three times with 5 mL of preheated LB medium to remove unadsorbed phage. The washed precipitate was resuspended in 5 mL of preheated LB medium and incubated at 37°C with shaking at 180 rpm. Samples of 200 μL were taken at time points of 0, 5, 10, 15, 20, 25, 30, 40, 60, 90, and 100 min, centrifuged at 12,000 g for 2 min at 4°C, and the supernatant was serially diluted 10-fold. The phage titer (PFU / mL) was determined using the double-layer agar plate method. Each time point was set up in triplicate, and the experiment was independently repeated 5 times. The average value was taken. The phage titer (log) was used as the statistical unit of measurement. 10 A one-step growth curve was plotted with PFU / mL as the ordinate and incubation time (min) as the abscissa, and the latent period and lysis period were analyzed accordingly. Lysis amount = (total PFU produced - initial PFU) / number of adsorbed bacteria.

[0055] The results are as follows Figure 6 As shown, the incubation period of Acinetobacter baumannii phage AB4P4 is about 20 minutes, and the burst amount is about 126 PFU / cell.

[0056] Test Example 4: Stability determination of bacteriophage AB4P4 Thermal stability tests were conducted. Take 1 mL of phage storage solution (10 9 Phage stock solutions (PFU / mL) were incubated in water baths at 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃ for 1 hour. Immediately after incubation, the solutions were rapidly cooled in an ice bath, followed by serial dilutions. The phage titer (PFU / mL) was determined using the double-layer agar plate method. The relative survival rate (%) was calculated using phage stock solutions incubated at 37℃ as a control.

[0057] The results are as follows Figure 7 As shown, the Acinetobacter baumannii phage AB4P4 maintained high lytic activity (titer >8.0 log) at temperatures ranging from 30 to 50°C. 10 (PFU / mL). The potency at 60℃ was 7.820.06 log₂O. 10 PFU / mL.

[0058] pH stability was measured. Prepare SM buffer solutions with different pH values ​​(2.0-12.0, in 1.0 increments), and adjust the pH using 1M HCl or 1M NaOH. Take 100L of phage storage solution (10 9 Phage stock solutions (PFU / mL) were mixed with 900 μL of SM buffer at different pH values ​​(volume ratio 1:9) and incubated at 37 °C for 1 h. After incubation, the solutions were diluted and the phage titer (PFU / mL) was determined using the double-layer agar plate method. A phage stock solution incubated with SM buffer at pH 7.0 was used as a control, and the relative survival rate (%) was calculated. Both thermal stability and pH stability experiments were performed in triplicate, with five replicates per experiment, and the average value was taken.

[0059] The results are as follows Figure 8 As shown, the Acinetobacter baumannii phage AB4P4 maintained high lytic activity (titer >8.0 log) across pH ranges from 3.0 to 11.0. 10 (PFU / mL), and performed exceptionally well under extreme pH conditions, with potencies of 5.73 and 0.24 log at pH 2.0 and 12.0, respectively. 10 PFU / mL and 5.870.18 log 10 PFU / mL.

[0060] Test Example 5: Inhibition and Scavenging Effects of Acinetobacter baumannii Bacteriophage on Biofilms Biofilm inhibition was assessed using a 12-well plate crystal violet staining method and a viable cell count method. Take 200L of logarithmic growth phase B4 bacterial culture (OD) 600 0.5, approximately 10 8 CFU / mL), add 100L of phage P4 stock solution (10 9 PFU / mL was mixed with the optimal multiple of infection (MOI) and brought to a final volume of 2 mL with fresh LB medium. The mixture was then inoculated into 12-well polystyrene plates and incubated at 37°C for 48 h. The control group was treated with 100 μL of PBS instead of phages. After incubation, the supernatant was removed, and the cells were gently washed three times with PBS. The OD was measured following the fixation, staining, and lysis procedures. 590 Absorbance. The inhibitory effect of bacteriophages on bacteria in biofilms was evaluated using a viable cell count method. After cultivation, the biofilm formed in each well was retained and resuspended in 2 mL of sterile PBS, repeatedly pipetting. The resulting suspension was transferred to 1.5 mL EP tubes and serially diluted 10-fold. 100 μL of each dilution was inoculated onto LB agar plates and incubated overnight at 37°C. Colony counts were performed on plates with colony counts ranging from 30 to 300, and the viable cell count (CFU / mL) was calculated.

[0061] The result of inhibition is as follows Figure 9 and Figure 10 As shown in the figure. CFU count results showed that the viable bacterial count in the biofilm of the control group (untreated) was 2.66710. 15 CFU / mL, decreased to 1.8410 in the treatment group. 9 CFU / mL (p<0.001), showing significant antibacterial effect. OD 590 The test results also showed that the biofilm content in the treatment group (3.247) was reduced by about 17.2% compared with that in the control group (3.921) (p<0.001).

[0062] The scavenging effect of bacteriophage P4 on mature biofilms was assessed using crystal violet staining and viable cell counting in 12-well plates. 200 μL of logarithmic growth phase B4 bacterial culture (OD200) was collected. 600 0.5, approximately 10 8 Add 2 mL of LB medium to each well of a 12-well polystyrene plate (Corning, USA) and incubate at 37°C for 48 h to form a mature biofilm. Remove the supernatant and wash three times with PBS, then invert and dry in a 60°C oven for 15 min. Subsequently, add 2 mL of fresh LB medium and 100 μL of phage P4 stock solution (10 CFU / mL) to each well. 9 PFU / mL (at optimal MOI), incubated at 37℃ for 4, 8, 12, and 16 h. The control group was treated with 100 μL of PBS instead of phage. After each time point, the supernatant was removed, the cells were washed three times with PBS, and the OD was determined using the crystal violet staining method. 590 Absorbance. Meanwhile, another biofilm sample was taken, resuspended in 1 mL of PBS and thoroughly vortexed, serially diluted 10-fold, and then plated onto LB agar plates. The plates were incubated at 37°C for 16 h, and the viable bacterial count (CFU / mL) was determined.

[0063] The results of the cleansing action are as follows Figure 11 and Figure 12 As shown. The results show that at 12h, OD 590 The value decreased by 91% (from 3.785 to 0.356, p<0.001), and CFU decreased by 3.45 log. 10 (From 10.74 to 7.29, p<0.05), indicating that P4 had the most significant scavenging effect on both biofilm biomass and viable bacteria at this time point.

[0064] Test Example 6: Validation of the antibacterial effect of bacteriophage AB4P4 in food samples To evaluate the antibacterial potential of bacteriophage AB4P4 in a real food environment, a validation experiment was conducted to assess its ability to control multidrug-resistant Acinetobacter baumannii (MDR-AB) biofilms in fresh chicken breast and pasteurized milk. Fresh chicken breast and commercially available pasteurized milk were purchased from a local supermarket and transported on ice in aseptic polyethylene bags. Chicken pieces (approximately 1g each) were wiped with 75% ethanol and treated with UV irradiation for 30 min. Milk samples were centrifuged at 10000g for 10 min to remove the fat layer and filtered through a 0.22µm filter membrane.

[0065] Adjust the MDR-AB4 bacterial culture to 10. 5 CFU / mL, 100 L of bacterial culture was added to each piece of chicken or 10 mL of milk, and incubated at room temperature for 30 min to promote bacterial adhesion. Then, phage AB4P4 (MOI=1000) was added, while the control group received an equal volume of SM buffer. Samples were incubated at 4℃ or 25℃, and collected at 0, 3, 6, 9, 12, and 24 h. Chicken samples were homogenized in sterile tubes containing 9 mL of PBS and then serially diluted for plate counting; milk samples were directly serially diluted and then plated. Colony counts were recorded after 24 h of incubation, expressed as log CFU / g (chicken) or log CFU / mL (milk). All experiments were performed in triplicate.

[0066] like Figure 13 , Figure 14 As shown, at 4°C, the bacterial count in the phage-treated group decreased by approximately 1.6 log CFU / g and 1.15 log CFU / mL in chicken and milk samples, respectively, within 24 hours, while the bacterial count in the control group remained relatively stable. This indicates that the phage can continuously inhibit MDR-AB under refrigeration conditions. Figure 15 , Figure 16 As shown, at 25℃, the bacterial count in the control group increased, while the count in the phage-treated group showed a continuous decreasing trend. Ultimately, the bacterial count in the chicken sample decreased by approximately 1.6 log CFU / g within 24 hours, and the bacterial concentration in the milk sample decreased rapidly within 9 hours and remained at a low level. These results indicate that phage AB4P4 can effectively inhibit the growth of MDR-AB in different food matrices under both refrigeration and mildly heated conditions. In summary, phage AB4P4 can stably exert antibacterial effects in typical food matrices such as chicken and milk, and has potential application value as a food preservative, biofilm control agent, or food safety intervention technology.

[0067] Appendix: The amino acid sequence of the tail filament protein is SEQ ID NO.1: MALYRRGTASMDADG. The amino acid sequence of the perforin is SEQ ID NO.2: MNIIDQFYAVLIYVWSGLDKLIVGAAATSFVVALLRTKKEDNKFSFIEALLCGIFTAIALVGMSFLGTLTGIIVPATLTAGAAHVVAGFIGWYGTVRTMKYLEGKVSNDSD.

Claims

1. A strain of Acinetobacter baumannii phage, characterized in that, It was named Acinetobacter baumannii phage AB4P4 and is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 20252580.

2. The Acinetobacter baumannii phage according to claim 1, characterized in that, The phage of Acinetobacter baumannii has a titer of 4.010 11 PFU / mL, the head is spherical or spheroidal icosahedral structure with a compact short tail structure, no obvious tail fiber, classified as the Caudovirales, the Podoviridae, the head diameter is 32 nm, and the tail length is 11 nm.

3. The Acinetobacter baumannii phage according to claim 1, characterized in that, The genome of the Acinetobacter baumannii phage is a double-stranded linear DNA. The genome size of the Acinetobacter baumannii phage is 41020 bp, the G+C content is 39.37%, and a total of 50 open reading frames with clear functions are annotated, all of which are located on the justice chain. The Genbank accession number is PX270320.

4. The Acinetobacter baumannii phage according to claim 3, characterized in that, The nucleotide sequence of the DNA polymerase of the Acinetobacter baumannii phage is SEQ ID NO.

1.

5. The Acinetobacter baumannii phage according to claim 4, characterized in that, The nucleotide sequence of the major capsid protein of the Acinetobacter baumannii phage is SEQ ID NO.

2.

6. An application of Acinetobacter baumannii bacteriophage, characterized in that, The use of the Acinetobacter baumannii phage according to any one of claims 1 to 5 in lysing Acinetobacter baumannii or in preparing compositions for lysing Acinetobacter baumannii.

7. The application of Acinetobacter baumannii phage according to claim 6, characterized in that, Lysed Acinetobacter baumannii include Acinetobacter baumannii resistant to aminoglycosides, penicillins, cephalosporins, carbapenems, tetracyclines, fluoroquinolones, or sulfonamides.

8. A bactericidal and disinfectant preparation, characterized in that, The Acinetobacter baumannii bacteriophage according to any one of claims 1 to 5, wherein the content of the Acinetobacter baumannii bacteriophage in the disinfectant is at least 10 7 PFU / mL.

9. A bactericidal and disinfectant preparation according to claim 8, characterized in that, The bactericidal and disinfectant preparation is used to remove biofilms formed by Acinetobacter baumannii.

10. An application of Acinetobacter baumannii bacteriophage in the field of food safety, characterized in that, The application of Acinetobacter baumannii phage according to any one of claims 1 to 5 in a food matrix for reducing or inhibiting Acinetobacter baumannii contamination, wherein the food matrix includes meat, dairy products or other foods susceptible to Acinetobacter baumannii contamination.

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