Adhaeribacter dacunhae bacteriophage vB_adhP_l12 and application thereof

Abstract

The application belongs to the technical field of microorganisms, and provides a daka aeromonas bacteriophage vB_AdhP_L12 and an application thereof. The taxonomic name of a daka aeromonas bacteriophage vB_AdhP_L12 is daka aeromonas bacteriophage vB_AdhP_L12 Aeromonas dhakensis The daka aeromonas bacteriophage vB_AdhP_L12 has a preservation number of CCTCC NO: M 2026249, and was preserved in the China Center for Type Culture Collection (CCTCC) on January 26, 2026. The application enriches the treatment strategy for daka aeromonas, and lays a foundation for inhibiting daka aeromonas disease infection in crocodile breeding.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to a strain of Aeromonas dacca bacteriophage vB_AdhP_L12 and its applications. Background Technology

[0002] In recent years, the large-scale development of my country's crocodile farming industry has accelerated. Acute septicemia has become a major disease restricting the high-quality development of this industry. Its rapid onset, rapid spread, and high mortality rate have caused huge economic losses to crocodile farming and have become a core focus of disease prevention and control in crocodile farming. Previous isolation, purification, and molecular biological identification of the pathogen have confirmed that outbreaks of this type of acute septicemia are caused by bacterial infection, with Aeromonas dacarbazina (…) being a key pathogen. Aeromonas dhakensis It is one of the main pathogens that induce acute septicemia in crocodiles.

[0003] Currently, the prevention and control of bacterial septicemia in farmed crocodiles still relies primarily on antibiotics. However, the Ministry of Agriculture and Rural Affairs has approved a limited range of antibiotics specifically for aquaculture. Furthermore, the non-standard and irrational use of antibiotics in production practices not only easily induces drug resistance in pathogens, leading to a significant decrease in drug efficacy and a continuous increase in dosage, but also causes a series of problems such as pollution of aquatic environments and excessive drug residues in aquaculture products. This not only violates the development requirements of green aquaculture but also poses a potential threat to the quality and safety of aquatic products and the ecological environment.

[0004] Therefore, developing efficient, safe, and environmentally friendly green prevention and control technologies to replace traditional antibacterial drugs for the prevention and treatment of acute septicemia caused by Aeromonas dilatatus in farmed crocodiles has become a key issue that the crocodile farming industry urgently needs to address. Summary of the Invention

[0005] This invention aims to provide a strain of Aeromonas dacarinae bacteriophage vB_AdhP_L12 and its applications, enriching the treatment strategies for Aeromonas dacarinae and laying the foundation for inhibiting Aeromonas dacarinae infection in crocodile farming.

[0006] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows:

[0007] This invention provides a strain of Aeromonas dacarbazin vB_AdhP_L12, taxonomically named Aeromonas dacarbazin vB_AdhP_L12. Aeromonas dhakensis Phage vB_AdhP_L12, with accession number CCTCC NO: M2026249, was deposited at the China Center for Type Culture Collection (CCTCC) on January 26, 2026.

[0008] Furthermore, the head of the Dacao Aeromonas phage vB_AdhP_L12 exhibits icosahedral symmetry, with an average head diameter of 50.03 ± 2.50 nm. The phage contains a non-contractile tail with an average tail length of 13.39 ± 2.05 nm and an average diameter of 12.55 ± 1.68 nm.

[0009] Furthermore, the nucleic acid type of the Aeromonas dacca phage vB_AdhP_L12 is linear double-stranded DNA, with a full length of 38749 bp, containing 42 open reading frames, and without antibiotic resistance genes, virulence factors, or lysogen-related genes.

[0010] The present invention also provides a phage preparation comprising the above-mentioned Aeromonas dacarbazin vB_AdhP_L12 phage.

[0011] The present invention further provides the use of the above-mentioned phage preparation in the preparation of a drug for the prevention and treatment of Aeromonas dacarba infection.

[0012] The beneficial effects of this invention are as follows:

[0013] This invention utilizes previously isolated and enriched bacteriophages, and through a series of biological characteristic analyses and gene sequencing, identifies a novel highly lytic bacteriophage, vB_AdhP_L12, which specifically infects *Aeromonas dacca*. Morphological results show that bacteriophage vB_AdhP_L12 can form distinct plaques on double-layer plates; transmission electron microscopy results show that this bacteriophage belongs to the Brachyceridae family; the optimal multiplicity of infection is 0.01; one-step growth experiments show that the bacteriophage has an extremely short latency period of less than 15 min, exhibits good stability under conditions of -20°C to 50°C and pH 4 to 11, and can withstand treatment with 0.2% chloroform. The bacteriophage's nucleic acid type is double-stranded DNA, and whole-genome sequencing results show that the bacteriophage is 38749 bp in length, contains 42 open reading frames, and has no antibiotic resistance genes, virulence factors, or lysogen-related genes, indicating high biocompatibility. Phylogenetic analysis revealed that this phage belongs to a new branch of the genus *Teseptimavirus*. Furthermore, zebrafish animal model experiments showed that treatment with phage vB_AdhP_L12 improved zebrafish survival by 63.33%, indicating that phage vB_AdhP_L12 has significant application value and therapeutic potential. Simultaneously, heatmaps demonstrated effective repair of intestinal tissue pathological damage. vB_AdhP_L12 also modulates the host immune response, inhibiting the overexpression of pro-inflammatory factors and upregulating the levels of anti-inflammatory factors and specific antibodies. In conclusion, phage vB_AdhP_L12 possesses advantages such as strong host specificity, good environmental adaptability, biosafety, and significant in vitro and in vivo therapeutic effects. It provides a novel technological prototype for the green control of *Aeromonas daca* infection in crocodile farming and represents a highly valuable antibiotic alternative for translational applications, playing a crucial role in alleviating the antibiotic resistance crisis and ensuring the safety of aquaculture. This invention enriches the treatment strategies for Aeromonas dacca, laying the foundation for the safe and efficient suppression of bacterial disease outbreaks in crocodile farming. Attached Figure Description

[0014] Figure 1 Plaque morphology of bacteriophage vB_AdhP_L12.

[0015] Figure 2 Transmission electron microscopy image of bacteriophage vB_AdhP_L12.

[0016] Figure 3 Determination of the optimal multiplicity of infection for bacteriophage vB_AdhP_L12.

[0017] Figure 4 Determination of the in vitro antibacterial activity of bacteriophage vB_AdhP_L12.

[0018] Figure 5 One-step growth curve determination of bacteriophage vB_AdhP_L12.

[0019] Figure 6 Determination of the thermal stability of bacteriophage vB_AdhP_L12.

[0020] Figure 7 pH stability determination of bacteriophage vB_AdhP_L12.

[0021] Figure 8 Chemical reagent sensitivity determination of bacteriophage vB_AdhP_L12.

[0022] Figure 9 A circos diagram of the genome of bacteriophage vB_AdhP_L12.

[0023] Figure 10 A circular proteomic phylogenetic tree representing the genome of bacteriophage vB_AdhP_L12.

[0024] Figure 11 Genome homology alignment analysis: Figure A shows the genome alignment results of vB_AdhP_L12 with Escherichia phage Ebrios (NC_047942.1); Figure B shows the genome alignment results of vB_AdhP_L12 with Stenotrophomonas phage IME15 (NC_019416.1); Figure C shows the genome alignment results of vB_AdhP_L12 with Aeromonas phage vB_AroA_KFSA9 (PQ824399.1); Figure D shows the genome alignment results of vB_AdhP_L12 with Aeromonas phage PZL-Ah1 (Aeromonas phage PZL-Ah1). The genome alignment results of PZL-Ah1 (MT681669.1) are shown in Figure E; the genome alignment results of vB_AdhP_L12 and Aeromonas phage avDM11-UST (OP380607.1) are shown in Figure E.

[0025] Figure 12 Heatmap of the evolutionary relationship between bacteriophage vB_AdhP_L12 and Aeromonas bacteriophage.

[0026] Figure 13 Phylogenetic evolutionary tree of the large subunit of the terminal enzyme of bacteriophage vB_AdhP_L12.

[0027] Figure 14 Phylogenetic evolutionary tree of major tail tube proteins of bacteriophage vB_AdhP_L12.

[0028] Figure 15 Phylogenetic evolutionary tree of major capsid proteins of bacteriophage vB_AdhP_L12.

[0029] Figure 16 Fluorescence scanning electron microscopy results of Caco-2 cells treated with bacteriophage vB_AdhP_L12.

[0030] Figure 17 Fluorescence scanning electron microscopy results of EPC cells treated with bacteriophage vB_AdhP_L12.

[0031] Figure 18 Survival curve results of zebrafish infected with Aeromonas dacca.

[0032] Figure 19 Survival curve results of zebrafish infected with Aeromonas dacca treated with bacteriophage vB_AdhP_L12.

[0033] Figure 20 The content of free bacteria in Caco-2 cells after treatment with bacteriophage vB_AdhP_L12, **** indicates p<0.0001.

[0034] Figure 21 The content of free bacteria in EPC cells after treatment with bacteriophage vB_AdhP_L12, **** indicates p<0.0001.

[0035] Figure 22 Zebrafish intestinal tissue sections treated with bacteriophage vB_AdhP_L12. Figure A represents group C4-1 (magnification × 100), Figure B represents group C4-1 (magnification × 400); Figure C represents the blank control group (magnification × 100), Figure D represents the blank control group (magnification × 400); Figure E represents the C4-1+ΦL12 treatment group (magnification × 100), Figure F represents the C4-1+ΦL12 treatment group (magnification × 400).

[0036] Figure 23 Expression of various immune factors after treatment with bacteriophage vB_AdhP_L12. Detailed Implementation

[0037] The present invention will now be described in detail with reference to specific embodiments. The following specific embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any way.

[0038] Example

[0039] 1. Materials and Methods

[0040] 1.1 Bacterial strains and culture conditions

[0041] The host bacterium used in this study, Aeromonas daca C4-1, was isolated from a crocodile farm in Hainan Province, China. It had been identified as Aeromonas daca in a previous study by 16S rDNA and gyrB (Pu, W., Guo, G., Yang, N., Li, Q., Yin, F., Wang, P., Zheng, J., & Zeng, J. (2019). Three species of Aeromonas (A. dhakensis, A. hydrophila and A. jandaei) isolated from freshwater crocodiles (Crocodylus siamensis) with pneumonia and septicemia. Lett Appl Microbiol, 68(3), 212–218. https: / / doi.org / 10.1111 / lam.13112).

[0042] The remaining Aeromonas dacca strains were also isolated from crocodiles that died during an outbreak of disease at a fish farm in Hainan Province, China. All bacterial cultures used in the experiments were cultured in LB liquid medium at 30°C.

[0043] 1.2 Isolation and Identification of Bacteriophages

[0044] Water samples were collected from Haikou People's Hospital and several crocodile farms in Dongfang and Chengmai cities and counties in Hainan Province for phage isolation.

[0045] Sample pretreatment: 25 mL of sewage sample was placed into a 50 mL centrifuge tube and centrifuged at 8000 rpm for 10 min at 4℃ to remove fibers and other impurities. The supernatant was coarsely filtered through a 0.45 μm microporous membrane using a 50 mL disposable syringe, and then filtered again with a 25 mL disposable syringe and a 0.22 μm filter membrane for sterilization and further aliquoting.

[0046] Take 20 mL of the obtained filtrate and mix it with the logarithmic phase culture (OD) of Aeromonas dacarbazin C4-1, the bacteriophage indicator host bacterium. 600nmThe mixture (≈0.6) was mixed and cultured at 37℃ with shaking at 160 rpm for 12 h for the first enrichment. The next day, the enriched solution was centrifuged at 8000 rpm for 10 min, and 20 mL of the supernatant was collected. 20 mL of LB liquid medium and 2 mL of enrichment broth of the host bacterium Aeromonas daca were added, and the mixture was cultured at 37℃ with shaking at 160 rpm for 10 h. The mixed culture was centrifuged at 8000 rpm for 10 min, and 10 mL of the supernatant was collected. 10 mL of LB liquid medium and 2 mL of Aeromonas daca broth were added, and the mixture was cultured at 37℃ with shaking at 160 rpm for 10 h. The mixed culture solution that had undergone three enrichments was centrifuged at 10000 rpm for 30 min, and the supernatant was filtered through a 0.22 μm microporous membrane to remove bacteria, thus obtaining the stock solution containing Aeromonas daca phage.

[0047] Phages were tested using the double-layer agar plate method: 200 μL of Aeromonas dacarbazin bacterial suspension (OD200) was taken. 600nm Add 100 μL of filtrate to ≈0.6), gently vortex to mix, then add 3.5 mL of 0.6% semi-solid soft agar medium (pre-warmed in a water bath) cooled to 55℃, gently vortex to mix, pour onto 1.5% solid LB agar plates to make a double-layer plate, let stand at room temperature for about 10 min, and after the upper culture medium has completely solidified, invert the plate and incubate overnight in a 37℃ constant temperature incubator.

[0048] The following day, the plates were observed. A single phage plaque with neat edges, large size, and clear color was collected from the plate and resuspended in 1 mL of sterile SM buffer (R21985-500 mL). The resuspended solution was added to 5.0 mL of logarithmic-phase *Aeromonas dacarinae* culture and mixed thoroughly. The plates were then incubated overnight at 37°C and 160 rpm with shaking. The following day, a new double-layer agar plate was prepared following the work of Kropinski et al. (Kropinski, AM, Mazzocco, A., Waddell, TE, Lingohr, E., & Johnson, RP (2009). Enumeration of bacteriophages by double agar overlay plaque assay. Methods Mol Biol, 501, 69–76. https: / / doi.org / 10.1007 / 978-1-60327-164-6_7). A purification cycle consisting of "single plaque picking → amplification culture → double-layer plate separation" was used, and this cycle was repeated at least three times until single-strain phage plates with similar plaque morphology were obtained. The upper layer of soft agar from the purified phage plates was scraped into a test tube containing 1.5 mL of SM buffer, resuspended by pipetting, and vortexed to mix. The suspension was centrifuged at 10,000 rpm for 10 min, filtered through a 0.22 μm microporous membrane to prepare a pure phage suspension. A portion was stored at 4°C for later use, while the remainder was mixed with an equal volume of 30% glycerol and stored at -80°C.

[0049] 100 μL of logarithmic-phase *Aeromonas dacarina* suspension was spread onto LB agar plates. Phage suspension concentration was determined using the double-layer plate method. The phage suspension was diluted with SM buffer at multiple gradients (10⁻⁶). -2 10 -3 10 -4 10 -5 10 -6 The antibacterial ability of bacteriophages was detected using the droplet method.

[0050] 1.3 Observation using transmission electron microscopy (TEM)

[0051] Phage suspensions were prepared according to the established protocol (Wu, Y., Ye, S., Liu, S., Yu, J., Gao, H., Feng, W., Cheng, X., Zhao, X., Zhao, J., Zeng, J., Zhang, X., Wang, X., Yang, N., Fan, L., Guo, G., Li, X., & Zheng, J. (2026). Characterization and biocontrolpotential of two novel lytic phages vB_EtaS_2 and vB_EtaS_6 against Edwardsiella tarda in aquaculture. Aquaculture, 612, 743245. https: / / doi.org / https: / / doi.org / 10.1016 / j.aquaculture.2025.743245). Lay the sealing film flat on a glass plate. Use tweezers to hold a copper mesh (400 mesh) on the sealing film. Transfer 10 μL of sample and drop it onto the copper mesh. Let it settle for 10 min, then use a small piece of filter paper to absorb the excess liquid. Transfer 10 μL of 3% uranium acetate and add it to the sample for staining. After staining for 2 min, use a small piece of filter paper to absorb the excess staining liquid. Observe using a transmission electron microscope (JEM-1200 EX, JEOL Ltd.).

[0052] 1.4 Host range determination

[0053] To determine the host range of this newly isolated bacteriophage, four strains of Aeromonas dacca and four common opportunistic pathogens were used for testing: Edwardsiella tarda, Aeromonas vera, Salmonella, and Escherichia coli standard strains. All strains other than the standard strains were isolated from dead crocodiles in different crocodile farms in Hainan Province. 2.5 μL of purified bacteriophage solution (10... 8 PFU (pFU / mL) was dropped onto 100 μL of each test strain on an LB plate, and the lysis effect was evaluated after overnight incubation at 30 °C.

[0054] 1.5 In vitro antibacterial effect

[0055] To evaluate the in vitro antibacterial activity of vB_AdhP_L12, Aeromonas dacarbazina C4-1 strain was cultured overnight, centrifuged at 12,000 rpm for 2 min, resuspended in sterile LB medium, and diluted to 10⁻⁶. 8CFU / mL. Subsequently, 100 μL of bacterial suspension and 100 μL of phage lysis buffer were added to 96-well microtiter plates with multiplicity of infection (MOI) values ​​of 10, 1, 0.1, 0.01, 0.001, 0.0001, and 0.00001, respectively. The mixtures were incubated at 30°C for 13 hours, and the optical density (OD) at 600 nm was measured every 30 minutes. 600nm Each experiment was repeated three times.

[0056] 1.6 Determination of Optimal MOI

[0057] Assess the in vitro antibacterial activity of vB_AdhP_L12: Culture Aeromonas dacarbamate C4-1 strain to the logarithmic growth phase (OD200). 600nm (≈0.6), adjust the bacterial concentration to 10. 7 CFU / mL, phages and host bacteria were added according to phage titer and different MOIs (0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10), so that each tube contained 10 CFU / mL. 2 10 3 10 4 10 5 10 6 10 7 10 8 PFU / mL phage was added to LB liquid medium to ensure a uniform total volume in each tube. The culture was incubated at 37°C and 160 rpm for 6 h on a shaker. A sample of the mixed culture was centrifuged at 10,000 rpm for 10 min. The supernatant was filtered through a 0.22 μm filter, and its titer was determined using the double-layer agar plate method, with three replicates and the average value. The MOI (Multiple of Infection) producing the highest phage titer was considered the optimal MOI.

[0058] 1.7 One-step growth curve

[0059] Following the established experimental procedure (Wu, M., Hu, K., Xie, Y., Liu, Y., Mu, D., Guo, H., Zhang, Z., Zhang, Y., Chang, D., & Shi, Y. (2018). A Novel Phage PD-6A3, and Its Endolysin Ply6A3, With Extended Lytic Activity Against Acinetobacter baumannii. Front Microbiol, 9, 3302. https: / / doi.org / 10.3389 / fmicb.2018.03302), phage lysate was added to 1 mL of Aeromonas dacarbazin C4-1 bacterial culture at an MOI of 0.01 and incubated at room temperature for 15 min. The mixture was then centrifuged at 12000 rpm for 2 min, resuspended in 1 mL of LB broth, and washed three times. The resuspension was added to 9 mL of LB medium and incubated at 30 °C with shaking at 180 rpm. Samples were taken every 10 minutes from 0 min to 260 min (samples were collected at the following time points: 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260). Phage titers at each time point were determined using the double-layer agar plate method. Three replicates were set up for each time point.

[0060] Outbreak size and incubation period can be calculated or read using the following formula:

[0061] Outbreak volume = (final total phage count [PFU / mL] - initial total phage count [PFU / mL]) / total number of bacteria infecting the host (CFU / mL).

[0062] The incubation period is determined by plotting a phage growth curve and reading the starting point of the upward segment of the straight line in the graph.

[0063] 1.8 Stability to heat, pH and chemical reagents

[0064] Following the experimental methods of previous studies (He, P., Cao, F., Qu, Q., Geng, H., Yang, X., Xu, T., Wang, R., Jia, X., Lu, M., Zeng, P., & Luan, G. (2024). Host range expansion of Acinetobacter phage vB_Ab4_Hep4 driven by a spontaneous tailtubular mutation. Front Cell Infect Microbiol, 14, 1301089. https: / / doi.org / 10.3389 / fcimb.2024.1301089 ; Ture, M., Cebeci, A., Altinok, I., Aygur, E., &Caliskan, N. (2022). Isolation and characterization of Aeromonas hydrophila-specific lytic bacteriophages. Aquaculture, 558, 738371. (https: / / doi.org / https: / / doi.org / 10.1016 / j.aquaculture.2022.738371), to determine the thermal and pH stability of the phage. For thermal stability, an initial titer of 10 was used. 8 Phage solutions of PFU / mL were incubated at different temperatures: -20℃, 4℃, 25℃, 37℃, 50℃, 60℃, and 70℃. Samples were collected at 2, 4, and 6 hours. The phage titers after treatment at each temperature were determined using the double-layer agar plate method.

[0065] To ensure pH stability, the pH of the physiological saline was adjusted to 4, 5, 6, 7, 8, 9, 10, and 11 using HCl and NaOH solutions. Subsequently, 900 μL of the pH-adjusted physiological saline was mixed with an initial titer of 10. 8 A phage solution of 100 μL with PFU / mL was mixed and incubated at 4°C, with samples taken after 6 hours. The phage titer was determined using the double-layer agar plate method.

[0066] Commonly used disinfectants in aquaculture include high-concentration ethanol, chloroform, and sodium dodecyl sulfate (SDS). Therefore, testing the sensitivity of bacteriophages to these chemical reagents is particularly important for practical applications. An initial titer of 10... 8Phage stock solution of PFU / mL was mixed with 2% chloroform, 0.2% SDS, and 75% ethanol, respectively. After thorough vortexing, the mixture was incubated at 4°C for 1 hour. Phage titers were determined using the double-layer agar plate method.

[0067] 1.9 Genome Extraction and Sequencing

[0068] Whole-genome DNA of bacteriophages was extracted using a phage DNA Isolation Kit (Phage DNA Isolation Kit 46800, Norgen Biotek, Canada) following the manufacturer's instructions. To minimize degradation caused by temperature fluctuations, all extraction steps were performed at 4 °C.

[0069] Whole-genome sequencing was performed on the Illumina NovSeq platform (Shanghai Paisenuo Biotechnology Co., Ltd. and Wuhan Bena Technology Co., Ltd.), and the required genome libraries were constructed using the standard Illumina TruSeq Nano DNA LT library preparation experimental procedure (Illumina TruSeq DNA Sample Preparation Guide).

[0070] The second-generation data was assembled from scratch using SPAdes software. Sequences were extracted based on the sequencing depth of the assembled sequences. The high-sequencing-depth sequences were compared with the NT library on NCBI using blastn. Viral genome sequences from each assembly were selected. Then, single-base correction was performed using Pilon software to obtain the final viral genome sequence.

[0071] GeneMarkS software was used to predict open reading frames (ORFs) in the genome. Non-coding RNA (NRNA) prediction was performed using the Rfam database, but no NRNAs were predicted. For the predicted protein-coding genes, sequence alignment and functional annotation were performed using Diamond software. EggNOG annotation of the gene-coding protein sequences was performed using the eggNOG database from Diamond BlastoP. KO and pathway annotation of protein-coding genes were completed using the KEGG KAAS automated annotation system. GO annotation of protein-coding genes was performed using InterPro software.

[0072] Virulence factors and antibiotic resistance genes were identified by comparison with VFDB and CARD databases. Genomic sequences, gene predictions, and non-coding RNA predictions were integrated into a standard GBK (GenBank) format file. A circular map of the genome was then drawn using CGView, and finally, the map was annotated using Photoshop CS.

[0073] 1.10 Phylogenetic Tree Construction

[0074] A viral proteomic phylogenetic tree was constructed using the tBLASTx algorithm on the ViPTree server. Genomic sequence homology was calculated using the BLASTN algorithm on NCBI (https: / / www.ncbi.nlm.nih.gov), and the five most genetically closely related bacteriophages were selected for multiple sequence alignment analysis in DigAlign. Furthermore, genomic distances between the genomes of 20 Aeromonas bacteriophages were calculated using VirClust software from the NCBI database.

[0075] To investigate the taxonomic classification of vB_AdhP_L12, highly conserved protein sequences were selected: the terminal enzyme large subunit (TerL), the major capsid protein (MCP), and the tail tube protein TTP (non-contractile microtubule protein). The protein sequences were then subjected to a BLASTp homology search and compared with protein databases. The 30 individuals with the highest homology were selected from each sequence, and a phylogenetic tree was constructed using the maximum likelihood method in MEGA12 software with 1000 repeated bootstrap calculations. The visualization and optimization of the phylogenetic tree were completed on the iTOL website.

[0076] 1.11 Evaluation of the efficacy of in vitro cell therapy

[0077] Human colon cancer cells Caco-2 and carp epithelioma cells (EPC cells) were selected.

[0078] With 1×10 per hole 4 Cells were seeded at a density of approximately 1 × 10⁶ cells per well in 12-well plates and cultured in Caco-2 for 2 days until the cell count reached approximately 1 × 10⁶ cells per well. 6 After culturing on EPC for 3 days, the cell count per well reached approximately 1 × 10⁶ cells. 6 Cells were washed three times with phosphate-buffered saline (PBS) to remove residual antibiotics. The PBS group was supplemented with 900 μL of antibiotic-free culture medium and 100 μL of PBS; the phage control group was adjusted to 1×10⁻⁶ cells / mL. 9Phage suspension with a PFU / mL titer was co-incubated with Caco-2 cells / EPC cells at 37°C for 3 hours. In the bacterial control group, 900 μL of antibiotic-free medium and 100 μL of log-phase Aeromonas dacarpa C4-1 bacterial suspension were added to each well and incubated at 37°C for 3 hours. In the ΦL12 treatment group, 900 μL of antibiotic-free medium and 100 μL of log-phase Aeromonas dacarpa C4-1 bacterial suspension were added and incubated at 37°C for 1 hour. Then, phage vB_AdhP_L12 was added according to the optimal multiplicity of infection and incubated at 37°C for 3 hours. After 3 hours, 100 μL of the supernatant was taken and spread on LB agar to determine the Aeromonas dacarpa C4-1 strain load. Another 100 μL of the supernatant was taken and the phage titer was determined using the double-layer plate method. Finally, each well was washed along the plate wall with PBS, and 10 μL of Hoechst staining solution and PI (propidium iodide) dye were added to each well for incubation (Hoechst dye can penetrate the cell membrane, insert into double-stranded DNA and release blue fluorescence; PI dye can stain necrotic cells). After standing for 15 min, the wells were washed along the plate wall with PBS 3 times, 1 mL each time, and observed by SIM fluorescence confocal microscopy.

[0079] 1.12 Determination of lethal dose in zebrafish

[0080] Referring to previous research (Feng, W., Li, X., Yang, N., Fan, L., Guo, G., Xie, J., Cai, X., Meng, Y., Zeng, J., Han, Y., & Zheng, J. (2025). The PhoBR two-component system upregulates virulence in Aeromonas dhakensis C4–1. Aquaculture, 595, 741665. https: / / doi.org / https: / / doi.org / 10.1016 / j.aquaculture.2024.741665), healthy adult zebrafish were randomly divided into seven groups of 30 each. Aeromonas dhakensis C4-1 strain was cultured overnight, centrifuged at 12,000 × g for 2 minutes at 4°C, and resuspended in PBS by pipetting. The bacterial concentration was adjusted to 5 × 10⁻⁶. 9 CFU / mL, 1×10 9 CFU / mL, 5×10 8 CFU / mL, 1×10 8 CFU / mL, 5×10 7 CFU / mL and 1×10 7CFU / mL. One group served as a blank control group, with each fish receiving an intraperitoneal injection of 10 μL of PBS buffer. The remaining groups were injected with 10 μL of the corresponding Aeromonas dacarba suspension, with doses increasing by 5 × 10⁻⁶. 7 CFU / tail, 1×10 7 CFU / tail, 5×10 6 CFU / tail, 1×10 6 CFU / tail, 5×10 5 CFU / tail and 1×10 5 CFU / tail. The bacterial suspension was diluted and counted after injection to verify concentration. The test period was 7 days, with records of surviving zebrafish from each group taken every 12 hours. Simultaneously, bacteria were re-isolated and identified from dead zebrafish.

[0081] 1.13 Zebrafish bacteriophage impregnation treatment

[0082] Following the method of Yunkun Wu et al. (Wu, Y., Ye, S., Liu, S., Yu, J., Gao, H., Feng, W., Cheng, X., Zhao, X., Zhao, J., Zeng, J., Zhang, X., Wang, X., Yang, N., Fan, L., Guo, G., Li, X., & Zheng, J. (2026). Characterization and biocontrol potential of two novel lytic phages vB_EtaS_2 and vB_EtaS_6 against Edwardsiella tarda in aquaculture. Aquaculture, 612, 743245. https: / / doi.org / https: / / doi.org / 10.1016 / j.aquaculture.2025.743245), healthy adult zebrafish were randomly divided into three groups of 30 each: a blank control group, a C4-1 group, and a C4-1+ΦL12 treatment group. Aeromonas dacca C4-1 strain overnight culture was centrifuged at 12,000 × g for 2 minutes, then resuspended in PBS buffer and administered at 10 × LD50 according to the challenge dose. 50 The standard has been adjusted to 3.50×10 6 CFU / mL concentration. The phage enrichment solution was centrifuged at 8000 rpm for 10 min at 4 °C and filtered through a 0.22 μm filter membrane. The phage titer was determined using the double-layer plate method.

[0083] In the blank control group, each zebrafish was injected with 10 μL of PBS buffer. In the C4-1 group, each zebrafish was injected with 10 μL of mixed bacterial suspension (3.50 × 10⁶ cells per fish). 6 CFU C4-1). In the C4-1+ΦL12 treatment group, zebrafish were first injected with 10 μL of mixed bacterial suspension (3.50 × 10⁻⁶ per fish). 6 CFU C4-1), after 1 hour, were immersed in an MOI of 10 (3.50 × 10). 7 The zebrafish were immersed in a phage suspension containing PFU / mL vB_AdhP_L12 for 24 hours. The experiment lasted for 8 days, and the survival status of each group of zebrafish was recorded every 12 hours. Simultaneously, bacteria were re-isolated and identified from the dead zebrafish.

[0084] 1.14 Histological evaluation of zebrafish

[0085] The histological evaluation of zebrafish was completed at Chengdu Lilai Biotechnology Co., Ltd.

[0086] Table 1. Lesion Grading System

[0087] Scoring lesion degree Definition of grading 0（-） Normal range Under experimental conditions, and considering factors such as age, sex, and pedigree, changes may occur, but in other cases, they may be considered deviations from the normal state. 1（+） slight The changes that occur are almost within the normal range (i.e., the minimum possible changes). 2（++） Mild The lesions are easily identifiable but of limited severity; they may not cause any functional impairment; the affected tissue comprises 11%–20% of the examined tissue. 3（+++） moderate The lesion is prominent and has a high probability of progressing to a more serious condition. It may cause limited tissue or organ dysfunction; 21%–40% of tissues are involved. 4（++++） Severe The lesion is severe and has formed a complete lesion, which is expected to cause significant tissue or organ dysfunction; the lesion involves 41% to 100% of the examined tissue area.

[0088] Intestinal tissues from the blank control group, C4-1 group, and C4-1+ΦL12 treatment group were fixed with 4% paraformaldehyde for 24 hours.

[0089] Fixed tissues were dehydrated using a fully automated dehydrator (dehydration time: 75% ethanol 3 h, 85% ethanol 2 h, 95% ethanol 1 h, 100% ethanol I 20 min, 100% ethanol II 20 min, 100% ethanol III 20 min, 100% ethanol IV 20 min, clearing agent I (Wuxi Jiangyuan Industrial Technology and Trade Corporation 240131) 25 min, clearing agent II (Wuxi Jiangyuan Industrial Technology and Trade Corporation 240131) 30 min, paraffin I 30 min, paraffin II 1 h, paraffin III 2 h), embedded, and sectioned as follows:

[0090] ① Dewax sections; ② Stain with hematoxylin for 5-10 min, rinse with running tap water until colorless; ③ Differentiate with hydrochloric acid-alcohol solution for about 3 s, rinse with tap water; ④ Revert to blue with weakly alkaline aqueous solution, rinse with tap water; ⑤ Immerse in alcohol-soluble eosin for 3 min; Dehydrate with graded alcohols, clear with a clearing agent, and mount with neutral resin.

[0091] They were then observed under a microscope and qualitatively evaluated according to Table 1.

[0092] 1.15 Analysis of the Immune Response in Zebrafish to Determine the Effectiveness of Phage Therapy

[0093] A zebrafish blank control group and an infection group (10×LD) were set up. 50 ), treatment group (LD) 50 (Soaked at &MOI=10), 50 samples per group. Samples were taken on days 1, 3, and 7. Five surviving individuals from each treatment group were dissected to obtain intestinal tissue, which was then placed in a 2 mL centrifuge tube and ground with 100 μL of physiological saline and 3 steel balls.

[0094] RNA was extracted from the homogenate according to the manufacturer's (FastPure Cell / Tissue TotalRNA Isolation Kit V2, Nanjing Vazyme Biotech Co., Ltd.) instructions. Then, RNA was reverse transcribed into cDNA using HP All-in-one qRT Master Mix II according to the manufacturer's (YUNBIO) instructions. Finally, qPCR was performed using the SYBR Green dye method according to the manufacturer's (YUNBIO) 2×Universal SYBR qPCR Master Mix instructions.

[0095] According to Wei Feng The study by Wei Feng, Xuesong Li, Nuo Yang, Lixia Fan, GuiyingGuo, Jun Xie, Xiuqing Cai, Yuqi Meng, Jifeng Zeng, Yu Han, Jiping Zheng, The PhoBR two-component system upregulates virulence in Aeromonas dhakensis C4–1, Aquaculture, Volume 595, Part 2, 2025, 741665, ISSN 0044-8486, https: / / doi.org / 10.1016 / j.aquaculture.2024.741665. designed 5 pairs of primers for immune factors and 1 pair of primers for internal reference genes (Table 2), with three replicates for each primer in each treatment group.

[0096] Table 2. Primers for pro-immune factors, anti-immune factors, chemokines, and internal reference genes.

[0097]

[0098] 2. Results

[0099] 2.1 Isolation and purification of bacteriophages

[0100] A highly lytic bacteriophage, named vB_AdhP_L12, was isolated from a water sample collected from Haikou People's Hospital using Aeromonas daca C4-1 as the host bacterium.

[0101] Bacteriophage vB_AdhP_L12 formed well-defined, transparent plaques. No halos were observed around the plaques on double-layer agar plates, indicating strong lytic activity and the potential production of very little or no diffusive extracellular enzymes. After 4 hours of incubation on 0.6% LB agar plates, vB_AdhP_L12 formed plaques approximately 1.3 mm in diameter. Figure 1 ).

[0102] 2.2 Transmission electron microscopy observation of bacteriophages

[0103] TEM showed that the vB_AdhP_L12 head has an icosahedral structure. Figure 2 The average diameter of the head is 50.03±2.50nm; the tail is short and lacks a retractable tail sheath structure, and has a tail filament structure at the end, with an average length of 13.39±2.05nm; the average diameter of the tail is 12.55±1.68nm.

[0104] Based on the above, it can be preliminarily classified into the family Podoviridae within the order Caudovirales.

[0105] 2.3 Phage Host Range

[0106] A total of 4 strains of Aeromonas daca and 4 common opportunistic pathogens in aquatic organisms were included in the host range determination experiment (Table 3).

[0107] vB_AdhP_L12 lysed four tested Aeromonas dacarinae strains, but did not produce lysis reactions against Edwardsiella tarda, Aeromonas vera, Salmonella, or Escherichia coli standard strains. This indicates that this bacteriophage strain has high specificity in lysing Aeromonas dacarinae.

[0108] Table 3. Determination of host range of vB_AdhP_L12

[0109]

[0110] (+) indicates cleavage, and (-) indicates no cleavage.

[0111] 2.4 Best MOI

[0112] The optimal MOI (multiple of infections) results are as follows Figure 3As shown, when the MOI was 10, the phage titers after host bacterial lysis were significantly lower in vB_AdhP_L12 than under other MOI conditions. Conversely, the phage yield was highest when the MOI was 0.01. These results are consistent with the in vitro antibacterial effect experiments. Therefore, these results indicate that an MOI of 0.01 achieves optimal results in both bacterial killing and phage production, and the optimal multiplicity of infection (MMO) is determined to be 0.01.

[0113] 2.5 Evaluation of in vitro antibacterial effect

[0114] In vitro antibacterial test ( Figure 4 The results showed that *Aeromonas daca* strains grew normally without phage treatment. However, after co-culturing with phage, the growth efficiency of *Aeromonas daca* strain C4-1 decreased by 40-60%. vB_AdhP_L12 showed the best antibacterial effect at an MOI of 0.01.

[0115] 2.6 One-step growth curve

[0116] The one-step growth curve of vB_AdhP_L12 showed that the phage began to proliferate immediately after infecting the host. It then exhibited logarithmic growth, with a rapid increase in titer between 20 and 100 minutes, entering the first plateau phase at 100–200 minutes, but then proliferating to another plateau and maintaining a stable number between 200–210 minutes. The calculated burst size was 2.28 PFU / cell, with an extremely short latency period, less than the 15-minute incubation time at room temperature. Figure 5 ).

[0117] 2.7 Stability to heat, pH and chemical reagents

[0118] Thermal stability results are as follows Figure 6 As shown in the figure, after 6 hours of incubation, the survival rate of vB_AdhP_L12 in the temperature range of -20℃ to 37℃ showed no significant difference compared with the initial titer, and the activity remained stable; after incubation at 50℃ and 60℃, a downward trend began to appear, but some lytic activity was still retained after 6 hours of treatment. At 70℃, no phages survived after 2 hours.

[0119] pH stability such as Figure 7 As shown in the figure, a pH range of 4 to 11 was selected. The results showed that the survival rate of bacteriophages was close to 100% at pH 6 to 8. As the acidity or alkalinity of the environment increased, the bacteriophage titer only showed a slight decreasing trend. Especially at pH 4 and pH 11, the bacteriophages maintained high activity with no significant difference from the initial activity.

[0120] To assess the survival of bacteriophage vB_AdhP_L12 in aquaculture environments, its chemical sensitivity to commonly used disinfectants was tested. After treatment with 2% chloroform, vB_AdhP_L12 maintained relatively stable activity. However, when exposed to 75% ethanol and 0.2% sodium dodecyl sulfate, its survival rate decreased significantly by three orders of magnitude, and its activity was almost completely lost. Figure 8 This indicates that these two chemical reagents have a very strong killing effect on bacteriophages.

[0121] 2.8 Genome sequencing analysis

[0122] The phage whole-genome sequencing results are shown in SEQ ID NO:13. Regarding the accuracy of the phage genome sequencing, the results show that the percentage of bases with a base recognition accuracy of 99.9% or higher (Q20_rate) in vB_AdhP_L12 was 95.82%. In terms of quality, the average quality score of the full reads remained consistently above 35, far exceeding the industry-standard Q30 qualification (error rate ≤0.1%), indicating extremely high base recognition accuracy and strong data reliability in this sequencing. Figure 9 As shown, vB_AdhP_L12 has a linear double-stranded DNA genome, 38749 bp in length, with a GC content of 52.87%. This genome contains 42 open reading frames (ORFs), accounting for 90.41% of the total genome length, with an average GC content of 53.61%. Twelve of the 42 ORFs are putative protein-coding sequences, and no non-coding RNA was detected. Genome annotation results show that this phage lacks genes encoding integrase, recombinase, and repressor proteins. The detection of numerous lysis-related proteins (perforin, amidase), replication-proliferation-related enzymes (DNA polymerase, primase, and helicase), and structural proteins (capsid protein, tail fimbriae) further corroborates that this phage is lytic. Furthermore, no genes related to drug resistance or virulence were detected, nor were any homologous sequences of virulence transfer or lysogen genes found.

[0123] 2.9 Phylogenetic Analysis

[0124] A circular proteomic phylogenetic tree of the phage genome was constructed using ViPTree. Figure 10 The results showed that vB_AdhP_L12 belongs to the family Autographiviridae, and its host range is within the phylum Pseudomonadota. Multiple genome alignment was performed between vB_AdhP_L12 and the five bacteriophages with the highest similarity scores using the BLAST algorithm. Figure 11As shown in Figures A, B, and C, vB_AdhP_L12 has many homologous regions and good collinearity with *Escherichia phage Ebrios* (NC_047942.1), *Stenotrophomonas phage IME15* (NC_019416.1), and *Aeromonas phage vB_AroA_KFSA9* (PQ824399.1), but there are also low similarity or deletions in certain segments, and some regions have different transcription directions (NC_047942.1 is about 1 kb longer than vB_AdhP_L12, mainly distributed at both ends of the genome (0-4 kb, 32-36 kb), which is a unique insertion sequence of this strain; NC_019416.1 has a 32-36 kb...). Large blank areas appear in the region; PQ824399.1 has a large number of scattered offset points, corresponding to multiple reverse bands and cross bands in the comparison figure, indicating that this strain has multiple local genome rearrangements and fragment inversions, and the variant sites are scattered throughout the genome (0-4 kb, 8-16 kb, and 28-36 kb are all distributed, and a large blank area also appears in the 32-36 kb region). Figure 11 Figures D and E indicate that Aeromonas phage PZL-Ah1 (MT681669.1) and Aeromonas phage avDM11-UST (OP380607.1) have rearranged genome structures compared to vB_AdhP_L12, sharing only some homologous genes and belonging to different evolutionary branches.

[0125] In a recent research report, Farhat Ansari et al. (Farhat Ansari, Vandan Nagar, Characterization and biocontrol potential of two novel lytic Aeromonasdhakensis bacteriophages vB_AdhaM_G2 and P5 on basa fish fillets, FoodControl, Volume 182, 2026, 111847, ISSN 0956-7135, https: / / doi.org / 10.1016 / j.foodcont.2025.111847.) discovered that the Aeromonas dacarina phage vB_AdhaM_G2 belongs to the genus Ceceduovirus of the family Straboviridae, while strain P5 was not classified. Like vB_AdhP_L12, it was isolated from Aeromonas dacarina, but their phylogenetic relationships are significantly different. Because only two strains of Aeromonas dacca bacteriophage, vB_AdhaM_G2 and P5, reported by Farhat Ansari et al., were previously isolated bacteriophages using Aeromonas as a host and subjected to genetic analysis when constructing evolutionary relationships, NCBI was used to retrieve and analyze these phages. Evolutionary relationship analysis ( Figure 12 The results showed that none of these phages exhibited proteomic similarity to vB_AdhP_L12. Previous reports had not investigated populations of Aeromonas dacarba as hosts that are highly homologous to vB_AdhP_L12. Within the Aeromonas dacarbacarba phage population, the vB_AdhP_L12 individual warrants further investigation. To further confirm the classification of vB_AdhP_L12, a phylogenetic tree was constructed using homologous protein sequences of core genes specific to the Autographiviridae family of phages: the terminal enzyme large subunit TerL, the non-contractile microtubule tail protein TTP, and the major capsid protein MCP. TerL is considered the most conserved key core gene among phages. Figure 13 The TerL of vB_AdhP_L12 is shown to form the same evolutionary branch as the TerL of Ebriosvirus phage, and is located next to the branch containing Teseptimavirus, indicating a close evolutionary relationship. Figure 14 The TTP of vB_AdhP_L12 forms the same branch as Teseptimavirus and is located next to the branch containing Rambovirus (Yersinia phage vB_YenP_Rambo). Figure 15The MCP of vB_AdhP_L12 shows that it forms the same clade as Teseptimavirus and is also closely related to the genus Rambovirus. Therefore, based on the phylogenetic relationships of conserved genes, vB_AdhP_L12 can be preliminarily classified as a newly isolated bacteriophage from the genus Teseptimavirus in the family Autographiviridae.

[0126] Aeromonas dacarina phage vB_AdhP_L12 was deposited on January 26, 2026, at the China Center for Type Culture Collection (CCTCC), accession number M2026249, located at Wuhan University, Wuhan, Hubei Province, China. Its taxonomic name is: Aeromonas dacarina phage vB_AdhP_L12. Aeromonas dhakensis phage vB_AdhP_L12.

[0127] 2.10 Evaluation of in vitro treatment efficacy

[0128] Aeromonas daca, as an extracellular bacterium, primarily disrupts cell membrane integrity, induces apoptosis, and triggers inflammatory responses through different mechanisms via cytotoxic enterotoxins (Act), hemolysin (Ahh1), and aerolysin (AerA), leading to tissue damage and disease. Furthermore, Dacamonas daca uniquely encodes a heat-stable cytotoxic enterotoxin (AST). Based on this, the extracellular therapeutic effects of vB_AdhP_L12 cells were analyzed using Caco-2 cells and EPC cells as experimental materials.

[0129] Hoechst is a blue fluorescent dye that can penetrate the cell membrane to stain the cell nucleus. Propidium iodide (PI), on the other hand, requires pre-treatment to permeate the cell membrane before it can penetrate intact cell membranes, allowing it to penetrate late-stage apoptotic or dead cells; it appears red under a fluorescence microscope. Regarding the assessment of phage cytotoxicity, experimental results show that high concentrations of phage have no toxic effect on Caco-2 cells (e.g., Figure 16 ), and has no toxic effect on EPC cells (e.g. Figure 17 In the treatment experiment, staining results showed that both the PBS group and the phage control group exhibited high survival rates and low mortality rates. In contrast, the bacterial control group cells showed a high mortality rate, characterized by strong red fluorescence. Figure 16 and Figure 17In the ΦL12 treatment group following C4-1 infection and phage treatment, the viral load of Aeromonas dacarbazin C4-1 in the cell supernatant showed a significant decrease at both 1 h and 3 h, indicating that ΦL12 is effective against Caco-2 cells (such as...). Figure 20 ) and EPC cells (such as Figure 21 All of them have highly effective antibacterial protective effects.

[0130] 2.11 Evaluation of the therapeutic effect of phage therapy

[0131] First, the survival curve of zebrafish infected with Aeromonas dacca was determined, and the results are as follows: Figure 18 As shown: All infection doses ≥5×10 6 All zebrafish infected with CFU / tail died during the experimental period; when the infection dose was ≥1×10 5 At CFU / tail, zebrafish exhibited significant mortality rates of varying degrees, with mortality increasing in a dose-dependent manner with increasing infectious dose. Based on survival curve data, the median lethal dose (LD50) of Aeromonas dacarbazide C4-1 for zebrafish was calculated. 50 The value is 3.60 × 10 5 CFU / tail.

[0132] To evaluate the therapeutic effect of this bacteriophage, zebrafish infected with Aeromonas dacarba were immersed in a bacteriophage suspension, and their survival rate was measured. Figure 19 As shown, the cumulative survival rate of the blank control group reached 100%. In contrast, the C4-1 group experienced a large number of deaths from day 1, with a final survival rate of 0%. The results of the C4-1+ΦL12 treatment group showed that vB_AdhP_L12 treatment significantly improved the survival rate, maintaining it at 63.33%.

[0133] In addition, pathological analysis was performed on the zebrafish intestinal tissues of the blank control group, C4-1 group, and C4-1+ΦL12 treatment group. The results are detailed in Table 4. Experimental data showed that the C4-1 group ( Figure 22 Figures A and B in the diagram show that the intestinal mucosa exhibits atrophy and thinning, a decrease in the number of goblet cells (marked by green arrows), and mucosal degeneration / necrosis (marked by yellow arrows). In contrast, the C4-1+ΦL12 treatment group ( Figure 22 Intestinal tissue samples (Figures E and F) showed no obvious mucosal damage or histological abnormalities, and the degree of damage was significantly reduced compared to the C4-1 infection group, and was superior to the blank control group which only showed very mild lamina propria degeneration and necrosis (+). Figure 22 (Figures C and D in the diagram) show that the intestinal pathological condition has been effectively improved.

[0134] Table 4 Qualitative Grading by Light Microscopy

[0135]

[0136] Note: A four-level lesion grading system (Table 1) was used, with lesions recorded as normal (—), mild (+), slight (++), moderate (+++), and severe (++++). The presence of slight lamina propria necrosis in the submitted blank control group slides is due to experimental error, possibly caused by damage during sample processing. The severity of this lesion differs significantly from the obvious lesion results in group C4-1, and therefore also has reference value.

[0137] Meanwhile, to systematically analyze the effectiveness, mechanism of action, and safety of phage therapy from the perspective of host immune response, a blank control group (PBS only) and an infection group (only Aeromonas dacarbazoidea C4-1, LD50) were set up. 50 The treatment group (treated with host bacteria after incubation followed by phage inoculation) was selected. Pro-inflammatory cytokines (IL-1β, TNF-α, IL-6), anti-inflammatory factors (IL-10), chemokines (IgM was a specific antibody against Aeromonas dacarbazini), and the internal reference gene (β-actin) were also included. (Heatmap) Figure 23 The results showed that the expression of all factors in the blank group was close to 0, representing the baseline level of immunity. In the infection group, significant upregulation of pro-inflammatory factors such as IL-1β, TNF-α, and IL-6 was observed on day 1, with a slight increase in IgM. On day 3, the expression of pro-inflammatory factors remained high, accompanied by a significant upregulation of IgM. On day 7, residual expression of pro-inflammatory factors was still visible, IL-10 was at a low level, and IgM expression remained high. In contrast, the treatment group showed a slightly higher increase in the expression of pro-inflammatory factors than the infection group on day 1, while IgM expression was similar to that of the infection group. On day 3, the expression level of pro-inflammatory factors was significantly lower than that of the infection group, IL-10 expression was slightly lower than that of the infection group, and IgM expression increased. By day 7, the expression of pro-inflammatory factors in the treatment group had returned to near the baseline level of the blank group, IL-10 expression reached its peak, and IgM expression remained at a high level but slightly lower than that of the infection group. The above results indicate that bacteriophage vB_AdhP_L12 efficiently lyses the pathogen Aeromonas dacarina from day 1 to day 3, and after day 3, it promotes the secretion of IL-10 anti-inflammatory factor and downregulates pro-inflammatory levels.

[0138] Obviously, the above embodiments of the present invention are merely examples to illustrate the present invention more clearly, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all implementation methods here. Any obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A strain of Aeromonas dacca bacteriophage vB_AdhP_L12, characterized in that, Taxonomous name: Aeromonas dacca phage vB_AdhP_L12 Aeromonas dhakensis Phage vB_AdhP_L12, with accession number CCTCC NO: M2026249, was deposited at the China Center for Type Culture Collection on January 26, 2026.

2. A phage preparation, characterized in that, Includes the Dacarya aeromonas phage vB_AdhP_L12 as described in claim 1.

3. The use of the phage preparation of claim 2 in the preparation of a medicament for the prevention and treatment of Aeromonas dacarba infection.

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