Aeromonas hydrophila bacteriophage, bacteriophage preparation and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and discloses a bacteriophage which comprises a bacteriophage strain M01 with the preservation number of CCTCC M 20251724 or a variant strain of the bacteriophage strain M01. The variant strain and the bacteriophage strain M01 at least have 95% of average nucleotide identity on the genome. The bacteriophage M01 or the variant strain thereof provided by the invention has strong adsorption and lysis effects on aeromonas hydrophila, does not have genes for coding toxin, virulence factor or antibiotic resistance in a genome, and has good temperature stability and acid-base tolerance; the aeromonas hydrophila can be well applied to aquaculture to prevent and / or treat bacterial septicemia and other diseases caused by aeromonas hydrophila infection.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and particularly relates to an Aeromonas hydrophila phage, phage preparations and their applications. Background Technology

[0002] Aeromonas hydrophila, belonging to the family Aeromonaceae and genus Aeromonas, is a Gram-negative bacterium widely found in freshwater, sewage, silt, and soil. It is one of the most common primary pathogens affecting aquatic animals. Aeromonas hydrophila can cause septicemia and high mortality rates in important farmed fish species such as carp, crucian carp, tilapia, bass, catfish, salmon, and eel, resulting in millions of dollars in direct economic losses to the global freshwater aquaculture industry. Particularly in intensive eel farming systems, Aeromonas hydrophila is considered one of the main pathogens causing serious bacterial diseases, often leading to highly contagious diseases such as hemorrhagic septicemia and gill rot in species like European eels, Japanese eels, and American eels during the spring and summer seasons, severely impacting survival rates and yields.

[0003] Currently, aquaculture mainly relies on antibiotics, vaccines, and chemical disinfectants to control Aeromonas hydrophila. While antibiotics can control disease in the short term when used to prevent and treat bacterial infections, overuse can lead to the widespread transmission of antibiotic resistance genes, the emergence of multidrug-resistant strains, and antibiotic residues in aquatic products, posing serious threats to the sustainable development of aquaculture systems, food safety, and public health. Although vaccine research is progressing, it is mostly in the experimental stage, limiting its practical application. While chemical disinfectants have antibacterial effects, they lack specificity, easily causing stress in farmed animals and damaging aquatic ecosystems.

[0004] Therefore, there is an urgent need to develop a novel control strategy for Aeromonas hydrophila that is efficient, safe, and highly specific. Summary of the Invention

[0005] The first objective of this invention is to provide a bacteriophage comprising bacteriophage strain M01 or its variant strain with accession number CCTCC M20251724; the variant strain has at least 95% average nucleotide identity in its genome with bacteriophage strain M01. The bacteriophage strain M01 or its variant strain exhibits strong adsorption and lysis activity against Aeromonas hydrophila, lacks genes encoding toxins, virulence factors, or antibiotic resistance in its genome, and possesses good temperature stability and acid-base tolerance, making it well-suited for application in aquaculture to prevent and / or treat diseases such as bacterial septicemia caused by Aeromonas hydrophila infection.

[0006] Furthermore, the bacteriophage strain M01 or its variant strains infect and lyse one or more Aeromonas hydrophila.

[0007] A second objective of the present invention is to provide a phage formulation comprising the aforementioned phage or its derivatives.

[0008] Furthermore, the phage preparation is in the form of a solution, and the titer of the phage is 10. 5 PFU / mL ~10 10 PFU / mL.

[0009] A third objective of this invention is to provide the use of the above-mentioned bacteriophage or bacteriophage preparation in the preparation of Aeromonas hydrophila inhibitors.

[0010] A fourth objective of this invention is to provide the use of the above-mentioned bacteriophage or bacteriophage preparation in the preparation of products for the prevention and / or treatment of Aeromonas hydrophila infections.

[0011] A fifth objective of the present invention is to provide the use of the above-mentioned bacteriophage or bacteriophage preparation in the preparation of products for the prevention and / or treatment of bacterial diseases in fish caused by Aeromonas hydrophila infection.

[0012] Furthermore, the product for preventing and / or treating bacterial diseases in fish is one or more of a bactericide, feed additive, and water conditioner.

[0013] The sixth objective of this invention is to provide the application of the above-mentioned bacteriophage or bacteriophage preparation in aquaculture.

[0014] Biological Preservation Instructions

[0015] Biological material: Bacteriophage strain M01, classified as Aeromonas hydrophila phage M01, was deposited on July 30, 2025, at the China Center for Type Culture Collection (CCTCC), located at School of Life Sciences, Wuhan University, No. 16 Luojia Mountain Road, Wuchang District, Wuhan, Hubei Province, China; accession number: CCTCC NO.M 20251724. Attached Figure Description

[0016] Figure 1 The electron microscope image (scale bar is 100 nm) of phage M01 provided in Example 1 of this invention;

[0017] Figure 2 The figure shows the experimental results of the temperature stability test of bacteriophage M01 provided in Example 1 of this invention;

[0018] Figure 3 The figure shows the experimental results of pH stability testing of bacteriophage M01 provided in Example 1 of this invention;

[0019] Figure 4 This is a diagram showing the experimental results of whole-genome sequence homology comparison of phage M01 provided in Example 1 of this invention;

[0020] Figure 5 This is a one-step growth curve of phage M01 provided in Example 2 of the present invention;

[0021] Figure 6 This is a graph showing the experimental results of the optimal multiplicity of infection test for phage M01 provided in Example 2 of this invention;

[0022] Figure 7 The figure shows the experimental results of the in vitro antibacterial effect test of phage M01 provided in Example 2 of this invention;

[0023] Figure 8 This is a diagram showing the experimental results of challenging American eels with Aeromonas hydrophila 2408, as provided in Example 3 of this invention.

[0024] Figure 9 This is one of the experimental results (survival rate) of treating a fish model infected with Aeromonas hydrophila using bacteriophage M01, as provided in Example 3 of this invention.

[0025] Figure 10 The second figure shows the experimental results of treating a fish model infected with Aeromonas hydrophila using bacteriophage M01, as provided in Example 3 of this invention (bacteriophage concentration in muscle tissue). Detailed Implementation

[0026] Based on the goal of controlling Aeromonas hydrophila infection in existing aquaculture, and considering the problems existing in the practical application of existing Aeromonas hydrophila control methods such as antibiotics, the inventors of this invention chose bacteriophages as a means to control Aeromonas hydrophila. However, the reported Aeromonas hydrophila bacteriophages are not ideal in terms of lytic ability, stability, safety, and in vivo efficacy, which limits their application in aquaculture.

[0027] Therefore, in order to obtain a bacteriophage with excellent temperature stability and acid-base tolerance, and strong lytic ability against Aeromonas hydrophila, the inventors first isolated a highly pathogenic Aeromonas hydrophila 2408 strain from the skin, abdominal cavity, and congested head of diseased eels. Then, using this Aeromonas hydrophila 2408 as a host bacterium, they creatively isolated and screened a bacteriophage strain M01 from aquaculture water in Fujian Province. This bacteriophage strain M01 exhibited excellent adsorption, lysis, and inhibition of Aeromonas hydrophila 2408 in both in vitro and in vivo experiments. Furthermore, it maintained high activity under temperature conditions of 10℃–50℃ and solution environments with pH values ​​of 4–11, demonstrating excellent temperature stability and acid-base tolerance, strong environmental adaptability, and promising practical application prospects in aquaculture. Based on this, the technical solution of this invention was obtained.

[0028] First, this invention provides a bacteriophage. This bacteriophage includes bacteriophage strain M01 or its variant strain with accession number CCTCC M20251724. The variant strain shares at least 95% average nucleotide identity with bacteriophage strain M01 in its genome. Specifically, the variant strain can be a strain of the same species but a different subtype as bacteriophage strain M01, or it can be a progeny obtained by culturing the preserved bacteriophage strain M01. The variant strain and bacteriophage strain M01 may have some differences in their genomes, but they share the same phenotypic characteristics—morphologically, both have a regular polyhedral head with a diameter of approximately 53±2 nm and a contracted tail with a length of approximately 85±5 nm, and the tail fibers are distinctly curled, making them a typical myovirus; in terms of culture characteristics, they can form small, clear spots with halos on solid culture media, with a spot diameter of approximately 1 mm to 1.6 mm.

[0029] In this invention, the bacteriophage strain M01 or its variant strains can infect and lyse Aeromonas hydrophila, and specific examples of Aeromonas hydrophila include, but are not limited to, Aeromonas hydrophila 2408. That is, the host spectrum of the bacteriophage is not limited to the single strain Aeromonas hydrophila 2408, and it can also act on other isolates of Aeromonas hydrophila.

[0030] In this invention, the incubation period of the bacteriophage strain M01 or its variant strain for infecting Aeromonas hydrophila is about 10 min to 30 min, the outbreak time is about 20 min to 100 min, and the outbreak dose is about 100 PFU / cell to 300 PFU / cell. The bacteriophage strain M01 or its variant strain has a short incubation period, which can quickly suppress Aeromonas hydrophila and shorten the inhibition period, showing significant advantages in rapidly controlling and suppressing Aeromonas hydrophila.

[0031] Second, the present invention also provides a phage formulation. This phage formulation includes the aforementioned phage or its derivatives. The derivatives refer to substances constituting the aforementioned phage or their expression products, specifically including, but not limited to, one or more of the following: phage nucleic acid, phage structural proteins, subviral particles assembled from nucleic acids and proteins, and phage expression products. More specifically, the phage nucleic acid may be, but is not limited to, a complete genome or a fragment thereof, a truncated form, or a mutant; the phage structural proteins may specifically be, but are not limited to, capsid proteins, tail tube proteins, tail fimbriae, or substrate proteins; the subviral particles assembled from nucleic acids and proteins may specifically be, but are not limited to, phage precursors lacking partial structures or empty capsids.

[0032] In this invention, the phage formulation further includes biologically acceptable excipients and adjuvants. These biologically acceptable excipients and adjuvants are conventionally used techniques in the prior art, and those skilled in the art can make adaptive selections according to actual needs. This invention does not impose any particular limitations on them. More specifically, specific examples of the biologically acceptable excipients and adjuvants include, but are not limited to, one or more of the following: solid carriers, liquid carriers, surfactants, binders, stabilizers, and pH adjusters.

[0033] In this invention, the bacteriophage preparation preferably further includes an antibiotic; specifically, the antibiotic is a β-lactam antibiotic, including, but not limited to, one or more of penicillin, ampicillin, and amoxicillin. In this case, the antibiotic and the bacteriophage can synergistically work to achieve a better antibacterial effect.

[0034] In this invention, specific examples of dosage forms of the phage preparation include, but are not limited to, one or more of the following: tablets, granules, solutions, suspensions, emulsions, and gels.

[0035] In some specific embodiments, when the phage preparation is specifically in the form of a solution, the phage titer is preferably 10. 5 PFU / mL ~10 10 PFU / mL, specifically 10 5 PFU / mL, 2×10 5 PFU / mL, 4×10 5 PFU / mL, 8×10 5 PFU / mL, 10 6 PFU / mL, 5×10 6 PFU / mL, 10 7 PFU / mL, 10 8 PFU / mL, 10 9 PFU / mL, 1010 PFU / mL or any value between them. At this point, the phage preparation is better able to achieve infection and lysis of Aeromonas hydrophila.

[0036] Third, the present invention provides the application of the above-mentioned bacteriophage or bacteriophage preparation in the preparation of Aeromonas hydrophila inhibitors.

[0037] In this invention, the bacteriophage or bacteriophage preparation has high affinity and lytic ability for Aeromonas hydrophila, enabling rapid adsorption and lysis of Aeromonas hydrophila. Within 1 hour of co-culturing with Aeromonas hydrophila, a concentration of 10-1 can be achieved. 9 Titers above PFU / mL show great potential for rapid killing and proliferation inhibition of Aeromonas hydrophila.

[0038] Fourth, the present invention provides the use of the above-mentioned bacteriophage or bacteriophage preparation in the preparation of products for the prevention and / or treatment of Aeromonas hydrophila infection.

[0039] In this invention, the bacteriophage or bacteriophage preparation reduces the density of Aeromonas hydrophila in the environment or host by strongly lysing Aeromonas hydrophila, thereby reducing the number of Aeromonas hydrophila below the pathogenic threshold and achieving the effect of preventing Aeromonas hydrophila infection; and / or, reducing the Aeromonas hydrophila load in the host with Aeromonas hydrophila infection to alleviate infection symptoms and achieve the effect of treating Aeromonas hydrophila infection.

[0040] Fifth, this invention provides the use of the above-mentioned bacteriophage or bacteriophage preparation in the preparation of products for the prevention and / or treatment of bacterial diseases in fish. The bacterial diseases in fish are caused by Aeromonas hydrophila infection.

[0041] In this invention, the bacteriophage or bacteriophage preparation still has a strong lytic effect on Aeromonas hydrophila in the fish body, and can exist in the fish tissue at a high titer for more than 9 days. It has a very good therapeutic effect on fish infected with Aeromonas hydrophila, and will not damage the physiological state of the fish or the ecological environment of the aquatic body.

[0042] In this invention, the products for preventing and / or treating bacterial diseases in fish can be further subdivided into various types depending on the actual application scenario. Specific examples include, but are not limited to, one or more of bactericides, feed additives, and water quality conditioners.

[0043] In this invention, specific examples of the fish species include, but are not limited to, one or more of the following: carp, crucian carp, tilapia, perch, catfish, salmon, and eel.

[0044] Sixth, the present invention provides the application of the above-mentioned bacteriophage or bacteriophage preparation in aquaculture.

[0045] In this invention, the bacteriophage or bacteriophage preparation achieves the prevention and control of Aeromonas hydrophila throughout the entire aquaculture cycle through its strong lytic effect on Aeromonas hydrophila, thereby reducing the economic losses caused by Aeromonas hydrophila infection to aquaculture.

[0046] The embodiments of the present invention are described in detail below. These embodiments are intended to explain the present invention and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0047] Example 1

[0048] This embodiment illustrates the screening, identification, and biopreservation of bacteriophage M01, specifically including:

[0049] 1. Isolation and identification of Aeromonas hydrophila 2408

[0050] (1) Under sterile conditions, 1g of skin disease material, 1g of abdominal disease material and 1g of head congestion disease material of diseased eels were collected, resuspended in an appropriate amount of sterile water, and shaken at 30℃ and 180rpm for 1h to obtain bacterial solution.

[0051] (2) After serial dilution of the bacterial solution, it was spread on LB plates and incubated at 30°C for 12 hours. Single colonies were picked and purified by streaking five times on LB plates. Single colonies were then sent to Shanghai Sangon Biotech for identification. After molecular identification and biochemical characterization, it was identified as Aeromonas hydrophila and named Aeromonas hydrophila 2408.

[0052] 2. Isolation of bacteriophage M01

[0053] (1) Collect aquaculture water from Fujian Province and filter it through a 0.45 μm pore membrane to obtain aquaculture water samples to be screened; take Aeromonas hydrophila 2408 bacterial suspension (LB liquid medium, cell density approximately 10⁻¹⁰) in logarithmic growth phase at a 1:1 volume ratio. 7 After mixing CFU / mL with the culture water sample to be screened, the mixture was shaken at 37℃ and 180 rpm for 8 hours to obtain the enriched culture medium.

[0054] (2) The enriched culture medium was filtered through a 0.45 μm pore membrane. The enriched culture medium was cultured using the double-layer plate method until phage plaques appeared. A single phage plaque was picked and purified 5 times to obtain phage M01. The phage was then resuspended in an appropriate amount of SM buffer (pH=7.5) to obtain the phage solution.

[0055] (3) The phage solution was centrifuged at 200,000×g and 4℃ for 24h using CsCl gradient ultracentrifugation to obtain phage bands. The target band was then extracted using a sterile syringe and dialyzed 5 times with SM buffer (pH=7.5) to obtain phage concentrate. The concentrate was stored at 4℃ in the dark for later use.

[0056] 3. Determination of the biological characteristics of bacteriophage M01

[0057] (1) Appearance and morphology: 10 μL of phage concentrate was added to a 200-mesh copper grid and allowed to adsorb in the dark for 30 min. Then, it was stained with 1% phosphotungstic acid for 20 min and air-dried for 30 min. The morphology of phage M01 was observed using a transmission electron microscope HT-7800 (Hitachi) at 80 kV. The results are as follows: Figure 1 As shown.

[0058] Depend on Figure 1 The results show that phage M01 has a regular polyhedral head with a diameter of 53±2nm and a contractile tail with a length of approximately 85±5nm, and the tail fibers are obviously curled, which is a typical myovirus.

[0059] (2) Temperature stability: 1 mL of phage concentrate was incubated at 10℃, 20℃, 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃ for 1 h. The phage titer was then determined using the double-layer plate method. Three biological replicates were performed at each temperature, and the average value was taken. All reagents and conditions were kept consistent. The results are as follows: Figure 2 As shown.

[0060] Depend on Figure 2 The results show that bacteriophage M01 has good stability in the temperature range of 10℃ to 50℃, with a lethal temperature of 60℃, and can maintain good activity under the conventional temperature conditions of aquaculture.

[0061] (3) pH stability: 100 μL of phage concentrate was mixed with 0.9 mL of PBS, and the pH of each mixture was adjusted to 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0 using HCl or NaOH. The mixtures were incubated at 37°C for 1 h, and the phage titer was then determined using the double-layer plate method. Three biological replicates were performed for each pH value, and the average value was taken. All reagents and conditions were kept consistent. The results are shown below. Figure 3 As shown.

[0062] Depend on Figure 3The results show that bacteriophage M01 maintains high activity at pH values ​​of 4–11 with no significant change in titer. Furthermore, bacteriophage M01 maintains strong activity at pH 12, demonstrating excellent acid and alkali tolerance and adapting well to the complex and changing aquatic environment in aquaculture.

[0063] 4. Genome sequencing of bacteriophage M01

[0064] (1) The genomic DNA of phage M01 was extracted using a viral genome RNA / DNA extraction kit (Takara, catalog number 9766) and the instructions were followed. The DNA was then sent to Shanghai Ruixing for whole-genome sequencing. The sequencing results showed that the genome of phage M01 did not contain genes encoding toxins, virulence factors, or antibiotic resistance.

[0065] (2) The whole genome sequence of phage M01 was compared with existing phage sequences in the NCBI database for homology analysis. The results are as follows: Figure 4 As shown.

[0066] Depend on Figure 4 The results show that Aeromonas phage AVP1 (OP889247.1) has the highest identity with phage M01, with an identity of 85.3%, which means that phage M01 is a new species.

[0067] 5. Biopreservation of bacteriophage M01

[0068] Bacteriophage M01 was deposited at the China Center for Type Culture Collection (CCTCC) on July 30, 2025, with accession number CCTCC M 20251724.

[0069] Example 2

[0070] This embodiment illustrates the biological activity of phage M01 provided in Example 1, specifically including:

[0071] 1. One-step growth curve of bacteriophage M01

[0072] According to a multiplicity of infection of 0.01, the phage concentrate provided in Example 1 was inoculated into a culture of Aeromonas hydrophila 2408 in logarithmic growth phase (LB liquid medium, cell density approximately 10⁻¹¹). 7In a solution of CFU / mL, the phage was adsorbed in the dark for 10 min, centrifuged at 8000×g and 4℃ for 5 min, the precipitate was collected and resuspended in 1 mL of LB liquid medium. The centrifugation and resuspending process was repeated once to obtain a suspension. 1 mL of the suspension was mixed with 5 mL of LB liquid medium and incubated at 180 rpm and 37℃ for 3 h. Samples were taken every 20 min to determine the phage titer using the double-layer plate method, and the reagents and conditions were kept consistent. The results are as follows: Figure 5 As shown.

[0073] Depend on Figure 5 The results show that the incubation period for phage M01 infection of the host is approximately 20 minutes, the outbreak time is 20 to 100 minutes, and the outbreak dose is approximately 270 ± 42 PFU / cell. This indicates that phage M01 has a short incubation period and a large outbreak dose.

[0074] 2. Optimal multiplicity of infection for bacteriophage M01

[0075] According to different multiplicity of infection (10) -3 ~10 2 Take the phage concentrate provided in Example 1 and inoculate it into a culture of Aeromonas hydrophila 2408 in the logarithmic growth phase (LB liquid medium, cell density approximately 10⁻⁶). 7 The phage titer was determined by centrifuging at 8000×g at 4℃ for 5 min in CFU / mL, and then incubated in 1 mL of LB liquid medium in the dark for 10 min. The precipitate was collected, resuspended in 1 mL of LB liquid medium, and incubated at 37℃ for 4 h. The culture medium was then centrifuged at 8000×g for 5 min, and the supernatant was collected and filtered through a 0.45 μm syringe filter. The phage titer was determined using the double-layer plate method, with all reagents and conditions kept consistent. The results are as follows: Figure 6 As shown.

[0076] Depend on Figure 6 The results show that the multiplicity of infection is 10. -3 ~10 2 Within this range, the phage titer shows a trend of first increasing and then decreasing, with the optimal multiplicity of infection being 1, at which point the phage titer is approximately 10. 7 PFU / mL.

[0077] 3. In vitro antibacterial effect of bacteriophage M01

[0078] Take 100 μL of the phage concentrate provided in Example 1 and 100 μL of Aeromonas hydrophila 2408 bacterial culture in logarithmic growth phase (LB liquid medium, cell density approximately 10⁻⁶). 7After mixing thoroughly with CFU / mL, the mixture was added to a 96-well plate. The 96-well plate was placed in a Varioskan LUX multimode microplate reader (Thermo Fisher Scientific, USA) and incubated with shaking at 180 rpm and 37°C for 12 h. OD was measured every 0.5 h during the incubation process. 600 An equal volume of PBS solution (150 mM, pH = 7.4) was used as a blank control instead of the phage concentrate. Three biological replicates were performed for each group, and the average value was taken. All reagents and conditions were kept consistent. Results are as follows: Figure 7 As shown.

[0079] Depend on Figure 7 The results show that during the shaking culture period from 0 to 1 hour, the OD of the culture medium... 600 The decrease indicates that bacteriophage M01 lyses Aeromonas hydrophila 2408, and that the lysis rate of bacteriophage M01 is greater than the proliferation rate of Aeromonas hydrophila 2408; while with the extension of shaking culture time, the OD of the culture medium decreases. 600 The value initially rose slightly and then stabilized at around 0.4, indicating that the rate at which bacteriophage M01 lysed Aeromonas hydrophila 2408 was comparable to the rate at which Aeromonas hydrophila 2408 proliferated.

[0080] Example 3

[0081] This embodiment illustrates the application of the bacteriophage M01 provided in Example 1 in the preparation of a drug for treating or alleviating Aeromonas hydrophila infection, specifically including:

[0082] 1. Construction of a fish model infected with Aeromonas hydrophila

[0083] Twenty American eels, each approximately 20cm in length, were randomly divided into four groups and subjected to the following treatments:

[0084] (1) Low-dose group (N=5): administered via intraperitoneal injection at a dose of 10 5 The dosage of CFU / tail was determined by challenging American eels with Aeromonas hydrophila 2408 (injection volume of 100 μL / tail);

[0085] (2) Medium-dose group (N=5): Administered via intraperitoneal injection at a dose of 10... 6 The dosage of CFU / tail was determined by challenging American eels with Aeromonas hydrophila 2408 (injection volume of 100 μL / tail);

[0086] (3) High-dose group (N=5): Administered via intraperitoneal injection at a dose of 10... 7 The dosage of CFU / tail was determined by challenging American eels with Aeromonas hydrophila 2408 (injection volume of 100 μL / tail);

[0087] (4) Control group (N=5): American eels were treated with PBS solution (150mM, pH=7.4) by intraperitoneal injection at a volume of 100μL / tail (i.e., the dose of Aeromonas hydrophila 2408 was 0PFU / tail).

[0088] The day of intraperitoneal injection of Aeromonas hydrophila 2408 was designated as day 0 of rearing. American eels in each group were reared for 7 days under the same conditions. During this period, the physical signs and survival status of the eels were observed, and the survival rate was calculated. The results are as follows: Figure 8 As shown.

[0089] In the experiment, it was observed that infected American eels exhibited head congestion, abdominal edema, gallbladder swelling and filling with bile, and reduced activity. The symptoms of the American eels worsened with increasing injection doses of Aeromonas hydrophila 2408.

[0090] Depend on Figure 8 The results show that the minimum lethal dose of Aeromonas hydrophila 2408 for American eels is 10. 6 CFU / tail, highly pathogenic.

[0091] 2. Therapeutic effect of bacteriophage M01

[0092] Fifty American eels, each approximately 20cm in length, were randomly divided into 5 groups and subjected to the following treatments:

[0093] (1) Negative control group (N=10): 10 mg / L was administered via intraperitoneal injection. 6 The dosage of CFU / tail (injection volume of 100 μL / tail) was used to challenge American eels with Aeromonas hydrophila 2408, and American eels were treated with PBS solution (150 mM, pH=7.4) by intraperitoneal injection at a dose of 0.1 mL / tail / day 2 hours after challenge.

[0094] (2) Antibiotic group (N=10): 10 mg of antibiotics was administered via intraperitoneal injection. 6 The dosage of CFU / tail (injection volume of 100μL / tail) was used to challenge American eels with Aeromonas hydrophila 2408, and then ampicillin solution (2.5mg / mL) was administered to the American eels via intraperitoneal injection at a dose of 0.1mL / tail / day 2 hours after the challenge.

[0095] (3) Phage injection group (N=10): 10 phages were injected intraperitoneally. 6 The dosage was 100 μL / tail (CFU / tail). Aeromonas hydrophila 2408 was used to challenge American eels, and phage concentrate (100 μL / tail) was administered intraperitoneally at a dose of 0.1 mL / tail / day 2 hours post-challenge. 8Treatment of American eels with PFU / mL;

[0096] (4) Phage immersion group (N=10): 10 phages were injected intraperitoneally. 6 The dosage was 100 μL / fish (CFU / fish). American eels were challenged with Aeromonas hydrophila 2408, and phage concentrate was added to the culture water 2 hours after challenge to maintain the phage M01 titer at 10. 8 PFU / mL, used to treat American eels;

[0097] (5) Blank control group (N=10): No treatment was given to the American eels;

[0098] The day of intraperitoneal injection of Aeromonas hydrophila 2408 was designated as day 0 of rearing. American eels in each group were reared for 7 days under the same conditions. During this period, the physical signs and survival status of the eels were observed, and the survival rate was calculated. The results are as follows: Figure 9 As shown in the figure. American eels that died during the experiment in the phage immersion group were retrieved, and the phage content in their muscle tissue was determined using the double-layer plate method. On day 9 of rearing, surviving eels in both the phage injection and phage immersion groups were euthanized, and the phage content in their muscle tissue was determined using the double-layer plate method. The results are shown in the figure. Figure 10 As shown.

[0099] In the experiment, infected American eels were observed to exhibit significantly slowed behavior and weak avoidance response to stimuli.

[0100] Depend on Figure 9 The results show that in the negative control group, large numbers of eels began to die from Aeromonas hydrophila 2408 challenge treatment on day 3, reaching 100% mortality by day 5. In the phage immersion group, the survival rate of eels was greater than 70%, demonstrating a therapeutic effect comparable to the antibiotic group. Notably, no eels in the phage injection group died during the rearing period, achieving a 100% survival rate, indicating an excellent therapeutic effect.

[0101] Depend on Figure 10 The results show that phage M01 can effectively enter the body of the American eel through immersion, and the titer of phage M01 in the muscle tissue of the American eel in the phage injection group reached 10 on day 9. 6 The PFU / g indicates that phage M01 can survive at a high titer in American eels, making it a highly promising drug for treating or alleviating Aeromonas hydrophila infections.

[0102] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. A bacteriophage, characterized in that, The bacteriophage includes bacteriophage strain M01 or its variant strain with accession number CCTCC M20251724; wherein the variant strain has at least 95% average nucleotide identity with bacteriophage strain M01 in the genome.

2. The bacteriophage according to claim 1, characterized in that, The bacteriophage strain M01 or its variant strains infect and lyse one or more Aeromonas hydrophila.

3. A phage preparation, characterized in that, The phage formulation includes the phage or its derivatives as described in claim 1 or 2.

4. The phage preparation according to claim 3, characterized in that, The phage preparation is in the form of a solution, and the titer of the phage is 10. 5 PFU / mL ~10 10 PFU / mL.

5. The use of the bacteriophage according to claim 1 or 2 or the bacteriophage preparation according to claim 3 or 4 in the preparation of Aeromonas hydrophila inhibitors.

6. The use of the bacteriophage of claim 1 or 2 or the bacteriophage preparation of claim 3 or 4 in the preparation of products for the prevention and / or treatment of Aeromonas hydrophila infections.

7. The use of the bacteriophage according to claim 1 or 2 or the bacteriophage preparation according to claim 3 or 4 in the preparation of products for the prevention and / or treatment of bacterial diseases in fish, characterized in that, The bacterial disease in the fish was caused by Aeromonas hydrophila infection.

8. The application according to claim 7, characterized in that, The product used for the prevention and / or treatment of bacterial diseases in fish is one or more of bactericides, feed additives, and water conditioners.

9. The application of the bacteriophage according to claim 1 or 2 or the bacteriophage preparation according to claim 3 or 4 in aquaculture.

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