Aeromonas hydrophila broad-host-range bacteriophage and application thereof

Abstract

This invention relates to the field of microbial technology, and more particularly to a broad-spectrum Aeromonas hydrophila phage and its applications. The Aeromonas hydrophila phage AhP2505006 is classified as follows: Aeromonas hydrophila Phage AhP2505006, with accession number CCTCC NO: M20251307, exhibits cross-species lytic activity. It can effectively lyse *Aeromonas*, *Vibrio parahaemolyticus* and *Vibrio alginolyticus*, and *Citrobacter freundii*. It also possesses excellent targeting ability against multidrug-resistant strains and broad host spectrum characteristics, achieving a lysis rate of up to 80% against multidrug-resistant *Aeromonas hydrophila*. In the prevention and control of diseases caused by various pathogens, or in practical scenarios facing the limitations of traditional antibiotics and highly specific bacteriophages, it has become a highly promising solution.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a broad host spectrum Aeromonas hydrophila phage and its applications. Background Technology

[0002] Aeromonas hydrophila, a Gram-negative bacterium widely distributed in natural water bodies, is one of the most serious pathogens in aquaculture. It can infect almost all common farmed species, including carp, bass, and whiteleg shrimp, causing diseases such as hemorrhagic septicemia and ulcer disease. In high-density aquaculture settings, the mortality rate often exceeds 50%, and the economic losses caused account for 30% to 50% of the total economic losses from aquatic diseases. More alarmingly, this bacterium is a zoonotic pathogen, capable of causing acute gastroenteritis and septicemia in humans through contaminated aquatic products or contact infection, posing a dual threat to food safety and public health.

[0003] Bacteriophages, as viruses that specifically lyse bacteria, possess natural advantages such as high specificity, no residue, and low likelihood of developing drug resistance, making them a hot topic for antibiotic alternatives. While several Aeromonas hydrophila phage strains have been reported in existing technologies, they generally face significant application bottlenecks: First, their host spectrum is narrow; most phages can only lyse sensitive strains in specific regions, with lysis rates of less than 50% against multidrug-resistant strains, lacking specificity. Second, they have poor environmental tolerance; existing strains are mostly sensitive to high temperatures, and their activity drops sharply in the temperature fluctuations or pH imbalances common in aquaculture, making them difficult to adapt to actual aquaculture scenarios.

[0004] To address the shortcomings of existing technologies, developing environmentally adaptable, drug-resistant strains of freshwater-cultured Litopenaeus vannamei that target the disease, as well as Aeromonas hydrophila phages with highly efficient disease control capabilities, and constructing an efficient prevention and control system, is of great practical significance for promoting the green transformation of aquaculture and ensuring food safety. Summary of the Invention

[0005] In view of this, the present invention proposes a broad-spectrum Aeromonas hydrophila phage and its applications. The Aeromonas hydrophila phage AhP2505006 of the present invention exhibits cross-species lytic activity, effectively lysing *Vibrio parahaemolyticus* and *Vibrio alginolyticus* of the *Aeromonas* genus and *Citrobacter* genus. It also possesses excellent targeting ability against multidrug-resistant strains and a broad-spectrum characteristic, providing an effective control method for multiple infectious diseases and a new solution to the challenge of controlling multidrug-resistant *Aeromonas hydrophila*. Furthermore, the Aeromonas hydrophila phage AhP2505006 also exhibits high fermentation activity and high-temperature tolerance, ensuring high efficiency and stability during application.

[0006] The technical solution of this invention is implemented as follows: In a first aspect, the present invention provides a *Aeromonas hydrophila* phage AhP2505006 with cross-species lytic activity, wherein the *Aeromonas hydrophila* phage AhP2505006 is classified as... Aeromonas hydrophila Phage AhP2505006, accession number CCTCC NO: M20251307, was deposited on June 9, 2025 at the China Center for Type Culture Collection (CCTCC) (address: Wuhan University, Wuhan, China).

[0007] Secondly, the application of Aeromonas hydrophila phage AhP2505006 as described above in the lysis of aquatic pathogens is provided.

[0008] Based on the above technical solutions, preferably, the aquatic pathogenic bacteria include bacteria of the genus *Citrobacter*.

[0009] Based on the above technical solutions, a further preferred embodiment is that the Citrobacter genus includes Citrobacter freundii.

[0010] Based on the above technical solutions, preferably, the aquatic pathogenic bacteria also include one or more of the genera Aeromonas or Vibrio.

[0011] Based on the above technical solutions, a further preferred embodiment is that the Aeromonas genus bacteria includes one or more of Aeromonas hydrophila, Aeromonas vesiculosus, or Aeromonas vulgaris; and the Vibrio genus bacteria includes at least one of Vibrio parahaemolyticus or Vibrio alginolyticus.

[0012] Aeromonas hydrophila phage AhP2505006 exhibits cross-species lytic activity, which may be related to the phage genome carrying a broad-spectrum anti-restriction enzyme system (such as genes that produce modified bases) or genes that inhibit multiple bacterial defense mechanisms, or it may be related to the presence of a similar receptor on the surface of Aeromonas, Vibrio, or Citric Acid Bacteria. Coincidentally, the tail fibers or tail filaments of Aeromonas hydrophila phage AhP2505006 contain the corresponding receptor-binding proteins, thereby enabling the lysis of multiple genera of hosts.

[0013] Based on the above technical solutions, and even more preferably, the Aeromonas hydrophila is resistant to antibiotics, including one or more of colistin sulfate, enrofloxacin, florfenicol, doxycycline or amoxicillin.

[0014] Thirdly, the application of Aeromonas hydrophila phage AhP2505006 as described above in the preparation of products for the prevention or treatment of enteritis / ulceration in aquatic animals is provided.

[0015] Fourthly, a phage formulation is provided, comprising Aeromonas hydrophila phage AhP2505006 as described above.

[0016] The Aeromonas hydrophila phage AhP2505006 of the present invention has the following advantages over the prior art: 1. Aeromonas hydrophila phage AhP2505006 possesses cross-species lytic activity, making it a highly promising solution in many practical scenarios where traditional antibiotics or highly specific phages have limitations. For example, when facing enteritis and ulcer diseases in aquatic animals caused by various Aeromonas, Vibrio, or Citrobacter freundii, spraying a phage with cross-species activity can simultaneously control diseases caused by multiple related pathogens, improving control efficiency and reducing the use of chemical pesticides.

[0017] 2. Cross-species lytic activity enhances the broad-spectrum and practicality of phage therapy, combating mixed infections. Clinical infections are often caused by multiple bacteria, and a phage with cross-species activity can simultaneously target several pathogens, improving treatment efficiency. It also addresses infections with uncertain or rapidly changing pathogens. When the type of pathogen cannot be immediately identified, or when the infective flora is dynamically changing, phages with broad-spectrum lytic activity provide a more comprehensive and reliable approach. Furthermore, it reduces the complexity of phage cocktail formulations. An ideal phage cocktail requires multiple phages to cover the target strain and prevent drug resistance. A single phage that can cover multiple species simplifies cocktail composition and reduces the complexity of preparation, testing, and approval.

[0018] 3. Aeromonas hydrophila phage AhP2505006 exhibits high fermentation activity; at an MOI of 0.01, it can ferment to 5.7 × 10⁻⁶ ppm after 8 hours of cultivation. 10 With a fermentation efficiency of PFU / mL, it is 2-3 times higher than the existing Aeromonas hydrophila phage, which greatly reduces the time and economic cost of large-scale production and provides production feasibility for its large-scale promotion in freshwater shrimp farming.

[0019] 4. Aeromonas hydrophila phage AhP2505006 has the characteristic of being resistant to high temperatures of 50℃, and it can maintain its activity for 7 days under 50℃ conditions. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a phage plaque image of Aeromonas hydrophila phage AhP2505006 of the present invention; Figure 2 This is the drug sensitivity diagram of the host bacterium Ah250401 of the present invention; Figure 3 This is an MOI data diagram of Aeromonas hydrophila phage AhP2505006 of the present invention; Figure 4 The graph shows the thermal stability of Aeromonas hydrophila phage AhP2505006, which is the subject of this invention. Detailed Implementation

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0023] The host bacteria involved in this invention are all strains preserved by the inventors themselves.

[0024] Example 1 Isolation and identification of bacteriophage AhP2505006 and its corresponding host bacterium Ah250401.

[0025] Isolation and identification of the host bacterium: Water samples were taken from a Litopenaeus vannamei farm in Hubei Province, thoroughly shaken, and 100 μL was transferred to a TCBS agar plate (standard composition). The plate was spread evenly using a sterile spreader, air-dried, and incubated overnight at 37°C. Yellow single colonies were picked from the plate and transferred to sterile LB broth, incubated overnight at 37°C and 200 rpm. 16S RNA molecular identification confirmed the bacteria as Aeromonas hydrophila, designated as the host bacterium Ah250401.

[0026] Phage AhP2505006 screening and isolation: After water samples were allowed to stand for a sufficient period, the supernatant was filtered through a 0.45 μm filter membrane, followed by filtration through a 0.22 μm filter membrane. The resulting supernatant was added to sterilized LB broth containing the host bacteria and incubated overnight at 37°C and 200 rpm in a shaker. After filtration through a 0.22 μm filter membrane, the presence and titer of phages were determined using the double-layer plate method. For plates containing phage plaques, a single plaque was picked and immersed in sterile PBS buffer solution, then filtered through a 0.22 μm filter membrane. The titer of the phage was determined using the double-layer plate method. This step was repeated 3-4 times until the size and morphology of the plaques on the plates were uniform, yielding the purified phage. The plaque pattern is shown in the figure. Figure 1 As shown.

[0027] Example 2 Antimicrobial susceptibility test of host bacterium Ah250401.

[0028] The procedure was performed according to the method described in SN / T.1944-2016 Determination of Antimicrobial Resistance in Animals and Their Products - Paper Disc Diffusion Method. The results are shown in Table 1 and... Figure 2 .

[0029] Table 1. Antimicrobial resistance data of host bacterium Ah250401

[0030] Note: S represents sensitive, I represents intermediate, and R represents drug resistance.

[0031] According to Table 1 and Figure 2 It is known that this host bacterium is only sensitive to neomycin and amikacin, and exhibits resistance to colistin sulfate (CS), enrofloxacin (ENR), florfenicol (FFC), doxycycline (DXT), and amoxicillin (AML). The Aeromonas hydrophila phage AhP2505006 of this invention, isolated from this resistant strain, can efficiently lyse and ferment this highly resistant host bacterium, achieving targeted control of drug-resistant bacteria. This provides a specific solution to the dilemma of "drug resistance leading to no cure" in freshwater shrimp farming caused by Aeromonas hydrophila resistance.

[0032] Example 3 Optimal multiplicity of infection assay for bacteriophage AhP2505006.

[0033] Host bacteria and bacteriophages were added to sterile LB broth at ratios of 1:1, 10:1, 100:1, and 1000:1 (host bacteria: phage), respectively, and incubated at 37°C and 200 rpm for 8 hours. After centrifugation and filtration, the filtrate was plated on double-layer plates to determine the titer. The results are as follows: Figure 3 As shown.

[0034] As can be seen, bacteriophage AhP2505006 exhibits high fermentation activity; at an MOI of 0.01, it can ferment to 5.7 × 10⁻⁶ ppm after 8 hours of cultivation. 10 With a fermentation efficiency of PFU / mL, it is 2-3 times higher than the existing Aeromonas hydrophila phage, which greatly reduces the time and economic cost of large-scale production and provides production feasibility for its large-scale promotion in freshwater shrimp farming.

[0035] Example 4 Thermal stability test of bacteriophage AhP2505006.

[0036] Four temperature gradients were set up: 4℃, 25℃, 37℃, and 50℃. 4℃ represents the general refrigeration temperature for biological materials; 25℃ is room temperature, used to investigate the decay rate of phage titers at room temperature; 37℃ is the optimal growth temperature for most bacteria (hosts); and 50℃ is a higher temperature, often used to accelerate stability experiments of biologically active substances, where the activity of various active substances decays more rapidly. 5*10 8 Phage solution at pfu / ml was aliquoted into different sterile centrifuge tubes and placed under different temperature conditions. Phage titers were measured at different time points within 7 days. The results are as follows: Figure 4 As shown.

[0037] The results showed that the phage could maintain stability at temperatures ranging from 4 to 37°C and could maintain activity for 7 days at 50°C. It can be widely used in extreme environments such as summer high temperatures (water temperature often reaches 30-35°C) and various aquaculture sites in freshwater shrimp farming, solving the technical defects of poor environmental adaptability and limited application scenarios of existing phages, and ensuring the activity and stability during application.

[0038] Example 5 Validation of the AhP2505006 bacteriophage targeting multidrug-resistant strains and its broad host spectrum.

[0039] Test strains: Multidrug-resistant Aeromonas hydrophila: 20 strains, all of which were clinical isolates from aquatic animals. Drug susceptibility tests confirmed their resistance to commonly used aquatic antibiotics such as florfenicol, doxycycline, and enrofloxacin. Sensitive Aeromonas hydrophila: 10 strains, isolated from the intestines or water bodies of farmed animals from different regions and different farmed species. Closely related species of Aeromonas: 5 strains, including Aeromonas vernix and Aeromonas guinea pig, all of which were isolates from aquaculture environments.

[0040] Bacteriophage: The Aeromonas hydrophila phage AhP2505006 of this invention, with a titer adjusted to 1×10⁻⁶. 9 PFU / mL.

[0041] The above 35 bacterial strains (20 drug-resistant strains, 10 susceptible strains, and 5 closely related strains) were activated to the logarithmic growth phase. Using the double-layer agar plate method, each bacterial suspension was mixed with sterilized LB semi-solid agar at a suitable temperature and poured into LB solid plates. Prepared Aeromonas hydrophila phage AhP2505006 was then sequentially spotted onto each double-layer plate. After drying, the plates were inverted and incubated overnight in a 37°C shaker. Plaque formation was observed, and the lysis rate was calculated (lysis rate = number of lysed strains / total number of strains × 100%). The lysis results are shown in Tables 2, 3, and 4.

[0042] Table 2. Lysis of multidrug-resistant Aeromonas hydrophila (20 strains)

[0043] Note: "+" indicates cleavage, "-" indicates no cleavage.

[0044] Table 3. Lysis of susceptible Aeromonas hydrophila (10 strains)

[0045] Note: "+" indicates cleavage, "-" indicates no cleavage.

[0046] Table 4. Lysis of five closely related species of Aeromonas.

[0047] Note: "+" indicates cleavage, "-" indicates no cleavage.

[0048] According to Tables 2-4, among the 20 multidrug-resistant strains, 16 could form plaques with a lysis rate of 80%; among the 10 susceptible strains, 9 could form plaques with a lysis rate of 90%; among the closely related species of Aeromonas, 2 could form plaques, namely Aeromonas vernix and Aeromonas guinea pig.

[0049] The bacteriophage AhP2505006 of this invention possesses excellent targeting capabilities against multidrug-resistant strains and a broad host spectrum: it achieves an 80% lysis rate against clinically isolated multidrug-resistant Aeromonas hydrophila from aquatic organisms, and its host spectrum covers Aeromonas hydrophila from different regions and different aquaculture species. It can also lyse two closely related species of the Aeromonas genus. This characteristic distinguishes it from the limitations of existing bacteriophages with "single drug resistance spectrum and narrow host spectrum," making it adaptable to the prevention and control needs of Aeromonas hydrophila diseases in different regions and different aquaculture species, significantly improving the versatility and industrial application value of the technology.

[0050] Example 6 Determination of cross-species lysis activity of bacteriophage AhP2505006.

[0051] All strains used in the experiment were self-preserved aquatic isolates, and specific information is shown in Table 5: Table 5

[0052] The cross-species lytic activity of bacteriophages was determined using the double-layer agar plate method: Each pathogenic bacterium was activated to the logarithmic growth phase. Using the double-layer agar plate method, each bacterial suspension was mixed with a sterilized, appropriately heated semi-solid medium and poured onto a solid agar plate. Prepared Aeromonas hydrophila phage AhP2505006 was then sequentially spotted onto each double-layer plate. After drying, the plates were inverted and incubated overnight in a 37°C shaker. Plaque formation was observed, and the results are shown in Table 6.

[0053] Table 6. Lysis results of bacteriophage AhP2505006 on various tested aquatic strains.

[0054] Note: "+" indicates cleavage, "-" indicates no cleavage.

[0055] Table 6 shows that for Aeromonas spp.: 1 strain of Aeromonas verrucosa Aer-02 and 1 strain of Aeromonas guinea pig Aer-03 were successfully lysed; Aeromonas enterica Aer-04, Aeromonas temperate As-1, and Aeromonas verrucosa Aer-01 were not lysed; for Vibrio spp.: 2 strains of Vibrio parahaemolyticus Vp-1 and Vp-2 and 1 strain of Vibrio alginolyticus Va-3 were successfully lysed; 3 strains of Vibrio parahaemolyticus Vp-3 and Vp-4 and 3 strains of Vibrio alginolyticus Va-1, Va-2, and Va-4 were not lysed; for Citrobacter spp.: 1 strain of Citrobacter freundii Cf-1 was successfully lysed; Citrobacter freundii Cf-2 was not lysed.

[0056] In summary, Aeromonas hydrophila phage AhP2505006 exhibits cross-species lytic activity and can effectively lyse various aquatic pathogens from the genera Aeromonas, Vibrio (Vibrio parahaemolyticus and Vibrio alginolyticus), and Citrobacter (Citrobacter freundii).

[0057] Example 7 Determination of the efficacy of bacteriophage AhP2505006 in preventing Aeromonas hydrophila infection in Litopenaeus vannamei.

[0058] Nine sterilized 100L tanks were prepared, each stocked with approximately 30 healthy Litopenaeus vannamei shrimp (approximately 5g each). The tanks were divided into three groups: a treatment group, a challenge group, and a control group, with three replicates per group. The water temperature was maintained at 30℃ using heaters, and aeration was achieved using a small air pump connected to an aerator. The water conditions were: dissolved oxygen ≥5mg / L, pH 7.0–8.5, ammonia nitrogen <0.2mg / L, and nitrite <0.1mg / L. A final concentration of 1×10⁻⁶ shrimp was added to the water in the treatment and challenge groups. 7 A drug-resistant strain of Aeromonas hydrophila, Ah250401, with a titer of approximately CFU / mL, was added to the control group along with an equal volume of sterile PBS buffer. After 6 hours, the titer was increased to 2 × 10⁻⁶ CFU / mL. 8 Phage AhP2505006 at approximately PFU / mL was added to the water in the treatment group.

[0059] The number of shrimp deaths was recorded daily, and the relative survival rate (RPS) was calculated using the formula: RPS = (1 - mortality rate of the infected control group and mortality rate of the treated group) × 100%.

[0060] Three shrimp from each group were randomly selected at 24h, 48h, and 72h post-infection. Hepatopancreatic tissue was collected, and the pathogen load was determined using the plate count method. Pathological sections were prepared to observe tissue damage. The results are shown in Table 7.

[0061] Table 7. Efficacy of bacteriophages in preventing Aeromonas hydrophila infection in Litopenaeus vannamei.

[0062] In an artificial infection experiment with Aeromonas hydrophila in Litopenaeus vannamei, the bacteriophage was administered 6 hours post-infection (dose: 1×10⁻⁶). 8 The relative survival rate (RPS) of shrimp treated with this invention (PFU / mL) can reach over 75% within 7 days, reducing mortality by over 60% compared to the untreated group. For shrimp exhibiting red body and ulcer symptoms, continuous application for 3 days reduces bacterial load at lesions by 3 orders of magnitude. This invention's phage-based treatment significantly improves the survival rate of farmed Litopenaeus vannamei. The phage poses no chemical residue risk and, when applied to freshwater shrimp farming water, does not lyse beneficial bacteria such as Bacillus subtilis and nitrifying bacteria, thus maintaining the ecological balance of the water body. Simultaneously, it avoids the spread of antibiotic resistance and food safety hazards caused by antibiotic overuse, fully aligning with the current policy direction of "reducing and replacing antibiotics" in aquaculture, and providing technical support for the green and sustainable development of freshwater Litopenaeus vannamei farming.

[0063] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A hydrophilic Aeromonas phage AhP2505006 with cross-species lytic activity, characterized in that: The classification name of the Aeromonas hydrophila phage AhP2505006 is... Aeromonas hydrophila Phage AhP2505006, with accession number CCTCC NO: M20251307.

2. The application of Aeromonas hydrophila phage AhP2505006 as described in claim 1 in the lysis of aquatic pathogens.

3. The application as described in claim 2, characterized in that: The aquatic pathogens include bacteria of the genus *Citrobacter*.

4. The application as described in claim 3, characterized in that: The bacteria in question include Citrobacter freundii.

5. The application as described in claim 2, characterized in that: The aquatic pathogens also include one or more bacteria from the genera Aeromonas or Vibrio.

6. The application as described in claim 5, characterized in that: The Aeromonas genus bacteria include one or more of Aeromonas hydrophila, Aeromonas vernix, or Aeromonas vulgaris; the Vibrio genus bacteria include at least one of Vibrio parahaemolyticus or Vibrio alginolyticus.

7. The application as described in claim 6, characterized in that: The Aeromonas hydrophila is resistant to antibiotics, including one or more of colistin sulfate, enrofloxacin, florfenicol, doxycycline, or amoxicillin.

8. The use of Aeromonas hydrophila phage AhP2505006 as described in claim 1 in the preparation of products for the prevention or treatment of enteritis / ulceration in aquatic animals.

9. A bacteriophage preparation, characterized in that: Includes Aeromonas hydrophila phage AhP2505006 as described in claim 1.

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