Aeromonas hydrophila bacteriophage and application thereof

Abstract

The application discloses an Aeromonas hydrophila bacteriophage and application thereof, and the taxonomic name of the bacteriophage is Aeromonas hydrophila The application discloses an Aeromonas hydrophila bacteriophage and application thereof, and the taxonomic name of the bacteriophage is PTS0531-2, which is preserved in the Guangdong Microbial Culture Collection Center, and the preservation date is March 18, 2026, the preservation number is GDMCC No: 67971-B1, and the preservation address is No. 59, Block 5, 100, Martyrs' Road, Guangzhou. The Aeromonas hydrophila bacteriophage has a strong lysis effect on Aeromonas hydrophila derived from Chinese soft-shelled turtles, and can be used for preventing and controlling Aeromonas hydrophila in a Chinese soft-shelled turtle breeding environment or in vivo.

Description

Technical Field

[0001] This invention relates to the field of aquaculture technology, and in particular to an Aeromonas hydrophila phage and its applications. Background Technology

[0002] Aeromonas hydrophila is a Gram-negative, mesophilic bacterium widely distributed in freshwater and aquaculture environments worldwide. It is also an opportunistic pathogen, causing mass mortality in farmed animals when environmental conditions deteriorate or host immunity weakens. Aeromonas hydrophila has been reported to harm several important economically farmed species, such as grass carp, common carp, tilapia, whiteleg shrimp, and Chinese mitten crab. The pathogenic process of Aeromonas hydrophila generally includes steps such as adhesion, invasion, colonization, proliferation, toxin production, and evasion of host immunity. The virulence varies considerably among strains, and they can be classified into highly pathogenic and low-pathogenic strains. Pathogenicity is closely related to the types and expression levels of virulence factors carried (such as hemolysins, enterotoxins, and proteases). Currently, antibiotics remain the first-line treatment for the prevention and treatment of Aeromonas hydrophila infections. However, with the widespread use of antibiotics, drug-resistant strains are constantly emerging and spreading, and the number of available antibiotics is decreasing, necessitating the development of antibiotic alternatives.

[0003] Bacteriophages are a class of viruses with extremely high specificity, capable of infecting microorganisms such as bacteria, fungi, actinomycetes, or spirochetes. They were first discovered by a British biologist in 1915. It is estimated that there are approximately 10 million bacteriophages worldwide. 32 Bacteriophages play a crucial role in regulating the environment and the gut microbiota of animals. Bacteriophages are classified into virulent phages and temperate phages. Virulent phages can proliferate and lyse susceptible host bacteria, and are also known as virulent phages, specifically killing pathogens. Virulent phages invade bacteria primarily by utilizing the host's synthetic mechanisms, applying pressure to the bacterial cell wall, disrupting bacterial metabolism, leading to bacterial lysis and rupture, and subsequently releasing the phage. Compared to other treatment methods, phage therapy is safe, reliable, highly effective, rapid, economical, and practical, and shows promise as a superior antibacterial agent, especially in treating drug-resistant bacterial infections. However, currently, there are relatively few phages targeting *Aeromonas hydrophila*. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a novel Aeromonas hydrophila phage and its application, which exhibits a strong lytic effect against Aeromonas hydrophila derived from Chinese soft-shelled turtles and can be used for the prevention and control of Aeromonas hydrophila in the breeding environment or within the Chinese soft-shelled turtle.

[0005] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows: A type of Aeromonas hydrophila bacteriophage, its taxonomic name is Aeromonas hydrophilaPhage, abbreviated as PTS0531-2, is deposited at the Guangdong Provincial Center for Microbial Culture Collection on March 18, 2026, with accession number GDMCC No: 67971-B1. The deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0006] PTS0531-2 exhibits high alkali resistance and maintains high activity even at pH=12.

[0007] The application of the aforementioned Aeromonas hydrophila bacteriophage in the preparation of biological agents for the prevention or treatment of diseases caused by Aeromonas hydrophila. The Aeromonas hydrophila is derived from the Chinese soft-shelled turtle.

[0008] The biological agent is a water purifier, antibacterial drug, or feed additive.

[0009] A biological agent for the prevention and treatment of Aeromonas hydrophila, comprising a therapeutically effective amount of the Aeromonas hydrophila phage.

[0010] The beneficial effects of this invention are: This invention discovers a new Aeromonas hydrophila phage that has a strong lytic effect on Aeromonas hydrophila derived from Chinese soft-shelled turtles, and has high alkali resistance. It can be used for the prevention and control of Aeromonas hydrophila in the breeding environment or in the body of Chinese soft-shelled turtles, providing a new approach for the prevention and control of Aeromonas hydrophila. Attached Figure Description

[0011] Figure 1 This is an image showing the effect of phage plaques formed by the phages of this invention; Figure 2 This is an electron microscope image of the bacteriophage of the present invention; Figure 3 This is a sensitivity curve of the bacteriophage of the present invention to different temperatures; Figure 4 This is a sensitivity curve of the bacteriophage of the present invention to different pH values; Figure 5 This is a diagram showing the in vitro growth kinetics of the bacteriophage of the present invention; Figure 6 This is a diagram showing the lysis results of the bacteriophage of the present invention on different bacteria. Detailed Implementation

[0012] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0013] Example 1: Screening and purification of Aeromonas hydrophila bacteriophage (1) Sample collection: The intestinal contents of Chinese soft-shelled turtles were collected in September 2023 from a breeding farm in Yuhang District, Hangzhou City, Zhejiang Province.

[0014] Isolation of Aeromonas hydrophila: A disposable sterile inoculation loop was used to collect intestinal contents of a diseased Chinese soft-shelled turtle and immediately streaked onto the surface of LB solid medium. The medium was then incubated overnight at 37°C. The next day, single colonies were picked and immediately expanded in LB liquid medium. After the culture was completed, the colonies were streaked again for isolation. Colony morphology was observed, and after confirming that uniform single colonies were obtained, 16S rRNA sequencing was performed. The sequencing results were compared with the NCBI database, and Aeromonas hydrophila was identified.

[0015] (2) Specific amplification of Aeromonas hydrophila phage in the sample The intestinal contents of the diseased Chinese soft-shelled turtle were added to 20 mL of LB liquid culture medium, followed by 500 μL (1.34 × 10⁻⁶) of Aeromonas hydrophila from the intestinal contents of the diseased Chinese soft-shelled turtle. 7 (CFU / mL), mix well and incubate overnight at 28°C with shaking at 220 rpm.

[0016] (3) Detection of the presence of bacteriophages using the double-layer plate method Prepare LB solid medium with an agar content of 1.5%. After autoclaving, let it stand at room temperature for about 40-60°C. Then, take 10-15 mL and pour it into a petri dish, spreading it evenly on the bottom of the petri dish. Let it stand at room temperature for 30 minutes to solidify. Use this LB solid medium as the bottom culture medium.

[0017] Take the mixed culture broth of *Aeromonas hydrophila* and intestinal contents, centrifuge at 12000 rpm for 10 min, and filter the supernatant through a 0.22 μm filter membrane for sterilization. Mix 100 μL of the filtrate with 100 μL of *Aeromonas hydrophila* and incubate at 28°C for 10 min. Add 5 mL of 0.7% LB medium (semi-solid state), which has been autoclaved and cooled to 50°C. Mix well by pipetting with a Pasteur pipette and quickly pour the mixture onto the prepared bottom layer medium. Rotate the plate to distribute the mixture evenly into the top layer of agar. After the agar solidifies, incubate overnight at 28°C and observe for the presence of plaques.

[0018] The results of plaque observation were as follows: plaques were round, translucent spots, approximately 1-2 mm in diameter, with distinct halos. Figure 1 .

[0019] (4) Purification of phage samples A single plaque was collected and placed in 1 mL of LB liquid medium. Several extinction ceramic beads were added, and the mixture was shaken at 28°C for 1 h. Then, it was centrifuged at 12000 rpm for 5 min. The supernatant was taken and diluted using a serial dilution method. The plaque was then purified using the double-layer agar plate method. The purification was repeated three times to obtain plaques of consistent size and morphology. The phage monomer was obtained and named PTS0531-2.

[0020] (5) Amplification of phage samples Collect the purified single spot from step (4) and place it in 1 mL of LB liquid medium. Add several extinction ceramic beads, shake at 28°C for 1 h, then centrifuge at 12000 rpm for 10 min. Filter the supernatant through a 0.22 µm filter membrane for sterilization and set aside. Take 100 µL of the supernatant and add 10 mL of medium that has been cultured to the early logarithmic growth phase (OD2). 600 In a culture of Aeromonas hydrophila with a concentration of 0.3~0.6, the culture was incubated at 28°C for 5 h in a shaker at 220 rpm until the culture broth became clear, thus obtaining the phage proliferation broth.

[0021] (6) Preparation of phage preservation solution The phage propagation solution was mixed with autoclaved distilled water containing 50% glycerol at a 1:1 ratio to obtain the phage preservation solution. The phage preservation solution was stored at -80℃.

[0022] Example 2: Morphological observation of Aeromonas hydrophila bacteriophage Phage concentration was achieved using the PEG8000 precipitation method. 100 mL of phage solution, sterilized by filtration through a 0.22 µm membrane, was taken and chloroform was added to a final concentration of 1% (v / v). The solution was incubated at 37°C for 30 min, then NaCl was added at a concentration of 5.84 g / 100 mL. After shaking to dissolve, the solution was incubated on ice for 1 h, centrifuged at 10000×g for 10 min, and the supernatant was collected. Solid PEG8000 was added to a final concentration of 10% (w / v), and the solution was thoroughly shaken to dissolve. The solution was incubated on ice for at least 1 h to allow for complete phage particle precipitation. The solution was then centrifuged at 4°C for 12000×g for 10 min, and the supernatant was discarded. SM buffer was added at a ratio of 2 mL per 100 mL of bacterial solution to suspend the precipitate. An equal volume of chloroform was added, the solution was shaken for 30 s, centrifuged at 5000×g for 10 min, and the upper aqueous phase was collected. The solution was then extracted once more with an equal volume of chloroform; the upper aqueous phase contained in this extracted solution was the concentrated phage solution.

[0023] Electron microscopy observation of bacteriophages was performed using the phosphotungstic acid negative staining method. 10 µL of concentrated bacteriophage solution was added to a copper grid for adsorption for 1 min. Excess liquid was blotted away with filter paper, followed by staining with 1 drop of 2% phosphotungstic acid for 1 min. Excess staining was then blotted away with filter paper, and the solution was allowed to air dry before observation using a Hitachi H7650 transmission electron microscope at 80 kV. Measurements of the bacteriophages were performed using Nano Measure 1.2 software.

[0024] Electron microscopy revealed that phage PTS0531-2 is a short-tailed phage. This phage possesses an icosahedral head with a diameter of approximately 67.4 nm and a short tail, approximately 17.3 nm in length. Its overall morphology conforms to the typical characteristics of the Podoviridae family; no retractable tail structures or long tail filaments were observed. Figure 2 ).

[0025] Example 3: Genomic determination and analysis of bacteriophages (a) Gene sequencing of bacteriophage PTS0531-2 After extracting the genome of phage PTS0531-2, whole-genome sequencing was performed. The sequencing results showed a full genome length of 41723 bp (SEQ ID No. 1), with a GC content of 48.59%. RAST alignment yielded 42 open reading frames, and no tRNA, virulence genes, or drug resistance genes were detected. The sequencing results indicate that its nucleic acid is double-stranded linear DNA, classifying it as a short-tailed phage.

[0026] (II) Genomic alignment of bacteriophage PTS0531-2 Sequence similarity analysis was performed using the online BLAST tool. The results showed that the phage with the highest homology was Aeromonas phage PS, with a homology of 91.06%, which is lower than 95%. This indicates that phage PTS0531-2 is a new Aeromonas hydrophila phage and a newly discovered species.

[0027] Furthermore, genome sequencing results show that this bacteriophage contains DNA packaging-related proteins, replication-related proteins, cleavage-related proteins, and structural proteins.

[0028] Example 4: Detection of biological characteristics of bacteriophages (a) Phage counting methods The phage proliferation broth was serially diluted 10-fold using LB liquid medium, with 100 μL diluted to 10⁻¹⁰. 5 Phage proliferation broth and 100 μL of Aeromonas hydrophila bacterial suspension (1.34 × 10⁻⁶) 7After mixing (CFU / mL), incubate at 28°C for 10 min. Prepare double-layer plates using the method in step (iii) of Example 1, with three replicates for each dilution. Observe the plaques in the plates, and use plates with 30-300 plaques to count and determine the phage titer. Phage titer (PFU / mL) = average number of plaques × 10 × dilution factor.

[0029] Record the average number of phage plaques in the three parallel samples at this dilution and calculate the phage titer of the proliferation solution. The phage titer of this strain is 2.87 × 10⁻⁶. 9 PFU / mL.

[0030] (ii) Detection of bacteriophage thermal stability Take 1.0 mL of phage fluid with known titer into a 1.5 mL centrifuge tube and incubate it in a water bath at 28, 40, 50, 60 and 70 °C for 60 min. After the incubation time is over, remove the tube and immediately place it in an ice bath to cool. Determine the phage titer using the double-layer plate method.

[0031] like Figure 3 As shown, the titer of bacteriophages remained essentially unchanged between 30℃ and 60℃, maintaining a level of 10. 9 The titer dropped to 0 after exposure to 70℃. These results indicate that the titer of this bacteriophage was not affected within a temperature range of 30℃ to 60℃, which is close to the water temperature in actual aquaculture production. Even in the hot summer, the phage titer will not be affected.

[0032] (III) Detection of pH stability of bacteriophages The pH of LB liquid medium was adjusted with HCl and NaOH to 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0, respectively. The medium was then filtered through a 0.22µm filter for sterilization before use. 100 µL of phage solution was placed in 1.5mL centrifuge tubes, and 900µL of LB liquid medium at different pH values ​​was added to each tube. After incubation at 28℃ for 2 hours, the titer of the phage in each tube was determined using the double-layer plate method.

[0033] like Figure 4 As shown, phage PTS0531-2 maintained high activity for 2 hours in an environment with pH 5-12, but its titer decreased significantly after 2 hours in an environment with pH 4. When the pH dropped to 3 or reached above 12, no phage was detected, indicating that the phage basically lost its activity when the pH was below 3 or above 12.

[0034] (iv) Determination of the one-step growth curve of bacteriophage Add 1 mL of 6×10 7 PFU / mL phage solution and 1 mL 6×10 5A mixture of CFU / mL Aeromonas hydrophila culture was incubated at 28°C for 18 min to allow adsorption, followed by centrifugation at 8000g for 2 min. The supernatant was discarded, and the precipitate was resuspended in 20 mL of LB liquid medium and incubated at 28°C with shaking at 220 rpm. Phage titer was measured every 10 min using the double-layer plate method, with each time point repeated three times. Finally, a one-step growth curve of the phage was plotted with time on the x-axis and lgPFU / mL on the y-axis to calculate the phage latency, outbreak phase, stationary phase, and lysis rate. Outbreak rate = phage titer at the end of the outbreak ÷ host bacterial concentration at the initial stage of infection.

[0035] The results are as follows Figure 5 As shown, the incubation period of bacteriophage PTS0531-2 was approximately 30 minutes, the outbreak time was approximately 130 minutes (from the incubation period to the stationary phase), and the outbreak amount was 6.3 × 10⁻⁶. 7 PFU / cell.

[0036] Example 5: Determination of phage lysis profile Twenty bacterial strains provided by the Institute of Hydrobiology, Zhejiang Academy of Agricultural Sciences, were used as host bacteria, namely Aeromonas EX2-B ( Aeromonas EX2-B, source: juvenile glossopteris), Citrobacter freundii KY-2-1 ( Citrobacter freundii KY-2-1, Source: Goldfish), Lysine Bacillus MY ( Lysinibacillus MY, Source: Glossoptera litura, Aeromonas guinea pig X2D ( Aeromonas caviae X2D, source: shrimp), *Pseudomonas fluorescens* PF1 ( Pseudomonas fluorescens PF1, source: large yellow croaker), Aeromonas vera G2 ( Aeromonas veronii G2, source: salmon), Aeromonas hydrophila I1-3 ( Aeromonas hydrophila I1-3, Source: Tilapia), Aeromonas vera LPD ( Aeromonas veronii LPD, source: *Aeromonas hydrophila*, Aeromonas hydrophila I1-1 ( Aeromonas hydrophila I1-1, Source: Tilapia), Aeromonas hydrophila DC ( Aeromonas hydrophila DC, source: grouper fry), Pseudomonas proteoglycans PF2 ( Pseudomonas plecoglossicida PF2, source: large yellow croaker), Vibrio cholerae M2-2 ( Vibrio cholera M2-2, source: Litopenaeus vannamei), Pseudomonas thermophila W3 ( Pseudomonas gessardii W3, source: salmon), Vibrio parahaemolyticus S1317 ( Vibrio parahaemolyticus S1317, source: sea sand), Aeromonas hydrophila 4022B ( Aeromonas hydrophila4022B, source: grass carp), Bacillus YM3 ( Bacillus YM3, source: *Scophila glabra*, *Vibrio azuraceae* X1317 ( Vibrio azureus X1317, source: mud crab), Aeromonas hydrophila 4122C ( Aeromonas hydrophila 4122C, Source: Carp), Bacillus belye JFB ( Bacillus velezensis JFB, source: sea bass), Vibrio vulnificus S3317 ( Vibrio vulnificus S3317 (Source: Sea Sand) was used to determine the lysis spectrum of the phage using the spot method. 6 mL of melted LB medium with agar content of 0.7% was mixed with 100 µL of fresh host bacteria. After mixing, the mixture was poured onto LB solid medium with agar content of 1.7%. After the surface dried and solidified, 7 µL of the phage preservation solution prepared in Example 1 was added. The mixture was incubated overnight at 28°C, and the formation of lysis zones was observed.

[0037] The test results showed that bacteriophage PTS0531-2 could not lyse the above-mentioned bacteria. Figure 6 Some results are shown; results for other bacteria are also presented. Figure 6 (Results were consistent), only able to lyse Aeromonas hydrophila in Example 1, see Figure 1 Plaques.

[0038] SEQ ID No. 1:

Claims

1. A type of Aeromonas hydrophila phage, characterized in that, Its taxonomic name is Aeromonas hydrophila Phage, abbreviated as PTS0531-2, is deposited at the Guangdong Provincial Center for Microbial Culture Collection on March 18, 2026, with accession number GDMCC No: 67971-B1, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The Aeromonas hydrophila phage according to claim 1, characterized in that, PTS0531-2 exhibits high alkali resistance and maintains high activity even at pH=12.

3. The use of the Aeromonas hydrophila phage as described in claim 1 in the preparation of biological agents for the prevention or treatment of diseases caused by Aeromonas hydrophila.

4. The application according to claim 3, characterized in that, The biological agent is a water purifier, antibacterial drug, or feed additive.

5. A biological agent for the prevention and control of Aeromonas hydrophila, characterized in that, The Aeromonas hydrophila phage of claim 1 comprises a therapeutically effective amount.

6. The biological agent according to claim 5, characterized in that, The biological agent is a water purifier, antibacterial drug, or feed additive.

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