Vibrio alginolyticus bacteriophage and application thereof

Abstract

The invention discloses a vibrio alginolyticus phage and application of the vibrio alginolyticus phage. The vibrio alginolyticus phage is named as PV48 (vibrio alginolyticus phage). The strain is preserved in Guangdong Microbial Culture Collection Center, the preservation date is January 2, 2025, and the preservation number is GDMCC No. 65705-B1. The method can be used for preventing and controlling vibrio alginolyticus in a breeding environment or an animal body, and also can be used for preventing and controlling aeromonas hydrophila at the same time.

Description

Technical Field

[0001] The present invention relates to the technical field of aquaculture, and particularly relates to a Vibrio alginolyticus phage and its application. Background Art

[0002] Vibrio alginolyticus is a Gram-negative mesophilic bacterium that widely exists in aquaculture environments around the world. It is also a conditional pathogen that can cause massive deaths of farmed animals when the environment reaches specific conditions. It is reported that Vibrio alginolyticus is harmful to a variety of important economic aquaculture species, such as fish, shrimp, crabs, and shellfish. The pathogenic process of Vibrio alginolyticus generally includes five steps: adhesion, invasion, colonization, proliferation, and toxin production. The virulence of this bacterial species varies greatly among strains, which can be divided into pathogenic and non-pathogenic strains, and its pathogenicity is related to the types and quantities of virulence factors carried. Currently, antibiotics are still the preferred option for preventing and treating Vibrio alginolyticus infections. However, with the reduction in available antibiotic varieties, the emergence and spread of drug-resistant bacteria, there is an urgent need to develop antibiotic alternative technologies.

[0003] Phages are a general term for a class of viruses with extremely strong specificity that can infect microorganisms such as bacteria, fungi, actinomycetes, or spirochetes. They were first discovered by British biologists in 1915. It is estimated that there are about 10 32 species of phages globally, which play a key role in regulating the microbial populations in the environment and the animal gut. Phages are divided into lytic phages and temperate phages. Lytic phages invade bacteria and mainly utilize the host's synthesis mechanism to exert pressure on the bacterial cell wall, disrupt the metabolism of bacteria, resulting in the lysis and rupture of bacteria, and then release phages. Temperate phages do not immediately proliferate after infecting the host bacteria, but integrate their nucleic acids into the host bacterial chromosome, replicate with the replication of the host nucleic acid, and are passed on with cell division. Compared with other treatment methods, phage therapy has the characteristics of safety, reliability, high efficiency, rapidity, economy, and practicality, and is expected to become a good antibacterial drug, especially in the treatment of drug-resistant bacterial infections. However, there are currently few phages targeting Vibrio alginolyticus. Summary of the Invention

[0004] The purpose of the present invention is to provide a Vibrio alginolyticus phage and its application, which can be used for the prevention and control of Vibrio alginolyticus in aquaculture environments or in animals, and can also be used for the prevention and control of Aeromonas hydrophila at the same time.

[0005] The technical solution adopted by the present invention to solve its technical problems is: A Vibrio alginolyticus phage named PV48 ( Vibrio alginolyticus phage ); it is deposited in the Guangdong Provincial Culture Collection Center of Microorganisms, the deposit date is January 2, 2025, and the deposit number is GDMCC No. 65705-B1.

[0006] The Vibrio alginolyticus phage is a temperate phage.

[0007] The Vibrio alginolyticus phage of the present invention has strong lytic activity against multiple strains of Vibrio alginolyticus. The present invention observes the morphology of the phage and the formed plaques, and combines the results of genome sequencing and comparison to determine it as a new temperate phage of Vibrio alginolyticus.

[0008] The present invention studies the biological characteristics of the phage such as temperature stability, pH stability, and one-step growth curve, providing a basis for subsequent large-scale production. The present invention determines the inhibition of the phage PV48 on the host bacteria, and determines that within 7 hours, the concentration of the host bacteria remains at a relatively low level. The phage PV48 is used as the object for 11 strains of Vibrio and 2 strains of Aeromonas hydrophila by the double-layer plate method, and it is determined that the phage PV48 can lyse 2 strains of Vibrio alginolyticus and 1 strain of Aeromonas hydrophila among them. This result shows that the phage has a wide lysis spectrum for Aeromonas hydrophila and has good application prospects in clinical treatment.

[0009] Application of the described Vibrio alginolyticus phage in the preparation of a biological agent for preventing and controlling Vibrio alginolyticus.

[0010] Application of the described Vibrio alginolyticus phage in the preparation of a biological agent for preventing and controlling Aeromonas hydrophila.

[0011] A biological agent for preventing and treating Vibrio alginolyticus infection, wherein the active ingredient of the biological agent is the described Vibrio alginolyticus phage.

[0012] The beneficial effects of the present invention are: it can reduce the use of antibiotics, can be used for the prevention and control of Vibrio alginolyticus in the aquaculture environment or in the animal body, and can also be used for the prevention and control of Aeromonas hydrophila at the same time. Description of the Drawings

[0013] Figure 1 It is the effect diagram of the PV48 plaque; Figure 2 It is the electron microscope image of the phage PV48; Figure 3 It is the sensitivity curve diagram of the phage PV48 to different temperatures; Figure 4 It is the sensitivity curve diagram of the phage PV48 to different pH values; Figure 5 It is the in vitro growth kinetics diagram of the phage PV48; Figure 6 It is the effect diagram of the phage PV48 lysing a strain of Vibrio alginolyticus (A) and a strain of Aeromonas hydrophila (B) in vitro; Figure 7 It is the genome sequence diagram of the phage PV48; Figure 8 It is the phylogenetic tree of bacteriophage PV48. Specific implementation manners

[0014] The technical solution of the present invention will be further specifically described below through specific embodiments.

[0015] In the present invention, unless otherwise specified, the raw materials, equipment, etc. used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are all conventional methods in the art unless otherwise specified.

[0016] Example 1: Screening and purification of Vibrio alginolyticus bacteriophage (I) Sampling: The water sample in the present invention was collected from a white shrimp hatchery in Ningbo, Zhejiang Province.

[0017] (II) Specific amplification of Vibrio alginolyticus bacteriophage in the sample Take 10 mL of the water sample, after centrifugal filtration, add it to 5 mL of 3×Trypticase Soy Broth (TSB broth), and then add 500 μL of Vibrio alginolyticus (provided by the Institute of Hydrobiology, Zhejiang Academy of Agricultural Sciences, Vibrio alginolyticus V48, V. alginolyticus V48, source: white shrimp), mix well, and incubate overnight at 28 °C with shaking at 200 rpm.

[0018] (III) Detection of the presence of bacteriophage by the double-layer plate method Prepare a TSB nutrient agar solid medium with an agar content of 1.5%. After autoclaving, place it at room temperature until it reaches about 40 - 60 °C, then take 10 - 15 mL and pour it into a petri dish, evenly spread it on the bottom of the petri dish, and let it stand at room temperature for 30 min to solidify. This TSB nutrient agar is used as the bottom agar. Take the mixed culture solution of the above-mentioned Vibrio alginolyticus and the water sample, centrifuge it at 12000 rpm for 5 min, and filter the supernatant through a 0.22 μm filter for sterilization and reserve it. Take 100 μL of the filtrate and mix it with 100 μL of Vibrio alginolyticus, then incubate at 28 °C for 5 min, and add 5 mL of a TSB semi-solid nutrient agar medium with an agar content of 0.7% that has been autoclaved and cooled to 55 °C. Mix well by hand rubbing the test tube, and quickly pour it onto the prepared bottom agar plate, rotate the petri dish to make it evenly distributed as the upper agar. After the agar solidifies, incubate it upside down at 28 °C overnight, and observe whether there are plaques. If there are, proceed to step (IV). (All the above operations are carried out under sterile conditions).

[0019] Observation result of plaques: The plaques are round and transparent spots with a diameter of about 1 - 2 mm, see Figure 1 .

[0020] (IV) Purification of the bacteriophage sample Pick out the single plaque obtained in step (3), place it in 1 mL of SM buffer, add several sterilized ceramic beads, shake at 28 °C for 1 h, then centrifuge at 12,000 rpm for 5 min. Take the supernatant filtrate and dilute it tenfold to a suitable dilution, and purify the plaque by the double-layer plate method. Purify it continuously for 3 times to obtain plaques with consistent size and morphology. Obtain a single clone of the phage and name this phage PV48.

[0021] Amplification of phage samples Pick out the purified single plaque in step (4), place it in 1 mL of SM buffer, add several sterilized ceramic beads, shake at 28 °C for 1 h, then centrifuge at 12,000 rpm for 5 min. The supernatant is filtered through a 0.22 μm filter for sterilization and reserved. Take 100 μL of the supernatant filtrate and add it to 10 mL of the Vibrio alginolyticus bacterial solution that has been cultured to the early logarithmic growth phase (OD600 = 0.3 - 0.6), and culture it in a constant temperature shaker at 28 °C and 200 rpm for 5 h. Wait until the culture solution becomes clear to obtain a phage proliferation solution.

[0022] Preparation of phage preservation solution Mix the phage proliferation solution with the autoclaved TSB medium containing 50% glycerol in a volume ratio of 1:1 to obtain the phage preservation solution. The phage preservation solution is stored at -80 °C.

[0023] Example 2: Morphological observation of Aeromonas hydrophila phage For the electron microscopy observation of the phage, the phosphotungstic acid negative staining method is used. Take 10 μL of the phage concentrate and drop it onto the copper mesh for adsorption for 1 min. Use filter paper to absorb the excess liquid, add 1 drop of 2% phosphotungstic acid for staining for 1 min, use filter paper to absorb the excess staining solution, and observe it with a Hitachi H7650 transmission electron microscope at 80 kV after natural drying. Use Nano Measure 1.2 software to measure the phage.

[0024] The results of electron microscopy observation are as follows: Phage PV48 is a myovirus, with an icosahedral symmetric head with a diameter of about 67.39 nm and a tail about 17.3 nm long, as shown in Figure 2 .

[0025] Example 3: Genome determination and analysis of phage (1) Gene sequencing of phage PV48 After extracting the genome of phage PV48, perform whole-genome sequencing. The sequencing results are as follows: The full length of the genome is 43,398 bp, and the GC content is 49.54%. After comparison with rast, a total of 44 open reading frames are obtained, and no tRNA, virulence genes, and drug resistance genes are detected.

[0026] (II) Genome Alignment of Bacteriophage PV48 Sequence similarity alignment analysis was performed using the BLAST online tool. The results showed that the bacteriophage with the highest homology to it was vB_VpaP_KF1, and the homology within 99% of the covered region was 97.58%, indicating that bacteriophage PV48 is a new Vibrio alginolyticus bacteriophage. From the sequencing results, its nucleic acid is double-stranded linear DNA, belonging to the Myoviridae bacteriophage.

[0027] In addition, according to the genome sequencing results, this bacteriophage contains DNA packaging-related proteins, replication-related proteins, lysis-related proteins, and structural proteins. For specific functional proteins, see Figure 7 (The lysis module is shown in red; the structural module is shown in yellow; the DNA replication module is shown in green; the packaging module is shown in blue; the specific functional module is shown in purple).

[0028] Example 4: Detection of Bacteriophage Biological Characteristics (I) Bacteriophage Counting Method The bacteriophage proliferation solution was serially diluted 10-fold using TSB nutrient broth medium. After taking 100 μL of the diluted bacteriophage proliferation solution and mixing it with 100 μL of Aeromonas hydrophila bacterial solution, it was incubated at 28°C for 8 min. Double-layer plates were prepared using the method in step (III) of Example 1, and 3 parallel samples were made for each dilution. Observe the plaques in the plates, and select the plates with the number of plaques between 30 - 300 for counting to determine the bacteriophage titer. Bacteriophage titer (PFU / mL) = average number of plaques × 10 × dilution factor.

[0029] Record the average number of plaques in 3 parallel samples at this dilution, and calculate the bacteriophage titer of the proliferation solution. The titer of this strain of bacteriophage is 1.29×10 9 PFU / mL.

[0030] (II) Detection of Bacteriophage Thermal Stability Take 1.0 mL of the known titer bacteriophage solution 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 ends, take it out and immediately place it in an ice bath to cool, and determine the bacteriophage titer using the double-layer plate method.

[0031] As Figure 3 shown, when the bacteriophage acts at 30°C - 50°C for 30 min, the titer remains basically unchanged, maintaining at 10 9Around, after acting at 70 °C for 30 min, the titer decreased by 5 orders of magnitude, and after acting above 70 °C for 30 min, the titer dropped to 0. The above results indicate that the titer of this phage strain was not affected in the temperature range of 30 °C - 50 °C, which is close to the water temperature in the actual aquaculture production process. Even in the hot summer, the phage titer will not be affected.

[0032] (III) Detection of phage pH stability Use HCl and NaOH to adjust the pH of the liquid medium to 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0 respectively. After filtering and sterilizing with a 0.22 µm filter, it is reserved for use. Take 100 µL of phage liquid into a 1.5 mL centrifuge tube, add 900 µL of liquid medium with different pH values respectively. After standing in a 28 °C constant temperature incubator for 2 h, the titer of phage in each tube is determined by the double-layer plate method.

[0033] As Figure 4 shown, the phage still maintained high activity after 2 h in the pH 4 - 10 environment, and the titer decreased by 3 orders of magnitude after 2 h in the pH 11 environment. 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 phage Mix 1 mL of phage liquid with 6×10 7 PFU / mL with 1 mL of Vibrio alginolyticus bacterial liquid with 6×10 7 CFU / mL. After standing and adsorbing at 28 °C for 12 min, centrifuge at 8000 g for 2 min, discard the supernatant, resuspend the precipitate with 1 mL of SM buffer, and dilute it 10 -5 times to 10 mL. Incubate with shaking at 28 °C and 220 rpm. Measure the titer at appropriate intervals. The phage titer is determined by the double-layer plate method, and each time point is repeated 3 times. Finally, with time as the abscissa and lgPFU / mL as the ordinate, draw the one-step growth curve of the phage, and calculate the latent period, burst period, stationary period, and burst size of the phage.

[0035] The results are as Figure 5 shown. The latent period of PV48 phage is about 30 min, the burst time is about 50 min (from the latent period to the stationary period), and the burst size is 412 PFU / cell. Burst size = phage titer at the end of the burst ÷ concentration of host bacteria at the initial stage of infection.

[0036] Example 5: Determination of the phage lysis spectrum Using 11 Vibrio strains (provided by the Institute of Hydrobiology, Zhejiang Academy of Agricultural Sciences) and 2 Aeromonas hydrophila strains (provided by the Institute of Hydrobiology, Zhejiang Academy of Agricultural Sciences) as the objects, these 11 Vibrio strains are respectively Vibrio mediterranei 3-5( V. mediterranei 3-5, source: Litopenaeus vannamei), Vibrio alginolyticus V48( V. alginolyticus V48, source: Litopenaeus vannamei), Vibrio alginolyticus 3-6( V. alginolyticus 3-6, source: Litopenaeus vannamei), Vibrio alginolyticus 3-7( V. alginolyticus 3-7, source: Litopenaeus vannamei), Vibrio alginolyticus 3-9( V. alginolyticus 3-9, source: Litopenaeus vannamei), Vibrio alginolyticus 4-4( V. alginolyticus 4-4, source: Macrobrachium rosenbergii), Vibrio alginolyticus 5-1( V. alginolyticus 5-1, source: Ctenopharyngodon idella), Vibrio alginolyticus 4-5( V. alginolyticus 4-5, source: Macrobrachium rosenbergii), Vibrio alginolyticus 4-6( V. alginolyticus 4-6, source: Artemia salina), Vibrio alginolyticus 4-14( V. alginolyticus 4-14, source: Macrobrachium rosenbergii), Vibrio alginolyticus DXD( V. alginolyticus DXD, source: Litopenaeus vannamei). The 2 Aeromonas hydrophila strains are Aeromonas hydrophila 5-4( A. hydrophila 5-4, source: Acrossocheilus fasciatus), Aeromonas hydrophila JX-1( A. hydrophila JX-1, source: Acrossocheilus fasciatus). The lysis spectrum of the phage was determined by the spot test method. Take 6 mL of melted semi-solid medium and mix it with 100 µL of fresh host bacteria. After mixing, pour it onto the solid medium. After the surface air dries and solidifies, add 7 µL of phage liquid and incubate overnight at 28 °C, and observe the formation of the lysis zone.

[0037] The measurement results show that ( Figure 1 ; Figure 6 ) the phage PV48 can lyse 2 Vibrio alginolyticus strains (Vibrio alginolyticus V48, Vibrio alginolyticus 3-6) and 1 Aeromonas hydrophila strain (Aeromonas hydrophila 5-4) among them. The Vibrio alginolyticus strains that can be lysed all come from shrimp, and the Aeromonas hydrophila comes from Acrossocheilus fasciatus. This result indicates that this phage strain is not only specific to Vibrio alginolyticus from shrimp but also specific to Aeromonas hydrophila from Acrossocheilus fasciatus.

[0038] Example 6: Construction of a phylogenetic tree A phylogenetic tree was constructed and analyzed using the amino acid sequence of the large subunit protein of the terminase ( Figure 8). The results showed that phage PV48 was closely genetically related to vB_VpP_KF2 and was a different phage species in the genus Maculvirus. Through BLAST sequence alignment, phage PV48 had the highest sequence similarity of 97.58% with Vibrio phage vB_VpaP_KF1. These two phages both belonged to different species in the genus Maculvirus of the family Autographiviridae and could infect Vibrio alginolyticus.

[0039] The above-described embodiments are only a preferred solution of the present invention and do not impose any form of limitation on the present invention. There are other variations and modifications without exceeding the technical solutions described in the claims.

Claims

1. A Vibrio alginolyticus phage, characterized in that, Named PV48 ( Vibrio alginolyticus phage ); deposited in the Guangdong Microbial Culture Collection Center on January 2, 2025, with the deposit number GDMCC No. 65705-B1.

2. The Vibrio alginolyticus phage according to claim 1, characterized in that, The Vibrio alginolyticus phage is a temperate phage.

3. Use of a Vibrio alginolyticus phage according to claim 1 in the preparation of a biological agent for preventing and treating Vibrio alginolyticus.

4. Use of a Vibrio alginolyticus phage according to claim 1 in the preparation of a biological agent for preventing and treating Aeromonas hydrophila.

5. A biological agent for preventing and treating Vibrio alginolyticus infection, characterized in that: The active ingredient of the biological agent is the Vibrio alginolyticus phage according to claim 1.

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