Vibrio alginolyticus bacteriophage and application thereof

Abstract

The application discloses a Vibrio alginolyticus bacteriophage and application thereof, which is named as PV48 ( Vibrio alginolyticus phage ) and is preserved in the Guangdong Microbial Culture Collection Center on January 2, 2025, with a preservation number of GDMCC No. 65705-B1. The application can be used for preventing and controlling Vibrio alginolyticus in a breeding environment or in an animal body, and can also be used for simultaneously preventing and controlling Aeromonas hydrophila.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of aquaculture, in particular to a Vibrio alginolyticus bacteriophage and application thereof. BACKGROUND

[0002] Vibrio alginolyticus is a gram-negative thermophilic bacteria that widely exists in the breeding environment all over the world. It is also a conditional pathogen that can cause mass death of breeding animals when the environment reaches a certain condition. It is reported that Vibrio alginolyticus has harm to many important economic breeding species such as fish, shrimp, crab and shellfish. The pathogenic process of Vibrio alginolyticus generally includes five steps of adhesion, invasion, colonization, proliferation and toxin production. The virulence of the bacteria is quite different among species, which can be divided into pathogenic and non-pathogenic strains, and the virulence is related to the type and quantity of virulence factors carried. At present, antibiotics are still the preferred solution for the prevention and treatment of Vibrio alginolyticus infection, but with the decrease of available antibiotic species, the emergence and spread of drug-resistant bacteria, it is urgent to develop antibiotic alternatives.

[0003] Bacteriophage is a kind of virus with strong specificity, which can infect bacteria, fungi, actinomycetes or spirochetes and other microorganisms. It was first discovered by a British biologist in 1915. It is estimated that there are about 10 32 billion species of bacteriophages in the world, which play a key role in regulating the environment and animal intestinal microbial population. Bacteriophages are divided into virulent bacteriophages and temperate bacteriophages. Virulent bacteriophages invade the bacterial body, mainly use the synthesis mechanism of the host to exert pressure on the bacterial cell wall, destroy the metabolism of the bacteria, cause the bacteria to dissolve and rupture, and then release the bacteriophage. Temperate bacteriophages do not proliferate immediately after infecting the host bacteria, but integrate the nucleic acid into the host bacterial chromosome, replicate with the host nucleic acid, and pass on with the cell division. Compared with other treatment methods, bacteriophage therapy has the characteristics of safety, reliability, high efficiency, rapidness, economy and practicality, and is expected to become a better antibacterial drug, especially in the treatment of drug-resistant bacterial infections. However, there are few bacteriophages for Vibrio alginolyticus at present. SUMMARY

[0004] The purpose of the present application is to provide a Vibrio alginolyticus bacteriophage and application thereof, which can be used for the prevention and control of Vibrio alginolyticus in breeding environment or animal body, and can also be used for the prevention and control of Aeromonas hydrophila.

[0005] The technical scheme adopted by the present application to solve its technical problems is:

[0006] A Vibrio alginolyticus bacteriophage, named PV48 ( Vibrio alginolyticus phage ), which is preserved in the Guangdong Microbial Culture Collection Center on January 2, 2025, and the preservation number is GDMCC No. 65705-B1.

[0007] The Vibrio alginolyticus phage is a temperate phage.

[0008] The Vibrio alginolyticus phage of the present invention has strong lytic activity against multiple strains of Vibrio alginolyticus. The present invention observed the morphology of the phage and the plaques it formed, and combined with genome sequencing and comparison results, determined that it is a new temperate phage of Vibrio alginolyticus.

[0009] The present invention studies the biological properties of the phage, such as temperature stability, pH stability, and one-step growth curve, to provide a basis for subsequent large-scale production. The present invention measures the inhibition of phage PV48 on host bacteria and determines that the host bacteria concentration remains at a low level within 7 hours. Phage PV48 was tested using a double-layer plate method on 11 Vibrio strains and 2 Aeromonas hydrophila strains. It was determined that phage PV48 could lyse 2 strains of Vibrio alginolyticus and 1 strain of Aeromonas hydrophila. This result indicates that the phage has a broad lysis spectrum against Aeromonas hydrophila and has good application prospects in clinical treatment.

[0010] The invention relates to an application of the Vibrio alginolyticus phage in the preparation of a biological agent for preventing and treating Vibrio alginolyticus.

[0011] The invention relates to an application of the Vibrio alginolyticus phage in the preparation of a biological preparation for preventing and treating Aeromonas hydrophila.

[0012] A biological preparation for preventing and treating Vibrio alginolyticus infection, wherein the active ingredient of the biological preparation is the Vibrio alginolyticus phage.

[0013] 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 aquaculture environments or animals, and can also be used for the prevention and control of Aeromonas hydrophila at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a picture of the PV48 plaque effect;

[0015] Figure 2 This is an electron micrograph of bacteriophage PV48;

[0016] Figure 3 This is the sensitivity curve of bacteriophage PV48 to different temperatures;

[0017] Figure 4 This is the sensitivity curve of bacteriophage PV48 to different pH;

[0018] Figure 5 is a graph of the in vitro growth kinetics of bacteriophage PV48;

[0019] Figure 6The diagram shows the effect of bacteriophage PV48 lysing a strain of Vibrio alginolyticus (A) and a strain of Aeromonas hydrophila (B) in vitro.

[0020] Figure 7 is the genome sequence of bacteriophage PV48;

[0021] Figure 8 is the phylogenetic tree of bacteriophage PV48. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is further described in detail below through specific embodiments.

[0023] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following examples, unless otherwise specified, are all conventional methods in the art.

[0024] Example 1: Screening and purification of Vibrio alginolyticus phage

[0025] (1) Sample collection: The water samples in the present invention were collected from a whiteleg shrimp hatchery in Ningbo, Zhejiang Province.

[0026] (II) Specific amplification of Vibrio alginolyticus phage in samples

[0027] 10 mL of water sample was centrifuged and filtered, then added to 5 mL of 3×Trypticase Soy Broth (TSB broth), and then Vibrio alginolyticus (provided by the Institute of Hydrobiology, Zhejiang Academy of Agricultural Sciences, Vibrio alginolyticus V48, V. alginolyticus V48 (source: whiteleg shrimp) 500 μL, mixed well, and cultured overnight at 28°C with shaking at 200 rpm.

[0028] (III) Double-layer plate method for detecting the presence of bacteriophages

[0029] Prepare TSB nutrient agar solid medium with a 1.5% agar content. After autoclaving, incubate at room temperature to approximately 40-60°C. Pour 10-15 mL of the agar into a Petri dish and evenly spread it across the bottom of the dish. Let it solidify at room temperature for 30 minutes. This TSB nutrient agar serves as the bottom agar. Centrifuge the mixed culture of Vibrio alginolyticus and the water sample at 12,000 rpm for 5 minutes. Filter the supernatant through a 0.22 μm filter to sterilize and set aside. Mix 100 μL of the filtrate with 100 μL of Vibrio alginolyticus and incubate at 28°C for 5 minutes. Add 5 mL of TSB semisolid nutrient agar medium with a 0.7% agar content, which has been autoclaved and cooled to 55°C. Mix by hand and quickly pour the agar onto the prepared bottom agar plate. Swirl the plate to evenly distribute the agar to form the top agar layer. After the agar solidifies, incubate it inverted overnight at 28°C and observe for the presence of plaques. If yes, proceed to step (IV). (All the above operations are performed under sterile conditions).

[0030] The results of plaque observation are as follows: plaques are round, transparent spots with a diameter of about 1 to 2 mm. Figure 1 .

[0031] (IV) Purification of phage samples

[0032] Remove a single plaque obtained in step (3) and place it in 1 mL of SM buffer. Add several sterilized ceramic beads and shake at 28°C for 1 hour. Centrifuge at 12,000 rpm for 5 minutes. Dilute the supernatant filtrate tenfold to the appropriate dilution. Purify the plaques using the double-layer plate method. Repeat purification three times to obtain plaques of consistent size and morphology. This monoclonal phage is designated PV48.

[0033] Amplification of phage samples

[0034] Remove the single plaque purified in step (4) and place it in 1 mL of SM buffer. Add several sterilized ceramic beads and shake at 28°C for 1 hour. Centrifuge at 12,000 rpm for 5 minutes. Filter the supernatant through a 0.22 μm filter to sterilize and set aside. Add 100 μL of the supernatant to 10 mL of Vibrio alginolyticus culture that has been cultured to the early logarithmic growth phase (OD600 = 0.3-0.6). Incubate at 28°C on a shaker at 200 rpm for 5 hours. Once the culture has clarified, obtain the phage growth medium.

[0035] Preparation of phage storage solution

[0036] The phage proliferation solution was mixed with autoclaved TSB medium containing 50% glycerol at a volume ratio of 1:1 to obtain the phage preservation solution. The phage preservation solution was stored at -80°C.

[0037] Example 2: Morphological observation of Aeromonas hydrophila bacteriophage

[0038] The electron microscope observation of the bacteriophage was performed by phosphotungstic acid negative staining method. 10 μL of bacteriophage concentrate was dropped onto a copper grid and adsorbed for 1 min, and the excess liquid was absorbed with filter paper. One drop of 2% phosphotungstic acid was added for 1 min, and the excess staining solution was absorbed with filter paper. After natural drying, the Hitachi H7650 transmission electron microscope was used to observe at 80 kV. The Nano Measure 1.2 software was used to measure the bacteriophage.

[0039] The electron microscope observation results were as follows: the bacteriophage PV48 was a myovirus with a head of icosahedral symmetry, a diameter of about 67.39 nm, and a tail of about 17.3 nm long, as shown in Figure 2 .

[0040] Example 3: Genome determination and analysis of the bacteriophage

[0041] (I) Gene sequencing of the bacteriophage PV48

[0042] After extracting the genome of the bacteriophage PV48, whole genome sequencing was performed, and the sequencing results were as follows: the full length of the genome was 43398 bp, and the GC content was 49.54%. After rast comparison, a total of 44 open reading frames were obtained, and no tRNA, virulence gene, and drug resistance gene were detected.

[0043] (II) Genome alignment of the bacteriophage PV48

[0044] Sequence similarity alignment analysis used the BLAST online tool, and the results showed that the bacteriophage with the highest homology was vB_VpaP_KF1, with a homology of 97.58% in a 99% coverage region, indicating that the bacteriophage PV48 was a new Vibrio alginolyticus bacteriophage. From the sequencing results, it can be known that its nucleic acid was double-stranded linear DNA, and it was a myovirus.

[0045] In addition, according to the genome sequencing results, it can be known that the bacteriophage contains DNA packaging related proteins, replication related proteins, lysis related proteins, and structural proteins, and the specific functional proteins are shown in Figure 7 (lysis module is represented in red; structural module is represented in yellow; DNA replication module is represented in green; packaging module is represented in blue; specific functional module is represented in purple).

[0046] Example 4: Detection of biological properties of the bacteriophage

[0047] (I) Bacteriophage counting method

[0048] The phage propagation liquid was diluted ten times in turn using TSB nutrient broth medium, 100 μL of the diluted phage propagation liquid was mixed with 100 μL of Aeromonas hydrophila bacterial liquid, and then incubated at 28°C for 8 min, and then a double-layer plate was prepared using the method in step (three) of Example 1, and three parallel samples were prepared for each dilution. The phage plaques in the plate were observed, and a plate with a plaque number of 30-300 was selected for counting and determining the phage titer. The phage titer (PFU / mL) = average plaque number × 10 × dilution factor.

[0049] The average plaque number of the three parallel samples at this dilution was recorded, and the phage titer of the propagation liquid was calculated. The titer of the phage strain was 1.29 × 10 9 PFU / mL.

[0050] (II) Detection of the thermal stability of the phage

[0051] 1.0 mL of the phage liquid with a known titer was taken in a 1.5 mL centrifuge tube, and was subjected to 60 min of action in a water bath at 28, 40, 50, 60, and 70°C, and then was taken out and immediately placed in an ice bath for cooling, and then the phage titer was determined by the double-layer plate method.

[0052] As shown in Figure 3 , the phage titer remained basically unchanged at about 10 9 PFU / mL after 30 min of action at 30-50°C, and the titer decreased by 5 orders of magnitude after 30 min of action at 70°C, and the titer decreased to 0 after 30 min of action at above 70°C. The above results show that the titer of the phage strain is not affected in the temperature range of 30-50°C, which is close to the water temperature in the actual aquaculture production process, and the phage titer will not be affected even in hot summer.

[0053] (III) Detection of the pH stability of the phage

[0054] The pH of the liquid medium was adjusted to 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0 using HCl and NaOH, and then the bacteria were removed by filtration using a 0.22 μm filter for standby use. 100 μL of the phage liquid was taken in a 1.5 mL centrifuge tube, 900 μL of the liquid medium with different pH values was added, and then the tube was placed in a 28°C constant temperature incubator for 2 h of standing, and then the titer of the phage in each tube was determined by the double-layer plate method.

[0055] As shown in Figure 4 , the phage remained high activity in the pH 4-10 environment for 2 h, and the titer decreased by 3 orders of magnitude in the pH 11 environment. When the pH was reduced to 3 or reached above 12, the phage was not detected, indicating that the phage basically lost activity when the pH was below 3 or above 12.

[0056] (IV) Determination of one-step phage growth curve

[0057] 1 mL of 6×10 7 PFU / mL phage solution and 1 mL 6×10 7 CFU / mL of Vibrio alginolyticus liquid was mixed and allowed to stand for 12 min at 28 °C for adsorption. The mixture was then centrifuged at 8000 g for 2 min, the supernatant was discarded, and the precipitate was resuspended in 1 mL of SM buffer and diluted 10 -5 Dilute to 10 mL and culture at 28°C with shaking at 220 rpm. Measure titer at appropriate intervals. Phage titer is determined using the double-layer plate method, with three replicates per time point. Finally, plot a one-step phage growth curve with time as the abscissa and lgPFU / mL as the ordinate. Calculate the incubation period, burst period, stable period, and lysis yield of the phage.

[0058] The results are as follows Figure 5 As shown, the incubation period of PV48 phage is approximately 30 minutes, the outbreak duration is approximately 50 minutes (from the incubation period to the stable period), and the outbreak volume is 412 PFU / cell. The outbreak volume = phage titer at the end of the outbreak divided by the host bacterial concentration at the beginning of infection.

[0059] Example 5: Determination of phage lysis spectrum

[0060] Eleven strains of Vibrio (provided by the Institute of Hydrobiology, Zhejiang Academy of Agricultural Sciences) and two strains of Aeromonas hydrophila (provided by the Institute of Hydrobiology, Zhejiang Academy of Agricultural Sciences) were used as the subjects. These 11 strains of Vibrio were Mediterranean Vibrio 3-5 ( V. mediterranei 3-5, source: whiteleg shrimp), Vibrio alginolyticus V48 ( V. alginolyticus V48, source: whiteleg shrimp), Vibrio alginolyticus 3-6 ( V. alginolyticus 3-6, source: white shrimp), Vibrio alginolyticus 3-7 ( V. alginolyticus 3-7, source: whiteleg shrimp), Vibrio alginolyticus 3-9 ( V. alginolyticus 3-9, source: whiteleg shrimp), Vibrio alginolyticus 4-4 ( V. alginolyticus 4-4, source: Macrobrachium spp.), Vibrio alginolyticus 5-1 ( V. alginolyticus 5-1, source: grass carp), Vibrio alginolyticus 4-5 ( V. alginolyticus 4-5, source: Macrobrachium spp.), Vibrio alginolyticus 4-6 ( V. alginolyticus 4-6, source: Artemia), Vibrio alginolyticus 4-14 ( V. alginolyticus 4-14, source: Macrobrachium spp.), Vibrio alginolyticus DXD ( V. alginolyticusDXD, source: whiteleg shrimp). The two Aeromonas hydrophila strains were Aeromonas hydrophila 5-4 ( A. hydrophila 5-4, source: military fish), Aeromonas hydrophila JX-1 ( A. hydrophila JX-1, source: Junyu). The phage lysis spectrum was determined using the dot blot assay. 6 mL of melted semisolid culture medium was mixed with 100 µL of fresh host bacteria. The mixture was then poured onto the solid culture medium. After the surface air-dried and solidified, 7 µL of phage solution was added dropwise. The culture was incubated at 28°C overnight, and the formation of lysis zones was observed.

[0061] The results showed that ( Figure 1 ; Figure 6 Bacteriophage PV48 was able to lyse two strains of Vibrio alginolyticus (V48 and V. alginolyticus 3-6) and one strain of Aeromonas hydrophila (A. hydrophila 5-4). The V. alginolyticus strains were all derived from shrimp, while the A. hydrophila strain was derived from trout. This result demonstrates that the phage is specific not only for the shrimp-derived V. alginolyticus but also for the trout-derived A. hydrophila.

[0062] Example 6: Construction of phylogenetic tree

[0063] A phylogenetic tree was constructed and analyzed using the amino acid sequence of the terminase large subunit protein ( Figure 8 The results showed that phage PV48 and vB_VpP_KF2 are closely related genetically, belonging to different species of the genus Maculvirus. BLAST sequence alignment revealed that phage PV48 shared the highest sequence similarity with the Vibrio phage vB_VpaP_KF1, reaching 97.58%. Both phages belong to the family Autographiviridae, different species of the genus Maculvirus, and can infect Vibrio alginolyticus.

[0064] The embodiment described above is only a preferred solution of the present invention and does not limit the present invention in any form. Other variations and modifications are possible without exceeding the technical solution described in the claims.

Claims

1. A Vibrio alginolyticus phage, characterized in that: Its Latin name is Vibrio alginolyticus phage , named PV48; deposited in Guangdong Provincial Microbiological Culture Collection Center, the preservation date is January 2, 2025, and the preservation number is GDMCC No. 65705-B1.

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

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

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

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