Vibrio candersonii bacteriophage composition with efficient antibacterial ability and application thereof

Abstract

The invention provides a vibrio candersonii bacteriophage, a bacteriophage composition and application thereof, and belongs to the technical field of microbial control. According to the invention, two new strains of strong vibrio canicola bacteriophages are obtained through screening, have strong host specificity and strong cracking and killing effects on vibrio canicola, and can effectively prevent vibrio canicola from generating resistance mutation when being mixed for use, so that an ideal pathogen removal effect is achieved, and the vibrio canicola bacteriophage has a good application prospect. Therefore, the method has important application value in the aspect of prevention and control of vibrio candersonii infectious diseases of marine aquaculture animals.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial control, and in particular relates to a Vibrio cannelliger phage composition with high-efficiency antibacterial ability and an application thereof. Background Art

[0002] Vibrio cannelligensis is a common Gram-negative, facultative anaerobic pathogen in the marine environment, widely distributed in coastal waters, estuaries and aquatic products. The bacteria can cause outbreaks of red leg disease in farmed shrimp and sepsis in abalone. In addition, Vibrio cannelligensis infection is also associated with the onset of snails and farmed juvenile fish, causing mass deaths of fish and shellfish, and causing huge economic losses to the marine aquaculture industry.

[0003] In aquaculture, antibiotics have traditionally been used to prevent and treat Vibrio diseases. However, the extensive use of antibiotics has led to the emergence of multidrug-resistant bacteria (MDR). Therefore, it is urgent to find a fast and effective alternative to antibiotics.

[0004] As a new type of antimicrobial microbial agent, bacteriophages have the following advantages over antibiotics: they are highly targeted and safe, and only target specific hosts for lysis; bacteriophages are widely present in nature and are environmentally friendly agents; bacteriophages have different bactericidal mechanisms from antibiotics, so they are not affected by the antibiotic resistance that bacteria have acquired. Bacteriophages have strong proliferation capabilities and can replicate autonomously. Therefore, in the past two decades, bacteriophages have become a new type of antimicrobial agent actively developed by the pharmaceutical industry.

[0005] However, phage therapy also has certain limitations. The most notable one is that pathogens cannot be completely eliminated by phages, but rather resistant bacteria can rebound over time. In addition, many phages have a narrow host range and can only lyse a limited range of Vibrio strains, making it difficult to cover a variety of pathogens in the actual environment. These limitations can be overcome through phage cocktail therapy. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention provides a Vibrio cannelliger phage and a phage composition and applications thereof.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] A Vibrio cannelliger phage composition with high-efficiency antibacterial ability comprises phage VcpOK and phage Vcp YR.

[0009] Among them, the wild strain phage Vcp OK of Vibrio camobellii has been sent to the China Center for Type Culture Collection (address: Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei Province), with a preservation date of July 22, 2024, a preservation number of CCTCC M20241645, and a classification name of Vibrio camobellii phage Vcp OK;

[0010] The mutant phage Vcp YR of Vibrio camobellii has been sent to the China Center for Type Culture Collection (address: Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei Province), with a preservation date of July 22, 2024, a preservation number of CCTCC M20241646, and a classification name of Vibrio camobellii phage VcpYR.

[0011] The titers of bacteriophage Vcp OK and Vcp YR in the composition are not less than 10 8 PFU / ml.

[0012] The multiplicity of infection (MOI) was 0.1.

[0013] An application of a Vibrio cannelliger phage composition, and an application of the Vibrio cannelliger phage composition in the preparation of a Vibrio cannelliger bactericide.

[0014] The Vibrio cannelliger phage composition is used in preparing a bacterial agent for controlling Vibrio infection in aquaculture.

[0015] A Vibrio cannenbergii bactericide, comprising the Vibrio cannenbergii bacteriophage composition.

[0016] The formulation of the Vibrio cannelligensis fungicide is wettable powder, water dispersible granules or water suspension.

[0017] The Vibrio cannelliger bactericide also includes auxiliary materials acceptable in aquaculture.

[0018] The auxiliary materials acceptable in aquaculture are selected from one or more of dispersants, stabilizers, fillers and solvents.

[0019] An application of a Vibrio cannelliger bactericide, and an application of the Vibrio cannelliger bactericide in the preparation of the Vibrio cannelliger bactericide.

[0020] Application of the Vibrio cannerii fungicide in controlling Vibrio infection in aquaculture

[0021] Beneficial effects of the present invention:

[0022] The present invention screened and obtained two new strains of virulent phages of Vibrio canneri. The cocktail composed of these two strains of phages has a highly efficient lysis effect on Vibrio canneri and can effectively avoid the "rebound" growth of pathogenic bacteria. In addition, the cocktail can lyse six types of Vibrio, including Vibrio canneri, Vibrio alginolyticus, and Vibrio parahaemolyticus. It has a wide host range and can be widely used in the aquaculture industry, so it has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0024] Figure 1 : A plate culture photograph of the wild-type bacteriophage Vcp OK of Vibrio cannibalis in Example 2;

[0025] Figure 2 : A plate culture photograph of the mutant phage Vcp YR of Vibrio cannibalis in Example 5;

[0026] Figure 3 : Transmission electron microscope photo of the wild-type bacteriophage Vcp OK of Vibrio cannelligensis in Example 8;

[0027] Figure 4 : Transmission electron micrograph of the mutant phage Vcp YR of Vibrio cannellii in Example 8;

[0028] Figure 5 : Result of the chloroform sensitivity test of the wild-type bacteriophage Vcp OK of Vibrio cannellii in Example 9;

[0029] Figure 6 : The result of the chloroform sensitivity test of the mutant phage Vcp YR of Vibrio cannelliger in Example 10;

[0030] Figure 7 : One-step growth curve of the wild-type phage Vcp OK of Vibrio cannelligensis in Example 13;

[0031] Figure 8 : One-step growth curve of the mutant phage Vcp YR of Vibrio cannelligensis in Example 14;

[0032] Fig. 9 :Evaluation of bacteriocidal effect of phage cocktail; DETAILED DESCRIPTION

[0033] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0034] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention.

[0035] As mentioned earlier, phage therapy has certain limitations. For example, pathogenic bacteria can easily develop resistance mutations to infection with a single phage, affecting its actual therapeutic effect.

[0036] The present invention screened and obtained two new virulent Vibrio campbellii phages. The phage cocktail composed of the two phages can lyse eight types of Vibrio, including Vibrio alginolyticus, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio mytilus, etc., and can effectively prevent the "rebound growth" of Vibrio campbellii, thereby achieving an ideal pathogen elimination effect. Therefore, the phage cocktail has important application value in the prevention and control of Vibrio campbellii infectious diseases in marine aquaculture animals.

[0037] In view of this, in a specific embodiment of the present invention, a Vibrio cannenbergii phage composition is provided, wherein the Vibrio cannenbergii phage is a wild-type phage Vcp OK of Vibrio cannenbergii or a mutant phage Vcp YR of Vibrio cannenbergii.

[0038] Among them, the wild strain phage Vcp OK of Vibrio campestris has been sent to the China Center for Type Culture Collection (address: Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei Province), with a preservation date of July 22, 2024 and a preservation number of CCTCC M20241645;

[0039] The biological characteristics of the Vcp OK of the present invention are as follows: under an electron microscope, Vcp OK appears to be composed of a regular hexahedron with a diameter of about 28 nm and a tail with a length of 197 nm, and belongs to the family Myotailed Bacteriophage.

[0040] The Vcp OK of the present invention is insensitive to both 2% and 20% chloroform.

[0041] The infection characteristics of Vcp OK of the present invention are as follows: the incubation period of Vcp OK infecting the wild strain of Vibrio campbellii 18-1 is 30 minutes, the lysis period is 3.5 hours, and the phage release amount is 25 PFU / cell.

[0042] The mutant phage Vcp YR of Vibrio campestris has been sent to the China Center for Type Culture Collection (address: Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei Province), with a preservation date of July 22, 2024 and a preservation number of: CCTCC M20241646.

[0043] The biological characteristics of the Vcp YR of the present invention are as follows: under an electron microscope, Vcp YR appears to be composed of a regular hexahedral head with a diameter of about 66 nm and a tail with a length of 302 nm, and belongs to the family Myocaudatus.

[0044] The infection characteristics of the Vcp YR described in the present invention are: the incubation period of Vcp YR infecting the wild strain of Vibrio campbellii 18-1 is 30 minutes, the lysis period is 3 hours, and the phage release amount is 30 PFU / cell.

[0045] The Vcp YR of the present invention is insensitive to both 2% and 20% chloroform.

[0046] Experiments have shown that the Vcp OK of the present invention can infect not only the wild strain V.campbellii18-1 of Vibrio campbellii, but also Vibrio neocaledonicus NC470(T), Vibrio parahaemolyticusNBRC 12711(T), Vibrio harveyi NBRC 15634(T), Vibrio alginolyticus NBRC15630(T), and Vibrio mytiliCAIM 528(T). Vcp YR can infect not only Vibrio campbellii18-1, but also the mutant strain V.campbellii 18-1M generated by the single bacteriophage Vcp OK infecting the wild strain V.campbellii18-1 of Vibrio campbellii;

[0047] In another specific embodiment of the present invention, a Vibrio cannenbergii phage composition is provided, wherein the composition comprises the above-mentioned Vibrio cannenbergii wild-type phage Vcp OK and Vibrio cannenbergii mutant phage Vcp YR.

[0048] The titers of the Vibrio cannerii phages Vcp OK and Vcp YR of the present invention are both not less than 10 9 PFU / ml.

[0049] When the Vibrio cannery phages Vcp OK and Vcp YR of the present invention are used in combination, the titer ratio of the two is 1:1; and the multiplicity of infection MOI is 0.1.

[0050] In another specific embodiment of the present invention, there is provided use of the above-mentioned Vibrio cannelligensis phage or phage composition in the preparation of a Vibrio cannelligensis bactericide.

[0051] In another specific embodiment of the present invention, a bactericide for Vibrio cannenbergii is provided, wherein the bactericide for Vibrio cannenbergii comprises the above-mentioned Vibrio cannenbergii bacteriophage or bacteriophage composition.

[0052] In another specific embodiment of the present invention, the formulation of the Vibrio cannelligensis fungicide is a wettable powder, a water dispersible granule or a water suspension, preferably a water suspension.

[0053] In another specific embodiment of the present invention, the Vibrio cannelligeri bactericide further comprises an auxiliary material acceptable in aquaculture, and the auxiliary material acceptable in aquaculture is selected from one or more of a dispersant, a stabilizer, a filler and a solvent. There is no special restriction on the source of the auxiliary material acceptable in aquaculture of the present invention, and generally commercially available products can be used.

[0054] Wherein, the dispersant is one or both of sodium tripolyphosphate and sodium pyrophosphate.

[0055] The stabilizer may be selected from one or more of sodium alginate, gelatin, xanthan gum or guar gum.

[0056] The filler can be selected from one or more of chitosan, diatomaceous earth, bentonite, and attapulgite.

[0057] The solvent may be selected from water (preferably deionized water).

[0058] In another specific embodiment of the present invention, there is provided use of the above-mentioned Vibrio cannelligensis phage, phage composition or Vibrio cannelligensis bactericide in controlling Vibrio infection in aquaculture.

[0059] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0060] In the following example, the formula of the 2216E medium is: 1 g yeast powder, 5 g peptone, 0.01 g ferric orthophosphate, 1000 ml seawater, pH 7.6.

[0061] The 2216E solid culture medium comprises: 1 g yeast powder, 5 g peptone, 0.01 g ferric orthophosphate, 15 g agar, 1000 ml seawater, pH 7.6.

[0062] The 2216E semi-solid culture medium comprises: 1 g yeast powder, 5 g peptone, 0.01 g ferric orthophosphate, 15 g agar, 1000 ml seawater, pH 7.6.

[0063] Example 1 Isolation of wild strain phage Vcp OK of Vibrio cannelli

[0064] Take a nearshore water sample, filter it through a 0.22μm sterile filter membrane, add it to the exponential growth phase of the wild strain V.campbellii 18-1 of Vibrio campbellii at a volume ratio of 10%, and shake it at 28℃. Take samples on the 1st, 3rd, 5th, and 7th days, filter and sterilize the samples through a 0.22μm sterile filter head, dilute different concentration gradients with SM Buffer, and then mix 1ml of the filtrate with 1ml of the logarithmic phase wild strain V.campbellii 18-1 of Vibrio campbellii at 28℃ and shake it for 30min, then pour it into the melted pre-cooled 2216E semi-solid at about 48℃, mix it and pour it on the 2216E solid plate, shake it evenly until the semi-solid solid solidifies. Place the semi-solid plate in a 28℃ constant temperature incubator and culture it overnight. If plaques appear, it is the phage obtained by preliminary separation.

[0065] Example 2 Purification of wild-type bacteriophage Vcp OK of Vibrio cannelli

[0066] The plaques obtained in Example 1 were soaked in SM Buffer overnight, and then the soaking solution was filtered through a 0.22 μm sterile filter and diluted with SM Buffer in a gradient manner. A double-layer plate was prepared according to the method described in Example 1 to obtain plaques uniformly dispersed on the double-layer plate. The above operation was repeated 3-5 times until the size and morphology of the plaques on the double-layer plate obtained in the last step were consistent. The purified phage double-layer plate was shown in FIG. Figure 1 .

[0067] Example 3 Isolation of the anti-wild strain phage Vcp OK of Vibrio campbellii mutant V. campbellii18-1M

[0068] Mix 100ul of the wild strain V.campbellii 18-1 bacterial liquid in the logarithmic phase with 100ul of the wild strain phage liquid Vcp OK of Vibrio campbellii and incubate in a shaker at 28℃ for 30min, then pour into the melted 2216E semi-solid precooled to about 48℃, mix and pour on the 2216E solid plate, shake evenly horizontally until the semi-solid solidifies. Place the semi-solid plate in a 28℃ constant temperature incubator and culture overnight. After the bacteria on the plate grow out, use a 100ul pipette to pick different single colonies (determine whether they are different single colonies by color, morphology, size, transparency, etc.), and use the three-zone line method to purify the colonies. Purify 2-3 times to basically purify the single colonies. Pick out a single colony from the plate and place it in a 5ml 2216E liquid test tube. Culture it at 28°C and 150rpm for 2-3 days. Take out 1.4ml of the cultured bacterial solution and place it in a 1.5ml centrifuge tube. Use the freeze-thaw method to extract bacterial DNA.

[0069] The extracted DNA was used as a template for PCR amplification and sent for testing.

[0070] Example 4 Isolation of mutant phage Vcp YR of Vibrio cannelli

[0071] The specific implementation method is the same as that described in Example 1. The host bacteria is 18-1M.

[0072] Example 5 Purification of mutant phage Vcp YR of Vibrio cannelli

[0073] The specific implementation method is the same as in Example 2. The purified phage double-layer plate is shown in Figure 2 . Phage plaques of uniform size and shape are formed on the plate.

[0074] Example 6 Determination of the titer of wild strain phage Vcp OK of Vibrio cannelli

[0075] The solutions of different phages obtained in the above-mentioned embodiment after filtration of the above-mentioned cultured phages through a 0.22 μm sterile filter were diluted with SM Buffer at different times, mixed with the bacterial solution of the logarithmic phase wild strain V. campbellii 18-1, and then made into double-layer plates. The double-layer plates were cultured at 28°C overnight to grow plaques. The plaques on the double-layer plates were counted, and the plates with 30-300 plaques on the plates were selected to calculate the titer. The number of plaques on the plate was converted to the corresponding dilution multiple, which was the titer of the phage. For example, the Vcp OK phage solution was diluted 10 -6 There are 100 plaques on the double-layer plate that has been poured out, so the titer of the Vcp OK phage solution is 1*10 8 PFU / ml.

[0076] Example 7 Determination of the titer of the mutant phage Vcp YR of Vibrio cannelli

[0077] The specific implementation method of the titer determination of Vcp YR is the same as that of Example 6. The titer of Vcp YR determined by the experiment is 1*10 8 PFU / ml.

[0078] Example 8 Electron Microscopic Morphological Observation of Vibrio cannerii Phages Vcp OK and Vcp YR

[0079] The wild strain phage Vcp OK of Vibrio campbellii and the mutant phage VcpYR of Vibrio campbellii were added to the bacterial solution of the wild strain V. campbellii 18-1 of Vibrio campbellii in the logarithmic growth phase at a ratio of MOI = 0.1, and then placed in a shaker at 28°C and 150rpm for 2-3 days. The co-culture solution obtained was filtered with a 0.22um sterile filter to obtain an enriched phage solution. Then DNase and RNase with a final concentration of 2ng / L were added thereto, and the mixture was gently mixed and allowed to stand at room temperature for 1h. After standing, NaCl with a final concentration of 1M was added to the solution, and the mixture was gently mixed and allowed to stand for 30min. The above solution was centrifuged (8000rpm, 30min) to collect the supernatant, and PEG 8000 with a final concentration of 10% was added to the supernatant solution, and it was placed in the dark at 4°C for 24h. Then the precipitate was collected by centrifugation at 10000g, and the precipitate was resuspended in SMBuffer. Add 7 ml of 1.5 g / ml cesium chloride solution to the ultracentrifuge tube, then add 5 ml of the phage suspension resuspended in SM Buffer, ultra-high speed vacuum centrifuge (200000 g, 4 ° C, 8 h), and absorb the light blue phage band obtained by centrifugation. Replace the buffer with SM Buffer using a 30KD ultrafiltration tube, and use the sample to observe the phage morphology under a transmission electron microscope after negative staining with uranyl acetate. The electron microscopic morphology of Vcp OK is shown in Figure 3 The electron microscopic morphology of VcpYR is shown in Figure 4 Vcp OK has a regular hexahedron head with a diameter of 28 nm and a tail length of 197 nm. The hexahedron head of Vcp YR is a regular hexahedron with a diameter of 66 nm and a tail length of about 302 nm. Both belong to the family Myoviridae.

[0080] Example 9 Chloroform sensitivity test of wild strain Vibrio cannerii bacteriophage Vcp OK

[0081] A double-layer plate was prepared by mixing the logarithmic phase bacterial liquid of the wild strain of Vibrio campbellii 18-1 with a heated and pre-cooled semi-solid culture medium (48°C). Different phage solutions were obtained using the above embodiment to prepare chloroform solutions with volume fractions of 2% and 20%, respectively. The control group was a phage solution without any treatment. After vortexing, each experimental group was allowed to stand at room temperature for 30 minutes, and then centrifuged at 8000rpm. 10μl of the upper aqueous phase was taken and dropped onto the above double-layer plate. The plates were cultured overnight in a constant temperature incubator at 28°C, and the plaque size of the experimental group was observed to see if there was any difference between that of the control group. Chloroform sensitivity test double-layer plate of Vcp OK is shown in Figure 2. Figure 5 There was no difference in the size of plaques formed by chloroform-treated phages and those in the control group, indicating that the phages were insensitive to chloroform and had no lipid structure.

[0082] Example 10 Chloroform sensitivity test of the mutant phage Vcp YR of Vibrio cannelli

[0083] The specific implementation method is the same as in Example 9. The chloroform sensitivity test plate of Vcp YR is shown in Figure 6 There was no difference in the size of plaques formed by chloroform-treated phages and those in the control group, indicating that the phages were insensitive to chloroform and had no lipid structure.

[0084] Example 11 Host range detection of Vibrio cannelligensis mutant phage Vcp OK

[0085] In this experiment, 18 strains of Vibrio that are known and preserved in this laboratory and listed in Table 1 were selected for phage host range detection. That is, different Vibrio that have grown to the logarithmic phase were mixed with the pre-cooled 2216E semi-solid that was heated (about 48°C) to make a double-layer plate. After the upper semi-solid solidified, 10μl of phage Vcp OK filtered through a 0.22μm sterile filter membrane was added dropwise. The plate was placed in a 28°C incubator overnight to observe whether plaques appeared. If so, it indicates that the host can be lysed by phage Vcp OK; if not, it indicates that the host cannot be lysed by phage Vcp OK. The host range test results of Vcp OK are shown in Table 1.

[0086] Example 12 Detection of host range of mutant phage Vcp YR of Vibrio cannelli

[0087] The specific implementation method is the same as that of Example 11. The host range detection results of Vcp YR are shown in Table 1.

[0088] Example 13 One-step growth curve determination of wild-type Vibrio cannelligensis phage Vcp OK

[0089] According to the titer of Vcp OK phage determined in Example 6 and Example 7, when the wild strain V. campbellii 18-1 of Vibrio campbellii grows to the logarithmic phase, add phage Vcp OK filtered by 0.22 μm sterile filter membrane according to MOI=0.01, incubate at 28°C shaking table for 20 minutes, then centrifuge at 6000rpm for 5 minutes, resuspend and wash the bacterial precipitate with fresh 2216E medium for 3 times, add 2216E medium and incubate at 28°C shaking table. Starting from 0h, take samples every 15 minutes, take 1ml sample and directly add glutaraldehyde (final concentration 0.5%) to fix, which is the bacterial sample; take 1ml sample and filter with 0.22μm sterile filter head and add glutaraldehyde (final concentration 0.5%) to fix, which is the phage sample. After each group of samples is fixed with glutaraldehyde in the dark for 30 minutes, the amount of bacteria or phage is determined by flow cytometry, and the change curve of each parameter over time is plotted. The one-step growth curve of wild-type phage Vcp OK of Vibrio cannibalis is shown in Figure 7The incubation period of wild-type Vibrio campbellii phage Vcp OK lysing wild-type Vibrio campbellii 18-1 was 30 minutes, and the lysis period was about 3.5 hours.

[0090] Example 14 One-step growth curve determination of the mutant phage Vcp YR of Vibrio cannelli

[0091] The specific implementation method is the same as in Example 13. The one-step growth curve of the mutant phage Vcp YR of Vibrio cannelligensis is shown in Figure 8 The incubation period of the mutant phage Vcp YR of Vibrio campbellii to lyse the wild strain V. campbellii 18-1 was 30 minutes, and the lysis period was about 3 hours.

[0092] Example 15 Evaluation of bacteriocidal effect of phage cocktail

[0093] According to the titers of bacteriophages Vcp OK and Vcp YR determined in Examples 6 and 7, three experimental groups were set up, in which bacteriophages Vcp OK, Vcp YR, and Vcp OK+Vcp YR were mixed with V. campbellii 18-1 bacteria at a ratio of MOI=0.1, and then placed in a shaking incubator at 28°C. The control group was V. campbellii 18-1 bacteria cultured alone. Three parallel experiments were set up for each group of experiments. Starting from 0h, samples were taken every 1h to measure the OD 600 The bactericidal effect of phage cocktail under MOI=0.1 is shown in Fig. 9 As can be seen from the figure, phage Vcp OK has a good antibacterial effect in the first 13 hours (OD 600 <0.3), and then the pathogen growth "rebound" phenomenon appeared. After the addition of bacteriophage Vcp YR, the host OD 600 The value is maintained between 0.8-1.5, and the antibacterial effect is poor. However, the phage cocktail Vcp OK+Vcp YR can maintain the OD 600 <0.3 for up to 29h, with significant bactericidal effect and significantly prolonged inhibition time. Under MOI=0.1, 34h after adding bacteriophage, the bactericidal rates of Vcp OK, Vcp YR, and Vcp OK+YR were 34.7%, 69.3%, and 93.3%, respectively.

[0094] Table 1

[0095]

[0096] Note: “+” indicates that the host bacteria is sensitive to the bacteriophage; “-” indicates that the host bacteria is not sensitive to the bacteriophage.

[0097] It should be noted that the above examples are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the given examples, those skilled in the art may modify or replace the technical solution of the present invention as needed without departing from the spirit and scope of the technical solution of the present invention.

[0098]

[0099] The 16sRNA sequence of Vibrio campbellii mutant strain V.campbellii 18-1M is as follows: >V.campbellii18-1M

[0100] GTCGAGCGGAAACGAGTTATCTGAACCTTCGGGGAACGATAACGGCG

[0101] TCGAGCGGCGGACGGGTGAGTAATGCCTAGGAAATTGCCCTGATGTG

[0102] GGGGATAACCATTGGAAACGATGGCTAATACCGCATAATGCCTACGGG

[0103] CCAAAGAGGGGGACCTTCGGGCCTCTCGCGTCAGGATATGCCTAGGT

[0104] GGGATTAGCTAGTTGGTGAGGTAAGGGCTCACCAAGGCGACGATCCC

[0105] TAGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTGAGACACGGT

[0106] CCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCG

[0107] CAAGCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTT

[0108] GTAAAGCACTTTCAGTCGTGAGGAAGGTAGTGTAGTTAATAGCTGCAT

[0109] TATTTGACGTTAGCGACAGAAGAAGCACCGGCTAACTCCGTGCCAGC

[0110] AGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGC

[0111] GTAAAGCGCATGCAGGTGGTTTGTTAAGTCAGATGTGAAAGCCCGGG

[0112] GCTCAACCTCGGAATTGCATTTGAAACTGGCAGACTAGAGTACTGTAG

[0113] AGGGGGGTAGAATTTCAGGTGTAGCGGTGAAATGCGTAGAGATCTGA

[0114] AGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAGATACTGACACTC

[0115] AGATGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTC

[0116] CACGCCGTAAACGATGTCTACTTGGAGGTTGTGGCCTTGAGCCGTGGC

[0117] TTTCGGAGCTAACGCGTTAAGTAGACCGCCTGGGGAGTACGGTCGCA

[0118] AGATTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAG

[0119] CATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGAC

[0120] ATCCAGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCTGA

[0121] GACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGT

[0122] TAAGTCCCGCAACGAGCGCAACCCTTATCCTTGTTTGCCAGCGAGTAA

[0123] TGTCGGGAACTCCAGGGAGACTGCCGGTGATAAACCGGAGGAAGGT

[0124] GGGGACGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACAC

[0125] GTGCTACAATGGCGCATACAGAGGGCAGCCAACTTGCGAGAGTGAGC

[0126] GAATCCCAAAAAGTGCGTCGTAGTCCGGATCGGAGTCTGCAACTCGA

[0127] CTCCGTGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGT

[0128] GAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGT

[0129] GGGCTGCAAAAGAAGTAGGTAGTTTAACCTTCGGGAGGACGCTTACC

Claims

1. A Vibrio cannerii phage composition with high antibacterial ability, characterized in that: The composition contains bacteriophage VcpOK and bacteriophage Vcp YR; Among them, the wild strain phage Vcp OK of Vibrio campestris has been sent to the China Center for Type Culture Collection (address: Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei Province), with a preservation date of July 22, 2024 and a preservation number of CCTCC M20241645; The mutant phage Vcp YR of Vibrio campestris has been sent to the China Center for Type Culture Collection (address: Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei Province), with a preservation date of July 22, 2024 and a preservation number of: CCTCC M 20241646.

2. The Vibrio cannerii phage composition according to claim 1, characterized in that The titers of bacteriophage VcpOK and Vcp YR in the composition are not less than 10 8 PFU / ml.

3. The Vibrio cannerii phage composition according to claim 1, characterized in that The multiplicity of infection (MOI) was 0.

1.

4. An application of a Vibrio cannelliger phage composition, characterized in that: The use of the Vibrio cannereysi phage composition described in claim 1 in the preparation of a Vibrio cannereysi bactericide.

5. The use according to claim 4, characterized in that: The use of the Vibrio cannelliger phage composition described in claim 1 in the preparation of a bacterial agent for controlling Vibrio infections in aquaculture.

6. A Vibrio cannelligensis bactericide, characterized in that: The bactericide for Vibrio cannerii contains the Vibrio cannerii bacteriophage composition according to claim 1.

7. The Vibrio cannerii bactericide according to claim 6, characterized in that The formulation of the Vibrio cannelligensis fungicide is wettable powder, water dispersible granules or water suspension.

8. An application of a Vibrio cannelligensis bactericide, characterized in that: Application of Vibrio canneri fungicide in the preparation of Vibrio canneri fungicide.

9. The use according to claim 5, characterized in that: The Vibrio cannelligensis fungicide is used in controlling Vibrio infection in aquaculture.

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