Application of a Vibrio alginolyticus phage and its liposome-coated preparation for drug-resistant Vibrio

Abstract

The present invention provides a Vibrio alginolyticus phage acting on drug-resistant Vibrio and an application of its liposome-coated preparation, belonging to the field of biotechnology. The phage was sent to the China Center for Type Culture Collection for preservation on March 6, 2024. The preservation address is: Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 2024391. The bacteriophage VAP2312001 has a lytic effect on Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi and Vibrio cannelli. The liposome coating technology used improves the encapsulation rate of the phage, and the liposome-coated phages improve the survival rate of the mouse model infected with resistant Vibrio alginolyticus. The phage has the advantages of high value-added titer, safety and high efficiency. The liposome coating technology improves the tolerance of the phage VAP2312001 to temperature and pH values. The phage can prevent diseases caused by resistant Vibrio in various environments, and provide excellent strain resources and large-scale fermentation production biological prevention measures for phage therapy of resistant Vibrio infection problems.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to an application of a Vibrio alginolyticus phage applied to drug-resistant Vibrio and a liposome-coated preparation thereof. Background Art

[0002] Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi and Vibrio cannelli are not only pathogens that are extremely harmful to aquaculture, but are also sources of foodborne pathogens for humans. Vibrio can cause diseases such as sea cucumber rotten skin syndrome, shrimp enteritis, fish sepsis, and human diarrhea. Among them, Vibrio parahaemolyticus that can cause acute hepatopancreatic necrosis (AHPND) in shrimp and highly pathogenic Vibrio that causes shrimp vitrification disease (HLVD) have caused immeasurable losses to aquaculture.

[0003] Since 2023, some scholars have monitored antibiotic residues in Chinese waters. The levels of oxytetracycline, enrofloxacin and anhydroerythromycin in Chinese waters have exceeded the national standard, especially in southern Fujian. The oxytetracycline content has far exceeded the national standard limit. Later, some scholars reported the drug resistance of Vibrio in Chinese waters. At present, the drug resistance of Vibrio is very serious. Phages are nano-scale microorganisms that "eat" bacteria. Compared with antibiotics, phages have the following outstanding advantages: (1) they can replicate and proliferate at the site of bacterial infection and kill pathogenic bacteria in a targeted manner; (2) they are species-specific and can specifically lyse target bacteria without causing an imbalance in the body's microecology; (3) phages are widely present in nature and animals, with no toxic side effects and no residue; (4) the development time and cost of phage preparations are much lower than the development cycle and cost of new antibiotics. Phages are one of the most promising alternatives to antibiotics.

[0004] Liposome coating technology is a spherical nanoparticle surrounded by a lipid bilayer, which has high biocompatibility and is a directional drug carrier. The method of using liposome coating materials, also known as the "Trojan Horse" method, can improve the stress resistance of phages, prolong their storage period, and change the distribution of coated phages in animals. Due to the interaction between cationic liposomes and negatively charged bacteria, liposome-encapsulated phages can adhere to and diffuse into lesions or mucosa containing pathogenic bacteria, and prolong the retention time of phages at the infection site, thereby improving the phage therapeutic index, which is particularly suitable for respiratory tract, local wounds and gastrointestinal infections. It is necessary to develop a green, safe, highly targeted, and stable phage product that acts on drug-resistant Vibrio; based on this, the present invention provides a strain of Vibrio alginolyticus phage and its liposome-encapsulated preparation for drug-resistant Vibrio. Summary of the invention

[0005] In order to prevent diseases or breeding problems caused by drug-resistant Vibrio and reduce the morbidity caused by the problems, one of the objects of the present invention is to provide a multivalent Vibrio alginolyticus phage VAP2312001 for preventing and treating drug-resistant Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi and Vibrio cannelli.

[0006] The second purpose of the present invention is to provide parameters with important value and their application in the liposome coating process of Vibrio alginolyticus phage VAP2312001, so as to improve the phage encapsulation rate.

[0007] The third purpose of the present invention is to provide Vibrio alginolyticus phage VAP2312001 liposomes that effectively improve the phage's tolerance to temperature and pH value.

[0008] A fourth object of the present invention is to provide the use of Vibrio alginolyticus phage VAP2312001 in the preparation of drugs for treating or preventing diseases caused by Vibrio alginolyticus infection.

[0009] In order to achieve the above-mentioned purpose, the present invention is implemented through the following technical scheme: a Vibrio alginolyticus phage used for drug-resistant Vibrio, the classification name of the phage is Vibrio alginolyticus phage VAP2312001, the applicant isolated a Vibrio alginolyticus phage VAP2312001 from the water of a farm in Zhangzhou, Fujian, and the phage was sent to the China Center for Type Culture Collection for preservation on March 6, 2024, and the preservation address is: Wuhan University, Wuhan, China; the preservation number is CCTCCNO: M 2024391.

[0010] Furthermore, it also includes a compound of the Vibrio alginolyticus phage VAP2312001, including but not limited to compounding with other phages and other probiotics.

[0011] Furthermore, it is used in industries such as environmental disinfection, animal husbandry, poultry farming, aquaculture and biomedicine to prepare Vibrio for prevention and treatment.

[0012] Furthermore, the invention is also used for preparing drugs for treating or preventing diseases caused by Vibrio infection.

[0013] Furthermore, the Vibrio include but are not limited to drug-resistant Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi and Vibrio cannelli.

[0014] An application of the liposome-coated preparation of bacteriophage VAP2312001 as mentioned above, wherein the ingredients of the liposome-coated preparation of bacteriophage VAP2312001 include soybean lecithin, cholesterol, sodium dodecyl dimethyleneaminodicarboxylate and sodium alginate.

[0015] Furthermore, it is used to improve the phage encapsulation rate and enhance the phage tolerance to temperature and pH value.

[0016] Furthermore, liposome preparations effectively enhance the therapeutic effects of phages in animals.

[0017] Beneficial effects of the present invention:

[0018] 1. The bacteriophage VAP2312001 has lytic activity against drug-resistant Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi and Vibrio cannelli. The bacteriophage VAP2312001 has a remarkable inhibitory effect on different Vibrio species, which has important reference significance for the prevention, control and treatment of Vibrio species in the future. The bacteriophage VAP2312001 has a high fermentation rate. When the temperature is 30°C and the optimal multiplicity of infection MOI is 0.1, the titer can reach 3.3×10 after fermentation for 5 hours. 12 PFU / mL; in the industrial fermentation process, the fermentation titer can reach 4.1×10 12 PFU / mL.

[0019] 2. The Vibrio alginolyticus phage VAP2312001 has good environmental tolerance. The potency of the Vibrio alginolyticus phage VAP2312001 does not change significantly in the order of magnitude after being treated in the pH range of 4-11 for 6 hours. After liposome coating, the potency of VAP2312001 does not change significantly in the pH range of 3-12 for 6 hours. Liposome coating can effectively improve the temperature tolerance of the phage.

[0020] 3. The phage titer of the Vibrio algae phage VAP2312001 had no obvious order of magnitude difference after 16 weeks at 4°C, had no obvious order of magnitude difference after 12 weeks at 25°C, and had no obvious order of magnitude difference after 2 weeks at 37°C; after liposome coating, the titer of VAP2312001 had no obvious order of magnitude change when treated at 4°C for 16 weeks, had no obvious order of magnitude change when treated at 25°C for 16 weeks, and had no obvious order of magnitude difference when treated at 37°C for 4 weeks; it has good stability at 4-37°C and can be effectively preserved.

[0021] 4. The Vibrio alginolyticus phage VAP2312001 has lytic properties against multiple drug-resistant Vibrio (Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi and Vibrio cannenbergii). The phage VAP2312001 can completely lyse 7 out of 10 strains of drug-resistant Vibrio parahaemolyticus, 9 out of 10 strains of drug-resistant Vibrio alginolyticus, 8 out of 10 strains of drug-resistant Vibrio harveyi and 6 out of 10 strains of drug-resistant Vibrio cannenbergii. The experimental results confirmed that the phage VAP2312001 has a very outstanding effect in dealing with the lytic properties of one or more of the drug-resistant Vibrio, and is of great value for the targeted prevention, control and treatment of drug-resistant Vibrio in the future.

[0022] 5. The Vibrio alginolyticus phage VAP2312001 can effectively treat the enteritis problem of mice caused by drug-resistant Vibrio alginolyticus. The survival rate of mice in both the prevention and treatment groups was effectively improved. The survival rate of the blank group was 100%, and the survival rate of mice in the challenge group was 10%; the survival rate of the phage liposome-coated treatment group was 90%, the survival rate of the common phage treatment group was 70%, and the survival rate of the enrofloxacin group was 80%. It provides important reference value for the prevention and treatment of Vibrio alginolyticus in the future, and can be used as a biological antibacterial agent for the prevention and treatment of Vibrio alginolyticus diseases in mice and other animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The plaques are from the Vibrio alginolyticus phage VAP2312001;

[0024] Figure 2 is the titer of Vibrio alginolyticus phage VAP2312001 under different infection multiplicity conditions;

[0025] Figure 3 To determine the stability of liposome preparation of Vibrio alginolyticus phage VAP2312001 at different pH values;

[0026] Figure 4 To determine the stability of uncoated Vibrio alginolyticus phage VAP2312001 at different temperatures;

[0027] Figure 5 To determine the stability of liposome preparation of Vibrio alginolyticus phage VAP2312001 at different temperatures;

[0028] Figure 6 Schematic diagram of the fermentation dynamics of Vibrio alginolyticus phage VAP2312001. DETAILED DESCRIPTION

[0029] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0030] See also Figures 1 to 6 , the present invention provides the following technical solutions:

[0031] Embodiment 1:

[0032] Isolation and Purification of Vibrio alginolyticus Phage VAP2312001

[0033] 120 water samples were collected from a breeding site in Zhangzhou, Fujian, each with 50 mL. The sewage was centrifuged at 6000 rpm for 10 min, and the supernatant was sterilized with a 0.22 μm filter. 10 mL of filtrate, 10 mL of double 2% sodium chloride TSB medium and 1 mL of Vibrio alginolyticus (concentration of about 5×10 8CFU / mL) were mixed evenly and cultured overnight in a shaker at 37°C at 200 rpm. The fermentation broth was centrifuged at 6000 rpm for 10 min, the supernatant was sterilized with a 0.22 μm filter, and 100 μL of the filtrate was mixed with 300 μL of Vibrio alginolyticus (concentration of about 5×10 8 CFU / mL) and mix well, add 2% sodium chloride TSB semi-solid (containing 0.65% agar), gently invert and pour evenly on the 2% sodium chloride TSA bottom plate, and after solidification, place the culture dish in a 37°C incubator for overnight culture. Pick up the transparent and bright plaque spots, shake and desorb in 1mL SM liquid, and sterilize through a 0.22μm microporous filter to obtain the phage filtrate, inoculate it in 5mL 2% sodium chloride TSB liquid culture medium, add 500μL of the corresponding host Vibrio alginolyticus bacterial liquid and mix well, shake and culture at 200rpm overnight at 37°C, centrifuge at 6000rpm for 10min, take the supernatant and sterilize through a 0.22μm microporous filter to obtain the phage filtrate, and observe the plaque morphology by double-layer plate method. After repeating the operation 3-5 times, plaques of uniform shape and size can be obtained.

[0034] A strain of Vibrio alginolyticus phage VAP2312001 was isolated from the water sample. Figure 1 As shown, the plaques formed by it and the host bacteria are transparent, round, and have a diameter of 0.5 mm. The phage was sent to the China Center for Type Culture Collection for preservation on March 6, 2024, and the classification name is: Vibrio alginolyticus phage (Vibrio alginolyticus phage) VAP2312001, address: Wuhan University, Wuhan, China, preservation number: CCTCC NO: M 2024391.

[0035] Embodiment 2:

[0036] Determination of the titer of Vibrio alginolyticus phage VAP2312001 under different infection multiplicity conditions

[0037] The host bacterium of the Vibrio alginolyticus phage VAP2312001, VAFJ005, was inoculated into an EP tube with 3 mL of 2% sodium chloride TSB, and cultured at 37°C for 5-8 hours to obtain a VAFJ005 bacterial solution. The bacterial solution was inoculated into 100 mL of 2% sodium chloride TSB medium at a 1.5% inoculum volume, and when VAFJ005 grew to the initial logarithmic phase, the phage VAP2312001 was inoculated, and the infection was carried out according to different multiplicity of infection (MOI; MOI = number of phages / number of bacteria, see Figure 2 Each experiment was performed in triplicate. The temperature was set at 30°C and the shaking was performed at 200 rpm for 12 h.

[0038] Results Figure 2 When the temperature was 30°C and MOI = 0.01, the fermentation titer reached a maximum of 3.3 × 10 12 PFU / mL.

[0039] Embodiment 3:

[0040] Determination of encapsulation efficiency of bacteriophage VAP2312001 by different liposome formulations

[0041] In this part, the thin film dispersion-ultrasound method was used to prepare the liposome coating solution of bacteriophage VAP2312001. The concentration of bacteriophage was 2.0×10 10 PFU / mL. The specific steps are as follows: dissolve soybean lecithin, cholesterol and sodium dodecyl dimethylene aminodicarboxylate in a round-bottom beaker filled with ether solution, use a rotary evaporator to decompress and spin-dry the organic solvent at 50°C, so that the liposome forms a transparent film at the bottom of the bottle; mix the phage solution and the 0.5% sodium alginate solution in equal volumes, and after mixing, take the same volume of ether and the phage mixture into the above round-bottom flask, and ultrasonicate in a water bath at 35°C for 30 minutes to obtain phage liposome-coated particles of uniform size.

[0042] Determination of phage encapsulation efficiency: The original phage concentration is recorded as W1, and the phage liposome liquid after encapsulation is taken for phage titer determination, recorded as W2. The encapsulation efficiency (EE) is calculated as EE% = [(W1-W2) / W1]*100%.

[0043] The liposome process formula was explored using 3 factors and 3 levels. The 3 factors were soybean lecithin, cholesterol, and sodium dodecyl dimethylene amino dicarboxylate. Each factor had 3 different concentrations. Soybean lecithin was set at 150 mg (A1), 100 mg (A2), and 50 mg (A3), cholesterol was set at 50 mg (B1), 30 mg (B2), and 10 mg (B3), and sodium dodecyl dimethylene amino dicarboxylate was set at 50 mg (C1), 30 mg (C2), and 10 mg (C3). In addition, the orthogonal experimental table based on 3 factors and 3 levels was set as follows.

[0044] The experimental results were analyzed by range analysis based on the orthogonal experimental table, and the encapsulation efficiency of bacteriophage VAP2312001 under different parameter conditions was obtained. The influence of process parameters from large to small was soybean lecithin>cholesterol>sodium dodecyl dimethyleneaminodicarboxylate (A>B>C), and the optimal parameter ratio was A1B1C1, and the optimal encapsulation efficiency could reach 94.9%.

[0045] Table 1 Determination of encapsulation efficiency of bacteriophage VAP2312001 by different liposome process formulations

[0046]

[0047] Embodiment 4:

[0048] Stability of liposomal preparations of Vibrio alginolyticus phage VAP2312001 at different pH values

[0049] 0.1 mL of liposome preparation of Vibrio alginolyticus phage VAP2312001 and phage solution not coated by liposomes were added into 9.9 mL of 2% sodium chloride TSB liquid medium with pH values ​​of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0, respectively (titer after dilution was 5*10 8 pfu / mL), and incubated in a 25°C incubator for 6 hours before sampling. The pH of the culture medium was adjusted to Ph=7.4 with hydrochloric acid and sodium hydroxide solutions, respectively, and then diluted in multiples. The phage titer was determined by the double-layer plate method. Duplicate cultures were performed at each point to take the average value, and the experiment was repeated 3 times.

[0050] The results are as follows Figure 3 As shown, the Vibrio alginolyticus phage VAP2312001 has good environmental tolerance, and the potency of the Vibrio alginolyticus phage VAP2312001 has no obvious order of magnitude change when treated in the pH range of 4-11 for 6 hours; after liposome encapsulation, the potency of VAP2312001 has no obvious order of magnitude change when treated in the pH range of 3-12 for 6 hours.

[0051] Embodiment 5:

[0052] Stability of liposome preparation of Vibrio alginolyticus phage VAP2312001 at different temperatures

[0053] Take 1 mL of liposome-coated and uncoated Vibrio alginolyticus phage VAP2312001 and dispense them into 1.5 mL centrifuge tubes (initial titer is 5*10 8 pfu / mL). The centrifuge tubes containing phages were placed in a constant temperature of 4, 25, 37, 45, 55, and 65°C for 0h, 24h, 92h, 1w, 2w, 4w, 8w, 12w, and 16w. After the action time, the sample tubes were taken out and immediately placed in an ice bath for cooling. After appropriate dilution, the phage titer was determined by the double-layer plate method. Duplicate culture was performed at each point to obtain the average value, and the experiment was repeated 3 times.

[0054] The results are as follows Figure 4 and Figure 5As shown in the comparison, the Vibrio alginolyticus phage VAP2312001 has a longer survival time at temperatures of 45°C and below. At 4°C, there is no obvious order of magnitude difference in the phage titer after 16 weeks, at 25°C, there is no obvious order of magnitude difference in the phage titer after 12 weeks, and at 37°C, there is no obvious order of magnitude difference in the phage titer after 2 weeks. It has good stability at 4-37°C and can be effectively preserved. After liposome coating, the phage's temperature tolerance can be effectively improved.

[0055] Embodiment 6:

[0056] Host range of bacteriophage VAP2312001

[0057] The Vibrio alginolyticus selected in the experiment is divided into three types of Vibrio, namely Vibrio alginolyticus, Vibrio parahaemolyticus and Vibrio harveyi. The selected Vibrio were isolated from shrimp farms in southern Fujian. According to the monitoring of antibiotic residues in Chinese waters by scientific researchers in 2023, it was found that oxytetracycline residues in southern Fujian far exceeded the national antibiotic residue standards. The selected Vibrio were isolated from the breeding area in southern Fujian, and Vibrio containing tet-A and tet-C (tetracycline resistance genes) were selected for subsequent experiments.

[0058] Ten strains of each type of Vibrio were inoculated into 3 mL of 2% sodium chloride TSB EP tubes, and the tubes were shaken at 37°C and 200 rpm for 8 h to obtain a Vibrio alginolyticus solution. A sufficient amount of 2% sodium chloride TSA bottom plate was prepared, and 0.5 ml of Vibrio solution was mixed with 6 mL of 2% sodium chloride TSB semi-solid medium (containing 0.65% agar) at about 50°C, and then slowly spread on the 2% sodium chloride TSA bottom plate. After the plate medium solidified to room temperature, 5×10 8 5 μL of phage VAP2312001 with a PFU / ml was dropped onto the plate, and after air drying, the plate was inverted and placed in a 37°C constant temperature biochemical incubator for about 8 hours, and the results were observed. Each group of experiments was repeated 3 times.

[0059] As shown in Table 2, bacteriophage VAP2312001 can lyse 7 out of 10 strains of drug-resistant Vibrio parahaemolyticus, 9 out of 10 strains of drug-resistant Vibrio alginolyticus, 8 out of 10 strains of drug-resistant Vibrio harveyi and 6 out of 10 strains of drug-resistant Vibrio cannelli. The experimental results confirmed that bacteriophage VAP2312001 has a very outstanding effect in dealing with one or more lytic properties of drug-resistant Vibrio, and is of great value for the targeted prevention, control and treatment of drug-resistant Vibrio in the future.

[0060] Table 2 Lysis effect of bacteriophage VAP2312001 on different capsule types of Vibrio alginolyticus

[0061]

[0062]

[0063] Note: “-” indicates no lysis, “+” indicates lysis and the plaques are relatively transparent; “++” indicates lysis and the plaques are transparent.

[0064] Embodiment 7:

[0065] Fermentation preparation of Vibrio alginolyticus phage VAP2312001

[0066] A single colony of Vibrio alginolyticus VAFJ005 was picked from the plate and inoculated into 3 mL of 2% sodium chloride TSB medium, and cultured at 37°C and 200 rpm for 8 h to obtain the host bacterial solution. The bacterial suspension was inoculated into 120 mL of 2% sodium chloride TSB medium at a 2% inoculum amount, and cultured at 37°C and 150 rpm until the early logarithmic phase, and the bacterial solution OD600 value was 0.1.

[0067] The industrial fermentation is a 10L fermentation tank, the fermentation system is 6L, the medium used is 2% sodium chloride TSB, the fermentation temperature is 28°C, the initial pH value is 7.4, the flame method is used to inoculate Vibrio alginolyticus at 1% inoculation amount, and the phage VAP2312001 is added to the fermentation system at the optimal infection condition MOI=0.01. Sterile air is introduced during the fermentation process, and 3‰ defoamer is added. The fermentation preparation time is 12h. Since the start of fermentation, 20ml of fermentation liquid is taken from the sampling port in a sterile container at 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, and 12h, and centrifuged at 6000rpm for 10min. The supernatant is sterilized through a 0.22μm microporous filter membrane to obtain a filtrate containing phage and its titer is determined. The method is referred to Example 2.

[0068] After the fermentation is completed, the entire mixed solution of phage and host bacteria is taken out from the sampling port and placed in a sterile container, centrifuged at 6000 rpm for 10 min, and the supernatant is filtered through a vacuum pump into a sterile filter device to obtain the phage fermentation solution and stored at 4°C.

[0069] Depend on Figure 6 It can be seen that the titer of Vibrio alginolyticus phage VAP2312001 reached a maximum of 4.1×10 12 PFU / mL, in the period of 6h to 12h, the titer is stable at 10 12 PFU / mL; The results show that it is feasible to use fermentation method for industrial production of bacteriophages, which provides effective reference data for the preparation of bacteriophage products.

[0070] Embodiment 8:

[0071] Animal experiment on the treatment of Vibrio alginolyticus infection in mice by bacteriophage VAP2312001 and its liposome preparation

[0072] The mice selected for the experiment were normal and healthy in appearance, weighing about 18g; the mice were uniformly placed in cages for 7 days before the experiment, marked, and then randomly divided into groups according to weight order so that mice of different weights could be evenly distributed to each group, with 10 mice in each group and 5 mice in each cage. Fasting for 12 hours before the experiment, free drinking water; normal feeding during the experiment. The Vibrio alginolyticus used in this example is VAFJ002, and the selected phage is the Vibrio alginolyticus phage VAP2312001 of the present invention.

[0073] The experimental design is as follows: the mice were randomly divided into 5 groups on average, and the experiment lasted for 10 days:

[0074] 1) Control group: Each mouse was gavaged with an equal volume of normal saline for 3 consecutive days;

[0075] 2) Challenge group: Each mouse was injected orally with 1×10 9 1 mL of bacterial solution with a CFU / mL was administered orally for 3 consecutive days.

[0076] 3) Uncoated phage treatment group: Each mouse was injected orally with 1×10 9 CFU / mL of bacterial solution 1mL; continuous gavage for 3 days, treatment on the 4th and 5th days, bacteriophage VAP23120011×10 9 PFU / drug therapy only.

[0077] 4) Liposome preparation treatment group: Each mouse was injected orally with 1×10 9 CFU / mL of bacterial solution 1mL; continuous gavage for 3 days, treatment on the 4th and 5th days, phage VAP2312001 liposome preparation 1×10 9 PFU / drug therapy only.

[0078] 5) Enrofloxacin treatment group: Each mouse was injected orally with 1×10 9 1 mL of bacterial solution with a CFU / mL was administered orally for 3 consecutive days. Treatment was performed on the 4th and 5th days, and enrofloxacin was added according to the dosage specified for treatment.

[0079] The experiment lasted for 10 days, and the symptoms and number of deaths of mice were recorded. The survival rate of mice in each group was calculated as follows.

[0080]

[0081] ATotal: the total number of mice in each group of the experiment, mice;

[0082] A Death: The total number of mice that died in each group of the experiment, mice;

[0083] C: Survival rate of mice in each group of the experiment, %.

[0084] The experimental results are shown in Table 3. The survival rate of the blank group was 100%, and the survival rate of the mice in the challenge group was 10%; the survival rate of the uncoated phage treatment group was 70%, the survival rate of the phage liposome preparation treatment group was 90%, and the enrofloxacin group was 80%. The experimental results revealed that the Vibrio alginolyticus phage VAP2312001 can effectively treat the enteritis problem of mice caused by Vibrio alginolyticus. Both the prevention and treatment groups effectively improved the survival rate of mice. This example provides an important reference value for the prevention and treatment of drug-resistant Vibrio alginolyticus in the future, and can be used as a biological antibacterial agent for the prevention and treatment of Vibrio alginolyticus diseases in mice and other animals.

[0085] Table 3 The control effect of bacteriophage VAP2312001 on Vibrio alginolyticus in mice

[0086]

[0087] The basic principles and main features of the present invention and the advantages of the present invention are shown and described above. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention.

[0088] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A strain of Vibrio alginolyticus phage (V ibrio alginolyticus phage ), characterized in that: The phage was classified and named as Vibrio alginolyticus phage VAP2312001. The phage was sent to the China Center for Type Culture Collection for preservation on March 6, 2024. The preservation address is: Wuhan University, Wuhan City; the preservation number is CCTCC NO: M2024391.

2. A compound comprising the Vibrio alginolyticus phage VAP2312001 according to claim 1, characterized in that: It also includes other bacteriophages and probiotics.

3. The use of the bacteriophage according to claim 1, characterized in that: The invention is used for preparing a medicine for treating or preventing Vibrio infection; the Vibrio are drug-resistant Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi and Vibrio cannelli.

4. A liposome-coated preparation comprising the bacteriophage VAP2312001 as claimed in claim 1, characterized in that: The ingredients of the liposome-encapsulated formulation of bacteriophage VAP2312001 include soybean lecithin, cholesterol, sodium dodecyl dimethyleneaminodicarboxylate and sodium alginate.

5. The use of the liposome-coated preparation according to claim 4, characterized in that: The invention is used for preparing medicine for treating Vibrio infection, wherein the Vibrio are drug-resistant Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi and Vibrio cannelli.

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