Vibrio harveyi phage v-ydf132 and application thereof

Abstract

The application discloses a Vibrio harveyi bacteriophage V-YDF132 and application thereof. The bacteriophage is preserved in the Guangdong Microbial Culture Collection Center on July 5, 2022, and has a preservation number of GDMCC No: 62599-B1. A new virulent Vibrio harveyi bacteriophage V-YDF132 is screened in the application, the host specificity of the bacteriophage is strong, the bacteriophage has strong lysis and killing effects on Vibrio harveyi. The bacteriophage is not sensitive to chloroform, has high thermal stability and PH stability, has strong tolerance to temperatures below 60 DEG C, can maintain high lysis activity in a pH value of 5-11, and has an optimal infection multiple of 0.1-0.01. The Vibrio harveyi bacteriophage V-YDF132 provided in the application can be widely used in various links of water production and breeding processes which are prone to losses caused by Vibrio harveyi infection, daily disinfection of breeding environments and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial prevention and treatment, and particularly relates to a Vibrio harveyi phage V-YDF132 and application thereof. BACKGROUND

[0002] Vibrio harveyi is a gram-negative, luminescent marine bacteria, widely distributed in the coastal warm marine environment, and is one of the normal flora of marine biota. It was not recognized until the late 20th century, and is an important pathogenic bacterium of aquatic animals, mainly causing infection in shrimps and fish. Vibrio harveyi has a wide range of infection, and can infect various aquatic animals, such as sea bass, Epinephelus coioides, Scophthalmus maximus, Crassostrea gigas, and Pseudosciaena crocea. In particular, Vibrio harveyi is the main pathogenic bacterium of Pseudosciaena crocea vibrio disease. Vibrio harveyi disease can cause symptoms such as exophthalmos, muscle ulceration, and internal organ necrosis, and lead to death, causing great losses to the aquaculture industry.

[0003] Due to the rapid transition of the aquaculture mode from extensive to high-density intensive, in the process of aquaculture, bacteria and fungi infection often occurs, leading to the failure of batch culture. Although people can reduce the risk of bacterial infection through various means, such as maintaining the water quality of the culture water, controlling the temperature and salinity of the culture water, reducing the culture density, and improving the sanitary conditions of the culture environment, the effect of these preventive measures is very small and does not meet people's expectations. Therefore, the dependence on antibiotics and other chemical drugs is increasing, and this form of antibiotic abuse, on the one hand, leads to a large amount of antibiotic residues in cultured meat products, and people unknowingly ingest antibiotics through the consumption of such meat products, causing food safety problems; on the other hand, the residues of antibiotics in the environment or in the animal body not only have adverse effects on aquatic animals and the environment, but also select many drug-resistant bacteria, which makes the treatment of such bacteria more difficult when they are infected again.

[0004] To overcome the above problems, phage therapy is expected to be a reliable means, and specific phages can be added to the breeding environment to prevent and control bacterial infection, thereby avoiding long-term addition of antibiotics in feed, reducing the accumulation of antibiotics in the environment, and reducing the generation of drug-resistant bacteria. The above problems show that disease prevention and control in the field of aquaculture is also urgently in need of a new alternative strategy, so phage therapy can provide a new alternative measure for the prevention and control of aquaculture diseases. Because of its low cost, it greatly reduces the cost of breeding and improves the yield of aquaculture. At the same time, the phage therapy process is green and environmentally friendly. If used reasonably, it can make up for the deficiency of antibiotic prevention and control, greatly improve the breeding environment and improve the quality of aquatic products. For example, patent CN111676197A discloses a Vibrio harveyi phage vB_KaS_PK08, and patent CN111705041A discloses a Vibrio harveyi phage vB_KaS_PK22. Although they have strong lytic effect on Vibrio harveyi, their pH stability and environmental adaptability need to be further improved. At present, there are still few Vibrio harveyi phages disclosed, which is difficult to meet the actual application requirements. SUMMARY

[0005] The purpose of the present application is to overcome the above-mentioned defects and deficiencies in the prior art, and to provide a new virulent Vibrio harveyi phage V-YDF132 which has strong lytic and killing effect on Vibrio harveyi.

[0006] The second purpose of the present application is to provide the application of the new virulent Vibrio harveyi phage V-YDF132.

[0007] The above-mentioned purpose of the present application is realized by the following technical scheme:

[0008] The application isolates and screens a new virulent Vibrio phage V-YDF132. The phage V-YDF132 was preserved in the Guangdong Microbial Culture Collection Center (GDMCC) on July 5, 2022, and the preservation number is GDMCC No: 62599-B1. Using a projection electron microscope shows that the phage V-YDF132 has a regular icosahedral head with a diameter of about 69 nm, a tail about 160 nm long, and a soft and bendable tail. According to the virus classification standard of ICTV, it is identified as a virus of the Siphoviridae family. The phage V-YDF132 has high host specificity for Vibrio harveyi and strong lytic and killing effects on Vibrio harveyi. V-YDF132 has good thermal stability and pH stability, can maintain a high titer at 60℃ and pH 4-12, and has a short latent period, which can burst at 20 min of infection. Therefore, the Vibrio harveyi V-YDF132 screened in the application can efficiently kill and lyse Vibrio harveyi V-YDF132 and has strong applicability, and can be used as an excellent biological antibacterial means for Vibrio harveyi infection in aquaculture.

[0009] Further, the phage V-YDF132 has a significant ability to degrade Vibrio harveyi V-YDF132. Therefore, the application of the phage V-YDF132 in killing and lysing Vibrio harveyi V-YDF132 should be within the protection scope of the application.

[0010] Further, the application of the phage V-YDF132 in preparing products for killing and lysing Vibrio harveyi V-YDF132 should also be within the protection scope of the application. The product can be a disinfectant or other forms of biological products related to Vibrio harveyi prevention and control.

[0011] Further, the application also provides the application of the phage V-YDF132 in preparing a medicine for preventing and treating Vibrio harveyi infectious diseases.

[0012] Preferably, the Vibrio harveyi infectious disease is a disease caused by Vibrio harveyi infection of aquaculture animals.

[0013] Further preferably, the aquaculture animals include one or more of fish, crustaceans, shellfish, frogs, or turtles and tortoises.

[0014] Further, the Vibrio harveyi includes one or both of V-YDF132 and YF.42.

[0015] Further preferably, the Vibrio harveyi phage V-YDF132 is mainly directed against Vibrio harveyi V-YDF132.

[0016] A medicine for preventing and treating Vibrio harveyi infectious disease, comprising the Vibrio harveyi bacteriophage V-YDF132.

[0017] Preferably, the concentration of the Vibrio harveyi bacteriophage V-YDF132 is 1×10 8 ~ 1×10 9 PFU / mL.

[0018] Preferably, it further comprises a pharmaceutically acceptable adjuvant.

[0019] Therefore, the method for killing and lysing Vibrio harveyi and preventing and treating Vibrio harveyi infectious disease of aquatic animals by using the Vibrio harveyi bacteriophage V-YDF132 should also be within the protection scope of the present application.

[0020] Preferably, the optimal multiplicity of infection of the Vibrio harveyi bacteriophage V-YDF132 is between 0.1 and 0.01.

[0021] In order to control the effect better and more stably, preferably, the degradation or treatment condition of the bacteriolytic liquid is controlled as follows: the temperature is 30-60℃, the pH is 4-12, and the multiplicity of infection is 0.1-0.01.

[0022] Further, when killing and lysing Vibrio harveyi or preventing and treating Vibrio harveyi infectious disease of aquatic animals, the use temperature of the bacteriophage V-YDF132 is 30-60℃, the pH is 4-12, and the multiplicity of infection is 0.1-0.01.

[0023] Further preferably, the use temperature of the bacteriophage V-YDF132 is 30-40℃, the pH is 5-9, and the multiplicity of infection of the bacteriolytic liquid is 0.01.

[0024] Most preferably, the use temperature of the bacteriophage V-YDF132 is 40℃, the pH is 6, and the multiplicity of infection is 0.1.

[0025] Compared with the prior art, the present application has the following beneficial effects:

[0026] The application provides a new virulent harveyi bacteriophage V-YDF132, the bacteriophage V-YDF132 is preserved in the Guangdong Microbial Culture Collection Center on July 5, 2022, and the preservation number is GDMCC No: 62599-B1. The bacteriophage V-YDF132 has high host specificity for harveyi, has strong lysis and killing effects on harveyi, and V-YDF132 has good thermal stability and pH stability, can maintain a high titer at 60 DEG C and in a pH value of 4-12, has a short latent period, and has a burst volume of 298 PFU / cell. The harveyi V-YDF132 of the application can efficiently kill and lyse harveyi V-YDF132 and has strong applicability, and can be used as an excellent biological bacteriostatic means for harveyi infection in aquaculture. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 It is a plate culture photo of the harveyi bacteriophage V-YDF132 in Example 2.

[0028] Figure 2 It is a transmission electron microscope photo of the harveyi bacteriophage V-YDF132 in Example 4.

[0029] Figure 3 It is a chloroform sensitivity experiment result graph of the harveyi bacteriophage V-YDF132 in Example 5.

[0030] Figure 4 It is a thermal stability experiment result graph of the harveyi bacteriophage V-YDF132 in Example 6.

[0031] Figure 5 It is a pH stability experiment result graph of the harveyi bacteriophage V-YDF132 in Example 7.

[0032] Figure 6 It is an optimal infection multiple experiment result graph of the harveyi bacteriophage V-YDF132 in Example 8.

[0033] Figure 7 It is a one-step growth curve graph of the harveyi bacteriophage V-YDF132 in Example 10.

[0034] Figure 8 It is an effect curve graph of the harveyi bacteriophage V-YDF132 in Example 11. DETAILED DESCRIPTION

[0035] The present application is further described in conjunction with the accompanying drawings and specific examples, which do not limit the present application in any manner. Unless otherwise specified, the reagents, methods and apparatus employed in the following examples are of a type commonly employed in the art.

[0036] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0037] Vibrio harveyi YDF132 (YDF.132), YF.42 were preserved in the laboratory of Department of Aquatic Biomedicine, College of Oceanography, South China Agricultural University.

[0038] LB Broth Medium: 10 g tryptone, 5 g yeast, 10 g NaCl, add ddH2O to 1000 mL, adjust pH to 7.2 with 5 mol / L NaOH (about 0.2 ml), sterilize at 121℃ for 30 min.

[0039] SM buffer: purchased from Beijing Regen Biotechnology Co., Ltd.

[0040] 2216E medium: purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0041] Example 1 Isolation of Vibrio harveyi phage V-YDF132

[0042] Vibrio harveyi YDF132 was inoculated into 100 mL of LB broth medium supplemented with 2% NaCl and shaken at 28℃ to the logarithmic phase. The bacterial solution was divided into 2 mL in sterile EP tubes and stored at 4℃. The remaining 98 mL of bacterial solution was added to the aquaculture water sample, shaken well, and placed in a 28℃ incubator for 24 h, with occasional shaking. After the culture was completed, 10 mL of the culture was centrifuged at 10000 rpm for 5 min, and the supernatant was filtered with a 0.22 μm filter to remove residual bacteria and obtain the phage stock solution, which was stored at 4℃ for later use. The separation was carried out using the spot method, 200 μL of host bacterial solution was taken and evenly spread on NA plates, and left to stand for 20 min; then 20 μL of phage stock solution was carefully dropped 3-5 drops on the NA plate, and cultured at 28℃ for 6-24 h, and the presence of phage plaques on the plate was observed in real time. If plaques appeared on the plate, it indicated that there might be corresponding Vibrio harveyi phage in the water sample. The double-layer plate method was then used to verify the phage. If a single phage plaque appeared, it was determined that the phage was obtained.

[0043] Example 2 Purification of Vibrio harveyi phage V-YDF132

[0044] Phages were purified using SM buffer via a double-layer plate method. If more than one morphology of phage plaques appeared on the double-layer plate, each plaque was purified separately. The specific purification procedure was as follows: the largest, most transparent plaque with a different shape was selected, and a 1 mL sterile filter tip was used to transfer the plaque to a 1 mL agar plate. In SM buffer, shake for 15 seconds and incubate overnight at 4°C. The next day, remove the phage SM solution and dilute it 10-fold with sterile physiological saline to a suitable concentration gradient. Add 100 μL of host fluid in the logarithmic phase and 100 μL of phage dilution to a screw-cap centrifuge tube containing 5 mL of LB broth semi-solid medium (0.65% agar) cooled to about 50°C. Tighten the cap, rotate horizontally to mix, and then quickly pour it onto the prepared NA solid medium (1.5% agar). Rotate the plate to spread it evenly and let it stand for 15 minutes. Invert the solidified bilayer plate and incubate it in a 28°C incubator for 6–24 hours. After plaques appear, use a 1 mL filter tip to remove the largest, most transparent plaque from the bilayer plate, pipette it into 1 mL of SM buffer, shake for 15 seconds, and incubate overnight at 4°C. Continue to prepare bilayer plates. Repeat the above steps 3-5 times until phage plaques of uniform size are obtained, indicating that purification is complete. See the purified phage bilayer plate. Figure 1 It can be seen that the V-YDF132 phage plaque is about 2 mm in diameter, with a bright, translucent center and a halo around it.

[0045] Example 3: Titer determination of Vibrio harveyi bacteriophage V-YDF132

[0046] Phage solutions filtered through a 0.22 μL sterile filter were diluted with SM buffer at different dilutions and mixed with logarithmic-phase Vibrio haveyi YDF132 bacterial suspension to prepare double-layer plates. After incubation at 28°C overnight, phage plaques grew. Plaques on the double-layer plates were counted, and plates with 30-300 plaques were selected for titer calculation. The number of plaques on the plate was converted to the corresponding dilution factor to determine the phage titer. For example, a 10-fold dilution of Vibrio haveyi phage V-YDF132... -7 If there are 100 plaques on the double-layered plate after inverting, then the titer of Vibrio harveyi phage V-YDF132 solution is 1*10. 9 PFU / ml. The titer of Vibrio harveyi phage V-YDF132 was determined to be 2.38*10. 8 PFU / ml.

[0047] Example 4: Electron microscopic morphological observation of Vibrio harveyi bacteriophage V-YDF132

[0048] First, the cultured phage lysate was centrifuged at 4000 r / min for 15 min (4°C), and the supernatant was filtered through a 0.22 μm sterile filter to obtain a phage solution. Rnase A enzyme and Dnase I enzyme were added to the phage solution to a final concentration of 1 mg / mL each, and the mixture was incubated at room temperature for 30 min. NaCl (solid) was added to a final concentration of 1 M, and after the NaCl was dissolved, the mixture was placed in an ice bath for 1 h. After centrifugation at 8300 r / min for 10 min (4°C), the supernatant was transferred to a new centrifuge tube and the volume was measured. Solid polyethylene glycol (PEG 8000) was added to a final concentration of 100 mg / mL, and the mixture was dissolved in a shaker at room temperature. After the solid polyethylene glycol was dissolved, the mixture was placed in an ice-water mixture overnight. The next day, the mixture was centrifuged at 10000 r / min for 15 min (4°C), and the supernatant was discarded and the precipitate was recovered. The precipitate was washed with PBS three times and resuspended in SM buffer. After the above steps, the purified phage particles were obtained, and 10 mL of the purified phage solution (>10 9 PFU / mL) was stained with 2% uranyl acetate and observed under a transmission electron microscope for its morphological characteristics. The electron micrograph of the phage V-YDF132 is shown in Figure 2 , which shows that the phage V-YDF132 has a regular icosahedral head with a diameter of about 69 nm and a tail about 160 nm long. The tail is flexible and bendable. According to the ICTV virus classification standard, V-YDF132 belongs to the Siphoviridae family. The phage was deposited at the Guangdong Microbial Culture Collection Center (GDMCC) on July 05, 2022, with the classification name Vibrio phage V-YDF132 and the deposit number GDMCC No: 62599-B1, and the address is No. 100, Martyrs' Road, Guangzhou, Guangdong Province.

[0049] Example 5 Chloroform sensitivity detection of Vibrio phage V-YDF132

[0050] Three parallel controls were set up. 30 μL and 300 μL of chloroform were added to 1 mL of phage suspension, mixed well, and incubated at room temperature in the dark for 30 min. After centrifugation at 5000 r for 5 min, 1 mL of host bacteria in the logarithmic growth phase and 5 mL of LB broth semi-solid medium (agar concentration 0.65%) at about 50°C were mixed quickly and evenly poured onto NA plates. After the plates were solidified, 10 μL of the supernatant from the centrifuged upper aqueous phase was titrated onto the plates, the edges of the plates were sealed with parafilm, and the plates were incubated in a 28°C incubator. The size of the phage plaques in the experimental group and the control group was observed. The double-layer plate for chloroform sensitivity detection of phage V-YDF132 is shown in Figure 3 , which shows that there is no difference in the size of the plaques formed by the phage treated with chloroform and the control phage, indicating that the phage is not sensitive to chloroform and has no lipid structure.

[0051] Example 6 Detection of the thermal stability of Vibrio harveyi phage V-YDF132

[0052] 500 μL of the phage solution was placed in a 1.5 mL EP tube and treated in a water bath at 37°C, 40°C, 50°C, 60°C, 70°C and 80°C for 20 min, 40 min and 60 min. After the end of the treatment, the solution was immediately removed and placed on ice for cooling. The phage titer was determined by double-layer plate method, and the test was repeated three times. The thermal stability of the phage was determined according to the phage titer at different temperatures. The results of the thermal stability of the phage V-YDF132 are shown in Table 1. Figure 4 It can be seen that the phage V-YDF132 has high thermal stability and is resistant to temperatures below 60°C, but is very sensitive to high temperatures above 70°C.

[0053] Example 7 Detection of the pH stability of Vibrio harveyi phage V-YDF132

[0054] 11 2 mL centrifuge tubes were each added with 900 μL of LB medium with different pH (pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12). After temperature equilibration in a 37°C water bath, 100 uL of the phage solution was added, and the phage titer was determined. The test was repeated three times. The pH stability of the phage was determined according to the phage titer at different pH. The results of the pH stability of the phage V-YDF132 are shown in Table 2. Figure 5 It can be seen that the phage V-YDF132 has a wide pH adaptation range, and still has high lytic activity at pH 4-12, and can maintain high lytic activity at pH 5-11.

[0055] Example 8 Determination of the optimal multiplicity of infection of Vibrio harveyi phage V-YDF132

[0056] The phage and host bacteria solution were added to 900 μL of LB medium at a ratio of MOI 100, 10, 1, 0.1, 0.01, 0.001, 0.0001 and 0.00001, respectively, to make the total volume 1 mL. The mixture was shaken at 37°C for 4-6 h until it became clear. The mixture was centrifuged at 12000 r / min for 3 min, and the supernatant was filtered through a 0.22 μm filter. The titer was determined by double-layer plate method, and the MOI corresponding to the highest titer was the optimal multiplicity of infection. The results of the optimal multiplicity of infection of the phage V-YDF132 are shown in Table 3. Figure 6 It can be seen that the efficiency of V-YDF132 in infecting the host is very small when the multiplicity of infection is 0.1 and 0.01, and the efficiency of the phage in infecting the host is basically saturated when the multiplicity of infection is lower than 0.01. Therefore, the optimal multiplicity of infection of V-YDF132 is between 0.1 and 0.01.

[0057] Example 9 Host range detection of Vibrio harveyi phage V-YDF132

[0058] In this experiment, 5 strains of bacteria preserved in the laboratory were selected for host range detection of the phage. That is, the different Vibrio bacteria grown to the logarithmic phase were mixed with heated pre-cooled (about 50°C) LB semi-solid to make double-layer plates. After the upper semi-solid solidified, 10 μL of phage V-YDF132 filtered through a 0.22 μm sterile filter was added, and the plates were placed in a 28°C incubator overnight. Whether or not phage plaques appeared was observed. If yes, it indicated that the host could be lysed by phage V-YDF132; if no, it indicated that the host could not be lysed by phage V-YDF132. The host range detection results of V-YDF132 are shown in Table 1. Among the hosts detected, V-YDF132 could lyse two strains of Vibrio harveyi including YDF.132, but could not lyse Photobacterium damselae, Vibrio alginolyticus, Aeromonas hydrophila and other non-Vibrio harveyi, and had strong host specificity.

[0059] Table 1 Detection results of phage V-YDF132 lysis of different hosts

[0060]

[0061] Note: "+" indicates that the host bacteria are sensitive to the phage; "-" indicates that the host is not sensitive to the phage.

[0062] Example 10 One-step growth curve determination of Vibrio harveyi phage V-YDF132

[0063] According to the phage titer of Vibrio harveyi phage V-YDF132 determined in Example 3, the phage solution and host bacteria were added at the optimal MOI ratio, and were placed at 28°C for 10 min, centrifuged at 8000 r / min for 1 min, washed with SM buffer for 3 times to remove the unabsorbed phage, and the precipitate was placed in a 10 ml centrifuge tube and mixed thoroughly. It was immediately placed in a 28°C shaker (150 rpm) and the timing was started. At 0 min, 100 uL was sampled, and every 10 min thereafter, a sample was taken for a total of 70 min. The sampled phage titer was immediately determined by the double-layer plate method, and 3 parallel samples were taken at each time point. The horizontal coordinate was the infection time, and the vertical coordinate was the phage titer, and a one-step growth curve graph was drawn. The one-step growth curve of Vibrio harveyi phage V-YDF132 is shown in Figure 1. Figure 7 It can be seen that the latent period of Vibrio harveyi phage V-YDF132 lysis of Vibrio harveyi YDF132 is about 20 min, and the burst size is 298 PFU / cell.

[0064] Example 11 Bactericidal effect determination of Vibrio harveyi phage V-YDF132

[0065] Single colonies were picked from the streaked overnight TCBS solid plate and inoculated into 2216E liquid medium, incubated at 28°C, 120rmp to early logarithmic phase; the concentration of the bacterial solution was measured and diluted to 10 8 CFU / mL, centrifuged at 8000xg for 5min at 4°C, resuspended with equal volume of 2216E medium after discarding the supernatant; take cell culture plates, add 100μL of resuspended bacteria solution to each well, then add 100μL of phage diluent to make the multiplicity of infection 0.1, at the same time, doxycycline (100mg / L) was used as a positive control group, and each group had 3 parallel groups; add 100μL of 2216E medium to 100μL of resuspended bacteria solution as a blank control group; the cell culture plates were incubated at 28°C, and the absorbance value at 600nm was detected every hour using a microplate reader, and the data was recorded. The bactericidal effect of Vibrio harveyi phage V-YDF132 is shown in Figure 8 , V-YDF132 infects the host Vibrio harveyi YDF132 very effectively, and there is almost no bacterial growth after infection.

Claims

1. A bacteriophage of Vibrio harveyi V-YDF132, which is characterized by, The bacteriophage is preserved in Guangdong Microbial Culture Collection Center on July 05, 2022, and the preservation number is GDMCC No: 62599-B1.

2. Use of the bacteriophage V-YDF132 of claim 1 in the preparation of a product for lysing and killing Vibrio harveyi.

3. Use of the bacteriophage V-YDF132 of claim 1 in the preparation of a medicine for preventing and treating Vibrio harveyi infectious disease.

4. Use according to claim 3, characterized in that, The Vibrio harveyi infectious disease is a disease caused by Vibrio harveyi infecting aquatic animals.

5. Use according to claim 4, characterized in that, The aquatic animals include one or more of fish, crustaceans, shellfish, frogs, or turtles and tortoises.

6. A medicament for the prevention and treatment of Vibrio harveyi infectious disease, characterized by, The Vibrio harveyi bacteriophage V-YDF132 of claim 1 is included.

7. The medicament according to claim 6, characterized in that, The concentration of the Vibrio harveyi phage V-YDF132 is 1 x 10 8 ~1 x 10 9 PFU / mL.

8. The medicament according to claim 7, characterized in that, A pharmaceutically acceptable adjuvant is also included.

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