Vibrio Ewana bacteriophage RKP-VP23071 and application thereof

Abstract

The invention belongs to the technical field of microorganisms. The invention provides a strain of vibrio edulis bacteriophage RKP-VP23071, the bacteriophage is preserved in China General Microbiological Culture Collection Center on September 27, 2024, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No. 46194. The invention provides a bacteriophage composition, which comprises the vibrio Ewissi bacteriophage disclosed by the invention. The invention provides application of a vibrio Ewanii bacteriophage or a bacteriophage composition in preparation of a medicine for preventing and / or treating vibrio Ewanii infection diseases, an aquatic feed additive, a water body environment disinfectant or a biological bacteriostatic agent. The Vibrio Ewissi bacteriophage provided by the invention has a strong lysis effect on Vibrio Ewissi, can prevent part of shrimp hepatopancreas necrosis caused by Vibrio Ewissi, can efficiently and reliably treat aquatic animal diseases caused by Vibrio Ewissi, improves the water body environment, and has a good application prospect. And a healthy and safe new way is provided for aquaculture medication.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a Vibrio owensii phage RKP-VP23071 and its application. Background Art

[0002] Vibrio is a common group of halophilic Gram-negative bacteria, and its main habitat is seawater. It is widely present in seawater and seafood, with a very wide distribution. It is a common pathogenic bacterium of marine fish and shellfish in the coastal areas of China, usually causing gastrointestinal infections and also capable of causing extraintestinal infections, leading to hemorrhagic diseases in marine fish.

[0003] Acute hepatopancreatic necrosis disease (AHPND), also known as "early mortality syndrome (EMS)", is prevalent in many Southeast Asian countries and has caused huge losses to the shrimp farming industry. It has currently been confirmed that some Vibrio parahaemolyticus and Vibrio owensii can cause necrosis of shrimp hepatopancreas.

[0004] Currently, the main method for the prevention and control of Vibrio owensii is the use of antibiotics. Although antibiotics have the advantages of being convenient to use, having a broad-spectrum antibacterial effect, and quick results, the abuse of antibiotics has also led to serious bacterial drug resistance, and the effect is getting worse and worse when preventing and treating diseases caused by Vibrio. There is an urgent need to develop new anti-Vibrio drugs.

[0005] Phage is a kind of bacterial virus that does not damage the microecological environment. Moreover, the action target of Vibrio owensii phage is on the cell wall of prokaryotic microorganisms, and there is no corresponding action site on animal and plant cells, which is extremely safe. Using the natural enemy of bacteria, phage, to solve bacteria is a biological control approach with broad prospects.

[0006] Currently, there is no Vibrio owensii phage with a long bactericidal time and a high lysis rate that can be used to prevent and treat various diseases caused by Vibrio owensii infection. Therefore, the existing technology needs to be improved. Summary of the Invention

[0007] The present invention aims to address the deficiencies of the existing technology and provides a Vibrio owensii phage RKP-VP23071 and its application. The provided Vibrio owensii phage RKP-VP23071 of the present invention not only has strong lytic ability against Vibrio owensii, but can be used as an effective active ingredient to prepare drugs for preventing and treating diseases caused by Vibrio owensii infection, aquatic feed additives, water environment disinfectants, or biological bacteriostatic agents for treating fresh or frozen aquatic products, effectively preventing Vibrio owensii disease in aquatic products.

[0008] The above object of the present invention is achieved by the following technical solutions:

[0009] In a first aspect, the present invention provides a Vibrio owensiibacteriophage RKP-VP23071, which was deposited with the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on September 27, 2024, with the deposit number CGMCC No. 46194, and the deposit address: Beijing, China. The Vibrio owensi VP23071 used in the present invention was deposited together with the Vibrio owensiibacteriophage RKP-VP23071.

[0010] Observation by electron microscopy revealed that: the phage has a polyhedral head with cubic symmetry, encapsulating nucleic acid, with a diameter of approximately 50 nm, and a tail approximately 150 nm long.

[0011] Furthermore, the phage RKP-VP23071 has a high titer, up to 10 12 pfu / mL or higher.

[0012] Furthermore, the optimal multiplicity of infection of the phage RKP-VP23071 is 10:1.

[0013] Furthermore, the lysis rate of the phage RKP-VP23071 reaches 98%, showing a good lysis effect on the host, and is suitable for use in the aquaculture process.

[0014] In a second aspect, the present invention provides a phage composition, which comprises the above-mentioned Vibrio owensiibacteriophage.

[0015] In a third aspect, the present invention provides the application of the above-mentioned Vibrio owensiibacteriophage or the above-mentioned phage composition in the preparation of drugs for preventing and / or treating diseases caused by Vibrio owensi infection, aquaculture feed additives, water environment disinfectants, or biological bacteriostatic agents for treating fresh or frozen aquatic products. As used herein, the term "prevention" refers to all actions including inhibiting or delaying the disease by administering the phage or bactericidal composition. As used herein, the term "treatment" refers to all actions including improving or ameliorating the disease by administering the phage or bactericidal composition.

[0016] Preferably, the present invention provides the application of the Vibrio owensii phage or the phage composition as described above in the preparation of a drug for preventing diseases caused by Vibrio owensii infection, an aquatic feed additive, a water environment disinfectant, or a biological bacteriostatic agent for treating fresh or frozen aquatic products. More preferably, the present invention provides the application of the Vibrio owensii phage or the phage composition as described above in the preparation of a drug for preventing diseases caused by Vibrio owensii infection. More preferably, the diseases caused by Vibrio owensii infection are selected from the diseases caused by Vibrio owensii infection from shrimp sources.

[0017] In the fourth aspect, the present invention provides a phage pharmaceutical preparation, and the active ingredient of the phage pharmaceutical preparation includes the Vibrio owensii phage or the phage composition as described above. Preferably, the titer of the phage in the phage pharmaceutical preparation is at least 1×108 PFU / g.

[0018] Preferably, the phage pharmaceutical preparation further includes a pharmaceutically acceptable carrier, and its dosage form is powder, solution, emulsion, gel, granule or lyophilized product. Preferably, the dosage form of the phage pharmaceutical preparation is solution.

[0019] In the fifth aspect, the present invention provides an aquatic feed additive, and the active ingredient of the aquatic feed additive includes the Vibrio owensii phage or the phage composition as described above. After being mixed with the aquatic feed and fed, the aquatic feed additive can play a role in preventing or treating diseases caused by Vibrio owensii infection.

[0020] In the sixth aspect, the present invention provides a water environment disinfectant, and the active ingredient of the water environment disinfectant includes the Vibrio owensii phage or the phage composition as described above.

[0021] In the seventh aspect, the present invention provides a biological bacteriostatic agent for treating fresh or frozen aquatic products, and the active ingredient of the biological bacteriostatic agent includes the Vibrio owensii phage or the phage composition as described above.

[0022] The beneficial effects of the present invention are as follows:

[0023] 1. The Vibrio owensii phage RKP-VP23071 provided by the present invention can, on the one hand, prevent a part of the shrimp hepatopancreas necrosis caused by Vibrio owensii, and on the other hand, can efficiently and reliably treat the diseases of aquatic animals caused by Vibrio owensii, improve the water environment, provide a new healthy and safe way for the use of drugs in aquaculture, and can reduce the abuse of antibiotics.

[0024] 2. The phage RKP-VP23071 provided by the present invention has a relatively high titer, up to 1012 Above pfu / mL, it has high - efficient bactericidal ability; it is easy to dilute and use, with stronger application flexibility; it is environmentally friendly and has broad application prospects in the aquaculture field.

[0025] 3. The lysis rate of the phage RKP - VP23071 provided by the present invention reaches 98%, and it has a strong lysis effect on Vibrio owensii, with good lysis effect and is suitable for use in the breeding process.

[0026] 4. The graph showing the effect of Vibrio owensii phage RKP - VP23071 on the growth curve of Vibrio owensii indicates that its bactericidal duration is long. After 3 h of adding the host bacteria, the number of lysed host bacteria is greater than the proliferation rate of the host bacteria, and the mixed bacterial liquid reaches the clearest. After continuous action for 11 h, the mixed bacterial liquid becomes turbid again, and the original sensitive bacteria are lysed by the phage and replaced by mutated tolerant bacteria, showing a kind of bacterial flora alternation. Therefore, to obtain a high - titer phage, it is necessary to control the mixed bacterial liquid within the time range from reaching the highest clarity to the start of turbidity, so as to avoid affecting the phage titer, which provides a scientific basis for the practical application of phages.

[0027] 5. The animal experiments of the present invention show that the mortality rate of the protection group is significantly lower than that of the challenge group. It can be judged that adding phage RKP - VP23071 to the feed of Litopenaeus vannamei can effectively reduce the mortality rate of Litopenaeus vannamei, indicating that phage RKP - VP23071 has a good effect on preventing and treating diseases caused by Vibrio owensii infection. Using it in actual production can improve the survival rate of shrimp larvae and increase the breeding efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a plaque picture of Vibrio owensii phage RKP - VP23071;

[0029] Figure 2 It is an electron microscope photograph of Vibrio owensii phage RKP - VP23071;

[0030] Figure 3 It is a graph showing the effect of Vibrio owensii phage RKP - VP23071 on the growth curve of Vibrio owensii. DETAILED DESCRIPTION OF THE INVENTION

[0031] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the content in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0032] In the present invention, unless otherwise specified, the equipment, raw materials, etc. used can be purchased from the market or are commonly used in the art. The methods in the following examples are all conventional methods in the art unless otherwise specified. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. Vibrio owensii phage RKP-VP23071, or is simply referred to as "phage RKP-VP23071" or "RKP-VP23071".

[0033] Example 1 Isolation and purification of Vibrio owensii

[0034] Samples were taken from the diseased parts of the diseased Litopenaeus vannamei, and streaked on TCBS medium by aseptic operation. After culturing at 37°C for 24 h, round green colonies with neat edges appeared on the TCBS agar plate, with a diameter of about 2-4 mm. Typical colonies were picked and streaked and purified 3 times, and then single colonies were picked and inoculated into 5 mL of 2216E liquid medium, and cultured at 37°C with shaking at 140 rpm for 7 h to obtain a uniformly turbid bacterial suspension. The 16S fragment was amplified, and after sequencing the PCR product and aligning it with BLAST, it was identified as Vibrio owensii, and one of the strains was named VP23071.

[0035] Example 2 Isolation and purification of Vibrio owensii phage

[0036] (1) Isolation of phage

[0037] In 2023, sewage samples were collected from the sewage around the aquaculture farmers in Liangcheng Sub-district, Rizhao, as the water samples for phage isolation.

[0038] Take 10 mL of sewage, centrifuge at 10000 rpm for 5 minutes to remove larger impurities and most bacteria, and then filter and sterilize with a 0.22 μm microporous filter membrane. Take 3 mL of the filtrate and 3 mL of the host bacteria suspension, add them together to 20 mL of autoclaved 2216E liquid medium, and then place them in a 37°C incubator for overnight culture. After culturing, take 5 mL, centrifuge at 10000 rpm for 5 minutes, and then filter and sterilize with a 0.22 μm microporous filter. The filtrate is the original solution presumably containing phage. Then, the double-plate method was used to identify whether there was phage. If there were plaques, there was phage, otherwise it indicated that no phage was isolated and re-screening was required.

[0039] (2) Purification of phage

[0040] The sizes and shapes of the initially isolated plaques are often inconsistent. Therefore, further purification is required. Pick single, independent, uniformly shaped, clear and transparent plaques on the double-layer plate with plaques, place them in 1 mL of physiological saline, soak for 10 minutes, take the supernatant, filter it through a 0.22 μm microporous filter, and then appropriately dilute the filtrate (there is a single plaque when spreading the double-layer plate), and spread it on a double-layer plate with the host bacterial suspension. Repeat this step 3-4 times. When the sizes, shapes, and clarity of the plaques on the double-layer plate are consistent, the purified phage is obtained.

[0041] (3) Determination of phage titer

[0042] Spread the purified phage on a double-layer plate, then pick a single plaque, add it to 2216E, and at the same time add the host bacterial suspension. After the phage multiplies, centrifuge the proliferated liquid at 10000 rpm for 5 minutes and then filter it through a 0.22 μm microporous filter. Dilute the filtrate in a 10-fold gradient to 10 10 times. Take 0.1 mL of the phage diluent of the last 3 dilution degrees and 0.1 mL of the host bacterial suspension, spread them on a double-layer plate, and determine the phage titer.

[0043] Phage titer (pfu / mL) = number of plaques × dilution factor ÷ 0.1

[0044] Figure 1 Figure of the plaque of Vibrio owensii phage RKP-VP23071. The results show that, as Figure 1 shown, the plaque morphology, size, and clarity are consistent, with typical characteristics of lytic phages. The titer of this phage can reach 10 1 2 pfu / mL or more.

[0045] Example 3 Phage genome sequencing

[0046] After enrichment culture of a single phage, centrifuge at 8000 g for 15 minutes at 4°C, add 10% PEG8000 and 0.5 M NaCl, let it stand overnight, then add an equal volume of chloroform and mix well. After standing and separating layers, centrifuge at 5000 g for 10 min. After removing the chloroform layer and PEG layer, add restriction endonuclease for digestion treatment, and suspend the phage under gradient density cesium chloride conditions. Later, dialyze 3 times with TM buffer, 30 min each time. Finally, reserve a part of the dialyzed phage for electron microscopy observation and send a part to a gene sequencing company for whole-genome sequencing. Its whole gene sequence is shown in SEQ ID NO.1.

[0047] Example 4 Electron microscopy observation of Vibrio owensii phage RKP-VP23071

[0048] 1. Experimental method:

[0049] Using phosphotungstic acid negative staining method: Drop 100 μL of phage solution (titer 10 10 pfu / mL) on the paraffin section. Place the side of the copper grid with the film on the phage solution drop. After 10 min, remove it and let it air-dry naturally in the air for 2 - 3 min. Then, drop a drop of 2% phosphotungstic acid (PTA) aqueous solution on the copper grid for staining. After 10 min, remove it and dry it in the air for 10 - 15 min. Observe with an electron microscope and select clear phage images for photography. Figure 2 It is the electron micrograph of Vibrio owensii phage RKP-VP23071.

[0050] 2. Experimental results and analysis:

[0051] From Figure 2 it can be seen that through electron microscope observation, the phage has a polyhedral icosahedral head that encapsulates nucleic acid, with a diameter of about 50 nm, and a tail about 150 nm long.

[0052] This Vibrio owensii phage was deposited in the General Microbiological Center of the China National Center for Biotechnology Development on September 27, 2024, with the deposit number CGMCC No. 46194, and it was named RKP-VP23071.

[0053] Example 5 Determination of the optimal multiplicity of infection of Vibrio owensii phage RKP-VP23071

[0054] 1. Experimental method:

[0055] Add the host bacterial solution and phage to the 2216E liquid medium according to the ratios of multiplicity of infection of 1:1, 1:10, 10:1, 100:1, and 1000:1. Incubate with shaking at 37 °C and 140 rpm for 7 h, then centrifuge at 12000 r / min for 5 min at room temperature. Take the supernatant and spread it on a double plate to determine its titer. The multiplicity of infection with the highest titer is the optimal multiplicity of infection. The optimal multiplicity of infection of phage RKP-VP23071 was determined by the above method. The results are shown in Table 1.

[0056] Table 1 Results of the optimal multiplicity of infection

[0057]

[0058] 2. Experimental results and analysis:

[0059] As can be seen from Table 1, the optimal multiplicity of infection of phage RKP-VP23071 is 10:1.

[0060] Example 6 Effect of Vibrio owensii phage RKP-VP23071 on the growth curve of Vibrio owensii

[0061] 1. Experimental method:

[0062] Phage was added to the bacterial suspension. Due to the lysis of the host by the phage, the value of OD600 changed, and thus the lysis curve of the phage against the host was obtained. The host bacterial suspension cultured for 8 h was inoculated into 100 mL of 2216E medium at a ratio of 3%, and phage RKP-VP23071 was added at the optimal multiplicity of infection, denoted as the "experimental group". At the same time, the one without phage was used as a control, denoted as the "control group", and 3 sets of parallel controls were made, and the results were averaged. The results are as Figure 3 shown.

[0063] 2. Experimental results and analysis:

[0064] As Figure 3 shown, for the host bacterial suspension without phage added, i.e., the control group, as the host proliferated, its OD600 gradually increased and basically reached equilibrium at 9 h. For the host bacterial suspension with phage proliferation solution added, i.e., the experimental group, in the first 2 h, the phage was in the adsorption and infection stage of the host, and the increase in OD600 of the control group and the experimental group was similar. As the number of phages increased exponentially, after 2 h, the phage lysed the host. Compared with the control group, the increase in OD600 of the experimental group was smaller, but as time extended, its OD600 also gradually increased.

[0065] When Vibrio owensii phage RKP-VP23071 was added to the host bacterial suspension of Vibrio owensii VP23071, a certain lysis effect appeared after 2 h of action. The increase in OD600 of the one without phage added was obvious, indicating that this phage RKP-VP23071 had strong lysis ability against the host Vibrio owensii VP23071 and could continuously lyse the host bacteria.

[0066] Analyzing the reason, in the first 3 h, the phage was in the adsorption and infection stage of the host bacteria, and only a small part of the phages began to lyse the host. Therefore, the amount of host bacteria lysed by it was far less than the growth amount of the host. Therefore, OD600 still showed a certain upward trend within the first 3 h. Along with the exponential increase in the number of phages, after 3 h, the number of host bacteria lysed by it was greater than the proliferation rate of the host, so the value of OD600 began to gradually decrease, and the mixed bacterial suspension became the clearest. After continuous action for 11 h, the mixed bacterial suspension became turbid again, which was a kind of bacterial flora alternation where the original sensitive bacteria were lysed by the phage and replaced by the mutated resistant bacteria. Therefore, to obtain a high titer of phage, the mixed bacterial suspension should be controlled within the time range from reaching the highest clarity to the start of turbidity phenomenon, so as to avoid affecting the phage titer.

[0067] Example 7 Lysis rate experiment of Vibrio owensii phage RKP-VP23071

[0068] 1. Experimental method:

[0069] Under aseptic conditions, 1 mL of the sample and 1 mL of the host bacterial solution (1×10 5 cfu / mL) were taken respectively, incubated at 37°C for 15 minutes, mixed well and diluted to 10 -1 -10 -3 gradients with normal saline. 100 μL of each gradient was taken and spread on 2216E agar plates, and incubated at 37°C for 24 hours. Each gradient was repeated twice. At the same time, 1 mL of normal saline and 1 mL of the host bacterial solution (1×10 5 cfu / mL) were taken as a blank control, and the above steps were repeated. Plates with 30 - 300 colony counts were selected for counting. This experiment was repeated 3 times, and the average value was taken. Bacteriophage lysis rate = (1 - number of colonies in the treatment group / number of colonies in the control group) × 100%.

[0070] 2. Experimental results and analysis:

[0071] The lysis rate of bacteriophage RKP-VP23071 reached 98%, showing a good lysis effect on the host and being suitable for use in the breeding process.

[0072] Animal experiment of Vibrio owensii bacteriophage RKP-VP23071 in Example 8

[0073] 1. Experimental method:

[0074] Healthy white shrimp with a body weight of about 5 g (body length about 7 cm) were prepared and fasted overnight, with 60 shrimp in each group.

[0075] Protection group: According to the addition amount of the bacteriophage proliferation solution and the feed at a volume-mass ratio of 5% (that is, 5 mL of the bacteriophage RKP-VP23071 proliferation solution was added to 100 g of feed), the bacteriophage RKP-VP23071 proliferation solution was evenly mixed with the shrimp feed and fed at a dose of 3% of the shrimp body weight. The content of bacteriophage RKP-VP23071 was 2.37×10 8 pfu / mL.

[0076] Challenge group: According to the addition amount of the 2216E medium and the feed at a volume-mass ratio of 5% (that is, 5 mL of the 2216E medium was added to 100 g of feed), the 2216E medium was evenly mixed with the shrimp feed and fed at a dose of 3% of the shrimp body weight.

[0077] Blank control group: According to the addition amount of the 2216E medium and the feed at a volume-mass ratio of 5% (that is, 5 mL of the 2216E medium was added to 100 g of feed), the 2216E medium was evenly mixed with the shrimp feed and fed at a dose of 3% of the shrimp body weight.

[0078] After 1 h, 50 μL of Vibrio owensii VP23071 was injected into the shrimp in the protection group and the challenged group, and the actual dose was 1.13×10 7 cfu per shrimp. The blank control group was not injected with Vibrio owensii VP23071. The number of dead shrimp in each group within 48 h was recorded. The two experiments were repeated, and the specific results are shown in Table 2.

[0079] Table 2 Results of the challenge protection test

[0080]

[0081] 2. Experimental results and analysis:

[0082] As can be seen from Table 2, in the two experimental results, the mortality rate of the protection group was significantly lower than that of the challenged group. It can be judged that adding phage RKP-VP23071 to the feed of Litopenaeus vannamei can effectively reduce the mortality rate of Litopenaeus vannamei, improve the survival rate of shrimp fry in actual production, and increase the breeding efficiency.

[0083] It should be noted that the specific implementation manners are only relatively representative examples of the present invention. Obviously, the technical solutions of the present invention are not limited to the above embodiments and there can be many variations. Those of ordinary skill in the art who obtain, without any doubt, from what is clearly disclosed in the present invention or from the written description of the document should be considered as the scope to be protected by this patent.

Claims

1. A Vibrio owensii bacteriophage RKP-VP23071, characterized in that, The Vibrio owensii phage RKP-VP23071 was deposited with the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on September 27, 2024, and the deposit number is CGMCC No. 46194.

2. A bacteriophage composition, characterized in that, The phage composition comprises the Vibrio owensii phage as described in claim 1.

3. Use of the Vibrio owensii phage as described in claim 1 or the phage composition as described in claim 2 in the preparation of a drug for preventing and / or treating diseases caused by Vibrio owensii infection, an aquatic feed additive, a water environment disinfectant, or a biological bacteriostatic agent for treating fresh or frozen aquatic products.

4. A phage pharmaceutical preparation, characterized in that, The active ingredient of the phage pharmaceutical preparation comprises the Vibrio owensii phage as described in claim 1 or the phage composition as described in claim 2.

5. A phage pharmaceutical preparation according to claim 4, characterized in that, The titer of the phage in the phage pharmaceutical preparation is at least 1×10 8 PFU / g.

6. The phage pharmaceutical preparation according to claim 4, wherein The phage pharmaceutical preparation further comprises a pharmaceutically acceptable carrier, and its dosage form is powder, solution, emulsion, gel, granule or freeze-dried preparation.

7. A phage pharmaceutical preparation according to claim 4, characterized in that, The dosage form of the phage pharmaceutical preparation is solution.

8. An aquatic feed additive, characterized in that, The active ingredient of the aquatic feed additive comprises the Vibrio owensii phage as described in claim 1 or the phage composition as described in claim 2.

9. A water environment disinfectant, characterized in that, The active ingredient of the water environment disinfectant comprises the Vibrio owensii phage as described in claim 1 or the phage composition as described in claim 2.

10. A biological bacteriostatic agent for processing fresh or frozen aquatic products, characterized in that, The active ingredient of the biological bacteriostatic agent comprises the Vibrio owensii phage as described in claim 1 or the phage composition as described in claim 2.

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