High-temperature-resistant vibrio harveyi bacteriophage, composition thereof, shrimp feed and application

Abstract

The invention belongs to the field of microorganisms, and particularly relates to a high-temperature-resistant vibrio harveyi bacteriophage, a composition thereof, a shrimp feed and application. The high-temperature-resistant vibrio harveyi bacteriophage Ph037 can play a very good cracking effect on vibrio harveyi, has very strong tolerance to high temperature, can be stably stored in transportation and preservation processes in different environments, solves the problem that the bacteriophage is easy to inactivate in a high-temperature environment, provides a new means for treating, preventing and controlling vibriosis, and has good application prospects. And an alternative scheme is provided for treatment of multi-drug-resistant bacterial infection.

Description

Technical Field

[0001] The present invention belongs to the field of microorganisms, and in particular relates to a high-temperature resistant Vibrio harveyi phage, a composition thereof, a shrimp feed and an application thereof. Background Art

[0002] Vibrio harveyi is a Gram-negative bacterium widely found in aquaculture environments. It is a significant pathogen of aquatic invertebrates (especially shrimp) and fish, and can also cause human infection, posing a significant threat to the aquaculture industry. Vibrio harveyi is commonly associated with eye disease and gastroenteritis in fish, as well as acute hepatopancreatic necrosis in shrimp. It has a very high mortality rate and can suppress the shrimp immune system, allowing it to reproduce rapidly and further contaminate the aquaculture environment.

[0003] Antibiotics are one of the primary means of controlling Vibrio harveyi. However, the overuse of antibiotics has led to the increasing emergence of multidrug-resistant bacteria, primarily tetracyclines, macrolides, and β-lactams among Vibrio species. The development of resistance reduces the effectiveness of drug control, indirectly leading to increased aquaculture costs and other issues. Excessive and continuous antibiotic use can lead to drug residues, threatening food safety. Because shrimp lack the well-developed adaptive immune systems of mammals, existing vaccines cannot be used in shrimp farming. Therefore, there is an urgent need to find alternative control methods to antibiotics.

[0004] Bacteriophages are viruses that infect bacteria, replicate and proliferate within the bacteria, and then lyse the bacteria. Compared to antibiotic treatment, bacteriophages are highly specific, have fewer side effects, are safer, and are highly effective and require low doses. In recent years, as an alternative to antibiotics, bacteriophages have been used in the prevention and control of many bacterial diseases with excellent results. Their application has also been expanded to many similar fields, such as as preservatives for food transportation and preservation, and as disinfectants for environmental and equipment disinfection.

[0005] In aquaculture, bacteriophages also have great potential for the prevention and control of bacterial pathogens. However, there are few existing Vibrio harveyi phage preparations, and their tolerance to high temperatures is poor. Summary of the Invention

[0006] The present invention aims to provide a Vibrio harveyi bacteriophage (Vibrio phage) with high titer, which can effectively prevent acute hepatopancreatic necrosis of shrimp caused by Vibrio harveyi and eliminate pathogenic bacteria. It also has high temperature tolerance and can be stably stored in a high temperature environment. Therefore, it has good application prospects in the prevention and treatment of Vibrio harveyi disease.

[0007] The present invention is achieved through the following technical solutions: The first aspect of the present invention provides a high-temperature resistant Vibrio harveyi phage Ph037, which was isolated from a shrimp pond water in Sheyang County, Yancheng City, Jiangsu Province, and deposited in the China Center for Type Culture Collection on April 2, 2025, with the deposit address in Wuhan, China, with the deposit number CCTCC NO: M 2025677, and the classification name Vibrio phage Ph037.

[0008] Electron microscopic observations revealed that the head of the thermostable Vibrio harveyi phage Ph037 is 100 nm in diameter, with a multifaceted, three-dimensionally symmetrical structure that encapsulates nucleic acid. The tail, 120 nm in diameter, is connected to the head by a neck. According to the Ninth Report on Virus Taxonomy of the International Organization for Taxonomy of Viruses, this phage can be classified within the family Myocaudaphaga, order Caudovirales.

[0009] The thermostable Vibrio harveyi phage Ph037 provided by the present invention exhibits strong lysis ability against Vibrio harveyi, and has lysis ability against 36 of 40 strains of Vibrio harveyi preserved in the laboratory, with a lysis rate of about 90%, reflecting its wide lysis spectrum. It also has good acid and alkali tolerance, maintaining a pH of not less than 4.7 × 10 8 The optimal multiplicity of infection is 0.01 and the minimum is 10 -5 , reflecting good reproductive ability.

[0010] The high-temperature-resistant Vibrio harveyi phage Ph037 provided by the present invention has good temperature stability, can stably survive for 30-60 minutes in a temperature environment below 80° C., and maintain a high level of titer.

[0011] The present invention also provides use of the high-temperature resistant Vibrio harveyi phage Ph037 in preparing medicines, and the medicines are used for treating or preventing Vibrio harveyi infection in aquatic products.

[0012] The present invention also provides the use of the high-temperature resistant Vibrio harveyi phage Ph037 in the preparation of a feed additive or an environmental disinfectant, wherein the feed additive or the environmental disinfectant is used for treating or preventing Vibrio harveyi infection in aquatic products.

[0013] In some embodiments, the aquatic product is shrimp, fish, crab, or shellfish.

[0014] The present invention also provides a phage composition, comprising the thermostable Vibrio harveyi phage Ph037.

[0015] In some embodiments, the phage composition further comprises other phages, such as PP270, PH018, HP16, HP8, and HP21, in complex with phage Ph037.

[0016] In some embodiments, the phage composition is a pharmaceutical preparation for treating or preventing Vibrio harveyi infection in aquatic products.

[0017] In some embodiments, the pharmaceutical preparation is in the form of a gel, granules, solution, powder or lyophilized preparation.

[0018] In some embodiments, the phage composition is a purified solution of thermostable Vibrio harveyi phage.

[0019] In some embodiments, the titer of the phage in the purified solution is not less than 8.47×10 9 PFU / mL.

[0020] Furthermore, the present invention provides a shrimp feed comprising the thermostable Vibrio harveyi bacteriophage Ph037 or the bacteriophage composition.

[0021] Compared with the prior art, the beneficial effects of the present invention include at least: 1. The Vibrio harveyi phage Ph037 has strong thermal stability and can be stably stored and transported in high-temperature environments, effectively solving the problem of phage inactivation at high temperatures. It also has a broad lysis spectrum, high titer, and strong reproductive capacity, providing a new and effective treatment option for Vibrio harveyi disease.

[0022] 2. The phage composition or shrimp feed has no toxic effect on shrimp, will not produce drug resistance, is safe and has no residue, and has no impact on the survival of shrimp. It can replace antibiotics as a means of preventing and treating Vibrio harveyi disease.

[0023] 3. The Vibrio harveyi phage Ph037 is rapidly produced and readily available, and can be widely used in various situations in the shrimp aquaculture industry that are susceptible to contamination by Vibrio harveyi.

[0024] 4. Compared with the most similar phage VhaP_PG11, Ph037 of Vibrio harveyi showed 76.55% similarity and 6% coverage, indicating that Ph037 is a new species. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a photo of the phage plaque of bacteriophage Ph037; Figure 2 This is an electron microscope photo of bacteriophage Ph037; Figure 3 The optimal multiplicity of infection of phage Ph037 is determined; Figure 4 The results of one-step growth curve determination of bacteriophage Ph037; Figure 5 The results of the temperature stability test of bacteriophage Ph037 are shown; Figure 6 The pH stability test results of bacteriophage Ph037. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solutions and beneficial effects of the present invention more clear, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0027] The endpoints of ranges and any values disclosed herein are not limited to the exact range or value, and these ranges or values should be understood to include approximations to the range.

[0028] Example 1 Isolation and Purification of Vibrio harveyi Phage Ph037 1. Preparation of fresh bacterial solution Vibrio harveyi was streaked and activated on a TCBS plate, and a single colony was picked and inoculated into 5 mL of 2216E liquid culture medium, and cultured at 37°C with shaking at 180 rpm for 16-18 h.

[0029] 2. Isolation and purification of bacteriophage Water samples collected from a shrimp pond in Sheyang County, Yancheng City, Jiangsu Province were centrifuged at 12,000 rpm for 5 minutes to remove impurities in the water. The supernatant was collected and then filtered through a 0.22 μm sterile filter to further remove microorganisms and obtain the supernatant filtrate that may contain bacteriophages.

[0030] 200 μL of the supernatant filtrate was mixed with 100 μL of Vibrio harveyi host bacterial solution, cultured in liquid culture medium at 37°C and 180 rpm for 12-14 h, centrifuged at 12000 rpm for 5 min, and the supernatant was collected and then further filtered with a 0.22 μm sterile filter to obtain the phage stock solution.

[0031] 0.1 mL of phage stock solution was diluted 10-fold with PBS buffer, and 100 μL of each was mixed with 100 μL of host bacterial solution. Then 3 mL of upper agar was added, mixed well, and quickly poured onto the lower culture medium. After the upper agar solidified, it was allowed to stand at 37°C overnight to observe whether plaques were formed.

[0032] If plaque formation is observed, select a single plaque with neat edges and place it in 1 mL of PBS buffer. Centrifuge at 12,000 rpm for 5 minutes, and then filter it through a 0.22 μm sterile filter. Dilute the filtrate 10-fold with PBS buffer and culture it on a double-layer plate again. After determining the appropriate concentration, repeat the operation 3-5 times until plaques with uniform morphology, size, and transparency are obtained. This is the phage isolate. Figure 1 shown.

[0033] 3. Phage proliferation and titer determination 100 μL of purified phage liquid was mixed with 100 μL of host bacterial liquid, cultured in liquid culture medium at 37°C and 180 rpm until the liquid was clear, centrifuged at 12000 rpm for 5 min, and the supernatant was collected. It was then further filtered with a 0.22 μm sterile filter to obtain phage proliferation liquid.

[0034] The phage proliferation solution was diluted 10-fold with PBS buffer and the phage titer was determined by double-layer plate culture. Phage titer (PFU / mL) = number of plaques × dilution factor × 10. The measured titer was approximately 8.47 × 10 9 PFU / mL.

[0035] Example 2 Electron microscopic observation of bacteriophage 20 μL of phage solution was added to the copper mesh and allowed to precipitate naturally for 15 min. The excess liquid was then removed from the edge with filter paper. The mesh was stained with 2% phosphotungstic acid staining solution for 10 min. The staining solution was then removed from the edge with filter paper again. After the sample dried naturally, the phage morphology was observed under an electron microscope.

[0036] like Figure 2 As shown, the head of Vibrio harveyi phage Ph037 has a diameter of 100 nm, a multi-faceted three-dimensional symmetrical structure, encapsulated nucleic acid, and a tail length of 120 nm, connected to the head by a neck; according to the ninth report on virus classification of the International Organization for Taxonomy of Viruses, this phage can be classified as the Caudovirales family of the Caudoviridae.

[0037] Example 3: Experiment on the Lysis Range of Vibrio harveyi Phage Ph037 Phage lysis profiles were determined using the spot method. 1 mL of bacterial culture was mixed with semi-solid culture medium and plated onto a plate. 3 μL of phage culture was then spot-plated onto the plate and incubated overnight at 37°C. The results were observed and the lysis rate was recorded. The lysis rate was scored as 0: no lysis; 1: incomplete lysis; and 2: lysis.

[0038] The results, shown in Table 1, showed that phage Ph037 was able to lyse 36 of 40 strains of Vibrio harveyi isolated from laboratory samples of dead fish, shrimp, and water collected from various cities in Shandong Province, achieving a lysis rate of 90%. This suggests that phage Ph037 has a broad lytic spectrum and holds great potential for the treatment and prevention of Vibrio harveyi disease.

[0039] Table 1: Results of the lysis spectrum of Vibrio harveyi phage Ph037 against Vibrio harveyi Example 4 Determination of the optimal multiplicity of infection (MOI) of Vibrio harveyi bacteriophage Ph037 Phage dilutions were mixed with host bacteria cultured to the logarithmic growth phase at infection ratios of 0.0000001, 0.000001, 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100. The mixture was then added to 5 mL of 2216E liquid medium and incubated at 37°C and 180 rpm until the liquid became clear. The supernatant was collected and the phage titer was determined using the double-layer plate method. The optimal multiplicity of infection (MOI) is the ratio of phage to bacteria that achieves optimal growth conditions.

[0040] The titer of phage Ph037 was highest when the multiplicity of infection was 0.01. Figure 3 shown.

[0041] Example 5 One-step growth curve determination of Vibrio harveyi bacteriophage Ph037 Phage Ph037 was mixed with host bacteria in the logarithmic growth phase at the optimal multiplicity of infection (MOI). After incubation at 37°C for 10 minutes, the cells were centrifuged at 12,000 rpm for 30 seconds, and the supernatant was discarded to remove any phage that had not bound to the host bacteria. The cells were then resuspended in 10 mL of liquid culture medium and incubated at 37°C and 180 rpm. Phage titers were determined using a double-layer plate assay, with 100 μL of the culture medium aspirated every 10 minutes.

[0042] Phage suspension and host bacteria in the logarithmic growth phase were mixed at a multiplicity of infection (MOI) of 0.01 and added to liquid culture medium. The mixture was incubated at 37°C for 10 minutes, followed by centrifugation at 12,000 rpm for 30 seconds. The supernatant was removed and the pellet was resuspended and washed once with liquid culture medium to remove unadsorbed phage. The pellet was then resuspended in 10 mL of liquid culture medium and incubated at 37°C with shaking at 180 rpm. This time was defined as time 0. Every 10 minutes, 100 μL of the mixture was aspirated and titered using the double-layer plate method. The experiment was repeated three times, and the average value was used for analysis.

[0043] The results are as follows Figure 4 As shown, the incubation period of bacteriophage Ph037 after infection with the host bacteria is 10 minutes, during which the titer does not change significantly. The titer then rises sharply over the next 10 to 80 minutes before stabilizing. This indicates that the lytic period of bacteriophage Ph037 is approximately 70 minutes. The calculated lytic yield of bacteriophage Ph037 is 32 PFU / infected cell. Combined with the high lytic yield and short incubation and lytic periods of this phage, it suggests potential therapeutic applications.

[0044] Example 6 Temperature Stability Experiment of Vibrio harveyi Phage Ph037 1 mL of phage Ph037 phage solution was added to a centrifuge tube and incubated in a water bath at 50°C, 60°C, 70°C, 80°C, and 90°C for 30 min and 60 min, respectively. It was then mixed with the host bacteria, and the phage titer was determined using the double-layer plate method.

[0045] The results are as follows Figure 5 As shown, the titer of bacteriophage Ph037 can be maintained at 80°C for 30 to 60 minutes, and begins to decrease at 80°C. After being placed at 90°C for 30 minutes, the titer only decreases by about 2 orders of magnitude, indicating that bacteriophage Ph037 is highly tolerant to high temperature conditions.

[0046] Example 7 pH Stability Experiment of Vibrio harveyi Phage Ph037 100 μL of phage solution was added to 900 μL of SM buffer adjusted to pH 2-13 with HCL and NaOH, incubated in a 37°C water bath for 1 h, and then adjusted to pH 7 again. The solution was then mixed with the host bacteria, and the phage titer was determined using the double-layer plate method.

[0047] The results are as follows Figure 6 As shown in the results, under the conditions of pH 4-10, the bacteriophage Ph037 can maintain stable activity and its titer does not change significantly. At pH 3-13, it can still survive and has a good ability to adapt to acid-base environment, which is beneficial to the treatment of bacterial diseases.

[0048] Example 8 Determination of the efficacy of Vibrio harveyi bacteriophage Ph037 in treating shrimp infections The experiment was divided into 4 groups: blank group, control group 1, control group 2, and experimental group, with 20 healthy shrimp in each group. The blank group was the untreated group, control group 1 was the group that was challenged with Vibrio harveyi without treatment, control group 2 was the group that was not challenged with Vibrio harveyi but only treated, and the experimental group was the group that was challenged with Vibrio harveyi and treated. The challenge dose of Vibrio harveyi was uniformly 5×10 6 CFU / mL. The treatment consisted of mixing the phage solution with shrimp feed at a volume-to-weight ratio of 5%. Treatment efficacy was calculated 7 days after challenge, with mortality recorded daily during this period.

[0049] The results are shown in Table 2. All the animals in the control group 1 died; only 2 animals died in the experimental group. Phage Ph037 had an excellent therapeutic effect of 90%. There was no death in the control group 2, which also demonstrated the safety of phage Ph037.

[0050] Table 2: Results of therapeutic trials of Vibrio harveyi bacteriophage Ph037 Example 9 Whole genome sequencing and genome sequence analysis of bacteriophage Ph037 The genomic DNA of bacteriophage Ph037 was extracted using the TIANamp viral DNA / RNA extraction kit (Tiangen, Beijing, China) for whole-genome sequencing. The sequencing data were assembled using Unicycler software, annotated using Bakta software, and subsequently the genome map was drawn using Proksee software.

[0051] The Ph037 phage sequence is 74,846 bp in length, has a GC content of 44.1%, and contains 121 open reading frames. BLASTn comparison of the Ph037 genome sequence with other phage sequences in public databases revealed that the phage with the highest similarity to Ph037 in the public database was VhaP_PG11 (Genbank accession number OP745480.1), showing 76.55% similarity and 6% coverage, indicating that Ph037 is a new species.

[0052] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and do not constitute a limitation of the present invention. Within the technical concept of the present invention, the technical solutions of the present invention may be subjected to various simple modifications, including combining the various technical features in any other appropriate manner. These simple modifications and combinations should also be considered as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A thermostable Vibrio harveyi phage, characterized in that: The phage was deposited in the China Center for Type Culture Collection on April 2, 2025, with the deposit address in Wuhan, China, with the deposit number being CCTCC NO: M 2025677, and the classification name being Vibrio phage Ph037.

2. The use of the thermostable Vibrio harveyi phage according to claim 1 in the preparation of medicines, characterized in that: The medicine is used for treating or preventing Vibrio harveyi infection in aquatic products.

3. Use of the thermostable Vibrio harveyi phage according to claim 1 in the preparation of a feed additive or an environmental disinfectant, characterized in that: The feed additive or environmental disinfectant is used for treating or preventing Vibrio harveyi infection of aquatic products.

4. A phage composition, characterized in that The invention comprises the heat-resistant Vibrio harveyi phage according to claim 1.

5. The phage composition according to claim 4, wherein The phage composition further comprises other phages, which form a complex with the phage Ph037.

6. The phage composition according to claim 4, wherein The bacteriophage composition is a pharmaceutical preparation for treating or preventing Vibrio harveyi infection in aquatic products.

7. The phage composition according to claim 6, wherein The dosage form of the pharmaceutical preparation is gel, granule, solution, powder or lyophilized agent.

8. The phage composition according to claim 4, wherein The phage composition is the purified solution of the thermostable Vibrio harveyi phage according to claim 1.

9. The phage composition according to claim 8, wherein The titer of the phage in the purified solution is not less than 8.47×10 9 PFU / mL.

10. A shrimp feed additive, characterized in that: The method comprises the heat-resistant Vibrio harveyi phage according to claim 1 or the phage composition according to claim 4.

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