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

Abstract

The application belongs to the field of microorganisms, and particularly relates to a high-temperature-resistant Vibrio harveyi bacteriophage, a composition thereof and application. The high-temperature-resistant Vibrio harveyi bacteriophage Ph037 can achieve a good lysis effect on Vibrio harveyi, and has strong resistance to high temperature. The bacteriophage can be stably stored in different environmental transportation and storage processes, solves the problem that the bacteriophage is easy to be inactivated in a high-temperature environment, provides a new means for treatment and prevention of vibriosis, and provides an alternative scheme for treatment of multi-drug resistant bacterial infection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microorganisms, and particularly relates to a high-temperature-resistant Vibrio phage, a composition thereof and application thereof. BACKGROUND

[0002] Vibrio Harveyi is a gram-negative bacterium widely existing in aquaculture environment, is an important pathogen of aquatic invertebrates (especially prawns) and fish, and can cause infection of human beings, and is a major threat to aquaculture industry. Vibrio Harveyi is usually associated with fish eye disease and gastroenteritis, and acute hepatopancreas necrosis disease of shrimps. It has extremely high mortality and can inhibit the immunity of shrimps, so as to realize mass propagation and further pollute the aquaculture environment.

[0003] Using antibiotic drugs is one of the main means for preventing and controlling Vibrio Harveyi, however, the abuse of antibiotics leads to the emergence of more and more multi-drug resistant bacteria, mainly tetracyclines, macrolides and beta-lactams in Vibrio. The generation of drug resistance makes the drug prevention and control efficiency gradually decrease, indirectly causing problems such as increase of aquaculture cost. Excessive and continuous use of antibiotics will lead to drug residues, threatening food safety. Since shrimps do not have a developed adaptive immune system like mammals, existing vaccine means cannot be applied in shrimp farming, therefore, it is urgent to find another alternative means for preventing and controlling antibiotics.

[0004] Phage is a kind of virus that can infect bacteria, and after infecting bacteria, it replicates in the bacteria, and then lyses the bacteria. Compared with antibiotic treatment, phage has high specificity, smaller side effects, higher safety, and also has the characteristics of high efficiency and low dosage. In recent years, as an alternative to antibiotics, phage has been applied in the prevention and control of many bacterial diseases and has achieved good results, and has also been applied in many similar fields, such as being used as a preservative for food transportation and preservation, and being used as a disinfectant for environmental and instrument disinfection.

[0005] In aquaculture, phage has great potential for the prevention and control of bacterial pathogens, and there are not many existing Vibrio phage preparations, and the tolerance to high temperature is poor. SUMMARY

[0006] The purpose of the present application is to provide a Vibrio phage, which has high titer, can effectively prevent acute hepatopancreas necrosis disease of shrimps caused by Vibrio Harveyi, clear pathogenic bacteria, and has high temperature resistance, can be stably stored in high temperature environment, and has good application prospect in the prevention and control of Vibrio Harveyi disease.

[0007] The present application is realized by the following technical scheme:

[0008] The application provides a high-temperature-resistant Vibrio harveyi bacteriophage Ph037, which is isolated from a shrimp pond in Sheyang County, Yancheng City, Jiangsu Province, and is preserved in the China Center for Type Culture Collection on April 2, 2025, has a preservation address of Wuhan, China, a preservation number of CCTCC NO: M 2025677, and a classification name of Vibrio phage Ph037.

[0009] The high-temperature-resistant Vibrio harveyi bacteriophage Ph037 has a head diameter of 100 nm, a polyhedral symmetrical structure, and a tail of 120 nm, and the tail is linked with the head through a neck. According to the ninth report on virus taxonomy of the International Society for Virology, the bacteriophage can be classified into the Caudovirales order and the Myoviridae family.

[0010] The high-temperature-resistant Vibrio harveyi bacteriophage Ph037 provided by the application has strong lytic ability to Vibrio harveyi, and has lytic ability to 36 of 40 Vibrio harveyi strains preserved in a laboratory, with a lytic rate of about 90%, which reflects a wide lytic spectrum; the bacteriophage has good acid and alkali tolerance, and can maintain a titer of not less than 4.7 x 10 8 PFU / mL in a pH range of 4-10; the optimal infection multiple is 0.01, and the minimum is 10 -5 , which reflects good reproduction ability.

[0011] The high-temperature-resistant Vibrio harveyi bacteriophage Ph037 provided by the application has good temperature stability, and can stably survive for 30-60 min in a temperature environment below 80 DEG C, and maintain a high level of titer.

[0012] The application further provides application of the high-temperature-resistant Vibrio harveyi bacteriophage Ph037 in preparation of a medicine for treating or preventing Vibrio harveyi infection of aquatic products.

[0013] The application further provides application of the high-temperature-resistant Vibrio harveyi bacteriophage Ph037 in preparation of an environmental disinfectant for treating or preventing Vibrio harveyi infection of aquatic products.

[0014] In some embodiments, the aquatic product is shrimp, fish, crab or shellfish.

[0015] The application further provides a bacteriophage composition comprising the high-temperature-resistant Vibrio harveyi bacteriophage Ph037.

[0016] In some embodiments, the bacteriophage composition further comprises other bacteriophages, which form a complex with the bacteriophage Ph037, for example, PP270, PH018, HP16, HP8 and HP21.

[0017] In some embodiments, the bacteriophage composition is a pharmaceutical preparation for treating or preventing Vibrio harveyi infection of aquatic products.

[0018] In some embodiments, the dosage form of the pharmaceutical preparation is a gel, granules, solution, powder or lyophilized agent.

[0019] In some embodiments, the bacteriophage composition is a purified solution of thermostable Vibrio harveyi bacteriophage.

[0020] In some embodiments, the titer of the bacteriophage in the purified solution is not less than 8.47 x 10 9 PFU / mL.

[0021] Compared with the prior art, the beneficial effects of the present application at least include:

[0022] 1. The Vibrio harveyi bacteriophage Ph037 has strong heat stability, can be stably stored and transported in a high-temperature environment, effectively solves the problem of easy inactivation of bacteriophage at high temperature, has a wide lytic spectrum, high titer and strong reproduction ability, and provides a new effective treatment scheme for Vibrio harveyi disease.

[0023] 2. The bacteriophage composition has no toxic effect on shrimps, does not produce drug resistance, is safe and residue-free, does not affect the survival of shrimps, and can replace antibiotics as a prevention and control means for Vibrio harveyi disease.

[0024] 3. The Vibrio harveyi bacteriophage Ph037 is rapidly produced and easily obtained, and can be widely applied to various situations in the shrimp breeding industry that are easily contaminated by Vibrio harveyi.

[0025] 4. Compared with the bacteriophage VhaP_PG11 with the highest similarity, the Vibrio harveyi bacteriophage Ph037 shows 76.55% similarity and 6% coverage, indicating that the bacteriophage Ph037 is a new species. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is a plaque photograph of the bacteriophage Ph037;

[0027] Figure 2 is an electron microscope photograph of the bacteriophage Ph037;

[0028] Figure 3 is the determination result of the optimal infection multiple of the bacteriophage Ph037;

[0029] Figure 4 is the determination result of the one-step growth curve of the bacteriophage Ph037;

[0030] Figure 5 is the determination result of the temperature stability of the bacteriophage Ph037;

[0031] Figure 6 Results of pH stability assay for bacteriophage Ph037. DETAILED DESCRIPTION

[0032] In order to make the objectives, technical solutions and beneficial effects of the present application clearer, the present application will be further described in detail below with reference to the drawings and specific embodiments.

[0033] The endpoints of the ranges and any values disclosed herein are not limited to the precise values recited as the exact range or value should be understood as being encompassed by the ranges.

[0034] Example 1 Isolation and purification of Vibrio harveyi bacteriophage Ph037

[0035] 1. Preparation of fresh bacterial solution

[0036] Vibrio harveyi was streaked on TCBS plates and single colonies were picked and inoculated into 5 mL 2216E liquid medium and incubated at 37°C with 180 rpm shaking for 16-18 h.

[0037] 2. Isolation and purification of bacteriophage

[0038] A water sample collected from a shrimp pond in Sheyang County, Yancheng City, Jiangsu Province was centrifuged at 12000 rpm for 5 min to remove impurities in the water, and the supernatant was collected. Then, the supernatant was further filtered through a 0.22 μm sterile filter to remove microorganisms, etc., to obtain a supernatant filtrate that may contain bacteriophages.

[0039] 200 μL of the supernatant filtrate was mixed with 100 μL of Vibrio harveyi host bacterial solution, and incubated in liquid medium at 37°C with 180 rpm shaking for 12-14 h. Then, the mixture was centrifuged at 12000 rpm for 5 min, and the supernatant was collected. The supernatant was then further filtered through a 0.22 μm sterile filter to obtain a bacteriophage stock solution.

[0040] 0.1 mL of the bacteriophage stock solution was diluted 10-fold with PBS buffer, and 100 μL of the diluted solution was mixed with 100 μL of the host bacterial solution. Then, 3 mL of top agar was added, and the mixture was quickly poured onto the lower medium. After the top agar solidified, the mixture was incubated at 37°C overnight, and whether plaques were formed was observed.

[0041] If plaques were observed, a single plaque with a neat edge was selected and picked into 1 mL of PBS buffer, and centrifuged at 12000 rpm for 5 min. Then, the mixture was further filtered through a 0.22 μm sterile filter. The filtrate was diluted 10-fold with PBS buffer, and double-layer plate culture was performed again. After the appropriate concentration was determined, the operation was repeated 3-5 times until plaques with uniform morphology, size and transparency were obtained, i.e., a bacteriophage isolate was obtained, as shown in FIG. 1. Figure 1 ​

[0042] 3. Determination of phage proliferation and titer

[0043] 100 μL of purified phage fluid was mixed with 100 μL of host bacterial fluid and cultured in liquid culture medium at 37 °C and 180 rpm until the liquid was clear. The mixture was then centrifuged at 12,000 rpm for 5 min, and the supernatant was collected. The supernatant was then further filtered through a 0.22 μm sterile filter to obtain the phage proliferation solution.

[0044] Phage proliferation broth was serially diluted 10-fold with PBS buffer, and phage titer was determined by double-layer plate culture. Phage titer (PFU / mL) = number of phage plaques × dilution factor × 10. The measured titer was approximately 8.47 × 10⁻⁶. 9 PFU / mL.

[0045] Example 2: Electron microscopic observation of bacteriophages

[0046] 20 μL of phage solution was added to a copper grid and allowed to settle naturally for 15 min. Then, excess liquid was absorbed from the edge with filter paper. The sample was stained with 2% phosphotungstic acid solution for 10 min. The staining solution was then absorbed from the edge again with filter paper. After the sample dried naturally, the morphology of the phage was observed under an electron microscope.

[0047] like Figure 2 As shown, Vibrio harveyi phage Ph037 has a head diameter of 100 nm, a multifaceted three-dimensional symmetrical structure, encapsulates nucleic acid, and has a tail length of 120 nm, which is linked to the head through the neck. According to the Ninth Report on the Classification of Viruses by the International Organization for Taxonomy of Viruses, this phage can be classified as belonging to the order Myocaudidae of the order Tail Phages.

[0048] Example 3: Test on the lysis range of Vibrio harveyi bacteriophage Ph037 against Vibrio harveyi

[0049] The phage lysis profile was determined using the spot drop method. 1 mL of bacterial culture was mixed with a semi-solid culture medium and spread onto a plate. Then, 3 μL of phage culture was spot-dropped onto the plate. The plate was then incubated overnight at 37°C. The results were observed and the lysis effect was recorded. The lysis effect was scored as follows: 0: no lysis; 1: incomplete lysis; 2: lysis.

[0050] The results are shown in Table 1. Phage Ph037 demonstrated lytic activity against 36 out of 40 Vibrio harveyi strains from diseased and dead fish, shrimp, and water samples from various cities in Shandong Province, with a lysis rate of 90%. This indicates that phage Ph037 has a broad lytic spectrum and good potential in the treatment and prevention of Vibrio harveyi infection.

[0051] Table 1: Results of lysis spectrum determination of Vibrio harveyi bacteriophage Ph037 against Vibrio harveyi

[0052] Bacterial name Lysis effect Bacterial name Lysis effect VH01 2 VH26 2 VH02 2 VH27 1 VH03 2 VH28 2 VH04 2 VH29 2 VH05 2 VH30 2 VH06 1 VH31 0 VH07 2 VH32 2 VH08 2 VH33 2 VH09 0 VH34 2 VH10 2 VH35 2 VH11 1 VH36 2 VH12 2 VH37 1 VH13 2 VH38 2 VH14 2 VH39 2 VH15 1 VH40 2 VH16 2 VH41 1 VH17 2 VH42 2 VH18 1 VH43 2 VH19 2 VH44 0 VH20 2 VH45 2 VH21 2 VH46 2 VH22 1 VH47 2 VH23 2 VH48 1 VH24 2 VH49 2 VH25 0 VH50 1

[0053] Example 4 Determination of optimal multiplicity of infection MOI of Vibrio harveyi phage Ph037

[0054] The phage dilution liquid was mixed with the host bacteria liquid cultured to logarithmic growth phase at the infection ratio of 0.0000001, 0.000001, 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, then added into 5 mL 2216E liquid medium, and cultured in the liquid medium at 37°C, 180 rpm until the liquid was clear, centrifuged at 12000 rpm for 5 min, the supernatant was collected, and the phage titer was determined by double-layer plate method. The optimal multiplicity of infection was the ratio of the number of phage to bacteria when the phage reached the optimal growth condition.

[0055] The titer of phage Ph037 was the highest when the multiplicity of infection was 0.01, and the results are shown in Table 1. Figure 3

[0056] Example 5 One-step growth curve determination of Vibrio harveyi phage Ph037

[0057] The phage Ph037 was mixed with the host bacteria in logarithmic growth phase at the optimal multiplicity of infection ratio, incubated at 37°C for 10 min, then centrifuged at 12000 rpm for 30 s, the supernatant was discarded to remove the phage that did not bind to the host bacteria. Then resuspended with 10 mL liquid medium, and cultured at 37°C, 180 rpm. Then every 10 min, 100 μL of liquid was taken, and the phage titer was determined by double-layer plate method.

[0058] The phage liquid was mixed with the host bacteria in logarithmic growth phase at the multiplicity of infection of 0.01, added into the liquid medium, incubated at 37°C for 10 min, then centrifuged at 12000 rpm for 30 s, the supernatant was removed, and then resuspended and washed once with liquid medium to remove the unabsorbed phage. Then resuspended the precipitate with 10 mL liquid medium, and cultured at 37°C, 180 rpm, which was defined as 0 time, then every 10 min, 100 μL of the mixture was taken, and the titer was determined by double-layer plate method. The experiment was repeated three times, and the average value was analyzed.

[0059] The results are shown in Table 2. Figure 4 The latent period of phage Ph037 after infecting the host bacteria was 10 min, and the titer did not change significantly during this period. The phage titer increased sharply within 10-80 min, and then tended to be stable. It was shown that the lysis period of phage Ph037 was about 70 min. The calculated lysis amount of phage Ph037 was 32 PFU / infective cell. Combined with the high lysis amount, short latent period and lysis period of the phage, it was known that the phage had potential for therapeutic application. ​

[0060] Temperature stability experiment of Vibrio harveyi phage Ph037

[0061] 1 mL of phage liquid of phage Ph037 was added into centrifuge tubes, incubated in water bath at 50°C, 60°C, 70°C, 80°C and 90°C for 30 min and 60 min respectively, then mixed with host bacteria, and the phage titer was determined by double-layer plate method.

[0062] The results are shown in Table 1. Figure 5 As shown in Table 1, the phage Ph037 could maintain its titer for 30 min to 60 min at 80°C or below, and started to decrease at 80°C, and only decreased by about 2 orders of magnitude after 30 min at 90°C, indicating that the phage Ph037 was highly resistant to high temperature conditions.

[0063] pH stability experiment of Vibrio harveyi phage Ph037

[0064] 100 μL of phage liquid was added into 900 μL of SM buffer adjusted to pH 2-13 with HCL and NaOH, incubated in a 37°C water bath for 1 h, then the pH was adjusted to 7 again, then mixed with host bacteria, and the phage titer was determined by double-layer plate method.

[0065] The results are shown in Table 2. Figure 6 As shown in Table 2, the phage Ph037 could maintain stable activity at pH 4-10, and its titer did not change significantly, and it could still survive at pH 3-13, and had good ability to adapt to acid and alkaline environment, which was beneficial to the treatment of bacterial diseases.

[0066] Effect determination of Vibrio harveyi phage Ph037 treatment on shrimp infection

[0067] The experiment was divided into 4 groups: blank group, control group 1, control group 2, and experimental group, with 20 healthy shrimps in each group. The blank group was not treated, the control group 1 was only infected with Vibrio harveyi without treatment, the control group 2 was not infected with Vibrio harveyi but only treated, and the experimental group was infected with Vibrio harveyi and treated. The infection dose of Vibrio harveyi was uniformly 5 x 10 6 CFU / mL. The treatment method was to mix and stir the phage liquid with shrimp feed and then feed it, and the addition amount was 5% (volume to mass ratio). The treatment effect was calculated 7 days after infection, and the death was recorded daily during the period.

[0068] As shown in Table 2, all shrimps in the control group 1 died; only 2 shrimps in the experimental group died, and the phage Ph037 had excellent treatment effect, reaching 90%; no death in the control group 2 also reflected the safety of the phage Ph037.

[0069] Table 2: Therapeutic test results of Vibrio harveyi phage Ph037

[0070] Group Number of death / each Death rate % Protection rate % Blank group 0 0 / Control group 1 20 100 / Control group 2 0 0 / Test group 2 10 90

[0071] Example 9 Whole genome sequencing of phage Ph037 and genome sequence analysis

[0072] The genomic DNA of phage Ph037 was extracted using TIANamp Virus DNA / RNA Extraction Kit (Tiangen, Beijing, China) for whole genome sequencing. The sequencing data was assembled using Unicycler software, annotated using Bakta software, and then the genome map was drawn using Proksee software.

[0073] The phage Ph037 sequence was 74846 bp in size with a GC content of 44.1%, and contained 121 open reading frames. The whole genome sequence of phage Ph037 was compared with other phage sequences in the public database using BLASTn, and the results showed that the phage with the highest similarity to Ph037 in the public database was VhaP_PG11 (Genbank number OP745480.1), showing 76.55% similarity and 6% coverage, indicating that phage Ph037 was a new species.

[0074] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application and do not constitute a limitation on the content of the present application. Within the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, including the combination of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as disclosed by the present application and fall within the protection scope of the present application.

Claims

1. A heat-resistant Vibrio harveyi bacteriophage, characterized in that, The phage was deposited on April 2, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan, China, with accession number CCTCC NO: M 2025677 and classified as Vibrio phage.

2. The application of the thermoresistant Vibrio harveyi phage according to claim 1 in the preparation of drugs, characterized in that, The drug is used to treat Vibrio harveyi infection in aquatic products.

3. The application of the heat-resistant Vibrio harveyi bacteriophage according to claim 1 in the preparation of environmental disinfectants, characterized in that, The environmental disinfectant is used to treat Vibrio harveyi infection in aquatic products.

4. A bacteriophage composition, characterized in that, It includes the thermoresistant Vibrio harveyi bacteriophage as described in claim 1.

5. The phage composition according to claim 4, characterized in that, The phage composition is a pharmaceutical preparation for treating Vibrio harveyi infection in aquatic products.

6. The phage composition according to claim 5, characterized in that, The dosage form of the pharmaceutical preparation is a gel, granules, solution, powder, or lyophilized agent.

7. The phage composition according to claim 4, characterized in that, The phage composition is a purified solution of the thermoresistant Vibrio harveyi phage according to claim 1, wherein the phage titer in the purified solution is not less than 8.47 × 10⁻⁶. 9 PFU / mL.

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