A Vibrio parahaemolyticus phage with multivalent lysis spectrum and its application in sea cucumber farming

Abstract

The present invention discloses a Vibrio parahaemolyticus phage with a multivalent lysis spectrum and its application in sea cucumber farming, belonging to the field of biotechnology. The phage has been sent to the China Center for Type Culture Collection for preservation on January 18, 2024. The preservation address is: Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 2024134. The phage VPP19 has a good lysis effect on common pathogenic Vibrio in sea cucumber farming, such as Vibrio parahaemolyticus, Vibrio splendidus, Vibrio alginolyticus, Vibrio harveyi and Vibrio robustus. In addition, the Vibrio parahaemolyticus phage VPP19 has high value-added titer, wide lysis spectrum, safety and high efficiency, and good tolerance to temperature and pH. The present invention can effectively prevent diseases caused by common pathogenic Vibrio in sea cucumber farming, and provide excellent strain resources and biological prevention measures for large-scale fermentation production for phage therapy of Vibrio problems in sea cucumbers.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a Vibrio parahaemolyticus phage with multivalent lysis spectrum and application thereof in sea cucumber breeding. Background Art

[0002] At present, the scale and density of sea cucumber farming in my country are getting larger and larger. However, the rapid development of farming and unscientific operation, insufficient attention to disease problems, and uneven farming technology levels have inevitably led to more serious sea cucumber disease problems, with the emergence of a variety of obvious symptoms and large-scale deaths of sea cucumbers, causing huge economic losses to farmers and also having a serious impact on the stable development of the industry. Sea cucumber rotten skin syndrome, also known as sea cucumber dermatosis and skin ulcer disease, is the "number one killer" in the sea cucumber farming process. Research on rotten skin syndrome mainly focuses on etiology. Becker et al. first reported skin ulcer disease and believed that the occurrence of the disease was the result of multiple events and multiple pathogenic factors (including bacteria) co-infection.

[0003] At present, the prevention and control of Vibrio in sea cucumber farming is mainly treated with antibiotics and probiotics. According to various literature and related reports, the antibiotic resistance of sea cucumbers is becoming more and more serious. In November 2023, Xiapu County, Fujian Province, conducted a survey on sea cucumber skin erosion. The main pathogens of sea cucumber skin erosion and enteritis were Vibrio robustus, Vibrio alginolyticus and Vibrio harveyi. The isolated Vibrio were highly resistant to enrofloxacin. Therefore, it is necessary to develop a green and effective biological control method.

[0004] However, there are no phage preparations reported to prevent and treat one or more Vibrio species, such as Vibrio parahaemolyticus, Vibrio splendidus, Vibrio alginolyticus, Vibrio harveyi and Vibrio robustus, in the process of sea cucumber farming. Based on this, the present invention provides a Vibrio parahaemolyticus phage with a multivalent lysis spectrum and its application in sea cucumber farming. Summary of the invention

[0005] In order to prevent skin rot syndrome or other diseases caused by one or more Vibrio species such as Vibrio parahaemolyticus, Vibrio splendidus, Vibrio alginolyticus, Vibrio harveyi and Vibrio robustus in sea cucumber farming, and reduce the mortality rate caused by the disease, one of the objects of the present invention is to provide a Vibrio parahaemolyticus phage with a multivalent lysis spectrum;

[0006] The second object of the present invention is to provide the use of Vibrio parahaemolyticus phage VPP19 in the preparation of drugs for treating or preventing diseases caused by one or more Vibrio infections such as Vibrio parahaemolyticus, Vibrio splendidus, Vibrio alginolyticus, Vibrio harveyi and Vibrio robustus.

[0007] In order to achieve the above-mentioned object, the present invention is realized by the following technical scheme: a Vibrio parahaemolyticus phage with a multivalent lysis spectrum, including Vibrio parahaemolyticus phage VPP19, the applicant isolated a Vibrio parahaemolyticus phage VPP19 from the waters of a certain place in Jiangsu Province, and the phage morphology is as follows Figure 1 As shown, the plaques formed by it and the host bacteria are relatively transparent, round, and have a diameter of 0.5 mm. The phage was sent to the China Center for Type Culture Collection on January 18, 2024. The collection address is: Wuhan University, Wuhan, China; the collection number is CCTCC NO: M 2024134, and the classification name is: Vibrio parahaemolyticus phage VPP19.

[0008] Furthermore, it also includes a complex of Vibrio parahaemolyticus phage VPP19.

[0009] An application of the Vibrio parahaemolyticus phage VPP19 or phage complex as described above is applied in the sea cucumber breeding industry to prepare an antibacterial agent for Vibrio parahaemolyticus, Vibrio splendidus, Vibrio alginolyticus, Vibrio harveyi and Vibrio robustus.

[0010] Furthermore, it is also used to prepare drugs for treating or preventing diseases caused by infection with Vibrio parahaemolyticus, Vibrio splendidus, Vibrio alginolyticus, Vibrio harveyi and Vibrio robustus, wherein the diseases are sea cucumber rot syndrome or Vibrio diseases caused by Vibrio parahaemolyticus, Vibrio splendidus, Vibrio alginolyticus, Vibrio harveyi and Vibrio robustus.

[0011] Furthermore, the feed or feed additive can be used to prepare antagonistic Vibrio parahaemolyticus, Vibrio splendidus, Vibrio alginolyticus, Vibrio harveyi and Vibrio robustus.

[0012] Beneficial effects of the present invention:

[0013] 1. The present invention screened out for the first time the bacteriophage VPP19 that can polyvalently lyse Vibrio parahaemolyticus, Vibrio splendidus, Vibrio alginolyticus, Vibrio harveyi and Vibrio robustus that cause sea cucumber rot skin syndrome. The bacteriophage VPP19 has a high lysis rate against Vibrio. The bacteriophage VPP19 is very effective in inhibiting different Vibrio, and has great reference significance for the prevention, control and treatment of Vibrio in sea cucumber farming or aquaculture in the future.

[0014] 2. The Vibrio parahaemolyticus phage VPP19 with a multivalent lysis spectrum has a high fermentation rate. At the optimal multiplicity of infection MOI = 0.01, the titer can reach 8.7×10 after 4 hours of fermentation. 13 PFU / mL, providing a theoretical basis for industrial fermentation.

[0015] 3. The Vibrio parahaemolyticus phage VPP19 with multivalent lysis spectrum has good environmental tolerance. The titer of the Vibrio parahaemolyticus phage VPP19 does not change significantly by an order of magnitude when treated in the pH range of 4-11 for 2 to 24 hours. The Vibrio parahaemolyticus phage VPP19 has a long survival time at temperatures of 45°C and below, and the phage titer does not change significantly by an order of magnitude after 24 hours at 4°C, and has good stability at 4-37°C. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The plaques are of Vibrio parahaemolyticus phage VPP19;

[0017] Figure 2 is the titer of Vibrio parahaemolyticus phage VPP19 under different infection multiplicity conditions;

[0018] Figure 3 To test the stability of Vibrio parahaemolyticus phage VPP19 at different pH values;

[0019] Figure 4 To test the stability of Vibrio parahaemolyticus phage VPP19 at different temperatures;

[0020] Figure 5 It is a schematic diagram of the fermentation dynamics of Vibrio parahaemolyticus phage VPP19;

[0021] Figure 6 This is an annotation diagram of the DNA gene sequence information of Vibrio parahaemolyticus phage VPP19. DETAILED DESCRIPTION

[0022] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0023] See also Figures 1 to 6 , the present invention provides the following technical solutions:

[0024] Embodiment 1:

[0025] Isolation and Purification of Vibrio parahaemolyticus Phage VPP19

[0026] 30 water samples were collected from a certain aquaculture area in Dongying City, Shandong Province, each with 30 mL, centrifuged at 6000 rpm for 10 min, the supernatant was sterilized with a 0.22 μm filter, 5 mL of the filtrate was mixed with 0.5 mL of Vibrio parahaemolyticus (concentration was about 5×108 CFU / mL), and fermented overnight at 200 rpm in a 37°C shaker. The fermentation broth was centrifuged at 6000 rpm for 10 min, the supernatant was sterilized with a 0.22 μm filter, 100 μL of the filtrate was mixed with 300 μL of Vibrio parahaemolyticus (concentration was about 5×108 CFU / mL), 2% sodium chloride TSB semi-solid (containing 0.65% agar) was added, gently inverted and poured on a 2% sodium chloride TSA bottom plate, and after solidification, the culture dish was placed in a 37°C incubator for overnight culture. Pick up the transparent and bright plaque spots, shake and desorb in 1mL SM solution, and sterilize through a 0.22μm microporous filter to obtain the phage filtrate, inoculate it in 5mL 2% sodium chloride TSB liquid culture medium, add 500μL of the corresponding host Vibrio parahaemolyticus liquid and mix, shake and culture at 200rpm overnight at 37℃, centrifuge at 6000rpm for 10min, take the supernatant and sterilize through a 0.22μm microporous filter to obtain the phage filtrate, and observe the morphology of the plaques by the double-layer plate method. After repeating the operation 3-5 times, plaques of the same shape and size can be obtained.

[0027] A strain of Vibrio parahaemolyticus phage VPP19 was isolated from the water sample. Figure 1 As shown, the plaques formed by it and the host bacteria are transparent, round, and 0.5 mm in diameter. The phage was sent to the China Center for Type Culture Collection on January 18, 2024, and the classification name is: Vibrio parahaemolyticus phage (Vibrio parahaemolyticus phage) VPP19, address: Wuhan University, Wuhan, China, and the collection number is CCTCC NO: M 2024134.

[0028] Embodiment 2:

[0029] Determination of the titer of Vibrio parahaemolyticus phage VPP19 under different infection multiplicity conditions

[0030] The host bacteria of Vibrio parahaemolyticus phage VPP19, VPFJ3, was inoculated into 3 mL of 2% sodium chloride TSB in an EP tube and cultured at 37°C for 5-8 hours to obtain VPFJ3 bacterial solution. The bacterial solution was inoculated into 100 mL of TSB medium at a 2% inoculum volume, and VPGX12 was grown to the initial logarithmic phase, and then phage VPP19 was inoculated. The infection was carried out according to different multiplicity of infection (MOI; MOI = number of phages / number of bacteria, see Figure 2), each experiment was performed in triplicate. The cells were cultured in a 37°C shaker at 200 rpm for 12 h.

[0031] Results Figure 2 When MOI = 0.01, the fermentation titer reached a maximum of 8.7 × 10 13 PFU / mL.

[0032] Embodiment 3:

[0033] pH Stability Test of Vibrio parahaemolyticus Phage VPP19

[0034] 0.1 mL of Vibrio parahaemolyticus phage VPP19 fermentation broth was added into 0.9 mL of TSB liquid culture medium with pH values ​​of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0, respectively (the titer after dilution was 5*10 8 pfu / mL), and incubated in a 25°C incubator for 0h, 1h, 2h, and 24h. Then, samples were taken, and the pH of the original culture medium was adjusted to Ph=7.4 with hydrochloric acid and sodium hydroxide solutions, respectively. Then, multiple dilutions were performed, and the phage titer was determined by the double-layer plate method. Duplicate cultures were performed at each point to take the average value, and the experiment was repeated 3 times.

[0035] The results are as follows Figure 3 As shown, the titer of Vibrio parahaemolyticus phage VPP19 did not change significantly in the order of magnitude when treated in the pH range of 4-11 for 1 h to 24 h.

[0036] Embodiment 4:

[0037] Temperature stability test of Vibrio parahaemolyticus bacteriophage VPP19

[0038] The fermentation broth of Vibrio parahaemolyticus phage VPP19 was centrifuged and filtered, and 1 mL was dispensed into 1.5 mL centrifuge tubes (the initial titer was 3.5*10 8 pfu / mL). The centrifuge tubes containing phages were placed in 4, 25, 37, 45, 55, 65, and 75°C and kept at constant temperature for 0h, 1h, 2h, 24h, 48h, 92h, 1w, 2w, 4w, 8w, 12w, 16w, 20w, 24w, 28w, 32w, 36w, 40w, 44w, 48w, and 52w. After the action time, the sample tubes were taken out and immediately placed in an ice bath for cooling. After appropriate dilution, the phage titer was determined by the double-layer plate method. Each point was cultured in duplicate to obtain the average value, and the experiment was repeated 3 times.

[0039] The results are as follows Figure 4As shown, the Vibrio parahaemolyticus phage VPP19 has a longer survival time at temperatures of 45°C and below. At 4°C, the phage titer has no obvious order of magnitude difference after 48 hours. It has good stability at 4-37°C and can be effectively preserved.

[0040] Embodiment 5:

[0041] Host range of bacteriophage VPP19

[0042] The Vibrio used in the experiment were Vibrio parahaemolyticus, Vibrio splendidus, Vibrio alginolyticus, Vibrio harveyi and Vibrio robustus.

[0043] In the experiment, 20 strains of Vibrio parahaemolyticus, Vibrio splendidus, Vibrio alginolyticus, Vibrio harveyi and Vibrio robustus were selected and inoculated into 3 mL of 2% sodium chloride TSB EP tubes respectively, and shaken at 37 ° C at 200 rpm for 8 h to obtain Vibrio liquid; prepare a sufficient amount of TSA bottom plate, mix 0.5 ml of Vibrio liquid with 6 mL of 2% sodium chloride TSB semi-solid medium (containing 0.65% agar) at about 50 ° C, and slowly spread it on the 2% sodium chloride TSA bottom plate; after the plate medium solidifies to room temperature, take 5×10 8 5 μL of phage VPP19 with a PFU / ml was dropped onto the plate, and after air drying, the plate was inverted and placed in a 37°C constant temperature biochemical incubator for about 8 hours, and the results were observed. Each group of experiments was repeated 3 times.

[0044] As shown in Table 1, the Vibrio parahaemolyticus phage VPP19 can lyse 19 of 20 strains of Vibrio parahaemolyticus with a lysis rate of 95%; 17 of 20 strains of Vibrio robustus with a lysis rate of 85%; 18 of 20 strains of Vibrio splendidus with a lysis rate of 90%; 18 of 20 strains of Vibrio alginolyticus with a lysis rate of 90%; 18 of 20 strains of Vibrio harveyi with a lysis rate of 90%. This indicates that the Vibrio parahaemolyticus phage VPP19 has great application value in the prevention and control of Vibrio in the rotten skin syndrome during sea cucumber farming. In the lysis experiment of all 100 strains of Vibrio, VPP19 can lyse 90 of them with a lysis rate of 90%. The experimental results confirm that this phage has a very outstanding effect in the inhibition of Vibrio parahaemolyticus, Vibrio splendidus, Vibrio alginolyticus, Vibrio harveyi and Vibrio robustus, and has great value for the prevention and control of Vibrio in sea cucumbers and related aquaculture in the future.

[0045] Table 1 Lysis effect of bacteriophage VPP19 on 100 strains of Vibrio

[0046]

[0047]

[0048]

[0049] Note: “-” indicates no lysis, “+” indicates lysis and the plaques are relatively transparent; “++” indicates lysis and the plaques are transparent.

[0050] Embodiment 6:

[0051] Fermentation preparation of Vibrio parahaemolyticus bacteriophage VPP19

[0052] A single colony of Vibrio parahaemolyticus VPFJ3 was picked from the plate and inoculated into 3 mL of 2% sodium chloride TSB medium, and cultured at 37°C and 200 rpm for 8 h to obtain the host bacterial solution. The bacterial suspension was inoculated into 120 mL of 2% sodium chloride TSB medium at a 2% inoculum amount and cultured at 37°C and 200 rpm until the early logarithmic phase. The bacterial solution OD 600 The value is 0.1.

[0053] The industrial fermentation is a 10L fermentation tank, the fermentation system is 6L, the medium used is TSB, the initial pH value is 7.4, the flame method is used to inoculate Vibrio parahaemolyticus at a 1% inoculation amount, and the phage VPP19 is added to the fermentation system at the optimal infection condition MOI=0.01. Sterile air is introduced during the fermentation process, and 3‰ defoamer is added. The fermentation preparation time is 12h. Since the start of fermentation, 20ml of fermentation liquid is taken from the sampling port in a sterile container at 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, and 12h, centrifuged at 6000rpm for 10min, and the supernatant is sterilized through a 0.22μm microporous filter membrane to obtain a filtrate containing phage and determine its titer, and the method is referred to Example 2.

[0054] After the fermentation is completed, the entire mixed solution of phage and host bacteria is taken out from the sampling port and placed in a sterile container, centrifuged at 6000 rpm for 10 min, and the supernatant is filtered through a vacuum pump into a sterile filter device to obtain the phage fermentation solution and stored at 4°C.

[0055] Depend on Figure 6 It can be seen that the titer of Vibrio parahaemolyticus phage VPP19 reached a maximum of 7.4×10 13 PFU / mL, in the period of 5h to 12h, the titer is stable at 10 13 PFU / mL; The results show that it is feasible to use fermentation method for industrial production of bacteriophages, which provides effective reference data for the preparation of bacteriophage products.

[0056] Embodiment 7:

[0057] Experiment on the prevention of Vibrio parahaemolyticus phage VPP19 against Vibrio complex infection in sea cucumber

[0058] This section refers to Jing Kailin's (Jing Kailin. Research on the protective effect of specific yolk antibodies on sea cucumbers infected with Vibrio splendens [D]. Dalian University of Technology, 2016.) experiments on the attack and clinical reference basis of Vibrio splendens in sea cucumbers. Sea cucumbers infected with Vibrio will shake their heads and vomit intestines. In the late stage, large areas of epidermal ulcers will occur, and finally they will autolyze and die.

[0059] The imitation sea cucumbers of this experiment were purchased from a farm in Xiapu, Fujian. The imitation sea cucumbers purchased had normal appearance, normal feeding conditions, an average body weight of 7.8 ± 0.33 g, and a total of 250. Before the experiment, the imitation sea cucumbers were grouped and placed in an aquarium to adapt to feeding for 7 days. The water body had a salinity of 2.5%, a pH value of 7.6 ± 0.1, a temperature of 17 ° C, and a dissolved oxygen of 6.6 ± 0.1 mg / L. After the mortality rate stabilized at the rear (extremely low value), the experiment was carried out. The Vibrio parahaemolyticus, Vibrio robustus, Vibrio splendens, Vibrio alginolyticus, and Vibrio harveyi used in this embodiment are VPFJ47, VFFJ10, VSFJ1, VAJS178, and VHFJ25, respectively, and the selected phage is the Vibrio parahaemolyticus phage VPP19 of the present invention.

[0060] The experimental design is as follows: the sea cucumbers are divided into 5 groups:

[0061] 1) Control group: Each sea cucumber was intraperitoneally injected with an equal volume of saline (the following challenge methods were the same);

[0062] 2) Poisoning group: Each sea cucumber was injected with a concentration of 1×10 6 100 μL of bacterial solution with CFU / mL (mixed with VPFJ47, VFFJ10, VSFJ1, VAJS178, and VHFJ25, the same below);

[0063] 3) Treatment group: Each sea cucumber was injected with a concentration of 1×10 6 CFU / mL of bacterial solution 100μL; after 6h, inject phage VPP191×10 6 PFU / treatment only;

[0064] 4) Prevention group: 1 day before the experiment, 1×10 6 PFU / each, and then injected 1×10 6 CFU / mL bacterial solution 100μL;

[0065] 5) Antibiotic group: Each sea cucumber was injected with a final concentration of 1×10 6 CFU / mL of bacterial solution 100 μL. After 12 hours, add doxycycline according to the dosage instructions for treatment.

[0066] The experiment lasted for 7 days, and the symptoms and death of the sea cucumbers were recorded. After the observation period ended, the tissues of each group of sea cucumbers were mixed and ground, and the total number of Vibrio was counted using TCBS plates. The survival rate of each group of sea cucumbers was calculated as shown below.

[0067]

[0068] ATotal: the total number of sea cucumbers in each group of the experiment, pieces;

[0069] A dead: the total number of dead sea cucumbers in each group of the experiment, pieces;

[0070] C: Survival rate of each group of sea cucumbers in the experiment, %.

[0071] The experimental results are shown in Table 3. The survival rate of the blank group was 98%, the survival rate of the sea cucumber in the challenge group was 0%, the survival rate of the treatment group was 88%, the survival rate of the prevention group was 92%, and the survival rate of the antibiotic group was 90%. The experimental results revealed that the Vibrio parahaemolyticus phage VPP19 can effectively kill Vibrio, and both the prevention and treatment groups effectively reduce the content of Vibrio and improve the survival rate of sea cucumber. This example provides an important reference value for the prevention and treatment of sea cucumber rot syndrome in the future, and can be used as a biological antibacterial agent in the prevention and treatment of Vibrio parahaemolyticus diseases in sea cucumbers and aquatic animals.

[0072] Table 2 The control effect of bacteriophage VPP19 on Vibrio in sea cucumber

[0073]

[0074] Embodiment 8:

[0075] Genomic functional annotation of the complete genome of Vibrio parahaemolyticus bacteriophage VPP19

[0076] The phage VPP19 was annotated using the RAST online annotation tool. The results showed that the genome length was 46284bp, the GC content was 49.2%, there were 83 ORFs (Open Reading Frames, ORFs) and 64 CDSs (coding sequences), the longest ORFs was 2730bp long, and the shortest ORFs was 303bp long. After BLAST comparison, 55 of the 64 CDSs detected were CDSs of confirmed functional proteins, such as 26 phage protein CDSs and 13 hypothetical protein CDSs; the gene family tree was Figure 6 shown.

[0077] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the attached claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention. Any figure mark in the claims should not be regarded as limiting the claims involved.

[0078] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A Vibrio parahaemolyticus phage having a multivalent lysis spectrum, characterized in that: The phage was sent to the China Center for Type Culture Collection for preservation on January 18, 2024. The preservation address is: Wuhan University, Wuhan, China; the preservation number is CCTCC NO: M 2024134, and the classification name is: Vibrio parahaemolyticus phage VPP19.

2. A use of the Vibrio parahaemolyticus phage VPP19 as claimed in claim 1, characterized in that: It is used in the sea cucumber breeding industry to prepare antibacterial agents for Vibrio parahaemolyticus, Vibrio splendidus, Vibrio alginolyticus, Vibrio harveyi and Vibrio robustus.

3. A use of the Vibrio parahaemolyticus phage VPP19 as claimed in claim 1, characterized in that: The invention is used for preparing medicines for treating or preventing diseases caused by infection with Vibrio parahaemolyticus, Vibrio splendidus, Vibrio alginolyticus, Vibrio harveyi and Vibrio robustus, wherein the disease is sea cucumber rotten skin syndrome.

4. A use of the Vibrio parahaemolyticus phage VPP19 as claimed in claim 1, characterized in that: The invention is used for preparing medicines for treating or preventing diseases caused by infection with Vibrio parahaemolyticus, Vibrio splendidus, Vibrio alginolyticus, Vibrio harveyi and Vibrio robustus, wherein the diseases are Vibrio diseases caused by Vibrio parahaemolyticus, Vibrio splendidus, Vibrio alginolyticus, Vibrio harveyi and Vibrio robustus.

5. A use of the Vibrio parahaemolyticus phage VPP19 as claimed in claim 1, characterized in that: The invention is used for preparing feed or feed additives for antagonizing Vibrio parahaemolyticus, Vibrio splendidus, Vibrio alginolyticus, Vibrio harveyi and Vibrio robustus.

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