A bacteriophage of edwardsiella tarda and use thereof

Abstract

The application belongs to the technical field of microorganisms, and particularly relates to a bacteriophage of Edwardsiella tarda and application thereof. Edwardsiella tarda virus The bacteriophage of Edwardsiella tarda is classified and named as The bacteriophage of Edwardsiella tarda is classified and named as The bacteriophage of Edwardsiella tarda is classified and named as The bacteriophage of Edwardsiella tarda is classified and named as The bacteriophage of Edwardsiella tarda is classified and named as The bacteriophage of Edwardsiella tarda is classified and named as The bacteriophage of Edwardsiella tarda is classified and named as The bacteriophage of Edwardsiella tarda is classified and named as The bacteriophage of Edwardsiella tarda is classified and named as The bacteriophage of Edwardsiella tarda is classified and named as The bacteriophage of Edwardsiella tarda is classified and named as The bacteriophage of Edwardsiella tarda is classified and named as The bacteriophage of Edwardsiella tarda is classified and named as The bacteriophage of Edwardsiella tarda is classified and named as The bacteriophage of Edwardsiella tarda is classified and named as The bacteriophage of Edwardsiella tarda is classified and named as The bacteriophage of Edwardsiella tarda is classified and named as The bacteriophage of Edwardsiella tarda is classified and named as The bacteriophage of Edwardsiella tarda is classified and named as The bacteriophage of Edwardsiella tarda is classified and named as The bacteriophage of Edwardsiella tarda is classified and named as The bacteriophage of Edwardsiella tarda is classified and named as The bacteriophage of Edwardsiella tarda is classified and named as The bacteriophage of Edwardsiella tarda is classified and named as The bacteriophage of Edwardsiella tarda is classified and named as The bacteriophage of Edwardsiella tarda is classified and

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganisms, and particularly relates to an Edwardsiella tarda bacteriophage and application thereof. BACKGROUND

[0002] Edwardsiella tarda is a pathogenic bacterium widely existing in aquatic environments, which can cause systemic bacterial septicemia in many aquatic animals including Paralichthys olivaceus, Scophthalmus maximus, Ictalurus punctatus and Micropterus salmoides, causing huge economic losses to the aquaculture industry. Edwardsiella tarda also has a facultative intracellular parasitic property, that is, it can survive and reproduce in host cells, and is also an important pathogenic bacterium of human and fish co-infection, which can cause human meningitis, gastrointestinal infection, cellulitis, septicemia and muscle necrosis and other diseases. With the large-scale development of the aquaculture industry and the increase in consumption of fresh aquatic products, the risk of human infection with Edwardsiella tarda is increasingly prominent.

[0003] The main means for preventing and controlling Edwardsiella infection at present is to use antibiotics and chemical disinfectants. Due to the long-term and extensive use of antibiotics, most Edwardsiella tarda isolated from marine and freshwater fish in recent years exhibit multiple drug resistance, which increases the difficulty of preventing and controlling Edwardsiella infection, and antibiotic residues pollute the water environment, further enriching the food chain and endangering human health.

[0004] As natural bacterial predators, bacteriophages have high abundance in nature. Bacteriophage therapy provides a breakthrough solution for the prevention and control of Edwardsiella tarda in aquaculture due to its specificity and high efficiency, safety and environmental friendliness. However, the existing reported Edwardsiella tarda bacteriophages have weak bacteriostatic effect at the early stage of infection, resulting in low lysis efficiency and difficulty in achieving rapid pathogen clearance. Even if the bacteriophages have high lysis capacity, the delay in the early stage of bacteriostasis will lead to poor treatment effect and low protection effect on the host. SUMMARY

[0005] The present application provides a kind of Edwardsiella tarda bacteriophage and its application to solve the problems of existing Edwardsiella tarda bacteriophage, such as bacteriostatic delay and poor lysis capacity. The Edwardsiella tarda bacteriophage is a bacteriophage with high lysis, rapid killing of Edwardsiella tarda, acid and alkali resistance, strong temperature stability, wide strain spectrum and high safety, which can be used to prepare drugs, environmental bactericides or feed additives and other products for inhibiting or killing Edwardsiella tarda.

[0006] To achieve the above purpose, the specific technical solutions of the present application are as follows:

[0007] The application provides a bacteriophage of Edwardsiella tarda, which is preserved in the China General Microbiological Culture Collection Center and has a classification name of Edwardsiella tardavirus .

[0008] The application provides application of the bacteriophage of Edwardsiella tarda in preparation of a product for inhibiting or killing Edwardsiella tarda.

[0009] Further, the product is a medicine, a feed additive or an environmental sterilizing agent.

[0010] Further, the effective component of the medicine is the bacteriophage of Edwardsiella tarda.

[0011] Further, the medicine further comprises a pharmaceutically acceptable carrier.

[0012] Further, the dosage form of the medicine is a powder, a solution, an emulsion, a gel, a granule or a freeze-dried agent.

[0013] Further, the dosage form of the medicine is a solution.

[0014] Further, the titer of the bacteriophage of Edwardsiella tarda in the medicine is greater than or equal to 1.2x10 4 PFU / mL.

[0015] Further, the titer of the bacteriophage of Edwardsiella tarda in the feed additive is greater than or equal to 10 7 PFU / g.

[0016] Further, the feed additive is a feed additive for aquatic feed.

[0017] Further, the feed additive can be added into basic feed for aquatic animals at a proportion of 0.1wt%-0.5wt%.

[0018] Further, the environmental sterilizing agent comprises the bacteriophage of Edwardsiella tarda.

[0019] Further, the environmental sterilizing agent is an environmental sterilizing agent for aquatic products.

[0020] Further, the environmental sterilizing agent for aquatic products further comprises SM buffer or sterilized water for breeding of aquatic animals.

[0021] Further, the titer of the bacteriophage in the environmental sterilizing agent for aquatic products is greater than or equal to 10 9 PFU / mL.

[0022] Further, the pH value of the environmental sterilizing agent for aquatic products is 4-12.

[0023] Compared with the prior art, the present application has the beneficial effects that:

[0024] The present application takes pathogenic Edwardsiella tarda as a host, and a bacteriophage of Edwardsiella tarda is isolated, which is named vB-EtM_GDYR5 in the present application. The vB-EtM_GDYR5 has the characteristics of rapid and high lysis of Edwardsiella tarda, so as to solve the problems of common bacteriostasis lag and weak bacteriostasis in the early stage of the existing Edwardsiella tarda bacteriophage. The vB-EtM_GDYR5 can be used for preparing products such as drugs for inhibiting or killing Edwardsiella tarda, environmental bactericides or feed additives.

[0025] (1) Strong bactericidal ability: the existing reports of Edwardsiella tarda bacteriophage have a bacteriostasis lag in the early stage of infection, for example, the Edwardsiella tarda bacteriophage PKP-ET-2022001 has obvious bacteriostatic effect after 2.5 hours. The vB-EtM_GDYR5 provided by the present application has rapid and strong lysis ability to Edwardsiella tarda. In vitro experiments show that the vB-EtM_GDYR5 can significantly inhibit the proliferation of Edwardsiella tarda within 1 hour, and can continuously inhibit the proliferation of Edwardsiella tarda within 0h~14h. Therefore, the vB-EtM_GDYR5 has shorter bacteriostasis time, better bactericidal effect, and can continuously inhibit the proliferation of pathogenic bacteria. At the same time, the animal experiment results of the present application show that the injection of vB-EtM_GDYR5 can significantly reduce the load of Edwardsiella tarda in the liver and spleen within 2 days, and has obvious protective effect on animals.

[0026] (2) Wide strain spectrum: the lysis coverage of vB-EtM_GDYR5 to Edwardsiella tarda strains from different sources is 92%, and has a wide strain spectrum.

[0027] (3) Good stability: the titer of vB-EtM_GDYR5 is stable within the pH value range of 4.0~12.0, and the titer still remains at 1.05×10 4 PFU / mL after 20min under the condition of 65℃ high temperature, which shows that the vB-EtM_GDYR5 has good acid and alkali resistance and temperature resistance, and can maintain activity under different environmental conditions.

[0028] (4) High safety: the vB-EtM_GDYR5 genome does not contain antibiotic resistance genes and virulence factors, and will not cause adverse effects on the environment and other organisms. The vB-EtM_GDYR5 can be safely used in aquaculture environment, avoiding the problems of antibiotic residues, and is conducive to protecting the quality of aquatic products and ecological environment.

[0029] The Edwardsiella tarda bacteriophage vB-EtM_GDYR5 is named vB-EtM_GDYR5 in the present application, and the classification name is Edwardsiella tardavirus, the depositary: China General Microbiological Culture Collection Center, the depositary address: No. 1, Beichen West Road, Yuanmingyuan District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, the preservation number: CGMCC No. 46408, and the preservation date is March 21, 2025. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0031] Figure 1 The plaque formed by the bacteriophage vB-EtM_GDYR5 of the present application; Figure 1 The A figure of is a plate figure, Figure 1 The black box selected in the A figure of is the region where the plaque is formed; Figure 1 The B figure of is Figure 1 The A figure of is an enlarged view of the black box selected, Figure 1 The two arrows in the B figure of indicate two plaques formed.

[0032] Figure 2 The plaque formed by the bacteriophage vB-EtM_GDYR5, Figure 2 The A figure of is a plaque formed by 5 μL, 1.0×10 2 PFU / mL bacteriophage, Figure 2 The B figure of is a plaque formed by 5 μL, 1.0×10 4 PFU / mL bacteriophage, Figure 2 The C figure of is a plaque formed by 5 μL, 1.0×10 6 PFU / mL bacteriophage.

[0033] Figure 3 The electron microscope photograph of the bacteriophage vB-EtM_GDYR5 of the present application.

[0034] Figure 4 The optimal infection multiple of the bacteriophage vB-EtM_GDYR5 of the present application.

[0035] Figure 5 The acid-base stability of the bacteriophage vB-EtM_GDYR5 of the present application.

[0036] Figure 6 The temperature stability of the bacteriophage vB-EtM_GDYR5 of the present application.

[0037] Figure 7 One-step growth curve of the bacteriophage vB-EtM_GDYR5 of the present application Figure 7 The two dotted lines in the figure distinguish the first two major phases of the bacteriophage life cycle, namely the latent phase and the burst phase.

[0038] Figure 8 Genome map of the bacteriophage vB-EtM_GDYR5 of the present application

[0039] Figure 9 In vitro killing curve of the bacteriophage vB-EtM_GDYR5 of the present application, MOI stands for multiplicity of infection.

[0040] Figure 10 Survival curve of the Micropterus salmoides in each group of the animal protection test of the bacteriophage vB-EtM_GDYR5 of the present application, E stands for the infection group, A+E stands for the antibiotic treatment group after infection, P+E stands for the bacteriophage treatment group after infection, and PBS stands for the blank group.

[0041] Figure 11 In vivo killing effect of the bacteriophage vB-EtM_GDYR5 of the present application in the animal protection test Figure 11 The A figure is the Edwardsiella tarda load in the spleen Figure 11 The B figure is the Edwardsiella tarda load in the liver Figure 11 E stands for the infection group, A+E stands for the antibiotic treatment group after infection, and P+E stands for the bacteriophage treatment group after infection; different letters a, b, and c indicate that there is a significant difference between groups. DETAILED DESCRIPTION

[0042] In order to facilitate the understanding of the present application, the present application will be described more fully below, and preferred embodiments of the present application will be given. However, the present application can be realized in many different forms, and is not limited to the embodiments described in the present application. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive.

[0043] Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terms used in the present application in the specification of the present application are only for the purpose of describing the specific embodiments, and are not intended to limit the present application.

[0044] The beneficial effects of the present application will be illustrated below through specific embodiments.

[0045] Edwardsiella tarda is an important pathogen in aquaculture, which has multiple drug resistance, leading to poor effect of antibiotic treatment. Although phage therapy has potential, the existing reported E. tarda phages have weak bacteriostatic effect in the early stage of infection, resulting in low lysis efficiency and difficulty in quickly removing the pathogen. Even if the phage has high lysis ability, the delay of the early bacteriostasis will lead to poor treatment effect and low protection effect on the host. These problems seriously restrict the effective prevention and control of E. tarda.

[0046] The present application provides an Edwardsiella tarda bacteriophage and its application. The Edwardsiella tarda bacteriophage vB-EtM_GDYR5 is obtained by screening from the water for breeding of Micropterus salmoides. The results of host screening, in vitro experiment, in vivo experiment and genome analysis show that the Edwardsiella tarda bacteriophage vB-EtM_GDYR5 provided by the present application can quickly inhibit the proliferation of Edwardsiella tarda and has high lysis rate on Edwardsiella tarda. The bacteriophage has strong alkali resistance and certain acid resistance, certain temperature stability, and the genome of the bacteriophage does not contain drug resistance genes and virulence genes, and the strain spectrum is wide, and the bacteriophage can be used for preparing products such as drugs, environmental bactericides or feed additives for inhibiting or killing Edwardsiella tarda.

[0047] In the following examples, unless otherwise specified, all are conventional methods.

[0048] In the following examples, unless otherwise specified, all materials, reagents, etc. can be obtained from commercial channels.

[0049] The strains GDYR1, GDYR2, GDYR4, GDYR5, GDZB1, GDZB2, GDZB3, GDFP1, GDFP2, GDFP3, GDFP4, GDFP5, GDZS1, GDZS2, GDTS1 and GDTS2 in the present application are purchased from Shandong Xingde Technology Co., Ltd.

[0050] Sample and source: Micropterus salmoides and water samples were collected from a Micropterus salmoides breeding farm in Yangjiang, Guangdong, and the sampling time was May 2024.

[0051] Example 1: Isolation and purification of bacteriophage vB-EtM_GDYR5

[0052] 2mL of breeding water collected from a Micropterus salmoides breeding farm in Yangjiang, Guangdong was filtered to remove bacteria, then added to a sterile 10mL centrifuge tube, followed by adding 5mL of LB broth medium and 1mL of 1×10 8The Edwardsiella tarda bacterial solution with 2.8 x 1010CFU / mL was incubated at 37°C for 4 h on a constant temperature shaker. The culture solution was filtered to remove bacteria using a 0.22 μm filter membrane, and the filtrate was stored at 4°C. 5 mL of LB solid culture medium was added to a sterile plate to obtain an LB solid culture medium plate. Then, 200 μL of the Edwardsiella tarda bacterial solution with 1 x 105CFU / mL was added to the LB solid culture medium plate, and the plate was incubated at 37°C for 8 h. The number of colonies on the plate was counted to determine the number of viable bacteria. 8 The Edwardsiella tarda bacterial solution with 2.8 x 1010CFU / mL was mixed with 5 mL of LB semi-solid culture medium cooled to 55°C, and then poured onto the prepared LB solid plate medium. After the medium was allowed to solidify, a double-layer plate was obtained. 10 μL of the filtrate was dropped onto the medium of the double-layer plate, and after the filtrate was air-dried, the plate was incubated at 37°C for 8 h. The generation of plaques was observed. Then, a plaque was picked up using a sterile gun tip and placed in SM buffer to shake off the phage. After filtration using a 0.22 μm filter membrane, the supernatant obtained was the phage solution.

[0053] The phage solution was purified by a double-layer plate method. The phage solution was diluted 1000-fold, and 100 μL of the diluted phage solution was mixed with 100 μL of the Edwardsiella tarda bacterial solution and 5 mL of LB semi-solid culture medium cooled to 55°C. The mixture was poured onto the prepared LB solid plate medium to obtain a double-layer plate. After incubation at 37°C for 8 h, a single plaque was repeatedly picked up and plated on a double-layer plate for purification for 6 times, until plaques with uniform morphology and size were formed on the plate.

[0054] The results are shown in Figure 1 FIG. and the plaque diameter was 2 mm to 3 mm. The edge of the plaque was smooth and transparent, and no halo was observed. As shown in Figure 2 FIG., the plaque appeared transparent, and no resistant colonies were generated. According to the international naming rules, the phage was named vB-EtM_GDYR5.

[0055] Example 2: Electron microscope observation of the phage

[0056] The Edwardsiella tarda bacterial solution with 2.8 x 1010CFU / mL was incubated at 37°C for 4 h on a constant temperature shaker. The culture solution was filtered to remove bacteria using a 0.22 μm filter membrane, and the filtrate was stored at 4°C. 5 mL of LB solid culture medium was added to a sterile plate to obtain an LB solid culture medium plate. Then, 200 μL of the Edwardsiella tarda bacterial solution with 1 x 105CFU / mL was added to the LB solid culture medium plate, and the plate was incubated at 37°C for 8 h. The number of colonies on the plate was counted to determine the number of viable bacteria. 9 The phage solution with 2.8 x 1010PFU / mL was dropped onto a copper grid, and allowed to stand for 15 min. Then, 50 μL of 2% phosphotungstic acid solution with a pH value of 7.0 was added to the copper grid to negatively stain the phage particles for 10 min. Then, the excess stain was absorbed from the side using dry filter paper, and the phage vB-EtM_GDYR5 was observed under a transmission electron microscope after natural standing for 30 min.

[0057] As shown in Figure 3It can be seen that the head of the bacteriophage vB-EtM_GDYR5 is icosahedral, with a spherical head and a retractable tail, the head length is about 9.4 nm, the cross diameter is about 8.6 nm, and the tail length is about 13.7 nm. According to the 9th Report of the International Committee on the Taxonomy of Viruses, it can be judged that the bacteriophage belongs to the Caudovirales order and the Myoviridae family.

[0058] Example 3: Determination of the optimal multiplicity of infection of the bacteriophage vB-EtM_GDYR5

[0059] The Edwardsiella tarda GDYR5 was inoculated into 5 mL of LB liquid medium and cultured at 37°C with 200 rpm shaking until the bacterial solution concentration was 10 8 CFU / mL. The bacteriophage titer was enriched to 10 9 PFU / mL, and after sterilization by 0.22 μm filter membrane, the bacteriophage titer in the solution was diluted to 10 6 PFU / mL with SM buffer. Bacteriophage-Edwardsiella tarda mixed solutions with a multiplicity of infection of 10, 1, 0.1, 0.01, 0.001, and 0.0001 were prepared, respectively, and after incubation at 37°C for 15 min, 5 mL of preheated LB medium was added to the mixed solutions with different multiplicities of infection, and the mixture was cultured at 37°C with 200 rpm shaking for 4 h. After sterilization by 0.22 μm filter membrane, the bacteriophage titer was determined by double-layer plate method. The experiment was repeated three times and the average value was taken.

[0060] The multiplicity of infection = the number of bacteriophages / the number of host cells.

[0061] The results are shown in Figure 4 When the multiplicity of infection was 0.001, the bacteriophage titer was the highest, which was 1.47×10 10 PFU / mL, so the optimal multiplicity of infection was determined to be 0.001.

[0062] Example 4: Acid-base stability of the bacteriophage vB-EtM_GDYR5

[0063] 1 mol / L HCl solution and 1 mol / L NaOH solution were prepared, and LB liquid medium with pH values of 1.0-14.0 was prepared using the HCl solution and the NaOH solution. 100 μL of bacteriophage liquid with a titer of 10 9 PFU / mL was added to 900 μL of LB liquid medium with each pH value and vortexed for 20 s, and then incubated at 37°C for 2 h in a water bath. Immediately after, the mixture was ice-bathed for 5 min to terminate the reaction. The titer of the bacteriophage under different pH values was determined by double-layer plate method, and each sample was repeated three times.

[0064] The results are shown in Figure 5 Within the pH range of 4.0-12.0, the bacteriophage titer was stable, all >106 PFU / mL; but when the pH value was 2.0, the phage titer decreased to 1.66 x 10 4 PFU / mL; when the pH value was < 2.0 or the pH value was > 12.0, the phage titer decreased to < 10 4 PFU / mL.

[0065] The above results show that the phage vB-EtM_GDYR5 can tolerate weak acid and weak base conditions and has good acid and alkali tolerance. The phage vB-EtM_GDYR5 has an optimal growth pH value of 4.0-12.0 in a neutral environment.

[0066] Example 5: Temperature stability of the phage vB-EtM_GDYR5

[0067] The phage liquid was divided into centrifuge tubes and incubated in a constant temperature water bath at 4°C, 37°C, 55°C, 65°C and 75°C, respectively, for 20 min, 40 min and 60 min, respectively. The control group was placed on ice. After incubation, the phage liquid was diluted with sterile PBS solution, and the phage titer was determined by double-layer plate method. Each group had 3 replicates.

[0068] The results are shown in Table 3. Figure 6 As shown in Table 3, at 4°C-55°C, the phage titer did not change significantly, all > 10 8 PFU / mL; at 65°C for 20 min, the phage titer was 1.05 x 10 4 PFU / mL; after 40 min at 65°C, the phage titer decreased to 0; after treatment at 75°C, the phage titer decreased to 0.

[0069] Example 6: Determination of host spectrum of the phage vB-EtM_GDYR5

[0070] Twelve strains of Edwardsiella tarda and four strains of Edwardsiella ictaluri were selected, and double-layer plate method was used to detect whether the phage vB-EtM_GDYR5 had lytic activity on different strains and to determine the host spectrum of the phage vB-EtM_GDYR5. Edwardsiella tarda GDYR1, GDYR2, GDYR4 and GDYR5 were isolated from Yangjiang, Guangdong; Edwardsiella tarda GDZB1, GDZB2 and GDZB3 and Edwardsiella ictaluri GDZS1 and GDZS2 were isolated from Zhuhai, Guangdong; Edwardsiella tarda GDFP1, GDFP2, GDFP3, GDFP4 and GDFP5 were isolated from Foshan, Guangdong; and Edwardsiella ictaluri GDTS1 and GDTS2 were isolated from Jining, Shandong.

[0071] The criteria for determining the lysis of the bacteriophage to the strain is as follows: +4 represents large and transparent plaque, and the bacteria are completely lysed; +3 represents transparent plaque, but with weak hazy background; +2 represents incomplete lysis, and the turbidity of the spotted area is large; +1 represents that there are individual bacteriophage plaques in the spotted area; and - represents no plaque.

[0072] The results are shown in Table 1. The lysis coverage of the bacteriophage vB-EtM_GDYR5 of the present application to Edwardsiella tarda strains is 92%, and the strain spectrum is wide; but the bacteriophage vB-EtM_GDYR5 has no lysis ability to Edwardsiella piscicida strains.

[0073] Table 1 Lysis spectrum of the bacteriophage vB-EtM_GDYR5 of Edwardsiella tarda

[0074]

[0075] Example 7: Determination of one-step growth curve of the bacteriophage vB-EtM_GDYR5

[0076] S1, according to the optimal infection multiple of 0.001, the host bacteria liquid with a concentration of 10 8 CFU / mL is mixed with the bacteriophage liquid with a titer of 10 4 PFU / mL, the total volume of the mixed solution is 1 mL, and the mixed solution is incubated at 37°C for 5 min.

[0077] S2, the mixed solution after incubation is centrifuged at 4°C and 8000 rpm for 5 min, and then the centrifugal supernatant is removed, and 1 mL of LB liquid medium at 37°C is resuspended to resuspend the centrifugal precipitate. This step is repeated twice again to remove the unabsorbed bacteriophage.

[0078] S3, the mixed solution is transferred to 100 mL of LB medium, and is cultured at 37°C and 200 rpm. In order to optimize the monitoring of bacteriophage growth, 200 μL of culture sample is taken at 0 min, 10 min, 15 min and 20 min in the initial mixed culture, and then 1 mL of culture sample is taken every 10 min, and the culture sample taken each time needs to be immediately terminated by ice bath, and after being filtered by a 0.22 μm filter membrane, it is stored at -4°C. The one-step growth curve of the bacteriophage vB-EtM_GDYR5 is drawn with time as the horizontal axis and the titer of the bacteriophage as the vertical axis.

[0079] The burst size = the total number of bacteriophages at the end of the burst to the total number of bacteria at the beginning of the bacteriophage.

[0080] The results are shown in Table 1. The lysis coverage of the bacteriophage vB-EtM_GDYR5 of the present application to Edwardsiella tarda strains is 92%, and the strain spectrum is wide; but the bacteriophage vB-EtM_GDYR5 has no lysis ability to Edwardsiella piscicida strains. Figure 7As shown, the latent period of the phage vB-EtM_GDYR5 was about 40 min, which was roughly the same as that of most Edwardsiella tarda phages. The number of phages increased rapidly from 40 min to 260 min after the host bacteria were infected, which was the burst period of the phage, and the burst size was 14 PFU / mL.

[0081] Example 8: Phage vB-EtM_GDYR5 lysis rate experiment

[0082] Under sterile conditions, 1 mL of the phage solution and 1 mL of the host bacteria solution with a concentration of 1 x 10 6 CFU / mL were mixed, and then incubated at 37°C for 15 min. The mixed solution after incubation was diluted to 10 -1 , 10 -2 , and 10 -3 times of the original concentration of the mixed solution with 1 mL of PBS solution, respectively. 50 μL of each of the three gradient dilutions was spread on LB agar plates, and the plates were incubated at 37°C for 24 h. Each gradient was repeated three times. The mixed solution obtained by mixing 1 mL of PBS solution and 1 mL of the host bacteria solution with a concentration of 1 x 10 6 CFU / mL was used as a blank control, and the above operation was repeated. The gradient dilution samples were counted by selecting plates containing 30 to 300 colonies.

[0083] The phage lysis rate = (1 - number of colonies in the treatment group / number of colonies in the control group) x 100%.

[0084] According to the calculation, the lysis rate of the phage vB-EtM_GDYR5 on the strain GDYR5 was as high as 99%, which was suitable for use in aquaculture.

[0085] Example 9: Genome analysis of the phage vB-EtM_GDYR5

[0086] The phage obtained by the double-layer plate amplification method was centrifuged at 12000 g at 4°C for 10 min to obtain the supernatant. After sterilization by filtration, the genomic DNA of the phage vB-EtM_GDYR5 was extracted by the phenol-chloroform method, and the qualified DNA was sent to a sequencing company for sequence determination.

[0087] According to the sequencing, the full-length genome of the phage vB-EtM_GDYR5 was 147345 bp, and the GC content was 47.84%. The genome circle diagram is shown in Figure 8The results show that the phage genome is annotated with 180 open reading frames, and the sequence alignment of protein-coding genes is completed by using diamond software, and 140 proteins are annotated. This provides the possibility for the expression of broad-spectrum lysozyme and perforin, so as to expand the application range of vB-EtM_GDYR5 and improve the bacteriostatic effect. It can be predicted through the CARD database and the VFDB database that the phage vB-EtM_GDYR5 genome does not contain antibiotic resistance genes and virulence factors, and can be safely used in aquaculture environment.

[0088] Example 10: In vitro bactericidal effect of the phage vB-EtM_GDYR5

[0089] The Edwardsiella tarda GDYR5 was inoculated into LB culture medium and cultured at 37°C to the logarithmic growth phase. Different titers of phage solution were mixed with the GDYR5 bacterial solution to obtain mixed solutions with a multiplicity of infection of 10, 1, 0.1, and 0.01, which were then placed in 5 mL of LB broth medium and cultured at 37°C with 200 rpm shaking; three repeated experiments were set for each experimental group, and the bacterial solution without phage was used as the control group. Sampling was performed every 1 h, and the OD 600 value of the bacterial solution was determined by using a spectrophotometer to draw a Figure 9 .

[0090] As can be seen from Figure 9 , the phage vB-EtM_GDYR5 has a strong inhibitory effect on the growth of Edwardsiella tarda. The addition of the phage vB-EtM_GDYR5 can significantly inhibit the proliferation of Edwardsiella tarda within 1 h, and the OD 600 value of the GDYR5 bacterial solution remains at the initial OD 600 value from 0 h to 14 h after phage infection, and has a downward trend, which indicates that the phage can lyse Edwardsiella tarda in a very short time and has a strong lytic effect on Edwardsiella tarda. The existing reported Edwardsiella tarda lytic phages have weak bacteriostatic effects in the early stage of infection, resulting in low lytic efficiency and difficulty in achieving rapid pathogenic bacteria removal. For example, the Edwardsiella tarda phage PRE03 starts to have obvious bacteriostatic effect relative to the control group after 5 h of addition, the Edwardsiella tarda phage EPP-1 starts to have obvious bacteriostatic effect after 9 h of addition, the Edwardsiella tarda phage VB_EpM_ZHS starts to have obvious bacteriostatic effect after 3 h of addition, and the Edwardsiella tarda phage PKP-ET-2022001 starts to have obvious bacteriostatic effect after 2.5 h of addition. Therefore, compared with other Edwardsiella tarda lytic phages, the phage vB-EtM_GDYR5 provided by the present application has shorter bactericidal time, better bactericidal effect, and can continuously inhibit the proliferation of Edwardsiella tarda, and has more application potential.

[0091] Example 11: A medicine

[0092] The bacteriophage solution was diluted with sterile PBS buffer to a titer of 1.2 x 10 4 PFU / mL of bacteriophage solution, filtered through a 0.22 μm filter membrane, and packaged to obtain a medicine.

[0093] Example 12: In vivo bactericidal effect of bacteriophage vB-EtM_GDYR5

[0094] Largemouth bass were purchased from a breeding factory in Liaocheng, Shandong, and 120 healthy largemouth bass with a body length of about 12 cm were selected for the test. The water temperature was controlled at 27°C during cultivation, and commercial feed was normally fed.

[0095] The largemouth bass were divided into 4 groups, 30 in each group: the blank group was marked as PBS, the infection group was marked as E, the antibiotic treatment group after infection was marked as A+E, and the bacteriophage treatment group after infection was marked as P+E. The treatment methods of each group were as follows, and the number of deaths of each group of largemouth bass was counted daily.

[0096] The blank group: 100 μL of PBS solution was injected intraperitoneally, and 3 h later, 100 μL of SM buffer was injected intraperitoneally.

[0097] The infection group: 100 μL of Edwardsiella tarda bacterial solution with a concentration of 1.2 x 10 7 CFU / mL was injected intraperitoneally, and 3 h after the bacterial solution, 100 μL of SM buffer was injected intraperitoneally.

[0098] The antibiotic treatment group after infection: 100 μL of Edwardsiella tarda bacterial solution with a concentration of 1.2 x 10 7 CFU / mL was injected intraperitoneally, and 3 h after the bacterial solution, 100 μL of florfenicol solution diluted with SM buffer to a concentration of 10 mg / mL was injected intraperitoneally.

[0099] The bacteriophage treatment group after infection: 100 μL of Edwardsiella tarda bacterial solution with a concentration of 1.2 x 10 7 CFU / mL was injected intraperitoneally, and 3 h after the bacterial solution, 100 μL of the medicine prepared in Example 11 was injected intraperitoneally.

[0100] The number of deaths of each group of largemouth bass was counted as shown in Figure 10 The blank group of largemouth bass grew normally; however, by the 7th day, the cumulative mortality rate of the E group was 100%; the cumulative mortality rate of the A+E group was 76.7%, and the survival rate was 23.3%; and the cumulative mortality rate of the P+E group was 16.7%, and the survival rate was 83.3%.

[0101] The above results show that the relative protection rate of the bacteriophage vB-EtM_GDYR5 on the largemouth bass is 83.3%. And the survival protection effect is stronger than that of the florfenicol antibiotic.

[0102] Again, the same experimental grouping and experimental treatment as described above were set up. From 0d to 7d after infection, 3 largemouth bass were randomly sampled from each group every day, and the spleen and liver tissue samples of the largemouth bass were collected under sterile conditions, in duplicate: one for bacteriophage load detection, and the other for Edwardsiella tarda load detection. The spleen and liver tissue samples of the largemouth bass were ground thoroughly in sterile PBS, and the ground tissue homogenate was evenly divided into two parts: one was filtered with a 0.22 μm filter membrane to remove bacteria, and the double-layer plate method was used to measure the phage titer; the other was gradient diluted and plated to calculate the number of colonies. Each sample was tested in triplicate.

[0103] As shown in FIG. A of the drawings, Figure 11 In the spleen, there was no difference in the relative Edwardsiella tarda load among the groups on the first day after infection; on the second day, the relative bacterial load of the E group was 5.52×10 8 CFU / g, and the relative bacterial load of the P+E group was 3.84×10 8 CFU / g; on the third day, the relative bacterial load of the E group was 8.26×10 9 CFU / g, and the relative bacterial load of the P+E group was 2.8×10 6 CFU / g.

[0104] It can be seen that the Edwardsiella tarda load decreased significantly after injection of the drug prepared in Example 11.

[0105] As shown in FIG. B of the drawings, Figure 11 In the liver, on the first day after infection, the relative bacterial load of the E group was 2.73×10 9 CFU / g, and the relative bacterial load of the P+E group was 5.63×10 6 CFU / g, and the drug prepared in Example 11 significantly inhibited the proliferation of Edwardsiella tarda in the liver within 1 day after injection, and rapidly killed Edwardsiella tarda; from 1 day to 3 days after infection, Edwardsiella tarda slowly proliferated in the E group, while Edwardsiella tarda in the P+E group continued to decrease to 4.11×10 4 CFU / g.

[0106] It can be seen that, compared with the untreated and antibiotic-treated groups, the Edwardsiella tarda load in the liver and spleen can be rapidly reduced within 1 day to 2 days after infection with Edwardsiella tarda after treatment with the drug provided by the application.

[0107] Example 13: A water product feed additive

[0108] 100 mL Edwardsiella tarda bacteriophage liquid is mixed with 50 g trehalose, vortexed to completely dissolve to obtain a mixed solution; the inlet temperature is set to 45 DEG C, the outlet temperature is set to 30 DEG C, and the mixed solution is spray dried to obtain a bacteriophage freeze-dried powder; 15 g of the bacteriophage freeze-dried powder is mixed with 850 g of microcrystalline cellulose in a three-dimensional mixer for 30 min to ensure uniformity, then 100 g / bag is packed into an aluminum foil bag, sealed with nitrogen, and stored at 4 DEG C to obtain a water product feed additive. The Edwardsiella tarda bacteriophage titer in the water product feed additive is 10 7 PFU / g.

[0109] Example 14: An environmental disinfectant

[0110] Water for aquaculture is sterilized; 3 kg of sodium alginate is added to 50 L of sterilized water, stirred in a 40 DEG C water bath for 1 hour until completely dissolved to obtain a stabilizer solution; the Edwardsiella tarda bacteriophage liquid is filtered through a 0.22 μm filter membrane to obtain an Edwardsiella tarda bacteriophage suspension; the Edwardsiella tarda bacteriophage suspension, 50 L of the stabilizer solution, and the sterilized water for aquaculture are mixed to 1 m 3 L, and then stirred uniformly with a blender to obtain an environmental disinfectant, wherein the bacteriophage titer in the environmental disinfectant is 10 9 PFU / mL, and the pH value is 7.0.

[0111] The use method of the environmental disinfectant is whole pond spraying; the preventive dosage is 2 m 3 / acre, twice a month; the treatment dosage is 5 m 3 / acre, and is used continuously for 3 days.

[0112] It should be noted that when numerical ranges are involved in the present application, both endpoints of each numerical range and any number between the two endpoints can be selected. Since the same steps and examples are used, the preferred embodiments are described in the present application to prevent redundancy. Although the preferred embodiments of the present application have been described, those skilled in the art can make additional changes and modifications to the embodiments once they know the basic inventive concept. Therefore, the appended claims are intended to include the preferred embodiments and all changes and modifications falling within the scope of the present application.

[0113] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application is also intended to include these modifications and variations.

Claims

1. A bacteriophage of Edwardsiella tarda, characterized in that, The Edwardsiella tarda bacteriophage is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 46408, and the classification name is Edwardsiella tarda virus; the Edwardsiella tarda bacteriophage can inhibit the proliferation of Edwardsiella tarda within 1 hour, and continuously inhibit the proliferation of Edwardsiella tarda within 0-14 hours.

2. Use of the Edwardsiella tarda bacteriophage in the preparation of a product for inhibiting or killing Edwardsiella tarda according to claim 1.

3. Use of a bacteriophage of Edwardsiella tarda according to claim 2 for the preparation of a product for inhibiting or killing Edwardsiella tarda, characterized in that, The product is a medicine, a feed additive or an environmental bactericide.

4. Use of a bacteriophage of Edwardsiella tarda according to claim 3 for the preparation of a product for inhibiting or killing Edwardsiella tarda, characterized in that, The effective component of the medicine is the Edwardsiella tarda bacteriophage.

5. Use of a bacteriophage of Edwardsiella tarda according to claim 4 for the preparation of a product for inhibiting or killing Edwardsiella tarda, characterized in that, The dosage form of the medicine is powder, solution, emulsion, gel, granule or freeze-dried agent.

6. Use of a bacteriophage of Edwardsiella tarda according to claim 5 for the preparation of a product for inhibiting or killing Edwardsiella tarda, characterized in that, The dosage form of the drug is a solution; the titer of Edwardsiella tarda bacteriophage in the drug is ≥ 1.2 × 10 4 PFU / mL.

7. Use of the bacteriophage of Edwardsiella tarda according to claim 3 in the manufacture of a product for inhibiting or killing Edwardsiella tarda, characterized in that, The feed additive has a bacteriophage of Edwardsiella tarda with a titer of ≥10 7 PFU / g.

8. Use of the bacteriophage of Edwardsiella tarda according to claim 3 in the manufacture of a product for inhibiting or killing Edwardsiella tarda, characterized in that, The phage titer in the environmental sterilant is ≥ 10 9 PFU / mL.

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