Edwardsiella tarda bacteriophage and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to an edwardsiella tarda bacteriophage and application thereof, the edwardsiella tarda bacteriophage is classified and named as Edwardsiella tarda bacteriophage, and is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.46408. The invention further discloses a preparation method of the edwardsiella tarda bacteriophage. The edwardsiella tarda bacteriophage is infected with a plurality of gt; the bacteriophage can inhibit the growth of edwardsiella tarda within 1h when the temperature is 0.01, has the cracking rate of 99% for edwardsiella tarda GDYR5, is high in cracking property, can rapidly kill edwardsiella tarda, and is acid and alkali resistant, high in temperature stability, wide in strain spectrum and high in safety. The compound can be used for preparing products such as drugs for inhibiting or killing edwardsiella tarda, environmental bactericides or feed additives.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to an Edwardsiella tarda phage and its application. Background Art

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

[0003] The current main means of preventing and controlling Edwardsiella infection is the use of 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 show multidrug resistance, which exacerbates the difficulty of preventing and controlling Edwardsiella infection, and the antibiotic residues will pollute the water environment and further harm human health through food chain enrichment.

[0004] As natural bacterial predators, phages have a high abundance in nature. Phage therapy provides a breakthrough solution for the prevention and control of Edwardsiella tarda in aquaculture due to its specificity, high efficiency, safety, and environmental friendliness. However, the reported Edwardsiella tarda phages have a weak antibacterial effect in the initial stage of infection, resulting in low lysis efficiency and difficulty in rapidly clearing pathogenic bacteria. Even if the phage has a high lysis ability, the delayed antibacterial effect in the early stage will lead to poor treatment effect and low protection effect on the host. Summary of the Invention

[0005] Aiming at the problems of delayed antibacterial effect and poor lysis ability of existing Edwardsiella tarda phages, the present invention provides an Edwardsiella tarda phage and its application. The Edwardsiella tarda phage is a phage with high lysis ability, which can rapidly kill Edwardsiella tarda, is acid and alkali resistant, has strong temperature stability, a wide strain spectrum, and high safety, and can be used to prepare products such as drugs for inhibiting or killing Edwardsiella tarda, environmental fungicides, or feed additives.

[0006] To achieve the above object, the specific technical solutions of the present invention are as follows: In the first aspect of the present invention, a phage of Edwardsiella tarda is provided. The phage of Edwardsiella tarda is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number of CGMCC No. 46408, and the taxonomic name is Edwardsiella tardavirus .

[0007] In the second aspect of the present invention, an application of the above-mentioned phage of Edwardsiella tarda in preparing a product for inhibiting or killing Edwardsiella tarda is provided.

[0008] Furthermore, the product is a drug, a feed additive or an environmental germicide.

[0009] Furthermore, the active ingredient of the drug is the above-mentioned phage of Edwardsiella tarda.

[0010] Furthermore, the drug also contains a pharmaceutically acceptable carrier.

[0011] Furthermore, the dosage form of the drug is powder, solution, emulsion, gel, granule or lyophilized product.

[0012] Furthermore, the dosage form of the drug is solution.

[0013] Furthermore, the titer of the phage of Edwardsiella tarda in the drug is ≥1.2×10 4 PFU / mL.

[0014] Furthermore, the titer of the phage of Edwardsiella tarda in the feed additive is ≥10 7 PFU / g.

[0015] Furthermore, the feed additive is a feed additive for aquatic feed.

[0016] Furthermore, the feed additive can be added to the basic feed for aquatic animals at a ratio of 0.1wt% - 0.5wt%.

[0017] Furthermore, the environmental germicide contains the above-mentioned phage of Edwardsiella tarda.

[0018] Furthermore, the environmental germicide is an environmental germicide for aquatic products.

[0019] Furthermore, the environmental germicide for aquatic products also contains SM buffer solution or sterilized water for aquaculture of aquatic animals.

[0020] Furthermore, the titer of the phage in the environmental germicide for aquatic products is ≥10 9 PFU / mL.

[0021] Furthermore, the pH value of the environmental germicide for aquatic products is 4 - 12.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: Using pathogenic Edwardsiella tarda as the host, a phage of Edwardsiella tarda was isolated, which is named vB-EtM_GDYR5 in the present invention. vB-EtM_GDYR5 has the characteristics of rapidly and highly lysing Edwardsiella tarda, so as to solve the problems of generally lagging antibacterial effect and relatively weak antibacterial effect in the early stage existing in existing Edwardsiella tarda phages. It can be used to prepare products such as drugs for inhibiting or killing Edwardsiella tarda, environmental bactericides or feed additives.

[0023] (1) Potent bactericidal ability: Existing reports show that there is a phenomenon of delayed antibacterial effect in the initial stage of infection by Edwardsiella tarda phages. For example, an obvious antibacterial effect began to appear 2.5 hours after the addition of Edwardsiella tarda phage PKP-ET-2022001. However, vB-EtM_GDYR5 provided by the present invention has a rapid and powerful lysing ability against Edwardsiella tarda. In vitro experiments show that the addition of vB-EtM_GDYR5 can significantly inhibit the proliferation of Edwardsiella tarda within 1 hour, and can continuously inhibit the proliferation of Edwardsiella tarda between 0h and 14h. Therefore, vB-EtM_GDYR5 has a shorter antibacterial time, better bactericidal effect, and can continuously inhibit the proliferation of pathogenic bacteria. At the same time, the animal experiment results of the present invention show that injecting vB-EtM_GDYR5 can significantly reduce the load of Edwardsiella tarda in the liver and spleen within 2 days, and has an obvious protective effect on animals.

[0024] (2) Wide strain spectrum: The lysis coverage rate of vB-EtM_GDYR5 against Edwardsiella tarda strains from different sources is 92%, showing a relatively wide strain spectrum.

[0025] (3) Good stability: The titer of vB-EtM_GDYR5 is stable in the range of pH value from 4.0 to 12.0. After acting at 65°C for 20 minutes, the titer still remains above 1.05×10 4 PFU / mL, indicating that it has good acid-base tolerance and temperature tolerance, and can maintain its activity under different environmental conditions.

[0026] (4) High safety: The genome of vB-EtM_GDYR5 does not contain antibiotic resistance genes and virulence factors, will not cause adverse effects on the environment and other organisms, and can be safely used in the aquaculture environment, avoiding problems such as antibiotic residues, which is beneficial to ensuring the quality of aquatic products and the ecological environment.

[0027] The Edwardsiella tarda phage vB-EtM_GDYR5, named vB-EtM_GDYR5 in the present invention, is classified as Edwardsiella tardavirus, Preservation unit: China General Microbiological Culture Collection Center, Address of the preservation unit: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Preservation number: CGMCC No. 46408, Preservation date: March 21, 2025. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0029] Figure 1 The plaque formed by the phage vB-EtM_GDYR5 of the present invention; Figure 1 Figure A of is a flat plate diagram, Figure 1 The area where the formed plaque is located is selected by the black frame in Figure A of ; Figure 1 Figure B of is Figure 1 An enlarged view of the area selected by the black frame in Figure A of, Figure 1 The two arrows in Figure B of indicate the two formed plaques.

[0030] Figure 2 The plaque formed by spotting the phage vB-EtM_GDYR5, Figure 2 Figure A of is the plaque formed by spotting 5 μL, 1.0×10 2 PFU / mL phage, Figure 2 Figure B of is the plaque formed by spotting 5 μL, 1.0×10 4 PFU / mL phage, Figure 2 Figure C of is the plaque formed by spotting 5 μL, 1.0×10 6 PFU / mL phage.

[0031] Figure 3 The electron microscope photograph of the phage vB-EtM_GDYR5 of the present invention.

[0032] Figure 4 The optimal multiplicity of infection of the phage vB-EtM_GDYR5 of the present invention.

[0033] Figure 5 The acid-base stability of the phage vB-EtM_GDYR5 of the present invention.

[0034] Figure 6 The temperature stability of the phage vB-EtM_GDYR5 of the present invention.

[0035] Figure 7 This is the one-step growth curve of the phage vB-EtM_GDYR5 of the present invention. Figure 7 The two dashed lines in it distinguish the first two main stages of the phage life cycle, namely the latent period and the burst period.

[0036] Figure 8 This is the circular map of the genome of the phage vB-EtM_GDYR5 of the present invention.

[0037] Figure 9 This is the in vitro bactericidal curve of the phage vB-EtM_GDYR5 of the present invention, and MOI represents the multiplicity of infection.

[0038] Figure 10 This is the survival curve of each group of largemouth bass in the animal protection test of the phage vB-EtM_GDYR5 of the present invention. E represents the infection group, A+E represents the group treated with antibiotics after infection, P+E represents the group treated with phage after infection, and PBS represents the blank group.

[0039] Figure 11 This is the in vivo bactericidal effect of the phage vB-EtM_GDYR5 in the animal protection test of the present invention; Figure 11 Figure A of is the load of Edwardsiella tarda in the spleen; Figure 11 Figure B of is the load of Edwardsiella tarda in the liver; Figure 11 In it, E represents the infection group, A+E represents the group treated with antibiotics after infection, P+E represents the group treated with phage after infection; different letters a, b, c indicate significant differences between groups. Detailed implementation manners

[0040] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are given. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0041] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the description of the present invention in this specification are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0042] The beneficial effects of the present invention are illustrated below through specific embodiments.

[0043] Edwardsiella tarda is an important pathogenic bacterium in aquaculture, with multidrug resistance, resulting in poor antibiotic treatment effects. Although phage therapy has potential, the reported Edwardsiella tarda phages have weak antibacterial effects in the initial stage of infection, resulting in low lysis efficiency and difficulty in quickly clearing pathogenic bacteria. Even if the phage has a high lysis ability, the delayed antibacterial effect in the early stage will lead to poor treatment effects and low protection effects on the host. These problems severely restrict the effective prevention and control of Edwardsiella tarda.

[0044] The present invention provides an Edwardsiella tarda phage and its application. The Edwardsiella tarda phage vB-EtM_GDYR5 was isolated and screened from the culture water of largemouth bass. Through host screening, in vitro experiments, in vivo experiments and genome analysis, the results show that: the Edwardsiella tarda phage vB-EtM_GDYR5 provided by the present invention can quickly inhibit the proliferation of Edwardsiella tarda and has a high lysis rate for Edwardsiella tarda; the phage has strong alkalinity resistance and certain acid resistance, and has certain temperature stability. At the same time, the phage genome does not contain drug resistance genes and virulence genes, and has a relatively wide strain spectrum, and can be used to prepare products such as drugs, environmental fungicides or feed additives for inhibiting or killing Edwardsiella tarda.

[0045] In the following examples, unless otherwise specified, all are conventional methods.

[0046] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0047] The strains GDYR1, GDYR2, GDYR4, GDYR5, GDZB1, GDZB2, GDZB3, GDFP1, GDFP2, GDFP3, GDFP4, GDFP5, GDZS1, GDZS2, GDTS1 and GDTS2 in the present invention were purchased from Shandong Xind Technology Co., Ltd.

[0048] Samples and sources: Largemouth bass and water samples were from a largemouth bass farm in Yangjiang, Guangdong. The sampling time was May 2024.

[0049] Example 1: Isolation and purification of phage vB-EtM_GDYR5 Take 2 mL of the culture water collected from the largemouth bass farm in Yangjiang, Guangdong. After filtration and sterilization, add it to a sterile 10 mL centrifuge tube, and then add 5 mL of LB broth medium and 1 mL of live bacteria with a count of 1×10 8Edwardsiella tarda bacterial solution at CFU / mL was cultured in a constant temperature shaker at 37°C for 4 h. The culture solution was filtered through a 0.22 μm filter membrane to remove bacteria, and the filtrate was obtained and stored at 4°C. 5 mL of LB solid medium was added to a sterile petri dish to obtain an LB solid medium plate. Then, 200 μL of Edwardsiella tarda bacterial solution with a viable count of 1×10 8 CFU / mL was mixed with 5 mL of LB semi-solid medium cooled to 55°C and poured onto the prepared LB solid plate medium. After standing and solidifying, a double-layer plate was obtained. 10 μL of the filtrate was pipetted onto the medium of the double-layer plate, and after standing and waiting for the filtrate to air-dry, it was placed in a constant temperature incubator at 37°C for 8 h to observe the formation of plaques. Then, a sterile pipette tip was used to pick out the plaques and placed in SM buffer and shaken to precipitate the phages. After filtration through a 0.22 μm filter membrane, the supernatant obtained was the phage solution.

[0050] Purify the phages by the double-layer plate method: After diluting the above phage solution 1000-fold, 100 μL of the diluted phage solution was mixed with 100 μL of the host Edwardsiella tarda bacterial solution and 5 mL of LB semi-solid medium cooled to 55°C, and poured onto the prepared LB solid plate medium. A double-layer plate was prepared again. After culturing at 37°C for 8 h, single plaques were repeatedly picked out and the double-layer plate was laid for purification operation 6 times until plaques with uniform morphology and size were formed on the plate.

[0051] The results are as Figure 1 shown in the figure. The plaque diameter is 2 mm - 3 mm, with a smooth and translucent edge and no halo. As Figure 2 shown, the plaques that appeared as drops were translucent and no resistant colonies were produced. According to the international naming rules, this phage was named vB-EtM_GDYR5.

[0052] Example 2: Electron microscopy observation of phages 20 μL of phage solution at 2.8×10 9 PFU / mL was pipetted onto a copper mesh and left to air-dry for 15 min. Subsequently, 50 μL of phosphotungstic acid solution with a pH value of 7.0 and a mass concentration of 2% was added to the copper mesh to negatively stain the phage particles for 10 min. Then, a dry filter paper was used to absorb the excess stain from the side, and after naturally air-drying for 30 min, the morphology of phage vB-EtM_GDYR5 was observed under a transmission electron microscope.

[0053] By Figure 3It can be seen that the head of phage vB-EtM_GDYR5 is icosahedral, with a spherical head and a contractile tail. The major axis of the head is about 9.4 nm, the minor axis is about 8.6 nm, and the tail length is about 13.7 nm. According to the 9th Report of the International Committee on Taxonomy of Viruses, this phage can be judged to belong to the order Caudovirales and the family Myoviridae.

[0054] Example 3: Determination of the optimal multiplicity of infection of phage vB-EtM_GDYR5 Inoculate Edwardsiella tarda GDYR5 into 5 mL of LB liquid medium and incubate it at 37 °C with shaking at 200 rpm until the bacterial concentration reaches 10 8 CFU / mL. Enrich the phage titer to 10 9 PFU / mL. After filtering and sterilizing through a 0.22 μm filter membrane, dilute the phage titer in the solution to 10 6 PFU / mL with SM buffer. Prepare phage-Edwardsiella tarda mixed solutions with multiplicities of infection of 10, 1, 0.1, 0.01, 0.001, and 0.0001 respectively. After incubating at 37 °C for 15 min, add 5 mL of pre-warmed LB medium to the mixed solutions with different multiplicities of infection, and incubate at 37 °C with shaking at 200 rpm for 4 h. After filtering and sterilizing through a 0.22 μm filter membrane, determine the phage titer by the double-layer plate method. The experiment was repeated three times and the average value was taken.

[0055] Multiplicity of infection = number of phages / number of host cells.

[0056] The results are as Figure 4 shown. When the multiplicity of infection is 0.001, the phage titer is the highest, which is 1.47×10 10 PFU / mL. Therefore, the optimal multiplicity of infection is determined to be 0.001.

[0057] Example 4: Acid-base stability of phage vB-EtM_GDYR5 Prepare 1 mol / L HCl solution and 1 mol / L NaOH solution, and use the HCl solution and NaOH solution to prepare LB liquid medium with pH values ranging from 1.0 to 14.0. Take 100 μL of 10 9 PFU / mL phage liquid and add it to 900 μL of LB liquid medium with each pH value, vortex for 20 s, incubate in a water bath at 37 °C for 2 h, and immediately ice-bath the mixture for 5 min to terminate the reaction. Determine the phage titer under different pH values by the double-layer plate method, and repeat each sample three times.

[0058] The results are as Figure 5 shown: within the range of pH values from 4.0 to 12.0, the phage titer is stable, all > 10 6PFU / mL; However, when the pH value is 2.0, the phage titer drops to 1.66×10 4 PFU / mL; When the pH value < 2.0 or pH value > 12.0, the phage titer drops to < 10 4 PFU / mL.

[0059] The above results show that the phage vB-EtM_GDYR5 can tolerate weak acid and weak base conditions and has good acid-base tolerance. The optimal growth pH value of the phage vB-EtM_GDYR5 is a neutral environment of 4.0 - 12.0.

[0060] Example 5: Temperature stability of phage vB-EtM_GDYR5 The phage liquid was aliquoted into centrifuge tubes and incubated in a constant temperature water bath at 4°C, 37°C, 55°C, 65°C, and 75°C for 20 min, 40 min, and 60 min respectively, and the control group was placed on ice. After the incubated phage liquid was serially diluted with sterile PBS solution, the phage titer was determined by the double-layer plate method. Three replicates were set for each group.

[0061] The results are as Figure 6 shown. When treated at 4°C - 55°C for 20 min, 40 min, and 60 min, the phage titer showed no significant change and was all > 10 8 PFU / mL; When treated at 65°C for 20 min, the phage titer was 1.05×10 4 PFU / mL. After treatment at 65°C for 40 min, the phage titer dropped to 0; after treatment at 75°C, the phage titer all dropped to 0.

[0062] Example 6: Determination of the host spectrum of phage vB-EtM_GDYR5 Twelve strains of Edwardsiella tarda and four strains of Edwardsiella piscicida were selected, and the double-layer plate method was used to detect whether the phage vB-EtM_GDYR5 was lytic to different strains, and the host spectrum of the phage vB-EtM_GDYR5 was determined. Edwardsiella tarda GDYR1, GDYR2, GDYR4, GDYR5 were isolated from Yangjiang, Guangdong; Edwardsiella tarda GDZB1, GDZB2, GDZB3 and Edwardsiella piscicida GDZS1, GDZS2 were isolated from Zhuhai, Guangdong; Edwardsiella tarda GDFP1, GDFP2, GDFP3, GDFP4, GDFP5 were isolated from Foshan, Guangdong; Edwardsiella piscicida GDTS1 and GDTS2 were isolated from Jining, Shandong.

[0063] The criteria for determining the lytic ability of phages against strains are as follows: +4 indicates large and clear plaques and complete lysis of bacteria; +3 indicates clear plaques but with a faint hazy background; +2 indicates incomplete lysis and high turbidity in the spotting area; +1 indicates individual phage plaques in the spotting area; - indicates no plaques.

[0064] As shown in Table 1, the lysis coverage rate of the phage vB-EtM_GDYR5 of the present invention against Edwardsiella tarda strains is 92%, showing a broad strain spectrum; however, the phage vB-EtM_GDYR5 has no lytic ability against Edwardsiella piscicida strains.

[0065] Table 1 Lysis spectrum of Edwardsiella tarda phage vB-EtM_GDYR5

[0066] Example 7: Determination of the one-step growth curve of phage vB-EtM_GDYR5 S1. According to the optimal multiplicity of infection of 0.001, mix the host bacterial liquid with a concentration of 10 8 CFU / mL and the phage liquid with a titer of 10 4 PFU / mL. The total volume of the mixed solution is 1 mL, and incubate at 37 °C for 5 min with static.

[0067] S2. Centrifuge the incubated mixed solution at 4 °C and 8000 rpm for 5 min, then remove the centrifuged supernatant, and then aspirate 1 mL of LB liquid medium at 37 °C to resuspend the centrifuged precipitate. Repeat this step two more times to remove unadsorbed phages.

[0068] S3. Transfer the mixed solution to 100 mL of LB medium and culture it with shaking at 37 °C and 200 rpm. To optimize the monitoring of phage growth, 200 μL of culture solution samples are respectively aspirated at 0 min, 10 min, 15 min, and 20 min at the initial stage of mixed culture, and then 1 mL of culture solution samples are aspirated every 10 min. Each aspirated culture solution sample needs to be immediately terminated by ice bath, filtered through a 0.22 μm filter membrane, and stored at -4 °C. Draw the one-step growth curve of phage vB-EtM_GDYR5 with time as the horizontal axis and phage titer as the vertical axis.

[0069] Burst size = total number of phages at the end of phage burst / total number of bacteria at the initial stage of phages.

[0070] The results are as Figure 7As shown, the latent period of phage vB-EtM_GDYR5 is about 40 min, which is roughly the same as that of most Edwardsiella tarda phages. From 40 min to 260 min after infecting the host bacteria, the number of phages increases rapidly. This period is the burst period of the phages, and the burst size is 14 PFU / mL.

[0071] Example 8: Lysis rate experiment of phage vB-EtM_GDYR5 Under sterile conditions, 1 mL of phage solution and 1 mL of host bacteria solution with a concentration of 1×10 6 CFU / mL were mixed and incubated at 37°C for 15 min. Then, the incubated mixed solution was diluted to 10 -1 、10 -2 、10 -3 times with sterile PBS solution. 50 μL of the three gradient dilutions were respectively pipetted and spread on LB agar plates, and cultured at 37°C for 24 h. Each gradient was repeated 3 times. A mixed solution obtained by mixing 1 mL of PBS solution and host bacteria solution with a concentration of 1×10 6 CFU / mL was used as a blank control, and the above operations were repeated. The samples were serially diluted, and plates with 30 to 300 colony counts were selected for counting. The average value was calculated for the experiment.

[0072] Phage lysis rate = (1 - number of colonies in treatment group / number of colonies in control group) × 100%.

[0073] After calculation, the lysis rate of phage vB-EtM_GDYR5 against strain GDYR5 is as high as 99%, which is suitable for use in the process of aquaculture.

[0074] Example 9: Genome analysis of phage vB-EtM_GDYR5 The phage obtained by the double-layer plate amplification method was centrifuged at 12,000 g and 4°C for 10 min to obtain the centrifuged supernatant. After filtering and sterilizing the supernatant, the genomic DNA of phage vB-EtM_GDYR5 was extracted using the phenol-chloroform method, and the DNA with qualified quality was sent to a sequencing company for sequence determination.

[0075] After sequencing, the full length of the genome of phage vB-EtM_GDYR5 is 147,345 bp, and the GC content is 47.84%. The genome circular map is shown in Figure 8The results showed that a total of 180 open reading frames were annotated in the phage genome, and the sequence alignment of protein-coding genes was completed using the diamond software, with a total of 140 proteins annotated. This provides the possibility for the expression of lytic enzymes and perforins with broad-spectrum properties, so as to expand the application range of vB-EtM_GDYR5 and enhance the antibacterial effect. Through prediction by the CARD database and VFDB database, it can be known that the phage vB-EtM_GDYR5 genome does not contain antibiotic resistance genes and virulence factors, and can be safely used in the aquaculture environment.

[0076] Example 10: In vitro bactericidal effect of phage vB-EtM_GDYR5 Edwardsiella tarda GDYR5 was inoculated into LB medium and cultured at 37 °C until the logarithmic growth phase. Phage solutions with different titers were mixed with the GDYR5 bacterial solution to obtain mixed solutions with a multiplicity of infection of 10, 1, 0.1, and 0.01, and then they were respectively placed in 5 mL of LB broth medium and cultured with shaking at 37 °C and 200 rpm; each experimental group was set with 3 replicate experiments, and the bacterial solution without phage was used as the control group. Samples were taken every 1 h, and the OD 600 value of the bacterial solution was measured using a spectrophotometer to plot Figure 9 .

[0077] It can be seen from Figure 9 that phage vB-EtM_GDYR5 has a strong inhibitory effect on the growth of Edwardsiella tarda. The addition of phage vB-EtM_GDYR5 can significantly inhibit the proliferation of Edwardsiella tarda within 1 h, and within 0 h to 14 h of phage infection, the OD 600 value of the GDYR5 bacterial solution remained at the initial OD 600 value and showed a downward trend, indicating that the phage can lyse Edwardsiella tarda in a very short time and has a strong lytic effect on Edwardsiella tarda. The reported phages that can lyse Edwardsiella have a weak antibacterial effect in the initial stage of infection, resulting in low lysis efficiency and difficulty in achieving rapid pathogen clearance. For example, the bacteriophage PRE03 of Edwardsiella piscicida showed an obvious antibacterial effect 5 h after addition compared with the control group, the bacteriophage EPP-1 of Edwardsiella piscicida showed an obvious antibacterial effect 9 h after addition, the bacteriophage VB_EpM_ZHS of Edwardsiella piscicida showed an obvious antibacterial effect 3 h after addition, and the bacteriophage PKP-ET-2022001 of Edwardsiella tarda showed an obvious antibacterial effect 2.5 h after addition. Therefore, compared with other phages that can lyse Edwardsiella, the phage vB-EtM_GDYR5 provided by the present invention has a shorter bactericidal time, better bactericidal effect, and can continuously inhibit the proliferation of Edwardsiella tarda, and has greater application potential.

[0078] Example 11: A drug Dilute the phage liquid with sterile PBS buffer to a phage solution with a titer of 1.2×10 4 PFU / mL. After filtering through a 0.22 μm filter membrane, it is encapsulated to obtain a drug.

[0079] Example 12: In vivo bactericidal effect of phage vB-EtM_GDYR5 Purchase Micropterus salmoides from a breeding farm in Liaocheng, Shandong. Select 120 healthy Micropterus salmoides with a body length of about 12 cm for the experiment. During the breeding period, the water temperature is controlled at 27°C, and commercial feed is fed normally.

[0080] Divide the Micropterus salmoides into 4 groups, with 30 tails in each group: the blank group is labeled as PBS, the infection group is labeled as E, the antibiotic treatment group after infection is labeled as A+E, and the phage treatment group after infection is labeled as P+E. The treatment methods for each group are as follows, and the number of dead Micropterus salmoides in each group is counted daily.

[0081] Blank group: Intraperitoneally inject 100 μL of PBS solution. After 3 h, intraperitoneally inject 100 μL of SM buffer.

[0082] Infection group: Intraperitoneally inject 100 μL of Edwardsiella tarda bacterial solution with a concentration of 1.2×10 7 CFU / mL. After 3 h of bacterial solution infection, intraperitoneally inject 100 μL of SM buffer.

[0083] Antibiotic treatment group after infection: Intraperitoneally inject 100 μL of Edwardsiella tarda bacterial solution with a concentration of 1.2×10 7 CFU / mL. After 3 h of bacterial solution infection, intraperitoneally inject 100 μL of florfenicol solution with a concentration of 10 mg / mL diluted with SM buffer.

[0084] Phage treatment group after infection: Intraperitoneally inject 100 μL of Edwardsiella tarda bacterial solution with a concentration of 1.2×10 7 CFU / mL. After 3 h of bacterial solution infection, intraperitoneally inject 100 μL of the drug prepared in Example 11.

[0085] The statistical results of the number of dead Micropterus salmoides in each group are as Figure 10 shown. The Micropterus salmoides in the blank group grew normally; but 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%; the cumulative mortality rate of the P+E group was 16.7%, and the survival rate was 83.3%.

[0086] The above results show that the relative protection rate of phage vB-EtM_GDYR5 against Micropterus salmoides is 83.3%. And the survival protection effect is stronger than that of florfenicol antibiotic.

[0087] Set the same experimental groups and experimental treatments as above again. From 0 d to 7 d after infection, 3 largemouth bass were randomly sampled from each group every day. The spleen and liver tissue samples of largemouth bass were collected under sterile conditions in duplicate: one for detecting phage load and one for detecting the load of Edwardsiella tarda. The spleen and liver tissue samples of largemouth bass were ground thoroughly in sterile PBS. The ground tissue homogenate was evenly divided into two parts: one was filtered through a 0.22 μm filter membrane to remove bacteria, and the phage titer was measured using the double-layer plate method; the other was diluted serially and spread on plates to calculate the number of colonies. Each sample was subjected to three repeated experiments.

[0088] As Figure 11 shown in Figure A of 8 , in the spleen, there was no difference in the relative load of Edwardsiella tarda among groups on the 1st day after infection; on the 2nd day, the relative bacterial load in group E was 5.52×10 8 CFU / g, and the relative bacterial load in group P+E was 3.84×10 9 CFU / g; on the 3rd day, the relative bacterial load in group E was 8.26×10 9 CFU / g, and the relative bacterial load in group P+E was 2.8×10 6 CFU / g.

[0089] Thus, it can be seen that the load of Edwardsiella tarda decreased significantly after injecting the drug prepared in Example 11.

[0090] As Figure 11 shown in Figure B of 9 , in the liver, on the 1st day after infection, the relative bacterial load in group E was 2.73×10 6 CFU / g, and the relative bacterial load in group P+E was 5.63×10 6 CFU / g. The proliferation of Edwardsiella tarda in the liver was significantly inhibited within 1 day after injecting the drug prepared in Example 11, and Edwardsiella tarda was rapidly killed; from 1 day to 3 days after infection, Edwardsiella tarda slowly proliferated in group E, while in group P+E, Edwardsiella tarda continued to decline to 4.11×10 4 CFU / g.

[0091] Thus, it can be seen that compared with the untreated and antibiotic-treated groups, after treatment with the drug provided by the present invention, the load of Edwardsiella tarda in the liver and spleen can be rapidly reduced from the 1st day to the 2nd day after infection with Edwardsiella tarda.

[0092] Example 13: An aquatic feed additive Mix 100 mL of Edwardsiella tarda phage liquid with 50 g of trehalose and vortex until completely dissolved to obtain a mixed solution; set the inlet temperature at 45 °C and the outlet temperature at 30 °C, and spray-dry the mixed solution to obtain phage lyophilized powder; mix 15 g of phage lyophilized powder with 850 g of microcrystalline cellulose in a three-dimensional mixer for 30 min to ensure uniformity, then pack it into aluminum foil bags at 100 g / bag, fill with nitrogen and seal, and store at 4 °C to obtain an aquatic feed additive. The titer of Edwardsiella tarda phage in this aquatic feed additive is 10 7 PFU / g.

[0093] Example 14: An environmental germicide Sterilize the water for aquaculture animals; add 3 kg of sodium alginate to 50 L of sterilized water and stir in a water bath at 40 °C for 1 hour until completely dissolved to obtain a stabilizer solution; filter the Edwardsiella tarda phage liquid through a 0.22 μm filter membrane to obtain an Edwardsiella tarda phage suspension; mix the Edwardsiella tarda phage suspension, 50 L of the stabilizer solution and the sterilized water for aquaculture animals to 1 m 3 , stir evenly with a blender to obtain an environmental germicide. The titer of phage in this environmental germicide is 10 9 PFU / mL, and the pH value is 7.0.

[0094] The usage method of the environmental germicide is to sprinkle it all over the pond; the preventive dosage is 2 m 3 / mu, twice a month; the treatment dosage: 5 m 3 / mu, continuously used for 3 days.

[0095] It should be noted that when the present invention involves numerical ranges, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the adopted step methods are the same as those in the examples, in order to prevent repetition, the present invention describes the preferred examples. Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0096] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A Edwardsiella tarda phage, characterized in that, The Edwardsiella tarda phage is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number of CGMCC No. 46408, and the taxonomic name is Edwardsiella tardavirus .

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

3. Use of the Edwardsiella tarda phage according to claim 2 in the preparation of a product for inhibiting or killing Edwardsiella tarda, characterized in that, The product is a drug, feed additive or environmental germicide.

4. Use of the Edwardsiella tarda phage according to claim 3 in the preparation of a product for inhibiting or killing Edwardsiella tarda, characterized in that, The active ingredient of the drug is the Edwardsiella tarda phage.

5. Use of the Edwardsiella tarda phage according to claim 4 in the preparation of a product for inhibiting or killing Edwardsiella tarda, characterized in that, The dosage form of the drug is powder, solution, emulsion, gel, granule or lyophilized product.

6. Use of the Edwardsiella tarda phage according to claim 5 in 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 phage in the drug ≥ 1.2×10 4 PFU / mL.

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

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

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