Edwardsiella bacteriophage, and bacteriophage composition and application thereof

Abstract

The invention belongs to the technical field of bacteriophages, and discloses an edwardsiella bacteriophage, a bacteriophage composition and application thereof, the bacteriophage is named as edwardsiella bacteriophage PREE 50, the bacteriophage is preserved in China General Microbiological Culture Collection Center on May 26, 2025, and the preservation number of the bacteriophage is CGMCC NO. The preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.46488. The bacteriophage PREE 50 is wide in lysis spectrum, high in lysis rate for various types of edwardsiella, good in acid-base stability and temperature stability and capable of being used for preparing drugs, water disinfectants, aquatic feed additives, detection kits and the like for treating or preventing edwardsiella infection, and the bacteriophage PREE 50 can be applied to preparation of drugs, water disinfectants, aquatic feed additives, detection kits and the like while solving edwardsiella infection. And the problems of antibiotic residues and pathogen drug resistance caused by the use of antibiotics are avoided.

Description

Technical Field

[0001] This invention relates to the field of bacteriophage technology, and more particularly to an Edwardsiella edodes bacteriophage, its bacteriophage composition, and its applications. Background Technology

[0002] Edwardsiella is an important pathogenic bacterium in fish. It is a Gram-negative bacterium belonging to the Enterobacteriaceae family and possessing peritrichous flagella. The genus *Edwardella* currently mainly includes five species: *Edwardella tarda*, *Edwardella basilicata*, *Edwardella catfish*, *Edwardella pisciformis*, and *Edwardella eel*. Based on the different fish hosts they infect, these five species are divided into two categories: one with weaker pathogenicity to fish hosts, including *Edwardella tarda* and *Edwardella basilicata*, while *Edwardella catfish*, *Edwardella pisciformis*, and *Edwardella eel* are highly pathogenic to fish hosts.

[0003] Edwardsiella faecium is one of the major pathogens seriously affecting aquaculture. High-density farming of commercially important fish such as turbot and flounder easily leads to outbreaks of Edwardsiella faecium disease in fish. Flounder infected with this bacterium exhibit symptoms such as pigmentation, cloudy and protruding eyes, petechiae and hemorrhages on the skin, kidney and liver necrosis, and ascites. To avoid the adverse effects of antibiotic resistance on aquaculture and public health, my country enacted relevant laws in 2020 to prohibit or restrict the use of antibiotics and actively sought appropriate antibiotic alternatives to promote animal gut health. Against this backdrop of antibiotic prohibition, bacteriophages have received widespread attention due to their antibacterial properties.

[0004] Bacteriophages are obligate parasites with high specificity, destroying only their corresponding host bacteria without affecting other bacterial groups, and dying as the host bacteria are eliminated. When bacteriophages infect host bacteria, their self-encoded depolymerases and lysins degrade polysaccharides and peptidoglycans, disrupting extracellular polymeric structures and lysing biofilms. The successful isolation and application of numerous lysing bacteriophages of pathogenic bacteria in aquaculture animals demonstrate the great potential of using bacteriophages to replace antibiotics in the control of bacterial diseases in aquaculture animals.

[0005] However, there are currently no bacteriophages with high lytic performance specifically targeting Edwardsiella tarda. Existing bacteriophage products are difficult to use effectively to prevent and control diseases caused by Edwardsiella tarda, so the existing technology needs further improvement. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides an Edwardsiella edodes phage PRE50, its phage composition, and its applications. The Edwardsiella edodes phage PRE50 not only has a broad lytic spectrum, capable of lysing multiple Edwardsiella strains, but also exhibits heat resistance, making it effective in preventing and controlling Edwardsiella edodes infections in aquatic environments. This phage can be used as an active ingredient in the preparation of drugs for treating or preventing Edwardsiella edodes infections, as well as environmental disinfectants, effectively controlling diseases caused by Edwardsiella edodes in aquaculture farms.

[0007] To address the above problems, this application provides the following technical solution: In the first aspect, this application provides an Edwardsiella phage strain, named Edwardsiella phage PRE50, with accession number CGMCC No. 46488.

[0008] The Edwardsiella phage was isolated from a farm in Shandong Province and named Edwardsiella phage PRE50. It was deposited on May 26, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo. 46488.

[0009] In this application, phage PRE50 also includes mutant strains of the phage with accession number CGMCC No. 46488 that exhibit point mutations, deletion mutations, or addition mutations in their genome and maintain substantially the same bactericidal activity, with homology exceeding 98% or 99%. Since phages are highly susceptible to mutation during replication, mutants of the aforementioned phage are also within the scope of protection claimed in this application. The sequence of phage PRE50 can be obtained by sequencing the biological material deposited according to this invention using known methods. For those skilled in the art, screening for mutants with extremely similar characteristics to the phage provided by this invention requires no inventive effort.

[0010] Under a transmission electron microscope, phage PRE50 was observed to have a polyhedral head structure and a contractile tail. The head is 88-94 nm wide and 82-88 nm long, and the tail is about 95-105 nm long. According to the classification method of the International Committee on Taxonomy of Viruses (ICTV), the morphology of this phage is consistent with the characteristics of the Longtail Phagesidae family and belongs to Myotail Phage.

[0011] The described Edwardsiella edodes phage PRE50 exhibits broad-spectrum lytic activity against Edwardsiella bacteria, showing specific lytic advantages against various species, including Edwardsiella yucca, Edwardsiella eelii, Edwardsiella tarda, Edwardsiella campestris, and Edwardsiella catfishii. Experiments have demonstrated that this phage can lyse 34 out of 40 different types of Edwardsiella strains, achieving a lysis rate as high as 85%. Notably, the lysis rate against the major pathogen, Edwardsiella yucca, is as high as 94.73%, indicating promising application prospects in the prevention and control of Edwardsiella yucca disease in aquatic animals.

[0012] The Edwardsiella edodes phage PRE50 exhibits good acid-base stability and certain temperature stability. Its activity remains stable within a pH range of 5.0–10.0. After 2 hours of treatment at pH 2.0, the titer remains at 10. 5 It has a concentration of pfu / mL, which can resist certain strong acid environments and has strong stability in acidic environments, and can adapt to a certain range of strong acid environments.

[0013] Furthermore, the phage PRE50 exhibits good storage stability. Experiments have shown that its titer did not change significantly after 12 months of storage at 4°C, decreased by only one grade after 12 months of storage at 18°C, and decreased by only two grades after 12 months of storage at 30°C.

[0014] Secondly, this application also provides a phage composition comprising the aforementioned Edwardsiella edodes phage PRE50 and other phages.

[0015] In practical applications, in order to further broaden the lysis spectrum of phage preparations, give full play to the differences in the lysis spectra of different phages, and complement each other's advantages, Edwardsiella edodes phage PRE50 can be used in combination with other phages.

[0016] When used in combination with Edwardsiella tarda phage PRE03 or other Edwardsiella tarda phages, its bactericidal spectrum can be broadened to kill as many Edwardsiella tarda bacteria in the environment as possible, thus preventing and controlling Edwardsiella tarda disease. Furthermore, the aforementioned Edwardsiella tarda phage PRE50 can also be combined with other types of phages for the simultaneous prevention and control of multiple aquatic diseases.

[0017] Thirdly, this application also provides the use of the above-mentioned Edwardsiella phage or the above-mentioned phage composition in the preparation of drugs, environmental disinfectants and test kits for preventing and treating aquatic diseases caused by Edwardsiella infection.

[0018] The prevention and treatment include both prevention and treatment. The term "prevention" as used herein refers to all behaviors that suppress or delay the disease by administering the composition. The term "treatment" as used herein refers to all behaviors that improve or alleviate the disease by administering the composition.

[0019] Optionally, the aquatic products include: zebrafish, turbot, flounder and other flatfish, tilapia and amphibians.

[0020] Optionally, in the application, the diseases caused by Edwardsiella infection include: visceral white spot disease and acute sepsis.

[0021] Fourthly, this application also provides a phage drug formulation, the active ingredient of which includes the above-mentioned Edwardsiella edodes phage PRE50 or the above-mentioned phage composition.

[0022] Optionally, in the phage drug formulation, the drug formulation is in the form of an oral dosage form, a topical dosage form, or a parenteral dosage form.

[0023] Optionally, the phage drug formulation is a spray-dried powder, which is mainly composed of the following components mixed and spray-dried: phage PRE50 proliferation solution, gelatin, sodium glutamate and mannitol.

[0024] Preferably, the mass ratio of phage PRE50 proliferation medium, gelatin, monosodium glutamate, and mannitol is 100:4:2:3. Experiments have shown that, compared to phage proliferation medium, the temperature and pH stability of this phage spray-dried powder are significantly improved.

[0025] This application also provides a method for preparing the above-mentioned phage drug formulation, wherein the preparation method comprises: mixing phage PRE50 proliferation solution, gelatin, sodium glutamate and mannitol in a certain mass ratio and then spray drying.

[0026] Preferably, the spray drying method is as follows: a nano spray dryer is used for drying, and the process conditions are: inlet temperature 80℃, airflow speed 120L / min, spray rate 100%, nozzle cap size 4.0μm, spray drying for about 2 hours, and finally the spray-dried particles are collected, which is the bacteriophage drug preparation.

[0027] Optionally, the phage drug formulation may further comprise a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not cause significant irritation to the organism and does not eliminate the biological activity and properties of the administered active ingredient. For the pharmaceutical composition to be formulated into a liquid formulation, the pharmaceutically acceptable carrier must be suitable for sterility and biocompatibility. Examples include saline, sterile water, Ringer's solution, buffered physiological saline, albumin infusion, glucose solution, maltodextrin solution, glycerol, ethanol, and various culture media. They can be used alone or in any combination thereof. Other conventional additives, such as antioxidants, buffers, and antibacterial agents, may be added as needed.

[0028] When the phage is combined with diluents, dispersants, surfactants, binders and / or lubricants, the phage or phage composition of the present invention can also be prepared into various dosage forms, such as injections and oral dosage forms (e.g., aqueous solutions, suspensions and emulsions, pills, capsules, granules) and other intermediate dosage forms, such as lyophilized agents.

[0029] When applying, the above-mentioned bacteriophage drug preparations can be mixed into aquatic feed or aquaculture water.

[0030] Fifthly, this application also provides an environmental disinfectant, the effective ingredient of which includes the aforementioned Edwardsiella edodes phage PRE50 or the aforementioned phage composition.

[0031] Optionally, the environmental disinfectant may also contain other active ingredients for inhibiting or eliminating bacteria in the environment.

[0032] Sixthly, this application also provides the application of the above-mentioned environmental disinfectant in the disinfection of aquaculture environments, wherein the environmental disinfectant can disinfect the aquaculture environment and feeding equipment with Edwardsiella pneumoniae by spraying or soaking.

[0033] Preferably, the environments in which environmental disinfectants can be applied include feed, water bodies, and aquaculture environments, including ponds, feeding platforms, feeding equipment, and recirculating aquaculture systems. The application methods include, but are not limited to, disinfecting and decontaminating the water distribution system, aquaculture facilities, feeding equipment, or other environmental surfaces of the aquaculture farm through liquid immersion, spraying, or combined use with water-based carriers, and disinfecting and preserving feed. This disinfectant can be used to replace antibiotics or traditional disinfection products and will not cause harm to humans or aquatic animals.

[0034] Seventhly, this application also provides a detection kit comprising the aforementioned Edwardsiella phage or the aforementioned phage composition. The Edwardsiella phage PRE50 of this invention can be used for the rapid detection of Edwardsiella, including but not limited to detecting Edwardsiella in samples (such as water or aquatic tissues) in the form of test strips or test kits, effectively ensuring detection sensitivity, and can also be used to detect diseases caused by specific infection with Edwardsiella.

[0035] The present invention has the following beneficial effects: 1. This invention is the first to isolate a virulent Edwardsiella phage strain PRE50, which has broad-spectrum lytic activity against Edwardsiella spp. It exhibits lytic advantages against different species of Edwardsiella spp., including Edwardsiella spp. ichthyophthirius multifiliis, ... and Edwardsiella spp. ichthyophthirius multifiliis, especially against the main pathogen Edwardsiella spp. ichthyophthirius multifiliis, with a lytic rate as high as 94.73%. It shows great promise for the prevention and control of Edwardsiella spp. disease in aquatic animals.

[0036] 2. Based on the aforementioned biological characteristics, the bacteriophage PRE50 can effectively prevent and control Edwardsiella pneumoniae infection. It can be used to prepare drugs for treating or preventing Edwardsiella pneumoniae infection and environmental disinfectants, thereby effectively controlling diseases caused by Edwardsiella pneumoniae in aquaculture farms, greatly reducing the incidence of Edwardsiella pneumoniae, and benefiting the healthy development of the aquaculture industry. Furthermore, this bacteriophage is easy to industrialize, and the drugs or disinfectants prepared from it not only reduce costs but also have the advantages of being green and environmentally friendly, avoiding the problems of antibiotic residues and pathogen resistance caused by the use of antibiotics.

[0037] 3. The study of the biological characteristics of the bacteriophages isolated by this invention provides theoretical guidance for the next step of developing new antibacterial drug biocontrol agents. Attached Figure Description

[0038] Figure 1 Image of a plaque from Edwardsiella tarda phage PRE50; Figure 2 Electron micrograph of Edwardsiella phage PRE50; Figure 3 The results of pH stability test for Edwardsiella edema phage PRE50; Figure 4 The results of the temperature stability test of Edwardsiella tarda phage PRE50; Figure 5 This is the one-step growth curve of Edwardsiella edodes phage PRE50. Detailed Implementation

[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0040] Example 1: Isolation and purification of Edwardsiella edodes phage PRE50 1. Resuscitation and culture of host bacteria Eight Edwardsiella tarda strains preserved in our laboratory were selected. Streaking was performed on TSA medium with a sterile inoculating tube dipped in the cryopreservation solution. The cultures were then incubated at 37°C for 18-24 h to obtain single colonies. A single colony was picked and inoculated into 5 mL of TSB broth. The cultures were then incubated at 37°C with shaking at 170 rpm for 16 h to obtain fresh Edwardsiella tarda culture.

[0041] 2. Isolation of bacteriophages Thirty water samples from shrimp farms in Shandong Province were placed in a sample flask. An appropriate amount of TSB broth medium was added, along with eight strains of Edwardsiella tarda. The mixture was incubated at 37°C with shaking at 170 rpm / min for 12 hours, centrifuged at 11,000 rpm for 5 minutes, and then filtered through a 0.22 μm sterile microporous membrane to obtain phage proliferation solution. The phage stock solution was diluted 10-fold. Appropriate gradient phage dilutions were taken and mixed with the eight strains of Edwardsiella tarda in a 1:1 ratio. After incubation at 37°C for 5 minutes, 200 μL of the mixture was placed in the upper agar (0.7% concentration). After mixing, the mixture was quickly poured onto a regular lower agar (1.5% concentration) plate, shaken well, and allowed to solidify. The plate was then incubated upside down at 37°C for 4-6 hours to obtain a double-layer plate with phage plaques.

[0042] 3. Purification of bacteriophages A single phage plaque was picked from a double-layer agar medium containing phage and placed in 1 mL of PBS. The plate was incubated at 37°C and 170 rpm for approximately 30 min to obtain phage extract. The phage extract was mixed 1:1 with the corresponding Edwardsiella edodes (hereinafter referred to as the host bacterium) proliferation solution (incubated at 37°C for 5 min). 200 μL of this mixture was transferred to the upper agar layer, mixed thoroughly, and then quickly poured onto the lower agar plate. The plate was shaken well and allowed to solidify. It was then incubated upside down at 37°C for 4–6 h to obtain another double-layer agar plate containing phage plaque. A single phage plaque was picked from the plaque-forming plate using sterile forceps and placed in 1 mL of PBS. The plate was incubated at 37°C and 170 rpm for approximately 30 min to obtain phage extract. This process was repeated three times to obtain purified phage extract.

[0043] 4. Phage proliferation and titer determination Equal volumes of purified phage extract and host bacterial proliferation solution were added to 5 mL of liquid TSB medium and cultured at 37°C and 170 rpm with shaking until the liquid became clear. The clear liquid was centrifuged at 11,000 rpm for 10 min, and the supernatant was collected and filtered through a 0.22 μm sterile microporous membrane to obtain the phage proliferation solution. The titer of the newly isolated phage was determined using the double-layer plate method.

[0044] 5. Experimental Results and Analysis Following the experimental method described above, four Edwardsiella edodes phages were screened from eight Edwardsiella edodes isolates, numbered PRE47 to PRE50. All five Edwardsiella edodes phages formed clear plaques on double-layer agar plates, without surrounding halos, with clearly visible edges, and a diameter of approximately 0.1 mm to 1 mm. The titers of these four phages were 2.7 × 10⁻⁶. 8 PFU / mL 8.4×10 10 PFU / mL (results are shown in Table 1). Among them, phage PRE50 had the highest titer and was used as the experimental subject for various experiments in subsequent examples.

[0045] Table 1. Phage titers (PFU / mL) of the four newly isolated Edwardsiella tarda strains.

[0046] Example 2: Morphological observation and identification of Edwardsiella edodes phage PRE50 1. Copper mesh fabrication and electron microscopic observation Take 20 µL of phage sample and drop it onto a copper grid with a carbon-coated film. Allow it to settle naturally for 15 min. After slightly blotting it with filter paper, stain it with 2% (w / v) phosphotungstic acid (PTA) for 1-2 min. After slightly blotting it with filter paper and allowing it to dry, observe and photograph it under a transmission electron microscope.

[0047] 2. Appraisal Results like Figure 2 As shown, phage PRE50 has a polyhedral head structure and a contractile tail. The head is 88-94 nm wide and 82-88 nm long, and the tail is about 95-105 nm long. According to the classification method of the International Committee on Taxonomy of Viruses (ICTV), the morphology of the phage in this application conforms to the characteristics of the Myotail Phagesidae family and belongs to Myotail Phage.

[0048] Example 3: Determination of PRE50 lysis spectrum of Edwardsiella edodes bacteriophage 1. Experimental Method: Forty Edwardsiella tarda strains of different types isolated from aquaculture farms across the country were selected and preserved in the laboratory. The lysis rate of bacteriophage PRE50 against these 40 Edwardsiella tarda strains was determined by the double-layer plate method. Detailed information on the strains and lysis profiles are shown in Table 2 below.

[0049] Method for detecting lysis rate: Double-layer plate test was performed using the bacterial strain and bacteriophage, and the culture was carried out overnight at 37°C to observe whether lysis was observed.

[0050] 2. Experimental Results and Analysis The results of the cleavage spectrum experiments in Table 2 show that: (1) Edwardsiella phage PRE50 has broad-spectrum lytic activity, and it can lyse 34 of 40 strains of 5 different types of Edwardsiella, with a lysis rate of up to 85%.

[0051] Specifically, this bacteriophage was able to lyse 18 out of 19 strains of Edwardsiella tarda, with a lysis rate of 94.73%; 81.81% against 11 strains of Edwardsiella tarda; 90% against 5 strains of Edwardsiella eel; 66.67% against 3 strains of Edwardsiella basilidis; and 50% against 2 strains of Edwardsiella catfish. Compared with the lysis results of bacteriophage PRE03 disclosed in the previously granted patent CN114736877B, PRE50 showed a higher lysis rate against different types of Edwardsiella tarda than PRE03.

[0052] The results indicate that phage PRE50 has a specific lytic advantage against different types of Edwardsiella, especially against the major pathogen Edwardsiella piscicola, and has a high lytic rate, making it a promising candidate for the prevention and control of Edwardsiella piscicola infection in aquatic animals.

[0053] Table 2. Lysis patterns of Edwardsiella edodes phage PRE50 against 40 Edwardsiella strains.

[0054] Note: "++" indicates a clear and bright spot, "++" indicates a slightly blurred spot, "+" indicates a relatively blurred spot, and "-" indicates no lysis. Example 4: Lysis test of Edwardsiella tarda phage PRE50 against non-host bacteria. 1. Experimental Methods Twenty-five non-host bacteria of different types were selected, including ten Escherichia coli strains, five Staphylococcus strains, five Proteus strains, and five Clostridium perfringens strains. The lysis performance of Edwardsiella phage PRE50 was tested according to the lysis spectrum determination method in Example 3.

[0055] 2. Experimental Results and Analysis No clear plaques were found in the double-layer plates of Edwardsiella phage PRE50 and the above 25 non-host bacteria, indicating that the phage could not recognize the 10 Escherichia coli strains, 5 Staphylococcus strains, 5 Proteus strains, and 5 Clostridium perfringens strains. This shows that the tested phage PRE50 has extremely strong host specificity for Edwardsiella phage and has no damaging effect on other microbial communities. Its specificity can be used to prepare detection kits.

[0056] Example 5: pH tolerance of Edwardsiella edodes phage PRE50 1. Experimental Methods Add 4.5 mL of PBS at different pH values ​​(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) to three sterile test tubes. Then place the test tubes in a 37°C water bath. After the temperature stabilizes, add 500 μL of 1×10⁻⁶ PBS to each tube. 10 Pfu / mL phage proliferation solution was mixed and incubated in a water bath at 37°C for 1 h, 2 h, and 3 h. Immediately after incubation, an appropriate amount of 1 mol / L HCl or NaOH was added to the mixture to adjust the pH to approximately 7. The mixture was then serially diluted 10-fold, and the titer was determined using appropriate dilution gradients. Two replicates were set up for each pH value. A pH stability curve of the phage was plotted with pH value on the x-axis and the logarithm of the phage titer on the y-axis.

[0057] 2. Experimental Results and Analysis from Figure 3 It can be seen that the titer of bacteriophage PRE50 remains at 10 within the pH range of 5.0 to 10.0. 9 The activity was stable at pfu / mL. After 2 hours of treatment at pH 2.0, the potency remained at 10. 5The phage has a pfu / mL concentration, enabling it to resist certain strongly acidic environments; after 3 hours of treatment at pH 11, the titer of this phage remains at 10. 7 PFU / mL, at pH 13, for 3 hours, with a potency not lower than 10. 2 With a concentration of pfu / mL, phage PRE50 exhibits strong stability in acidic environments and can adapt to a certain range of strongly acidic environments.

[0058] Example 6: Temperature tolerance of Edwardsiella edodes phage PRE50 (1) Experimental methods 3×10 10 PFU / mL phage PRE50 proliferation solution was incubated at 65℃, 75℃, and 85℃ (three replicates at each temperature) for 20 min, 40 min, and 60 min respectively. After incubation, the samples were immediately cooled in an ice bath, and the phage titer at different temperatures was determined using the double-layer plate method. Thermostability curves of phage PRE50 were plotted with temperature on the x-axis and the logarithm of the phage titer on the y-axis.

[0059] (2) Experimental results and analysis The results are as follows Figure 4 It is evident that phage PRE50 retains its original activity after being exposed to temperatures below 65°C for 60 minutes; even after exposure to temperatures of 75°C for 40 minutes, the phage still contains 10... 9 PFU / mL viable phage, after incubation at 85℃ for 60 min, still retains some activity at PRE50, with a titer of approximately 1×10⁻⁶. 6 PFU / mL. This shows that Edwardsiella edodes phage PRE50 has good temperature stability and can adapt to high temperature environments.

[0060] Example 7: One-step growth curve of Edwardsiella tarda phage PRE50 1. Experimental Methods Mix 1 mL each of phage proliferation medium with a multiplicity of infection (MOI) of 10 and fresh host bacteria proliferation medium thoroughly (start timing at this point), incubate at 37°C for 5 min, centrifuge at 13000 rpm for 30 s, remove as much supernatant as possible using a micropipette, wash once with 5 mL PBS (centrifuge at 13000 rpm for 30 s), and discard the supernatant. Resuspend the precipitate (total volume 5 mL) in preheated PBS and mix thoroughly, then quickly place in a 37°C shaker at 170 rpm for incubation. At time 0 and every 5 min, take 150 μL and centrifuge at 10000 rpm for 1 min. Perform a 10-fold serial dilution with PBS and determine the phage titer using the double-layer plate method. Perform three replicates and take the average value. Plot a one-step growth curve with infection time as the x-axis and phage titer in the infection system as the y-axis to obtain the phage latency period and outbreak period, and calculate the outbreak quantity.

[0061] Outbreak size = Total number of bacteriophages at the end of the outbreak / Total number of bacteria at the beginning of the outbreak 2. Experimental Results and Analysis from Figure 5 The results showed that after phage PRE50 infected the host bacteria, the phage lysis cycle lasted approximately 90 minutes, the latency period was approximately 20 minutes, and the phage outbreak period was approximately 75 minutes. After 90 minutes, the phage number remained essentially unchanged, entering a stationary phase, at which point the titer could reach 10. 10 pfu / mL, the burst amount of phage PRE50 was 145.

[0062] Example 8: Determination of the optimal multiple of infection (MOI) of Edwardsiella edodes phage PRE50 1. Experimental Methods Colonies were picked and inoculated into 5 ml of TSB medium and incubated at 37°C and 170 rpm for 12–16 h to obtain bacterial suspension. The bacterial concentration was determined using the pour method, and the suspension concentration was adjusted to 1 × 10⁻⁶ based on the results. 9 cfu / mL, 1×10 8 cfu / mL...1×10 5 cfu / mL. The concentration of the bacteriophage isolated in Example 1 was adjusted to 1×10⁻⁶. 5 ~1×10 7 PFU / mL. Phages and bacterial culture were added to TSB medium according to the phage-to-bacterial ratio in Table 3. The medium was incubated at 37°C and 170 rpm in a shaker until the liquid became clear. The incubation time was recorded. An appropriate amount of the clear liquid was centrifuged at 11000 rpm for 10 min. The supernatant was collected and filtered through a 0.22 μm sterile microporous membrane. The phage titer in the filtrate was determined using the double-layer plate method. The MOI (phage count / bacterial count) with the highest phage titer was the optimal multiple of infection (MOI) for that phage.

[0063] 2. Experimental Results Table 3 shows that the phage PRE50 titer reached its highest value (2.3 × 10⁻⁶) at an MOI of 0.001:1. 11 At a concentration of PFU / mL, the initial amount of phage introduced is relatively small, yet the highest reproduction yield can be achieved, which is beneficial for large-scale industrial production.

[0064] Table 3. Titer of Edwardsiella phage PRE50 at different multiplicity of infection

[0065] Example 9: Survival stability test of Edwardsiella tarda phage PRE50 1. Experimental Methods 4×1010 Phage PRE50 proliferation medium with PFU / mL was stored at 4℃, 18℃, and 30℃, and the titer of the phage stored at different temperatures was detected periodically using the double-layer plate method after 1 month, 3 months, 6 months, and 12 months of storage.

[0066] 2. Experimental Results and Analysis The results are shown in Table 4. The results show that the titer of Edwardsiella edodes phage PRE50 did not change significantly after 12 months of storage at 4℃, decreased by one gradient after 12 months of storage at 18℃, and decreased by two gradients after 12 months of storage at 30℃, indicating that Edwardsiella edodes phage PRE50 has good storage stability.

[0067] Table 4. Survival stability of Edwardsiella tarda PRE50

[0068] Example 10: Application of Edwardsiella phage PRE50 in the prevention and treatment of Edwardsiella dysenteriae infection. 1. Experimental Methods Two hundred healthy zebrafish, approximately 3±0.5 cm in length, were selected. Four groups were established: a prevention group, a treatment group, a virus challenge group, and a control group. Each group underwent the following treatments: Prevention group: Bacteriophage PRE50 powder (preparation method as described in Example 12) was mixed evenly with zebrafish feed as a feed additive, and the content of bacteriophage PRE50 in the feed was 1×10⁻⁶. 8 PFU / g.

[0069] Challenge group: Feeded with the same amount of feed, all zebrafish were intraperitoneally injected with Edwardsiella tarda var. tarda 1 hour later at a dose of 3.5 × 10⁻⁶. 2 CFU / tail; the blank control group was given the same dose of conventional feed.

[0070] Treatment group: All zebrafish were intraperitoneally injected with Edwardsiella tarda at a dose of 3.5 × 10⁻⁶. 2 CFU / tail, 1 hour after challenge, the bacteriophage was evenly mixed with feed and fed to the animal. The bacteriophage content in the feed was 1×10⁻⁶. 8 PFU / g.

[0071] Record the number of zebrafish that died within 48 hours in each group and calculate the phage protection rate. After 15 days, samples were taken from the water to test for Edwardsiella tarda and the data were recorded. The specific results are shown in Table 5 below.

[0072] 2. Experimental Results and Analysis As shown in Table 5, the feed containing PRE50 bacteriophage powder significantly reduced the amount of Edwardsiella tarda in the water, effectively preventing fish infection. The PRE50 bacteriophage can effectively treat infected fish and improve the survival rate of zebrafish.

[0073] The above experimental results show that PRE50 phage powder, as a feed additive, has the potential to reduce the number of Edwardsiella tarda in water and protect fish growth, and can provide a new biological control method for aquaculture.

[0074] Table 5. Preventive and therapeutic effects of Edwardsiella edodes phage PRE50 in feed on Edwardsiella edodes.

[0075] Example 11 Safety test of Edwardsiella tarda phage PRE50 1. Experimental Methods Sixty healthy zebrafish, approximately (3±0.5) cm in length, were selected. Experimental and control groups were established. Phage PRE50 was uniformly mixed with the feed, resulting in a phage titer of 1×10⁻⁶. 8 The feed was air-dried and fed twice daily, morning and evening. The experimental group of zebrafish was fed this feed, while the control group was fed the same dose of feed soaked in TSB medium. The phage content in the viscera and in the control group (normal zebrafish) was measured at 0.5, 6, 24, 48, 72, 96, 120, and 144 hours after feeding. 2. Experimental Results and Analysis After feeding zebrafish with feed soaked in PRE50 phage for 0.5 hours, the presence of phage was detected in the viscera of the experimental group of zebrafish, with a titer of up to 3.0 × 10⁻⁶. 6 The PFU / g concentration of bacteriophages allows them to enter fish bodies through feeding and persist for more than 2 days, providing guidance for their use as aquatic feed additives and for their potential application in the prevention and treatment of Edwardsiella pneumoniae. Experimental results also indicate that the bacteriophages have no impact on the health of zebrafish and do not affect their normal activity, demonstrating excellent safety.

[0076] Example 12 Preparation and performance determination of bacteriophage drug formulation 1. Experimental Methods (1) Preparation of bacteriophage drug formulations The bacteriophage drug formulation is a spray-dried powder, prepared by: mixing a powder with a concentration of 3×10⁻⁶... 10 Phage PRE50 proliferation medium (PFU / mL), gelatin, monosodium glutamate, and mannitol were mixed evenly in a mass ratio of 100:4:2:3, and then spray-dried using a Swiss BUCHIB... The 90-type nano-spray dryer was used for drying. The process parameters were: inlet temperature 80℃, airflow speed 120L / min, spray rate 100%, nozzle cap size 4.0μm, and spray drying for about 2 hours. Finally, the spray-dried particles (i.e. spray-dried powder) were collected, which is the phage drug preparation.

[0077] Set up a control group: Perform the following simultaneous temperature and pH stability tests using the same concentration of phage PRE50 proliferation medium.

[0078] (2) Temperature stability test of bacteriophage drug formulations Before the stability experiment, three sample tubes were randomly selected, immediately reconstituted, and their phage titers were determined as the baseline value for "Day 0". One sample tube was then removed, physiological saline was added, and the mixture was vortexed until completely dissolved, then allowed to stand for 5 minutes. After serial dilution, the titer was determined using the bilayer plate method.

[0079] (3) pH stability test of phage drug formulations The titer was calculated using the reconstitution test at pH=7 as the baseline. One sample tube was taken out, physiological saline was added, and the sample was vortexed to dissolve it completely. The tube was then allowed to stand for 5 minutes. After serial dilution, the sample was incubated in solutions of different pH values. After incubation, the titer was determined using the bilayer plate method.

[0080] (4) The phage titers of Edwardsiella phage PRE50 proliferation solution and its drug preparation were determined after treatment at pH 4 for 3 h, pH 7 for 3 h, storage at 30℃ for 6 months and storage at 85℃ for 1 h, respectively, so as to compare the activity stability of Edwardsiella phage PRE50 proliferation solution and its drug preparation.

[0081] 2. Experimental Results and Analysis (1) Temperature stability test results: As shown in Table 6, the phage drug preparation retained 86.7% of its phages after being stored at 4°C for 60 days.

[0082] Table 6 Temperature stability test

[0083] (2) pH stability test results The results are shown in Table 7. The phage drug preparation retained 96% of the phage after incubation at pH 4 for 2 hours.

[0084] Table 7 pH stability test

[0085] (3) Comparison of the stability of Edwardsiella edodes phage PRE50 proliferation broth and its drug formulation The results are shown in Table 8. After Edwardsiella edodes phage PRE50 was prepared into the above-mentioned phage drug formulation, its temperature resistance, acid and alkali resistance and other properties were improved.

[0086] Table 8 Performance determination of PRE50 after preparation into pharmaceutical formulations

[0087] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solutions and concepts of this invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.

Claims

1. A bacteriophage of Edwardsiella, characterized in that, Edwardsiella phage PRE50, and its preservation number is CGMCC No. 46488.

2. A bacteriophage composition, characterized in that, The application also discloses a phage composition comprising the Edwardsiella phage PRE50 and other phages.

3. Use of the Edwardsiella phage according to claim 1 or the phage composition according to claim 2 in the preparation of a medicament, an environmental disinfectant and a detection kit for preventing and treating aquaculture diseases caused by Edwardsiella infection.

4. Use according to claim 3, characterized in that, The diseases caused by Edwardsiella infection include visceral white spot disease and acute septicemia.

5. A bacteriophage pharmaceutical formulation, characterized in that, The effective component comprises the Edwardsiella phage PRE50 according to claim 1 or the phage composition according to claim 2.

6. The bacteriophage pharmaceutical preparation of claim 1, wherein, The medicament preparation is a spray dry powder which is prepared by mixing and spray drying the following components: the phage PRE50 proliferation solution, gelatin, sodium glutamate and mannitol.

7. The method of preparing a bacteriophage pharmaceutical formulation of claim 6, wherein, The preparation method comprises the following steps: uniformly mixing the phage PRE50 proliferation solution, gelatin, sodium glutamate and mannitol in a certain mass ratio, and then performing spray drying to prepare the phage medicament preparation.

8. An environmental disinfectant, characterized by, The effective component comprises the Edwardsiella phage PRE50 according to claim 1 or the phage composition according to claim 2.

9. Use of the environmental disinfectant according to claim 8 for disinfecting a farming environment, characterized in that, The environmental disinfectant is used for disinfecting Edwardsiella in feed, water and breeding environment, and the breeding environment comprises a pool, a feeding table, a feeding appliance and a recirculating aquaculture system.

10. A test kit comprising, The application also discloses a phage composition comprising the Edwardsiella phage according to claim 1 or the phage composition according to claim 2.

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