Newly isolated pseudomonas phage, its preparation method and application

Abstract

The application provides a newly isolated Pseudomonas plecoglossicida phage, a preparation method and application thereof. The phage is preserved in the China Center for Type Culture Collection on February 15, 2023, and the preservation number is CCTCC NO:M 2023124. The application discloses a phage which can efficiently inhibit the growth of Pseudomonas plecoglossicida, and can only infect Pseudomonas plecoglossicida, and has high specificity. The phage can be applied to the fields of agriculture, medicine, environment and the like to resist the bacterial host, and has a good development prospect as an antibiotic substitute.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and microbiology. More specifically, this invention relates to a newly isolated Pseudomonas aeruginosa phage, its preparation method, and its application. Background Technology

[0002] Bacteriophages are viruses capable of infecting bacteria and are widely distributed in nature. As a type of virus, bacteriophages have a structure similar to viruses, mainly composed of a nucleic acid core and a protein capsid. Bacteriophage therapy utilizes bacteriophages to kill or inhibit the reproduction of pathogenic bacteria, thereby treating bacterial infectious diseases. Bacteriophages only infect and lyse host bacteria and reproduce themselves without harming human cells and tissues, thus exhibiting high specificity and low toxicity. Furthermore, compared to traditional antibiotic therapy, bacteriophages have the ability to clear biofilms and show excellent clearance effects against multidrug-resistant bacteria. Because bacteriophage therapy can inhibit various drug-resistant bacteria, it is considered a potential antibiotic alternative. Currently, bacteriophages have shown good antibacterial effects in the human body, animal husbandry, aquaculture, and food industries, demonstrating promising application prospects. However, screening, isolating, and identifying bacteriophages with ideal killing effects and specific killing activity against pathogenic bacteria remains a bottleneck in this field. This process relies on the research design and experience of technical personnel and requires extensive screening and analytical research. At the same time, whether or not a valuable bacteriophage can be obtained is also somewhat random and involves uncertainties.

[0003] *Pseudomonas aeruginosa* is a Gram-negative bacillus with peritrichous flagella. It is an important pathogenic bacterium in fish, primarily causing symptoms such as blackening and swelling of the spleen, white rice-grain-like nodules, and kidney edema. It is a major pathogen of visceral white spot disease. *Pseudomonas aeruginosa* can infect a wide range of hosts, including large yellow croaker, sweetfish, and sea bream. Large-scale infections of *Pseudomonas aeruginosa* can cause mass fish mortality, resulting in significant losses to the aquaculture industry.

[0004] In the existing technology of this field, adding antibiotics to feed remains the main means of preventing and controlling visceral white spot disease in fish, such as large yellow croaker. However, the overuse of antibiotics has led to the continuous improvement of drug resistance in pathogens, poor drug efficacy, and antibiotic residue problems.

[0005] Therefore, there is an urgent need in this field to develop new bacterial control strategies to prevent and treat visceral white spot disease caused by *Pseudomonas aeruginosa*, and to reduce the emergence of multidrug-resistant bacteria. Summary of the Invention

[0006] The purpose of this invention is to provide a newly isolated Pseudomonas aeruginosa phage, its preparation method, and its application.

[0007] In a first aspect of the invention, an isolated Pseudomonas aeruginosa phage is provided, which has the accession number CCTCC NO:M 2023124 at the China Center for Type Culture Collection.

[0008] In one or more embodiments, it includes features selected from the group consisting of:

[0009] (a) Includes a head and a tail, the head being icosahedral; preferably, the head diameter of the bacteriophage is 88±10nm and the tail length is 13±6nm; more preferably, the head length of the bacteriophage is 88±5nm and the tail length is 13±3nm.

[0010] (b) Belongs to the family Brachyphageidae in the order Caudataphages;

[0011] (c) The plaques are transparent and round, with a diameter of approximately 0.7–1.1 mm;

[0012] (d) The incubation period in the host is 10–30 minutes; preferably 15–25 minutes; more preferably 18–22 minutes;

[0013] (e) The lysis rate against Pseudomonas aeruginosa is 130–220 PFU / cell; preferably 150–190 PFU / cell;

[0014] (f) The lysis rate against *Pseudomonas aeruginosa* exceeds 95%; preferably exceeds 98%.

[0015] In another aspect of the invention, the application of the bacteriophage is provided for: (1) inhibiting its bacterial host; or (2) preparing a composition that inhibits its bacterial host.

[0016] In one or more embodiments, the bacterial host of the bacteriophage includes *Pseudomonas plecoglossicida*.

[0017] In one or more embodiments, the application is a non-therapeutic application; for example, inhibition of the host of the bacteriophage in the environment (e.g., *Pseudomonas aeruginosa*).

[0018] In one or more embodiments, the composition is a pharmaceutical composition, a food composition, a feed composition, a cleaning agent, and / or a disinfectant.

[0019] In one or more embodiments, the pharmaceutical composition is a vaccine composition; more preferably, the vaccine composition prevents fish diseases associated with bacterial host infection of the bacteriophage.

[0020] In one or more embodiments, the pharmaceutical composition is a therapeutic composition.

[0021] In one or more embodiments, the therapeutic composition treats fish diseases associated with bacterial host infection of the bacteriophage.

[0022] In one or more embodiments, the fish disease includes: visceral white spot disease.

[0023] In one or more embodiments, the bacteriophage is used as a feed additive in the feed composition.

[0024] In one or more embodiments, the cleaning agent or disinfectant is an environmental cleaning agent or an environmental disinfectant.

[0025] In one or more embodiments, the fish is a marine fish.

[0026] In another aspect of the invention, a composition for inhibiting bacteria is provided, comprising any of the isolated bacteriophages described above.

[0027] In one or more embodiments, it further comprises a biologically acceptable vector.

[0028] In one or more embodiments, the composition contains 10 or more bacteriophages. 3 PFU / mL.

[0029] In one or more embodiments, the composition contains 10 bacteriophages. 3 ~10 10 PFU / mL.

[0030] In one or more embodiments, it is further preferred to be 10. 4 ~10 9 PFU / mL (e.g., 10) 5 10 6 10 7 10 8 PFU / mL).

[0031] In another aspect of the invention, a method for preparing a composition using any of the aforementioned bacteriophages is provided, comprising: amplifying and culturing any of the aforementioned bacteriophages; and mixing them with a vector, wherein the vector is a biologically acceptable vector.

[0032] In one or more embodiments, the carrier includes, but is not limited to: solvent, adjuvant, buffer solution, lyophilization protectant, wetting agent, penetrant, dispersant, emulsifier, stabilizer, adhesive, filler, additive, surfactant, or controlled-release agent.

[0033] In one or more embodiments, the amplification culture includes: inoculating the bacteriophage into its bacterial host culture (culture medium), thereby allowing the bacteriophage to infect its host and replicate.

[0034] In one or more embodiments, the bacterial host includes: *Pseudomonas aeruginosa*.

[0035] In one or more embodiments, the composition is a pharmaceutical composition, a food composition, a feed composition, a cleaning agent and / or a disinfectant;

[0036] In one or more embodiments, the pharmaceutical composition is a vaccine composition; more preferably, the vaccine composition prevents fish diseases associated with bacterial host infection of the bacteriophage.

[0037] In one or more embodiments, the pharmaceutical composition is a therapeutic composition; more preferably, the therapeutic composition treats fish diseases associated with bacterial host infection of the bacteriophage; more preferably, the fish diseases include inflammatory diseases.

[0038] In one or more embodiments, the bacteriophage is used as a feed additive in the feed composition.

[0039] In one or more embodiments, the cleaning agent or disinfectant is an environmental cleaning agent or an environmental disinfectant.

[0040] In one or more embodiments, the dosage form of the phage preparation includes (but is not limited to): liquid formulation or solid formulation; more preferably: lyophilized agent, aqueous solution, emulsion, sprayable solution, oily or aqueous dispersion, suspension, powder, granules, wettable powder, emulsifiable concentrate or microcapsule.

[0041] In another aspect of the present invention, a method for inhibiting pathogenic microorganisms is provided, the method comprising: treating an object to be inhibited by bacteria with any of the bacteriophages or the compositions described above; wherein the bacteria are bacteria that can be infected (infected) and subsequently lysed by the bacteriophages; preferably, the bacteria include: *Pseudomonas aeruginosa*.

[0042] In one or more embodiments, the application and method are non-therapeutic applications and methods that do not directly target humans or animals for prevention or treatment (e.g., disinfection of areas (e.g., public places, fish farming water) / equipment containing pathogens, or disinfection of food, feed, etc. that may be attached to pathogens).

[0043] In another aspect of the invention, a kit / pharmaceutical kit for inhibiting bacteria is provided, the kit / pharmaceutical kit comprising: any of the bacteriophages described above; or the composition described above.

[0044] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0045] The above and other features, properties and advantages of the present invention will become more apparent from the following description taken in conjunction with the accompanying drawings and embodiments:

[0046] Figure 1 Phage plaque diagram of phage vB_PpS_SYPE1 of the present invention.

[0047] Figure 2 Electron micrograph of the bacteriophage vB_PpS_SYPE1 of the present invention.

[0048] Figure 3 A one-step growth curve of the bacteriophage vB_PpS_SYPE1 of the present invention.

[0049] Figure 4 The lysis curve results of the host strain XSDHY-P of the bacteriophage vB_PpS_SYPE1 of the present invention are shown in the figure. Detailed Implementation

[0050] The inventors of this invention are dedicated to the research and control of fish diseases caused by pathogens without resistance. Using *Pseudomonas aeruginosa* XSDHY-P as the host bacterium, extensive screening was conducted, resulting in the isolation of a *Pseudomonas aeruginosa* bacteriophage. The bacteriophage of this invention exhibits strong infectivity and lytic activity, high host specificity, and excellent inhibitory effect on the host bacterium. This bacteriophage can be applied in agriculture, medicine, and environmental fields to combat its bacterial host, and it shows promising development prospects as an antibiotic alternative.

[0051] the term

[0052] As used in this invention, the term "pathogen" refers to a microorganism that is harmful to humans, animals, plants, or the environment, particularly a microorganism harmful to fish. More specifically, "pathogen" refers to a microorganism that can be lysed / damaged by the bacteriophage of this invention, including the bacterial host of the bacteriophage described in this invention; preferably, the bacterial host includes *Pseudomonas aeruginosa*, which lyses / damages when the bacteriophage of this invention acts on the bacterial host.

[0053] In this invention, the term "containing" indicates that various ingredients may be used together in the mixtures or compositions of this invention. Therefore, the terms "consistent with..." and "composed of..." are included in the term "containing".

[0054] As used in this invention, a "biologically acceptable carrier" is a carrier used to deliver the bacteriophages of this invention to the object requiring treatment (including the pathogen host, the environment in which the pathogen resides (location, equipment, food, feed, etc.) or the environment in which the pathogen host resides). The carrier may be, but is not limited to, a pharmaceutically acceptable carrier, a food-acceptable carrier, a feed-acceptable carrier, and / or a chemically acceptable carrier. The carrier is typically a solvent, suspending agent, or excipient that is controllable in terms of toxicity and side effects, and is environmentally friendly or harmless to humans and animals. The carrier may be liquid or solid, preferably a carrier capable of maintaining the biological activity of the bacteriophages of this invention to a high degree.

[0055] As used in this invention, "combination" means the use of more than one active substance in combination or together.

[0056] As used in this invention, when referring to bacteriophages or compositions containing them, the terms “lysis rate,” “antibacterial activity,” and “antipathogenic activity” are used interchangeably to refer to the ability to kill and / or inhibit the growth or reproduction of pathogens (especially bacterial hosts).

[0057] Bacteriophages and their applications

[0058] The inventors used *Pseudomonas aeruginosa*, derived from large yellow croaker, as the host bacterium. Through extensive screening and research analysis, they isolated a *Pseudomonas aeruginosa* bacteriophage from large yellow croaker farming wastewater in Zhoushan City, Zhejiang Province. The inventors named this naturally isolated bacteriophage vB_PpS_SYPE1, with accession number CCTCC NO: M 2023124.

[0059] Further analysis by the inventors shows that the bacteriophage includes features selected from the group consisting of: (a) a head and a tail, the head being icosahedral; preferably, the head diameter of the bacteriophage is 88±10 nm and the tail length is 13±6 nm; more preferably, the head length of the bacteriophage is 88±5 nm and the tail length is 13±3 nm.

[0060] (b) They belong to the order Caudataphages and the family Brachyphages;

[0061] (c) The plaques are transparent and round, with a diameter of approximately 0.7–1.1 mm;

[0062] (f) The incubation period in the host is 10–30 minutes; preferably 15–25 minutes; more preferably 18–22 minutes;

[0063] (h) The lysis rate against Pseudomonas aeruginosa is 130–220 PFU / cell; preferably 150–190 PFU / cell;

[0064] (i) The lysis rate against *Pseudomonas aeruginosa* exceeds 95%; preferably exceeds 98%.

[0065] The bacteriophages of this invention are living organisms that can be preserved and propagated. Therefore, once the bacteriophages of this invention are obtained, they can be prepared in large quantities using methods known in the art. This typically involves contacting them with a bacterial host, allowing them to invade the host cell and replicate extensively, and finally releasing them by lysing the host cell.

[0066] The bacteriophage of the present invention may be the naturally isolated vB_PpS_SYPE1, as well as its variants, such as mutants obtained by performing molecular genetic operations (e.g., genome modification) to adjust / improve certain aspects of its performance, such as by further promoting its ability to infect the host, expanding its host species, shortening its latency in the host and / or enhancing its lysis ability in the host through molecular genetic operations (changing the activity of one or more of its proteins).

[0067] That is, the bacteriophage vB_PpS_SYPE1 of the present invention can be used as a starting phage, and further improved through laboratory domestication, genetic breeding, molecular genetic manipulation, and other means to obtain derivative phages with higher yields or stronger activity. Phages obtained by further screening and optimization using vB_PpS_SYPE1 of the present invention as the starting phage through these artificial manipulation methods are also included within the overall scope of the present invention.

[0068] Alternatively, the present invention also provides active biomolecules isolated from the phages of the present invention (e.g., isolated phage polypeptides or their active fragments, variants, or derivatives). Since the phages of the present invention are novel, their genomes and the active polypeptides encoded by their genomes can also be included in the present invention, and potentially one or more polypeptides can be used for purposes such as inhibiting bacterial hosts or other uses. The inhibition can be partial or complete. For example, one, two, or more polypeptides isolated from said phages can infect cells; while other one, two, or polypeptides can lyse cells.

[0069] The bacteriophage of the present invention exhibits strong lytic activity against pathogenic bacterial hosts in aquaculture (such as *Pseudomonas aeruginosa*). According to embodiments of the present invention, its lysis rate against *Pseudomonas aeruginosa* can reach 98%. Therefore, it can provide a source of bacteriophages for large-scale industrial production and for the inhibition of pathogenic bacteria (especially *Pseudomonas aeruginosa*) in aquaculture.

[0070] Based on the characteristics of the bacteriophage according to the present invention, it has a wide range of applications, including but not limited to the following:

[0071] (1) Inhibits and suppresses the growth of bacterial hosts (such as Pseudomonas aeruginosa);

[0072] (2) Prepare products that kill bacterial hosts (such as killing Pseudomonas aeruginosa);

[0073] (3) Prepare products that inhibit the growth of bacterial hosts (such as Pseudomonas aeruginosa);

[0074] (4) Prepare products for the prevention and / or treatment of fish diseases caused by bacterial hosts (such as Pseudomonas aeruginosa);

[0075] (5) Prepare products for the prevention and / or treatment of inflammatory responses caused by bacterial hosts (such as Pseudomonas aeruginosa);

[0076] (6) The bacteriophage of the present invention can be used as a feed additive or to prepare an environmental disinfectant as an effective bactericidal component.

[0077] In the described application, the fish can be any fish that can be infected by the pathogen (bacterial host). For example, it can be a marine fish or a freshwater fish, preferably a marine fish.

[0078] The bacteriophage of the present invention can be used alone or in combination with one or more other types of bacteriophages.

[0079] In application, the phages or polypeptides of the present invention can be applied alone or mixed with a carrier to form a composition for application.

[0080] Composition / Formulation / Reagent Kit

[0081] This invention provides a composition comprising an effective amount of bacteriophage and the remainder of a biologically acceptable vector. The compositions of this invention may additionally contain excipients or stabilizers.

[0082] The dosage form of the composition can be diverse, including but not limited to: lyophilized agents, aqueous solutions, emulsions, sprayable solutions, oily or aqueous dispersions, suspensions, powders, granules, wettable powders, emulsifiable concentrates, or microcapsules.

[0083] It should be understood that any dosage form capable of delivering the bacteriophages described in this invention to the object to be treated while retaining all or part of their activity is desirable. Preferred dosage forms are those that are easy to deliver, and as some preferred methods, the composition may be a lyophilized form, a liquid oral / injectable formulation, a spray, or an aerosol.

[0084] Concentrated compositions contain a higher content of active ingredients (peptides), such as 10... 8 ~10 9PFU / mL phage content; while the diluted composition has a lower content of active ingredient, for example, it may contain 10 3 ~10 6 PFU / mL phage content; a moderate content could be, for example, 10. 6 ~10 8 PFU / mL. In addition, other suitable components may be included, such as the various biologically acceptable carriers listed above. According to embodiments of the invention, in the phage composition of the invention, the dose of the phage is greater than or equal to 10. 3 PFU / mL is relatively suitable, but the present invention is not limited thereto, considering some specific applications.

[0085] If necessary, the composition of the present invention may also contain other active biocides (which may be biological biocides or chemical biocides such as antibiotics) to achieve the simultaneous killing of the bacteriophage-specific pathogens of the present invention and other harmful organisms with a single use.

[0086] The bacteriophages, host cells containing them, or compositions containing them of the present invention may also be included in containers or kits. Preferably, the kits also include instructions for use, etc., to facilitate application by those skilled in the art.

[0087] The phage preparations described herein are in the form of oral solid dosage forms, liquid dosage forms, or lyophilized dosage forms, and can be used for the prevention and / or treatment of *Pseudomonas aeruginosa* via oral administration, medicated baths, and injections.

[0088] The main advantages of this invention are:

[0089] (1) A bacteriophage capable of efficiently lysing bacterial hosts was screened and isolated. This bacteriophage has a strong lysing effect on pathogenic bacterial hosts in aquaculture environments, providing a new source of bacteriophages for large-scale production and use in the prevention and control of pathogenic bacterial hosts in aquaculture environments.

[0090] (2) The bacteriophage provided by this invention has strong infectivity; the incubation period against *Pseudomonas aeruginosa* is 15-25 minutes, and the burst dose is 150-190 PFU / cell; its short incubation period and high lysis rate make it a preferred bacteriophage for inhibiting *Pseudomonas aeruginosa*. It inhibits the growth of more than 98% of *Pseudomonas aeruginosa* within 12 hours. This bacteriophage only has a lytic effect on *Pseudomonas aeruginosa* from fish sources and cannot infect other bacteria; its high specificity is beneficial to environmental safety.

[0091] (3) The bacteriophage provided by the present invention can be used for large-scale industrial production and can be specifically amplified by the host bacterium Pseudomonas aeruginosa; the bacteriophage can also be used as a disinfectant for disinfection of aquaculture water and aquaculture ponds, thereby treating pollution in animal farms; the bacteriophage can also be used to prepare drugs, and then used for the prevention and treatment of diseases caused by its bacterial host, especially Pseudomonas aeruginosa.

[0092] In summary, the bacteriophage provided by this invention has the characteristics of high infection efficiency, strong lysis, and high specificity, and has a very promising application prospect in aquaculture.

[0093] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, or according to the manufacturer's recommendations.

[0094] Example 1: Isolation and identification of *Pseudomonas aeruginosa* phage vB_PpS_SYPE1

[0095] (1) Sample pretreatment

[0096] Wastewater samples were collected from a large yellow croaker farm in Zhoushan City, Zhejiang Province. The samples were centrifuged at 8000×g for 5 min. The supernatant was filtered through a microporous membrane with a pore size of 0.22 μm and stored at 4℃ for later use.

[0097] (2) Enrichment culture of phage samples

[0098] Take 40 mL of filtrate, 10 mL of 5×TSB medium and 1 mL of logarithmic growth phase Pseudomonas plecoglossicida XSDHY-P culture medium, add them to a 250 mL shake flask and incubate overnight at 28 °C at 100 rpm.

[0099] Take 1 mL of culture medium, centrifuge at 8000×g for 5 min, take the supernatant after centrifugation, and filter it with a microporous membrane with a pore size of 0.22 μm for sterilization.

[0100] (3) Phage isolation

[0101] The filtrate was serially diluted with SM buffer. 0.5 mL of the diluted solution was mixed with 0.1 mL of *Pseudomonas aeruginosa* XSDHY-P suspension and incubated at room temperature for 10 min. Then, semi-solid TSB medium (approximately 55°C) was added, mixed thoroughly, and poured onto pre-prepared TSA solid plates. The plates were incubated at 28°C for 12-24 h. The presence of plaques on the double-layer plates was observed; plaques indicated the presence of bacteriophages.

[0102] (4) Purification and screening of phage plaques to obtain target phages

[0103] Different sized phage plaques were picked up with a sterilized pipette tip and repeatedly pipetted into 1 mL of SM buffer. The plaques were then incubated overnight at 4°C. The phage extract was then serially diluted, and the morphology of the plaques was observed using the bilayer plate method.

[0104] The above purification steps were repeated three times to obtain phages with plaques of the same morphology and size. After repeated screening and experimental observation, the inventors obtained a high-performance phage strain, which they named vB_PpS_SYPE1.

[0105] A schematic diagram of the plaques formed by vB_PpS_SYPE1 is shown below. Figure 1 .

[0106] according to Figure 1 It can be seen that the phage plaques formed by the Pseudomonas aeruginosa phage vB_PpS_SYPE1 are transparent and round, with a diameter of approximately 0.7–1.1 mm.

[0107] (5) Filtration, sterilization and preservation

[0108] The purified phage culture was centrifuged at 8000×g for 5 min. The supernatant was filtered through a 0.22μm microporous membrane for sterilization. Glycerol was added to the filtrate to make the final concentration 30%, and the solution was stored at -80℃.

[0109] (6) Determining phage titer

[0110] Centrifuge the phage culture at 8000×g for 5 min, filter the supernatant through a 0.22μm filter membrane, serially dilute the filtrate with SM buffer, and use the dilution to prepare a double-layer plate with *Pseudomonas aeruginosa* XSDHY-P as the host bacterium, and incubate at 28℃ inverted.

[0111] Phage titer (pfu / mL) = number of phage plaques × dilution factor.

[0112] The titer of this bacteriophage was determined to be 10. 9 PFU / mL.

[0113] Example 2: Morphological observation of bacteriophages

[0114] Phage samples were pretreated using phosphotungstic acid negative staining. 20 μL of phage culture, filtered through a 0.22 μm filter, was added dropwise onto a copper grid. After 10 min, excess liquid was blotted off with absorbent paper. The sample was allowed to stand at room temperature for 2 min, then 20 μL of 2% phosphotungstic acid staining was added dropwise onto the copper grid. After staining for 30 s, excess phosphotungstic acid solution was immediately blotted off with absorbent paper. The sample was allowed to stand at room temperature for 5 min, and phage morphology was observed using a transmission electron microscope.

[0115] Electron micrograph of Pseudomonas aeruginosa phage vB_PpS_SYPE1 is shown below. Figure 2 Electron microscopy revealed that the head of bacteriophage vB_PpS_SYPE1 was icosahedral. Based on the rules for bacteriophage isolation and nomenclature, and considering the morphology of this bacteriophage, it belongs to the order Tailed Phages. This bacteriophage is from the family Short-tailed Phagesaceae, with its tail consisting solely of tail filaments. The head diameter of this bacteriophage is 88 nm, and the tail filament length is 13 nm.

[0116] Example 3: Determination of phage growth curve

[0117] The bacteriophage was mixed with 1 mL of *Pseudomonas aeruginosa* XSDHY-P suspension at a 1:100 multiplicity of infection, incubated for 10 minutes, and centrifuged at 12,000 × g at 4 °C for 2 minutes. The pellet was resuspended in 1 mL of TSB medium, and this process was repeated three times. The suspension was then added to 10 mL of TSB and incubated with shaking at 28 °C. The bacteriophage titer was determined using the double-layer plate method, with the titer measured every 10 minutes. The outbreak size was calculated by dividing the final bacteriophage titer by the initial bacteriophage titer.

[0118] The one-step growth curve of the *Pseudomonas aeruginosa* bacteriophage vB_PpS_SYPE1 is shown below. Figure 3 As shown, the incubation period of bacteriophage vB_PpS_SYPE1 is approximately 20 minutes. The titer of the bacteriophage gradually increases within 20-40 minutes after infection, indicating that the lysis period is approximately 20 minutes. Calculations show that the lysis yield of bacteriophage vB_PpS_SYPE1 is approximately 178 PFU / cell, demonstrating that this bacteriophage has strong replication and lysis capabilities.

[0119] Example 4: Determination of the host spectrum of bacteriophages

[0120] The bacterial suspension was spread onto a TSA plate, and 10 μl of *Pseudomonas aeruginosa* phage vB_PpS_SYPE1 was added to the plate. The plates were incubated at 28°C for 12 hours. The presence of an inhibition zone indicated that the phage had lytic ability against the bacterium; the absence of an inhibition zone indicated that the phage could not infect the bacterium. The results are shown in Table 1.

[0121] Table 1. Host spectrum of Pseudomonas aeruginosa bacteriophage vB_PpS_SYPE1

[0122] Bacterial species strain Bacterial source Phage sensitivity Pseudomonas plecoglossicida XSDHY-P Large yellow croaker + Pseudomonas plecoglossicida △homP Large yellow croaker + Pseudomonas putida KT2440 ATCC - Pseudomonas aeruginosa PAO1 ATCC - Pseudomonas fluorescens PFDLP-1 turbot - Edwardsiella piscicida XSDHY-P turbot - Edwardsiella anguillarum ET080729 Flower eel - Edwardsiella tarda ATCC15947 ATCC - Aeromonas salmonicida AS01 turbot - Vibrio alginolyticus EPGS turbot - Vibrio harveyi HS-H1-1 Large yellow croaker - Escherichia coli ATCC 25922 ATCC -

[0123] According to Table 1, this bacteriophage can infect *Pseudomonas aeruginosa* and its *homP* gene-deleted strain derived from large yellow croaker, but it has no ability to infect *Pseudomonas aeruginosa*, *Pseudomonas putida*, *Pseudomonas fluorescens*, and other types of bacteria.

[0124] This result demonstrates that the bacteriophage vB_PpS_SYPE1 of the present invention has high specificity.

[0125] Example 5: Pyrolysis Rate Experiment

[0126] Take 1 mL of bacteriophage vB_PpS_SYPE1 and 1 mL of host bacteria XSDHY-P and incubate at 28°C for 20 minutes. Dilute serially with SM buffer, spread the diluted solutions on plates for colony counting, and incubate at 28°C for 24 hours. Simultaneously, use 1 mL of SM buffer and 1 mL of host bacteria XSDHY-P incubated at 28°C for 20 minutes as a control group.

[0127] The lysis rate of bacteriophages = (1 - number of colonies in the experimental group / number of colonies in the control group) × 100%.

[0128] The test results showed that the lysis rate of the bacteriophage vB_PpS_SYPE1 of *Pseudomonas aeruginosa* reached 98%, which has an excellent lysis effect on the host and is suitable for use in aquaculture to specifically kill *Pseudomonas aeruginosa*.

[0129] Example 6: Phage lysis curve against host

[0130] In a 96-well plate, 2 μL of bacterial host suspension in logarithmic growth phase and phage solution were added to 100 μL of 2×TSB medium at a multiplicity of infection (MOI) of 10:1. The final volume was then adjusted to 200 μL with deionized water, and the plate was incubated at 28°C with shaking for 60 h. A control without phage solution was used. OD600 was measured every minute using a microplate reader. The results are shown below. Figure 4 As shown.

[0131] according to Figure 4 When no bacteriophage was added to the system, the bacteria immediately entered the exponential growth phase, reaching an OD600 of 0.9 within 12 hours. When the bacteriophage vB_PpS_SYPE1 was added to the system, the OD600 increased significantly within 12 hours, and the phage's inhibition rate against the bacteria exceeded 98%.

[0132] Preservation of biological materials

[0133] The *Pseudomonas plecoglossicidaphage* phage vB_PpS_SYPE1 of this invention has been deposited at the China Center for Type Culture Collection (Wuhan, Wuhan University, China) on February 15, 2023, with accession number CCTCC NO: M 2023124. It has been confirmed as a viable phage by the collection center.

[0134] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference.

Claims

1. An isolated *Pseudomonas aeruginosa* bacteriophage ( Pseudomonas plecoglossicida phage The accession number of the bacteriophage is CCTCC NO: M 2023124 at the China Center for Type Culture Collection; the bacterial host of the bacteriophage is *Pseudomonas aeruginosa*, and the bacteriophage is lysed by *Pseudomonas aeruginosa*.

2. The bacteriophage as described in claim 1, characterized in that, It includes features selected from the following group: (a) Includes a head and a tail, the head being icosahedral; the head diameter of the bacteriophage is 88±10nm and the tail length is 13±6nm; (b) Belongs to the family Brachyphageidae in the order Caudatephages; (c) The plaques are transparent and round, with a diameter of 0.7–1.1 mm; (d) The incubation period within the host is 10–30 minutes; (e) The lysis rate against Pseudomonas aeruginosa was 130–220 PFU / cell; (f) The lysis rate against Pseudomonas aeruginosa exceeds 95%.

3. The bacteriophage as described in claim 2, characterized in that, In (a), the head of the bacteriophage is 88±5 nm long and the tail is 13±3 nm long.

4. The bacteriophage as described in claim 2, characterized in that, In (d), the incubation period within the host is 15–25 minutes.

5. The bacteriophage as described in claim 2, characterized in that, In (e), the lysis rate against Pseudomonas aeruginosa was 150–190 PFU / cell.

6. The bacteriophage as described in claim 2, characterized in that, In (f), the lysis rate against Pseudomonas aeruginosa exceeded 98%.

7. The application of the bacteriophage according to any one of claims 1 to 6, for preparing a composition that inhibits its bacterial host, wherein the bacterial host is *Pseudomonas aeruginosa*.

8. The application of the bacteriophage as described in claim 7, characterized in that, The composition is a pharmaceutical composition, a cleaning agent, and / or a disinfectant.

9. The application of the bacteriophage as described in claim 8, characterized in that, The cleaning agent or disinfectant is an environmental cleaning agent or environmental disinfectant.

10. A composition for inhibiting bacteria, comprising the isolated bacteriophage as described in any one of claims 1 to 6.

11. The composition for inhibiting bacteria as described in claim 10, characterized in that, It also contains biologically acceptable vectors.

12. The composition for inhibiting bacteria as claimed in claim 11, characterized in that, The composition contains 10 or more bacteriophages. 3 PFU / mL.

13. The composition for inhibiting bacteria as claimed in claim 12, characterized in that, The composition contains 10 bacteriophages. 3 ~10 10 PFU / mL.

14. The composition for inhibiting bacteria as described in claim 13, characterized in that, The composition contains 10 bacteriophages. 4 ~10 9 PFU / mL.

15. A method for preparing a composition using the bacteriophage according to any one of claims 1 to 6, comprising: Amplify and culture any one of the bacteriophages described in 1 to 6; It is mixed with a biologically acceptable carrier.

16. The method as described in claim 15, characterized in that, The amplification culture includes: inoculating the bacteriophage into its bacterial host culture, thereby allowing the bacteriophage to infect its host and replicate; the bacterial host is *Pseudomonas aeruginosa*.

17. The method as described in claim 15 or 16, characterized in that, The composition is a pharmaceutical composition, a cleaning agent, and / or a disinfectant.

18. The method as described in claim 17, characterized in that, The cleaning agent or disinfectant is an environmental cleaning agent or environmental disinfectant.

19. The method as described in claim 17, characterized in that, The dosage form of the composition includes: liquid formulation or solid formulation.

20. The method as described in claim 19, characterized in that, The dosage forms of the composition include: lyophilized agents, aqueous solutions, emulsions, suspensions, powders, granules, or microcapsules.

21. The method as described in claim 20, characterized in that, The aqueous solution is a sprayable solution.

22. The method as described in claim 20, characterized in that, The powder is a wettable powder.

23. The method as described in claim 19, characterized in that, The composition is in the form of an oil-based or water-based dispersion.

24. A non-therapeutic method for inhibiting pathogenic microorganisms, characterized in that, The method includes treating an object requiring bacterial inhibition with a bacteriophage according to any one of claims 1 to 6 or a composition according to any one of claims 10 to 14; wherein the bacteria are bacteria that can be infected and subsequently lysed by the bacteriophage; and wherein the bacteria are *Pseudomonas aeruginosa*.

25. A reagent kit or cassette for inhibiting bacteria, characterized in that, The kit or medicine box contains: The bacteriophage according to any one of claims 1 to 6; or The composition according to any one of claims 10 to 14.

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