Mycobacterium abscessus phage WST2, preparations and applications

Abstract

This application discloses Mycobacterium abscessus phage WST2, its formulation, and applications, belonging to the field of microbial technology. This phage, named as a phage belonging to the family Long-tailed Phagecaceae in the order Caudata, is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2026261. The genome of phage WST2 is a full-length double-stranded DNA of 39,800 bp–40,300 bp, with a G+C content of 60%–65% and containing 50–70 open reading frames. The formulation includes the aforementioned Mycobacterium abscessus phage WST2. Applications include drug preparation, inhibition or elimination of Mycobacterium abscessus, addition to liquids, attachment to object surfaces, or placement inside objects. This application can accurately identify and bind to the surface receptors of Mycobacterium abscessus, effectively lysing clinical strains of Mycobacterium abscessus, thereby inhibiting or eliminating clinical strains of Mycobacterium abscessus.

Description

Technical Field

[0001] This application belongs to the field of microbial technology, and in particular relates to Mycobacterium abscessus phage WST2, its formulation and application. Background Technology

[0002] Mycobacterium abscessus, a widely distributed opportunistic pathogen in the environment, has become a significant cause of hospital-acquired infections. This bacterium can cause a variety of serious diseases, including respiratory infections, skin and soft tissue infections, and bacteremia, posing a particularly fatal threat to immunocompromised patients, critically ill patients, and patients with chronic lung disease. In livestock farming, this bacterium also causes diseases such as necrotic enteritis, resulting in significant economic losses.

[0003] Currently, the main treatment for Mycobacterium abscessus infection in clinical practice still relies on antibiotic therapy, but its effectiveness is increasingly limited. This bacterium exhibits high-frequency resistance to many commonly used antibiotics, including β-lactams, macrolides, and aminoglycosides. Its resistance mechanisms include the production of β-lactamases, activation of drug efflux pumps, and modification of drug targets. More seriously, this bacterium easily forms biofilms at the site of infection. These biofilm structures can significantly enhance bacterial resistance to antibiotics, making conventional doses of antibiotics ineffective.

[0004] Current methods for inhibiting Mycobacterium abscessis mainly employ polymyxins and tigecycline, but these ingredients have significant limitations. Polymyxins exhibit significant nephrotoxicity and neurotoxicity, while tigecycline concentrations in lung tissue are insufficient for effective treatment. Furthermore, long-term use has led to the emergence of drug-resistant strains, creating a vicious cycle of "treatment-resistance-retreatment."

[0005] Bacteriophages have attracted attention in recent years as a potential alternative to antibiotics, but their application against Mycobacterium abscessis faces significant bottlenecks. Currently reported Mycobacterium abscessis phage resources are scarce, and most suffer from narrow host spectrum and poor environmental stability. Existing phages have limited coverage of clinically prevalent drug-resistant strains and are ineffective at clearing bacteria within biofilms. Furthermore, the lack of unified production and quality control standards for phage formulations further limits their clinical translation and application. Summary of the Invention

[0006] This application aims to solve the technical problem of effectively combating Mycobacterium abscessus. To this end, this application provides Mycobacterium abscessus phage WST2, its formulation and application, which can accurately identify and bind to the surface receptors of Mycobacterium abscessus, effectively lyse clinical strains of Mycobacterium abscessus, thereby inhibiting or eliminating clinical strains of Mycobacterium abscessus.

[0007] In the first aspect, this application provides a Mycobacterium abscessus phage WST2, which is named a phage belonging to the family Long-tailed Phagesaceae in the order Caudataphages. It is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M2026261. The genome of phage WST2 is a full-length double-stranded DNA of 39,800 bp to 40,300 bp, with a G+C content of 60% to 65% and containing 50 to 70 open reading frames.

[0008] In some embodiments, it remains active at pH 5-8 and is inactivated at pH ≤ 4 and pH ≥ 10.

[0009] In some embodiments, it remains active at temperatures ≤42°C and is deactivated at temperatures ≥60°C.

[0010] In some embodiments, it has an icosahedral head and a non-contracting tail, the diameter of the icosahedral head being 60nm-65nm and the length of the non-contracting tail being 160nm-180nm, and the end of the non-contracting tail having a tail spike structure.

[0011] Secondly, embodiments of this application provide a formulation of Mycobacterium abscessus phage WST2, which includes the aforementioned Mycobacterium abscessus phage WST2, and the formulation is in solid or liquid form.

[0012] In some implementations, the pH of the formulation is 5-8.

[0013] In some embodiments, the potency of the formulation is greater than or equal to 10. 9 PFU / mL.

[0014] In some embodiments, the formulation also includes a buffer, stabilizer, or diluent that serves as a phage carrier.

[0015] Thirdly, this application provides an application of Mycobacterium abscessus phage WST2, which uses the aforementioned Mycobacterium abscessus phage WST2 to prepare drugs that inhibit or eliminate Mycobacterium abscessus.

[0016] Fourthly, this application provides an application of Mycobacterium abscessus phage WST2, which uses the aforementioned Mycobacterium abscessus phage WST2 to prepare biomaterials that inhibit or eliminate Mycobacterium abscessus.

[0017] Fifthly, embodiments of this application provide an application of Mycobacterium abscessus phage WST2, which is a product prepared by one or more methods, such as adding liquid, attaching to the surface of an object, or placing it inside an object, using Mycobacterium abscessus phage WST2 as described above. The product includes disinfectant, cleaning agent, antibacterial coating, or antibacterial material.

[0018] As can be seen from the above technical solution, the beneficial effects of this application are as follows: 1. The bacteriophage of this application, based on the biological characteristics of the family Long-tailed Phages, can accurately identify and bind to the surface receptor of Mycobacterium abscessus, effectively lyse clinical strains of Mycobacterium abscessus, thereby inhibiting or eliminating clinical strains of Mycobacterium abscessus, solving the problem of lack of effective antibacterial agents against Mycobacterium abscessus in the prior art due to the use of antibiotics.

[0019] 2. The formulation of this application extends the above-mentioned phage to a pharmaceutical product, which can enhance the stability and delivery efficiency of the phage, facilitate storage and use, and improve the convenience of phage application.

[0020] 3. Application 1 of this application provides the use of drugs to inhibit or eliminate Mycobacterium abscesses, expands the application of bacteriophages, and facilitates their use in organisms.

[0021] 4. Application 2 of this application provides the use of biomaterials for inhibiting or eliminating clinical strains of Mycobacterium abscessus, providing a carrier for bacteriophages, breaking through the limitations of bacteriophage use, and enabling active and controllable application in multiple fields of anti-clinical strains of Mycobacterium abscessus or elimination of Mycobacterium abscessus.

[0022] 5. Application 3 of this application provides a flexible and targeted use of bacteriophages, which can be used as antibacterial agents on the surface and inside of various carriers such as liquids and solids to effectively inhibit or eliminate clinical strains of Mycobacterium abscessis. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced one by one below. Obviously, the accompanying drawings described below are some embodiments of this application. For those skilled in the art, other embodiments and drawings can be obtained based on these drawings without creative effort. Various schematic diagrams according to the embodiments of this application are shown in the accompanying drawings. These drawings are not necessarily drawn to scale. For the purpose of clarity, some details have been enlarged and some details may have been omitted.

[0024] Figure 1 A schematic diagram of an embodiment of the abscess mycobacterium phage WST2 of the present invention on a double-layer plate is shown; Figure 2 A schematic diagram of an embodiment of the abscess mycobacterium phage WST2 of the present invention on a monolayer plate is shown; Figure 3 The experimental schematic diagram of the lysis of clinical strains of Mycobacterium abscessus by the WST2 bacteriophage of the present invention is shown. Figure 4 The stability test results of the abscess mycobacterium phage WST2 of the present invention at different temperatures are shown in the figure; Figure 5 The figure shows the stability test results of the Mycobacterium abscessus phage WST2 of the present invention at different pH values; Figure 6 The figure shows the stability test results of the Mycobacterium abscessus phage WST2 of the present invention at different distances under ultraviolet light. Figure 7 The diagram shows the lysis activity detection of the Mycobacterium abscessus phage WST2 after 20 consecutive passages according to the present invention; Figure 8 A transmission electron microscope (TEM) image (negative staining) of the abscess mycobacterium phage WST2 of the present invention is shown as an example. Figure 9 This invention demonstrates the culture of Mycobacterium abscesses for 0, 1, and 2 days (Day 0, 1, and 2) with the addition of ten dilutions of Mycobacterium abscesses phage (10... 1 ~10 10 The result of culturing for another 6 days (PFU / mL); Figure 10 The present invention is shown. Figure 9 Crystal violet staining results of biofilm after aspiration of planktonic culture; Figure 11 The present invention is shown. Figure 9-10 Statistical chart of crystal violet staining results for biological membranes.

[0025] Figure 12 An example of the complete genome map of the abscess mycobacterium phage WST2 of the present invention is shown.

[0026] Figure 13 The diagram shows the ANI correlation analysis of the Mycobacterium abscessis phage WST2 of this invention. The gray bars represent the comparison of genome lengths of the phages involved in the analysis; the upper right triangular area of ​​the heatmap represents the ANI value (%) between two corresponding phages, with color intensity indicating the degree of similarity; the lower left triangular area represents gene collinearity, indicating the similarity of the linear arrangement of genes on the chromosomes between two corresponding phages.

[0027] Figure 14 The survival curves of the in vivo therapeutic effect of the drug made from the Mycobacterium abscessus phage of the present invention on the larvae of the JZ11-infected giant wax moth are shown. Detailed Implementation

[0028] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. The detailed description of the embodiments of this application provided in the drawings is not intended to limit the scope of the claimed application. The described embodiments are only a part of the embodiments of this application, not all of them. Based on the embodiments in this application, they can be arranged and designed in various different configurations. All other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0029] This application is described below with reference to the accompanying drawings and specific embodiments: The first aspect of this application provides a Mycobacterium abscess phage WST2, named as a phage belonging to the family Long-tailed Phagecetidae in the order Caudataphages. It is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M2026261, located at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, on January 28, 2026. Its taxonomic name is *Mycobacterium phage* WST2, and its Chinese taxonomic name is “Mycobacterium abscess phage WST2”. Its genome is a full-length double-stranded DNA of 39,800 bp-40,300 bp, with a G+C content of 60%-65%, and contains 50-70 open reading frames. The DNA sequence of Mycobacterium abscessus phage WST2 is shown in SEQ ID NO:1. The full genome length is 40,004 bp, the G+C content is 63.27%, and it contains 60 open reading frames (ORFs) encoding structural proteins, DNA replication and packaging modules, lysins (perforin and endosomalin), and lysogenic genes (such as tyrosine integrase).

[0030] The bacteriophage in this application targets clinical strains of *Mycobacterium abscessus*, not standard strains. Clinical strains are "real pathogens" directly isolated from patients; their origin determines their characteristics: high genetic and phenotypic diversity, complex and variable drug resistance, and they reflect the pathogen's behavior in the real world. Therefore, their direct use is to guide clinical diagnosis, develop individualized treatment plans, and conduct epidemiological surveys. Standard strains, on the other hand, are "standard rulers" from strain banks; their genetic background is clear, and their characteristics are stable and uniform. Their core use is as a benchmark for scientific research, for validating experimental methods, or for quality control.

[0031] The bacteriophage of this application, based on the biological characteristics of the family Long-tailed Phages, can accurately identify and bind to the surface receptors of Mycobacterium abscessus, effectively lysing clinical strains of Mycobacterium abscessus, thereby inhibiting or eliminating clinical strains of Mycobacterium abscessus, solving the problem of the lack of effective antibacterial agents against Mycobacterium abscessus in the prior art due to the use of antibiotics.

[0032] In some embodiments, the Mycobacterium abscessus phage WST2 (hereinafter referred to as the phage) has an icosahedral head and a non-contractile tail. The diameter of the icosahedral head is 55nm-65nm, such as 55nm, 60nm, or 65nm, and the length of the non-contractile tail is 160nm-180nm, such as 160nm, 170nm, or 180nm. The end of the non-contractile tail has a pinhead structure. The phage's unique morphology (icosahedral head and non-contractile tail) and genomic characteristics (40,004 bp of full-length DNA) enable it to accurately recognize and bind to the surface receptors of Mycobacterium abscessus, thereby specifically lysing 41 clinical isolates (including the subspecies *Abscessus* and *Massai*). This specificity stems from the molecular interaction between the phage and the host bacterium.

[0033] In some embodiments, it remains active at pH 5-8 and temperature ≤42°C, and is inactivated at pH ≤4, pH ≥10, or temperature ≥60°C. By avoiding high-temperature storage and strong acid / alkali environments, the application of drugs based on this phage ensures stable action of the phage in the human body (e.g., intestinal pH approximately 5-7, body temperature 37°C).

[0034] A second aspect of this application provides a formulation of Mycobacterium abscessis phage WST2, comprising the aforementioned Mycobacterium abscessis phage WST2, in either solid or liquid form. The formulation is based on the biological characteristics of the phage (such as pH and temperature stability) to ensure activity retention and applicability. It can be a liquid phage solution or a solid phage aggregate. If the formulation is liquid, such as an injection, spray, or drop, it can be dispensed into sterile glass vials, metal bottles, or syringes and stored at 4°C. If the formulation is solid, such as a lyophilized powder, tablet, or capsule, it can be stored in a sealed bag or box filled with nitrogen.

[0035] In some implementations, the formulation has a pH of 5-8, such as pH 5, 6.5, or 8. The potency remains constant within a pH range of 5-8, and the formulation controls the pH within this range (e.g., PBS pH 7.0), which allows the formulation to function more effectively.

[0036] In some embodiments, the potency of the formulation is greater than or equal to 10. 9 PFU / mL, e.g., potency 10 9 PFU / mL, 10 10 PFU / mL, etc. The preferred concentration range of the active ingredient in the bacteriophage is 10. 8 ~10 11 Based on the above potency (PFU / mL), the formulation can better exert the antibacterial effect of bacteriophages.

[0037] In some embodiments, the formulation also includes a buffer, stabilizer, or diluent serving as a phage carrier. This expands the applicability of phages and provides more phage formulation products suitable for different environmental applications.

[0038] A third aspect of this application provides an application of Mycobacterium abscessus phage WST2, which uses the aforementioned Mycobacterium abscessus phage WST2 to prepare drugs that inhibit or eliminate Mycobacterium abscessus. For example, it can be used to prepare drugs for respiratory diseases such as pneumonia and bronchial infections caused by clinical strains of Mycobacterium abscessus. This application is based on existing drug preparation processes, in which the culture medium or formulation of the phage is added.

[0039] The fourth aspect of this application provides an application of Mycobacterium abscessus phage WST2, which uses the aforementioned Mycobacterium abscessus phage WST2 to prepare biomaterials that inhibit or eliminate Mycobacterium abscessus, such as biofilms containing the phage (as filter membranes), organic soft membranes (as antibacterial product materials), etc. This application is based on the existing biomaterial preparation process, in which the phage is added as a production raw material.

[0040] A fifth aspect of this application provides an application of the Mycobacterium abscessus phage WST2. This phage, as described above, is used by adding it to liquids, attaching it to object surfaces, or placing it inside objects to inhibit or eliminate clinical strains of Mycobacterium abscessus. For example, in operating rooms, research facilities, or other environments requiring cleanliness, it can be used for disinfection via spray to inhibit clinical strains of Mycobacterium abscessus. This embodiment provides a flexible and targeted use of the phage, enabling its antibacterial application in various scenarios to effectively inhibit or eliminate clinical strains of Mycobacterium abscessus.

[0041] The following method describes the preparation of Mycobacterium abscessus phage WST2, which includes: A1. A sterile filtrate was obtained from the treated wastewater sample. This filtrate was then mixed with Mycobacterium abscessis culture for enrichment culture, followed by centrifugation and filtration to obtain a mixed phage filtrate. This step used Michaelis 7H9 liquid medium for bacterial culture.

[0042] A2. Phage initial screening: The phage mixture filtrate is spotted onto a solid medium coated with Mycobacterium abscessus (host bacteria) and incubated until lysis zones appear to complete the initial screening. The solid medium used in this step is 7H9 solid plates (double-layer plates). The lower plate and the dot method test use 7H9 solid plates, while the upper plate uses a semi-solid medium, i.e., 0.6% LB solid medium containing 0.002 M CaCl2.

[0043] A3, please refer to Figure 1 and Figure 2 The phage is then mixed with Mycobacterium abscessus culture in a semi-solid medium and cultured to obtain individual plaques for isolation. Alternatively, 1 mL of phage filtrate and 1 mL of washed and resuspended bacterial culture can be added, mixed thoroughly, and poured onto a 7H9 thin-layer plate for incubation.

[0044] A4. Prepare a phage stock solution from a single plaque. Dilute the phage stock solution and perform the initial screening and separation operations again. Repeat this process until the obtained plaques have a consistent morphology to complete purification and obtain the diluted solution of Mycobacterium abscessis phage WST2. Repeat the above operation more than three times, repeatedly confirming that the size and transparency of the formed plaques are consistent, to complete the phage purification.

[0045] The specific steps for preparing Mycobacterium abscessus phage WST2 are as follows: S1. Sample Pretreatment: Collect wastewater samples, centrifuge, filter, and sterilize to obtain treated water samples. If samples are collected from a wastewater treatment plant, aliquot them into 50 mL centrifuge tubes. Centrifuge the aliquoted wastewater at 8000 rpm for 30 min. Collect the supernatant, pass it through coarse filter paper, and then through a 0.22 μm microporous membrane for sterilization. Store at 4℃ for immediate use. The pore size of the microporous membrane is 0.22 μm ± 0.02 μm, such as 0.2 μm, 0.22 μm, or 0.24 μm. This membrane effectively removes impurities, bacteria, and other particles.

[0046] S2. Bacterial suspension pretreatment: Mycobacterium abscessis bacterial suspension is added to the treated water sample and incubated with shaking to obtain the treated solution; the Mycobacterium abscessis bacterial suspension meets the following requirements: OD 600 =1.0±0.01, this value is an approximation, OD 600 Approximately 1.0 is sufficient. For example, weigh 10 mL of the treated water sample into a 50 mL centrifuge tube and add OD... 600 10 mL of Mycobacterium abscessus culture with a concentration of approximately 1.0. The incubation process with shaking includes: incubating at 37℃±2℃ with shaking at 220 rpm±20 rpm for at least 24 h, then adding more Mycobacterium abscessus culture; and then incubating again at 30℃±2℃ with shaking at 220 rpm±20 rpm for at least 24 h.

[0047] S3. Enrichment: Centrifuge the treated solution at room temperature, and filter the supernatant through a micron-sized microporous membrane to obtain a phage mixed filtrate; steps S1-S3 correspond to step A1 above. Centrifuge at 8000 rpm at room temperature for 10 min, and use a 10 mL disposable sterile syringe to draw up the supernatant, filter it through a 0.22 μm microporous membrane into a 50 mL sterile centrifuge tube to obtain the phage mixed filtrate, which is stored at 4℃.

[0048] S4. Spotting: Spread the Mycobacterium abscessis bacterial suspension onto a semi-solid culture medium and spot it with the phage mixture filtrate. Incubate until a lysis zone forms to obtain the phage intermediate solution. This step corresponds to step A2 above. For example, spread the Mycobacterium abscessis bacterial suspension evenly on a 7H9 solid plate, spot 5 μL of the enriched phage mixture filtrate, and incubate at 37℃ and 30℃ respectively to preliminarily determine whether lysis has occurred. Incubate until a culture medium with a clear lysis zone is selected.

[0049] S5. Initial Screening: Mix the intermediate phage solution with the Mycobacterium abscessus bacterial solution in a specific ratio into a semi-solid culture medium and place it in a solid culture dish for incubation until single, non-adherent phage plaques appear. When incubating in a solid culture dish, maintain the incubation temperature within the range of 37℃±3℃, ideally at 37℃, with slight fluctuations allowed. This step corresponds to step A3 above. For example, if culturing the Mycobacterium abscessus bacterial solution in the stable phase, wash and resuspend to OD. 600 ≈1.0, heat to melt the upper layer of agar semi-solid culture medium, dispense into 5 mL tubes while hot, place in a 45℃ water bath for incubation, add 1 mL of phage intermediate solution and 1 mL of washed and resuspended bacterial solution (Mycobacterium abscessus bacterial solution), mix well and pour onto 7H9 thin layer plates, solidify and incubate upside down for 48 h.

[0050] S6. Preliminary Purification: Individual phage plaques are fragmented, leached with buffer, and filtered to obtain the phage stock solution. This stock solution is then serially diluted (serial dilution is a standard procedure; the dilution ratio is determined as needed) to obtain the phage dilution. For example, a single phage plaque with no adhering edges can be picked up from a plate using a pipette tip, crushed in a centrifuge tube, and 1 mL of MP buffer added. The mixture is then incubated at 16°C and 100 rpm for 48 h. After vortexing and filtering through a 0.22 μm microporous membrane, the serially diluted solution is spotted onto a plate evenly coated with host bacterial culture and incubated at 37°C for 48 h. If the buffer solution is MP Buffer (50 mM Tris-Base, 150 mM NaCl, 10 mM MgSO4, 1.5 mM CaCl2; pH 7.5), each liter contains 6.055 g Tris-Base, 8.766 g NaCl, 2.4647 g MgSO4, 0.22196 g CaCl2, and pH 7.5.

[0051] S7. Secondary Purification: Spot the phage dilution onto a semi-solid culture medium coated with Mycobacterium abscessis bacterial suspension, and repeat the incubation to obtain phage intermediate solution, single phage plaques, and phage dilution (repeat steps S4-S6) until the size and transparency of single phage plaques from the same batch are consistent. Steps S6 and S7 correspond to step A4 above.

[0052] The above preparation method involves centrifuging and filtering wastewater samples to remove bacteria, reducing contamination, adding host bacterial culture of *Mycobacterium abscessus* for enrichment culture, specifically amplifying the target phage, and then obtaining a pure phage suspension through centrifugation and secondary filtration. Initial screening using spotting is employed to observe lysis zones, rapidly verifying phage activity. Individual phage plaques are then isolated. The preparation and dilution of the phage stock solution are repeated until purification is complete, ensuring a genetically homogeneous phage population. This method enables the efficient and reproducible production of high-purity phage products, reducing variability and improving product reliability and safety.

[0053] The solutions prepared with the aforementioned phages, or the product solutions cultured using the aforementioned methods, were tested. The clinical isolates of *Mycobacterium abscessus* were provided by the Microbiology Laboratory of the Department of Laboratory Medicine, West China Fourth Hospital, Sichuan University, while *Mycobacterium smegmatis* was provided by the West China Public Health Laboratory Center, Sichuan University. The standard strain of *Mycobacterium abscessus*, ATCC19977, was purchased from the American Type Culture Collection. *Mycobacterium abscessus* bacterial suspension was spread evenly on 7H9 plates, dried, and then 5 μL of phage solution was added. The plates were incubated at 37°C for 2–7 days, and plaque formation was observed. *Mycobacterium smegmatis* mc was also tested. 2 155. A total of 116 strains, including standard and clinical strains of Mycobacterium abscessus.

[0054] Please refer to Figure 3 The lytic activity of clinical strains of *Mycobacterium abscessus* against a variety of mycobacteria was systematically evaluated using a dot assay. Within the *Mycobacterium abscessus* range, the phages exhibited broad lytic activity against a wide range of clinical strains, such as JZ11-JZ20, 230387, 211707, and 211839. In addition to the experimental host JZ11, the phages could lyse 40 other clinical isolates of *Mycobacterium abscessus*, covering the two main subspecies of *Mycobacterium abscessus* prevalent in my country (*Mycobacterium abscessus* subspecies and *Mycobacterium masei* subspecies). Meanwhile, the phage of clinical strains of *Mycobacterium abscessum* exhibited lytic ability against strains with different colony morphologies. Among the 41 clinical strains of *Mycobacterium abscessum* that could be lysed, 32 were rough (R) strains and 9 were smooth (S) strains, with lysis rates of 32 / 56 and 9 / 60 for the two colony morphologies, respectively. This indicates that it has lytic ability against both phenotypes, although the lysis rate is higher for the rough strains. The results suggest that the host specificity of the phage of clinical strains of *Mycobacterium abscessum* is not clearly and singularly associated with bacterial subspecies classification or colony morphology (R / S), and its recognition may rely on more specific surface receptors.

[0055] 1. Temperature stability test: Incubation environments of 4℃, 25℃, 37℃, 42℃, 50℃, 60℃, 70℃, 80℃, 90℃, and 100℃ were prepared in a refrigerator, dry thermostat, and water bath, respectively. 500 μL of phage stock solution was taken in centrifuge tubes and placed in different temperatures. The titer of each group of phages was determined by monolayer coating spot method after incubation for 30 min and 1 h.

[0056] like Figure 4 As shown, the phage titer of clinical strains of Mycobacterium abscessus remained at a high level at 42℃ and below, decreased significantly after treatment at 50℃ for 30 min, and was completely inactivated after treatment at 60℃ and above for more than 30 min, indicating that it is sensitive to high temperature.

[0057] 2. pH stability test: The pH value of MP Buffer was adjusted with 1 mol / L HCl and NaOH to expose the bacteriophages to different pH values ​​(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13). The bacteriophages were incubated in a 37 ℃ water bath for 1 h, and the titer of each group of bacteriophages under different pH conditions was determined by the monolayer coating spot method.

[0058] like Figure 5 As shown, the clinical strain of Mycobacterium abscessus phage exhibits excellent stability within the pH range of 5–8, with its titer remaining essentially unchanged. The titer decreases significantly at pH ≥ 9, and it becomes completely inactive at pH ≤ 4 or pH ≥ 10. This indicates that the clinical strain of Mycobacterium abscessus phage can tolerate the pH of the human internal environment, but its tolerance to extreme pH environments is limited.

[0059] 3. Ultraviolet tolerance test: The phage stock solution was taken from centrifuge tubes and tested for tolerance to irradiation by a 30 W UVC ultraviolet lamp with a wavelength of 254 nm when placed directly under an ultraviolet lamp (0 cm) and on a biosafety counter surface (60 cm). Samples were taken at 0, 5, 10, 15, 20, 25, 30, 60, 90 and 120 min for potency determination.

[0060] like Figure 6 As shown, under direct irradiation at a distance of 0 cm, the potency rapidly decreased to undetectable levels within 10 minutes. Under irradiation at a distance of 60 cm, simulating biosafety cabinet conditions, the inactivation rate slowed down, and the time to complete inactivation was extended to 120 minutes.

[0061] 4. Passage stability test: The phage was passaged 20 times consecutively, and bacterial culture was plated after each passage. The stability of the original host after multiple passages was observed. Figure 7 The lytic activity of bacteriophages from clinical strains of Mycobacterium abscessis after 20 consecutive passages was tested. The results showed that the bacteriophages still had obvious lysis zones, indicating that they could stably lyse the host after 20 consecutive passages.

[0062] 5. Potency Determination: Testing showed that the phage of the clinical strain of *Mycobacterium abscessus* did not amplify in liquid; only semi-solid culture medium was used for enrichment. Phage morphology was confirmed to be uniform in size and was poured into double-layer plates. After 48 h of incubation, an appropriate amount of MPBuffer was added, and the plates were shaken at 16°C and 100 rpm for 48 h on a horizontal shaker to leach the phage. The supernatant was aspirated using a sterile syringe and filtered through a 0.22 μm microporous membrane to obtain the phage enrichment solution. The phage enrichment titer obtained in this manner can reach 10. 10 PFU / mL. For titer determination, phage titer = number of phage plaques on the plate × dilution factor (PFU / mL). The phage solution for which titer needs to be determined is serially diluted with MP Buffer. 5 μL of the solution (Mycobacterium abscessus) is spotted onto a 7H9 thin-layer plate. After 48 h, the phage is counted, and the actual titer is calculated as 200 × number of plaques / dilution factor (PFU / mL).

[0063] 6. Morphological characterization of bacteriophages: Observation was performed using negative staining transmission electron microscopy, conducted by Wuhan Maispu Biotechnology Co., Ltd. Absorption titer > 10 9 10–20 μL of PFU / mL phage suspension was dropped onto an electron microscope grid and allowed to adsorb for 10 min at room temperature. Excess droplets were then aspirated and the mixture was allowed to air dry. 20 μL of 2% uranium acetate staining solution was dropped onto a 200-mesh electron microscope grid and allowed to stain negatively for 5 min. Excess staining solution was aspirated, and the mixture was allowed to air dry under an incandescent lamp. Images were then observed and acquired under a transmission electron microscope.

[0064] The embedding and sectioning observation of the interaction between the bacteriophage of clinical strains of Mycobacterium abscessus and the host bacteria was performed by Wuhan Luochuang Biotechnology Co., Ltd. The method is as follows: (1) Prefixation: The host bacterial suspension in the logarithmic growth phase was diluted to 1×10⁻⁶. 8 CFU / mL was mixed with phage enrichment solution at an MOI of 10, incubated on a shaker at 37°C for 15 min, and centrifuged at 1000 rpm for 10 min to collect the bacterial pellet. The bacterial cells were about the size of a mung bean. Most of the supernatant was removed, leaving about 1 mL. The bacteria in the centrifuge tube were gently dispersed and transferred to a 1.5 mL centrifuge tube. An appropriate amount of pre-chilled 2.5% glutaraldehyde (prepared with phosphate buffer) was added, and the tube was allowed to settle vertically for 2 h. The supernatant was then gently aspirated, and 1 mL of freshly pre-chilled 2.5% glutaraldehyde was slowly added along the tube wall for fixation and storage before delivery.

[0065] (2) Post-fixation: After removing the sample from glutaraldehyde, wash the sample with 0.1 M phosphate buffer for 15 min 3 times, and then completely immerse the sample in 1% osmium tetroxide for fixation at room temperature for 2 h. Remove the sample and wash the sample with 0.1 M phosphate buffer for 15 min 3 times.

[0066] (3) Dehydration: Immerse the sample in 50% alcohol, 75% alcohol, 80% alcohol, 95% alcohol, 100% alcohol (twice), and 100% acetone (twice) for 15 min to fully dehydrate.

[0067] (4) Infiltration and embedding curing: Immerse the tissue in infiltration agent A (acetone: 812 embedding agent = 2:1) at room temperature for 2 h, then in infiltration agent B (acetone: 812 embedding agent = 1:2) at room temperature overnight. Immerse twice more in the embedding agent, each time at room temperature for 2 h. Finally, place the tissue in a mold and add embedding agent again, adjusting the sample orientation according to experimental requirements. Place the mold in an oven at 37℃ and 60℃ for 12 h respectively to promote the hardening of the embedding agent.

[0068] (5) Section staining: After finding the ideal anatomical location, use an ultramicrotome to cut sections at 60-80 nm, and use a copper mesh to collect the ultrathin section samples. Immerse the copper mesh containing the sample in 2% uranium acetate staining solution for 30 min in the dark, rinse with deionized water, and then immerse in lead citrate staining solution for 15 min to complete lead-uranium double staining. After rinsing and drying the copper mesh, images can be taken using a transmission electron microscope.

[0069] like Figure 8 The test results are as follows: Phages from the clinical strain of *Mycobacterium abscessum* formed clear, rough-edged patches after incubation at 37°C for 48 h in a single layer of bacterial moss of *Mycobacterium abscessum* clinical strain JZ11, and formed turbid patches after 72 h in a double-layer plate. Electron microscopy with negative staining revealed that the phages from the clinical strain of *Mycobacterium abscessum* possessed a typical icosahedral head and a long, non-contractile tail. The average diameter of the head was approximately 60 nm, and the tail was approximately 170 nm. A clear, small, spherical bulge was observed at the end of the tail, a typical pincer structure characteristic of long-tailed phages. Based on morphological characteristics, the phages from the clinical strain of *Mycobacterium abscessum* can be classified into the order Caudovirales and the family Siphoviridae.

[0070] 7. Effects of clinical strains of Mycobacterium abscessis bacteriophage on Mycobacterium abscessis biofilm: The bacterial culture in the logarithmic growth phase was washed twice with Sauton's medium to remove dispersants such as Tween, and the culture was adjusted to OD. 600 ≈1, 1:100 dilution was added to 96-well plates, the plates were sealed with sealing film, and incubated statically in a 37°C 5% CO2 incubator. New biofilm was cultured daily. On the third day, while completing biofilm inoculation, ten dilutions of clinical strains of Mycobacterium abscessis phage (10T) were simultaneously added to the wells cultured for 0, 1, and 2 days. 1 ~10 10 (PFU / mL). To avoid the influence of phages on adjacent wells, only the growth control wells separated from the experimental wells by one well were included in the statistics.

[0071] The inhibitory effect of bacteriophages on the total biomass of Mycobacterium abscess formation was quantified by crystal violet staining. After culturing for 7-10 days, the biofilm in the control wells was allowed to form at the gas-liquid interface. The liquid culture beneath the biofilm was gently aspirated using a 1 mL microinsulin syringe, and the wells were gently washed once with sterile PBS to remove airborne bacteria. The biofilm was fixed with 4% (w / v) paraformaldehyde at room temperature for 15 min. After aspirating excess liquid, the wells were dried at 37°C and stained with 0.01% (w / v) crystal violet solution at room temperature for 5 min. The wells were gently washed once with sterile PBS to remove excess dye, and then gently shaken (60 r / min) with 33% (v / v) acetic acid for 30 min to dissolve the dye. Finally, 100 μL of crystal violet dissolution solution from each well was transferred to a new 96-well plate, and the absorbance was read at 575 nm using a microplate reader.

[0072] like Figure 9 and Figure 10 As shown, crystal violet can bind to bacteria and extracellular polymers within the biofilm. The effect of drug treatment on the biofilm can be quantitatively examined by measuring changes in absorbance. It can be observed that *Mycobacterium abscessum* can form a smooth and dense biofilm at the air-liquid interface. However, after phage intervention with clinical strains of *Mycobacterium abscessum*, biofilm formation was significantly affected regardless of phage concentration. After removing the lower layer of planktonic culture, the bacteria that were previously floating on the surface appeared as scattered debris. Quantitative results from crystal violet staining indicate that phage treatment with clinical strains of *Mycobacterium abscessum* significantly reduced biofilm biomass.

[0073] like Figure 11 Mycobacterium abscesses were cultured for 0, 1, and 2 days (Day 0, 1, and 2), and ten dilutions of clinical Mycobacterium abscesses phage were added simultaneously. 1 ~10 10 Results of further incubation for 6 days (PFU / mL). (All groups and controls met the requirements) P <0.0001).

[0074] For the phage solution prepared as described above, or the product cultured using the preparation method, phage DNA is extracted and analyzed. Details are as follows: 1. Genome extraction DNA extraction: The sterilized phage filtrate was treated with 200 U Benzo DNase (NOVOPROTEINsciinetific INC) and 0.1 mg / mL RNase A (Sangon Biotech). The DNase was heat-inactivated at 65°C for 10 min. Phage genomic DNA was extracted using the Qiagen MinElute Virus Spin Kit according to the manufacturer's instructions.

[0075] Quality control of extracted DNA: For extracted nucleic acid samples, the nucleic acid concentration and A260 / 280 ratio were initially determined using a Nanodrop spectrophotometer to identify the concentration and purity of the nucleic acid; then, 0.8% agarose gel electrophoresis was performed to detect the size of genomic DNA, and iBright 1500 gel imaging was used to rule out bacterial genomic contamination. Electrophoresis parameters: constant voltage 120 V electrophoresis for 30 min.

[0076] 2. Whole genome sequencing The whole genome sequencing and gene function annotation of the clinical strain of Mycobacterium abscessus were completed by Chengdu Life Baseline Technology Co., Ltd. (Genebang).

[0077] For qualified nucleic acid samples verified by DNA quantification and agarose gel electrophoresis, shotgun library construction was performed: using the VAHTS Universal Plus DNA Library Prep Kit, DNA fragmentation, end repair, and dA tailing were combined into one step, followed by adapter ligation, library enrichment, and sorting to form a complete library. Quality checks were performed using an Agilent 4200 bioanalyzer; the library fragments should exhibit a single-peak distribution, with no extraneous peaks, adapters, or primer dimers, and the insert fragment length should be at least 100 bp. Paired-end 150 bp (PE150) high-throughput sequencing was then performed on the DNBSEQ-T7 platform.

[0078] Sequencing data quality control: FastP is used to filter and control the obtained raw sequencing data, cut and connect, remove low-quality sequences and sequences with a high proportion of N, and obtain the net sequence.

[0079] Genome assembly: The net sequence was assembled de novo using metaSPAdes software. Different fixed-length nucleotide strings were selected for testing to obtain the best assembly results. Then, bwa software was used to align the net sequence to the assembled genome sequence for statistical coverage.

[0080] 3. Genomics analysis This includes genome annotation, virulence factor analysis, drug resistance analysis, phage lifestyle prediction, and correlation heatmaps.

[0081] Genome annotation: Prokka was used to annotate coding genes and tRNAs in the genome. Then, blastp was used to align protein sequences with the NR database to obtain sequence information with high similarity for each gene in the NR database. EggNOG-mapper was used for gene functional annotation, which included annotation information from COG, GO, KEGG, CAZy, BiGG, and PFAM databases.

[0082] Virulence factor analysis: BLAST homology comparison of gene sequences with the VFDB database is performed to predict virulence genes.

[0083] Drug resistance analysis: Resfinder is used to analyze and predict whether there are drug-resistant genes on the genome.

[0084] Phage lifestyle prediction: DeePhage was used to predict phage lifestyles. Based on the DeePhage score, phages were classified as lytic (lifestyle_score≥0.5) or temperate (lifestyle_score<0.5).

[0085] Correlation heatmap: BLASTn was compared with the Vira (taxid:10239) sub-library, and a correlation heatmap was drawn using VIRIDIC.

[0086] The test results for this embodiment are as follows: like Figure 12 The genome of the clinical strain of *Mycobacterium abscessus* phage is a 40,004 bp double-stranded DNA with a G+C content of 63.27%. The genome contains 60 open reading frames (ORFs), 20 of which have been identified as having specific functions, including structural modules, DNA replication and packaging modules, lysis modules, lysogenic modules, and regulatory and transcriptional modules. The *Mycobacterium abscessus* clinical strain phage genome lacks tRNA genes, relies on host translation, and can express perforin and endosomalin, possessing lysogenic functional genes (tyrosine integrase and immunomodulatory factors). No virulence genes or drug resistance genes were predicted in the whole genome. With a DeePhage score of 0.05979, combined with the annotated functional genes, the *Mycobacterium abscessus* clinical strain phage is a temperate phage.

[0087] To assess the relationship between clinical strains of *Mycobacterium abscessus* phages and other known mycobacterial phages, this application calculated genome-wide average nucleotide identity. For example... Figure 13As shown, the phage of the clinical strain of *Mycobacterium abscessus* exhibited high genomic relevance at the whole-genome level with multiple *Mycobacterium abscessus* phages (including phiGD21-1, phiGD43A-2, prophiGD16-1, and P3MA), with ANI values ​​ranging from 82.2% to 91.8%. P3MA was isolated from the environment, while the other phages were prophages from the *Mycobacterium abscessus* sequence. phiGDxxx was spontaneously released from the *Mycobacterium abscessus* strain, while prophiGDxxx and prophiTxxx were prophages manually extracted from the prophage region of the strain's whole genome using PHASTER. The *Mycobacterium abscessus* clinical strain phage of this application showed the highest ANI value (91.8%) with phage prophiGD89-1. According to the species definition threshold (ANI ≥ 95%) recommended by the International Committee on Taxonomy of Viruses, the clinical strain of Mycobacterium abscessus phage in this application belongs to a different species from the aforementioned phages, but they constitute a closely related evolutionary cluster with highly similar genomes.

[0088] In this embodiment, the Mycobacterium abscessus phage WST2 was used to prepare a drug. To evaluate the drug's effect on the larval model of the large wax moth, the following tests were conducted: A concentration of bacteria exhibiting a significant lethal effect on the larvae within 120 hours (4 × 10⁻⁶) was selected. 6 CFU / larva was used as the infectious dose in the test experiment. A 0.3 mL Viagra disposable insulin syringe was used to slowly inject 10 μL of the test solution 2 mm into the left hind tarsus of the larva to evaluate the in vivo protective effect of the clinical strain of Mycobacterium abscessis. Healthy fifth-instar larvae of the large wax moth were randomly divided into four groups, with an initial number of 12-15 larvae in each group, as follows: ① PBS control group (n=12), injected with 10 μL of sterile PBS; ② Phage control group (n=12), injected with 10 μL of phage with a titer of 1×10⁻⁶. 9 PFU / mL of clinical strains of Mycobacterium abscessus phage; ③ JZ11 infection group n=15, injected with 10 μL 4×10 8 JZ11 bacterial suspension at CFU / mL (i.e., 4×10⁻⁶ CFU / mL) 6 CFU / larva); ④ JZ11 infection + Mycobacterium abscess clinical strain phage test group n=15, inject 10 μL of Mycobacterium abscess clinical strain phage and 10 μL of JZ11 bacterial suspension.

[0089] After injection, the larvae were transferred to 9 mm sterile culture dishes and incubated at 37°C in the dark. Survival was recorded every 12 hours. Larvae were considered dead if they turned black, became stiff, and did not respond to light touch. The observation period was 120 hours (5 days), and the number of dead larvae at each time point and the number of surviving larvae in each group after 120 hours were recorded. Survival analysis was performed using GraphPad Prism 10.1.2 software, and Kaplan-Meier survival curves were plotted. The Log-rank test was used to compare the differences in larval survival rates among the groups to evaluate the in vivo testing effect of the clinical strain of Mycobacterium abscessis bacteriophage. Pairwise comparisons between groups were considered statistically significant (P < 0.05).

[0090] like Figure 14 The test results showed that the 120-hour survival rates of the PBS control group and the phage control group of *Mycobacterium abscessus* clinical strain were 100% and 91.7%, respectively, with no significant difference between the two groups (P=0.3175, P>0.05), ruling out the toxicity of the phage itself to the larvae of *Mycobacterium abscessus*. The 120-hour larval survival rate of the JZ11 infection group was 6.7%, while the survival rate of the JZ11 + *Mycobacterium abscessus* clinical strain phage test group was 66.7%. The median survival time of the JZ11 infection group was 84 hours, while the cumulative mortality rate of the JZ11 + *Mycobacterium abscessus* clinical strain phage test group at the observation endpoint was only 33.3%, with a median survival time exceeding 120 hours, extending by more than 36 hours. Pairwise comparisons using the Log-rank test showed that the survival rate of the infection + test group was significantly higher than that of the infection-only group (P=0.0029, P<0.05), indicating that the bacteriophage of the clinical strain of Mycobacterium abscessus has a significant in vivo protective effect against the larvae of the large wax moth infected with the rough clinical isolate JZ11 of Mycobacterium abscessus, effectively delaying larval death and improving survival rate.

[0091] In summary, based on the above experimental data, this application isolated and identified a novel long-tailed Mycobacterium abscess phage with specific lytic activity against clinical strains of Mycobacterium abscessus. This phage possesses the potential to serve as a biological agent, drug, or biomaterial against clinical strains of Mycobacterium abscessus. The results show that the Mycobacterium abscessus clinical strain phage is a newly identified, stable, and mild-tempered long-tailed phage. Its specific lytic ability against Mycobacterium abscessus, biofilm clearance activity, and well-defined genomic background make it of significant application value and development potential in the development of novel antibacterial methods (such as phage cocktails and phage-derived enzyme preparations) against drug-resistant Mycobacterium abscessus infections.

[0092] 1. The clinical strain of *Mycobacterium abscessus* is a morphologically typical and well-defined temperate long-tailed phage. Transmission electron microscopy revealed that the phage possesses a typical icosahedral head (average diameter approximately 60 nm) and a long, non-contracted tail (approximately 170 nm in length), with a pinhead structure visible at the tail end, consistent with the characteristics of long-tailed phages. The genome is a full-length 40,004 bp double-stranded DNA with a GC content of 63.27%, containing 60 ORFs. Functional annotation showed that it encodes structural proteins, DNA replication and packaging proteins, lysins (perforin and endosomalin), and lysogenic genes; no virulence or resistance genes were predicted. The DeePhage score (0.05979) combined with gene annotation confirms its temperate phage nature. The clinical strain of Mycobacterium abscessis bacteriophage is stable within a pH range of 5-8 and can adapt to the human internal environment; it maintains high activity at temperatures of 42°C and below; however, it is easily inactivated under extreme pH (≤4 or ≥10), high temperature (≥60°C), and direct ultraviolet radiation. It can still stably lyse the original host after 20 consecutive passages.

[0093] 2. The bacteriophage of clinical strains of *Mycobacterium abscessum* exhibits specific lytic activity against clinical isolates of *Mycobacterium abscessum* and effectively inhibits its biofilm formation. However, it also shows no effect on the standard strain of *Mycobacterium abscessum* and the model bacterium *Mycobacterium smegma* mc. 2 155 samples showed no lytic activity. The phage of the clinical strain of *Mycobacterium abscessum* exhibited lytic ability against strains with different colony morphologies. Besides the experimental host JZ11, the phage of the clinical strain of *Mycobacterium abscessum* could lyse another 40 clinical isolates of *Mycobacterium abscessum*. Among the 41 strains of *Mycobacterium abscessum* that could be lysed, there were 32 rough (R) strains and 9 smooth (S) strains, with lysis rates of 32 / 56 and 9 / 60 for the two colony morphologies, respectively, indicating lytic ability against both phenotypes, although the lysis rate was higher for the rough strains. The lysis spectrum included the major subspecies *Mycobacterium abscessum* and *Masses*, which are prevalent in my country. In the biofilm model, phage treatment of the clinical strain of *Mycobacterium abscessum* significantly disrupted the biofilm structure formed at the air-liquid interface, changing it from a dense membrane to a dispersed, fragmented structure. Crystal violet staining quantitatively confirmed that phage treatment of clinical strains of Mycobacterium abscessus with different multiples of infection (MOI) significantly reduced biofilm biomass (P<0.0001 compared with the control group).

[0094] 3. The clinical strain of *Mycobacterium abscessus* phage is highly closely related to the genomes of known *Mycobacterium abscessus* phages / prephages, belonging to a new species. Mean nucleotide identity (ANI) analysis showed that the clinical strain of *Mycobacterium abscessus* phage is highly similar to the genomes of known *Mycobacterium abscessus* phages (e.g., phiGD21-1) and prephage sequences (e.g., prophiGD89-1) (ANI values ​​82.2%-91.8%), with the highest ANI value (91.8%) compared to prophiGD89-1. According to the International Committee on Taxonomy of Viruses (ICTV) species definition criteria (ANI ≥ 95%), the clinical strain of *Mycobacterium abscessus* phage belongs to a different species from these phages, forming a unique closely related evolutionary cluster.

[0095] Regarding the specific implementation methods of this application, it should be noted that: In the description of this application, unless otherwise expressly specified and limited, the terms "connection," "fixed," "connected," etc., should be interpreted broadly. For example, "fixed" can refer to a fixed connection, a detachable connection, or an integral molding; "connection" can refer to a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium; it can refer to the internal connection of two components or the interaction between two components, unless otherwise expressly limited; "connected" can refer to the internal connection of two parts and the connection between two parts, or the spatial connection between them, whereby the two parts are directly or indirectly connected through the part forming the space. The terms "set," "installed," "equipped with," "configured," etc., should also be interpreted broadly. For those skilled in the art, the specific meaning of the above terms in this application can be understood according to the specific circumstances.

[0096] In the description of this application, the terms "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," and "counterclockwise," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the system or component referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this application. All directional indications are only used to explain the relative positional relationship and movement between components in a specific orientation. If the specific orientation changes, the directional indication will also change accordingly.

[0097] In the description of this application, the use of terms such as "some embodiments," "optional embodiments," "example," "specific example," "optional example," or "optional embodiment," etc., indicates that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application, but does not imply that these embodiments illustrate and describe all possible forms of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Furthermore, those skilled in the art can combine and integrate the different embodiments or examples described in this specification.

[0098] The present invention has been described in detail above with reference to specific embodiments and exemplary examples. The above description is exemplary and not exhaustive, and is not limited to the disclosed embodiments; the above description should not be construed as a limitation of the present invention. Technical solutions between various embodiments can be combined with each other, but must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed in this application. Although embodiments of the present application have been shown and described, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present application. Those skilled in the art will understand that various other specific changes and combinations of embodiments based on the technical teachings disclosed in this application, without departing from the essence of the present application, are still within the scope of protection defined by the claims of the present invention and their equivalent technical solutions.

Claims

1. A bacteriophage WST2 of Mycobacterium abscessus, characterized in that, It is named a bacteriophage belonging to the family Long-tailed Phages in the order Caudataphages. It is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2026261. The genome of the bacteriophage WST2 is a full-length double-stranded DNA of 39,800 bp to 40,300 bp. The bacteriophage genome does not contain tRNA genes, and the G+C content is 60%-65%, and it contains 50-70 open reading frames.

2. A formulation of Mycobacterium abscessis bacteriophage WST2, characterized in that, The preparation includes the Mycobacterium abscessus phage WST2 as described in claim 1, wherein the preparation is in solid or liquid form and the pH of the preparation is 5-8.

3. The formulation of Mycobacterium abscessis phage WST2 according to claim 2, characterized in that, The potency of the formulation is greater than or equal to 10. 9 PFU / mL.

4. The formulation of Mycobacterium abscessis phage WST2 according to claim 2, characterized in that, The formulation also includes a buffer, stabilizer, or diluent that serves as a phage carrier.

5. An application of Mycobacterium abscessis bacteriophage WST2, characterized in that, The abscess-inhibiting or eliminating Mycobacterium abscessus phage WST2 as described in claim 1 is used to prepare drugs.

6. An application of Mycobacterium abscessis phage WST2, characterized in that, The abscess-inhibiting or eliminating mycobacterium phage WST2 as described in claim 1 is used to prepare biomaterials.

7. An application of Mycobacterium abscessis phage WST2, characterized in that, Products prepared using Mycobacterium abscessis bacteriophage WST2 as described in claim 1 by one or more methods, such as adding to a liquid, attaching to the surface of an object, or placing it inside an object, include disinfectants, cleaning agents, antibacterial coatings, or antibacterial materials.

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