Mycobacterium phage and application thereof

Abstract

The invention discloses a mycobacteriophage and application thereof, and relates to the technical field of bacteriophages. The mycobacteriophage is named as Mycobacter phage BZNK001, is preserved in Guangdong Microbial Culture Collection Center, has a preservation number of GDMCC No: 67907-B1, does not contain antibiotic drug-resistant genes and integrase genes, has excellent biological safety and good environmental tolerance, can maintain high biological activity in a temperature range of 37-50 DEG C under an acid-base condition of pH 5-9, and can be used for preparing the antibiotic drug-resistant gene and integrase gene-containing mycobacteriophage for preventing and treating the antibiotic drug-resistant gene and integrase gene-containing mycobacteriophage for preventing and treating the antibiotic drug-resistant gene and integrase gene-containing mycobacteriophage. The bacillus subtilis has broad-spectrum and high-efficiency non-tuberculous mycobacterium cracking activity, and can effectively crack four clinical key mycobacteria such as mycobacterium abscessus, mycobacterium avium, mycobacterium intracellularis and mycobacterium smegmatis. According to the invention, a mycobacteriophage resource library is enriched, a novel and stable biological prevention and control means is provided for efficient prevention and control of mycobacteria, and the mycobacteriophage has good clinical application and industrialization values.

Description

Technical Field

[0001] This invention relates to the field of bacteriophage technology, and more particularly to a mycobacterial bacteriophage and its applications. Background Technology

[0002] Mycobacterium is a genus of Gram-positive, acid-fast bacilli, including many highly pathogenic strains in humans and animals. Among them, tuberculosis caused by *Mycobacterium tuberculosis* has long been a threat to public health. Meanwhile, non-tuberculous mycobacteria (NTM), represented by *Mycobacterium abscessum*, *Mycobacterium avium*, and *Mycobacterium intracellulare*, have also seen a continuous increase in infection rates in recent years, becoming a significant challenge in clinical anti-infective therapy. These strains can be transmitted through various routes, including the respiratory tract, digestive tract, and skin / mucous membranes, causing a variety of diseases such as lung infections, lymphadenitis, and soft tissue infections, with particularly severe harm to immunocompromised individuals.

[0003] With the widespread use and even abuse of antibiotics, the problem of drug resistance in mycobacteria is becoming increasingly prominent. The incidence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) is rising year by year. Meanwhile, non-tuberculous mycobacteria such as Mycobacterium abscessus and Mycobacterium avium have resistance rates exceeding 60% to commonly used antimicrobial drugs, with some strains even exhibiting pan-drug resistance. This leads to limited clinical treatment options and a significantly increased treatment failure rate, posing a severe bottleneck to traditional chemotherapy systems. Furthermore, mycobacteria readily form biofilms, further enhancing their drug resistance and resistance to the host's immune system, significantly increasing the difficulty of infection control and eradication. Therefore, there is an urgent need to develop novel and highly effective anti-infection strategies.

[0004] Phage therapy, an ancient yet resurgent biocontrol technology, has become an important direction for overcoming the limitations of traditional antibiotic treatment due to its advantages such as high specificity, high bactericidal efficiency, and low likelihood of inducing drug resistance. Bacteriophages are viruses that specifically infect bacteria, accurately identifying and lysing host bacteria. Their proliferation depends on the host bacteria's metabolic system, causing no direct damage to normal human cells and exhibiting high biosafety. In recent years, phage therapy has achieved several breakthroughs in the treatment of multidrug-resistant pathogen infections. For example, engineered mycobacterial phage preparations have successfully cured patients with disseminated drug-resistant Mycobacterium abscessus infection of cystic fibrosis, providing new insights into the treatment of mycobacterial infections.

[0005] However, the application of existing mycobacterial phages still faces four core problems: First, most isolated mycobacterial phages have a narrow host spectrum, often only capable of lysing a single or a few types of mycobacteria, making it difficult to cope with complex scenarios of mixed infections of multiple highly drug-resistant non-tuberculous mycobacteria such as abscesses, birds, and intracellular mycobacteria in clinical practice; Second, about 60% of mycobacterial phages are lysogenic, with weak lytic ability, and some carry integrase genes that are prone to genomic integration, making it impossible to quickly and efficiently eliminate host bacteria; Third, some phage genomes carry antibiotic resistance genes, posing a biosafety risk of drug resistance gene spread; Fourth, there is a scarcity of broad-spectrum virulent phage resources targeting common non-tuberculous mycobacteria in clinical practice, a lack of industrially viable strains, and existing phages have poor environmental tolerance, maintaining activity only within a narrow temperature, acidity, and alkalinity range, making them unsuitable for practical application scenarios.

[0006] Therefore, screening for mycobacterial phages that do not carry antibiotic resistance genes and integrase genes, have good biosafety, excellent environmental tolerance, and a broad host spectrum is of great significance for enriching the mycobacterial phage resource library, breaking through existing treatment bottlenecks, and improving the prevention and control of mycobacterial infections.

[0007] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0008] In view of the shortcomings of the prior art, the purpose of this invention is to provide a mycobacterial phage and its application, aiming to provide a mycobacterial phage that does not carry antibiotic resistance genes and integrase genes, has good biosafety, excellent environmental tolerance, and a broad host spectrum.

[0009] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a mycobacterial phage, wherein the mycobacterial phage is named Mycobacterio phage BZNK001 and is deposited at the Guangdong Provincial Center for Microbial Culture Collection with accession number GDMCC No: 67907-B1.

[0010] In a second aspect, the present invention provides a microbial preparation comprising a first mycobacterial phage, wherein the first mycobacterial phage is the mycobacterial phage described above in this invention.

[0011] Optionally, the microbial preparation may further include a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable additive.

[0012] Optionally, the pharmaceutically acceptable carrier includes at least one of a flow aid, diluent, wetting agent, suspending agent, solvent, and emulsifier; the pharmaceutically acceptable adjuvant includes at least one of a preservative, colorant, flavoring agent, stabilizer, and isotonic agent.

[0013] Optionally, the dosage form of the microbial preparation is selected from one of lyophilized powder, gel, ointment, solution, spray, pill, tablet, capsule and lozenge.

[0014] Optionally, the microbial preparation further includes at least one of Mycobacterium graft, an antimicrobial drug, and probiotics.

[0015] A third aspect of the present invention provides a sustained-release drug comprising a matrix and a mycobacterial phage of the present invention as described above and / or a microbial preparation of the present invention as described above, loaded on the matrix; The matrix includes medical excipients, and / or the matrix is ​​in the shape of microspheres or nanoparticles.

[0016] In a fourth aspect, the present invention provides the use of the mycobacterial phage, the microbial preparation, or the sustained-release drug as described above in lysing mycobacteria, wherein the use is for non-disease treatment purposes.

[0017] A fifth aspect of the present invention provides an application of the mycobacterial bacteriophage described above in the field of mycobacterial detection.

[0018] Optionally, the mycobacteria include nontuberculous mycobacteria, which include at least one of Mycobacterium abscessus, Mycobacterium avium, intracellular mycobacteria, and Mycobacterium smegmatis.

[0019] Beneficial Effects: The mycobacterial phage provided by this invention is a strain of the Longtail Phage family, free of antibiotic resistance genes and integrase genes, exhibiting excellent biosafety and good environmental tolerance. It maintains high biological activity within a temperature range of 37℃ to 50℃ and a pH range of 5 to 9, perfectly adapting to practical applications in human physiology, food processing, and animal husbandry. It possesses broad-spectrum and highly efficient lytic activity against non-tuberculous mycobacteria, effectively lysing four key clinical mycobacteria: Mycobacterium abscessum, Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium smegmatis. This invention enriches the mycobacterial phage resource library, broadens the application scenarios and scope of phages in mycobacterial control, and provides a novel and stable biological control technology for efficient mycobacterial control, possessing significant clinical application and industrialization value. Attached Figure Description

[0020] Figure 1 This is a morphological diagram of plaques from bacteriophage BZNK001.

[0021] Figure 2 This is an electron micrograph of the morphology of bacteriophage BZNK001.

[0022] Figure 3 This is a complete genome loop of bacteriophage BZNK001.

[0023] Figure 4 The graph shows the thermal stability results of bacteriophage BZNK001.

[0024] Figure 5 The figure shows the acid-base stability results of bacteriophage BZNK001. Detailed Implementation

[0025] This invention provides a mycobacterial bacteriophage and its applications. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0027] If the embodiments of the present invention involve descriptions such as "first" or "second", such descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated.

[0028] To address the problems of existing mycobacterial phages having a narrow host spectrum, weak lysogenic lysis, high biosafety risks, and poor environmental tolerance, this invention utilizes *Mycobacterium smegmatis* mc²155 as a host to isolate a novel, broad-spectrum, highly efficient, safe, and environmentally tolerant virulent mycobacterial phage from the aquatic environment of South China for the first time. This mycobacterial phage is named *Mycobacterium phage* BZNK001 and is deposited at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Road, Guangzhou, Guangdong Province, on March 9, 2026, with accession number GDMCC No: 67907-B1.

[0029] In this embodiment of the invention, the mycobacterial phage has a polyhedral head and long tail structure, with a head length of approximately 50 nm and a tail length of approximately 250 nm. According to the virus classification standards published by the International Committee on Taxonomy of Viruses in 2025, this phage belongs to the order Longtailophages and family Longtailophages. The mycobacterial phage can form clear, regular plaques with a diameter of 3–4 mm on a double-layer solid culture medium, which are its unique morphological characteristics. The genome of the described mycobacterial phage is 48,245 bp in length, is double-stranded DNA, has a GC content of 58.84%, and contains 76 open reading frames. Whole-genome sequencing and whole-genome feature analysis revealed that the mycobacterial phage does not contain any antibiotic resistance genes or integrase genes, and therefore will not integrate its own genome into the host bacterium. Its lysis efficiency is much higher than that of lysogenic phages, and it has no potential risk of spreading antibiotic resistance genes. It is an ideal candidate strain for phage therapy. Therefore, the described mycobacterial phage avoids the risk of antibiotic resistance gene spread at the genomic level, and has no lysogenic characteristics, with a lysis efficiency much higher than that of existing lysogenic phages.

[0030] The described mycobacterial phage exhibits excellent tolerance under conditions of 37 ℃ to 50 ℃ and pH 5 to 9, meaning it can maintain high biological activity under these conditions, inhibit mycobacterial proliferation, and block biofilm formation. This range is fully compatible with human physiological environments (around 37 ℃, pH 7.35 to 7.45), food processing environments (room temperature to 50 ℃, pH 5 to 9), and livestock farming environments (pH 6 to 8, room temperature to 40 ℃), thus solving the problem of poor environmental adaptability of existing bacteriophages.

[0031] Most bacteriophages have a narrow host spectrum, capable of lysing only 1-2 types of mycobacteria, and are unable to address mixed infections of highly drug-resistant NTMs such as clinical abscesses, avian mycobacteria, and intracellular mycobacteria. However, the mycobacterial bacteriophage described in this invention can efficiently lyse four key clinical mycobacteria, targeting three of the most prevalent and drug-resistant core nontuberculous mycobacteria with the highest clinical incidence (BNCC364330), avian mycobacteria (BNCC354724), and intracellular mycobacteria (BNCC359356). It can also lyse the model bacterium *Mycobacterium smegmaeella* mc²155, overcoming the limitations of existing bacteriophages with narrow host spectra, adapting to complex clinical scenarios of mixed nontuberculous mycobacterial infections, significantly improving the clearance efficiency of mycobacteria, and compensating for the single-function deficiency of existing bacteriophages.

[0032] In summary, the mycobacterial phage provided by this invention is a strain of the Longtail Phage family, free of antibiotic resistance genes and integrase genes, exhibiting excellent biosafety and good environmental tolerance. It maintains high biological activity within a temperature range of 37℃ to 50℃ and a pH range of 5 to 9, perfectly adapting to practical applications in human physiology, food processing, and animal husbandry. It possesses broad-spectrum and highly efficient lytic activity against non-tuberculous mycobacteria, effectively lysing four key clinical mycobacteria: Mycobacterium abscessum, Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium smegmatis. This invention enriches the mycobacterial phage resource library, broadens the application scenarios and scope of phages in mycobacterial control, and provides a novel and stable biological control technology for efficient mycobacterial control, possessing significant clinical application and industrialization value.

[0033] This invention also provides a method for screening the mycobacterial bacteriophage, comprising the following steps: Water samples were filtered through a 0.22 μm pore size filter membrane and then mixed with 2×7H9 broth medium (the volume ratio of water sample to 2×7H9 broth medium was 1:2 to 2:1, specifically 1:1, 1:2, 2:1, etc.). Logarithmic growth phase Mycobacterium smegmatis mc²155 (OD250) was then added. 600 (≈0.6~0.8), incubate at 37℃ and 100~150 rpm (e.g., 100 rpm, 110 rpm, 120 rpm, 130 rpm, 140 rpm or 150 rpm, etc.) for 2 days. After incubation, centrifuge the sample at 8000 rpm~12000 rpm (e.g., 8000 rpm, 9000 rpm, 10000 rpm, 11000 rpm or 12000 rpm) for 5 min~15 min (e.g., 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min or 15 min, etc.) and filter using a 0.22 μm pore size filter membrane to obtain the incubation solution.

[0034] Mix the incubation solution with logarithmically growing Mycobacterium smegmatis mc²155 (volume ratio can be 1:1), then mix with semi-solid agar, and pour onto a 7H10 solid plate to prepare a double-layer agar plate. Incubate at 37 °C upside down for 2 days. Then pick single plaques and culture them using the double-layer agar plate method. Repeat the plaque picking and purification 3 to 8 times (e.g., 3, 4, 5, 6, 7, or 8 times) to obtain plaques with uniform morphology and transparency. Enrich the plaques to obtain the mycobacterial phages.

[0035] The filter membrane can be made of materials such as cellulose acetate membrane, polyethersulfone membrane, or cellulose nitrate membrane.

[0036] This invention provides a microbial preparation, wherein the microbial preparation includes a first mycobacterial phage, and the first mycobacterial phage is the mycobacterial phage described above in this invention.

[0037] In this embodiment, the mycobacterial phage can be used as the sole active ingredient, or the mycobacterial phage can be used as one of the active ingredients.

[0038] The mycobacterial phage provided by this invention is a strain of the Longtail Phage family, free of antibiotic resistance genes and integrase genes, exhibiting excellent biosafety and good environmental tolerance. It maintains high biological activity within a temperature range of 37℃ to 50℃ and a pH range of 5 to 9, perfectly adapting to practical applications in human physiology, food processing, and animal husbandry. It possesses anti-biofilm capabilities and broad-spectrum, highly efficient lytic activity against non-tuberculous mycobacteria, effectively lysing four key clinical mycobacteria: Mycobacterium abscessum, Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium smegmatis. Therefore, the microbial preparation provided by this invention can lyse mycobacteria, inhibit mycobacterial proliferation, and block mycobacterial biofilm formation, and can be used for the prevention or treatment of mycobacterial infections. Specifically, it can be used to treat mycobacterial infections of the lungs, skin and soft tissues, lymph nodes, and the digestive tract. Furthermore, it can be used for the prevention and control of mycobacterial infections in specific populations such as immunocompromised individuals (e.g., AIDS patients, cancer chemotherapy patients), infants, and the elderly. Furthermore, the aforementioned microbial preparations can also be used for the prevention and treatment of mycobacterial infections in animals. For example, they can be used in livestock farming, specifically for the prevention and treatment of mycobacterial infections in livestock and poultry, and more specifically, for the control of bovine mycobacterial infections.

[0039] In some embodiments, the microbial preparation further includes a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable additive.

[0040] In some embodiments, the pharmaceutically acceptable carrier includes, but is not limited to, at least one of a flow aid, diluent, wetting agent, suspending agent, solvent, and emulsifier.

[0041] In some embodiments, the pharmaceutically acceptable adjuvant includes, but is not limited to, at least one of preservatives, colorants, flavoring agents, stabilizers, and isotonic agents.

[0042] The present invention does not limit the specific dosage form of the microbial preparation. In some embodiments, the dosage form of the microbial preparation may be selected from one of the following: lyophilized powder, gel, ointment, solution, spray, pill, tablet, capsule, and lozenge, but is not limited thereto.

[0043] Among them, freeze-dried powders can be supplemented with freeze-drying protectants such as mannitol, sucrose, and trehalose, while sprays can be supplemented with surfactants such as Tween 80 and sodium dodecyl sulfate.

[0044] In this invention, the administration method of the microbial preparation can be at least one of liquid infusion, nebulized inhalation, topical application, intraperitoneal injection, nasal drops, or oral administration, suitable for infections at different sites. The specific concentration used can be 1×10⁻⁶. 9 pfu / mL~1×10 11 PFU / mL high concentration potency. The frequency of use can be adjusted according to actual application needs. For example, high-frequency short-term dosing is used for acute infections, while low-frequency long-term sustained release is used for chronic infections.

[0045] In some embodiments, the microbial preparation further includes at least one of Mycobacterium virginiae bacteriophage, an antimicrobial agent (such as a natural antimicrobial peptide), and probiotics. In this embodiment, the microbial preparation is a compound microbial preparation, which can further enhance its broad-spectrum activity and lysis efficiency. The antimicrobial agent is a low-concentration, non-drug-resistant antimicrobial agent.

[0046] This invention also provides a sustained-release drug (specifically a targeted sustained-release drug), comprising a matrix and the mycobacterial phage and / or the microbial preparation of this invention as described above, loaded on the matrix. The matrix includes medical excipients (e.g., a biodegradable carrier), and the matrix is ​​shaped as microspheres or nanoparticles. In this embodiment, the sustained-release drug is suitable for clinical mycobacterial infections of the skin and mucous membranes, and the control of mycobacteria on the surface of medical devices.

[0047] The present invention also provides the application of the mycobacterial bacteriophage, the microbial preparation, or the sustained-release drug as described above in lysing mycobacteria, wherein the application is for non-disease treatment purposes.

[0048] The mycobacterial phages provided by this invention have both clinical and non-clinical applications, such as the lysis (or elimination) of mycobacterial phages for non-disease treatment purposes. Specifically, the mycobacterial phages provided by this invention can be used for the elimination of mycobacteria in the environment, food, or medical devices. For the elimination of mycobacteria in the environment, for example, in hospitals, livestock farms, sewage treatment plants, laboratories, etc. For the elimination of mycobacteria in food, for example, in milk, meat products, fresh food, etc. For the elimination of mycobacteria in medical devices, for example, in removing mycobacterial biofilms from the surfaces of medical catheters, ventilators, dialysis equipment, etc. Specific application methods include spraying, soaking, coating, slow release, etc.

[0049] For environmental disinfection, the concentration of the mycobacteria used can be 1×10⁻⁶. 8 pfu / mL~1×10 10 The potency is at a medium concentration of pfu / mL. For foodborne disease control, the concentration of the mycobacterium used can be 1×10⁻⁶. 6 pfu / mL~1×10 8 low concentration potency of pfu / mL.

[0050] This invention also provides an application of the mycobacterial phage described above in the field of mycobacterial detection. In this invention, the specific lytic activity of the mycobacterial phage is utilized to prepare rapid detection reagents or kits for mycobacteria. For example, qualitative or quantitative detection of mycobacteria in samples can be achieved by using markers released after the phage lyses the mycobacteria, thus expanding its application scope.

[0051] In some embodiments, the mycobacteria include nontuberculous mycobacteria, which include at least one of Mycobacterium abscessus, Mycobacterium avium, intracellular mycobacteria, and Mycobacterium smegmatis.

[0052] Furthermore, to further improve the lysis efficiency, host spectrum, and environmental tolerance of the mycobacterial phages, performance upgrades can be achieved through targeted genetic engineering, specifically including: Enhance lysis and anti-biofilm capabilities: Site-directed mutations or overexpression of the lysozyme, perforin, and endolysin genes of the aforementioned bacteriophages can further improve their lysis efficiency against mycobacteria; or insertion of anti-biofilm-related genes can enhance their ability to clear mature biofilms. Broadening the host spectrum: Inserting receptor-binding protein genes of other broad-spectrum mycobacterial phages into the genome of the mycobacterial phage, enabling it to cleave more types of mycobacteria (such as Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium marinum, etc.). Enhanced environmental tolerance: Modification of the capsid protein gene of bacteriophages to enhance their stability under conditions such as high temperature (>50 ℃), extreme acid and alkalinity (pH<5 / pH>9), ultraviolet light, and chloroform, making them suitable for more demanding application scenarios; Targeted modification: Inserting targeted peptide genes into the phage genome enables the phage to specifically recognize mycobacteria at the site of infection, improving the targeting of clinical applications.

[0053] The present invention will be further described below through specific embodiments.

[0054] Example 1: Isolation and purification of bacteriophages A 15 mL water sample from a park in Shenzhen, Guangdong Province, was filtered through a 0.22 μm pore size membrane (cellulose acetate membrane) and mixed with 15 mL of 2×7H9 broth culture medium. 1 mL of logarithmic growth phase Mycobacterium smegmatis mc²155 (OD200) was then added. 600 (≈0.6-0.8), and incubated at 37 ℃ and 120 rpm for 2 days. After incubation, the sample was centrifuged at 10000 r / min for 10 min and filtered through a 0.22 μm pore size filter to obtain the incubation solution. 500 μL of the incubation solution was mixed with 500 μL of logarithmic growth phase Mycobacterium smegmae mc²155 (OD2000). 600 Mix the sample with approximately 0.6-0.8 mL of agar (approximately 0.6-0.8 mL), then mix with 10 mL of semi-solid agar and pour onto a 7H10 solid plate to prepare a double-layer agar plate. Incubate at 37 °C upside down for 2 days. Single plaques were then picked and cultured using the double-layer agar plate method. The plaque picking and purification process was repeated 5 times to obtain plaques with uniform morphology and transparency. These plaques were then enriched to obtain a mycobacterial phage (named *Mycobacterio phage* BZNK001), which is deposited at the Guangdong Provincial Microbial Culture Collection Center, accession number GDMCC No: 67907-B1, on March 9, 2026.

[0055] The mycobacterial phage obtained in this embodiment is referred to as phage BZNK001.

[0056] The morphology of phage plaque BZNK001 is shown in the figure below. Figure 1 As shown, phage BZNK001 can form clear, empty plaques on a double-layer solid culture medium with clear and regular edges and a diameter of about 3–4 nm, which are its unique morphological characteristics.

[0057] Example 2: Morphological identification of bacteriophage BZNK001 Phage BZNK001 was observed by transmission electron microscopy (TEM) using phosphotungstic acid (PTA) negative staining. Concentrated phage particles of BZNK001 were dropped onto a Formvar carbon support membrane and allowed to dry for 10 min. Excess liquid was removed with paper. 10 μL of PTA was added to the membrane for staining for 10 min. Excess PTA was washed away with deionized water, and the sample was dried again for 10 min. The morphology of phage BZNK001 was observed under TEM. The electron micrograph of phage BZNK001 is shown below. Figure 2 As shown, bacteriophage BZNK001 exhibits a polyhedral head and long tail structure, with a head length of approximately 50 nm and a tail length of approximately 250 nm. It belongs to the order Caudataphages and the family Longtailphages.

[0058] Example 3: Whole genome sequencing analysis of bacteriophage BZNK001 After amplifying the purified phage BZNK001 from Example 1, 1 mL of phage BZNK001 (1.0 × 10⁻⁶) was taken. 10 Genomic DNA was extracted using a TIANGEN viral genome extraction kit (pfu / mL). Whole-genome sequencing was performed using an Illumina NextSeq sequencer. The whole-genome annotation results of phage BZNK001 are shown in Table 1, and the whole-genome diagram of phage BZNK001 is shown below. Figure 3 As shown in the figure. Whole-genome analysis revealed that bacteriophage BZNK001 belongs to the genus Mycobacteriophage, with a genome of dsDNA, a size of 48245 bp, a GC content of 58.84%, and 76 open reading frames (ORFs) annotated. The genome contains no antibiotic resistance genes or integrase genes, ensuring biosafety at the genomic level. Furthermore, it lacks lysogenic genes, making it a highly virulent bacteriophage with excellent lysis efficiency.

[0059] Table 1. Complete genome annotation of phage BZNK001

[0060] Example 4: Thermal stability of bacteriophage BZNK001 Several sterile 2 mL EP tubes were used, and 500 μL of phage BZNK001 was added to each tube. The tubes were incubated at 4 ℃, 25 ℃, 37 ℃, 50 ℃, 60 ℃, 70 ℃, and 80 ℃ for 1 h, respectively. The phage titer after incubation at different temperatures was determined using the double-layer agar method. Results are as follows: Figure 4As shown, phage BZNK001 maintains high biological activity and no significant decrease in titer within a temperature range of 37 ℃ to 50 ℃. This temperature range is fully compatible with practical application scenarios such as human physiology and food processing, demonstrating excellent thermal stability and solving the problem of rapid activity drop of existing phages above 45 ℃.

[0061] Example 5 pH stability of bacteriophage BZNK001 The pH of LB liquid medium was adjusted with HCl and NaOH to obtain solutions with pH values ​​ranging from 2 to 13, which were then filtered through a 0.22 μm microporous membrane. 100 μL of bacteriophage BZNK001 was mixed with 900 μL of each solution at a different pH, and incubated in a 37 ℃ water bath for 1 h. The titer of the bacteriophage at different pH values ​​was determined using the double-layer agar method. The results are as follows: Figure 5 As shown, phage BZNK001 maintains high biological activity and stable titer under acidic and alkaline conditions of pH 5 to 9. This pH range is suitable for most clinical, food, and animal husbandry applications, and its environmental tolerance is significantly better than that of existing mycobacterial phages that can only survive in environments of pH 7 to 8.

[0062] Example 6 Host profile determination of bacteriophage BZNK001 Phage lysis profiles were determined using the standard dot assay. 500 µL of different mycobacteria in mid-logarithmic growth phase (Mycobacterium smegmatis mc²155, Mycobacterium abscessus subsp. BNCC364330, Mycobacterium avium BNCC354724, Mycobacterium intracellulare BNCC359356, Mycobacterium marineum BNCC360708, and Mycobacterium guillochénei BNCC364223; these mycobacteria were known strains and commercially available) were mixed with 7H9 semi-solid medium, spread onto double-layer agar plates, and after the agar plates dried and solidified, 5 µL of phage BZNK001 was added to the surface of the plates and incubated at 37 °C for 2–7 days. After incubation, phage plaques were observed to assess phage lysis activity. The results are shown in Table 2. Phage BZNK001 can efficiently lyse Mycobacterium smegmatis mc²155, Mycobacterium abscessus BNCC364330, Mycobacterium avium BNCC354724, and Mycobacterium intracellularis BNCC359356. Among them, Mycobacterium abscessus, Mycobacterium avium, and Mycobacterium intracellularis are clinically highly drug-resistant nontuberculous mycobacteria, which breaks through the technical limitations of existing phages with narrow host spectrum and inability to cope with mixed infections of nontuberculous mycobacteria.

[0063] Table 2. Results of lysis spectrum experiment of mycobacterial bacteriophage BZNK001

[0064] Note: "+" indicates cleavage; "-" indicates no cleavage.

[0065] In summary, the mycobacterial phage provided by this invention is a strain of the Longtail Phage family, free of antibiotic resistance genes and integrase genes, exhibiting excellent biosafety and good environmental tolerance. It maintains high biological activity within a temperature range of 37℃ to 50℃ and a pH range of 5 to 9, perfectly adapting to practical applications in human physiology, food processing, and animal husbandry. It possesses broad-spectrum and highly efficient lytic activity against non-tuberculous mycobacteria, effectively lysing four key clinical mycobacteria: Mycobacterium abscessum, Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium smegmatis. This invention enriches the mycobacterial phage resource library, broadens the application scenarios and scope of phages in mycobacterial control, and provides a novel and stable biological control technology for efficient mycobacterial control, possessing significant clinical application and industrialization value.

[0066] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A mycobacterial bacteriophage, characterized in that, The mycobacterial phage was named Mycobacteriophage BZNK001 and is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 67907-B1.

2. A microbial preparation, characterized in that, The microbial preparation includes a first mycobacterial phage, which is the mycobacterial phage as described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, The microbial preparation also includes pharmaceutically acceptable carriers and / or pharmaceutically acceptable additives.

4. The microbial preparation according to claim 3, characterized in that, The pharmaceutically acceptable carrier includes at least one of the following: a gliding agent, a diluent, a wetting agent, a suspending agent, a solvent, and an emulsifier; the pharmaceutically acceptable adjuvant includes at least one of the following: a preservative, a coloring agent, a flavoring agent, a stabilizer, and an isotonic agent.

5. The microbial preparation according to claim 2, characterized in that, The dosage form of the microbial preparation is selected from one of the following: lyophilized powder, gel, ointment, solution, spray, pill, tablet, capsule, and lozenge.

6. The microbial preparation according to claim 2, characterized in that, The microbial preparation also includes at least one of Mycobacterium graft, antimicrobial drugs, and probiotics.

7. A sustained-release drug, characterized in that, The substrate includes a mycobacterial bacteriophage of claim 1 and / or a microbial preparation of any one of claims 2-6 loaded on the substrate; the substrate includes medical excipients, and / or the substrate is in the shape of microspheres or nanoparticles.

8. The use of a mycobacterial phage according to claim 1, a microbial preparation according to any one of claims 2-6, or a sustained-release drug according to claim 7 in lysing mycobacteria, wherein the use is for non-disease treatment purposes.

9. The application of the mycobacterial phage according to claim 1 in the field of mycobacterial detection.

10. The application according to claim 8 or 9, characterized in that, The mycobacteria include nontuberculous mycobacteria, which include at least one of Mycobacterium abscessus, Mycobacterium avium, intracellular mycobacteria, and Mycobacterium smegmatis.

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