bacteriophage

Abstract

The present invention addresses the problem of providing a bacteriophage having bacteriolysis activity against Mycobacterium avium and / or Mycobacterium intracellulare. The present invention provides a bacteriophage having bacteriolysis activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of said bacteriophage containing a nucleic acid sequence shown in the genome of a bacteriophage identified by one of deposition numbers NITE BP-03513 to 03521.

Description

Bacteriophage 【0001】 The present invention relates to bacteriophages and uses of said bacteriophages. 【0002】 Nontuberculous mycobacteria (NTM) disease is an infectious disease caused by acid-fast bacteria other than Mycobacterium tuberculosis and Mycobacterium leprae. NTM bacteria are common in the environment, such as lakes and soil, and can cause respiratory symptoms when inhaled as aerosols. Examples of NTM include Mycobacterium avium (M. avium), Mycobacterium intracellulare (M. intracellulare), Mycobacterium fortuitum (M. fortuitum), Mycobacterium chelonae (M. chelonae), Mycobacterium gordonae (M. gordonae), Mycobacterium szulgai (M. kansasii), and Mycobacterium genavense (M. genavense) (Infection. 2004; 32: 257.). Among these, infections caused by the Mycobacterium avium complex (MAC), which includes Mycobacterium avium and Mycobacterium intracellulare, are called MAC diseases, and among these, pulmonary infections are particularly called pulmonary MAC diseases. 【0003】 Treatment methods for bacterial infections, including NTM disease, are generally multidrug combination therapies including macrolide antibiotics. In recent years, phage therapy utilizing the lytic activity of bacteriophages (hereinafter also referred to as phages) has become known (Microorganisms. 2021; 9 (3): 596.). For example, D29, TM4, and ZoeJ have been reported as phages that exhibit lytic activity against Mycobacterium avium (Journal of Medical Microbiology 2006; 55 (1): 37., Microorganisms. 2021; 9 (3): 596., Microb. Drug Resist. 2006; 12: 1.). 【0004】Bacteriophages are viruses that use bacteria as their host. When a phage infects a specific bacterium, it injects its genome into the bacterium and uses the host bacterium's metabolic machinery to propagate progeny phages. Subsequently, the bacterial cell wall is destroyed by a lytic enzyme contained in the phage genome, releasing the progeny phages and causing the host bacterium to die. 【0005】 Phages can be broadly classified into non-temperate phages (lytic phages) and temperate phages (lysogenic phages) (Scientifica (Cairo) 2014; 2014: 581639). Non-temperate phages replicate without integrating into the host bacterial DNA. They grow progeny phages within the bacteria, lyse the bacteria, and release mature phage particles. On the other hand, temperate phages are integrated into the host bacterial DNA after infecting the bacteria and replicate together with the host bacterial DNA. Simply being integrated into the host bacterial DNA is harmless to the host bacteria, and they are able to maintain a lysogenic state (a state in which the phage DNA is integrated into the host bacterial DNA and replicates together with the host bacterial DNA). However, when temperate phages are exposed to an external stimulus such as ultraviolet light, they release lytic enzymes like non-temperate phages, lyse the host bacteria, and release mature phage particles. 【0006】 Phages lacking genes involved in lysogenization have also been reported, such as phages lacking the integrase gene (Nat Med. 2019; 25(5): 730.) and phages lacking the integrase gene and the immunity repressor gene (mBio. 2021; 12(3): e00973.). 【0007】 An object of the present invention is to provide a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare. Another object of the present invention is to provide means and methods, particularly bacteriophages and pharmaceutical compositions, for preventing or treating nontuberculous mycobacterial diseases such as pulmonary mycobacterial infection (MAC) disease. 【0008】As a result of considerable inventive and inventive investigation into the creation of bacteriophages, the present inventors obtained novel bacteriophages and gene-deleted strains thereof in which their lysogeny genes had been deleted (Example 1), and discovered that the bacteriophages have lytic activity against Mycobacterium avium and Mycobacterium intracellulare (Example 2), thereby completing the present invention. Furthermore, the present inventors discovered that pharmaceutical compositions containing the bacteriophages identified by accession numbers NITE BP-03513, NITE BP-03514, and NITE BP-03519 have superior bactericidal activity compared to the individual bacteriophages (Example 3). 【0009】That is, the present invention may include the following inventions as medically or industrially useful substances or methods: [1] A bacteriophage selected from the following (1) to (9): (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and (b) is deficient in an integrase gene and / or an immunity repressor gene; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513; (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03515, and (b) is deficient in the integrase gene and / or the immunity repressor gene; (4) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03515, and (b) is deficient in the integrase gene and / or the immunity repressor gene; (4) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03516, and (b) is deficient in the integrase gene and / or the immunity repressor gene;(5) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03517, and (b) is deficient in the integrase gene and / or the immunity repressor gene; (6) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03518; (7) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03519; (8) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03520; and (9) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03521. 【0010】[2] A bacteriophage according to [1], selected from the following (1) to (9): (1) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which comprises the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513; (2) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which comprises the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03514; (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03515; (4) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03516; (5) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03517; (6) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by NITE Accession No. BP-03518;(7) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03519; (8) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03520; and (9) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03520. A bacteriophage comprising the nucleic acid sequence of the genome of the bacteriophage identified as BP-03521; 【0011】[3] A bacteriophage according to [1] or [2], selected from the following (1) to (9): (1) a bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513; (2) a bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03514; (3) a bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03515; (4) a bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03516; (5) A bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03517; (6) A bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03518; (7) A bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03519; (8) A bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03520; and (9) A bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03521. A bacteriophage comprising the nucleic acid sequence of the genome of the bacteriophage identified as BP-03521. 【0012】[4] A bacteriophage selected from the following (1) to (9): (1) a bacteriophage identified by accession number NITE BP-03513, or a passage strain thereof; (2) a bacteriophage identified by accession number NITE BP-03514, or a passage strain thereof; (3) a bacteriophage identified by accession number NITE BP-03515, or a passage strain thereof; (4) a bacteriophage identified by accession number NITE BP-03516, or a passage strain thereof; (5) a bacteriophage identified by accession number NITE BP-03517, or a passage strain thereof; (6) a bacteriophage identified by accession number NITE BP-03518, or a passage strain thereof; (7) A bacteriophage identified by accession number NITE BP-03519, or a subculture thereof; (8) A bacteriophage identified by accession number NITE BP-03520, or a subculture thereof; and (9) A bacteriophage identified by accession number NITE BP-03521, or a subculture thereof. 【0013】 [5] A pharmaceutical composition comprising the bacteriophage according to any one of [1] to [4] and a pharmaceutically acceptable excipient. [6] A pharmaceutical composition comprising at least two types of bacteriophage according to any one of [1] to [4] and a pharmaceutically acceptable excipient. [7] A pharmaceutical composition comprising three types of bacteriophage according to any one of [1] to [4] and a pharmaceutically acceptable excipient. [8] The pharmaceutical composition according to any one of [5] to [7], which is a pharmaceutical composition for the prevention or treatment of nontuberculous mycobacteriosis. [9] The pharmaceutical composition according to [8], wherein the nontuberculous mycobacteriosis is pulmonary MAC disease. 【0014】

[10] A pharmaceutical composition comprising a bacteriophage of any one of (1) to (3) below and a pharmaceutically acceptable excipient: (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and (b) is deficient in an integrase gene and / or an immunity repressor gene; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, (b) a bacteriophage comprising a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03514, and which is deficient in the integrase gene and / or the immunity repressor gene; and (3) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03519. 【0015】

[11] The pharmaceutical composition according to

[10] , which contains the bacteriophage of (1) to (3) below: (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which comprises the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which comprises the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03514; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03519. 【0016】

[12] The pharmaceutical composition according to

[10] or

[11] , comprising a bacteriophage of any one of the following (1) to (3): (1) a bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513; (2) a bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03514; and (3) a bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03519. 【0017】

[13] A pharmaceutical composition comprising a bacteriophage of any one of the following (1) to (3) and a pharmaceutically acceptable excipient: (1) a bacteriophage identified by accession number NITE BP-03513, or a subculture strain thereof; (2) a bacteriophage identified by accession number NITE BP-03514, or a subculture strain thereof; and (3) a bacteriophage identified by accession number NITE BP-03519, or a subculture strain thereof. 【0018】

[14] The pharmaceutical composition according to any one of

[10] to

[13] , which is a pharmaceutical composition for preventing or treating nontuberculous mycobacterial disease.

[15] The pharmaceutical composition according to

[14] , wherein the nontuberculous mycobacterial disease is pulmonary MAC disease. 【0019】 The bacteriophage and pharmaceutical composition of the present invention exhibit lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, and it is expected that the bacteriophage and pharmaceutical composition of the present invention can be used for the prevention or treatment of nontuberculous mycobacterial diseases such as pulmonary MAC disease. 【0020】 Specific examples of plaques are shown below. By visual inspection, transparent plaques with little or no turbidity and clear outlines were rated as +++, turbid plaques with clear outlines as ++, and turbid plaques with unclear outlines as +. This is a graph showing the results of the lytic activity of bacteriophages over time. The vertical axis shows the colony-forming unit concentration (CFU / mL), and the horizontal axis shows the time after the start of culture. LOQ represents the lower limit of quantification. This is a graph showing the results of the bactericidal activity of bacteriophages in a mouse infection model. The vertical axis shows colony-forming units in the lungs (CFU / lung). LOQ represents the lower limit of quantification. 【0021】 The present invention will be described in detail below. The following embodiments are merely examples for explaining the present invention, and are not intended to limit the present invention to these embodiments. The present invention can be embodied in various forms without departing from the gist of the present invention. 【0022】 1. Bacteriophage of the Present Invention In one embodiment, the present invention provides a novel bacteriophage having lytic activity against nontuberculous mycobacteria (NTM), particularly Mycobacterium avium and / or Mycobacterium intracellulare, which cause pulmonary MAC disease (hereinafter, sometimes referred to as the "bacteriophage of the present invention"). 【0023】The D29 lysogen gene-deficient strain D29Δ, an example of the bacteriophage of the present invention, has been deposited by the applicant with the Patent Microorganisms Deposit Center of the National Institute of Technology and Evaluation (ROI 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818), an international depositary authority under the provisions of the Budapest Treaty for the Deposit of Patent Microorganisms, on August 26, 2021 (Accession No. NITE BP-03513). 【0024】 The B1 lysogen gene-deficient strain B1Δ, an example of the bacteriophage of the present invention, was deposited by the applicant at the National Institute of Technology and Evaluation, Patent Microorganisms Deposit Center (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818) on August 26, 2021 (Accession No. NITE BP-03514). 【0025】 The Y2 lysogen gene-deficient strain Y2Δ, an example of the bacteriophage of the present invention, was deposited by the applicant at the National Institute of Technology and Evaluation, Patent Microorganisms Deposit Center (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818) on August 26, 2021 (Accession No. NITE BP-03515). 【0026】 The #33 lysogen gene-deficient strain #33Δ, an example of the bacteriophage of the present invention, was deposited by the applicant at the National Institute of Technology and Evaluation, Patent Microorganisms Deposit Center (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818, Room 122) on August 26, 2021 (Accession No. NITE BP-03516). 【0027】 The #63 lysogen gene-deficient strain #63Δ, an example of the bacteriophage of the present invention, was deposited by the applicant at the National Institute of Technology and Evaluation, Patent Microorganisms Deposit Center (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818, Room 122) on August 26, 2021 (Accession No. NITE BP-03517). 【0028】The #105 parent strain, an example of the bacteriophage of the present invention, was deposited by the applicant at the National Institute of Technology and Evaluation, Patent Microorganisms Deposit Center (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818, Room 122) on August 26, 2021 (Accession No. NITE BP-03518). 【0029】 The #123 parent strain, an example of the bacteriophage of the present invention, was deposited by the applicant at the National Institute of Technology and Evaluation, Patent Microorganisms Deposit Center (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818, Room 122) on August 26, 2021 (Accession No. NITE BP-03519). 【0030】 The #33 parent strain, an example of the bacteriophage of the present invention, was deposited by the applicant at the National Institute of Technology and Evaluation, Patent Microorganisms Deposit Center (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818, Room 122) on August 26, 2021 (Accession No. NITE BP-03520). 【0031】 The #63 parent strain, an example of the bacteriophage of the present invention, was deposited by the applicant at the National Institute of Technology and Evaluation, Patent Microorganisms Deposit Center (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818, Room 122) on August 26, 2021 (Accession No. NITE BP-03521). 【0032】In one embodiment, the bacteriophage of the present invention includes a bacteriophage selected from the group consisting of the following (1) to (9): (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and (b) is deficient in a lysogeny gene; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of a bacteriophage specified under Accession No. NITE BP-03515, and (b) is deficient in a lysogenic gene; (4) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of a bacteriophage specified under Accession No. NITE BP-03516, and (b) is deficient in a lysogenic gene; (5) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03517, and (b) is deficient in a lysogenic gene; (6) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03517, and (b) is deficient in a lysogenic gene;(6) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03518; (7) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03519; (8) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03520; and (9) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03521. 【0033】 The term "identity" as used herein refers to the value obtained by searching using the NEEDLE program (J. Mol. Biol., 1970, Vol. 48, p. 443.) using the default parameters. The parameters are as follows: Gap penalty = 10, Extend penalty = 0.5, Matrix = EBLOSUM62. 【0034】As used herein, the term "lysogenic gene" refers to a gene involved in inducing and / or maintaining a lysogenic state. Examples of "lysogenic genes" include the integrase gene, the immunity repressor gene, and the Cro gene. Specific lysogenic genes and their sequence information are also publicly known (Nat Med. 2019; 25(5): 730., mBio. 2021; 12(3): e00973., Cell. 2018; 172(6): 1260.). 【0035】 As used herein, "lysogenic gene deletion" means 1) the deletion of the entire length of at least one lysogenic gene from the genome sequence of the bacteriophage, or 2-1) the bacteriophage has become a lytic bacteriophage by modifying at least one lysogenic gene region from the genome sequence of the bacteriophage by deletion, substitution, insertion, addition, or a combination thereof, or 2-2) the function of the lysogenic gene is deleted. Lysogenic gene deletion can be performed by methods known in the art depending on the type of lysogenic gene to be deleted (e.g., Nat Med. 2019; 25(5): 730., mBio. 2021; 12(3): e00973., PLoS One. 2008; 3(12): e3957.). 【0036】 As used herein, "having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare" means that it shows lytic plaques against at least one strain of bacteria belonging to Mycobacterium avium or Mycobacterium intracellulare. The lytic activity against these bacteria can be confirmed using methods and means known in the art. For example, 10 ９ By determining whether pfu / mL of bacteriophage show lytic plaques against Mycobacterium avium or Mycobacterium intracellulare, it can be determined whether the bacteriophage has lytic activity. 【0037】In one embodiment, the bacteriophage of the present invention includes a bacteriophage selected from the group consisting of the following (1) to (9): (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and (b) is deficient in an integrase gene and / or an immunity repressor gene; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03515, and (b) is deficient in the integrase gene and / or the immunity repressor gene; (4) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03515, and (b) is deficient in the integrase gene and / or the immunity repressor gene; (4) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03516, and (b) is deficient in the integrase gene and / or the immunity repressor gene;(5) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03517, and (b) is deficient in the integrase gene and / or the immunity repressor gene; (6) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03518; (7) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03519; (8) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03520; and (9) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03521. 【0038】"Deficient in integrase gene and / or immunity repressor gene" means 1) that the entire integrase gene and / or immunity repressor gene is deleted from the genome sequence of the bacteriophage, or 2-1) that the bacteriophage has become a lytic bacteriophage by modifying the genome sequence of the bacteriophage by deletion, substitution, insertion, addition, or a combination thereof of a portion of the integrase gene and / or the immunity repressor gene, or 2-2) that the function of the integrase gene and / or the immunity repressor gene is defective. As described above, deletion of lysogenic genes such as the integrase gene and the immunity repressor gene can be performed by methods known in the art depending on the type of lysogenic gene to be deleted (e.g., Nat Med. 2019; 25(5): 730., mBio. 2021; 12(3): e00973., PLoS One. 2008; 3(12): e3957.). 【0039】 In one embodiment, the bacteriophage of the present invention includes a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and (b) is deficient in an integrase gene and / or an immunity repressor gene. 【0040】In one embodiment, the bacteriophage of the present invention includes a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03514, and (b) is deficient in an integrase gene and / or an immunity repressor gene. 【0041】 In one embodiment, the bacteriophage of the present invention includes a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03515, and (b) is deficient in an integrase gene and / or an immunity repressor gene. 【0042】 In one embodiment, the bacteriophage of the present invention includes a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03516, and (b) is deficient in an integrase gene and / or an immunity repressor gene. 【0043】In one embodiment, the bacteriophage of the present invention includes a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03517, and (b) is deficient in an integrase gene and / or an immunity repressor gene. 【0044】 In one embodiment, the bacteriophage of the present invention includes a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises a nucleic acid sequence that has 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03518. 【0045】 In one embodiment, the bacteriophage of the present invention includes a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises a nucleic acid sequence that has 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03519. 【0046】 In one embodiment, the bacteriophage of the present invention includes a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises a nucleic acid sequence that has 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03520. 【0047】 In one embodiment, the bacteriophage of the present invention includes a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises a nucleic acid sequence that has 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03521. 【0048】The bacteriophage of the present invention includes a bacteriophage selected from the following (1) to (9): (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which comprises the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03513; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which comprises the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-03514; (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03515; (4) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03516; (5) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03517; (6) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by NITE Accession No. BP-03518;(7) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03519; (8) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03520; and (9) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03520. A bacteriophage comprising the nucleic acid sequence of the genome of the bacteriophage identified as BP-03521; 【0049】The bacteriophage of the present invention includes bacteriophages selected from the following (1) to (9): (1) a bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513; (2) a bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03514; (3) a bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03515; (4) a bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03516; (5) A bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03517; (6) A bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03518; (7) A bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03519; (8) A bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03520; and (9) A bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03521. A bacteriophage comprising the nucleic acid sequence of the genome of the bacteriophage identified as BP-03521. 【0050】The bacteriophage of the present invention also includes bacteriophages selected from the following (1) to (9): (1) a bacteriophage identified by accession number NITE BP-03513; (2) a bacteriophage identified by accession number NITE BP-03514; (3) a bacteriophage identified by accession number NITE BP-03515; (4) a bacteriophage identified by accession number NITE BP-03516; (5) a bacteriophage identified by accession number NITE BP-03517; (6) a bacteriophage identified by accession number NITE BP-03518; (7) a bacteriophage identified by accession number NITE BP-03519; (8) A bacteriophage identified by accession number NITE BP-03520; and (9) A bacteriophage identified by accession number NITE BP-03521. 【0051】The bacteriophage of the present invention also includes a bacteriophage selected from the following (1) to (9): (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence in which 1 to 5,000 (e.g., 1 to 1,000, 1 to 500, or 1 to 100) bases have been deleted, substituted, inserted, or added, or a combination thereof, in the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and (b) is deficient in an integrase gene and / or an immunity repressor gene; (2) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence in which 1 to 5,000 (e.g., 1 to 1,000, 1 to 500, or 1 to 100) bases have been deleted, substituted, inserted, or added, or a combination thereof has been modified in the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03514, and (b) the bacteriophage is deficient in an integrase gene and / or an immunity repressor gene; (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence in which 1 to 5,000 (e.g., 1 to 1,000, 1 to 500, or 1 to 100) bases have been deleted, substituted, inserted, or added, or a combination thereof has been modified in the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03514. a bacteriophage (b) comprising a nucleic acid sequence in which 1 to 5,000 (e.g., 1 to 1,000, 1 to 500, or 1 to 100) bases have been deleted, substituted, inserted, or added, or a combination thereof, in the nucleic acid sequence of the genome of the bacteriophage identified as BP-03515, and which is deficient in an integrase gene and / or an immunity repressor gene;(4) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence in which 1 to 5,000 (e.g., 1 to 1,000, 1 to 500, or 1 to 100) bases have been deleted, substituted, inserted, or added, or a combination thereof has been modified in the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03516, and (b) the bacteriophage is deficient in an integrase gene and / or an immunity repressor gene; (5) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence in which 1 to 5,000 (e.g., 1 to 1,000, 1 to 500, or 1 to 100) bases have been deleted, substituted, inserted, or added, or a combination thereof has been modified in the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03516. (b) a bacteriophage having a lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage is designated by NITE accession number BP-03517 and the nucleic acid sequence is modified by deleting, substituting, inserting, or adding 1 to 5,000 (e.g., 1 to 1,000, 1 to 500, or 1 to 100) bases, or by a combination thereof; and (c) a bacteriophage having a lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage is designated by NITE accession number BP-03517 and the nucleic acid sequence is modified by deleting, substituting, inserting, or adding 1 to 5,000 (e.g., 1 to 1,000, 1 to 500, or 1 to 100) bases, or by a combination thereof. (7) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which comprises a nucleic acid sequence modified by deletion, substitution, insertion or addition of 1 to 5,000 (e.g., 1 to 1,000, 1 to 500, or 1 to 100) bases, or a combination thereof, in the nucleic acid sequence of the genome of a bacteriophage specified by accession number NITE BP-03519;(8) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage is identified by accession number NITE BP-03520 and the genome of the bacteriophage is identified by a nucleic acid sequence in which 1 to 5,000 (e.g., 1 to 1,000, 1 to 500, or 1 to 100) bases have been deleted, substituted, inserted, or added, or a combination thereof has been modified; and (9) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage is identified by accession number NITE BP-03520 and ... a nucleic acid sequence in which 1 to 5,000 (e.g., 1 to 1,000, 1 to 500, or 1 to 100) bases have been deleted, substituted, inserted, or added, or a combination thereof has been modified. A bacteriophage comprising a nucleic acid sequence in which 1 to 5,000 (e.g., 1 to 1,000, 1 to 500, or 1 to 100) bases have been deleted, substituted, inserted, or added, or a combination thereof, in the nucleic acid sequence of the genome of the bacteriophage identified as BP-03521; 【0052】 With regard to the above-mentioned modifications such as deletion, substitution, insertion or addition of bases, multiple modifications may be consecutive, or multiple modifications may be present at different positions. 【0053】 Bacteriophage mutants may be generated in which the nucleic acid sequences of the genome contained in the bacteriophage are partially deleted, substituted, inserted, and / or added during the process of subculture, production, and / or replication of the bacteriophage, and such mutants are also included in the bacteriophage of the present invention as long as they have lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare. 【0054】 The bacteriophage of the present invention also includes passaged strains of the bacteriophages identified by any of the above-mentioned NITE BP-03513 to 03521, so long as they have lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare. 【0055】 As used herein, the term "passaged strain" refers to a bacteriophage obtained by subculturing a released bacteriophage. 【0056】 The present invention also includes a polynucleotide consisting of a genome contained in a bacteriophage of the present invention (hereinafter, sometimes referred to as the "genome of the present invention"). Accordingly, the present invention provides a polynucleotide of a bacteriophage genome comprising a nucleic acid sequence having 90% or more identity to the nucleic acid sequence of the genome of any one of the bacteriophages identified by accession numbers NITE BP-03513 to 03517 and lacking an integrase gene and / or an immunity repressor gene, and a polynucleotide of a bacteriophage genome comprising a nucleic acid sequence having 90% or more identity to the nucleic acid sequence of the genome of any one of the bacteriophages identified by accession numbers NITE BP-03518 to 03521. In one embodiment, the polynucleotide encodes a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare. 【0057】 In one embodiment, the genome of the present invention includes a genome that (a) comprises a nucleic acid sequence having 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and (b) is deficient in an integrase gene and / or an immunity repressor gene. 【0058】 In one embodiment, the genome of the present invention includes a genome that (a) comprises a nucleic acid sequence having 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03514, and (b) is deficient in an integrase gene and / or an immunity repressor gene. 【0059】In one embodiment, the genome of the present invention includes a genome that (a) comprises a nucleic acid sequence having 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03515, and (b) is deficient in an integrase gene and / or an immunity repressor gene. 【0060】 In one embodiment, the genome of the present invention includes a genome that (a) comprises a nucleic acid sequence having 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03516, and (b) is deficient in an integrase gene and / or an immunity repressor gene. 【0061】 In one embodiment, the genome of the present invention includes a genome that (a) comprises a nucleic acid sequence having 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03517, and (b) is deficient in an integrase gene and / or an immunity repressor gene. 【0062】 In one embodiment, the genome of the present invention includes a genome comprising a nucleic acid sequence having 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03518. 【0063】 In one embodiment, the genome of the present invention includes a genome comprising a nucleic acid sequence having 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03519. 【0064】In one embodiment, the genome of the present invention includes a genome comprising a nucleic acid sequence having 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03520. 【0065】 In one embodiment, the genome of the present invention includes a genome comprising a nucleic acid sequence having 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03521. 【0066】 The genome of the present invention includes genomes selected from the following (1) to (9): (1) a genome comprising the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513; (2) a genome comprising the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03514; (3) a genome comprising the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03515; (4) a genome comprising the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03516; (5) a genome comprising the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03517; (6) a genome comprising the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03518; (7) A genome comprising the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03519; (8) A genome comprising the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03520; and (9) A genome comprising the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03521. 【0067】The genome of the present invention includes genomes selected from the following (1) to (9): (1) a genome consisting of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513; (2) a genome consisting of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03514; (3) a genome consisting of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03515; (4) a genome consisting of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03516; (5) a genome consisting of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03517; (6) a genome consisting of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03518; (7) A genome consisting of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03519; (8) A genome consisting of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03520; and (9) A genome consisting of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03521. 【0068】 The genomes of the present invention can be produced using common techniques known in the art, such as recombinant DNA techniques (e.g., polymerase chain reaction (PCR) amplification, cloning), enzymatic or chemical synthesis, or a combination thereof. For example, the genomes of the present invention can be produced by linking multiple polynucleotides containing partial base sequences of the genomes of the present invention using genetic engineering techniques. In one embodiment, the full-length or partial sequence of the genomes of the present invention may be contained in a vector known in the art. 【0069】 The bacteriophage of the present invention can be obtained by requesting it from the above-mentioned depository center. 【0070】Furthermore, the bacteriophage of the present invention can be produced by deciphering the nucleic acid sequence of the genome of the provided bacteriophage using common techniques known in the art and based on the sequence information. For example, the genome of the present invention produced by the above-described method is introduced into a host bacterium by electroporation (e.g., Mycobacterium is used as the host bacterium to obtain a bacteriophage that exhibits lytic activity against Mycobacterium). The bacteria into which the genome has been introduced are then placed on a plate overlaid with soft agar and cultured. Single plaques are then obtained by plaque assay. The single plaques are added to a host bacterium culture medium and cultured. The culture supernatant obtained by standing or centrifuging is then filtered, whereby the bacteriophage of the present invention can be produced. Furthermore, to prepare the bacteriophage of the present invention, the lysogen gene can be deleted from the phage genome using Bacteriophage Recombineering of Electroporated DNA (BRED) (PLoS One. 2008; 3(12): e3957.). 【0071】 The bacteriophage of the present invention can be prepared by common culture, isolation, and purification methods known in the art. For example, host bacteria (e.g., Mycobacterium smegmatis, Mycobacterium avium, or Mycobacterium intracellulare) are cultured in advance, infected with the bacteriophage of the present invention, and cultured at 37°C. After culture, the culture supernatant obtained by standing or centrifugation is filtered to obtain purified bacteriophage. The medium can be selected appropriately depending on the bacterium used. For example, 7H9 medium can be used when culturing Mycobacterium smegmatis. When preparing multiple bacteriophages, they may be grown in different host bacteria or the same host bacteria. 【0072】 Furthermore, the bacteriophage of the present invention can be stored in various forms (liquid, lyophilized, etc.) by following appropriate methods known in the art. 【0073】Based on the present invention, those skilled in the art can also prepare fusions of the bacteriophage of the present invention with peptides, proteins, or modifying agents, and these bacteriophage fusions are also included in the bacteriophage of the present invention. The peptides, proteins, and modifying agents used for fusion are not particularly limited as long as the bacteriophage fusion has lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, and examples thereof include cell membrane-permeable peptides, tag peptides, toxins, antibodies or antigen-binding fragments thereof, liposomes, lipids, lipid nanoparticles, polyethylene glycol, sugar chains, and low-molecular-weight compounds. The peptides, proteins, and modifying agents may be fused directly or indirectly to the bacteriophage of the present invention. In the case of indirect fusion, for example, a linker or tag peptide can be used. 【0074】 Bacteriophage fusion proteins can be easily prepared by those skilled in the art using methods known in the art based on the sequence information of the bacteriophage of the present invention and information on the peptide, protein, or modifier used in the fusion protein. For example, bacteriophage particles fused with a protein of interest can be prepared by infecting a host bacterium transformed with a plasmid carrying a foreign gene encoding the protein of interest with a bacteriophage. 【0075】 2. Uses of the Pharmaceutical Composition and Bacteriophage of the Present Invention The bacteriophage of the present invention has lytic activity against nontuberculous mycobacteria (NTM), particularly Mycobacterium avium and / or Mycobacterium intracellulare, which cause pulmonary MAC disease. Therefore, the bacteriophage of the present invention is expected to be effective in treating or preventing nontuberculous mycobacterial disease (NTM disease), particularly pulmonary MAC disease. 【0076】 In another aspect, the present invention provides uses of the bacteriophage of the present invention, for example, a pharmaceutical composition comprising the bacteriophage of the present invention as an active ingredient (hereinafter, sometimes referred to as the "pharmaceutical composition of the present invention"). 【0077】Pharmaceutical compositions of the present invention include those comprising a bacteriophage of the present invention and a pharmaceutically acceptable excipient. 【0078】 The pharmaceutical compositions of the present invention can be prepared by commonly used methods using excipients commonly used in the art, i.e., pharmaceutical excipients and pharmaceutical carriers. Examples of dosage forms of these pharmaceutical compositions include parenteral preparations such as injections, infusions, powder inhalants, and nebulizers, which can be administered intravenously or via the lungs. When formulating the compositions, excipients, carriers, or additives appropriate for these dosage forms can be used within a pharmaceutically acceptable range. For example, the pharmaceutical compositions of the present invention can be produced by mixing the bacteriophage of the present invention with a pharmaceutically acceptable excipient, or by suspending the bacteriophage of the present invention in a pharmaceutically acceptable excipient. 【0079】 Pharmaceutical compositions of the present invention include those containing at least two types of bacteriophages of the present invention and a pharmaceutically acceptable excipient. 【0080】 The pharmaceutical compositions of the present invention include those containing three types of bacteriophages of the present invention and a pharmaceutically acceptable excipient. 【0081】In one embodiment, the pharmaceutical composition of the present invention includes a pharmaceutical composition comprising the bacteriophage of any one of (1) to (3) below and a pharmaceutically acceptable excipient: (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and (b) is deficient in a lysogeny gene; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, (b) a bacteriophage that (a) comprises a nucleic acid sequence that is 90% or more identical to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03514, and (b) is deficient in a lysogeny gene; and (3) a bacteriophage that has lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of the bacteriophage comprising a nucleic acid sequence that is 90% or more identical to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03519. 【0082】In one embodiment, the pharmaceutical composition of the present invention includes the following (1) to (3): (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and (b) is deficient in an integrase gene and / or an immunity repressor gene; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, (b) a bacteriophage comprising a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03514, and which is deficient in the integrase gene and / or the immunity repressor gene; and (3) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03519. 【0083】In one embodiment, the pharmaceutical composition of the present invention comprises the following (1) to (3): (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513; (2) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03514; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03519. 【0084】 In one embodiment, the pharmaceutical composition of the present invention comprises the following (1) to (3): (1) a bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513; (2) a bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03514; and (3) a bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03519. 【0085】 In one embodiment, the pharmaceutical composition of the present invention comprises the following (1) to (3): (1) a bacteriophage identified by accession number NITE BP-03513; (2) a bacteriophage identified by accession number NITE BP-03514; and (3) a bacteriophage identified by accession number NITE BP-03519. 【0086】In one embodiment, the pharmaceutical composition of the present invention comprises the following (1) to (3): (1) a bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, the genome of which (a) comprises a nucleic acid sequence in which 1 to 5,000 (e.g., 1 to 1,000, 1 to 500, or 1 to 100) bases have been deleted, substituted, inserted, or added, or a combination thereof, in the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03513, and (b) is deficient in an integrase gene and / or an immunity repressor gene; (2) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence in which 1 to 5,000 (e.g., 1 to 1,000, 1 to 500, or 1 to 100) bases have been deleted, substituted, inserted, or added, or a combination thereof has been modified in the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03514, and (b) the bacteriophage is deficient in an integrase gene and / or an immunity repressor gene; and (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence in which 1 to 5,000 (e.g., 1 to 1,000, 1 to 500, or 1 to 100) bases have been deleted, substituted, inserted, or added, or a combination thereof has been modified in the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-03514. A bacteriophage comprising a nucleic acid sequence in which 1 to 5,000 (e.g., 1 to 1,000, 1 to 500, or 1 to 100) bases have been deleted, substituted, inserted, or added, or a combination thereof has been modified in the nucleic acid sequence of the genome of the bacteriophage identified as BP-03519. 【0087】 With regard to the above-mentioned modifications such as deletion, substitution, insertion or addition of bases, multiple modifications may be consecutive, or multiple modifications may be present at different positions. 【0088】The pharmaceutical composition of the present invention also includes a passaged strain of the bacteriophage identified by any of the above-mentioned accession numbers NITE BP-03513, NITE BP-03514, and NITE BP-03519, so long as it has lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare. 【0089】 The effective dose, number of doses, and duration of administration vary depending on the purpose of administration (therapeutic or preventive), the severity and age of the subject, the dosage form of the preparation used, the titer of the bacteriophage, etc. For example, the effective dose of a single bacteriophage or the total effective dose of two or more bacteriophages is 10 ４ ~10 １４ The dosage ratio of two or more bacteriophages can be adjusted appropriately depending on the severity of symptoms and age of the patient, the dosage form of the preparation used, the titer of the bacteriophage, etc. For example, when a pharmaceutical composition contains three bacteriophages, each bacteriophage is used in approximately equal amounts (e.g., 3.3 × 10 ３ ~3.3 x 10 １３ pfu), or each bacteriophage may be contained in different proportions. 【0090】 The pharmaceutical composition of the present invention can be used as an agent for preventing or treating NTM diseases, such as MAC disease or pulmonary MAC disease. 【0091】 As used herein, "treatment" means at least partial improvement of symptoms of NTM disease (e.g., cough, sputum, bloody sputum, fever, dyspnea, fatigue, pulmonary nodules, bronchiectasis, etc.), arrest of the progression or worsening of NTM disease including negative sputum culture, complete cure, etc. As used herein, "prevention" means preventing a subject not suffering from NTM disease from contracting NTM disease, preventing the recurrence of NTM disease, etc. 【0092】 The present invention includes a pharmaceutical composition for preventing or treating NTM diseases, such as MAC disease or pulmonary MAC disease, which comprises the bacteriophage of the present invention or the pharmaceutical composition of the present invention. 【0093】The present invention also includes a method for preventing or treating an NTM disease, e.g., MAC disease or pulmonary MAC disease, in a subject, comprising administering a therapeutically effective amount of a bacteriophage of the present invention or a pharmaceutical composition of the present invention. In one embodiment, at least two, e.g., three, types of bacteriophages of the present invention can be administered to a subject, and the multiple bacteriophages can be administered simultaneously or separately. 【0094】 The present invention further includes the bacteriophage of the present invention or the pharmaceutical composition of the present invention for use in the prevention or treatment of an NTM disease, such as MAC disease or pulmonary MAC disease. The present invention further includes use of the bacteriophage of the present invention or the pharmaceutical composition of the present invention in the manufacture of a pharmaceutical composition for the prevention or treatment of an NTM disease, such as MAC disease or pulmonary MAC disease. 【0095】 In the present invention, the subject to which the bacteriophage is administered is not limited as long as it is a mammal, and examples thereof include mice, rats, dogs, pigs, monkeys, humans, etc. For example, the bacteriophage is administered to subjects diagnosed with NTM disease or subjects at risk of contracting NTM disease. 【0096】 Furthermore, the bacteriophage of the present invention or the pharmaceutical composition of the present invention may be used or administered in combination with other ingredients that are effective in treating or preventing NTM disease, and the bacteriophage of the present invention or the pharmaceutical composition of the present invention may be provided as a combined medicine with such other active ingredients. 【0097】 Having generally described the invention, reference is now made to specific examples to provide a further understanding thereof, which are intended for purposes of illustration and not as limitations of the invention. 【0098】 Unless otherwise specified, experiments using commercially available kits or reagents were conducted according to the accompanying protocols. For convenience, concentrations are expressed as M (mol / L). For example, a 1M sodium hydroxide solution means a 1 mol / L sodium hydroxide solution. 【0099】Example 1: Preparation of bacteriophage Bacteriophage was isolated from a natural environment sample by the following method. A natural environment sample (soil in Japan) was diluted with 1 mM CaCl ２ , 10 mM MgSO ４ The bacteria were suspended in 10 mM Tris-HCl (pH 7.5) containing 68.4 mM NaCl. The suspension was centrifuged, and the supernatant was separated. The supernatant was filtered through a 0.22 μm filter. The filtrate was mixed with Mycobacterium smegmatis and cultured with shaking in 7H9 medium for several hours to one day. The culture was centrifuged, and the supernatant was separated. The supernatant was filtered through a 0.22 μm filter. The filtrate was mixed with Mycobacterium smegmatis and 7H9 medium containing 0.6% soft agar, and the mixture was layered on a 7H10 plate. Culture was continued at 37°C for several days. Plaques that appeared on the medium were collected and used as parent phages. The parent phages obtained are referred to as parent strains #33, #63, #105, and #123. 【0100】 Next, genomes were extracted from phages D29, B1, and Y2 (also referred to as the D29 parent strain, B1 parent strain, and Y2 parent strain, respectively) provided by Okayama University, and from phages #33 and #63 parent strains obtained by the above method, and the entire nucleotide sequences were determined. Lysogenic genes were identified through annotation. Phages lacking the integrase gene and the immunity repressor gene, which are lysogenic genes, were obtained by one of the following methods: 1) The lysogenic genes were deleted from the parent phages obtained above using the method of Marinelli et al. (PLoS One. 2008; 3(12): e3957.) to obtain gene-deficient phages of interest. Alternatively, 2) a target phage genome (having a base sequence from which the lysogen gene has been deleted) was constructed by genetic engineering techniques from multiple polynucleotides having a partial base sequence of the phage genome, and the phage genome was introduced into Mycobacterium smegmatis by electroporation to obtain the target gene-deficient phages. The resulting bacteriophages are referred to as the D29 lysogen gene-deficient strain, the B1 lysogen gene-deficient strain, the Y2 lysogen gene-deficient strain, the #33 lysogen gene-deficient strain, and the #63 lysogen gene-deficient strain, respectively. 【0101】The genome of the bacteriophage obtained above was extracted and the entire nucleotide sequence was determined. By comparing the nucleotide sequence with that of the parent phage, it was confirmed that the integrase gene and the immunity repressor gene had been deleted. 【0102】 The accession numbers of the D29 lysogen gene-deficient strain, B1 lysogen gene-deficient strain, Y2 lysogen gene-deficient strain, #33 lysogen gene-deficient strain, #63 lysogen gene-deficient strain, #105 parent strain, #123 parent strain, #33 parent strain, and #63 parent strain obtained above are accession number NITE BP-03513, accession number NITE BP-03514, accession number NITE BP-03515, accession number NITE BP-03516, accession number NITE BP-03517, accession number NITE BP-03518, accession number NITE BP-03519, accession number NITE BP-03520, and accession number NITE BP-03521, respectively. 【0103】Example 2: Lytic activity of bacteriophage As target bacteria, clarithromycin-resistant Mycobacterium avium clinical isolate KCH-ASGF-MAC-53 or KCH-ASGF-MAC-376 (provided by the Kinki Central Respiratory Center) or clarithromycin-resistant Mycobacterium intracellulare clinical isolate KCH-ASGF-MAC-27 or KCH-ASGF-MAC-90 (provided by the Kinki Central Respiratory Center) were grown on 7H10 plates (19 g / L Middlebrook 7H10 Agar (Difco), 6.3 g / L glycerol, 100 mL / L Middlebrook OADC Enrichment (Difco)). Bacterial colonies were scraped with a cotton swab and suspended in 7H9 medium (4.7 g / L Middlebrook 7H9 Broth (Difco)). The bacterial suspension was adjusted with 7H9 medium to an OD600 of approximately 1.5 to 2.0 using a spectrophotometer. Seven hundred microliters of the bacterial suspension was mixed with 7 mL of 0.6% soft agar-containing 7H9 medium (4.7 g / L Middlebrook 7H9 Broth (Difco), 6 g / L agarose, 100 mL / L Middlebrook ADC Enrichment (Difco) or 4.7 g / L Middlebrook 7H9 Broth (Difco), 6 g / L agarose, 100 mL / L Middlebrook OADC Enrichment (Difco)), and the mixture was layered on a 7H10 plate (100 mm long x 140 mm wide x 14.5 mm high). 【0104】 After confirming that the soft agar had completely solidified, the D29 parent strain, the D29 lysogen gene-deficient strain, the B1 parent strain, the B1 lysogen gene-deficient strain, the Y2 parent strain, the Y2 lysogen gene-deficient strain, the #33 parent strain, the #33 lysogen gene-deficient strain, the #63 parent strain, the #63 lysogen gene-deficient strain, the #105 parent strain, and the #123 parent strain described in Example 1 were added to a phage buffer (1 mM CaCl ２ , 10 mM MgSO ４ 10 mM Tris-HCl (pH 7.5) containing 68.4 mM NaCl ９The solution was diluted to pfu / mL and 2.5 μL was dropped onto the plate. The plate was cultured at 37°C for approximately 3 to 7 days. The presence or absence of plaques was confirmed by visual inspection. When plaques were observed, those that were transparent with little or no turbidity and showed clear contours were rated as +++, those that were turbid and showed clear contours were rated as ++, and those that were turbid and showed unclear contours were rated as + (see Figure 1). Note that preliminary studies have confirmed that plaques do not form when only phage buffer is added to solidified soft agar. 【0105】 The results are shown in Table 1. The bacteriophage D29 parent strain, the D29 lysogen gene-deficient strain, the B1 parent strain, the B1 lysogen gene-deficient strain, the Y2 parent strain, the Y2 lysogen gene-deficient strain, the #33 parent strain, the #33 lysogen gene-deficient strain, the #63 parent strain, the #63 lysogen gene-deficient strain, the #105 parent strain, and the #123 parent strain were found to produce lytic plaques against Mycobacterium avium and Mycobacterium intracellulare. 【0106】 【0107】 From the above, it was confirmed that the D29 parent strain, the D29 lysogen gene-deficient strain, the B1 parent strain, the B1 lysogen gene-deficient strain, the Y2 parent strain, the Y2 lysogen gene-deficient strain, the #33 parent strain, the #33 lysogen gene-deficient strain, the #63 parent strain, the #63 lysogen gene-deficient strain, the #105 parent strain, and the #123 parent strain have lytic activity against Mycobacterium avium and Mycobacterium intracellulare. 【0108】Example 3: Time-dependent lytic activity of bacteriophage alone or a phage cocktail. Approximately three colonies of Mycobacterium smegmatis grown on a 7H10 plate were scraped and suspended in 5 mL of 7H9 medium containing 0.05% Tween 80 (Nacalai Tesque). The bacterial suspension was then shaken at 37°C until the OD600 reached 3 or higher using a spectrophotometer. After incubation, the bacterial suspension was centrifuged, the supernatant removed, and the precipitate resuspended in 7H9 medium. This procedure was repeated three times to obtain a bacterial suspension. The bacterial suspension was mixed with 7H9 medium containing 0.6% soft agar, and the D29 lysogen gene-deficient strain, the B1 lysogen gene-deficient strain, or the #123 parent strain (three strains) described in Example 1 was added. The mixture was then mixed by inversion and layered on a 7H10 plate. After the soft agar solidified, the mixture was incubated at 37°C for approximately one day. 10 mL of phage buffer was added to the plate, and the plate was left to stand at room temperature for approximately 4 hours. The supernatant of the plate was filtered through a 0.22 μm filter to obtain phage lysates of each of the three strains. The pfu of the phage lysates was determined and stored at 4°C. 【0109】 The phage lysates of each of the three strains were diluted to 1 × 10 １０ pfu / mL, or 3 x 10 １０ The phage was diluted with phage buffer to a total of 1 × 10 pfu / mL. １０ pfu / mL, or a total of 3 x 10 １０Equal amounts of each were mixed to achieve a concentration of pfu / mL. 10 μL of these phage solutions or phage buffers were added to each well of a 96-well plate. Two Mycobacterium avium strains (KCH-ASGF-MA-05 (distributed by the Kinki Central Respiratory Center) or ATCC 700898 (distributed by ATCC)) grown on a 7H10 plate at 37°C for 6 days were scraped and suspended in 7H9 medium. A bacterial suspension was adjusted in 7H9 medium using a spectrophotometer to a final OD600 of 0.0025, and 90 μL was added to each well of a 96-well plate. A plate seal was affixed to the 96-well plate as a lid, and the plate was incubated at 37°C. The colony-forming unit concentration (CFU / mL) was calculated at 0, 48, 96, and 240 hours after incubation. Three trials were performed to evaluate the phage. At each time point, the colony-forming unit concentration (CFU / mL) for each of the three trials was converted to a logarithmic value, and the arithmetic mean ± standard error was calculated (Figure 2). The CFU / mL after 240 hours was tested using one-way analysis of variance and Tukey's multiple comparison test (Table 2). 【0110】 Table 2 shows the results of the colony-forming unit concentration (CFU / mL) test 240 hours after culture. * indicates that a significant difference was observed between any two groups when tested by Tukey's multiple comparison test at a significance level of less than 5% (p<0.05), ** indicates that a significant difference was observed between any two groups by the same test (p<0.01), *** indicates that a significant difference was observed between any two groups by the same test (p<0.001), and n.s. (not significant) indicates that no significant difference was observed by the same test. 【0111】 From the above, a comparison of colony-forming unit concentrations (CFU / mL) at 0 and 96 hours after incubation confirmed that each of the three phages and the phage cocktail had the activity to reduce the bacterial count of Mycobacterium avium at 0 hours after incubation (Figure 2). Furthermore, it was confirmed that this activity was maintained for a longer period when the phage cocktail was used compared to when each phage was used alone (Figure 2 and Table 2). 【0112】 【0113】 Example 4: Efficacy of phage cocktail in a mouse infection model Bacterial colonies of Mycobacterium smegmatis grown on a 7H10 plate were scraped and suspended in LB (Luria-Bertani) medium, followed by expansion culture at 37°C. After culture, the D29 lysogen gene-deficient strain, the B1 lysogen gene-deficient strain, or the #123 parent strain, all obtained by culture using the method described in Example 3, was added, and culture was continued at 37°C for approximately one day. The supernatant after culture was filtered through a 0.22 μm filter to obtain phage lysates for each of the three strains. Next, each lysate was purified by ultracentrifugation to obtain a phage solution. 【0114】 Purification by ultracentrifugation: DNase I and RNase A were added to each phage lysate to a final concentration of 1 μg / mL, and the mixture was incubated at room temperature for 30 minutes. NaCl was added to a final concentration of 1 M, and the mixture was then incubated on ice or in a refrigerator for 1 hour. Polyethylene glycol 8000 was added to a final concentration of 10% w / v, and the mixture was then incubated on ice or in a refrigerator for 2 hours. The mixture was centrifuged at 11,000 g for 10 minutes at 4°C, the supernatant was removed, and the precipitate was suspended in phage buffer and collected. The collected solution was gently dissolved in 0.5 g of cesium chloride per mL and transferred to an ultracentrifugation container. Cesium chloride solutions at concentrations of 1.45 g / mL, 1.5 g / mL, and 1.7 g / mL were sequentially injected into the bottom of the container using a syringe. The phage-containing band was collected after centrifugation at 22,000 g for 2 hours at 4°C. The solution containing the recovered phages was replaced with a phage buffer by dialysis to obtain a phage solution. The pfu of the phage solution was determined and stored at 4°C. 【0115】 The phage solutions of the D29 lysogen gene-deficient strain, the B1 lysogen gene-deficient strain, and the #123 parent strain, which were cultured by the method described above, were each diluted to 5 × 10 １１ After adjustment, the phage solutions were mixed in a ratio of 1:1:1 to prepare a phage cocktail administration solution. 【0116】Clarithromycin-resistant Mycobacterium intracellulare KCH-ASGF-MAC-475 strain (provided by the Kinki Central Respiratory Center) grown on a 7H10 plate at 37°C for approximately 4 days was scraped and suspended in saline. After suspension, the bacterial suspension was adjusted with saline to a final OD600 of 0.005 using a spectrophotometer. Twenty nude mice (Jackson Laboratory Japan, BALB / c-nu (nu / nu), 7-week-old, female) were intranasally administered with 40 μL of the bacterial suspension per mouse to infect the lungs. Phage buffer or phage cocktail administration solution (20 μL per mouse) was administered intranasally once per day (19 times in total) on days 6-10, 13-17, 20-23, and 27-31 after infection. Mice were euthanized 34 days after infection, and lung tissue was collected. ＴＭ Lung tissue was disrupted in saline using an Octo Dissociator (Miltenyi Biotec) to obtain a disruption solution. The colony-forming unit concentration (CFU / mL) of the disruption solution was calculated and expressed as the number of bacteria in the lung (CFU / lung) (Figure 3). 【0117】 Figure 3 shows the results of testing the bacterial count in the lungs (CFU / lung). The difference between the two groups was tested by unpaired t-test at a significance level of less than 5%, and it was found that the phage cocktail group had a significant difference from the phage buffer group (p<0.001). 【0118】 These results demonstrate that the bacteriophage cocktail has bactericidal activity in an in vivo pulmonary infection model using mice. 【0119】 The bacteriophage and pharmaceutical composition of the present invention are expected to be useful in the prevention or treatment of nontuberculous mycobacterial diseases, such as MAC disease or pulmonary MAC disease. 【0120】[Accession number] Accession number NITE BP-03513 (bacteriophage D29 lysogen gene-deficient strain D29Δ, deposited on August 26, 2021) Accession number NITE BP-03514 (bacteriophage B1 lysogen gene-deficient strain B1Δ, deposited on August 26, 2021) Accession number NITE BP-03515 (bacteriophage Y2 lysogen gene-deficient strain Y2Δ, deposited on August 26, 2021) Accession number NITE BP-03516 (bacteriophage #33 lysogen gene-deficient strain #33Δ, deposited on August 26, 2021) Accession number NITE BP-03517 (bacteriophage #63 lysogen gene-deficient strain #63Δ, deposited on August 26, 2021) Accession number: NITE BP-03518 (bacteriophage #105 parent strain, deposited on August 26, 2021) Accession number: NITE BP-03519 (bacteriophage #123 parent strain, deposited on August 26, 2021) Accession number: NITE BP-03520 (bacteriophage #33 parent strain, deposited on August 26, 2021) Accession number: NITE BP-03521 (bacteriophage #63 parent strain, deposited on August 26, 2021)

Claims

[Claim 1] A pharmaceutical composition comprising the bacteriophages (1) to (3) below and pharmaceutically acceptable excipients: (1) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE BP-03513; (2) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE BP-03514; and, (3) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage contains the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE BP-03519. [Claim 2] The pharmaceutical composition according to claim 1, further comprising at least one bacteriophage selected from (4) to (7) below: (4) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE BP-03515; (5) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE BP-03516; (6) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE BP-03517; and (7) A bacteriophage having lytic activity against Mycobacterium avium and / or Mycobacterium intracellulare, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE BP-03518. [Claim 3] The pharmaceutical composition according to claim 1, comprising the bacteriophages (1) to (3) below: (1) A bacteriophage whose genome consists of the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE BP-03513; (2) A bacteriophage whose genome consists of the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE BP-03514; and, (3) A bacteriophage whose genome consists of the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE BP-03519. [Claim 4] The pharmaceutical composition according to claim 3, further comprising at least one bacteriophage selected from (4) to (7) below: (4) A bacteriophage whose genome consists of the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE BP-03515; (5) A bacteriophage whose genome consists of the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE BP-03516; (6) A bacteriophage whose genome consists of the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE BP-03517; and (7) A bacteriophage whose genome consists of the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE BP-03518. [Claim 5] A pharmaceutical composition comprising the bacteriophages (1) to (3) below and pharmaceutically acceptable excipients: (1) Bacteriophages identified by accession number NITE BP-03513, or their subcultivated strains; (2) Bacteriophages identified by accession number NITE BP-03514, or their subcultivated strains; and, (3) A bacteriophage identified by accession number NITE BP-03519, or its subcultivated strain. [Claim 6] The pharmaceutical composition according to claim 5, further comprising at least one bacteriophage selected from (4) to (7) below: (4) Bacteriophages identified by accession number NITE BP-03515, or their subcultivated strains; (5) Bacteriophages identified by accession number NITE BP-03516, or their subcultivated strains; (6) Bacteriophages identified by accession number NITE BP-03517, or their subcultivated strains; and (7) A bacteriophage identified by accession number NITE BP-03518, or its subcultivated strain. [Claim 7] A pharmaceutical composition according to any one of claims 1 to 6, which is a pharmaceutical composition for the prevention or treatment of nontuberculous mycobacterial disease. [Claim 8] The pharmaceutical composition according to claim 7, wherein the nontuberculous mycobacterial disease is pulmonary MAC disease. [Claim 9] The pharmaceutical composition according to any one of claims 1 to 6, which is in the form of an injection, an intravenous infusion, a powder inhalation, or a nebulizer. [Claim 10] A pharmaceutical composition according to any one of claims 1 to 6, which is administered intravenously or pulmonaryly. [Claim 11] The pharmaceutical composition according to any one of claims 1 to 6, wherein the bacteriophage is administered simultaneously or separately.