Pseudomonas aeruginosa bacteriophage

Abstract

The present invention provides a bacteriophage having bacteriolytic activity against Pseudomonas aeruginosa, the bacteriophage genome comprising a nucleic acid sequence represented in the genome of a bacteriophage identified by accession numbers NITE BP-04040, NITE BP-04031, NITE BP-04033, NITE BP-04036, NITE BP-04034, NITE BP-04035, accession number NITE ABP-04200, or accession number NITE ABP-04201. The present invention also provides a bacteriophage having bacteriolytic activity against Pseudomonas aeruginosa, the bacteriophage including a nucleic acid sequence encoding a tail fiber gene including at least a portion of a tail fiber gene derived from another bacteriophage and / or a nucleic acid sequence encoding at least one protein or active domain selected from the group consisting of (a) Pyocin G, (b) PslGh, and (c) PelAh.

Description

Pseudomonas aeruginosa bacteriophage

[0001] The present invention relates to novel bacteriophages and modified bacteriophages having lytic activity against Pseudomonas aeruginosa, and uses of these bacteriophages.

[0002] Pseudomonas aeruginosa is a species of bacteria belonging to the genus Pseudomonas and is a Gram-negative bacterium known to cause hospital-acquired infections. It is a common cause of pneumonia, infections in immunocompromised patients, and infections in patients with respiratory symptoms, such as cystic fibrosis. Antibiotics are primarily used to treat Pseudomonas aeruginosa infections. Pseudomonas aeruginosa produces an extracellular matrix containing polysaccharides and forms a biofilm. Biofilm-forming Pseudomonas aeruginosa is protected from phagocytosis by neutrophils and antibiotics, which can pose a therapeutic challenge (Drugs. 2021; 81(18): 2117-2131).

[0003] Bacteriophages (hereinafter also referred to as phages) are viruses that use bacteria as hosts. Phages infect specific bacteria, use the metabolic machinery of the host bacteria to grow progeny phages, and then lyse the host bacteria using lytic enzymes encoded in the phage genome. In recent years, phage therapy using bacteriophages for Pseudomonas aeruginosa infections has been attempted (Antibiotics. 2021; 10(5): 556).

[0004] It has been reported that the host range can be changed by creating a bacteriophage in which part of the tail fiber gene of a bacteriophage that infects Pseudomonas aeruginosa has been exchanged with the tail fiber gene of another bacteriophage (WO 2016 / 055585).

[0005] Bacteriophages can be genetically modified using synthetic biology techniques, such as by deleting or inserting specific genes, including those that infect Pseudomonas aeruginosa (Proceedings of the National Academy of Sciences of the United States of America. 2022; 119(48): e2206739119).

[0006] Proteins known to inhibit and destroy P. aeruginosa biofilm formation include glycoside hydrolases PelA and PslG, and their active domains PelAh and PslGh, derived from P. aeruginosa. These hydrolyze Pel and Psl, respectively, which are exopolysaccharides produced by P. aeruginosa (Science Advances. 2016; 2(5): e1501632). The degree of dependence of biofilms formed by P. aeruginosa strains on Pel and Psl varies among strains, with some strains forming matrices dominated by Pel, others dominated by Psl, and others showing redundancy in Pel and Psl (Environmental microbiology. 2012; 14(8): 1913-1928). Therefore, it is expected that the ability of PelA, PelAh, PslG, and PslGh to degrade Pseudomonas aeruginosa biofilms varies depending on the Pseudomonas aeruginosa strain.

[0007] Bacteriocins are proteins produced by bacteria that usually kill the same bacterial species. Pyocin G (hereinafter also referred to as PyoG), a bacteriocin derived from Pseudomonas aeruginosa, is known to exhibit antibacterial activity against Pseudomonas aeruginosa (Journal of molecular biology. 2020; 432(13): 3869-3880).

[0008] The following are known examples of reports that change the activity of bacteriophages by inserting specific genes into their genomes. It has been reported that a genetically engineered bacteriophage, in which a gene encoding an enzyme that breaks down extracellular polysaccharides has been artificially inserted into the genome of bacteriophage T7 that infects Escherichia coli, can remove biofilms formed by Escherichia coli (Proceedings of the National Academy of Sciences of the United States of America. 2007; 104 (27): 11197-11202). It has been reported that inserting an alginate-degrading enzyme (alginate lyase) into the genome of a bacteriophage that infects Pseudomonas aeruginosa can decompose alginic acid, a component of biofilms (International Publication No. WO 2023 / 015195). It has been reported that by inserting a bacteriocin into the genome of a bacteriophage that infects Escherichia coli, Klebsiella, or Enterococcus, the bacteriocin can also exert bactericidal activity against other corresponding bacterial species (WO 2023 / 052628, Nat Commun 2023;14:4337), however, there have been no reported cases of inserting a bacteriocin into the genome of a bacteriophage that infects Pseudomonas aeruginosa.

[0009] In general, in bacteriophage therapy, a cocktail containing multiple types of bacteriophages is sometimes used for the purpose of expanding the host range and preventing the development of resistance to the bacteriophage. It has also been shown that the bactericidal activity of bacteriophages that infect Pseudomonas aeruginosa can be improved by mixing bacteriophages of different genetic lineages (Scientific Reports. 2023; 13(1): 8921).

[0010] In bacteria including Pseudomonas aeruginosa, temperate bacteriophages may lysogenize and exist inserted into the genome of the host bacterium (lysogenized bacteriophages are also called prophage). For example, two filamentous phages, Pf4 and Pf6, are lysogenized in the Pseudomonas aeruginosa PAO1 strain, and a method for obtaining a strain from which these phages have been removed from the host bacterium has been reported (Journal of Bacteriology. 2024; 206(5): e0040223).

[0011] Prophages that lysogenize bacteria can encode defensive genes to protect the host bacteria from infection by competing bacteriophages, which have been reported to inhibit the growth of competing bacteriophages (Nat. Commun. 2024; 15(1): 1644).

[0012] International Publication No. WO 2016 / 055585 International Publication No. WO 2023 / 015195 International Publication No. WO 2023 / 052628

[0013] Science Advances. 2016;2(5):e1501632Journal of molecular biology. 2020;432(13):3869-3880 Proceedings of the National Academy of Sciences of the United States of America. 2007;104(27):11197-11202 Nat Commun 2023;14:4337Scientific Reports. 2023;13(1):8921Journal of bacteriology. 2024;206(5):e0040223Nat Commun. 2024;15(1):1644

[0014] An object of the present invention is to provide a bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, including Pseudomonas aeruginosa. Another object of the present invention is to provide means and methods, particularly bacteriophages and pharmaceutical compositions, for preventing or treating infections caused by bacteria of the genus Pseudomonas, such as Pseudomonas aeruginosa infections.

[0015] As a result of considerable ingenuity and investigation into the creation of bacteriophages, the present inventors obtained novel bacteriophages and found that these bacteriophages had lytic activity against Pseudomonas aeruginosa (Examples 1, 8, 9, and 10). They also created bacteriophages with an exchanged tail fiber gene and found that these bacteriophages had lytic activity against a wider range of Pseudomonas aeruginosa strains (Example 2). They created bacteriophages with introduced glycoside hydrolases (Example 3) and bacteriocins (Example 4), and confirmed that each had lytic activity against Pseudomonas aeruginosa (Examples 3 and 4). They found that novel bacteriophages, modified bacteriophages, and cocktails containing multiple types of bacteriophages had biofilm-degrading activity (Examples 5, 6, and 12), and that they also had lytic activity against Pseudomonas aeruginosa (Examples 7 to 9, 11, and 13). The present inventors created a host bacterium in which a gene derived from a prophage was deleted, and found that the host bacterium amplifies bacteriophages with high efficiency (Example 14). Based on these findings, the present invention was completed.

[0016] 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 (8): (1) a bacteriophage having lytic activity against Pseudomonas aeruginosa, 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 specified by accession number NITE BP-04040; (2) a bacteriophage having lytic activity against Pseudomonas aeruginosa, 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 specified by accession number NITE BP-04031; (3) a bacteriophage having lytic activity against Pseudomonas aeruginosa, 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 specified by accession number NITE BP-04031; (4) A bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of which 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-04036; (5) A bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of which 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-04034; (6) A bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of which 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-04036; (7) A bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of which comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of a bacteriophage specified by accession number NITE ABP-04200;and (8) a bacteriophage having lytic activity against Pseudomonas aeruginosa, 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 ABP-04201. [2] A bacteriophage according to [1], selected from the following (1) to (8): (1) A bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of which comprises the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-04040; (2) A bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of which comprises the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-04031; (3) A bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of which comprises the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-04033; (4) A bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04036; (5) A bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04034; (6) A bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04035; (7) A bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04036; a bacteriophage comprising the nucleic acid sequence of the genome of the bacteriophage identified in ABP-04200;and (8) a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE ABP-04201. [3] A bacteriophage according to [1] or [2], selected from the following (1) to (8): (1) a bacteriophage whose genome consists of the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE BP-04040; (2) a bacteriophage whose genome consists of the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE BP-04031; (3) a bacteriophage whose genome consists of the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE BP-04033; (4) a bacteriophage whose genome consists of the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE BP-04036; (5) A bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04034; (6) A bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04035; (7) A bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE ABP-04200; and (8) A bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE ABP-04201. [4] A bacteriophage selected from the following (1) to (8): (1) a bacteriophage identified by accession number NITE BP-04040, or a passage strain thereof; (2) a bacteriophage identified by accession number NITE BP-04031, or a passage strain thereof; (3) a bacteriophage identified by accession number NITE BP-04033, or a passage strain thereof; (4) a bacteriophage identified by accession number NITE BP-04036, or a passage strain thereof;(5) A bacteriophage identified by accession number NITE BP-04034, or a subculture thereof; (6) A bacteriophage identified by accession number NITE BP-04035, or a subculture thereof; (7) A bacteriophage identified by accession number NITE ABP-04200, or a subculture thereof; and (8) A bacteriophage identified by accession number NITE ABP-04201, or a subculture thereof.

[0017] [5] A bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage (a) comprises a nucleic acid sequence having 90% or more identity to the nucleic acid sequence of the genome of the bacteriophage specified by Accession No. NITE BP-04032, excluding a portion of the tail fiber gene, and (b) comprises a nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa. [6] A bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage (a) comprises the nucleic acid sequence of the genome of the bacteriophage specified by Accession No. NITE BP-04032, excluding a portion of the tail fiber gene, and (b) comprises a nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa, the bacteriophage according to [5]. [7] A bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage (a) consists of the nucleic acid sequence of the genome of the bacteriophage identified by Accession No. NITE BP-04032, excluding a portion of the tail fiber gene, and (b) comprises a nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa. [8] The bacteriophage of [5], wherein the tail fiber gene derived from another bacteriophage comprises the nucleic acid sequence set forth in SEQ ID NO:6 or a nucleic acid sequence having 90% or more identity to SEQ ID NO:6.

[0018] [9] A bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises a nucleic acid sequence encoding at least one protein or active domain selected from the following (a) to (c): (a) Pyocin G; (b) PslGh; and (c) PelAh.

[10] The bacteriophage according to [9], wherein the genome of the bacteriophage comprises a nucleic acid sequence encoding at least two proteins or active domains selected from the following (a) to (c): (a) Pyocin G; (b) PslGh; and (c) PelAh.

[11] The bacteriophage according to [9], wherein the genome of the bacteriophage comprises a nucleic acid sequence encoding the proteins or active domains selected from the following (a) to (c): (a) Pyocin G; (b) PslGh; and (c) PelAh.

[0019]

[12] A pharmaceutical composition comprising the bacteriophage according to any one of [1] to

[11] and a pharmaceutically acceptable excipient.

[13] A pharmaceutical composition comprising at least two types of bacteriophage according to any one of [1] to

[11] and a pharmaceutically acceptable excipient.

[14] A pharmaceutical composition comprising three types of bacteriophage according to any one of [1] to

[11] and a pharmaceutically acceptable excipient.

[15] A pharmaceutical composition comprising four types of bacteriophage according to any one of [1] to

[11] and a pharmaceutically acceptable excipient.

[16] A pharmaceutical composition comprising five types of bacteriophage according to any one of [1] to

[11] and a pharmaceutically acceptable excipient.

[17] The pharmaceutical composition according to any one of

[12] to

[16] , which is a pharmaceutical composition for preventing or treating Pseudomonas aeruginosa infection.

[18] The pharmaceutical composition according to

[17] , wherein the Pseudomonas aeruginosa infection is a respiratory infection.

[0020]

[19] A bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage 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-04032, excluding a portion of the tail fiber gene, and the bacteriophage comprises the following nucleic acid sequences (A) and (B): (A) a nucleic acid sequence encoding a tail fiber derived from bacteriophage JG024, and (B) a nucleic acid sequence encoding PslGh.

[20] A bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04032, excluding a portion of the tail fiber gene, and the bacteriophage comprises the following nucleic acid sequences (A) and (B): (A) the nucleic acid sequence shown in SEQ ID NO: 6, or a nucleic acid sequence having 90% or more identity to SEQ ID NO: 6, (B) the nucleic acid sequence shown in SEQ ID NO: 4, or a nucleic acid sequence having 90% or more identity to SEQ ID NO: 4 and encoding a protein having Psl degrading activity.

[21] A bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04032, excluding a portion of the tail fiber gene, and the bacteriophage comprises the following nucleic acid sequences (A) and (B): (A) the nucleic acid sequence shown in SEQ ID NO: 6, and (B) the nucleic acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 11.

[0021]

[22] A pharmaceutical composition comprising a bacteriophage and a pharmaceutically acceptable excipient, wherein the bacteriophage is a bacteriophage selected from the following (i) to (iv): (i) a bacteriophage identified by accession number NITE BP-04037 or accession number NITE ABP-04201, or a passaged strain thereof, (ii) a bacteriophage identified by accession number NITE BP-04031, or a passaged strain thereof, (iii) a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04032, excluding a portion of the tail fiber gene, and wherein the bacteriophage has the nucleic acid sequences of the following (A) and (B): (A) the nucleic acid sequence shown in SEQ ID NO: 6, (B) a bacteriophage or a passaged strain thereof comprising the nucleic acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 11, (iv) a bacteriophage identified by accession number NITE BP-04040 or accession number NITE ABP-04200, or a passaged strain thereof.

[23] The pharmaceutical composition according to

[22] , which is a pharmaceutical composition for preventing or treating Pseudomonas aeruginosa infection.

[24] The pharmaceutical composition according to

[23] , wherein the Pseudomonas aeruginosa infection is a respiratory infection.

[0022]

[25] A method for producing a bacteriophage having lytic activity against Pseudomonas aeruginosa, comprising a step of culturing the bacteriophage in a host bacterium in which a gene derived from the prophage has been deleted.

[0023] The bacteriophages and modified bacteriophages, as well as the pharmaceutical compositions of the present invention, exhibit lytic activity against Pseudomonas aeruginosa, and are expected to be useful for the prevention or treatment of Pseudomonas aeruginosa infections, including pulmonary infections.

[0024]

[0023] Figure 1 shows photographs illustrating the lytic activity of natural bacteriophages. Images of lytic plaques that appeared after Pseudomonas aeruginosa ATCC15692, NBRC3080, or NBRC13746 strains were layered on an agar plate as hosts, and serial dilutions of each bacteriophage were dropped onto the plate.

[0024] Figure 1 shows photographs illustrating the lytic activity of PAi23-JG024 tail and PAi23 phages. Images of lytic plaques that appeared after Pseudomonas aeruginosa ATCC15692 strain or clinically isolated strains 3-55-PA or 9-8-PA were layered on an agar plate as hosts, and serial dilutions of each bacteriophage were dropped onto the plate.

[0033] Figure 1 shows photographs illustrating the lytic activity of PAi23-JG024 tail::Plac-PelAh(AB), PAi23-JG024 tail::Plac-PslGh(AB), PAi23-JG024 tail::Plac-PslG(AB), PAi23-JG024 tail::Plac-PslGh(AB)_v3, and PAi23-JG024 tail::Plac-PslGh-PelAh(AB)_v3 phages. Images of lytic plaques that appeared after Pseudomonas aeruginosa ATCC15692 strain was layered on an agar plate as a host and a dilution series of bacteriophage was dropped onto the plate.

[0034] Figure 1 shows photographs illustrating the lytic activity of PAi23::PyoG(CD) phage. The images show images of plaques that appeared after Pseudomonas aeruginosa ATCC15692 strain or an ATCC15692 strain expressing the ImG gene (ATCC15692-ImG) was layered on an agar plate as a host, and a dilution series of each bacteriophage was dropped onto the plate. These graphs show the activity of φLCX, PAPT1, and PAi239 to reduce biofilm mass. After allowing a biofilm to form for 24 hours using Pseudomonas aeruginosa ATCC15692 strain, media containing each bacteriophage was contacted with the biofilm, and after 6 hours, the biofilm mass was quantified by crystal violet staining. Error bars indicate standard error. (A) These graphs show the activity of PAi23-JG024 tail::Plac-PslGh(AB) and PAi23-JG024 tail::Plac-PslG(AB) phages to reduce biofilm mass.Biofilms formed by culturing Pseudomonas aeruginosa ATCC15692 for 24 hours were contacted with media containing each bacteriophage for 6 hours, and the amount of biofilm in the medium was quantified using crystal violet staining. Error bars indicate standard error. (B) A graph showing the activity of the PAi23-JG024 tail::Plac-PelAh (AB) phage in reducing biofilm volume. Biofilms formed by culturing the respiratory clinical isolate 28-42-PA for 24 hours were contacted with media containing each bacteriophage for 6 hours, and the amount of biofilm in the medium was quantified using crystal violet staining (left). A graph of the amount of biofilm converted to a relative value is also shown (right). Error bars indicate standard error. (C) A graph showing the activity of PAi23-JG024 tail::Plac-PslGh(AB)_v3 and PAi23-JG024 tail::Plac-PslGh-PelAh(AB)_v3 phages in reducing biofilm mass. Biofilms formed by culturing Pseudomonas aeruginosa ATCC15692 for 24 hours were contacted with media containing each bacteriophage for 6 hours, and the amount of biofilm in the medium was quantified using crystal violet staining. Error bars indicate standard error. A graph showing the growth curve of Pseudomonas aeruginosa in liquid culture. The graph shows that addition of PAi23 phage inhibits the growth of the respiratory clinical isolate 27-12-PA strain, and that addition of PAi23::PyoG(CD) phage maintains its growth inhibitory effect on the respiratory clinical isolate 27-12-PA strain for a longer period than PAi23 phage. Error bars indicate standard error. This graph shows the bactericidal activity of φLCX phage against Pseudomonas aeruginosa after overnight culture. Pseudomonas aeruginosa ATCC15692 strain was cultured in a glass test tube, and φLCX was added after 18 hours. This graph shows the change in absorbance at 600 nm. Error bars indicate standard deviation. This graph shows the bactericidal activity of a single bacteriophage in a mouse Pseudomonas aeruginosa infection model. Mice were intranasally infected with Pseudomonas aeruginosa ATCC15692 strain, and 30 minutes later, PAi23 or φBrmt was intranasally administered.The figures show the number of viable bacteria (log CFU / lung) in the lungs 24 hours after infection (CFU: Colony Forming Unit). Error bars indicate standard error. (A) Photographs showing the results of quantifying the plaque-forming units per mL (PFU / mL) of each phage for φBrkr and φBrkr_FTR2, a freeze-thaw-resistant strain of φBrkr, before and after freeze-thawing. Images of plaques appearing after Pseudomonas aeruginosa ATCC15692ΔPf4 / Pf6 strain was layered on an agar plate as a host, and serial dilutions of each bacteriophage were dropped onto the plate. (B) Graph showing that while φBrkr activity was significantly lost before and after freeze-thawing, φBrkr_FTR2 acquired resistance to freeze-thawing. Error bars indicate standard error.

[0023] Figure 1 shows a graph demonstrating that the growth inhibitory effect against Pseudomonas aeruginosa is prolonged by using a bacteriophage cocktail. The graph shows representative examples of growth curves after infecting Pseudomonas aeruginosa ATCC15692 strain with a single bacteriophage, a cocktail of two or four bacteriophages adjusted to an MOI of 0.0001. Error bars indicate standard error.

[0024] Figure 1 shows a graph demonstrating the activity of bacteriophage cocktails to reduce biofilm mass. Biofilms formed by culturing Pseudomonas aeruginosa ATCC15692 strain for 24 hours were contacted with media containing each bacteriophage cocktail (Cocktail A or B) for 6 hours, and the amount of biofilm in the medium was quantified using crystal violet staining. Error bars indicate standard error.

[0025] Figure 1 shows a graph demonstrating the bacteriophage cocktail's bactericidal effect in a mouse Pseudomonas aeruginosa infection model. Mice were infected with the respiratory clinical isolate 27-12-PA strain by oropharyngeal aspiration, and one hour later, the respective phage cocktail (cocktail A, B, or G) was administered. The figures show the number of viable bacteria (log CFU / lung) in the lungs approximately 22.5 hours after infection. Error bars indicate standard error. (A) Images of plaques that appeared after P. aeruginosa ATCC15692 or ATCC15692ΔPf4 / Pf6, a prophage-cured strain of ATCC15692, were layered on an agar plate as hosts, and serial dilutions of PAi242 phage were dropped onto the plate.(B) A graph showing the titer values ​​of bacteriophage solutions obtained approximately 6 hours after culturing each bacteriophage with Pseudomonas aeruginosa ATCC 15692 strain or a prophage-cured strain. Error bars indicate standard error.

[0025] This application claims priority from Japanese Patent Application No. 2023-221078, filed December 27, 2023, the entire contents of which are incorporated herein by reference.

[0026] 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.

[0027] <1. Bacteriophage of the Present Invention> In one aspect, the present invention provides a novel bacteriophage (hereinafter, may be referred to as "bacteriophage of the present invention"). The bacteriophage of the present invention is a bacteriophage that has lytic activity against bacteria belonging to the genus Pseudomonas, particularly Pseudomonas aeruginosa.

[0028] The bacteriophage of the present invention, the φLCX strain, was deposited by the applicant at the National Institute of Technology and Evaluation (NITE) Patent Microorganism Depositary Center (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan), an international depositary authority under the provisions of the Budapest Treaty for the Deposit of Patent Microorganisms, on December 7, 2023 (Accession No. NITE BP-04040).

[0029] The bacteriophage PAPT1 strain of the present invention was sent by the applicant for international deposit to the National Institute of Technology and Evaluation (NIET) Patent Microorganisms Depositary Center (Room 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, and was deposited on December 7, 2023 (Accession No. NITE BP-04031).

[0030] The bacteriophage PAi140 strain of the present invention was sent by the applicant for international deposit to the National Institute of Technology and Evaluation (NIET) Patent Microorganisms Depositary Center (Room 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, and was deposited on December 7, 2023 (Accession No. NITE BP-04033).

[0031] The bacteriophage PAi242 strain of the present invention was sent by the applicant for international deposit to the National Institute of Technology and Evaluation (NIET) Patent Microorganisms Depositary Center (Room 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, and was deposited on December 7, 2023 (Accession No. NITE BP-04036).

[0032] The PAi228 strain, which is a bacteriophage of the present invention, was sent by the applicant for international deposit to the National Institute of Technology and Evaluation (NITE) Patent Microorganisms Depositary Center (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818), which is an international depositary authority under the provisions of the Budapest Treaty for the Deposit of Patent Microorganisms, and was deposited on December 7, 2023 (Accession No. NITE BP-04034).

[0033] The bacteriophage PAi239 strain of the present invention was sent by the applicant for international deposit to the National Institute of Technology and Evaluation (NIET) Patent Microorganisms Depositary Center (Room 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, and was deposited on December 7, 2023 (Accession No. NITE BP-04035).

[0034] The bacteriophage of the present invention, strain φLCX2, was sent by the applicant for international deposit to the National Institute of Technology and Evaluation (NIET) Patent Microorganisms Depositary, an international depository authority under the provisions of the Budapest Treaty for the Deposit of Patent Microorganisms (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818), and received on November 13, 2024 (Receipt No. NITE ABP-04200).

[0035] The φBrkr_FTR2 strain, which is the bacteriophage of the present invention, was sent by the applicant for international deposit to the National Institute of Technology and Evaluation (NITE) Patent Microorganisms Depositary, an international depository authority under the provisions of the Budapest Treaty for the Deposit of Patent Microorganisms (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan), and received on November 13, 2024 (Receipt No. NITE ABP-04201).

[0036] In one embodiment, the bacteriophage of the present invention includes the following bacteriophages: (1) a bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, 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-04040; (2) a bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, 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-04031; (3) a bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, 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-04031; (4) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, 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 specified by accession number NITE BP-04036; (5) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, 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 specified by accession number NITE BP-04034; (6) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage contains 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-04035;(7) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, 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 the accession number NITE ABP-04200; and (8) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, 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 the accession number NITE ABP-04201.

[0037] As used herein, "bacteriophage" or "phage" refers to a phage particle comprising a genome packaged within an envelope or capsid, and includes whole phage particles, functionally equivalent phage parts (e.g., phage heads), and aggregates of phage components. In particular, an isolated phage refers to a phage that has been separated from its natural environment and grown, purified, or cultivated.

[0038] As used herein, "identity" refers to the identity of a nucleotide sequence or an amino acid sequence determined by techniques known in the art. As a method for aligning sequences, publicly available alignment software can be used. For example, the CLUSTAL W program (Nucleic Acids Research. 1994; 22(22): 4673-80), the FASTA program (Proceedings of the National Academy of Sciences of the United States of America. 1988; 85(8): 2444-8), the BLAST program (including BLASTP, BLASTN, BLASTX, TBLASTN, TBLASTX, etc.) (Journal of Molecular Biology. 1990; 215(3): 403-10) and the like can be used, but are not limited to these. A specific sequence alignment method is, for example, the value "Identity" obtained by searching with the NEEDLE program (Journal of Molecular Biology. 1970; 48(3): 443-53) using the default parameters. The parameters are as follows: Gap penalty = 10 Extend penalty = 0.5 Matrix = EBLOSUM62

[0039] As used herein, "bacteria belonging to the genus Pseudomonas" (also referred to herein as Pseudomonas bacteria) are bacteria classified as gram-negative aerobic bacilli, and are known in the art. In one embodiment, Pseudomonas bacteria are bacteria that cause or are the cause of infectious diseases, such as Pseudomonas aeruginosa (P. aeruginosa), Pseudomonas paucimobilis, Pseudomonas putida, Pseudomonas fluorescens, and Pseudomonas acidovorans. Pseudomonas bacteria are known to form biofilms by secreting exopolysaccharides such as alginic acid, which protect them from external enemies and physical stress.

[0040] As used herein, "having bacteriolytic activity against bacteria belonging to the genus Pseudomonas" or "having bacteriolytic activity against Pseudomonas aeruginosa" means that a bacteriolytic plaque is formed against at least one strain of bacteria belonging to the genus Pseudomonas or at least one strain of bacteria belonging to Pseudomonas aeruginosa, respectively. The bacteriolytic activity against these bacteria can be confirmed using methods and means known in the art. For example, the bacteriolytic activity against approximately 10 １０ PFU (plaque-forming unit) / mL ~ 10 ５ Whether or not a bacteriophage has lytic activity can be determined by determining whether or not PFU / mL of the bacteriophage (or a mixture of multiple bacteriophages) produces lytic plaques against bacteria belonging to the genus Pseudomonas or Pseudomonas.

[0041] In one embodiment, the bacteriophage of the present invention is resistant to freezing and thawing. For example, the bacteriophage of the present invention includes the following bacteriophage: a bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, such as Pseudomonas aeruginosa, 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 ABP-04201, and the bacteriophage is resistant to freezing and thawing.

[0042] As used herein, "freeze-thaw resistant" means that, although it is known that some bacteriophages are killed or damaged by freeze-thaw treatment, the bacteriophage are maintained to a desired degree even after freeze-thaw treatment. For example, the number of bacteriophages after freeze-thaw treatment is approximately the same as that before freeze-thaw treatment, or the number of bacteriophages after freeze-thaw treatment is maintained at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more compared to that before freeze-thaw treatment. Freeze-thaw resistance can be confirmed using methods and means known in the art. For example, whether a bacteriophage is resistant to freeze-thaw can be confirmed by determining whether the number of bacteriophages before and after freeze-thaw treatment is reduced using the method described in Example 10 of the present specification. Alternatively, resistance to freeze-thawing can be confirmed by determining whether the bacteriophage retains a desired activity (e.g., lytic activity against Pseudomonas aeruginosa) after the freeze-thaw treatment. For example, if the bacteriophage after the freeze-thaw treatment retains approximately the same activity as that before the freeze-thaw treatment, or retains at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more of the activity before the freeze-thaw treatment, the bacteriophage can be said to be resistant to freeze-thawing.

[0043] In one embodiment, the bacteriophage of the present invention includes the following bacteriophages: (1) a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04040; (2) a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04031; (3) a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04033; (4) A bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04036; (5) A bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04034; (6) A bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04035; (7) A bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04036; (8) A bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE ABP-04201.

[0044] In one embodiment, the bacteriophage of the present invention includes the following bacteriophages: (1) a bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04040; (2) a bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04031; (3) a bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04033; (4) a bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04036; (5) A bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04034; (6) A bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04035; (7) A bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE ABP-04200; and (8) A bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE ABP-04201.

[0045] The bacteriophage of the present invention also includes the following bacteriophages: (1) a bacteriophage identified by accession number NITE BP-04040, or a passage strain thereof; (2) a bacteriophage identified by accession number NITE BP-04031, or a passage strain thereof; (3) a bacteriophage identified by accession number NITE BP-04033, or a passage strain thereof; (4) a bacteriophage identified by accession number NITE BP-04036, or a passage strain thereof; (5) a bacteriophage identified by accession number NITE BP-04034, or a passage strain thereof; (6) a bacteriophage identified by accession number NITE BP-04035, or a passage strain thereof; (7) a bacteriophage identified by accession number NITE BP-04036, or a passage strain thereof; (8) A bacteriophage identified by accession number NITE ABP-04201, or a subculture thereof.

[0046] The bacteriophage of the present invention also includes the following bacteriophages: (1) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises a nucleic acid sequence in which 1 to 5,000 (for example, 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-04040; (2) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises a nucleic acid sequence in which 1 to 5,000 (for example, 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; (3) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, such as Pseudomonas aeruginosa, in which the genome of the bacteriophage specified by Accession No. NITE BP-04033 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 the bacteriophage specified by Accession No. NITE BP-04031; (4) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, such as Pseudomonas aeruginosa, in which the genome of the bacteriophage 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 the bacteriophage specified by Accession No. NITE BP-04033; (4) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage is 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-04036;(5) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage is a bacteriophage identified by accession number NITE BP-04034, and ... (7) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, such as Pseudomonas aeruginosa, in which the genome of the bacteriophage specified by Accession Number NITE ABP-04200 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 specified by Accession Number NITE ABP-04035; (8) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, such as Pseudomonas aeruginosa, in which the genome of the bacteriophage 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 specified by Accession Number NITE ABP-04200; and (8) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage 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 ABP-04201;

[0047] 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. Such modifications are included in the bacteriophage of the present invention as long as they have lytic activity against bacteria belonging to the genus Pseudomonas, such as Pseudomonas aeruginosa.

[0048] Bacteriophage mutants may be generated in the process of subculture, production, and / or replication of bacteriophages, in which the nucleic acid sequence of the genome contained in the bacteriophage is partially deleted, substituted, inserted, and / or added. Such mutants are also included in the bacteriophages of the present invention, so long as they have bacteriolytic activity against bacteria belonging to the genus Pseudomonas, such as Pseudomonas aeruginosa.

[0049] The bacteriophage of the present invention also includes passaged strains of bacteriophages identified by the above-mentioned accession numbers or accession numbers, so long as they have bacteriolytic activity against bacteria belonging to the genus Pseudomonas, such as Pseudomonas aeruginosa.

[0050] As used herein, the term "passaged strain" refers to a bacteriophage obtained by subculturing a provided bacteriophage.

[0051] The present invention also includes a polynucleotide consisting of the genome contained in the bacteriophage of the present invention (hereinafter sometimes referred to as the "genome of the present invention"). Therefore, the present invention also encompasses the following: (1) a polynucleotide of a bacteriophage genome comprising a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified under accession number NITE BP-04040; (2) a polynucleotide of a bacteriophage genome comprising a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified under accession number NITE BP-04031; (3) a polynucleotide of a bacteriophage genome comprising a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified under accession number NITE BP-04033; (4) a polynucleotide of a bacteriophage genome comprising a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified under accession number NITE BP-04036; (5) A polynucleotide of a bacteriophage genome 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-04034; (6) A polynucleotide of a bacteriophage genome 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-04035; (7) A polynucleotide of a bacteriophage genome 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 ABP-04200; and (8) A polynucleotide of a bacteriophage genome 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 ABP-04201. In one embodiment, the polynucleotide is a polynucleotide encoding a bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, such as Pseudomonas aeruginosa.

[0052] In one embodiment, the genome of the present invention includes the following: (1) a polynucleotide of a bacteriophage 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-04040; (2) a polynucleotide of a bacteriophage 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-04031; (3) a polynucleotide of a bacteriophage 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-04031; (4) A polynucleotide of a bacteriophage 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 with the nucleic acid sequence of the genome of a bacteriophage identified under Accession No. NITE BP-04036; (5) A polynucleotide of a bacteriophage 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 with the nucleic acid sequence of the genome of a bacteriophage identified under Accession No. NITE BP-04034; (6) A polynucleotide of a bacteriophage 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 with the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE BP-04035; (7) A polynucleotide of a bacteriophage 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 with the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE ABP-04200;and (8) a polynucleotide of a bacteriophage 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 a bacteriophage identified by accession number NITE ABP-04201.

[0053] In one embodiment, the genome of the present invention includes the following genomes: (1) a genome comprising the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04040; (2) a genome comprising the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04031; (3) a genome comprising the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04033; (4) a genome comprising the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04036; (5) a genome comprising the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04034; (6) a genome comprising the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04035; (7) A genome comprising the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE ABP-04200; and (8) A genome comprising the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE ABP-04201.

[0054] The genomes of the present invention include the following genomes: (1) a genome consisting of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04040; (2) a genome consisting of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04031; (3) a genome consisting of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04033; (4) a genome consisting of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04036; (5) a genome consisting of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04034; (6) a genome consisting of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04035; (7) a genome consisting of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04036; A genome consisting of the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE ABP-04200; and (8) A genome consisting of the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE ABP-04201.

[0055] 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.

[0056] The bacteriophage of the present invention can be obtained by requesting it from the above-mentioned depository center.

[0057] 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 (e.g., Pseudomonas aeruginosa is used as the host bacterium to obtain a bacteriophage that exhibits lytic activity against Pseudomonas aeruginosa) by electroporation. 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.

[0058] The bacteriophage of the present invention can be prepared by common culture, isolation, and purification methods known in the art. For example, a host bacterium (a bacterium of the genus Pseudomonas, such as Pseudomonas aeruginosa) is cultured in advance, and the host bacterium is 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, LB medium can be used when culturing Pseudomonas aeruginosa. When preparing multiple bacteriophages, they may be grown in different host bacteria or the same host bacterium.

[0059] Furthermore, the bacteriophage of the present invention can be stored in various forms (liquid, lyophilized, etc.) by following appropriate methods known in the art.

[0060] 2. Engineered Bacteriophage of the Present Invention In another aspect, the present invention provides an engineered bacteriophage (hereinafter, sometimes referred to as "engineered bacteriophage of the present invention"). The engineered bacteriophage of the present invention comprises: [1] modification of the tail fiber gene, and / or [2] introduction of a glycoside hydrolase and / or a bacteriocin.

[0061] The bacteriophage to be modified is not particularly limited as long as it has lytic activity against Pseudomonas bacteria, such as Pseudomonas aeruginosa. In one embodiment, the bacteriophage to be modified is a bacteriophage that has lytic activity against Pseudomonas bacteria that cause or are the cause of an infectious disease, such as Pseudomonas aeruginosa.

[0062] For example, the modified bacteriophage of the present invention is a bacteriophage obtained by modifying any one of the following bacteriophages (1) to (12): (1) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, such as Pseudomonas aeruginosa, 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-04040; (2) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, such as Pseudomonas aeruginosa, 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-04031; (3) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, 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-04033; (4) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, 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-04036; (5) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, 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-04036. (6) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, such as Pseudomonas aeruginosa, 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-04035;(7) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, 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-04032; (8) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, 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-04037; (9) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, 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-04037. (10) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, 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 specified by accession number NITE BP-04038; (11) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, 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 specified by accession number NITE ABP-04200; (12) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage contains a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE ABP-04201.

[0063] The bacteriophage PAi23 strain was deposited by the applicant at the National Institute of Technology and Evaluation, Patent Microorganism Depositary Center (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, 292-0818), an international depository authority under the provisions of the Budapest Treaty for the Deposit of Patent Microorganisms. 122) It was sent for international deposit and deposited on December 7, 2023 (accession number NITE BP-04032).

[0064] The bacteriophage φBrkr strain was sent by the applicant for international deposit to the National Institute of Technology and Evaluation (NIET) Patent Microorganisms Depositary Center (Room 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, and was deposited on December 7, 2023 (Accession No. NITE BP-04037).

[0065] The bacteriophage φ30-1 strain was sent by the applicant to 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), an international depositary authority under the provisions of the Budapest Treaty for the Deposit of Patent Microorganisms, for international deposit, and was deposited on December 7, 2023 (Accession No. NITE BP-04039).

[0066] The bacteriophage φBrmt strain was sent by the applicant for international deposit to the National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818), which is an international depositary authority under the provisions of the Budapest Treaty for the Deposit of Patent Microorganisms, and was deposited on December 7, 2023 (Accession No. NITE BP-04038).

[0067] In one embodiment, the modified bacteriophage of the present invention is a bacteriophage obtained by modifying any of the following bacteriophages (1) to (12): (1) a bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, such as Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04040; (2) a bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, such as Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04031; (3) a bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, such as Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04031; (4) a bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-04036; (5) a bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-04034; and (6) a bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-04036. (7) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, such as Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04032;(8) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04037; (9) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04039; (10) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04038; (11) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE ABP-04200; (12) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE ABP-04201;

[0068] In one embodiment, the modified bacteriophage of the present invention is a bacteriophage obtained by modifying any one of the following bacteriophages (1) to (12): (1) a bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04040; (2) a bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04031; (3) a bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04033; (4) a bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04034; (5) a bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-04034; and (6) a bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-04035; (7) a bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-04032; (8) a bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-04037; (9) A bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04039; (10) A bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04038; (11) A bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE ABP-04200; (12) A bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE ABP-04201.

[0069] The bacteriophage to be modified also includes passaged strains of the bacteriophage identified by the above-mentioned accession number or accession number, so long as they have bacteriolytic activity against bacteria belonging to the genus Pseudomonas, such as Pseudomonas aeruginosa.

[0070] In one embodiment, the modified bacteriophage of the present invention is a bacteriophage obtained by modifying any of the bacteriophages (1) to (12) below: (1) a bacteriophage identified by accession number NITE BP-04040 or a passage strain thereof; (2) a bacteriophage identified by accession number NITE BP-04031 or a passage strain thereof; (3) a bacteriophage identified by accession number NITE BP-04033 or a passage strain thereof; (4) a bacteriophage identified by accession number NITE BP-04036 or a passage strain thereof; (5) a bacteriophage identified by accession number NITE BP-04034 or a passage strain thereof; (6) a bacteriophage identified by accession number NITE BP-04035 or a passage strain thereof; (7) a bacteriophage identified by accession number NITE BP-04032 or a passage strain thereof; (8) a bacteriophage identified by accession number NITE BP-04037 or a passage strain thereof; (9) a bacteriophage identified by accession number NITE BP-04039 or a passage strain thereof; (10) a bacteriophage identified by accession number NITE BP-04038 or a passage strain thereof; (11) a bacteriophage identified by accession number NITE ABP-04200 or a passage strain thereof; (12) a bacteriophage identified by accession number NITE ABP-04201 or a passage strain thereof.

[0071] Among the bacteriophages to be modified, those identified by accession numbers or receipt numbers can be obtained by requesting the above-mentioned depository center.

[0072] The bacteriophage to be modified can be prepared and cultured in the same manner as in the previous section <1. Bacteriophage of the present invention>.

[0073] [1] Modification of Tail Fiber Gene In one aspect, the present invention relates to a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the tail fiber gene of the bacteriophage has been modified.

[0074] The tail fiber gene and tail fiber protein of a bacteriophage are related to the host range (host spectrum) and host specificity of infection (WO 2016 / 055585, WO 2017 / 174810). Host range or host specificity refers to the types or range of bacterial species or strains that a bacteriophage can infect. By modifying the tail fiber gene of a bacteriophage, the host range or host specificity of the bacteriophage can be changed or expanded. For example, by exchanging it with a tail fiber gene from another bacteriophage, the host range or host specificity of another bacteriophage can be acquired. The presence or absence of host range or host specificity for a specific species or strain of Pseudomonas bacteria (e.g., Pseudomonas aeruginosa) can be determined by examining the lytic activity of the bacteriophage against that specific species or strain of bacteria.

[0075] In one embodiment, the engineered bacteriophage of the invention is a bacteriophage with lytic activity against Pseudomonas aeruginosa, in which the tail fiber gene of the bacteriophage has been replaced with a tail fiber gene from another bacteriophage with lytic activity against Pseudomonas aeruginosa.

[0076] The other bacteriophage having lytic activity against Pseudomonas aeruginosa is not particularly limited as long as it is different from the bacteriophage to be modified, and examples of the other bacteriophage include JG024, Phi33, PTP92, PTP47, LBL3, SPM-1, F8, PB1, KPP12, LMA2, SN, 14-1, NH-4, PTP93, PTP47, and C36.

[0077] Such bacteriophage tail fiber genes and proteins are known (e.g., WO 2016 / 055585, WO 2017 / 174810). One example is the tail fiber gene of phage JG024, the deduced sequence of which has been registered as NCBI Reference Sequence NC_017674.1 and is shown as SEQ ID NO: 6. Furthermore, a person skilled in the art could isolate all or part of a tail fiber gene from a bacteriophage that has lytic activity against Pseudomonas aeruginosa, exchange it for a tail fiber gene in a bacteriophage to be modified, and then confirm that the resulting bacteriophage has lytic activity against Pseudomonas aeruginosa, thereby screening for a tail fiber gene associated with a desired host range or host specificity.

[0078] The tail fiber gene may be exchanged by exchanging the entire gene, or by exchanging a portion of it (a hybrid gene), or by exchanging it with a gene that combines portions of multiple tail fiber genes from multiple other bacteriophage strains. The tail fiber protein contains a C-terminal receptor-binding region for binding to bacteria and an N-terminal region that links the C-terminal receptor-binding region to the body of the bacteriophage. Of these, it is believed that the C-terminal receptor-binding region is associated with host range or host specificity, so it is preferable to exchange at least this region.

[0079] Replacement of the tail fiber gene can be carried out by genetic recombination methods known in the art, and the method is not particularly limited. For example, the tail fiber gene in the bacteriophage can be replaced by inserting the tail fiber gene to be replaced and a sequence for homologous recombination into a vector or plasmid suitable for the Pseudomonas aeruginosa host, introducing this into the Pseudomonas aeruginosa host together with the bacteriophage genome to be modified, and performing homologous recombination. Alternatively, a modified bacteriophage can be prepared by preparing a genome of the bacteriophage to be modified containing a nucleic acid sequence in which the original tail fiber gene is replaced with the tail fiber gene to be replaced, and then introducing the resulting bacteriophage genome into a Pseudomonas aeruginosa host.

[0080] In one embodiment, the modified bacteriophage of the present invention includes the following: (1) a bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage (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-04040, except for a portion of the tail fiber gene, and (b) comprises a nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene from another bacteriophage having lytic activity against Pseudomonas aeruginosa; (2) a bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage (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-04040, except for a portion of the tail fiber gene, and (a) a bacteriophage having a nucleic acid sequence that is 90% or more identical to the nucleic acid sequence of the genome of a bacteriophage specified under Accession No. BP-04031, and (b) a nucleic acid sequence of a tail fiber gene that includes at least a portion of a tail fiber gene derived from another bacteriophage that has lytic activity against Pseudomonas aeruginosa; (3) a bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of which includes (a) a nucleic acid sequence that is 90% or more identical to the nucleic acid sequence of the genome of a bacteriophage specified under Accession No. NITE BP-04033, excluding the tail fiber gene portion, and (b) a nucleic acid sequence of a tail fiber gene that includes at least a portion of a tail fiber gene derived from another bacteriophage that has lytic activity against Pseudomonas aeruginosa; (4) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage (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-04036, excluding a portion of the tail fiber gene, and (b) comprises a nucleic acid sequence of a tail fiber gene that includes at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa;(5) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, 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-04034, except for the tail fiber gene portion, and (b) comprises a nucleic acid sequence of a tail fiber gene that includes at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa; (6) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, 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-04034, except for the tail fiber gene portion, and (b) comprises a nucleic acid sequence of a tail fiber gene that includes at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa; (7) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, such as Pseudomonas aeruginosa, 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-04032, excluding the tail fiber gene portion, and (b) comprises a nucleic acid sequence of a tail fiber gene that includes at least a portion of a tail fiber gene from another bacteriophage that has lytic activity against Pseudomonas aeruginosa; (8) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, such as Pseudomonas aeruginosa, 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-04032, excluding the tail fiber gene portion, and (b) comprises a nucleic acid sequence of a tail fiber gene that includes at least a portion of a tail fiber gene from another bacteriophage that has lytic activity against Pseudomonas aeruginosa; (8) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage (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-04037, excluding a portion of the tail fiber gene, and (b) comprises a nucleic acid sequence of a tail fiber gene that comprises at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa;(9) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, 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-04039, except for the tail fiber gene portion, and (b) comprises a nucleic acid sequence of a tail fiber gene that includes at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa; (10) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, 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-04039, except for the tail fiber gene portion, and (b) comprises a nucleic acid sequence of a tail fiber gene that includes at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa; (11) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, such as Pseudomonas aeruginosa, 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 by Accession Number NITE ABP-04200, excluding the tail fiber gene portion, and (b) comprises a nucleic acid sequence of a tail fiber gene that includes at least a portion of a tail fiber gene from another bacteriophage having lytic activity against Pseudomonas aeruginosa; (12) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, such as Pseudomonas aeruginosa, 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 by Accession Number NITE ABP-04200, excluding the tail fiber gene portion, and (b) comprises a nucleic acid sequence of a tail fiber gene that includes at least a portion of a tail fiber gene from another bacteriophage having lytic activity against Pseudomonas aeruginosa; (12) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, 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 ABP-04201, excluding a portion of the tail fiber gene, and (b) comprises a nucleic acid sequence of a tail fiber gene that includes at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa;

[0081] In one embodiment, the modified bacteriophage of the present invention includes the following: (1) a bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage (a) comprises the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE BP-04040, except for a portion of the tail fiber gene, and (b) comprises the nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene from another bacteriophage having lytic activity against Pseudomonas aeruginosa; (2) a bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage (a) comprises the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE BP-04040, except for a portion of the tail fiber gene, and (3) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of which (a) comprises the nucleic acid sequence of the genome of a bacteriophage specified under Accession No. NITE BP-04033, excluding a portion of the tail fiber gene, and (b) comprises the nucleic acid sequence of a tail fiber gene that includes at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa; (4) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage (a) comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04036, excluding a portion of the tail fiber gene, and (b) comprises the nucleic acid sequence of a tail fiber gene that includes at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa;(5) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage (a) comprises the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE BP-04034, excluding a portion of the tail fiber gene, and (b) comprises the nucleic acid sequence of a tail fiber gene containing at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa; (6) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage (a) comprises the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE BP-04034, excluding a portion of the tail fiber gene, and (b) comprises the nucleic acid sequence of a tail fiber gene containing at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa; (7) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of which (a) comprises the nucleic acid sequence of the genome of a bacteriophage specified under Accession No. NITE BP-04032, excluding a portion of the tail fiber gene, and (b) comprises the nucleic acid sequence of a tail fiber gene that includes at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa; (8) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of which (a) comprises the nucleic acid sequence of the genome of a bacteriophage specified under Accession No. NITE BP-04032, excluding a portion of the tail fiber gene, and (b) comprises the nucleic acid sequence of a tail fiber gene that includes at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa; (8) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage (a) comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04037, excluding a portion of the tail fiber gene, and (b) comprises the nucleic acid sequence of a tail fiber gene that includes at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa;(9) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage (a) comprises the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE BP-04039, excluding a portion of the tail fiber gene, and (b) comprises the nucleic acid sequence of a tail fiber gene containing at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa; (10) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage (a) comprises the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE BP-04039, excluding a portion of the tail fiber gene, and (b) comprises the nucleic acid sequence of a tail fiber gene containing at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa; (11) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of which (a) comprises the nucleic acid sequence of the genome of a bacteriophage specified by Accession Number NITE ABP-04200, excluding a portion of the tail fiber gene, and (b) comprises the nucleic acid sequence of a tail fiber gene that includes at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa; (12) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of which (a) comprises the nucleic acid sequence of the genome of a bacteriophage specified by Accession Number NITE ABP-04200, excluding a portion of the tail fiber gene, and (b) comprises the nucleic acid sequence of a tail fiber gene that includes at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa; (12) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage (a) comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE ABP-04201, excluding a portion of the tail fiber gene, and (b) comprises the nucleic acid sequence of a tail fiber gene that includes at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa;

[0082] In one embodiment, the modified bacteriophage of the present invention includes the following: (1) a bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage (a) consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04040, except for a portion of the tail fiber gene, and (b) comprises the nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa; (2) a bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage (a) consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04040, except for a portion of the tail fiber gene, and (b) comprises the nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa; (3) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of which (a) consists of the nucleic acid sequence of the genome of a bacteriophage specified under NITE Accession No. BP-04033, excluding a portion of the tail fiber gene, and (b) comprises the nucleic acid sequence of a tail fiber gene that includes at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa; (4) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage (a) consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04036, excluding a portion of the tail fiber gene, and (b) comprises the nucleic acid sequence of a tail fiber gene that includes at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa;(5) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage (a) consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04034, excluding the tail fiber gene portion, and (b) comprises the nucleic acid sequence of a tail fiber gene containing at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa; (6) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage (a) consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04034, excluding the tail fiber gene portion, and (b) comprises the nucleic acid sequence of a tail fiber gene containing at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa; (7) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of which (a) consists of the nucleic acid sequence of the genome of a bacteriophage specified under Accession No. NITE BP-04032, excluding a portion of the tail fiber gene, and (b) comprises the nucleic acid sequence of a tail fiber gene that includes at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa; (8) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of which (a) consists of the nucleic acid sequence of the genome of a bacteriophage specified under Accession No. NITE BP-04032, excluding a portion of the tail fiber gene, and (b) comprises the nucleic acid sequence of a tail fiber gene that includes at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa; (8) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage (a) consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04037, excluding a portion of the tail fiber gene, and (b) comprises the nucleic acid sequence of a tail fiber gene that includes at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa;(9) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage (a) consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04039, excluding the tail fiber gene portion, and (b) comprises the nucleic acid sequence of a tail fiber gene containing at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa; (10) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage (a) consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04039, excluding the tail fiber gene portion, and (b) comprises the nucleic acid sequence of a tail fiber gene containing at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa; (11) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of which (a) consists of the nucleic acid sequence of the genome of a bacteriophage specified by Accession Number NITE ABP-04200, excluding a portion of the tail fiber gene, and (b) comprises the nucleic acid sequence of a tail fiber gene that includes at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa; (12) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of which (a) consists of the nucleic acid sequence of the genome of a bacteriophage specified by Accession Number NITE ABP-04200, excluding a portion of the tail fiber gene, and (b) comprises the nucleic acid sequence of a tail fiber gene that includes at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa; (12) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage (a) consists of the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE ABP-04201, excluding a portion of the tail fiber gene, and (b) comprises the nucleic acid sequence of a tail fiber gene that includes at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa;

[0083] In one embodiment, an engineered bacteriophage of the invention comprises the nucleic acid sequence of a tail fiber gene from another bacteriophage that has lytic activity against Pseudomonas aeruginosa. In one embodiment, an engineered bacteriophage of the invention comprises a nucleic acid sequence encoding a tail fiber from bacteriophage JG024. In one embodiment, the tail fiber gene from another bacteriophage is the tail fiber gene of bacteriophage JG024, for example, comprising the nucleic acid sequence set forth in SEQ ID NO:6, or 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 SEQ ID NO:6. In one embodiment, an engineered bacteriophage of the invention comprises the nucleic acid sequence set forth in SEQ ID NO:6.

[0084] In one embodiment, the engineered bacteriophage of the present invention is a bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of which (a) consists of the nucleic acid sequence of the genome of the bacteriophage identified by NITE BP-04032, excluding a portion of the tail fiber gene, and (b) comprises a nucleic acid sequence encoding a tail fiber derived from bacteriophage JG024.

[0085] In one embodiment, the engineered bacteriophage of the present invention is a bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of which (a) consists of the nucleic acid sequence of the genome of the bacteriophage identified by NITE BP-04032, excluding a portion of the tail fiber gene, and (b) comprises the nucleic acid sequence set forth in SEQ ID NO:6.

[0086] [2] Introduction of glycoside hydrolase and / or bacteriocin In one aspect, the present invention relates to a bacteriophage having lytic activity against Pseudomonas aeruginosa, into which a glycoside hydrolase and / or a bacteriocin has been introduced.

[0087] Glycoside hydrolases derived from Pseudomonas aeruginosa are proteins that inhibit and destroy biofilm formation in Pseudomonas aeruginosa (Science Advances. 2016; 2(5): e1501632). By modifying the bacteriophage of the present invention to incorporate glycoside hydrolases, the ability to degrade Pseudomonas aeruginosa biofilms is imparted, the lytic activity of the bacteriophage is enhanced, and when used in combination with antibiotics, the effectiveness of the antibiotics is expected to be enhanced. Examples of such glycoside hydrolases include PelA and PslG, and their active domains PelAh and PslGh, which degrade Pel (a polysaccharide composed of N-acetylgalactosamine and N-acetylglucosamine) and Psl (a polysaccharide composed of a pentasaccharide repeating unit of d-mannose, D-glucose, and L-rhamnose), which are extracellular polysaccharides that constitute biofilms. As used herein, the term "PelA" includes the PelA protein and its active domain PelAh, as well as fragments thereof, so long as they have the activity of degrading Pel. Furthermore, as used herein, the term "PslG" includes the PslG protein and its active domain PslGh, as well as fragments thereof, so long as they have the activity of degrading Psl.

[0088] PelA, PslG, and their active domains are known in the art. For example, the amino acid sequence of PelA derived from the Pseudomonas aeruginosa PAO1 strain is registered under GenBank accession number AAG06452.1 (Science Advances. 2016; 2(5): e1501632), and the nucleic acid sequence encoding PelA comprises the sequence shown in SEQ ID NO: 1. Furthermore, the nucleic acid sequence encoding the active domain PelAh present at the N-terminus of PelA comprises the sequence shown in SEQ ID NO: 3. Furthermore, for example, the amino acid sequence of PslG derived from the Pseudomonas aeruginosa PAO1 strain is registered under GenBank accession number AAG05625.1 (J. Biol. Chem. 2015; 290(47): 28374-28387), and the nucleic acid sequence encoding PslG comprises the sequence shown in SEQ ID NO: 2. Furthermore, the nucleic acid sequence encoding the active domain PslGh obtained by removing the transmembrane domain present at the N-terminus of PslG includes the sequence shown in SEQ ID NO:4.

[0089] In one embodiment, the engineered bacteriophage of the present invention is a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises a nucleic acid sequence encoding PelA or an active domain thereof. In one embodiment, the engineered bacteriophage of the present invention is a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises a nucleic acid sequence encoding PelA derived from Pseudomonas aeruginosa or an active domain thereof (e.g., PelAh). In one embodiment, the engineered bacteriophage of the present invention is a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises a nucleic acid sequence encoding PelA or PelAh derived from Pseudomonas aeruginosa, wherein the nucleic acid sequence encoding PelA or PelAh (a) comprises the nucleic acid sequence set forth in SEQ ID NO: 1 or 3, or (b) 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 SEQ ID NO: 1 or 3 and encoding a protein having Pel degrading activity.

[0090] In one embodiment, the engineered bacteriophage of the present invention is a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises a nucleic acid sequence encoding PslG or an active domain thereof. In one embodiment, the engineered bacteriophage of the present invention is a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises a nucleic acid sequence encoding PslG derived from Pseudomonas aeruginosa or an active domain thereof (e.g., PslGh). In one embodiment, the engineered bacteriophage of the present invention is a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises a nucleic acid sequence encoding PslG or PslGh derived from Pseudomonas aeruginosa, wherein the nucleic acid sequence encoding PslG or PslGh (a) comprises the nucleic acid sequence set forth in SEQ ID NO: 2 or 4, or (b) comprises a nucleic acid sequence encoding a protein having 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to SEQ ID NO: 2 or 4 and having Psl-degrading activity.

[0091] Bacteriocins are proteins produced by bacteria that have antibacterial activity against bacteria of the same or related species, and Pyocin G (PyoG), a bacteriocin derived from Pseudomonas aeruginosa, is known to be a factor that exhibits antibacterial activity against Pseudomonas aeruginosa (Journal of molecular biology. 2020; 432(13): 3869-3880). By modifying the bacteriophage of the present invention to introduce a bacteriocin, the bactericidal activity against Pseudomonas aeruginosa is enhanced.

[0092] PyoG is known in the art. For example, the amino acid sequence of PyoG derived from Pseudomonas aeruginosa BWHPSA046 is registered in GenBank under accession number ETV05907.1 (Journal of molecular biology. 2020; 432(13): 3869-3880) and is encoded by the sequence shown in SEQ ID NO: 5.

[0093] In one embodiment, the engineered bacteriophage of the present invention is a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises a nucleic acid sequence encoding Pyocin G. In one embodiment, the engineered bacteriophage of the present invention is a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises a nucleic acid sequence encoding Pyocin G derived from Pseudomonas aeruginosa. In one embodiment, the engineered bacteriophage of the present invention is a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises a nucleic acid sequence encoding Pyocin G derived from Pseudomonas aeruginosa, wherein the nucleic acid sequence encoding Pyocin G (a) comprises the nucleic acid sequence set forth in SEQ ID NO: 5, or (b) 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 SEQ ID NO: 5 and encoding a protein having bacteriocin activity.

[0094] The nucleic acid sequence encoding the glycoside hydrolase and / or bacteriocin is introduced into a bacteriophage so that it is under the control of an appropriate regulatory sequence so that it can be expressed as a protein. For example, the nucleic acid sequence encoding the glycoside hydrolase and / or bacteriocin is operably linked to an appropriate promoter sequence, for example, a promoter sequence active in Pseudomonas aeruginosa (such as the Pseudomonas aeruginosa rpsB gene promoter, the Pseudomonas aeruginosa fda gene promoter, or the Escherichia coli lac promoter).

[0095] In one embodiment, a spacer (1 to 10 bases) may be provided between a regulatory sequence (such as a promoter sequence) and another regulatory sequence, or between a regulatory sequence and a nucleic acid sequence encoding a glycoside hydrolase and / or a bacteriocin. In a specific embodiment, the engineered bacteriophage of the present invention can use a sequence (SEQ ID NO: 10) in which a portion of the lac operon and a Shine-Dalgarno sequence-like sequence (SEQ ID NO: 8) as a regulatory sequence are linked to a nucleic acid sequence encoding PslGh, or a sequence (SEQ ID NO: 11) in which a portion of the lac operon and a spacer (5 bases: TTAGA) as a regulatory sequence are linked to a Shine-Dalgarno sequence-like sequence and a nucleic acid sequence encoding PslGh.

[0096] Introduction of a glycoside hydrolase and / or a bacteriocin into a bacteriophage can be carried out by genetic recombination methods known in the art, and the method is not particularly limited. For example, a nucleic acid sequence encoding the glycoside hydrolase and / or bacteriocin to be introduced and a sequence for homologous recombination can be inserted into a vector or plasmid suitable for a Pseudomonas aeruginosa host, and the vector or plasmid can be introduced into a Pseudomonas aeruginosa host together with the bacteriophage genome to be modified, followed by homologous recombination to introduce the nucleic acid sequence encoding the glycoside hydrolase and / or a bacteriocin into the bacteriophage. Alternatively, a modified bacteriophage can be prepared by preparing a genome of the bacteriophage to be modified into which a nucleic acid sequence encoding the glycoside hydrolase and / or a bacteriocin has been inserted, and then introducing the resulting bacteriophage genome into a Pseudomonas aeruginosa host.

[0097] When producing bacteriophages incorporating Pyocin G as the bacteriocin, it is preferable to introduce into the host bacterium used for amplification a gene encoding an immunity protein (ImG), which is produced as a multidomain protein together with Pyocin G. This allows the immunity protein to inhibit the activity of the bacteriocin Pyocin G produced by the bacteriophage, thereby protecting the host bacterium (Journal of molecular biology. 2020; 432(13): 3869-3880).

[0098] In one embodiment, the engineered bacteriophage of the present invention is a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises a nucleic acid sequence encoding at least one protein or active domain selected from the following (a) to (c): (a) Pyocin G; (b) PslG or PslGh; and (c) PelA or PelAh.

[0099] In one embodiment, the engineered bacteriophage of the present invention is a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises nucleic acid sequences encoding at least two proteins or active domains selected from the following (a) to (c): (a) Pyocin G; (b) PslG or PslGh; and (c) PelA or PelAh.

[0100] In one embodiment, the engineered bacteriophage of the present invention is a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises nucleic acid sequences encoding the following proteins or active domains (a) to (c): (a) Pyocin G; (b) PslG or PslGh; and (c) PelA or PelAh.

[0101] In one embodiment, the engineered bacteriophage of the present invention is a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises a nucleic acid sequence encoding at least one protein or active domain derived from Pseudomonas aeruginosa selected from the following (a) to (c): (a) Pyocin G; (b) PslG or PslGh; and (c) PelA or PelAh.

[0102] In one embodiment, the engineered bacteriophage of the present invention is a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises at least one nucleic acid sequence selected from the following (a) to (c): (a) the nucleic acid sequence set forth in SEQ ID NO: 5, or 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 SEQ ID NO: 5, and encoding a protein having bacteriocin activity; (b) the nucleic acid sequence set forth in SEQ ID NO: 2 or 4, or 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 SEQ ID NO: 2 or 4, and encoding a protein having Psl-degrading activity; and (c) A nucleic acid sequence comprising the nucleic acid sequence shown in SEQ ID NO: 1 or 3, or having 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to SEQ ID NO: 1 or 3, and encoding a protein having Pel degrading activity.

[0103] In one embodiment, the engineered bacteriophage of the present invention includes the following: (1) a bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage 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-04040 (excluding the portion of the tail fiber gene in the case of (A) below), and comprising the following nucleic acid sequences (A) and / or (B): (A) a nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa, and / or (B) a nucleic acid sequence encoding at least one protein or active domain selected from the following (a) to (c): (a) Pyocin G; (b) PslG or PslGh; and (c) PelA or PelAh; (2) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage 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-04031 (excluding the tail fiber gene portion in the case of (A) below), and comprising the following nucleic acid sequences (A) and / or (B): (A) a nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa, and / or (B) a nucleic acid sequence encoding at least one protein or active domain selected from the following (a) to (c): (a) Pyocin G; (b) PslG or PslGh; and (c) PelA or PelAh;(3) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage 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-04033 (excluding the tail fiber gene portion in the case of (A) below), and comprising the following nucleic acid sequences (A) and / or (B): (A) a nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa, and / or (B) a nucleic acid sequence encoding at least one protein or active domain selected from the following (a) to (c): (a) Pyocin G; (b) PslG or PslGh; and (c) PelA or PelAh; (4) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage 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-04036 (excluding the tail fiber gene portion in the case of (A) below), and comprising the following nucleic acid sequences (A) and / or (B): (A) a nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa, and / or (B) a nucleic acid sequence encoding at least one protein or active domain selected from the following (a) to (c): (a) Pyocin G; (b) PslG or PslGh; and (c) PelA or PelAh;(5) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage 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-04034 (excluding the tail fiber gene portion in the case of (A) below), and comprising the following nucleic acid sequences (A) and / or (B): (A) a nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa, and / or (B) a nucleic acid sequence encoding at least one protein or active domain selected from the following (a) to (c): (a) Pyocin G; (b) PslG or PslGh; and (c) PelA or PelAh; and (6) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage 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-04035 (excluding the tail fiber gene portion in the case of (A) below), and the bacteriophage comprises the following nucleic acid sequence (A) and / or (B): (A) a nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa, and / or (B) a nucleic acid sequence encoding at least one protein or active domain selected from the following (a) to (c): (a) Pyocin G; (b) PslG or PslGh; and (c) PelA or PelAh;(7) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage 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-04032 (excluding the tail fiber gene portion in the case of (A) below), and comprising the following nucleic acid sequences (A) and / or (B): (A) a nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa, and / or (B) a nucleic acid sequence encoding at least one protein or active domain selected from the following (a) to (c): (a) Pyocin G; (b) PslG or PslGh; and (c) PelA or PelAh; (8) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage 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-04037 (excluding the tail fiber gene portion in the case of (A) below), and comprising the following nucleic acid sequences (A) and / or (B): (A) a nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa, and / or (B) a nucleic acid sequence encoding at least one protein or active domain selected from the following (a) to (c): (a) Pyocin G; (b) PslG or PslGh; and (c) PelA or PelAh;(9) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage 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-04039 (excluding the tail fiber gene portion in the case of (A) below), and comprising the following nucleic acid sequences (A) and / or (B): (A) a nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa, and / or (B) a nucleic acid sequence encoding at least one protein or active domain selected from the following (a) to (c): (a) Pyocin G; (b) PslG or PslGh; and (c) PelA or PelAh; (10) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage 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-04038 (excluding the tail fiber gene portion in the case of (A) below), and the bacteriophage comprises the following nucleic acid sequences (A) and / or (B): (A) a nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa, and / or (B) a nucleic acid sequence encoding at least one protein or active domain selected from the following (a) to (c): (a) Pyocin G; (b) PslG or PslGh; and (c) PelA or PelAh.

[0104] In one embodiment, the modified bacteriophage of the present invention includes the following: (1) a bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage 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 the bacteriophage identified by accession number NITE BP-04040 (excluding the tail fiber gene portion in the case of (A) below), and the following nucleic acid sequences (A) and / or (B): (A) a nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa, and / or (B) the following (a) to (c): (a) Pyocin G; (b) PslG or PslGh; and (c) a nucleic acid sequence encoding at least one protein or active domain selected from PelA or PelAh; (2) a bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage 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-04031 (excluding the tail fiber gene portion in the case of (A) below), and the following nucleic acid sequence (A) and / or (B): (A) the nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa, and / or (B) a bacteriophage comprising a nucleic acid sequence encoding at least one protein or active domain selected from the following (a) to (c): (a) Pyocin G; (b) PslG or PslGh; and (c) PelA or PelAh;(3) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage 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-04033 (excluding the tail fiber gene portion in the case of (A) below), and the nucleic acid sequence is (A) and / or (B) below: (A) a nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa, and / or (B) the following (a) to (c): (a) Pyocin G; (b) PslG or PslGh; and (c) a nucleic acid sequence encoding at least one protein or active domain selected from PelA or PelAh; (4) a bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage 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-04036 (excluding the tail fiber gene portion in the case of (A) below), and wherein the nucleic acid sequence is the following (A) and / or (B): (A) a nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa, and / or (B) the following (a) to (c): A bacteriophage comprising a nucleic acid sequence encoding at least one protein or active domain selected from: (a) Pyocin G; (b) PslG or PslGh; and (c) PelA or PelAh;(5) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage 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-04034 (excluding the tail fiber gene portion in the case of (A) below), and the nucleic acid sequence is (A) the nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa, and / or (B) the following (a) to (c): (a) Pyocin G; (b) PslG or PslGh; and (c) a nucleic acid sequence encoding at least one protein or active domain selected from PelA or PelAh; and (6) a bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage 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-04035 (excluding the tail fiber gene portion in the case of (A) below), and the nucleic acid sequence is (A) a nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa, and / or (B) the following (a) to (c): A bacteriophage comprising a nucleic acid sequence encoding at least one protein or active domain selected from: (a) Pyocin G; (b) PslG or PslGh; and (c) PelA or PelAh;(7) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage 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-04032 (excluding the tail fiber gene portion in the case of (A) below), and the nucleic acid sequence is (A) the nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa, and / or (B) the following (a) to (c): (a) Pyocin G; (b) PslG or PslGh; and (c) a nucleic acid sequence encoding at least one protein or active domain selected from PelA or PelAh; (8) a bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage 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-04037 (excluding the tail fiber gene portion in the case of (A) below), and wherein the nucleic acid sequence is the following (A) and / or (B): (A) a nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa, and / or (B) the following (a) to (c): A bacteriophage comprising a nucleic acid sequence encoding at least one protein or active domain selected from: (a) Pyocin G; (b) PslG or PslGh; and (c) PelA or PelAh;(9) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage 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-04039 (excluding the tail fiber gene portion in the case of (A) below), and the nucleic acid sequence is (A) the nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa, and / or (B) the following (a) to (c): (a) Pyocin G; (b) PslG or PslGh; and (c) a nucleic acid sequence encoding at least one protein or active domain selected from PelA or PelAh; (10) a bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage 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-04038 (excluding the tail fiber gene portion in the case of (A) below), and wherein the nucleic acid sequence is the following (A) and / or (B): (A) a nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa, and / or (B) the following (a) to (c): A bacteriophage comprising a nucleic acid sequence encoding at least one protein or active domain selected from: (a) Pyocin G; (b) PslG or PslGh; and (c) PelA or PelAh.

[0105] 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.

[0106] In one embodiment, the engineered bacteriophage of the present invention includes the following: (1) a bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04040 (excluding the portion of the tail fiber gene in the case of (A) below), and the bacteriophage comprises the following nucleic acid sequence (A) and / or (B): (A) a nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa, and / or (B) a nucleic acid sequence encoding at least one protein or active domain selected from the following (a) to (c): (a) Pyocin G; (b) PslG or PslGh; and (c) PelA or PelAh; (2) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04031 (excluding the tail fiber gene portion in the case of (A) below), and comprises the following nucleic acid sequences (A) and / or (B): (A) a nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa, and / or (B) a nucleic acid sequence encoding at least one protein or active domain selected from the following (a) to (c): (a) Pyocin G; (b) PslG or PslGh; and (c) PelA or PelAh;(3) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04033 (excluding the tail fiber gene portion in the case of (A) below), and comprises the following nucleic acid sequence (A) and / or (B): (A) a nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa, and / or (B) a nucleic acid sequence encoding at least one protein or active domain selected from the following (a) to (c): (a) Pyocin G; (b) PslG or PslGh; and (c) PelA or PelAh; (4) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein a bacteriophage, the genome of which comprises the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE BP-04036 (excluding the tail fiber gene portion in the case of (A) below), and which comprises the following nucleic acid sequence (A) and / or (B): (A) a nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa, and / or (B) a nucleic acid sequence encoding at least one protein or active domain selected from the following (a) to (c): (a) Pyocin G; (b) PslG or PslGh; and (c) PelA or PelAh; (5) a bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04034 (excluding the tail fiber gene portion in the case of (A) below), and the nucleic acid sequence of (A) and / or (B) below: (A) a nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa, and / or (B) the following (a) to (c): (a) Pyocin G; (b) PslG or PslGh;and (c) a nucleic acid sequence encoding at least one protein or active domain selected from PelA or PelAh; and (6) a bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04035 (excluding the tail fiber gene portion in the case of (A) below), and the following nucleic acid sequence (A) and / or (B): (A) a nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa, and / or (B) the following (a) to (c): (a) Pyocin G; (b) PslG or PslGh; and (c) a nucleic acid sequence encoding at least one protein or active domain selected from PelA or PelAh. (7) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04032 (excluding the tail fiber gene portion in the case of (A) below), and comprises the following nucleic acid sequence (A) and / or (B): (A) a nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa, and / or (B) a nucleic acid sequence encoding at least one protein or active domain selected from the following (a) to (c): (a) Pyocin G; (b) PslG or PslGh; and (c) PelA or PelAh;(8) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04037 (excluding the tail fiber gene portion in the case of (A) below), and comprises the following nucleic acid sequence (A) and / or (B): (A) a nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa, and / or (B) a nucleic acid sequence encoding at least one protein or active domain selected from the following (a) to (c): (a) Pyocin G; (b) PslG or PslGh; and (c) PelA or PelAh; (9) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, a bacteriophage, the genome of which comprises the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE BP-04039 (excluding the tail fiber gene portion in the case of (A) below), and which comprises the following nucleic acid sequence (A) and / or (B): (A) a nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa, and / or (B) a nucleic acid sequence encoding at least one protein or active domain selected from the following (a) to (c): (a) Pyocin G; (b) PslG or PslGh; and (c) PelA or PelAh; (10) a bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04038 (excluding the tail fiber gene portion in the case of (A) below), and the nucleic acid sequence of (A) and / or (B) below: (A) a nucleic acid sequence of a tail fiber gene comprising at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa, and / or (B) the following (a) to (c): (a) Pyocin G; (b) PslG or PslGh;and (c) a nucleic acid sequence encoding at least one protein or active domain selected from PelA or PelAh;

[0107] In one embodiment, the engineered bacteriophage of the present invention includes: a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises a nucleic acid sequence having 90% or greater identity to the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04032, excluding a portion of the tail fiber gene; and the bacteriophage comprises the following nucleic acid sequences (A) and / or (B): (A) a nucleic acid sequence of a tail fiber gene comprising at least a portion of the tail fiber gene derived from bacteriophage JG024, and / or (B) a nucleic acid sequence encoding at least one protein or active domain selected from the following (a) to (c): (a) Pyocin G; (b) PslG or PslGh; and (c) PelA or PelAh.

[0108] In one embodiment, the engineered bacteriophage of the present invention includes: a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04032, and comprises a nucleic acid sequence encoding at least one protein or active domain derived from Pseudomonas aeruginosa selected from the following (a) to (c): (a) Pyocin G; (b) PslG or PslGh; and (c) PelA or PelAh.

[0109] In one embodiment, the engineered bacteriophage of the invention includes the following: a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04032, and comprises at least one nucleic acid sequence selected from the following (a) to (c): (a) the nucleic acid sequence set forth in SEQ ID NO: 5, or 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 SEQ ID NO: 5, and encoding a protein having bacteriocin activity; (b) the nucleic acid sequence set forth in SEQ ID NO: 2 or 4, or 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 SEQ ID NO: 2 or 4, and encoding a protein having Psl-degrading activity; and (c) a nucleic acid sequence set forth in SEQ ID NO: 1 or 3, or 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 SEQ ID NO: 1 or 3 and encoding a protein having Pel degrading activity.

[0110] In one embodiment, the engineered bacteriophage of the present invention includes the following: a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises a nucleic acid sequence having 90% or more identity to the genome of the bacteriophage identified by accession number NITE BP-04032, excluding a portion of the tail fiber gene, and the bacteriophage comprises the following nucleic acid sequences (A) and (B): (A) a nucleic acid sequence encoding a tail fiber derived from bacteriophage JG024, (B) a nucleic acid sequence encoding at least one protein or active domain selected from the following (a) to (c) derived from Pseudomonas aeruginosa: (a) Pyocin G; (b) PslG or PslGh; and (c) PelA or PelAh.

[0111] In one embodiment, the engineered bacteriophage of the present invention includes: a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage 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-04032, excluding a portion of the tail fiber gene; and the bacteriophage comprises the following nucleic acid sequences (A) and (B): (A) a nucleic acid sequence encoding a tail fiber derived from bacteriophage JG024, and (B) a nucleic acid sequence encoding PslGh.

[0112] In one embodiment, the engineered bacteriophage of the present invention includes the following: a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises a nucleic acid sequence having 90% or more identity to the genome of the bacteriophage identified by accession number NITE BP-04032, except for a portion of the tail fiber gene, and the bacteriophage comprises the following nucleic acid sequences (A) and (B): (A) a nucleic acid sequence encoding the tail fiber derived from bacteriophage JG024, and (B) a nucleic acid sequence encoding PslGh and PelAh.

[0113] In one embodiment, the engineered bacteriophage of the present invention includes the following: a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04032, excluding a portion of the tail fiber gene, and the bacteriophage comprises the following nucleic acid sequences (A) and (B): (A) a nucleic acid sequence encoding a tail fiber derived from bacteriophage JG024, (B) a nucleic acid sequence encoding at least one protein or active domain selected from the following (a) to (c) derived from Pseudomonas aeruginosa: (a) Pyocin G; (b) PslG or PslGh; and (c) PelA or PelAh.

[0114] In one embodiment, the engineered bacteriophage of the invention includes: a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by Accession No. NITE BP-04032, excluding a portion of the tail fiber gene, and the following nucleic acid sequences (A) and (B): (A) a nucleic acid sequence encoding a tail fiber from bacteriophage JG024, (B) at least one nucleic acid sequence selected from the following (a) to (c): (a) the nucleic acid sequence set forth in SEQ ID NO: 5, or 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 SEQ ID NO: 5 and encoding a protein having bacteriocin activity; (b) a nucleic acid sequence set forth in SEQ ID NO: 2 or 4, or 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 SEQ ID NO: 2 or 4 and encoding a protein having Psl degrading activity; and (c) a nucleic acid sequence set forth in SEQ ID NO: 1 or 3, or 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 SEQ ID NO: 1 or 3 and encoding a protein having Pel degrading activity.

[0115] In one embodiment, the engineered bacteriophage of the invention includes: a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04032, excluding a portion of the tail fiber gene, and the following nucleic acid sequences (A) and (B): (A) the nucleic acid sequence set forth in SEQ ID NO: 6, or a nucleic acid sequence having 90% or more identity to SEQ ID NO: 6, (B) at least one of the nucleic acid sequences selected from the following (a) to (c): (a) the nucleic acid sequence set forth in SEQ ID NO: 5, or a nucleic acid sequence having 90% or more identity to SEQ ID NO: 5 and encoding a protein having bacteriocin activity; (b) the nucleic acid sequence set forth in SEQ ID NO: 2 or 4, or a nucleic acid sequence having 90% or more identity to SEQ ID NO: 2 or 4 and encoding a protein having Psl-degrading activity; and (c) A bacteriophage comprising the nucleic acid sequence shown in SEQ ID NO: 1 or 3, or a nucleic acid sequence having 90% or more identity to SEQ ID NO: 1 or 3 and encoding a protein having Pel degrading activity.

[0116] In one embodiment, the engineered bacteriophage of the present invention includes the following: a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04032, excluding a portion of the tail fiber gene, and the bacteriophage comprises the following nucleic acid sequences (A) and (B): (A) the nucleic acid sequence set forth in SEQ ID NO: 6, or a nucleic acid sequence having 90% or more identity to SEQ ID NO: 6, (B) the nucleic acid sequence set forth in SEQ ID NO: 4, or a nucleic acid sequence having 90% or more identity to SEQ ID NO: 4 and encoding a protein having Psl degrading activity.

[0117] In one embodiment, the engineered bacteriophage of the present invention includes the following: a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04032, excluding a portion of the tail fiber gene; and the bacteriophage comprises the following nucleic acid sequences (A) and (B): (A) the nucleic acid sequence set forth in SEQ ID NO:6, or a nucleic acid sequence having 90% or more identity to SEQ ID NO:6, (B) the nucleic acid sequence set forth in SEQ ID NO:4, or a nucleic acid sequence having 90% or more identity to SEQ ID NO:4 and encoding a protein having Psl degrading activity, and (C) the nucleic acid sequence set forth in SEQ ID NO:3, or a nucleic acid sequence having 90% or more identity to SEQ ID NO:3 and encoding a protein having Pel degrading activity.

[0118] In one embodiment, the engineered bacteriophage of the present invention includes the following: a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04032, excluding a portion of the tail fiber gene, and the bacteriophage comprises the following nucleic acid sequences (A) and (B): (A) the nucleic acid sequence shown in SEQ ID NO: 6, (B) at least one nucleic acid sequence selected from the following (a) to (c): (a) the nucleic acid sequence shown in SEQ ID NO: 5; (b) the nucleic acid sequence shown in SEQ ID NO: 2 or 4; and (c) the nucleic acid sequence shown in SEQ ID NO: 1 or 3.

[0119] In one embodiment, the engineered bacteriophage of the present invention includes the following: a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04032, excluding a portion of the tail fiber gene, and the bacteriophage comprises the following nucleic acid sequences (A) and (B): (A) the nucleic acid sequence shown in SEQ ID NO:6, (B) the nucleic acid sequence shown in SEQ ID NO:4, SEQ ID NO:10, or SEQ ID NO:11.

[0120] 3. Uses of the Pharmaceutical Composition and Bacteriophage of the Present Invention The bacteriophage of the present invention and / or the modified bacteriophage of the present invention have lytic activity against Pseudomonas bacteria, particularly Pseudomonas aeruginosa, which cause infectious diseases. Therefore, the bacteriophage of the present invention and / or the modified bacteriophage of the present invention are expected to be effective in treating or preventing infectious diseases.

[0121] In another aspect, the present invention provides uses of the bacteriophage of the present invention and / or the engineered bacteriophage of the present invention, for example, pharmaceutical compositions containing the bacteriophage of the present invention and / or the engineered bacteriophage of the present invention as an active ingredient (hereinafter sometimes referred to as the "pharmaceutical composition of the present invention").

[0122] Pharmaceutical compositions of the present invention include those comprising at least one bacteriophage, including a bacteriophage of the present invention and / or an engineered bacteriophage of the present invention, and a pharmaceutically acceptable excipient.

[0123] The pharmaceutical composition of the present invention may further comprise one or more strains of bacteriophage known to have lytic activity against Pseudomonas bacteria, such as Pseudomonas aeruginosa, etc. Examples of such strains include, but are not limited to, the bacteriophage of the present invention, the modified bacteriophage of the present invention, other known bacteriophages, etc.

[0124] 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 pulmonary administration. 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 a bacteriophage with a pharmaceutically acceptable excipient, or by suspending a bacteriophage in a pharmaceutically acceptable excipient.

[0125] The pharmaceutical compositions of the present invention include those containing at least two types of bacteriophages and a pharmaceutically acceptable excipient.

[0126] The pharmaceutical compositions of the present invention include those containing three types of bacteriophages and a pharmaceutically acceptable excipient.

[0127] The pharmaceutical compositions of the present invention include those containing four types of bacteriophages and a pharmaceutically acceptable excipient.

[0128] The pharmaceutical compositions of the present invention include those containing five types of bacteriophages and a pharmaceutically acceptable excipient.

[0129] The pharmaceutical composition of the present invention can contain a combination of any type of bacteriophage. In one embodiment, the pharmaceutical composition of the present invention contains a combination of bacteriophages with different properties. For example, bacteriophages include phages that recognize pili (Pili-recognizing phages) and phages that recognize LPS (LPS-recognizing phages).

[0130] Examples of pilus-recognizing phages include, but are not limited to, the bacteriophage identified by accession number NITE BP-04040, the bacteriophage identified by accession number NITE ABP-04200, the bacteriophage identified by accession number NITE BP-04037, the bacteriophage identified by accession number NITE ABP-04201, the bacteriophage identified by accession number NITE BP-04039, and the bacteriophage identified by accession number NITE BP-04036, as well as passage strains thereof.

[0131] Examples of LPS-recognizing phages include, but are not limited to, the bacteriophage identified by accession number NITE BP-04031; a bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of which comprises a nucleic acid sequence having the genome of the bacteriophage identified by accession number NITE BP-04032, excluding the tail fiber gene portion, and which comprises the following nucleic acid sequences (A) and (B): (A) a nucleic acid sequence encoding the tail fiber derived from bacteriophage JG024, and a nucleic acid sequence encoding PslGh; and the bacteriophage identified by accession number NITE BP-04033, as well as passaged strains thereof.

[0132] The pharmaceutical composition of the present invention may comprise at least one bacteriophage from the group of pilus-recognizing phages and at least one bacteriophage from the group of LPS-recognizing phages.

[0133] In one embodiment, the pharmaceutical composition of the present invention includes a pharmaceutical composition comprising the bacteriophages of (i) and (ii) below and a pharmaceutically acceptable excipient: (i) a bacteriophage identified by accession number NITE BP-04037 or a passaged strain thereof, (ii) a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04032, excluding a portion of the tail fiber gene, and the bacteriophage or a passaged strain thereof comprises the following nucleic acid sequences (A) and (B): (A) the nucleic acid sequence shown in SEQ ID NO:6, (B) the nucleic acid sequence shown in SEQ ID NO:4 or SEQ ID NO:10.

[0134] In one embodiment, the pharmaceutical composition of the present invention includes a pharmaceutical composition comprising the bacteriophage of (i) and (ii) below and a pharmaceutically acceptable excipient: (i) a bacteriophage identified by accession number NITE BP-04037 or a subcultured strain thereof, (ii) a bacteriophage identified by accession number NITE BP-04031 or a subcultured strain thereof.

[0135] In one embodiment, the pharmaceutical composition of the present invention includes a pharmaceutical composition comprising the bacteriophages of (i) and (ii) below and a pharmaceutically acceptable excipient: (i) a bacteriophage identified by accession number NITE BP-04040 or a passaged strain thereof, (ii) a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04032, excluding a portion of the tail fiber gene, and the bacteriophage or a passaged strain thereof comprises the following nucleic acid sequences (A) and (B): (A) the nucleic acid sequence shown in SEQ ID NO:6, (B) the nucleic acid sequence shown in SEQ ID NO:4 or SEQ ID NO:10.

[0136] In one embodiment, the pharmaceutical composition of the present invention includes a pharmaceutical composition comprising the bacteriophage of (i) and (ii) below and a pharmaceutically acceptable excipient: (i) a bacteriophage identified by accession number NITE BP-04040 or a subcultured strain thereof, (ii) a bacteriophage identified by accession number NITE BP-04031 or a subcultured strain thereof.

[0137] In one embodiment, the pharmaceutical composition of the present invention includes a pharmaceutical composition comprising the bacteriophages of (i) and (ii) below and a pharmaceutically acceptable excipient: (i) a bacteriophage identified by accession number NITE BP-04039 or a passaged strain thereof, (ii) a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04032, excluding a portion of the tail fiber gene, and the bacteriophage or a passaged strain thereof comprises the following nucleic acid sequences (A) and (B): (A) the nucleic acid sequence shown in SEQ ID NO:6, (B) the nucleic acid sequence shown in SEQ ID NO:4 or SEQ ID NO:10.

[0138] In one embodiment, the pharmaceutical composition of the present invention includes a pharmaceutical composition comprising a bacteriophage of any one of the following (i) to (iv) and a pharmaceutically acceptable excipient: (i) a bacteriophage identified by accession number NITE BP-04037 or accession number NITE ABP-04201, or a passaged strain thereof, (ii) a bacteriophage identified by accession number NITE BP-04031, or a passaged strain thereof, (iii) a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04032, excluding a portion of the tail fiber gene, and wherein the bacteriophage has the nucleic acid sequences of the following (A) and (B): (A) the nucleic acid sequence shown in SEQ ID NO: 6, (B) the nucleic acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 11 (iv) A bacteriophage identified by accession number NITE BP-04040 or accession number NITE ABP-04200, or a subcultured strain thereof.

[0139] In one embodiment, the pharmaceutical composition of the present invention includes a pharmaceutical composition comprising a bacteriophage of any one of the following (i) to (iv) and a pharmaceutically acceptable excipient: (i) a bacteriophage identified by accession number NITE BP-04037 or a passaged strain thereof, (ii) a bacteriophage identified by accession number NITE BP-04040 or a passaged strain thereof, (iii) a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04032, excluding a portion of the tail fiber gene, and comprising the following nucleic acid sequences (A) and (B): (A) the nucleic acid sequence shown in SEQ ID NO: 6, (B) the nucleic acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 10, or a passaged strain thereof, (iv) A bacteriophage identified by accession number NITE BP-04031 or a subcultured strain thereof.

[0140] In one embodiment, the pharmaceutical composition of the present invention includes a pharmaceutical composition comprising a bacteriophage of any one of the following (i) to (iv) and a pharmaceutically acceptable excipient: (i) a bacteriophage identified by accession number NITE BP-04037 or a passaged strain thereof, (ii) a bacteriophage identified by accession number NITE ABP-04200 or a passaged strain thereof, (iii) a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04032, excluding a portion of the tail fiber gene, and comprising the following nucleic acid sequences (A) and (B): (A) the nucleic acid sequence shown in SEQ ID NO: 6, (B) the nucleic acid sequence shown in SEQ ID NO: 11, or a passaged strain thereof, (iv) A bacteriophage identified by accession number NITE BP-04031 or a subcultured strain thereof.

[0141] In one embodiment, the pharmaceutical composition of the present invention includes a pharmaceutical composition comprising a bacteriophage of any one of the following (i) to (iv) and a pharmaceutically acceptable excipient: (i) a bacteriophage identified by accession number NITE ABP-04201 or a passaged strain thereof, (ii) a bacteriophage identified by accession number NITE ABP-04200 or a passaged strain thereof, (iii) a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04032, excluding a portion of the tail fiber gene, and comprising the following nucleic acid sequences (A) and (B): (A) the nucleic acid sequence shown in SEQ ID NO: 6, (B) the nucleic acid sequence shown in SEQ ID NO: 11, or a passaged strain thereof, (iv) A bacteriophage identified by accession number NITE BP-04031 or a subcultured strain thereof.

[0142] In one embodiment, the pharmaceutical composition of the present invention includes a pharmaceutical composition comprising a bacteriophage of any one of the following (i) to (iv) and a pharmaceutically acceptable excipient: (i) a bacteriophage identified by accession number NITE BP-04037 or a passaged strain thereof, (ii) a bacteriophage identified by accession number NITE ABP-04200 or a passaged strain thereof, (iii) a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04032, excluding a portion of the tail fiber gene, and comprising the following nucleic acid sequences (A) and (B): (A) the nucleic acid sequence shown in SEQ ID NO: 6, (B) the nucleic acid sequence shown in SEQ ID NO: 11, or a passaged strain thereof, (iv) A bacteriophage identified by accession number NITE BP-04033 or a subcultured strain thereof.

[0143] In one embodiment, the pharmaceutical composition of the present invention includes a pharmaceutical composition comprising a bacteriophage of any one of the following (i) to (iv) and a pharmaceutically acceptable excipient: (i) a bacteriophage identified by accession number NITE BP-04037 or a passaged strain thereof, (ii) a bacteriophage identified by accession number NITE BP-04036 or a passaged strain thereof, (iii) a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04032, excluding a portion of the tail fiber gene, and comprising the following nucleic acid sequences (A) and (B): (A) the nucleic acid sequence shown in SEQ ID NO: 6, (B) the nucleic acid sequence shown in SEQ ID NO: 11, or a passaged strain thereof, (iv) A bacteriophage identified by accession number NITE BP-04031 or a subcultured strain thereof.

[0144] In one embodiment, the pharmaceutical composition of the present invention includes a pharmaceutical composition comprising a bacteriophage of any one of the following (i) to (iv) and a pharmaceutically acceptable excipient: (i) a bacteriophage identified by accession number NITE BP-04039 or a passaged strain thereof, (ii) a bacteriophage identified by accession number NITE ABP-04200 or a passaged strain thereof, (iii) a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04032, excluding a portion of the tail fiber gene, and comprising the following nucleic acid sequences (A) and (B): (A) the nucleic acid sequence shown in SEQ ID NO: 6, (B) the nucleic acid sequence shown in SEQ ID NO: 11, or a passaged strain thereof, (iv) A bacteriophage identified by accession number NITE BP-04031 or a subcultured strain thereof.

[0145] In one embodiment, the pharmaceutical composition of the present invention includes a pharmaceutical composition comprising a bacteriophage of any one of the following (i) to (iv) and a pharmaceutically acceptable excipient: (i) a bacteriophage identified by accession number NITE BP-04039 or a passaged strain thereof, (ii) a bacteriophage identified by accession number NITE BP-04036 or a passaged strain thereof, (iii) a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04032, excluding a portion of the tail fiber gene, and comprising the following nucleic acid sequences (A) and (B): (A) the nucleic acid sequence shown in SEQ ID NO: 6, (B) the nucleic acid sequence shown in SEQ ID NO: 11, or a passaged strain thereof, (iv) A bacteriophage identified by accession number NITE BP-04033 or a subcultured strain thereof.

[0146] In one embodiment, the pharmaceutical composition of the present invention includes a pharmaceutical composition comprising a bacteriophage of any one of the following (i) to (iv) and a pharmaceutically acceptable excipient: (i) a bacteriophage identified by accession number NITE ABP-04200 or a passaged strain thereof, (ii) a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04032, excluding a portion of the tail fiber gene, and the bacteriophage or a passaged strain thereof comprises the following nucleic acid sequences (A) and (B): (A) the nucleic acid sequence shown in SEQ ID NO:6, (B) the nucleic acid sequence shown in SEQ ID NO:11, (iii) a bacteriophage identified by accession number NITE BP-04031 or a passaged strain thereof. (iv) A bacteriophage identified by accession number NITE BP-04033 or a subcultured strain thereof.

[0147] 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 if the pharmaceutical composition contains four bacteriophages, each bacteriophage may be present in approximately equal amounts (e.g., 2.5 x 10 ３ ~2.5 x 10 １３ PFU), or each bacteriophage may be contained in a different ratio.

[0148] The pharmaceutical composition of the present invention can be used as an agent for preventing or treating Pseudomonas bacterial infections, such as Pseudomonas aeruginosa infections. As used herein, "infectious disease" refers to an infectious disease caused by or resulting from infection with Pseudomonas bacteria, such as Pseudomonas aeruginosa, and includes, for example, infections of the lungs, respiratory tract, skin, subcutaneous tissue, bones, ears, eyes, urinary tract, heart valves, blood, and the whole body. Symptoms of infectious diseases include, but are not limited to, cough, sputum, bloody sputum, fever, dyspnea, fatigue, pulmonary nodules, bronchiectasis, etc. in the case of lung or respiratory tract infection; purulent discharge, fever, inflammation, etc. in the case of skin, subcutaneous tissue, bones, ears, eyes, or urinary tract infection; and bacteremia, sepsis, septic shock, fever, hypotension, anuria, etc. in the case of blood or systemic infection.

[0149] As used herein, "treatment" means at least partial improvement of symptoms of an infectious disease, halting the progression or worsening of an infectious disease, complete cure, etc. As used herein, "prevention" means preventing a subject who is not currently infected with an infectious disease from contracting the infectious disease, preventing the recurrence of the infectious disease, etc.

[0150] The present invention includes a pharmaceutical composition for preventing or treating an infection caused by a bacterium of the genus Pseudomonas, such as a Pseudomonas aeruginosa infection, comprising the bacteriophage or modified bacteriophage of the present invention or the pharmaceutical composition of the present invention. In one embodiment, the Pseudomonas aeruginosa infection is a respiratory infection. In one embodiment, the Pseudomonas aeruginosa infection is a pulmonary infection.

[0151] The present invention also includes a method for preventing or treating an infection caused by Pseudomonas bacteria, such as a Pseudomonas aeruginosa infection, in a subject, comprising the step of administering a therapeutically effective amount of a bacteriophage or modified bacteriophage of the present invention or a pharmaceutical composition of the present invention. In one embodiment, at least two, for example, three, four, or five, of the bacteriophages or modified bacteriophages of the present invention can be administered to a subject, and the multiple bacteriophages may be administered simultaneously or separately.

[0152] The present invention further includes the bacteriophage or modified bacteriophage of the present invention, or the pharmaceutical composition of the present invention for use in the prevention or treatment of infections caused by bacteria of the genus Pseudomonas, such as Pseudomonas aeruginosa infections. The present invention further includes use of the bacteriophage or modified 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 infections caused by bacteria of the genus Pseudomonas, such as Pseudomonas aeruginosa infections.

[0153] In the present invention, the subject to which the bacteriophage is administered is not limited as long as it is a mammal, and examples include mice, rats, dogs, pigs, monkeys, humans, etc. For example, the bacteriophage is administered to a subject diagnosed with an infection with a Pseudomonas bacterium, such as Pseudomonas aeruginosa, or a subject at risk of contracting an infection with a Pseudomonas bacterium, such as Pseudomonas aeruginosa (e.g., hospitalized patients, immunocompromised patients, patients with trauma, etc.).

[0154] Furthermore, the bacteriophage or modified 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 infections caused by Pseudomonas bacteria, such as Pseudomonas aeruginosa, and the bacteriophage or modified bacteriophage of the present invention or the pharmaceutical composition of the present invention may be provided as a combination drug with such other active ingredients. Such active ingredients include, but are not limited to, antibiotics such as fluoroquinolones, carbapenems, aminoglycosides, ansamycins, cephalosporins, penicillins, beta-lactams, beta-lactamase inhibitors, folate pathway inhibitors, fusidans, glycopeptides, glycylcyclines, lincosamides, lipopeptides, macrolides, quinolones, oxazolidinones, phenicol phosphonates, streptogramins, tetracyclines, sulfonamides, imipenem, meropenem, amikacin, ciprofloxacin, levofloxacin, tobramycin, azithromycin, aztreonam, colistin, and the like.

[0155] 4. Use of Prophage-Cut Strains as Host Bacteria The present invention also relates to the use of a Pseudomonas aeruginosa strain in which a prophage-derived gene has been deleted as a host bacterium for use in producing a bacteriophage having lytic activity against Pseudomonas aeruginosa. Accordingly, in one aspect, the present invention provides a method for producing a bacteriophage having lytic activity against Pseudomonas aeruginosa, the method comprising the step of culturing the bacteriophage in a host bacterium in which a prophage-derived gene has been deleted.

[0156] In the present invention, a prophage refers to a lysogenized, temperate bacteriophage that exists in a genomic integrated state in a host bacterium (Pseudomonas aeruginosa), and one type of prophage may exist in one host bacterium, or two or more types of prophage may exist in one host bacterium. By deleting prophage-derived genes from Pseudomonas aeruginosa and removing the prophage, it becomes possible to produce bacteriophages with high efficiency using Pseudomonas aeruginosa as a host bacterium.

[0157] As used herein, the term "prophage-derived gene" refers to a gene present in a prophage, particularly a gene involved in prophage maintenance. Examples of "prophage-derived genes" include the pflM gene (Journal of Bacteriology. 2024; 206(5): e0040223).

[0158] As used herein, "deleting a prophage-derived gene" means 1) the deletion of the entire length of at least one gene from the genomic sequence of the prophage, or 2-1) the removal of the prophage from the host bacterium by modifying the genomic sequence of at least one gene by deletion, substitution, insertion, addition, or a combination thereof, or 2-2) the deletion of the function of the prophage-derived gene. Deletion of a prophage-derived gene can be performed by methods known in the art depending on the type of host bacterium used and the type of gene to be deleted (e.g., Journal of Bacteriology. 2024; 206(5): e0040223).

[0159] In the method of the present invention, a host bacterium in which a prophage-derived gene has been deleted as described above is inoculated with a target bacteriophage, i.e., a bacteriophage having lytic activity against Pseudomonas aeruginosa, or the genome of the bacteriophage is introduced into the host bacterium, and the bacteriophage is then cultured. Culture conditions are appropriately selected depending on the host bacterium and culture method used. After culture, the culture supernatant obtained by standing or centrifugation can be filtered to obtain purified bacteriophage.

[0160] 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.

[0161] 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.

[0162] Regarding the strains, those designated as ATCC can be obtained from the American Type Culture Collection, and those designated as NBRC can be obtained from the National Institute of Technology and Evaluation (NITE). In addition, the PA14 strain (catalog number NR-50573), MRSN 321 strain (catalog number NR-51517), and MRSN 4841 strain can be obtained from BEI Resources, NIAID, and NIH. The MRSN 321 and MRSN 4841 strains are strains included in the Pseudomonas aeruginosa Diversity Panel provided by the Multidrug-Resistant Organism Repository and Surveillance Network (MRSN) at the Walter Reed Army Institute for Research (WRAIR).

[0163] Example 1: Isolation of bacteriophages exhibiting lytic activity against Pseudomonas aeruginosa Bacteriophages were isolated from environmental samples (sewage and wastewater in Japan) using the following method. The host used for isolation was either Pseudomonas aeruginosa (Schroeter) Migula ATCC15692 strain (a strain related to the PAO1 strain), NBRC3080 strain, PA14 strain, MRSN 321 strain, or MRSN 4841 strain. The environmental sample was centrifuged, and the supernatant was separated and filtered through a 0.22 μm or 0.45 μm filter. The filtered sample was mixed with equal volumes of LB liquid medium (20 g / L LB medium, Lennox (Nacalai Tesque)) adjusted to twice the final concentration. To the mixture was added a Pseudomonas aeruginosa culture solution cultured overnight in LB liquid medium, and the mixture was cultured with shaking at 37°C. The culture solution was centrifuged, and the supernatant was separated. This supernatant and a Pseudomonas aeruginosa culture solution cultured overnight in LB liquid medium were mixed with LB liquid medium containing 0.3 to 0.6% soft agar, and the mixture was layered on an LB agar plate. After culturing at 37°C for several hours to several days, plaques that appeared on the medium were collected, and bacteriophages were isolated. The resulting bacteriophages are designated PAPT1, PAi140, PAi228, PAi239, and PAi242, respectively. Plaques were collected from a bacteriophage stock provided by Rakuno Gakuen University in the same manner as above, and bacteriophages φLCX and φLCX2 (closely related strains of bacteriophage φLP identified by NCBI Reference Sequence LC727700) were isolated. These bacteriophages were cultured in soft agar-containing LB liquid medium in the same manner as above using Pseudomonas aeruginosa strain ATCC15692, NBRC3080, or NBRC13746 as a host, to prepare bacteriophage lysates.

[0164] Bacteriophage PAi23 (a passaged strain of PaGU11 identified by NCBI Reference Sequence NC_050145) provided by Gifu University, and bacteriophages φBrkr (a passaged strain of bacteriophage identified by NCBI Reference Sequence LC765218), φBrmt (a passaged strain of bacteriophage identified by NCBI Reference Sequence LC727695), and φ30-1 (a passaged strain of bacteriophage identified by NCBI Reference Sequence LC727695) provided by Rakuno Gakuen University. A subculture of a bacteriophage identified as LC727696) was co-cultured with Pseudomonas aeruginosa ATCC15692 strain to prepare bacteriophage lysates.

[0165] The lytic activity of PAPT1, PAi140, PAi228, PAi239, PAi242, φLCX, φLCX2, PAi23, φBrkr, φBrmt, and φ30-1 against Pseudomonas aeruginosa was confirmed by the following method. Pseudomonas aeruginosa culture solution cultured overnight in LB liquid medium was mixed with LB liquid medium containing 0.3% or 0.6% soft agar, and layered on an LB agar plate. Each bacteriophage lysate was lysed in phage buffer (1 mM CaCl ２ , 10 mM MgSO ４ 2.5 μL of 10-fold serial dilutions prepared using 10 mM Tris-HCl (pH 7.5) containing 68.4 mM NaCl were dropped onto the plate and incubated at 37°C. Plaques appeared due to each bacteriophage, confirming the lysis of Pseudomonas aeruginosa (Figure 1).

[0166] The accession numbers or accession numbers of the thus obtained PAPT1, PAi140, PAi228, PAi239, PAi242, PAi23, φBrkr, φBrmt, φ30-1, φLCX, and φLCX2 are respectively Accession No. NITE BP-04031, Accession No. NITE BP-04033, Accession No. NITE BP-04034, Accession No. NITE BP-04035, Accession No. NITE BP-04036, Accession No. NITE BP-04032, Accession No. NITE BP-04037, Accession No. NITE BP-04038, Accession No. NITE BP-04039, and Accession No. NITE BP-04040, respectively. BP-04040, receipt number NITE ABP-04200.

[0167] Example 2: Construction of a bacteriophage with a tail fiber gene exchange A recombinant bacteriophage was constructed in which the tail fiber gene of PAi23 phage was exchanged with the tail fiber gene of the known Pseudomonas aeruginosa bacteriophage JG024 (a bacteriophage identified by NCBI Reference Sequence NC_017674.1). Multiple polynucleotides containing partial base sequences of the PAi23 phage genome and the artificially synthesized JG024 phage tail fiber gene (SEQ ID NO: 6) were amplified by PCR. The amplified polynucleotides were ligated using Gibson assembly master mix (New England Biolabs, catalog number E2611). As a result, bases 31081-33975 encoding the tail fiber gene of PAi23 phage were deleted, and the deleted phage genome was recombined with the tail fiber gene (SEQ ID NO: 6) of JG024 phage to construct a phage genome. The phage genome was introduced into Pseudomonas aeruginosa strain ATCC15692 by electroporation to obtain the desired bacteriophage. The resulting bacteriophage is designated PAi23-JG024 tail.

[0168] The bacteriolytic activity of the PAi23-JG024 tail obtained above against Pseudomonas aeruginosa was confirmed. The P. aeruginosa strain used was the ATCC15692 strain and the respiratory clinical isolates 3-55-PA and 9-8-PA strains (provided by the Joint Committee on Antimicrobial Susceptibility Surveillance of Three Academic Societies). P. aeruginosa cultures grown overnight in LB liquid medium were mixed with LB liquid medium containing 0.6% soft agar and layered on an LB agar plate. Ten-fold serial dilutions of the PAi23 phage and PAi23-JG024 tail phage lysates were prepared using phage buffer. 2.5 μL of each dilution was dropped onto the plate and incubated at 37°C. Plaques appeared due to the presence of each bacteriophage, confirming the bacteriolytic activity against P. aeruginosa (Figure 2). As shown in Table 1, it was confirmed that PAi23-JG024 tail was able to infect strains that were not infected by PAi23.

[0169]

[0170] Example 3: Construction of PAi23-JG024 tail phage carrying PelAh, PslGh, and PslG Phages were constructed by inserting the PelAh gene (SEQ ID NO: 3), PslGh gene (SEQ ID NO: 4), and PslG gene (SEQ ID NO: 2) derived from Pseudomonas aeruginosa into the PAi23 phage genome. PelAh is a fragment sequence of the PelA gene (SEQ ID NO: 1), and PslGh is a fragment sequence of the PslG gene (SEQ ID NO: 2) (Science Advances. 2016; 2(5): e1501632). The sequences of the PelAh gene, PslGh gene, and PslG gene were amplified by PCR from the genome of the NBRC106052 (PAO1) strain. The ampicillin resistance gene of the pUCP18 plasmid vector (NCBI Reference Sequence U07164) was replaced with a gentamicin resistance gene to create the pUCP18-GmR plasmid vector. A Shine-Dalgarno sequence (SEQ ID NO: 8) was added to the 5' side of the start codons of the obtained PelAh gene, PslGh gene, and PslG gene, and each was inserted into the EcoRI / XbaI restriction enzyme sites so as to be linked to the lac operon of the pUCP18-GmR plasmid vector. Similar to the method shown in Example 2, the PelAh gene, PslGh gene, and PslG gene, each with a portion of the lac operon (SEQ ID NO: 7) and a Shine-Dalgarno sequence-like sequence (SEQ ID NO: 8) added, were inserted between bases 12081 and 12082 of the PAi23 phage genome (the sequence obtained by linking a portion of the lac operon, the Shine-Dalgarno sequence-like sequence (SEQ ID NO: 8), and the PslGh gene is SEQ ID NO: 10). Furthermore, similar to Example 2, a phage genome was constructed in which the tail fiber gene was exchanged for that of JG024 phage. The constructed phage genomes were introduced into Pseudomonas aeruginosa ATCC15692 strain by electroporation to obtain recombinant bacteriophages, which are designated PAi23-JG024 tail::Plac-PelAh(AB), PAi23-JG024 tail::Plac-PslGh(AB), and PAi23-JG024 tail::Plac-PslG(AB), respectively.

[0171] Using the same procedure as described above, a phage was constructed in which the Shine-Dalgarno sequence-like sequence (SEQ ID NO: 8), which does not encode a gene 5' from the start codon, was replaced with a spacer-added sequence, Shine-Dalgarno sequence-like sequence_version 3 (v3) (SEQ ID NO: 12). A PslGh gene (SEQ ID NO: 4) to which a portion of the lac operon (SEQ ID NO: 7) and the Shine-Dalgarno sequence-like sequence_v3 (SEQ ID NO: 12) had been added was inserted between bases 12081 and 12082 of the PAi23 phage genome (the sequence consisting of a portion of the lac operon, the Shine-Dalgarno sequence-like sequence_v3 (SEQ ID NO: 12), and the PslGh gene is SEQ ID NO: 11). Furthermore, a phage genome was constructed in which the tail fiber gene was replaced with that of JG024 phage, as in Example 2. The constructed phage genome was introduced into Pseudomonas aeruginosa ATCC15692 strain by electroporation to obtain a recombinant bacteriophage, designated PAi23-JG024 tail::Plac-PslGh(AB)_v3.

[0172] Furthermore, a phage was constructed into which the PslGh gene (SEQ ID NO: 4) and PelAh gene (SEQ ID NO: 3) were inserted. A nucleotide sequence was inserted between bases 12081 and 12082 of the PAi23 phage genome, linking the PslGh gene (SEQ ID NO: 4) to which a portion of the lac operon (SEQ ID NO: 7) and a Shine-Dalgarno sequence-like sequence_v3 (SEQ ID NO: 12) had been added, and the PelAh gene (SEQ ID NO: 3) to which a Shine-Dalgarno sequence-like sequence (SEQ ID NO: 8) had been added. Furthermore, a phage genome was constructed in which the tail fiber gene had been replaced with that of JG024 phage, as in Example 2. The constructed phage genome was introduced into Pseudomonas aeruginosa ATCC15692 strain by electroporation, and the resulting recombinant bacteriophage is designated PAi23-JG024 tail::Plac-PslGh-PelAh(AB)_v3.

[0173] The lytic activity of the above-obtained PAi23-JG024 tail::Plac-PelAh(AB), PAi23-JG024 tail::Plac-PslGh(AB), PAi23-JG024 tail::Plac-PslG(AB), PAi23-JG024 tail::Plac-PslGh(AB)_v3, and PAi23-JG024 tail::Plac-PslGh-PelAh(AB)_v3 against Pseudomonas aeruginosa was confirmed. The Pseudomonas aeruginosa strain used was ATCC15692. A Pseudomonas aeruginosa culture solution cultured overnight in LB liquid medium was mixed with LB liquid medium containing 0.3% soft agar and layered on an LB agar plate. Each phage lysate was serially diluted 10-fold using phage buffer. 2.5 μL of each diluted solution was dropped onto a plate and incubated at 37°C. Plaques appeared due to each bacteriophage, confirming its lytic activity against Pseudomonas aeruginosa (Figure 3).

[0174] Example 4: Construction of PyoG-Equipped PAi23 Phage A bacteriophage was constructed by inserting the PyoG gene derived from Pseudomonas aeruginosa into the PAi23 phage genome. The bacteriocin PyoG has bactericidal activity against Pseudomonas aeruginosa. To produce a bacteriophage stably inserted with the PyoG gene, a Pseudomonas aeruginosa ATCC15692 strain was previously constructed that constitutively expresses the ImG gene (Journal of molecular biology. 2020; 432(13): 3869-3880), which is known as the immunity gene of PyoG. The ImG gene (SEQ ID NO: 9) with a Shine-Dalgarno sequence-like sequence (SEQ ID NO: 8) added was inserted into the EcoRI / XbaI site of the pUCP18-GmR plasmid vector, and the vector was introduced into the Pseudomonas aeruginosa ATCC15692 strain by electroporation. As a result, a Pseudomonas aeruginosa ATCC15692-ImG strain was obtained that constitutively expresses the ImG gene under the control of the lac promoter.

[0175] Next, a phage genome was constructed in which the artificially synthesized PyoG gene (SEQ ID NO: 5) was inserted between bases 35068 and 35069 of the PAi23 phage genome in the same manner as in Example 2. The recombinant bacteriophage obtained by introducing the artificially synthesized PyoG gene (SEQ ID NO: 5) into the Pseudomonas aeruginosa ATCC15692-ImG strain is designated PAi23::PyoG(CD).

[0176] The lytic activity of the PAi23::PyoG(CD) obtained above against Pseudomonas aeruginosa was confirmed by the following method. The Pseudomonas aeruginosa strains ATCC15692 and ATCC15692-ImG were used. Pseudomonas aeruginosa cultures grown overnight in LB liquid medium were mixed with LB liquid medium containing 0.3% soft agar and layered on LB agar plates. Ten-fold serial dilutions of each phage lysate of PAi23 and PAi23::PyoG(CD) were prepared using phage buffer. 2.5 μL of each dilution was dropped onto the plate and incubated at 37°C. Plaques formed by the bacteriophage confirmed the lytic activity against Pseudomonas aeruginosa (Figure 4). Compared to the PAi23 phage, PAi23::PyoG(CD) formed more distinct plaques against the Pseudomonas aeruginosa ATCC15692 strain.

[0177] Example 5: Biofilm-degrading activity of natural bacteriophages We investigated whether natural bacteriophages have the activity to reduce the amount of biofilm formed by Pseudomonas aeruginosa. Pseudomonas aeruginosa ATCC15692 strain was inoculated into a 96-well plate (Nunc, Thermo Scientific) containing LB liquid medium, and the plate was covered with a pinned lid (Nunc Immuno TSP Lids, Thermo Scientific). Static culture was performed at 37°C for 24 hours to form a biofilm on the pin. The formed biofilm was washed with physiological saline and then added to a phage buffer with a final concentration of 1.29 x 10 ８ Phage φLCX adjusted to PFU / mL in LB liquid medium, final concentration 1 × 10 ８PAPT1 or PAi239, which had been adjusted to PFU / mL in LB liquid medium, was contacted with biofilms in a 96-well plate at 37°C for 6 hours. Each well was washed with saline and then stained with crystal violet. After staining, each well was washed with saline. After washing, the crystal violet in each well was eluted with ethanol, and the absorbance at 590 nm of each eluate was measured. Higher absorbance indicates a larger amount of biofilm. Three trials were performed in three wells per condition. The average of the three trials was calculated, and one-way analysis of variance and Dunnett's multiple comparison test were performed. The results showed that the amount of biofilm was significantly reduced under conditions in which φLCX, PAPT1, or PAi239 was added compared to the phage buffer group (control) (Figure 5, ＊ p<0.05).

[0178] Example 6: Biofilm degradation activity of modified bacteriophages We investigated whether PAi23-JG024 tail::Plac-PslGh(AB) and PAi23-JG024 tail::Plac-PslG(AB) have the activity to reduce the amount of biofilm formed by Pseudomonas aeruginosa. As in the method of Example 5, PAi23-JG024 tail, PAi23-JG024 tail::Plac-PslGh(AB), and PAi23-JG024 tail::Plac-PslG(AB) were each added to a final concentration of 1 × 10 ８ The mixture was added to LB liquid medium at a concentration of 100 PFU / mL and contacted with biofilms in a 96-well plate. The experiment was performed in 6 wells per condition, with three trials. The average of the three trials was calculated, and one-way analysis of variance and Dunnett's multiple comparison test were performed. The results showed that PAi23-JG024 tail did not reduce the amount of biofilm, whereas PAi23-JG024 tail::Plac-PslGh(AB) (in Figure 6, "::PslGh") and PAi23-JG024 tail::Plac-PslG(AB) (in Figure 6, "::PslG") significantly reduced the amount of biofilm (Figure 6A, ＊p<0.05).

[0179] Next, we investigated whether PAi23-JG024 tail::Plac-PelAh(AB) has the activity to reduce the amount of biofilm formed by Pseudomonas aeruginosa. Using the same method as above, a biofilm was formed on a pin using the respiratory clinical isolate 28-42-PA strain (provided by the Joint Committee on Antimicrobial Susceptibility Surveillance of Three Academic Societies). PAi23-JG024 tail and PAi23-JG024 tail::Plac-PelAh(AB) were each added to a final concentration of 1 x 10 ８ The PAi23-JG024 tail was added to LB liquid medium at a concentration of PFU / mL and contacted with biofilms in a 96-well plate. Eight trials were performed, with three wells per condition (graph on the left in Figure 6B). The relative biofilm amount was calculated for each trial, with the control group set at 1, and a Kruskal-Wallis test and a Dunn's multiple comparison test were performed. The results showed that PAi23-JG024 tail did not reduce the biofilm amount, whereas PAi23-JG024 tail::Plac-PelAh(AB) ("::PelAh" in Figure 6) significantly reduced the biofilm amount (Figure 6B, right). ＊ p<0.05).

[0180] Next, we investigated whether PAi23-JG024 tail::Plac-PslGh(AB)_v3 and PAi23-JG024 tail::Plac-PslGh-PelAh(AB)_v3 have the activity to reduce the amount of biofilm formed by Pseudomonas aeruginosa. Biofilms were formed on pins using the Pseudomonas aeruginosa ATCC15692 strain in the same manner as described above. PAi23-JG024 tail, PAi23-JG024 tail::Plac-PslGh(AB)_v3, and PAi23-JG024 tail::Plac-PslGh-PelAh(AB)_v3 were added to a final concentration of 1 x 10 ８The mixture was added to LB liquid medium so that the concentration was PFU / mL, and contacted with biofilms in a 96-well plate. The experiment was performed three times, with three wells per condition. The average of the three trials was calculated, and one-way analysis of variance and Dunnett's multiple comparison test were performed. As a result, PAi23-JG024 tail did not reduce the amount of biofilm, whereas PAi23-JG024 tail::Plac-PslGh(AB)_v3 (in FIG. 6, "::PslGh_v3") and PAi23-JG024 tail::Plac-PslGh-PelAh(AB)_v3 (in FIG. 6, "::PslGh-PelAh_v3") significantly reduced the amount of biofilm (FIG. 6C, ＊ p<0.05).

[0181] Example 7: Activity of modified bacteriophages in inhibiting growth of Pseudomonas aeruginosa The growth inhibitory activity of PAi23 and PAi23::PyoG(CD) against Pseudomonas aeruginosa was investigated. Respiratory clinical isolate 27-12-PA strain (provided by the Joint Committee on Antimicrobial Susceptibility Surveillance of Three Academic Societies) was diluted with LB liquid medium to an OD600 of 0.1, and 90 μL of the diluted solution was inoculated into a 96-well plate at a final concentration of 1 × 10. Phage buffer (Control), or PAi23 and PAi23::PyoG(CD) were added to each well. ８ 10 μL of the culture medium was added to each well to achieve a PFU / mL concentration, and the mixture was incubated at 37°C. The absorbance at 600 nm of the culture medium was measured over time using a plate reader. Four trials were performed, with three wells per condition. The average absorbance at 12 hours for the four trials was calculated, and one-way analysis of variance and Tukey's multiple comparison test were performed. The results showed that PAi23::PyoG(CD) exhibited a longer inhibitory effect on the growth of Pseudomonas aeruginosa than PAi23 (FIG. 7, ＊ p<0.05).

[0182] Example 8: Lytic activity of bacteriophage φLCX against Pseudomonas aeruginosa The lytic activity of bacteriophage φLCX against Pseudomonas aeruginosa that had been cultured overnight and grown sufficiently was examined. Pseudomonas aeruginosa ATCC15692 strain was inoculated into LB liquid medium and cultured at 37°C in a glass test tube with shaking. Starting from 15 hours of culture, the absorbance at 600 nm of the culture solution was measured over time. After 18 hours of culture, phage buffer (Control) or bacteriophage φLCX was added to the culture at a final concentration of 1 x 10 ７ PFU / mL was added, and the culture was continued with shaking at 37°C. This experiment was performed in triplicate using glass test tubes per condition. The average absorbance of the three tubes was calculated. The results showed that φLCX had bacteriolytic activity against Pseudomonas aeruginosa that had passed the logarithmic growth phase (Figure 8).

[0183] Example 9: Lytic activity of a single bacteriophage in a mouse infection model Pseudomonas aeruginosa ATCC15692 strain was inoculated into tryptic soy broth (Merck) and cultured with shaking at 37°C for 2.5 hours. The culture was centrifuged, the supernatant discarded, and suspended in physiological saline. The inoculum was diluted with physiological saline to an absorbance at 600 nm (OD600) of 0.5, and used as an inoculum. C57BL / 6Jms Slc mice (Japan SLC, Inc., female, approximately 5 weeks old) were infected by inoculating 40 μL of the bacterial solution into the nasal cavity of each mouse. After 30 minutes, phage buffer, PAi23, or φBrmt phage was added to five mice each at 5 × 10 per mouse. ８PFU was administered intranasally. 24 hours after infection, lungs were removed and homogenized in 2 mL of Hank's Balanced Salt Solution (Life Technologies Corporation) to prepare a tissue stock solution. Ten-fold serial dilutions of the tissue stock solution were prepared with physiological saline. The tissue was plated on tryptosoy agar medium (Eiken Chemical) to measure viable bacterial counts. The viable bacterial counts were converted to common logarithms, and the arithmetic mean was calculated. One-way analysis of variance and Dunnett's multiple comparison test were then performed. Figure 9 shows the viable bacterial counts in the lungs 24 hours after infection (log CFU / lung) (N=5, mean ± standard error). The results showed that administration of PAi23 or φBrmt significantly reduced the number of live bacteria in the lungs compared to the phage buffer group (Figure 9, ＊ p<0.05).

[0184] Example 10: Preparation of freeze-thaw resistant φBrkr phage A freeze-thaw resistant φBrkr strain (referred to as φBrkr_FTR2) was prepared by the following method using φBrkr (accession number NITE BP-04037) as a parent phage.

[0185] Ethyl methanesulfonate (Nacalai Tesque) was added to φBrkr in phage buffer to a final concentration of approximately 60 mM, and the mixture was allowed to react at 37°C for 1 hour. A lysate of bacteriophage in which gene mutations had been induced after the reaction was prepared using the same method as in Example 1. The prepared lysate was diluted 20-fold with phage buffer and then subjected to a freeze-thaw process (freeze-thaw) twice in a -80°C freezer. The remaining bacteriophage was then amplified using the same method as in Example 1, and a lysate was prepared again. This process constitutes one cycle, and nine cycles were performed. The lysate after nine cycles was cultured using the same method as above, and purified φBrkr_FTR2 was obtained from a single plaque. The accession number for φBrkr_FTR2 is NITE ABP-04201.

[0186] The freeze-thaw resistance of φBrkr_FTR2 was confirmed by the following method. φBrkr and φBrkr_FTR2 were each diluted to 1 × 10 ８The titer values ​​before and after freeze-thawing were calculated using the following method. Pseudomonas aeruginosa culture solution cultured overnight in LB liquid medium was mixed with LB liquid medium containing 0.3% soft agar and layered on an LB agar plate. 10-fold serial dilutions were prepared for each bacteriophage solution using phage buffer. 2.5 μL of each dilution was dropped onto the plate and allowed to stand at 37°C. Plaques appeared for each bacteriophage, confirming their lytic activity against Pseudomonas aeruginosa (FIG. 10A). Titer values ​​were calculated from the number of plaques, and the phage recovery rates before and after freeze-thawing were calculated (FIG. 10B). As a result, it was confirmed that φBrkr_FTR2 had significantly improved freeze-thaw resistance compared to φBrkr (unpaired t-test, ＊ p<0.05).

[0187] Example 11 Extension of the Pseudomonas aeruginosa Growth Inhibitory Effect by Bacteriophage Cocktailing In this example and thereafter, bacteriophage lysates were prepared using ATCC15692ΔPf4 / Pf6 (prophage-removed strain) described later in Example 14 as a culture host for PAPT1, PAi242, φBrkr, φBrkr_FTR2, φ30-1, φLCX, φLCX2, PAi23-JG024 tail::Plac-PslGh(AB), and PAi23-JG024 tail::Plac-PslGh(AB)_v3.

[0188] We investigated whether the inhibitory effect on the growth of Pseudomonas aeruginosa could be extended by using a bacteriophage cocktail. ８ The bacteriophage solution was then added to each well to a total of 100 μL. The single bacteriophage, the cocktail of two bacteriophages, and the cocktail of four bacteriophages were each seeded at a final concentration of 1 × 10 ４The bacteriophages were added to each well so that the final concentration of each bacteriophage was 0.25 x 10 PFU / mL (i.e., the multiplicity of infection (MOI) was approximately 0.0001). The ratio of the amount of each bacteriophage in the cocktail was equal. For example, in a cocktail of four bacteriophages, the final concentration of each bacteriophage was 0.25 x 10 ４ The cells were cultured at 37°C, and the absorbance at 600 nm of each well was measured every hour.

[0189] As representative examples of growth curves for Pseudomonas aeruginosa, graphs for cocktail a (a cocktail of φBrkr, PAi23-JG024 tail::Plac-PslGh(AB)) and each of the individual bacteriophages that make up cocktail a, and graphs for cocktail A (a cocktail of φBrkr, φLCX, PAi23-JG024 tail::Plac-PslGh(AB), PAPT1) and each of the individual bacteriophages that make up cocktail A are shown in Figure 11. Similar graphs were created for combinations of cocktails a to e and cocktails A to H, and the area under the curve (AUC) was calculated for each. The AUC under conditions without phage addition was set to 100%, and the relative AUC (%) ± standard error was calculated. The results are shown in Tables 2 and 3 below. One-way analysis of variance and Tukey's multiple comparison test were performed on each of the cocktails, and the results showed that all cocktails significantly prolonged the growth inhibitory effect on Pseudomonas aeruginosa compared to the single bacteriophages that comprised each cocktail ( ＊ p<0.05).

[0190]

[0191]

[0192] Example 12: Biofilm-degrading activity of bacteriophage cocktail We investigated whether a bacteriophage cocktail has the activity to reduce the amount of biofilm formed by Pseudomonas aeruginosa. As in the method of Example 5, a biofilm was formed on a pin using Pseudomonas aeruginosa ATCC 15692 strain. The bacteriophage cocktail was added at a final concentration of 1 x 10 ８The phages were added to LB liquid medium at a concentration of 0.01 PFU / mL (the ratio of each single phage was adjusted to be equal), and contacted with biofilms in a 96-well plate. The experiment was performed in triplicate with each condition, for a total of four trials. The average of the four trials was calculated, and one-way analysis of variance and Dunnett's multiple comparison test were performed. The results showed that cocktail A (a cocktail of φBrkr, φLCX, PAi23-JG024 tail::Plac-PslGh(AB), PAPT1) and cocktail B (a cocktail of φBrkr, φLCX2, PAi23-JG024 tail::Plac-PslGh(AB)_v3, PAPT1) significantly reduced the amount of biofilm (Figure 12, ＊ p<0.05).

[0193] Example 13: Lytic Activity of Bacteriophage Cocktail in a Mouse Infection Model. Ultracentrifugation-grade bacteriophage solutions were used in mouse infection experiments using density gradient centrifugation. Ultracentrifugation purification: DNase I and RNase A were added to each phage lysate to a final concentration of 1 μg / mL, and the mixture was left to stand at room temperature for 30 minutes. Bacteriophages were precipitated using polyethylene glycol 8000, and the precipitate was collected by centrifugation. The collected bacteriophage solution was gently dissolved by adding 0.4 g of cesium chloride per mL and transferred to an ultracentrifugation container. Cesium chloride solutions with densities of 1.3 g / mL, 1.4 g / mL, 1.5 g / mL, and 1.7 g / mL were sequentially injected into the bottom of the container using a syringe. The mixture was centrifuged at 59,000 g for 3 hours at 4°C, and the band containing the bacteriophage was collected. The solution containing the recovered bacteriophage was replaced with a phage buffer by dialysis to obtain an ultracentrifugally purified grade bacteriophage solution.

[0194] Respiratory clinical isolate 27-12-PA strain was inoculated into LB liquid medium and cultured with shaking at 37°C for 2.5 hours. The culture medium was centrifuged, the supernatant was discarded, and the precipitate was suspended in physiological saline. The suspension was diluted with physiological saline to an absorbance at 600 nm (OD600) of 0.25, and used as an inoculum. C57BL / 6Jms Slc mice (Japan SLC Co., Ltd., female, 5 weeks old) were infected by oropharyngeal aspiration with 50 μL of the bacterial solution per mouse. One hour after infection, phage buffer or bacteriophage cocktail was added at a total of 1 x 10 per mouse. ９ Bacteriophage cocktails were administered by oropharyngeal aspiration to achieve PFU. The bacteriophage cocktails were cocktail A (φBrkr, φLCX, PAi23-JG024 tail::Plac-PslGh(AB), PAPT1), cocktail B (φBrkr, φLCX2, PAi23-JG024 tail::Plac-PslGh(AB)_v3, PAPT1), or cocktail G (φ30-1, PAi242, PAi23-JG024 tail::Plac-PslGh(AB)_v3, PAi140). Approximately 22.5 hours after infection, lungs were removed. The lungs were homogenized in 2 mL of saline and washed twice with saline to prepare a tissue stock solution. Ten-fold serial dilutions of the tissue stock solution were prepared with physiological saline. These were applied to LB agar plates, and viable bacterial counts were measured. When the viable bacterial count was below the detection threshold, it was calculated as the detection threshold value. The viable bacterial counts were converted to common logarithms, and the arithmetic mean was calculated. One-way analysis of variance and Dunnett's multiple comparison test were then performed. Figure 13 shows the viable bacterial counts in the lungs (log CFU / lung) (N = 10, mean ± standard error). As a result, it was shown that the amount of viable bacteria in the lungs was significantly reduced in the cocktail A, cocktail B, and cocktail G groups compared to the phage buffer group ( ＊ p<0.05).

[0195] Example 14: Cultivation of bacteriophages using a prophage-cured strain of Pseudomonas aeruginosa A method for removing lysogenizing filamentous phages (e.g., Pf4 and Pf6) from Pseudomonas aeruginosa strains such as the PAO1 strain has been reported (Journal of Bacteriology. 2024; 206(5): e0040223). Briefly, this method removes filamentous phages by deleting the pflM gene, which is involved in the maintenance of filamentous phages. Following this method, Pf4 and Pf6 were deleted from the genome of Pseudomonas aeruginosa strain ATCC15692 (a strain related to the PAO1 strain) as follows. First, the pflM genes in Pf4 and Pf6 were replaced with kanamycin resistance genes using the pUCP18-RedS vector. After obtaining a strain lacking the pflM gene, a strain lacking both Pf4 and Pf6 was obtained. Furthermore, a strain from which the pUCP18-RedS vector had been removed was obtained by sucrose treatment. The prophage-removed strain thus obtained is designated ATCC15692ΔPf4 / Pf6.

[0196] The bacteriolytic activity of PAi242 against Pseudomonas aeruginosa ATCC15692 strain and ATCC15692ΔPf4 / Pf6 strain was confirmed in the same manner as in Example 1. The culture solution of Pseudomonas aeruginosa ATCC15692 strain or ATCC15692ΔPf4 / Pf6 strain cultured overnight in LB liquid medium was mixed with LB liquid medium containing 0.3% soft agar and layered on an LB agar plate. For the bacteriophage lysate of PAi242, 1 × 10 １０ A PFU / mL solution was prepared, and 10-fold serial dilutions were prepared using phage buffer. 2.5 μL of each dilution was dropped onto a plate and incubated at 37°C (dilution factor 1 in Figure 14A is 1 × 10 １０(Figure 14A shows images of lytic plaques that appeared after 10-fold serial dilutions of PAi242 phage were spotted onto a plate. PAi242 showed lytic activity against the P. aeruginosa ATCC15692 strain but did not form visible plaques. However, it formed discernible plaques against the ATCC15692ΔPf4 / Pf6 strain. This indicates that the sensitivity of PAi242 to P. aeruginosa ATCC15692 strain was increased by removing Pf4 and Pf6 from its genome.

[0197] Next, Pseudomonas aeruginosa ATCC15692 strain and ATCC15692ΔPf4 / Pf6 strain were used to carry out liquid culture of bacteriophages. Each strain was inoculated into LB liquid medium, and after 2.5 hours, φBrkr, φ30-1, PAPT1, and PAi242 were added to a final concentration of 1 × 10 ６ PFU / mL of bacteriophage was added to the culture medium and allowed to infect. After approximately 6 hours of shaking culture at 37°C, each bacteriophage lysate was obtained. Three liquid cultures of each bacteriophage were performed, and the titer values ​​of the resulting lysates were calculated using the method of Example 10. The results are shown in Figure 14B. When bacteriophages were cultured using the ATCC15692ΔPf4 / Pf6 strain (prophage-removed strain) compared to the Pseudomonas aeruginosa ATCC15692 strain, lysates with titers of PAPT1 or more were obtained with titers 10 times higher, and those of PAi242 were obtained with titers 10,000 times higher (unpaired t-test). ＊ p<0.05). These results demonstrate that PAPT1 and PAi242 can be amplified with high efficiency by culturing them using the ATCC15692ΔPf4 / Pf6 strain as the host bacterium.

[0198] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

[0199] [Accession number (receipt number)] Accession number NITE BP-04040 (bacteriophage φLCX strain, deposited on December 7, 2023) Accession number NITE BP-04031 (bacteriophage PAPT1 strain, deposited on December 7, 2023) Accession number NITE BP-04033 (bacteriophage PAi140 strain, deposited on December 7, 2023) Accession number NITE BP-04036 (bacteriophage PAi242 strain, deposited on December 7, 2023) Accession number NITE BP-04034 (bacteriophage PAi228 strain, deposited on December 7, 2023) Accession number NITE BP-04035 (bacteriophage PAi239 strain, deposited on December 7, 2023) Accession number NITE BP-04032 (bacteriophage PAi23 strain, deposited on December 7, 2023) Accession number NITE BP-04037 (bacteriophage φBrkr strain, deposited on December 7, 2023) Accession number NITE BP-04039 (bacteriophage φ30-1 strain, deposited on December 7, 2023) Accession number NITE BP-04038 (bacteriophage φBrmt strain, deposited on December 7, 2023) Accession number NITE ABP-04200 (bacteriophage φLCX2 strain, received on November 13, 2024) Receipt number NITE ABP-04201 (bacteriophage φBrkr_FTR2 strain, received on November 13, 2024)

Claims

1. A bacteriophage selected from the following (1) to (8): (1) A bacteriophage having lytic activity against Pseudomonas aeruginosa, 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 specified by the accession number NITE BP-04040; (2) A bacteriophage having lytic activity against Pseudomonas aeruginosa, 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 specified by the accession number NITE BP-04031; (3) A bacteriophage having lytic activity against Pseudomonas aeruginosa, 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 specified by the accession number NITE BP-04033; (4) A bacteriophage having lytic activity against Pseudomonas aeruginosa, 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 specified by the accession number NITE BP-04036; (5) A bacteriophage having lytic activity against Pseudomonas aeruginosa, 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 specified by the accession number NITE BP-04034; (6) A bacteriophage having lytic activity against Pseudomonas aeruginosa, 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 specified by the accession number NITE BP-04035; (7) A bacteriophage having lytic activity against Pseudomonas aeruginosa, 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 specified by the accession number NITE ABP-04200;And (8) a bacteriophage having lytic activity against Pseudomonas aeruginosa, 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 specified by accession number NITE ABP-04201.; 2. The bacteriophage according to claim 1, selected from the following (1) to (8): (1) A bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage contains the nucleic acid sequence of the genome of the bacteriophage specified by the accession number NITE BP-04040; (2) A bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage contains the nucleic acid sequence of the genome of the bacteriophage specified by the accession number NITE BP-04031; (3) A bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage contains the nucleic acid sequence of the genome of the bacteriophage specified by the accession number NITE BP-04033; (4) A bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage contains the nucleic acid sequence of the genome of the bacteriophage specified by the accession number NITE BP-04036; (5) A bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage contains the nucleic acid sequence of the genome of the bacteriophage specified by the accession number NITE BP-04034; (6) A bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage contains the nucleic acid sequence of the genome of the bacteriophage specified by the accession number NITE BP-04035; (7) A bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage contains the nucleic acid sequence of the genome of the bacteriophage specified by the accession number NITE ABP-04200; and (8) A bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage contains the nucleic acid sequence of the genome of the bacteriophage specified by the accession number NITE ABP-04201.

3. The bacteriophage according to claim 1 or claim 2, selected from the following (1) to (8): (1) a bacteriophage comprising a nucleic acid sequence of a bacteriophage genome specified by accession number NITE BP-04040; (2) a bacteriophage comprising a nucleic acid sequence of a bacteriophage genome specified by accession number NITE BP-04031; (3) a bacteriophage comprising a nucleic acid sequence of a bacteriophage genome specified by accession number NITE BP-04033; (4) a bacteriophage comprising a nucleic acid sequence of a bacteriophage genome specified by accession number NITE BP-04036; (5) a bacteriophage comprising a nucleic acid sequence of a bacteriophage genome specified by accession number NITE BP-04034; (6) a bacteriophage comprising a nucleic acid sequence of a bacteriophage genome specified by accession number NITE BP-04035; (7) a bacteriophage comprising a nucleic acid sequence of a bacteriophage genome specified by accession number NITE ABP-04200; and (8) a bacteriophage comprising a nucleic acid sequence of a bacteriophage genome specified by accession number NITE ABP-04201.

4. A bacteriophage selected from the following (1) to (8): (1) the bacteriophage identified by the accession number NITE BP-04040, or a subculture strain thereof; (2) the bacteriophage identified by the accession number NITE BP-04031, or a subculture strain thereof; (3) the bacteriophage identified by the accession number NITE BP-04033, or a subculture strain thereof; (4) the bacteriophage identified by the accession number NITE BP-04036, or a subculture strain thereof; (5) the bacteriophage identified by the accession number NITE BP-04034, or a subculture strain thereof; (6) the bacteriophage identified by the accession number NITE BP-04035, or a subculture strain thereof; (7) the bacteriophage identified by the accession number NITE ABP-04200, or a subculture strain thereof; and (8) the bacteriophage identified by the accession number NITE ABP-04201, or a subculture strain thereof.

5. A bacteriophage having lytic activity against Pseudomonas aeruginosa, 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 the accession number NITE BP-04032, excluding the part of the tail fiber gene, and (b) a nucleic acid sequence of a tail fiber gene comprising at least a part of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa.

6. The bacteriophage according to claim 5, which is a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage comprises (a) the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04032, excluding the part of the tail fiber gene, and (b) a nucleic acid sequence of a tail fiber gene comprising at least a part of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa.

7. A bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage consists of the nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-04032, except for the part of the tail fiber gene, and (b) contains the nucleic acid sequence of a tail fiber gene containing at least a part of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa. The bacteriophage according to claim 5.

8. The bacteriophage according to claim 5, wherein the tail fiber gene derived from another bacteriophage contains the nucleic acid sequence shown in SEQ ID NO: 6, or contains a nucleic acid sequence having 90% or more identity with SEQ ID NO:

6.

9. A bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage contains a nucleic acid sequence encoding at least one protein or active domain selected from the following (a) to (c): (a) Pyocin G; (b) PslGh; and (c) PelAh.

10. The bacteriophage according to claim 9, wherein the genome of the bacteriophage contains nucleic acid sequences encoding at least two proteins or active domains selected from the following (a) to (c): (a) Pyocin G; (b) PslGh; and (c) PelAh.

11. The bacteriophage according to claim 9, wherein the genome of the bacteriophage contains nucleic acid sequences encoding the proteins or active domains of the following (a) to (c): (a) Pyocin G; (b) PslGh; and (c) PelAh.

12. A pharmaceutical composition comprising the bacteriophage according to any one of claims 1 to 11 and a pharmaceutically acceptable excipient.

13. A pharmaceutical composition comprising at least two kinds of the bacteriophage according to any one of claims 1 to 11 and a pharmaceutically acceptable excipient.

14. A pharmaceutical composition comprising three kinds of the bacteriophage according to any one of claims 1 to 11 and a pharmaceutically acceptable excipient.

15. A pharmaceutical composition comprising four kinds of the bacteriophage according to any one of claims 1 to 11 and a pharmaceutically acceptable excipient.

16. A pharmaceutical composition comprising five kinds of the bacteriophage according to any one of claims 1 to 11 and a pharmaceutically acceptable excipient.

17. The pharmaceutical composition according to any one of claims 12 to 16, which is a pharmaceutical composition for preventing or treating Pseudomonas aeruginosa infection.

18. The pharmaceutical composition according to claim 17, wherein the Pseudomonas aeruginosa infection is a respiratory tract infection.

19. A bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage contains 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-04032, except for the part of the tail fiber gene, and contains the following nucleic acid sequences (A) and (B): (A) a nucleic acid sequence encoding a tail fiber derived from bacteriophage JG024, (B) a nucleic acid sequence encoding PslGh.

20. A bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage contains a nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-04032, except for the part of the tail fiber gene, and contains the following nucleic acid sequences (A) and (B): (A) the nucleic acid sequence shown in SEQ ID NO: 6, or a nucleic acid sequence having 90% or more identity with SEQ ID NO: 6, (B) the nucleic acid sequence shown in SEQ ID NO: 4, or a nucleic acid sequence having 90% or more identity with SEQ ID NO: 4 and encoding a protein having Psl-degrading activity.

21. A bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage contains a nucleic acid sequence of the genome of the bacteriophage specified by accession number NITE BP-04032, except for the part of the tail fiber gene, and contains the following nucleic acid sequences (A) and (B): (A) the nucleic acid sequence shown in SEQ ID NO: 6, (B) the nucleic acid sequence shown in SEQ ID NO: 10 or SEQ ID NO:

11.

22. A pharmaceutical composition comprising a bacteriophage and a pharmaceutically acceptable excipient, wherein the bacteriophage is one of the following bacteriophages (i) to (iv): (i) a bacteriophage identified by accession number NITE BP-04037 or receipt number NITE ABP-04201, or a subculture strain thereof; (ii) a bacteriophage identified by accession number NITE BP-04031 or a subculture strain thereof; (iii) a bacteriophage having lytic activity against Pseudomonas aeruginosa, wherein the genome of the bacteriophage contains the nucleic acid sequence of the genome of the bacteriophage identified by accession number NITE BP-04032 except for the part of the tail fiber gene, and contains the following nucleic acid sequences (A) and (B): (A) the nucleic acid sequence shown in SEQ ID NO: 6; (B) the nucleic acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 11, or a subculture strain thereof; (iv) a bacteriophage identified by accession number NITE BP-04040 or receipt number NITE ABP-04200, or a subculture strain thereof.

23. The pharmaceutical composition according to claim 22, which is a pharmaceutical composition for preventing or treating Pseudomonas aeruginosa infection.

24. The pharmaceutical composition according to claim 23, wherein the Pseudomonas aeruginosa infection is a respiratory tract infection.

25. A method for producing a bacteriophage having lytic activity against Pseudomonas aeruginosa, comprising the step of culturing in a host bacterium deficient in a prophage-derived gene.

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