WO2025143108A1 - Pseudomonas aeruginosa bacteriophage - Google Patents

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

The present invention relates to novel bacteriophages and modified bacteriophages that have lytic activity against Pseudomonas aeruginosa, as well as uses of these bacteriophages.

Pseudomonas aeruginosa is a type 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 mainly used to treat Pseudomonas aeruginosa infections. Pseudomonas aeruginosa produces an extracellular matrix containing polysaccharides and forms a biofilm. Pseudomonas aeruginosa that forms a biofilm is protected from phagocytosis by neutrophils and antibiotics, which can pose a therapeutic challenge (Drugs. 2021; 81 (18): 2117-2131).

Bacteriophages (hereafter also referred to as phages) are viruses that use bacteria as hosts. Phages infect certain bacteria, use the metabolic mechanisms 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).

It has been reported that it is possible to change the host range by creating a bacteriophage in which part of the tail fiber gene of a bacteriophage that infects Pseudomonas aeruginosa is exchanged with the tail fiber gene of another bacteriophage (WO 2016/055585).

Bacteriophages can be genetically modified using synthetic biology techniques. For example, specific genes can be deleted or inserted. The same is true for bacteriophages that infect Pseudomonas aeruginosa (Proceedings of the National Academy of Sciences of the United States of America. 2022;119(48):e2206739119).

PelA and PslG, glycoside hydrolases derived from P. aeruginosa, and their active domains PelAh and PslGh are known to inhibit and destroy the formation of P. aeruginosa biofilms. These degrade Pel and Psl, which are extracellular polysaccharides produced by P. aeruginosa, respectively (Science Advances. 2016; 2(5): e1501632). The degree of dependency of biofilms formed by P. aeruginosa strains on Pel and Psl varies from strain to strain, with some strains forming a matrix dominated by Pel, others forming a matrix dominated by Psl, and others with 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.

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 have antibacterial activity against Pseudomonas aeruginosa (Journal of molecular biology. 2020; 432 (13): 3869-3880).

The following are known examples of reports that change the activity of bacteriophages by inserting a specific gene into the genome of the bacteriophage. It has been reported that a genetically modified 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 by inserting an alginate-degrading enzyme (alginate lyase) into the genome of a bacteriophage that infects Pseudomonas aeruginosa, it is possible to break down 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 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.

In general, in bacteriophage therapy, cocktails containing multiple types of bacteriophages are sometimes used to expand the host range and prevent resistance to bacteriophages. 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).

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

Prophages that lysogenize bacteria can encode defensive genes to protect the host bacteria from infection by competing bacteriophages. Examples have been reported in which this has led to inhibition of the proliferation of competing bacteriophages (Nat. Commun. 2024; 15(1): 1644).

International Publication No. 2016/055585 International Publication No. 2023/015195 International Publication No. 2023/052628

Science Advances. 2016;2(5):e1501632 Journal 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;14:4337 Scientific Reports. 2023;13(1):8921 Journal of bacteriology. 2024;206(5):e0040223 Nat Commun. 2024;15(1):1644

The 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 a means and method, particularly a bacteriophage and a pharmaceutical composition, for preventing or treating infections caused by bacteria of the genus Pseudomonas, such as Pseudomonas aeruginosa infections.

　As a result of considerable inventive and creative investigations in the creation of bacteriophages, the inventors obtained novel bacteriophages and found that the bacteriophages had lytic activity against Pseudomonas aeruginosa (Examples 1, 8, 9, and 10). In addition, they created bacteriophages with an exchanged tail fiber gene and found that the bacteriophages had lytic activity against a wider range of Pseudomonas aeruginosa (Example 2). They created bacteriophages into which glycoside hydrolase had been introduced (Example 3), and also created bacteriophages into which bacteriocin had been introduced (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 decomposition activity (Examples 5, 6, and 12), and had lytic activity against Pseudomonas aeruginosa (Examples 7 to 9, 11, and 13). They 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.

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

[5] A bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of the bacteriophage (a) comprising a nucleic acid sequence having 90% or greater identity to 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) comprising 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.
[6] A bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of the bacteriophage comprising (a) 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) 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, according to [5].
[7] 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 Accession No. NITE BP-04032, excluding a portion of the tail fiber gene, and (b) contains 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, according to [5].
[8] The bacteriophage described in [5], wherein the tail fiber gene derived from another bacteriophage comprises 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.

[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 following proteins or active domains (a) to (c):
(a) Pyocin G;
(b) PslGh; and (c) PelAh.

[12] A pharmaceutical composition comprising the bacteriophage according to any one of [1] to [11] and a pharma- ceutical acceptable excipient.
[13] A pharmaceutical composition comprising at least two types of bacteriophage according to any one of [1] to [11] and a pharma- ceutical acceptable excipient.
[14] A pharmaceutical composition comprising three types of bacteriophages according to any one of [1] to [11] and a pharma- ceutical acceptable excipient.
[15] A pharmaceutical composition comprising four types of bacteriophages according to any one of [1] to [11] and a pharma- ceutical acceptable excipient.
[16] A pharmaceutical composition comprising five kinds of bacteriophages according to any one of [1] to [11] and a pharma- ceutical acceptable excipient.
[17] The pharmaceutical composition according to any one of [12] to [16], which is a pharmaceutical composition for preventing or treating a Pseudomonas aeruginosa infection.
[18] The pharmaceutical composition according to [17], wherein the Pseudomonas aeruginosa infection is a respiratory infection.

[19] A bacteriophage having lytic activity against Pseudomonas aeruginosa,
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 the accession number NITE BP-04032, except for a portion of the tail fiber gene, and
(A) a nucleic acid sequence encoding the tail fiber from bacteriophage JG024;
(B) A bacteriophage comprising a nucleic acid sequence encoding PslGh.
[20] A bacteriophage having lytic activity against Pseudomonas aeruginosa,
The genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04032, except for a portion of the tail fiber gene, and
(A) a nucleic acid sequence as set forth in SEQ ID NO:6, or a nucleic acid sequence having 90% or more identity to SEQ ID NO:6;
(B) A bacteriophage comprising 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,
The genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04032, except for a portion of the tail fiber gene, and
(A) a nucleic acid sequence as set forth in SEQ ID NO:6;
(B) A bacteriophage comprising the nucleic acid sequence shown in SEQ ID NO:10 or SEQ ID NO:11.

[22] A pharmaceutical composition comprising a bacteriophage and a pharma- ceutically acceptable excipient,
The bacteriophage is any one of the following bacteriophages (i) to (iv):
(i) a bacteriophage identified by accession number NITE BP-04037 or accession number NITE ABP-04201, or a passage strain thereof;
(ii) a bacteriophage identified by accession number NITE BP-04031 or a passage strain thereof;
(iii) a bacteriophage having lytic activity against Pseudomonas aeruginosa,
The genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04032, except for a portion of the tail fiber gene, and
(A) a nucleic acid sequence as set forth in SEQ ID NO:6;
(B) a bacteriophage, or a subculture thereof, comprising the nucleic acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 11;
(iv) A pharmaceutical composition comprising a bacteriophage identified by accession number NITE BP-04040 or accession number NITE ABP-04200, or a passage strain thereof.
[23] The pharmaceutical composition according to [22], which is a pharmaceutical composition for preventing or treating a Pseudomonas aeruginosa infection.
[24] The pharmaceutical composition according to [23], wherein the Pseudomonas aeruginosa infection is a respiratory infection.

[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 a prophage has been deleted.

The bacteriophage and modified bacteriophage, as well as the pharmaceutical composition of the present invention, exhibit lytic activity against Pseudomonas aeruginosa, and it is expected that the bacteriophage and modified bacteriophage, as well as the pharmaceutical composition of the present invention can be used to prevent or treat Pseudomonas aeruginosa infections, including pulmonary infections.

Photographs showing the lytic activity of natural bacteriophages, in which Pseudomonas aeruginosa ATCC15692, NBRC3080, or NBRC13746 strains were layered on an agar plate as hosts, and a dilution series of each bacteriophage was dropped onto the plate, showing images of lytic plaques that appeared. Photographs showing the bacteriolytic activity of PAi23-JG024 tail and PAi23 phages. Pseudomonas aeruginosa ATCC15692 strain, and clinically isolated 3-55-PA or 9-8-PA strains were layered on an agar plate as hosts, and serial dilutions of each bacteriophage were dropped onto the plate, resulting in images of lytic plaques that appeared. Photographs showing the bacteriolytic 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. Photographs showing the bacteriolytic activity of PAi23::PyoG(CD) phage, in which Pseudomonas aeruginosa ATCC15692 strain or ATCC15692 strain expressing ImG gene (ATCC15692-ImG) was layered on an agar plate as a host, and serial dilutions of each bacteriophage were dropped onto the plate, showing images of lytic plaques that appeared. 1 is a graph showing that φLCX, PAPT1 and PAi239 have activity to reduce biofilm amount. After forming a biofilm for 24 hours using Pseudomonas aeruginosa ATCC15692 strain, a medium containing each bacteriophage was contacted with the biofilm, and the biofilm amount was quantified by crystal violet staining after 6 hours. Error bars indicate standard error. (A) Graph showing that PAi23-JG024 tail::Plac-PslGh (AB) and PAi23-JG024 tail::Plac-PslG (AB) phages have activity to reduce biofilm amount. Pseudomonas aeruginosa ATCC15692 strain was cultured for 24 hours to form a biofilm, and the medium containing each bacteriophage was contacted for 6 hours, and the amount of biofilm in the medium was quantified using crystal violet staining. Error bars indicate standard error. (B) Graph showing that PAi23-JG024 tail::Plac-PelAh (AB) phages have activity to reduce biofilm amount. Respiratory clinical isolate 28-42-PA strain was cultured for 24 hours to form a biofilm, and the medium containing each bacteriophage was contacted for 6 hours, and the amount of biofilm in the medium was quantified using crystal violet staining (left). A graph showing the amount of biofilm converted to a relative value is also shown (right). Error bars indicate standard error. (C) A graph showing that PAi23-JG024 tail::Plac-PslGh(AB)_v3 and PAi23-JG024 tail::Plac-PslGh-PelAh(AB)_v3 phages have activity in reducing the amount of biofilm. After contacting a medium containing each bacteriophage with a biofilm formed by culturing Pseudomonas aeruginosa ATCC15692 strain for 24 hours for 6 hours, the amount of biofilm in the medium was quantified using crystal violet staining. Error bars indicate standard error. 1 is a graph showing the growth curve of Pseudomonas aeruginosa in liquid culture, which shows that the addition of PAi23 phage has an inhibitory effect on the growth of the respiratory clinical isolate 27-12-PA strain, and that the addition of PAi23::PyoG(CD) phage maintains the inhibitory effect on the growth of the respiratory clinical isolate 27-12-PA strain for a longer period of time than the addition of PAi23 phage. Error bars indicate standard error. 1 is a graph showing 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. A graph of the change in absorbance at 600 nm is shown. Error bars indicate standard deviation. This is a graph showing the bactericidal effect 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 administered intranasally. The number of live bacteria in the lungs (log CFU/lung) 24 hours after infection is shown (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 lytic plaques that appeared after Pseudomonas aeruginosa ATCC15692ΔPf4/Pf6 strain was layered on an agar plate as a host and a dilution series of each bacteriophage was dropped onto the plate. (B) Graphs showing that while φBrkr activity was largely lost before and after freeze-thawing, φBrkr_FTR2 acquired resistance to freeze-thawing. Error bars indicate standard error. 1 is a graph showing that cocktailing bacteriophages extends the growth inhibitory effect against Pseudomonas aeruginosa. The graph shows representative growth curves after infection of Pseudomonas aeruginosa ATCC 15692 with a single bacteriophage, a cocktail of two or four bacteriophages at an MOI of 0.0001. Error bars indicate standard error. 1 is a graph showing that the cocktail of bacteriophages has activity in reducing the amount of biofilm. The biofilm formed by culturing Pseudomonas aeruginosa ATCC15692 strain for 24 hours was contacted with a medium containing each cocktail of bacteriophages (cocktail A or B) for 6 hours, and then the amount of biofilm in the medium was quantified using crystal violet staining. Error bars indicate standard error. This is a graph showing the bactericidal effect of bacteriophage cocktails 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, each phage cocktail (cocktail A, B, or G) was administered. The number of viable bacteria in the lungs (log CFU/lung) approximately 22.5 hours after infection is shown. Error bars indicate standard error. (A) Images of plaques that appeared after a Pseudomonas aeruginosa ATCC15692 strain or a prophage-removed strain of the ATCC15692 strain, ATCC15692ΔPf4/Pf6 strain, was layered on an agar plate as a host, and a dilution series of PAi242 phage was dropped onto the plate. (B) A graph showing the titer values of bacteriophage solutions obtained approximately 6 hours after culturing each bacteriophage using the Pseudomonas aeruginosa ATCC15692 strain or a prophage-removed strain. Error bars indicate standard error.

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

The present invention will be described in detail below. The following embodiments are merely examples for the purpose of explaining the present invention, and are not intended to limit the present invention to these embodiments. The present invention can be implemented in various forms without departing from the gist of the invention.

1. Bacteriophage of the present invention
In one aspect, the present invention provides a novel bacteriophage (hereinafter, may be referred to as "the bacteriophage of the present invention"). The bacteriophage of the present invention is a bacteriophage having a lytic activity against bacteria belonging to the genus Pseudomonas, particularly against Pseudomonas aeruginosa.

The φLCX 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, Patent Microorganisms Depositary Center (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818), which is the international depositary authority under the provisions of the Budapest Treaty for the Deposit of Patent Microorganisms, and was deposited on December 7, 2023 (deposit number NITE BP-04040).

The PAPT1 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, Patent Microorganisms Depositary Center (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818), which is the international depositary authority under the provisions of the Budapest Treaty for the Deposit of Patent Microorganisms, and was deposited on December 7, 2023 (deposit number NITE BP-04031).

The PAi140 strain, which is the bacteriophage of the present invention, has been sent by the applicant for international deposit 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), which is the international depository authority under the provisions of the Budapest Treaty for the Deposit of Patent Microorganisms, and has been deposited on December 7, 2023 (deposit number NITE BP-04033).

The PAi242 strain, which is the bacteriophage of the present invention, has been sent by the applicant for international deposit 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), which is the international depositary authority under the provisions of the Budapest Treaty for the Deposit of Patent Microorganisms, and has been deposited on December 7, 2023 (Deposit No. NITE BP-04036).

The PAi228 strain, which is the bacteriophage of the present invention, has been sent by the applicant for international deposit 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), which is the international depositary authority under the provisions of the Budapest Treaty for the Deposit of Patent Microorganisms, and has been deposited on December 7, 2023 (deposit number NITE BP-04034).

The PAi239 strain, which is the bacteriophage of the present invention, has been 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 the international depositary authority under the provisions of the Budapest Treaty for the Deposit of Patent Microorganisms, and has been deposited on December 7, 2023 (Deposit No. NITE BP-04035).

The bacteriophage of the present invention, strain φLCX2, has been sent by the applicant for international deposit 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), which is the international depositary authority under the provisions of the Budapest Treaty for the Deposit of Patent Microorganisms, and was received on November 13, 2024 (received number NITE ABP-04200).

The bacteriophage of the present invention, φBrkr_FTR2 strain, was sent by the applicant for international deposit 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), which is the international depositary authority under the provisions of the Budapest Treaty for the Deposit of Patent Microorganisms, and was received on November 13, 2024 (received number NITE ABP-04201).

In one embodiment, the bacteriophage of the invention includes the following bacteriophage:
(1) A bacteriophage having lytic activity against a bacterium belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of which 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 BP-04040;
(2) A bacteriophage having lytic activity against a bacterium belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of which 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 BP-04031;
(3) A bacteriophage having lytic activity against a bacterium belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of which 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 BP-04033;
(4) A bacteriophage having lytic activity against a bacterium 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 BP-04036;
(5) A bacteriophage having lytic activity against a bacterium belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of which 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 BP-04034;
(6) A bacteriophage having lytic activity against a bacterium 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 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 specified 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 specified by the accession number NITE ABP-04201.

As used herein, "bacteriophage" or "phage" refers to a phage particle that contains a genome packaged within an envelope or capsid, and includes the entire phage particle, a portion of a phage having an equivalent function (e.g., the head of a phage), and an aggregate of phage components. In particular, as an isolated phage, it refers to a phage that has been separated from its natural environment and grown, purified, or cultured.

As used herein, "identity" refers to the identity of a base sequence or an amino acid sequence determined by a technique known in the art. As a method for aligning sequences, generally 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. As a specific sequence alignment method, for example, the value "Identity" obtained by searching with the NEEDLE program (Journal of Molecular Biology. 1970; 48(3): 443-53) using parameters provided as defaults is meant. The parameters are as follows.
Gap penalty=10
Extend penalty=0.5
Matrix=EBLOSUM62

In this specification, "bacteria belonging to the genus Pseudomonas" (also referred to as Pseudomonas bacteria in this specification) are bacteria classified as gram-negative aerobic bacilli, and are known in the art. In one embodiment, the 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. It is known that Pseudomonas bacteria form biofilms by secreting exopolysaccharides such as alginic acid, which protect them from external enemies and physical stress.

As used herein, "having lytic activity against bacteria belonging to the genus Pseudomonas" or "having lytic activity against Pseudomonas aeruginosa" means showing lytic plaques against at least one strain of bacteria belonging to the genus Pseudomonas or at least one strain of bacteria belonging to Pseudomonas aeruginosa, respectively. Confirmation of lytic activity against these bacteria can be performed using methods and means known in the art. For example, by using the method described in Example 1 of the present specification, it can be confirmed whether or not a bacteriophage has lytic activity by confirming whether or not a bacteriophage of approximately 10 ¹⁰ PFU (plaque forming units)/mL to 10 ⁵ PFU/mL (or a mixture of multiple bacteriophages) shows lytic plaques against Pseudomonas aeruginosa or bacteria belonging to the genus Pseudomonas.

In one embodiment, the bacteriophage of the present invention is resistant to freeze-thawing. For example, the bacteriophage of the present invention includes the following bacteriophages:
A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of the bacteriophage containing a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of a bacteriophage identified by the accession number NITE ABP-04201, and the bacteriophage having resistance to freezing and thawing.

In this specification, "resistant to freezing and thawing" means that the bacteriophage is maintained to a desired degree even after the freezing and thawing treatment, although it is known that some of the bacteriophage are killed or damaged by the freezing and thawing treatment. For example, the bacteriophage after the freezing and thawing treatment maintains approximately the same number as before the freezing and thawing treatment, or maintains 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 number before the freezing and thawing treatment. Whether or not the bacteriophage is resistant to freezing and thawing can be confirmed using methods and means known in the art. For example, whether or not the bacteriophage is resistant to freezing and thawing can be confirmed by confirming whether or not the bacteriophage shows a decrease in number before and after the freezing and thawing treatment using the method described in Example 10 of this specification. Alternatively, resistance to freeze-thawing can be confirmed by checking whether the bacteriophage activity (e.g., lytic activity against Pseudomonas aeruginosa) is maintained after the freeze-thawing treatment. For example, if the bacteriophage after the freeze-thawing treatment maintains approximately the same activity as the activity before the freeze-thawing treatment, or maintains 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-thawing treatment, the bacteriophage can be said to be resistant to freeze-thawing.

In one embodiment, the bacteriophage of the invention includes the following bacteriophage:
(1) A bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of the bacteriophage comprising a nucleic acid sequence of the genome of the bacteriophage identified 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 of the genome of the bacteriophage identified by the 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 the 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 the 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 the 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 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 of the genome of a bacteriophage identified by 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 of the genome of a bacteriophage identified by accession number NITE ABP-04201.

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

The bacteriophages of the present invention also include the following bacteriophages:
(1) A bacteriophage identified by the accession number NITE BP-04040, or a passage strain thereof;
(2) A bacteriophage identified by the accession number NITE BP-04031, or a passage strain thereof;
(3) A bacteriophage identified by the accession number NITE BP-04033, or a passage strain thereof;
(4) A bacteriophage identified by the 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 the accession number NITE BP-04035, or a passage strain thereof;
(7) A bacteriophage identified by accession number NITE ABP-04200, or a subcultured strain thereof; and (8) a bacteriophage identified by accession number NITE ABP-04201, or a subcultured strain thereof.

The bacteriophages of the present invention also include 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 (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 the 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 (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 the 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 includes 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 the 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 includes 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 the 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 includes 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 the 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 includes 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 the accession number NITE BP-04035;
(7) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, such as Pseudomonas aeruginosa, wherein the genome of the bacteriophage includes 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 the accession number NITE ABP-04200; and (8) a bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, such as Pseudomonas aeruginosa, wherein the genome of the bacteriophage includes a nucleic acid sequence in which 1 to 5,000 (e.g., 1 to 1,000, 1 to 500, or 1 to 100) bases have been deleted, substituted, inserted or added, or a combination thereof has been modified; A bacteriophage comprising a nucleic acid sequence in which 1 to 5,000 (e.g., 1 to 1,000, 1 to 500, or 1 to 100) bases have been deleted, substituted, inserted, or added, or a combination thereof has been modified in the nucleic acid sequence of the genome of the bacteriophage identified in ABP-04201.

　Regarding the above 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.

During the process of subculture, production and/or replication of a bacteriophage, a bacteriophage mutant may be generated 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 bacteriophage of the present invention, so long as they have lytic activity against bacteria belonging to the genus Pseudomonas, such as Pseudomonas aeruginosa.

The bacteriophage of the present invention also includes passaged strains of the bacteriophage identified by the above accession number or accession number, so long as they have lytic activity against bacteria belonging to the genus Pseudomonas, such as Pseudomonas aeruginosa.

In this specification, the term "passaged strain" refers to a bacteriophage obtained by subculturing a distributed bacteriophage.

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"). Thus, the present invention also includes:
(1) A polynucleotide of a bacteriophage genome comprising a nucleic acid sequence having 90% or more identity to the nucleic acid sequence of the genome of a bacteriophage identified by the accession number NITE BP-04040;
(2) A polynucleotide of a bacteriophage genome comprising a nucleic acid sequence having 90% or more identity to the nucleic acid sequence of the genome of the bacteriophage identified by the 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 by the 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 by the 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 the 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 a bacteriophage identified by the accession number NITE BP-04035;
(7) A polynucleotide of a bacteriophage genome, comprising a nucleic acid sequence having 90% or more identity to the nucleic acid sequence of the genome of a bacteriophage specified by accession number NITE ABP-04200; and (8) a polynucleotide of a bacteriophage genome, comprising a nucleic acid sequence having 90% or more identity to the nucleic acid sequence of the genome of a bacteriophage specified 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, for example, Pseudomonas aeruginosa.

In one embodiment, a genome of the invention includes:
(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 the 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 the 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 the accession number NITE BP-04033;
(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 to the nucleic acid sequence of the genome of the bacteriophage identified by the accession number 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 to the nucleic acid sequence of the genome of the bacteriophage identified by the accession number 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 to the nucleic acid sequence of the genome of the bacteriophage identified by the 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 to the nucleic acid sequence of the genome of a bacteriophage specified 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 specified by accession number NITE ABP-04201.

In one embodiment, the genome of the invention includes the following genome:
(1) A genome comprising the nucleic acid sequence of the genome of a 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 the accession number NITE BP-04033;
(4) A genome comprising the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04036;
(5) A genome comprising the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04034;
(6) A genome comprising the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04035;
(7) A genome comprising the nucleic acid sequence of the genome of a bacteriophage identified under Accession No. NITE ABP-04200; and (8) A genome comprising the nucleic acid sequence of the genome of a bacteriophage identified under Accession No. NITE ABP-04201.

Genomes of the present invention include the following genomes:
(1) A genome consisting of the nucleic acid sequence of the genome of a 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 the accession number NITE BP-04031;
(3) A genome consisting of the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04033;
(4) A genome consisting of the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04036;
(5) A genome consisting of the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04034;
(6) A genome consisting of the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04035;
(7) 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.

The genome 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 genome of the present invention can be produced by linking multiple polynucleotides containing partial base sequences of the genome of the present invention using genetic engineering techniques. In one embodiment, the genome of the present invention, either in full length or in partial sequence, may be contained in a vector known in the art.

The bacteriophage of the present invention can be obtained by making a request to the above-mentioned depository center.

The bacteriophage of the present invention can be produced by deciphering the nucleic acid sequence of the genome of the bacteriophage provided by a general technique known in the art, and based on the sequence information. For example, the genome of the present invention produced by the above method is introduced into a host bacterium by electroporation (for example, Pseudomonas aeruginosa is used as the host bacterium to obtain a bacteriophage that exhibits lytic activity against Pseudomonas aeruginosa). The bacteria into which the genome has been introduced are then added to a plate overlaid with soft agar and cultured. Thereafter, a single lytic plaque is obtained by a plaque assay. The single lytic plaque is added to a host bacterium culture liquid and cultured, and the culture supernatant obtained by standing or centrifuging is filtered, whereby the bacteriophage of the present invention can be produced.

The bacteriophage of the present invention can be prepared by general culture, isolation and purification methods known in the art. For example, a host bacterium (such as a bacterium of the genus Pseudomonas, e.g., Pseudomonas aeruginosa) is cultured in advance, and the bacteriophage of the present invention is infected into the host bacterium 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 appropriately selected 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.

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

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

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.

For example, the modified bacteriophage of the present invention is a modified bacteriophage of any one of the following bacteriophages (1) to (12):
(1) A bacteriophage having lytic activity against a bacterium belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of which 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 BP-04040;
(2) A bacteriophage having lytic activity against a bacterium belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of which 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 BP-04031;
(3) A bacteriophage having lytic activity against a bacterium belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of which 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 BP-04033;
(4) A bacteriophage having lytic activity against a bacterium 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 BP-04036;
(5) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, 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 the accession number NITE BP-04034; and (6) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, 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 the accession number NITE BP-04035;
(7) A bacteriophage having lytic activity against a bacterium 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 BP-04032;
(8) A bacteriophage having lytic activity against a bacterium 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 BP-04037;
(9) A bacteriophage having lytic activity against a bacterium 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 BP-04039;
(10) A bacteriophage having lytic activity against a bacterium 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 BP-04038;
(11) A bacteriophage having lytic activity against a bacterium 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-04200;
(12) A bacteriophage having lytic activity against a bacterium 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.

The bacteriophage PAi23 strain was sent by the applicant for international deposit 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), which is the international depository authority under the provisions of the Budapest Treaty for the Deposit of Patent Microorganisms, and was deposited on December 7, 2023 (deposit number NITE BP-04032).

The bacteriophage φBrkr 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 the international depositary authority under the provisions of the Budapest Treaty for the Deposit of Patent Microorganisms, and was deposited on December 7, 2023 (deposit number NITE BP-04037).

The bacteriophage φ30-1 strain was sent by the applicant for international deposit 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), which is the international depositary authority under the provisions of the Budapest Treaty for the Deposit of Patent Microorganisms, and was deposited on December 7, 2023 (deposit number NITE BP-04039).

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 the international depositary authority under the provisions of the Budapest Treaty for the Deposit of Patent Microorganisms, and was deposited on December 7, 2023 (deposit number NITE BP-04038).

In one embodiment, the engineered bacteriophage of the present invention is a bacteriophage obtained by engineering any one of the following bacteriophages (1) to (12):
(1) A bacteriophage having lytic activity against a bacterium belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage contains the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04040;
(2) A bacteriophage having lytic activity against a bacterium belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage contains the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04031;
(3) A bacteriophage having lytic activity against a bacterium belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage contains the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04033;
(4) A bacteriophage having lytic activity against a bacterium belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage contains the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04036;
(5) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of which comprises the nucleic acid sequence of the genome of the bacteriophage specified by the accession number NITE BP-04034; and (6) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of which comprises 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 a bacterium belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage contains the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04032;
(8) A bacteriophage having lytic activity against a bacterium belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage contains the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04037;
(9) A bacteriophage having lytic activity against a bacterium belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage contains the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04039;
(10) A bacteriophage having lytic activity against a bacterium belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage contains the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04038;
(11) A bacteriophage having lytic activity against a bacterium belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage contains 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 a bacterium belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, wherein the genome of the bacteriophage contains a nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE ABP-04201.

In one embodiment, the engineered bacteriophage of the present invention is a bacteriophage obtained by engineering any one of the following bacteriophages (1) to (12):
(1) A bacteriophage, the genome of which consists of the nucleic acid sequence of the genome of a bacteriophage identified by the accession number NITE BP-04040;
(2) A bacteriophage, the genome of which consists of the nucleic acid sequence of the genome of a bacteriophage identified by the accession number NITE BP-04031;
(3) A bacteriophage, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04033;
(4) A bacteriophage, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04036;
(5) A bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04034; and (6) A bacteriophage whose genome consists of the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04035;
(7) A bacteriophage, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04032;
(8) A bacteriophage, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04037;
(9) A bacteriophage, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04039;
(10) A bacteriophage, the genome of which consists of the nucleic acid sequence of the genome of a bacteriophage identified by the accession number NITE BP-04038;
(11) A bacteriophage, the genome of which consists of the nucleic acid sequence of the genome of a bacteriophage identified by the accession number NITE ABP-04200;
(12) A bacteriophage, the genome of which consists of the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE ABP-04201.

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

In one embodiment, the engineered bacteriophage of the present invention is a bacteriophage obtained by engineering any one of the following bacteriophages (1) to (12):
(1) A bacteriophage identified by the accession number NITE BP-04040 or a subcultured strain thereof;
(2) A bacteriophage identified by the accession number NITE BP-04031 or a subcultured strain thereof;
(3) A bacteriophage identified by the accession number NITE BP-04033 or a subcultured strain thereof;
(4) A bacteriophage identified by the accession number NITE BP-04036 or a subcultured strain thereof;
(5) A bacteriophage identified by the accession number NITE BP-04034 or a subcultured strain thereof;
(6) A bacteriophage identified by the 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 the accession number NITE BP-04037 or a passage strain thereof;
(9) A bacteriophage identified by the accession number NITE BP-04039 or a passage strain thereof;
(10) A bacteriophage identified by the 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 subcultured strain thereof.

Among the bacteriophages to be modified, those identified by their accession number or receipt number can be obtained by submitting a request to the above-mentioned depository center.

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

[1] Modification of the 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.

The tail fiber gene and tail fiber protein of a bacteriophage are related to the host range (host spectrum) and host specificity of the host it infects (WO 2016/055585, WO 2017/174810). Host range or host specificity refers to the type 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 the other bacteriophage can be acquired. The host range or host specificity can be determined by examining the lytic activity of the bacteriophage against a specific species or strain of Pseudomonas bacteria (e.g., Pseudomonas aeruginosa) to determine whether the bacteriophage has a host range or host specificity for that specific bacterial species or strain.

In one embodiment, the modified bacteriophage of the present invention is a bacteriophage having 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 having lytic activity against Pseudomonas aeruginosa.

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. For example, other bacteriophages include JG024, Phi33, PTP92, PTP47, LBL3, SPM-1, F8, PB1, KPP12, LMA2, SN, 14-1, NH-4, PTP93, PTP47, and C36.

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 is registered as NCBI Reference Sequence NC_017674.1 and set forth as SEQ ID NO:6. In addition, a person skilled in the art can screen for a tail fiber gene associated with a desired host range or host specificity by isolating all or a portion of a tail fiber gene from a bacteriophage having lytic activity against Pseudomonas aeruginosa, exchanging it with a tail fiber gene in a bacteriophage to be modified, and confirming the lytic activity of the resulting bacteriophage against Pseudomonas aeruginosa.

The tail fiber gene may be replaced by a complete or partial replacement (hybrid gene), or may be replaced with a gene that combines portions of multiple tail fiber genes derived 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, the C-terminal receptor binding region is thought to be associated with host range or host specificity, so it is preferable to replace at least this region.

　The tail fiber gene can be replaced by any recombinant gene method known in the art, and the method is not particularly limited. For example, the tail fiber gene to be replaced and a sequence for homologous recombination can be inserted into a vector or plasmid suitable for a Pseudomonas aeruginosa host, and then introduced into a Pseudomonas aeruginosa host together with the bacteriophage genome to be modified, and homologous recombination can be performed to replace the tail fiber gene in the bacteriophage. Alternatively, a modified bacteriophage can be prepared by preparing a genome of the bacteriophage to be modified that contains a nucleic acid sequence in which the original tail fiber gene is replaced with the tail fiber gene to be replaced, and introducing the resulting bacteriophage genome into a Pseudomonas aeruginosa host.

In one embodiment, an engineered bacteriophage of the invention includes:
(1) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, 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 the bacteriophage identified by the 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 that comprises 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, the genome of which (a) comprises a nucleic acid sequence having 90% or more identity with the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04031, except for 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;
(3) A bacteriophage having lytic activity against a bacterium belonging to the genus Pseudomonas, for example, 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 the bacteriophage identified by Accession No. NITE BP-04033, except for 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;
(4) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, 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 the bacteriophage identified by the accession number NITE BP-04036, except for 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;
(5) A bacteriophage having lytic activity against a bacterium belonging to the genus Pseudomonas, for example, 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 the bacteriophage identified by Accession No. NITE BP-04034, except for 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;
(6) A bacteriophage having lytic activity against a bacterium belonging to the genus Pseudomonas, for example, 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 the bacteriophage identified by the accession number NITE BP-04035, except for 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;
(7) A bacteriophage having lytic activity against a bacterium belonging to the genus Pseudomonas, for example, 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 the bacteriophage identified by the accession number NITE BP-04032, except for 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;
(8) A bacteriophage having lytic activity against a bacterium belonging to the genus Pseudomonas, for example, 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 the bacteriophage identified by the accession number NITE BP-04037, except for 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 a bacterium belonging to the genus Pseudomonas, for example, 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 the bacteriophage identified by Accession No. NITE BP-04039, except for 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;
(10) A bacteriophage having lytic activity against a bacterium belonging to the genus Pseudomonas, for example, 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 the bacteriophage identified by the accession number NITE BP-04038, except for 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;
(11) A bacteriophage having lytic activity against bacteria belonging to the genus Pseudomonas, for example, 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 the bacteriophage identified by Accession Number NITE ABP-04200, except for 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;
(12) A bacteriophage having lytic activity against a bacterium belonging to the genus Pseudomonas, for example, 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 identified by Accession Number NITE ABP-04201, except for 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.

In one embodiment, an engineered bacteriophage of the invention includes:
(1) 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 the bacteriophage identified by the accession number NITE BP-04040, excluding a portion of the tail fiber gene, and (b) comprises the 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;
(2) 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 the bacteriophage identified by the accession number NITE BP-04031, excluding a portion of the tail fiber gene, and (b) comprises the 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;
(3) A bacteriophage having lytic activity against a bacterium belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of which (a) comprises the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04033, excluding a portion of the tail fiber gene, and (b) comprises the 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;
(4) A bacteriophage having lytic activity against a bacterium belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of which (a) comprises the nucleic acid sequence of the genome of the bacteriophage identified by the 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 comprises 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 a bacterium belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of which (a) comprises the nucleic acid sequence of the genome of the bacteriophage identified by the 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 that comprises 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 a bacterium belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of which (a) comprises the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04035, excluding a portion of the tail fiber gene, and (b) comprises the 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;
(7) A bacteriophage having lytic activity against a bacterium belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of which (a) comprises the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04032, excluding a portion of the tail fiber gene, and (b) comprises the 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;
(8) A bacteriophage having lytic activity against a bacterium belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of which (a) comprises the nucleic acid sequence of the genome of the bacteriophage identified by the 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 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 a bacterium belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of which (a) comprises the nucleic acid sequence of the genome of the bacteriophage identified by the 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 that comprises 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 a bacterium belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of which (a) comprises the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04038, excluding a portion of the tail fiber gene, and (b) comprises the 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;
(11) A bacteriophage having lytic activity against a bacterium 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 identified 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 comprises 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 a bacterium 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 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 comprises at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa.

In one embodiment, an engineered bacteriophage of the invention includes:
(1) 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 the bacteriophage identified by the accession number NITE BP-04040, excluding a portion of the tail fiber gene, and (b) contains 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;
(2) 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 the bacteriophage identified by the accession number NITE BP-04031, except for a portion of the tail fiber gene, and (b) contains 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;
(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 the bacteriophage identified by the accession number NITE BP-04033, except for a portion of the tail fiber gene, and (b) contains 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, the genome of which (a) consists of the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04036, except for a portion of the tail fiber gene, and (b) contains 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, the genome of which (a) consists of the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04034, except for a portion of the tail fiber gene, and (b) contains 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;
(6) A bacteriophage having lytic activity against a bacterium belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of which (a) consists of the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04035, except for a portion of the tail fiber gene, and (b) contains 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;
(7) A bacteriophage having lytic activity against a bacterium belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of which (a) consists of the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04032, except for a portion of the tail fiber gene, and (b) contains 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 a bacterium belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of which (a) consists of the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04037, except for a portion of the tail fiber gene, and (b) contains 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 a bacterium belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of which (a) consists of the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04039, except for a portion of the tail fiber gene, and (b) contains 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;
(10) A bacteriophage having lytic activity against a bacterium belonging to the genus Pseudomonas, for example, Pseudomonas aeruginosa, the genome of which (a) consists of the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04038, except for a portion of the tail fiber gene, and (b) contains 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;
(11) A bacteriophage having lytic activity against a bacterium 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 identified by Accession Number NITE ABP-04200, except for a portion of the tail fiber gene, and (b) contains 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 a bacterium 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 identified by Accession Number NITE ABP-04201, excluding a portion of the tail fiber gene, and (b) contains 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.

In one embodiment, the modified bacteriophage of the invention comprises a nucleic acid sequence of a tail fiber gene from another bacteriophage that has lytic activity against Pseudomonas aeruginosa. In one embodiment, the modified 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 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 SEQ ID NO:6. In one embodiment, the modified bacteriophage of the invention comprises the nucleic acid sequence set forth in SEQ ID NO:6.

In one embodiment, the engineered bacteriophage of the invention is the following bacteriophage:
A bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of the bacteriophage (a) consisting of the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04032, excluding a portion of the tail fiber gene, and (b) comprising a nucleic acid sequence encoding a tail fiber derived from bacteriophage JG024.

In one embodiment, the engineered bacteriophage of the invention includes the following bacteriophage:
A bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of the bacteriophage (a) consisting of the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04032, except for a portion of the tail fiber gene, and (b) comprising the nucleic acid sequence shown in SEQ ID NO:6.

[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.

Glycoside hydrolases derived from Pseudomonas aeruginosa are proteins that inhibit and destroy the formation of biofilms of Pseudomonas aeruginosa (Science Advances. 2016; 2(5): e1501632). By modifying the bacteriophage of the present invention to introduce glycoside hydrolases, the ability to degrade Pseudomonas aeruginosa biofilms is imparted, the bacteriophage's lytic activity is enhanced, and when used in combination with antibiotics, the effectiveness of the antibiotics is expected to be increased. Examples of such glycoside hydrolases include PelA and PslG, which degrade Pel (a polysaccharide composed of N-acetylgalactosamine and N-acetylglucosamine) and Psl (a polysaccharide composed of pentasaccharide repeating units of d-mannose, D-glucose, and L-rhamnose), which are extracellular polysaccharides that constitute biofilms, and their active domains PelAh and PslGh. In this specification, 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. In addition, in this specification, 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.

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

In one embodiment, the modified bacteriophage of the present invention is a bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of the bacteriophage comprising a nucleic acid sequence encoding PelA or an active domain thereof. In one embodiment, the modified bacteriophage of the present invention is a bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of the bacteriophage comprising a nucleic acid sequence encoding PelA or an active domain thereof (e.g., PelAh) derived from Pseudomonas aeruginosa. In one embodiment, the modified bacteriophage of the present invention is a bacteriophage having lytic activity against Pseudomonas aeruginosa, and 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 shown 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.

In one embodiment, the modified bacteriophage of the present invention is a bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of the bacteriophage comprising a nucleic acid sequence encoding PslG or an active domain thereof. In one embodiment, the modified bacteriophage of the present invention is a bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of the bacteriophage comprising a nucleic acid sequence encoding PslG or an active domain thereof (e.g., PslGh) derived from Pseudomonas aeruginosa. In one embodiment, the modified bacteriophage of the present invention is a bacteriophage having lytic activity against Pseudomonas aeruginosa, and 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 shown in SEQ ID NO: 2 or 4, 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: 2 or 4 and encoding a protein having Psl degrading activity.

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 and introducing a bacteriocin, the bactericidal activity against Pseudomonas aeruginosa is enhanced.

PyoG is known in the art. As an 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.

In one embodiment, the modified bacteriophage of the present invention is a bacteriophage having lytic activity against Pseudomonas aeruginosa, and the genome of the bacteriophage includes a nucleic acid sequence encoding Pyocin G. In one embodiment, the modified bacteriophage of the present invention is a bacteriophage having lytic activity against Pseudomonas aeruginosa, and the genome of the bacteriophage includes a nucleic acid sequence encoding Pyocin G derived from Pseudomonas aeruginosa. In one embodiment, the modified bacteriophage of the present invention is a bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of the bacteriophage comprising a nucleic acid sequence encoding Pyocin G derived from Pseudomonas aeruginosa, wherein the nucleic acid sequence encoding Pyocin G comprises (a) the nucleic acid sequence shown in SEQ ID NO: 5, or (b) 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.

The nucleic acid sequence encoding the glycoside hydrolase and/or the bacteriocin is introduced under the control of an appropriate control sequence so that it is expressed as a protein after being introduced into the bacteriophage. For example, the nucleic acid sequence encoding the glycoside hydrolase and/or the bacteriocin is functionally 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, the Escherichia coli lac promoter, etc.).

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

Introduction of glycoside hydrolases and/or bacteriocins into bacteriophages can be performed by genetic recombination methods known in the art, and the method is not particularly limited. For example, a sequence for homologous recombination with a nucleic acid sequence encoding a glycoside hydrolase and/or bacteriocin to be introduced 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, and homologous recombination can be performed to introduce the nucleic acid sequence encoding glycoside hydrolase and/or bacteriocin into the bacteriophage. Alternatively, a genome in which a nucleic acid sequence encoding a glycoside hydrolase and/or bacteriocin is inserted into the genome of the bacteriophage to be modified is prepared, and the genome of the resulting bacteriophage can be introduced into a Pseudomonas aeruginosa host to prepare a modified bacteriophage.

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

In one embodiment, the engineered bacteriophage of the present invention is a bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of the 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.

In one embodiment, the engineered bacteriophage of the present invention is a bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of the bacteriophage comprising a nucleic acid sequence 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.

In one embodiment, the engineered bacteriophage of the present invention is a bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of the bacteriophage comprising 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.

In one embodiment, the engineered bacteriophage of the present invention is a bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of the bacteriophage comprising 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.

In one embodiment, the modified bacteriophage of the present invention is a bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of the bacteriophage comprising at least one nucleic acid sequence selected from the following (a) to (c):
(a) a nucleic acid sequence as 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 as 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 as 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.

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

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

　Regarding the above modifications such as deletion, substitution, insertion or addition of bases, multiple modifications may be consecutive, or multiple modifications may be present at different positions.

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

In one embodiment, an engineered bacteriophage of the invention includes:
A bacteriophage having lytic activity against Pseudomonas aeruginosa,
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 the accession number NITE BP-04032, except for a portion of the tail fiber gene, and
(A) a nucleic acid sequence of a tail fiber gene comprising at least a portion of the tail fiber gene from bacteriophage JG024; and/or (B) the following:
(a) Pyocin G;
(b) PslG or PslGh; and (c) PelA or PelAh
A bacteriophage comprising a nucleic acid sequence encoding at least one protein or active domain selected from the group consisting of:

In one embodiment, an engineered bacteriophage of the invention includes:
A bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of the bacteriophage comprising a nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE BP-04032, and comprising 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.

In one embodiment, an engineered bacteriophage of the invention includes:
A bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of the bacteriophage comprising a nucleic acid sequence of a genome of a bacteriophage identified by accession number NITE BP-04032, and comprising at least one nucleic acid sequence selected from the following (a) to (c):
(a) a nucleic acid sequence as 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 as 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 as 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.

In one embodiment, an engineered bacteriophage of the invention includes:
A bacteriophage having lytic activity against Pseudomonas aeruginosa,
The genome of the bacteriophage comprises a nucleic acid sequence having 90% or more identity to the genome of the bacteriophage identified by the accession number NITE BP-04032, except for a portion of the tail fiber gene, and
(A) a nucleic acid sequence encoding the tail fiber 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
bacteriophage, including

In one embodiment, an engineered bacteriophage of the invention includes:
A bacteriophage having lytic activity against Pseudomonas aeruginosa,
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 the accession number NITE BP-04032, except for a portion of the tail fiber gene, and
(A) a nucleic acid sequence encoding the tail fiber from bacteriophage JG024;
(B) A bacteriophage comprising a nucleic acid sequence encoding PslGh.

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

In one embodiment, an engineered bacteriophage of the invention includes:
A bacteriophage having lytic activity against Pseudomonas aeruginosa,
The genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04032, except for a portion of the tail fiber gene, and
(A) a nucleic acid sequence encoding the tail fiber 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
bacteriophage, including

In one embodiment, an engineered bacteriophage of the invention includes:
A bacteriophage having lytic activity against Pseudomonas aeruginosa,
The genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04032, except for a portion of the tail fiber gene, and
(A) a nucleic acid sequence encoding the tail fiber from bacteriophage JG024;
(B) at least one nucleic acid sequence selected from the following (a) to (c):
(a) a nucleic acid sequence as 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 as 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 as 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.

In one embodiment, an engineered bacteriophage of the invention includes:
A bacteriophage having lytic activity against Pseudomonas aeruginosa,
The genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04032, except for a portion of the tail fiber gene, and
(A) a 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) at least one nucleic acid sequence selected from the following (a) to (c):
(a) a nucleic acid sequence as set forth in SEQ ID NO:5, or a nucleic acid sequence having 90% or greater identity to SEQ ID NO:5 and encoding a protein having bacteriocin activity;
(b) a nucleic acid sequence as 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 nucleic acid sequence as set forth 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.

In one embodiment, an engineered bacteriophage of the invention includes:
A bacteriophage having lytic activity against Pseudomonas aeruginosa,
The genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04032, except for a portion of the tail fiber gene, and
(A) a 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) A bacteriophage comprising 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.

In one embodiment, an engineered bacteriophage of the invention includes:
A bacteriophage having lytic activity against Pseudomonas aeruginosa,
The genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04032, except for a portion of the tail fiber gene, and
(A) a nucleic acid sequence as set forth in SEQ ID NO:6, or a nucleic acid sequence having 90% or more identity to SEQ ID NO:6;
(B) a bacteriophage comprising a 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, and a nucleic acid sequence shown 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.

In one embodiment, an engineered bacteriophage of the invention includes:
A bacteriophage having lytic activity against Pseudomonas aeruginosa,
The genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04032, except for a portion of the tail fiber gene, and
(A) a nucleic acid sequence as set forth in SEQ ID NO:6;
(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;
(b) a nucleic acid sequence as set forth in SEQ ID NO:2 or 4; and (c) a bacteriophage comprising the nucleic acid sequence as set forth in SEQ ID NO:1 or 3.

In one embodiment, an engineered bacteriophage of the invention includes:
A bacteriophage having lytic activity against Pseudomonas aeruginosa,
The genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04032, except for a portion of the tail fiber gene, and
(A) a nucleic acid sequence as set forth in SEQ ID NO:6;
(B) A bacteriophage comprising the nucleic acid sequence shown in SEQ ID NO:4, SEQ ID NO:10 or SEQ ID NO:11.

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, and are therefore expected to be effective in treating or preventing infectious diseases.

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

The pharmaceutical composition of the present invention includes a pharmaceutical composition comprising at least one bacteriophage, including a bacteriophage of the present invention and/or a modified bacteriophage of the present invention, and a pharma- ceutical acceptable excipient.

The pharmaceutical composition of the present invention may further contain one or more strains of bacteriophage known to have lytic activity against Pseudomonas bacteria, such as Pseudomonas aeruginosa. 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.

The pharmaceutical compositions of the present invention can be prepared by a commonly used method using excipients commonly used in the field, i.e., pharmaceutical excipients and pharmaceutical carriers. Examples of dosage forms of these pharmaceutical compositions include parenteral preparations such as injections, drip infusions, powder inhalants, and nebulizers, and can be administered intravenously or via the lungs. In formulating the compositions, excipients, carriers, or additives appropriate for these dosage forms can be used within a pharmaceutical acceptable range. For example, the pharmaceutical compositions of the present invention can be produced by mixing the bacteriophage with a pharmaceutical acceptable excipient, or by suspending the bacteriophage in a pharmaceutical acceptable excipient.

The pharmaceutical composition of the present invention includes a pharmaceutical composition that contains at least two types of bacteriophages and a pharma- ceutical acceptable excipient.

The pharmaceutical composition of the present invention includes a pharmaceutical composition that contains three types of bacteriophages and a pharma- ceutical acceptable excipient.

The pharmaceutical composition of the present invention includes a pharmaceutical composition that contains four types of bacteriophages and a pharma- ceutical acceptable excipient.

The pharmaceutical composition of the present invention includes a pharmaceutical composition that contains five types of bacteriophages and a pharma- ceutical acceptable excipient.

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

Pili-recognition 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 their passage strains.

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 contains a nucleic acid sequence having the genome of the bacteriophage identified by accession number NITE BP-04032, except for the tail fiber gene portion, and which contains 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; a bacteriophage identified by accession number NITE BP-04033, and passage strains thereof.

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

In one embodiment, the pharmaceutical composition of the invention includes a pharmaceutical composition comprising a bacteriophage of (i) and (ii) below and a pharmaceutically acceptable excipient:
(i) a bacteriophage identified by accession number NITE BP-04037 or a passage strain thereof;
(ii) a bacteriophage having lytic activity against Pseudomonas aeruginosa,
The genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04032, except for a portion of the tail fiber gene, and
(A) a nucleic acid sequence as set forth in SEQ ID NO:6;
(B) A bacteriophage, or a subsequence thereof, comprising the nucleic acid sequence shown in SEQ ID NO:4 or SEQ ID NO:10.

In one embodiment, the pharmaceutical composition of the invention includes a pharmaceutical composition comprising a bacteriophage of (i) and (ii) below and a pharma- ceutical acceptable excipient:
(i) a bacteriophage identified by accession number NITE BP-04037 or a passage strain thereof;
(ii) A bacteriophage identified by accession number NITE BP-04031 or a subcultured strain thereof.

In one embodiment, the pharmaceutical composition of the invention includes a pharmaceutical composition comprising a 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 having lytic activity against Pseudomonas aeruginosa,
The genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04032, except for a portion of the tail fiber gene, and
(A) a nucleic acid sequence as set forth in SEQ ID NO:6;
(B) A bacteriophage, or a subsequence thereof, comprising the nucleic acid sequence shown in SEQ ID NO:4 or SEQ ID NO:10.

In one embodiment, the pharmaceutical composition of the invention includes a pharmaceutical composition comprising a bacteriophage of (i) and (ii) below and a pharma- ceutical 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.

In one embodiment, the pharmaceutical composition of the invention includes a pharmaceutical composition comprising a bacteriophage of (i) and (ii) below and a pharmaceutically acceptable excipient:
(i) a bacteriophage identified by accession number NITE BP-04039 or a passage strain thereof;
(ii) a bacteriophage having lytic activity against Pseudomonas aeruginosa,
The genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04032, except for a portion of the tail fiber gene, and
(A) a nucleic acid sequence as set forth in SEQ ID NO:6;
(B) A bacteriophage, or a subsequence thereof, comprising the nucleic acid sequence shown in SEQ ID NO:4 or SEQ ID NO:10.

In one embodiment, the pharmaceutical composition of the present invention includes a pharmaceutical composition comprising a bacteriophage of any one of (i) to (iv) below and a pharma- ceutical acceptable excipient:
(i) a bacteriophage identified by accession number NITE BP-04037 or accession number NITE ABP-04201, or a passage strain thereof;
(ii) a bacteriophage identified by accession number NITE BP-04031 or a passage strain thereof;
(iii) a bacteriophage having lytic activity against Pseudomonas aeruginosa,
The genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04032, except for a portion of the tail fiber gene, and
(A) a nucleic acid sequence as set forth in SEQ ID NO:6;
(B) a bacteriophage, or a subculture 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 passage strain thereof.

In one embodiment, the pharmaceutical composition of the present invention includes a pharmaceutical composition comprising a bacteriophage of any one of (i) to (iv) below and a pharma- ceutical acceptable excipient:
(i) a bacteriophage identified by accession number NITE BP-04037 or a passage strain thereof;
(ii) a bacteriophage identified by the accession number NITE BP-04040 or a passage strain thereof;
(iii) a bacteriophage having lytic activity against Pseudomonas aeruginosa,
The genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04032, except for a portion of the tail fiber gene, and
(A) a nucleic acid sequence as set forth in SEQ ID NO:6;
(B) a bacteriophage, or a subculture thereof, comprising the nucleic acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 10;
(iv) A bacteriophage identified by accession number NITE BP-04031 or a subcultured strain thereof.

In one embodiment, the pharmaceutical composition of the present invention includes a pharmaceutical composition comprising a bacteriophage of any one of (i) to (iv) below and a pharma- ceutical acceptable excipient:
(i) a bacteriophage identified by accession number NITE BP-04037 or a passage strain thereof;
(ii) a bacteriophage identified by accession number NITE ABP-04200 or a passage strain thereof;
(iii) a bacteriophage having lytic activity against Pseudomonas aeruginosa,
The genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04032, except for a portion of the tail fiber gene, and
(A) a nucleic acid sequence as set forth in SEQ ID NO:6;
(B) a bacteriophage, or a subculture thereof, comprising the nucleic acid sequence shown in SEQ ID NO:11;
(iv) A bacteriophage identified by accession number NITE BP-04031 or a subcultured strain thereof.

In one embodiment, the pharmaceutical composition of the present invention includes a pharmaceutical composition comprising a bacteriophage of any one of (i) to (iv) below and a pharma- ceutical acceptable excipient:
(i) a bacteriophage identified by accession number NITE ABP-04201 or a passage strain thereof;
(ii) a bacteriophage identified by accession number NITE ABP-04200 or a passage strain thereof;
(iii) a bacteriophage having lytic activity against Pseudomonas aeruginosa,
The genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04032, except for a portion of the tail fiber gene, and
(A) a nucleic acid sequence as set forth in SEQ ID NO:6;
(B) a bacteriophage, or a subculture thereof, comprising the nucleic acid sequence shown in SEQ ID NO:11;
(iv) A bacteriophage identified by accession number NITE BP-04031 or a subcultured strain thereof.

In one embodiment, the pharmaceutical composition of the present invention includes a pharmaceutical composition comprising a bacteriophage of any one of (i) to (iv) below and a pharma- ceutical acceptable excipient:
(i) a bacteriophage identified by accession number NITE BP-04037 or a passage strain thereof;
(ii) a bacteriophage identified by accession number NITE ABP-04200 or a passage strain thereof;
(iii) a bacteriophage having lytic activity against Pseudomonas aeruginosa,
The genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04032, except for a portion of the tail fiber gene, and
(A) a nucleic acid sequence as set forth in SEQ ID NO:6;
(B) a bacteriophage, or a subculture thereof, comprising the nucleic acid sequence shown in SEQ ID NO:11;
(iv) A bacteriophage identified by accession number NITE BP-04033 or a subcultured strain thereof.

In one embodiment, the pharmaceutical composition of the present invention includes a pharmaceutical composition comprising a bacteriophage of any one of (i) to (iv) below and a pharma- ceutical acceptable excipient:
(i) a bacteriophage identified by accession number NITE BP-04037 or a passage strain thereof;
(ii) a bacteriophage identified by accession number NITE BP-04036 or a passage strain thereof;
(iii) a bacteriophage having lytic activity against Pseudomonas aeruginosa,
The genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04032, except for a portion of the tail fiber gene, and
(A) a nucleic acid sequence as set forth in SEQ ID NO:6;
(B) a bacteriophage, or a subculture thereof, comprising the nucleic acid sequence shown in SEQ ID NO:11;
(iv) A bacteriophage identified by accession number NITE BP-04031 or a subcultured strain thereof.

In one embodiment, the pharmaceutical composition of the present invention includes a pharmaceutical composition comprising a bacteriophage of any one of (i) to (iv) below and a pharma- ceutical acceptable excipient:
(i) a bacteriophage identified by accession number NITE BP-04039 or a passage strain thereof;
(ii) a bacteriophage identified by accession number NITE ABP-04200 or a passage strain thereof;
(iii) a bacteriophage having lytic activity against Pseudomonas aeruginosa,
The genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04032, except for a portion of the tail fiber gene, and
(A) a nucleic acid sequence as set forth in SEQ ID NO:6;
(B) a bacteriophage, or a subculture thereof, comprising the nucleic acid sequence shown in SEQ ID NO:11;
(iv) A bacteriophage identified by accession number NITE BP-04031 or a subcultured strain thereof.

In one embodiment, the pharmaceutical composition of the present invention includes a pharmaceutical composition comprising a bacteriophage of any one of (i) to (iv) below and a pharma- ceutical acceptable excipient:
(i) a bacteriophage identified by accession number NITE BP-04039 or a passage strain thereof;
(ii) a bacteriophage identified by accession number NITE BP-04036 or a passage strain thereof;
(iii) a bacteriophage having lytic activity against Pseudomonas aeruginosa,
The genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04032, except for a portion of the tail fiber gene, and
(A) a nucleic acid sequence as set forth in SEQ ID NO:6;
(B) a bacteriophage, or a subculture thereof, comprising the nucleic acid sequence shown in SEQ ID NO:11;
(iv) A bacteriophage identified by accession number NITE BP-04033 or a subcultured strain thereof.

In one embodiment, the pharmaceutical composition of the present invention includes a pharmaceutical composition comprising a bacteriophage of any one of (i) to (iv) below and a pharma- ceutical acceptable excipient:
(i) a bacteriophage identified by accession number NITE ABP-04200 or a passage strain thereof;
(ii) a bacteriophage having lytic activity against Pseudomonas aeruginosa,
The genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04032, except for a portion of the tail fiber gene, and
(A) a nucleic acid sequence as set forth in SEQ ID NO:6;
(B) a bacteriophage, or a subculture thereof, comprising the nucleic acid sequence shown in SEQ ID NO:11;
(iii) A bacteriophage identified by accession number NITE BP-04031 or a subcultured strain thereof.
(iv) A bacteriophage identified by accession number NITE BP-04033 or a subcultured strain thereof.

The effective dose, number of doses and duration of administration vary depending on the purpose of administration (therapeutic or preventive), the severity of symptoms and age of the subject to be administered, the dosage form of the preparation used, the titer of the bacteriophage, etc. For example, an effective dose of a single bacteriophage or a total effective dose of two or more bacteriophages can be about 10 ⁴ to 10 ¹⁴ plaque forming units (PFU). The ratio of the doses of two or more bacteriophages can be appropriately adjusted 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, if the pharmaceutical composition contains three bacteriophages, each bacteriophage may be contained in approximately equal amounts (e.g., 3.3× ¹⁰ to 3.3× ¹⁰ PFU), and if the pharmaceutical composition contains four bacteriophages, each bacteriophage may be contained in approximately equal amounts (e.g., 2.5× ¹⁰ to 2.5× ¹⁰ PFU), or each bacteriophage may be contained in a different ratio.

The pharmaceutical composition of the present invention can be used as a preventive or therapeutic agent for Pseudomonas bacteria infections, for example, Pseudomonas aeruginosa infections. In this specification, "infectious disease" refers to an infection caused or resulting from an infection with Pseudomonas bacteria, for example, Pseudomonas aeruginosa, and examples of such infections include infections of the lungs, airways, 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, and bronchiectasis in the case of infection of the lungs or airways; pus discharge, fever, and inflammation in the case of infection of the skin, subcutaneous tissue, bones, ears, eyes, or urinary tract; and bacteremia, sepsis, septic shock, fever, hypotension, and anuria in the case of infection of the blood or the whole body.

In this specification, "treatment" means at least partial improvement of the symptoms of an infectious disease, halting the progression or worsening of an infectious disease, complete cure, etc. In this specification, "prevention" means preventing a subject not currently infected with an infectious disease from contracting an infectious disease, preventing the recurrence of an infectious disease, etc.

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 a bacteriophage or modified bacteriophage of the present invention or a 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.

The present invention also includes a method for preventing or treating an infection caused by a bacteriophage of the genus Pseudomonas, such as a Pseudomonas aeruginosa infection, in a subject, comprising 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 can be administered simultaneously or separately.

Furthermore, the present invention includes the bacteriophage or modified bacteriophage of the present invention, or the pharmaceutical composition of the present invention for use in preventing or treating infections caused by bacteria of the genus Pseudomonas, such as Pseudomonas aeruginosa infections.Furthermore, the present invention includes the 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 preventing or treating infections caused by bacteria of the genus Pseudomonas, such as Pseudomonas aeruginosa infections.

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

In addition, 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 the treatment or prevention of 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 medicine 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.

4. Use of the prophage-cured strain as a host bacterium
The present invention also relates to the use of a strain of Pseudomonas aeruginosa 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. Thus, 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.

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

In this specification, "prophage-derived gene" refers to a gene present in a prophage, particularly a gene involved in the maintenance of the prophage. An example of a "prophage-derived gene" is the pflM gene (Journal of bacteriology. 2024; 206(5): e0040223).

In this specification, "deleting a gene derived from a prophage" means 1) that at least one full-length gene is deleted from the genomic sequence of the prophage, or 2-1) that at least one part of the genomic sequence of the prophage is modified by deletion, substitution, insertion, addition, or a combination thereof, thereby removing the prophage from the host bacterium, or 2-2) that the function of the gene derived from the prophage is deleted. Deletion of a gene derived from a prophage 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 (for example, Journal of Bacteriology. 2024; 206(5): e0040223).

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

Having generally described the invention, reference is now made to specific examples for a further understanding. These are intended to be illustrative and not limiting of the invention.

In cases where commercially available kits or reagents were used, the experiments were conducted according to the attached protocols unless otherwise noted. For convenience, the concentration in mol/L is expressed as M. For example, 1M sodium hydroxide solution means that the sodium hydroxide solution is 1 mol/L.

As for 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. MRSN 321 and MRSN 4841 strains are included in the Pseudomonas aeruginosa Diversity Panel provided by the Multidrug-Resistant Organism Repository and Surveillance Network (MRSN) at the Walter Reed Army Institute of Research (WRAIR).

Example 1: Isolation of bacteriophage exhibiting lytic activity against Pseudomonas aeruginosa
Bacteriophages were isolated from environmental samples (sewage and wastewater in Japan) by the following method. As the host for isolation, Pseudomonas aeruginosa (Schroeter) Migula ATCC15692 strain (related strain of PAO1 strain), NBRC3080 strain, PA14 strain, MRSN 321 strain, or MRSN 4841 strain was used. The environmental sample was centrifuged, the supernatant was separated, and then filtered through a 0.22 μm or 0.45 μm filter. The filtered sample was mixed with equal amounts of LB liquid medium (20 g/L LB medium, Lennox (Nacalai Tesque)) adjusted to twice the final concentration. A Pseudomonas aeruginosa culture solution cultured overnight in LB liquid medium was added to the mixture, and the mixture was shaken and cultured at 37°C. The culture was centrifuged, and the supernatant was separated. This supernatant and a Pseudomonas aeruginosa culture solution cultured overnight in LB liquid medium were mixed with 0.3 to 0.6% soft agar-containing LB liquid medium, and 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 called PAPT1, PAi140, PAi228, PAi239, and PAi242, respectively. In addition, 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, respectively) 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.

Bacteriophage PAi23 (a passage strain of PaGU11 identified by NCBI Reference Sequence NC_050145) provided by Gifu University, and bacteriophage φBrkr (a passage strain of bacteriophage identified by NCBI Reference Sequence LC765218) provided by Rakuno Gakuen University, 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 LC727696) were co-cultured with Pseudomonas aeruginosa ATCC15692 strain to prepare bacteriophage lysates.

The bacteriolytic 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 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. For each bacteriophage lysate, 2.5 μL of a 10-fold serial dilution prepared using phage buffer (1 mM CaCl ₂ , 10 mM MgSO ₄ , 10 mM Tris-HCl (pH 7.5) containing 68.4 mM NaCl) was dropped onto the plate and cultured at 37° C. The appearance of lytic plaques by each bacteriophage confirmed the bacteriolysis against Pseudomonas aeruginosa (FIG. 1).

The accession numbers or receipt numbers of the above obtained PAPT1, PAi140, PAi228, PAi239, PAi242, PAi23, φBrkr, φBrmt, φ30-1, φLCX, and φLCX2 are respectively NITE BP-04031, NITE BP-04033, NITE BP-04034, The accession numbers are NITE BP-04035, NITE BP-04036, NITE BP-04032, NITE BP-04037, NITE BP-04038, NITE BP-04039, NITE BP-04040, and receipt number NITE ABP-04200.

Example 2: Construction of tail fiber gene-exchanged bacteriophage
A recombinant bacteriophage was constructed in which the tail fiber gene of PAi23 phage was replaced 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 reaction. Each amplified polynucleotide was ligated using Gibson assembly master mix (New England Biolabs, catalog number E2611). As a result, a phage genome was constructed in which bases 31081-33975 encoding the tail fiber gene of PAi23 phage were deleted and recombined with the tail fiber gene (SEQ ID NO: 6) of JG024 phage. The phage genome was introduced into Pseudomonas aeruginosa ATCC15692 strain by electroporation to obtain the desired bacteriophage. The obtained bacteriophage is referred to as PAi23-JG024 tail.

The bacteriolytic activity of the PAi23-JG024 tail obtained above against Pseudomonas aeruginosa was confirmed. The Pseudomonas aeruginosa strain used was the ATCC15692 strain and the respiratory clinical isolates 3-55-PA strain and 9-8-PA strain (provided by the Joint Committee on Antimicrobial Susceptibility Surveillance of Three Academic Societies). The Pseudomonas aeruginosa culture solution cultured overnight in LB liquid medium was mixed with LB liquid medium containing 0.6% soft agar and layered on an LB agar plate. 10-fold serial dilutions were prepared for the PAi23 phage and PAi23-JG024 tail phage lysate using phage buffer. 2.5 μL of each dilution was dropped onto the plate and incubated at 37°C. Lytic plaques appeared due to each bacteriophage, confirming the bacteriolytic activity against Pseudomonas aeruginosa (Figure 2). As shown in Table 1, it was confirmed that PAi23-JG024 tail can infect strains that are not infected by PAi23.

<Example 3: Construction of PelAh, PslGh, and PslG-equipped PAi23-JG024 tail phage>
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, the PslGh gene and the PslG gene were amplified by PCR reaction 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 obtain the pUCP18-GmR plasmid vector. A Shine-Dalgarno sequence-like sequence (SEQ ID NO: 8) was added to the 5' side of the start codon of the obtained PelAh gene, PslGh gene, and PslG gene, and each was inserted into the EcoRI/XbaI restriction enzyme site so as to be linked to the lac operon of the pUCP18-GmR plasmid vector. As in the method shown in Example 2, the PelAh gene, PslGh gene, and PslG gene to which a part of the lac operon (SEQ ID NO: 7) and a Shine-Dalgarno sequence-like sequence (SEQ ID NO: 8) were added were inserted between the 12081st and 12082nd bases of the PAi23 phage genome (the sequence in which a part of the lac operon, the Shine-Dalgarno sequence-like sequence (SEQ ID NO: 8), and the PslGh gene were linked is SEQ ID NO: 10). Furthermore, as in Example 2, a phage genome was constructed in which the tail fiber gene was replaced with the tail fiber gene 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.

Using the same procedure as above, a phage was constructed in which the Shine-Dalgarno sequence-like sequence (SEQ ID NO: 8), which is a sequence that does not code for a gene on the 5' side of the start codon, was replaced with the Shine-Dalgarno sequence-like sequence_version 3 (v3) (SEQ ID NO: 12), which is a sequence with a spacer added. A PslGh gene (SEQ ID NO: 4) to which a part of the lac operon (SEQ ID NO: 7) and the Shine-Dalgarno sequence-like sequence_v3 (SEQ ID NO: 12) were added was inserted between the 12081st and 12082nd bases of the PAi23 phage genome (the sequence in which a part of the lac operon, the Shine-Dalgarno sequence-like sequence_v3 (SEQ ID NO: 12), and the PslGh gene are linked is SEQ ID NO: 11). Furthermore, a phage genome was constructed in which the tail fiber gene was replaced with the tail fiber gene 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.

Furthermore, a phage was constructed in which the PslGh gene (sequence number 4) and the PelAh gene (sequence number 3) were inserted. A base sequence was inserted between bases 12081 and 12082 of the PAi23 phage genome, linking the PslGh gene (sequence number 4) to which a part of the lac operon (sequence number 7) and the Shine-Dalgarno sequence-like sequence_v3 (sequence number 12) had been added, and the PelAh gene (sequence number 3) to which a Shine-Dalgarno sequence-like sequence (sequence number 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 to obtain a recombinant bacteriophage, designated PAi23-JG024 tail::Plac-PslGh-PelAh(AB)_v3.

The lytic activity of the above-obtained 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 ATCC15692 strain was used as Pseudomonas aeruginosa. The Pseudomonas aeruginosa culture solution cultured overnight in LB liquid medium was mixed with 0.3% soft agar-containing LB liquid medium and layered on an LB agar plate. For each phage lysate, 10-fold serial dilutions were made using phage buffer. 2.5 μL of each dilution 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).

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. Bacteriocin PyoG has bactericidal activity against Pseudomonas aeruginosa. In order to produce a bacteriophage with a stable PyoG gene inserted therein, a Pseudomonas aeruginosa ATCC15692 strain was constructed in advance 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) to which a Shine-Dalgarno sequence-like sequence (SEQ ID NO: 8) was added was inserted into the EcoRI / XbaI site of the pUCP18-GmR plasmid vector, and introduced into the Pseudomonas aeruginosa ATCC15692 strain by electroporation. As a result, a Pseudomonas aeruginosa ATCC15692-ImG strain that constitutively expresses the ImG gene under the control of the lac promoter was obtained.

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

The bacteriolytic activity of the PAi23::PyoG(CD) obtained above against Pseudomonas aeruginosa was confirmed by the following method. ATCC15692 and ATCC15692-ImG strains were used as Pseudomonas aeruginosa. Pseudomonas aeruginosa 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 phage lysate of PAi23 and PAi23::PyoG(CD) using phage buffer. 2.5 μL of each dilution was dropped onto the plate and incubated at 37°C. Bacteriophage-induced lytic plaques appeared, confirming the lytic activity against Pseudomonas aeruginosa (Figure 4). PAi23::PyoG(CD) formed clearer lytic plaques in Pseudomonas aeruginosa ATCC15692 strain compared to PAi23 phage.

Example 5: Biofilm decomposition activity of natural bacteriophages
We investigated whether natural bacteriophages have the activity of reducing 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 a pin-equipped lid (Nunc Immuno TSP Lids, Thermo Scientific) was placed on the plate. The plate was incubated at 37°C for 24 hours to form a biofilm on the pin. The formed biofilm was washed with physiological saline, and contacted with a phage buffer, phage φLCX adjusted to a final concentration of 1.29 x 10 ⁸ PFU/mL in LB liquid medium, and PAPT1 or PAi239 adjusted to a final concentration of 1 x 10 ⁸ PFU/mL in LB liquid medium for 6 hours at 37°C in a 96-well plate. Each well was washed with saline and then stained with crystal violet. After staining, each well was washed with saline. After washing, crystal violet from each well was eluted with ethanol, and the absorbance of each eluate at 590 nm was measured. The higher the absorbance, the greater the amount of biofilm. The experiment was performed in three wells per condition, and three trials were performed. 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, it was shown that the amount of biofilm was significantly reduced under the conditions where φLCX, PAPT1, or PAi239 was added compared to the phage buffer group (Control) (FIG. 5, ^* p<0.05).

Example 6: Biofilm decomposition activity of engineered bacteriophages
It was examined whether PAi23-JG024 tail::Plac-PslGh (AB) and PAi23-JG024 tail::Plac-PslG (AB) have the activity of reducing 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 added to LB liquid medium so that the final concentration was 1 x 10 ⁸ PFU / mL, and contacted with the biofilm in a 96-well plate. The experiment was carried out in 6 wells per condition, and three trials were performed. The average of 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) (in FIG. 6, "::PslGh") and PAi23-JG024 tail::Plac-PslG(AB) (in FIG. 6, "::PslG") significantly reduced the amount of biofilm (FIG. 6A, ^* p<0.05).

Next, we investigated whether PAi23-JG024 tail::Plac-PelAh (AB) has the activity of reducing the amount of biofilm formed by Pseudomonas aeruginosa. In the same manner as above, a biofilm was formed on a pin using the respiratory clinical isolate 28-42-PA strain (distributed by the Joint Committee on Antimicrobial Susceptibility Surveillance of Three Academic Societies). PAi23-JG024 tail and PAi23-JG024 tail::Plac-PelAh (AB) were added to LB liquid medium to a final concentration of 1 x 10 ⁸ PFU/mL, and contacted with the biofilm in a 96-well plate. The experiment was performed in 3 wells per condition, and 8 trials were performed (graph on the left in Figure 6B). In each trial, the relative biofilm amount was calculated when the control group was set to 1, and the Kruskal-Wallis test and Dunn's multiple comparison test were performed. As a result, PAi23-JG024 tail did not reduce the biofilm amount, whereas PAi23-JG024 tail::Plac-PelAh (AB) (in Figure 6, "::PelAh") significantly reduced the biofilm amount (Figure 6B right, ^* p<0.05).

Next, PAi23-JG024 tail:: Plac-PslGh (AB) _v3 and PAi23-JG024 tail:: Plac-PslGh-PelAh (AB) _v3 were examined for their activity in reducing the amount of biofilm formed by Pseudomonas aeruginosa. A biofilm was formed on a pin 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 each added to LB liquid medium to a final concentration of 1 x 10 ⁸ PFU / mL, and contacted with the biofilm in a 96-well plate. The experiment was carried out in three wells per condition, and three trials were performed. 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).

Example 7: Growth-inhibitory activity of modified bacteriophages against Pseudomonas aeruginosa
The growth inhibitory effect of PAi23 and PAi23::PyoG (CD) against Pseudomonas aeruginosa was examined. Respiratory clinical isolate 27-12-PA strain (distributed by the Joint Committee on Antimicrobial Susceptibility Surveillance of Three Academic Societies) was diluted with LB liquid medium to OD600 = 0.1, and 90 μL was inoculated into a 96-well plate per well. Phage buffer (Control), or PAi23 and PAi23::PyoG (CD) were added to each well at a final concentration of 1 x 10 ⁸ PFU/mL at 10 μL each, and the plate was incubated at 37°C. The absorbance of the culture solution at 600 nm was measured over time using a plate reader. The experiment was performed in 3 wells per condition, for a total of 4 trials. 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 growth inhibitory effect on P. aeruginosa than PAi23 ( FIG. 7 , ^* p<0.05).

Example 8: Lytic activity of bacteriophage φLCX against Pseudomonas aeruginosa
The bacteriophage φLCX was examined for its bacteriolytic activity against Pseudomonas aeruginosa that had been cultured overnight and grown sufficiently. Pseudomonas aeruginosa ATCC15692 strain was inoculated into LB liquid medium and cultured in a glass test tube with shaking at 37°C. The absorbance of the culture solution at 600 nm was measured over time from 15 hours after culture. After 18 hours of culture, phage buffer (Control) or bacteriophage φLCX was added to a final concentration of 1 x ¹⁰⁷ PFU/mL, and the culture was continued with shaking at 37°C. This experiment was performed in three glass test tubes per condition. The average absorbance of the three tubes was calculated. As a result, it was shown that φLCX has bacteriolytic activity against Pseudomonas aeruginosa that has passed the logarithmic growth phase (Figure 8).

Example 9: Lytic activity of a single bacteriophage in a mouse infection model
Tryptosoy broth (Merck) was inoculated with Pseudomonas aeruginosa ATCC15692 strain and cultured at 37°C for 2.5 hours with shaking. The culture was centrifuged, the supernatant was discarded, and suspended in physiological saline. The culture was diluted with physiological saline to adjust the absorbance at 600 nm (OD600) to 0.5, and used as an inoculum. C57BL/6Jms Slc mice (Japan SLC Co., Ltd., female, about 5 weeks old) were infected by inoculating 40 μL of the bacterial solution per mouse into the nasal cavity. After 30 minutes, phage buffer, PAi23 or φBrmt phage was administered to 5 mice each at 5×10 ⁸ PFU per mouse into the nasal cavity. 24 hours after infection, the lungs were removed and homogenized in 2 mL of Hank's Balanced Salt Solution (Life Technologies Corporation) to prepare a tissue stock solution. The tissue stock solution was serially diluted 10-fold with physiological saline. The tissue stock solution was spread on a tryptosoy agar medium (Eiken Chemical) to measure the viable cell count. The viable cell count was converted to a common logarithm, and the arithmetic mean was calculated, followed by one-way analysis of variance and Dunnett's multiple comparison test. FIG. 9 shows the viable cell count 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 viable bacterial load in the lungs compared to the phage buffer group (FIG. 9, ^* p<0.05).

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

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

It was confirmed that φBrkr_FTR2 had acquired freeze-thaw resistance by the following method. φBrkr and φBrkr_FTR2 were adjusted to 1×10 ⁸ PFU/mL using phage buffer, and subjected to freeze-thawing. The titer values before and after freeze-thawing were calculated by the following method. Pseudomonas aeruginosa culture solution cultured overnight in LB liquid medium was mixed with 0.3% soft agar-containing LB liquid medium and layered on an LB agar plate. For each bacteriophage solution, 10-fold serial dilutions were prepared using phage buffer. 2.5 μL of each dilution was dropped onto the plate and cultured at 37° C. The bacteriophage caused the appearance of lytic plaques, and the lytic activity against Pseudomonas aeruginosa was confirmed (FIG. 10A). The titer value was calculated from the number of lytic plaques, and the recovery rate of the phage before and after freeze-thawing was 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).

Example 11: Prolongation of Pseudomonas aeruginosa growth inhibitory effect by bacteriophage cocktail
In this example and thereafter, PAPT1, PAi242, φBrkr, φBrkr_FTR2, φ30-1, φLCX, φLCX2, PAi23-JG024 tail::Plac-PslGh(AB), and PAi23-JG024 tail::Plac-PslGh(AB)_v3 were cultured using ATCC15692ΔPf4/Pf6 (prophage-removed strain) described later in Example 14 as a culture host, and each bacteriophage lysate was prepared.

It was examined whether the inhibition of Pseudomonas aeruginosa growth could be extended by making a bacteriophage cocktail. Pseudomonas aeruginosa ATCC15692 strain was inoculated to about 10 ⁸ CFU/mL in a 96-well plate containing LB liquid medium. Furthermore, a bacteriophage solution was added to each well to make a total of 100 μL. A single bacteriophage, a cocktail of two bacteriophages, and a cocktail of four bacteriophages were added to each well to make a final concentration of 1×10 ⁴ PFU/mL (i.e., multiplicity of infection (MOI) = about 0.0001). The ratio of the amount of each bacteriophage in the cocktail was equal. For example, in the cocktail of four bacteriophages, each bacteriophage was added to make a final concentration of 0.25×10 ⁴ PFU/mL. The plates were cultured at 37° C., and the absorbance at 600 nm of each well was measured every hour.

As representative examples of the growth curves of Pseudomonas aeruginosa, graphs of cocktail a (a cocktail of φBrkr, PAi23-JG024 tail::Plac-PslGh (AB)) and each of the single bacteriophages constituting cocktail a, and graphs of cocktail A (a cocktail of φBrkr, φLCX, PAi23-JG024 tail::Plac-PslGh (AB), PAPT1) and each of the single bacteriophages constituting cocktail A are shown in FIG. 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. The AUC under conditions without phage addition was set as 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 for each of the cocktails, and the results showed that all of the cocktails significantly prolonged the growth inhibitory effect on Pseudomonas aeruginosa compared to the single bacteriophages that constituted each cocktail ( ^* p<0.05).

Example 12: Biofilm decomposition activity of bacteriophage cocktail
It was examined whether the cocktail of bacteriophages has the activity of reducing 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 ATCC15692 strain. The cocktail of bacteriophages was added to LB liquid medium to a final concentration of 1 x 10 ⁸ PFU/mL (the ratio of each single phage was adjusted to be equal), and contacted with the biofilm in a 96-well plate. The experiment was performed in 3 wells per condition, and 4 trials were performed. The average of the 4 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).

Example 13: Lytic activity of bacteriophage cocktail in a mouse infection model
In the mouse infection experiment, a bacteriophage solution of ultracentrifugation purification grade by density gradient centrifugation was used. Purification by ultracentrifugation: DNase I and RNase A were added to each phage lysate to a final concentration of 1 μg/mL, and the mixture was left to stand at room temperature for 30 minutes. Bacteriophage was precipitated using polyethylene glycol 8000, and the precipitate was collected by centrifugation. 0.4 g of cesium chloride per mL was added to the collected bacteriophage solution, which was gently dissolved and transferred to a container for ultracentrifugation. Cesium chloride solutions of each density 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 a band containing bacteriophage was collected. The solution containing the recovered bacteriophage was replaced with a phage buffer by dialysis to obtain an ultracentrifugation-purification grade bacteriophage solution.

Respiratory clinical isolate 27-12-PA strain was inoculated into LB liquid medium and cultured at 37°C for 2.5 hours with shaking. The culture was centrifuged to discard the supernatant, 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 to prepare an inoculum. C57BL/6Jms Slc mice (Japan SLC Co., Ltd., female, 5 weeks old) were infected by inoculation with 50 μL of the bacterial solution per mouse using the oropharyngeal aspiration method. One hour after infection, phage buffer or bacteriophage cocktail was administered by oropharyngeal aspiration to a total of 1 x 10 ⁹ PFU per mouse. The bacteriophage cocktail was cocktail A (φBrkr, φLCX, PAi23-JG024 tail::Plac-PslGh (AB), PAPT1 cocktail), cocktail B (φBrkr, φLCX2, PAi23-JG024 tail::Plac-PslGh (AB)_v3, PAPT1 cocktail), or cocktail G (φ30-1, PAi242, PAi23-JG024 tail::Plac-PslGh (AB)_v3, PAi140 cocktail). Approximately 22.5 hours after infection, the lungs were removed. The lungs were homogenized in 2 mL of saline and washed twice with saline to prepare a tissue stock solution. A 10-fold serial dilution was made with saline for the tissue stock solution. The viable cell count was measured by applying the solution to an LB agar plate. When the viable cell count was below the detection threshold, it was calculated as the detection threshold. The viable cell count was converted to a common logarithm, and the arithmetic mean was calculated, and one-way analysis of variance and Dunnett's multiple comparison test were performed. Figure 13 shows the viable cell count in the lung (log CFU/lung) (N=10, mean±standard error). As a result, it was shown that the amount of viable cells in the lung was significantly reduced in all of the cocktail A, cocktail B, and cocktail G groups compared to the phage buffer group ( ^* p<0.05).

Example 14: Cultivation of bacteriophages using a prophage-removed strain of Pseudomonas aeruginosa
A method for removing filamentous phages (e.g., Pf4 and Pf6) that lysogenize from P. aeruginosa such as the PAO1 strain has been reported (Journal of bacteriology. 2024; 206(5): e0040223). Briefly, this method is a method for removing filamentous phages by deleting the pflM gene involved in the maintenance of filamentous phages. Following this method, Pf4 and Pf6 were removed from the genome of P. aeruginosa ATCC15692 strain (a related strain 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 this 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-cured strain thus obtained is designated ATCC15692ΔPf4/Pf6.

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

Next, liquid culture of bacteriophage was carried out using Pseudomonas aeruginosa ATCC15692 strain and ATCC15692ΔPf4/Pf6 strain. 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, respectively, for infection. After shaking culture at 37° C. for about 6 hours, each bacteriophage lysate was obtained. Liquid culture of each bacteriophage was carried out in triplicate, and the titer value of the obtained lysate was calculated by the method of Example 10, and the result is shown in FIG. 14B. When bacteriophages were cultured using the ATCC15692ΔPf4/Pf6 strain (prophage-removed strain) compared to the P. aeruginosa ATCC15692 strain, the titer of PAPT1 was 10 times higher and that of PAi242 was 10,000 times higher (unpaired t-test, ^* p<0.05). These results indicate that PAPT1 and PAi242 can be amplified with high efficiency by culturing them using the ATCC15692ΔPf4/Pf6 strain as the host bacterium.

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

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

Claims (25)

A bacteriophage selected from the following (1) to (8):
(1) A bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of the bacteriophage comprising 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-04040;
(2) A bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of the bacteriophage comprising 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-04031;
(3) A bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of the bacteriophage comprising 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-04033;
(4) A bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of the bacteriophage comprising 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-04036;
(5) A bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of the bacteriophage comprising 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-04034;
(6) A bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of the bacteriophage comprising 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-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 the accession number NITE ABP-04201. The bacteriophage according to claim 1, selected from the following (1) to (8):
(1) A bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of the bacteriophage comprising a nucleic acid sequence of the genome of the bacteriophage identified 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 of the genome of the bacteriophage identified by the 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 the 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 the 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 the 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 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 of the genome of a bacteriophage identified by 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 of the genome of a bacteriophage identified by accession number NITE ABP-04201. The bacteriophage according to claim 1 or 2, selected from the following (1) to (8):
(1) A bacteriophage, the genome of which consists of the nucleic acid sequence of the genome of a bacteriophage identified by the accession number NITE BP-04040;
(2) A bacteriophage, the genome of which consists of the nucleic acid sequence of the genome of a bacteriophage identified by the accession number NITE BP-04031;
(3) A bacteriophage, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04033;
(4) A bacteriophage, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04036;
(5) A bacteriophage, the genome of which consists of the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04034;
(6) A bacteriophage, the genome of which consists of the nucleic acid sequence of the genome of a bacteriophage identified by the accession number NITE BP-04035;
(7) A bacteriophage, the genome of which consists of the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE ABP-04200; and (8) a bacteriophage, the genome of which consists of the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE ABP-04201. A bacteriophage selected from the following (1) to (8):
(1) A bacteriophage identified by the accession number NITE BP-04040, or a passage strain thereof;
(2) A bacteriophage identified by the 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 the 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 the accession number NITE BP-04035, or a passage strain thereof;
(7) A bacteriophage identified by accession number NITE ABP-04200, or a subcultured strain thereof; and (8) a bacteriophage identified by accession number NITE ABP-04201, or a subcultured strain thereof. A bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of which (a) contains a nucleic acid sequence that has 90% or more identity with the nucleic acid sequence of the genome of a bacteriophage identified by accession number NITE BP-04032, except for a portion of the tail fiber gene, and (b) contains a nucleic acid sequence of a tail fiber gene that contains at least a portion of a tail fiber gene derived from another bacteriophage having lytic activity against Pseudomonas aeruginosa. The bacteriophage according to claim 5, which is a bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of the bacteriophage (a) containing the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04032, excluding a portion of the tail fiber gene, and (b) containing 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. The bacteriophage according to claim 5, which is a bacteriophage having lytic activity against Pseudomonas aeruginosa, the genome of the bacteriophage (a) consisting of the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04032, excluding a portion of the tail fiber gene, and (b) containing 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. The bacteriophage of claim 5, wherein the tail fiber gene from another bacteriophage comprises the nucleic acid sequence shown in SEQ ID NO:6 or comprises a nucleic acid sequence having 90% or more identity to SEQ ID NO:6. 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 of claim 9, wherein the genome of the bacteriophage comprises nucleic acid sequences encoding at least two proteins or active domains selected from (a) to (c):
(a) Pyocin G;
(b) PslGh; and (c) PelAh. The bacteriophage of claim 9, wherein the genome of the bacteriophage comprises nucleic acid sequences encoding the following proteins or active domains:
(a) Pyocin G;
(b) PslGh; and (c) PelAh. A pharmaceutical composition comprising the bacteriophage according to any one of claims 1 to 11 and a pharma- ceutical acceptable excipient. A pharmaceutical composition comprising at least two types of bacteriophages according to any one of claims 1 to 11 and a pharma- ceutical acceptable excipient. A pharmaceutical composition comprising three types of bacteriophages according to any one of claims 1 to 11 and a pharma- ceutical acceptable excipient. A pharmaceutical composition comprising four types of bacteriophages according to any one of claims 1 to 11 and a pharma- ceutical acceptable excipient. A pharmaceutical composition comprising five types of bacteriophages according to any one of claims 1 to 11 and a pharma- ceutical acceptable excipient. The pharmaceutical composition according to any one of claims 12 to 16, which is a pharmaceutical composition for preventing or treating Pseudomonas aeruginosa infection. The pharmaceutical composition according to claim 17, wherein the Pseudomonas aeruginosa infection is a respiratory infection. A bacteriophage having lytic activity against Pseudomonas aeruginosa,
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 the accession number NITE BP-04032, except for a portion of the tail fiber gene, and
(A) a nucleic acid sequence encoding the tail fiber from bacteriophage JG024;
(B) A bacteriophage comprising a nucleic acid sequence encoding PslGh. A bacteriophage having lytic activity against Pseudomonas aeruginosa,
The genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04032, except for a portion of the tail fiber gene, and
(A) a nucleic acid sequence as set forth in SEQ ID NO:6, or a nucleic acid sequence having 90% or more identity to SEQ ID NO:6;
(B) A bacteriophage comprising 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. A bacteriophage having lytic activity against Pseudomonas aeruginosa,
The genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04032, except for a portion of the tail fiber gene, and
(A) a nucleic acid sequence as set forth in SEQ ID NO:6;
(B) A bacteriophage comprising the nucleic acid sequence shown in SEQ ID NO:10 or SEQ ID NO:11. 1. A pharmaceutical composition comprising a bacteriophage and a pharma- ceutically acceptable excipient,
The bacteriophage is any one of the following bacteriophages (i) to (iv):
(i) a bacteriophage identified by accession number NITE BP-04037 or accession number NITE ABP-04201, or a passage strain thereof;
(ii) a bacteriophage identified by accession number NITE BP-04031 or a passage strain thereof;
(iii) a bacteriophage having lytic activity against Pseudomonas aeruginosa,
The genome of the bacteriophage comprises the nucleic acid sequence of the genome of the bacteriophage identified by the accession number NITE BP-04032, except for a portion of the tail fiber gene, and
(A) a nucleic acid sequence as set forth in SEQ ID NO:6;
(B) a bacteriophage, or a subculture thereof, comprising the nucleic acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 11;
(iv) A pharmaceutical composition comprising a bacteriophage identified by accession number NITE BP-04040 or accession number NITE ABP-04200, or a passage strain thereof. The pharmaceutical composition according to claim 22, which is a pharmaceutical composition for preventing or treating Pseudomonas aeruginosa infection. The pharmaceutical composition according to claim 23, wherein the Pseudomonas aeruginosa infection is a respiratory infection. A method for producing a bacteriophage having lytic activity against Pseudomonas aeruginosa, comprising a step of culturing the bacteriophage in a host bacterium lacking a gene derived from the prophage.

Images