Bacteriophage, salmonella bacteria lytic agent, composition, and salmonella bacteria control method

Abstract

One purpose of the present disclosure is to (i) provide a novel bacteriophage having lytic activity with respect to Salmonella bacteria such as S. Enteritidis, or a lytic agent comprising the same, (ii) provide a bacteriophage that has a wide host range with respect to Salmonella bacteria, or a lytic agent comprising the same, (iii) provide a host-specific bacteriophage, or an effective Salmonella bacteria lytic agent comprising the same, or (iv) provide a bacteriophage that can effectively control S. Typhimurium, particularly S. Typhimurium having multiple drug resistance, or a lytic agent comprising the same.　The present disclosure provides a bacteriophage having a specific genomic DNA sequence, a Salmonella bacteria lytic agent comprising the same, and a composition comprising the same.

Description

Bacteriophage, Salmonella lysing agent, composition, and method for controlling Salmonella

[0001] The present invention relates to a bacteriophage, a lytic agent comprising the bacteriophage, a composition containing the same, and a method for controlling Salmonella bacteria using the same.

[0002] Salmonella bacteria are one of the main causes of food poisoning, infecting humans and animals such as livestock and causing salmonellosis, including diarrhea. Salmonella bacteria are present in the digestive tracts of humans and animals such as livestock, and cause contamination by being excreted in feces. Infection with Salmonella bacteria often occurs through the ingestion of food, drink, or feed contaminated with Salmonella bacteria.

[0003] Conventionally, low-molecular-weight compounds have been used as antibacterial agents against Salmonella bacteria, but their continued use has negative effects such as the emergence of multidrug-resistant bacteria, so new control methods have been sought. In recent years, bacteriophages have attracted attention as a new means of controlling Salmonella bacteria because of their high target specificity, lack of damage to the microflora, and low toxicity (Non-Patent Document 1).

[0004] Bacteriophage (often abbreviated simply as "phage" herein) is a general term for viruses that infect only bacteria. After adsorbing to their target host bacteria, many phages inject their own DNA into the bacteria and self-amplify using the bacterial translation machinery. They then lyse the bacteria, disseminating the amplified phages and repeatedly infecting new target bacteria (Non-Patent Document 2).

[0005] Examples of phages that are lytic to Salmonella bacteria are described in, for example, Patent Documents 1 and 2. Phages that lyse Salmonella bacteria can be used, for example, to control Salmonella bacteria in poultry and pig farming, and to detect and control Salmonella bacteria in the food industry (Non-Patent Document 3). In fact, a product containing a phage that is lytic to Salmonella bacteria is BAFASAL, a feed additive for preventing Salmonella infection in chickens. Ｒ(Proteon Pharmaceuticals), SalmoFresh, a food processing formulation that kills Salmonella in foods; ＴＭ (intralytix) and PhageGuard (Micros), etc., have already been put on the market (Non-Patent Document 4).

[0006] WO2013-027146 JP2014-217336

[0007] Jun-Hyun Oh et al. , 2017, J. Microbiol. Biotechnol. , 27(12), 2075-2088 Sharma S. et al. , Folia Microbiol. , 2017, 62:17-55 Shuai Wei et al. , Microorganisms, 2019, 7, 570 Katarzyna Zbikowska et al. , Animals, 2020, 10, 872

[0008] As mentioned above, phages that are lytic to Salmonella bacteria have been discovered, and products using these phages are on the market. However, if a specific phage is used excessively, it is expected that Salmonella bacteria that are resistant to the phage will emerge. Therefore, there is still a need to discover new phages.

[0009] For example, since S. Enteritidis is the serotype of Salmonella bacteria most frequently detected in chickens (Non-Patent Document 1), a phage that exhibits bacteriolytic activity against a wide range of strains of this serotype is desirable.

[0010] Furthermore, one of the properties required for phages used in the bacteriolytic composition is a wide host range for Salmonella bacteria, which is desirable because phages with a wide host range can be applied to various Salmonella bacteria and therefore have a wide range of applications.

[0011] Furthermore, a lytic composition using a phage with high host specificity that targets a specific Salmonella bacterium is also desirable because it is useful for identifying the serotype of bacteria that cause food poisoning.

[0012] Furthermore, among Salmonella bacteria, S. Typhimurium in particular has become plagued by multidrug resistance, which is a problem for bacteria that are resistant to multiple antibacterial agents, and there are bacterial strains that are resistant to five typical drugs: ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline. Such multidrug-resistant S. Typhimurium has been confirmed to be spreading throughout the world, and one of the reasons for this is thought to be the increased use of antibiotics in animal husbandry and hospitals. Therefore, there is a demand for a technology that can effectively control S. Typhimurium.

[0013] Therefore, one object of the present disclosure is to (i) provide a novel bacteriophage having lytic activity against Salmonella bacteria such as S. Enteritidis or a lytic agent comprising the same, (ii) provide a bacteriophage having a broad host range against Salmonella bacteria or a lytic agent comprising the same, (iii) provide a host-specific bacteriophage or an effective lytic agent for Salmonella bacteria comprising the same, or (iv) provide a bacteriophage or a lytic agent comprising the same that can effectively control S. Typhimurium, particularly multidrug-resistant S. Typhimurium.

[0014] The present inventors isolated novel phages from natural wastewater and soil using a method for detecting lytic plaques formed on soft agar medium cultured with Salmonella bacteria, evaluated the lytic activity of the phages against various Salmonella bacteria, and analyzed their genome sequences.

[0015] As a result, it was revealed that seven bacteriophages (corresponding to the first phage in this specification) having a specific genomic DNA sequence have lytic activity against specific Salmonella bacteria.

[0016] Furthermore, it was revealed that three specific bacteriophages (corresponding to the second phage in this specification) have broad-spectrum lytic activity against Salmonella bacteria, specifically, lytic activity against S. Enteritidis, S. Typhimurium, S. Infantis, S. Montevideo, and S. Javiana.

[0017] Furthermore, it has been revealed that one particular bacteriophage (corresponding to the third phage in this specification) has lytic activity against S. Typhimurium, particularly against various strains of S. Typhimurium that exhibit multiple drug resistance to antibiotics.

[0018] It has also been revealed that a bacteriophage having a specific genomic DNA sequence (corresponding to the fourth phage in this specification) has lytic activity against specific Salmonella bacteria.

[0019] Furthermore, it has been revealed that a bacteriophage (corresponding to the fifth phage in this specification) having genomic DNA containing a gene encoding an endonuclease consisting of a specific amino acid sequence has lytic activity against specific Salmonella bacteria.

[0020] Furthermore, it has been revealed that a specific bacteriophage (corresponding to the sixth phage in this specification) is a novel bacteriophage having lytic activity against bacteria of the genus Salmonella, specifically, lytic activity against S. Enteritidis, S. Typhimurium, and S. Javiana.

[0021] It has also been revealed that a bacteriophage having a specific genomic DNA sequence (corresponding to the seventh phage in this specification) has lytic activity against specific Salmonella bacteria.

[0022] The present invention has been completed based on the results of the above research and development, and specifically provides the following exemplary embodiments: [1] A bacteriophage exhibiting lytic activity against bacteria of the genus Salmonella, which has genomic DNA including a gene encoding a tail fiber protein having the activity of recognizing a target bacterium, and which consists of an amino acid sequence shown in any of the following (a) to (c): (a) the amino acid sequence shown in SEQ ID NO: 18; (b) the amino acid sequence shown in SEQ ID NO: 18 with one or more amino acids added, deleted, and / or substituted; (c) an amino acid sequence having 99% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 18. [2] The bacteriophage according to [1], wherein the gene encoding the tail fiber protein contains a nucleotide sequence shown in any of the following (d) to (f): (d) the nucleotide sequence shown in SEQ ID NO: 19; (e) the nucleotide sequence shown in SEQ ID NO: 19 with one or more bases added, deleted, and / or substituted; (f) a nucleotide sequence having 97% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 19. [3] The bacteriophage according to [1] or [2], wherein the genomic DNA sequence comprises any of the nucleotide sequences shown in (g) to (k) below: (g) the nucleotide sequence shown in SEQ ID NO: 20; (h) the nucleotide sequence shown in SEQ ID NO: 20, in which one or more bases have been added, deleted, and / or substituted in a nucleotide sequence other than the nucleotide sequence of the gene; (i) the nucleotide sequence shown in SEQ ID NO: 20, in which a nucleotide sequence other than the nucleotide sequence of the gene has 90% or more sequence identity; (j) the nucleotide sequence shown in SEQ ID NO: 20, in which one or more bases have been added, deleted, and / or substituted; (k) a nucleotide sequence that has 95% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 20.[4] A bacteriophage having a genomic DNA sequence containing any of the nucleotide sequences shown in (a) to (c) below: (a) a nucleotide sequence shown in any of SEQ ID NOs: 1 to 7; (b) a nucleotide sequence in which one or more bases have been added, deleted, and / or substituted in the nucleotide sequence shown in any of SEQ ID NOs: 1 to 7; (c) a nucleotide sequence having 99% or more sequence identity to the nucleotide sequence shown in any of SEQ ID NOs: 1 to 7. [5] A bacteriophage having lytic activity against bacteria of the genus Salmonella, having a genomic DNA sequence containing any of the nucleotide sequences shown in (a) to (c) below: (a) a nucleotide sequence shown in SEQ ID NO: 13; (b) a nucleotide sequence in which one or more bases have been added, deleted, and / or substituted in the nucleotide sequence shown in SEQ ID NO: 13; (c) a nucleotide sequence having 90% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 13. [6] A lytic agent for Salmonella bacteria, comprising a bacteriophage having a genomic DNA sequence containing any of the following nucleotide sequences (a) to (c): (a) the nucleotide sequence shown in SEQ ID NO: 14; (b) the nucleotide sequence shown in SEQ ID NO: 14, in which one or more nucleotides have been added, deleted, and / or substituted; (c) a nucleotide sequence having 95% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 14. [7] A lytic agent for Salmonella bacteria, comprising a bacteriophage having genomic DNA containing a gene encoding an endonuclease having endonuclease activity, comprising any of the following amino acid sequences (a) to (c): (a) the amino acid sequence shown in SEQ ID NO: 15; (b) the amino acid sequence shown in SEQ ID NO: 15, in which one or more amino acids have been added, deleted, and / or substituted; (c) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 15.[8] The bacteriolytic agent according to [7], wherein the gene encoding the endonuclease contains any of the following base sequences (d) to (f): (d) the base sequence shown in SEQ ID NO: 16; (e) a base sequence in which one or more bases have been added, deleted, and / or substituted in the base sequence shown in SEQ ID NO: 16; (f) a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO: 16. [9] The bacteriolytic agent according to [7] or [8], wherein the genomic DNA sequence comprises any of the nucleotide sequences shown in (g) to (k) below: (g) the nucleotide sequence shown in SEQ ID NO: 17; (h) the nucleotide sequence shown in SEQ ID NO: 17, in which one or more nucleotides have been added, deleted, and / or substituted in a nucleotide sequence other than the nucleotide sequence of the gene; (i) the nucleotide sequence shown in SEQ ID NO: 17, in which a nucleotide sequence other than the nucleotide sequence of the gene has 80% or more sequence identity; (j) the nucleotide sequence shown in SEQ ID NO: 17, in which one or more nucleotides have been added, deleted, and / or substituted; (k) a nucleotide sequence that has 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 17.

[10] A bacteriolytic agent according to [7] or [8], wherein the genomic DNA sequence comprises a gene encoding a tailspike protein consisting of the amino acid sequence shown in SEQ ID NO: 21. A B. Enteritidis bacteriolytic agent, wherein the genomic DNA sequence comprises any of the following nucleotide sequences (a) to (e): (a) the nucleotide sequence shown in SEQ ID NO: 23; (b) the nucleotide sequence shown in SEQ ID NO: 23, in which one or more bases have been added, deleted, and / or substituted to a nucleotide sequence other than the nucleotide sequence of the gene; (c) the nucleotide sequence shown in SEQ ID NO: 23, in which a nucleotide sequence other than the nucleotide sequence of the gene has 99% or more sequence identity; (d) the nucleotide sequence shown in SEQ ID NO: 23, in which one or more bases have been added, deleted, and / or substituted; (e) a nucleotide sequence that has 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 23.

[11] The lytic agent according to

[10] , wherein the gene encoding the tailspike protein comprises the nucleotide sequence shown in SEQ ID NO: 22.

[12] The bacteriophage according to any one of [1] to [3], wherein the Salmonella bacteria are S. Enteritidis, S. Typhimurium, or S. Javana; the bacteriophage according to [5], wherein the Salmonella bacteria are S. Typhimurium; the lytic agent according to [6], wherein the Salmonella bacteria are S. Montevideo; or the lytic agent according to any one of [7] to [9], wherein the Salmonella bacteria are S. Typhimurium.

[13] A composition comprising the bacteriophage according to any one of [1] to [3], the lytic agent according to [4], the bacteriophage according to [5], the lytic agent according to [6], the lytic agent according to any one of [7] to [9], the lytic agent according to

[10] or

[11] , or the bacteriophage or lytic agent according to

[12] .

[14] A composition for controlling S. Enteritidis, S. Typhimurium, and S. Javana, comprising the bacteriophage according to any one of [1] to [3]; a composition for controlling S. Enteritidis, comprising the lytic agent according to [4]; a composition for controlling S. Typhimurium, comprising the bacteriophage according to [5]; a composition for controlling Salmonella bacteria or S., comprising the lytic agent according to [6]. a composition for controlling Montevideo; a composition for controlling Salmonella bacteria or S. Typhimurium, comprising the lytic agent according to any one of [7] to [9]; or a composition for controlling S. Enteritidis, comprising the lytic agent according to

[10] or

[11] .

[15] The composition according to

[13] or

[14] , which is a pharmaceutical composition.

[16] The composition according to

[13] or

[14] , which is a food or drink additive, a feed additive, or a drinking water additive.

[17] The composition according to

[13] or

[14] , which is a food or drink additive or a feed.

[18] The composition according to

[13] or

[14] , which is a cleaning agent, a disinfectant, a bactericide, or a sanitizer.

[19] The composition according to any one of

[13] to

[18] , further comprising another bacteriophage that exhibits lytic activity against Salmonella bacteria.

[20] A method for controlling Salmonella bacteria, comprising a contacting step of contacting an object of application with the bacteriophage according to any one of [1] to [3], the lytic agent according to [4], the bacteriophage according to [5], the lytic agent according to [6], the lytic agent according to any one of [7] to [9], the lytic agent according to

[10] or

[11] , the bacteriophage or lytic agent according to

[12] , or the composition according to any one of

[13] to

[19] .

[21] A method for treating or preventing an infection caused by Salmonella bacteria in a subject, comprising administering to the subject the bacteriophage according to any one of [1] to [3], the lytic agent according to [4], the bacteriophage according to [5], the lytic agent according to [6], the lytic agent according to any one of [7] to [9], the lytic agent according to

[10] or

[11] , the bacteriophage or lytic agent according to

[12] , or the composition according to any one of

[13] to

[19] .

[22] A method for identifying bacteria of the genus Salmonella, comprising: a culturing step of culturing test bacteria isolated from a specimen suspected of containing bacteria of the genus Salmonella to obtain a culture; a mixing step of mixing the culture with the bacteriophage described in any one of [1] to [3], the lytic agent described in [4], the bacteriophage described in [5], the lytic agent described in [6], the lytic agent described in any one of [7] to [9], the lytic agent described in

[10] or

[11] , the bacteriophage or lytic agent described in

[12] , or the composition described in any one of

[13] to

[19] to obtain a mixture; a mixture culturing step of culturing the mixture under predetermined conditions; and a determination step of determining that the test bacteria are bacteria of the genus Salmonella if the test bacteria have been lysed after the mixture culturing step.

[23] The method according to

[22] , wherein in the mixture culturing step, the mixture further comprises a soft agar-containing liquid medium, and the mixture is cultured on a solid medium.

[24] The method according to

[22] , wherein in the culturing step, the culture contains a soft agar-containing liquid medium and the culture is cultured on a solid medium.

[25] The method according to

[22] , further comprising an isolation step of isolating the test bacterium from a specimen suspected of containing Salmonella bacteria before the culturing step.This specification includes the disclosures of Japanese Patent Application Nos. 2023-057024, 2023-057035, 2023-057554, 2023-057617, 2023-057261, 2023-057268, 2023-057565, and 2023-057568, from which the present application claims priority.

[0023] The present invention can provide novel bacteriophages, lytic agents, or compositions containing them that have lytic activity against Salmonella bacteria. Alternatively, the present invention can provide bacteriophages, lytic agents, or compositions containing them that have a broad host range against Salmonella bacteria. Alternatively, the present invention can provide bacteriophages, lytic agents, or compositions containing them that can lyse specific target Salmonella bacteria. Alternatively, the present invention can provide bacteriophages, lytic agents, or compositions containing them that can effectively control S. Typhimurium, particularly multidrug-resistant S. Typhimurium.

[0024] 7 is a diagram showing the lytic activity of a first bacteriophage obtained in Example 1. FIG. 1A shows a photograph of an agar plate after culture, in which Salmonella bacteria were spread on the agar plate, and a first phage purified solution was added dropwise and statically cultured. FIG. 1B is a plate diagram corresponding to FIG. 1A, showing the strain ID of the Salmonella bacteria spread on each plate and the position of the added purified phage solution. In FIG. 1B, "a" shows the position of the purified solution of the phage having the genomic DNA sequence of SEQ ID NO: 7. FIG. 1A shows the lytic activity of a first bacteriophage obtained in Example 1. FIG. 1A shows a photograph of an agar plate after culture, in which Salmonella bacteria were spread on the agar plate, and a first phage purified solution was added dropwise and statically cultured. FIG. 1B is a plate diagram corresponding to FIG. 1A, showing the strain ID of the Salmonella bacteria spread on each plate and the position of the added purified phage solution. 1B, a, b, c, d, e, f, and g indicate the positions of the purified solution of the phage having the genomic DNA sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, and 7, respectively. This figure shows the lytic activity of the second bacteriophage obtained in Example 2. A shows a photograph of an agar plate after culture, in which Salmonella bacteria were spread on the agar plate, and the second phage purified solution was added dropwise and allowed to stand. B is a plate diagram corresponding to A, showing the strain ID of the Salmonella bacteria spread on each plate and the position of the added purified phage solution. In B, a indicates the position of the purified solution of the phage having the genomic DNA sequence of SEQ ID NO: 10. Following FIG. 3, this figure shows the lytic activity of the second bacteriophage obtained in Example 2. This figure shows the lytic activity of the second bacteriophage obtained in Example 2. 1A shows a photograph of an agar plate after static culture in which Salmonella bacteria were spread on the agar plate and the second phage purified solution was added dropwise. 1B shows a plate diagram corresponding to 1A, showing the strain ID of the Salmonella bacteria spread on each plate and the position of the added purified phage solution. 1C shows the positions of the purified phage solution having the genomic DNA sequences of SEQ ID NOs: 10, 11, and 12, respectively. 1D shows the lytic activity of the third bacteriophage obtained in Example 3.1A shows a photograph of an agar plate after culture in which Salmonella bacteria (S. Typhimurium) were spread on the plate, and the third phage purified solution was added dropwise and allowed to stand. B is a plate diagram corresponding to A, showing the strain ID of the Salmonella bacteria (S. Typhimurium) spread on each plate and the position of the added purified phage solution. In B, "a" indicates the position of the purified solution of the phage having the genomic DNA sequence of SEQ ID NO: 13. This shows the lytic activity of the fourth bacteriophage obtained in Example 4. A shows a photograph of an agar plate after culture in which Salmonella bacteria were spread on the plate, and the fourth phage purified solution was added dropwise and allowed to stand. B is a plate diagram corresponding to A, showing the strain ID of the Salmonella bacteria spread on each plate and the position of the added purified phage solution. 1B, "a" indicates the position of the purified solution of the phage having the genomic DNA sequence of SEQ ID NO: 14. This figure shows the lytic activity of the fifth bacteriophage obtained in Example 5. A shows a photograph of an agar plate after culture, in which Salmonella bacteria were spread on the agar plate, and the first phage purified solution was added dropwise and allowed to stand. B is a plate diagram corresponding to A, showing the strain ID of the Salmonella bacteria spread on each plate and the position of the added purified phage solution. B is a plate diagram corresponding to A, showing the strain ID of the Salmonella bacteria spread on each plate and the position of the added purified phage solution. B is a diagram showing the lytic activity of the sixth bacteriophage obtained in Example 6. A shows a photograph of an agar plate after culture, in which Salmonella bacteria were spread on the agar plate, and the sixth phage purified solution was added dropwise and allowed to stand. B is a plate diagram corresponding to A, showing the strain ID of the Salmonella bacteria spread on each plate and the position of the added purified phage solution. In B, a indicates the position of the purified solution of the phage having the genomic DNA sequence of SEQ ID NO: 20. It is a diagram showing the lytic activity of the seventh bacteriophage obtained in Example 7. A shows a photograph of an agar plate after culture, in which Salmonella bacteria were spread on the agar plate, and the seventh purified phage solution was added dropwise and allowed to stand. B is a plate diagram corresponding to A, showing the strain ID of the Salmonella bacteria spread on each plate and the position of the added purified phage solution.In Figure 12B, "a" indicates the position in the purified solution of the phage having the genomic DNA sequence of SEQ ID NO: 23. An alignment of the query sequence (the amino acid sequence of the tailtip protein of the obtained second phage (SEQ ID NO: 8)) and the searched sequence in Example 2 is shown. A multiple alignment performed in Example 6 is shown following Figure 12A. A multiple alignment performed in Example 6 is shown following Figure 12B.

[0025] The present invention will be described in detail below.

[0026] [Definitions] Terms used in this specification are defined below.

[0027] As used herein, "lysis" refers to the phenomenon of destroying the bacterial cell membrane. Bacteria die as a result of lysis. Lysis begins when a phage specifically adsorbs to a target bacterium and injects its own DNA into the target bacterium's cells via its tail. The phage then uses the bacterial translation mechanism to replicate itself and produce a large amount of progeny phages, which are then lysed and released into the outside world.

[0028] As used herein, the term "lytic agent" refers to a drug comprising a bacteriophage that has lytic activity against a target bacterium. The lytic agent may be a lytic agent for specifically lysing a target bacterium (a target bacterium-specific lytic agent). The lytic agent may be the bacteriophage itself.

[0029] As used herein, "bacteria" refers to one of the major lineages of organisms that divide the entire kingdom of life into three parts, along with archaea and eukaryotes. Bacteria are composed of cells without a nucleus and can self-replicate if they have a nutrient source.

[0030] As used herein, the term "target bacteria" refers to host bacteria that can be targeted by the phage constituting the bacteriolytic agent of the present invention or the phage contained in the composition of the present invention. Specifically, for example, the target bacteria are bacteria having a membrane surface receptor on the outer cell membrane that is recognized by the phage. Alternatively, for example, the target bacteria are bacteria having a membrane surface receptor on the outer cell membrane that is recognized by a tail fiber protein, tail tip protein, tail spike protein, or tail tube protein consisting of a specific amino acid sequence. The "membrane surface receptor" is a site where, for example, the tail and tail fibers of the phage bind, and is composed of proteins, lipopolysaccharides, pili, etc. present in the outer layer of the bacterial outer membrane. The target bacteria in this specification are particularly Salmonella bacteria.

[0031] As used herein, "Salmonella bacteria" refers to bacteria belonging to the genus Salmonella. Salmonella bacteria are classified into two species, Salmonella enterica and Salmonella bongori, and the former is further divided into six subspecies: ssp. enterica, ssp. salamae, ssp. arizonae, ssp. diarizonae, ssp. houtenae, and ssp. indica. Salmonella bacteria are also serotyped based on two types of surface structures: somatic antigens (also called O antigens) and flagellar antigens (also called H antigens). The subspecies and serotype of Salmonella bacteria are indicated by adding "subspecies" (ssp.) and "serovar" (or "serotype") after the name of the bacteria. The name of Salmonella bacteria may be abbreviated by adding the serotype after "S." For example, S. enterica ssp. enterica serovar Typhimurium may be abbreviated as S. Typhimurium. The smallest unit of classification is the strain, which refers to a population of cells that is considered to be genetically uniform.

[0032] Specific serotypes of Salmonella bacteria include, for example, S. Enteritidis (Salmonella enterica ssp. enterica serovar Enteritidis), S. Typhimurium (Salmonella enterica ssp. enterica serovar Typhimurium), S. Newport, S. I 4,[5],12:i:-, S. Javiana (Salmonella enterica ssp. enterica serovar Javiana), S. Heidelberg, S. Infantis (Salmonella enterica ssp. enterica serovar Infantis), S. Saintpaul, S. Muenchen, S. Montevideo (Salmonella enterica ssp. enterica serovar Montevideo), S. Braenderup, S. Oranienburg, S. Thompson, S. Mississippi, S. Agona, S. Typhi, S. Bareilly, S. Paratyphi B, S. Poona, S. Bertha, S. Abony, S. Anatum, S. Baird, S. Bredeney, S. Chester, S. Gamirara, S. Hartford, S. Kentucky, S. Kiambu, S. Mbandaka, S. Nchanga, S. Reading, S. Senftenberg, S. Stanley, S. Virchow, S. Urbana, etc.

[0033] As used herein, the term "Salmonella lytic agent" refers to a lytic agent for lysing Salmonella bacteria. Similarly, the term "S. Enteritidis lytic agent" refers to a lytic agent for lysing S. Enteritidis. The S. Enteritidis lytic agent may be a lytic agent for specifically lysing S. Enteritidis (an S. Enteritidis-specific lytic agent). The term "S. Montevideo lytic agent" refers to a lytic agent for lysing S. Montevideo. The S. Montevideo lytic agent may be a lytic agent for specifically lysing S. Montevideo (an S. Montevideo-specific lytic agent). "S. Typhimurium lytic agent" refers to a lytic agent for lysing S. Typhimurium. The S. Typhimurium lytic agent may be a lytic agent for specifically lysing S. Typhimurium (an S. Typhimurium-specific lytic agent).

[0034] As used herein, "controlling" bacteria means killing bacteria and / or inhibiting bacterial growth.

[0035] As used herein, the term "multidrug resistance" refers to resistance to multiple antibacterial agents (e.g., antibiotics). Examples of antibacterial agents include, but are not limited to, ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline, kanamycin, sulfamethoxazole / trimethoprim, cefazolin, cefotaxime, nalidixic acid, and gentamicin.

[0036] As used herein, "bacteriophage" (as mentioned above, often simply referred to as "phage" herein) is a general term for viruses that infect bacteria. A typical phage is composed of three parts: a head, a tail, and tail fiber. The head is composed of a capsomere, which is an outer coat protein, and consists of a capsid (virus shell) with an icosahedral structure, encapsulating the phage's genomic DNA in its internal space. The tail has a tubular structure composed of a tail tube protein and a sheath protein that covers it. One end of the tail is connected to the head, and the other end is connected to the tail fiber. The tail functions as an introduction tube that injects the genomic DNA of the head into the cell of the host bacterium. The tail fiber is composed of several fibrous structures made of tail fiber protein. The tail and tail fibers are responsible for host recognition and adsorption functions, recognizing receptors present on the outer membrane surface of the host bacterium and adsorbing to the cell surface. Phages have extremely high host specificity, a characteristic of which is based on the function of the tail and tail fibers. More specifically, any of the following proteins—tail fiber protein, tail tube protein, tail tip protein, and tail spike protein—plays a central role in this function.

[0037] As used herein, the term "tail fiber protein" refers to a protein that constitutes the tail fiber of a phage, as described above. It is known that tail fiber proteins play an important role in the specificity of the host recognition and adsorption ability of the tail and tail fiber (Nobrega F.L. et al., Nat. Rev. Microbiol., 2018, 16:760-773). Therefore, even if the host bacterium is the same as that of a known phage, novel phages having characteristics in a tail fiber protein have a different host recognition site, making them highly useful, as they can exhibit bacteriolytic activity even against bacteria that are resistant to infection by the known phages.

[0038] As used herein, the term "tail fiber gene" refers to a gene contained in the genomic DNA of a phage and encoding the tail fiber protein.

[0039] As used herein, "tail tube protein" refers to a protein that constitutes the tubular structure of the phage tail, as described above. It is known that tail tube proteins interact with the tail fibers and, together with the tail fibers, play an important role in the specificity of host recognition and adsorption (Maozhi Hu, et al., 2020, 9:1, 855-867). Known tail tube proteins include tail tube fiber protein A and tail tube protein B. "Tail tube protein A" is a protein that forms a ring at the lower part of the tubular structure of the tail and interacts with the tail fiber. "Tail tube protein B" is a protein that forms the lower end of the tubular structure of the tail and binds to a receptor present on the outer membrane surface of the host bacterium.

[0040] As used herein, the term "tailtube gene" refers to a gene contained in the genomic DNA of a phage and encoding the tailtube protein. The term "tailtube protein A gene" refers to the gene encoding tailtube protein A, and the term "tailtube protein B gene" refers to the gene encoding tailtube protein B.

[0041] As used herein, the term "tailtip protein" refers to a protein that constitutes the tip of the tail of a phage. Its sharp structure plays a role in penetrating the cell wall of a host bacterium, but as described above, it also has the function of binding to a receptor on the host bacterium. Because it has the function of binding to a receptor on the host bacterium, the tailtip protein is known to play an important role in host recognition and adsorption (Nobrega F.L. et al., Nat. Rev. Microbiol., 2018, 16:760-773).

[0042] As used herein, the term "tailtip gene" refers to a gene contained in the genomic DNA of a phage and encoding the tailtip protein.

[0043] As used herein, the term "tail spike protein" refers to a protein that constitutes the tip of the phage tail and, as described above, has the function of binding to a receptor on the host bacterium. When a dish-shaped structure (tail plate) is present at the tip of the phage tail, the tail spike protein forms a spike-like structure at the bottom of the plate. Because the tail spike protein has the function of binding to a receptor on the host bacterium, it is known to play an important role in host recognition and adsorption (Nobrega F.L. et al., Nat. Rev. Microbiol., 2018, 16:760-773).

[0044] As used herein, the term "tailspike gene" refers to a gene contained in the genomic DNA of a phage and encoding the tailspike protein.

[0045] Note that a phage does not necessarily have all of the above-mentioned tail fiber gene, tail tube gene, tail tip gene, and tail spike gene, but may include one, two, three, or all four of the tail fiber gene, tail tube gene, tail tip gene, and tail spike gene.

[0046] As used herein, the term "endonuclease" refers to an enzyme that cleaves a polynucleotide chain within a polynucleotide chain. Upon infection, lytic phages take over the life support mechanisms of the host bacterium by various means, allowing only their own replication, but are known to shut down replication of the host genome at the same time. Although the details of this mechanism are still unclear, it has long been known that degradation of the host genome by phage-derived nuclease is involved (Warren et al., Journal of Virology, Vol. 2, No. 4, 1968). Therefore, it is thought that lytic phage endonucleases are involved in the mechanism that shuts down replication of the host genome.

[0047] Since phages do not infect eukaryotes, drugs using phages are harmless to humans, animals, and plants. The life cycle of a phage is broadly divided into a "lytic cycle," a "lysogenic cycle," and a "lytic / lysogenic cycle." In the lysogenic cycle, the phage integrates its own DNA into the bacterial chromosome without lysing the target bacterium, and grows along with the growth of the bacterium. On the other hand, in the lytic cycle, the phage self-multiplies within the cells of the host bacterium, then lyses the host bacterium and releases a large amount of progeny phages. The phage of the present invention may be a phage that undergoes a lytic cycle or a lytic / lysogenic cycle.

[0048] As used herein, "plurality" refers to 2 to 10, for example, 2 to 7, 2 to 5, 2 to 4, or 2 to 3.

[0049] As used herein, "nucleotide sequence identity" refers to a numerical value indicating the proportion of sites with the same type of base within the comparison range of two nucleotide sequences. Even when the lengths of the two nucleotide sequences are different, nucleotide sequence identity can be calculated by aligning the sequences so that the degree of base identity within the comparison range is highest. A representative algorithm for such analysis is BLAST, although not limited thereto. BLAST can be used in various software and web services. For example, nucleotide sequence identity can be easily calculated using the genetic information processing software GENETYX (https: / / www.genetyx.co.jp / ) or the NCBI BLAST server (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). In addition to BLAST, there are also algorithms such as FASTA, which can be used if they can calculate reasonable identity. Analysis algorithms such as MUMmer can also be used to analyze nucleotide sequence identity. Depending on the software or analysis server, an index of sequence identity may be expressed as Average Nucleotide Identity (ANI), and these may also be used. When the above-mentioned software or web service aligns long base sequences such as phage genomic DNA, the comparison range may be automatically determined and the sequence identity within the comparison range may be calculated. Therefore, the above-mentioned sequence identity may be present within the range automatically aligned by the above-mentioned software or web service. For example, in analysis using the BLAST server provided by NCBI, the query sequence and subject sequence are automatically aligned within the maximum possible range to determine the comparison range, and the sequence identity within the comparison range is calculated. In addition, the ratio of the comparison range to the entire range of the query sequence may be calculated as a value called Query Cover. In such cases, the sequence identity within the entire range of the aligned base sequences may also be estimated based on the results. For example, the Query Cover value may be multiplied by the sequence identity value in the comparison range to obtain an estimate of sequence identity in the entire range.In this case, to improve the accuracy of the estimate, further corrections may be made, such as by calculating expected sequence identity in a range outside the alignment range. Phage genomic DNA packaging can be linear or circular. Furthermore, in next-generation genome sequencer analysis, the genomic DNA is fragmented, the base sequences of the individual fragments are read, and the sequence is determined through analysis that connects them. In the case of phages, the DNA is often connected without a reference genomic DNA sequence (de novo assembly). Therefore, it is difficult to unambiguously determine the start and end of the analyzed genome (Merrill, B.D., et al. BMC Genomics, 2016 17, 679). Therefore, the start and end of the genome sequences to be compared may be different, and this is automatically taken into account in analysis using software or analysis servers.

[0050] As used herein, "highly stringent conditions" refers to environmental conditions that make it difficult for nonspecific hybridization to occur. Under highly stringent conditions, a hybrid can be formed with a nucleic acid having a target nucleotide sequence, but a hybrid cannot be substantially formed with a nucleic acid having a nonspecific nucleotide sequence. Generally, highly stringent conditions refer to conditions with a low salt concentration and a high temperature. A low salt concentration refers to, for example, 15 to 750 mM, preferably 15 to 500 mM, 15 to 300 mM, or 15 to 200 mM. Furthermore, a high temperature refers to, for example, 50 to 68°C or 55 to 70°C. A specific example of highly stringent conditions is a condition in which post-hybridization washing is performed at 65°C with 0.1xSSC and 0.1% SDS.

[0051] As used herein, "amino acid sequence identity" refers to a numerical value indicating the proportion of sites in which the type of amino acid residue is the same within the comparison range of two amino acid sequences. Even when the lengths of the two amino acid sequences are different, amino acid sequence identity can be calculated by aligning the sequences so that the degree of amino acid identity within the comparison range is highest. A representative algorithm for such analysis is BLAST, although not limited thereto. BLAST can be used in various software and web services. For example, amino acid sequence identity can be easily calculated using the genetic information processing software GENETYX (https: / / www.genetyx.co.jp / ) or the NCBI BLAST server (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). In addition to BLAST, there is also an algorithm called FASTA, which can be used if it can calculate a reasonable identity.

[0052] As used herein, "(amino acid) substitution" preferably refers to a substitution within a conservative amino acid group that has similar properties, such as charge, side chain, polarity, and aromaticity, among the 20 types of amino acids that constitute natural proteins. Examples include substitutions within the group of uncharged polar amino acids with low polarity side chains (Gly, Asn, Gln, Ser, Thr, Cys, Tyr), branched-chain amino acids (Leu, Val, Ile), neutral amino acids (Gly, Ile, Val, Leu, Ala, Met, Pro), neutral amino acids with hydrophilic side chains (Asn, Gln, Thr, Ser, Tyr, Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), and aromatic amino acids (Phe, Tyr, Trp). Substitutions may occur singly or in combination. Amino acid substitutions within these groups are preferred because they are known to be less likely to cause changes in the properties of the polypeptide.

[0053] 1. Bacteriophage / Lysic Agent A first aspect of the present invention relates to the following bacteriophage that exhibits lytic activity against bacteria of the genus Salmonella and a lytic agent comprising the same.

[0054] <First Phage / Lysic Agent> (Summary) The present invention provides a phage (sometimes referred to herein as a "first phage") having the following configuration and having lytic activity against bacteria of the genus Salmonella, and a lytic agent (sometimes referred to herein as a "first lytic agent") comprising the same. The first lytic agent is a lytic agent that exhibits specific lytic activity against particular bacteria of the genus Salmonella, for example, a lytic agent for S. Enteritidis. The first lytic agent comprises a bacteriophage having a genomic DNA sequence that includes a specific base sequence.

[0055] The first lytic agent can lyse and control the target bacteria.

[0056] (Configuration) The first lytic agent is a lytic agent for bacteria of the genus Salmonella, particularly a lytic agent for S. Enteritidis. The first lytic agent comprises a first phage having lytic activity against bacteria of the genus Salmonella and having the following configuration:

[0057] The first phage has a genomic DNA sequence that includes a specific base sequence.

[0058] The present inventors discovered seven phages that have bacteriolytic activity specific to S. Enteritidis bacterial strains, and found that the genomic DNA sequences of these phages (SEQ ID NOS: 1 to 7, respectively) have extremely high sequence identity. For example, using the genetic information processing software GENETYX (https: / / www.genetyx.co.jp / ), the sequence identity of the shortest genomic DNA sequence, SEQ ID NOS: 7, to the genomic DNA sequences, SEQ ID NOS: 1 to 6, was calculated to be 100% across the entire range.

[0059] The first phage has a genomic DNA sequence that includes or consists of any of the nucleotide sequences shown in (a) to (c) below: (a) a nucleotide sequence shown in any of SEQ ID NOs: 1 to 7; (b) a nucleotide sequence in which one or more nucleotides have been added, deleted, and / or substituted in the nucleotide sequence shown in any of SEQ ID NOs: 1 to 7; (c) a nucleotide sequence that has 99% or more sequence identity with the nucleotide sequence shown in any of SEQ ID NOs: 1 to 7.

[0060] Preferably, the sequence identity defined in (c) is 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.

[0061] The first phage is capable of exhibiting broad lytic activity against S. Enteritidis, the serotype most frequently detected in human food poisoning, and is therefore useful for treating or preventing food poisoning. The first phage is also capable of exhibiting lytic activity specifically against S. Enteritidis, and is therefore useful, for example, for identifying the serotype of bacteria causing food poisoning.

[0062] <Second Phage / Lysic Agent> (Summary) The present invention provides a phage (sometimes referred to herein as a "second phage") having the following configuration and having lytic activity against bacteria of the genus Salmonella, and a lytic agent (sometimes referred to herein as a "second lytic agent") comprising the same. The second phage can exhibit lytic activity against S. Enteritidis, S. Typhimurium, S. Infantis, S. Montevideo, and S. Javiana. The second phage has a genomic DNA sequence including a gene encoding a tailtip protein consisting of a specific amino acid sequence.

[0063] The second phage can lyse and control Salmonella bacteria as target bacteria.

[0064] (Configuration) The second phage has the following configuration and has lytic activity against bacteria of the genus Salmonella.

[0065] The second phage has genomic DNA containing a gene encoding a tail tip protein consisting of a specific amino acid sequence and having the activity of recognizing a target bacterium.

[0066] The present inventors discovered three phages that have lytic activity against Salmonella bacteria, and identified the tailtip protein (SEQ ID NO: 8) and tailtip gene (SEQ ID NO: 9) from the genomic DNA sequences of these phages (SEQ ID NOs: 10 to 12, respectively). The genomic sequences of the three phages share 99% sequence identity, and the amino acid sequences of the tailtip proteins are shown in SEQ ID NO: 8, which are completely identical to each other.

[0067] The tailtip protein consists of 637 amino acid residues and has the amino acid sequence shown in SEQ ID NO: 8. In the present invention, the tailtip protein consisting of the amino acid sequence shown in SEQ ID NO: 8 can achieve extremely useful host specificity, which is specific to bacteria of the genus Salmonella and also exhibits bacteriolytic activity against a wide range of bacterial species within the genus Salmonella.

[0068] (1) Tailtip protein The tailtip protein in the second phage consists of an amino acid sequence shown in any of the following (a) to (c): (a) the amino acid sequence shown in SEQ ID NO: 8; (b) an amino acid sequence in which one or more amino acids have been added, deleted, and / or substituted in the amino acid sequence shown in SEQ ID NO: 8; (c) an amino acid sequence having 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 8.

[0069] The sequence identity defined in (c) is preferably 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.

[0070] In the amino acid sequence defined in (b) or (c), it is preferred that the amino acid at the position corresponding to the 258th amino acid in SEQ ID NO: 8 of the tailtip protein is phenylalanine and / or the amino acid at the position corresponding to the 617th amino acid in SEQ ID NO: 8 is serine. Note that the position numbers are expressed with the initiating methionine as the first position.

[0071] (2) Tailtip gene The gene encoding the tailtip protein includes, for example, any of the nucleotide sequences shown in (d) to (f) below: (d) the nucleotide sequence shown in SEQ ID NO: 9; (e) a nucleotide sequence in which one or more nucleotides have been added, deleted, and / or substituted in the nucleotide sequence shown in SEQ ID NO: 9; (f) a nucleotide sequence having 95% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 9.

[0072] Alternatively, the base sequence may be a base sequence that hybridizes under highly stringent conditions to a base sequence complementary to the base sequence shown in SEQ ID NO: 9.

[0073] The sequence identity defined in (f) is preferably 96% or more, 97% or more, 98% or more, or 99% or more.

[0074] (3) Genomic DNA The second phage has genomic DNA containing a gene encoding a tailtip protein.

[0075] The genomic DNA sequence may, for example, comprise or consist of any of the nucleotide sequences shown in (g) to (k) below: (g) a nucleotide sequence shown in any of SEQ ID NOs: 10 to 12; (h) a nucleotide sequence in which one or more bases have been added, deleted, and / or substituted in a nucleotide sequence other than the nucleotide sequence of the gene in the nucleotide sequence shown in any of SEQ ID NOs: 10 to 12; (i) a nucleotide sequence in which a nucleotide sequence other than the nucleotide sequence of the gene in the nucleotide sequence shown in any of SEQ ID NOs: 10 to 12 has 80% or more sequence identity; (j) a nucleotide sequence in which one or more bases have been added, deleted, and / or substituted in the nucleotide sequence shown in any of SEQ ID NOs: 10 to 12; (k) a nucleotide sequence that has 90% or more sequence identity to the nucleotide sequence shown in any of SEQ ID NOs: 10 to 12.

[0076] The sequence identity defined in (i) is 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 90.5% or more, 91.0% or more, 91.5% or more, 92.0% or more, 92.5% or more, 93.0% or more, 93.5% or more, 94.0% or more, 94.5% or more, 95.0% or more, 96.0% or more, 97.0% or more, 98.0% or more, 99.0% or more, 100% or more, 101% or more, 102% or more, 103% or more, 104% or more, 105% or more, 106% or more, 107% or more, 108% or more, 109% or more, 110% or more, 111% or more, 112% or more, 113% or more, 114% or more, 115% or more, 116% or more, 117% or more, 118% or more, 119% or more, 120% or more, 121% or more, 122% or more, 123% or more, 124% or more, 125% or more, 126% or more, 127% or more, 128% or more, 129% or more, 130% or more, 131% or more, 132% or more, 133% or more, 134% or more, 135% or more, 136% or more, 137% or more, 1 % or more, 95.5% or more, 96.0% or more, 96.5% or more, 97.0% or more, 97.5% or more, 98.0% or more, 98.5% or more, 99.0% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more is preferred.

[0077] The base sequence defined in (i) is, in other words, a base sequence in which the sequence identity of the base sequence other than the gene corresponding to the gene in the base sequence shown in any one of SEQ ID NOs: 10 to 12 is 80% or more.

[0078] The sequence identity defined in (k) is preferably 90.5% or more, 91.0% or more, 91.5% or more, 92.0% or more, 92.5% or more, 93.0% or more, 93.5% or more, 94.0% or more, 94.5% or more, 95.0% or more, 95.5% or more, 96.0% or more, 96.5% or more, 97.0% or more, 97.5% or more, 98.0% or more, 98.5% or more, 99.0% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.

[0079] In one embodiment, the second phage is characterized by having a genomic DNA sequence containing a specific nucleotide sequence and exhibits bacteriolytic activity against target bacteria. Examples of the genomic DNA sequence of the second phage include a nucleotide sequence shown in any of SEQ ID NOs: 10 to 12 (113946 bp, 113936 bp, and 113949 bp, respectively), a nucleotide sequence in which one or more nucleotides have been added, deleted, and / or substituted in the nucleotide sequence shown in any of SEQ ID NOs: 10 to 12, and a nucleotide sequence that is 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, or 90.5% or more of the nucleotide sequence shown in any of SEQ ID NOs: 10 to 12. , 91.0% or more, 91.5% or more, 92.0% or more, 92.5% or more, 93.0% or more, 93.5% or more, 94.0% or more, 94.5% or more, 95.0% or more, 95.5% or more, 96.0% or more, 96.5% or more, 97.0% or more, 97.5% or more, 98.0% or more, 98.5% or more, 99.0% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more sequence identity.

[0080] (4) Effects The second phage can exhibit bacteriolytic activity against a wide range of Salmonella species, thereby effectively controlling Salmonella bacteria. The second phage is also useful for treating or preventing food poisoning. The second phage has a wide host range, so it can effectively cover the diversity of target bacteria. Therefore, phages that exhibit bacteriolytic activity against a wide range of bacterial species, such as the second phage, are extremely useful.

[0081] <Third Phage / Lysic Agent> (Summary) The present invention provides a phage (sometimes referred to herein as a "third phage") having the following configuration and having lytic activity against bacteria of the genus Salmonella, and a lytic agent (sometimes referred to herein as a "third lytic agent") comprising the same. The third phage exhibits lytic activity against at least one bacterium selected from the group consisting of S. Typhimurium, and preferably exhibits lytic activity against multidrug-resistant S. Typhimurium. The third phage has a specific genomic DNA sequence.

[0082] The target bacterium of the third phage is not limited to S. Typhimurium. The third phage can effectively control S. Typhimurium, particularly multidrug-resistant S. Typhimurium, but may also be effective against other serotypes of target bacteria, such as S. Enteritidis, S. Infantis, or S. Javiana.

[0083] The third phage can effectively lyse and control target bacteria, such as S. Typhimurium, particularly multidrug-resistant S. Typhimurium.

[0084] (Configuration) The third phage has the following configuration and has bacteriolytic activity against bacteria of the genus Salmonella.

[0085] The third phage has genomic DNA containing a specific base sequence.

[0086] The present inventors have discovered a phage having lytic activity against bacteria of the genus Salmonella, and identified the genomic DNA sequence of this phage (SEQ ID NO: 13).

[0087] The third phage is highly useful because it is specific to bacteria of the genus Salmonella and exhibits broad bacteriolytic activity against S. Typhimurium, which is particularly problematic among Salmonella bacteria due to the large number of multidrug-resistant strains.

[0088] The third phage has genomic DNA that includes or consists of any of the following nucleotide sequences (a) to (c): (a) the nucleotide sequence shown in SEQ ID NO: 13; (b) a nucleotide sequence in which one or more nucleotides have been added, deleted, and / or substituted in the nucleotide sequence shown in SEQ ID NO: 13; (c) a nucleotide sequence that has 90% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 13.

[0089] The sequence identity defined in (c) is preferably 90.5% or more, 91.0% or more, 91.5% or more, 92.0% or more, 92.5% or more, 93.0% or more, 93.5% or more, 94.0% or more, 94.5% or more, 95.0% or more, 95.5% or more, 96.0% or more, 96.5% or more, 97.0% or more, 97.5% or more, 98.0% or more, 98.5% or more, 99.0% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.

[0090] The third phage can exhibit broad bacteriolytic activity against Salmonella bacteria, particularly S. Typhimurium, which is problematic due to the large number of multidrug-resistant strains, and is therefore useful for treating or preventing food poisoning.

[0091] <Fourth Phage / Lysing Agent> (Summary) The present invention provides a phage (sometimes referred to herein as a "fourth phage") having the following configuration and having lytic activity against bacteria of the genus Salmonella, and a lytic agent (sometimes referred to herein as a "fourth lytic agent") comprising the same. The fourth lytic agent is a lytic agent that exhibits specific lytic activity against particular bacteria of the genus Salmonella, for example, a lytic agent for S. Montevideo. The fourth lytic agent comprises a bacteriophage having a genomic DNA sequence that includes a specific base sequence.

[0092] The fourth bacteriolytic agent can lyse and control target bacteria.

[0093] (Configuration) The fourth bacteriolytic agent is particularly a bacteriolytic agent for bacteria of the genus Salmonella, particularly a bacteriolytic agent for S. Montevideo. The fourth bacteriolytic agent comprises a phage having the following configuration and having bacteriolytic activity against bacteria of the genus Salmonella:

[0094] The fourth phage has a genomic DNA sequence that includes a specific base sequence.

[0095] The present inventors discovered a phage having lytic activity specific to bacterial strains of S. Montevideo, and identified the genomic DNA sequence of the phage (SEQ ID NO: 14).

[0096] The fourth phage has a genomic DNA sequence that includes or consists of any of the following nucleotide sequences (a) to (c): (a) the nucleotide sequence shown in SEQ ID NO: 14; (b) the nucleotide sequence shown in SEQ ID NO: 14 in which one or more nucleotides have been added, deleted, and / or substituted; (c) a nucleotide sequence that has 95% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 14.

[0097] Preferably, the sequence identity defined in (c) is 95.5% or more, 96.0% or more, 96.5% or more, 97.0% or more, 97.5% or more, 98.0% or more, 98.5% or more, 99.0% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.

[0098] The fourth phage can exhibit bacteriolytic activity specifically against bacteria of the genus Salmonella, particularly S. Montevideo, and is therefore useful, for example, for identifying the serotype of bacteria that cause food poisoning.

[0099] <Fifth Phage / Lysing Agent> (Summary) The present invention provides a phage (sometimes referred to herein as a "fifth phage") having the following configuration and having lytic activity against bacteria of the genus Salmonella, and a lytic agent comprising the same (sometimes referred to herein as a "fifth lytic agent"). The fifth lytic agent is a lytic agent that exhibits specific lytic activity against particular bacteria of the genus Salmonella, for example, a lytic agent for S. Typhimurium. The fifth lytic agent comprises a bacteriophage having genomic DNA that includes a gene encoding an endonuclease consisting of a specific amino acid sequence.

[0100] The fifth bacteriolytic agent can lyse and control target bacteria.

[0101] (Configuration) The fifth bacteriolytic agent is a bacteriolytic agent for bacteria of the genus Salmonella, particularly a bacteriolytic agent for S. Typhimurium. The fifth bacteriolytic agent comprises a phage having lytic activity against bacteria of the genus Salmonella and having the following configuration:

[0102] The fifth phage has genomic DNA containing a gene encoding an endonuclease having a specific amino acid sequence and endonuclease activity.

[0103] The present inventors discovered a phage having lytic activity specific to S. Typhimurium and identified the genomic DNA sequence of the phage (SEQ ID NO: 17). The present inventors further identified a novel endonuclease gene from the genomic DNA sequence of the phage. The amino acid sequence of the endonuclease and the nucleotide sequence encoding it are shown in SEQ ID NOs: 15 and 16, respectively. The endonuclease is involved in shutting down replication of the host genome, thereby enhancing lytic activity.

[0104] (1) Endonuclease The endonuclease in the fifth phage consists of any of the following amino acid sequences (a) to (c): (a) the amino acid sequence shown in SEQ ID NO: 15; (b) an amino acid sequence in which one or more amino acids have been added, deleted, and / or substituted in the amino acid sequence shown in SEQ ID NO: 15; (c) an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 15.

[0105] The sequence identity defined in (c) is preferably 90.5% or more, 91.0% or more, 91.5% or more, 92.0% or more, 92.5% or more, 93.0% or more, 93.5% or more, 94.0% or more, 94.5% or more, 95.0% or more, 95.5% or more, 96.0% or more, 96.5% or more, 97.0% or more, 97.5% or more, 98.0% or more, 98.5% or more, 99.0% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.

[0106] (2) Endonuclease Gene The gene encoding the endonuclease includes, for example, any of the nucleotide sequences shown in (d) to (f) below: (d) the nucleotide sequence shown in SEQ ID NO: 16; (e) a nucleotide sequence in which one or more nucleotides have been added, deleted, and / or substituted in the nucleotide sequence shown in SEQ ID NO: 16; (f) a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 16. Alternatively, the gene may be a nucleotide sequence that hybridizes under highly stringent conditions to a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 16.

[0107] The sequence identity defined in (f) is preferably 90.5% or more, 91.0% or more, 91.5% or more, 92.0% or more, 92.5% or more, 93.0% or more, 93.5% or more, 94.0% or more, 94.5% or more, 95.0% or more, 95.5% or more, 96.0% or more, 96.5% or more, 97.0% or more, 97.5% or more, 98.0% or more, 98.5% or more, 99.0% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.

[0108] (3) Genomic DNA The fifth phage has genomic DNA containing a gene encoding an endonuclease.

[0109] The genomic DNA sequence comprises or consists of, for example, any of the nucleotide sequences shown in (g) to (k) below: (g) the nucleotide sequence shown in SEQ ID NO: 17; (h) a nucleotide sequence in which one or more bases have been added, deleted, and / or substituted in a nucleotide sequence other than the nucleotide sequence of the gene in the nucleotide sequence shown in SEQ ID NO: 17; (i) a nucleotide sequence in which the nucleotide sequence other than the nucleotide sequence of the gene in the nucleotide sequence shown in SEQ ID NO: 17 has 80% or more sequence identity; (j) a nucleotide sequence in which one or more bases have been added, deleted, and / or substituted in the nucleotide sequence shown in SEQ ID NO: 17; (k) a nucleotide sequence that has 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 17.

[0110] The sequence identity defined in (i) is 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 90.5% or more, 91.0% or more, 91.5% or more, 92.0% or more, 92.5% or more, 93.0% or more, 93.5% or more, 94.0% or more, 94.5% or more, 95.0% or more, 96.0% or more, 97.0% or more, 98.0% or more, 99.0% or more, 100% or more, 101% or more, 102% or more, 103% or more, 104% or more, 105% or more, 106% or more, 107% or more, 108% or more, 109% or more, 110% or more, 111% or more, 112% or more, 113% or more, 114% or more, 115% or more, 116% or more, 117% or more, 118% or more, 119% or more, 120% or more, 121% or more, 122% or more, 123% or more, 124% or more, 125% or more, 126% or more, 127% or more, 128% or more, 129% or more, 130% or more, 131% or more, 132% or more, 133% or more, 134% or more, 135% or more, 136% or more, 137% or more, 1 % or more, 95.5% or more, 96.0% or more, 96.5% or more, 97.0% or more, 97.5% or more, 98.0% or more, 98.5% or more, 99.0% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more is preferred.

[0111] The base sequence defined in (i) is, in other words, a base sequence in which the sequence identity of the base sequence other than the gene corresponding to the gene in the base sequence shown in SEQ ID NO: 17 is 80% or more to the base sequence other than the gene base sequence.

[0112] The sequence identity defined in (k) is preferably 90.5% or more, 91.0% or more, 91.5% or more, 92.0% or more, 92.5% or more, 93.0% or more, 93.5% or more, 94.0% or more, 94.5% or more, 95.0% or more, 95.5% or more, 96.0% or more, 96.5% or more, 97.0% or more, 97.5% or more, 98.0% or more, 98.5% or more, 99.0% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.

[0113] In one embodiment, the fifth phage has a genomic DNA sequence containing a specific base sequence and exhibits bacteriolytic activity against target bacteria. Examples of the genomic DNA sequence of the fifth phage include the base sequence shown in SEQ ID NO: 17 (47638 bp), a base sequence in which one or more bases are added, deleted, and / or substituted in the base sequence shown in SEQ ID NO: 17, and a base sequence which is 90% or more, 90.5% or more, 91.0% or more, 91.5% or more, 92.0% or more, 92.5% or more, 93.0% or more, 93.5% or more, or 94.0% or more similar to the base sequence shown in SEQ ID NO: 17. , 94.5% or more, 95.0% or more, 95.5% or more, 96.0% or more, 96.5% or more, 97.0% or more, 97.5% or more, 98.0% or more, 98.5% or more, 99.0% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more sequence identity.

[0114] The fifth phage can exhibit bacteriolytic activity specifically against bacteria of the genus Salmonella, particularly S. Typhimurium, and is therefore useful, for example, for identifying the serotype of bacteria that cause food poisoning.

[0115] <Sixth Phage / Lysic Agent> (Summary) The present invention provides a phage (sometimes referred to herein as the "sixth phage") having the following configuration and having lytic activity against bacteria of the genus Salmonella, and a lytic agent (sometimes referred to herein as the "sixth lytic agent") comprising the same. The sixth phage can exhibit lytic activity against S. Enteritidis, S. Typhimurium, and S. Javana. The sixth phage has a genomic DNA sequence including a gene encoding a tail fiber protein consisting of a specific amino acid sequence.

[0116] The sixth phage can lyse and control Salmonella bacteria as target bacteria.

[0117] (Configuration) The sixth phage has the following configuration and has bacteriolytic activity against bacteria of the genus Salmonella.

[0118] The sixth phage has genomic DNA containing a gene encoding a tail fiber protein consisting of a specific amino acid sequence and having the activity of recognizing a target bacterium.

[0119] The present inventors discovered a phage that has lytic activity against Salmonella bacteria, and identified a tail fiber protein (SEQ ID NO: 18) and a tail fiber gene (SEQ ID NO: 19) from the genomic DNA sequence (SEQ ID NO: 20) of this phage.

[0120] The tail fiber protein consists of 684 amino acid residues and has the amino acid sequence shown in SEQ ID NO: 18. In the present invention, the tail fiber protein consisting of the amino acid sequence shown in SEQ ID NO: 18 is specific to bacteria of the genus Salmonella and exhibits bacteriolytic activity against bacteria of the genus Salmonella, particularly S. Enteritidis, S. Typhimurium, and S. Javana, thereby achieving extremely useful host specificity.

[0121] (1) Tail fiber protein The tail fiber protein in the sixth phage consists of any of the following amino acid sequences (a) to (c): (a) the amino acid sequence shown in SEQ ID NO: 18; (b) an amino acid sequence in which one or more amino acids have been added, deleted, and / or substituted in the amino acid sequence shown in SEQ ID NO: 18; (c) an amino acid sequence having 99% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 18.

[0122] The sequence identity defined in (c) is preferably 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.

[0123] The amino acid sequence of the tail fiber protein of the sixth phage contains multiple unique amino acid residues that differ from the amino acid sequences of known tail fiber proteins. In the amino acid sequence of the tail fiber protein of the sixth phage (SEQ ID NO: 18), the amino acid corresponding to position 211 is Val, the amino acid corresponding to position 321 is Val, the amino acid corresponding to position 485 is Val, the amino acid corresponding to position 533 is Ala, the amino acid corresponding to position 577 is Ser, and the amino acid corresponding to position 583 is Ser. It is surprising that many of these positions are highly conserved in the tail fiber proteins of other phages, yet contain different amino acid residues, and this is thought to be related to the distinctive host range of the sixth phage. Therefore, in the amino acid sequence defined by (b) or (c), it is preferred that the amino acid corresponding to position 211 is Val, the amino acid corresponding to position 321 is Val, the amino acid corresponding to position 485 is Val, the amino acid corresponding to position 533 is Ala, the amino acid corresponding to position 577 is Ser, and / or the amino acid corresponding to position 583 is Ser. Note that the position numbers are expressed with the initiating methionine as the first position.

[0124] (2) Tail Fiber Gene The gene encoding the tail fiber protein includes, for example, any of the nucleotide sequences shown in (d) to (f) below: (d) the nucleotide sequence shown in SEQ ID NO: 19; (e) a nucleotide sequence in which one or more nucleotides have been added, deleted, and / or substituted in the nucleotide sequence shown in SEQ ID NO: 19; (f) a nucleotide sequence having 97% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 19.

[0125] Alternatively, the base sequence may be a base sequence that hybridizes under highly stringent conditions to a base sequence complementary to the base sequence shown in SEQ ID NO: 19.

[0126] The sequence identity defined in (f) is preferably 97.5% or more, 98.0% or more, 98.5% or more, 99.0% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.

[0127] (3) Genomic DNA The sixth phage has genomic DNA containing a gene encoding a tail fiber protein. The genomic DNA sequence includes, for example, any of the nucleotide sequences shown in (g) to (k) below: (g) the nucleotide sequence shown in SEQ ID NO:20; (h) a nucleotide sequence in which one or more bases have been added, deleted, and / or substituted in a nucleotide sequence other than the nucleotide sequence of the gene in the nucleotide sequence shown in SEQ ID NO:20; (i) a nucleotide sequence in which a nucleotide sequence other than the nucleotide sequence of the gene in the nucleotide sequence shown in SEQ ID NO:20 has 90% or more sequence identity; (j) a nucleotide sequence in which one or more bases have been added, deleted, and / or substituted in the nucleotide sequence shown in SEQ ID NO:20; (k) a nucleotide sequence in which a nucleotide sequence in which one or more bases have been added, deleted, and / or substituted in the nucleotide sequence shown in SEQ ID NO:20 has 95% or more sequence identity.

[0128] Preferably, the sequence identity defined in (i) is 90.5% or more, 91.0% or more, 91.5% or more, 92.0% or more, 92.5% or more, 93.0% or more, 93.5% or more, 94.0% or more, 94.5% or more, 95.0% or more, 95.5% or more, 96.0% or more, 96.5% or more, 97.0% or more, 97.5% or more, 98.0% or more, 98.5% or more, 99.0% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.

[0129] The base sequence defined in (i) is, in other words, a base sequence in which the sequence identity of the base sequence other than the gene corresponding to the gene in the base sequence shown in SEQ ID NO: 20 is 90% or more.

[0130] The sequence identity defined in (k) is preferably 95.5% or more, 96.0% or more, 96.5% or more, 97.0% or more, 97.5% or more, 98.0% or more, 98.5% or more, 99.0% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.

[0131] In one embodiment, the sixth phage is characterized by having a genomic DNA sequence containing a specific base sequence and exhibits bacteriolytic activity against target bacteria. The genomic DNA sequence of the sixth phage includes the base sequence shown in SEQ ID NO: 20 (each 40784 bp), a base sequence in which one or more bases are added, deleted, and / or substituted in the base sequence shown in SEQ ID NO: 20, a base sequence that is 90% or more, 90.5% or more, 91.0% or more, 91.5% or more, 92.0% or more, 92.5% or more, 93.0% or more, 93.5% or more, 94.0% or more, or a base sequence that is 90% or more, 90.5% or more, 91.0% or more, 91.5% or more, 92.0% or more, 92.5% or more, 93.0% or more, 93.5% or more, or 94.0% or more of the base sequence shown in SEQ ID NO: 20. Examples of such sequences include genomic DNA sequences containing base sequences with sequence identity of 0% or more, 94.5% or more, 95.0% or more, 95.5% or more, 96.0% or more, 96.5% or more, 97.0% or more, 97.5% or more, 98.0% or more, 98.5% or more, 99.0% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.

[0132] (4) Effects The present invention provides a novel bacteriophage having lytic activity against bacteria of the genus Salmonella. In the present invention, the tail fiber protein consisting of the amino acid sequence set forth in SEQ ID NO: 18 can achieve highly useful host specificity, being specific to bacteria of the genus Salmonella and exhibiting lytic activity against Salmonella species, particularly S. Enteritidis, S. Typhimurium, and S. Javana. Therefore, the sixth phage is also useful for treating or preventing food poisoning.

[0133] <Seventh Phage / Lysing Agent> (Summary) The present invention provides a phage (sometimes referred to herein as the "seventh phage") having the following configuration and having lytic activity against bacteria of the genus Salmonella, and a lytic agent comprising the same (sometimes referred to herein as the "seventh lytic agent"). The seventh lytic agent is a lytic agent that exhibits specific lytic activity against particular bacteria of the genus Salmonella, for example, a lytic agent for S. Enteritidis. The seventh lytic agent comprises a bacteriophage having a genomic DNA sequence that includes a specific base sequence. The seventh lytic agent can lyse and control target bacteria.

[0134] (Configuration) The seventh lytic agent is a lytic agent for bacteria of the genus Salmonella, particularly a lytic agent for S. Enteritidis. The seventh lytic agent comprises a seventh phage having the following configuration and having lytic activity against bacteria of the genus Salmonella:

[0135] The seventh phage has genomic DNA containing a gene encoding a tail spike protein consisting of a specific amino acid sequence and having the activity of recognizing target bacteria.

[0136] The present inventors discovered a phage with lytic activity specific to S. Enteritidis and identified the genomic DNA sequence of the phage (SEQ ID NO: 23). From the genomic DNA sequence of the phage, the present inventors further identified a gene encoding a tailspike protein that is thought to determine the host range of the phage. The amino acid sequence of the tailspike protein and the nucleotide sequence encoding it are shown in SEQ ID NOs: 21 and 22, respectively.

[0137] (1) Tailspike Protein The tailspike protein of the present invention consists of the amino acid sequence shown in SEQ ID NO:21.

[0138] (2) Tailspike Gene The gene encoding the tailspike protein comprises, for example, the base sequence shown in SEQ ID NO: 22.

[0139] (3) Genomic DNA The seventh bacteriophage has genomic DNA that includes a gene encoding a tailspike protein.

[0140] The genomic DNA sequence comprises or consists of, for example, any of the nucleotide sequences shown in (a) to (e) below: (a) the nucleotide sequence shown in SEQ ID NO: 23; (b) the nucleotide sequence shown in SEQ ID NO: 23, in which one or more bases have been added, deleted, and / or substituted in a nucleotide sequence other than the nucleotide sequence of the gene; (c) the nucleotide sequence shown in SEQ ID NO: 23, in which a nucleotide sequence other than the nucleotide sequence of the gene has 99% or more sequence identity; (d) the nucleotide sequence shown in SEQ ID NO: 23, in which one or more bases have been added, deleted, and / or substituted; (e) a nucleotide sequence that has 99% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 23.

[0141] Preferably, the sequence identity defined in (c) is 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.

[0142] The base sequence specified in (c) is, in other words, a base sequence in which the sequence identity of the base sequence other than the gene corresponding to the gene in the base sequence shown in SEQ ID NO: 23 is 99% or more.

[0143] Preferably, the sequence identity defined in (e) is 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.

[0144] In one embodiment, the seventh phage has a genomic DNA sequence containing a specific nucleotide sequence and exhibits bacteriolytic activity against a target bacterium. Examples of the genomic DNA sequence of the seventh phage include the nucleotide sequence shown in SEQ ID NO:23 (39,162 bp), a nucleotide sequence in which one or more nucleotides have been added, deleted, and / or substituted in the nucleotide sequence shown in SEQ ID NO:23, and a genomic DNA sequence containing a nucleotide sequence having 99.0% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more sequence identity to the nucleotide sequence shown in SEQ ID NO:23.

[0145] The seventh phage is useful for treating or preventing food poisoning because it can exhibit lytic activity against S. Enteritidis, the serotype most frequently detected in human food poisoning. The seventh phage is also useful for identifying the serotype of bacteria causing food poisoning, for example, because it can exhibit lytic activity specifically against S. Enteritidis.

[0146] 2. Compositions 2-1. Overview A second aspect of the present invention is a composition, particularly a composition for controlling target bacteria. The composition of the present invention is characterized by comprising the bacteriophage or lytic agent described in the first aspect. In the composition of the present invention, the target bacterium is particularly a Salmonella bacterium. For example, in the case of a composition comprising a first phage or a first lytic agent, the target bacterium is particularly S. Enteritidis. In the case of a composition comprising a second phage or a second lytic agent, the target bacterium is particularly S. Enteritidis, S. Typhimurium, S. Infantis, S. Montevideo, and S. Javiana. In the case of a composition comprising a third phage or a third lytic agent, the target bacterium is particularly S. Typhimurium. In the case of a composition comprising a fourth phage or a fourth lytic agent, the target bacterium is particularly S. Montevideo. For compositions comprising a fifth phage or a fifth lytic agent, the target bacterium is particularly S. Typhimurium. For compositions comprising a sixth phage or a sixth lytic agent, the target bacterium is particularly S. Enteritidis, S. Typhimurium, and S. Javana. For compositions comprising a seventh phage or a seventh lytic agent, the target bacterium is particularly S. Enteritidis.

[0147] The compositions of the present invention can provide pharmaceutical compositions, additives (e.g., food and drink additives, feed additives, drinking water additives), foods and drinks, feed, cleaning agents, disinfectants, bactericides, sanitizers, and the like that are safe for the human body, non-toxic to the environment, and capable of controlling target bacteria.

[0148] 2-2. Composition (1) Essential Active Ingredient The composition of the present invention contains at least one of the bacteriophages and lytic agents described in the first aspect (i.e., the first phage, the second phage, the third phage, the fourth phage, the fifth phage, the sixth phage, the seventh phage, the first lytic agent, the second lytic agent, the third lytic agent, the fourth lytic agent, the fifth lytic agent, the sixth lytic agent, the seventh lytic agent, or a combination thereof) as an essential active ingredient. The composition of the present invention can lyse and control target bacteria by using this active ingredient.

[0149] The specific configurations of the bacteriophage and the lytic agent are described in detail in the first embodiment, and therefore will not be described here.

[0150] The amount of bacteriophage or lytic agent in the composition of the present invention depends on various conditions such as the use of the composition, the subject of use, the method of use, the dosage form, and the type of bacteria to be lysed, but it is preferable that the amount is sufficient for the bacteriophage to contact and infect the target bacteria in the subject of use. The amount of bacteriophage or lytic agent in the composition of the present invention can be an amount effective for the bacteriophage or lytic agent in the composition of the present invention to control the target bacteria, within the scope of common technical knowledge in the field. The titer of the phage in the composition of the present invention can be, for example, 1 x 10 １ ~1 x 10 １５ pfu / mL, 1×10 ３ ~1 x 10 １３ pfu / mL, 1×10 ５ ~1 x 10 １１ pfu / mL, or 1 x 10 ７ ~1 x 10 ９ It may be pfu / mL.

[0151] In the composition of the present invention, the first to seventh phages or lytic agents may be used singly or in combination of two or more. The composition of the present invention may, for example, contain, in addition to the first phage or lytic agent, at least one phage or lytic agent selected from the group consisting of the second to seventh phages or lytic agents, in combination as an active ingredient. The composition of the present invention may, for example, contain, in addition to the second phage or lytic agent, at least one phage or lytic agent selected from the group consisting of the first and third to seventh phages or lytic agents, in combination as an active ingredient. The composition of the present invention may, for example, contain, in addition to the third phage or lytic agent, at least one phage or lytic agent selected from the group consisting of the first, second, and fourth to seventh phages or lytic agents, in combination as an active ingredient. The composition of the present invention may, for example, contain, in addition to the fourth phage or lytic agent, at least one phage or lytic agent selected from the group consisting of the first to third and fifth to seventh phages or lytic agents, in combination as an active ingredient. The composition of the present invention can contain, for example, the fifth phage or lytic agent in combination with at least one phage or lytic agent selected from the group consisting of the above-mentioned phages or lytic agents Nos. 1 to 4, 6, and 7, as an active ingredient. The composition of the present invention can contain, for example, the sixth phage or lytic agent in combination with at least one phage or lytic agent selected from the group consisting of the above-mentioned phages or lytic agents Nos. 1 to 5, and 7, as an active ingredient. The composition of the present invention can contain, for example, the seventh phage or lytic agent in combination with at least one phage or lytic agent selected from the group consisting of the above-mentioned phages or lytic agents Nos. 1 to 6, as an active ingredient.

[0152] For example, when phages that target different bacteria are combined, or when phages that target the same bacteria but recognize different cell surface receptors are combined, a synergistic or complementary effect in bacteriolytic activity can be expected.

[0153] (2) Other Active Ingredients In addition to the bacteriophage or lytic agent described in the first aspect, the composition of the present invention may contain one or more other active ingredients having the same pharmacological action as the bacteriophage or lytic agent and / or a different pharmacological action from that of the bacteriophage or lytic agent, provided that the lytic activity of the phage is not affected.

[0154] The type of the other active ingredient is not limited. The other active ingredient may be, for example, a phage having lytic activity against the same bacteria as the target bacteria of the bacteriophage or lytic agent described in the first aspect and / or a different bacteria. Such a phage may be, for example, a phage having lytic activity against Salmonella bacteria.

[0155] Other examples of the active ingredient include known antibiotics.

[0156] (3) Inactive Ingredients The composition of the present invention may further contain inactive ingredients, such as carriers (such as solid carriers or liquid carriers), excipients, surfactants, emulsifiers, binders, disintegrants, lubricants, solubilizers, suspending agents, coating agents, colorants, flavoring agents, preservatives, stabilizers, isotonicity agents, chelating agents, thickening agents, viscosity enhancers, buffers, pH adjusters, etc., to the extent that they do not affect the lytic activity of the bacteriophage or lytic agent described in the first aspect.

[0157] 2-3. Targets for Application Targets for application of the composition of the present invention (often abbreviated herein simply as "target") include, but are not limited to, livestock breeding facilities such as poultry farms, pig farms, ranches, and dairy farms (including, for example, houses, cages, soil, etc.); food, drink, or feed; food, drink, or feed processing plants or feed manufacturing plants; food, drink, or feed processing equipment; food, drink, or feed containers; and any vertebrate including humans, livestock (horses, cows, sheep, goats, pigs, chickens, etc.), pets (dogs, cats, rabbits, birds, etc.), and laboratory animals (mice, rats, monkeys, etc.).

[0158] The composition of the present invention may be in the form of a pharmaceutical composition, an additive (e.g., a food or drink additive, a feed additive, or a drinking water additive), a food or drink, a feed, a cleaning agent, a disinfectant, a bactericide, or a sanitizer. Each form is described in detail below.

[0159] (1) Pharmaceutical Composition The composition of the present invention may be a pharmaceutical composition.

[0160] The pharmaceutical composition of the present invention can be used, for example, to control target bacteria in a subject. The pharmaceutical composition of the present invention can also be used, for example, to treat or prevent infection caused by the target bacteria. In the pharmaceutical composition of the present invention, the target bacteria is as described in "2-1. Overview," and is particularly a Salmonella bacterium.

[0161] As used herein, "infection caused by bacteria of the genus Salmonella" refers to a disease caused by bacteria of the genus Salmonella, and is also called salmonellosis or salmonellosis. Symptoms of infection caused by bacteria of the genus Salmonella (including S. Enteritidis, S. Montevideo, and S. Typhimurium) include fever, abdominal pain, diarrhea, nausea, retching, vomiting, bacteremia, and the like. Infection caused by bacteria of the genus Salmonella can be, for example, food poisoning.

[0162] The pharmaceutical composition of the present invention may further comprise the above-mentioned pharmaceutically acceptable non-active ingredients (i.e., pharmaceutical excipients) in addition to the bacteriophage or lytic agent described in the first aspect.

[0163] The pharmaceutical composition of the present invention may be formulated into any dosage form, including solid preparations such as tablets, granules, powders, pills, and capsules; liquid preparations such as solutions, suspensions, and syrups; gels; and aerosols. When the pharmaceutical composition is used as a liquid preparation, it can also be formulated as a dry product intended to be reconstituted with, for example, physiological saline immediately before use. Furthermore, the amount of the bacteriophage or lytic agent described in the first aspect of the pharmaceutical composition can be appropriately determined, and the amount can be changed depending on the dosage form, the severity of the disease in the subject, and the like.

[0164] The subjects to which the pharmaceutical composition of the present invention is administered include any vertebrate including humans, livestock (horses, cows, sheep, goats, pigs, chickens, etc.), pets (dogs, cats, rabbits, birds, etc.), and laboratory animals (mice, rats, monkeys, etc.), and is preferably humans.

[0165] The routes of administration of the pharmaceutical composition of the present invention include, but are not limited to, oral, intravenous, rectal, vaginal, and topical administration.

[0166] The dosage of the pharmaceutical composition of the present invention can be appropriately determined taking into consideration various factors such as the route of administration, the age, weight, symptoms, etc. The pharmaceutical composition of the present invention may be administered once or multiple times at intervals of several hours to several months.

[0167] (2) Food and Drink Additive The composition of the present invention may be a food and drink additive.

[0168] The food and beverage additive of the present invention can be used, for example, to control target bacteria in foods and beverages. The food and beverage additive of the present invention can also be used to impart a specific effect to foods and beverages (a control effect on target bacteria or an effect of treating or preventing infections caused by target bacteria) by adding it to foods and beverages. In the food and beverage additive of the present invention, the target bacteria are as described in "2-1. Overview", and are particularly bacteria of the genus Salmonella.

[0169] The food and drink additive of the present invention may further contain the above-mentioned inactive ingredients that are acceptable for the production of foods and drinks, in addition to the bacteriophage or lytic agent described in the first aspect.

[0170] The food and beverage additive of the present invention may be in the form of a liquid, gel, or dry powder. The types of food and beverage to which the food and beverage additive of the present invention is to be added are as described in "(4) Food and beverage."

[0171] The food and beverage additive of the present invention can be added to, applied to, or sprayed onto a food or beverage by any suitable method available to those skilled in the art. For example, the food and beverage additive of the present invention may be mixed into the ingredients of the food or beverage during production of the food or beverage.

[0172] (3) Feed Additive / Drinking Water Additive The composition of the present invention can be a feed additive or a drinking water additive. The feed additive or drinking water additive of the present invention can be used, for example, when raising livestock.

[0173] The feed additive or drinking water additive of the present invention can be used, for example, to control target bacteria in feed or drinking water. The feed additive or drinking water additive of the present invention can also be used to impart a specific effect to feed or drinking water (a control effect against target bacteria or a therapeutic or preventive effect against infection caused by target bacteria) by adding it to feed or drinking water. In the feed additive or drinking water additive of the present invention, the target bacteria are as described in "2-1. Overview," and are particularly bacteria of the genus Salmonella.

[0174] The feed additive of the present invention may further contain the above-mentioned inactive ingredients that are acceptable for use in the production of feed, in addition to the bacteriophage or lytic agent described in the first aspect.

[0175] The feed additive of the present invention may be in the form of a liquid, gel, or dry powder. The types of feed to which the feed additive of the present invention is added are as described in "(5) Feed."

[0176] The feed additive of the present invention can be added to, applied to, or sprayed onto feed by any suitable method available to those skilled in the art. For example, the feed additive of the present invention may be mixed into the feed ingredients during the production of the feed.

[0177] The drinking water additive of the present invention may be in the form of a liquid, gel, or dry powder. The drinking water to which the drinking water additive of the present invention is added may be, for example, tap water, well water, groundwater, rainwater, etc., but is not particularly limited. The drinking water may contain other ingredients (e.g., antibiotics, etc.).

[0178] The drinking water additive of the present invention can be added to drinking water by any suitable method available to those skilled in the art, for example, the drinking water additive of the present invention can be mixed with drinking water in a suitable container or in a water supply system.

[0179] (4) Food and Drink The composition of the present invention may be a food or drink.

[0180] The food and drink of the present invention can be used, for example, to control target bacteria in a subject. The food and drink of the present invention can also be used, for example, to treat or prevent infections caused by target bacteria. In the food and drink of the present invention, the target bacteria are as described in "2-1. Overview," and are particularly bacteria of the genus Salmonella. The infection caused by the target bacteria can be, for example, food poisoning.

[0181] The food and drink of the present invention may further contain, in addition to the bacteriophage or lytic agent according to the first aspect, the above-mentioned inactive ingredients that are acceptable for the production of food and drink.

[0182] The food and beverage products of the present invention may be in any form, such as fresh foods (vegetables, fruits, meat, seafood, grains, etc.), processed foods, prepared dishes, confectioneries, condiments, beverages, functional foods, etc. Functional foods include, for example, foods for specified health uses (including conditionally designated foods for specified health uses [FOSHU]), foods with functional claims, health functional foods including foods with nutrient functions, special dietary foods, nutritional supplements, health supplements, supplements (e.g., tablets, coated tablets, sugar-coated tablets, capsules, liquids, etc.), beauty foods (e.g., diet foods), etc. The food and beverage products may also be prepared in any form, such as solids, liquids, mixtures, suspensions, pastes, gels, powders, granules, capsules, etc. The food and beverage products of the present invention may contain the bacteriophage or lytic agent described in the first aspect by any suitable method available to those skilled in the art. Specifically, the food and beverage products of the present invention may contain a bacteriophage or lytic agent encapsulated in an edible film or edible coating, or may be formed into any desired form, such as a tablet, after incorporating (adding) a suitable excipient or other additive into the bacteriophage or lytic agent. The food and beverage products of the present invention may also be produced by processing a composition containing the bacteriophage or lytic agent of the present invention and other food ingredients. Furthermore, the food and beverage products of the present invention can also be produced, for example, by incorporating (adding) the bacteriophage or lytic agent into various foods (such as beverages, liquid foods, foods for the sick, nutritional foods, frozen foods, processed foods, and other commercially available foods).

[0183] (5) Feed The composition of the present invention may be a feed.

[0184] The feed of the present invention can be used, for example, to control target bacteria in a subject. The feed of the present invention can also be used, for example, to treat or prevent infections caused by target bacteria. In the feed of the present invention, the target bacteria are as described in "2-1. Overview," and are particularly bacteria of the genus Salmonella. The infection caused by the target bacteria can be, for example, food poisoning.

[0185] The feed of the present invention may further comprise, in addition to the bacteriophage or lytic agent described in the first aspect, the above-mentioned inactive ingredients that are acceptable for the manufacture of feed.

[0186] Examples of the feed of the present invention include, but are not limited to, grass, straw, Japanese silver grass, hay, silage, grains (corn, barley, wheat, rice, etc.), compound feed, food by-products (soybean pulp, beer lees, bread crumbs, etc.), etc. The feed may also be prepared in any form, such as a solid, liquid, mixture, suspension, paste, gel, powder, granules, capsule, etc.

[0187] The feed of the present invention may contain the bacteriophage or lytic agent described in the first aspect by any suitable method available to those skilled in the art. Specifically, the feed of the present invention may contain the bacteriophage or lytic agent encapsulated in a capsule, or may contain the bacteriophage or lytic agent wrapped in an edible film or edible coating agent, or may be formed into any desired form, such as a tablet, after blending (adding) a suitable excipient to the bacteriophage or lytic agent. The feed of the present invention may also be produced by processing a composition containing the bacteriophage or lytic agent of the present invention and other feed ingredients. Furthermore, the feed of the present invention can also be produced, for example, by blending (adding) the bacteriophage or lytic agent to various feeds.

[0188] (6) Cleaning Agent, Disinfectant, Bactericide, Sanitizer The composition of the present invention may be a cleaning agent, disinfectant, bactericide, or sanitizer. As used herein, the term "cleaning agent" refers to a composition intended to remove dirt from an application target. As used herein, the term "disinfectant" refers to a composition intended to reduce pathogenic microorganisms in an application target to a harmless level. As used herein, the term "sterilizer" refers to a composition intended to kill bacteria in an application target. As used herein, the term "sanitizer" refers to a composition intended to reduce bacteria in an application target.

[0189] The cleaning agent, disinfectant, bactericide, or sanitizer of the present invention can be used, for example, to control target bacteria in an application target. In the cleaning agent, disinfectant, bactericide, or sanitizer of the present invention, the target bacteria is as described in "2-1. Overview," and in particular, bacteria of the genus Salmonella.

[0190] The cleaners, disinfectants, sanitizers or sanitizers of the present invention may be in the form of a liquid, gel or dry powder.

[0191] Examples of target objects to which the cleaning agent, disinfectant, bactericide, or sanitizer of the present invention is applied include, but are not limited to, livestock breeding facilities such as poultry farms, pig farms, ranches, and dairy farms (including, for example, houses, cages, soil, etc.); food, drink, or feed; food, drink, or feed processing factories or feed manufacturing factories; food, drink, or feed processing equipment; food, drink, or feed containers; and any vertebrate including humans, livestock (horses, cows, sheep, goats, pigs, chickens, etc.), pets (dogs, cats, rabbits, birds, etc.), and laboratory animals (mice, rats, monkeys, etc.).

[0192] The cleaning agent, disinfectant, bactericide, or sanitizer of the present invention can be used, for example, by adding, applying, spraying, or sprinkling it on an object to be applied. The cleaning agent, disinfectant, bactericide, or sanitizer of the present invention can also be used, for example, by immersing the object to be applied.

[0193] 3. Target Bacteria Control Method 3-1. Overview A third aspect of the present invention is a method for controlling target bacteria. The target bacteria control method of the present invention is characterized in that the bacteriophage or lytic agent described in the first aspect or the composition described in the second aspect is used to control the target bacteria. In the target bacteria control method of the present invention, the target bacteria is particularly bacteria of the genus Salmonella. For example, when a composition containing a first phage or a first lytic agent is used, the target bacteria is particularly S. Enteritidis. When a composition containing a second phage or a second lytic agent is used, the target bacteria is particularly S. Enteritidis, S. Typhimurium, S. Infantis, S. Montevideo, and S. Javiana. When a composition containing a third phage or a third lytic agent is used, the target bacteria is particularly S. When a composition comprising a fourth phage or a fourth lytic agent is used, the target bacterium is particularly S. Montevideo. When a composition comprising a fifth phage or a fifth lytic agent is used, the target bacterium is particularly S. Typhimurium. When a composition comprising a sixth phage or a sixth lytic agent is used, the target bacterium is particularly S. Enteritidis, S. Typhimurium, and S. Javana. When a composition comprising a seventh phage or a seventh lytic agent is used, the target bacterium is particularly S. Enteritidis.

[0194] According to the control method of the present invention, it is possible to control the target bacteria in the subject of application.

[0195] 3-2. Method The method for controlling a target bacterium of the present invention includes a contact step as an essential step.

[0196] The "contacting step" is a step of contacting the bacteriophage or lytic agent according to the first aspect or the composition according to the second aspect with an application target.

[0197] In this aspect, "contact" refers to direct contact between the bacteriophage or lytic agent described in the first aspect or the composition described in the second aspect and the target of application. More specifically, it refers to contact between the bacteriophage or lytic agent described in the first aspect or the phage in the composition described in the second aspect and the target of application, preferably a site that may be contaminated with the target bacterium. The purpose of this step is to infect the target bacterium with the phage, which is the active ingredient, thereby lysing the target bacterium. As a result, the target bacterium can be controlled effectively.

[0198] In the method for controlling a target bacterium of the present invention, the subject of application is as described in the second embodiment.

[0199] In the target bacterium control method of the present invention, the contacting step can be carried out by, for example, adding, applying, spraying or sprinkling the bacteriophage or lytic agent described in the first aspect or the composition described in the second aspect (particularly pharmaceutical compositions, food and drink additives, feed additives, drinking water additives, detergents, disinfectants, bactericides and sanitizers) to the target of application, or by immersing the target of application in the bacteriophage or lytic agent described in the first aspect or the composition described in the second aspect (particularly pharmaceutical compositions, food and drink additives, feed additives, drinking water additives, detergents, disinfectants, bactericides and sanitizers).

[0200] The contacting step can also be carried out by administering the composition according to the second aspect (particularly a pharmaceutical composition, food or drink, or feed) to the subject of application.

[0201] 4. Method for treating or preventing an infection caused by a target bacterium 4-1. Overview A fourth aspect of the present invention is a method for treating or preventing an infection caused by a target bacterium. The method for treating or preventing an infection caused by a target bacterium is characterized by using the bacteriophage or lytic agent described in the first aspect or the composition described in the second aspect for treating or preventing an infection caused by a target bacterium.

[0202] 4-2. Methods The therapeutic or preventive methods of the present invention include an administration step as an essential step. The "administration step" is a step of administering the bacteriophage or lytic agent described in the first aspect or the composition described in the second aspect to a subject. In the therapeutic or preventive methods of the present invention, the target bacterium is particularly a Salmonella bacterium. For example, when a composition containing a first phage or a first lytic agent is used, the target bacterium is particularly S. Enteritidis. When a composition containing a second phage or a second lytic agent is used, the target bacterium is particularly S. Enteritidis, S. Typhimurium, S. Infantis, S. Montevideo, and S. Javiana. When a composition containing a third phage or a third lytic agent is used, the target bacterium is particularly S. Typhimurium. When a composition containing a fourth phage or a fourth lytic agent is used, the target bacterium is particularly S. Montevideo. When a composition containing a fifth phage or a fifth lytic agent is used, the target bacterium is particularly S. Typhimurium. When a composition containing a sixth phage or a sixth lytic agent is used, the target bacterium is particularly S. Enteritidis, S. Typhimurium, and S. Javana. When a composition containing a seventh phage or a seventh lytic agent is used, the target bacterium is particularly S. Enteritidis. In the therapeutic or preventive methods of the present invention, the administration subjects and administration methods (dosage, administration route, administration frequency) are as described above in "2-4. (1) Pharmaceutical Compositions."

[0203] 5. Method for Identifying Bacteria of the Genus Salmonella 5-1. Overview A fifth aspect of the present invention is a method for identifying bacteria of the genus Salmonella. The identification method of the present invention is characterized by identifying bacteria of the genus Salmonella by utilizing the host specificity of the phage constituting the bacteriophage or lytic agent described in the first aspect. When a first phage or a first lytic agent is used, the identification method of the present invention may be a method for identifying S. Enteritidis. When a second phage or a second lytic agent is used, the identification method of the present invention may be a method for identifying S. Enteritidis, S. Typhimurium, S. Infantis, S. Montevideo, and S. Javiana. When a third phage or a third lytic agent is used, the identification method of the present invention may be a method for identifying S. Typhimurium. The identification method of the present invention may be a method for identifying S. Montevideo when a fourth phage or a fourth lytic agent is used. The identification method of the present invention may be a method for identifying S. Typhimurium when a fifth phage or a fifth lytic agent is used. The identification method of the present invention may be a method for identifying S. Enteritidis, S. Typhimurium, and S. Javiana when a sixth phage or a sixth lytic agent is used. The identification method of the present invention may be a method for identifying S. Enteritidis when a seventh phage or a seventh lytic agent is used.

[0204] According to the present invention, it is possible to determine whether or not an unidentified bacterium is a bacterium of the genus Salmonella, and to identify it.

[0205] The identification method of the present invention includes a culturing step, a mixing step, a mixture culturing step, and a determination step as essential steps, and an isolation step as a selection step. Each step will be explained below.

[0206] (1) Isolation Step The "isolation step" is a step of isolating test bacteria from a specimen suspected of containing Salmonella bacteria. This step is a selection step and may be performed as needed.

[0207] The term "test bacterium" refers to a bacterium that is subjected to the identification method of the present invention, and whose species has not been identified.

[0208] The specimen may be feces, food or drink, or feed, or may be a swab specimen collected from livestock farms, food processing plants, feed manufacturing plants, etc.

[0209] When the amount of Salmonella bacteria in the sample is expected to be high (e.g., when the sample is stool from a subject exhibiting salmonellosis), the sample can be streaked directly onto an agar medium for isolation and culture. After isolation and culture, the test bacteria can be isolated by picking a single colony. When the amount of Salmonella bacteria in the sample is expected to be low (e.g., when food or swab samples are used), the sample can be placed in a medium for enrichment culture, and the culture can then be streaked onto an agar medium for isolation and culture. After isolation and culture, the test bacteria can be isolated in the same manner as above. When the Salmonella bacteria are expected to be damaged or dormant (e.g., when processed foods are used as samples), a pre-enrichment culture can be further performed before the enrichment culture.

[0210] (2) Culturing Step The "culturing step" is a step of culturing the isolated test bacterium to obtain a culture. The test bacterium may be cultured by a method known in the art.

[0211] The term "culture" refers to a substance obtained by culturing a test bacterium, and may be either liquid or solid.

[0212] In this step, the test bacterium is unidentified, so it is desirable to use a medium that can cultivate a wide range of bacteria. A medium that can cultivate at least the Salmonella bacteria that are the target bacteria of the present invention is used. Such a medium may contain, for example, one or more components selected from the group consisting of proteolytic enzyme hydrolysates such as peptone and tryptone, biological extracts such as potato dextrose and yeast extract, amino acids such as glutamic acid or salts thereof, sugars such as glucose, sucrose, and lactose, and inorganic salts such as sodium chloride, magnesium chloride, potassium dihydrogen phosphate, and sodium thiosulfate. Specific media and compositions include LB medium (Lysogeny Broth medium; a standard medium containing tryptone, yeast extract, and sodium chloride), DHL medium (Desoxycholate Hydrogen Sulfide Lactose medium; a medium for Enterobacteriaceae bacteria containing desoxycholate, etc.), SS medium (Salmonella-Shigella medium; a selective medium for Salmonella and Shigella bacteria containing meat extract, peptone, etc.), and RV medium (Rappaport-Vassiliadis medium; a Salmonella bacteria enrichment medium containing peptone, etc.).

[0213] The isolated test bacterium is inoculated into the medium and cultured under appropriate culture conditions. Culture conditions include, for example, 20-40°C, 20-30°C, 22-28°C, or 24-26°C. In the case of a liquid medium, a culture can be obtained by culturing with stirring. The culture time is not limited, but may be, for example, cultured until the turbidity at 600 nm reaches approximately 1.0. This step yields a culture of the test bacterium. The culture may also be a multi-stage culture of two or more stages. For example, a soft agar-containing liquid medium can be added to the culture solution obtained after culture in a liquid medium, poured onto a solid medium such as an agar medium, and allowed to solidify, after which further culture can be performed.

[0214] (3) Mixing Step The "mixing step" is a step of mixing the culture obtained in the culturing step with the bacteriophage or lytic agent according to the first aspect to obtain a mixture.

[0215] The "mixture" refers to a mixture of a culture and a bacteriophage or a lytic agent, and may be in either a liquid or solid form.

[0216] The method of mixing the culture and the bacteriophage or lytic agent is not particularly limited as long as it allows the bacteriophage or lytic agent to be mixed. The bacteriophage or lytic agent according to the first aspect may be administered in a solid state or in a liquid state suspended in water or a liquid medium.

[0217] If the culture and the bacteriophage or lytic agent are both liquid, the volume ratio of the culture to the lytic agent may be 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, or 9:1. After administration, the culture and the bacteriophage or lytic agent may be thoroughly mixed by stirring or the like. On the other hand, when a soft agar-containing liquid medium is layered as described above, the culture is solid. In this case, the bacteriophage or lytic agent may be dropped onto a solid culture such as a gel surface, and the two may be mixed on the solid medium to obtain a mixture.

[0218] (4) Mixture Culturing Step The "mixture culturing step" is a step of culturing the mixture under predetermined conditions. In culturing the mixture, a soft agar-containing liquid medium may be added to the mixture, poured onto a solid medium such as an agar medium, and allowed to solidify, followed by further culturing.

[0219] The basic procedure of this step is similar to that of the culturing step described above. In this step, although not limited thereto, it is preferable to carry out culturing based on the so-called plaque assay method so as to facilitate confirmation of the presence or absence of bacteriophage lysis of the test bacterium in the determination step described below. For example, a portion of the mixture may be mixed with a soft agar medium of the same composition, and then, before the soft agar medium solidifies, the mixture may be poured onto an agar medium of the same composition and spread over the entire medium. The mixture may then be cultured under the same conditions as in the culturing step described above.

[0220] (5) Determination Step The "determination step" is a step of determining that the test bacterium is a bacterium of the genus Salmonella when the test bacterium is lysed after the culture step.

[0221] The determination of the presence or absence of bacteriolysis is not limited, but for example, in the case of a plaque assay, the determination can be made based on the presence or absence of plaque formation. If plaques are present on the solidified soft agar medium spread on the agar medium after the aforementioned mixture culture step, this indicates that the test bacterium has been lysed by infection with the bacteriophage of the present invention. Therefore, the test bacterium can be determined to be a Salmonella bacterium. On the other hand, if the test bacterium grows entirely on the agar medium and no plaques are present at all, the test bacterium can be determined not to be a Salmonella bacterium.

[0222] For more accurate determination, a negative control may be prepared in which the mixture is mixed with a medium containing neither bacteriophage nor a lytic agent in the mixture culture step, and / or a positive control may be prepared in which identified Salmonella bacteria are used in the culture step instead of the test bacteria, and it may be confirmed that no plaques form in the negative control, and that plaques are observed in the positive control.

[0223] 5-3. Effects The method for identifying Salmonella bacteria of the present invention makes it possible to identify whether or not Salmonella bacteria are the cause of, for example, food poisoning, diarrhea, vomiting, etc. Furthermore, the method for identifying Salmonella bacteria of the present invention makes it possible to detect the presence or absence of contamination with Salmonella bacteria.

[0224] The present invention will be described in more detail below using examples, although the technical scope of the present invention is not limited to these examples.

[0225] [Obtaining and culturing Salmonella bacteria] The bacterial strains used in the Examples (Examples 1 to 7) are listed in the tables accompanying each Example. The strains owned by Rakuno Gakuen University shown in the tables are Salmonella strains isolated from animals in Japan. The strains obtained from the National Agriculture and Food Research Organization (NARO) Animal Health Institute shown in the tables are Salmonella strains isolated from chickens and poultry farms in Japan.

[0226] The strain IDs in the table are identification numbers assigned in this specification. The serotypes of each strain in the table were identified based on the results of agglutination tests using Salmonella diagnostic immune serum (Denka) and the Kaufmann-White antigen structure table. If necessary, the serotypes were also confirmed by genetic analysis techniques such as PFGE (pulsed-field gel electrophoresis) and PCR (polymerase chain reaction).

[0227] The drug susceptibility and PFGE type of the strains ST1 to 6 in the table have been investigated (Tamura Yukino, "Molecular epidemiological study on bovine-derived Salmonella enterica subsp. enterica serovar Typhimurium," doctoral thesis, School of Veterinary Medicine, Rakuno Gakuen University (2015)).

[0228] For the cultivation of various Salmonella bacteria, 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride were mixed in H ２ A liquid medium (LB Broth) was used, which was prepared by dissolving the agar in 1 L of LB Broth and autoclaving it. Furthermore, an agar medium (hereinafter referred to as "LB Agar") was used as the agar medium, which was prepared by adding 15 g of agar per 1 L to the above-mentioned LB Broth and autoclaving it. Furthermore, a soft agar medium (hereinafter referred to as "LB Top Agar") was used as the soft agar medium to be layered on top of the agar medium, which was prepared by adding 5 g of agarose per 1 L to the above-mentioned LB Broth and autoclaving it. The soft agar medium was stored at approximately 50°C and used as needed.

[0229] Each of the above strains in a dry powder state was suspended in 0.1 mL of LB broth, and then streak culture was performed in LB agar at 25°C to isolate a single colony. The isolated colony was inoculated into LB broth and cultured with shaking at 25°C, and this was used as a preculture solution. For main culture, the preculture solution was inoculated into LB broth and cultured at 25°C for 10 to 30 hours until the turbidity (optical density 600 nm) reached approximately 1.0. The culture solution after culture was used as it was as a bacterial solution.

[0230] [Phage Isolation and Purification] Novel phages were isolated from natural wastewater or soil obtained in Japan. Phage isolation was performed using a conventional plaque assay method. First, wastewater from ponds or lakes, or wastewater obtained by suspending soil in water, was filtered through a 0.45 μm filter to prepare a phage-containing solution. Equal amounts of the bacterial solution and the phage-containing solution were then mixed and left at room temperature for approximately 10 minutes. Next, 0.2 mL of the bacteria / phage mixture was added to 3 mL of LB Top Agar, quickly mixed using a vortex mixer, and then poured onto the LB Agar. After the LB Top Agar solidified, the mixture was allowed to stand at 25°C for approximately 12 hours. Lytic plaques were formed on the bacterial lawns formed by the culture. The gel from the plaques was then aspirated using a cut-off tip, and phages with lytic activity against Salmonella bacteria were isolated. Thereafter, the phage-containing liquid containing the isolated phages at a high concentration was used instead of the wastewater, and the procedure was repeated to purify the phages.

[0231] The isolated phages were suspended in SM Buffer and passed through a 0.2 μm filter to recover a phage-containing solution. This phage-containing solution was mixed with the bacterial solution under the above conditions, and the phages were isolated again. This procedure was repeated several times to further purify the phages. The composition of SM Buffer is shown in the table below.

[0232]

[0233] [Phage Amplification and Purification] The isolated and purified phages were amplified and purified by the plate lysate (PL) method, an amplification method using a plaque assay. A bacteria / phage mixture was prepared so that many plaques would form on LB agar, and then mixed with LB Top Agar and spread on the LB agar for cultivation. 3 mL of SM buffer was then added to the LB Top Agar on which plaques had formed, and the mixture was shaken at 25°C for approximately 30 minutes. The supernatant was then passed through a 0.2 μm filter to recover the phage-containing recovery solution.

[0234] To 10 mL of the recovered solution, 1 g of PEG 6000 (final concentration: 10%) and 0.4 g of NaCl (final concentration: 4%) were added and dissolved, and the solution was rotated overnight at 4°C using a rotator. The solution was then centrifuged at 15,000 x g / 4°C / 60 minutes, and the supernatant was removed. The recovered pellet was resuspended in 0.5 mL of SM Buffer. 0.5 mL of chloroform was then added, the solution was vigorously stirred, and the solution was left on ice for 6 hours. After centrifugation at 8,000 x g / 4°C / 10 minutes, the upper layer was carefully collected to obtain a purified phage solution. The concentration of the purified phage solution is generally expressed as a titer [PFU / mL] based on the number of plaques (Plaque Forming Units, PFU) in a plaque assay, and is an index of bacteriolytic activity. The titer of the prepared purified phage solution was determined by plaque assay using an appropriately diluted solution.

[0235] [Evaluation of Phage Host Range] The host range of the phage was evaluated by the spot test method. 0.1 mL of the bacterial solution alone was added to 3 mL of LB Top Agar, mixed, and then poured into LB Agar, allowing it to spread over the entire plate and solidify. The bacterial solution used was a bacterial solution of each Salmonella bacterium prepared in each Example. Approximately 5 μL of the purified phage solution was then dropped onto the plate, followed by static culture at 25°C for approximately 12 hours. If the area where the phage was dropped on the plate with a bacterial lawn formed became clear and circular (approximately 1 cm in diameter), the dropped phage was determined to have bacteriolytic activity against that bacterial strain.

[0236] [Preparation and sequencing of phage genomic DNA] TURBO DNA-free ＴＭThe phage genome was extracted using a kit (Thermo Fisher Scientific). Genomic DNA derived from the host bacteria, which acts as a contaminant, was removed by treatment according to the manual provided with the kit. Subsequently, the phage coat molecules were degraded by proteinase K treatment using NucleoSpin (registered trademark) Virus (Machery-Nagel) according to the manual provided. After genomic DNA purification using a silica spin column, a phage genomic DNA solution was prepared. The concentration of genomic DNA was then measured using a Qubit dsDNA HS Assay kit (Thermo Fisher Scientific), and 50 μL of genomic DNA solution was prepared to a final concentration of 0.2 ng / μL. Subsequently, using Nextera XT DNA Library Prep (Illumina), the phage genome was fragmented and an adapter sequence was added by PCR according to the attached manual. Next, using Agilent High Sensitivity DNA Kit (Agilent Technologies), electrophoresis was performed using a Bioanalyzer (Agilent Technologies), the average bp size of the sample was measured, and the concentration of the DNA fragment was determined. Finally, using Miseq Reagent kit (Illumina), a measurement sample was prepared by processing according to the attached manual, and measurements were performed using a next-generation sequencer Miseq (Illumina). Using CLC genomics workbench (Qiagen), the obtained data was preprocessed (trimmed, etc.) and then de novo assembled to obtain a contig sequence corresponding to the phage genome sequence.

[0237] Example 1: Isolation of a first bacteriophage and its lytic activity (Objective) A novel bacteriophage having lytic activity against bacteria of the genus Salmonella was isolated, and its lytic activity against bacteria of the genus Salmonella was verified.

[0238] (Methods and Results) (1) Obtaining and Cultivating Salmonella Bacteria The bacterial strains used in Example 1 are listed in the table below.

[0239]

[0240] (2) Isolation and Purification of the First Phage Seven new phages were isolated and purified from natural wastewater and soil according to the method described in the above section [Isolation and Purification of Phages] (corresponding to the first phages).

[0241] (3) Amplification and Purification of First Phage A first phage purified solution was prepared and its titer was measured according to the method described in the above section [Amplification and Purification of Phage]. ８ It was confirmed that the number of PFU / mL or more was higher.

[0242] (4) Evaluation of host range of first phage The host range of the first phage was evaluated by spot test method according to the method described in the above section [Evaluation of host range of phage].

[0243] Examples of the results are shown in Figures 1 and 2. The seven first phages obtained in this example exhibited lytic activity against various strains of S. Enteritidis, but did not exhibit lytic activity against S. Typhimurium, S. Infantis, S. Montevideo, or S. Javiana.

[0244] S. Enteritidis is the serotype most frequently detected in human food poisoning (Oh and Park, J. Microbiol. Biotechnol. (2017), 27(12), 2075-2088). Therefore, the first phage is particularly useful for treating or preventing human food poisoning, for example. Furthermore, since the first phage exhibited bacteriolytic activity specific to S. Enteritidis, it is particularly useful for identifying S. Enteritidis.

[0245] (5) Genome Analysis of the First Phage The genomic DNA sequence of the first phage was determined and analyzed.

[0246] (i) Preparation and sequencing of genomic DNA of first phages The genomic DNA sequences of the first phages were determined according to the method described in the above section [Preparation and sequencing of phage genomic DNA]. The determined genomic DNA sequences of the seven first phages are shown in SEQ ID NOs: 1 to 7.

[0247] (ii) Bioinformatics analysis based on genome sequence information The genome DNA sequences of the first phage (SEQ ID NOS: 1 to 7) had high sequence identity to each other. Using the genetic information processing software GENETYX (https: / / www.genetyx.co.jp / ), the sequence identity of the shortest genome DNA sequence (SEQ ID NOS: 7) to the genome DNA sequences (SEQ ID NOS: 1 to 6) was calculated to be 100% over the entire range.

[0248] Similar DNA sequences were searched for on the NCBI BLAST server (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) using the genomic DNA sequences of SEQ ID NOs: 2 and 7 as query sequences. For phages having genomic DNA sequences with high overall sequence identity to SEQ ID NO: 2 or 7, prior literature regarding host range was further investigated. As a result, no phages were found that had genomic DNA sequences estimated to have an overall sequence identity of 99% or more to SEQ ID NO: 2 or 7 and were known to have the same host range as the first phage.

[0249] Example 2: Isolation of a second bacteriophage and its lytic activity (Objective) A novel bacteriophage having lytic activity against bacteria of the genus Salmonella was isolated, and its lytic activity against bacteria of the genus Salmonella was verified.

[0250] (Methods and Results) (1) Obtaining and Cultivating Salmonella Bacteria The bacterial strains used in Example 2 are listed in the table below.

[0251]

[0252] (2) Isolation and purification of the second phage Three new phages were isolated and purified from natural wastewater and soil according to the method described in the above section [Isolation and purification of phages] (corresponding to the second phages).

[0253] (3) Amplification and purification of second phage A second phage purified solution was prepared according to the method described in the above section [Amplification and purification of phage], and the titer was measured. ８ It was confirmed that the number of PFU / mL or more was higher.

[0254] (4) Evaluation of Host Range of Second Phage The host range of the second phage was evaluated by spot test according to the method described in the above section [Evaluation of Host Range of Phage].

[0255] Examples of the results are shown in Figures 3 to 5. The three types of second phages obtained in this example exhibited lytic activity against various bacterial strains, and exhibited lytic activity against all of the tested bacterial strains, including S. Enteritidis, S. Typhimurium, S. Infantis, S. Montevideo, and S. Javiana. All of these bacterial strains are serotypes that are frequently detected in human food poisoning. Therefore, the second phages are particularly useful for treating or preventing human food poisoning, for example.

[0256] (5) Genome Analysis of the Second Phage The genomic DNA sequence of the second phage was determined and analyzed.

[0257] (i) Preparation and sequencing of genomic DNA of second phages The genomic DNA sequences of the second phages were determined according to the method described in the above section [Preparation and sequencing of phage genomic DNA]. The determined genomic DNA sequences of the three second phages are shown in SEQ ID NOs: 10 to 12.

[0258] (ii) Bioinformatics analysis based on genome sequence information The sequence identity of the obtained genome DNA sequences (SEQ ID NOs: 10 to 12) of the three phages was 99%, and the amino acid sequence of each tailtip protein was as shown in SEQ ID NO: 8, which was completely identical to each other.

[0259] The obtained genomic DNA sequences of the three phages (SEQ ID NOS: 10 to 12) were searched for similar DNA sequences and sequence identity was confirmed using the BLAST server provided by NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). As a result of the search, the base sequence with the highest identity was the genome sequence of Salmonella phage S124 (GenBank accession number: NC_048013.1), with an overall sequence identity of 79.14% (Query Cover / Per. Ident values: 83% / 95.36%).

[0260] To verify the cause of the difference in host range between the three phages obtained and S124, the tailtip protein sequences of both phages were compared. The tailtip protein genes were identified from the three phages obtained. The RAST server (https: / / rast.nmpdr.org / ) and the PHASTER server (https: / / phaster.ca / ) were used for gene identification. As a result, the nucleotide sequence shown in SEQ ID NO:9 was identified as the tailtip protein gene.

[0261] Furthermore, when the amino acid sequence (SEQ ID NO: 8) encoded by the gene containing the nucleotide sequence shown in SEQ ID NO: 9 was compared with the amino acid sequence of a related protein of S124 (access code: YP_009806053.1), the sequence identity was 97.49%. It is highly likely that this sequence difference is responsible for the difference in host range. Furthermore, when the amino acid sequences (SEQ ID NO: 8) of the tailtip proteins of the three phages obtained were used as query sequences and searched on the NCBI-provided BLAST server, four known sequences with sequence identity of 95% or more were detected. An alignment of the query sequence and the searched sequence is shown in Figure 11. In Figure 11, the amino acid sequence of "HCH9411546.1" is shown in SEQ ID NO: 24, the amino acid sequence of "YP_009966103.1" is shown in SEQ ID NO: 25, the amino acid sequence of "YP_009194791.1" is shown in SEQ ID NO: 26, and the amino acid sequence of "YP_009806053.1" is shown in SEQ ID NO: 27. Of these four known sequences, the sequences other than S124 either lack detailed information regarding the host range or are prophage-derived sequences. It can be seen that F (Phe) at position 258 and S (Ser) at position 617 in the query sequence (the amino acid sequences of the tailtip proteins of the three phages obtained) are unique amino acid residues found only in the query sequence, unlike the corresponding residues in the four known sequences. These sequence features are presumed to be responsible for the lytic activity of the second phage against a wide range of serotypes.

[0262] Example 3: Isolation of a third bacteriophage and its lytic activity (Objective) A novel bacteriophage having lytic activity against bacteria of the genus Salmonella was isolated, and its lytic activity against bacteria of the genus Salmonella was verified.

[0263] (Methods and Results) (1) Obtaining and Cultivating Salmonella Bacteria The bacterial strains used in Example 3 are listed in the table below.

[0264]

[0265] (2) Isolation and purification of the third phage According to the method described in the above section [Isolation and purification of phage], a new phage was isolated from natural wastewater and soil and purified (corresponding to the third phage).

[0266] (3) Amplification and Purification of the Third Phage According to the method described in the above section [Amplification and Purification of Phage], a purified solution of the third phage was prepared and the titer was measured. ８ It was confirmed that the number of PFU / mL or more was higher.

[0267] (4) Evaluation of Host Range of Third Phage The host range of the third phage was evaluated by spot test according to the method described in the above section [Evaluation of Host Range of Phage].

[0268] An example of the results is shown in Figure 6. One type of third phage obtained in this example exhibited lytic activity against various strains of S. Typhimurium, a serotype frequently detected in human food poisoning cases.

[0269] In addition, S. Typhimurium is known to be a bacterium that is resistant to drugs and resistant to antibiotics. For example, the S. Typhimurium used in this example has been confirmed to exhibit multidrug resistance to various antibiotics. Specifically, ST1 is known to be resistant to S / Su, ST4 to A / C / S / Su / T, ST2 to A / C / Su, and ST3 to A / S / Su / T (Yukino Tamamura, "Molecular Epidemiological Study on Bovine Salmonella enterica subsp. enterica serovar Typhimurium", Doctoral Thesis, Rakuno Gakuen University, 2015). A represents ampicillin, C represents chloramphenicol, S represents streptomycin, Su represents sulfonamide, and T represents tetracycline. Therefore, the third phage can effectively control S. Typhimurium that is resistant to antibiotics due to its drug resistance, and is particularly useful for treating or preventing food poisoning in humans.

[0270] Furthermore, although not shown, it was confirmed that the third phage also exhibited bacteriolytic activity against ST5 (S. Typhimurium HRS-KST-203, School of Veterinary Medicine, Rakuno Gakuen University) and ST6 (S. Typhimurium HRS-U1, School of Veterinary Medicine, Rakuno Gakuen University).

[0271] (5) Genome Analysis of the Third Phage The genomic DNA sequence of the third phage was determined and analyzed.

[0272] (i) Preparation and sequencing of genomic DNA of third phage The genomic DNA sequence of the third phage was determined according to the method described in the above section [Preparation and sequencing of phage genomic DNA]. The determined genomic DNA sequence of one third phage is shown in SEQ ID NO: 13.

[0273] (ii) Bioinformatics analysis based on genome sequence information Using the NCBI BLAST server (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), a search for similar DNA sequences and confirmation of sequence identity were carried out using the genomic DNA sequence of the third phage (SEQ ID NO: 13) as a query sequence. As a result of the search, the closest base sequence was the genome sequence of Escherichia phage vB_EcoM-RPN242 (GenBank accession number: OL656110.1), and the overall sequence identity was 87.85% (Query Cover / Per. Ident values ​​were 89% / 98.71%). The closest nucleotide sequence with a sequence identity of about 85% is the genome sequence of Escherichia phage vB_EcoM-ZQ1 (GenBank accession number: MW650886.1), with a total sequence identity of 84.35% (Query Cover / Per. Ident values ​​of 86% / 98.09%). Since the host of neither phage is a Salmonella bacterium, this suggests that the third phage is a phage with a novel genome sequence with no known related genome sequences.

[0274] Example 4: Isolation of a fourth bacteriophage and its lytic activity (Objective) A novel bacteriophage having lytic activity against bacteria of the genus Salmonella was isolated, and its lytic activity against bacteria of the genus Salmonella was verified.

[0275] (Methods and Results) (1) Obtaining and Cultivating Salmonella Bacteria The bacterial strains used in Example 4 are listed in the table below.

[0276]

[0277] (2) Isolation and Purification of the Fourth Phage According to the method described in the above section [Isolation and Purification of Phage], a new phage was isolated from natural wastewater and soil and purified (corresponding to the fourth phage).

[0278] (3) Amplification and Purification of the Fourth Phage According to the method described in the above section [Amplification and Purification of Phage], a purified solution of the fourth phage was prepared and the titer was measured. ８ It was confirmed that the number of PFU / mL or more was higher.

[0279] (4) Evaluation of Host Range of the Fourth Phage The host range of the fourth phage was evaluated by spot test according to the method described in the above section [Evaluation of Host Range of Phage].

[0280] An example of the results is shown in Figure 7. The fourth phage obtained in this example exhibited lytic activity against S. Montevideo, but did not exhibit lytic activity against S. Enteritidis, S. Typhimurium, S. Infantis, or S. Javiana.

[0281] (5) Genome Analysis of the Fourth Phage The genomic DNA sequence of the fourth phage was determined and analyzed.

[0282] (i) Preparation and sequencing of genomic DNA of the fourth phage The genomic DNA sequence of the fourth phage was determined according to the method described in the above section [Preparation and sequencing of phage genomic DNA]. The determined genomic DNA sequence of the fourth phage is shown in SEQ ID NO: 14.

[0283] (ii) Bioinformatics analysis based on genome sequence information Similar DNA sequences were searched for on the NCBI-provided BLAST server (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) using the genome DNA sequence of the fourth phage as a query sequence. As a result of the search, the closest DNA sequence was the genome sequence of Escherichia coli bacteriophage esc-cop-9 (SEQ ID NO: 1 in U.S. Patent Application Publication No. 2019 / 0321423), with an estimated overall sequence identity of 90.68%. However, no phages were found that had an overall sequence identity of 95% or more and that used Salmonella bacteria as a host.

[0284] Example 5: Isolation of a fifth bacteriophage and its lytic activity (Objective) A novel bacteriophage having lytic activity against bacteria of the genus Salmonella was isolated, and its lytic activity against bacteria of the genus Salmonella was verified.

[0285] (Methods and Results) (1) Obtaining and Cultivating Salmonella Bacteria The bacterial strains used in Example 5 are listed in the table below.

[0286]

[0287] (2) Isolation and Purification of the Fifth Phage According to the method described in the above section [Isolation and Purification of Phage], a new phage was isolated from natural wastewater and soil and purified (corresponding to the fifth phage).

[0288] (3) Amplification and purification of the fifth phage A purified solution of the fifth phage was prepared according to the method described in the above section [Amplification and purification of the phage], and the titer was measured. ８ PFU / mL or more. (4) Evaluation of host range of fifth phage The host range of the fifth phage was evaluated by spot test method according to the method described in the above section [Evaluation of host range of phage].

[0289] An example of the results is shown in Figure 8. The fifth phage obtained in this example exhibited very high bacteriolytic activity against S. Typhimurium. The bacteriolytic activity against other serotypes of Salmonella, including S. Enteritidis, was examined using a similar method, but the fifth phage did not exhibit bacteriolytic activity against other serotypes of Salmonella.

[0290] (5) Genome Analysis of the Fifth Phage The genomic DNA sequence of the fifth phage was determined and analyzed.

[0291] (i) Preparation and sequencing of genomic DNA of fifth phage The genomic DNA sequence of the fifth phage was determined according to the method described in the above section [Preparation and sequencing of phage genomic DNA]. The determined genomic DNA sequence of the fifth phage is shown in SEQ ID NO: 17.

[0292] (ii) Bioinformatics analysis based on genome sequence information The NCBI-provided BLAST server (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) was used to search for similar DNA sequences using the genomic DNA sequence of the fifth phage as a query sequence. As a result of the search, the closest base sequence was the genomic sequence of Salmonella phage Skate (GenBank accession number: NC 054639.1), and the overall sequence identity was estimated to be 86.61%. A detailed comparison of the genomic DNA sequences of both phages revealed that the region corresponding to positions 2385 to 3606 of the genomic DNA sequence of the fifth phage was deleted in Skate. This region contains a gene encoding an endonuclease (positions 2434 to 3000 of SEQ ID NO: 17). The amino acid sequence of the endonuclease and the nucleotide sequence encoding it are shown in SEQ ID NOs: 15 and 16, respectively. Nucleases are known to be involved in the mechanism of shutting down host genome replication. Therefore, it was suggested that the fifth phage carrying the above-mentioned endonuclease gene can efficiently shut down host genome replication, thereby having high bacteriolytic activity.

[0293] Using the amino acid sequence of the endonuclease as a query sequence, a search for similar amino acid sequences was performed on the BLAST server. As a result, no phage genome sequence with a nuclease sequence with a sequence identity of 50% or more was found. Therefore, the fifth phage was shown to be a novel phage carrying a novel endonuclease gene.

[0294] Example 6: Isolation of a sixth bacteriophage and its lytic activity (Objective) A novel bacteriophage having lytic activity against bacteria of the genus Salmonella was isolated, and its lytic activity against bacteria of the genus Salmonella was verified.

[0295] (Methods and Results) (1) Obtaining and Cultivating Salmonella Bacteria The bacterial strains used in Example 6 are listed in the table below.

[0296]

[0297] (2) Isolation and purification of the sixth phage According to the method described in the above section [Isolation and purification of phage], a new phage was isolated from natural wastewater and soil and purified (corresponding to the sixth phage).

[0298] (3) Amplification and Purification of the Sixth Phage A purified solution of the sixth phage was prepared according to the method described in the above section [Amplification and Purification of Phage], and the titer was measured. ８ It was confirmed that the number of PFU / mL or more was higher.

[0299] (4) Evaluation of Host Range of the Sixth Phage The host range of the sixth phage was evaluated by the spot test method according to the method described in the above section [Evaluation of Host Range of Phage].

[0300] An example of the results is shown in Figure 9. One type of sixth phage obtained in this example exhibited lytic activity against multiple bacterial strains tested, specifically against S. Enteritidis, S. Typhimurium, and S. Javiana. All of these bacterial strains are serotypes frequently detected in human food poisoning. Therefore, the sixth phage is particularly useful for treating or preventing human food poisoning, for example.

[0301] (5) Genome Analysis of the Sixth Phage The genomic DNA sequence of the sixth phage was determined and analyzed.

[0302] (i) Preparation and sequencing of genomic DNA of sixth phage The genomic DNA sequence of the sixth phage was determined according to the method described in the above section [Preparation and sequencing of phage genomic DNA]. The determined genomic DNA sequence of one sixth phage is shown in SEQ ID NO:20.

[0303] (ii) Bioinformatics Analysis Based on Genome Sequence Information Analysis of the genomic DNA sequence (SEQ ID NO: 20) of one of the obtained phages revealed that the amino acid sequence encoded by bases 32468 to 34522 (CDS) was the tail fiber protein. Using the amino acid sequence of this tail fiber protein as a query sequence, a search for similar amino acid sequences and confirmation of sequence identity was performed using the NCBI BLAST server (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). As a result, the amino acid sequences of many phages' tail fiber proteins against Salmonella bacteria were detected, but none with 100% sequence identity were found. Therefore, sequences with an amino acid sequence length of 684 residues, the same as that of the tail fiber protein of the sixth phage, and with a sequence identity of 95% or more were extracted by search, and multiple alignment was performed. The results are shown in Figures 12A, 12B, and 12C. The table below also shows the phage names, sequence identity, Genbank access codes, sequence numbers assigned in this specification, and reactive serotypes (particularly focusing on Enteritidis and Typhimurium) of the sequences used in the alignment, which were confirmed from registration information and literature information.

[0304]

[0305] As shown in Table 8, even if we focus only on the two species, Enteritidis and Typhimurium, it is highly likely that the host ranges of the respective phages are different, despite the sequence identity of the tail fiber proteins being 95% or more.

[0306] Furthermore, as can be seen from the amino acid sequence alignment, the tail fiber protein of the sixth phage contains several unique amino acid residues that differ from all other sequences: Val at position 211 (all others are Ile), Val at position 321 (all others are Ile), Val at position 485 (all others are Ile or Met), Ala at position 533 (all others are Ser), Ser at position 577 (all others are Gly), and Ser at position 583 (all others are Gly). It is surprising that many of these sites are highly conserved in the tail fiber proteins of other phages, yet contain different amino acid residues, and this is thought to be related to the distinctive host range of the sixth phage.

[0307] The genomic DNA sequence of phage No. 6 was also searched on a BLAST server, and the highest sequence identity was found to be with Salmonella phage GRNsp27, with a sequence identity of 94.64% (Cover 95% / Ident 99.62%). However, when the sequence similarity between the two was compared using MUMmer with the genetic analysis software GENETYX (https: / / www.genetyx.co.jp / ), the identity of the region 29733-34770, which corresponds to the area around the tail fiber protein gene (32468-34522), was found to be low at 87% (see table below). Again, this suggests that phage No. 6 is a novel phage whose gene region involved in host recognition is significantly different from that of known phages.

[0308]

[0309] Example 7: Isolation of a seventh bacteriophage and its lytic activity (Objective) A novel bacteriophage having lytic activity against bacteria of the genus Salmonella was isolated, and its lytic activity against bacteria of the genus Salmonella was verified.

[0310] (Methods and Results) (1) Obtaining and Cultivating Salmonella Bacteria The bacterial strains used in Example 7 are listed in the table below.

[0311]

[0312] (2) Isolation and Purification of the Seventh Phage According to the method described in the above section [Isolation and Purification of Phage], a new phage was isolated from natural wastewater and soil and purified (corresponding to the seventh phage).

[0313] (3) Amplification and Purification of the Seventh Phage A purified solution of the seventh phage was prepared according to the method described in the above section [Amplification and Purification of Phage], and the titer was measured. ８ It was confirmed that the number of PFU / mL or more was higher.

[0314] (4) Evaluation of Host Range of Seventh Phage The host range of the seventh phage was evaluated by spot test according to the method described in the above section [Evaluation of Host Range of Phage].

[0315] An example of the results is shown in Figure 10. The seventh phage obtained in this example exhibited lytic activity against S. Enteritidis, but did not exhibit lytic activity against S. Typhimurium, S. Infantis, S. Montevideo, or S. Javiana. S. Enteritidis is the serotype most frequently detected in human food poisoning (Oh and Park, J. Microbiol. Biotechnol. (2017), 27(12), 2075-2088). Therefore, the seventh phage is particularly useful, for example, for treating or preventing human food poisoning. Furthermore, since the seventh phage exhibited lytic activity specifically against S. Enteritidis, it is believed to be effective against S. It is particularly useful for identifying S. Enteritidis.

[0316] (5) Genome Analysis of the Seventh Phage The genomic DNA sequence of the seventh phage was determined and analyzed.

[0317] (i) Preparation and sequencing of genomic DNA of seventh phage The genomic DNA sequence of the seventh phage was determined according to the method described in the above section [Preparation and sequencing of phage genomic DNA]. The determined genomic DNA sequence of the seventh phage is shown in SEQ ID NO:23.

[0318] (ii) Bioinformatics analysis based on genome sequence information Similar DNA sequences were searched for using the genomic DNA sequence of the seventh phage as a query sequence on the NCBI BLAST server (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). As a result of the search, the closest base sequence was the genome sequence of Salmonella phage SPN9CC (GenBank accession number: JF900176.1). Shin et al., Applied and Environmental Microbiology, 2014, vol. 80, No. 1, 374-384, reports that SPN9CC is S. It has been reported that SPN9CC exhibited lytic activity against a total of seven strains of S. Typhimurium. Therefore, the host range of SPN9CC is clearly different from that of the seventh phage, which exhibits lytic activity specifically against S. Enteritidis. Therefore, comparison of the amino acid sequences of proteins important for host recognition between the seventh phage and SPN9CC revealed differences in the amino acid sequences of the tailspike protein. Therefore, it was demonstrated that the difference in the amino acid sequence of the tailspike protein is the cause of the difference in the host range between the two phages. The gene encoding the tailspike protein was located at positions 30879 to 32882 of the genomic DNA sequence of the seventh phage. The amino acid sequence of the tailspike protein possessed by the seventh phage is shown in SEQ ID NO: 21, and the nucleotide sequence encoding it is shown in SEQ ID NO: 22.

[0319] In the search for similar DNA sequences, no phage was found that has a gene encoding a tailspike protein consisting of the amino acid sequence shown in SEQ ID NO: 21 and a genomic DNA sequence with an overall sequence identity of 99% or more to the nucleotide sequence shown in SEQ ID NO: 23. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

1. A bacteriophage exhibiting lytic activity against bacteria of the genus Salmonella, which has genomic DNA including a gene encoding a tail fiber protein having an activity of recognizing a target bacterium and which has an amino acid sequence shown in any one of (a) to (c) below: (a) the amino acid sequence shown in SEQ ID NO: 18; (b) an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence represented by SEQ ID NO: 18; (c) an amino acid sequence having 99% or more sequence identity to the amino acid sequence shown in SEQ ID NO:

18.

2. The bacteriophage according to claim 1, wherein the gene encoding the tail fiber protein comprises any of the nucleotide sequences shown in (d) to (f) below: (d) the base sequence represented by SEQ ID NO: 19; (e) a base sequence in which one or more bases are added, deleted, and / or substituted in the base sequence shown in SEQ ID NO: 19; (f) A base sequence having 97% or more sequence identity to the base sequence shown in SEQ ID NO:

19.

3. The bacteriophage according to claim 1, wherein the genomic DNA sequence comprises any of the nucleotide sequences shown in (g) to (k) below: (g) a base sequence represented by SEQ ID NO: 20; (h) a base sequence in which one or more bases have been added, deleted, and / or substituted in a base sequence other than the base sequence of the gene in the base sequence represented by SEQ ID NO: 20; (i) a base sequence having 90% or more sequence identity with a base sequence other than the gene base sequence in the base sequence shown in SEQ ID NO: 20; (j) a base sequence in which one or more bases are added, deleted, and / or substituted in the base sequence represented by SEQ ID NO: 20; (k) A base sequence having 95% or more sequence identity to the base sequence shown in SEQ ID NO:

20.

4. 2. The bacteriophage according to claim 1, wherein the Salmonella bacteria are S. Enteritidis, S. Typhimurium, and S. Javiana.

5. A composition comprising the bacteriophage of claim 1.

6. A composition for controlling S. Enteritidis, S. Typhimurium, and S. Javana, comprising the bacteriophage of claim 1.

7. The composition of claim 5 which is a pharmaceutical composition.

8. The composition according to claim 5, which is a food and drink additive, a feed additive, or a drinking water additive.

9. The composition according to claim 5, which is a food, drink or feed.

10. The composition of claim 5 which is a cleaning agent, disinfectant, sanitizer, or sanitizer.

11. The composition according to claim 5, further comprising another bacteriophage that exhibits lytic activity against Salmonella bacteria.

12. A method for controlling bacteria of the genus Salmonella, comprising a contacting step of contacting a target of application with the bacteriophage according to claim 1.

13. A method for treating or preventing an infection caused by Salmonella bacteria in a subject, comprising administering the bacteriophage according to claim 1 to the subject.

14. A method for identifying Salmonella bacteria, comprising: a culturing step of culturing a test bacterium isolated from a sample suspected of containing Salmonella bacteria to obtain a culture; a mixing step of mixing a culture with the bacteriophage of claim 1 to obtain a mixture; a mixture culturing step of culturing the mixture under predetermined conditions; and a determining step of determining that the test bacterium is a Salmonella bacterium when the test bacterium is lysed after the mixture culturing step; A method comprising:

15. The method according to claim 14, wherein in the mixture culturing step, the mixture further comprises a soft agar-containing liquid medium, and the mixture is cultured on a solid medium.

16. 15. The method of claim 14, wherein in the culturing step, the culture comprises a soft agar-containing liquid medium and the culture is cultivated on a solid medium.

17. The method according to claim 14, further comprising an isolation step of isolating the test bacterium from a specimen suspected of containing Salmonella bacteria before the culturing step.