Phage, salmonella bacteria lysozyme, composition and method for controlling salmonella bacteria

Abstract

One of the purposes of the present disclosure is to: (i) provide a novel bacteriophage having a lysolytic activity against a Salmonella bacterium such as S.Enteridis, or a lysolytic agent comprising the same; (ii) providing a bacteriophage having a broad range of hosts for Salmonella bacteria or a lysozyme comprising the same; (iii) providing a host-specific bacteriophage or an effective bacterial lysozyme of the Salmonella genus comprising the same; or (iv) providing a bacteriophage capable of effectively controlling S.Typhimurium, in particular, S.Typhimurium having a multidrug resistance, or a bacteriolytic agent containing the bacteriophage, which is capable of effectively controlling S.Typhimurium, in particular, S.Typhimurium having a multidrug resistance. The present disclosure provides a bacteriophage having a specific genomic DNA sequence, a Salmonella bacterial lysozyme comprising the same, and a composition comprising the same.

Description

Technical Field

[0001] This invention relates to bacteriophages, lysing agents containing bacteriophages, compositions containing the same, and methods for controlling Salmonella bacteria using the same. Background Technology

[0002] Salmonella bacteria are one of the main pathogens causing food poisoning, infecting humans, livestock, and other animals to cause salmonellosis, including diarrhea. Salmonella bacteria reside in the digestive tract of humans and livestock, are contained in feces, and are excreted, thus causing contamination. Most Salmonella infections occur through the ingestion of food or feed contaminated with Salmonella.

[0003] Previously, low-molecular-weight compounds were used as antibacterial agents against Salmonella bacteria. However, their continued use has led to negative consequences such as the emergence of multidrug-resistant bacteria. Therefore, new control methods are being explored. In recent years, bacteriophages have attracted attention as a new control method against Salmonella bacteria due to their high targeting specificity without damaging the microbiome and their low toxicity (Non-Patent Literature 1).

[0004] Bacteriophages (often abbreviated as "Phage" in this specification) are a general term for viruses that infect only bacteria. Most bacteriophages, after attaching to a target bacterium as a host, inject their own DNA into the bacteria and reproduce using the bacteria's translation mechanism. In addition, the proliferating bacteriophages can be spread by lysing the bacteria and repeatedly infect new target bacteria (Non-Patent Document 2).

[0005] As bacteriophages exhibiting lytic activity against Salmonella spp., reported examples are documented, for instance, in Patent Documents 1 and 2. Bacteriophages that lyse Salmonella spp. can be used, for example, for the control of Salmonella spp. in chicken and pig farming, and for the detection and control of Salmonella spp. in the food industry (Non-Patent Document 3). In fact, products containing bacteriophages exhibiting lytic activity against Salmonella spp. include BAFASALR (Proteon Pharmaceuticals), a feed additive for preventing Salmonella spp. infection in chickens, and SalmoFresh, a food processing formulation for sterilizing Salmonella spp. in food. TM (intralytix) and PhageGuard (Micreos), etc. (Non-patent literature 4).

[0006] Existing technical documents

[0007] Patent documents

[0008] Patent Document 1: WO2013-027146

[0009] Patent Document 2: Japanese Patent Application Publication No. 2014-217336

[0010] Non-patent literature

[0011] Non-patent literature 1: Jun-Hyun Oh et al., 2017, J. Microbiol. Biotechnol., 27(12), 2075-2088

[0012] Non-patent literature 2: Sharma S. et al., Folia Microbiol., 2017, 62: 17-55

[0013] Non-patent literature 3: Shuai Wei et al., Microorganisms, 2019, 7, 570

[0014] Non-patent literature 4: Katarzyna Zbikowska et al., Animals, 2020, 10, 872 Summary of the Invention

[0015] The problem that the invention aims to solve

[0016] As mentioned above, bacteriophages exhibiting lytic activity against Salmonella have been discovered, and products using them are already on the market. However, it is conceivable that with the large-scale use of a particular bacteriophage, drug-resistant Salmonella bacteria may emerge from that bacteriophage, thus necessitating the discovery of new bacteriophages.

[0017] For example, since *Salmonella enteritidis* is the most frequently detected serotype of *Salmonella* in chickens (Non-Patent Literature 1), it is desirable to develop bacteriophages that exhibit broad-spectrum lytic activity against this serotype.

[0018] In addition, one of the required characteristics of bacteriophages used in lysing compositions is a broad host range for Salmonella bacteria. A bacteriophage with a broad host range can be applied to various Salmonella species, making it a desirable bacteriophage for expanding its adaptability.

[0019] In addition, since it is useful for identifying the serotype of bacteria that cause food poisoning, a lysing composition using a host-specific bacteriophage that targets a specific Salmonella species is a desirable lysing composition.

[0020] Furthermore, multidrug resistance, particularly to multiple antibiotics, is becoming a problem among Salmonella bacteria, especially *Salmonella Typhimurium*. Representative strains are resistant to five antibiotics: ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline. The global spread of this multidrug-resistant *S. Typhimurium* is attributed, in part, to increased antibiotic use in animal products and hospitals. Therefore, there is an urgent need for effective technologies to control *S. Typhimurium*.

[0021] Therefore, one of the objectives of this disclosure is to: (i) provide novel bacteriophages or lysing agents containing them that have lytic activity against Salmonella bacteria such as S. Enteritidis; (ii) provide bacteriophages or lysing agents containing them that have a broad host range against Salmonella bacteria; (iii) provide host-specific bacteriophages or effective Salmonella lysing agents containing them; or (iv) provide bacteriophages or lysing agents containing them that can effectively control S. Typhimurium, especially multidrug-resistant S. Typhimurium.

[0022] Problem Solving Methods

[0023] The inventors isolated a novel bacteriophage from natural sewage / soil by detecting lysing plaques formed on soft agar culture medium containing Salmonella bacteria, evaluated the lysing activity of the bacteriophage against various Salmonella bacteria, and analyzed its genome sequence.

[0024] As a result, it was determined that seven bacteriophages with specific genomic DNA sequences (corresponding to bacteriophage No. 1 in this specification) have lytic activity against specific Salmonella bacteria.

[0025] In addition, three specific bacteriophages (corresponding to bacteriophage No. 2 in this specification) were identified as having broad-spectrum lytic activity against Salmonella bacteria, specifically against S. Enteritidis, S. Typhimurium, S. Infantis, S. Montevideo, and S. Javiana.

[0026] In addition, a specific phage (corresponding to phage 3 in this specification) was identified as having lytic activity against S. Typhimurium, particularly against various S. Typhimurium species exhibiting multidrug resistance to antibiotics.

[0027] In addition, it has been confirmed that bacteriophages with specific genomic DNA sequences (corresponding to bacteriophage No. 4 in this specification) have lytic activity against specific Salmonella bacteria.

[0028] In addition, it has been confirmed that bacteriophages containing genomic DNA that encodes a nuclease with a specific amino acid sequence (corresponding to bacteriophage No. 5 in this specification) have lytic activity against specific Salmonella bacteria.

[0029] In addition, it was clarified that a specific bacteriophage (corresponding to bacteriophage No. 6 in this specification) is a novel bacteriophage with lytic activity against Salmonella bacteria, specifically against S. Enteritidis, S. Typhimurium and S. Javiana.

[0030] In addition, it has been confirmed that bacteriophages with specific genomic DNA sequences (corresponding to bacteriophage No. 7 in this specification) have lytic activity against specific Salmonella bacteria.

[0031] The present invention is based on the above research and development results, and specifically, the following embodiments are provided.

[0032] [1] A S. enteritidis lysing agent, comprising:

[0033] A bacteriophage having a genomic DNA sequence containing any of the base sequences shown in sequence numbers 1 to 7.

[0034] [2] A bacteriophage having genomic DNA containing a gene encoding a tail tip protein and exhibiting lytic activity against Salmonella bacteria, the tail tip protein containing the amino acid sequence shown in sequence number 8.

[0035] [3] According to the bacteriophage described in [2], wherein,

[0036] The gene encoding the tail tip protein contains the base sequence shown in sequence number 9.

[0037] [4] According to the bacteriophage described in [2] or [3], wherein,

[0038] The genomic DNA sequence contains the base sequence shown in any of the sequence numbers 10 to 12.

[0039] [5] A bacteriophage having a genomic DNA sequence containing the base sequence shown in sequence number 13 and exhibiting lytic activity against Salmonella bacteria.

[0040] [6] A Salmonella lysing agent comprising: a bacteriophage having a genomic DNA sequence containing the base sequence shown in sequence number 14.

[0041] [7] A Salmonella lysing agent comprising: a bacteriophage having genomic DNA containing a gene encoding a nuclease, said nuclease containing the amino acid sequence shown in sequence number 15.

[0042] [8] According to the lysozyme described in [7], wherein,

[0043] The gene encoding the endonuclease contains the base sequence shown in sequence number 16.

[0044] [9] According to the lysozyme described in [7] or [8], wherein,

[0045] The genomic DNA sequence contains the base sequence shown in sequence number 17.

[0046]

[10] A bacteriophage having genomic DNA containing a gene encoding a tail fibrin and exhibiting lytic activity against Salmonella bacteria, the tail fibrin containing the amino acid sequence shown in sequence number 18.

[0047]

[11] According to the bacteriophage described in

[10] , wherein,

[0048] The gene encoding tail fibroin contains the base sequence shown in sequence number 19.

[0049]

[12] According to the bacteriophage described in

[10] or

[11] , wherein,

[0050] The genomic DNA sequence contains the base sequence shown in sequence number 20.

[0051]

[13] An S. Enteritidis lysing agent comprising: a bacteriophage having a genomic DNA sequence containing the base sequence shown in sequence number 23.

[0052]

[14] A composition comprising any one of the lysing agents described in [1], [6] to [9] and

[13] , or any one of the bacteriophages described in [2] to [5] and

[10] to

[12] .

[0053]

[15] The composition according to

[14] is a pharmaceutical composition.

[0054]

[16] The composition according to

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

[0055]

[17] The composition according to

[14] is a food or feed.

[0056]

[18] The composition according to

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

[0057]

[19] The composition according to any one of

[14] to

[18] further comprises other bacteriophages that exhibit lytic activity against Salmonella bacteria.

[0058]

[20] A method for preventing and controlling Salmonella bacteria, comprising:

[0059] A contacting process in which the lysing agent, the bacteriophage, or the composition of any one of [1], [6] to [9] and

[13] , the bacteriophage, or the composition of any one of

[14] to

[19] comes into contact with the object to be applied.

[0060]

[21] A method for treating or preventing an infection caused by Salmonella bacteria, comprising:

[0061] A drug administration process of administering to a subject the lysing agent described in any one of [1], [6] to [9] and

[13] , the bacteriophage described in any one of [2] to [5] and

[10] to

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

[14] to

[19] .

[0062]

[22] A method for identifying Salmonella bacteria, the method comprising:

[0063] The culture process of culturing test bacteria isolated from a specimen suspected of containing Salmonella to obtain a culture;

[0064] A mixing process in which a culture is mixed with any one of the lysing agents [1], [6] to [9] and

[13] or any one of the bacteriophages [2] to [5] and

[10] to

[12] to obtain a mixture;

[0065] A mixture cultivation process for culturing a mixture under given conditions; and

[0066] The determination process after the mixture culture step, when the test bacteria are lysed, is to identify the test bacteria as Salmonella bacteria.

[0067]

[23] According to the method described in

[22] , wherein,

[0068] In the mixture culture process, the mixture further includes a liquid culture medium containing soft agar, and the mixture is cultured on a solid culture medium.

[0069]

[24] According to the method described in

[22] , wherein,

[0070] In the cultivation process, the culture contains a liquid culture medium containing soft agar, and the culture is also cultured on a solid culture medium.

[0071]

[25] The method according to any one of

[22] to

[24] further comprises:

[0072] The isolation process, which involves separating the test bacteria from a specimen suspected of containing Salmonella bacteria, prior to the culture process.

[0073] This specification contains the disclosure of Japanese Patent Application No. 2023-057568, which forms the basis of the priority claim of this application.

[0074] The effects of the invention

[0075] This invention can provide novel bacteriophages, lysing agents, or compositions comprising them that have lytic activity against Salmonella spp. Alternatively, this invention can provide bacteriophages, lysing agents, or compositions comprising them that have a broad host range against Salmonella spp. Alternatively, this invention can provide bacteriophages, lysing agents, or compositions comprising them capable of lysing specific target Salmonella spp. bacteria. Alternatively, this invention can provide bacteriophages, lysing agents, or compositions comprising them that are effective against S. typhimurium, particularly effective against multidrug-resistant S. typhimurium. Attached Figure Description

[0076] Figure 1 Figure A shows the lysing activity of the first phage obtained in Example 1. Figure B is a photograph of the agar plate after Salmonella bacteria were spread on it, the first phage purification solution was added, and the plate was incubated. Figure B is the plate diagram corresponding to A, showing the strain ID of the spread Salmonella bacteria and the position of the added phage purification solution on each plate. In B, 'a' indicates the position of the phage purification solution with the genomic DNA sequence number 7.

[0077] Figure 2 Figure A shows the lysing activity of the first phage obtained in Example 1. Figure B is a photograph of the agar plate after Salmonella bacteria were spread on it, the first phage purification solution was added, and the plate was incubated. Figure B is the plate diagram corresponding to A, showing the strain ID of the spread Salmonella bacteria and the position of the added phage purification solution on each plate. In B, a, b, c, d, e, f, and g represent the positions of the phage purification solutions with genomic DNA sequences numbered 1, 2, 3, 4, 5, 6, and 7, respectively.

[0078] Figure 3Figure A shows the lysing activity of the second phage obtained in Example 2. Figure A is a photograph of the agar plate after Salmonella bacteria were spread on it, the second phage purification solution was added, and the plate was incubated. Figure B is the plate diagram corresponding to A, showing the strain ID of the spread Salmonella bacteria and the position of the added phage purification solution on each plate. In B, 'a' indicates the position of the phage purification solution with the genomic DNA sequence number 10.

[0079] Figure 4 Continued Figure 3 Figure 1 shows the lysing activity of the second phage obtained in Example 2.

[0080] Figure 5 Figure A shows the lysing activity of the second phage obtained in Example 2. Figure A is a photograph of the agar plate after Salmonella bacteria were spread on it, the second phage purification solution was added, and the plate was incubated. Figure B is the plate diagram corresponding to A, showing the strain ID of the spread Salmonella bacteria and the position of the added phage purification solution on each plate. In B, a, b, and c represent the positions of the phage purification solutions with genomic DNA sequences numbered 10, 11, and 12, respectively.

[0081] Figure 6 Figure A shows the lysing activity of the third phage obtained in Example 3. Figure A is a photograph of the agar plate after incubation, showing the growth of Salmonella Typhimurium on an agar plate, the addition of the third phage purification solution, and static incubation. Figure B is the plate diagram corresponding to A, showing the strain ID of the Salmonella Typhimurium bacteria grown on each plate and the position of the added phage purification solution. In B, 'a' indicates the position of the purification solution of the phage with genomic DNA sequence number 13.

[0082] Figure 7 Figure A shows the lysing activity of the fourth phage obtained in Example 4. Figure A is a photograph of the agar plate after Salmonella bacteria were spread on it, the fourth phage purification solution was added, and the plate was incubated. Figure B is the plate diagram corresponding to A, showing the strain ID of the spread Salmonella bacteria and the position of the added phage purification solution on each plate. In B, 'a' indicates the position of the purification solution of the phage with genomic DNA sequence number 14.

[0083] Figure 8Figure A shows the lysing activity of the fifth phage obtained in Example 5. Figure A is a photograph of an agar plate after Salmonella bacteria were spread on it, the first phage purification solution was added, and the plate was incubated. Figure B is the plate diagram corresponding to A, showing the strain ID of the spread Salmonella bacteria and the position of the added phage purification solution on each plate. In B, 'a' indicates the position of the phage purification solution with the genomic DNA sequence number 17.

[0084] Figure 9 Figure A shows the lysing activity of the 6th phage obtained in Example 6. Figure B is a photograph of an agar plate after Salmonella bacteria were spread on it, the 6th phage purification solution was added, and the plate was incubated. Figure B is the plate diagram corresponding to A, showing the strain ID of the spread Salmonella bacteria and the position of the added phage purification solution on each plate. In B, 'a' indicates the position of the phage purification solution with the genomic DNA sequence of sequence number 20.

[0085] Figure 10 This is a diagram showing the lysis activity of the 7th phage obtained in Example 7. A is a photograph of the agar plate after incubation, showing the Salmonella spp. bacteria spread on the agar plate, the addition of the 7th phage purification solution, and static incubation. B is the corresponding plate diagram to A, showing the strain ID of the spread Salmonella spp. bacteria and the position of the added phage purification solution on each plate. In B, 'a' indicates the position of the phage purification solution containing the genomic DNA sequence with sequence number 23.

[0086] Figure 11 The comparison between the query sequence (the amino acid sequence of the tail tip protein of the second phage obtained (sequence number 8)) and the search sequence in Example 2 is shown.

[0087] Figure 12A The multiple sequence alignment implemented in Example 6 is shown.

[0088] Figure 12B Continued Figure 12A This illustrates the multiple sequence alignment implemented in Example 6.

[0089] Figure 12C Continued Figure 12B This illustrates the multiple sequence alignment implemented in Example 6. Detailed Implementation

[0090] The present invention will now be described in detail.

[0091] [definition]

[0092] The terms used in this specification are defined below.

[0093] In this specification, "lysis" refers to the phenomenon of disrupting the bacterial cell membrane. Through lysis, bacteria die. Lysis begins with the specific adsorption of a bacteriophage onto a target bacterium, injecting its own DNA into the target bacterium's cell via its tail. Subsequently, it replicates itself using the bacterial translation mechanism, producing a large number of progeny bacteriophages, which are then lysed and released into the environment.

[0094] In this specification, "lysozyme" refers to a reagent containing a bacteriophage that has lysing activity against target bacteria. The lysozyme can be a lysozyme for specifically lysing target bacteria (target-bacterial specific lysozyme). The lysozyme can also be the bacteriophage itself.

[0095] In this specification, "bacteria" refers to one of the major biological systems that, along with archaea and eukaryotes, divide the entire biosphere into three parts. Bacteria are cells without a nucleus and can self-replicate as long as there is a source of nutrients.

[0096] In this specification, "target bacteria" refers to host bacteria that can serve as a target for the bacteriophage constituting the lysing agent of the present invention, or for the bacteriophage included in the composition of the present invention. Specifically, this includes, for example, bacteria having membrane surface receptors on their outer membranes that are recognized by the aforementioned bacteriophages. Alternatively, it includes, for example, bacteria having membrane surface receptors on their outer membranes that are recognized by tail fiber protein, tail tip protein, tail spike protein, or tail tube protein containing specific amino acid sequences. "Membrane surface receptors" are binding sites for bacteriophages, such as the tail and tail fibers, and are composed of proteins, lipopolysaccharides, or pili present in the outer layer of the bacterial outer membrane. In particular, "target bacteria" in this specification refers to Salmonella bacteria.

[0097] In this specification, "Salmonella bacteria" refers to bacteria belonging to the genus *Salmonella*. *Salmonella* bacteria are classified into two species: *Salmonella enterica* and *Salmonella bongori*. The former is further divided into six subspecies: *ssp. enterica*, *ssp. salamae*, *ssp. arizonae*, *ssp. diarizonae*, *ssp. houtenae*, and *ssp. Indica*. *Salmonella* bacteria can also be classified into serotypes based on two surface structures: somatic antigens (also known as O antigens) and flagellar antigens (also known as H antigens). The subspecies name and serotype of *Salmonella* bacteria are indicated by adding "subspecies (ssp.)" or "serover (or serotype)" after the bacterial name, respectively. The name of *Salmonella* bacteria is sometimes abbreviated by listing the serotype after "S." For example, *S. enterica* sp. *enterica* serovar Typhimurium is sometimes simply referred to as *S. Typhimurium*. The smallest unit of classification is the strain, which refers to a genetically homogeneous population of cells.

[0098] As specific serotypes of Salmonella bacteria, examples include: S. Enteritidis (Salmonella enterica ssp.enterica serovar Enteritidis), S. Typhimurium (Salmonella enterica ssp.enterica serovar Typhimurium), S. Newport, SI 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 Infantis) serovarMontevideo), S.Braenderup, S.Oranienburg, S.Thompson, S.Mississippi, S.Agona, S.Typhi, S.Bareilly, S.Paratyphi B. S.Poona, S.Berta, S.Abony, S.Anatum, S.Baildon, S.Bredeney, S.Chester, S.Gaminara, S.Hartford, S.Kentucky, S.Kiambu, S.Mbandaka, S.Nchanga, S.Reading, S.Senftenberg, S.Stanley, S.Virchow, S.Urbana, etc.

[0099] In this specification, "Salmonella spp. lysing agent" refers to a lysing agent used for lysing Salmonella spp. bacteria. Similarly, "S. enteritidis lysing agent" refers to a lysing agent used for lysing S. enteritidis. The S. enteritidis lysing agent can be a lysing agent specifically for lysing S. enteritidis (S. enteritidis specific lysing agent). "S. Montevideo lysing agent" refers to a lysing agent used for lysing S. Montevideo. The S. Montevideo lysing agent can be a lysing agent specifically for lysing S. Montevideo (S. Montevideo specific lysing agent). "S. Typhimurium lysing agent" refers to a lysing agent used for lysing S. Typhimurium. The S. Typhimurium lysing agent can be a lysing agent specifically for lysing S. Typhimurium (S. Typhimurium specific lysing agent).

[0100] In this specification, "prevention and control" of bacteria means causing bacterial death and / or inhibiting bacterial proliferation.

[0101] In this specification, "multidrug resistance" refers to resistance to multiple antimicrobial agents (such as antibiotics). There are no particular limitations on the antimicrobial agents that can be cited, such as ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline, kanamycin, sulfamethoxazole / trimethoprim, cefazolin, cefotaxime, nalidixic acid, or gentamicin.

[0102] In this specification, "phage" (as mentioned above, often simply referred to as "phage") refers to the general term for viruses that infect bacteria. A typical bacteriophage consists of three parts: a head, a tail, and a tail fiber. The head is composed of a capsid, which is a protein coat and contains an icosahedral capsid (viral capsid) containing the phage's genomic DNA. The tail has tail tube proteins and a tubular structure covered by sheath proteins. One end of the tail connects to the head, and the other end connects to the tail fiber. The tail functions as an introductory tube for injecting the genomic DNA from the head into the host bacterial cell. The tail fiber consists of multiple fibrous structures formed by tail fiber proteins. The tail and tail fiber recognize receptors on the outer membrane surface of the host bacteria, performing host recognition and adsorption functions on the cell surface. Bacteriophages exhibit very high host specificity, a characteristic based on the function of the tail and tail fiber. More specifically, any one of the following proteins—tail fibrin, tail tube, tail tip, and tail nail—plays a central role in this function.

[0103] In this specification, "tail fibrin" refers to the protein that constitutes the tail fibrils of a bacteriophage as described above. Tail fibrin is known to play a crucial role in the specificity of host recognition and adsorption capabilities of the tail and tail fibrils (Nobrega Flet al., Nat. Rev. Microbiol., 2018, 16: 760-773). Therefore, novel bacteriophages possessing tail fibrin characteristics can exhibit bacteriolytic activity even against bacteria resistant to known bacteriophages, due to differences in host recognition sites, even if the host bacteria are the same. This makes them highly valuable for utilization.

[0104] In this specification, "tail fiber gene" refers to the gene encoding the aforementioned tail fiber protein contained in the genomic DNA of the bacteriophage.

[0105] In this specification, "tail tube protein" refers to the protein that constitutes the tubular structure of the tail of a bacteriophage as described above. Tail tube proteins are known to interact with tail filaments and, together with the tail filaments, play an important role in the specificity of host recognition and adsorption capabilities (Maozhi Hu, et al., 2020, 9:1, 855-867). Tail tube proteins include tail tube fibrin A and tail tube protein B. "Tail tube protein A" refers to the protein that forms a ring at the lower part of the tubular structure of the tail and interacts with the tail filaments. "Tail tube protein B" refers to the protein that forms the lower end of the tubular structure of the tail and binds to receptors present on the outer membrane surface of the host bacteria.

[0106] In this specification, "tail tube gene" refers to the gene in the genomic DNA of a bacteriophage that encodes the aforementioned tail tube protein. "Tail tube protein A gene" refers to the gene encoding tail tube protein A; "tail tube protein B gene" refers to the gene encoding tail tube protein B.

[0107] In this specification, "tail tip protein" refers to the protein that constitutes the tail tip of a bacteriophage. Its sharp structure allows it to penetrate the cell wall of the host bacteria, and as mentioned above, it also has the function of binding to receptors in the host bacteria. Tail tip proteins are known to bind to receptors in the host bacteria, and therefore play an important role in host recognition and adsorption capabilities (Nobrega Flet al., Nat. Rev. Microbiol., 2018, 16: 760-773).

[0108] In this specification, "tail tip gene" refers to the gene in the genomic DNA of a bacteriophage that encodes the aforementioned tail tip protein.

[0109] In this specification, "pin protein" refers to the protein that constitutes the tip of the tail of a bacteriophage, and as described above, it has the function of binding to receptors of the host bacteria. When a disc-shaped structure (tail plate) exists at the tip of the bacteriophage tail, the pin protein forms a pin-like structure at the bottom of the plate. Pin proteins are known to have the function of binding to receptors of the host bacteria, and therefore play an important role in host recognition and adsorption capabilities (Nobrega Flet al., Nat. Rev. Microbiol., 2018, 16: 760-773).

[0110] In this specification, "tail pin gene" refers to the gene in the genomic DNA of a bacteriophage that encodes the aforementioned tail pin protein.

[0111] It should be noted that bacteriophages do not necessarily possess all of the aforementioned tail fiber genes, tail tube genes, tail tip genes, and tail nail genes simultaneously. Bacteriophages may contain one, two, three, or all four of the tail fiber genes, tail tube genes, tail tip genes, and tail nail genes.

[0112] In this specification, "endonuclease" refers to an enzyme that cleaves polynucleotide chains within a polynucleotide chain. It is known that lysophages, while infecting, disrupt the host bacteria's life-sustaining mechanisms through various methods, allowing only their own replication and thus shutting down the replication of the host genome. The detailed nature of this mechanism remains unclear, but it was previously established that phage-derived nucleases are involved in the degradation of the host genome (Warren et al., Journal of Virology, Vol. 2, No. 4, 1968). Therefore, it can be considered that lysophage endonucleases participate in the mechanism of shutting down host genome replication.

[0113] Because bacteriophages do not infect eukaryotes, reagents using bacteriophages are harmless to humans, animals, and plants. It should be noted that the life cycle of a bacteriophage is roughly divided into a "lysis cycle," a "lysogen cycle," and a "lysis / lysogen cycle." During the lysogen cycle, the bacteriophage does not lyse the target bacteria but instead integrates its own DNA into the bacterial chromosome, multiplying alongside the bacteria. Conversely, during the lysis cycle, the bacteriophage self-reproduces within the host bacterial cell, then lyses the host bacteria, releasing a large number of progeny bacteriophages. The bacteriophage of this invention can be a bacteriophage that has undergone either a lysis cycle or a lysis / lysogen cycle.

[0114] In this specification, "multiple" refers to 2 to 10, such as 2 to 7, 2 to 5, 2 to 4, or 2 to 3.

[0115] In this specification, "base sequence identity" refers to the proportion of bases that are identical within the alignment range of two base sequences. Even when the lengths of the two base sequences differ, base sequence identity can be calculated by aligning the entire sequence to achieve the highest possible base consistency within the alignment range. While not limited to a single algorithm, BLAST is a representative algorithm for such analysis. BLAST can be utilized through various software and web services. For example, base sequence identity can be easily calculated using genetic information processing software GENTYX (https: / / www.genetyx.co.jp / ) or the BLAST server provided by NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). In addition to BLAST, algorithms such as FASTA can also be used, as long as a reasonable identity can be calculated. Furthermore, base sequence identity analysis can also be performed using analysis algorithms such as MUMmer. It should be noted that, depending on the software and analysis server, some use Average Nucleotide Identity (ANI) or similar metrics to represent sequence identity, and these can also be used. It should also be noted that, in the aforementioned software and web services, when arranging long-chain base sequences such as bacteriophage genomic DNA, the alignment range is sometimes automatically determined, and the sequence identity within that alignment range is calculated. Therefore, within the range automatically arranged and configured by the aforementioned software and web services, the aforementioned sequence identity can be present. For example, in analysis using the BLAST server provided by NCBI, the query sequence and subject sequence are automatically arranged within the maximum permutable range to determine the alignment range, and the sequence identity within the alignment range is calculated. Simultaneously, sometimes the ratio of the alignment range to the entire range of the query sequence is calculated as a value called QueryCover. In this case, based on the result, sequence identity can be inferred across the entire range of the arranged base sequences. For example, the value obtained by multiplying the QueryCover value by the sequence identity value within the alignment range can be used as the inferred value of sequence identity across the entire range. At this point, to improve the accuracy of the inferred values, further corrections can be added, such as taking into account expected sequence identity in ranges other than the permutation range. It should be noted that during the packaging of bacteriophage genomic DNA, both linear and circular patterns exist. Furthermore, in next-generation genome sequencing analysis, the base sequences of the fragmented genomic DNA are read, and the sequences are determined by splicing them together.In the case of bacteriophages, a reference genomic DNA sequence is usually not set for de novo assembly. Therefore, it is difficult to uniquely determine the start / end point of the genome analysis (Merrill, BD, et al. BMC Genomics, 2016, 17, 679). Therefore, the start / end point of the paired genome sequence can be different, and this is automatically taken into account in analyses using software and analysis servers.

[0116] In this specification, "high stringency conditions" refers to environmental conditions that make it difficult to produce nonspecific hybridization. Under high stringency conditions, hybrids can be formed with nucleic acids having the target base sequence, but essentially cannot be formed with nucleic acids having nonspecific base sequences. Typically, high stringency conditions refer to conditions with low salt concentration and high temperature. Low salt concentration is, for example, 15–750 mM, preferably 15–500 mM, 15–300 mM, or 15–200 mM. High temperature refers to, for example, 50–68°C or 55–70°C. A specific example of high stringency conditions is washing after hybridization at 65°C, 0.1×SSC, and 0.1% SDS.

[0117] In this specification, "amino acid sequence identity" refers to the proportion of sites where the types of amino acid residues are the same within the alignment range of two amino acid sequences. Even when the lengths of two amino acid sequences differ, amino acid sequence identity can be calculated by aligning them in a manner that maximizes the amino acid matching within the alignment range. Although not limited, BLAST is a representative algorithm for performing such analysis. BLAST can be used using various software and web services. For example, the genetic information processing software GENTYX (https: / / www.genetyx.co.jp / ) and the BLAST server provided by NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) can be used to easily calculate amino acid sequence identity. In addition to BLAST, there are algorithms such as FASTA, which can be used as long as a reasonable identity can be calculated.

[0118] In this specification, "(amino acid) substitution" preferably refers to substitution within a conserved group of amino acids that are similar in properties such as charge, side chain, polarity, and aromaticity, among the 20 amino acids that constitute natural proteins. Examples include substitutions within groups of charge-free polar amino acids with low-polarity side chains (Gly, Ash, Gln, Ser, Thr, Cys, Tyr), branched-chain amino acids (Leu, Val, Ile), neutral amino acids (Gly, Ile, Va1, 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). A single substitution or two or more substitutions may be present. It is known that substitutions within these groups do not significantly alter the properties of the polypeptide, and are therefore preferred.

[0119] 1. Bacteriophage / lysing agent

[0120] The first aspect of the present invention is the following bacteriophages exhibiting lytic activity against Salmonella bacteria and a lysing agent containing the bacteriophage.

[0121] <First phage / lysing agent>

[0122] (summary)

[0123] This invention provides a bacteriophage (sometimes referred to as "first phage" in this specification) having the following composition and exhibiting lytic activity against Salmonella bacteria, and provides a lysing agent comprising the first phage (sometimes referred to as "first lysing agent" in this specification). The first lysing agent is particularly a lysing agent exhibiting specific lytic activity against specific Salmonella bacteria, such as a lysing agent for S. enteritidis. The first lysing agent comprises a bacteriophage having a genomic DNA sequence containing a specific base sequence.

[0124] According to the first lysosomal agent, the target bacteria can be lysed to achieve prevention and control.

[0125] (constitute)

[0126] The first lysing agent is a lysing agent for Salmonella bacteria, particularly S. enteritidis. The first lysing agent contains a first bacteriophage having the following composition, which has lysing activity against Salmonella bacteria.

[0127] Phage 1 has a genomic DNA sequence containing a specific base sequence.

[0128] The inventors discovered seven bacteriophages with specific lytic activity against strains of S. enteritidis, and found that the genomic DNA sequences of these bacteriophages (sequence numbers 1 to 7) 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 sequence number 7 relative to the genomic DNA sequences numbered 1 to 6 was calculated, and the results showed that the identity was 100% across the entire range.

[0129] The first phage contains any one of the base sequences shown in (a) to (c) below, or has a genomic DNA sequence containing it.

[0130] (a) The base sequence shown in any one of sequence numbers 1 to 7;

[0131] (b) A base sequence in any of the base sequences shown in sequence numbers 1 to 7 in which one or more bases have been added, deleted, and / or substituted;

[0132] (c) A base sequence that has more than 99% sequence identity with any of the base sequences shown in sequence numbers 1 to 7.

[0133] (c) The sequence identity specified is preferably 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher, 99.5% or higher, 99.6% or higher, 99.7% or higher, 99.8% or higher, or 99.9% or higher.

[0134] Phage 1 can exhibit broad-spectrum lytic activity, particularly against *S. enteritidis*, the serotype most frequently detected in human food poisoning, and is therefore useful for the treatment or prevention of food poisoning. Phage 1 can also exhibit specific lytic activity against *S. enteritidis*, and is therefore useful, for example, for the identification of serotypes of pathogens causing food poisoning.

[0135] <Second phage / lysing agent>

[0136] (summary)

[0137] This invention provides a bacteriophage (sometimes referred to as "second phage" in this specification) having the following composition and exhibiting lytic activity against Salmonella bacteria, and a lytic agent comprising the phage (sometimes referred to as "second lytic agent" in this specification). 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 comprising a gene encoding a tail tip protein containing a specific amino acid sequence.

[0138] Based on the second bacteriophage, Salmonella bacteria that serve as target bacteria can be lysed to achieve prevention and control.

[0139] (constitute)

[0140] Phage 2 is a bacteriophage with the following composition that has lytic activity against Salmonella bacteria.

[0141] The second phage contains a specific amino acid sequence and genomic DNA that includes a gene encoding a tail tip protein that has recognition activity against target bacteria.

[0142] The inventors discovered three bacteriophages with lytic activity against Salmonella bacteria, and identified the tail tip protein (sequence number 8) and tail tip gene (sequence number 9) from the genomic DNA sequences of each bacteriophage (sequence numbers 10-12). The genomic sequences of the three bacteriophages were 99% identical, and the amino acid sequences of their respective tail tip proteins, as shown in sequence number 8, were completely identical.

[0143] The tail tip protein contains the amino acid sequence shown in Sequence Number 8, consisting of 637 amino acid residues. In this invention, by using the tail tip protein containing the amino acid sequence shown in Sequence Number 8, it is possible to achieve host specificity with high availability, exhibiting specificity against Salmonella bacteria and broad-spectrum lytic activity against various species within the Salmonella genus.

[0144] (1) Tail tip protein

[0145] The tail tip protein in phage 2 contains any of the following amino acid sequences (a) to (c):

[0146] (a) The amino acid sequence shown in sequence number 8;

[0147] (b) An amino acid sequence in which one or more amino acids have been added, deleted, and / or replaced in the amino acid sequence shown in Sequence Number 8;

[0148] (c) An amino acid sequence that has more than 99% sequence identity with the amino acid sequence shown in sequence number 8.

[0149] (c) The sequence identity specified is preferably 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher, 99.5% or higher, 99.6% or higher, 99.7% or higher, 99.8% or higher, or 99.9% or higher.

[0150] In the amino acid sequence specified in (b) or (c), the amino acid at position 258 of sequence number 8 of the tail tip protein is preferably phenylalanine, and / or the amino acid at position 617 of sequence number 8 is preferably serine. It should be noted that the position numbering is indicated with methionine as the first position.

[0151] (2) Tail tip gene

[0152] The gene encoding the tail tip protein contains, for example, the base sequence shown in any of (d) to (f) below:

[0153] (d) The base sequence shown in sequence number 9;

[0154] (e) A base sequence in which one or more bases are added, deleted, and / or replaced in the base sequence shown in sequence number 9;

[0155] (f) A base sequence that has more than 95% sequence identity with the base sequence shown in sequence number 9.

[0156] Alternatively, examples could be given of base sequences that hybridize under highly stringent conditions to base sequences complementary to the base sequence shown in sequence number 9.

[0157] (f) The sequence identity specified is preferably 96% or more, 97% or more, 98% or more, or 99% or more.

[0158] (3) Genomic DNA

[0159] Phage 2 has genomic DNA containing a gene encoding a tail tip protein.

[0160] The genomic DNA sequence contains, or consists of, any of the base sequences shown in (g) to (k) below:

[0161] (g) The base sequence shown in any one of sequence numbers 10 to 12;

[0162] (h) In any of the base sequences shown in sequence numbers 10 to 12, a base sequence in which one or more bases are added, deleted, and / or substituted in a base sequence other than the base sequence of the above-mentioned gene;

[0163] (i) Among the base sequences shown in any one of sequence numbers 10 to 12, the base sequences other than the above-mentioned gene base sequences have more than 80% sequence identity;

[0164] (j) A base sequence in which one or more bases are added, deleted, and / or replaced in any of the base sequences shown in sequence numbers 10 to 12;

[0165] (k) A base sequence that has more than 90% sequence identity with any of the base sequences shown in sequence numbers 10 to 12.

[0166] (i) The specified sequence identity is preferably 81% or higher, 82% or higher, 83% or higher, 84% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, 90.5% or higher, 91.0% or higher, 91.5% or higher, 92.0% or higher, 92.5% or higher, 93.0% or higher, 93.5% or higher, 94.0% or higher, or 94.5% or higher. Above, 95.0%, 95.5%, 96.0%, 96.5%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%.

[0167] In other words, (i) refers to a base sequence that has a sequence identity of more than 80% with respect to the base sequence shown in any of the sequence numbers 10 to 12, excluding the base sequence of the gene mentioned above.

[0168] (k) The sequence identity specified is preferably 90.5% or higher, 91.0% or higher, 91.5% or higher, 92.0% or higher, 92.5% or higher, 93.0% or higher, 93.5% or higher, 94.0% or higher, 94.5% or higher, 95.0% or higher, 95.5% or higher, 96.0% or higher, 96.5% or higher, 97.0% or higher, 97.5% or higher, 98.0% or higher, 98.5% or higher, 99.0% or higher, 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher, 99.5% or higher, 99.6% or higher, 99.7% or higher, 99.8% or higher, or 99.9% or higher.

[0169] In one embodiment, the second phage has a genomic DNA sequence containing a specific base sequence that exhibits lytic activity against target bacteria. Examples of the genomic DNA sequence of the second phage include: a base sequence represented by any one of sequence numbers 10-12 (113946 bp, 113936 bp, and 113949 bp, respectively); a base sequence in which one or more bases are added, deleted, and / or substituted in any one of the base sequences represented by sequence numbers 10-12; or a base sequence containing 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, 90.5% or more, or 90% or more of the base sequence represented by sequence numbers 10-12. Genomic DNA sequences with a sequence identity of 1.0% or higher, 91.5% or higher, 92.0% or higher, 92.5% or higher, 93.0% or higher, 93.5% or higher, 94.0% or higher, 94.5% or higher, 95.0% or higher, 95.5% or higher, 96.0% or higher, 96.5% or higher, 97.0% or higher, 97.5% or higher, 98.0% or higher, 98.5% or higher, 99.0% or higher, 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher, 99.5% or higher, 99.6% or higher, 99.7% or higher, 99.8% or higher, or 99.9% or higher.

[0170] (4) Effect

[0171] Because phage 2 exhibits broad-spectrum lytic activity against various species of Salmonella, it can effectively control Salmonella bacteria. Additionally, phage 2 is useful for treating or preventing food poisoning. Due to its broad host range, phage 2 can effectively cover the diversity of target bacteria. Therefore, phages like phage 2, which exhibit broad-spectrum lytic activity against a wide range of bacterial species, are highly usable.

[0172] <Third phage / lysing agent>

[0173] (summary)

[0174] This invention provides a bacteriophage (sometimes referred to as "third phage" in this specification) having the following composition and exhibiting lytic activity against Salmonella bacteria, and a lytic agent comprising the third phage (sometimes referred to as "third lytic agent" in this specification). The third phage exhibits lytic activity against at least one bacterium selected from *S. typhimurium*, preferably against multidrug-resistant *S. typhimurium*. The third phage has a specific genomic DNA sequence.

[0175] It should be noted that the target bacteria of bacteriophage 3 are not limited to S. Typhimurium. Bacteriophage 3 can effectively control S. Typhimurium, especially multidrug-resistant S. Typhimurium, but as a target bacterium, it can also be effective against other serotypes, such as S. Enteritidis, S. Infantis, or S. Javiana.

[0176] Based on the third phage, S. typhimurium, a target bacterium, can be effectively lysed to achieve prevention and control, especially S. typhimurium with multidrug resistance.

[0177] (constitute)

[0178] The third bacteriophage is a bacteriophage with the following composition that has lytic activity against Salmonella bacteria.

[0179] Phage 3 has genomic DNA containing a specific base sequence.

[0180] The inventors have discovered a bacteriophage with lytic activity against Salmonella bacteria and identified the genomic DNA sequence of the bacteriophage (sequence number 13).

[0181] The third phage is specific to Salmonella bacteria, and exhibits broad-spectrum lytic activity, particularly against S. typhimurium, a strain of Salmonella that is problematic due to its high prevalence of multidrug-resistant strains, thus making it highly usable.

[0182] The third phage contains the base sequence shown in any one of (a) to (c) below, or has genomic DNA containing it.

[0183] (a) The base sequence shown in sequence number 13;

[0184] (b) A base sequence in which one or more bases are added, deleted, and / or replaced in the base sequence shown in sequence number 13;

[0185] (c) A sequence of more than 90% sequence identity with respect to the sequence shown in sequence number 13.

[0186] (c) The specified sequence identity is preferably 90.5% or higher, 91.0% or higher, 91.5% or higher, 92.0% or higher, 92.5% or higher, 93.0% or higher, 93.5% or higher, 94.0% or higher, 94.5% or higher, 95.0% or higher, 95.5% or higher, 96.0% or higher, 96.5% or higher, 97.0% or higher, 97.5% or higher, 98.0% or higher, 98.5% or higher, 99.0% or higher, 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher, 99.5% or higher, 99.6% or higher, 99.7% or higher, 99.8% or higher, or 99.9% or higher.

[0187] Because phage 3 exhibits broad-spectrum lytic activity against Salmonella bacteria, especially S. typhimurium, which is problematic due to its many multidrug-resistant strains, it is useful for the treatment or prevention of food poisoning.

[0188] <4th phage / lysing agent>

[0189] (summary)

[0190] This invention provides a bacteriophage (sometimes referred to as "fourth phage" in this specification) having the following composition and exhibiting lytic activity against Salmonella bacteria, and a lysing agent comprising the phage (sometimes referred to as "fourth lysing agent" in this specification). The fourth lysing agent is a lysing agent that exhibits specific lytic activity against a particular Salmonella spp., for example, a lysing agent for S. Montevideo. The fourth lysing agent comprises a bacteriophage having a genomic DNA sequence containing a specific base sequence.

[0191] According to the fourth lysosomal agent, it can lyse the target bacteria to achieve prevention and control.

[0192] (constitute)

[0193] The fourth lysing agent is a lysing agent for Salmonella bacteria, particularly S. Montevideo. This fourth lysing agent contains a bacteriophage with the following composition that has lysing activity against Salmonella bacteria.

[0194] Phage 4 has a genomic DNA sequence containing a specific base sequence.

[0195] The inventors discovered a bacteriophage with strain-specific lysing activity of S. Montevideo and identified the genomic DNA sequence of the bacteriophage (sequence number 14).

[0196] Phage 4 contains the base sequence shown in any one of (a) to (c) below, or has a genomic DNA sequence containing it.

[0197] (a) The base sequence shown in sequence number 14;

[0198] (b) A base sequence in which one or more bases have been added, deleted, and / or substituted in the base sequence shown in sequence number 14;

[0199] (c) A base sequence that has more than 95% sequence identity with the base sequence shown in sequence number 14.

[0200] (c) The specified sequence identity is preferably 95.5% or higher, 96.0% or higher, 96.5% or higher, 97.0% or higher, 97.5% or higher, 98.0% or higher, 98.5% or higher, 99.0% or higher, 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher, 99.5% or higher, 99.6% or higher, 99.7% or higher, 99.8% or higher, or 99.9% or higher.

[0201] Because phage 4 can exhibit specific lytic activity against Salmonella bacteria, especially S. Montevideo, it is useful, for example, for the identification of serotypes of pathogens causing food poisoning.

[0202] <5th phage / lysing agent>

[0203] (summary)

[0204] This invention provides a bacteriophage (sometimes referred to in this specification as "Phage 5") having the following composition and exhibiting lytic activity against Salmonella bacteria, and a lysing agent comprising the phage (sometimes referred to in this specification as "Lysing Agent 5"). The Lysing Agent 5 is a lysing agent that exhibits specific lytic activity against a particular Salmonella spp., for example, a lysing agent for S. Typhimurium. The Lysing Agent 5 comprises a bacteriophage having genomic DNA containing a gene encoding a nuclease having a specific amino acid sequence.

[0205] According to the fifth lysosomal agent, it can lyse the target bacteria to achieve prevention and control.

[0206] (constitute)

[0207] The fifth type of lysing agent is a lysing agent for Salmonella bacteria, particularly S. typhimurium. This fifth type of lysing agent contains a bacteriophage with the following composition that has lysing activity against Salmonella bacteria.

[0208] Phage 5 has a specific amino acid sequence and genomic DNA containing a gene encoding a nuclease with endonuclease activity.

[0209] The inventors discovered a bacteriophage with specific lytic activity against *S. typhimurium* and identified its genomic DNA sequence (Sequence No. 17). The inventors further identified a novel endonuclease gene derived from the genomic DNA sequence of this bacteriophage. The amino acid sequence of this endonuclease and the base sequence encoding it are shown in Sequence Nos. 15 and 16, respectively. This endonuclease can participate in the shutdown of host genome replication, thereby enhancing lytic activity.

[0210] (1) Endonuclease

[0211] The endonuclease in phage 5 contains any of the amino acid sequences shown in (a) to (c) below:

[0212] (a) The amino acid sequence shown in sequence number 15;

[0213] (b) An amino acid sequence in which one or more amino acids have been added, deleted, and / or replaced in the amino acid sequence shown in sequence number 15;

[0214] (c) An amino acid sequence that has more than 90% sequence identity with the amino acid sequence shown in sequence number 15.

[0215] (c) The specified sequence identity is preferably 90.5% or higher, 91.0% or higher, 91.5% or higher, 92.0% or higher, 92.5% or higher, 93.0% or higher, 93.5% or higher, 94.0% or higher, 94.5% or higher, 95.0% or higher, 95.5% or higher, 96.0% or higher, 96.5% or higher, 97.0% or higher, 97.5% or higher, 98.0% or higher, 98.5% or higher, 99.0% or higher, 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher, 99.5% or higher, 99.6% or higher, 99.7% or higher, 99.8% or higher, or 99.9% or higher.

[0216] (2) Endonuclease gene

[0217] Genes encoding endonucleases contain, for example, the base sequences shown in any of (d) to (f) below:

[0218] (d) The base sequence shown in sequence number 16;

[0219] (e) A base sequence in which one or more bases are added, deleted, and / or replaced in the base sequence shown in sequence number 16;

[0220] (f) A base sequence that has more than 90% sequence identity with the base sequence shown in sequence number 16.

[0221] Alternatively, examples could be given of base sequences that hybridize with base sequences complementary to the base sequence shown in sequence number 16 under highly stringent conditions.

[0222] (f) The sequence identity specified is preferably 90.5% or higher, 91.0% or higher, 91.5% or higher, 92.0% or higher, 92.5% or higher, 93.0% or higher, 93.5% or higher, 94.0% or higher, 94.5% or higher, 95.0% or higher, 95.5% or higher, 96.0% or higher, 96.5% or higher, 97.0% or higher, 97.5% or higher, 98.0% or higher, 98.5% or higher, 99.0% or higher, 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher, 99.5% or higher, 99.6% or higher, 99.7% or higher, 99.8% or higher, or 99.9% or higher.

[0223] (3) Genomic DNA

[0224] Phage 5 has genomic DNA containing genes encoding endonucleases.

[0225] The genomic DNA sequence contains, or consists of, any of the base sequences shown in (g) to (k) below:

[0226] (g) The base sequence shown in sequence number 17;

[0227] (h) In the base sequence shown in sequence number 17, a base sequence in which one or more bases are added, deleted, and / or substituted in a base sequence other than the base sequence of the above-mentioned gene;

[0228] (i) In the base sequence shown in sequence number 17, the base sequence other than the above-mentioned gene base sequence has more than 80% sequence identity;

[0229] (i) A base sequence in which one or more bases have been added, deleted, and / or replaced in the base sequence shown in sequence number 17;

[0230] (k) A base sequence that has more than 90% sequence identity with the base sequence shown in sequence number 17.

[0231] (i) The specified sequence identity is preferably 81% or higher, 82% or higher, 83% or higher, 84% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, 90.5% or higher, 91.0% or higher, 91.5% or higher, 92.0% or higher, 92.5% or higher, 93.0% or higher, 93.5% or higher, 94.0% or higher, or 94.5% or higher. 95.0% or above, 95.5% or above, 96.0% or above, 96.5% or above, 97.0% or above, 97.5% or above, 98.0% or above, 98.5% or above, 99.0% or above, 99.1% or above, 99.2% or above, 99.3% or above, 99.4% or above, 99.5% or above, 99.6% or above, 99.7% or above, 99.8% or above, or 99.9% or above.

[0232] In other words, (i) refers to a base sequence that has a sequence identity of more than 80% with respect to the base sequence shown in sequence number 17, excluding the base sequence of the gene mentioned above.

[0233] (k) The sequence identity specified is preferably 90.5% or higher, 91.0% or higher, 91.5% or higher, 92.0% or higher, 92.5% or higher, 93.0% or higher, 93.5% or higher, 94.0% or higher, 94.5% or higher, 95.0% or higher, 95.5% or higher, 96.0% or higher, 96.5% or higher, 97.0% or higher, 97.5% or higher, 98.0% or higher, 98.5% or higher, 99.0% or higher, 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher, 99.5% or higher, 99.6% or higher, 99.7% or higher, 99.8% or higher, or 99.9% or higher.

[0234] In one embodiment, the fifth phage has a genomic DNA sequence containing a specific base sequence and exhibits lytic activity against target bacteria. Examples of the genomic DNA sequence of the fifth phage include: the base sequence shown in sequence number 17 (47638 bp); a base sequence in which one or more bases are added, deleted, and / or substituted in the base sequence shown in sequence number 17; or a sequence containing 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 sequence number 17. Genomic DNA sequences with a sequence identity of 94.5% or higher, 95.0% or higher, 95.5% or higher, 96.0% or higher, 96.5% or higher, 97.0% or higher, 97.5% or higher, 98.0% or higher, 98.5% or higher, 99.0% or higher, 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher, 99.5% or higher, 99.6% or higher, 99.7% or higher, 99.8% or higher, or 99.9% or higher.

[0235] Because phage 5 can exhibit specific lytic activity against Salmonella bacteria, especially S. typhimurium, it is useful, for example, in the identification of serotypes of pathogens causing food poisoning.

[0236] <6th phage / lysing agent>

[0237] (summary)

[0238] This invention provides a bacteriophage (sometimes referred to as "S. 6" in this specification) having the following composition and exhibiting lytic activity against Salmonella bacteria, and a lytic agent containing the phage (sometimes referred to as "S. 6 lytic agent" in this specification). Phage 6 exhibits lytic activity against S. enteritidis, S. typhimurium, and S. javiana. Phage 6 has a genomic DNA sequence containing a gene encoding a tail fibrin protein containing a specific amino acid sequence.

[0239] Based on the 6th phage, Salmonella bacteria that serve as target bacteria can be lysed to achieve prevention and control.

[0240] (constitute)

[0241] Phage 6 is a bacteriophage with the following composition that has lytic activity against Salmonella bacteria.

[0242] Phage 6 contains a specific amino acid sequence and has genomic DNA that includes a gene encoding a tail fibrin that has target bacterial recognition activity.

[0243] The inventors discovered a bacteriophage with lytic activity against Salmonella bacteria, and identified tail fibrin (sequence number 18) and tail fibrin gene (sequence number 19) from the genomic DNA sequence (sequence number 20) of the bacteriophage.

[0244] The tail fibroin contains the amino acid sequence shown in Sequence Number 18, consisting of 684 amino acid residues. In this invention, based on the tail fibroin containing the amino acid sequence shown in Sequence Number 18, it is possible to achieve highly specific host availability, exhibiting lytic activity against Salmonella bacteria, particularly against *S. enteritidis*, *S. typhimurium*, and *S. javiana*.

[0245] (1) Tail fibrous protein

[0246] The tail fibrin in phage 6 contains the amino acid sequence shown in any one of (a) to (c) below:

[0247] (a) The amino acid sequence shown in sequence number 18;

[0248] (b) An amino acid sequence in which one or more amino acids have been added, deleted, and / or replaced in the amino acid sequence shown in sequence number 18;

[0249] (c) An amino acid sequence that has more than 99% sequence identity with respect to the amino acid sequence shown in sequence number 18.

[0250] (c) The sequence identity specified is preferably 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher, 99.5% or higher, 99.6% or higher, 99.7% or higher, 99.8% or higher, or 99.9% or higher.

[0251] The amino acid sequence of the tail fibrin of bacteriophage 6 contains several unique amino acid residues that differ from those of known tail fibrin sequences. In the amino acid sequence of the tail fibrin of bacteriophage 6 (Sequence number 18), the amino acid at position 211 is Val, the amino acid at position 321 is Val, the amino acid at position 485 is Val, the amino acid at position 533 is Ala, the amino acid at position 577 is Ser, and the amino acid at position 583 is Ser. Surprisingly, these sites, despite being highly conserved in the tail fibrins of other bacteriophages, are different amino acid residues, which can be attributed to the specific host range of bacteriophage 6. Therefore, in the amino acid sequence specified in (b) or (c), it is preferable that the amino acid at position 211 is Val, the amino acid at position 321 is Val, the amino acid at position 485 is Val, the amino acid at position 533 is Ala, the amino acid at position 577 is Ser, and / or the amino acid at position 583 is Ser. It should be noted that the position number is indicated with the starting methionine as the first position.

[0252] (2) Tail fiber gene

[0253] The gene encoding tail fibroin contains, for example, the base sequence shown in any of (d) to (f) below:

[0254] (d) The base sequence shown in sequence number 19;

[0255] (e) A base sequence in which one or more bases are added, deleted, and / or replaced in the base sequence shown in sequence number 19;

[0256] (f) A base sequence that has more than 97% sequence identity with respect to the base sequence shown in sequence number 19.

[0257] Alternatively, examples could be given of base sequences that hybridize with base sequences complementary to the base sequence shown in sequence number 19 under highly stringent conditions.

[0258] (f) The sequence identity specified is preferably 97.5% or higher, 98.0% or higher, 98.5% or higher, 99.0% or higher, 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher, 99.5% or higher, 99.6% or higher, 99.7% or higher, 99.8% or higher, or 99.9% or higher.

[0259] (3) Genomic DNA

[0260] Phage 6 has genomic DNA containing a gene encoding tail fibrin.

[0261] The genomic DNA sequence contains, for example, the base sequence shown in any of the following (g) to (k):

[0262] (g) The base sequence shown in sequence number 20;

[0263] (h) In the base sequence shown in sequence number 20, a base sequence in which one or more bases are added, deleted, and / or substituted in a base sequence other than the base sequence of the above-mentioned gene;

[0264] (i) In the base sequence shown in sequence number 20, the base sequence other than the above-mentioned gene base sequence has more than 90% sequence identity;

[0265] (j) A base sequence in which one or more bases have been added, deleted, and / or replaced in the base sequence shown in sequence number 20;

[0266] (k) A base sequence in sequence number 20 that has more than 95% sequence identity.

[0267] (i) The specified sequence identity is preferably 90.5% or higher, 91.0% or higher, 91.5% or higher, 92.0% or higher, 92.5% or higher, 93.0% or higher, 93.5% or higher, 94.0% or higher, 94.5% or higher, 95.0% or higher, 95.5% or higher, 96.0% or higher, 96.5% or higher, 97.0% or higher, 97.5% or higher, 98.0% or higher, 98.5% or higher, 99.0% or higher, 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher, 99.5% or higher, 99.6% or higher, 99.7% or higher, 99.8% or higher, or 99.9% or higher.

[0268] In other words, (i) refers to a base sequence that has a sequence identity of more than 90% with respect to the base sequence shown in sequence number 20, excluding the gene base sequence mentioned above and other base sequences that are equivalent to the gene mentioned above.

[0269] (k) The sequence identity specified is preferably 95.5% or higher, 96.0% or higher, 96.5% or higher, 97.0% or higher, 97.5% or higher, 98.0% or higher, 98.5% or higher, 99.0% or higher, 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher, 99.5% or higher, 99.6% or higher, 99.7% or higher, 99.8% or higher, or 99.9% or higher.

[0270] In one embodiment, the sixth phage has a genomic DNA sequence containing a specific base sequence that exhibits lytic activity against target bacteria. Examples of the genomic DNA sequence of the sixth phage include: the base sequence shown in sequence number 20 (40784 bp); a base sequence in which one or more bases are added, deleted, and / or substituted in the base sequence shown in sequence number 20; or a sequence containing a base sequence containing 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 sequence number 20. Genomic DNA sequences with a sequence identity of 94.5%, 95.0%, 95.5%, 96.0%, 96.5%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or higher.

[0271] (4) Effect

[0272] According to the present invention, a novel bacteriophage with lytic activity against Salmonella bacteria can be provided. In this invention, specificity against Salmonella bacteria, particularly exhibiting lytic activity against *S. enteritidis*, *S. typhimurium*, and *S. javiana*, can be achieved through the tail fibrous protein containing the amino acid sequence shown in sequence number 18. Therefore, this sixth bacteriophage is also useful for the treatment or prevention of food poisoning.

[0273] <7th phage / lysing agent>

[0274] (summary)

[0275] This invention provides a bacteriophage (sometimes referred to as "the 7th phage" in this specification) having the following composition and exhibiting lytic activity against Salmonella bacteria, and a lysing agent comprising the phage (referred to as "the 7th lysing agent" in this specification). The 7th lysing agent is a lysing agent that exhibits specific lytic activity against certain Salmonella bacteria, such as the lysing agent used for S. enteritidis. The 7th lysing agent comprises a bacteriophage having a genomic DNA sequence containing a specific base sequence.

[0276] According to the 7th lysosomal agent, it can lyse the target bacteria to achieve prevention and control.

[0277] (constitute)

[0278] The seventh lysin is a bacteriophage, particularly a Salmonella lysin, especially a S. enteritidis lysin. The seventh lysin contains a seventh bacteriophage with the following composition that has lysing activity against Salmonella spp.

[0279] Phage 7 contains a specific amino acid sequence and genomic DNA that includes a gene encoding a caustic protein that has target bacterial recognition activity.

[0280] The inventors discovered a bacteriophage with specific lytic activity against *S. enteritidis* and identified its genomic DNA sequence (Sequence No. 23). The inventors further identified a gene encoding a pinnated protein from the genomic DNA sequence of this bacteriophage, which is believed to determine the host range of the bacteriophage. The amino acid sequence of this pinnated protein and the base sequence encoding it are shown in Sequence Nos. 21 and 22, respectively.

[0281] (1) Tail nail protein

[0282] The tail nail protein in this invention contains the amino acid sequence shown in sequence number 21.

[0283] (2) Tail nail gene

[0284] Genes encoding tail nail proteins, for example, contain the base sequence shown in sequence number 22.

[0285] (3) Genomic DNA

[0286] Phage 7 has genomic DNA containing a gene encoding a caustic protein.

[0287] The genomic DNA sequence contains, or is formed from, any of the base sequences shown in (a) to (e) below:

[0288] (a) The base sequence shown in sequence number 23;

[0289] (b) In the base sequence shown in sequence number 23, a base sequence in which one or more bases are added, deleted, and / or substituted in a base sequence other than the base sequence of the gene described above;

[0290] (c) In the base sequence shown in sequence number 23, the base sequence other than the base sequence of the above-mentioned gene is a base sequence with more than 99% sequence identity;

[0291] (d) A base sequence in which one or more bases are added, deleted, and / or replaced in the base sequence shown in sequence number 23;

[0292] (e) A base sequence that has more than 99% sequence identity with the base sequence shown in sequence number 23.

[0293] (c) The sequence identity specified is preferably 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher, 99.5% or higher, 99.6% or higher, 99.7% or higher, 99.8% or higher, or 99.9% or higher.

[0294] In other words, the base sequence specified in (c) refers to a base sequence other than the gene base sequence shown in sequence number 23, and whose sequence identity is 99% or more with the base sequence other than the gene equivalent to the gene mentioned above.

[0295] (e) The sequence identity specified 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.

[0296] In one embodiment, the 7th phage has a genomic DNA sequence containing a specific base sequence and exhibits lytic activity against target bacteria. Examples of the genomic DNA sequence of the 7th phage include: the base sequence shown in sequence number 23 (39162 bp); a base sequence in which one or more bases are added, deleted, and / or substituted in the base sequence shown in sequence number 23; or a genomic DNA sequence containing a base sequence having 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or more sequence identity with respect to the base sequence shown in sequence number 23.

[0297] Phage 7 exhibits lytic activity against *S. enteritidis*, a serotype most frequently detected in human food poisoning, and is therefore useful for the treatment or prevention of food poisoning. Phage 7 also exhibits specific lytic activity against *S. enteritidis*, and is therefore useful, for example, for the identification of serotypes of pathogens causing food poisoning.

[0298] 2. Composition

[0299] 2-1. Overview

[0300] The second aspect of the present invention is a composition, particularly a composition for the control of target bacteria. The composition of the present invention comprises the bacteriophage or lysing agent described in the first aspect. In the composition of the present invention, the target bacteria particularly refers to bacteria of the genus *Salmonella*. For example, in the case of a composition comprising a first bacteriophage or a first lysing agent, the target bacteria are particularly *S. enteritidis*. In the case of a composition comprising a second bacteriophage or a second lysing agent, the target bacteria are particularly *S. enteritidis*, *S. typhimurium*, *S. infantis*, *S. Montevideo*, and *S. javiana*. In the case of a composition comprising a third bacteriophage or a third lysing agent, the target bacteria are particularly *S. typhimurium*. In the case of a composition comprising a fourth bacteriophage or a fourth lysing agent, the target bacteria are particularly *S. Montevideo*. In the case of a composition comprising a fifth bacteriophage or a fifth lysing agent, the target bacteria are particularly *S. typhimurium*. In the case of a composition containing a 6th bacteriophage or a 6th lysing agent, the target bacteria are particularly S. Enteritidis, S. Typhimurium, and S. Javiana. In the case of a composition containing a 7th bacteriophage or a 7th lysing agent, the target bacteria are particularly S. Enteritidis.

[0301] The compositions according to the present invention can provide pharmaceutical compositions, additives (e.g., food additives, feed additives, drinking water additives), food products, feeds, cleaning agents, disinfectants, bactericides, and sterilizing agents that are safe for human use and environmentally friendly, and can prevent and treat target bacteria.

[0302] 2-2. Composition

[0303] (1) Essential active ingredients

[0304] The composition of the present invention contains at least one of the bacteriophages and lysing agents described in the first embodiment (i.e., the first bacteriophage, the second bacteriophage, the third bacteriophage, the fourth bacteriophage, the fifth bacteriophage, the sixth bacteriophage, the seventh bacteriophage, the first lysing agent, the second lysing agent, the third lysing agent, the fourth lysing agent, the fifth lysing agent, the sixth lysing agent, the seventh lysing agent, or a combination thereof) as an essential active ingredient. The composition of the present invention can achieve prevention and control by lysing the target bacteria with this active ingredient.

[0305] The specific composition of the bacteriophage and lysing agent is described in detail in Method 1, so its description is omitted here.

[0306] The amount of bacteriophage or lysozyme in the composition of the present invention varies depending on various conditions such as the intended use of the composition, the target organism, the method of use, the dosage form, and the type of bacteria targeted for lysis. However, it is preferable that the amount of bacteriophage is sufficient when it comes into contact with and infects the target bacteria in the target organism. The amount of bacteriophage or lysozyme in the composition of the present invention can be within the range of common knowledge in the art, representing an effective amount of bacteriophage or lysozyme in the composition of the present invention for the prevention and control of target bacteria. The titer of the bacteriophage in the composition of the present invention can be, for example, 1 × 10⁻⁶. 1 ~1×10 15 pfu / mL, 1×10 3 ~1×10 13 pfu / mL, 1×10 5 ~1×10 11 pfu / mL or 1×10 7 ~1×10 9 pfu / mL.

[0307] In the compositions of the present invention, the first to seventh phages or lysins can be used alone or in combination of two or more. For example, in addition to the first phage or lysin, the compositions of the present invention may contain at least one phage or lysin selected from the second to seventh phages or lysins as an active ingredient. For example, in addition to the second phage or lysin, the compositions of the present invention may contain at least one phage or lysin selected from the first and third to seventh phages or lysins as an active ingredient. For example, in addition to the third phage or lysin, the compositions of the present invention may contain at least one phage or lysin selected from the first, second, and fourth to seventh phages or lysins as an active ingredient. For example, in addition to the fourth phage or lysin, the compositions of the present invention may contain at least one phage or lysin selected from the first to third and fifth to seventh phages or lysins as an active ingredient. For example, in addition to the fifth phage or lysozyme, the composition of the present invention may contain at least one phage or lysozyme selected from the first to fourth, sixth, and seventh phages or lysozymes described above as an active ingredient. For example, in addition to the sixth phage or lysozyme, the composition of the present invention may contain at least one phage or lysozyme selected from the first to fifth and seventh phages or lysozymes described above as an active ingredient. For example, in addition to the seventh phage or lysozyme, the composition of the present invention may contain at least one phage or lysozyme selected from the first to sixth phages or lysozymes described above as an active ingredient.

[0308] For example, when combining bacteriophages that target bacteria that are different from each other, or bacteriophages that target bacteria that are the same but recognize different cell surface receptors, synergistic and complementary effects of lysis activity can be expected.

[0309] (2) Other active ingredients

[0310] In addition to the bacteriophage or lysing agent described in the first embodiment, the composition of the present invention may contain one or more other active ingredients that have the same and / or different pharmacological effects as the bacteriophage or lysing agent, without affecting the lysing activity of the bacteriophage.

[0311] There are no restrictions on the types of other active ingredients. Other active ingredients may be, for example, bacteriophages that have lytic activity against the same and / or different bacteria as the target bacteria of the bacteriophage or lysin described in the first embodiment. Such bacteriophages may be, for example, bacteriophages that have lytic activity against Salmonella spp.

[0312] Other examples include well-known antibiotics as other effective ingredients.

[0313] (3) Non-effective ingredients

[0314] Within the scope of not affecting the lysing activity of the bacteriophage or lysin described in the first embodiment, the composition of the present invention may further include non-active ingredients, such as carriers (solid carriers, liquid carriers, etc.), excipients, surfactants, emulsifiers, binders, disintegrants, lubricants, solubilizers, suspending agents, coating agents, colorants, flavoring and odor-correcting agents, preservatives, stabilizers, isotonic agents, chelating agents, thickeners, viscous agents, buffers, pH adjusters, etc.

[0315] 2-3. Target of application

[0316] The objects to which the compositions of the present invention are applied (often referred to as "objects" in this specification) are not limited, and can include, for example, livestock breeding farms such as chicken farms, pig farms, pastures, and dairy farms (including, for example, houses, cages, soil, etc.); food or feed; food processing plants or feed manufacturing plants; food or feed processing equipment; food or feed containers; and any vertebrate including humans, livestock (horses, cattle, sheep, goats, pigs, chickens, etc.), pets (dogs, cats, rabbits, birds, etc.), laboratory animals (mice, rats, monkeys, etc.).

[0317] 2-4. Morphology

[0318] The compositions of the present invention can be in the form of pharmaceutical compositions, additives (e.g., food additives, feed additives, drinking water additives), food products, feed, cleaning agents, disinfectants, bactericides, and sterilizing agents. Each form is described in detail below.

[0319] (1) Pharmaceutical Composition

[0320] The compositions of the present invention can be pharmaceutical compositions.

[0321] The pharmaceutical compositions of the present invention can be used, for example, for the prevention and treatment of target bacteria. The pharmaceutical compositions of the present invention can also be used to treat or prevent, for example, infections caused by target bacteria. In the pharmaceutical compositions of the present invention, the target bacteria are as described in “2-1. Summary”, particularly Salmonella bacteria.

[0322] In this instruction manual, "infections caused by Salmonella bacteria" refers to diseases caused by Salmonella bacteria, also known as salmonellosis or salmonellosis. Symptoms of infections caused by Salmonella bacteria (including S. Enteritidis, S. Montevideo, and S. Typhimurium) include: fever, abdominal pain, diarrhea, nausea, vomiting, and bacteremia. Infections caused by Salmonella bacteria can also include, for example, food poisoning.

[0323] In addition to the bacteriophage or lysin described in the first embodiment, the pharmaceutical composition of the present invention may further contain the aforementioned non-effective ingredients (i.e., pharmaceutical additives) that are permitted in pharmaceutical manufacturing.

[0324] The pharmaceutical composition of the present invention can be formulated into: solid dosage forms such as tablets, granules, powders, pills, and capsules; liquid dosage forms such as liquids, suspensions, and syrups; and any dosage form such as gels and aerosols. It should be noted that when the pharmaceutical composition is used as a liquid dosage form, it can also be formulated as a dried product intended to be reconstituted with physiological saline before use. Furthermore, the dosage of the bacteriophage or lysozyme described in the first embodiment of the pharmaceutical composition of the present invention can be appropriately set, and its dosage can be changed according to the dosage form, the severity of the disease of the target patient, etc.

[0325] As the target of administration of the pharmaceutical composition of the present invention, any vertebrate can be included, such as humans, livestock (horses, cattle, sheep, goats, pigs, chickens, etc.), pets (dogs, cats, rabbits, birds, etc.), and laboratory animals (mice, rats, monkeys, etc.), with humans being preferred.

[0326] The route of administration for the pharmaceutical composition of the present invention is not limited, and examples include oral, intravenous, rectal, vaginal, and local administration.

[0327] The dosage of the pharmaceutical composition of the present invention can be appropriately set taking into account various factors such as the route of administration, the age, weight, and symptoms of the test subject. The pharmaceutical composition of the present invention can be administered as a single dose or multiple doses at intervals of several hours to several months.

[0328] (2) Food additives

[0329] The compositions of the present invention can be food additives.

[0330] The food additives of the present invention can be used, for example, to prevent or control target bacteria in food and beverages. The food additives of the present invention can also be used to impart specific effects to food and beverages by addition (prevention or control of target bacteria or treatment or prevention of infections caused by target bacteria). In the food additives of the present invention, the target bacteria are as described in “2-1. Summary”, particularly Salmonella bacteria.

[0331] In addition to the bacteriophages or lysins described in the first embodiment, the food additives of the present invention may further contain the aforementioned ineffective ingredients permitted in the manufacture of food products.

[0332] The food additives of the present invention may be in the form of liquid, gel or dry powder. The types of food products to which the food additives of the present invention are added are as described in “(4) Food Products”.

[0333] The food additives of the present invention can be added, coated, or sprayed onto food products using any suitable method that can be utilized by those skilled in the art. For example, the food additives of the present invention can be mixed into the raw materials of food products during the manufacture of food products.

[0334] (3) Feed additives / drinking water additives

[0335] The compositions of this invention can be feed additives or drinking water additives. These feed additives or drinking water additives can be used, for example, when feeding livestock.

[0336] The feed additives or drinking water additives of the present invention can be used, for example, to prevent or control target bacteria in feed or drinking water. The feed additives or drinking water additives of the present invention can also be used to impart specific effects (prevention or control of target bacteria or treatment or prevention of infections caused by target bacteria) to feed or drinking water by addition. In the feed additives or drinking water additives of the present invention, the target bacteria are as described in “2-1. Summary”, particularly Salmonella spp.

[0337] In addition to the bacteriophages or lysozymes described in the first embodiment, the feed additives of the present invention may further contain the aforementioned ineffective ingredients permitted in the manufacture of feed.

[0338] The feed additive of the present invention may be in the form of 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".

[0339] The feed additive of the present invention can be added to, coated, or sprayed onto feed using any suitable method that can be utilized by those skilled in the art. For example, the feed additive of the present invention can be mixed into the feed ingredients during feed manufacturing.

[0340] The drinking water additive of the present invention can be in liquid, gel, or dry powder form. The drinking water to which the drinking water additive of the present invention is added can be, for example, tap water, well water, groundwater, or rainwater, without particular limitation. The drinking water may contain other components (e.g., antibiotics).

[0341] The drinking water additive of the present invention can be added to drinking water by any suitable method that can be used by those skilled in the art. For example, the drinking water additive of the present invention can be mixed into drinking water in a suitable container or into drinking water in a water supply device.

[0342] (4) Food and beverages

[0343] The compositions of the present invention can be food or beverages.

[0344] The food and beverage products of the present invention can be used, for example, to prevent or control target bacteria in a subject. The food and beverage products of the present invention can also be used, for example, to treat or prevent infections caused by target bacteria. In the food and beverage products of the present invention, the target bacteria are as described in “2-1. Summary”, particularly Salmonella bacteria. Infections caused by target bacteria can be, for example, food poisoning.

[0345] In addition to the bacteriophages or lysins described in the first embodiment, the food and beverage products of the present invention may further contain the aforementioned ineffective ingredients permitted in the manufacture of the food and beverage products.

[0346] The food and beverage products of the present invention can be in any form, including fresh foods (vegetables, fruits, meat, aquatic products, grains, etc.), processed foods, home-cooked dishes, snacks, seasonings, beverages, and functional foods. Functional foods include, for example, foods for specific health purposes (including conditionally formulated foods for specific health purposes), foods with functional labeling, health foods containing nutritional functions, foods for special purposes, nutritional supplements, health supplements, supplements (e.g., tablets, coated tablets, sugar-coated tablets, capsules, liquids, and other dosage forms), and beauty foods (e.g., weight-loss foods). The food and beverage products can also be prepared in any form, such as solids, liquids, mixtures, suspensions, pastes, gels, powders, granules, and capsules. The food and beverage products of the present invention can contain the bacteriophage or lysin described in the first embodiment by any suitable method that can be utilized by those skilled in the art. Specifically, the food and beverage products of the present invention can encapsulate bacteriophages or lysozymes into capsules, or encapsulate bacteriophages or lysozymes with edible films, edible coating agents, etc., or be formed into tablets or any other form after combining (adding) bacteriophages or lysozymes with appropriate excipients, etc. The food and beverage products of the present invention can be manufactured by processing a composition containing the bacteriophages or lysozymes of the present invention with other food ingredients. Then, the food and beverage products of the present invention can be manufactured by combining (adding) bacteriophages or lysozymes to, for example, various foods (beverages, liquid foods, patient foods, nutritional foods, frozen foods, processed foods, other commercially available foods, etc.).

[0347] (5) Feed

[0348] The composition of the present invention can be used as feed.

[0349] The feed of the present invention can be used, for example, to control target bacteria in an organism. 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. Summary”, particularly Salmonella bacteria. Infections caused by target bacteria can be, for example, food poisoning.

[0350] In addition to the bacteriophages or lysozymes described in the first embodiment, the feed of the present invention may further contain the aforementioned ineffective ingredients permitted in the manufacture of the feed.

[0351] The feed used in this invention is not limited and can include, for example, pasture, straw, miscanthus, hay, silage, grains (corn, barley, wheat, rice, etc.), compound feed, food by-products (tofu residue, brewer's grains, breadcrumbs, etc.). The feed can also be prepared in any form, such as solid, liquid, mixture, suspension, paste, gel, powder, granules, or capsules.

[0352] The feed of the present invention can contain the bacteriophage or lysozyme described in the first embodiment by any suitable method available to those skilled in the art. Specifically, the feed of the present invention can encapsulate the bacteriophage or lysozyme, or encapsulate the bacteriophage or lysozyme with an edible film, edible coating agent, etc., or form it into tablets or any other form after combining (adding) the bacteriophage or lysozyme with appropriate excipients, etc. The feed of the present invention can be manufactured by processing it into a composition containing the bacteriophage or lysozyme of the present invention and other feed ingredients. Furthermore, the feed of the present invention can also be manufactured by, for example, combining (adding) the bacteriophage or lysozyme to various feeds.

[0353] (6) Cleaning agents, disinfectants, bactericides, and germicides

[0354] The compositions of this invention can be cleaning agents, disinfectants, bactericides, or sterilizing agents. In this specification, "cleaning agent" refers to a composition intended to remove dirt from an object to which it is applied. In this specification, "disinfectant" refers to a composition intended to reduce pathogenic microorganisms in an object to a harmless level. In this specification, "bactericide" refers to a composition intended to kill bacteria in an object to which it is applied. In this specification, "sterilizing agent" refers to a composition intended to reduce bacteria in an object to which it is applied.

[0355] The cleaning agents, disinfectants, bactericides, or disinfectants of the present invention can be used, for example, to control target bacteria in the object of application. In the cleaning agents, disinfectants, bactericides, or disinfectants of the present invention, the target bacteria are as described in “2-1. Summary”, particularly Salmonella bacteria.

[0356] The cleaning agent, disinfectant, bactericide, or sterilizing agent of the present invention may be in the form of liquid, gel, or dry powder.

[0357] The objects to which the cleaning agents, disinfectants, bactericides, or sterilizing agents of the present invention are applied are not limited, and examples include: livestock breeding farms such as chicken farms, pig farms, pastures, and dairy farms (including, for example, houses, cages, soil, etc.); food or feed; food processing plants or feed manufacturing plants; food or feed processing equipment; food or feed containers; and any vertebrate including humans, livestock (horses, cattle, sheep, goats, pigs, chickens, etc.), pets (dogs, cats, rabbits, birds, etc.), and laboratory animals (mice, rats, monkeys, etc.).

[0358] The cleaning agents, disinfectants, bactericides, or disinfectants of the present invention can be used, for example, by adding, coating, spraying, or dispersing to the object to which they are applied. The cleaning agents, disinfectants, bactericides, or disinfectants of the present invention can also be used, for example, by impregnating the object to which they are applied.

[0359] 3. Target bacteria control methods

[0360] 3-1. Overview

[0361] The third aspect of the present invention is a method for controlling target bacteria. This method uses the bacteriophage or lysing agent described in the first aspect or the composition described in the second aspect to control target bacteria. In this method, the target bacteria are particularly Salmonella bacteria. For example, when using a composition containing a first bacteriophage or a first lysing agent, the target bacteria are particularly *S. enteritidis*. When using a composition containing a second bacteriophage or a second lysing agent, the target bacteria are particularly *S. enteritidis*, *S. typhimurium*, *S. infantis*, *S. Montevideo*, and *S. javiana*. When using a composition containing a third bacteriophage or a third lysing agent, the target bacteria are particularly *S. typhimurium*. When using a composition containing a fourth bacteriophage or a fourth lysing agent, the target bacteria are particularly *S. Montevideo*. When using a composition containing a fifth bacteriophage or a fifth lysing agent, the target bacteria are particularly *S. typhimurium*. When using a composition containing a 6th bacteriophage or a 6th lysin, the target bacteria are particularly *S. enteritidis*, *S. typhimurium*, and *S. javiana*. When using a composition containing a 7th bacteriophage or a 7th lysin, the target bacteria are particularly *S. enteritidis*.

[0362] According to the prevention and control method of the present invention, target bacteria can be prevented and controlled in the application object.

[0363] 3-2. Methods

[0364] The target bacteria control method of the present invention includes a contact step as an essential step.

[0365] "Contact process" refers to the process of bringing the bacteriophage or lysozyme described in Method 1 or the composition described in Method 2 into contact with the object to which it is applied.

[0366] In this method, "contact" refers to direct contact between the bacteriophage or lysing agent described in Method 1 or the composition described in Method 2 and the target organism. More specifically, the bacteriophage in the bacteriophage or lysing agent described in Method 1 or the composition described in Method 2 comes into contact with the target organism, preferably at a site where there is a risk of contamination due to the target bacteria. This process aims to infect the target bacteria with the bacteriophage, which is the active ingredient, thereby lysing the target bacteria. As a result, a preventive effect against the target bacteria can be achieved.

[0367] In the target bacterial control method of the present invention, the application target is as described in the second embodiment.

[0368] In the target bacteria control method of the present invention, the contact step can be carried out in the following ways: for example, adding, coating, spraying, or distributing the bacteriophage or lysozyme described in the first method or the composition described in the second method (especially pharmaceutical compositions, food additives, feed additives, drinking water additives, cleaning agents, disinfectants, bactericides, and disinfectants) to the target object; or immersing the target object in the bacteriophage or lysozyme described in the first method or the composition described in the second method (especially pharmaceutical compositions, food additives, feed additives, drinking water additives, cleaning agents, disinfectants, bactericides, and disinfectants).

[0369] The contact process can also be carried out by administering the composition described in Method 2 (especially pharmaceutical compositions, food and beverages, feed) to the recipient.

[0370] 4. Methods for treating or preventing infections caused by target bacteria

[0371] 4-1. Overview

[0372] The fourth aspect of the present invention is a method for treating or preventing infections caused by target bacteria. The method of treatment or prevention of the present invention involves using the bacteriophage or lysin described in the first aspect or the composition described in the second aspect for the treatment or prevention of infections caused by target bacteria.

[0373] 4-2. Methods

[0374] The treatment or prevention method of the present invention includes an essential drug administration step. "Drug administration step" refers to the step of administering a drug to the bacteriophage or lysin described in the first method or the composition described in the second method. In the treatment or prevention method of the present invention, the target bacteria are particularly Salmonella bacteria. For example, when using a composition containing a first bacteriophage or a first lysin, the target bacteria are particularly *S. enteritidis*. When using a composition containing a second bacteriophage or a second lysin, the target bacteria are particularly *S. enteritidis*, *S. typhimurium*, *S. infantis*, *S. Montevideo*, and *S. javiana*. When using a composition containing a third bacteriophage or a third lysin, the target bacteria are particularly *S. typhimurium*. When using a composition containing a fourth bacteriophage or a fourth lysin, the target bacteria are particularly *S. Montevideo*. When using a composition containing a fifth bacteriophage or a fifth lysin, the target bacteria are particularly *S. typhimurium*. When using a composition containing a 6th bacteriophage or a 6th lysin, the target bacteria are particularly *S. enteritidis*, *S. typhimurium*, and *S. javiana*. When using a composition containing a 7th bacteriophage or a 7th lysin, the target bacteria are particularly *S. enteritidis*.

[0375] In the treatment or prevention method of the present invention, the target of administration and the method of administration (dosage, route of administration, frequency of administration) are as described in “2-4.(1) Pharmaceutical Composition” above.

[0376] 5. Methods for identifying Salmonella bacteria

[0377] 5-1. Summary

[0378] The fifth aspect of the present invention is a method for identifying Salmonella bacteria. The identification method of the present invention utilizes the host specificity of the bacteriophage constituting the bacteriophage or lysing agent described in the first aspect to identify Salmonella bacteria. When using the first bacteriophage or the first lysing agent, the identification method of the present invention can be the identification method for *S. enteritidis*. When using the second bacteriophage or the second lysing agent, the identification method of the present invention can be the identification method for *S. enteritidis*, *S. typhimurium*, *S. infantis*, *S. Montevideo*, and *S. javiana*. When using the third bacteriophage or the third lysing agent, the identification method of the present invention can be the identification method for *S. typhimurium*. When using the fourth bacteriophage or the fourth lysing agent, the identification method of the present invention can be the identification method for *S. Montevideo*. When using the fifth bacteriophage or the fifth lysing agent, the identification method of the present invention can be the identification method for *S. typhimurium*. When using a sixth phage or a sixth lysing agent, the identification method of the present invention can be the identification method for S. enteritidis, S. typhimurium, and S. Javiana. When using a seventh phage or a seventh lysing agent, the identification method of the present invention can be the identification method for S. enteritidis.

[0379] According to the present invention, it is possible to determine whether an unidentified bacterium is a Salmonella bacterium and to perform identification.

[0380] 5-2. Methods

[0381] The identification method of the present invention includes a cultivation step, a mixing step, a mixture cultivation step, and a determination step as mandatory steps, and includes a separation step as an optional step. The following describes each step.

[0382] (1) Separation process

[0383] The "isolation process" is the process of isolating test bacteria from specimens suspected of containing Salmonella. This process is optional and can be performed as needed.

[0384] "Test bacteria" refers to the bacteria used in the identification method of this invention, which are bacteria whose species have not yet been identified.

[0385] The specimen can be feces, food or feed, or a swab specimen collected from livestock farms, food processing plants, feed manufacturing plants, etc.

[0386] When the predicted amount of Salmonella spp. in the sample is high (e.g., using feces from an individual showing signs of salmonellosis), the sample can be directly streaked onto agar medium for isolation and culture. After isolation and culture, the test bacteria can be isolated by selecting a single colony. When the predicted amount of Salmonella spp. in the sample is low (e.g., using food or swab samples), the sample can be placed in a culture medium for enrichment culture, and then the culture can be streaked onto agar medium for isolation and culture. After isolation and culture, the test bacteria can be isolated in the same manner as described above. When the predicted Salmonella spp. are damaged or dormant (e.g., using processed food samples), a pre-enrichment culture can be performed before further enrichment culture.

[0387] (2) Cultivation process

[0388] The "culturing process" is the process of culturing isolated test bacteria to obtain a culture. The culturing method for test bacteria can be carried out using methods known in the field.

[0389] "Culture" refers to the substance obtained by culturing test bacteria, which can be either liquid or solid.

[0390] In this process, since the tested bacteria are in an unidentified state, it is desirable to use a culture medium that can widely culture bacteria. At least a culture medium capable of culturing Salmonella bacteria, the bacteria targeted for identification in this invention, should be used. Such a culture medium may contain one or more components selected from, for example, protease digests such as peptone and tryptone; biological extracts such as potato glucose and yeast extract; amino acids such as glutamic acid or their salts; sugars such as glucose, sucrose, and lactose; and inorganic salts such as sodium chloride, magnesium chloride, potassium dihydrogen phosphate, and sodium thiosulfate. Specific examples of culture media and their 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 deoxycholate, 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; an enrichment medium for Salmonella bacteria containing peptone, etc.).

[0391] The isolated test bacteria are inoculated into the above-mentioned culture medium and cultured under appropriate conditions. Culture conditions include, for example, 20–40°C, 20–30°C, 22–28°C, or 24–26°C. When using liquid culture medium, cultures can be obtained by stirring while culturing. There is no limit to the culture time; for example, culturing can continue until the turbidity at 600 nm reaches approximately 1.0. This process yields a culture of the test bacteria. Furthermore, culturing can be performed in multiple stages, such as two or more. For example, after culturing in liquid culture medium, a liquid culture medium containing soft agar can be added to the resulting culture, which is then poured onto a solid culture medium like agar medium and solidified before further culturing.

[0392] (3) Mixing process

[0393] The “mixing process” is a process of mixing the culture obtained in the above-mentioned culture process with the bacteriophage or lysin described in the first method to obtain a mixture.

[0394] A "mixture" refers to a substance that is formed by mixing a culture with a bacteriophage or a lysozyme, and can be either liquid or solid.

[0395] If the above culture can be mixed with bacteriophages or lysing agents, the mixing method is not particularly limited. The bacteriophages or lysing agents described in Method 1 can be in solid form or in liquid form suspended in water or liquid culture medium for administration.

[0396] If both the culture and the phage or lysozyme are liquids, the volume ratio of culture to lysozyme can be set to 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, or 9:1. After addition, the culture and phage or lysozyme can be thoroughly mixed by stirring. On the other hand, as mentioned earlier, in the case of layered liquid culture media containing soft agar, the culture is solid. In this case, a mixture can be obtained by dripping the phage or lysozyme onto the solid culture, which acts like a gel surface, and mixing the two on the solid culture medium.

[0397] (4) Mixture culture process

[0398] The “mixture cultivation process” is the process of culturing the above mixture under given conditions.

[0399] It should be noted that when culturing the mixture, liquid culture medium containing soft agar can be added to the mixture, and then poured onto a solid culture medium such as agar medium and solidified before further cultivation.

[0400] The basic steps of this process are based on the above-described culture process. While there are no limitations in this process, it is preferable to use a plaque assay to easily determine whether the bacteriophage has lysed the test bacteria in the following determination step. For example, a portion of the mixture can be mixed with a soft agar medium of the same composition, and before the soft agar medium hardens, it can be poured onto agar medium of the same composition and spread out over the entire medium. Subsequently, culture can be performed under the same conditions as the above-described culture process.

[0401] (5) Judgment process

[0402] The "judgment process" is the process of determining whether the test bacteria are Salmonella bacteria when the test bacteria are lysed after the above-mentioned culture process.

[0403] There is no limitation on the determination of whether lysis has occurred. For example, in the case of plaque assay, it can be determined by whether plaques have formed. If plaques are present in the soft agar medium after the above-described mixture culture step, once the medium has been developed and solidified, it indicates that the test bacteria have been lysed by the bacteriophage of the present invention. Therefore, the test bacteria can be determined to be Salmonella. On the other hand, if the test bacteria proliferate completely on the agar medium and no plaques are present, the test bacteria can be determined not to be Salmonella.

[0404] To make a more accurate determination, a negative control can be prepared simultaneously in the mixed culture step, mixed with a culture medium that does not contain bacteriophages and lysing agents, and / or a positive control can be prepared from the beginning of the culture step, using identified Salmonella bacteria instead of the test bacteria, to confirm that no plaques are produced in the negative control and that plaques are observed in the positive control.

[0405] 5-3. Effects

[0406] According to the Salmonella identification method of the present invention, it is possible to determine whether the cause of diseases such as food poisoning, diarrhea, and vomiting is Salmonella. Furthermore, according to the Salmonella identification method of the present invention, it is possible to detect the presence of contamination caused by Salmonella.

[0407] Example

[0408] The present invention will be further described in detail below using examples. However, the technical scope of the present invention is not limited to these examples.

[0409] [Obtaining and culturing Salmonella bacteria]

[0410] The bacterial strains used in these examples (Examples 1-7) are listed in the table shown in each example. However, all the strains from Daigo Gakuen University shown in the table are Salmonella strains isolated from animals in Japan. In addition, the strains obtained from the National Institute for Animal Health (NARO) shown in the table are Salmonella strains isolated from chickens and chicken farms in Japan.

[0411] In addition, the strain IDs in the table are the identification numbers provided in this instruction manual. The serotypes of each strain in the table were identified based on the Kaufmann-White antigen structure table, using agglutination assays with Salmonella diagnostic immune serum (Denka). Serotypes were also confirmed using gene analysis methods such as PFGE (pulsed-field gel electrophoresis) and PCR (polymerase chain reaction).

[0412] Regarding the ST1-6 strains listed in the table, their drug susceptibility, PFGE type, etc. have been investigated (Yukino Tamura, “Molecular Epidemiological Study of Bovine Salmonella enterica subsp. enterica serovar Typhimurium”, Doctoral Dissertation, Department of Veterinary Medicine, Daigo Gakuen University (2015)).

[0413] In the cultivation of various Salmonella species, a liquid culture medium (LB Broth) was used, which consisted of 10g tryptone, 5g yeast extract, and 10g sodium chloride dissolved in 1L of H2O and then autoclaved. Additionally, an agar medium (referred to as "LB Agar") was used, which consisted of 15g agar added to each 1L of the above LB Broth and then autoclaved. Furthermore, a soft agar medium (referred to as "LB Top Agar") was used as a layering medium on top of the agar medium, which consisted of 5g agarose added to each 1L of the above LB Broth and then autoclaved. The soft agar medium was stored at approximately 50°C and used as needed.

[0414] The above-mentioned bacterial strains in their dry powder form were suspended in 0.1 mL of LB Broth and streaked at 25°C on LB Agar to isolate single colonies. The isolated colonies were inoculated into LB Broth and cultured with shaking at 25°C to obtain the pre-culture medium. For this main culture, the pre-culture medium was inoculated into LB Broth until the turbidity (Optical Density 600 nm) reached approximately 1.0, and then incubated at 25°C for 10–30 hours. The culture medium after incubation was used directly as the bacterial culture.

[0415] [Isolation and purification of bacteriophages]

[0416] The novel bacteriophages were isolated from natural wastewater or soil samples obtained domestically in Japan. The isolation method was based on conventional plaque assays. First, wastewater from ponds, lakes, or other sources, or wastewater containing soil suspended in water, was filtered through a 0.45 μm filter to prepare a liquid containing the bacteriophages. Next, an equal volume of the bacterial solution and the phage-containing liquid were mixed and allowed to stand at room temperature for approximately 10 minutes. Then, 0.2 mL of the bacterial / phage mixture was added to 3 mL of an LB Top Agar, rapidly mixed using a vortex mixer, and poured onto the LB Agar. After the LB Top Agar solidified, it was incubated at 25°C for approximately 12 hours. Plaques formed on the bacterial lawn that developed through the culture. Subsequently, the gel from the plaques was aspirated using a tip-cutting connector, separating the bacteriophages exhibiting lytic activity against Salmonella. Then, the phages were purified by repeating this step, replacing the wastewater with a phage-containing liquid containing a high concentration of isolated phages.

[0417] The isolated phages were suspended in SM Buffer and recovered as a phage-containing solution passing through a 0.2 μm filter. This phage-containing solution was mixed with the bacterial culture under the above conditions, and the phages were isolated again. This step was repeated multiple times to further purify the phages. The composition of the SM Buffer is shown in the table below.

[0418] [Table 1]

[0419] SM Buffer Final Add to 1L NaCl 0.1M 5.8g <![CDATA[Mg2SO4·7H2O]]> 10mM 1g 1M Tris-HCl pH 8.0 50mM 50mL gelatin 0.1％ 0.1g

[0420] [Bacteriophage proliferation and purification]

[0421] To propagate and purify the isolated and purified phages, the plate lysate (PL) method, a propagation method used for plaque assays, was employed. A bacterial / phage mixture was prepared, mixed with LB top agar plates, and incubated on LB agar plates to generate numerous plaques. Then, 3 mL of SM Buffer was added to the plaque-bearing LB top agar plates, 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.

[0422] Add 1g of PEG 6000 (final concentration 10%) and 0.4g of NaCl (final concentration 4%) to 10mL of the recovered solution and dissolve. Rotate the solution overnight at 4°C. Then, centrifuge at 15000g / 4°C / 60min to remove the supernatant. Resuspend the recovered particles in 0.5mL of SM Buffer. Next, add 0.5mL of chloroform, stir vigorously, and incubate on ice for 6 hours. After centrifugation at 8000g / 4°C / 10min, carefully recover the supernatant to obtain the phage purification solution. The concentration of the phage purification solution is usually expressed as a titer [PFU / mL] based on the number of plaques (PFU) in plaque assays, serving as an indicator of lytic activity. The titer of the prepared phage purification solution can be determined using plaque assays with an appropriately diluted solution.

[0423] [Evaluation of the host range of bacteriophages]

[0424] The host range of bacteriophages was evaluated using a plaque assay. Only 0.1 mL of bacterial suspension was added to 3 mL of LB TopAgar and mixed, then injected into an LB Agar plate, spread throughout the plate, and allowed to solidify. Bacterial suspensions of various Salmonella species prepared in each example were used as the bacterial suspension. Subsequently, approximately 5 μL of phage purification solution was added to the plate, and the plate was incubated at 25°C for approximately 12 hours. If the location of the added phage on the plate where a bacterial colony formed became a clear, circular area (approximately 1 cm in diameter), the added phage was considered to have lytic activity against that strain.

[0425] [Preparation and Sequencing of Bacteriophage Genomic DNA]

[0426] The phage genome was extracted using the TURBO DNA-free™ kit (Thermo Fisher Scientific). Genomic DNA from the host bacteria was removed as contaminants by processing according to the kit's instructions. Subsequently, the phage coat molecules were degraded using Proteinase K with NucleoSpin Virus (Machery-Nagel) according to the kit's instructions. A phage genomic DNA solution was prepared by purification using a silica column. The concentration of the genomic DNA was then determined using the Qubit dsDNA HS Assay kit (Thermo Fisher Scientific), and 50 μL of the genomic DNA solution was prepared to a final concentration of 0.2 ng / μL. Next, the phage genome was fragmented and ligand sequences were added via PCR using the Nextera XT DNA Library Prep (Illumina) according to the kit's instructions. Next, using the Agilent High Sensitivity DNA Kit (Agilent Technologies), electrophoresis was performed using a Bioanalyzer (Agilent Technologies) to determine the average bp size of the samples and calculate the concentration of DNA fragments. Finally, samples were prepared using the Miseq Reagent kit (Illumina) according to the accompanying instructions, and sequencing was performed using next-generation DNA sequencing technology, Miseq (Illumina). The obtained data were preprocessed (modified, etc.) using the CLCgenomics workbench (Qiagen), followed by de novo assembly to obtain contig sequences equivalent to the phage genome sequence.

[0427] <Example 1: Isolation and lysing activity of the first bacteriophage>

[0428] (Purpose)

[0429] A novel bacteriophage with lytic activity against Salmonella was isolated and its lytic activity against Salmonella was verified.

[0430] (Methods and Results)

[0431] (1) Acquisition and culture of Salmonella bacteria

[0432] The bacterial strains used in Example 1 are listed in the table below.

[0433] [Table 2]

[0434] strain ID serotype strain name Supply source SE6 S.Entertidis L-2728 Agricultural Research Institute Animal Health SE8 S.Entertidis L-2844 Agricultural Research Institute Animal Health SE12 S.Entertidis L-3164 Agricultural Research Institute Animal Health SE17 S.Entertidis L-3782 Agricultural Research Institute Animal Health SE18 S.Entertidis L-5104 Agricultural Research Institute Animal Health ST1 S.Typhimurium HRS-TST-129 Dairy Farm University Veterinary Medicine Group ST4 S.Typhimurium HRS-KST-31 Dairy Farm University Veterinary Medicine Group ST6 S.Typhimurium HRS-U1 Dairy Farm University Veterinary Medicine Group SI1 S.Infantis O7: Hr70A Dairy Farm University Veterinary Medicine Group SI3 S.Infantis O7: Hr1.5 Dairy Farm University Veterinary Medicine Group SM S.Montevideo S.Montevideo No.1 Dairy Farm University Veterinary Medicine Group SJ S.Javiana L-750 Dairy Farm University Veterinary Medicine Group

[0435] (2) Isolation and purification of the first bacteriophage

[0436] Following the methods described in the [Isolation and Purification of Bacteriophages] section above, seven new bacteriophages were isolated from natural sewage / soil and purified (corresponding to phage No. 1).

[0437] (3) Propagation and purification of the first phage

[0438] Following the method described in the section on [phage proliferation and purification] above, the first phage purification solution was prepared, and its titer was determined. The titer was confirmed to be 10. 8 PFU / mL or higher.

[0439] (4) Evaluation of the host range of the first phage

[0440] The host range of bacteriophage 1 was evaluated using plaque assay, following the method described in the [Evaluation of Host Range of Bacteriophages] section above.

[0441] An example of the result is shown below. Figure 1 and Figure 2 The seven phages obtained through this embodiment showed lytic activity against various strains of S. Enteritidis, but did not show lytic activity against S. Typhimurium, S. Infantis, S. Montevideo, and S. Javiana.

[0442] *S. enteritidis* is the serotype detected with the highest frequency in human food poisoning (Oh and Park, J. Microbiol. Biotechnol. (2017), 27(12), 2075-2088). Therefore, phage 1 is particularly useful, for example, for the treatment or prevention of human food poisoning. Furthermore, phage 1 is particularly useful in the identification of *S. enteritidis* because it exhibits specific lytic activity against it.

[0443] (5) Genome analysis of the first phage

[0444] The genomic DNA sequence of phage 1 was determined and analyzed.

[0445] (i) Preparation and sequencing of genomic DNA of phage #1

[0446] The genomic DNA sequence of bacteriophage 1 was determined according to the method described in the section on [Preparation and Sequencing of Genomic DNA of Bacteriophages]. The determined genomic DNA sequences of the seven bacteriophage 1 species are shown in sequence numbers 1 to 7.

[0447] (ii) Bioinformatics analysis based on genome sequence information

[0448] The genomic DNA sequences of bacteriophage 1 (sequence numbers 1–7) exhibit high sequence identity. Using the genetic information processing software GENTYX (https: / / www.genetyx.co.jp / ), the sequence identity of the shortest genomic DNA sequence (sequence number 7) relative to the genomic DNA sequences (sequence numbers 1–6) was calculated, and the result was 100% across the entire range.

[0449] Similar DNA sequences were retrieved using the genomic DNA sequences of sequence numbers 2 and 7 as query sequences on the NCBI BLAST server (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). For phages with genomic DNA sequences exhibiting high sequence identity across the entire range relative to sequence numbers 2 or 7, further literature regarding host range was investigated. The results showed that no phages with genomic DNA sequences presumed to have more than 99% sequence identity across the entire range relative to sequence numbers 2 or 7, and known to have the same host range as phage #1, were found.

[0450] <Example 2: Isolation and lysing activity of bacteriophage #2>

[0451] (Purpose)

[0452] A novel bacteriophage with lytic activity against Salmonella was isolated and its lytic activity against Salmonella was verified.

[0453] (Methods and Results)

[0454] (1) Acquisition and culture of Salmonella bacteria

[0455] The bacterial strains used in Example 2 are listed in the table below.

[0456] [Table 3]

[0457] strain ID serotype strain name Supply source SE1 S.Entertidis L-2596 Agricultural Research Institute Animal Health SE2 S.Entertidis L-2653 Agricultural Research Institute Animal Health SE3 S.Entertidis L-2602 Agricultural Research Institute Animal Health SE4 S.Entertidis L-2685 Agricultural Research Institute Animal Health SE5 S.Entertidis L-2712 Agricultural Research Institute Animal Health SE6 S.Entertidis L-2728 Agricultural Research Institute Animal Health SE7 S.Entertidis L-2777 Agricultural Research Institute Animal Health SE8 S.Entertidis L-2844 Agricultural Research Institute Animal Health SE9 S.Entertidis L-2916 Agricultural Research Institute Animal Health SE10 S.Entertidis L-2917 Agricultural Research Institute Animal Health SE11 S.Entertidis L-3080 Agricultural Research Institute Animal Health SE12 S.Entertidis L-3164 Agricultural Research Institute Animal Health SE13 S.Entertidis L-3241 Agricultural Research Institute Animal Health SE14 S.Entertidis L-3244 Agricultural Research Institute Animal Health SE15 S.Entertidis L-3246 Agricultural Research Institute Animal Health SE16 S.Entertidis L-3247 Agricultural Research Institute Animal Health SE17 S.Entertidis L-3782 Agricultural Research Institute Animal Health SE18 S.Entertidis L-5104 Agricultural Research Institute Animal Health ST3 S.Typhimurium HRS-TST-219 Dairy Farm University Veterinary Medicine Group SI1 S.Infantis O7: Hr70A Dairy Farm University Veterinary Medicine Group SI2 S.Infantis O7: Hd70B Dairy Farm University Veterinary Medicine Group SI3 S.Infantis O7: Hr1.5 Dairy Farm University Veterinary Medicine Group SM S.Montevideo S.Montevideo No.1 Dairy Farm University Veterinary Medicine Group SJ S.Javiana L-750 Dairy Farm University Veterinary Medicine Group

[0458] (2) Isolation and purification of the second phage

[0459] Following the method described in the [Isolation and Purification of Bacteriophages] section above, three new bacteriophages were isolated from natural sewage / soil and purified (corresponding to bacteriophage No. 2).

[0460] (3) Proliferation and purification of the second phage

[0461] Following the method described in the section on [phage proliferation and purification] above, the second phage purification solution was prepared, and its titer was determined. The titer was confirmed to be 10. 8 PFU / mL or higher.

[0462] (4) Evaluation of the host range of the second phage

[0463] The host range of bacteriophage 2 was evaluated using plaque assay, following the method described in the [Evaluation of Host Range of Bacteriophages] section above.

[0464] An example of the result is shown below. Figures 3-5 The three second phages obtained through this embodiment exhibited lytic activity against various bacterial strains, including *S. enteritidis*, *S. typhimurium*, *S. infantis*, *S. Montevideo*, and *S. javiana*. These bacterial strains are all serotypes frequently detected in human food poisoning. Therefore, second phages are particularly useful for, for example, treating or preventing human food poisoning.

[0465] (5) Genome analysis of the second phage

[0466] The genomic DNA sequence of phage 2 was determined and analyzed.

[0467] (i) Preparation and sequencing of genomic DNA from phage #2

[0468] The genomic DNA sequence of bacteriophage 2 was determined according to the method described in the section on [Preparation and Sequencing of Bacteriophage Genomic DNA]. The determined genomic DNA sequences of the three bacteriophage 2 species are shown in sequence numbers 10–12.

[0469] (ii) Bioinformatics analysis based on genome sequence information

[0470] The genomic DNA sequences of the three bacteriophages (sequence numbers 10-12) were 99% identical. As shown in sequence number 8, the amino acid sequences of their respective tail tip proteins were completely identical.

[0471] For the obtained genomic DNA sequences of the three bacteriophages (sequence numbers 10–12), a search for similar DNA sequences and confirmation of sequence identity were performed using the BLAST server provided by NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The search results showed that the sequence with the highest identity was the genomic sequence of Salmonella phage S124 (GenBank accession number: NC_048013.1), with a sequence identity of 79.14% across the entire range (QueryCover / Per.Ident values ​​of 83% / 95.36%).

[0472] To verify the reason for the difference in host range between the three obtained bacteriophages and S124, the sequences of the tail tip protein were compared between the two. The tail tip protein gene was identified from the three obtained bacteriophages. In gene identification, the RAST server (https: / / rast.nmpdr.org / ) and the PHASTER server (https: / / phaster.ca / ) were used. The result was that the tail tip protein gene was identified as the base sequence shown in sequence number 9.

[0473] Furthermore, the amino acid sequence encoded by the gene containing the base sequence shown in Sequence Number 9 (Sequence Number 8) was compared with the amino acid sequence of the similar protein S124 (accession number: YP_009806053.1), and the sequence identity was 97.49%. It can be considered that the difference in this sequence is highly likely related to differences in host range. It should be noted that when only the amino acid sequences of the tail tip proteins of the three bacteriophages obtained (Sequence Number 8) were used as query sequences and a search was performed using the BLAST server provided by NCBI, four known sequences with a sequence identity of over 95% were detected. The comparison between the query sequence and the retrieved sequences is shown below. Figure 11 .exist Figure 11In the sequence, the amino acid sequence of "HCH9411546.1" is designated as sequence number 24, the amino acid sequence of "YP_009966103.1" as sequence number 25, the amino acid sequence of "YP_009194791.1" as sequence number 26, and the amino acid sequence of "YP_009806053.1" as sequence number 27. Of these four known sequences, except for S124, no detailed information on host range or sequence originating from prophages has been reported. It is known that, unlike the corresponding residues in the above four known sequences, the F (Phe) at position 258 and the S (Ser) at position 617 in the query sequence (the amino acid sequences of the tail tip proteins of the three phages obtained) are unique amino acid residues visible only in the query sequence. It is presumed that the characteristics in these sequences are related to the lytic activity of phage 2 against a wide range of serotypes.

[0474] <Example 3: Isolation and lysing activity of bacteriophage #3>

[0475] (Purpose)

[0476] A novel bacteriophage with lytic activity against Salmonella was isolated and its lytic activity against Salmonella was verified.

[0477] (Methods and Results)

[0478] (1) Acquisition and culture of Salmonella bacteria

[0479] The bacterial strains used in Example 3 are listed in the table below.

[0480] [Table 4]

[0481] strain ID serotype strain name Supply source ST1 S.Typhimurium HRS-TST-129 Dairy Farm University Veterinary Medicine Group ST2 S.Typhimurium HRS-TST-139 Dairy Farm University Veterinary Medicine Group ST3 S.Typhimurium HRS-TST-219 Dairy Farm University Veterinary Medicine Group ST4 S.Typhimurium HRS-KST-31 Dairy Farm University Veterinary Medicine Group

[0482] (2) Isolation and purification of the third phage

[0483] Following the method described in the [Isolation and Purification of Bacteriophages] section above, a new bacteriophage was isolated from natural sewage / soil and purified (corresponding to bacteriophage No. 3).

[0484] (3) Propagation and purification of the third phage

[0485] Following the method described in the section on [phage proliferation and purification] above, the purified phage solution was prepared, and its titer was determined. The titer was confirmed to be 10. 8 PFU / mL or higher.

[0486] (4) Evaluation of the host range of the third phage

[0487] The host range of bacteriophage 3 was evaluated using plaque assay, following the method described in the [Evaluation of Host Range of Bacteriophages] section above.

[0488] An example of the result is shown below. Figure 6 The third phage obtained through this embodiment exhibited lytic activity against various bacterial strains of *S. typhimurium*, a serotype frequently detected in human food poisoning.

[0489] Furthermore, *S. typhimurium* is known to be a chemically resistant bacterium, making it difficult for antibiotics to work. For example, the *S. typhimurium* used in this embodiment 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 of Bovine Salmonella enterica subsp. enterica serovar Typhimurium", Doctoral Dissertation, Aurono Gakuen University, 2015). It should be noted that A represents ampicillin, C represents chloramphenicol, S represents streptomycin, Su represents sulfonamides, and T represents tetracycline. Therefore, phage number 3 can effectively control *S. typhimurium*, which is resistant to chemical drugs and therefore difficult for antibiotics to work, making it particularly useful for treating or preventing food poisoning in humans.

[0490] Furthermore, although not shown in the figure, it was confirmed that phage 3 also exhibited lytic activity against ST5 (S. Typhimurium HRS-KST-203, Veterinary Medicine Group, Daigo Gakuen University) and ST6 (S. Typhimurium HRS-U1, Veterinary Medicine Group, Daigo Gakuen University).

[0491] (5) Genome analysis of the third phage

[0492] The genomic DNA sequence of phage 3 was determined and analyzed.

[0493] (i) Preparation and sequencing of genomic DNA from phage #3

[0494] The genomic DNA sequence of bacteriophage 3 was determined according to the method described in the section on [Preparation and Sequencing of Bacteriophage Genomic DNA]. The determined genomic DNA sequence of one bacteriophage 3 is shown in sequence number 13.

[0495] (ii) Bioinformatics analysis based on genome sequence information

[0496] Using the BLAST server provided by NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), the genomic DNA sequence of phage 3 (sequence number 13) was used as the query sequence to perform a search for similar DNA sequences and confirm sequence identity. The search results showed that the closest base sequence was the genome sequence of *Escherichia phage vB_EcoM-RPN242* (GenBank accession number: OL656110.1), with a sequence identity of 87.85% across the entire range (Query Cover / Per.Ident value: 89% / 98.71%). Another closest base sequence with approximately 85% sequence identity was the genome sequence of *Escherichia phage vB_EcoM-ZQ1* (GenBank accession number: MW650886.1), with a sequence identity of 84.35% across the entire range (Query Cover / Per.Ident value: 86% / 98.09%). Since none of the hosts of any of the bacteriophages are Salmonella bacteria, this indicates that bacteriophage 3 is a bacteriophage with a novel genome sequence that is completely unknown.

[0497] <Example 4: Isolation and lysing activity of bacteriophage #4>

[0498] (Purpose)

[0499] A novel bacteriophage with lytic activity against Salmonella was isolated and its lytic activity against Salmonella was verified.

[0500] (Methods and Results)

[0501] (1) Acquisition and culture of Salmonella bacteria

[0502] The bacterial strains used in Example 4 are listed in the table below.

[0503] [Table 5].

[0504] strain ID serotype strain name Supply source SE1 S.Entertidis L-2596 Agricultural Research Institute Animal Health SE2 S.Entertidis L-2653 Agricultural Research Institute Animal Health SE8 S.Entertidis L-2844 Agricultural Research Institute Animal Health SE10 S.Entertidis L-2917 Agricultural Research Institute Animal Health SE11 S.Entertidis L-3080 Agricultural Research Institute Animal Health ST2 S.Typhimurium HRS-TST-139 Dairy Farm University Veterinary Medicine Group ST3 S.Typhimurium HRS-TST-219 Dairy Farm University Veterinary Medicine Group ST6 S.Typhimurium HRS-U1 Dairy Farm University Veterinary Medicine Group SI1 S.Infantis O7: Hr70A Dairy Farm University Veterinary Medicine Group S13 S.Infantis O7: Hr1.5 Dairy Farm University Veterinary Medicine Group SJ S.Javiana L-750 Dairy Farm University Veterinary Medicine Group SM S.Montevideo S.Montevideo No.1 Dairy Farm University Veterinary Medicine Group

[0505] (2) Isolation and purification of the fourth phage

[0506] Following the method described in the [Isolation and Purification of Bacteriophages] section above, new bacteriophages were isolated from natural sewage / soil and purified (corresponding to phage number 4).

[0507] (3) Proliferation and purification of the fourth phage

[0508] Following the method described in the section on [phage proliferation and purification] above, the purified solution of phage #4 was prepared, and its titer was determined. The titer was confirmed to be 10. 8 PFU / mL or higher.

[0509] (4) Evaluation of the host range of the fourth phage

[0510] The host range of bacteriophage 4 was evaluated using plaque assay, following the method described in the [Evaluation of Host Range of Bacteriophages] section above.

[0511] An example of the result is shown below. Figure 7 The fourth phage obtained in this embodiment showed lytic activity against S. Montevideo, but not against S. Enteritidis, S. Typhimurium, S. Infantis, and S. Javiana.

[0512] (5) Genome analysis of phage #4

[0513] The genomic DNA sequence of phage 4 was determined and analyzed.

[0514] (i) Preparation and sequencing of genomic DNA from phage #4

[0515] The genomic DNA sequence of bacteriophage 4 was determined according to the method described in the section on [Preparation and Sequencing of Bacteriophage Genomic DNA]. The determined genomic DNA sequence of bacteriophage 4 is shown in sequence number 14.

[0516] (ii) Bioinformatics analysis based on genome sequence information

[0517] Using the genomic DNA sequence of bacteriophage 4 as the query sequence, a search for similar DNA sequences was conducted on the NCBI BLAST server (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The search results showed that the closest DNA sequence was the genomic sequence of *Escherichia coli* bacteriophage esc-cop-9 (Sequence No. 1 of U.S. Patent Application Publication No. 2019 / 0321423), with an estimated sequence identity of 90.68% across the entire range. However, no bacteriophage with a sequence identity greater than 95% across the entire range and a host of *Salmonella* was found.

[0518] <Example 5: Isolation and lysing activity of bacteriophage #5>

[0519] (Purpose)

[0520] A novel bacteriophage with lytic activity against Salmonella was isolated and its lytic activity against Salmonella was verified.

[0521] (Methods and Results)

[0522] (1) Acquisition and culture of Salmonella bacteria

[0523] The bacterial strains used in Example 5 are listed in the table below.

[0524] [Table 6]

[0525] strain ID serotype strain name Supply source ST3 S.Typhimurium HRS-TST-219 Dairy Farm University Veterinary Medicine Group ST4 S.Typhimurium HRS-KST-31 Dairy Farm University Veterinary Medicine Group ST5 S.Typhimurium HRS-KST-203 Dairy Farm University Veterinary Medicine Group ST6 S.Typhimurium HRS-U1 Dairy Farm University Veterinary Medicine Group

[0526] (2) Isolation and purification of phage #5

[0527] Following the method described in the section on [Isolation and Purification of Bacteriophages] above, new bacteriophages were isolated from natural sewage / soil and purified (corresponding to phage number 5).

[0528] (3) Propagation and purification of phage #5

[0529] Following the method described in the section on [phage proliferation and purification] above, the purified solution of phage #5 was prepared, and its titer was determined. The titer was confirmed to be 10. 8 PFU / mL or higher.

[0530] (4) Evaluation of the host range of phage 5

[0531] The host range of bacteriophage 5 was evaluated using plaque assay, following the method described in the [Evaluation of Host Range of Bacteriophages] section above.

[0532] An example of the result is shown below. Figure 8 The fifth phage obtained in this embodiment exhibited very high lytic activity against *S. typhimurium*. It should be noted that the same method was used to investigate the lytic activity against other serotypes of *Salmonella* containing *S. enteritidis*, but the fifth phage did not exhibit lytic activity against these other serotypes.

[0533] (5) Genome analysis of phage #5

[0534] The genomic DNA sequence of phage 5 was determined and analyzed.

[0535] (i) Preparation and sequencing of genomic DNA from phage #5

[0536] The genomic DNA sequence of bacteriophage 5 was determined according to the method described in the section on [Preparation and Sequencing of Bacteriophage Genomic DNA]. The sequenced genomic DNA sequence of bacteriophage 5 is shown in sequence number 17.

[0537] (ii) Bioinformatics analysis based on genome sequence information

[0538] Using the genomic DNA sequence of bacteriophage 5 as the query sequence, a search for similar DNA sequences was conducted on the BLAST server provided by NCBI (https: / / blast.ncbi.nlm.nih.gov / B1ast.cgi). The search results showed that the closest base sequence was the genomic sequence of *Salmonella phage* Skate (GenBank accession number: NC 054639.1), with an estimated sequence identity of 86.61% across the entire range. A detailed alignment of the genomic DNA sequences of the two bacteriophages revealed that the region corresponding to positions 2385–3606 of the genomic DNA sequence of bacteriophage 5 was deleted in Skate. Within this region, a gene encoding a nuclease (positions 2434–3000 of sequence number 17) is present. The amino acid sequence of this nuclease and the base sequence encoding it are shown in sequence numbers 15 and 16, respectively. Nucleases are known to be associated with mechanisms that shut down host genome replication. Therefore, it is shown that the fifth phage with the above-mentioned endonuclease gene can effectively shut down the replication of the host genome, thus exhibiting high lysis activity.

[0539] Using the amino acid sequences of the aforementioned endonucleases as query sequences, a BLAST search was conducted to find similar amino acid sequences. The results showed that no phage genome sequence with more than 50% sequence identity was found. Therefore, this indicates that phage #5 is a novel phage with a new endonuclease gene.

[0540] <Example 6: Isolation and lysing activity of bacteriophage #6>

[0541] (Purpose)

[0542] A novel bacteriophage with lytic activity against Salmonella was isolated and its lytic activity against Salmonella was verified.

[0543] (Methods and Results)

[0544] (1) Acquisition and culture of Salmonella bacteria

[0545] The bacterial strains used in Example 6 are listed in the table below.

[0546] [Table 7]

[0547] strain ID serotype strain name Supply source SE1 S.Entertidis L-2596 Agricultural Research Institute Animal Health SE4 S.Entertidis L-2685 Agricultural Research Institute Animal Health SE6 S.Entertidis L-2728 Agricultural Research Institute Animal Health SE8 S.Entertidis L-2844 Agricultural Research Institute Animal Health ST1 S.Typhimurium HRS-TST-129 Dairy Farm University Veterinary Medicine Group ST4 S.Typhimurium HRS-KST-31 Dairy Farm University Veterinary Medicine Group ST5 S.Typhimurium HRS-KST-203 Dairy Farm University Veterinary Medicine Group ST6 S.Typhimurium HRS-U1 Dairy Farm University Veterinary Medicine Group SJ S.Javiana L-750 Dairy Farm University Veterinary Medicine Group SI1 S.Infantis O7: Hr70A Dairy Farm University Veterinary Medicine Group SI3 S.Infantis O7: Hr1.5 Dairy Farm University Veterinary Medicine Group SM S.Montevideo S.Montevideo No.1 Dairy Farm University Veterinary Medicine Group

[0548] (2) Isolation and purification of phage No. 6

[0549] Following the method described in the [Isolation and Purification of Bacteriophages] section above, a new bacteriophage was isolated from natural sewage / soil and purified (corresponding to bacteriophage No. 6).

[0550] (3) Propagation and purification of phage #6

[0551] Following the method described in the section on [phage proliferation and purification] above, the purified solution of phage No. 6 was prepared, and its titer was determined. The titer was confirmed to be 10. 8 PFU / mL or higher.

[0552] (4) Evaluation of the host range of phage 6

[0553] The host range of bacteriophage 6 was evaluated using plaque assay, following the method described in the [Evaluation of Host Range of Bacteriophages] section above.

[0554] An example of the result is shown below. Figure 9 The sixth phage obtained in this embodiment exhibited lytic activity against multiple bacterial strains tested, specifically against *S. enteritidis*, *S. typhimurium*, and *S. javiana*. These strains are all serotypes frequently detected in human food poisoning. Therefore, the sixth phage is particularly useful for, for example, treating or preventing human food poisoning.

[0555] (5) Genome analysis of phage 6

[0556] The genomic DNA sequence of phage 6 was determined and analyzed.

[0557] (i) Preparation and sequencing of genomic DNA from phage #6

[0558] The genomic DNA sequence of bacteriophage 6 was determined according to the method described in the section on [Preparation and Sequencing of Bacteriophage Genomic DNA]. The determined genomic DNA sequence of one bacteriophage 6 is shown in sequence number 20.

[0559] (ii) Bioinformatics analysis based on genome sequence information

[0560] The genomic DNA sequence of one bacteriophage obtained (P) 序Analysis was performed on column number 20, and the results showed that the amino acid sequence encoded by the base sequence (CDS) from position 32468 to 34522 was tail fibrin. Using this tail fibrin amino acid sequence as the query sequence, a search for approximate amino acid sequences and confirmation of sequence identity were performed using the NCBI BLAST server (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The results showed that amino acid sequences resembling those of tail fibrin from most Salmonella phages were detected, but no amino acid sequence with 100% sequence identity was found. Therefore, a sequence with an amino acid length of 684 residues, identical to the tail fibrin of phage 6, and with a sequence identity of over 95% was extracted and multiple sequence alignment was performed. The results are shown below. Figure 12A , Figure 12B and Figure 12C Additionally, the phage name, sequence identity, Genbank accession number, sequence number assigned in this specification, and serotype of the reaction (with particular attention to Enteritidis and Typhimurium) that can be confirmed based on registration and literature information for the sequences used in the alignment are shown in the table below.

[0561] [Table 8]

[0562]

[0563] >75%: O, 75~50%: Δ, 50%>: x, P: Pullorum, U: UnKnown

[0564] As shown in Table 8, focusing only on Enteritidis and Typhimurium reveals that even with over 95% sequence identity of tail fibrous proteins, the host range of each phage is likely to differ.

[0565] Furthermore, amino acid sequence alignment revealed that the tail filament protein of phage 6 contains several unique amino acid residues that differ from all other sequences. These are 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). Surprisingly, these multiple sites, despite being highly conserved in the tail filament proteins of other phages, are distinct amino acid residues, suggesting a connection to the characteristic host range of phage 6.

[0566] Regarding the genomic DNA sequence of phage 6, BLAST server searches showed that the highest sequence identity was with *Salmonella phage* GRNsp27, at 94.64% (Cover 95% / Ident 99.62%). However, when comparing the sequence similarity of the two using the gene analysis software GENTYX (https: / / www.genetyx.co.jp / ) via MUMmer, the sequence identity near the tail fibrin gene (positions 32468–34522) at positions 29733–34770 dropped to 87% (see table below). This indicates that phage 6 is a novel phage whose host-recognized gene region is significantly different from that of known phages.

[0567] [Table 9]

[0568]

[0569] <Example 7: Isolation and lysing activity of bacteriophage #7>

[0570] (Purpose)

[0571] A novel bacteriophage with lytic activity against Salmonella was isolated and its lytic activity against Salmonella was verified.

[0572] (Methods and Results)

[0573] (1) Acquisition and culture of Salmonella bacteria

[0574] The bacterial strains used in Example 7 are listed in the table below.

[0575] [Table 10]

[0576] strain ID serotype strain name Supply source SE11 S.Entertidis L-3080 Agricultural Research Institute Animal Health SE13 S.Entertidis L-3241 Agricultural Research Institute Animal Health SE14 S.Entertidis L-3244 Agricultural Research Institute Animal Health SE15 S.Entertidis L-3246 Agricultural Research Institute Animal Health SE16 S.Entertidis L-3247 Agricultural Research Institute Animal Health ST3 S.Typhimurium HRS-TST-219 Dairy Farm University Veterinary Medicine Group ST4 S.Typhimurium HRS-KST-31 Dairy Farm University Veterinary Medicine Group SI1 S.Infantis O7: Hr70A Dairy Farm University Veterinary Medicine Group SI2 S.Infantis O7: Hd70B Dairy Farm University Veterinary Medicine Group SI3 S.Infantis O7: Hr1.5 Dairy Farm University Veterinary Medicine Group SM S.Montevideo S. Montevede No. 1 Dairy Farm University Veterinary Medicine Group SJ S.Javiana L-750 Dairy Farm University Veterinary Medicine Group

[0577] (2) Isolation and purification of the 7th phage

[0578] Following the method described in the [Isolation and Purification of Bacteriophages] section above, new bacteriophages were isolated from natural sewage / soil and purified (corresponding to phage number 7).

[0579] (3) Propagation and purification of the 7th phage

[0580] Following the method described in the section on [phage proliferation and purification] above, the purified solution of phage #7 was prepared, and its titer was determined. The titer was confirmed to be 10. 8 PFU / mL or higher.

[0581] (4) Evaluation of the host range of the 7th phage

[0582] The host range of bacteriophage 7 was evaluated using plaque assay, following the method described in the [Evaluation of Host Range of Bacteriophages] section above.

[0583] An example of the result is shown below. Figure 10 The 7th phage obtained in this embodiment exhibited lytic activity against *S. enteritidis*, but not against *S. typhimurium*, *S. infantis*, *S. Montevideo*, or *S. javiana*. *S. enteritidis* is the serotype detected with the highest frequency in human food poisoning (Oh and Park, J. Microbiol. Biotechnol. (2017), 27(12), 2075-2088). Therefore, the 7th phage is particularly useful for, for example, treating or preventing human food poisoning. Furthermore, because the 7th phage exhibits specific lytic activity against *S. enteritidis*, it is particularly useful in the identification of *S. enteritidis*.

[0584] (5) Genome analysis of the 7th phage

[0585] The genomic DNA sequence of phage 7 was determined and analyzed.

[0586] (i) Preparation and sequencing of genomic DNA from phage #7

[0587] The genomic DNA sequence of bacteriophage 7 was determined according to the method described in the section on [Preparation and Sequencing of Bacteriophage Genomic DNA]. The sequenced genomic DNA sequence of bacteriophage 7 is shown in sequence number 23.

[0588] (ii) Bioinformatics analysis based on genome sequence information

[0589] Using the genomic DNA sequence of bacteriophage 7 as the query sequence, a search was conducted on the BLAST server provided by NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to find similar DNA sequences. The search results showed that the closest base sequence was the genomic sequence of Salmonellaphage SPN9CC (GenBank accession number: JF900176.1). Shin et al., Applied and Environmental Microbiology, 2014, vol.80, No.1, 374-384, states that SPN9CC exhibited lytic activity against all seven strains of S. Typhimurium. Therefore, the host range of SPN9CC is significantly different from that of bacteriophage 7, which exhibits S. Enteritidis-specific lytic activity. Consequently, the amino acid sequences of important proteins involved in host recognition in bacteriophage 7 and SPN9CC were compared, revealing differences in the amino acid sequence of the caustic protein. This difference in the amino acid sequence of the caustic protein is the reason for the different host ranges of the two bacteriophages. It should be noted that the gene encoding the caustic protein is located at positions 30879–32882 of the genomic DNA sequence of bacteriophage 7. The amino acid sequence of the caustic protein of bacteriophage 7 is shown in sequence number 21, and the base sequence encoding it is shown in sequence number 22.

[0590] In the search of the above-mentioned approximate DNA sequences, no phage was found that has a gene encoding a tail pin protein containing the amino acid sequence shown in sequence number 21, and has a genomic DNA sequence with more than 99% sequence identity with the base sequence shown in sequence number 23 across the entire range.

[0591] All publications, patents and patent applications referenced in this specification are incorporated herein by direct reference.

Claims

1. A S. enteritidis lysing agent, comprising: A bacteriophage having a genomic DNA sequence containing any of the base sequences shown in sequence numbers 1 to 7.

2. A bacteriophage having genomic DNA containing a gene encoding a tail tip protein and exhibiting lytic activity against Salmonella bacteria. The tail tip protein contains the amino acid sequence shown in sequence number 8.

3. The bacteriophage according to claim 2, wherein, The gene encoding the tail tip protein contains the base sequence shown in sequence number 9.

4. The bacteriophage according to claim 2, wherein, The genomic DNA sequence contains the base sequence shown in any of the sequence numbers 10 to 12.

5. A bacteriophage having a genomic DNA sequence comprising the base sequence shown in sequence number 13, and exhibiting lytic activity against Salmonella bacteria.

6. A Salmonella lysing agent comprising: a bacteriophage having a genomic DNA sequence containing the base sequence shown in sequence number 14.

7. A Salmonella lysing agent comprising: a bacteriophage having genomic DNA containing a gene encoding a nuclease, The endonuclease contains the amino acid sequence shown in sequence number 15.

8. The lysozyme according to claim 7, wherein, The gene encoding the endonuclease contains the base sequence shown in sequence number 16.

9. The lysozyme according to claim 7, wherein, The genomic DNA sequence contains the base sequence shown in sequence number 17.

10. A bacteriophage having genomic DNA containing a gene encoding tail fibrin and exhibiting lytic activity against Salmonella bacteria. The tail fibroin contains the amino acid sequence shown in sequence number 18.

11. The bacteriophage according to claim 10, wherein, The gene encoding tail fibroin contains the base sequence shown in sequence number 19.

12. The bacteriophage according to claim 10, wherein, The genomic DNA sequence contains the base sequence shown in sequence number 20.

13. An S. enteritidis lysing agent comprising: a bacteriophage having a genomic DNA sequence containing the base sequence shown in sequence number 23.

14. A composition comprising the lysing agent of any one of claims 1, 6 to 9 and 13, or the bacteriophage of any one of claims 2 to 5 and 10 to 12.

15. The composition according to claim 14, wherein it is a pharmaceutical composition.

16. The composition according to claim 14, wherein it is a food additive, feed additive, or drinking water additive.

17. The composition according to claim 14, wherein it is a food or feed.

18. The composition according to claim 14, wherein it is a cleaning agent, disinfectant, bactericide, or sterilizing agent.

19. The composition of claim 14, further comprising other bacteriophages exhibiting lytic activity against Salmonella bacteria.

20. A method for preventing and controlling Salmonella bacteria, comprising: A contacting process that brings the lysing agent of any one of claims 1, 6 to 9 and 13, or the bacteriophage of any one of claims 2 to 5 and 10 to 12, into contact with the target of application.

21. A method for treating or preventing an infection caused by Salmonella bacteria, comprising: A drug delivery process of administering the lysing agent of any one of claims 1, 6 to 9 and 13, or the bacteriophage of any one of claims 2 to 5 and 10 to 12, to a target.

22. A method for identifying Salmonella bacteria, the method comprising: The culture process of culturing test bacteria isolated from a specimen suspected of containing Salmonella to obtain a culture; A mixing step of mixing a culture with a lysing agent according to any one of claims 1, 6 to 9 and 13, or a bacteriophage according to any one of claims 2 to 5 and 10 to 12 to obtain a mixture; A mixture cultivation process for culturing a mixture under given conditions; and The determination process after the mixture culture step, when the test bacteria are lysed, is to identify the test bacteria as Salmonella bacteria.

23. The method according to claim 22, wherein, In the mixture culture process, the mixture further includes a liquid culture medium containing soft agar, and the mixture is cultured on a solid culture medium.

24. The method according to claim 22, wherein, In the cultivation process, the culture contains a liquid culture medium containing soft agar, and the culture is also cultured on a solid culture medium.

25. The method of claim 22, further comprising: The isolation process, which involves separating the test bacteria from a specimen suspected of containing Salmonella bacteria, prior to the culture process.

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