Bacteriophage exhibiting broad antibacterial spectrum against escherichia coli

Abstract

The purpose is to provide a novel bacteriophage having a wider range of bacterial strains that serve as hosts for infection than conventional bacteriophages and having bacteriolytic activity against Escherichia coli, including drug-resistant E. coli. The present inventors have discovered a novel bacteriophage having a broad antibacterial spectrum against E. coli, including drug-resistant E. coli. The phage was named ΦWec 277 phage and deposited with the Patent Microorganism Depositary (NPMD) of the National Institute of Technology and Evaluation (NITE) (accession number: NITE P-04067). It was discovered that E. coli can be effectively eliminated by using this bacteriophage, and the present invention was completed. Furthermore, genomic analysis of the ΦWec 277 phage was performed to clarify the full-length sequence. This ΦWec 277 phage was identified as a bacteriophage belonging to the Caudoviricetes Straboviridae as a result of phylogenetic analysis.

Description

A bacteriophage with a broad antibacterial spectrum against Escherichia coli

[0001] The present invention relates to a bacteriophage that exhibits a broad antibacterial spectrum against Escherichia coli, including drug-resistant Escherichia coli, as well as a bacteriophage composition, an antibacterial bacteriophage composition, a pharmaceutical composition, and an antibacterial pharmaceutical composition containing the same, and nucleic acids constituting the genome of the bacteriophage.

[0002] Antibiotics have traditionally been used to treat infectious diseases caused by bacteria, but in recent years, excessive use of antibiotics has led to the problem of bacteria acquiring drug resistance. The emergence of drug-resistant bacteria not only narrows treatment options and leads to the worsening of patients' conditions, but also discourages pharmaceutical companies from developing antibiotics. The use of bacteriophages (hereinafter sometimes referred to as "phages"), which are viruses that infect bacteria, has been proposed as a new method for treating infections caused by drug-resistant bacteria (Non-Patent Documents 1 to 3).

[0003] Non-Patent Document 1 describes a method for phage concentration of 10 11 Phages that exhibit a lysis rate of 51 to 73.6% under PFU / mL conditions have been described. However, the lysis phenomenon caused by high concentrations of phages described in this document does not necessarily indicate a lytic cycle that involves the phage proliferation process.

[0004] Non-Patent Document 2 describes a method for phage concentration of 10 8 A phage showing a 100% lysis rate under PFU / mL conditions is described. However, the number of E. coli strains tested in this paper is small, and their diversity has not been verified. Furthermore, the effectiveness of the phage against drug-resistant bacteria has not been verified.

[0005] Non-Patent Document 3 describes a phage that exhibits a 100% lysis rate against 39 Shiga toxin-producing Escherichia coli (STEC) O157:H7 strains and 12 O26 strains. On the other hand, the lysis activity against 25 drug-resistant (ESBL-producing) strains was only 16 strains (64%), which poses a problem of low lysis rate.

[0006] Furthermore, because phage genes undergo mutations at a certain rate, just like bacteria, even if the host bacteria acquire phage resistance through mutation, the phage can also mutate and acquire the ability to infect resistant bacteria. Therefore, compared to antibiotics, phage can provide a treatment method that can flexibly respond to bacterial mutations.

[0007] Phages have a strict host recognition mechanism and infect and lyse only specific strains of bacteria. Therefore, they have the advantage of being safe for administration to mammals such as humans. However, when a patient shows symptoms of an infectious disease and the causative bacterium has not yet been identified, it is difficult to select a phage that will be effective in treating the disease due to its extremely narrow host range, making it difficult to use as a first-line drug for infectious diseases. To widely disseminate bacteriophage-based treatment, a phage that can infect a wider range of bacterial strains than conventional bacteriophages was needed.

[0008] L. Goodridge, et al. , Appl Environ Microbiol. 2003 Sep; 69(9): 5364-5371. Y. Zhou, et al. , Front Microbiol. 2022 Aug 5:13:929005H. M. Son, et al. , Appl Microbiol Biotechnol. 2018 Dec; 102(23):10259-10271

[0009] An object of the present invention is to provide a novel bacteriophage that can infect a wider range of bacterial strains than conventional bacteriophages and has bacteriolytic activity against E. coli, including drug-resistant E. coli.

[0010] The present inventors conducted extensive research and discovered a novel bacteriophage that has a broad antibacterial spectrum against Escherichia coli, including drug-resistant Escherichia coli. This phage was named ΦWec277 phage and deposited with the National Patent Microorganisms Depositary (NPMD) of the National Institute of Technology and Evaluation (NITE) (Accession No. NITE P-04067). The inventors then discovered that Escherichia coli can be effectively eradicated by utilizing this bacteriophage, leading to the completion of the present invention. Furthermore, the ΦWec277 phage genome was analyzed and its full-length sequence was determined. Phylogenetic analysis of this ΦWec277 phage identified it as a bacteriophage belonging to the Caudoviricetes Straboviridae family.

[0011] Specifically, the present invention provides a bacteriophage having lytic activity against Escherichia coli and having a genome comprising the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 95%, more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, and even more preferably at least 99% identity thereto.

[0012] In the bacteriophage of the present invention, the bacteriophage may be an isolated bacteriophage.

[0013] The bacteriophage of the present invention may be bacteriophage ΦWec277 deposited with the National Institute of Technology and Evaluation under accession number NITE P-04067.

[0014] In the bacteriophage of the present invention, the E. coli strain may be a drug-resistant strain.

[0015] In the bacteriophage of the present invention, the drug-resistant bacterial strain may be a carbapenem-resistant bacterial strain or an ESBL (Extended Spectrum β-lactamase)-producing bacterium.

[0016] The present invention also provides a bacteriophage composition containing the bacteriophage.

[0017] The present invention also provides an antibacterial bacteriophage composition containing the bacteriophage.

[0018] The present invention also provides a pharmaceutical composition containing the bacteriophage as an active ingredient.

[0019] The present invention also provides an antibacterial pharmaceutical composition containing the bacteriophage as an active ingredient.

[0020] The present invention also provides a disinfectant characterized by containing the bacteriophage as an active ingredient.

[0021] The present invention also provides a cleaning agent characterized by containing the bacteriophage as an active ingredient.

[0022] The present invention also provides a food additive containing the bacteriophage as an active ingredient.

[0023] The present invention also provides an isolated nucleic acid consisting of a nucleotide sequence selected from SEQ ID NO: 1 or a sequence having at least 95%, more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99% identity thereto, wherein the nucleic acid is capable of producing a bacteriophage having lytic activity against E. coli.

[0024] In the nucleic acid of the present invention, the E. coli strain may be a drug-resistant strain.

[0025] In the nucleic acid of the present invention, the drug-resistant bacterial strain may be a carbapenem-resistant bacterial strain or an ESBL-producing bacterium.

[0026] The present invention also provides a method for preventing the proliferation of Escherichia coli, which comprises applying a bacteriophage composition containing the bacteriophage as an active ingredient to a mammal in need thereof.

[0027] In the method for preventing the proliferation of E. coli of the present invention, the mammal may be a mammal other than a human.

[0028] The present invention also provides a method for use in the prevention or treatment of infectious diseases, which method uses a bacteriophage having lytic activity against Escherichia coli and having a genome comprising the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 95%, more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99% identity thereto.

[0029] The present invention also provides a method for lysis using a bacteriophage having a genome that has lytic activity against E. coli and comprises the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 95%, more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, and even more preferably at least 99% identity thereto.

[0030] The present invention also provides a method for the prevention or treatment of an infectious disease, comprising administering to a patient in need thereof an effective amount of a bacteriophage having lytic activity against Escherichia coli and having a genome comprising the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 95%, more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99% identity thereto.

[0031] The present invention also provides use of a bacteriophage having lytic activity against Escherichia coli and having a genome comprising the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 95%, more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99% identity thereto, for the prevention or treatment of an infectious disease.

[0032] The present invention also provides a composition for preventing or treating an infectious disease, which comprises a bacteriophage having a genome that has lytic activity against Escherichia coli and includes the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 95%, more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, and even more preferably at least 99% identity thereto.

[0033] In the composition of the present invention, the composition may be a disinfectant or a pharmaceutical composition.

[0034] The composition of the present invention may be a composition comprising a bacteriophage having lytic activity against Escherichia coli and having a genome comprising the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 95%, more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, and even more preferably at least 99% identity thereto, and a pharmaceutically acceptable additive or carrier.

[0035] In the composition of the present invention, the additive may be a preservative.

[0036] In the composition of the present invention, the preservative may be a cryopreservative.

[0037] In the composition of the present invention, the cryopreservation agent may be sucrose and / or glycerol.

[0038] The present invention also provides a bacteriophage composition for use in the prevention of infectious diseases, which contains as an active ingredient a bacteriophage having a genome comprising the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 95%, more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, and even more preferably at least 99% identity thereto, and which has lytic activity against Escherichia coli.

[0039] The bacteriophage composition of the present invention may be a pharmaceutical composition.

[0040] In the bacteriophage composition, the bacteriophage composition may be a disinfectant.

[0041] In the bacteriophage composition, the bacteriophage composition may be a detergent.

[0042] The bacteriophage composition may be a food additive.

[0043] The present invention also provides use of a bacteriophage having a genome comprising the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 95%, more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99% identity thereto, which has lytic activity against Escherichia coli, in the manufacture of a medicament for the prevention or treatment of an infectious disease.

[0044] The present invention also provides a bacteriophage having lytic activity against Escherichia coli, and having a genome comprising the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 95%, more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99% identity thereto, for use in the prevention or treatment of infectious diseases.

[0045] The present invention also provides a method for preventing or treating an infectious disease, which comprises administering to a patient in need thereof a bacteriophage having a genome comprising the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 95%, more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, and even more preferably 99% identity thereto, and other bacteriophages having lytic activity.

[0046] By using the ΦWec277 phage of the present invention, among 144 clinically isolated E. coli strains whose diversity was verified by whole genome analysis, minimum inhibitory antimicrobial concentration (MIC), and susceptibility testing against 46 phages including the ΦWec277 phage, the ΦWec277 phage of the present invention was found to be effective at a phage concentration of 7×10 9 The ΦWec277 phage was able to infect and lyse 113 strains under conditions of PFU / mL (infection rate: 78.5%). The ΦWec277 phage exhibited a broad antibacterial spectrum not only against clinically isolated strains but also against multiple E. coli strains that have acquired resistance to carbapenems, a drug that is effective against many bacteria and is widely used in treatment, as well as ESBL-producing bacteria, making it a potential therapeutic tool for drug-resistant bacteria.

[0047] A diagram showing the results of a simple bacteriophage lytic zone test using a spot test (undiluted). A diagram showing a comparison of the infection rates of collected bacteriophages. A diagram showing the turbidity curves of ΦWec277 phage for E. coli clinical isolates (14 strains) and a laboratory strain (MG1655 strain) stored at Waseda University. A diagram showing the alignment of ΦWec277 phage and T6 phage.

[0048] Known pathogenic E. coli include enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (EHEC), and enteroaggregative-adherent E. coli (EAggEC).

[0049] The present invention relates to a bacteriophage having a broad antibacterial spectrum lytic activity against clinical isolates of Escherichia coli, and having a genome comprising the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 95%, more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, and even more preferably at least 99% identity thereto.

[0050] The bacteriophage of the present invention may be an isolated bacteriophage.

[0051] As used herein, "identity" refers to the value obtained by multiplying the average nucleotide identity (ANI), which indicates the proportion of bases that actually match in the sequence region of two nucleotides being compared, by the query coverage, which indicates the proportion of the sequence length of the region used in the comparison calculation to the entire sequence length of the two nucleotides.

[0052] As used herein, the term "infection" refers to the establishment of infection when a bacteriophage invades a host cell such as a bacterium and multiplies within the host cell.

[0053] Unless otherwise specified, "bacteriolysis" as used herein refers to the phenomenon in which bacteria are lysed by the action of a bacteriophage. This phenomenon is not limited to cases in which a bacteriophage infects a host bacterium and lyses the bacterium, but also includes cases in which a bacteriophage acts externally to lyse bacteria without infecting the host bacterium.

[0054] As used herein, the term "isolated bacteriophage" refers to a bacteriophage that has been removed from its natural environment and / or separated from components of its natural environment. This term particularly refers to, for example, phage grown in vitro, purified phage, and / or phage formulated with any suitable diluent or excipient.

[0055] A bacteriophage having a genome with the nucleotide sequence of SEQ ID NO: 1 was obtained from sewage in Japan, isolated, and purified by the method described in the Examples below. It was named ΦWec277 phage and has been deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, postal code: 292-0818) under accession number: NITE P-04067. Phylogenetic analysis of ΦWec277 phage identified it as a bacteriophage belonging to the Caudoviricetes Straboviridae family.

[0056] The nucleotide sequence of the genome of the ΦWec277 phage of the present invention was analyzed by the method shown in the Examples and is identified as SEQ ID NO: 1. The bacteriophage of the present invention is not limited to the ΦWec277 phage identified by SEQ ID NO: 1, but is a bacteriophage having a genome comprising the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 95%, more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, and even more preferably at least 99% identity thereto.

[0057] The bacteriophage of the present invention may be isolated.

[0058] The ΦWec277 phage was found to be the most abundant phage in the 144 clinically isolated E. coli strains whose diversity was verified through whole genome analysis, minimum inhibitory concentrations (MICs), and susceptibility tests against 46 phages, including the ΦWec277 phage, at a phage concentration of 7 × 10 9 The ΦWec277 phage was able to infect and lyse 113 strains under conditions of PFU / mL (infection rate: 78.5%). The ΦWec277 phage exhibited a broad antibacterial spectrum not only against clinically isolated strains but also against multiple E. coli strains that had acquired resistance to carbapenems, a drug that is effective against many bacteria and is widely used for treatment, and it could be a powerful therapeutic tool for drug-resistant bacteria.

[0059] The bacteriophage of the present invention can be grown by a common bacteriophage growth method such as the plate lysate method. For example, after culturing and sufficiently growing host Escherichia coli, the bacteriophage is inoculated with the deposited ΦWec277 phage of the present invention or a bacteriophage having a genome containing a nucleotide sequence having at least 95%, more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, and even more preferably at least 99% identity thereto, and the plaques that appear can be collected and further cultured to obtain a large amount of bacteriophage solution.

[0060] Alternatively, a large amount of bacteriophage solution can be obtained by preparing a vector or a plasmid containing nucleic acid having SEQ ID NO: 1 or a nucleotide sequence having at least 95%, more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, and even more preferably at least 99% identity thereto, and inserting the nucleic acid into E. coli, and culturing the E. coli into which the nucleic acid has been inserted.

[0061] The ΦWec277 phage of the present invention, or a bacteriophage having a genome containing a nucleotide sequence that is at least 95%, more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, and even more preferably at least 99% identical thereto, can be used as a bacteriophage composition. The bacteriophage composition may be any composition containing the bacteriophage of the present invention. For example, a bacteriophage solution obtained by the above-described propagation method may be used. Furthermore, other suitable bacteriophages (note: bacteriophages for E. coli as well as other suitable bacteria may also be used) or pharmaceuticals such as antibacterial agents composed of low-molecular-weight compounds may also be appropriately mixed with the bacteriophage.

[0062] The concentration of bacteriophage in the bacteriophage composition of the present invention varies depending on the form of use. When used as a liquid bactericide, disinfectant, sanitizer, cleaner, food additive, or the like, the concentration of bacteriophage is 1 x 10 4 ~1 x 10 12 It is preferably expressed as PFU / mL (PFU: plaque forming unit), and 1 x 10 7 ~1 x 10 11 More preferably, it is expressed as PFU / mL.

[0063] The E. coli host for the bacteriophage of the present invention may be cultured according to a standard method. For example, the E. coli may be cultured at a temperature of 27 to 37°C using LB (Lysogeny Broth) medium, Brain Heart Infusion medium, or the like, which are commonly used for culturing E. coli. The culture method may be liquid culture or solid culture. The timing for inoculating the host medium with the bacteriophage is not particularly limited as long as the E. coli is sufficiently grown, but it is preferable that the bacterial cell concentration is 1 x 10 7 ~1 x 10 8 CFU / mL (CFU: colony forming units) (OD 660 It is desirable to inoculate the bacteriophage at a pH of about 0.01 to 0.1. After culturing for about 6 to 12 hours from inoculation, almost all of the bacteria will be lysed and a bacteriophage solution can be obtained.

[0064] Other materials constituting the bacteriophage composition of the present invention may be selected appropriately from those conventionally used for the respective applications, such as bactericides, disinfectants, preventive agents for infectious diseases, therapeutic agents, disinfectants, cleaning agents, food additives, etc., depending on the mode of use, as long as they do not impair the infectivity and bacteriolytic activity of the bacteriophage of the present invention against E. coli.

[0065] The bacteriophage composition of the present invention can be used as an antibacterial bacteriophage composition.

[0066] Furthermore, the ΦWec277 phage of the present invention, or a bacteriophage having a genome comprising a nucleotide sequence having at least 95%, more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, and even more preferably at least 99% identity thereto, can be used as a pharmaceutical composition, particularly an antibacterial pharmaceutical composition, containing the bacteriophage. The antibacterial pharmaceutical composition of the present invention can be used in the medical field, including human or veterinary fields, for example, to prevent or treat infections in mammals or to improve the condition of a subject.

[0067] Furthermore, the antibacterial pharmaceutical composition of the present invention can be used for the prevention or treatment of mammalian infections caused by drug-resistant Escherichia coli. For example, as shown in the examples below, the antibacterial pharmaceutical composition can be used as an antibacterial pharmaceutical composition such as an agent for treating or preventing infections caused by E. coli including carbapenem-resistant bacteria or ESBL-producing bacteria. In this case, the composition is prepared into a dosage form suitable for administration. For example, the composition may be prepared into a liquid formulation such as an injection, a powder, a capsule, a liposome formulation, a topical formulation, or a transdermal patch.

[0068] Each formulation can be prepared with a suitable carrier commonly used in the pharmaceutical field. For example, when used as a liquid formulation such as an injection or oral agent, examples of the carrier or diluent include buffer solutions such as phosphate buffer, physiological saline, distilled water or other aqueous media, and injectable non-aqueous media such as polyethylene glycol, propylene glycol, and ethyl oleate. These formulations may also contain stabilizers, buffers, isotonicity agents, chelating agents, pH adjusters, surfactants, preservatives, antioxidants, etc.

[0069] When used as a drug such as a therapeutic agent, it can be administered to humans and other mammals infected with pathogenic E. coli, for example, by intravascular or local injection, oral, nasal, topical, subcutaneous, transdermal, intradermal, and other routes, but is not limited to these.

[0070] The concentration of the bacteriophage in the injection containing the bacteriophage of the present invention varies depending on the type of mammalian target, but is, for example, 1 x 10 5 ~1 x 10 12 It is sufficient if it is on the order of PFU / mL.

[0071] Mammals that can be the target of prevention or treatment using the bacteriophage of the present invention include, but are not limited to, humans, cows, pigs, horses, sheep, dogs, cats, etc.

[0072] When an injection containing the bacteriophage of the present invention is administered locally, the dosage may vary depending on the type of mammalian subject, the condition of the affected area, etc., but may be, for example, 1 x 10 4 ~1 x 1012 About PFU, preferably 1 x 10 5 ~1 x 10 12 The amount of the agent may be administered to the affected area by injection or orally as an enteric-coated preparation, but is not limited thereto.

[0073] When the bacteriophage of the present invention is administered as an injectable solution or an enteric-coated formulation for oral administration into the bloodstream, for example, through the median cutaneous vein of the forearm, the dosage may vary depending on the type of mammal and the condition of the affected area, but may be, for example, 1 x 10 per body weight. 4 ~1 x 10 12 It is sufficient to inject approximately PFU / kg into the blood vessel, but the present invention is not limited to this.

[0074] As used herein, "PFU" refers to plaque-forming units, as is well defined in the art. Lytic bacteriophages lyse host cells, initiating zones of exclusion (or plaques) on culture plates. Theoretically, each plaque is formed by one phage, and the number of plaques multiplied by the dilution factor equals the total number of phages in the test preparation.

[0075] Furthermore, bacteriophages are highly host-specific and are viruses that only infect host bacteria, so they are thought to have almost no toxicity when administered to animals. Therefore, depending on the type of mammal and the condition of the affected area, it is possible to administer larger amounts of bacteriophages. For example, if the animal to be administered is large, it is possible to administer larger amounts of bacteriophages.

[0076] A method for preventing the proliferation of E. coli using a bacteriophage composition containing the bacteriophage of the present invention as an active ingredient includes applying the bacteriophage composition of the present invention to an object to be prevented from proliferating, for example by contacting the bacteriophage composition with the object.

[0077] In another embodiment, a disinfectant containing the bacteriophage of the present invention as an active ingredient is provided. In yet another embodiment, a cleaning agent containing the bacteriophage as an active ingredient is provided. In yet another embodiment, a food additive containing the bacteriophage as an active ingredient is provided, which is intended to prevent the growth of and / or kill E. coli in foods such as meat that may be contaminated with E. coli.

[0078] Specifically, for example, when a bacteriophage composition containing the bacteriophage of the present invention as an active ingredient is used as a disinfectant, it can be brought into contact with the surface of an object to prevent the growth of E. coli by spraying, atomizing, dipping, wiping, applying, or the like.

[0079] The bacteriophage is as described above.

[0080] The formulation of the disinfectant, cleaning agent, or food additive is not particularly limited, and may be prepared in a formulation known in the art. The disinfectant, cleaning agent, or food additive may be prepared by a manufacturing method commonly used in the art.

[0081] The disinfectant is applied to eliminate E. coli and is applied to, but not limited to, animal activity areas, slaughterhouses, killing areas, kitchens or food preparation facilities.

[0082] The detergent can be used for cleaning the skin surface or various parts of the body of an animal that has been exposed or may be exposed to E. coli, but is not limited thereto.

[0083] The food additive can be used for, but is not limited to, washing or spraying the surface or interior of food such as meat that is contaminated or may be contaminated with E. coli.

[0084] The disinfectant, cleaning agent, or food additive contains an additive in an effective amount sufficient to reduce deterioration of the disinfectant, cleaning agent, or food additive, and the additive may include a preservative, stabilizer, excipient, or cryoprotectant. By including the additive in the disinfectant, cleaning agent, or food additive, the bacteriophage of the present invention survives or maintains its activity for a longer period within the disinfectant, cleaning agent, or food additive, unlike bacteriophages present in their natural state. The preservative, stabilizer, excipient, or cryoprotectant may be any preservative commonly used in the art, as long as it can reduce deterioration of the disinfectant, cleaning agent, or food additive.

[0085] The time for which the bacteriophage composition of the present invention is brought into contact with the subject in which the proliferation of E. coli is to be prevented may be long enough to lyse the subject E. coli and prevent proliferation.

[0086] The genome nucleic acid of the isolated ΦWec277 phage of the present invention can be isolated and purified according to a conventional method. This isolated nucleic acid was identified as a nucleic acid having the nucleotide sequence of SEQ ID NO:1.

[0087] A nucleic acid having SEQ ID NO:1 or a sequence having at least 95%, more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, and even more preferably at least 99% identity thereto can be isolated from a deposited bacteriophage or can be produced using recombinant DNA techniques (e.g., polymerase chain reaction (PCR) amplification, cloning), enzymatic synthesis, chemical synthesis, or a combination thereof, according to techniques known in the art. The nucleic acid of the present invention can be in a free form or cloned into a vector such as a plasmid, a viral vector, an expression cassette, or a cosmid. For example, a ΦWec277 phage having lytic activity against E. coli or a bacteriophage having a genome comprising a nucleotide sequence having at least 95%, more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, and even more preferably at least 99% identity thereto can be produced by using this nucleic acid to prepare a plasmid, inserting the plasmid into E. coli, and culturing the cells. The E. coli on which the ΦWec277 phage exerts bacteriolytic activity may be a drug-resistant E. coli, for example, a carbapenem-resistant or ESBL-producing E. coli.

[0088] The present invention also relates to a method for treating an infection in a mammal, comprising administering a composition or bacteriophage or nucleic acid or polypeptide as described above to a mammal in need thereof.

[0089] The present invention will be described in more detail below with reference to examples, but modifications can be made as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed as being limited to the specific examples shown below.

[0090] Example 1: Simple test for bacteriophage lytic zone using spot test (undiluted solution) 1-1. Experimental method 1-1-1. Bacterial strains used In this experiment, 144 strains of E. coli stored at Jichi Medical University were used. All of these were isolated from clinical settings and possess drug resistance, with 50 of these strains possessing carbapenem resistance. Furthermore, the identity of the strains was confirmed by 16S rRNA gene sequencing or complete genome sequencing.

[0091] 1-1-2. Pre-culturing of bacterial strains Each strain was picked, suspended in LB (Difco) medium, and cultured overnight (approximately 9 hours) in an incubator (37°C). After overnight culture, the bacterial solution was diluted 100-fold with LB medium and cultured with shaking to allow regrowth in the logarithmic growth phase, which was used in the experiment. The composition of LB medium is as follows: NaCl 1 w / v%, yeast extract 0.5 w / v%, polypeptone 1 w / v%. LB agar medium further contains 1.5% agar in addition to the above composition. Soft agar medium contains 0.5% agar.

[0092] 1-1-3. Isolation and purification of ΦWec277 phage Sewage concentrate was prepared as follows: Sewage was centrifuged (6000 g, 60 min, 4°C), and polyethylene glycol 6000 was added to the supernatant to a concentration of 10% w / v and NaCl to a concentration of 4% w / v. After allowing to stand overnight (4°C), the pellet obtained by centrifugation (6000 g, 60 min, 4°C) was dissolved by adding SM buffer dropwise. The composition of the SM buffer is as follows: 945 mL of pure water contains 5.8 g of NaCl, 2.0 g of MgSO4·7H2O, 50 mL of 1 M Tris-HCl (pH 7.5), and 5 mL of 2% gelatin. The lysate was mixed with an equal volume of chloroform and stirred using a vortex mixer. The supernatant was used as a phage concentrate. Bacteriophage screening was performed using the E. coli MG1655 strain as the host bacterium using the double-layer agar method. 100 μL each of E. coli culture solution and bacteriophage concentrate was added to 4 mL of soft agar heated to 50°C, stirred for 1 second using a vortex mixer, and then layered on the agar medium. The agar medium was cultured overnight at 37°C, and the presence or absence of plaques was confirmed. If there were many plaques, or if there were so many plaques that the soft agar was transparent, the bacteriophage concentrate was diluted 100-fold. Plaques were picked and suspended in SM buffer. The isolated bacteriophage was purified as follows: the culture solution from the plaque pick was centrifuged (9000 G, 5 minutes, 4°C), and the supernatant was diluted 100 times with SM buffer. 1 ~10 7 The bacteriophage was diluted 2x and subjected to a plaque assay using the double-layer agar method. A single plaque was picked from a plate containing >100 PFU / plate, inoculated into a 1.5 mL Eppendorf tube containing 1 mL of LB medium, and cultured overnight at 37°C. This process was repeated three times to obtain purified bacteriophage. The resulting bacteriophage stock was mixed with 40% glycerol containing 0.2 M sucrose in a 1 mL cryotube and stored at -80°C. The working solution was stored at 4°C and diluted with SM buffer.

[0093] A bacteriophage with a broad antibacterial spectrum, obtained from domestic sewage in 2022 and isolated and purified using the above-mentioned method, was named ΦWec277 phage.

[0094] 1-1-4. Preliminary Test of Bacteriolytic Activity of Phage Stock Solution A simple bacteriolytic activity evaluation was performed using 144 clinically isolated strains of E. coli stored at Jichi Medical University. Dissolved LB (Luria Broth) soft agar (agar concentration: 0.5%) was kept at 50°C using a heat block, and each enriched strain was added. After thorough mixing of the LB top agar and each strain, the mixture was poured evenly onto the LB agar medium and allowed to stand at room temperature for approximately 30 minutes to completely solidify. 1 μL of high-concentration phage stock solution was spotted onto the solidified LB top agar and incubated overnight (37°C, approximately 9 hours) in an incubator. Bacteriolytic activity was evaluated by scoring the transparency of the plaques at the spotted area. Bacteriolytic activity was evaluated on a four-point scale: complete transparency (score 3), lysis with transparency visible from the bottom (score 2), only discoloration (score 1), and no change (score 0).

[0095] 1-2. Experimental Results The lytic ability of individual phages varied greatly, with the number of strains that showed complete lysis (score 3) ranging from 1 to 105 (0.7% to 73%). ΦWec277 phage showed complete lysis (score 3) against 105 of 144 strains (Figure 1) and some lytic activity (score 1 or higher) against 135 of 144 strains, demonstrating the broadest lytic spectrum.

[0096] Example 2: Spot test (dilution series) to test for lysis zone by bacteriophage infection 2-1. Experimental method 2-1-1. Bacterial strains used The same bacterial strains as in 1-1-1 above were used.

[0097] 2-1-2. Pre-cultivation of bacterial strains Pre-cultivation of bacterial strains was carried out in the same manner as in 1-1-2.

[0098] 2-1-3. Isolation and purification of ΦWec277 phage ΦWec277 phage was isolated and purified in the same manner as in 1-1-3 above.

[0099] 2-1-4. Spot test for bacteriophage infection zone Melted LB soft agar (agar concentration: 0.5%) was kept at 50°C using a heat block, and each strain after bacterial proliferation was added. After thoroughly stirring the LB top agar and each strain, it was poured evenly onto the LB agar medium and left at room temperature for about 30 minutes to completely solidify. Bacteriophage was dissolved in SM buffer for 10 minutes. 2 double, 10 4 double, 10 6 The stock solution was diluted 2x and 1 μL of each of the four dilutions was spotted onto solidified LB top agar (soft agar medium) and cultured overnight in an incubator (37°C, approximately 9 hours). Plaques formed in each spot on the soft agar medium were then detected. Detection of plaques in the spots indicates bacteriophage-associated bacteriolysis. In other words, even if the spotted area is clear with the stock solution alone, if no plaques are formed in the diluted solution, this means that the host bacteria have been lysed but bacteriophage infection has not occurred, indicating the lack of bacteriophage infectivity. In this experiment, only when a spot was observed with any of the diluted solutions was it determined that the bacteriophage had infected and exhibited bacteriolytic activity.

[0100] 2-2. Experimental Results: Infection Range of ΦWec277 Phage ΦWec277 phage (hereinafter referred to as "ΦWec277") exhibited bacteriolytic activity upon infection of 113 strains, including carbapenem-resistant strains, out of 144 clinically isolated strains of E. coli stored at Jichi Medical University (Table 1, Figure 2). Furthermore, compared to other bacteriophages measured at the same time, namely ΦWec269 phage (hereinafter also referred to as "ΦWec269"), ΦWec207 phage (hereinafter also referred to as "ΦWec207"), ΦWec271 phage (hereinafter also referred to as "ΦWec271"), ΦWec273 phage (hereinafter also referred to as "ΦWec273"), ΦWec276 phage (hereinafter also referred to as "ΦWec276"), and ΦWec281 phage (hereinafter also referred to as "ΦWec281"), as well as T4 phage (hereinafter also referred to as "T4"), T6 phage (hereinafter also referred to as "T6"), and T7 phage (hereinafter also referred to as "T7"), it was found that ΦWec277 phage has a higher bacteriolytic activity upon infection, i.e., a broad antibacterial spectrum.

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[0102] Furthermore, 50 of the 144 strains of E. coli were carbapenem-resistant, and 38 of these strains were lysed by the ΦWec277 phage.

[0103] Furthermore, among the 144 strains of E. coli, at least 65 strains were ESBL-producing bacteria, and 53 of these strains could be lysed by ΦWec277 phage.

[0104] Furthermore, among the 144 strains of E. coli, there were at least 12 drug-resistant strains other than carbapenem-resistant strains and ESBL-producing strains, and 10 of these strains were able to be lysed by the ΦWec277 phage.

[0105] Among the 144 strains of E. coli, 17 strains were not drug-resistant, and 12 of these strains could be lysed by the ΦWec277 phage.

[0106] ​Example 3 3-1. Experimental Method 3-1-1. Bacterial Strains Used In this experiment, 18 clinically isolated strains of E. coli stored at Waseda University were used. Of these, one strain was an ampicillin-resistant strain, and 17 strains were ESBL-producing strains, and they were confirmed to be E. coli by 16S rRNA gene sequencing or complete genome sequencing. A laboratory strain (MG1655 strain) was also used for comparison.

[0107] 3-1-2. Pre-culture of bacterial strains As in Example 1, 18 clinically isolated strains of Escherichia coli stored at Waseda University were pre-cultured.

[0108] 3-1-3. Spot test for bacteriophage lysis zone Melted LB soft agar (agar concentration: 0.5%) was kept at 50°C using a heat block, and each enriched strain was added. After thoroughly stirring the LB top agar and each strain, it was poured evenly onto the LB agar medium and left at room temperature for approximately 30 minutes to completely solidify. 1 μL of the phage stock solution was dropped (spotted) onto the solidified LB top agar (soft agar medium) and cultured overnight in an incubator (37°C, approximately 9 hours). Then, traces of lysis were detected on each spot on the soft agar medium.

[0109] 3-1-4. Evaluation of lytic activity using turbidity curves The lytic activity of strains infectable with ΦWec277 phage and a laboratory strain (MG1655 strain) was investigated using a turbidity curve. Bacteriophage was tested under the following conditions, and lytic activity was assessed by measuring turbidity over time using a MULTISKA FC (Thermos Fisher Scientific). First, each strain regrowth in the logarithmic growth phase was diluted with LB medium to a final optical density (OD) of 0.01 at 595 nm, and added to a 96-well plate. In the same system, ΦWec277 phage was added at a concentration of 1.6 x 10 8The bacteriophage was diluted and mixed to a concentration of PFU / mL. A control group was prepared without the addition of bacteriophage. N=2 samples were prepared for each group. 200 μL of each sample was added to a 96-well plate (FALCON, Model 353072) and incubated with shaking at 37°C, during which the OD was measured at 15-minute intervals.

[0110] 3-2-1. Experimental results Lytic range of ΦWec277 phage (estimated by spot test) ΦWec277 phage produced clear plaques and was able to lyse 16 of the 18 clinical isolates of E. coli stored at Waseda University (lysis rate 89%). Of the remaining two strains, one showed a change in the spot but was not completely clear, and the remaining strain showed no change in the spot, indicating that it was unable to lyse at least strains that did not cause a change in the spot.

[0111] 3-2-2. Lytic Activity of ΦWec277 Phage (Confirmed by Turbidity Curve) Turbidity curves were measured for 14 of the 16 E. coli clinical isolates for which ΦWec277 phage showed lytic activity. Turbidity increased monotonically in the bacteriophage-free group, whereas an initial decrease in turbidity was observed in the bacteriophage-added group (Figure 3). A similar trend was observed for the laboratory strain (MG1655) used for comparison (Figure 3). When bacteriophage with strong lytic activity was added, the bacteriophage proliferated as the number of bacteria increased, and the proliferation rate of the bacteriophage exceeded that of the bacteria, resulting in a decrease in turbidity. These results demonstrate that ΦWec277 phage exhibited high lytic activity against all E. coli strains used in this experiment.

[0112] Example 4: Genome analysis of ΦWec277 phage 4-1. Experimental method The entire genome of the bacteriophage was extracted using the phenol-chloroform method. Shotgun sequencing (paired-end, 150 bp) was performed on the extracted genome, and a complete genome was constructed by de novo assembly using Velvet (ver. 1.2.10). Annotation of the constructed genome was performed using DFAST and RAST tk with default options. Comparison of genome similarity was performed using Average Nucleotide Identity (ANI) analysis and alignment. ANI analysis was performed using pyani, and alignment was performed using GenomeMatcher software (ver. 3.06, options: blast+, parameters: defaults) and MEGA X. Phylogenetic analysis was performed using vContact2.

[0113] 4-2. Experimental Results The complete base sequence of the genome of ΦWec277 phage is shown as SEQ ID NO: 1. Annotation revealed that ΦWec277 phage does not contain a lysogen gene integrase. Phylogenetic analysis identified it as a bacteriophage belonging to Caudoviricetes Straboviridae, but no consensus was reached at the genus level. The results of calculating the ANI (Average Nucleotide Identity) with the T2, T4, and T6 phages, which were suggested to be closely related, are shown in Tables 2-1, 2-2, and 2-3, respectively, along with the percent identity, query coverage, and identity value obtained by multiplying the ANI and coverage. Since T6 had the highest identity × coverage, it was suggested that among the T2, T4, and T6 phages, T6 phage was the most similar to ΦWec277 phage. The alignment results of ΦWec277 phage and T6 phage are shown in heat map format (higher homology is red, lower homology is blue, and no homology is black) (Figure 4). Although ΦWec277 phage and T6 phage share homology, several differences were observed.

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[0117] Example 5 5-1. Bacteriolytic activity of ΦWec277 against strains other than E. coli Experimental method The presence or absence of bacteriolytic activity of ΦWec277 against various strains other than E. coli was examined.

[0118] 5-2. Experimental Results As a result, the bacteriolytic activity of ΦWec277 was confirmed against the following strains: Salmonella enterica subspecies enterica serovar Minnesota NBRC15335 Salmonella enterica subspecies enterica serovar Pullorum NBRC3163

[0119] On the other hand, it did not show any bacteriolytic effect against the following bacterial strains: Salmonella enterica subspecies enterica serovar Enteritidis NBRC3313 Salmonella enterica subspecies enterica serovar Typhimurium NBRC13245 Escherichia fergusonii NBRC102419 Atlantibacter hermannii NBRC105704 Citrobacter amalonaticus (derived from mouse feces) Morganella morganii (clinical isolate)

[0120] Microorganism deposit (1) Identification: ΦWec277 (2) Accession number: NITE P-04067 (3) Date of deposit: January 18, 2024 (4) Depository institution: National Institute of Technology and Evaluation

[0121] [Rule 26, amended 28.03.2025]

Claims

1. A bacteriophage having lytic activity against Escherichia coli and having a genome comprising the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 95% identity thereto.

2. The bacteriophage according to claim 1, which is bacteriophage ΦWec277 deposited at the National Institute of Technology and Evaluation under accession number NITE P-04067.

3. The bacteriophage according to claim 1, wherein the E. coli strain is a drug-resistant strain.

4. The bacteriophage according to claim 3, wherein the drug-resistant bacterial strain is a carbapenem-resistant bacterial strain or an ESBL-producing bacterium.

5. A bacteriophage composition comprising the bacteriophage according to any one of claims 1 to 4 as an active ingredient.

6. An antibacterial bacteriophage composition, characterized in that it contains the bacteriophage according to any one of claims 1 to 4 as an active ingredient.

7. A pharmaceutical composition comprising the bacteriophage according to any one of claims 1 to 4 as an active ingredient.

8. An antibacterial pharmaceutical composition comprising the bacteriophage according to any one of claims 1 to 4 as an active ingredient.

9. A disinfectant characterized by containing the bacteriophage according to any one of claims 1 to 4 as an active ingredient.

10. A cleaning agent characterized by containing the bacteriophage according to any one of claims 1 to 4 as an active ingredient.

11. A food additive comprising the bacteriophage according to any one of claims 1 to 4 as an active ingredient.

12. An isolated nucleic acid consisting of a nucleotide sequence selected from SEQ ID NO: 1 or a sequence having at least 95% identity thereto, said nucleic acid being capable of producing a bacteriophage having lytic activity against E. coli.

13. The nucleic acid according to claim 12, wherein the E. coli strain is a drug-resistant strain.

14. The nucleic acid according to claim 13, wherein the drug-resistant bacterial strain is a carbapenem-resistant bacterial strain or an ESBL-producing bacterium.

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