WO2021157732A1 - Agent for use with bacteriophage - Google Patents

Abstract

A composition for lysing at least one type of staphylococcus bacteria selected from the group consisting of Staphylococcus hyicus, Staphylococcus chromogenes, Staphylococcus simulans, Staphylococcus schleiferi, and Staphylococcus felis, or for the prevention or treatment of diseases caused by said bacteria, said composition containing, as active ingredients: at least one bacteriophage selected from the group consisting of bacteriophages identified by accession number NITE BP-693 and bacteriophages identified by accession number NITE BP-694; and an endolysin, which is a protein specified by an amino acid sequence of SEQ ID NO: 1.

Description

Bacteriophage concomitant

The present invention includes bacteriophage and endolysin having the property of lysing staphylococci (Staphylococcus hycus, Staphylococcus chromodinas, Staphylococcus simulation, Staphylococcus shreiferi, Staphylococcus ferris). The present invention relates to a composition for lysing the bacterium and a composition for preventing or treating a disease caused by the staphylococcus.

Staphylococcus hycus, Staphylococcus chromogenes, Staphylococcus simulations, Staphylococcus schleiferi, Staphylococcus ferris Staphylococci such as (S. ferris) cause various diseases in humans and animals. Skin infections, dermatitis, greasy pig disease and the like are known as one of the skin diseases caused by these (Non-Patent Documents 1 to 4). Exudative epidermitis is called "soot disease", and in piglets, it becomes a state where the whole body is covered with soot, and it often develops in abdominal units, and 20 to 30% of cases die when it becomes severe. It also becomes. For the treatment, antibacterial agents, disinfectants, steroids and the like showing their effectiveness are used (Non-Patent Document 5). However, in recent years, there are many problems such as the increase of drug-resistant bacteria, the inability to exert the effect of antibacterial agents, the damage of the skin by disinfectants, and the worsening of symptoms, and there is no effective treatment method.

In this regard, the use of bacteriophage is being attempted. Bacteriophage is a general term for viruses that infect bacteria, and when infected with a host bacterium, it proliferates within the bacterium. The proliferated daughter phage is released from the cell wall of the bacterium to the outside of the bacterium, and the bacterium is killed by lysis. In addition, the bacteriophage has advantages that it does not affect the bacterial flora other than the host pathogen, that it is easy to grow, and that a large amount of bacteriophage can be prepared at low cost.

Therefore, efforts are being made to use bacteriophage as a fungicide for bacteria, and some research results on the sterilization of Staphylococcus aureus using bacteriophage have been reported. For example, in Patent Document 1, S.A. As bacteriophages having the property of lysing aureus, bacteriophages identified by accession numbers NITE BP-693 and accession number NITE BP-694 have been found and reported by the present inventors.

However, bacteriophage has a problem of high host specificity, such as not being infected if the strains are different even if the bacteria are of the same species (species). hyicus, S.A. Chromogenes, S.A. simulans, S.M. Schleiferi and S.M. There are no reports of felis, a bacteriophage that has lytic activity against these multiple bacteria. Therefore, an effective bacteriophage is required for preventing or treating diseases caused by the staphylococcus.

In addition, endolysin is a peptidoglycan hydrolase produced by bacteriophage and is known to cleave bacterial cell wall peptidoglycan. As described above, endolysin exhibits a lytic effect on bacteria by a mechanism different from that of antibiotics, and is also effective against bacteria showing high resistance to antibiotics and the like. So far, Staphylococcus aureus bacteriophage lysin such as Lys-phiK, Lys-GH15, and Lys-fiPort have been isolated and reported. These endolysin are multidomain enzymes composed of an N-terminal cysteine-histidine-dependent amide hydrolase / peptidase (CHAP) domain, an amidase (AMID) domain, and a C-terminal SH3b cell wall binding domain. Lysis and the like on aureus have been reported (Non-Patent Document 6). In particular, Lys-phiK has been reported to have an antibacterial effect on staphylococci clinically isolated from cattle and humans (Non-Patent Document 7).

Japanese Patent No. 57240045

The present invention is described in S.I. hyicus, S.A. Chromogenes, S. simulans, S. Schleiferi or S.M. It is an object of the present invention to provide a composition for lysing ferlis and a composition for preventing or treating a disease caused by these.

The present inventors have previously used S.A. As a bacteriophage having a property of lysing aureus, a bacteriophage specified by accession number NITE BP-693 and accession number NITE BP-694 was found (Patent Document 1), and endolysin (Lys-phiSA012) produced by the bacteriophage was found. ) Has been successfully isolated and identified (Non-Patent Document 8).

This time, when the bacteriophage and endolysin were used in combination, S. hyicus, S.A. Chromogenes, S. simulans, S. Schleiferi or S.M. The lytic activity on feris was detected, and the combined effect of these was evaluated. As a result, surprisingly, it was clarified that synergistically higher lytic activity was exhibited by using each of them in combination as compared with the case of using each of them alone.

The present invention has been completed based on such findings, and has the following configuration.
<1> Containing at least one staphylococcus selected from the group consisting of the staphylococcus specified by accession number NITE BP-693 and the staphylococcus specified by accession number NITE BP-694, and endolysin as active ingredients. However, the endolysin is at least one protein selected from the group consisting of the following (a) to (c), Staphylococcus hycus, Staphylococcus chromogenus, Staphylococcus simulation, and Staphylococcus. Composition for lysing at least one staphylococcus selected from the group consisting of Philococcus shreiferi and Staphylococcus ferris (a) Protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 (b) SEQ ID NO:: Protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in 1 (c) In the amino acid sequence shown in SEQ ID NO: 1, one or more amino acids are substituted, deleted, added, and / or inserted. A protein consisting of the resulting amino acid sequence.
<2> Contains at least one staphylococcus selected from the group consisting of the staphylococcus specified by accession number NITE BP-693 and the staphylococcus specified by accession number NITE BP-694, and endolysine as an active ingredient. However, the endolysin is at least one protein selected from the group consisting of the following (a) to (c), Staphylococcus hycus, Staphylococcus chromogenus, Staphylococcus simulation, and Staphylococcus. Composition for preventing or treating a disease caused by at least one staphylococcus selected from the group consisting of Philococcus shreiferi and Staphylococcus ferris (a) Protein consisting of the amino acid sequence set forth in SEQ ID NO: 1. (B) Protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1 (c) One or more amino acids are substituted or deleted in the amino acid sequence shown in SEQ ID NO: 1. A protein consisting of an added and / or inserted amino acid sequence.
<3> The disease is skin infection, urinary tract infection, blood infection, bone / joint infection, genital infection, breast infection, soft tissue infection, ear nose infection, wound infection due to the staphylococcus. The composition according to <2>, which is a disease, respiratory infection, eye infection, central nervous system infection or food poisoning.
<4> The composition according to <2> or <3>, which is an injectable composition, an oral composition, or an external composition.
<5> The composition according to <2> or <3>, which is a composition for external use and is administered to a subject by spraying, dipping, spreading or dropping.

According to the present invention, S. hyicus, S.A. Chromogenes, S.A. simulans, S.M. Schleiferi or S.M. It is possible to lyse ferlis and, by extension, prevent or treat diseases caused by the bacteria. In particular, when bacteriophage and endolysin, which are the active ingredients in the present invention, are used in combination, they exhibit high lytic activity against these staphylococci, and the bacteria can be sterilized efficiently. Therefore, for example, for various infectious diseases (epidermal infection, food poisoning, pneumonia, endocarditis, meningitis, arthritis, sepsis, mastitis, etc.) caused by infection of mammals and birds with these staphylococci. Therefore, the present invention can provide effective preventive agents, therapeutic agents and the like.

Bacteriophage, if a corresponding host exists, infects that host and proliferates explosively. However, bacteriophage cannot grow alone in the absence of a host. Therefore, if the particular host is completely lysed and killed by the bacteriophage, the bacteriophage cannot grow either. In addition, it does not affect other bacteria or the like by infecting them. Even in endolysin, if a specific bacterium does not exist, it does not affect other bacteria or the like. Therefore, it is not necessary to perform any treatment other than bacteriophage after performing the lysis treatment on the staphylococcus using the composition of the present invention. From the above points, the present invention also has an advantage that it is safe and easy to use without adversely affecting the environment and other bacteria.

S. It is a graph which showed the result of having evaluated the lytic activity with bacteriophage φSA012 and / or endolysin Lyas-phiSA012 with respect to a hyicus strain. In the figure, the vertical axis shows the absorbance of the staphylococcal culture solution at 660 nm 12 hours after the addition of the bacteriophage and / or endolysin. The amounts of bacteriophage and endolysin added are as shown in "φMOI" and "Lys" in the figure. "Mock" indicates the result in the condition that bacteriophage and endolysin are not added. S. It is a graph which showed the result of having evaluated the lytic activity with bacteriophage φSA012 and / or endolysin Lyas-phiSA012 with respect to a chromogenes strain. The notation in the figure is the same as in FIG. 1A. S. It is a graph which showed the result of having evaluated the lytic activity with bacteriophage φSA012 and / or endolysin Lyas-phiSA012 with respect to a simulans strain. The notation in the figure is the same as in FIG. 1A. S. It is a graph which showed the result of having evaluated the lytic activity with bacteriophage φSA012 and / or endolysin Lyas-phiSA012 with respect to a schleiferi strain. The notation in the figure is the same as in FIG. 1A. S. It is a graph which showed the result of having evaluated the lytic activity with bacteriophage φSA012 and / or endolysin Lyas-phiSA012 with respect to a feris strain. In the figure, the vertical axis shows the absorbance of the staphylococcal culture solution at 660 nm 15 hours after the addition of the bacteriophage and / or endolysin. Other notations in the figure are the same as in FIG. 1A. S. It is a graph which showed the result of having evaluated the lytic activity with bacteriophage φSA039 and / or endolysin Lyas-phiSA012 with respect to a hyicus strain. The notation in the figure is the same as in FIG. 1A. S. It is a graph which showed the result of having evaluated the lytic activity with bacteriophage φSA039 and / or endolysin Lyas-phiSA012 with respect to a chromogenes strain. The notation in the figure is the same as in FIG. 1A. S. It is a graph which showed the result of having evaluated the lytic activity with bacteriophage φSA039 and / or endolysin Lyas-phiSA012 with respect to a simulans strain. The notation in the figure is the same as in FIG. 1A. S. It is a graph which showed the result of having evaluated the lytic activity with bacteriophage φSA039 and / or endolysin Lyas-phiSA012 with respect to a schleiferi strain. The notation in the figure is the same as in FIG. 1A. S. It is a graph which showed the result of having evaluated the lytic activity with bacteriophage φSA039 and / or endolysin Lyas-phiSA012 with respect to a feris strain. In the figure, the vertical axis shows the absorbance of the staphylococcal culture solution at 660 nm 10 hours after the addition of the bacteriophage and / or endolysin. Other notations in the figure are the same as in FIG. 1A. S. It is a graph which showed the result of having evaluated the lytic activity with bacteriophage φI4α and / or endolysin Lyas-phiSA012 with respect to a hyicus strain. In the figure, the vertical axis shows the absorbance of the staphylococcal culture solution at 660 nm 20 hours after the addition of the bacteriophage and / or endolysin. Other notations in the figure are the same as in FIG. 1A. S. It is a graph which showed the result of having evaluated the lytic activity with bacteriophage φI4α and / or endolysin Lyas-phiSA012 with respect to the chromogenes strain. The notation in the figure is the same as in FIG. 1A. S. It is a graph which showed the result of having evaluated the lytic activity with bacteriophage φSAI4α and / or endolysin Lyas-phiSA012 with respect to a simulans strain. The notation in the figure is the same as in FIG. 1A. S. It is a graph which showed the result of having evaluated the lytic activity with bacteriophage φI4α and / or endolysin Lyas-phiSA012 with respect to a schleiferi strain. The notation in the figure is the same as in FIG. 1A.

As shown in the examples below, the combined use of bacteriophage and endolysin known to have lytic activity against Staphylococcus aureus includes Staphylococcus hycus, Staphylococcus chromodinas, Staphylococcus. -It was revealed that the lytic activity against Simulance, Staphylococcus shreiferi or Staphylococcus ferris was synergistically higher than when each was used alone. Therefore, the present invention relates to the prevention or treatment of diseases caused by staphylococci, which are lysed by using bacteriophage and endolysin, which will be described later, in combination.

(Staphylococcus)
Staphylococcus hyicus, Staphylococcus chromogenes, Staphylococcus chromogenes, S. chromogenes, Staphylococcus simulations Staphylococcus schleifferi (Sstaphylococcus schleiferi) and Staphylococcus ferris (S.felis) are all staphylococcus eubacteria belonging to the staphylococcus genus (Staphylococcus genus). It is one kind. In addition, "lysing" means that the cell membrane and cell wall of a bacterium are destroyed by bacteriophage and endolysin, the cytoplasm is released (eluting) to the outside of the cell, and the bacterium is killed.

(Bacteriophage)
One of the bacteriophages used in the present invention was dated December 25, 2008, and was the Patented Microorganism Depositary Center, Biotechnology Headquarters, National Institute of Technology and Evaluation (Postal code 292-0818, Kazusakamatari, Kisarazu City, Chiba Prefecture, Japan). It is a bacteriophage deposited under the accession number NITE BP-693 on foot 2-5-8). The present inventors have named this bacteriophage "φSA012".

The other bacteriophage according to the present invention is Kazusakamatari, Kisarazu City, Chiba Prefecture, Japan, on December 25, 2008, National Institute of Technology and Evaluation, Biotechnology Headquarters, Patented Microbial Deposit Center (postal code 292-0818). It is a bacteriophage deposited under the accession number NITE BP-694 in 2-5-8). The present inventors have named this bacteriophage "φSA039".

Observation using a transmission electron microscope revealed that both φSA012 and φSA039 are about the same size as T4 phage and slightly longer. Further, since φSA012 and φSA039 have a contractile sheath, it is presumed that they belong to the Myoviridae family. The family Myoviridae is based on the classical classification, and the family has recently been reorganized into the family Myoviridae, Podoviridae, Siphoviridae, Ackermanviridae, and Herelleviridae based on the genetic classification. In such a reorganized classification, both φSA012 and φSA039 belong to the family Herelleviridae. For other structures and properties of the bacteriophage according to the present invention, refer to the description in Patent Document 1.

The bacteriophage according to the present invention can be obtained by making a request for sale to the above depository institution where these are deposited. In addition, sewage running water of ordinary households, wastewater collected from sewage treatment facilities, etc., or S.I. Obtained from biological samples such as blood, nasal cavity, pharynx, skin, and intestinal tract derived from animals infected with aureus by, for example, separation and purification by a conventional method such as the plaque assay / separation method described in Patent Document 1. You can also do it. More specifically, a sample such as waste water is centrifuged at 10,000 to 15,000 rpm for about 10 to 15 minutes to remove impurities, and then, for example, PEG6000 and NaCl are added to precipitate phage particles, and then further 10,000 to 10000. Bacteriophage is collected by a conventional method of centrifuging at 15,000 rpm for about 10 to 15 minutes. The recovered bacteriophage and fully cultured S. cerevisiae. Mix aureus with soft agar and spread on agar medium. After culturing at 27 to 37 ° C. for 6 hours to 24 hours, the detected single plaque on the soft agar medium is collected, and for example, a density gradient centrifugation such as a conventional plaque formation method, filtration, or cesium chloride (CsCl) density gradient centrifugation is performed. Bacteriophage can be isolated by conventional methods of isolation. Further, the bacteriophage may be purified by a known method such as ultracentrifugation or ultrafiltration. Further, the bacteriophage according to the present invention can be grown by a general bacteriophage growth method such as the plate lysate method described in Patent Document 1.

The mode of the bacteriophage contained in the composition of the present invention is not particularly limited, and examples thereof include isolated and / or purified bacteriophage.

(End Lysin)
The endolysin according to the present invention is described in S.I. It is a peptidoglycan hydrolase that has an activity of cleaving cell wall peptidoglycan of staphylococci such as aureus and exhibits lytic activity against the bacterium. It is a multi-domain enzyme composed of an N-terminal cysteine-histidine-dependent amidohydrolase / peptidase (CHAP) domain, an amidase (AMID) domain, and a C-terminal SH3b cell wall binding domain, and more specifically, "SEQ ID NO:: "Protease consisting of the amino acid sequence according to 1" can be mentioned.

The protein is an endolysin produced by a bacteriophage (φSA012) deposited under the accession number NITE BP-693, and is named "Lys-phiSA012" by the present inventors (see Non-Patent Document 8). About). Lys-phiSA012 (the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence described in GenBank Accession No. BAO47098.1) is the genomic DNA of phiSA012 (the DNA sequence described in GenBank Accession No. AB903967.1). It is encoded by 27339-28826 (ORF51).

In addition, at the current state of the art, if a person skilled in the art obtains sequence information of a DNA encoding Lys-phiSA012, he / she makes various modifications to the DNA sequence to obtain lytic activity against various staphylococci. It is possible to produce a mutant gene encoding a protein having. Furthermore, even in nature, mutations in DNA sequences are possible.

Therefore, as long as the endolysin according to the present invention has the lytic activity, from the amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 1. Protein is also included.

Here, "plurality" generally means 100 amino acids or less (for example, 90 amino acids or less, 80 amino acids or less, 70 amino acids or less), preferably 60 amino acids or less (for example, within 90 amino acids) in the entire amino acid sequence of endolysin according to the present invention. 50 amino acids or less, 40 amino acids or less), more preferably 30 amino acids or less (for example, 20 amino acids or less, 10 amino acids or less), particularly preferably several amino acids or less (for example, 5 amino acids or less, 3 amino acids or less, 2 amino acids or less) Is.

Note that the introduction of a mutation into an amino acid sequence can be performed, for example, through the introduction of a mutation into a DNA sequence encoding the amino acid sequence. Mutation introduction into such a DNA sequence is carried out by a known method such as the Kunkel method or the Gapped duplex method, or a method similar thereto, for example, a mutation introduction kit using a site-specific mutagenesis method (for example, Mutant-K (TAKARA)). It can be performed using (manufactured by TAKARA), Mutant-G (manufactured by TAKARA), etc., or by using the LA PCR in vitro Mutagenesis series kit of TAKARA.

Furthermore, at the current state of the art, those skilled in the art can use the sequence information of a specific DNA to obtain a homologous gene encoding a protein having each activity from the same species or other phages. It is possible to separate. Examples of phages for acquiring such homologous genes include phages belonging to the family Myobilide (based on the classical classification. In the reorganized classification, phages belonging to the family Herelleviridae).

Methods for obtaining homologous genes include hybridization techniques (Southern, EM, J. Mol. Biol., 98: 503, 1975) and polymerase chain reaction (PCR) techniques (Saiki, RK. , Et al. Science, 230: 1350-1354, 1985, Saiki, RK et al. Science, 239: 487-491, 1988) and the like. In order to isolate a homologous gene, a hybridization reaction is usually carried out under stringent conditions. The stringent hybridization condition is, for example, about "1XSSC, 0.1% SDS, 37 ° C.", and the stricter condition is about "0.5XSSC, 0.1% SDS, 42 ° C.". As a more severe condition, there is a condition of about "0.2XSSC, 0.1% SDS, 65 ° C.". As described above, the more severe the hybridization conditions, the higher the identity of DNA can be expected to be isolated. However, the above combination of SSC, SDS and temperature conditions is an example, and the required stringency can be realized by appropriately combining the concentration of DNA, the length of DNA, the reaction time of hybridization and the like. be.

The protein encoded by the acquired homologous gene usually has high identity with the amino acid sequence shown in SEQ ID NO: 1. High identity means, for example, 80% or more, preferably 85% or more, more preferably 90% or more (for example, 91% or more, 92% or more, 93% or more, 94% or more), still more preferably 95% or more (for example, 91% or more, 92% or more, 93% or more, 94% or more). For example, 96% or more, 97% or more, 98% or more, 99% or more) sequence identity. Sequence identity can be determined by using BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information (basic local alignment search tool of the US National Center for Biological Information)) (for example, default, that is, default parameter). Can be determined (using).

Therefore, the endolysin according to the present invention also contains a protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1 as long as it has the lytic activity.

Whether or not a mutant, homologue, or the like of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 has lytic activity against various staphylococci is determined by, for example, a lytic activity test as shown in Examples described later. It can be determined by doing. Specifically, the mutant or the like is added to the bacterial culture solution, a change in the absorbance in the culture solution is detected, and if a decrease in the absorbance is observed after the addition, the mutant or the like is removed. It can be judged that it has lytic activity against various staphylococci. Those skilled in the art can appropriately set various conditions in such a test.

Further, as for the endolysin according to the present invention, if a person skilled in the art, the DNA encoding the endolysin is placed on an appropriate vector, and host cells such as Escherichia coli, animal cells, insect cells, and plant cells, or cell-free protein synthesis. It can be expressed as a recombinant protein by introducing it into a system (for example, reticulated erythrocyte extract, wheat germ extract). Furthermore, the recombinant protein expressed in a host cell or the like can be purified by a known peptide purification method. For example, it can be purified by chromatography (for example, ion exchange affinity chromatography, size exclusion column chromatography, affinity chromatography using an antibody that specifically recognizes endolysine according to the present invention), centrifugation or the like. Further, as a known method for purifying a recombinant protein expressed in a host cell or the like, the endolysin is used in a form in which a functional protein such as glutathione-S-transferase (GST) protein or His-tag protein is fused. Examples thereof include a method of synthesizing and purifying by binding to a GST-affinitive resin and a metal chelate resin (Smith, MC et al., J. Biol. Chem. 263, 7211-7215 (1988)). Further, for example, by cleaving between the functional protein and the endolysin with thrombin, blood coagulation factor Xa, or the like, only the endolysin can be separated and purified.

The endolysin according to the present invention can also be prepared by chemically synthesizing it with a commercially available polypeptide synthesizer based on its amino acid sequence. Further, the endolysin according to the present invention is S. lysin infected with φSA012. It can also be prepared from aureus culture or the like by the above-mentioned known peptide purification method.

(Composition for lysis)
In the present invention, S. cerevisiae containing the above-mentioned bacteriophage and endolysin as active ingredients. hyicus, S.A. Chromogenes, S.A. simulans, S.M. Schleiferi and S.M. Provided is a composition for lysing at least one staphylococcus selected from the group consisting of feris (hereinafter, also referred to as “composition for lysis”). More specifically, the active ingredient is at least one bacteriophage and endolysine selected from the group consisting of the bacteriophage identified by accession number NITE BP-693 and the bacteriophage identified by accession number NITE BP-694. Composition (a) for lysing the staphylococcus, wherein the endolysin is at least one protein selected from the group consisting of the following (a) to (c). Protein consisting of the amino acid sequence described (b) Protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 1 (c) One or more in the amino acid sequence set forth in SEQ ID NO: 1. The present invention provides a protein consisting of an amino acid sequence in which amino acids are substituted, deleted, added, and / or inserted.

In addition, when the composition of the present invention can be used in combination, and as a result, the staphylococcus is lysed, the bacteriophage and endolysin according to the present invention are contained in two or more kinds of compositions. It can also exist separately.

The bacteriophage according to the present invention contained as an active ingredient in the composition of the present invention is as described above, but the concentration thereof is not particularly limited and can be appropriately adjusted by those skilled in the art according to the mode of use thereof. .. For example, when used in a liquid dosage form (liquid antibacterial agent, etc.) described later, it is preferably 1 × 10 ³ to 1 × 10 ¹³ PFU / ml (PFU: plqueforming unit plaque forming unit). It is more preferably 1 × 10 ⁵ to 1 × 10 ¹¹ PFU / ml, and even more preferably 1 × 10 ⁸ to 1 × 10 ¹⁰ PFU / ml.

Further, the bacteriophage contained in the composition of the present invention may be one kind (strain, straight), and may further contain other bacteriophages other than the bacteriophage according to the present invention. The other bacteriophage is not particularly limited, and examples thereof include bacteriophage against bacteria different from the staphylococcus according to the present invention (for example, E. coli, P. aeruginosa). also. It may be not only lytic but also lysogenic bacteriophage.

The endolysin according to the present invention contained as an active ingredient in the composition of the present invention is as described above, but the concentration thereof is not particularly limited and can be appropriately adjusted by those skilled in the art according to the mode of use thereof. .. For example, when used in a liquid dosage form (liquid antibacterial agent, etc.) described later, it is preferably 0.16 to 500 μg / ml, more preferably 0.16 to 50 μg / ml1. It is more preferably .6 to 50 μg / ml.

Further, the endolysin contained in the composition of the present invention may be one kind or may further contain other endolysin other than the endolysin according to the present invention. The other endolysin is not particularly limited, and examples thereof include Lys-phiK, Lys-GH15, and Lys-phiTort (see Non-Patent Documents 6 and 7).

The composition for lysis of the present invention is S.I. hyicus, S.A. Chromogenes, S.A. simulans, S.M. Schleiferi and S.M. Since any staphylococcus of feris can be lysed, it can take the form of a pharmaceutical composition. The pharmaceutical composition includes a pharmaceutical composition for preventing or treating a disease caused by the staphylococcus (therapeutic agent, a preventive agent, a symptom-improving drug, etc.), and a pharmaceutical composition for killing or killing the staphylococcus. Items (disinfectants and the like, for example, pharmaceuticals such as disinfectants, quasi-drugs such as medicated soap) can be mentioned.

The lytic composition of the present invention is not limited to the pharmaceutical composition, but can also be used for the prevention or improvement of diseases caused by the staphylococcus in the form of food. Such food and drink compositions include, for example, foods for specified health use, foods with nutritional function and foods with functional claims, as well as animal feeds, nutritional supplements, health supplements, nutritionally prepared foods, and sick people. It can be a food product or a food additive.

In addition, the lytic composition of the present invention is an embodiment of a composition for reducing the number of staphylococci (disinfectant, etc.) and a composition for suppressing the growth of staphylococci (antibacterial agent, etc.). It can be taken. Furthermore, the lytic composition of the present invention may also take the form of a composition (reagent or the like) for lysing the staphylococcus for research purposes (for example, in vitro or in vivo experiments).

The form of the lytic composition of the present invention can be appropriately changed depending on the target (administration target, etc.), and for example, solid, semi-solid, and liquid are adopted.

For example, when the lytic composition of the present invention is used as a pharmaceutical composition, a sterilizing agent, an antibacterial agent, etc., it is prepared in a form (formulation form, dosage form) suitable for the subject. Examples of the dosage form include an oral composition (internal composition), an external composition, an injectable composition (including a breast injection composition and the like), and more specifically, as an oral composition. Tablets, chewable tablets, effervescent tablets, troches, drops, capsules (hard capsules, soft capsules), large pills, granules, powders, pills, granules, powders, dry syrups, dipping / decoctions , Solid agents such as liposome preparations, semi-solid agents such as licking agents, jelly agents and chewing gum agents, liquid agents such as syrup agents, drink agents, suspending agents, oil agents, emulsions and alcoholic preparations. The external composition includes solid preparations such as suppositories, poultices and plasters, ointments, creams, mousses, sheet preparations, resin preparations, gel preparations (Nazar gel preparations, etc.), inhaler preparations and the like. Semi-solid preparations, nasal drops, eye drops, liniments, lotions, sprays, aerosols, spot-on preparations, pore-on preparations, transdermal patches, sprays, suspensions, oils, emulsions and other liquids. Be done. Further, the liquid agent may take a form such as a woven fabric, a knitted fabric, a non-woven fabric or the like contained by a method such as immersion. Further, the disinfectant, the antibacterial agent and the like may also take the form of a wettable powder, a flowable agent, a heat transpiration agent, a fuming agent, a smoking agent, a ULV agent, a pelleting agent and the like.

These dosage forms can be appropriately prepared by those skilled in the art using pharmacologically acceptable auxiliary components (mediums and carriers (solid carriers, liquid carriers, gaseous carriers, etc.)). Auxiliary components include, for example, an aqueous medium such as a buffer solution (phosphate buffer solution, etc.), physiological saline, distilled water, a non-aqueous medium such as polyethylene glycol, propylene glycol, ethyl oleate, a surfactant, a vegetable oil, and a solvent. , Bases, emulsifiers, suspensions, matrix-forming agents, excipients, vehicles, thickeners, pressure-sensitive agents, spreading agents, binders, diluents, lubricants, protective agents, viscosity accelerators, wicking Agents, stabilizers, buffers, tonicity agents, chelating agents, disintegrants, pH regulators, solubilizers, wetting agents, colorants, dyes, pigments, flavoring agents, fragrances, flavoring agents, sweeteners, Commonly used substances such as flavoring agents, preservatives, thickeners, preservatives, and antioxidants can be mentioned. In addition, these auxiliary components must be compatible with the above-mentioned bacteriophage and endolysin which are active ingredients.

The lytic composition of the present invention may further contain other drugs, or may be used in combination. When used in combination, they may be administered at the same time or before or after. The drug can also be added to the bacteriophage and / or endolysin described above.

The "other agent" is not particularly limited as long as it does not lose the lytic activity of the bacteriophage and endolysin according to the present invention against the staphylococcus, but is not particularly limited, but is an antibacterial agent and an antiinflammatory agent. , Immunosuppressants, infectious disease preventive agents, infectious disease therapeutic agents, insecticides, anthelmintic agents, antifungal agents, antiviral agents, etc. Can be used.

Examples of the "antibacterial agent" include β-lactams, aminoglycosides, lincomycins, fosfomycins, tetracyclines, chloramphenicols, macrolides, ketrides, polypeptides, glycopeptides, and streptogramins. Systems, quinolones, and oxazolidinones are examples, but are not limited to these.

Examples of the "anti-inflammatory agent" include steroid agents and non-steroidal agents. Examples of the "steroid agent" include corticosteroids, and more specifically, prednisolone, beclomethasone, betamethasone, fluticasone, dexamethasone, hydrocortisone, prednisolone, mometasone and derivatives thereof. Not limited. Examples of the "non-steroidal agent" include, but are not limited to, salicylic acid-based, propionic acid-based, acetic acid-based, oxime-based, pyrin-based, non-pyrin-based compounds, and COX-2 inhibitors.

Examples of the "immunosuppressant" include, but are not limited to, antimetabolites, alkylating agents, microtubule synthesis inhibitors, lymphocyte signal transduction inhibitors, cytokine inhibitors, and antibodies.

(Lysis method)
In the present invention, S. hyicus, S.A. Chromogenes, S.A. simulans, S.M. Schleiferi or S.M. The lysis of ferlis (sterilization, sterilization, suppression of growth, etc.) is not particularly limited, and the bacteriophage and endolysin according to the present invention, or a composition for lysis containing them as active ingredients, is to be subjected to the lysis treatment. It can be done by contacting the above.

That is, a method for lysing the staphylococcus in the subject by contacting the bacteriophage and endolysin according to the present invention or the lytic composition of the present invention with the subject is provided.

The "target" is not particularly limited, and in addition to the animals exemplified in the following (treatment method, etc.), substances and environments in which any of the above-mentioned staphylococcus may be present (for example, food, food processing / cooking, etc.) Equipment and equipment used for storage, food processing factories, environments to which food is exposed, breeding facilities for mammals (pigs, etc.), breeding facilities for birds (chickens, etc.), etc. can be mentioned.

The "contact" method is not particularly limited, and for example, the bacteriophage and endolysin according to the present invention can be brought into contact with the subject by spraying, dipping, spreading, dropping, etc., which will be described later. The contact time with the subject is not particularly limited, and the bacteriophage and endolysin according to the present invention, or the lytic composition of the present invention lyse the staphylococcus in the subject, and the staphylococcus grows. It suffices if the time is sufficient to prevent the above.

Further, the lysis method of the present invention is to use the bacteriophage and the endolysin according to the present invention in combination, and the bacteriophage according to the present invention and the endolysin according to the present invention may be brought into contact with each other independently. That is, the bacteriophage according to the present invention and the endolysin according to the present invention may be brought into contact with each other at the same time or separately, and the contact order and the number of contacts may be different from each other.

(Treatment method, etc.)
The combined use of bacteriophage and endolysin according to the present invention is described in the examples described below in S. cerevisiae. hyicus, S.A. Chromogenes, S.A. simulans, S.M. Schleiferi and S.M. It exhibits high lytic activity against any staphylococcus of feelis and can kill the bacterium with high efficiency. Therefore, by using the bacteriophage and endolysin according to the present invention in combination, it is possible to prevent, treat or ameliorate the diseases caused by these staphylococci.

Therefore, the present invention prevents or treats diseases caused by the staphylococcus of the subject by administering the bacteriophage and endolysin according to the present invention, or a pharmaceutical composition containing them as an active ingredient, to the subject. It also provides a method.

The "target" for treatment or the like is not particularly limited as long as any of the staphylococci can be infected, and may be a human or a non-human animal. Non-human animals include livestock, pets and laboratory animals, more specifically pigs, cows, horses, sheep, goats, monkeys, rabbits, cats, dogs, minks, mice, rats, hamsters and guinea pigs. Examples include mammals such as guinea pigs and birds such as chickens.

The "disease caused by the staphylococcus" is not particularly limited, but examples thereof include staphylococcal infections, and more specifically, S. cerevisiae. hyicus, S.A. Chromogenes, S.A. simulans, S.M. Schleiferi or S.M. Skin infections (dermatitis (eg, edema dermatitis, buttery disease, toe aneurysm, toe bottom inflammation), epidermitis, folliculitis, pyoderma), urinary tract infections (cystitis, urinary tract infections) due to feels Etc.), blood infections (catheter-related fungalemia, mycemia, artificial joint infections, sepsis, septic polyarthritis, etc.), endocarditis, bone / joint infections (bone marrow / spondylitis (eg, suppuration) Myelitis, purulent spondylitis, bone fragility, sagging disease), arthritis, bone / arthritis, etc.), genital infections (vaginitis, uterine purulence, etc.) Soft tissue infection (soft tissue infection, etc.), ear and nose infection (ear inflammation, sinusitis, etc.), wound infection (wound infection, surgical wound infection, etc.), respiratory infection (airway infection, pneumonia, etc.), eye Infectious diseases (endophthalmitis, conjunctivitis, eye infections including eyelids and conjunctivitis, etc.), central nervous system infections (menelitis, etc.), food poisoning can be mentioned.

Examples of diseases caused by staphylococci in each animal are as follows.

In addition, the diseases caused by each staphylococcus are as follows.

The "literatures" shown in Tables 6 to 8 disclose the relationship between each staphylococcus and the disease caused by infecting each animal.

The method of "administration" of the bacteriophage and endolysin according to the present invention, or the pharmaceutical composition of the present invention containing them as an active ingredient is not particularly limited, and for example, transdermal administration, intradermal administration, subcutaneous administration, etc. Intravascular injection (intravenous administration, intraarterial administration), local administration (for example, local injection such as breast injection), oral administration, transmucosal (nasal injection, etc.) administration, intraperitoneal administration, intraairway administration, rectal administration and muscle Examples include oral administration and administration by infusion. Such administration can be carried out by appropriately adopting the above-mentioned dosage form. The dose is appropriately adjusted according to the type, age (week age), body weight, symptom, health condition, etc. of the subject (animal).

For example, when the pharmaceutical composition of the present invention is used as an external composition, as described above, although it depends on the type of the subject and the like, the external composition contains about 1 × 10 ⁶ to 1 × 10 ¹² PFU / ml. The bacteriophage according to the present invention and the endolysin according to the present invention of about 0.16 to 500 μg / ml can be contained and administered.

Examples of the method for administering the external composition include spraying, dipping, spreading, and dropping. More specifically, the immersion includes bathing of the target in the external composition or water containing the external composition. Examples of spraying include spraying, spraying, spraying, showering, and dusting (dusting) an external composition. For spreading, apply or wipe the external composition, or apply the external composition to strips, plates, bands, collars, earmarks, rim bands, labeling devices, etc. It is also possible to attach and disperse the external composition to the animal when the objects (animals) to which the molded product is attached come into contact with each other. Examples of the dropping include pore-on and spot-on on an external composition.

More specifically, when the external composition is applied or sprayed to a target, as described above, it depends on the type and condition of the target, but for example, 1 × 10 ⁴ to 1 × 10 ^{12 per 5 cm 2 of the affected area.} To the bacteriophage according to the present invention of about PFU (preferably about 1 × 10 ⁵ to 1 × 10 ¹² PFU) and the present invention of about 0.16 to 500 μg / ml (preferably about 0.16 to 50 μg / ml). A concomitant agent containing such endolysin may be applied or sprayed, but the present invention is not limited thereto.

Further, when the injection containing the bacteriophage of the present invention is locally injected, it varies depending on the type of the subject, the condition of the affected area, etc., but for example, about 1 × 10 ⁴ to 1 × 10 ¹² PFU (preferably 1 ×). A concomitant drug containing bacteriophage according to the present invention of about 10 ⁵ to 1 × 10 ¹² PFU) and endolysin according to the present invention of about 0.16 to 500 μg / ml (preferably about 0.16 to 50 μg / ml). Can be injected, but is not limited to this.

When the injection solution containing the bacteriophage and endolysin of the present invention is administered into the blood from a blood vessel such as the carotid artery, the bacteriophage of the present invention varies depending on the type of subject, the condition of the affected area, and the like. 1 × 10 ⁴ to 1 × 10 ¹² PFU / Kg, and the endolysin according to the present invention is about 0.16 to 500 μg / ml (preferably about 0.16 to 50 μg / ml). , Can be injected intravascularly, but is not limited to this.

Further, the therapeutic method of the present invention is to use the bacteriophage and endolysin according to the present invention in combination, and the bacteriophage according to the present invention and the endolysin according to the present invention may be administered independently. That is, the bacteriophage according to the present invention and the endolysin according to the present invention may be administered simultaneously or separately, and the order of administration and the number of administrations of each may be different.

Since phages and endolysin have high host specificity and show lytic activity only to host bacteria, they have almost no toxicity when administered to animals. Therefore, it is possible to administer a larger amount of bacteriophage depending on the type of subject, the condition of the affected area, and the like. For example, if the animal to be administered is a large animal, larger amounts of bacteriophage and endolysin can also be administered.

The present invention will be described in more detail with reference to Examples below, but the present invention is not limited to these Examples.

The present inventors have previously identified bacteriophage (φSA012) with accession number NITE BP-693 and bacteriophage specified with accession number NITE BP-694 as bacteriophages having the property of lysing Staphylococcus aureus. We have succeeded in isolating the phage (φSA039).

In this example, regarding these bacteriophages, in the test shown below, S. hyicus, S.A. Chromogenes, S.A. simulans, S.M. Schleiferi, S.M. The lytic activity on felts was evaluated. Furthermore, the combined effect of each bacteriophage and endolysin on the lytic activity against these staphylococci was also evaluated.

(1) Strain As a strain for examining bacteriolytic activity, staphylococcal exudative epidermitis pigs bred at multiple different locations stored at the Bioindustry Research Institute of Meiji Seika Pharma Co., Ltd. by a conventional method using mannitol agar medium Separated and selected Staphylococcus hycus strain, Staphylococcus chromogenes strain, Staphylococcus simulans strain, Staphylococcus shreiferi (S. Staphylococcus strains and Staphylococcus ferris strains were used.

(2) Bacteriophage Bacteriophage φSA012 (accession number NITE BP-693), φSA039 (accession number NITE BP-694) or φI4α (separated from S. simulans at Rakuno Gakuen University) in SM buffer solution (0.1 mM NaCl). _{, 8mM MgSO 4 · 7H 2 O} , 50mM Tris-HCl, and stirred with 0.1% gelatin), to obtain a suspension of bacteriophages.

(3) Endolysin Endolysin (Lys-phiSA012, a protein consisting of the amino acid sequence shown in SEQ ID NO: 1) was prepared by the method described in Non-Patent Document 8. More specifically, first, the DNA encoding Lys-phiSA012 was amplified, and a plasmid for expressing the endolysin in Escherichia coli was constructed. That is, using the DNA extracted from the bacteriophage φSA012 by a usual method as a template, the primers Lys012-1Fw (5'-TCCCCAGGATATTCCCATGGCTAGACTCAAGCAGA-3', SEQ ID NO: 2) and Lys012-495Rv (5'-CGCTCGAGTCGACCCTACTTGAACTGACTGATCCGCTACTCACTGAACTGAACTCAAGCAAGCAGA-3', SEQ ID NO: 2) The DNA encoding Lys-phiSA012 was amplified by PCR using 3: 3). The amplified fragment was purified and subcloned into pGEX-6P-2 (GE, Buckinghamshire, UK) using an In-Fusion HD cloning kit (Clontech, Palo Alto, CA, USA). Subcloning was performed with E. coli DH5α. For plasmid constructs, a DNA sequence analysis using the Big Dye Terminator V3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) and Applied Biosystems 3130 Genetic Analyzer containing DNA sequencer, Ly2 I confirmed that.

Next, Escherichia coli BL21 (DE3) was transformed using the obtained plasmid, and Lys was added by adding 0.1 mM isopropyl-β-thiogalactopyranoside (final concentration) (Nacalai Tesque, Kyoto, Japan). -Induced the expression of fiSA012. By inserting it into pGEX-6P-2, Lys-phiSA012 will be expressed in the form of a GST tag fused to its C-terminal side. Then, the E. coli was incubated in a bioshaker BR-40LF (Taitec, Saitama, Japan) at 25 ° C. with shaking overnight, centrifuged at 2300 xg at 4 ° C. for 5 minutes, and the supernatant was removed. HCl, 1M MgCl ₂ and 10% NP-40 (Wako, Osaka, Japan) were added to the pellet and the mixture was centrifuged using Bioruptor UCD-200 (Cosmo Bio, Tokyo, Japan). After centrifuging the mixture at 16,000 xg at 4 ° C. for 30 minutes, the supernatant containing the soluble protein was subjected to glutathione-filled Econo-Pac® chromatography columns (Bio-Rad Laboratories, Inc., Hercules, CA). , USA). At the time of the purification, the GST tag was excluded from the column by treating the column to which the GST tag fusion Lys-phiSA012 was bound with a protease for GST fusion protein cleavage (PreConfiguration Protein). Lys-phiSA012 was eluted. The protein thus purified was Lys-phiSA012, which was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protein concentration was also measured by SDS-PAGE. The obtained Lys-phiSA012 was stored at −30 ° C. until use.

(4) Evaluation of bacteriolytic activity 4 ml of LB medium was taken in an L-shaped test tube, and the above S. hyicus strain, S. cerevisiae. Chromogenes strain, S. cerevisiae. Simulans strain, S. Schleiferi strain or S. cerevisiae. The ferlis strain was inoculated with 40 μl of the day and night (16 hours) culture solution, and cultured at 37 ° C. with shaking at 40 rpm until the logarithmic growth phase was reached (OD660 = 0.1).

Next, the bacteriophage φSA012 or φSA039 is added to each strain culture solution thus prepared, and the MOI (Multiplicity of Infection: relative amount of the bacteriophage to the bacterial amount) becomes 10 ^-4 , 10 ^-3 or 10 ^-1 . Was added. In addition, endolysin Lys-phiSA012 was added to each of the above-mentioned strain culture solutions so as to have a concentration of 1.56, 3.125, or 6.25 μg / ml. Furthermore, in combination of these conditions, each bacteriophage and endolysin was added to each of the above-mentioned strain culture broths. Then, in this way, each strain culture solution to which bacteriophage and / or endolysin was added was cultured for 12 to 20 hours, and the bacteriolytic effect on each bacterium was measured using the absorbance at 660 nm as an index. The measurement was performed using TVS062CA Biophotocoder (Advantec, Tokyo, Japan).

The obtained results are shown in FIGS. 1A-1E, 2A-2E and 3A-3D. The results of evaluating the lytic activity of bacteriophage φSA012 (additional concentration: MOI 10 ^-4 or ^10-3 ) and / or endolysin Lys-phiSA012 (additional concentration: 1.56 or 3.125 μg / ml) are shown in S. .. hyicus strain, S. cerevisiae. Chromogenes strain, S. cerevisiae. Simulans strain, S. Schleiferi strain and S. cerevisiae. The feris strains are shown in FIGS. 1A to 1E, respectively. Evaluate the lytic activity of bacteriophage φSA039 (addition concentration: MOI 10 ^-4 , ^10-3 , or 10 ^-1 ) and / or endolysin (addition concentration: 1.56, 3.125, or 6.25 μg / ml) The result of S.I. hyicus strain, S. cerevisiae. Chromogenes strain, S. cerevisiae. Simulans strain, S. Schleiferi strain and S. cerevisiae. The feris strains are shown in FIGS. 2A to 2E, respectively. Also, the lytic activity of bacteriophage φI4α (additional concentration: MOI 10 ^-4 , ^10-3 , or 10 ^-1 ) and / or endolysin (additional concentration: 1.56, 3.125, or 6.25 μg / ml). The result of the evaluation of S.I. hyicus strain, S. cerevisiae. Chromogenes strain, S. cerevisiae. Simulans strain and S. The schleiferi strains are shown in FIGS. 3A to 3D, respectively.

As is clear from the results shown in FIGS. 1A to 1E and FIGS. 2A to 2E, the combined use of the bacteriophage φSA012 or φSA039 and the endolysin Lys-phiSA012 was compared with the case where these were added alone. It showed excellent lytic activity against hycius, S, chromogenes and the like. On the other hand, as shown in FIGS. 3A to 3D, such a combined effect was not observed for the bacteriophage φI4α.

As described above, according to the present invention, by using bacteriophage φSA012 or φSA039 in combination with endolysin, staphylococcus exhibits synergistically high bactericidal activity as compared with the case where each of them is used alone. Staphylococci such as Coccus hycus, Staphylococcus chromodinas, Staphylococcus simulation, Staphylococcus shreiferi and Staphylococcus ferris can be efficiently sterilized.

Therefore, the present invention is useful in eradication of these staphylococci, prevention or treatment of diseases caused by the bacteria, and the like.

(1) Identification display: 003 + φSA012
(2) Contract number: NITE BP-693
(3) Contract date: December 25, 2008 (4) Depositary organization: National Institute of Technology and Evaluation (1) Identification display: 0031 + φSA039
(2) Contract number: NITE BP-694
(3) Deposit date: December 25, 2008 (4) Depositary organization: National Institute of Technology and Evaluation

Claims (5)

1. It contains at least one bacteriophage selected from the group consisting of the bacteriophage specified by accession number NITE BP-693 and the bacteriophage specified by accession number NITE BP-694, and endolysin as an active ingredient. The endolysin is at least one protein selected from the group consisting of the following (a) to (c), Staphylococcus hycus, Staphylococcus chromodinas, Staphylococcus simulation, Staphylococcus. Composition for lysing at least one bacteriophage selected from the group consisting of Schleiferi and Staphylococcus ferris (a) Protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 (b) Set forth in SEQ ID NO: 1. Protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence of (c) Amino acid in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence shown in SEQ ID NO: 1. A protein consisting of a sequence.
2. It contains at least one staphylococcus selected from the group consisting of the staphylococcus identified by accession number NITE BP-693 and the staphylococcus identified by accession number NITE BP-694, and endolysine as an active ingredient. The endolysin is at least one protein selected from the group consisting of the following (a) to (c), Staphylococcus hycus, Staphylococcus chromodinas, Staphylococcus simulation, Staphylococcus. Composition for preventing or treating a disease caused by at least one staphylococcus selected from the group consisting of Schleiferi and Staphylococcus ferris (a) Protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 (b) Protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 1 (c) In the amino acid sequence set forth in SEQ ID NO: 1, one or more amino acids are substituted, deleted, added, and / Or a protein consisting of the inserted amino acid sequence.
3. Skin infection by at least one staphylococcus selected from the group consisting of Staphylococcus hycus, Staphylococcus chromodinas, Staphylococcus simulation, Staphylococcus shreiferi and Staphylococcus ferris. Diseases, urinary tract infections, blood infections, bone / joint infections, genital infections, breast infections, soft tissue infections, ear and nose infections, wound infections, respiratory infections, eye infections, central nervous system infections The composition according to claim 2, which is a disease or food poisoning.
4. The composition according to claim 2 or 3, which is an injection composition, an oral composition, or an external composition.
5. The composition according to claim 2 or 3, which is a composition for external use and is administered to a subject by spraying, dipping, spreading or dropping.

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