WO2023140723A1 - Bacteriophage kmsp1 specific to staphylococcus bacteria and antibacterial composition comprising same - Google Patents

Abstract

The present invention relates to novel bacteriophage KMSP1 having the ability to specifically kill Staphylococcus bacteria, an antibiotic composition comprising the bacteriophage, a feed additive composition, a feed, a disinfectant or a detergent, and a method for the prevention or treatment of infectious diseases caused by Staphylococcus bacteria, comprising a step of administering the bacteriophage to a subject. The bacteriophage KMSP1 of the present invention exhibits broad host infectivity and lytic activity against various Staphylococcus bacteria and exhibits high physicochemical stability, and thus can replace existing antibiotics and be widely used as an antibiotic composition for preventing or treating Staphylococcus infection in the fields of food, livestock industry, fisheries, and medicine.

Description

Staphylococcus specific bacteriophage KMSP1 and antibacterial composition containing the same

The present invention relates to a novel bacteriophage KMSP1 having a specific killing ability for Staphylococcus bacteria, an antibiotic composition containing the bacteriophage, a feed additive composition, a feed, a disinfectant or cleaning agent, and a method for preventing or treating an infectious disease caused by Staphylococcus bacteria comprising the step of administering the bacteriophage to a subject.

Bacteriophage refers to a bacterium-specific virus that infects a specific bacterium to inhibit and inhibit the growth of the infected bacterium. In 1915, British microbiologist Frederic Twart first observed the lysis of Staphylococcus aureus, and in 1917, French bacteriologist Felix Thererell also observed the same phenomenon in red bacteria, and found out that this was caused by a virus (bacterial virus) that infects bacteria. Since then, viruses that infect bacteria have been named bacteriophages, meaning "to eat" "bacteria", and are also simply called "phages".

Phages are largely classified into two types: virulent phage, which repeats only the lytic cycle, and temperamental phage, which undergoes both lytic and lysogenic cycles.

In the case of phages in the lytic life cycle, 100 to 200 new phages are formed about 30 minutes after infection with bacteria, destroy the membrane of bacteria, come out and lyse. Since the growth rate of phages is much faster than the growth rate of bacteria, it can be an important factor in limiting the growth of bacteria. The attenuated phage inserts the genetic information of the phage into the gene of the host bacterium, so the shape of the phage disappears, but the host bacterium is not destroyed, and this state persists as long as the host bacterium is in favorable environmental conditions.

Since a specific bacteriophage has a characteristic of infecting only bacteria of a specific category and specifically killing them, the utilization of bacteriophage has recently attracted great attention as a countermeasure against bacterial diseases. In particular, since the increase in antibiotic-resistant bacteria after 2000, the limitations of existing antibiotics have been pointed out, and the possibility of utilizing bacteriophages as a substitute for existing antibiotics has been highlighted, and bacteriophages are attracting attention as anti-bacterial agents again.

Accordingly, cases of successful prevention and treatment of various diseases such as calf Escherichia coli diarrhea, various diseases of fish, vancomycin-resistant enterococcal sepsis, tuberculosis bacillus, chicken paratyphoid and Escherichia coli O157: H7 have been successfully prevented and treated using toxic phages. In addition to the direct use of phage, drugs specific to anthrax and pneumococci are being developed using lysin, which acts to specifically destroy the cell walls of host bacteria. .

However, many bacteriophages capable of controlling Staphylococcus, a pathogenic bacterium that causes serious diseases, in a wider host range and antibacterial agents using the same have not yet been reported, and there is a need for research on this.

Accordingly, while the present inventors were studying to isolate and identify bacteriophages having lytic activity specific to various Staphylococcus bacteria, the bacteriophages isolated from unsterilized crude milk showed broad antibacterial activity against Staphylococcus aureus , Staphylococcus intermedius ( S. intermedius ) and methicillin-resistant Staphylococcus aureus infection (MRSA) confirmed and completed the present invention.

An object of the present invention is to provide a novel bacteriophage KMSP1 having a specific killing ability for Staphylococcus bacteria and various antibiotic compositions containing the same.

In order to achieve the above object, the present invention provides a bacteriophage KMSP1 (accession number: KCTC18975P) having a specific killing ability for Staphylococcus bacteria.

In addition, the present invention provides an antibiotic composition comprising the bacteriophage KMSP1.

In addition, the present invention provides a composition for adding feed, including the bacteriophage KMSP1.

In addition, the present invention provides a feed comprising the composition for adding the feed.

In addition, the present invention provides a disinfectant containing the bacteriophage KMSP1.

In addition, the present invention provides a detergent containing the bacteriophage KMSP1.

In addition, the present invention provides a method for preventing or treating infectious diseases caused by Staphylococcus bacteria, comprising administering the bacteriophage KMSP1 to the subject.

Since the bacteriophage KMSP1 of the present invention exhibits broad host infectivity and lytic activity against various Staphylococcus bacteria and exhibits high physicochemical stability, it can be widely used in the food, livestock industry, fishery, and pharmaceutical fields as an antibiotic composition for preventing or treating Staphylococcus infection by replacing existing antibiotics.

1 is a diagram showing the results of confirming the morphological characteristics of bacteriophage KMSP1 through an electron microscope.

Figure 2 is a diagram showing the results of confirming the bacteriophage KMSP1's lytic activity against Staphylococcus aureus (control group (●), experimental group (■, ▲, ▼, ◆)).

Figure 3 is a diagram showing the results of confirming the thermal stability of the bacteriophage KMSP1.

Figure 4 is a diagram showing the results of confirming the pH stability of the bacteriophage KMSP1.

The present invention relates to a bacteriophage KMSP1 (accession number: KCTC18975P) having a specific killing ability for Staphylococcus bacteria and an antibiotic composition using the same.

Bacteriophage KMSP1 having a specific killing ability for Staphylococcus bacteria of the present invention has a very high specificity for Staphylococcus bacteria and is strong against physicochemical stimulation compared to conventional chemical antibiotics.

Hereinafter, the present invention will be described in detail.

The bacteriophage KMSP1 according to the present invention is a novel bacteriophage isolated from unsterilized crude oil and belongs to the Myoviridae family of complex type having a contractile long tail and is classified as a type 1 virus having double-stranded DNA according to the Baltimore classification. It is a bacteriophage.

The bacteriophage KMSP1 of the present invention may consist of the nucleotide sequence represented by SEQ ID NO: 1. The entire gene of bacteriophage KMSP1 is represented by 138,528 base sequences.

Thus, the bacteriophage KMSP1 may include the nucleotide sequence represented by SEQ ID NO: 1 as all or part of the entire gene. In addition, the bacteriophage KMSP1 of the present invention may consist of the nucleotide sequence represented by SEQ ID NO: 1 and a functional equivalent of the nucleotide sequence. The functional equivalent refers to a sequence having at least 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more sequence homology with the nucleotide sequence represented by SEQ ID NO: 1 as a result of modification or substitution of the nucleotide sequence, and exhibiting substantially the same physiological activity as the nucleotide sequence represented by SEQ ID NO: 1.

The present inventors named the new bacteriophage isolated from non-sterilized crude oil as KMSP1, and deposited it with the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center on January 6, 2022, and was given accession number KCTC18975P.

The bacteriophage KMSP1 of the present invention may exhibit a specific killing ability for Staphylococcus bacteria, and the Staphylococcus bacteria are preferably at least one selected from the group consisting of Staphylococcus aureus , Staphylococcus intermedius ( S. intermedius ) and methicillin-resistant Staphylococcus aureus infection (MRSA) However, it is not limited thereto. Since the bacteriophage KMSP1 of the present invention has the advantage of simultaneously infecting and killing Staphylococcus aureus , Staphylococcus intermedius , and methicillin-resistant Staphylococcus aureus, it can be used as a biological control agent for the prevention or treatment of various diseases caused by the bacteria, thereby reducing the use of antibiotics.

The bacteriophage KMSP1 of the present invention has excellent stability to heat and pH. More specifically, bacteriophage KMSP1 may exhibit thermal stability at 3 to 60 °C, preferably at 4 to 55 °C. In addition, the bacteriophage KMSP1 of the present invention can be characterized in that it exhibits excellent stability even in a wide pH range such as pH5 to pH11. That is, since the bacteriophage KMSP1 of the present invention is stably maintained in a wide range of temperatures and pH, it can be used in various environments.

Staphylococcus-specific lytic activity, acid resistance, base resistance and heat resistance as described above make it possible to utilize the bacteriophage KMSP1 of the present invention in various compositions for Staphylococcus bacteria. Accordingly, the present invention provides an antibiotic composition, a composition for feed additives, a feed, a disinfectant and a detergent containing the bacteriophage KMSP1 having a specific killing activity for Staphylococcus bacteria.

In the present invention, the "antibiotic composition" means a preparation that is provided in the form of a drug and can kill bacteria, and is a generic term for microbial preparations for killing bacteria, preservatives, bactericides, antibiotics and antibacterial agents.

Since the bacteriophage KMSP1 of the present invention has very high specificity for Staphylococcus bacteria, it does not kill beneficial bacteria, preferably Staphylococcus aureus , Staphylococcus intermedius ( S. intermedius ) and methicillin-resistant Staphylococcus aureus (MRSA), and does not induce drug resistance or resistance, and thus has a longer life cycling than conventional antibiotics. Can be used as an antibiotic.

The antibiotic composition according to the present invention can be formulated by a conventional microbial preparation method. Although not particularly limited, when administered to the human body, it is formulated within the pharmaceutically acceptable range, and when administered to livestock or fish, it is formulated within the veterinary acceptable range, and the administration route can be determined accordingly.

The antibiotic composition of the present invention may include a carrier, other auxiliary agents, etc. as necessary in addition to the bacteriophage KMSP1 of the present invention, which is an active ingredient.

In addition, the antibiotic composition of the present invention can be formulated into a liquid, semi-solid or solid formulation, such as solid formulations such as powders, granules, and tablets, liquid formulations such as suspensions or injection solutions, and semi-solid formulations such as creams, lotions, gels, and pastes.

The composition of the present invention can be administered in a conventional manner through routes such as oral, intravenous, intraarterial, intraperitoneal, intramuscular, transdermal, intranasal, inhalation or rectal.

The dosage of the composition of the present invention refers to the amount required to achieve the effect of killing Staphylococcus aureus, S. intermedius and/or methicillin-resistant Staphylococcus aureus in the subject to be administered. Therefore, it can be adjusted according to various factors including the type of disease, severity of disease, type of dosage form and age, body weight, general health condition, sex and diet of the subject to be administered, route of administration, time and period of administration, and drugs used concurrently.

In addition, the present invention provides a feed composition and feed containing a bacteriophage KMSP1 having a specific killing ability for Staphylococcus bacteria.

Antibiotics for feed additives used in livestock and fisheries are used for the purpose of preventing diseases, but the administration of antibiotics for the purpose of prevention increases the possibility of resistant bacteria and has a problem that antibiotics remaining in livestock can be transmitted to humans. When antibiotics are absorbed into the body through meat, they can lead to antibiotic resistance in humans. In addition, since there is a problem that the probability of occurrence of multi-drug resistant bacteria increases when the number of antibiotics mixed with feed increases, a new antibiotic for feed addition that is more nature-friendly and solves problems that may occur or may occur in the use of existing antibiotics is required, and the bacteriophage KMSP1 of the invention can be used.

In addition, the present invention can provide a feed containing the composition for adding feed, and the feed of the present invention can be prepared by separately preparing bacteriophages in the form of feed additives and mixing them with feed, or by directly adding them during feed preparation. The bacteriophage in the feed of the present invention may be in a liquid or dry state, preferably in a dry powder form. Drying methods include air drying, natural drying, spray drying and freeze drying, but are not limited thereto. The bacteriophage of the present invention may be mixed in a powder form at a component ratio of 0.05 to 10% by weight, preferably 0.1 to 2% by weight of the feed weight. In addition, the feed may further include conventional additives capable of increasing the preservability of the feed in addition to the bacteriophage of the present invention.

Other non-pathogenic microorganisms may be additionally added to the feed additive composition of the present invention. Microorganisms that can be added include Bacillus subtilis, which can produce proteolytic enzymes, lipolytic enzymes, and sugar converting enzymes, such as Bacillus subtilis ; It may be selected from the group consisting of molds such as filamentous fungi and yeasts such as Saccharomyces cerevisiae .

The feed containing the bacteriophage KMSP1 of the present invention includes grains, root fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, meal, grain by-products, etc. as vegetable, and proteins, inorganic materials, oils, mineral oils, single cell proteins, zooplankton, leftover food, etc., but is not limited thereto.

The feed additive composition of the present invention may include binders, emulsifiers, preservatives, etc. added to prevent quality deterioration, amino acids, vitamins, enzymes, probiotics, flavors, non-protein nitrogen compounds, silicate agents, buffers, coloring agents, extractants, oligosaccharides, etc. added to feeds to increase efficacy, and may further include feed mixtures.

In addition, in the present invention, the bacteriophage KMSP1 may be included in the drinking water additive.

The drinking water additive of the present invention can be used in a way in which the bacteriophage KMSP1 or a composition containing the same is separately prepared in the form of a drinking water additive and mixed with feed or drinking water, or directly added during preparation of drinking water. By mixing and supplying drinking water as described above, there is an effect of continuously reducing the number of Staphylococcus bacteria. In the present invention, drinking water is not particularly limited, and drinking water commonly used in the art may be used.

In addition, the present invention provides a disinfectant containing a bacteriophage KMSP1 having a specific killing ability for Staphylococcus bacteria.

Staphylococcus bacteria, preferably Staphylococcus aureus ( Staphylococcus aureus ), Staphylococcus intermedius ( S. intermedius ) and methicillin-resistant Staphylococcus aureus (MRSA) A disinfectant containing the bacteriophage KMSP1 of the present invention having a specific killing activity for at least one Staphylococcus aureus selected from the group consisting of can be usefully used as a disinfectant for hospitals and health to prevent hospital infection and can be used as a disinfectant for general life It can be used for disinfection of food and cooking places and facilities, disinfection of buildings such as poultry farms and barns, livestock, drinking water, litter, egg seats, transportation vehicles, and disinfection of various growing products such as tableware.

In addition, the present invention provides a cleaning agent containing a bacteriophage KMSP1 having a specific killing ability for Staphylococcus bacteria.

Since the bacteriophage KMSP1 of the present invention has a specific killing ability for at least one species selected from the group consisting of Staphylococcus aureus, Staphylococcus intermedius ( S. intermedius ) and methicillin-resistant Staphylococcus aureus (MRSA), the skin surface or body parts of livestock exposed or likely to be exposed to the Staphylococcus bacteria, or Staphylococcus bacteria It can also be used to wash various tableware and foods that require infection prevention.

In addition, the present invention has a bacterial pharmaceutical composition for preventing or treating infectious diseases caused by Starpilococcus, comprising a bacteriophygic diseases caused by staphylococcus bacteria, the present invention, the bacteriococcus having specific killing functions for the pharmaceutical composition for the treatment or treatment of the staphylococcus ( staphylococcus ) bacteria by Star Philocaccus. It provides a method of preventing or treating infectious diseases by Star Philocaccus bacteria comprising administering phage KMSP1 to individuals except humans.

Since the bacteriophage KMSP1 of the present invention has a specific killing ability for Staphylococcus bacteria, it can be used in a pharmaceutical composition for preventing or treating infectious diseases caused by Staphylococcus bacteria. In the present invention, Staphylococcus bacteria, preferably Staphylococcus aureus ( Staphylococcus aureus ), Staphylococcus intermedius ( S. intermedius ) and methicillin-resistant Staphylococcus aureus (MRSA) Infectious diseases caused by at least one selected from the group consisting of Staphylococcus aureus is a concept that collectively refers to an epidemic or acutely onset infectious disease.

In the present invention, the infectious disease is not limited thereto, but may be at least one selected from the group consisting of food poisoning, sepsis, inflammation, abscess, endocarditis, toxin syndrome, skin erythema, skin pruritus and skin pustules caused by infection with Staphylococcus bacteria, and any infectious disease caused by infection with the Staphylococcus bacteria can be applied without limitation thereto.

The pharmaceutical composition of the present invention contains 1 × 10 ³ to 1 × 10 ¹⁰ PFU/mL of bacteriophages, preferably 1 × 10 ⁶ to 1 × 10 ⁹ PFU/mL of bacteriophages. As used in the present invention, the term PFU (plaque forming unit) is a unit that quantifies the formation of plaques by bacteriophages.

The term prevention of the present invention refers to any action that suppresses or delays the onset of a disease by administering a composition.

The term treatment of the present invention refers to all activities in which the symptoms of the disease are improved or the disease is suppressed or alleviated and beneficially changed by administration of the composition.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier.

In the present invention, the term pharmaceutically acceptable carrier means a carrier or diluent that does not stimulate organisms and does not inhibit the biological activity and properties of the administered compound. In the composition formulated as a liquid solution, acceptable pharmaceutical carriers are sterile and biocompatible, and saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare formulations for injections such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.

In addition to oral administration, the pharmaceutical composition of the present invention can be used by parenteral administration, nasal spray, coating or spraying on diseased areas, and in the case of parenteral administration, intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration or local administration.

Formulations for oral administration containing the pharmaceutical composition of the present invention as an active ingredient may be formulated into, for example, tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs. For formulation into tablets and capsules, a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin, an excipient such as dicalcium phosphate, a disintegrant such as corn starch or sweet potato starch, a lubricant such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax, and in the case of a capsule formulation, in addition to the above-mentioned substances, a liquid carrier such as fatty oil It may contain more sieves.

Formulations for parenteral administration containing the pharmaceutical composition of the present invention as an active ingredient include injection forms such as subcutaneous injection, intravenous injection, or intramuscular injection, suppository injection method, or spray such as aerosol to enable inhalation through the respiratory tract. It can be formulated for. In order to formulate an injectable formulation, the composition of the present invention may be mixed in water with a stabilizer or buffer to prepare a solution or suspension, which may be formulated for unit administration in an ampoule or vial. When formulated for spraying, such as aerosols, propellants and the like may be blended with additives so that the concentrated concentrate or wet powder dispersed in water is dispersed.

Appropriate application, spraying and dosage of the pharmaceutical composition of the present invention vary depending on factors such as formulation method, administration method, age, weight, sex, severity of disease symptoms, food, administration time, administration route, excretion rate and response sensitivity of animals and patients to be targeted, and usually a skilled doctor or veterinarian can easily determine and prescribe an effective dosage for the desired treatment.

In the present invention, the term subject refers to any subject capable of developing an infectious disease caused by Staphylococcus bacteria, and includes mammals, including humans, livestock or poultry, but is not limited thereto.

The term livestock is a concept that refers to useful animals that have been domesticated and improved by humans and live together with humans, and include, for example, pigs, cows, chickens, horses, ducks, or dogs, but are not limited thereto.

The term poultry is a concept that collectively refers to animals belonging to birds among livestock, and includes, for example, chickens, ducks, pheasants, quails, ostriches, geese or turkeys, but is not limited thereto.

In the method of the present invention, the bacteriophage KMSP1 or the composition may be administered to animals in the form of pharmaceutical preparations, or may be administered by mixing with feed or drinking water of livestock and feeding them.

In the method of the present invention, the route of administration of the bacteriophage KMSP1 or the composition may be administered through various oral or parenteral routes as long as it can reach the target tissue, and specifically, oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, intranasal, inhalation, etc. may be administered in a conventional manner.

It is obvious to those skilled in the art that an appropriate total daily amount of the bacteriophage KMSP1 administered in the method of the present invention can be determined by a treating physician within the scope of sound medical judgment. The specific therapeutically effective amount for a specific individual is preferably applied differently depending on the type and degree of response to be achieved, the patient's age, weight, general health condition, sex and diet, administration time, administration route and secretion rate of the composition, treatment period, various factors including drugs used or simultaneously used with the specific composition, and similar factors well known in the medical field.

Terms not defined otherwise in this specification have meanings commonly used in the technical field to which the present invention belongs.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

Example 1: Separation and identification of novel bacteriophages

1.1 Isolation of bacteriophage

Non-sterilized raw milk was centrifuged at 8000 rpm for 4 minutes to obtain a supernatant, which was then mixed with a liquid medium. Staphylococcus aureus bacteria are inoculated into this mixed solution, cultured at 37° C., and the supernatant obtained after centrifuging the culture solution is filtered through a filter having a size of 0.4 μm to obtain a filtrate. A suspension of 0.1 mL of the filtrate and 0.1 mL of bacteria (staphylococcus aureus) cultured for more than 12 hours was extracted with a pipette and mixed. This mixture was mixed with low agar (0.4%) medium and poured onto a plate containing 1.5% agar medium. The plates were incubated overnight at 37°C.

When the bacteriophage appeared on the plate, an independent and transparent plaque was observed, and the plaque area was separated using a pipette tip, and then put into a new bacterial culture medium and cultured at 37°C. After confirming that the culture solution became clear, the culture solution was centrifuged, and only the supernatant was filtered through a 0.22 μm membrane filter to remove all bacteria remaining in the lysate, and only the filtrate was taken. This filtrate contains only bacteriophages, and the bacteriophages were separated by performing plaque re-separation three times in which the bacteriophage solution obtained by the above method was differently diluted and injected on an agar medium.

1.2 Observation and classification of bacteriophage morphology

In order to examine the morphological characteristics of the bacteriophage isolated in Example 1.1, the morphology was confirmed through an electron microscope. A bacteriophage sample was mounted on a copper grid coated with carbon, and negatively stained with 2% liquid uranyl acetate. The dyed sample was observed using an energy filtration transmission electron microscope (LIBRA 120, Carl Zeiss) at a voltage of 80 kV, and the results are shown in FIG. 1 .

As shown in FIG. 1, the isolated bacteriophage has a size of about 300 nm, an icosahedral 104.6 nm head, a contractile 197.7 nm long tail, and a sheath, so it is classified as belonging to the complex type Myoviridae.

In addition, since the bacteriophage of the present invention has double-stranded DNA and was cut by restriction enzyme treatment, it was classified as a type 1 virus having a double-stranded DNA genome according to the Baltimore classification.

The isolated and identified bacteriophage was named KMSP1, and deposited at the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center on January 06, 2022, and was given accession number KCTC18975P.

1.3 Genetic analysis of bacteriophage

Sequencing was performed to analyze the entire gene of the isolated and identified bacteriophage KMSP1. The bacteriophage genome for genetic analysis was isolated through a general phenol-chloroform method using a culture medium of about 10 ¹⁰ PFU/mL. The isolated bacteriophage genome was quality checked through agarose gel electrophoresis and then a library was produced. Whole gene analysis was performed by gene sequencing using Miseq 300 bp paired end (Illumina, USA) (Senigen, Korea). The quality of raw data was verified using FastQC v0.11.9, and adapter sequences and low quality reads were removed using Trimmomatic v0.39. Finally, as a result of de novo assembly using the SPAdes v.3.13.0 program, a nucleotide sequence consisting of one contig was obtained. The entire gene of bacteriophage KMSP1 consists of a total of 138,528 bases, and the identified sequence is shown in SEQ ID NO: 1.

Example 2: Confirmation of Staphylococcus infectivity of bacteriophage KMSP1

In order to confirm infectivity for various Staphylococcus aureus, Staphylococcus intermedius ( S. intermedius ) and methicillin-resistant Staphylococcus aureus, infectivity for a total of 12 bacteria was confirmed using a serial dilution dropping method (Adams, MH 1959. Bacteriophage, pp27-34. Interscience Publishers Inc., New York). After mixing the bacterial culture medium cultured for 10 hours and 0.4% soft agar medium, it was poured onto an LB agar plate and dried at 37 ° C. for 15 minutes, and then the bacteriophage KMSP1 diluted by dilution factor was dropped thereon, dried at room temperature, and then incubated at 37 ° C. for 10 hours. When the strain is sensitive to the phage, plaques are visible on the plate, which indicates that the bacterial cells are completely lysed and killed. If the sensitivity is weak, a clouded area may occur, and an inhibition area may occur when an antibacterial enzyme capable of inhibiting the growth of bacteria such as lysostaphin is present. Table 1 shows the results of confirming susceptibility to Staphylococcus aureus and methicillin-resistant Staphylococcus aureus isolated from various isolates.

[Table 1]

(+: lytic activity present, -; lytic activity absent)

As can be seen in Table 1, the bacteriophage KMSP1 broadly lysed various Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus intermedius to show plaques. This is a result showing that the bacteriophage KMSP1 of the present invention has a broad host infection range that simultaneously kills Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and Staphylococcus intermedius.

Example 3: Confirmation of Staphylococcus aureus killing ability of bacteriophage KMSP1

Bacterial killing ability of bacteriophage KMSP1 was investigated. In order to analyze the killing ability of bacteriophage KMSP1 against Staphylococcus aureus, the lytic activity was further confirmed by dividing into five test groups. The host strain Staphylococcus aureus (food isolate 1) was cultured for 2 hours in a liquid medium (absorbance = about 0.2), and the amount of bacteriophage KMSP1 was infected by varying the multiplicity of infection (M.O.I) 10, 1, 0.1, 0.01. After infection, an experiment was performed to determine the degree of bacterial growth and death. As a sample without bacteriophage solution, only Staphylococcus aureus (food isolate 1) was cultured in a liquid medium as a control. Incubation was performed for 12 hours, and the culture of bacteria was performed at an absorbance of 600 nm every hour, and the absorbance was confirmed, and the results are shown in FIG.

As shown in FIG. 2, it was confirmed that in the experimental group infected with the bacteriophage KMSP1 of the present invention, excellent bacteria killing ability was exhibited within 4 hours with only 1/100 of the phage amount compared to the number of bacteria.

Example 4. Confirmation of thermal stability of bacteriophage KMSP1

In order to confirm the thermal stability of the bacteriophage KMSP1, the number of bacteriophages was measured after standing for a certain period of time in various temperature ranges. More specifically, 1 mL of a bacteriophage KMSP1 solution of 1.0x10 ⁸ PFU/mL present in the buffer was exposed to various temperature ranges of 4 °C, 25 °C, 35 °C, 45 °C 55 °C, 65 °C and 75 °C for 1 hour. Then, each reaction solution was diluted step by step to measure the number of bacteriophages using a soft agar overlay method, and the results are shown in FIG. 3 .

As shown in Figure 3, it was confirmed that the bacteriophage KMSP1 of the present invention is stable for up to 1 hour in a wide temperature range of 4-55 ° C and is widely applicable in various temperature environments.

Example 5: Investigation of pH stability of bacteriophage KMSP1

In order to confirm the pH stability of the bacteriophage KMSP1, the number of bacteriophages was measured in various pH ranges. More specifically, 100 μL of a 1.0x10 ⁹ PFU/mL KMSP1 solution present in the buffer was mixed with 900 μL of each buffer adjusted to a range of pH (3, 4, 5, 6, 7, 8, 9, 10, 11, 12), and then exposed at 25° C. for 1 hour. Then, each reaction solution was diluted step by step and the number of bacteriophages was measured using a soft agar overlay method. The measurement results are shown in FIG. 4 .

As shown in Figure 4, bacteriophage KMSP1 showed excellent stability in a wide pH range of pH 5 to 11, it was confirmed that it is applicable in various pH environments.

From the above experiments, it was confirmed that bacteriophage KMSP1 exhibits infectivity against Staphylococcus aureus and Staphylococcus intermedius among Staphylococcus bacteria ( S. intermedius ), as well as exhibits excellent infectivity against methicillin-resistant Staphylococcus aureus (MRSA). In addition, it was confirmed that it exhibits high lytic activity and at the same time has high stability at various temperatures and pH, so it is resistant to physicochemical stimulation and can be used in various ways to prevent or treat various infectious diseases that can be caused by these bacteria.

In the above, specific parts of the present invention have been described in detail, to those skilled in the art, it will be clear that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Using the bacteriophage of the present invention and the antibacterial composition containing the same, it can contribute to the prevention or treatment improvement of infectious diseases in the fields of food, livestock, fisheries, and medicine.

[Accession number]

Depository Institution Name: Korea Research Institute of Bioscience and Biotechnology Biological Resources Center

Accession number: KCTC18975P

Entrusted date: 20220106

Depository Address: 181 Ipsin-gil, Jeongeup-si, Jeollabuk-do (Sinjeong-dong), Biological Resources Center, Korea Research Institute of Bioscience and Biotechnology, 56212, Republic of Korea

Claims (13)

1. Staphylococcus ( Staphylococcus ) Bacteriophage KMSP1 (accession number: KCTC18975P) having a specific killing ability for bacteria.
2. According to claim 1, wherein the bacteriophage KMSP1 consists of the nucleotide sequence represented by SEQ ID NO: 1, bacteriophage KMSP1.
3. According to claim 1, wherein the Staphylococcus aureus ( Staphylococcus aureus ), Staphylococcus intermedius ( S. intermedius ) and methicillin-resistant Staphylococcus aureus (methicillin-resistant Staphylococcus aureus infection, MRSA) At least one selected from the group consisting of, bacteriophage KMSP1.
4. The bacteriophage KMSP1 according to claim 1, wherein the bacteriophage KMSP1 has stability at pH5 to pH11.
5. The bacteriophage KMSP1 according to claim 1, wherein the bacteriophage KMSP1 has stability at 3 to 60 °C.
6. An antibiotic composition comprising a bacteriophage KMSP1 (accession number: KCTC18975P) having a specific killing activity on Staphylococcus bacteria.
7. Staphylococcus ( Staphylococcus ) Having a specific killing ability for bacteria, bacteriophage KMSP1 (accession number: KCTC18975P) containing a composition for addition to feed.
8. A feed comprising the composition for adding feed according to claim 7.
9. A disinfectant containing a bacteriophage KMSP1 (accession number: KCTC18975P) having a specific killing ability for Staphylococcus bacteria.
10. A detergent containing a bacteriophage KMSP1 (accession number: KCTC18975P) having a specific killing ability for Staphylococcus bacteria.
11. A method for preventing or treating infectious diseases caused by Staphylococcus bacteria, comprising the step of administering bacteriophage KMSP1 (accession number: KCTC18975P), which has a specific killing ability to Staphylococcus bacteria, to a non-human subject.
12. The method of claim 11, wherein the Staphylococcus aureus ( Staphylococcus aureus ), Staphylococcus intermedius ( S. intermedius ) and methicillin-resistant Staphylococcus aureus (MRSA) is one or more species selected from the group consisting of, Staphylococcus Infectious disease prevention or treatment method.
13. The method of claim 11, wherein the infectious disease caused by Staphylococcus bacteria is at least one selected from the group consisting of food poisoning, sepsis, inflammation, abscess, endocarditis, toxin syndrome, skin erythema, skin pruritus and skin pustules caused by infection with Staphylococcus. Method for preventing or treating infectious diseases caused by Staphylococcus bacteria.

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