WO2023140723A1 - Bacteriophage kmsp1 specific to staphylococcus bacteria and antibacterial composition comprising same - Google Patents
Bacteriophage kmsp1 specific to staphylococcus bacteria and antibacterial composition comprising same Download PDFInfo
- Publication number
- WO2023140723A1 WO2023140723A1 PCT/KR2023/001149 KR2023001149W WO2023140723A1 WO 2023140723 A1 WO2023140723 A1 WO 2023140723A1 KR 2023001149 W KR2023001149 W KR 2023001149W WO 2023140723 A1 WO2023140723 A1 WO 2023140723A1
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- WO
- WIPO (PCT)
- Prior art keywords
- bacteriophage
- kmsp1
- staphylococcus
- bacteria
- present
- Prior art date
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/40—Viruses, e.g. bacteriophages
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/195—Antibiotics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/76—Viruses; Subviral particles; Bacteriophages
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
Definitions
- 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.
- 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.
- 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.
- 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.
- Staphylococcus intermedius S. intermedius
- MRSA methicillin-resistant Staphylococcus aureus infection
- 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.
- the present invention provides a bacteriophage KMSP1 (accession number: KCTC18975P) having a specific killing ability for Staphylococcus bacteria.
- the present invention provides an antibiotic composition comprising the bacteriophage KMSP1.
- the present invention provides a composition for adding feed, including the bacteriophage KMSP1.
- the present invention provides a feed comprising the composition for adding the feed.
- the present invention provides a disinfectant containing the bacteriophage KMSP1.
- the present invention provides a detergent containing the bacteriophage KMSP1.
- the present invention provides a method for preventing or treating infectious diseases caused by Staphylococcus bacteria, comprising administering the bacteriophage KMSP1 to the subject.
- 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.
- FIG. 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.
- 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.
- the bacteriophage KMSP1 may include the nucleotide sequence represented by SEQ ID NO: 1 as all or part of the entire gene.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- solid formulations such as powders, granules, and tablets
- liquid formulations such as suspensions or injection solutions
- semi-solid formulations such as creams, lotions, gels, and pastes.
- 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.
- 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.
- 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.
- the feed may further include conventional additives capable of increasing the preservability of the feed in addition to the bacteriophage 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.
- 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.
- 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.
- drinking water is not particularly limited, and drinking water commonly used in the art may be used.
- 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.
- the present invention provides a cleaning agent containing a bacteriophage KMSP1 having a specific killing ability for Staphylococcus bacteria.
- 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.
- Staphylococcus aureus Staphylococcus intermedius
- MRSA methicillin-resistant Staphylococcus aureus
- 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.
- 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.
- Staphylococcus bacteria preferably Staphylococcus aureus ( Staphylococcus aureus ), Staphylococcus intermedius ( S. intermedius ) and methicillin-resistant Staphylococcus aureus (MRSA)
- 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.
- 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.
- PFU plaque forming unit
- the term PFU is a unit that quantifies the formation of plaques by bacteriophages.
- prevention of the present invention refers to any action that suppresses or delays the onset of a disease by administering a composition.
- 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.
- 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.
- 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.
- diluents such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- Example 1 Separation and identification of novel bacteriophages
- 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 ⁇ m 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.
- Example 1.1 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 .
- LIBRA 120 energy filtration transmission electron microscope
- 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.
- the bacteriophage of the present invention 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.
- 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.
- Staphylococcus intermedius S. intermedius
- 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.
- 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.
- MRSA methicillin-resistant Staphylococcus aureus
- 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.
- M.O.I multiplicity of infection
- 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.
- 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 .
- 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.
- the number of bacteriophages was measured in various pH ranges. More specifically, 100 ⁇ L of a 1.0x10 9 PFU/mL KMSP1 solution present in the buffer was mixed with 900 ⁇ L 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 .
- 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.
- 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).
- MRSA methicillin-resistant Staphylococcus aureus
- 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.
- 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.
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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
본 발명은 스타필로코커스균에 특이적인 사멸능을 갖는 신규 박테리오파지 KMSP1, 상기 박테리오파지를 포함하는 항생용 조성물, 사료 첨가용 조성물, 사료, 소독제 또는 세척제 및 상기 박테리오파지를 개체에 투여하는 단계를 포함하는 스타필로코커스균에 의한 감염성 질병의 예방 또는 치료 방법에 관한 것이다.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.
박테리오파지(bacteriophage)는 특정 세균을 감염시켜 감염된 세균의 성장을 억제하고 저해하는 세균 특이적 바이러스를 의미한다. 1915년 영국의 미생물학자인 프레데릭 트워트가 처음으로 포도상구균의 용균 현상을 관찰하였고, 1917년 프랑스 세균학자인 펠릭스 데렐 역시 적리균에서 같은 현상을 관찰하게 되면서, 이것이 세균에 감염하는 바이러스(세균바이러스)에 의한 것임을 알아내었다. 이후, 세균을 감염하는 바이러스는 “세균(Bacteria)”을 “먹는다(Phage)”라는 의미에서 박테리오파지(bacteriophage)로 명명하였으며 간단히 “파지”로도 불리고 있다. 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".
파지는 크게 2가지로, 용균성 생활사(lytic cycle)만을 반복하는 독성 파지(virulent phage)와, 용균성 생활사와 용원성 생활사(lysogenic cycle)를 모두 거치는 약독성 파지(temperate phage)로 분류한다.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.
용균성 생활사에 있는 파지의 경우, 세균 감염 약 30분 후에 100~200개의 새로운 파지를 만들어 세균의 막을 파괴하며 밖으로 나와 용균시키는데, 세균의 증식속도보다 파지의 증식속도가 훨씬 빠르므로 세균의 증식을 제한하는 중요한 요인이 될 수 있다. 약독성 파지는 숙주세균의 유전자에 파지의 유전자 정보를 넣어 파지의 모양은 사라지지만 숙주세균이 파괴되지도 않으며, 이런 상태는 숙주세균이 좋은 환경조건에 있는 한 지속하는데, 숙주세균의 환경이 악화되면, 숙주세균의 유전자에 있던 일부의 파지 유전자는 용균 사이클로 바뀌어 새로운 파지를 만들어 숙주세균을 용균시킨 후, 숙주세포 밖으로 방출된다. 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.
특정 박테리오파지는 특정 범주의 박테리아에만 감염하고 이를 특이적으로 사멸시킬 수 있는 특징이 있으므로, 최근 세균성 질환의 대처 방안으로 박테리오파지(bacteriophage)의 활용이 크게 주목을 받고 있다. 특히 2000년 이후에 항생제 내성균의 증가로 인하여 기존 항생제의 한계가 지적되고, 기존 항생제의 대체 물질로 박테리오파지를 활용할 수 있다는 가능성이 부각되면서 다시 박테리오파지가 항-박테리아제로 주목을 받고 있다.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.
이에 따라 독성 파지를 이용하여 송아지의 대장균성 설사증, 어류의 다양한 질병, 반코마이신 내성 장구균 패혈증, 결핵균, 닭의 파라티푸스 및 대장균 O157:H7 등 다양한 질병을 성공적으로 예방 및 치료한 사례가 보고되었으며, 파지를 직접 사용하는 방법뿐만 아니라, 파지가 숙주세균의 세포벽을 특이적으로 파괴하는데 작용하는 리신(lysin)을 이용하여 탄저균, 폐렴구균 등에 특이적인 약제의 개발도 이루어지고 있는 실정이다. 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. .
그러나 아직까지 심각한 질병을 유발하는 병원성 세균인 스타필로코커스균에 대하여 보다 넓은 숙주 범위로 제어할 수 있는 박테리오파지 및 이를 이용한 항균제가 많이 보고되어 있지 않아, 이에 대한 연구의 필요성이 있다. 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.
이에 본 발명자들은 다양한 스타필로코커스균에 특이적인 용균활성을 갖는 박테리오파지를 분리 동정하기 위해 연구하던 중, 멸균되지 않은 원유로부터 분리된 박테리오파지가 황색포도상구균(Staphylococcus aureus), 스타필로코커스 인터메디우스 (S. intermedius) 및 메티실린 내성 황색포도상구균(methicillin-resistant Staphylococcus aureus infection, MRSA)에 대한 폭넓은 항균 활성을 나타냄을 확인하고 본 발명을 완성하였다. 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.
본 발명의 목적은 스타필로코커스(Staphylococcus)균에 특이적인 사멸능을 갖는, 신규한 박테리오파지 KMSP1 및 이를 포함하는 다양한 항생용 조성물을 제공하는 것이다.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.
상기 목적을 달성하기 위하여, 본 발명은 스타필로코커스(Staphylococcus)균에 특이적인 사멸능을 갖는, 박테리오파지 KMSP1 (수탁번호: KCTC18975P) 를 제공한다. 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.
또한, 본 발명은 상기 박테리오파지 KMSP1을 포함하는, 항생용 조성물을 제공한다. In addition, the present invention provides an antibiotic composition comprising the bacteriophage KMSP1.
또한, 본 발명은 상기 박테리오파지 KMSP1을 포함하는, 사료 첨가용 조성물을 제공한다. In addition, the present invention provides a composition for adding feed, including the bacteriophage KMSP1.
또한, 본 발명은 상기 사료 첨가용 조성물을 포함하는, 사료를 제공한다.In addition, the present invention provides a feed comprising the composition for adding the feed.
또한, 본 발명은 상기 박테리오파지 KMSP1을 포함하는, 소독제를 제공한다.In addition, the present invention provides a disinfectant containing the bacteriophage KMSP1.
또한, 본 발명은 상기 박테리오파지 KMSP1을 포함하는, 세척제를 제공한다.In addition, the present invention provides a detergent containing the bacteriophage KMSP1.
또한, 본 발명은 상기 박테리오파지 KMSP1을 개체에 투여하는 단계를 포함하는, 스타필로코커스(Staphylococcus)균에 의한 감염성 질병의 예방 또는 치료 방법을 제공한다.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.
본 발명의 박테리오파지 KMSP1은 다양한 스타필로코커스(Staphylococcus)균에 넓은 숙주 감염성 및 용균 활성을 나타내고, 높은 물리화학적 안정성을 나타내므로, 기존 항생제를 대체하여 스타필로코커스 감염 예방 또는 치료를 위한 항생용 조성물로 식품, 축산업, 어업, 의약 분야에서 널리 활용될 수 있다. 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.
도 1은 전자 현미경을 통해 박테리오파지 KMSP1의 형태학적 특성을 확인한 결과를 나타낸 도이다.1 is a diagram showing the results of confirming the morphological characteristics of bacteriophage KMSP1 through an electron microscope.
도 2는 박테리오파지 KMSP1의 황색포도상구균에 대한 용균 활성능을 확인한 결과를 나타낸 도이다 (대조군(●), 실험군(■, ▲, ▼, ◆)).Figure 2 is a diagram showing the results of confirming the bacteriophage KMSP1's lytic activity against Staphylococcus aureus (control group (●), experimental group (■, ▲, ▼, ◆)).
도 3은 박테리오파지 KMSP1의 열 안정성을 확인한 결과를 나타낸 도이다.Figure 3 is a diagram showing the results of confirming the thermal stability of the bacteriophage KMSP1.
도 4는 박테리오파지 KMSP1의 pH 안정성을 확인한 결과를 나타낸 도이다.Figure 4 is a diagram showing the results of confirming the pH stability of the bacteriophage KMSP1.
본 발명은 스타필로코커스(Staphylococcus)균에 특이적인 사멸능을 갖는, 박테리오파지 KMSP1 (수탁번호: KCTC18975P) 및 이를 이용한 항생용 조성물에 관한 것이다. 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.
본 발명의 스타필로코커스(Staphylococcus)균에 특이적인 사멸능을 갖는 박테리오파지 KMSP1는 기존의 화학물질 항생제에 비해 스타필로코커스균에 대한 특이성이 매우 높고, 물리화학적 자극에 강하다는 장점을 가지고 있다. 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.
이하, 본 발명에 대해 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명에 따른 박테리오파지 KMSP1은 멸균되지 않은 원유로부터 분리된 신규한 박테리오파지로 수축성의 긴 꼬리를 갖는 복합형의 마이오비리대(Myoviridae)에 속하며 볼티모어 분류법에 의하면 이중 나선 DNA를 갖는 제1형 바이러스로 분류되는 박테리오파지이다. 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.
본 발명의 박테리오파지 KMSP1는 서열번호 1로 표시되는 염기서열로 이루어진 것일 수 있다. 박테리오파지 KMSP1의 전체 유전자는 138,528개의 염기서열로 표시된다. 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.
따라서, 상기 박테리오파지 KMSP1은 서열번호 1로 표시되는 염기서열을 전체 유전자의 전체 또는 일부로서 포함할 수 있다. 또한, 본 발명의 박테리오파지 KMSP1는 서열번호 1로 표시되는 염기서열, 및 상기 염기서열의 기능적 동등물로 이루어질 수 있다. 상기 기능적 동등물이란 염기서열의 변형, 치환의 결과, 상기 서열번호 1로 표시되는 염기서열과 적어도 70% 이상, 바람직하게는 80% 이상, 더욱 바람직하게는 90% 이상, 더더욱 바람직하게는 95% 이상의 서열 상동성을 갖는 것으로, 서열번호 1로 표시되는 염기서열과 실질적으로 동질의 생리활성을 나타내는 서열을 의미한다. 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.
본 발명자는 멸균되지 않은 원유로부터 분리된 새로운 박테리오파지를 KMSP1 로 명명하고, 2022년 1월 6일자로 한국생명공학연구원 생물자원센터에 기탁하여, 수탁번호 KCTC18975P 를 부여받았다. 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.
본 발명의 박테리오파지 KMSP1 은 스타필로코커스 균에 특이적인 사멸능을 나타낼 수 있으며, 상기 스타필로코커스 균은 바람직하게는 황색포도상구균(Staphylococcus aureus), 스타필로코커스 인터메디우스 (S. intermedius) 및 메티실린 내성 황색포도상구균(methicillin-resistant Staphylococcus aureus infection, MRSA)으로 이루어진 군에서 선택된 1종 이상일 수 있으나, 이에 제한되는 것은 아니다. 본 발명의 박테리오파지 KMSP1 은 황색포도상구균(Staphylococcus aureus), 스타필로코커스 인터메디우스 (S. intermedius) 및 메티실린 내성 황색포도상구균에 동시에 감염하여 이들을 사멸시킬 수 있는 장점을 가지고 있으므로, 상기 세균들에 의한 다양한 질병의 예방 또는 치료를 위한 생물학적 조절제로 사용하여 항생제 사용 절감을 유도할 수 있다. 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.
본 발명의 박테리오파지 KMSP1은 열 및 pH에 대한 안정성이 우수한 특성을 가지고 있다. 보다 구체적으로 박테리오파지 KMSP1는 3 내지 60℃에서 열 안정성을 나타낼 수 있으며, 바람직하게는 4 내지 55℃ 에서 열 안정성을 나타낼 수 있다. 또한 본 발명의 박테리오파지 KMSP1은 pH5 내지 pH11 와 같은 넓은 pH 범위에서도 뛰어난 안전성을 나타내는 것을 특징으로 할 수 있다. 즉 본 발명의 박테리오파지 KMSP1 폭넓은 온도 및 pH 에서 안정하게 유지되므로 다양한 환경에서 활용될 수 있다. 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.
상기와 같은 스타필로코커스균 특이적 용균 활성, 내산성, 내염기성 및 내열성은 본 발명의 박테리오파지 KMSP1을 스타필로코커스균에 대한 다양한 조성물로 활용 가능하게 한다. 따라서 본 발명은 스타필로코커스(Staphylococcus)균에 특이적인 사멸능을 갖는 박테리오파지 KMSP1을 포함하는 항생용 조성물, 사료첨가용 조성물, 사료, 소독제 및 세척제를 제공한다.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.
본 발명에서, "항생용 조성물"은 약제 형태로 제공되어 균을 사멸시킬 수 있는 제제를 의미하며, 세균 사멸용 미생물 제제, 방부제, 살균제, 항생제 및 항균제를 총칭하는 것이다.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.
본 발명의 박테리오파지 KMSP1은 스타필로코커스균에 대한 특이성이 매우 높으므로, 유익균은 죽이지 않고, 바람직하게는 황색포도상구균(Staphylococcus aureus), 스타필로코커스 인터메디우스 (S. intermedius) 및 메티실린 내성 황색포도상구균(MRSA)만 사멸시킬 수 있고, 약물 내성 내지 저항성을 유도하지 않아, 기존의 항생물질에 비하여 제품수명(life cycling)이 긴 신규 항생제로서 이용될 수 있다.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.
본 발명에 의한 항생용 조성물은 통상적인 미생물 제제 제조방법으로 제제화할 수 있다. 특별히 한정되지 않으나, 인체에 투여하는 경우는 약제학적으로 허용하는 범위 내에서, 가축 또는 어류에 투여하는 경우는 수의학에서 허용하는 범위 내에서 제제화 하고 그에 따라 투여경로를 정할 수 있다.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.
상기 본 발명의 항생용 조성물은 유효성분인 본 발명의 박테리오파지 KMSP1 외에 필요에 따라 담체, 기타 보조제 등을 포함할 수 있는데, 상기 담체는 생리식염수, 증류수, 사료, 박테리아용 배지(예, Luria broth 등) 등이 될 수 있으며, 보조제는 pH 교정 등의 목적으로 완충액 등을 첨가하여 사용할 수 있다. 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.
또한, 본 발명의 항생용 조성물은 액상, 반 고체상 또는 고체상으로 제제 형태는 분말제, 과립제, 정제 등의 고체상 제제, 현탁제 또는 주사액제 등의 액상 제제 및 크림, 로션, 젤, 페이스트 등의 반 고체상 제제 등으로 제형화될 수 있다. 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.
본 발명의 조성물은 경구, 정맥내, 동맥내, 복강내, 근육내, 경피, 비측내, 흡입 또는 직장 등의 경로를 통해 통상적인 방식으로 투여할 수 있다. 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.
본 발명 조성물의 투여량은 투여대상에서 황색포도상구균, S. intermedius 및/또는 메티실린 내성 황색포도상구균을 사멸시키는 효과를 이루는데 요구되는 양을 의미한다. 따라서, 질환의 종류, 질환의 중증도, 제형의 종류 및 투여대상의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여 경로, 투여 시간 및 기간, 동시 사용되는 약물을 비롯한 다양한 인자에 따라 조절될 수 있다.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.
또한, 본 발명은 스타필로코커스(Staphylococcus)균에 특이적인 사멸능을 갖는 박테리오파지 KMSP1을 포함하는 사료 첨가용 조성물 및 사료를 제공한다. In addition, the present invention provides a feed composition and feed containing a bacteriophage KMSP1 having a specific killing ability for Staphylococcus bacteria.
축산, 수산업에서 사용되는 사료 첨가용 항생제는 질병의 예방 목적으로 사용되고 있는데, 예방 목적의 항생제 투여는 내성균 발생 가능성을 높이고 가축에 잔류하는 항생제가 사람에게 전달될 수 있다는 문제점이 있다. 항생제가 육류를 통해 인체에 흡수되면 사람에서 항생제 내성을 유발할 수도 있다. 또한, 사료에 섞어 먹이는 항생제의 종류가 많아지면 다제내성균 발생 확률이 높아지는 문제점이 있기 때문에 좀 더 자연 친화적이면서도 기존 항생제 사용에서 발생하거나 할 수 있는 문제를 해결할 새로운 사료 첨가용 항생물질이 필요하며, 발명의 상기 박테리오파지 KMSP1을 이용할 수 있다.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.
또한, 본 발명은 상기 사료 첨가용 조성물을 포함하는 사료를 제공할 수 있으며, 본 발명의 사료는 박테리오파지를 사료 첨가제 형태로 따로 제조하여 사료에 혼합시키거나, 사료 제조 시 직접 첨가시켜 제조할 수 있다. 본 발명의 사료 내 박테리오파지는 액상 또는 건조 상태일 수 있으며, 바람직하게는 건조된 분말형태이다. 건조방법은 통풍건조, 자연건조, 분무건조 및 동결건조가 가능하지만, 이에 제한되는 것은 아니다. 본 발명의 박테리오파지는 분말형태로 사료 중량의 0.05 내지 10 중량%, 바람직하게는 0.1 내지 2 중량%의 성분비로 혼합될 수 있다. 또한, 상기 사료는 본 발명의 박테리오파지 외에 사료의 보존성을 높일 수 있는 통상의 첨가제들을 추가로 포함할 수 있다.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.
본 발명의 사료 첨가용 조성물에는 비병원성의 다른 미생물이 추가로 첨가될 수 있다. 첨가될 수 있는 미생물로는 단백질 분해 효소, 지질 분해 효소 및 당 전환 효소를 생산할 수 있는 바실러스 서브틸리스(Bacillus subtilis)와 같은 고초균, 소의 위와 같은 혐기적 조건에서 생리적 활성 및 유기물 분해능이 있는 락토바실러스 균주(Lactobacillus sp.), 가축의 체중을 증가시키며 우유의 산유량을 늘리고 사료의 소화 흡수율을 높이는 효과를 보여주는 아스퍼질러스 오리자에(Aspergillus oryzae)와 같은 사상균 및 사카로미세스 세레비지에(Saccharomyces cerevisiae)와 같은 효모로 구성된 군으로부터 선택될 수 있다.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 .
본 발명의 박테리오파지 KMSP1을 포함하는 사료에는 식물성으로는 곡물류, 근과류, 식품가공 부산물류, 조류, 섬유질류, 제약 부산물류, 유지류, 전분류, 박류, 곡물부산물류 등이 있으며, 동물성으로는 단백질류, 무기물류, 유지류, 광물성류, 단세포 단백질, 동물성 플랑크톤류, 남은 음식물 등이 있으며 이에 제한되는 것은 아니다.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.
본 발명의 사료 첨가용 조성물에는 품질 저하를 방지하기 위하여 첨가하는 결착제, 유화제, 보존제 등이 포함될 수 있고, 효용 증대를 위하여 사료에 첨가하는 아미노산제, 비타민제, 효소제, 생균제, 향미제, 비단백태질소화합물, 규산염제, 완충제, 착색제, 추출제, 올리고당 등이 포함될 수 있으며, 그 외에도 사료 혼합제 등을 추가로 포함할 수 있다.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.
또한, 본 발명은 박테리오파지 KMSP1는 음용수 첨가제에 포함될 수 있다. In addition, in the present invention, the bacteriophage KMSP1 may be included in the drinking water additive.
본 발명의 음용수 첨가제는 상기 박테리오파지 KMSP1 또는 이를 포함하는 조성물을 음용수 첨가제 형태로 따로 제조하여 사료 또는 음용수에 혼합하는 방식으로 사용되거나, 음용수 제조 시 직접 첨가하는 방식으로 사용할 수 있다. 상기와 같이 음용수에 혼합하여 공급함으로써 지속적으로 스타필로코커스균의 숫자를 감소시킬 수 있는 효과가 있다. 본 발명에서 음용수는 특별히 제한되지 아니하며, 당해 기술 분야에서 통상적으로 사용되는 음용수를 사용할 수 있다.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.
또한, 본 발명은 스타필로코커스(Staphylococcus)균에 특이적인 사멸능을 갖는 박테리오파지 KMSP1을 포함하는, 소독제를 제공한다.In addition, the present invention provides a disinfectant containing a bacteriophage KMSP1 having a specific killing ability for Staphylococcus bacteria.
스타필로코커스균, 바람직하게는 황색포도상구균(Staphylococcus aureus), 스타필로코커스 인터메디우스 (S. intermedius) 및 메티실린 내성 황색포도상구균(MRSA) 으로 이루어진 군에서 선택된 1종 이상의 스타필로코커스 균에 특이적인 사멸능을 갖는 본 발명의 박테리오파지 KMSP1을 포함하는 소독제는 병원감염을 막기 위한 병원 및 보건용의 소독제로 유용하게 사용될 수 있고 일반 생활 소독제, 식품 및 조리 장소 및 설비의 소독제, 양계장, 축사 등의 건물, 축체, 음수, 깔짚, 난좌, 운반차량, 식기 등의 각종 생육 용품의 소독 등에 사용될 수 있다.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.
또한, 본 발명은 스타필로코커스(Staphylococcus)균에 특이적인 사멸능을 갖는 박테리오파지 KMSP1을 포함하는, 세척제를 제공한다.In addition, the present invention provides a cleaning agent containing a bacteriophage KMSP1 having a specific killing ability for Staphylococcus bacteria.
본 발명의 박테리오파지 KMSP1은 스타필로코커스균, 예컨대 황색포도상구균(Staphylococcus aureus), 스타필로코커스 인터메디우스 (S. intermedius) 및 메티실린 내성 황색포도상구균(MRSA)으로 이루어진 군에서 선택된 1종 이상에 특이적 사멸능을 가지므로, 상기 스타필로코커스균에 노출되었거나 노출될 가능성이 있는 가축의 피부 표면 또는 신체 각 부위 또는 스타필로코커스 균의 감염 예방이 필요한 각종 식기류 및 식품을 세척하는 용도로도 사용될 수 있다.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.
또한, 본 발명은 스타필로코커스(Staphylococcus)균에 특이적인 사멸능을 갖는 박테리오파지 KMSP1을 포함하는, 스타필로코커스에 의한 감염성 질병의 예방 또는 치료용 약학적 조성물 또는 스타필로코커스(Staphylococcus)균에 특이적인 사멸능을 갖는 박테리오파지 KMSP1를 인간을 제외한 개체에 투여하는 단계를 포함하는 스타필로코커스균에 의한 감염성 질병의 예방 또는 치료 방법을 제공한다. 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.
본 발명의 박테리오파지 KMSP1은 스타필로코커스균에 특이적인 사멸능을 가지므로, 스타필로코커스균에 의한 감염성 질병의 예방 또는 치료용 약학적 조성물에 사용될 수 있다. 본 발명에서, 스타필로코커스균, 바람직하게는 황색포도상구균(Staphylococcus aureus), 스타필로코커스 인터메디우스 (S. intermedius) 및 메티실린 내성 황색포도상구균(MRSA)으로 이루어진 군에서 선택된 1종 이상의 스타필로코커스 균에 의한 감염성 질병은 유행성 또는 급성으로 발병하는 전염성 질환을 통칭하는 개념이다.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.
본 발명에서, 상기 감염성 질병은 이에 제한되는 것은 아니나 스타필로코커스균 감염에 의한 식중독, 패혈증, 염증, 농양, 심장 내막염, 독소증후군, 피부 홍반, 피부 소양증 및 피부 농포로 이루어진 군으로부터 선택된 1종 이상일 수 있고, 상기 스타필로코커스균의 감염으로 인해 발병하는 감염성 질병이라면 이에 제한없이 적용 가능하다.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.
본 발명의 약학적 조성물은 1 × 103 내지 1 × 1010 PFU/mL의 박테리오파지를 포함하며, 바람직하게는 1 × 106 내지 1 × 109 PFU/mL의 박테리오파지를 포함한다. 본 발명에 사용된 용어, PFU(plaque forming unit)는 박테리오파지가 플라그를 형성하는 것을 수치화한 단위이다.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.
본 발명의 용어 예방이란 조성물의 투여로 질병을 억제시키거나 발병을 지연시키는 모든 행위를 의미한다.The term prevention of the present invention refers to any action that suppresses or delays the onset of a disease by administering a composition.
본 발명의 용어 치료란 조성물의 투여로 상기 질병의 증세가 호전되거나 상기 질병의 억제 또는 경감 및 이롭게 변경되는 모든 행위를 의미한다.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.
본 발명의 약학적 조성물은 약학적으로 허용 가능한 담체를 추가로 포함할 수 있다.The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier.
본 발명에서, 용어 약학적으로 허용 가능한 담체란 생물체를 자극하지 않고 투여 화합물의 생물학적 활성 및 특성을 저해하지 않는 담체 또는 희석제를 의미한다. 액상 용액으로 제제화되는 조성물에 있어서 허용되는 약제학적 담체로는, 멸균 및 생체에 적합한 것으로서, 식염수, 멸균수, 링거액, 완충 식염수, 알부민 주사용액, 덱스트로오스 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다.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.
본 발명의 약학적 조성물은 경구 투여 외에 비경구 투여, 비강분무, 질환 부위에의 도포 또는 분무하는 방법으로 이용할 수 있으며, 비경구 투여의 경우 정맥 내 투여, 복강 내 투여, 근육 내 투여, 피하 투여 또는 국부 투여를 이용하여 투여할 수도 있다.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.
본 발명의 약학적 조성물을 유효성분으로 포함하는 경구 투여용 제형으로는, 예를 들어 정제, 트로키제, 로렌지, 수용성 또는 유성현탁액, 조제분말 또는 과립, 에멀젼, 하드 또는 소프트 캡슐, 시럽 또는 엘릭시르제로 제제화할 수 있다. 정제 및 캡슐 등의 제형으로 제제화하기 위해, 락토오스, 사카로오스, 솔비톨, 만니톨, 전분, 아밀로펙틴, 셀룰로오스 또는 젤라틴과 같은 결합제, 디칼슘 포스페이트와 같은 부형제, 옥수수 전분 또는 고구마 전분과 같은 붕괴제, 스테아르산 마그네슘, 스테아르산 칼슘, 스테아릴푸마르산 나트륨 또는 폴리에틸렌글리콜 왁스와 같은 윤활유를 포함할 수 있으며, 캡슐 제형의 경우 상기 언급한 물질 외에도 지방유와 같은 액체 담체를 더 함유할 수 있다.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.
본 발명의 약학적 조성물을 유효성분으로 포함하는 비경구 투여용 제형으로는, 피하주사, 정맥주사 또는 근육 내 주사 등의 주사용 형태, 좌제 주입방식 또는 호흡기를 통하여 흡입이 가능하도록 하는 에어로졸제 등 스프레이용으로 제제화할 수 있다. 주사용 제형으로 제제화하기 위해서는 본 발명의 조성물을 안정제 또는 완충제와 함께 물에서 혼합하여 용액 또는 현탁액으로 제조하고, 이를 앰플 또는 바이알의 단위 투여용으로 제제화할 수 있다. 에어로졸제 등의 스프레이용으로 제형화하는 경우, 수분산된 농축물 또는 습윤 분말이 분산되도록 추진제 등이 첨가제와 함께 배합될 수 있다.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.
본 발명의 약학적 조성물의 적합한 도포, 분무 및 투여량은 제제화 방법, 투여 방식, 대상이 되는 동물 및 환자의 연령, 체중, 성, 질병 증상의 정도, 음식, 투여시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하며, 보통으로 숙련된 의사나 수의사는 목적하는 치료에 효과적인 투여량을 용이하게 결정 및 처방할 수 있다.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.
본 발명에서, 용어 개체는 스타필로코커스균에 의한 감염성 질병이 발병될 수 있는 모든 개체를 의미하는 것으로 인간을 포함한 포유동물, 가축류 또는 가금류를 포함하나, 이에 제한되는 것은 아니다. 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.
상기 용어 가축류는 인간에 의하여 순화, 개량되어 사람과 함께 공동생활을 하는 유용한 동물을 지칭하는 개념으로, 예를 들어, 돼지, 소, 닭, 말, 오리 또는 개 등을 포함하나, 이에 제한되는 것은 아니다.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.
상기 용어 가금류는 가축 중 조류에 속하는 동물을 총칭하여 이르는 개념으로, 예를 들어, 닭, 오리, 꿩, 메추리, 타조, 거위 또는 칠면조 등을 포함하나, 이에 제한되는 것은 아니다.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.
본 발명의 방법에서 상기 박테리오파지 KMSP1 또는 조성물은 약학적 제제의 형태로 동물에게 투여되거나, 가축의 사료 또는 음용수에 혼합하여 이를 섭식시키는 방법을 통해 투여될 수 있다.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.
본 발명의 방법에서 상기 박테리오파지 KMSP1 또는 조성물의 투여 경로는 목적 조직에 도달할 수 있는 한 경구 또는 비경구의 다양한 경로를 통하여 투여될 수 있으며, 구체적으로, 구강, 직장, 국소, 정맥 내, 복강 내, 근육 내, 동맥 내, 경피, 비측 내, 흡입 등을 통해 통상적인 방식으로 투여될 수 있다.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.
본 발명의 방법에 투여되는 상기 박테리오파지 KMSP1의 적합한 총 1일 사용량은 올바른 의학적 판단범위 내에서 처치의에 의해 결정될 수 있다는 것은 당업자에게 자명한 일이다. 특정 개체에 대한 구체적인 치료적 유효량은 달성하고자 하는 반응의 종류와 정도, 환자의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여시간, 투여 경로 및 조성물의 분비율, 치료기간, 구체적 조성물과 함께 사용되거나 동시 사용되는 약물을 비롯한 다양한 인자와 의약 분야에 잘 알려진 유사 인자에 따라 다르게 적용하는 것이 바람직하다.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.
본 명세서에서 달리 정의되지 않은 용어들은 본 발명이 속하는 기술분야에서 통상적으로 사용되는 의미를 갖는 것이다.Terms not defined otherwise in this specification have meanings commonly used in the technical field to which the present invention belongs.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.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.
실시예 1: 신규한 박테리오파지의 분리 동정 Example 1: Separation and identification of novel bacteriophages
1.11.1
박테리오파지의 분리Isolation of bacteriophage
멸균되지 않은 원유를 8000 rpm의 속도로 4분 간 원심분리하여 상등액을 취한 후 액체 배지와 혼합하였다. 이 혼합액에 포도상구균의 박테리아를 접종하여 37℃에서 배양하고, 배양액을 원심분리한 후 수득한 상등액을 0.4 μm 크기의 필터로 여과하여 여과액을 취한다. 여과액 0.1 mL와 12시간 이상 배양한 박테리아(포도상구균) 0.1 mL의 현탁액을 피펫으로 추출한 후에 섞어 주었다. 이 혼합액을 아가 농도가 낮은 아가(0.4%) 배지와 함께 섞어서 1.5% 아가 배지를 포함하는 플레이트에 부었다. 상기 플레이트룰 37℃에서 하룻밤 동안 배양하였다.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.
박테리오파지가 플레이트 상에 나타난 경우 독립되고 투명한 플라그(plaque)가 관찰되었으며, 이 플라그 영역을 파이펫 팁을 이용하여 분리해낸 다음, 새로운 박테리아 배양액에 넣어 37℃에서 배양하였다. 배양액이 맑아지는 것을 확인한 후, 배양액을 원심분리하고, 상등액만을 0.22 μm 막 필터로 여과시켜 용해물에 남아있는 모든 박테리아를 제거함으로써, 여과액만을 취하였다. 이 여과액은 박테리오파지만을 포함하는데, 상기의 방법으로 얻은 박테리오파지 용액을 아가 배지 상에서 다르게 희석하여 주입하는 플라그 재분리를 3회 수행하여 박테리오파지를 분리하였다. 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.
1.21.2
박테리오파지 형태 관찰 및 분류 Observation and classification of bacteriophage morphology
실시예 1.1 에서 분리된 박테리오파지의 형태학적 특성을 알아보기 위해, 전자현미경을 통해 형태를 확인하였다. 박테리오파지 샘플을 탄소를 입힌 구리 그리드(grid)에 올리고, 2% 액상 우라닐 아세테이트(uranyl acetate)로 음성 염색을 하였다. 염색된 시료를 80kV의 전압으로 에너지 여과 투과 전자현미경(LIBRA 120, Carl Zeiss)을 이용하여 관찰하였고, 도 1에 그 결과를 나타내었다. 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 .
도 1에 나타낸 바와 같이, 분리된 박테리오파지는 약 300 nm의 크기로, 정이십면체인 104.6nm 크기 머리와 수축성이 있는 197.7nm의 긴 꼬리를 갖고 시스(sheath)가 있으므로 복합형의 마이오비리데 (Myoviridae)에 속하는 것으로 분류하였다. 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.
또한 본 발명의 박테리오파지는 유전 정보가 이중가닥 DNA 로 되어 있어 제한효소 처리로 절단되었으므로, 볼티모어 분류법(Baltimore classification)에 따라, 이중가닥 DNA 유전체를 가진 제1형 바이러스로 분류하였다. 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.
분리 동정된 박테리오파지를 KMSP1으로 명명하고, 2022년 01월 06일에 한국생명공학연구원 생물자원센터에 기탁하고, 수탁번호 KCTC18975P를 부여받았다.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 박테리오파지의 유전자 분석 1.3 Genetic analysis of bacteriophage
분리 동정된 박테리오파지 KMSP1 의 전체 유전자를 분석하기 위한 시퀀싱을 수행하였다. 유전자 분석을 위한 박테리오파지 유전체는 약 1010 PFU/mL의 배양액을 사용하여 일반적인 페놀-클로로포름 방법을 통해 분리하였다. 분리된 박테리오파지 유전체는 agarose gel 전기영동을 통해 quality 확인 후 library를 제작하였다. 전체 유전자 분석은 Miseq 300 bp paired end (Illumina, USA)를 이용한 유전자 시퀀싱을 진행하여 수행하였다(세니젠, 대한민국). Raw data의 quality는 FastQC v0.11.9를 이용하여 검증되었으며 adapter 시퀀스와 low quality read의 경우 Trimmomatic v0.39를 통해 제거되었다. 최종적으로 SPAdes v.3.13.0 프로그램을 통해 de novo assembly를 진행한 결과, 하나의 콘티그(contig)로 이루어진 염기서열을 얻었다. 박테리오파지 KMSP1 의 전체 유전자는 총 138,528개의 염기로 이루어져 있으며, 확인된 서열을 서열번호 1로 나타내었다. 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.
실시예 2: 박테리오파지 KMSP1 의 스타필로코커스 균 감염성 확인 Example 2: Confirmation of Staphylococcus infectivity of bacteriophage KMSP1
다양한 황색포도상구균, 스타필로코커스 인터메디우스 (S. intermedius) 및 메티실린 내성 황색포도상구균에 대한 감염성을 확인하기 위하여, 총 12종의 균에 대한 감염성을 연속 희석 적하법 (serial dilution dropping method)을 사용하여 확인하였다(Adams, M.H. 1959. Bacteriophage, pp27-34. Interscience Publishers Inc., New York). 10시간 배양한 세균 배양액과 0.4%의 소프트 아가 배지를 섞은 후, LB 아가 플레이트 상에 부어서 37℃에서 15분간 건조하였고, 그 위에 희석 배수별로 희석된 박테리오파지 KMSP1을 떨어뜨리고, 상온에서 말린 후 37℃에서 10시간 정치 배양하였다. 균주가 파지에 대해 감수성을 갖는 경우 플레이트 상에 용균반이 보이게 되며, 이는 세균 세포가 완전하게 용해되어 사멸된 결과를 나타낸다. 만약 감수성이 약한 경우, 뿌옇게 나타나는 영역이 생길 수 있고, 리소스타핀(lysostaphin)과 같은 세균의 증식을 억제할 수 있는 항균성 효소가 존재하는 경우 억제 영역이 생길 수 있다. 다양한 분리원에서 분리한 황색포도상구균 및 메티실린 내성 황색포도상구균에 대한 감수성을 확인한 결과를 표 1에 나타내었다. 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.
[표 1][Table 1]
(+: 용균활성 있음, -; 용균활성 없음)(+: lytic activity present, -; lytic activity absent)
표 1에서 확인할 수 있는 바와 같이, 박테리오파지 KMSP1는 다양한 황색포도상구균, 메티실린 내성 황색포도상구균(MRSA) 및 스타필로코커스 인터메디우스를 폭넓게 용해시켜 용균반을 나타내었다. 이는 본 발명의 박테리오파지 KMSP1가 황색포도상구균, 메티실린 내성 황색포도상구균 및 스타필로코커스 인터메디우스를 동시에 사멸시키는 폭넓은 숙주 감염 범위를 갖는 것을 보여주는 결과이다. 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.
실시예 3: 박테리오파지 KMSP1 의 황색포도상구균 사멸 능력 확인Example 3: Confirmation of Staphylococcus aureus killing ability of bacteriophage KMSP1
박테리오파지 KMSP1의 세균 사멸 능력을 조사하였다. 박테리오파지 KMSP1의 황색포도상구균에 대한 사멸 능력을 분석하기 위하여, 5개의 시험군으로 나누어 용균 활성능을 추가 확인하였다. 숙주 균주인 황색포도상구균 (food isolate 1)를 액체 배지에서 2시간 동안 배양(흡광도= 약 0.2)하였고, 박테리오파지 KMSP1의 양을 M.O.I(Multiplicity of infection) 10, 1, 0.1, 0.01 로 달리하여 감염시킨 후 세균의 생장 및 사멸 정도를 확인하는 실험을 수행하였다. 박테리오파지액을 넣지 않은 시료로 황색포도상구균 (food isolate 1)만을 액체 배지에서 배양하여 대조군으로 하였다. 12시간 동안 배양하며 1시간 단위로 흡광도 600nm 에서 세균의 배양을 진행하였고, 흡광도를 확인하여 그 결과를 도 2에 나타내었다. 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.
도 2에 나타낸 바와 같이, 본 발명의 박테리오파지 KMSP1 를 감염시킨 실험군에서는 균 수 대비 1/100의 파지 양만으로도 4시간 이내에 우수한 균 사멸능을 나타냄을 확인하였다. 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.
실시예 4. 박테리오파지 KMSP1 의 열 안정성 확인 Example 4. Confirmation of thermal stability of bacteriophage KMSP1
박테리오파지 KMSP1의 열 안정성을 확인하기 위하여, 다양한 온도 범위에서 일정 시간 방치 후 박테리오파지의 수를 측정하였다. 보다 구체적으로, 버퍼 상에 존재하는 1.0x108 PFU/mL의 박테리오파지 KMSP1 용액 1 mL을 4℃, 25℃, 35℃, 45℃55℃, 65℃ 및 75℃의 다양한 온도 범위에 1시간 동안 노출시켰다. 그 후, 각 반응액을 단계 별 희석하여 소프트 아가 오버레이법을 이용하여 박테리오파지의 수를 측정하였으며, 그 결과를 도 3에 나타내었다. 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 .
도 3에 나타낸 바와 같이, 본 발명의 박테리오파지 KMSP1은 4-55℃의 넓은 온도 범위에서 1시간까지 안정하며 다양한 온도 환경에서 폭넓게 적용 가능함을 확인하였다. 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.
실시예 5: 박테리오파지 KMSP1의 pH 안정성 조사Example 5: Investigation of pH stability of bacteriophage KMSP1
박테리오파지 KMSP1의 pH에 대한 안정성을 확인하기 위하여, 다양한 pH 범위에서 박테리오파지의 수를 측정하였다. 보다 구체적으로 버퍼 상에 존재하는 1.0x109 PFU/mL의 KMSP1 용액 100 μL을 다양한 범위의 pH (3, 4, 5, 6, 7, 8, 9, 10, 11, 12)로 맞춰진 각 완충액 900 μL에 섞어준 후 25℃에서 1시간 동안 노출시켰다. 그 후, 각 반응액을 단계 별 희석하여 소프트 아가 오버레이법을 이용하여 박테리오파지의 수를 측정하였다. 측정 결과를 도 4에 나타내었다. 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 .
도 4에 나타낸 바와 같이, 박테리오파지 KMSP1는 pH 5 내지 11 의 넓은 pH 범위에서 뛰어난 안정성을 나타내었으며, 다양한 pH 환경에서 적용가능함을 확인하였다. 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.
상기 실험으로부터 박테리오파지 KMSP1은 스타필로코커스 균 중 황색포도상구균, 스타필로코커스 인터메디우스(S. intermedius)에 대한 감염성을 나타낼 뿐만 아니라, 메티실린 내성 황색포도상구균(MRSA)에 대해서도 우수한 감염성을 나타냄을 확인하였다. 또한, 높은 용균 활성을 나타냄과 동시에 다양한 온도 및 pH 에서 높은 안정성을 가지므로 물리화학적 자극에 강하며, 이들 균으로부터 유발될 수 있는 다양한 감염성 질병의 예방 또는 치료에 다양하게 활용될 수 있음을 확인하였다. 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.
이상, 본 발명내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적인 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의해 정의된다고 할 것이다. 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.
본 발명의 박테리오파지 및 이를 포함하는 항균 조성물을 이용하면, 식품, 축산업, 어업, 의약 분야에서 감염성 질병의 예방 또는 치료 향상에 기여할 수 있다. 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.
[수탁번호][Accession number]
기탁기관명 : 한국생명공학연구원 생물자원센터Depository Institution Name: Korea Research Institute of Bioscience and Biotechnology Biological Resources Center
수탁번호 : KCTC18975PAccession number: KCTC18975P
수탁일자 : 20220106Entrusted date: 20220106
기탁기관주소 : 대한민국, 한국생명공학연구원 생물자원센터, 56212 전라북도 정읍시 입신길 181(신정동)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)
- 스타필로코커스(Staphylococcus)균에 특이적인 사멸능을 갖는, 박테리오파지 KMSP1 (수탁번호: KCTC18975P). Staphylococcus ( Staphylococcus ) Bacteriophage KMSP1 (accession number: KCTC18975P) having a specific killing ability for bacteria.
- 제1항에 있어서, 상기 박테리오파지 KMSP1은 서열번호 1로 표시되는 염기서열로 이루어진 것인, 박테리오파지 KMSP1. According to claim 1, wherein the bacteriophage KMSP1 consists of the nucleotide sequence represented by SEQ ID NO: 1, bacteriophage KMSP1.
- 제1항에 있어서, 상기 스타필로코커스균은 황색포도상구균(Staphylococcus aureus), 스타필로코커스 인터메디우스(S. intermedius) 및 메티실린 내성 황색포도상구균(methicillin-resistant Staphylococcus aureus infection, MRSA) 으로 이루어진 군에서 선택된 1종 이상인, 박테리오파지 KMSP1. 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.
- 제1항에 있어서, 상기 박테리오파지 KMSP1는 pH5 내지 pH11 에서 안정성을 갖는 것인, 박테리오파지 KMSP1.The bacteriophage KMSP1 according to claim 1, wherein the bacteriophage KMSP1 has stability at pH5 to pH11.
- 제1항에 있어서, 상기 박테리오파지 KMSP1는 3 내지 60℃에서 안정성을 갖는 것인, 박테리오파지 KMSP1.The bacteriophage KMSP1 according to claim 1, wherein the bacteriophage KMSP1 has stability at 3 to 60 °C.
- 스타필로코커스(Staphylococcus)균에 특이적인 사멸능을 갖는 박테리오파지 KMSP1 (수탁번호: KCTC18975P) 를 포함하는, 항생용 조성물.An antibiotic composition comprising a bacteriophage KMSP1 (accession number: KCTC18975P) having a specific killing activity on Staphylococcus bacteria.
- 스타필로코커스(Staphylococcus)균에 특이적인 사멸능을 갖는, 박테리오파지 KMSP1 (수탁번호: KCTC18975P) 를 포함하는, 사료 첨가용 조성물.Staphylococcus ( Staphylococcus ) Having a specific killing ability for bacteria, bacteriophage KMSP1 (accession number: KCTC18975P) containing a composition for addition to feed.
- 제7항의 사료 첨가용 조성물을 포함하는, 사료.A feed comprising the composition for adding feed according to claim 7.
- 스타필로코커스(Staphylococcus)균에 특이적인 사멸능을 갖는, 박테리오파지 KMSP1 (수탁번호: KCTC18975P) 를 포함하는, 소독제.A disinfectant containing a bacteriophage KMSP1 (accession number: KCTC18975P) having a specific killing ability for Staphylococcus bacteria.
- 스타필로코커스(Staphylococcus)균에 특이적인 사멸능을 갖는, 박테리오파지 KMSP1 (수탁번호: KCTC18975P) 를 포함하는, 세척제.A detergent containing a bacteriophage KMSP1 (accession number: KCTC18975P) having a specific killing ability for Staphylococcus bacteria.
- 스타필로코커스(Staphylococcus)균에 특이적인 사멸능을 갖는, 박테리오파지 KMSP1 (수탁번호: KCTC18975P) 를 인간을 제외한 개체에 투여하는 단계를 포함하는, 스타필로코커스균에 의한 감염성 질병의 예방 또는 치료 방법.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.
- 제11항에 있어서, 상기 스타필로코커스균은 황색포도상구균(Staphylococcus aureus), 스타필로코커스 인터메디우스 (S. intermedius) 및 메티실린 내성 황색포도상구균(MRSA)으로 이루어진 군에서 선택된 1종 이상인, 스타필로코커스균에 의한 감염성 질병의 예방 또는 치료 방법.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.
- 제11항에 있어서, 상기 스타필로코커스 균에 의한 감염성 질병은 스타필로코커스균 감염에 의한 식중독, 패혈증, 염증, 농양, 심장 내막염, 독소증후군, 피부 홍반, 피부 소양증 및 피부 농포로 이루어진 군으로부터 선택된 1종 이상인, 스타필로코커스균에 의한 감염성 질병의 예방 또는 치료 방법.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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| KR1020220008990A KR102723118B1 (en) | 2022-01-21 | Staphylococcus specific bacteriophage KMSP1 and antibacterial composition comprising the same |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100068787A1 (en) * | 2008-09-16 | 2010-03-18 | Intralytix, Inc. | Novel Staphylococcus aureus: Bacteriophage and Uses Thereof |
| KR20110130285A (en) * | 2010-05-27 | 2011-12-05 | 경북대학교 산학협력단 | A bacteriophage killing staphylococcus aureus |
| KR101643235B1 (en) * | 2015-06-03 | 2016-07-27 | 서울대학교산학협력단 | Bacteriophage Siphoviridae family SAP4 against Staphylococcus aureus and composition thereof |
| KR20180042748A (en) * | 2016-10-18 | 2018-04-26 | 주식회사 옵티팜 | Novel Staphylococcus specific bacteriophage SA7 and antibacterial composition comprising the same |
| KR102003770B1 (en) * | 2016-12-23 | 2019-07-25 | 주식회사 옵티팜 | Novel Staphylococcus specific bacteriophage SA3 and antibacterial composition comprising the same |
-
2023
- 2023-01-25 WO PCT/KR2023/001149 patent/WO2023140723A1/en unknown
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100068787A1 (en) * | 2008-09-16 | 2010-03-18 | Intralytix, Inc. | Novel Staphylococcus aureus: Bacteriophage and Uses Thereof |
| KR20110130285A (en) * | 2010-05-27 | 2011-12-05 | 경북대학교 산학협력단 | A bacteriophage killing staphylococcus aureus |
| KR101643235B1 (en) * | 2015-06-03 | 2016-07-27 | 서울대학교산학협력단 | Bacteriophage Siphoviridae family SAP4 against Staphylococcus aureus and composition thereof |
| KR20180042748A (en) * | 2016-10-18 | 2018-04-26 | 주식회사 옵티팜 | Novel Staphylococcus specific bacteriophage SA7 and antibacterial composition comprising the same |
| KR102003770B1 (en) * | 2016-12-23 | 2019-07-25 | 주식회사 옵티팜 | Novel Staphylococcus specific bacteriophage SA3 and antibacterial composition comprising the same |
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