WO2022181891A1 - 장독소성 대장균에 대한 특이적인 사멸능을 가지는 신규한 박테리오파지 및 이를 포함하는 항균 조성물 - Google Patents
장독소성 대장균에 대한 특이적인 사멸능을 가지는 신규한 박테리오파지 및 이를 포함하는 항균 조성물 Download PDFInfo
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- WO2022181891A1 WO2022181891A1 PCT/KR2021/007481 KR2021007481W WO2022181891A1 WO 2022181891 A1 WO2022181891 A1 WO 2022181891A1 KR 2021007481 W KR2021007481 W KR 2021007481W WO 2022181891 A1 WO2022181891 A1 WO 2022181891A1
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- bacteriophage
- eco
- coli
- enterotoxin
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- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
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Definitions
- the present application relates to a novel bacteriophage having a specific killing ability against enterotoxin E. coli and an antibacterial composition comprising the same.
- Escherichia coli (hereinafter, referred to as ' E. coli ') is a gram-negative monococcus belonging to the genus Enterobacteriaceae and Escherichia , and is present in the intestinal tract of various animals including mammals. one of the bacterial flora. Escherichia coli may cause opportunistic infections in most cases non-pathogenically, but some highly pathogenic strains are known to cause various intestinal diseases and sepsis in animals, including humans.
- bacteriophages which are bacteria-specific viruses that inhibit bacterial growth by infecting specific bacteria, have stronger host specificity than antibiotics, and are being studied as alternatives to new antibiotics, but the number is still absolutely insufficient. . Therefore, in order to prevent and treat infectious diseases caused by enterotoxin E. coli, which is an important problem in livestock industry, including pigs, the development of new bacteriophages with a wide lysis spectrum and excellent acid resistance and heat resistance with high industrial application potential continues. is requested as
- Patent Document 1 US Registered Patent US 6,942,858 B1
- the object of the present application is the bacteriophage CJ_Eco_20-4 deposited with accession number KCCM12936P having a specific killing ability for enterotoxin E. coli, a composition for preventing or treating infectious diseases caused by enterotoxin E. coli as an active ingredient, antibiotic, feed
- a drinking water additive, a disinfectant, or a cleaning agent, or a feed including the feed additive, or drinking water including the drinking water additive is provided to provide an additive, a drinking water additive, a disinfectant, or a cleaning agent, or a feed including the feed additive, or drinking water including the drinking water additive.
- Another object of the present application is to provide a method for preventing or treating an infectious disease caused by enterotoxin E. coli using the bacteriophage CJ_Eco_20-4 or a composition for preventing or treating an infectious disease caused by enterotoxin E. coli comprising the same as an active ingredient.
- Another object of the present application is the use of the bacteriophage CJ_Eco_20-4, or a composition for preventing or treating an infectious disease caused by enterotoxin E. coli comprising the same as an active ingredient, for the preparation of a preventive or therapeutic agent for an infectious disease caused by enterotoxin E. coli will provide
- One aspect provides a bacteriophage CJ_Eco_20-4 deposited with accession number KCCM12936P, having a specific killing ability for enterotoxin E. coli.
- ETEC Enterotoxigenic Escherichia coli
- bacteriophage is a bacterium-specific virus that infects a specific bacterium and inhibits the growth of the bacterium, and refers to a virus containing single or double-stranded DNA or RNA as a genetic material. .
- compositions for preventing or treating infectious diseases caused by enterotoxin E. coli comprising the bacteriophage CJ_Eco_20-4 as an active ingredient.
- the bacteriophage CJ_Eco_20-4 is as described above.
- the bacteriophage CJ_Eco_20-4 Since the bacteriophage CJ_Eco_20-4 has a specific killing ability for enterotoxin E. coli, when administered before the onset of an infectious disease caused by enterotoxin E. coli, it kills enterotoxin E. Infectious diseases caused by E. coli can be prevented. In addition, when the bacteriophage CJ_Eco_20-4 is administered to an individual having an infectious disease caused by enterotoxin E. coli, it can alleviate or eliminate symptoms caused by this by killing enterotoxin E. coli present in the individual. Infectious diseases can be treated.
- prevention may refer to any action that inhibits or delays the onset of a disease in an individual by administration of a bacteriophage or a composition
- treatment is a bacteriophage or composition in an individual by administration. It may refer to any action that causes the symptoms of a disease to improve, alleviate, or disappear.
- the infectious disease caused by the enterotoxin E. coli may be, for example, E. coliosis, but is not limited thereto.
- colibacillosis refers to a disease caused by infection with pathogenic E. coli in livestock, and the symptoms include sepsis, diarrhea (diarrhoea in newborns and diarrhea after weaning), toxemia (edema and cerebrospinal cord). vasculopathy) may occur.
- sepsis is an acute systemic infection that occurs frequently during the neonatal period within 2 to 3 days after birth and has a high mortality rate.
- Diarrhea is an intestinal infection that occurs frequently during lactation within 1 to 2 weeks of age and immediately after weaning, and causes death or developmental disability.
- Toxemia occurs mainly in piglets of 8 to 12 weeks of age after weaning, and there are many cases of sudden death with swelling of the body and neurological symptoms.
- the composition for preventing or treating infectious diseases caused by enterotoxin E. coli is to include the bacteriophage CJ_Eco_20-4 at 1 X 10 2 to 1 X 10 20 PFU/mL or 1 X 10 2 to 1 X 10 20 PFU/g
- the bacteriophage CJ_Eco_20-4 may be 1 X 10 2 to 1 X 10 12 PFU / mL or 1 X 10 5 to 1 X 10 10 PFU / g comprising.
- composition for preventing or treating infectious diseases caused by enterotoxin E. coli may further include a pharmaceutically acceptable carrier, and may be formulated with the carrier and provided as a food, drug, feed additive or drinking water additive, etc. .
- the term "pharmaceutically acceptable carrier” may mean a carrier or diluent that does not stimulate the organism and does not inhibit the biological activity and properties of the administered compound.
- the type of carrier that may be included in the composition is not particularly limited, and any carrier commonly used in the art and pharmaceutically acceptable may be used.
- Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, and the like. These may be used alone or in mixture of two or more, and other conventional additives such as antioxidants, buffers, and bacteriostats may be added as needed.
- diluents, dispersants, binders and lubricants may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets.
- composition for preventing or treating infectious diseases caused by enterotoxin E. coli may be administered through oral administration or parenteral administration, and a method of applying or spraying to a diseased site may also be used.
- parenteral administration intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, or local administration may be used.
- Suitable application, spraying, and dosage of the composition include formulation method, administration mode, age, weight, sex, severity of disease symptoms, food, administration time, administration route, excretion rate and reaction sensitivity of the target animal and patient. This varies depending on factors, and an normally skilled physician or veterinarian can readily determine and prescribe an effective dosage for the desired treatment.
- Formulations for oral administration of the composition for preventing or treating infectious diseases caused by enterotoxin E. coli include, for example, tablets, troches, lozenges, aqueous or oily suspensions, powders or granules, emulsions, hard or It may be formulated as 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, Masene stearate Lubricating oils such as calcium, calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax may be included, and in the case of capsule formulations, a liquid carrier such as fatty oil may be further contained in addition to the above-mentioned substances.
- Formulations for parenteral administration of the composition for preventing or treating infectious diseases caused by enterotoxin E. coli include subcutaneous injection, intravenous injection or intramuscular injection, such as injection, suppository injection, or inhalation through the respiratory tract. It can be formulated for spraying, such as an aerosol.
- the composition of the present invention may be mixed with a stabilizer or buffer in water to prepare a solution or suspension, and it may be formulated for unit administration in ampoules or vials.
- a propellant or the like may be blended with the additive so that the water-dispersed concentrate or wet powder is dispersed.
- composition for preventing or treating an infectious disease caused by enterotoxin E. coli may include a preservative, a stabilizer, a wetting or emulsifying agent, a cryoprotectant, or an excipient.
- the preservative, stabilizer, or excipient may be included in the composition in an effective amount sufficient to reduce deterioration of the composition.
- the term "quality reduction” may include a decrease in the number of bacteria, a decrease in activity, or a combination thereof of the bacteriophage CJ_Eco_20-4 included in the composition.
- the composition contains an effective amount of a preservative, stabilizer, or excipient sufficient to reduce deterioration of the composition, so that, unlike bacteriophages existing in nature, the bacteriophage CJ_Eco_20-4 in the composition survives for a longer period of time Or the activity may be maintained.
- the preservative, stabilizing agent, or excipient may be used without limitation, as long as it can reduce deterioration of the composition.
- the stabilizer is alginate, sodium alginate, casein, sodium caseinate, cellulose, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, pectin, gelatin , gum arabic, xanthan gum, dextran, cyclodextrin, and may be any one or more selected from the group consisting of starch.
- the cryoprotectant may be included in the composition in an effective amount sufficient to reduce deterioration of the composition when the composition is in a lyophilized state.
- the composition may contain a cryoprotectant in an effective amount sufficient to reduce deterioration of the composition in the lyophilized state. It may be that the bacteriophage activity is maintained or the bacteriophage can survive even in the lyophilized composition.
- the cryoprotectant may be used without limitation, as long as it is intended to reduce the deterioration of the composition in a lyophilized state, those commonly used in the art.
- the cryoprotectant may be any one or more selected from the group consisting of glycerol, ethylene glycol, propylene glycol, dimethyl sulfoxide (DMSO), glucose, trehalose, maltodextrin, dextrin, skim milk and starch. have.
- Another aspect provides an antibiotic comprising the bacteriophage CJ_Eco_20-4 as an active ingredient.
- the bacteriophage CJ_Eco_20-4 is as described above.
- antibiotic means an agent capable of killing or inhibiting the growth of bacteria, and may be a generic term for preservatives, bactericides and antibacterial agents.
- the antibiotic containing the bacteriophage CJ_Eco_20-4 as an active ingredient has a very high specificity for enterotoxin E. coli compared to conventional antibiotics, so it can kill only specific pathogens without killing beneficial bacteria, and does not induce drug resistance. It can have the effect of extending the lifespan.
- the antibiotic may be prepared by a manufacturing method commonly used in the art.
- the antibiotic may include an additive in an effective amount sufficient to reduce deterioration in the quality of the antibiotic, and the additive may be a preservative, a stabilizer, an excipient, or a cryoprotectant.
- the antibiotic may be one in which the bacteriophage CJ_Eco_20-4 survives for a longer period of time or the activity is maintained in the antibiotic, unlike the bacteriophage existing in the natural state.
- the preservative, stabilizing agent, excipient, or cryoprotectant may be used without limitation, as long as it can reduce the deterioration of the antibiotic quality, those commonly used in the art.
- Another aspect provides a feed additive comprising the bacteriophage CJ_Eco_20-4 as an active ingredient. Another aspect provides a feed comprising the feed additive. Another aspect provides a drinking water additive comprising the bacteriophage CJ_Eco_20-4 as an active ingredient. Another aspect provides drinking water comprising the drinking water additive.
- the bacteriophage CJ_Eco_20-4 is as described above.
- the feed additive or drinking water additive is prepared in the form of a composition containing the bacteriophage CJ_Eco_20-4 and mixed with feed or drinking water, or the bacteriophage CJ_Eco_20-4 is directly added when preparing feed or drinking water. It may be used in a way.
- Bacteriophage CJ_Eco_20-4 included in the feed additive or drinking water additive may be in a liquid state or a dried solid state, and specifically, may be in a dried powder form.
- the drying method of the bacteriophage CJ_Eco_20-4 may be ventilation drying, natural drying, spray drying or freeze drying, but is not limited thereto.
- the bacteriophage CJ_Eco_20-4 may be mixed in a dry powder form in an amount of 0.05 to 10% by weight, specifically, 0.1 to 2% by weight of the weight of the feed additive or drinking water additive.
- the feed additive or drinking water additive containing the bacteriophage CJ_Eco_20-4 as an active ingredient may further include other additives as needed.
- the additives include, but are not limited to, binders, emulsifiers, preservatives, etc. added to prevent deterioration of feed or drinking water quality;
- binders binders, emulsifiers, preservatives, etc. added to prevent deterioration of feed or drinking water quality
- Raw materials other than the bacteriophage CJ_Eco_20-4 contained in the feed or drinking water may be used without limitation as commonly used in the art.
- Non-limiting examples of the feed include plant feeds such as grains, root fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, gourds or grain by-products; and animal feeds such as proteins, inorganic materials, oils and fats, minerals, oils and fats, single cell proteins, zooplankton, or food. These may be used alone or may be used in combination of two or more.
- the feed additive may be included in an amount of 0.05 to 10 parts by weight, for example, 0.1 to 2 parts by weight, based on 100 parts by weight of the feed.
- the drinking water additive may be included in an amount of 0.0001 to 0.01 parts by weight, for example, 0.001 to 0.005 parts by weight, based on 100 parts by weight of drinking water.
- the feed additive, feed, drinking water additive or drinking water may be prepared by a manufacturing method commonly used in the art.
- the feed additive, feed, drinking water additive or drinking water may include an additive in an effective amount sufficient to reduce deterioration thereof, and the additive may be a preservative, a stabilizer, an excipient, or a cryoprotectant.
- the feed additive, feed, drinking water additive or drinking water contains the additive, so that the bacteriophage CJ_Eco_20-4 survives or is active for a longer period of time in the feed additive, feed, drinking water additive or drinking water, unlike bacteriophages existing in nature. may be maintained.
- the preservative, stabilizing agent, excipient, or cryoprotectant may be used without limitation as long as it can reduce the deterioration of the quality of feed additives, feed, drinking water additives or drinking water.
- Another aspect provides a disinfectant comprising the bacteriophage CJ_Eco_20-4 as an active ingredient.
- a cleaning agent comprising the bacteriophage CJ_Eco_20-4 as an active ingredient.
- the bacteriophage CJ_Eco_20-4 is as described above.
- the formulation of the disinfectant or cleaning agent is not particularly limited, and may be prepared and used in a formulation commonly known in the art.
- the disinfectant or cleaning agent may be prepared by a manufacturing method commonly used in the art.
- the disinfectant may be sprayed to remove enterotoxin E. coli, and may be sprayed to an animal's active area, a slaughterhouse, a dead area, a cooking place, or a cooking facility, but is not limited thereto.
- the cleaning agent may be used for cleaning the skin surface or body parts of animals exposed or likely to be exposed to enterotoxin E. coli, but is not limited thereto.
- the disinfectant or cleaning agent may include an additive in an effective amount sufficient to reduce deterioration thereof, and the additive may be a preservative, a stabilizer, an excipient, or a cryoprotectant.
- the additive may be a preservative, a stabilizer, an excipient, or a cryoprotectant.
- the bacteriophage CJ_Eco_20-4 in the disinfectant or cleaning agent may survive or remain active for a longer period of time, unlike bacteriophages existing in nature.
- the preservative, stabilizing agent, excipient, or cryoprotectant may be used without limitation as long as it can reduce the deterioration of the quality of the disinfectant or cleaning agent.
- Another aspect is the bacteriophage CJ_Eco_20-4, or enterotoxin Escherichia coli, comprising administering a composition for the prevention or treatment of infectious diseases caused by enterotoxin E. coli comprising the same as an active ingredient to animals other than humans.
- a method of preventing or treating is provided.
- the bacteriophage CJ_Eco_20-4, a composition for preventing or treating an infectious disease caused by enterotoxin E. coli containing it as an active ingredient, and an infectious disease caused by enterotoxin E. coli are as described above.
- the infectious disease caused by the enterotoxin E. coli may be, for example, Escherichia coliosis.
- the prevention or treatment method is specifically, the bacteriophage CJ_Eco_20-4, or a composition comprising the same as an active ingredient, to an animal other than humans, which is infected or at risk of being infected by enterotoxin E. coli, in a pharmaceutically effective amount.
- the bacteriophage CJ_Eco_20-4 or a composition comprising the same as an active ingredient is administered to an animal in the form of a pharmaceutical formulation, or mixed with animal feed or drinking water in the form of a feed additive or drinking water additive to be administered through a method of feeding it can
- the route of administration of the bacteriophage CJ_Eco_20-4 or a composition comprising the same as an active ingredient may be administered through various routes, either oral or parenteral, as long as it can reach the target tissue, and specifically, oral, rectal, topical, intravenous, It can be administered in a conventional manner via intraperitoneal, intramuscular, intraarterial, transdermal, intranasal, inhalation and the like.
- the suitable total daily usage of the bacteriophage CJ_Eco_20-4 or a composition containing it as an active ingredient can be determined by the treating physician within the scope of correct medical judgment, which is obvious to those with ordinary skill in the art. .
- the specific pharmaceutically effective amount of the bacteriophage CJ_Eco_20-4 or a composition comprising it as an active ingredient for a specific animal is the type and extent of the reaction to be achieved, the age, weight, general health status, sex or diet of the subject, as well as,
- the bacteriophage CJ_Eco_20-4 or the composition containing the same as an active ingredient may be determined in consideration of the administration time, administration route and secretion rate of the composition, treatment period, etc. It may vary depending on several factors and similar factors well known in the medical arts.
- Animals other than humans are not particularly limited as long as they are animals that can be infected by enterotoxin E. coli.
- the animals may be birds and mammals, and specifically, may include chickens, ducks, pigs, and the like, but is not limited thereto.
- Another aspect provides the use of the bacteriophage CJ_Eco_20-4, or a composition for preventing or treating an infectious disease caused by enterotoxin E. coli, comprising the same as an active ingredient, for the preparation of a preventive or therapeutic agent for an infectious disease caused by enterotoxin E. coli.
- the bacteriophage CJ_Eco_20-4, a composition for preventing or treating an infectious disease caused by enterotoxin E. coli containing it as an active ingredient, and an infectious disease caused by enterotoxin E. coli are as described above.
- the infectious disease caused by the enterotoxin E. coli may be, for example, Escherichia coliosis.
- the novel bacteriophage CJ_Eco_20-4 has the effect of specifically killing enterotoxin E. coli, and has excellent acid resistance and heat resistance, so it is used for the prevention or treatment of infectious diseases caused by enterotoxin E. coli, antibiotics, feed additives, drinking water additives, feed, drinking water , can be widely used as a disinfectant or cleaning agent.
- Figure 2 is a graph of the result of confirming the pH stability of the novel bacteriophage CJ_Eco_20-4.
- Figure 3 is a graph of the results of confirming the stability at 60 °C of the novel bacteriophage CJ_Eco_20-4.
- Figure 4 is a graph of the result of confirming the lytic ability of the new bacteriophage CJ_Eco_20-4 enterotoxigenic Escherichia coli (ETEC) strain.
- Figure 5 is a graph of the result of confirming the lytic ability of the novel bacteriophage CJ_Eco_20-4 enterotoxigenic Escherichia coli (ETEC) strain.
- Example 1 Enterotoxigenic E. coli Escherichia coli : ETEC) Isolation of bacteriophage having apoptosis
- Example 1-1 Preparation of fecal sample preparation solution
- Fecal samples from pigs, poultry and cattle were collected from farms in Seoul, Gyeonggi, Chungcheong-do, and Gyeongsang-do, Korea, and 20 g of each sample was diluted in 80 mL of PBS and centrifuged at 10,000 rpm for 15 minutes. After filtering the supernatant through a 0.2 ⁇ m filter, 10% (w/v) aqueous sodium chloride solution was added to the filtrate and stored at 4° C. for 12 hours. Thereafter, 10% (w/v) polyethylene glycol 8000 (Sigma-Aldrich, Cat. No. P2139) was added thereto, stored at 4° C. for 12 hours, and centrifuged at 15,000 rpm for 1 hour for concentration.
- Example 1-2 Bacteriophage concentrate production
- ETEC strains isolated from fecal samples collected from livestock farms in Seoul, Gyeonggi, Chungcheong-do, and Gyeongsang-do, Republic of Korea, and ETEC strains distributed from the National Veterinary Science and Quarantine Service were treated with 2 mL of LB medium (Luria-Bertani medium; 10 g/L tryptone ( tryptone), 5 g/L yeast extract, 10 g/L sodium chloride) and incubated for 20 hours at 30° C. and 180 rpm, 1 mL of the ETEC strain culture solution in 50 mL of LB medium and in Example 1-1 1 mL of the obtained fecal sample pretreatment solution was inoculated and mixed and cultured at 30° C.
- LB medium Lia-Bertani medium
- tryptone tryptone
- yeast extract 5 g/L yeast extract
- Example 1-2 600 50 ⁇ L of the bacteriophage concentrate prepared in Example 1-2 0.7% (w/v) agar (BD DIFCO, Cat. No. 44164) 5 mL, and the same ETEC strain as used in Example 1-2 600
- the absorbance (O.D.) in nm was mixed with 50 ⁇ L of the shake culture medium to be 2, and double-layer agar plaque assay was performed using a plate medium with a diameter of 150 mm.
- the lysate formed on soft agar was eluted by punching it with a 200 ⁇ L tip and putting it in 0.5 mL of SM buffer.
- the solution containing the eluted bacteriophage was separated from the solution containing the pure bacteriophage by repeating the double-layer agar plaque assay until a single plaque of the same type was formed.
- the obtained bacteriophage-containing solution was filtered with a 0.2 ⁇ m filter, 10% (w/v) polyethylene glycol 8000 was added thereto, and stored at 4° C. for 12 hours. Thereafter, the supernatant was removed by centrifugation at 15,000 rpm for 1 hour for concentration, the precipitate was dissolved in 1 mL of SM buffer, filtered through a 0.2 ⁇ m filter, and the filtrate was stored at 4°C.
- Example 2 Whole genome sequencing (WGS) analysis of the isolated bacteriophage
- DNA was extracted from 1 mL of the bacteriophage concentrate purely isolated in Example 1-3 using the CsCl gradient method and the phage DNA isolation kit (Norgen Biotek-Corp. Kit, Cat. No. 46800). Whole genome analysis was performed by commissioning Macrogen Inc., and the genes were combined using De novo assembly software (SPAdes 3.13.0), and the open reading frame (ORF) was GeneMark.hmm and NCBI ORF This was done using a finder. The function of each ORF was annotated using BLASTP (E values of ⁇ 0.1) and PSI-BLAST (E value of ⁇ 0.005) programs.
- BLASTP E values of ⁇ 0.1
- PSI-BLAST E value of ⁇ 0.005
- the isolated bacteriophage was confirmed to have the nucleotide sequence of SEQ ID NO: 1 with 170,632 bp, 276 ORF, G + C content of 39.6%, which was previously reported Enterobacteria phage vB_EcoM_IME281 (MH051913.1) and 92% of the sequence Although it showed the identity, it was confirmed that there is no bacteriophage that matches 100% of all fragments, and it can be seen that the bacteriophage is a newly isolated bacteriophage.
- the new bacteriophage was named bacteriophage CJ_Eco_20-4, and was deposited with the Korean Culture Center of Microorganisms, an international depository under the Budapest Treaty as of January 18, 2021, and was given an accession number KCCM12936P.
- a CsCl gradient method was performed. Specifically, prepare a CsCl solution dissolved in SM buffer having a density of 1.7, 1.5, 1.45 or 1.3, and add the CsCl solution to a 15 mL ultracentrifuge tube (Beckman Coulter, Cat. No. Z00901SCA). 2 mL of the bacteriophage CJ_Eco_20-4 concentrate obtained in Example 1-3 was dispensed at the top by 2 mL to form a layer at a low density. After centrifugation at 4° C.
- bacteriophage CJ_Eco_20-4 is morphologically observed to have an icosahedral head and a contractile tail of about 100 nm in length, Caudovirales order, Myobiri It was found to belong to the Myoviridae family.
- a solution of pH 3, 4, 7, 7.5 or 10 (pH 3, 4: 0.2M sodium acetate solution; pH 7, 7.5: 0.2 M sodium phosphate solution; and pH 10: 0.2M Tris-HCl solution) was prepared. 450 ⁇ L of each pH solution and 50 ⁇ L of 2 X 10 10 PFU/mL of a bacteriophage solution were mixed and left at 4° C. for 2 hours, followed by double-layer agar plaque assay to evaluate titer increase or decrease.
- the bacteriophage CJ_Eco_20-4 is stable without losing activity in the range of pH 3 to 10, it was found that the bacteriophage having excellent acid resistance.
- bacteriophage CJ_Eco_20-4 In order to confirm that the bacteriophage CJ_Eco_20-4 is stable at high temperature, 500 ⁇ L of a bacteriophage solution of 2 X 10 8 PFU/mL was left at 60 ° C for 0, 3, 6 or 24 hours, then a Double-layer agar plaque assay was performed. Thus, titer increase and decrease were evaluated.
- the bacteriophage CJ_Eco_20-4 showed a decrease in activity of about 3.3 logs compared to the control exposed for 0 hours when exposed at 60 ° C. for 3 hours, and when exposed for 6 hours, compared to the control group of about 4.8 logs showed decreased activity. Therefore, it was found that the bacteriophage CJ_Eco_20-4 is a bacteriophage having excellent heat resistance.
- a total of 10 ETEC strains isolated from fecal samples collected from livestock farms in Seoul, Gyeonggi, Chungcheong, and Gyeongsang provinces in Korea and ETEC strains distributed from the National Veterinary Science and Quarantine Service were each LB After culturing in a liquid medium, 50 ⁇ L of each of the strain cultures was inoculated into 5 mL of 0.7% soft agar, poured into a Petri dish, and left for 5 minutes after plating. Then, 10 ⁇ L of the bacteriophage CJ_Eco_20-4 concentrate obtained in Example 1-3 was added dropwise on soft agar and then cultured at 30° C. for 20 hours stationary. The lysis range of the bacteriophage CJ_Eco_20-4 was evaluated according to the presence or absence of a lys plaque formed on the soft agar after the end of the culture.
- ETEC strain name Whether or not a plaque is formed ETEC strain name Whether or not a plaque is formed E.coli SNU273 + Philippines T5 + E.coli SNU287 + F18-31 + E.coli SNU337 + ETEC-3 + 12-194-1 + ETEC-4 + 12-364-1 + ETEC-11 +
- a Time-kill assay was performed. Specifically, the ETEC-11 strain isolated from fecal samples collected from pig farms in Gyeonggi-do, Korea was incubated for 3 hours at 30° C. and 180 rpm, and then diluted to have an absorbance (OD) of 0.2 at 600 nm to 2 X 10 Each of the 4 tubes was prepared by putting the culture solution of 8 CFU/mL into each tube by 30 mL.
- the concentrate of bacteriophage CJ_Eco_20-4 was prepared at 2 X 10 9 PFU/mL, and added to each tube containing the ETEC strain culture so that the multiplicity of infection (MOI) was 0.1, 1 or 10, and as a control, no bacteriophage was added.
- An ETEC strain culture medium was used. Each tube was incubated at 30° C. and 180 rpm for 6 hours, and 1 mL of the culture solution was taken at 1 hour intervals and absorbance was measured at 600 nm.
- control and the bacteriophage CJ_Eco_20-4 were cultured for 24 hours in the culture medium of the ETEC strain added at an MOI of 0.1, except that the absorbance of the culture was measured. As shown in 5, it was confirmed that the bacteriophage CJ_Eco_20-4 exhibited strong lytic activity up to 24 hours even when treated with a low MOI of 0.1.
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Abstract
Description
ETEC 균주명 | 용균반 형성 유무 | ETEC 균주명 | 용균반 형성 유무 |
E.coli SNU273 | + | 필리핀 T5 | + |
E.coli SNU287 | + | F18-31 | + |
E.coli SNU337 | + | ETEC-3 | + |
12-194-1 | + | ETEC-4 | + |
12-364-1 | + | ETEC-11 | + |
Claims (12)
- 장독소성 대장균(Enterotoxigenic Escherichia coli: ETEC)에 특이적인 사멸능을 가지고, 수탁번호 KCCM12936P로 기탁된 박테리오파지 CJ_Eco_20-4.
- 청구항 1의 박테리오파지 CJ_Eco_20-4를 유효성분으로 포함하는 장독소성 대장균에 의한 감염성 질환의 예방 또는 치료용 조성물.
- 청구항 2에 있어서, 상기 장독소성 대장균에 의한 감염성 질환은 대장균증인 것인, 조성물.
- 청구항 1의 박테리오파지 CJ_Eco_20-4를 유효성분으로 포함하는 항생제.
- 청구항 1의 박테리오파지 CJ_Eco_20-4를 유효성분으로 포함하는 사료 첨가제.
- 청구항 5의 사료 첨가제를 포함하는 사료.
- 청구항 1의 박테리오파지 CJ_Eco_20-4를 유효성분으로 포함하는 음용수 첨가제.
- 청구항 7의 음용수 첨가제를 포함하는 음용수.
- 청구항 1의 박테리오파지 CJ_Eco_20-4를 유효성분으로 포함하는 소독제.
- 청구항 1의 박테리오파지 CJ_Eco_20-4를 유효성분으로 포함하는 세척제.
- 청구항 1의 박테리오파지 CJ_Eco_20-4 또는 청구항 2의 조성물을 인간을 제외한 동물에 투여하는 단계를 포함하는, 장독소성 대장균에 의한 감염성 질환의 예방 또는 치료 방법.
- 청구항 11에 있어서, 상기 장독소성 대장균에 의한 감염성 질환은 대장균증인 것인, 방법.
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EP21928186.2A EP4299725A1 (en) | 2021-02-23 | 2021-06-15 | Novel bacteriophage having enterotoxigenic escherichia coli-specific bactericidal effect and antibacterial composition comprising same |
CN202180094511.2A CN117255854A (zh) | 2021-02-23 | 2021-06-15 | 具有产肠毒素性大肠杆菌特异性杀菌作用的新噬菌体及包含其的抗菌组合物 |
US18/278,567 US20240141302A1 (en) | 2021-02-23 | 2021-06-15 | Novel bacteriophage having enterotoxigenic escherichia coli-specific bactericidal effect and antibacterial composition comprising same |
JP2023551250A JP2024508813A (ja) | 2021-02-23 | 2021-06-15 | 腸管毒素原性大腸菌に対する特異的な死滅能を有する新規なバクテリオファージおよびこれを含む抗菌組成物 |
BR112023016870A BR112023016870A2 (pt) | 2021-02-23 | 2021-06-15 | Novo bacteriófago com efeito bactericida específico de escherichia coli enterotoxigênica e composição antibacteriana compreendendo o mesmo |
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KR1020210024235A KR102586835B1 (ko) | 2021-02-23 | 2021-02-23 | 장독소성 대장균에 대한 특이적인 사멸능을 가지는 신규한 박테리오파지 및 이를 포함하는 항균 조성물 |
KR10-2021-0024235 | 2021-02-23 |
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US6942858B1 (en) | 1996-04-15 | 2005-09-13 | Nymox Corporation | Compositions containing bacteriophages and methods of using bacteriophages to treat infections |
US8021657B2 (en) * | 2001-12-13 | 2011-09-20 | Nestec S.A. | Isolated phages and their use in food or pet food products |
KR20150118836A (ko) * | 2014-04-15 | 2015-10-23 | 씨제이제일제당 (주) | 신규 박테리오파지 및 이를 포함하는 조성물 |
KR20180068294A (ko) * | 2016-12-13 | 2018-06-21 | 주식회사 옵티팜 | 신규한 병원성 대장균 특이 박테리오파지 ec10 및 이를 포함하는 항균 조성물 |
KR101871338B1 (ko) * | 2016-12-22 | 2018-06-26 | 주식회사 옵티팜 | 신규한 병원성 대장균 특이 박테리오파지 ec14 및 이를 포함하는 항균 조성물 |
-
2021
- 2021-02-23 KR KR1020210024235A patent/KR102586835B1/ko active IP Right Grant
- 2021-06-15 US US18/278,567 patent/US20240141302A1/en active Pending
- 2021-06-15 WO PCT/KR2021/007481 patent/WO2022181891A1/ko active Application Filing
- 2021-06-15 BR BR112023016870A patent/BR112023016870A2/pt unknown
- 2021-06-15 CN CN202180094511.2A patent/CN117255854A/zh active Pending
- 2021-06-15 EP EP21928186.2A patent/EP4299725A1/en active Pending
- 2021-06-15 JP JP2023551250A patent/JP2024508813A/ja active Pending
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US6942858B1 (en) | 1996-04-15 | 2005-09-13 | Nymox Corporation | Compositions containing bacteriophages and methods of using bacteriophages to treat infections |
US8021657B2 (en) * | 2001-12-13 | 2011-09-20 | Nestec S.A. | Isolated phages and their use in food or pet food products |
KR20150118836A (ko) * | 2014-04-15 | 2015-10-23 | 씨제이제일제당 (주) | 신규 박테리오파지 및 이를 포함하는 조성물 |
KR20180068294A (ko) * | 2016-12-13 | 2018-06-21 | 주식회사 옵티팜 | 신규한 병원성 대장균 특이 박테리오파지 ec10 및 이를 포함하는 항균 조성물 |
KR101871338B1 (ko) * | 2016-12-22 | 2018-06-26 | 주식회사 옵티팜 | 신규한 병원성 대장균 특이 박테리오파지 ec14 및 이를 포함하는 항균 조성물 |
Non-Patent Citations (1)
Title |
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DATABASE NUCLEOTIDE 2 May 2018 (2018-05-02), ANONYMOUS : "Enterobacteria phage vB_EcoM_IME281. complete genome", XP055961143, retrieved from NCBI Database accession no. MH051913 * |
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KR20220120307A (ko) | 2022-08-30 |
BR112023016870A2 (pt) | 2023-12-05 |
EP4299725A1 (en) | 2024-01-03 |
CN117255854A (zh) | 2023-12-19 |
US20240141302A1 (en) | 2024-05-02 |
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