Novel bacteriophage kfsec11 having antibiotic-resistant and pathogenic escherichia coli-specific antibacterial activity and composition comprising the same

Abstract

The present invention relates to a novel bacteriophage KFSEC11 having specific antimicrobial activity against antibiotic-resistant and pathogenic Escherichia coli, and a composition containing the same. The bacteriophage KFSEC11 has the effect of specifically killing Escherichia coli and, in particular, exhibits specificity against antibiotic-resistant and pathogenic Escherichia coli and excellent lytic activity, thereby allowing it to be used in various ways, such as antimicrobial compositions, feed compositions, and compositions for treating infectious diseases caused by Escherichia coli. Furthermore, the bacteriophage KFSEC11 has excellent acid and heat resistance, allowing it to be utilized in various environments with a wide range of temperatures and pH values. It also offers the advantage of resolving issues regarding antibiotic-resistant bacteria, antibiotic residues in food, and broad host ranges.

Description

Technology Field

[0001] The present invention relates to antibiotic-resistant and pathogenic E. coli ( Escherichia coli The present invention relates to a novel bacteriophage KFSEC11 having specific antibacterial activity against ) and a composition containing the same. Background Technology

[0003] E. coli ( Escherichia coli ) is intestinal bacteria and ( Enterobacteriaceae It is a Gram-negative bacillus and facultative anaerobic bacterium belonging to the family, and is one of the normal bacterial flora present in the intestinal tract of various animals including mammals, and some Escherichia coli are pathogenic Escherichia coli that have acquired pathogenic factors (such as adhesion factors and heat-resistant toxins).

[0004] Depending on disease characteristics and the type of toxin, enterotoxigenic Escherichia coli are broadly classified E. coli , ETEC), Enteroinvasive Escherichia coli (Enteroinvasive E. coli , EIEC), enteropathogenic Escherichia coli (Enteropathogenic E. coli , EPEC), Enterohemorrhagic Escherichia coli (Enterohemorrhagic E. coli , EHEC), Enteroaggregative Escherichia coli (Enteroaggregative E. coli It can be classified into , EAEC, etc., and when pathogenic E. coli infects livestock, the main symptom is diarrhea, and the mortality rate due to severe dehydration is also very high.

[0005] In particular, enterohemorrhagic E. coli produce verotoxin, which causes ulcers on the colonic mucosa, leading to tissue abrasion and bleeding. Depending on the serotype, enterohemorrhagic E. coli includes O26, O103, O104, O146, and O157, with E. coli O157:H7 being the most representative. E. coli O157:H7 can exhibit pathogenicity with as few as 10 to 100 bacteria. The main symptoms are similar, including diarrhea, abdominal pain, fever, and vomiting; in severe cases, hemorrhagic colitis, hemolytic uremic syndrome, and thrombotic thrombocytopenia may occur.

[0006] Typically, the methods used to prevent and treat infectious diseases caused by the aforementioned intestinal bacteria include the use of vaccines and antibiotics. However, according to the U.S. Centers for Disease Control and Prevention (CDC), antibiotic resistance has recently been increasing in pathogenic E. coli O157:H7 against streptomycin, tetracycline, sulfisoxazole, ampicillin, and nalidixic acid; in Shigella against ciprofloxacin, azithromycin, tetracycline, and streptomycin; and in Salmonella against ciprofloxacin, azithromycin, and tetracycline. Consequently, the effectiveness of antibiotic treatment continues to decline. The cause of this increase in antibiotic resistance has been identified as the misuse and overuse of antibiotics, and regulations on antibiotic use have begun. Furthermore, as consumer demand for safe food and food safety increases, the use of antibiotics is gradually decreasing. Consequently, there is a growing demand for methods to control pathogenic bacteria without the use of antibiotics, and research utilizing bacteriophages that specifically and effectively kill bacteria is gaining attention as an alternative.

[0007] Meanwhile, bacteriophages are viruses that specifically infect bacteria to inhibit and suppress their growth. Compared to antibiotics, they have the advantages of high host specificity and relative stability across temperature and pH. Additionally, they can exhibit antimicrobial activity regardless of susceptibility to antibiotics, and they require shorter development times and lower development costs compared to antibiotics.

[0008] Accordingly, in order to solve the problems of antibiotic-resistant bacteria caused by the misuse and overuse of antibiotics, the problem of antibiotic residues in food, and the problem of a wide host range similar to that of past antibiotics, the inventors [developed] pathogenic E. coli ( Escherichia coli A novel bacteriophage capable of killing ) was isolated and purified from nature, and the present invention was completed through the analysis of its life cycle, bactericidal and bacteriostatic effects, stability against temperature, pH, UV, and agricultural antibiotics, and morphological characteristics. Prior art literature

[0010] Republic of Korea Registered Patent No. 10-0958139 The problem to be solved

[0011] The present invention aims to provide a novel bacteriophage that exhibits a specific killing ability against Escherichia coli.

[0012] In addition, the present invention aims to provide an antimicrobial composition comprising the above-mentioned bacteriophage.

[0013] In addition, the present invention aims to provide a disinfection composition comprising the above-mentioned bacteriophage.

[0014] In addition, the present invention aims to provide an animal feed composition comprising the above-mentioned bacteriophage.

[0015] In addition, the present invention aims to provide a composition for use as an auxiliary agent for agricultural antibiotics comprising the above-mentioned bacteriophage.

[0016] In addition, the present invention aims to provide a pharmaceutical composition for the prevention or treatment of infectious diseases caused by Escherichia coli comprising the above-mentioned bacteriophage.

[0017] In addition, the present invention aims to provide a method for preventing or treating an infectious disease caused by Escherichia coli, comprising the step of administering the bacteriophage to an individual other than a human. means of solving the problem

[0019] To achieve the above objective, the present invention relates to Escherichia coli ( Escherichia coli Provides a bacteriophage KFSEC11 (accession number: KCTC16165BP) having a specific killing ability for )

[0020] In addition, the present invention provides an antibiotic composition comprising the bacteriophage KFSEC11.

[0021] In addition, the present invention provides a disinfection composition comprising the bacteriophage KFSEC11.

[0022] In addition, the present invention provides an animal feed composition comprising the bacteriophage KFSEC11.

[0023] In addition, the present invention provides a composition for use as an auxiliary agent for agricultural antibiotics comprising the bacteriophage KFSEC11.

[0024] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of infectious diseases caused by Escherichia coli, comprising the bacteriophage KFSEC11.

[0025] In addition, the present invention provides a method for preventing or treating an infectious disease caused by Escherichia coli, comprising the step of administering the bacteriophage KFSEC11 to an individual other than a human.

[0026] The above E. coli may have antibiotic resistance or pathogenicity.

[0027] The above E. coli is E. coli ( Escherichia coli ) CMD01 or E. coli( Escherichia coli ) It could be O157:H7.

[0028] The above bacteriophage can exhibit lytic activity in the range of -20 to 50°C, lytic activity in the range of pH 3.0 to 11.0, and lytic activity at an ultraviolet wavelength of 365 nm.

[0029] The above bacteriophage can exhibit stable lytic activity when used in combination with an antibiotic, and the antibiotic may be an agronomic antibiotic of streptomycin, oxolinic acid, validamycin A, kasugamycin, oxytetracycline dehydrate, or polyoxin B.

[0030] The above bacteriophage can maintain lytic activity for more than 12 hours and can have excellent adsorption capacity for E. coli. Effects of the invention

[0032] The bacteriophage KFSEC11 of the present invention has the effect of specifically killing Escherichia coli, and by exhibiting specificity and excellent lytic activity against E. coli that is resistant to antibiotics and pathogenic, it can be used in various ways, such as in antimicrobial compositions, feed compositions, and compositions for treating infectious diseases caused by E. coli.

[0033] In addition, the above-mentioned bacteriophage KFSEC11 has excellent acid and heat resistance, allowing it to be utilized in various environments with a wide range of temperatures and pH levels. It also has the advantage of resolving issues such as antibiotic resistance, antibiotic residues in food, and broad host ranges. Brief explanation of the drawing

[0035] Figure 1 shows the results of confirming the antibiotic resistance and lytic activity of bacteriophage KFSEC11 against pathogenic E. coli CMD01. Figure 2 shows a transmission electron microscope image of bacteriophage KFSEC11. Figure 3 shows the results of the temperature stability experiment of bacteriophage KFSEC11. Figure 4 shows the pH stability test results of bacteriophage KFSEC11. Figure 5 shows the results of the UV stability experiment of bacteriophage KFSEC11. Figure 6 shows the results of the agricultural antibiotic stability test of bacteriophage KFSEC11. Figure 7 shows the results of the challenge assay analysis of bacteriophage KFSEC11. Figure 8 shows the results of the analysis of the adsorption capacity of bacteriophage KFSEC11 on E. coli. Figure 9 shows the results of the analysis of the first stage proliferation curve of bacteriophage KFSEC11. Specific details for implementing the invention

[0036] Hereinafter, embodiments and examples of the present invention will be described in detail with reference to the attached drawings so that those skilled in the art can easily implement the present invention. However, the present invention may be embodied in various forms and is not limited to the embodiments and examples described herein.

[0037] Throughout this specification, when a part is described as "comprising" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0038] The present invention relates to E. coli ( Escherichia coli Provides a bacteriophage KFSEC11 (accession number: KCTC16165BP) having a specific killing ability for )

[0039] The bacteriophage KFSEC11 of the present invention is a bacteriophage isolated from potato soil, named bacteriophage KFSEC11, and deposited at the Korea National Institute of Biological Resources on November 29, 2024, under accession number KCTC16165BP.

[0040] In the present invention, "bacteriophage" refers to a bacterium-specific virus that infects a specific bacterium and inhibits and suppresses the growth of said bacterium, and means a virus containing single or double stranded DNA (Deoxyribonucleic acid) or RNA (Ribonucleic acid) as genetic material.

[0041] The bacteriophage KFSEC11 of the present invention has excellent lytic activity against Escherichia coli and has a myovirium morphology having an angular head and a contractile tail ( Myoviridae It was confirmed that it belongs to the category, and that it has excellent stability against temperature, pH, ultraviolet rays, and agricultural antibiotics.

[0042] The above bacteriophage maintains lytic activity in the range of -20 to 50°C, but is not limited thereto.

[0043] The above bacteriophage maintains lytic activity in the pH range of 3.0 to 11.0, but is not limited thereto.

[0044] The above bacteriophage maintains lytic activity at an ultraviolet wavelength of 365 nm, but is not limited thereto.

[0045] The above bacteriophage maintains lytic activity for 12 hours or more, but is not limited thereto.

[0046] The above bacteriophage has excellent adsorption capacity for E. coli, but is not limited thereto.

[0047] In addition, the present invention provides an antibiotic composition comprising the bacteriophage KFSEC11.

[0048] The antibiotic composition of the present invention refers to a preparation that can kill bacteria when provided to animals in the form of a drug, and is a collective term for preservatives, disinfectants, antibiotics, and antimicrobial agents.

[0049] The bacteriophage KFSEC11 of the present invention exhibits specificity against infectious diseases of E. coli compared to existing antibiotics, so it can kill only specific pathogens without killing beneficial bacteria and can be used as a novel antibiotic with a longer life cycle compared to existing antibiotics because it does not induce drug resistance or intolerance.

[0050] The bacteriophage KFSEC11 of the present invention is Escherichia coli ( Escherichia coli Characterized by having antibacterial activity against strains belonging to ), wherein the E. coli may have antibiotic resistance or pathogenicity, preferably may have antibiotic resistance and pathogenicity, and more preferably E. coli ( Escherichia coli ) CMD01 or E. coli( Escherichia coli ) It may be O157:H7, but is not limited to this.

[0051] In addition, the present invention provides a disinfection composition comprising the bacteriophage KFSEC11.

[0052] The disinfection composition of the present invention can be usefully used as a disinfectant for hospitals and healthcare to prevent hospital infections, and can be used as a general household disinfectant, a disinfectant for food and cooking areas and facilities, and for disinfecting various growth supplies such as buildings, livestock, drinking water, bedding, egg trays, transport vehicles, and tableware.

[0053] In addition, the present invention provides an animal feed composition or an animal feed additive composition comprising the bacteriophage KFSEC11.

[0054] Antibiotics used as feed additives in the livestock or fisheries industries are used for the purpose of preventing disease. However, the administration of antibiotics for preventive purposes has become problematic because it increases the likelihood of antibiotic resistance and allows antibiotics remaining in livestock to be transferred to humans. Furthermore, if antibiotics are absorbed into the human body through meat, they can induce antibiotic resistance, potentially leading to the spread of disease. Additionally, there are many types of antibiotics mixed into feed, which increases the probability of multidrug-resistant bacteria. Therefore, the bacteriophage KFSEC11 of the present invention can be used as a new feed additive antibiotic that is environmentally friendly and solves the problems arising from the use of conventional antibiotics.

[0055] The feed additive composition of the present invention may additionally include other non-pathogenic microorganisms, and may include binders, emulsifiers, preservatives, etc. added to prevent quality degradation, and may include amino acid preparations, vitamin preparations, enzyme preparations, probiotics, flavoring agents, non-protein nitrogen compounds, silicate preparations, buffers, coloring agents, extractants, oligosaccharides, etc. added to the feed to increase utility, and may additionally include feed mixing agents, etc.

[0056] The feed composition of the present invention may be prepared by separately manufacturing bacteriophages in the form of feed additives and mixing them into the feed, or by directly adding them during feed production. The bacteriophages in the feed of the present invention may be in a liquid or dried state, preferably in the form of dried powder. Drying methods may include air drying, natural drying, spray drying, and freeze-drying, but are not limited thereto. The bacteriophages of the present invention may be mixed in powder form at a component ratio of 0.05 to 10 weight %, preferably 0.1 to 2 weight %, of the weight of the feed, but are not limited thereto. In addition, the feed may additionally include conventional additives that can enhance the shelf life of the feed in addition to the bacteriophages of the present invention.

[0057] The feed composition of the present invention includes, for plant-based uses, grains, root fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, meal, grain by-products, etc., and for animal-based uses, proteins, inorganic substances, oils and fats, mineral substances, oils and fats, single-cell proteins, zooplankton, leftover food, etc., but is not limited thereto.

[0058] In addition, the present invention provides a composition for use as an auxiliary agent for agricultural antibiotics comprising the bacteriophage KFSEC11.

[0059] The above bacteriophage KFSEC11 is characterized by maintaining lytic activity even in an agricultural antibiotic suspension and exhibiting stable lytic activity when used in combination with an agricultural antibiotic, and the above agricultural antibiotic may be streptomycin, oxolinic acid, validamycin A, kasugamycin, oxytetracycline dehydrate, or polyoxin B, but is not limited thereto.

[0060] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of infectious diseases caused by Escherichia coli, comprising the bacteriophage KFSEC11.

[0061] In the present invention, infectious diseases caused by E. coli include symptoms resulting from the disease, such as general symptoms of food poisoning like abdominal pain, diarrhea, vomiting, fever, and headache. In addition, in some patients, central nervous system symptoms such as renal failure and death due to complications like hemolytic uremic syndrome are included.

[0062] The pharmaceutical composition of the present invention may be used by applying or spraying it onto a diseased area, or it may also be administered via nasal spray, oral administration, or parenteral administration. In the case of parenteral administration, it may be administered via intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, or local administration.

[0063] Suitable application, spraying, and dosage of the pharmaceutical composition of the present invention vary depending on factors such as the formulation method, mode of administration, age, body weight, sex, severity of disease symptoms, food, time of administration, route of administration, excretion rate, and responsiveness, and a physician or veterinarian who is normally skilled can easily determine and prescribe a dosage effective for the intended treatment.

[0064] The pharmaceutical composition of the present invention may additionally include an adjuvant in addition to the active ingredient, and any adjuvant known in the art may be used without limitation.

[0065] In addition, the present invention provides a method for preventing or treating an infectious disease caused by Escherichia coli, comprising the step of administering the bacteriophage KFSEC11 to an individual other than a human.

[0066] In the method of the present invention, the bacteriophage or the composition containing it may be administered to animals in the form of a pharmaceutical preparation, or administered by mixing it into livestock feed or drinking water and feeding it. In the method of the present invention, the administration route of the bacteriophage or the composition containing it may be administered through various oral or parenteral routes as long as it can reach the target tissue.

[0067] It is obvious to those skilled in the art that the appropriate total daily dose of the bacteriophage administered in the method of the present invention can be determined by treatment within the scope of proper medical judgment. It is desirable to apply the specific therapeutically effective dose for a specific individual differently depending on various factors and similar factors well known in the pharmaceutical field, including the type and degree of response to be achieved, the age, body weight, general health condition, sex and diet, time of administration, route of administration and distribution ratio of the composition, duration of treatment, and drugs used together or simultaneously with the specific composition.

[0068] The term "individual" in this invention may include all animals excluding humans, preferably mammals or fish, but is not limited thereto.

[0069] The term "livestock" in this invention refers to a concept of useful animals that have been domesticated and improved by humans and live together with humans. Examples include, but are not limited to, pigs, cattle, chickens, horses, ducks, or dogs.

[0070] The term "PFU (plaque forming unit)" used in this invention is a unit that quantifies the lytic plaques that appear after bacteriophages kill bacteria.

[0071] As used in the present invention, the term "prevention" refers to any act of suppressing or delaying gastrointestinal diseases, etc. by administering a composition according to the present invention; "treatment" refers to any act of improving or beneficially changing the symptoms of an individual suspected of or suffering from a gastrointestinal disease by administering said composition; and "improvement" refers to any act of at least reducing parameters related to the state being treated, such as the degree of symptoms, by administering a composition containing the extract of the present invention.

[0072] In the present invention, the "%" used to indicate the concentration of a specific substance is (w / w) % for solid / solid, (w / v) % for solid / liquid, and (v / v) % for liquid / liquid, unless otherwise noted.

[0073] The present invention is to be explained in more detail through the following examples, but the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0075] [Example 1] Isolation and Identification of Antibiotic-Resistant and Pathogenic Escherichia Coli

[0076] 1-1. Isolation of E. coli from slaughterhouses, agricultural products, and the agricultural environment

[0077] During the period from 2019 to 2021 (3 years), agricultural environment samples such as wastewater from slaughterhouses and agricultural products, soil, and irrigation water from farms were collected and stored at 4℃, and the samples were applied to the test.

[0078] 25 g (or mL) of slaughterhouse, agricultural product, and agricultural environment samples were each suspended in 225 mL of EC medium (broth) and incubated at 37°C for 18 hours. Afterward, the culture medium was plated onto EMB agar (Eosin-Methylene Blue agar) and incubated at 37°C for 24 hours. Subsequently, colonies with a green sheen and a dark center that appeared on the agar medium were collected, and single colonies were isolated using TSA plates. The isolated single colonies were plated and incubated on m-Endo agar medium to confirm the formation of colonies with a red metallic sheen. Representative colonies were then selected and subcultured on the same medium to obtain pure isolation. For colonies showing a metallic luster on EMB agar medium, the characteristics and biochemical reactions of the strains were confirmed using the API20E kit (bioMerieux, France), and strains with an identification probability (identity %) of 90% or higher were selected and provisionally identified as Escherichia coli.

[0079] Five strains of Escherichia coli that were provisionally isolated and identified were two (PLC01, PLC02) from pumpkin leaves collected in Cheongdo, Gyeongsangbuk-do, one (PSC01) from soil in a peach cultivation field in Cheongdo, Gyeongsangbuk-do, and one (CMD01) from compost in a cattle slaughterhouse in Daegu, and were named PLC01, PLC02, PSC01, and CMD01, respectively.

[0080] Subsequently, the biochemical reactions of these four strain colonies were tested using the API20E test, and as shown in Table 1 below, all were identified as Escherichia coli with an identity probability (identity %) of 90% or higher.

[0081] Inspection Items / Strains PLC01 PLC02 PSC01 CMD01 ONPG + + + + ADH - - - - LDC + + + + ODC + - + - CIT - - - - H2S - - - - URE - - - - TDA - - - - IND + + + + VP - - - - GEL(gelatin) - - - - GLU(glucose) + + + + MAN(mannose) + + + + INO(inositol) - - - - SOR(sorbitol) + - + - RHA(rhamnose) + + + + SAC(sucrose) - - - - MEL(melibiose) + + + + AMY(amygdalin) - - - - ARA (arabinose) + + + + Oxidation of cytochrome - - - - Identity 99.9% 97.2% 99.9% 97.2% Result E. coli E. coli E. coli E. coli

[0083] 1-2. Analysis of Isolated E. coli Using 16S rRNA Sequencing

[0084] In order to perform 16S rRNA sequencing on the four strains provisionally identified in Example 1-1 above, genomic DNA of the strains was extracted using a DNA isolation kit (Qiagen). Subsequently, 16S rRNA sequencing was performed by Solgent Co. Using the 16S rRNA sequences obtained from the sequencing results, the nucleotide sequences were compared and analyzed with other E. coli strains registered in the database using the BLAST program (http: / / www.ncbi.nih.gov / BLAST), and homology values ​​were determined.

[0085] As a result, as shown in Table 2 below, all four isolated strains showed more than 99% homology, and Escherichia coli PLC01 is E. coli 44A and E. coli PLC02 are E. coli P15-385 and E. coli PSC01 are E. coli E2 and E. coli CMD01 are E. coli It was found to have the highest phylogenetic relationship with 84, and all were ultimately identified as E. coli.

[0086] strain The species that showed the highest degree of phylogenetic relationship Homology (Identity %) E. coli PLC01 E. coli 44A(KP789331.1) 99.79 E. coli PLC02 E. coli P15-385(CP075627.1) 100.00 E. coli PSC01 E. coli E2(CP048915.1) 99.93 E. coli CMD01 E. coli 84(MF399285.1) 100.00

[0088] 1-3. Antibiotic susceptibility testing of isolated and identified Escherichia coli

[0089] The four Escherichia coli strains finally identified in Examples 1-2 above E. coli PLC01, E. coli PLC02, E. coli PSC01, E. coli The susceptibility of CMD01 to a total of 16 antibiotics was investigated using the disk diffusion method according to the CLSI (Clinical and Laboratory Standards Institute) method. Single colonies of E. coli strains pure-cultured in TSA were suspended in MH medium (Mueller Hinton broth) (~10 8 A bacterial suspension was applied to a sterile cotton swab (CFU / mL) and spread onto the surface of an MH agar medium, after which an antibiotic disc was placed on top. After incubating at 37°C for 24 hours, the size of the inhibition zone was measured in mm, and antibiotic susceptibility and resistance were determined according to the cut-off value criteria provided by CLSI.

[0090] As a result, as shown in Table 3 below, E. coli PLC01 showed resistance to 9 antibiotics, and PLC02 and CMD01 showed resistance to 8 antibiotics, and E. coli PSC01 showed moderate resistance to four antibiotics. Therefore, subsequent bacteriophage isolation tests were conducted using the antibiotic-resistant E. coli.

[0091] antibiotics E. coli PLC01 E. coli PLC02 E. coli CMD01 E. coli PSC01 Amikacin S S S S Gentamicin R S S S Kanamycin R R R I Streptomycin R R R I Ceftriaxone S S S S Ciprofloxacin I I I I Nalidixic acid R I I S Ampicillin R R R S Amoxicillin-clavulanic acid R R R S Azithromycin S R R S Meropenem S S S S Chloramphenicol R S S S Trimethoprim-sulfamethazole S R R S Sulfisoxazole R R R I Cefoxitin S S S S Tetraccline R R R S S: Susteptible, R: Resistant, I: Intermediate

[0093] [Example 2] Isolation and culture of a novel bacteriophage

[0094] 2-1. Screening of bacteriophages infecting antibiotic-resistant and pathogenic E. coli and isolation of single bacteriophages

[0095] 25 mL of potato soil sample from a farm in Daegu was prepared as a sample solution, and the soft agar overlay method was performed using it. The soft agar overlay method is a method of observing bacteriophage lysis using host cells grown on top agar (attaching 0.4% agar to a solid medium).

[0096] Specifically, 25 mL of sample and the above antibiotic-resistant and pathogenic E. coli ( Escherichia coli ) CMD01 Shaking Culture Medium (OD 640 = 0.3) 1% was mixed and incubated at 37°C at 160 rpm for 16 hours, after which the culture medium was centrifuged at 8,000 rpm for 10 minutes, and the supernatant was filtered through a 0.25 µm filter. Next, 4 mL of TA soft agar [0.4%(w / v) agar, nutrient broth, NaCl, MgSO4, CaCl2] and E. coli CMD01 shaken culture medium (OD) were placed on a TSA plate. 640 = 0.3) After pouring 200 μl of the mixture and letting it solidify, 10 μl of the sample solution was added on top and incubated at 37°C for 16 hours to confirm that a lysis plate was formed (Fig. 1).

[0097] A single bacteriophage was to be isolated from the lysate formed above. Specifically, 900 μl of SM solution (50 mM Tris-HCl, 100 mM NaCl, 10 mM MgSO4) was added to the lysate and vortexed at room temperature for 1 hour to obtain a bacteriophage solution.

[0099] 2-2. Mass Culture and Purification of Bacteriophages

[0100] The bacteriophage obtained in Example 2-1 above was cultured in large quantities using E. coli CMD01, and the bacteriophage was purified therefrom.

[0101] Specifically, Escherichia coli CMD01 was cultured with shaking and dispensed into 3 ml of TA broth (nutrient broth, NaCl, MgSO4, CaCl2), and cultured for 2 hours. Then, the bacteriophage obtained in Example 2-1 was dispensed and cultured for 2 hours in the same manner, after which the bacteriophage was isolated by centrifugation and filtering. The same method was performed by adjusting the amount of TA broth in the order of 8, 20, 200, and 800 ml. Next, polyethylene glycol (PEG) and 1 M NaCl were added to achieve a final concentration of 10% (w / v), and the mixture was allowed to stand for an additional 16 hours at 4°C. The precipitate was obtained by centrifugation at 8,000 rpm for 20 minutes at 4°C.

[0102] The purified bacteriophages were subjected to CsCl density gradient ultracentrifugation (density gradients: 1.20, 1.30, 1.40, 1.45, 1.50, 1.70 g / mL) at 22,000 rpm for 2 hours at 4°C, and after dialysis using SM buffer (50 mM Tris-HCl, 100 mM NaCl, 10 mM MgSO4), were stored at 4°C.

[0103] The inventors isolated and purified the bacteriophage from potato soil that specifically kills antibiotic-resistant and pathogenic E. coli, and named it "KFSEC11".

[0105] [Example 3] Analysis of the infectivity of bacteriophage KFSEC11 against other pathogenic strains

[0106] In order to confirm whether the bacteriophage KFSEC11 purified in Example 2 above infects pathogenic strains other than E. coli, the range of host inhibition was determined using a dot assay.

[0107] Specifically, 24 types of pathogenic bacteria were cultured separately, and a dot assay was performed on each culture medium to confirm whether the purified KFSEC11 formed lytic spots, and the results are shown in Table 4 below.

[0108] strain name Whether lytic plaques are formed Escherichia coli CMD01 (host) + E. coli O157:H7 ATCC 10536 + E. coli O157:H7 ATCC 43895 + E. coli O157:H7 ATCC 700599 - E. coli ATCC BAA-2192 - E. coli ATCC BAA-2196 - Bacillus cereus ATCC 14579 - B. cereus ATCC 13061 - B. cereus ATCC 21768 - Listeria monocytogenes ATCC 7644 - L. monocytogenes NCCP 15743 - L. monocytogenes KBN10P05036 - L. monocytogenes KBN10P05037 - Salmonella Enteritidis ATCC 13076 - S. Typhimurium ATCC 13311 - S. Dublin NCCP 13700 - S. Heidelberg NCCP 13698 - S. Montevideo NCCP 13704 - S. Newport NCCP 13686 - S. Panama NCCP 13694 - Shigella sonnei ATCC 9290 - S. boydii NCCP 11190 - S.flexeneri 2457 - Staphylococcus aureus ATCC 25923 - Yersinia enterocolitica ATCC 55075 -

[0110] [Example 4] Analysis of Morphological Characteristics of Bacteriophage KFSEC11

[0111] Transmission electron microscopy (TEM) observations were performed for the morphological classification of bacteriophage KFSEC11.

[0112] Specifically, after absorbing bacteriophage KFSEC11 onto a copper TEM grid, it was negatively stained with 2% phosphotungstic acid and dried. The morphology of the bacteriophage KFSEC11 was observed using a bio-electron transmission microscope H-7100 (Hitachi Ltd., Japan) at a voltage of 100 kV.

[0113] As a result, the length and width of the head of bacteriophage KFSEC11 were confirmed to be 64.02 ± 3.66 nm and 55.37 ± 7.77 nm, respectively, and the length of the tail of bacteriophage KFSEC11 was 128.41 ± 16.54 nm. Through this, it was found that bacteriophage KFSEC11 has an icosahedral head and a contractile tail, and a myovirium band ( Myoviridae It was estimated to belong to ) (Fig. 2).

[0115] [Example 5] Investigation of Temperature Stability of Bacteriophage KFSEC11

[0116] To confirm the temperature stability of bacteriophage KFSEC11, a lysis plate test was performed after exposure to various temperatures (-20℃, 4℃, 10℃, 22℃, 37℃, 50℃, 60℃, and 70℃).

[0117] Specifically, 1.30 × 10 of each of the above temperatures 7 After leaving 1 mL of KFSEC11 with a PFU / mL concentration at room temperature for 1 hour, the bacteriophage titer was measured using the lysis plate test.

[0118] As a result, although the lytic activity of bacteriophage KFSEC11 decreased at 60℃, there was no significant difference between -20℃ and 50℃, confirming that the lytic activity was stably maintained over a wide temperature range (Fig. 3).

[0120] [Example 6] Investigation of pH stability of bacteriophage KFSEC11

[0121] To confirm the pH stability of bacteriophage KFSEC11, a lysis plaque test was performed after exposure to various pH values ​​(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12).

[0122] Specifically, 900 μl of each of the above pH solutions and 3.52 × 10 8 100 μl of KFSEC11 at a concentration of PFU / mL was mixed and left to stand at room temperature for 1 hour, after which the bacteriophage titer was measured using the lysis plate test.

[0123] As a result, bacteriophage KFSEC11 was found to have no significant difference from pH 3 to pH 11, thus confirming that lytic activity is maintained over a wide range of pH (Fig. 4).

[0125] [Example 7] Investigation of UV stability of bacteriophage KFSEC11

[0126] To confirm the stability of bacteriophage KFSEC11 against UV, the stability of bacteriophage KFSEC11 was tested by exposing it to UV light.

[0127] Specifically, 3.52 × 10⁻⁶ 8 After inoculating 2 ml of bacteriophage KFSEC11 solution at a concentration of PFU / ml into a 12-well plate and incubating under UV light (wavelength 365 nm) for 1 hour, the bacteriophage titer was measured using a lysis plate test.

[0128] As a result, bacteriophage KFSEC11 was found to maintain lytic activity without significant difference under UV (Fig. 5).

[0130] [Example 8] Investigation of the stability of bacteriophage KFSEC11 as an agrobiotic antibiotic

[0131] To confirm the stability of bacteriophage KFSEC11 against agricultural antibiotics, bacteriophage KFSEC11 was exposed to various agricultural antibiotics to test the stability of the bacteriophage's potency.

[0132] Specifically, 3.52 × 10⁻⁶ 8 100 μl of KFSEC11 solution at a concentration of PFU / ml was mixed with 900 μl of each of seven types of agrobiotic antibiotics [streptomycin (STR; 1 mg / ml), oxolinic acid (OXA; 1 mg / ml), validamycin A (VMA; 1 mg / ml), kasugamycin (KAG; 1 mg / ml), oxytetracycline dihydrate (OTC; 1 mg / ml), polyoxin B (POB; 1 mg / ml), oxytetracycline dihydrate (OTC; 0.5 mg / ml)], left to stand for 1 hour, and then the titer was measured by the lysis plate test.

[0133] As a result, it was confirmed that bacteriophage KFSEC11 maintained lytic activity against six types of antibiotics without significant difference (Fig. 6).

[0135] [Example 9] Analysis of the challenge assay of bacteriophage KFSEC11

[0136] E. coli CMD01 culture was inoculated into 100 mL of TSB at 1% (v / v), and when the absorbance value of the culture reached approximately 0.486 at 640 nm (10 8The culture was incubated at 190 rpm at 37°C until the CFU / mL level was reached. Subsequently, KFSEC11 and the bacterial culture medium were mixed and adjusted to MOI (Multiplicity of Infection) levels of 0.01, 0.1, 1.0, and 10, and incubated at 110 rpm at 37°C for 24 hours. An E. coli CMD01 culture medium without added KFSEC11 was used as a control. The lytic activity of the phage was confirmed by measuring the absorbance values ​​at 640 nm at 2-hour intervals to monitor bacterial growth.

[0137] As a result, compared to the control group in all cases regardless of MOI It was confirmed that E. coli CMD01 was significantly inhibited, thereby confirming that the lytic activity of KFSEC11 was sustained for 12 hours (Fig. 7). In particular, it was confirmed that lytic activity and inhibitory ability were maintained for more than 12 hours even at the lowest MOI of 0.001. Therefore, it was confirmed that bacteriophage KFSEC11 has excellent persistence with a duration of more than 12 hours.

[0139] [Example 10] Analysis of Adsorption Capacity and One-Step Growth Curve of Bacteriophage KFSEC11

[0140] After culturing Escherichia coli CMD01 until the absorbance value reached 0.5 at 640 nm (10 8 CFU / mL), E. coli culture medium and KFSEC11 were mixed with an MOI of 0.001. The mixture was statically cultured at 37°C for 10 minutes, and lysis plate analysis was performed on samples obtained at 2.5-minute intervals to measure the adsorption capacity of the bacteriophage.

[0141] As a result, it was confirmed that 90% of the bacteriophage KFSEC11 was adsorbed to pathogenic E. coli within 15 minutes of inoculation (Fig. 8).

[0142] In addition, for the analysis of the first growth curve, Escherichia coli CMD01 was cultured until the absorbance value at 640 nm became 0.5 (10 8(CFU / mL), centrifuged at 16,000 g for 1 minute at 4°C, and the supernatant was removed to precipitate only the bacterial cells. Subsequently, the precipitate was diluted in 0.5 mL of TSB medium, mixed with bacteriophage KFSEC11 adjusted to an MOI of 0.001, and incubated at 37°C for 10 minutes, the precipitation time confirmed in the previous experiment. The cultured mixed sample was centrifuged at 13,000 rpm for 1 minute, the precipitate was diluted in 50 mL of TSB medium, and incubated at 37°C for 1 hour. Samples were collected every 5 minutes during incubation for lysis plaque analysis. Additionally, to determine the eclipse period, 1% chloroform was added to the collected samples, and lysis plaque analysis was performed in the same manner as described above.

[0143] Based on the analysis of the first-stage proliferation curve, the burst size of KFSEC11 is 10 The PFU / infected cell, latent time was confirmed to be 10 minutes and the eclipse period was 10 minutes, confirming that the bacteriophage KFSEC11 of the present invention can adsorb to E. coli in a short time and is a phage with a high burst size (Fig. 9).

[0145] Depository Name: Korea Research Institute of Biotechnology and Bioengineering Biological Resource Center (KCTC) Trustee Number: KCTC16165BP Date of Deposit: 2024-11-29

Claims

Claim 1 E. coli ( Escherichia coli Bacteriophage KFSEC11 deposited under accession number KCTC16165BP, having a specific killing ability for ) Claim 2 In paragraph 1, the above E. coli is a bacteriophage KFSEC11 having antibiotic resistance. Claim 3 In paragraph 1, the above-mentioned E. coli is a pathogenic bacteriophage KFSEC11. Claim 4 In paragraph 1, the above E. coli is E. coli ( Escherichia coli ) CMD01 or E. coli( Escherichia coli ) Bacteriophage KFSEC11 that is O157:H7. Claim 5 In claim 1, the bacteriophage KFSEC11 is a bacteriophage that exhibits lytic activity in the range of -20 to 50℃. Claim 6 In claim 1, the bacteriophage KFSEC11 is a bacteriophage that exhibits lytic activity in the pH range of 3.0 to 11.

0. Claim 7 In claim 1, the bacteriophage is bacteriophage KFSEC11, which exhibits lytic activity at an ultraviolet wavelength of 365 nm. Claim 8 In claim 1, the bacteriophage KFSEC11 is a bacteriophage that exhibits stable lytic activity when used in combination with an antibiotic. Claim 9 In claim 8, the antibiotic is one or more agronomic antibiotics selected from the group consisting of streptomycin, oxolinic acid, validamycin A, kasugamycin, oxytetracycline dehydrate, and polyoxin B, bacteriophage KFSEC11. Claim 10 In claim 1, the bacteriophage is a bacteriophage KFSEC11 that maintains lytic activity for 12 hours or more. Claim 11 In claim 1, the bacteriophage is a bacteriophage KFSEC11 having excellent adsorption capacity for E. coli. Claim 12 Antibiotic composition comprising the bacteriophage KFSEC11 of claim 1. Claim 13 A disinfection composition comprising the bacteriophage KFSEC11 of claim 1. Claim 14 Animal feed composition comprising the bacteriophage KFSEC11 of claim 1. Claim 15 A composition for adjuvating agricultural antibiotics comprising the bacteriophage KFSEC11 of claim 1. Claim 16 A pharmaceutical composition for the prevention or treatment of infectious diseases caused by Escherichia coli, comprising the bacteriophage KFSEC11 of claim 1. Claim 17 A method for preventing or treating an infectious disease caused by Escherichia coli, comprising the step of administering the bacteriophage KFSEC11 of claim 1 to an individual other than a human.

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