Antibiotic-resistant and pathogenic escherichia coli-specific novel bacteriophage kfsec8 and antibacterial composition comprising the same

Abstract

The present invention relates to a novel bacteriophage KFSEC8 having a specific killing ability against antibiotic-resistant and pathogenic Escherichia coli, and an antimicrobial composition containing the same. The bacteriophage KFSEC8 has the effect of specifically killing Escherichia coli and, in particular, exhibits specificity against antibiotic-resistant Escherichia coli and excellent lytic activity; thereby, it can be used in various fields such as antibiotic compositions, compositions for the prevention or treatment of infectious diseases caused by Escherichia coli, feed additive compositions, disinfectants, or cleaning agents. Furthermore, the bacteriophage KFSEC8 exhibits excellent acid resistance, heat resistance, and stability against UV exposure, allowing it to be utilized in various environments with a wide range of temperatures and pH levels. It also offers the advantage of resolving issues related to antibiotic-resistant bacteria, antibiotic residues in food, and broad host ranges.

Description

Technology Field

[0001] The present invention relates to E. coli having antibiotic resistance and pathogenicity ( Escherichia coli The present invention relates to a novel bacteriophage KFSEC8 exhibiting specific killing ability against ) and an antimicrobial composition containing the same. Background Technology

[0002] E. coli ( Escherichia coli ) is of the Enterobacteriaceae Escherichia It is a Gram-negative bacillus belonging to the genus Escherichia coli. Escherichia coli can generally be classified into non-pathogenic E. coli, which normally exist in the large intestine, and pathogenic E. coli, which acquires specific pathogenic factors to cause disease. Although E. coli is a microorganism that constitutes a large portion of the intestines of warm-blooded animals, most are non-pathogenic bacteria that can cause opportunistic infections. Pathogenic E. coli that has acquired pathogenic factors exhibits symptoms such as diarrhea, abdominal pain, vomiting, fatigue, dehydration, and uremics; depending on the disease characteristics and the type of toxin, it is typically classified as enterohemorrhagic E. coli (EHEC). E . coli ), Enterotoxigenic Escherichia coli (ETEC, Enterotoxigenic E . coli ), Enteroinvasive Escherichia coli (EIEC, Enteroinvasive E . coli ), enteropathogenic Escherichia coli (EPEC, pathogenic E . coli ), Enteroaggregative Escherichia coli (EAEC, Enteroaggregative E . coli It is classified into five categories. Pathogenic E. coli can be transmitted through water contaminated with animal feces, food washed with contaminated water, or food ingredients contaminated during the slaughtering process. In particular, if groundwater or river water contaminated with feces or livestock wastewater is used as agricultural water without purification, there is a possibility that agricultural products may also be contaminated with pathogenic E. coli.

[0003] Generally, antibiotics and other drugs are used to control pathogenic E. coli and to prevent and treat infectious diseases caused by them; however, due to their recent misuse and overuse, antibiotic-resistant bacteria exhibiting resistance to various antibiotics are emerging. Accordingly, the World Health Organization (WHO) introduced the Global Antimicrobial Surveillance System (GLASS) to monitor the global status of antibiotic resistance and to respond rapidly. In Korea, the Global Antimicrobial Resistance Surveillance System in Korea (KorGLASS) was also established in 2016 to reflect domestic conditions. A recent survey of antibiotic resistance status among major resistant bacteria isolated from general hospitals collected through KorGLASS from 2016 to 2019 revealed that E. coli accounted for the highest proportion at 33,420 strains. In addition, according to a survey by the domestic "National Antibiotic Resistance Management Plan," more than 70% of domestic pig-derived E. coli were found to be resistant to ampicillin, chloramphenicol, sulfisoxazole, streptomycin, and tetracycline (Korea Food and Drug Administration, 2021). Therefore, given the high frequency of infections caused by antibiotic-resistant and pathogenic E. coli, there is a need to develop antibiotics with new mechanisms of action that can effectively control them.

[0004] Accordingly, the inventors sought to develop a bacteriophage capable of specifically and effectively killing bacteria existing in nature as a new alternative. A bacteriophage (phage) is a bacterial virus that uses bacteria as a host; in particular, lytic bacteriophages possess host specificity, selectively invading only specific bacteria to proliferate and cause lysis. After attaching to bacteria, lytic bacteriophages replicate their genetic material (DNA) inside the bacterial cell, and the generated genetic material and proteins are assembled to create new bacteriophages. Subsequently, these progeny bacteriophages are released outside the cell, killing the bacteria by destroying the host bacteria's cell membrane and cell wall using various enzymes such as holin and endolysin. In fact, the U.S. Food and Drug Administration (FDA) has recognized bacteriophages as Generally Recognized as Safe (GRAS) and has permitted their use on the surfaces of food or equipment for certain products. EcoShield, developed by Intralytix Inc., is a representative example of a bacteriophage mixture product approved for use in food for controlling E. coli.

[0005] However, research on novel bacteriophages for preventing or treating infections caused by antibiotic-resistant E. coli, which remain a public health issue, is still insufficient. Therefore, there is a need to develop technologies for bacteriophages with lytic activity specific to antibiotic-resistant pathogenic E. coli, as well as for their applications. The problem to be solved

[0006] The present invention aims to provide a novel bacteriophage KFSEC8 that exhibits specific killing ability against antibiotic-resistant and pathogenic Escherichia coli.

[0007] In addition, the present invention aims to provide an antimicrobial composition comprising the bacteriophage KFSEC8.

[0008] 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 bacteriophage KFSEC8 to an individual other than a human. means of solving the problem

[0009] To achieve the above objective, the present invention relates to Escherichia coli ( Escherichia coli Provides a bacteriophage KFSEC8 (accession number: KCTC15458BP) having a specific killing ability for )

[0010] In addition, the present invention provides an antibiotic composition comprising the bacteriophage KFSEC8.

[0011] In addition, the present invention provides a feed additive composition comprising the bacteriophage KFSEC8 and a feed composition comprising the feed additive composition.

[0012] In addition, the present invention provides a disinfectant comprising the bacteriophage KFSEC8.

[0013] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of infectious diseases caused by Escherichia coli, comprising the bacteriophage KFSEC8.

[0014] 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 KFSEC8 to an individual other than a human.

[0015] The above E. coli may be antibiotic-resistant, pathogenic, or both antibiotic-resistant and pathogenic.

[0016] The above E. coli is E. coli O157:H7 ATCC 10536( Escherichia coli O157:H7 ATCC 10536), E. coli O157:H7 ATCC 43895( Escherichia coli O157:H7 ATCC 43895), Escherichia coli ATCC BAA-2196( Escherichia coli ATCC BAA-2196), E. coli PLC01( Escherichia coli PLC01), E. coli PLC02( Escherichia coli PLC02), E. coli PSC01( Escherichia coli PSC01) and E. coli CMD01( Escherichia coli It may be one or more selected from the group consisting of CMD01).

[0017] The above bacteriophage may exhibit lytic activity at -80 to 60°C, lytic activity at pH 2 to 11, and lytic activity at an ultraviolet wavelength of 365 nm.

[0018] The above bacteriophage may include the nucleotide sequence of SEQ ID NO. 1. Effects of the invention

[0019] The bacteriophage KFSEC8 of the present invention has the effect of specifically killing Escherichia coli and, in particular, exhibits specificity against antibiotic-resistant Escherichia coli and excellent lytic activity, and can be used in various fields such as antibiotic compositions, compositions for the prevention or treatment of infectious diseases caused by Escherichia coli, feed additive compositions, disinfectants, or cleaning agents.

[0020] In addition, the above-mentioned bacteriophage KFSEC8 exhibits excellent acid resistance, heat resistance, and stability against UV exposure, 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

[0021] Figure 1 shows the results of confirming the lytic plaque-forming activity of bacteriophage KFSEC8 against Escherichia coli. Figure 2 shows the results of confirming the morphological characteristics of bacteriophage KFSEC8 through an electron microscope. Figure 3 shows the results of confirming the temperature stability of bacteriophage KFSEC8. Figure 4 shows the results of confirming the pH stability of bacteriophage KFSEC8. Figure 5 shows the results of confirming the UV stability of bacteriophage KFSEC8. Figure 6 shows the results of a challenge assay confirming the bactericidal and bacteriostatic effects of bacteriophage KFSEC8 against Escherichia coli. Specific details for implementing the invention

[0022] 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.

[0023] 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.

[0024] The present invention relates to E. coli ( Escherichia coli Provides bacteriophage KFSEC8 (accession number: KCTC15458BP) having a specific killing ability for ).

[0025] The bacteriophage KFSEC8 of the present invention is a bacteriophage isolated by collecting a sample from the soil of a field where tomatoes are grown, and was named bacteriophage KFSEC8 and deposited at the National Center for Biological Resources on May 25, 2023, under accession number KCTC15458BP.

[0026] 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 comprising single or double-stranded DNA or RNA as genetic material.

[0027] The bacteriophage KFSEC8 of the present invention has excellent lytic activity against Escherichia coli, and the bacteriophage KFSEC8 has a sifobiridae having an angular head and a non-contractile tail ( Siphoviridae It was confirmed that it belongs to the group, and that it has excellent stability against temperature, pH, ultraviolet rays, and agricultural antibiotics.

[0028] The bacteriophage KFSEC8 of the present invention maintains lytic activity within the range of -80˚C to 60˚C, but is not limited thereto.

[0029] The bacteriophage KFSEC8 of the present invention maintains lytic activity at pH 2.0 to pH 11.0, but is not limited thereto.

[0030] The bacteriophage KFSEC8 of the present invention maintains lytic activity after irradiation with ultraviolet light at a wavelength of 365 nm, but is not limited thereto.

[0031] In addition, the present invention provides an antibiotic composition comprising the bacteriophage KFSEC8.

[0032] In the present invention, "antibiotic composition" 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.

[0033] Since the bacteriophage KFSEC8 of the present invention has higher specificity for infectious diseases of E. coli compared to existing antibiotics, it can kill only specific pathogens without killing beneficial bacteria and does not induce drug resistance or intolerance, so it can be used as a novel antibiotic with a longer product life cycle compared to existing antibiotics.

[0034] The bacteriophage KFSEC8 of the present invention is Escherichia coli ( Escherichia coli It is characterized by having antibacterial activity against strains belonging to ). Specifically, the E. coli is characterized by having antibiotic resistance or pathogenicity, and more specifically, Escherichia coli O157:H7 ATCC 10536, E. coli O157:H7 ATCC 43895, E. coli ATCC BAA-2196, E. coli PLC01, E. coli PLC02, E. coli PSC01, E. coli It was confirmed that there is lytic activity against CMD01.

[0035] In addition, the present invention provides a feed additive composition comprising the bacteriophage KFSEC8 and a feed composition comprising the feed additive composition.

[0036] 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 KFSEC8 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.

[0037] Other non-pathogenic microorganisms may be additionally added to the feed additive composition of the present invention. Microorganisms that may be added include Bacillus subtilis, which is capable of producing proteolytic enzymes, lipidolytic enzymes, and sugar-converting enzymes ( Bacillus subtilis Lactobacillus strains having physiological activity and organic matter decomposition ability under anaerobic conditions such as Bacillus subtilis ( and bovine stomach) Lactobacillusspp.), Aspergillus oryzae, which shows effects of increasing livestock body weight, increasing milk production, and improving the digestibility and absorption rate of feed ( Aspergillus oryzae Filamentous fungi such as ) and Saccharomyces cerevisiae ( Saccharomyces cerevisiae It can be selected from a group consisting of yeasts such as ).

[0038] The feed additive composition of the present invention 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.

[0039] 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.

[0040] The feed composition of the present invention includes plant-based grains, root fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, meal, grain by-products, etc., and animal-based proteins, inorganic substances, oils and fats, minerals, oils and fats, single-cell proteins, zooplankton, leftover food, etc., but is not limited thereto.

[0041] In addition, the present invention provides a disinfectant comprising the bacteriophage KFSEC8.

[0042] The disinfectant 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, etc.

[0043] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of infectious diseases caused by Escherichia coli, comprising the bacteriophage KFSEC8.

[0044] 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.

[0045] The pharmaceutical composition of the present invention is 10 3 ~ 10 10 Contains bacteriophages of PFU / ml, preferably 10 6 ~ 10 9 It contains bacteriophages in PFU / ml. The term "PFU (plaque forming unit)" used in this invention is a unit that quantifies the lytic plaques that appear after bacterial death caused by bacteriophages.

[0046] In the present invention, the term "pharmaceuticalally acceptable carrier" refers to a carrier or diluent that does not irritate living organisms and does not impair the biological activity and properties of the administered compound. Acceptable pharmaceutical carriers for compositions formulated as liquid solutions include saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components, provided that they are sterile and biocompatible. Additionally, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets.

[0047] 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.

[0048] 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.

[0049] The pharmaceutical composition of the present invention may be formulated as an oral dosage form containing an active ingredient, for example, as a tablet, lozenge, water-soluble or oily suspension, prepared powder or granule, emulsion, hard or soft capsule, syrup, or elixir. To formulate into dosage forms such as tablets and capsules, the composition may include 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 capsule formulations, in addition to the aforementioned substances, a liquid carrier such as a fatty oil may be further included.

[0050] As a parenteral administration formulation containing the pharmaceutical composition of the present invention as an active ingredient, it can be formulated in an injectable form such as intravenous injection or intramuscular injection, or as a spray such as an aerosol that allows for inhalation through the respiratory system. To formulate it as an injectable formulation, the composition of the present invention may be mixed in water with a stabilizer or a buffer to prepare a solution or suspension, and this may be formulated for unit administration in ampoules or vials. When formulating it as a spray such as an aerosol, a propellant or the like may be combined with an additive to disperse the water-dispersed concentrate or wet powder.

[0051] 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 KFSEC8 to an individual other than a human.

[0052] In the method of the present invention, the bacteriophage KFSEC8 or a 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 KFSEC8 or a composition containing it may be administered through various oral or parenteral routes as long as it can reach the target tissue.

[0053] It is obvious to those skilled in the art that the appropriate total daily dose of the bacteriophage KFSEC8 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.

[0054] The term "individual" in this invention may include all animals excluding humans, preferably mammals or fish, but is not limited thereto.

[0055] 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.

[0056] 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.

[0057] Unless otherwise noted, the "%" used to indicate the concentration of a specific substance in this invention is (w / w) % for solid / solid, (w / v) % for solid / liquid, and (v / v) % for liquid / liquid.

[0058] 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.

[0060] [Example 1] Isolation and Identification of Antibiotic-Resistant and Pathogenic Escherichia Coli

[0061] 1-1. Isolation of E. coli from slaughterhouses, agricultural products, and the agricultural environment

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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

[0068] 1-2. Analysis of Isolated E. coli Using 16S rRNA Sequencing

[0069] 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.

[0070] 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.

[0071] 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

[0073] 1-3. Antibiotic susceptibility testing of isolated and identified Escherichia coli

[0074] 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 8A 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.

[0075] 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.

[0076] 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

[0078] [Example 2] Isolation and culture of a novel bacteriophage

[0079] 2-1. Screening of Bacteriophages Infecting Antibiotic-Resistant and Pathogenic Escherichia coli (E. coli) and Isolation of Single Bacteriophages

[0080] Antibiotic-resistant Escherichia coli isolated and identified in Example 1 above E. coli PLC01, E. coli PLC02, E. coli PSC01, E. coli Lytic plate analysis was performed using CMD01 to screen and isolate bacteriophages with lytic activity specific to it. 25 g of tomato soil obtained from a farm in Chilgok-gun, Gyeongsangbuk-do was prepared as a sample, and a soft agar overlay method was performed using top-agar and 0.4% agar on a solid medium.

[0081] Specifically, 25 g of tomato soil and E. coli shaking culture solution (OD) in 225 mL of TSB medium 640= 0.495) 1% was mixed and incubated at 37°C for 18 hours, after which the culture medium was centrifuged at 8,000 rpm for 10 minutes, and the supernatant was filtered through a 0.22 μm filter. Next, 4 mL of TA soft agar (0.4% (w / v) agar, nutrient medium, NaCl, MgSO4, CaCl2) and Escherichia coli PSC09011 shaken culture medium (OD) were placed on top of the TSA agar plates. 640 = 0.495) After pouring and solidifying the mixture containing 200 μL, 10 μL of the above tomato soil sample solution was added dropwise on top, and the mixture was incubated at 37°C for 18 hours to check for the formation of lysis spots.

[0082] We intended to isolate a single bacteriophage from the lysate formed above. Specifically, the lysate was added to 900 μL of SM buffer solution (50 mM Tris-HCl, 100 mM NaCl, 10 mM MgSO4) and vortexed at room temperature for 1 hour to obtain a bacteriophage solution. Then, 4 mL of TA soft agar and E. coli shaking culture medium (OD) were placed on top of TSA agar plates. 640 A mixture containing 200 μL of (= 0.495) and 100 μL of the obtained bacteriophage was poured and solidified, then incubated at 37°C for 18 hours to check for the formation of lysis plaques. The results of confirming the formation of lysis plaques are shown in Figure 1.

[0084] 2-2. Mass Culture and Purification of Bacteriophages

[0085] Among the bacteriophages specific to E. coli isolated in Example 2-1 above, the bacteriophage that formed the most transparent lytic plaque was selected, and to mass culture it, it was cultured in large quantities using the host strain E. coli PSC01, and the bacteriophage was purified therefrom.

[0086] Specifically, Escherichia coli PSC01 was cultured with shaking and dispensed into 3 mL of TA culture medium (nutrient broth, NaCl, MgSO4, CaCl2), and cultured for 2 hours. The bacteriophage obtained in Example 2-1 was dispensed and cultured for 2 hours, after which the bacteriophage was isolated by centrifugation and filtration through a 0.22 μm filter. The same procedure was carried out by increasing the amount of TA culture medium (in the order of 8, 20, 200, and 800 mL). Then, polyethylene glycol (PEG) and 1 M NaCl were added to achieve a final concentration of 10% (w / v), and the mixture was left to stand at 4°C for 16 hours. The precipitate was obtained by centrifugation at 8,000 rpm for 20 minutes at 4°C.

[0087] The purified phages were subjected to CsCl density gradient ultracentrifugation (density gradient 1.20, 1.30, 1.40, 1.45, 1.50, 1.70 g / mL; 22,000 rpm; 2 hours; 4℃), and after dialysis in SM buffer, were stored at 4℃.

[0088] The inventors collected a sample from tomato soil and named the bacteriophage that specifically kills antibiotic-resistant E. coli as "KFSEC8".

[0090] [Example 3] Investigation of Morphological Characteristics of Bacteriophage KFSEC8

[0091] Transmission electron microscope (TEM) observations were performed to investigate the morphological characteristics of bacteriophage KFSEC8.

[0092] Specifically, bacteriophage KFSEC8 was loaded onto a copper grid, then negatively stained with 2% phosphotungstic acid and dried. Electron microscopy was performed at the Kyungpook National University Joint Laboratory, and the morphology of bacteriophage KFSEC8 was observed using H-7100 (Hitachi, Japan) at a voltage of 100 kV, and the results are shown in Figure 2.

[0093] As a result, the length and width of the phage head were confirmed to be 117.31 ± 10.87 nm and 135.67 ± 7.22 nm, respectively, and the length and width of the phage tail were confirmed to be 122.80 ± 6.21 nm and 20.18 ± 3.21 nm, respectively. As shown in Figure 2, since an icosahedral head and a non-contractile tail were observed in bacteriophage KFSEC8, the sifobiridae ( Siphoviridae It was estimated to have the form of ).

[0095] [Example 4] Investigation of Temperature Stability of Bacteriophage KFSEC8

[0096] To confirm the temperature stability of bacteriophage KFSEC8, the activity of KFSEC8 was measured in various temperature ranges (-80˚C, -20˚C, -10˚C, 4˚C, 10˚C, 20˚C, 30˚C, 37˚C, 40˚C, 50˚C, 60˚C, 70˚C).

[0097] Specifically, 1.20 x 10 7 1 mL of bacteriophage KFSEC8 solution at a concentration of PFU / mL was mixed with TSB medium solution and left to stand for 1 hour under each temperature condition. After incubating the KFSEC8 reacted for 1 hour at 37°C for 18 hours using the soft agar overlay method, the titer of the remaining bacteriophage KFSEC8 was measured, and the results are shown in Figure 3.

[0098] As a result, it was confirmed that bacteriophage KFSEC8 maintained stability over a wide temperature range of -80 to 60°C.

[0099] Therefore, since it was confirmed that the bacteriophage KFSEC8 of the present invention maintains its activity over a wide range of temperatures, it was confirmed that it is a bacteriophage with excellent heat and cold resistance.

[0101] [Example 5] Investigation of pH stability of bacteriophage KFSEC8

[0102] To confirm the pH stability of bacteriophage KFSEC8, the activity of KFSEC8 was measured in various pH ranges (1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

[0103] Specifically, 6.60 x 10 6 1 mL of bacteriophage KFSEC8 solution at a concentration of PFU / mL was mixed with TSB medium solution and left to stand for 1 hour under each pH condition. After incubating the KFSEC8 reacted for 1 hour at 37°C for 18 hours using the soft agar overlay method, the titer of the remaining bacteriophage KFSEC8 was measured, and the results are shown in Figure 4.

[0104] As a result, it was confirmed that bacteriophage KFSEC8 maintained stability in the pH range of 2 to 11.

[0105] Therefore, since it was confirmed that the bacteriophage KFSEC8 of the present invention maintains its activity over a wide range of pH, it was confirmed that it is a bacteriophage with excellent pH stability.

[0107] [Example 6] Investigation of UV stability of bacteriophage KFSEC8

[0108] To confirm the UV stability of bacteriophage KFSEC8, the activity of KFSEC8 was measured after irradiating with UV light for 1 hour using a UV lamp at room temperature.

[0109] Specifically, 2.26 x 10⁶ diluted with TSB medium 72 mL of bacteriophage KFSEC8 solution at a concentration of PFU / mL was placed in a 12-well plate and incubated for 1 hour under UV irradiation of 365 nm wavelength at a distance of 15 cm from a UV lamp at room temperature. After incubating the KFSEC8 reacted for 1 hour at 37°C for 18 hours using the soft agar overlay method, the titer of the remaining bacteriophage KFSEC8 was measured, and the results are shown in Figure 5.

[0110] As a result, it was confirmed that bacteriophage KFSEC8 maintained 95.06% lytic activity after UV irradiation.

[0111] Therefore, since it was confirmed that the bacteriophage KFSEC8 of the present invention maintains its activity even after UV irradiation, it was confirmed that it is a bacteriophage with excellent stability against UV rays.

[0113] [Example 7] Investigation of the challenge assay of bacteriophage KFSEC8

[0114] To confirm the bactericidal and bacteriostatic effects of bacteriophage KFSEC8, Escherichia coli PSC09011 was inoculated into 100 mL of TSB and cultured at 37°C at 190 rpm for 2 hours. When the absorbance of the culture medium reached approximately 0.495 at 640 nm (10 8 1 mL of KFSEC8 suspension (CFU / mL) was adjusted to MOIs of 0.1, 1.0, 10, and 100, and cultured at 37°C at 110 rpm for 24 hours to confirm the lytic activity of bacteriophage KFSEC8. An E. coli PSC09011 culture that was not inoculated with bacteriophage KFSEC8 was used as a control. To determine whether E. coli PSC01 was inhibited from growing or killed by bacteriophage KFSEC8, culture samples were taken at 2-hour intervals and the viable cell count was measured, and the results are shown in Figure 6.

[0115] As a result, KFSEC8 maintained lytic activity for 4 hours at MOI 0.1 and 1.0, 6 hours at MOI 10, and 8 hours at MOI 100. It was confirmed that lytic activity was most active at 2 hours at MOI 0.1, 1.0, and 10, and most active at 4 hours at MOI 100.

[0117] [Example 8] Investigation of the specificity of bacteriophage KFSEC8 against pathogenic strains

[0118] Specificity testing was performed to confirm the lytic activity of bacteriophage KFSEC8 against foodborne pathogen strains other than the antibiotic-resistant E. coli isolated and identified in this invention.

[0119] Specifically, 28 strains of Salmonella and other pathogenic strains were cultured separately to obtain their culture solutions, and then the formation of lytic plaques was confirmed using a soft agar overlay method with each culture solution and bacteriophage KFSEC8. The results are shown in Table 4 below.

[0120] In addition, efficiency of plating (EOP) analysis was performed using lysis plate analysis on strains that formed lysis plates. The EOP value was calculated using the following Equation 1.

[0121]

[0123] Strain name Whether lytic plaques are formed EOP Escherichia coli O157:H7 ATCC 10536 O 1.21±0.26 E. coli O157:H7 ATCC 43895 O 1.81±0.29 E. coli ATCC BAA-2196 O 1.91±0.87 E. coli (PLC01) O <0.001 E. coli (PLC02) O 0.94±0.22 E. coli (PSC01) O 1.00±0.00 E. coli (CMD01) O <0.001 Bacillus cereus ATCC 14579 X NT B. cereus ATCC 13061 X NT B. cereus ATCC 21768 X NT Listeria monocytogenes NCCP 15743 X NT L. monocytogenes NCCP 15743 X NT Salmonella Typhimurium ATCC 13076 X NT S . Typhimurium ATCC 13311 X NT S . Typhimurium ATCC 15812 X NT S . Dublin NCCP 13700 X NT S . Heidelberg NCCP 13698 X NT S . Montevideo NCCP 13704 X NT S . Newport NCCP 13686 X NT S . Panama NCCP 13694 X NT Salmonella (JLS05211) X NT Salmonella (GLD09241) X NT Shigella sonnei ATCC 9290 X NT S . boydii NCCP 11190 X NT S. flexeneri 2457 X NT Staphylococcus aureus ATCC 25923 X NT Yersinia enterocolitica ATCC 55075 X NT Y. enterocolitica ATCC 23715 X NT

[0125] As a result, as shown in Table 4 above, it was confirmed that the bacteriophage KFSEC8 of the present invention exhibits lytic activity against a total of three wild-type E. coli strains (test strains) as well as all four isolated antibiotic-resistant E. coli strains. It was found that it did not exhibit lytic activity against other foodborne pathogenic strains, confirming that the bacteriophage KFSEC8 is a bacteriophage that specifically exhibits lytic activity against pathogenic E. coli. In particular, through the EOP results, it was found that it has a high EOP value of 0.9 or higher against not only the three test strains (pathogenic E. coli) but also antibiotic-resistant E. coli CMD01 and E. coli PLC02, indicating that it exhibits specific and high lytic activity against antibiotic-resistant and pathogenic E. coli.

[0127] [Example 9] Whole gene analysis of bacteriophage KFSEC8

[0128] To analyze the entire gene of the bacteriophage KFSEC8 purified in Example 2 above, DNA of the bacteriophage was extracted using the Phage DNA Isolation Kit (NORGEN Ltd., Canada). The genome sequence of KFSEC8 was analyzed using Illumina Nextseq (2X300, Sanigen) and assembled using the de novo method. Additionally, sequence trimming, assembly, and mapping were performed using Genomic v10.0.9.

[0129] As a result, it was confirmed that the entire gene sequence of bacteriophage KFSEC8 has the nucleotide sequence of SEQ ID NO. 1. In addition, it was confirmed that KFSEC8 has a genome sequence of 349,171 bp and a GC content of 37.06%, and has 597 CDS (coding domain sequences) and 7 tRNAs.

[0131] The following describes examples of pharmaceutical compositions for the prevention or treatment of infectious diseases comprising the bacteriophage KFSEC8 of the present invention; however, these examples are intended to illustrate examples of formulations that can be prepared according to the present invention, and the scope of the present invention is not limited to these examples.

[0133] [Preparation Example 1] Preparation of a pharmaceutical product

[0134] 1-1. Preparation of Injectables

[0135] Bacteriophage KFSEC8 1 X 10 6 PFU / ml

[0136] Appropriate amount of sterile distilled water for injection

[0137] Appropriate amount of pH adjuster

[0138] It was prepared according to the conventional method of preparing injectables with the above ingredient content per 1 ampoule (2 mL).

[0140] 1-2. Preparation of Liquid Formulations

[0141] Bacteriophage KFSEC8 1 X 10 6 PFU / ml

[0142] 20 g sugar

[0143] 20 g isomerized sugar

[0144] Appropriate amount of lemon flavor

[0145] Purified water was added to make the total volume 1,000 ml, and the above ingredients were mixed according to the standard method for preparing liquid preparations, then filled into a brown bottle and sterilized to prepare the liquid preparation.

[0147] 1-3. Preparation of Tablets

[0148] Bacteriophage KFSEC8 1 X 10 6 PFU / ml

[0149] 100 mg of corn starch

[0150] 100 mg lactose

[0151] Magnesium stearate 2 mg

[0152] After mixing the above ingredients, tablets were manufactured by compressing them according to the conventional method of manufacturing tablets.

[0154] Depository Name: Korea Research Institute of Biotechnology and Bioengineering Biological Resource Center (KCTC) Trustee Number: KCTC15458BP Date of Deposit: 2023-05-25

Claims

Claim 1 E. coli ( Escherichia coli Bacteriophage KFSEC8, deposited under accession number KCTC15458BP, having a specific killing ability for ) Claim 2 In paragraph 1, the above-mentioned E. coli is a bacteriophage KFSEC8 having antibiotic resistance. Claim 3 In paragraph 1, the above-mentioned E. coli is a pathogenic bacteriophage KFSEC8. Claim 4 In paragraph 1, the above E. coli is E. coli ( Escherichia coli ) O157:H7 ATCC 10536, Escherichia coli( Escherichia coli ) O157:H7 ATCC 43895, Escherichia coli( Escherichia coli ) ATCC BAA-2196, E. coli( Escherichia coli ) PLC01, E. coli( Escherichia coli ) PLC02, E. coli( Escherichia coli ) PSC01 and Escherichia coli ( Escherichia coli ) Bacteriophage KFSEC8, which is one or more selected from the group consisting of CMD01. Claim 5 In claim 1, the bacteriophage KFSEC8 is a bacteriophage that exhibits lytic activity at -80 to 60℃. Claim 6 In claim 1, the bacteriophage KFSEC8 is a bacteriophage that exhibits lytic activity at pH 2.0 to 11.

0. Claim 7 In claim 1, the bacteriophage KFSEC8 is a bacteriophage that exhibits lytic activity at an ultraviolet wavelength of 365 nm. Claim 8 In claim 1, the bacteriophage KFSEC8 is composed of the nucleotide sequence of SEQ ID NO.

1. Claim 9 Antibiotic composition comprising the bacteriophage KFSEC8 of claim 1. Claim 10 A feed additive composition comprising the bacteriophage KFSEC8 of claim 1. Claim 11 A feed composition comprising the feed additive composition of claim 10. Claim 12 A disinfectant comprising the bacteriophage KFSEC8 of claim 1. Claim 13 delete Claim 14 delete

Images