# Patent

## Title
Bacteriophage and antibacterial composition comprising the same

## Abstract

Provided is a novel bacteriophage ΦCJ19 (KCCM11361P). In addition, the present invention relates to an antibacterial composition including the bacteriophage ΦCJ19 (KCCM11361P) as an active ingredient. Further, the present invention is a method of preventing and/or treating infectious diseases by enterotoxigenic Escherichia coli in animals except for humans using the bacteriophage ΦCJ19 (KCCM11361P) or the antibacterial composition containing the bacteriophage ΦCJ19 (KCCM11361P) as an active ingredient.

## Background
TECHNICAL FIELD
The present invention relates to a novel bacteriophage having a specific bactericidal activity against Enterotoxigenic Escherichia coli (ETEC), and an antibacterial composition comprising the same. In addition, the present invention relates to a method of preventing or treating animal diseases using the novel bacteriophage or the antibacterial composition.
BACKGROUND ART
Escherichia coli (hereinafter referred to as ‘E. coli’ is a Gram-negative, short rod-shaped bacterium belonging to the genus Escherichia and the family Enterobacteriaceae, and is one of the normal flora existing in the intestines of various animals including mammals. It was known that most of the strains of E. coli are non-pathogenic and may cause opportunistic infections, but some highly pathogenic strains cause diverse intestinal diseases and sepsis in animals including humans.
An example of E. coli may include enterotoxigenic E. coil (ETEC), enteropathogenic E. coil (EPEC), enterohemorrhagic E. coil (EHEC), enteroaggregative E. coil (EAEC), enteroinvacive E. coil (EIEC), necrotoxigenic E. coil (NTEC), or the like. It is known that among them, particularly, the ETEC generates infectious disease associated with E. coli in swine.
Currently, as a large number of swine are collectively bred in a pork industry, colibacillosis has been in the spotlight as a most frequent and troublesome disease (Non-Patent Document 1). Recently, occurrence of swine colibacillosis has increased in Korea, which has caused growth retardation and death of young swine due to diarrhea, resulting in tremendous economic losses to farmers (Non-Patent Document 2).
In order to prevent and treat colibacillosis in swine, many antibiotics have been administered to swine in the prior art, but when antibiotics has been misused or overused, the misused or overused antibiotics may give rise to drug resistance and remain in bodies of the swine. Therefore, currently, the use of antibiotics has been restricted around the world (Non-Patent Document 3).
Meanwhile, bacteriophage is a specialized type of virus that infects and destroys only bacteria, and can self-replicate only inside host bacteria. The bacteriophage has strong host specificity as compared to antibiotics, and recently, a problem of emergence of strain resistant against antibiotics has been serious, such that an interest in practical use of the bacteriophage has increased (Non-Patent Documents 4 and 5).
Therefore, research into the bacteriophage has been actively conducted in various countries around the world, and in addition to a patent application for bacteriophage, an attempt to acquire Food and Drug Administration (FDA) approval for a composition containing the bacteriophage has been gradually increased.
As the prior art for the bacteriophage, 7 kinds of bacteriophages for controlling E. coli 0157:H have been disclosed in Patent Document 1, and a bacteriophage having killing activity specific to Staphylococcus aureus has been disclosed in Patent Document 2. Further, lytic protein derived from a bacteriophage specifically destroying a peptidoglycan structure of bacterial cell membrane, and bacteria lysates by the lytic protein have been disclosed in Patent Document 3.
However, in spite of presence of the following prior arts, a technology associated with the bacteriophage for preventing and/or treating infectious diseases by the ETEC that are a still important problem in an livestock industry including the pork industry is still insufficient, such that a bacteriophage and a technology associated with the bacteriophage should be developed.
PRIOR ART DOCUMENTS
Patent Documents
(Patent Document 1) U.S. Pat. No. 6,485,902
(Patent Document 2) Korea Patent No. 10-0910961 B1
(Patent Document 3) Korean Patent Application Publication No. 10-2009-0021475 A
Non-Patent Document
(Non-Patent Document 1) Young Il Park, Swine production science, Sunjin Publishing group, 353-359, 1998
(Non-Patent Document 2) Eu Chul Hong, master's thesis, Dankook University, Addition Effect of Egg Yolk in Early Weaned Piglets, 2001
(Non-Patent Document 3) Mason H S et al., Trends in Biotech, 13:388-392, 1995
(Non Patent Document 4) Cislo M, et al., Arch. Immunol. Ther. Exp. 2:175-183, 1987
(Non Patent Document 5) Sung Hun Kim et al, Bacteriophage, novel alternative antibiotics, BioWave Vol. 7 No. 15, 2005, BRIC
DISCLOSURE
Technical Problem
The present inventors conducted studies in order to solve problems such as resistant bacteria occurring upon the use of antibiotics, antibiotics remaining in meat, and the like, and efficiently prevent and treat infectious diseases by E. coli, and as a result, the present inventors isolated new bacteriophage ΦCJ19 (KCCM11361P) having a specific bactericidal activity against ETEC from the nature.
In addition, the present inventors identified morphological, biochemical, and genetic characteristics of the novel bacteriophage and confirmed that the bacteriophage had excellent acid resistance, heat resistance, and the like, thereby developing an antibiotic, a disinfectant, a feed additive, and other compositions using the novel bacteriophage.
Further, the present inventors developed a composition for preventing or treating infectious diseases by E. coli, and a method of preventing or treating the disease using the composition.
The present invention provides a novel bacteriophage ΦCJ19 (KCCM11361P) having a specific bactericidal activity against ETEC.
In addition, the present invention provides a composition for preventing and/or treating infectious diseases by ETEC containing the bacteriophage ΦCJ19 (KCCM11361P) as an active ingredient.
Further, the present invention provides an antibiotic, a feed additive, a drinking water additive, a disinfectant, or a cleaner containing the bacteriophage ΦCJ19 (KCCM11361P) as an active ingredient.
Furthermore, the present invention provides a method of preventing and/or treating infectious diseases by ETEC in animals except for humans using the bacteriophage ΦCJ19 (KCCM11361P) or a composition containing the bacteriophage ΦCJ19 (KCCM11361P) as an active ingredient.
Technical Solution
According to an exemplary embodiment of the present invention, there is provided a novel bacteriophage ΦCJ19 (KCCM11361P) having a specific bactericidal activity against enterotoxigenic Escherichia coli (ETEC).
According to another exemplary embodiment of the present invention, there is provided a composition for preventing or treating an infectious disease caused by ETEC, containing the bacteriophage ΦCJ19 (KCCM11361P) as described above as an active ingredient.
According to another exemplary embodiment of the present invention, there are provided an antibiotic, a feed additive, a drinking water additive, a disinfectant, or a cleaner containing the bacteriophage ΦCJ19 (KCCM11361P) as described above as an active ingredient.
According to another exemplary embodiment of the present invention, there is provided a method of preventing or treating an infectious disease caused by ETEC, comprising administering the bacteriophage ΦCJ19 (KCCM11361P), or the composition containing the bacteriophage ΦCJ19 as described above to animals except for humans.
Advantageous Effects
The bacteriophage ΦCJ19 (KCCM11361P) according to the present invention has an effect of specifically killing enterotoxigenic Escherichia coli (ETEC).
In addition, the bacteriophage ΦCJ19 (KCCM11361P) according to the present invention has excellent acid resistance and heat resistance, such that the bacteriophage ΦCJ19 (KCCM11361P) may be used as a material for preventing or treating infectious diseases by ETEC in various temperature or pH ranges and utilized as an antibiotic, a feed additive, a drinking water additive, a disinfectant, a cleaner, or the like.
Further, according to the present invention, infectious diseases by ETEC may be prevented or treated by administering the bacteriophage ΦCJ19 (KCCM11361P) or a composition containing the bacteriophage ΦCJ19 (KCCM11361P) as an active ingredient to animals except for humans.



DESCRIPTION OF DRAWINGS
FIG. 1 is an electron microscope photograph of a novel bacteriophage ΦCJ19 (KCCM11361P, hereinafter, referred to as ‘ΦCJ19’).
FIG. 2 shows a pulsed field gel electrophoresis (PFGE) result of the novel bacteriophage ΦCJ19.
FIG. 3 shows a sodiumdodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) result of the novel bacteriophage ΦCJ19.
FIG. 4 is a graph showing a result of an acid resistance test of the novel bacteriophage ΦCJ19.
FIG. 5 is a graph showing a result of a heat resistance test of the novel bacteriophage ΦCJ19.



BEST MODE
Hereinafter, the present invention will be described in detail. Since contents that are not described in the present specification may be sufficiently recognized and inferred by those skilled in the art or similar art, a description thereof will be omitted.
In detail, in an embodiment, the present invention provides a novel bacteriophage ΦCJ19 (KCCM11361P) having a specific bactericidal activity against enterotoxigenic Escherichia coli (ETEC).
The ETEC, which is a Gram-negative, rod-shaped bacterium, is an aerobic or facultative anaerobic bacterium decomposing lactose or fructose to produce acid and gas. The ETEC well grows in a general medium and may grow at about 7 to 48° C., and an optimal growth temperature is about 35 to 37° C. In addition, the ETEC may grow in a pH range of 4.5 to 9.
Since ETEC produces toxin similar to that of Vibrio cholerae, in the case of infection of ETEC, symptoms similar to those of cholera are exhibited. The produced toxins are divided into two kinds, that is, a heat-labile enterotoxin (LT) and heat-stable enterotoxin (ST). The heat-labile enterotoxin means an enterotoxin losing its activity in the case of heating at 60° C. for 10 minutes, and the heat-stable enterotoxin means an enterotoxin that does not lose its activity even in the case of heating at 100° C. for 30 minutes.
In the case in which a concentration of ETEC arrives at 107 cfu (colony formation unit) to 108 cfu per a unit volume (1 ml) of serous fluid while ETEC proliferates in an upper portion of intestine, ETEC causes infectious diseases by E. coli such as colibacillosis.
A bacteriophage is a bacteria-specific virus infecting specific bacteria to suppress and inhibit growth of the bacteria and means a virus including single or double stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) as a genetic material.
The bacteriophage ΦCJ19 according to the present invention, which is a bacteriophage species-selectively infecting ETEC, has a structure of an isometric capsid but a tail is not observed (FIG. 1), and morphologically belongs to Podoviridae.
The bacteriophage ΦCJ19, which was a bacteriophage newly isolated by the present inventors, was deposited at Korean Culture Center of Microorganisms (361-221, Hongjedong, Seodamun-gu, Seoul, Korea) as a deposition number KCCM11361P on Jan. 30, 2013.
In another embodiment, the present invention provides a composition for preventing or treating infectious diseases by ETEC containing the bacteriophage ΦCJ19 as an active ingredient.
Since the bacteriophage ΦCJ19 has an antibacterial activity capable of specifically killing ETEC, the bacteriophage ΦCJ19 may be used to prevent or treat diseases generated by infection of ETEC. An example of the infectious disease caused by ETEC that may be treated using the bacteriophage ΦCJ19 may include preferably colibacillosis, more preferably colibacillosis in swine, but is not limited thereto.
The term “colibacillosis” as used herein means a disease caused by infection of an animal with pathogenic E. coli, and shows symptoms such as sepsis, diarrhea (neonatal diarrhea and post-weaning diarrhea), toxemia (edema and cerebrospinal angiopathy), or the like. Among them, sepsis is an acute systemic infection that frequently occurs in 2 to 3 days after birth and has a high mortality rate. Diarrhea is the most common outcome of gastrointestinal tract infections that occur during the lactation period within 1-2 weeks after birth and immediately after the weaning period, and causes death or growth retardation. Toxemia mainly occurs in 8-12 week-old piglets after the weaning period, and is frequently accompanied by edema and neurologic signs, followed by sudden death.
The term “prevention” as used herein refers to all actions of providing the bacteriophage ΦCJ19 and/or the composition containing the bacteriophage ΦCJ19 as the active ingredient to animals except for humans to suppress the corresponding disease or retard disease occurring.
The term “treatment” as used herein refers to all actions of providing the bacteriophage ΦCJ19 and/or the composition containing the bacteriophage ΦCJ19 as the active ingredient to animals except for humans to thereby allow the symptom of the corresponding disease caused by infection to get better or be alleviated.
The composition for preventing or treating the infectious disease caused by ETEC according to the present invention may contain the bacteriophage ΦCJ19 in an amount of preferably 5×102 to 5×1012 pfu/ml, more preferably, 1×106 to 1×1010 pfu/ml.
The composition for preventing or treating the infectious disease caused by ETEC according to the present invention may further contain a pharmaceutically acceptable carrier and be formulated together with the carrier to thereby be provided as food, a drug, a feed additive, a drinking water additive, and the like. The term “pharmaceutically acceptable carrier” as used herein means a carrier or a diluent that does not stimulate living organism nor inhibit biological activity and properties of an administered compound.
A kind of carrier usable in the present invention is not particularly limited, and any carrier may be used as long as it is generally used in the art and is pharmaceutically acceptable. As a non-restrictive example of the carrier, there are normal saline, sterile water, buffered saline, Ringer's solution, an albumin injection solution, a dextrose solution, a maltodextrin solution, glycerol, ethanol, and the like. One or a mixture of at least two of these carriers may be used.
In addition, if necessary, another general additive such as an antioxidant, a buffer, a bacteriostatic agent, and/or the like, may be further added and used, and the composition may be formulated into an injection formulation such as an aqueous solution, suspension, emulsion, or the like, pills, capsules, granules, tablets, or the like by additionally adding a diluent, a dispersant, a surfactant, a binder, a lubricant, and/or the like, and then used.
An administration method of the composition for preventing or treating infectious diseases by ETEC is not particularly limited, and any method generally used in the art may be used. As a non-restrictive example of the administration method, the composition may be orally or parenterally administered.
As a non-restrictive example of the formulation for oral administration, there are troches, lozenge, tablets, aqueous suspensions, oily suspensions, prepared powder, granules, emulsions, hard capsules, soft capsules, syrups, elixirs, or the like.
In order to formulate the composition according to the present invention into a formulation such as a tablet, a capsule, or the like, the formulation may further contain a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose, gelatin; an excipient such as dicalcium phosphate, or the like; a disintegrant such as corn starch, sweet potato starch, or the like; a lubricant such as magnesium stearate, calcium stearate, sodium stearyl fumarate, polyethylene glycol wax, or the like. In the case of the capsule formulation, the formulation may additionally contain a liquid carrier such as fatty oil in addition to the above-mentioned materials.
As a parenteral administration method, an intravenous administration method, an intraperitoneal administration method, an intramuscular administration method, a subcutaneous administration method, a local administration method, or the like, may be used. In addition, a method of applying or spraying the composition onto a disease site may also be used, but the present invention is not limited thereto.
An example of the formulation for parenteral administration may include injection formulations for subcutaneous injection, intravenous injection, intramuscular injection, or the like; suppository formulations; spray formulations such as aerosol formulations capable of being inhaled through respiratory system, or the like, but the present invention is not limited thereto. In order to formulate the composition into the injection formulation, the composition according to the present invention may be mixed with a stabilizer or a buffer in water to thereby prepare a solution or suspension, and then, the prepared solution or suspension may be formulated in a unit dose for an ampoule or vial. In the case of formulating the composition into the spray formulation such as the aerosol formulation, or the like, a propellant, or the like, may be mixed together with an additive so that a water-dispersed condensate or wet powder is dispersed.
A suitable application, spray, or administration dose of the composition for preventing or treating infectious diseases by ETEC may be variously determined depending on factors such as age, weight, sex, degree of symptom of disease, a kind of food, excretion rate of administration target animals, or the like, as well as a method of formulating the composition, an administration method, an administration time and/or route. Generally, a veterinarian having ordinary skill in the art may easily determine and prescribe an effective dose for the desired treatment.
In another general aspect, the present invention may provide an antibiotic containing the bacteriophage ΦCJ19 as an active ingredient.
The term ‘antibiotic’ as used herein means an agent capable of being provided to animals including humans in a drug form to thereby kill bacteria, and corresponds to a concept collectively indicating a preservative, a disinfectant, and an antibacterial agent.
The antibiotic containing the bacteriophage ΦCJ19 according to the present invention as the active ingredient may have high specificity to ETEC as compared to an antibiotic according to the prior art to thereby not kill beneficial bacteria but kill specific pathogenic bacteria, and does not induce drug resistance, such that the antibiotic according to the present invention may be provided as a novel antibiotic having an elongated lifespan as compared to the antibiotic according to the prior art.
In another general aspect, the present invention may provide a feed additive and a drinking water additive containing the bacteriophage ΦCJ19 as an active ingredient.
The feed additive and the drinking water additive according to the present invention may be used in a manner in which the bacteriophage ΦCJ19 or the composition containing the bacteriophage ΦCJ19 is individually prepared in a feed additive or drinking water additive form and then mixed with a feed or drinking water, or in a manner in which the bacteriophage ΦCJ19 or the composition containing the bacteriophage ΦCJ19 is directly added at the time of preparing the feed or the drinking water.
The bacteriophage ΦCJ19 or the composition containing the bacteriophage ΦCJ19 used as the feed additive or drinking water additive according to the present invention may be in a liquid state or dried state, and preferably, in a dried powder form.
A drying method for preparing the feed additive and the drinking water additive according to the present invention in the dried powder form is not particularly limited, but a method generally used in the art may be used. As a non-restrictive example of the drying method, there is a natural air drying method, natural drying method, a spray drying method, a freeze-drying method, or the like. One method of these methods may be used alone or at least two methods may be used together with each other.
Another non-pathogenic microbe may be additionally added to the feed additive or drinking water additive. A non-restrictive example of the microbe capable of being added may be selected from a group consisting of bacillus sp. capable of producing protease, lipase, and/or sugar converting enzyme such as bacillus subtilis, or the like; Lactobacillus sp. having physiological activity and degradation activity for an organic material under anaerobic conditions such as cow's stomach; mold fungi having effects of increasing a weight of domestic animal, a milk yield, and digestibility of the feed such as Aspergillus oryzae, or the like; and yeasts such as Saccharomyces cerevisiae, or the like. One or a mixture of at least two of these microbes may be used.
The feed additive or the drinking water additive containing the bacteriophage ΦCJ19 according to the present invention as the active ingredient may further contain other additives, as needed. As a non-restrictive example of the usable additive, there are a binder, an emulsifier, a preservative, and the like, which are added in order to prevent quality of the feed or driving water from being deteriorated; amino acids, vitamins, enzymes, probiotics, flavoring agents, non-protein nitrogen compounds, silicates, buffers, coloring agents, extractants, oligosaccharides, and the like, which are added in order to increase utility of the feed or drinking water. Otherwise, the additive may further include a feed mixing agent, or the like. One or a mixture of at least two of these additives may be used.
The feed additive may be contained at a content of 0.05 to 10, more preferably 0.1 to 2 parts by weight based on 100 parts by weight of the feed. The drinking water additive may be contained at a content of 0.0001 to 0.01, more preferably 0.001 to 0.005 parts by weight based on 100 parts by weight of the drinking water. The activity of the bacteriophage ΦCJ19 against ETEC may be sufficiently exhibited in the above-mentioned range.
In another general aspect, the present invention provides a feed or drinking water prepared by adding a feed additive or a drinking water additive containing the bacteriophage ΦCJ19 as an active ingredient or directly adding the bacteriophage ΦCJ19.
The feed used in the present invention is not particularly limited, but any feed generally used in the art may be used. A non-restrictive example of the feed may include plant feeds such as grains, roots and fruit, food processing byproducts, algaes, fibers, pharmaceutical byproducts, fats, starches, cucurbitaceous, or grain byproducts; and animal feeds such as proteins, inorganic materials, fats, minerals, single cell proteins, animal planktons, or foods. One or a mixture of at least two of these feeds may be used.
The drinking water used in the present invention is not particularly limited, but any drinking water generally used in the present invention may be used.
In another general aspect, the present invention may provide a disinfectant or a cleaner containing the bacteriophage ΦCJ19 as an active ingredient. A formulation of the disinfectant or cleaner is not particularly limited, but the disinfectant or cleaner may be formulated into any formulation known in the art.
The disinfectant may be sprayed in order to remove ETEC onto a region in which animals live, a slaughterhouse, a mortality generation area, a cooking place or cooking equipment, or the like, but the present invention is not limited thereto.
The cleaner may be used to wash skin's surfaces or each of the sites of bodies of animals exposed or to be exposed to ETEC, but the present invention is not limited thereto.
In another general aspect, the present invention provides a method of preventing or treating infectious diseases by using the bacteriophage ΦCJ19 or the composition comprising the bacteriophage ΦCJ19 as an active ingredient.
In detail, the method of preventing or treating infectious diseases according to the present invention may include administering the bacteriophage ΦCJ19 or the composition containing the bacteriophage ΦCJ19 as the active ingredient to targets infected by ETEC or being at risk of infection of ETEC except for humans in a pharmaceutically effective dose.
It will be apparent to those skilled in the art that when the pharmaceutical composition is administered to patient, the suitable total daily dose may be determined by an attending physician or veterinarian within the scope of sound medical judgement.
A specific pharmaceutically effective dose of the bacteriophage ΦCJ19 or the composition containing the bacteriophage ΦCJ19 as the active ingredient for a specific animal may be determined by considering an administration time and an administration route of the bacteriophage ΦCJ19 or the composition containing the bacteriophage ΦCJ19, a secretion rate of the composition, a therapy duration period, or the like, in addition to a kind and a degree of the desired response, an age, a weight, a general healthy state, sex, or diet of the corresponding individual. In addition, the pharmaceutically effective dose may be variously changed according to various factors such as ingredients of drugs or other compositions simultaneously or separately used and similar factors well known in a medical field.
The bacteriophage ΦCJ19 according to the present invention or the composition containing the bacteriophage ΦCJ19 as the active ingredient may be administered as a pharmaceutical form (nasal spray) to animals or administered in a method of directly added to a feed or drinking water of the animals and then feeding the feed or drinking water. In addition, the bacteriophage ΦCJ19 or the composition containing the same may be mixed in a feed or drinking water in a form of a feed additive or drinking water additive and then administered.
The administration route and administration method of the bacteriophage ΦCJ19 according to the present invention or the composition containing the bacteriophage ΦCJ19 as the active ingredient are not particularly limited, but any administration route and administration method may be used as long as the bacteriophage ΦCJ19 or the composition containing the same may arrive at the corresponding target tissue. That is, the bacteriophage ΦCJ19 or the composition containing the bacteriophage ΦCJ19 as the active ingredient may be administered through various oral or parenteral routes. As a non-restrictive example of the administration route, oral, rectal, local, intravenous, intraperitoneal, intramuscular, intraarterial, subcutaneous, and nasal administration, inhalation, or the like, may be performed.
Hereinafter, the present invention will be described in detail through the Examples. However, these Examples are only to illustrate the present invention, and a scope of the present invention is not limited to these Examples.
Example 1
Isolation of Bacteriophage Infecting ETEC
Examples 1-1
Screening of Bacteriophage and Isolation of Single Bacteriophage
After 50 ml of a sample obtained from pig feces and environmental samples of Samwhaw Gps. Breeding Agri. in Gwangcheon area, Hong seong-gun, Chungchong Province was centrifuged at 4,000 rpm for 10 minutes, the supernatant was filtered with a 0.45 μm filter to prepare a sample solution, and then a soft agar overlay method was performed using the prepared sample solution. The soft agar overlay method is a method of observing a lysis action of bacteriophage using host cells growing in top agar (attached onto a solid medium using 0.7% agar).
In detail, 18 ml of the sample filtrates was mixed with 150 μl of a shake culture solution (OD600=2) of ETEC (SNUJG280) obtained from College of Veterinary Medicine, Seoul National University and 2 ml of 10× Luria Bertani (LB) medium (tryptone 10 g/l, yeast extract 5 g/l, and NaCl 10 g/l) and cultured at 30° C. for 18 hours. Then, the culture solution was centrifuged at 4,000 rpm for 10 minutes, and the supernatant was filtered with a 0.45 μm filter. Then, after a mixed solution of 3 ml of 0.7% (w/v) agar and 150 μl of the shake culture solution (OD600=2) of ETEC (SNUJG280) was poured and hardened onto a LB plate medium, 10 μl of the sample solution was dropped thereon, followed by culturing at 30° C. for 18 hours. Then, it was confirmed that a plaque was formed.
Since it is known that one kind of bacteriophage is present in a single plaque, separation of a single bacteriophage from the formed plaque was attempted. In detail, the plaque was added to 400 μl of a SM solution (NaCl (5.8 g/l), MgSO47H2O (2 g/l), 1M Tris-Cl (pH 7.5, 50 ml)) and left at room temperature for 4 hours, thereby obtaining a bacteriophage solution. Thereafter, 100 μl of the bacteriophage solution was mixed with 5 ml of 0.7% (w/v) agar and 150 μl of the shake culture solution (OD600=2) of ETEC (SNUJG280), followed by performing the soft agar overlay method using a LB medium having a diameter of 150 m. The culturing was performed until ETEC was completely lysed. After the culturing was terminated, 5 ml of the SM solution was added to the LB plate medium and left at room temperature for 4 hours, thereby obtaining a bacteriophage solution.
After the solution was recovered and 1% (v/v) chloroform was added thereto, the mixture was mixed for 10 minutes, followed by centrifugation at 4,000 rpm for 10 minutes, thereby obtaining a supernatant. The obtained supernatant was filtered with a 0.45 μm filter, thereby obtaining a final sample.
Examples 1-2
Large-Scale Culture and Purification of Bacteriophage
The bacteriophage obtained in Example 1-1 was cultured at large scale using ETEC (SNUJG280), and then the bacteriophage was purified therefrom.
In detail, after ETEC (SNUJG280) was shake-cultured, and an aliquot of 1.5×1010 cfu was centrifuged at 4,000 rpm for 10 minutes and then resuspended in 4 ml of the SM solution. The bacteriophage of 1.5×106 pfu was inoculated thereto (multiplicity of infection (MOI)=0.0001), and left at room temperature for 20 minutes. Thereafter, the solution was inoculated into 150 ml of the LB medium and cultured at 30° C. for 5 hours.
After the culturing was terminated, chloroform was added at an amount of 1% (v/v) of a final volume and stirred for 20 minutes. Then, restriction enzymes DNase I and RNase A were added so as to have a final concentration of 1 μg/ml, respectively, and the solution was left at 30° C. for 30 minutes. Then, NaCl and polyethylene glycol (PEG) were added so as to have final concentrations of 1M and 10% (w/v), respectively, and further left at 4° C. for 3 hours, followed by centrifugation at 4° C. and 12,000 rpm for 20 minutes, thereby obtaining precipitates.
The obtained precipitate was suspended in 5 ml of a SM solution and left at room temperature for 20 minutes. Then, 1 ml of chloroform was added thereto and stirred, followed by centrifugation at 4° C. and 4,000 rpm for 20 minutes, thereby obtaining a supernatant. Thereafter, the supernatant was filtered with a 0.45 μm filter, and ultracentrifugation (35,000 rpm, 1 hour, 4° C.) using a glycerol density gradient method (density: 40%, 5% glycerol) was performed, thereby purifying the bacteriophage.
The present inventor designated the bacteriophage obtained by extracting the sample from pig feces and having the specific bacteriocidal activity against ETEC as “Bacteriophage ΦCJ19” and deposited the bacteriophage at Korean Culture Center of Microorganisms (361-221, Hongjedong, Seodamun-gu, Seoul, Korea) as a deposition number KCCM11361P on Jan. 30, 2013.
Example 2
Examination of ΦCJ19 Infection on E. coli 
In order to confirm whether or not the bacteriophage ΦCJ19 purified in Example 1 has a lytic activity on E. coli species other than ETEC (SNUJG280), cross infection with other E. coli species was performed.
In detail, 2 kinds of ETEC (SNUJG280 and CANR08 (obtained from University of Guelph in Canada)) strains and 12 kinds of non-pathogenic E. coli strains (MC4100, BL21(DE3), Rosetta(DE3), 2616, 281, 1917, DH5a, GM2929, Tuner(DE3), W3110, K12G, and 0122ΔLT) were cultured, thereby obtaining culture solutions, respectively. Then, each of the culture solutions and the purified bacteriophage ΦCJ19 were used to perform the soft agar overlay method, and whether or not a plaque was formed was confirmed.
The results were shown in the following Table 1.











TABLE 1







Serotype
strain
Plaque formation









Non-pathogenic
MC4100
∘



E. coli
BL21(DE3)
∘




Rosetta(DE3)
∘




2616
x




 281
x




1917
x




DH5a
x




GM2929
∘




Turner
∘




W3110
∘




K12G
x




0122(ΔLT)
∘



ETEC
SNUJG280
∘




CANR08
∘










As shown in Table 1, it may be confirmed that the bacteriophage ΦCJ19 purified in Example 1 had the lytic activity on 7 kinds of non-pathogenic E. coli strains (MC4100, BL21(DE3), Rosetta(DE3), GM2929, Tuner(DE3), W3110, and 0122ΔLT) but did not have lytic activity on the residual 5 kinds of non-pathogenic E. coli strains (2616, 281, 1917, DH5a, and K12G) among the non-pathogenic E. coli strains.
Example 3
Morphology Observation of ΦCJ19
The bacteriophage ΦCJ19 purified in Example 1 was diluted in a 0.01% gelatin solution, and then fixed in a 2.5% glutaraldehyde solution. The fixed bacteriophage was dropped onto a carbon-coated mica plate (ca. 2.5×2.5 mm), adapted thereto for 10 minutes, and washed with sterile distilled water. A carbon film was mounted on a copper grid, stained with 2% uranyl acetate for 30 to 60 seconds, dried, and investigated using a transmission electron microscope (JEM-1011, 80 kV, magnification: ×120,000 to ×200,000) (FIG. 1).
FIG. 1 is an electron microscopy photograph of the bacteriophage ΦCJ19. It may be appreciated that the bacteriophage ΦCJ19 has an isometric capsid but does not have a tail, such that the bacteriophage ΦCJ19 morphologically belongs to Podoviridae.
Example 4
Genomic DNA Size Analysis of ΦCJ19
Genomic DNA was extracted from the bacteriophage ΦCJ19 purified in Example 1.
In detail, 20 mM ethylenediaminetetraacetic acid (EDTA), 50 μg/ml proteinase K, and 0.5% (w/v) sodium dodecyl sulfate (SDS) were added to a culture solution of the purified bacteriophage ΦCJ19 and left at 50° C. for 1 hour. An equal volume of phenol (pH 8.0) was added and stirred, followed by centrifugation at room temperature and 12,000 rpm for 10 minutes, thereby obtaining a supernatant.
The supernatant was mixed with an equal volume of PC (phenol:chloroform=1:1) and centrifuged at room temperature and 12,000 rpm for 10 minutes, thereby obtaining a supernatant. The supernatant was mixed with an equal volume of chloroform and centrifuged at room temperature and 12,000 rpm for 10 minutes, thereby obtaining a supernatant. The obtained supernatant was mixed with 10% (v/v) of 3M sodium acetate and then double volume of cold 95% ethanol, based on the total volume, and left at −20° C. for 1 hour. Subsequently, centrifugation was performed at 0° C. and 12,000 rpm for 10 minutes, and the precipitate was obtained by removing the supernatant. Then, 50 μl of Tris-EDTA (TE) buffer (pH 8.0) was added thereto to thereby dissolve the obtained precipitate. The extracted DNA was diluted 10 times, and a concentration was measured by measuring absorbance at OD260.
Next, 1 μg of DNA was loaded onto 1% pulse-field gel electrophoresis (PFGE) agarose gel, and electrophoresis was performed at room temperature for 20 hours using a BIORAD PFGE system program 7 (size range: 25-100 kb; switch time ramp: 0.4-2.0 seconds, linear shape; forward voltage: 180 V; reverse voltage: 120 V) (FIG. 2).
FIG. 2 is a pulsed field gel electrophoresis (PFGE) photograph of the genomic DNA of the bacteriophage ΦCJ19, and it may be confirmed that the genomic DNA of the bacteriophage ΦCJ19 has a size of about 54 kb. In FIG. 2, M is a protein that becomes a standard for measuring a molecular weight.
Example 5
Protein Pattern Analysis of ΦCJ19
15 μl of purified bacteriophage ΦCJ19 solution at a titer of 1010 pfu/ml was mixed with 3 μl of a 5×SDS sample solution, and heated for 5 minutes. Then, 12% SDS-PAGE was performed (FIG. 3).
FIG. 3 is an electrophoresis photograph showing a result of SDS-PAGE performed on the bacteriophage ΦCJ19, and main proteins having sizes of about 44 kDa, 40.5 kDa, 37 kDa, 20 kDa, and 17 kDa were observed.
Example 6
Genetic Property Analysis of ΦCJ19
In order to confirm genetic characteristics of the bacteriophage ΦCJ19 purified in Example 1, DNA of the bacteriophage ΦCJ19 was analyzed using a FLX titanium sequencer (Roche), which is a gene analysis apparatus. Genes was assembled at Macrogen INC. using GS and de novo assembler software (Roche). Sequence analysis of an open reading frame was performed using GeneMArk.hmm, Glimmer v3.02, and FGENESB software. Identification of the open reading frame was performed using BLASTP and InterProScan program.
The genome sequence of the bacteriophage had various similarities with that of the existing reported bacteriophage, but it was confirmed that a bacteriophage of which all of the fractions were completely (100%) equal to those of the bacteriophage of the present invention did not exist. Therefore, it may be confirmed that the bacteriophage was a newly isolated bacteriophage.
The following Table 2 shows results obtained by comparing homologues of the genome sequence of the bacteriophage ΦCJ19 and decoded genome sequences of other bacteriophages.










Table 2







Query
Subject
Score
Identities














Name
Length
Start
End
Description
E- Value
Match/Total
Pct. (%)

















contig00001_orf00001
159
1
144
hypothetical protein
8E-16
41/48
85






[Enterobacteria 









phage F20]





contig00001_orf00009
294
4
282
TfmS [Enterobacteria
2E-32
67/93
72






phage TLS]





contig00001_orf00018
231
1
228
gp32 [Escherichia 
8E-37
73/76
96






phage phiEB49]





contig00001_orf00013
726
1
717
gp48 [Enterobacteria 
 2E-109
179/240
74






phage TLS]





contig00001_orf00014
594
4
591
putative tail assembly
2E-76
157/196
80






protein [Enterobacteria 









phage T1]





contig00001_orf00017
918
1
915
gp33 [Escherichia 
 1E-162
284/305
93






phage phiEB49]





contig00001_orf00012
747
13
744
thmL[Enterobacteria 
 1E-108
188/244
77






phage TLS]





contig00001_orf00030
729
7
726
hypothetical protein
8E-90
170/241
70






[Enterobacteria 









phage F20]





contig00001_orf00043
375
1
330
JK_56P [Enterobacteria
3E-44
 80/110
72






phage JK06]





contig00001_orf00026
474
1
426
gp22 [Escherichia 
3E-72
129/142
90






phage phiEB49]





contig00001_orf00024
1920
1
423
putative tail fiber
7E-55
127/141
90






[Enterobacteria 









phage RTP]





contig00001_orf00036
1143
1
1134
hypothetical protein
 1E-170
290/380
76






Shf11p67 [Shigella 









phage Shfl1]





contig00001_orf00037
174
1
171
gp10 [Escherichia 
1E-23
52/57
91






phage phiEB49]





contig00001_orf00044
1584
1
1581
conserved protein
0
485/527
92






[Enterobacteria 









phage RTP]





contig00001_orf00025
924
1
918
putative DNA primase
 1E-162
275/306
89






[Enterobacteria 









phage RTP]





contig00001_orf00046
351
4
348
hypothetical protein
6E-51
 94/115
81






[Enterobacterla 









phage RTP]





contig00001_orr00075
546
1
537
putative polynucleotlde
3E-76
137/179
76






kinase/phosphatase









[Enterobacteria phage









RTP]





contig00001_orf00050
240
1
237
hypothetical protein
1E-36
72/79
91






[Enterobacteria phage









RTP]









please see the [partial genome Sequence of ΦCJ19] in attachment.
Example 7
Stability Test of ΦCJ19 Depending on pH
In order to confirm whether or not the bacteriophage ΦCJ19 may have stability in a low pH environment in stomach, stability test was performed over a wide pH range (pH 3.0, 3.5, 4.0, 5.5, 6.4, 7.5, 8.3, 9.2, and 11.0).
For test, various pH solutions (sodium acetate buffer (pH 4.0, pH 5.5, and pH 6.4), sodium citrate buffer (pH 3.0 and pH 3.5), sodium phosphate buffer (pH 6.9 and pH 7.4), and Tris-HCl solution (pH 8.2, pH 9.0, pH 9.8, and pH 11.0)) were prepared at a concentration of 0.2 M, respectively.
After 180 μl of each of the pH solutions was mixed with 20 μl of bacteriophage solution having a titer of 5.0×1010 pfu/ml so that a concentration of each of pH solution became 1 M, and each of the pH solutions was left at room temperature for 2 hours. In a control group, 20 μl of the bacteriophage solution (5.0×1010 pfu/ml) was mixed with 180 μl of SM solution and then left at room temperature for 2 hours. Then, the reaction solution was diluted step by step, 10 μl of the diluted solution at each step was dropped and cultured at 30° C. for 18 hours by a soft agar overlay method, and the titer was measured through the presence or absence of lysis (FIG. 4).
FIG. 4 shows a result of the acid resistance test of the bacteriophage ΦCJ19. As shown in FIG. 4, it may be confirmed that the bacteriophage ΦCJ19 did not lose its activity and was stable in a pH range of 4.0 to 11.0 as compared to the control group.
Example 8
Stability Test of ΦCJ19 Depending on Temperature
A test for confirming stability against heat generated during a formulating process of the bacteriophage in the case of using the bacteriophage as a feed additive formulation among formulations of the bacteriophage was performed.
In detail, 100 μl of bacteriophage ΦCJ19 solution having a concentration of 5.0×1010 pfu/ml was left at 37° C., 42° C., 53° C., and 60° C. for 0, 30, 60, and 120 minutes, respectively. Then, the solutions above were diluted step by step, 10 μl of each of the diluted solutions was dropped and cultured at 30° C. for 18 hours by a soft agar overlay method, and the titer was measured through the presence or absence of lysis (FIG. 5).
FIG. 5 shows a result of a heat resistance test of the bacteriophage ΦCJ19. As shown in FIG. 5, the bacteriophage ΦCJ19 maintained its activity even though exposed at 53° C. for up to 2 hours, but its activity was reduced according to the exposure time, after exposed at 60° C. for 60 minutes.
Example 9
Infection Spectrum Test of ΦCJ19 on Wild-Type Strains of ETEC
Whether or not the bacteriophage ΦCJ19 had a lytic activity was tested on 15 wild-type strains of ETEC obtained from College of Veterinary Medicine, Seoul National University and University of Guelph in Canada other than ETEC (SNUJG280) used in the experiment.
In detail, 10 μl of bacteriophage ΦCJ19 solution having a titer of 109 pfu/ml and mixed with 150 μl of a shake culture solution (OD600=2) of each of the strains was dropped and cultured at 30° C. for 18 hours by a soft agar overlay method. Then, whether or not a plaque was formed was observed.
The results were shown in the following Table 3.











TABLE 3







Serotype
Strain
Plaque formation









ETEC
SNU345
x




SNU105
x




SNU0122
∘




SNU0149
∘




SNUJG280
∘




SNUF4
∘




SNU162
∘




SNU160
∘




SNU107
∘




CANR08
∘




SNU2618
∘




SNU2617
x




SNU193
x




SNU274
∘




SNU3220
∘










As shown in Table 3, the bacteriophage showed infectivity on F-serotype K88 (SNU107, SNU160, SNU162, SNUF4, SNU3220, CANR08, SNUJG280) and K99 (SNU2618) as well as ETEC 0-serotype 0149 (SNU107, SNUF4, SNUJG280, SNU3220, and CANR08) which is the most common cause of diarrhea in swine. Therefore, it may be expected that the bacteriophage will show excellent efficiency at the time of actually applying the bacteriophage.

## Claims

1. A method of preparing an animal feed composition, the method comprising:
providing a bacteriophage composition comprising a bacteriophage having the nucleic acid sequence of SEQ ID No: 1; and
mixing the composition with an animal feed base to provide the animal feed composition such that the bacteriophage is in an amount of 1×106 to 5×1012 PFU/ml with reference to the total amount of the animal feed composition.

2. The method of claim 1, further comprising:
mixing, with the animal feed base, one or more additives selected from the group consisting of an amino acid, a vitamin, an enzyme, a probiotic, a flavoring agent, a non-protein nitrogen compound, a silicate, a buffer, a coloring agent, an extractant and an oligosaccharide.

3. A method of feeding, the method comprising:
preparing the animal feed composition according to the method of claim 1; and
feeding, to an animal, the animal feed composition as a feed.

4. A method of preparing a drinking water composition, the method comprising:
providing a bacteriophage composition comprising a bacteriophage having the nucleic acid sequence of SEQ ID No: 1; and
mixing the composition with drinking water to provide the drinking water composition such that the bacteriophage is in an amount of 1×106 to 5×1012 PFU/ml with reference to the total amount of the drinking water composition.

5. The method of claim 4, further comprising:
mixing, with the animal feed base, one or more additives selected from the group consisting of an amino acid, a vitamin, an enzyme, a probiotic, a flavoring agent, a non-protein nitrogen compound, a silicate, a buffer, a coloring agent, an extractant and an oligosaccharide.

6. A method of feeding, the method comprising:
preparing the drinking water composition according to the method of claim 4; and
feeding, to an animal, the drinking water composition as drinking water.

7. A method of treating an infectious disease caused by ETEC, the method comprising:
administering a bacteriophage composition to a subject in need of such treatment, wherein the bacteriophage composition comprises a pharmaceutically acceptable carrier and a bacteriophage having the nucleic acid sequence of SEQ ID No: 1 in an amount of 1×106 to 5×1012 PFU/ml with reference to the total amount of the bacteriophage composition.

8. A method of claim 7, wherein the infectious disease is colibacillosis.

9. A method of retarding progress of an infectious disease caused by ETEC, the method comprising:
feeding an animal feed composition to an animal, wherein the animal feed composition comprising an animal feed base and a bacteriophage having the nucleic acid sequence of SEQ ID No: 1 in an amount of 1×106 to 5×1012 PFU/ml with reference to the total amount of the animal feed composition.

10. A method of claim 9, wherein the infectious disease is colibacillosis.