KR101299179B1 - Novel bacteriophage and antibacterial composition comprising the same - Google Patents

Abstract

The present invention relates to novel bacteriophage ΦCJ18 (KCCM11272P).
In addition, the present invention relates to a composition for the prevention or treatment of infectious diseases caused by avian pathogenic E. coli, feed for poultry or drinking water, and a disinfectant or cleaning agent comprising ΦCJ18 (KCCM11272P) as an active ingredient.
In addition, the present invention relates to a method for preventing or treating infectious diseases caused by avian pathogenic E. coli of poultry using the ΦCJ18 (KCCM11272P) or a composition comprising the same as an active ingredient.

Description

A novel bacteriophage and an antibacterial composition comprising the bacteriophage and antibacterial composition comprising the same.

The present invention relates to a novel bacteriophage that exhibits specific killing ability against avian pathogenic E. coli, and an antimicrobial composition comprising the same, and a method for preventing or treating diseases of poultry using the same.

Escherichia coli is a Gram-negative simple bacillus belonging to the genus Enterobacteriaceae and Escherichia, and is one of the normal flora in the intestines of various animals including mammals. E. coli is mostly non-pathogenic and can cause opportunistic infections. However, some highly pathogenic strains are known to cause various intestinal diseases and sepsis in animals including humans.

Among these Escherichia coli, Avian Pathogenic E. coli (APEC) is known to infiltrate the body through the respiratory mucosa as E. coli, which is infected through the respiratory system of birds, for example, chickens, ducks, turkeys.

The avian pathogenic Escherichia coli causes a variety of diseases such as sepsis, granulomas, cystitis, ovitis, arthritis, etc. in birds, and causes serious economic damage to the poultry industry, especially in poultry with respect to respiratory diseases, which is a problem. .

On the other hand, bacteriophage refers to a bacterial specific virus that inhibits and inhibits the growth of bacteria by infecting certain bacteria. Bacteriophage not only has stronger host specificity than antibiotics, but has recently become increasingly interested in its application as the problem of the emergence of resistant bacteria against antibiotic use has increased (Cislo, M et al. Arch Immunol. Ther.Exp. 2: 175 -183, 1987; Kim Sung Hoon et al., Bacteriophage, a new alternative antibiotic, BioWave Vol. 7 No.15, 2005, BRIC).

Accordingly, research on bacteriophage is actively being conducted in various countries around the world, and the patent application and registration of bacteriophage, as well as the movement to obtain approval of the US Food and Drug Administration (FDA) for the composition using the bacteriophage This is an increasing trend.

Prior arts related to bacteriophages are disclosed in US Patent No. 6,485,902 for seven bacteriophages for controlling E. coli 0157: H, and specific for Staphylococcus aureus in Korean Patent No. 10-0910961. Disclosed is a bacteriophage having an aggressive killing ability. In addition, the Republic of Korea Patent Publication No. 10-2009-0021475 discloses a bacteriophage-derived lysate protein that specifically destroys the peptidoglycan structure of the bacterial cell membrane and thereby bacterial lysate.

US Patent No. 6,485,902 Republic of Korea Patent Publication B1 No. 10-0910961 (2009.08.05. Notification) Republic of Korea Publication No. A-1-2009-0021475 (published March 4, 2009)

Cislo, M et al. Arch Immunol. Ther. Exp. 2: 175-183, 1987 Sunghoon Kim et al. Bacteriophage, a new alternative antibiotic. BioWave Vol. 7 No.15, 2005, BRIC

The inventors of the present invention have repeatedly studied to solve the emergence of resistant bacteria and antibiotic residues in meat, and to effectively prevent and treat infectious diseases of birds, as a result of APEC Escherichia coli causing poultry respiratory diseases. The new bacteriophage ΦCJ18 (KCCM11272P), which has the ability to kill enemies, has been separated from the natural world.

In addition, by identifying the morphological, biochemical and genetic characteristics of the new bacteriophage and confirming that the bacteriophage is excellent in acid resistance, dry resistance and heat resistance, antibiotics, disinfectants, feed additives, and other compositions using the same were developed. A composition for preventing or treating an infectious disease that develops and a method for preventing or treating a disease using the same have been developed.

An object of the present invention is to provide a novel bacteriophage Φ CJ18 (KCCM11272P) having a specific killing ability for avian pathogenic E. coli.

In addition, an object of the present invention is to provide a composition for the prevention or treatment of infectious diseases caused by avian pathogenic E. coli, comprising the bacteriophage Φ CJ18 (KCCM11272P) as an active ingredient.

In addition, an object of the present invention is to provide an antibiotic, poultry feed or drinking water additives, and a disinfectant or cleaning agent containing the bacteriophage Φ CJ18 (KCCM11272P) as an active ingredient.

In addition, an object of the present invention is to provide a method for preventing or treating infectious diseases caused by avian pathogenic E. coli using the bacteriophage Φ CJ18 (KCCM11272P) or a composition comprising the same as an active ingredient.

One aspect of the present invention provides a novel bacteriophage ΦCJ18 (KCCM11272P) having a killing ability specific to Avian Pathogenic Escherichia coli .

According to another aspect of the present invention, the bacteriophage ΦCJ18 (KCCM11272P) comprising an active ingredient, provides a composition for the prevention or treatment of infectious diseases caused by avian pathogenic E. coli.

According to another aspect of the present invention, there is provided an antibiotic comprising the bacteriophage ΦCJ18 (KCCM11272P) as an active ingredient.

According to another aspect of the present invention, it provides a feed or drinking water additives for poultry comprising the bacteriophage Φ CJ18 (KCCM11272P) as an active ingredient.

According to another aspect of the present invention, a bacteriophage ΦCJ18 (KCCM11272P) comprising an active ingredient, disinfectant or cleaning agent is provided.

According to another aspect of the present invention, the bacteriophage Φ CJ18 (KCCM11272P) Or it provides a method for preventing or treating an infectious disease caused by avian pathogenic E. coli, comprising administering to the poultry a composition comprising the same as an active ingredient.

Bacteriophage ΦCJ18 (KCCM11272P) of the present invention has the effect of specifically killing avian pathogenic E. coli.

In addition, the bacteriophage ΦCJ18 (KCCM11272P) of the present invention is excellent in acid resistance, heat resistance and dry resistance, can be used as a material for the prevention or treatment of infectious diseases caused by avian pathogenic E. coli at various temperature and pH range, as well as antibiotics, Poultry feed or drinking water has an effect that can be utilized as additives, disinfectants or cleaning agents.

In addition, the present invention has the effect of preventing or treating infectious diseases caused by avian pathogenic E. coli of poultry by administering to the poultry the bacteriophage Φ CJ18 (KCCM11272P) or a composition comprising the same as an active ingredient.

1 is an electron micrograph of a novel bacteriophage Φ CJ18 (KCCM11272P).
Figure 2 is a graph showing the PFGE results of the novel bacteriophage Φ CJ18.
3 is a graph showing the SDS-PAGE results of the novel bacteriophage Φ CJ18.
Figure 4 is a graph showing the acid resistance test results of the novel bacteriophage Φ CJ18.
Figure 5 is a graph showing the heat resistance test results of the novel bacteriophage Φ CJ18.
Figure 6 is a graph showing the results of the dry resistance test of novel bacteriophage Φ CJ18.
Figure 7 shows part of the DNA base sequence of novel bacteriophage Φ CJ18.
Figure 8 shows the nucleotide sequence of the primer used in Example 7.

Hereinafter, the present invention will be described in more detail. Those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

One aspect of the present invention is a novel bacteriophage ΦCJ18 (KCCM11272P) (hereinafter, 'ΦCJ18') having a specific killing ability against Avian Pathogenic Escherichia coli (APEC). .

Avian Escherichia coli is an Escherichia coli that infects birds through the respiratory tract of birds such as chickens, ducks, and turkeys, and enters the body of birds through the respiratory mucosa and causes various diseases such as sepsis, granulomas, cystitis, meningitis and arthritis. .

The algal pathogenic Escherichia coli is a gram-negative bacillus like a common Escherichia coli and has a main hair flagella, which is motility, and is an aerobic or breathable anaerobic bacterium that decomposes lactose and fructose to generate acids and gases.

The avian pathogenic Escherichia coli grows well in a normal medium, and the developable temperature is about 7 to 48 ℃ and the optimum culture temperature is 35 to 37 ℃, especially the expression of the pathogenic factor is effective at about 42 ℃ close to the body temperature of algae. The developable pH range is about pH 4.5 to 9.0.

Bacteriophage is a bacterial specific virus that infects a particular bacterium and inhibits and inhibits the growth of the bacterium. The bacteriophage refers to a virus including a single or double chain DNA or RNA as a genetic material.

ΦCJ18 of the present invention is a bacteriophage having species specificity for selectively infecting avian pathogenic Escherichia coli, having a structure of an isometric capsid and a tail not observed (Fig. 1). It is a bacteriophage belonging to Poriviridae.

When the bacteriophage is infected with APEC, not only lytic plaques (clear zones formed by lysis of host cells by one bacteriophage in soft agar with phage plaques) are formed. Since the size and turbidity are the same, it was confirmed that the bacteriophage lysed APEC to inhibit the growth of APEC.

In addition, the stability of the bacteriophage in a variety of pH range and temperature, as a result, it was confirmed that exhibits a stable survival characteristics in a wide range of pH 5.5 to 11 (Fig. 4), especially those having heat resistance at high temperatures It was confirmed (FIG. 5). In addition, when used as a feed additive in the product formulation of the bacteriophage, it was confirmed that the stability to the drying occurring during the formulation process of the bacteriophage was high (Fig. 6).

Such APEC-specific lytic activity, acid resistance, heat resistance and dryness resistance, in applying the bacteriophage of the present invention to a composition for the prevention and treatment of diseases of APEC-derived diseases of birds, and various products including the bacteriophage as an active ingredient, a variety of temperature and pH range The application of is made possible.

The ΦCJ18 is a bacteriophage newly isolated by the present inventors, and was deposited on March 22, 2012 at the Korean Culture Center of Microorganisms (361-221, Hongje 1-dong, Seodaemun-gu, Seoul) under the accession number KCCM11272P.

According to another aspect of the present invention, a composition for preventing or treating infectious diseases caused by avian pathogenic E. coli, comprising Φ CJ18 as an active ingredient.

The infectious disease caused by the avian pathogenic E. coli is not particularly limited, but may preferably be avian coliosis (Colibacillosis).

The avian coliform disease is a disease caused by infecting the pathogen Escherichia coli with the bird, such as airsacculitis, perihepatitis, peritonitis, pericarditis, pericarditis, salpingitis ), A disease that causes various lesions such as oomphalitis, osteomyelitis, or septicemia, causing growth and death of infected birds.

As used herein, the term "prevention" means any action that provides a composition comprising the Φ CJ18 and / or the Φ CJ18 as an active ingredient to inhibit the disease or delay the onset of the disease.

As used herein, the term "treatment" refers to any action that provides a composition comprising the Φ CJ18 and / or the Φ CJ18 as an active ingredient to ameliorate the pathological condition of a pre-infected disease.

The composition for the prevention or treatment of infectious diseases caused by the avian pathogenic E. coli of the present invention may preferably contain the Φ CJ18 5 x 10 ² to 5 x 10 ¹² pfu / ml, more preferably the Φ CJ18 1 x 10 ⁶ to 1 x 10 ¹⁰ pfu / ml.

The composition for preventing or treating an infectious disease caused by the avian pathogenic E. coli of the present invention may further include a pharmaceutically acceptable carrier.

By "pharmaceutically acceptable carrier" is meant a carrier or diluent that does not stimulate the organism and does not inhibit the biological activity and properties of the administered compound.

The carrier is a carrier which can be used for the composition for the prevention or treatment of infectious diseases caused by the avian pathogenic E. coli, which is formulated as a liquid solution, and is suitable for sterilization and living body, including saline solution, sterile water, Ringer's solution, buffered saline, and albumin injection solution. , Dextrose solution, maltodextrin solution, glycerol or ethanol and the like. These can be used individually or in mixture of 2 or more types, and can add other conventional additives, such as antioxidant, buffer, and / or bacteriostatic agent, as needed.

Diluents, dispersants, surfactants, binders and / or lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

The mode of administration of the composition for the prevention or treatment of infectious diseases by the avian pathogenic E. coli of the present invention is not particularly limited, and may be in accordance with the method commonly used in the art.

For example, the composition of the present invention can be administered to birds via nasal spray, oral or parenteral administration, and can be used by application or spraying to a diseased site.

In the case of nasal spraying, a certain amount of Φ CJ18 may be diluted in water to be absorbed into the nasal cavity through a sprayer or a spray system. Examples of the nasal spray or respiratory formulation for nasal spray include aerosols and the like.

The nasal spray or respiratory formulation may further comprise a propellant and / or other additives to disperse the dispersed dispersion or the wet powder.

In the case of parenteral administration, intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration or topical administration may be used.

Non-limiting examples of the formulation for parenteral administration include injection formulations such as subcutaneous injection, intravenous injection or intramuscular injection, and the parenteral administration formulation is used for infectious diseases caused by the avian pathogenic E. coli of the present invention. Prophylactic or therapeutic compositions can be prepared in the form of solutions or suspensions by mixing in water with stabilizers and / or buffers.

Non-limiting examples of such oral dosage forms include tablets, water-soluble or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs, and the like.

For formulation into such tablets or capsules, lactose, saccharose, sorbitol, mannitol, starch, amylopectin, binders such as cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, stearic acid Lubricating oils such as magnesium, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax, and the like, and the capsule formulation may further contain a liquid carrier such as fatty oil in addition to the above-mentioned materials.

Suitable application, spraying or dosage of a composition for the prophylaxis or treatment of an infectious disease caused by the avian pathogenic E. coli of the present invention, as well as the formulation method, mode of administration, time of administration and / or route of administration of the composition is subject to administration It may vary depending on factors such as the age, weight, sex of the animal, the extent of the disease symptoms, the foods eaten, the rate of excretion, etc. The skilled veterinarian will normally determine and prescribe the effective dosage for the desired treatment. Can be.

According to another aspect of the present invention, there is provided an antibiotic comprising the Φ CJ18 as an active ingredient.

As used herein, the term "antibiotic" refers to an agent that is provided to an animal in the form of a drug and can kill bacteria, and corresponds to the concept of preservatives, fungicides and antimicrobials.

The animal means mammals and birds including humans.

According to still another aspect of the present invention, there is provided a feed or drinking water additive for poultry comprising the Φ CJ18 as an active ingredient.

The term "poultry" used in the present invention is a concept that collectively leads to animals belonging to birds in livestock.

The poultry is not particularly limited, but may preferably include one or more selected from the group consisting of chicken, duck, and turkey.

The feed or drinking water additives for poultry of the present invention may be prepared by separately preparing Φ CJ18 in the form of feed or drinking water additives and mixing the feed or drinking water, or directly adding the same when preparing feed or drinking water.

The composition comprising the Φ CJ18 and / or the Φ CJ18 used as the feed or drinking water additive of the present invention may be in a liquid or dry state, preferably in the form of a dry powder.

The drying method for preparing the feed or drinking water additive of the present invention in the form of a dried powder is not particularly limited, and a method commonly used in the art may be used.

Non-limiting examples of the drying method may be ventilation drying, natural drying, spray drying or freeze drying, and these may be carried out by using a single method or a combination of two or more methods.

The feed or drinking water additive of the present invention may further be added to other non-pathogenic microorganisms.

Non-limiting examples of the microorganisms that may be added include physiological conditions under anaerobic conditions such as Bacillus subtilis, Bacillus subtilis, cattle stomach, which can produce proteolytic enzymes, lipolytic enzymes and / or sugar converting enzymes. Lactobacillus sp. ( Lactobacillus sp.) With active and organic degradability, filamentous bacteria such as Aspergillus oryzae , which have the effect of increasing the weight of livestock, increasing the milk yield of milk, and increasing the digestion absorption rate of feed. 43: 910-926, 1976) and Saccharomyces cerevisiae ) such as yeast (J Anim Sci 56: 735-739, 1983). These may be used alone or in combination of two or more.

The poultry feed used in the present invention is not particularly limited and may be used a feed commonly used in the art.

Non-limiting examples of the feed for poultry include, but are not limited to, vegetable feeds such as cereals, fruit fruits, food processing by-products, algae, fibres, pharmaceutical by-products, oils, starches, gourds or grain by-products; And animal feeds such as proteins, minerals, fats and oils, minerals, fats and oils, single cell proteins, zooplankton or foods. These may be used alone or in combination of two or more.

Drinking water for poultry used in the present invention is not particularly limited, it is possible to use drinking water commonly used in the art.

Feed or drinking water additive containing the Φ CJ18 of the present invention as an active ingredient may further include other additives as necessary.

Non-limiting examples of the additives that can be used include binders, emulsifiers, preservatives, amino acids, vitamins, enzymes, probiotics, flavoring agents, nonprotein nitrogen compounds, silicates, buffers, coloring agents, Extractant or oligosaccharide, and the like, and may further include a feed admixture. These may be added alone or in combination of two or more.

The feed additive of the present invention may be contained in an amount of preferably 0.05 to 10 parts by weight, more preferably 0.1 to 2 parts by weight based on 100 parts by weight of feed.

The drinking water additive of the present invention may be contained with respect to 100 parts by weight of drinking water, preferably 0.0001 to 0.01 parts by weight, more preferably 0.001 to 0.005 parts by weight.

According to another aspect of the present invention, there is provided a disinfectant or cleaning agent comprising the Φ CJ18 as an active ingredient.

The formulation of the disinfectant or cleaning agent is not particularly limited and may be prepared and used in a formulation known in the art.

The disinfectant may be used in, but not limited to, an active area of poultry, a slaughterhouse, a dead zone, a cooking place, or a cooking facility.

The cleaning agent may be used for cleaning the surface of the poultry or each part of the body, which is exposed to or may be exposed to avian pathogenic E. coli, but is not limited thereto.

According to another aspect of the present invention, a method for preventing or treating an infectious disease caused by avian pathogenic E. coli is provided by using the composition comprising ΦCJ18 or ΦCJ18 as an active ingredient.

The prophylactic or therapeutic method of the present invention specifically includes administering to the individual infected with or at risk of being infected with avian pathogenic E. coli, the ΦCJ18 or a composition comprising the ΦCJ18 as an active ingredient in a pharmaceutically effective amount.

The composition comprising the Φ CJ18 or the Φ CJ18 of the present invention as an active ingredient may be administered by spraying nasal to poultry in the form of a pharmaceutical preparation, or directly added to feed or drinking water of poultry to be administered by a method of feeding it. It can be administered in admixture with feed or drinking water in the form of feed or drinking water additives.

The route of administration of the composition comprising ΦCJ18 or ΦCJ18 of the present invention as an active ingredient is not particularly limited as long as it can reach the target tissue of interest, and may be administered through various routes, for example, nasal, oral or parenteral. have.

Non-limiting examples of the route of administration of the composition comprising ΦCJ18 or ΦCJ18 as an active ingredient, oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, nasal or inhaled The way it works.

A suitable daily dose of the composition comprising the Φ CJ18 or the Φ CJ18 of the present invention as an active ingredient administered to the prophylactic or therapeutic method may be determined by the treating physician within the scope of correct medical judgment.

The specific therapeutically effective amount of the composition comprising ΦCJ18 or ΦCJ18 as an active ingredient for a particular poultry may vary depending on the type and extent of the reaction to be achieved, the age, weight, general state of health, sex or diet of the subject. It may be determined in consideration of the administration time of ΦCJ18 or the composition, the route of administration and the ratio of the composition, the duration of treatment, etc. It is preferable.

Hereinafter, the present invention will be described in more detail by describing Examples. However, the following embodiments are only examples of the present invention and should not be construed as limiting the scope of the present invention.

Example  Isolation of Bacteriophage Infected with Avian Pathogenic E. Coli

Example  1-1

Bacteriophage Screening and Single Bacteriophage Separation

After centrifugation of 50 ml of the sample obtained from the farming fecal and environmental samples from a duck farm in Namwon, Jeollabuk-do for 10 minutes at 4,000 rpm, the supernatant was filtered with a 0.45 μm filter to prepare a sample solution. A soft agar overlay method was performed.

Specifically, APEC (E10-5) shake culture (OD ₆₀₀ = 2) 18 ml of the sample filtrate was mixed with 150 µl and 2 ml of 10 X LB medium (tryptone 10 g / l; yeast extract 5 g / l; NaCl 10 g / l) and incubated at 30 ° C. for 18 hours. Next, the culture solution was centrifuged at 4,000 rpm for 10 minutes and the supernatant was filtered with a 0.45 μm filter. Then, 3 ml of 0.7% (w / v) agar and APEC (E10-5) shake culture (OD ₆₀₀ ) on top of the LB plate medium = 2) 150 μl of the mixed solution was poured and hardened. Then, 10 μl of the sample solution was added dropwise thereto, followed by incubation at 30 ° C. for 18 hours to confirm that lysate plaques were formed.

Since one type of bacteriophage is known to exist in one lysate, it was intended to separate a single bacteriophage from the formed lysate. Specifically, the lysate was added to 400 μl of SM solution (NaCl 5.8 g / L; MgSO ₄ 7H ₂ O 2 g / L; 1M Tris-Cl (pH 7.5) 50 mL) and left at room temperature for 4 hours to bacteriophage. A solution was obtained.

Next, 100 μl of the bacteriophage solution was added to 12 mL of 0.7% (w / v) agar and the APEC (E10-5) shake culture (OD _600). = 2) mixed with 500 μl, followed by a soft agar overlay method using 150 mm diameter LB plate medium and incubated until complete lysis. After the incubation was completed, 15 ml of SM solution was added to the LB plate medium and left at room temperature for 4 hours to obtain a bacteriophage solution.

The solution was recovered, 1% (v / v) chloroform was added, mixed for 10 minutes and centrifuged at 4,000 rpm for 10 minutes to obtain the supernatant, which was filtered through a 0.45 μm filter to obtain a final sample.

Example  1-2

Mass Cultivation and Purification of Bacteriophage

The bacteriophage obtained in Example 1-1 was incubated in large quantities in APEC (E10-5) and purified from the bacteriophage.

Specifically, APEC (E10-5) was shaken and cultured to 1.5 X 10 ¹⁰ cfu, centrifuged at 4,000 rpm for 10 minutes, and then resuspended in 4 ml SM solution. Here, the bacteriophage obtained in Example 1-1 was inoculated with 1.5 X 10 ⁶ pfu, titrated with MOI (multiplicity of infection) = 0.0001, and left to stand at room temperature for 20 minutes.

It was then inoculated in 150 ml LB medium and incubated at 30 ° C. for 6 hours. After the incubation was completed, chloroform was added to 1% (v / v) of the final volume and stirred for 20 minutes. The restriction enzymes DNase I and RNase A were respectively added to a final concentration of 1 μg / ml and the mixture was heated at 30 ° C. Allow to stand for 30 minutes. Then, NaCl and PEG (polyethylene glycol) were added to the final concentrations of 1 M and 10% (w / v), respectively, and further left at 4 ° C. for 3 hours, followed by centrifugation at 12,000 rpm for 20 minutes at 4 ° C. Obtained.

The obtained precipitate was suspended in 5 ml SM solution and left to stand at room temperature for 20 minutes. Then, 4 ml chloroform was added thereto, stirred, and centrifuged at 4,000 rpm for 20 minutes to obtain the supernatant. The supernatant was filtered with a 0.45 μm filter, followed by ultracentrifugation (35,000 rpm, 1 hour, 4 ° C.) using glycerol density gradient (density: 40%, 5% glycerol) to purify the bacteriophage.

The present inventors took a sample from the slaughtered meal, named the bacteriophage having a specific killing ability to APEC as "Bacteriophage Φ CJ18," and the Korean Culture Center of Microorganisms, Seodaemun-gu, Seoul, March 22, 2012. It was deposited with Accession No. KCCM11272P in Hongje-dong 361-221).

Example  2

Φ CJ18 Comparison of Avian Pathogenic E. Coli Infection

Cross infection was performed to confirm whether Φ CJ18 purified in Example 1 exhibits lytic activity against E. coli in addition to APEC (E10-5).

Specifically, two types of APEC (E10-5) and APEC (E09-35) and six types of non-pathogenic E. coli (E09-1, E09-10, E09-13, E09-14, E09-15, E09-16) After culturing each of them to obtain a culture solution, the formation of lysate plaques was confirmed by performing a soft agar overlay method using the respective culture solution and the purified Φ CJ18.

The results are shown in Table 1.

Strain name Formation of lytic plaque APEC (E10-5) O E. coli (E09-35) O E. coli (E09-1) X E. coli (E09-10) X E. coli (E09-13) X E. coli (E09-14) X E. coli (E09-15) X E. coli (E09-16) X

As shown in Table 1, ΦCJ18 purified in Example 1 did not show the lytic activity against non-pathogenic E. coli.

Example  3

Φ CJ18 Form observation

ΦCJ18 purified in Example 1 was diluted in 0.01% gelatin solution and fixed with 2.5% glutaraldehyde solution. This was added dropwise to a carbon-coated mica plate (ca. 2.5 X 2.5 mm) and then adapted for 10 minutes and then washed with sterile distilled water.

Next, the carbon film was placed on a copper grid, dyed in 4% uranyl acetate for 30 to 60 seconds, dried and then JEM-1011 transmission electron microscope (80 kV). And magnification X 120,000 to X 200,000) (FIG. 1).

Figure 1 shows an electron micrograph of ΦCJ18, which was found to have a icosahedron head of about 40 nm size and a tailless morphotype.

Example  4

Φ CJ18 Whole genome of DNA Size analysis

Genomic DNA was extracted from ΦCJ18 purified in Example 1 above.

Specifically, 20 mM EDTA, 50 μg / ml proteinase K and 0.5% (w / v) SDS were added to the purified ΦCJ18 culture, and allowed to stand at 50 ° C. for 1 hour, and the same volume of phenol (pH 8.0) was added and stirred, followed by centrifugation at 12,000 rpm for 10 minutes at room temperature to obtain the supernatant.

The supernatant was then mixed with an equal volume of PC (phenol: chloroform = 1: 1) and centrifuged at 12,000 rpm for 10 minutes at room temperature to obtain the supernatant. The supernatant was mixed with an equal volume of chloroform and centrifuged at 12,000 rpm for 10 minutes at room temperature to obtain a supernatant. Then, 3M sodium acetate was added to the supernatant so that 10% (v / v) of the total volume was mixed, and mixed with 2 times the volume of cold 95% ethanol, and then mixed at -20 ° C for 1 hour. Let it stand.

Then, the precipitate was obtained by centrifugation at 12,000 rpm for 10 minutes at 0 ° C., to which 50 µl TE buffer (Tris-EDTA, pH 8.0) was added to dissolve and the concentration thereof was measured. Next, 1 μg of DNA was loaded onto a 1% pulse-field gel electrophoresis (PFGE) agarose gel, and the BioORAD PFGE System No. 7 program (size range 25 to 100 kb; switch time ramp). ) 0.4 to 2.0 seconds, linear shape; forward voltage 180 V; reverse voltage 120 V) for 20 hours at room temperature (FIG. 2).

Figure 2 is an electrophoresis picture of genomic DNA of ΦCJ18, genomic DNA of ΦCJ18 was confirmed that the size of about 47 kbp.

Example  5

Φ CJ18 Protein pattern analysis

15 μl of 10 ¹⁰ pfu / ml titer purified ΦCJ18 solution and 3 μl of 5X SDS sample solution were mixed and boiled for 5 minutes, followed by 15% SDS-PAGE (FIG. 3).

Figure 3 is an electrophoresis picture showing the SDS-PAGE results performed on ΦCJ18, the major proteins of 45 kDa, 36 kDa, 35 kDa and 13 kDa size can be confirmed.

Example  6

Φ CJ18 Genetic characterization of

In order to determine the genetic properties of ΦCJ18 purified in Example 1, 5 μg of genomic DNA of ΦCJ18 was treated with EcoR V, a restriction enzyme, and pCL1920 (Promega) was cut with Sma I restriction enzyme as a vector. CIP (calf intestinal alkaline phosphatase) was used. After the reaction conditions were mixed so that the amount of the genomic DNA and the vector was 3: 1, ligation was performed at 16 ° C. for 5 minutes. It was then introduced into DH5α cells, a type of E. coli.

The transformant was converted into spectinomycin and X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal (5-bromo-)). Two colonies were selected by conventional blue-white colony selection in LB plate medium containing 4-chloro-3-indolyl-beta-D-galactopyranoside). Shake incubation for hours where plasmids were extracted using plasmid purification kit (Solgent).

The plasmids were cloned by PCR using M13 forward primer and M13 reverse primer set, and the insertion fragment size was 1 kb or more, and the M13 forward primer and M13 reverse primer set were used. Similarity of base sequences was analyzed (Table 2).

The base sequence of the bacteriophage showed various similarities to the base sequence of the previously reported bacteriophage, but it was confirmed that there was no bacteriophage in which all fragments were 100% identical. As a result, the bacteriophages could be identified as newly isolated bacteriophages.

Table 2 below shows the result of comparing the homology of the base sequence of Φ CJ18 and the translation base sequence of other bacteriophages.

Example  7

Φ CJ18  Specific primer  Construction of the base sequence

In order to identify ΦCJ18, a ΦCJ18 specific primer was prepared.

Specifically, primer sets of SEQ ID NOs: 7 and 8 based on SEQ ID NO: 1, and primer sets of SEQ ID NOs: 9 and 10 were prepared based on SEQ ID NO: 2. In addition, primer sets SEQ ID NO: 11 and 12, and SEQ ID NO: 13 and 14, respectively, based on SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 15 and 16, and SEQ ID NO: 15 based on SEQ ID NO: 5, SEQ ID NO: 6, respectively PCR was performed by preparing 17 and 18 primer sets.

Primers were added to Pre-mix (Bioneer) to 0.1 μg of bacteriophage whole genomic DNA and 0.5 pmol and adjusted to a final volume of 20 μl. This is then denatured; 94 ° C., 30 seconds, annealing; 60 ° C., 30 seconds, polymerization; PCR was carried out for 30 cycles under conditions of 72 ° C. and 1 minute.

As a result, when using the primer sets SEQ ID NO: 7 and 8, SEQ ID NO: 9 and 10, SEQ ID NO: 11 and 12, SEQ ID NO: 13 and 14, SEQ ID NO: 15 and 16, SEQ ID NO: 17 and 18, respectively, about 213 bp, PCR products of 306 bp, 220 bp, 500 bp, 1146 bp and 897 bp were obtained.

Example  8

pH Φ according to CJ18 Investigation of stability

Stability investigation experiments were conducted at various pH ranges (pH 2.1, 2.5, 3.0, 3.5, 4.0, 5.5, 6.4, 7.5, 8.3, 9.2 and 11.0) to ensure that ΦCJ18 can retain stability at low pH in the stomach. .

Various pH solutions (sodium acetate buffer solution (pH 2.1, pH 4.0, pH 5.5, and pH 6.4), sodium citrate buffer solution (pH 2.5, pH 3.0 and pH 3.5), sodium phosphate buffer solution (pH 6.9 and pH 7.4), Tris-HCl solution (pH 8.2, pH 9.0, pH 9.8 and pH 11.0) was prepared at 0.2 M each.

90 μl of each pH solution and 10 μl of bacteriophage solution of 1.0 × 10 ¹¹ PFU / ml titer were mixed to make the concentration of each pH solution 1M, and then allowed to stand at room temperature for 2 hours. Then, these were diluted in stages, and 10 μl of the diluted solution of each stage was dropped using a soft agar overlay method, followed by incubation at 37 ° C. for 18 hours to determine titer through lysis (FIG. 4).

Figure 4 shows the acid resistance test results of the bacteriophage Φ CJ18. As shown in FIG. 4, ΦCJ18 was stable without losing activity from pH 5.5 to pH 11.0 as compared to the control group.

Example  9

Φ according to temperature CJ18 Investigation of stability

When used as a feed additive in the formulation of the bacteriophage experiment was conducted to confirm the stability of the heat generated during the formulation of the bacteriophage.

Specifically, 100 μl of a solution of ΦCJ18 at a concentration of 4.0 × 10 ⁸ PFU / ml was allowed to stand at 60 ° C. for 0, 10, 30, 60 and 120 minutes, respectively, and then the experimental culture was diluted in steps to soft agar overlay. 10 μl of the diluted solution of each step was dropped by the method, followed by incubation at 30 ° C. for 18 hours to determine titer through lysis (FIG. 5).

Figure 5 shows the heat resistance test results of bacteriophage Φ CJ18. As shown in FIG. 5, ΦCJ18 was decreased by 1 log when exposed to 60 ° C. for 1 hour, and decreased by about 2 log after 2 hours of exposure.

Example  10

Φ due to drying CJ18 Investigation of stability

When used as a feed additive in the formulation of the bacteriophage experiment was carried out to confirm the stability of the drying occurs during the formulation of the bacteriophage.

Specifically, 100 μl of a solution of ΦCJ18 at a concentration of 6.0 × 10 ⁸ PFU / mL was allowed to stand at 60 ° C. for 0 minutes, 60 minutes, and 120 minutes, respectively. After dropping the ㎕ by incubation at 30 ℃ for 18 hours to determine the titer through the lysis (Fig. 6).

Figure 6 shows the results of the drying resistance of the bacteriophage Φ CJ18. As shown in FIG. 6, ΦCJ18 decreased by 1 log when dried at 60 ° C. for 1 hour, and decreased by about 3 logs after 2 hours of exposure.

Example  11

wild Separator APEC Φ for CJ18 The scope of infection

In addition to the APEC (E10-5) used for the experiment, ΦCJ18 was confirmed to have lytic activity against APEC 6 strains isolated from Konkuk University.

Specifically, 150 μl of shake culture (OD ₆₀₀ = 2) of each strain was mixed, followed by a soft agar overlay method, followed by 10 μl of a solution of ΦCJ18 of 10 ⁸ pfu / ml titer, followed by incubation at 30 ° C. for 18 hours. The formation of lytic plaques was observed (Table 3).

The results are shown in Table 3.

Strain name ΦCJ18 lytic plaque formation APEC (E09-6) O APEC (E09-11) O APEC (E09-35) O APEC (E10-03) O APEC (E10-04) O APEC (E10-05) O

As shown in Table 3, it was confirmed that the general poultry farms show an effective infectious ability against APEC (including O-78 serotype), which is the causative agent of avian Escherichia coli.

On the other hand, the O-78 serotype is generally known to be the most frequently appearing strain of bird pathogenic E. coli isolated from poultry farms.

Korea Microorganism Conservation Center (overseas) KCCM11272P 20120322

Attach an electronic file to a sequence list

Claims (10)

Avian Pathogenic Escherichia coli) New bacteriophage ΦCJ18 (KCCM11272P) having a specific ability to kill. A composition for preventing or treating infectious diseases caused by avian pathogenic E. coli, comprising the bacteriophage Φ CJ18 (KCCM11272P) according to claim 1 as an active ingredient. The composition for preventing or treating infectious diseases caused by avian pathogenic E. coli according to claim 2, wherein the infectious disease is avian coliform (Colibacillosis). An antibiotic comprising the bacteriophage ΦCJ18 (KCCM11272P) according to claim 1 as an active ingredient. A feed or drinking water additive for poultry comprising the bacteriophage ΦCJ18 (KCCM11272P) according to claim 1 as an active ingredient. The feed or drinking water additive for poultry according to claim 5, wherein the poultry comprises at least one selected from the group consisting of chicken, duck and turkey. A disinfectant or cleaning agent comprising the bacteriophage Φ CJ18 (KCCM11272P) according to claim 1 as an active ingredient. The bacteriophage Φ CJ18 (KCCM11272P) according to claim 1 Or administering the composition of claim 2 to poultry, the method for preventing or treating an infectious disease caused by avian pathogenic E. coli. The method of claim 8, wherein the infectious disease is avian coliform bacterium. The method of claim 8, wherein the poultry comprises at least one member selected from the group consisting of chicken, duck, and turkey.

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