KR102203670B1 - Novel Enterococcus specific bacteriophage EH11 and antibacterial composition comprising the same - Google Patents

Abstract

The present invention relates to a novel Enterococcus bacterium-specific bacteriophage EH11, and more particularly, to an antibacterial composition comprising the Enterococcus bacterium-specific bacteriophage EH11. The bacteriophage EH11 according to the present invention has very high specificity for Enterococcus bacteria, has excellent lytic activity by infecting Enterococcus bacteria and proliferates in Enterococcus bacteria, and has the advantage of being strong against physicochemical stimulation. In addition, since the bacteriophage EH11 of the present invention does not infect other hosts other than bacteria such as humans, animals, plants, etc., it can solve the problems of antibiotic-resistant bacteria due to misuse of antibiotics, the problem of remaining antibiotics in food, and problems of a wide host range. It can be used in a variety of fields for composition, composition for feed addition, feed, disinfectant or cleaning agent.

Description

Novel Enterococcus specific bacteriophage EH11 and antibacterial composition comprising the same}

The present invention relates to a novel Enterococcus bacterium-specific bacteriophage EH11, and more particularly, to an antibacterial composition comprising the Enterococcus bacterium-specific bacteriophage EH11.

In general, Enterococcus is a gram-positive bacterium as Enterococcus , and it is a major pathogen because of the concern about the transmission of antibiotic-resistant traits. It is an opportunistic infectious bacteria related to nosocomial and community infections and a causative agent of infectious endocarditis, accounting for 5 to 15%. It can also cause infections in the airways, wounds, liver, bile ducts, abdomen and urinary tract. Representative species are Enterococcus faecalis and Enterococcus faecium , and Enterococcus hirae occupies a low incidence rate of 1 to 3% in clinical samples, but mammals and poultry It is often found on the back and is known to cause disease. When samples were taken from more than 10 species of birds showing sepsis symptoms, Enterococcus hirae was detected at a rate of 70%. It also causes sepsis and brain necrosis in chicks and parrots. A common feature of Enterococcus hirae infection is the attachment of the fungus to the intestinal villi. It is associated with pathological conditions in animals and has been shown to be the cause of diseases such as sepsis, endocarditis and urinary tract infections.

In the case of bacteria carrying the antibiotic resistance gene on the farm, the antibiotic resistance factor can be transmitted to the human body due to consumption of meat or dairy products. According to the reported paper, Enterococcus hirae, which carries the antibiotic resistance gene vanA, was isolated from the antibiotic-treated flock. vanA is a resistant gene that is present in the plasmid and can be rapidly transferred. Resistance to antibiotics is further enhanced due to the biofilm-forming ability of Enterococcus bacteria. A biofilm is a structure in the form of a membrane produced by microorganisms, and microorganisms proliferating into a biofilm have higher resistance to antibiotics and host defense than the plankton form of the same microorganism. There are many difficulties in resolving most biofilm infections, and chronic or recurrent infections are common. Accordingly, as it becomes difficult to remove the bacteria, the development of effective therapeutic agents is required.

On the other hand, bacteriophage refers to a bacteria-specific virus that inhibits and inhibits the growth of infected bacteria by infecting specific bacteria. Bacteriophages have the ability to kill bacteria by proliferating inside bacterial cells after infection with bacteria, and destroying the cell walls of host bacteria when progeny bacteriophages come out of the bacteria after proliferation. The bacterial infection method of bacteriophage is very specific, so the types of bacteriophage that can infect specific bacteria are limited to some. That is, a specific bacteriophage can infect only a specific category of bacteria, and because of this, a specific bacteriophage kills only specific bacteria and does not affect other bacteria. Therefore, the use of bacteriophage as a countermeasure for recent bacterial diseases is receiving great attention. It can be said that interest in bacteriophage is higher than ever due to the preference of nature-friendly methods. As the possibility of development as a substitute for existing antibiotics has emerged, bacteriophage is again attracting attention as an anti-bacterial agent.

Accordingly, the present inventors have isolated the Enterococcus bacteriophage specific bacteriophage in order to utilize the bacteriophage as a substitute for the existing antibiotic, and confirmed that it can selectively kill Enterococcus bacteria through morphological and genetic analysis of the isolated bacteriophage. Thus, the present invention was completed.

Accordingly, an object of the present invention is to provide a bacteriophage EH11 consisting of a nucleotide sequence represented by SEQ ID NO: 1, which has a specific killing ability for Enterococcus bacteria.

Another object of the present invention is to provide an antibiotic composition comprising the bacteriophage EH11.

Another object of the present invention is to provide a feed additive containing the bacteriophage EH11.

Another object of the present invention is to provide a disinfectant comprising the bacteriophage EH11.

Another object of the present invention is to provide a cleaning agent containing the bacteriophage EH11.

Another object of the present invention is to provide a method for preventing or treating infectious diseases caused by Enterococcus bacteria comprising administering the bacteriophage EH11 to an individual.

In order to achieve the above object, the present invention provides a bacteriophage EH11 consisting of a nucleotide sequence represented by SEQ ID NO: 1, having a specific killing ability for Enterococcus bacteria.

In addition, the present invention provides an antibiotic composition comprising the bacteriophage EH11.

In addition, the present invention provides a feed additive containing the bacteriophage EH11.

In addition, the present invention provides a disinfectant comprising the bacteriophage EH11.

In addition, the present invention provides a cleaning agent containing the bacteriophage EH11.

In addition, the present invention provides a method for preventing or treating infectious diseases caused by Enterococcus bacteria, comprising administering the bacteriophage EH11 to an individual.

The bacteriophage EH11 according to the present invention has very high specificity for Enterococcus bacteria, has excellent lytic activity by infecting Enterococcus bacteria and proliferates in Enterococcus bacteria, and has the advantage of being strong against physicochemical stimulation. In addition, since the bacteriophage EH11 of the present invention does not infect other hosts other than bacteria such as humans, animals, plants, etc., it can solve the problems of antibiotic-resistant bacteria due to misuse of antibiotics, the problem of remaining antibiotics in food, and problems of a wide host range. It can be used in a variety of fields for composition, composition for feed addition, feed, disinfectant or cleaning agent.

Figure 1 is a diagram showing the result of confirming the lysis plaque formation activity for Enterococcus Hira of the bacteriophage EH11 according to the present invention.
Figure 2 is a diagram showing the result of analyzing the entire gene of the bacteriophage EH11 according to the present invention.
Figure 3 is a view showing the result of confirming the morphological characteristics of the bacteriophage EH11 according to the present invention.
Figure 4 is a diagram showing the result of analyzing the killing ability of the bacteriophage EH11 according to the present invention against Enterococcus Hira.
Figure 5 is a diagram showing the result of confirming the pH stability of the bacteriophage EH11 according to the present invention.
6 is a diagram showing the results of confirming the thermal stability of the bacteriophage EH11 according to the present invention.

Hereinafter, the present invention will be described in detail.

The present invention provides a bacteriophage EH11 consisting of a nucleotide sequence represented by SEQ ID NO: 1, which has a specific killing ability for Enterococcus bacteria.

In the present invention, "bacteriophage" is a bacterial-specific virus that inhibits and inhibits the growth of the bacterium by infecting a specific bacterium, and refers to a virus containing a single or double-stranded DNA or RNA as a genetic material.

In the present invention, "Enterococcus bacterium" is a streptococcus belonging to the serotype D group belonging to the genus Enterococcus, and is a gram-positive bacterium. The Enterococcus bacterium is a bacterium that coexists in the human intestine, and its infectivity is different depending on the specific species.

In the present invention, the Enterococcus bacterium is Enterococcus faecalis , Enterococcus faecium , Enterococcus hirae , Enterococcus durans , Enterococcus ralcedinis ( Enterococcus alcedinis ), Enterococcus aquimarinus , Enterococcus asini , Enterococcus avium , Enterococcus bulliens , Enterococcus cacae , Enterococcus cacae It is preferably at least one selected from the group consisting of caucus camelliae ( Enterococcus camelliae ) and Enterococcus canitestini , and more preferably Enterococcus hiraeina , but is not limited thereto.

The bacteriophage EH11 has excellent lytic activity against Enterococcus bacteria, particularly Enterococcus Hira. In addition, the bacteriophage EH11 has a size of 70 to 80 nm, and belongs to Podoviridae having a head and a short tail of an icosahedron. The bacteriophage EH11 has excellent antimicrobial activity against Enterococcus bacteria, as well as excellent stability against heat and pH. More specifically, the bacteriophage EH11 is stable in the range of pH 3 to 11, and in particular, it is preferably stable for 2 hours in the range of pH 4 to 11, but is not limited thereto. In addition, the bacteriophage EH11 is stable in a temperature range of 40 to 60° C., but is not limited thereto.

Very stable at a temperature of 40 to 60° C., but is not limited thereto.

The bacteriophage EH11 of the present invention may include the nucleotide sequence represented by SEQ ID NO: 1 as all or part of the entire gene. In addition, the bacteriophage EH11 of the present invention may consist of a nucleotide sequence represented by SEQ ID NO: 1, and a functional equivalent of the nucleotide sequence. The functional equivalent is at least 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% of the base sequence represented by SEQ ID NO: 1 as a result of modification or substitution of the base sequence. It has the above sequence homology, and refers to a sequence that exhibits substantially the same physiological activity as the nucleotide sequence represented by SEQ ID NO: 1.

As described above, the specific lytic activity of Enterococcus bacteria, acid resistance, base resistance and heat resistance are various products containing the bacteriophage EH11 of the present invention for the prevention and treatment of infectious diseases caused by Enterococcus bacteria, and the bacteriophage EH11 as an active ingredient. In application to, it enables the application of various temperature and pH ranges.

The bacteriophage EH11 of the present invention is a bacteriophage obtained by collecting samples from a slaughterhouse, and the present inventors named it bacteriophage EH11 and deposited with the Korean Culture Center of Microorganisms (Seoul, Korea) on August 14, 2018. , Has been assigned the accession number KFCC11783P.

In addition, the present invention provides a composition for antibiotics, including the bacteriophage EH11.

In the present invention, the "antibiotic composition" refers to an agent that is provided to an individual in the form of a drug and can kill bacteria, and refers to preservatives, fungicides, antibiotics and antibacterial agents.

The bacteriophage EH11 of the present invention has very high specificity for Enterococcus bacteria compared to conventional antibiotics, so it does not kill beneficial bacteria, can kill only specific pathogens, and does not induce drug resistance or resistance, compared to conventional antibiotics. It can be used as a novel antibiotic with long life cycling.

In addition, the present invention provides a composition for feed addition, including the bacteriophage EH11.

Feed-added antibiotics used in livestock and fishery industries are used for the purpose of preventing diseases, and administration of antibiotics for preventive purposes increases the likelihood of developing resistant bacteria and is a problem because antibiotics remaining in livestock can be delivered to humans. When antibiotics are absorbed into the body through meat, they can cause antibiotic resistance, which can lead to the spread of disease. In addition, since there are many kinds of antibiotics mixed with feed and this has a problem that the probability of multidrug resistant bacteria increases, the bacteriophage EH11 of the present invention is a new feed addition antibiotic that is more natural friendly and solves the problems arising from the use of existing antibiotics. You can use

In addition, the present invention may provide a feed containing the composition for feed addition, and the feed of the present invention may be prepared by separately preparing bacteriophage in the form of a feed additive and mixing it with the feed, or by directly adding it when preparing the feed. . The bacteriophage in the feed of the present invention may be in a liquid or dry state, preferably in a dried powder form. The drying method may be ventilated drying, natural drying, spray drying, and freeze drying, but is not limited thereto. The bacteriophage of the present invention may be mixed in a powder form in a component ratio of 0.05 to 10% by weight, preferably 0.1 to 2% by weight of the feed weight. In addition, the feed may further include conventional additives that can increase the preservability of the feed in addition to the bacteriophage of the present invention.

Other non-pathogenic microorganisms may be additionally added to the composition for feed addition of the present invention. Microorganisms that can be added include Bacillus subtilis , which can produce proteases, lipolytic enzymes, and sugar converting enzymes, and Lactobacillus strains that have physiological activity and ability to decompose organic matter under anaerobic conditions such as the stomach of cattle. sp.), selected from the group consisting of Aspergillus oryzae and Saccharomyces cerevisiae , which show the effect of increasing the weight of animals, increasing milk production and increasing the digestion and absorption rate of feed. Can be.

The feed containing the bacteriophage EH11 of the present invention includes cereals, root fruits, food processing by-products, algae, fiber, pharmaceutical by-products, oils and fats, starch, meals, grain by-products, etc. Proteins, inorganic logistics, oils and fats, minerals, single-cell proteins, zooplanktons, food leftovers, etc., but are not limited thereto.

The composition for feed addition of the present invention may contain binders, emulsifiers, preservatives, etc. added to prevent quality deterioration, and amino acids, vitamins, enzymes, probiotics, flavoring agents, non-proteinaceous agents added to the feed to increase utility Nitrogen compounds, silicates, buffers, colorants, extractants, oligosaccharides, etc. may be included, and in addition, feed mixing agents may be additionally included.

In addition, the present invention provides a disinfectant comprising the bacteriophage EH11.

The disinfectant containing the bacteriophage EH11 of the present invention having specific killing ability for various Enterococcus bacteria can be usefully used as a disinfectant for hospitals and health care to prevent hospital infection, and as a disinfectant for general life, disinfectant for food and cooking places and facilities It can be used for disinfection of various growth products such as, poultry farms, buildings such as barns, livestock, drinking water, litter, nanjwa, transport vehicles, and tableware.

In addition, the present invention provides a cleaning agent comprising the bacteriophage EH11.

Since the bacteriophage EH11 of the present invention has a specific killing ability for various Enterococcus bacteria, it can also be used to clean the skin surface or parts of the body of livestock exposed to or likely to be exposed to the Enterococcus bacteria.

In addition, the present invention provides a method for preventing or treating infectious diseases caused by Enterococcus bacteria, comprising administering the bacteriophage EH11 to an individual.

The Enterococcus faecalis ( Enterococcus faecalis ), Enterococcus faecium ( Enterococcus faecium ), Enterococcus hirae ( Enterococcus hirae ), Enterococcus durans ( Enterococcus durans ), Enterococcus alcedinis ( Enterococcus alcedinis ) , Enterococcus aquimarinus , Enterococcus asini , Enterococcus avium , Enterococcus bulliens , Enterococcus caccae , Enterococcus caccae , Enterococcus caccae Enterococcus camelliae ) and Enterococcus canitestini ( Enterococcus canitestini ) It is preferably at least one selected from the group consisting of, more preferably Enterococcus hiraena , but is not limited thereto.

The method of the present invention comprises the steps of administering bacteriophage EH11 to a disease caused by an infection of Enterococcus bacteria, more specifically sepsis, airway infection, hepatitis, cholangitis, peritonitis, endocarditis, or urinary tract infection. Include.

In the present invention, "individual" refers to any individual capable of developing an infectious disease caused by Enterococcus bacteria, and includes, but is not limited to, mammals, livestock, or poultry including humans. The bacteriophage EH11 of the present invention has a specific killing ability for Enterococcus hirae, and may be preferably administered to poultry.

The "livestock" is a concept that refers to a useful animal that has been purified and improved by humans to live in common with humans, and includes, for example, pigs, cows, chickens, horses, ducks or dogs, but is limited thereto. It is not.

The "poultry" is a concept that collectively refers to animals belonging to birds among livestock, and includes, for example, chicken, duck, pheasant, quail, ostrich, goose, parrot or turkey, but is not limited thereto.

In the method of the present invention, the bacteriophage EH11 or a composition containing the same may be administered to an individual in the form of a pharmaceutical preparation, or may be administered through a method of feeding it by mixing it with feed or drinking water of livestock.

In the method of the present invention, the route of administration of the bacteriophage EH11 or a composition containing the same may be administered through various routes, either orally or parenterally, as long as it can reach the target tissue, and specifically, oral, rectal, topical, intravenous, It can be administered in a conventional manner via intraperitoneal, intramuscular, intraarterial, transdermal, intranasal, inhalation, and the like.

It is obvious to those skilled in the art that the appropriate total daily use amount of the bacteriophage EH11 administered in the method of the present invention can be determined by the treating physician within the correct medical judgment range. The specific therapeutically effective amount for a specific individual is determined along with the type and extent of the reaction to be achieved, the patient's age, weight, general health status, sex and diet, administration time, route of administration and secretion rate of the composition, treatment period, and specific composition. It is desirable to apply differently depending on various factors including drugs used or used simultaneously and similar factors well known in the medical field.

Terms not otherwise defined in the present specification have the meanings commonly used in the technical field to which the present invention belongs.

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

Example 1. Isolation and culture of novel bacteriophage

1.1 Screening of bacteriophage and separation of single bacteriophage

In order to screen and isolate the bacteriophage specific to Enterococcus hirae, lysis spot analysis was performed. Samples were collected from slaughterhouses where bacteriophages are expected to exist, and the Enterococcus Hirae used to separate bacteriophages was separated and identified from outdoor samples collected from Optipharm Co., Ltd.

Specifically, the collected sample was homogenized and cultured with Enterococcus Hirae at 35° C. for 24 hours with shaking. After centrifuging the culture solution at 4,000 rpm for 20 minutes, the supernatant was filtered using a filter having a pore size of 0.2 μm to obtain a sample filtrate.

Take 150 μL of pre-cultured Enterococcus hira, put it in a sterilized tube, and put 3 mL of BHI top agar [brain heart leaching solution (brain leaching solids) 12.5 g, bovine heart leaching solids 5.0 g, proteospeptone 10.0 g, Glucose 2.0 g, sodium chloride 5 g, sodium phosphate 2.5 g/L) + 0.6% agar] were added, mixed well, and then layered on a 2% BHI top agar plate made in advance. After allowing the sample to stand at room temperature for 30 minutes, 10 μL of each of the sample filtrate was instilled, and then left at room temperature for 30 minutes, and then cultured at 35° C. for 24 hours to confirm the presence or absence of bacteriophage.

In order to purely separate the bacteriophage from the sample confirming the presence of the bacteriophage, a soft agar overlay method was used. Dilute the sample confirmed to contain bacteriophage properly with saline, take 100 μL of the sample dilution solution from each dilution stage, mix it with 150 μL of the target bacteria (Enterococcus hirae) culture solution, and add 3 mL of 0.6% BHI top agar. After mixing well, the mixture was layered on a pre-made 2% BHI agar plate, left to stand, and incubated at 35° C. for 24 hours. After confirming the lysing plaques formed in each cultured plate, select it and add 400 μL of SM buffer (NaCl 5.8 g/L; MgSO ₄ 7H ₂ O 2 g/L; 1M Tris-HCl (pH7.5) 50 mL). Suspended and left at room temperature for 4 hours. 100 μL of the bacteriophage solution was mixed with 500 μL of 0.6% BHI top agar and bacterial culture solution, then dispensed on a 150 mm diameter BHI plate, layered, and incubated at 35° C. until completely lysis occurred. After the cultivation was completed, 15 mL of SM buffer was added to the BHI plate and the bacteriophage solution was collected while slowly stirring at room temperature for 4 hours, and then centrifuged at 4,000 rpm for 20 minutes to take only the supernatant and filtered through a 0.2 μm filter to final bacteriophage. The solution was recovered. The results of confirming the lysis plate of the final bacteriophage thus obtained are shown in FIG. 1.

As shown in Figure 1, by confirming the lysis plate having a size of 2 to 3 mm, it was confirmed that the bacteriophage is a bacteriophage having a specific lysis ability for Enterococcus Hirae.

1.2 Mass culture and purification of bacteriophage

In order to mass-cultivate the Enterococcus hirae-specific bacteriophage identified in Example 1.1, the final bacteriophage culture solution recovered in Example 1.1 was diluted to 1.0x10 ⁸ PFU/mL and then pre-cultured Enterococcus Hirae 1.0x10 ⁹ CFU. The mixture was mixed with /mL, and the MOI (Multiplicity of Infection) was set to 0.1, followed by incubation at 35°C at 100 rpm for 6 hours. After the cultivation was completed, the entire amount of the culture solution was centrifuged at 6,000 rpm for 30 minutes to obtain a supernatant, which was filtered through a 0.2 μm filter to recover the final bacteriophage culture solution.

The isolated bacteriophage was named "Bacteriophage EH11", and was deposited with the Korean Culture Center of Microorganisms (Seoul, Korea) on August 14, 2018, and the accession number KFCC11783P was granted.

Example 2. Whole gene analysis of bacteriophage EH11

To analyze the entire gene of bacteriophage EH11, 20 mM EDTA, 50 μg/mL proteinase K, 0.5% (w/v) SDS were added to the bacteriophage EH11 culture, and then left at 50° C. for 1 hour. After mixing with the same amount of phenol-chloroform-isopropyl alcohol (25:24:1), the supernatant was recovered by centrifugation for 10 minutes at 12,000 rpm at room temperature. The recovered supernatant was mixed with the same volume of phenol-chloroform-isopropyl alcohol (25:24:1), centrifuged at 12,000 rpm for 10 minutes at room temperature, and then the supernatant was recovered, and 3 M sodium acetate was added to the supernatant. After adding so that it became 10% (v/v) of, it was mixed. Two times the volume of cold 95% ethanol was added thereto, mixed, and allowed to stand at -20°C for 1 hour. After the reaction was completed, a precipitate was obtained by centrifugation at 0° C. for 10 minutes at 12,000 rpm. After washing twice with cold 70% ethanol, the ethanol was completely removed to recover the DNA pellet. The recovered pellet was dissolved in TE buffer (Tris-EDTA, pH 8.0) so that the final volume was 50 μL, and the DNA concentration was measured. Gene sequencing was performed using HiSeq 2500 (Illumina) for full gene analysis (Macrogen, Korea). The gene map of the bacteriophage EH11 obtained as a sequencing result is shown in FIG. 2.

As shown in Figure 2, it was confirmed that the size of the total genes of the bacteriophage EH11 is 19,196 bp, the GC content is 35.5%, and the total number of genes is 25. The entire gene sequence of the analyzed bacteriophage EH11 is shown in SEQ ID NO: 1.

Example 3. Morphological properties of bacteriophage EH11

In order to investigate the morphological characteristics of the bacteriophage EH11, the culture medium 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), acclimated for 10 minutes, and washed with sterile distilled water. It was inserted into a copper grid, dyed in 2% uranyl acetate for 30 to 60 seconds, dried, and then dried, followed by a transmission electron microscope (Tecnai G ² Spirit Twin, Bio-Transmission electron microscope, 120 kV, 120,000~). 400,000). The results are shown in FIG. 3.

As shown in FIG. 3, it was confirmed that the bacteriophage EH11 has a size of 70 to 80 nm, and belongs to Podoviridae having a head and a short tail of an icosahedron.

Example 4. Infectivity and killing ability of bacteriophage EH11 for various bacteria

In order to confirm whether the bacteriophage EH11 exhibits lytic activity against various bacteria, infectivity was confirmed through the soft agar overlay method for strains isolated and possessed by Optipharm Co., Ltd. and strains distributed in microbial resource centers. The results are shown in Table 1.

As shown in Table 1, as a result of performing a susceptibility investigation on Enterococcus bacteria and other microorganisms, it was confirmed that bacteriophage EH11 exhibits lytic activity specific to Enterococcus bacteria, particularly Enterococcus hirae.

In addition, the ability of the bacteriophage EH11 to kill Enterococcus Hira was analyzed. Specifically, 10 mL of a bacterial culture solution having an absorbance of 0.4 at 600 nm was prepared. The experiment was performed by inoculating the bacteriophage solution of various conditions in the bacterial culture solution. Experimental group 1 (MOI 1) was inoculated with MOI 1 by putting 1 mL of bacteriophage solution having a concentration of 1.0x10 ⁹ PFU/mL. Experimental group 2 (MOI 0.1) was inoculated with a MOI of 0.1 by putting 1 mL of bacteriophage solution at a concentration of 1.0x10 ⁸ PFU/mL, and the bacteriophage solution was not added as a control group. After inoculation, the culture was performed for 2 hours, and the culture state of the bacteria was observed by measuring the absorbance at 600 nm in 1 hour increments. The analysis results of the killing ability for Enterococcus Hira using bacteriophage EH11 are shown in Table 2 and FIG. 4.

As shown in Table 2 and Figure 4, in the case of the control group to which the bacteriophage EH11 was not added, the absorbance at 600 nm was 2.79 after 2 hours, indicating that the bacteria grew well. On the other hand, in the case of the experimental group to which the bacteriophage EH11 was added, the absorbance at 600 nm was 0.06 (experimental group 1, MOI 1) and 0.05 (experimental group 2, MOI 0.1) after 2 hours. The result means that the bacteriophage EH11 has excellent killing ability against Enterococcus Hira.

Example 5. Investigation of pH stability of bacteriophage EH11

In order to confirm the stability of the bacteriophage EH11 against the pH, the number of bacteriophages in various pH ranges (pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) was measured. Sodium acetate buffer, sodium phosphate buffer, and Tris-HCl were used to adjust the pH to each pH, and then dispensed into 990 μL to prepare. The bacteriophage EH11 culture solution was prepared to be 5.0× ^{10 8} PFU/mL, and then 10 μL of the culture solution was added to each buffer prepared according to the pH, and left at room temperature for 2 hours. After standing, each reaction solution was diluted stepwise, and the result of performing titer measurement by a soft agar overlay method is shown in FIG. 5.

As shown in Figure 5, it was confirmed that the bacteriophage EH11 of the present invention is stable in the range of pH 3 to 11, and in particular, it was confirmed that it is very stable for 2 hours in the range of pH 4 to 11. Through this, it can be seen that the bacteriophage EH11 is a bacteriophage having excellent pH stability.

Example 6. Thermal stability investigation of bacteriophage EH11

In order to confirm the thermal stability of the bacteriophage EH11, the number of bacteriophages was measured after being left for a certain time at various temperature ranges (40, 50 and 60°C). More specifically, the bacteriophage EH11 culture solution was diluted to 5.0×10 ⁷ PFU/mL, and then 5 mL of the culture solution was dispensed into a sterilized tube and left at each temperature for 10, 30 and 60 minutes. After standing, each reaction solution was diluted stepwise to perform titer measurement by a soft agar overlay method, and the results are shown in FIG. 6.

As shown in Figure 6, it was confirmed that the bacteriophage EH11 of the present invention is stable in the range of 40 to 60 ℃. Through this, it can be seen that the bacteriophage EH11 is a bacteriophage having excellent heat resistance.

Overall, the present inventors have found that the bacteriophage EH11 has very high specificity for Enterococcus bacteria, in particular Enterococcus hira, and has excellent lytic activity by infecting Enterococcus bacteria and proliferating in Enterococcus bacteria, and is strong against physicochemical stimulation. It was confirmed that it has. In addition, since the bacteriophage EH11 of the present invention does not infect other hosts other than bacteria such as humans, animals, real objects, etc., it has the advantage of solving the problems of antibiotic-resistant bacteria due to misuse of antibiotics, the problem of residual antibiotics in food, and problems of a wide host range. have. Therefore, the bacteriophage EH11 of the present invention can be variously used in the field of antibiotic compositions, feed addition compositions, feed, disinfectants or cleaning agents.

Hereinafter, the present invention will be described in more detail through formulation examples. Formulation examples are for illustrative purposes only, and the scope of the present invention is not construed as being limited by formulation examples.

Formulation Example 1. Composition for feed addition

Bacteriophage EH11 1x10 ⁹ PFU/mL

100 mg of vitamin mix

100 mg of mineral mix

2 mg of magnesium stearate

It is prepared by mixing the above ingredients according to a conventional feed additive manufacturing method.

Formulation Example 2. Disinfectant Composition

Bacteriophage EH11 1x10 ⁹ PFU/mL

Appropriate amount of ethanol

Proper amount of sterile distilled water

proper amount of pH adjuster

It is prepared by mixing the above components according to a conventional disinfectant manufacturing method.

Formulation Example 3. Cleaning agent composition

Bacteriophage EH11 1x10 ⁹ PFU/mL

Appropriate amount of ethanol

Appropriate amount of enzyme complex

Proper amount of sterile distilled water

It is prepared by mixing the above components according to a conventional cleaning agent manufacturing method.

As described above, specific parts of the present invention have been described in detail, and for those of ordinary skill in the art, it is obvious that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Therefore, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

Korea Microorganism Conservation Center KFCC11783P 20180814

<110> Optipharm.CO.,LTD <120> Novel Enterococcus specific bacteriophage EH11 and antibacterial composition comprising the same <130> 1-78P <150> KR 10-2018-0166984 <151> 2018-12-21 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 19196 <212> DNA <213> Unknown <220> <223> bacteriophage EH11 <400> 1 taaatagagg tgaaccaccc tgttaaccta gaccccccac ccttggtaac gagatttcta 60 attaaactct aattaattta taactgtatt cacagtaaaa ctattcatat ccatggtata 120 cttaattcat caagtaaagg gagtgattaa atgattcaag ataaaataat tatcaataga 180 ctgggaatga gaattgaaga actaaaagca gaactaacct attggaaaga aacagcaaac 240 gaatggagag ataaatacaa agatgctgaa tctgatttac actactggca aaaatattct 300 tcagacttag aaaaagcttt aactaattca aacaaaacac ttgacaagta aaacaccaca 360 tgatatactt aattcaaggt taaggaataa cctaacaaac taaaaactta aacagtagag 420 gagaaataaa ttatgaataa cgtaacatta ctaggacgcg taactaaaga ctttgcaggt 480 actaaaacag ataaagggtt agtaatcgct cgtaacagct tagcagttaa ccgaccaaca 540 gaaggcgcag acttcatcaa tattgtagca tttggtaaaa cagctgaaac actaggaaaa 600 tttgttaaga aaggtcatcg tgttcttatt caaggtcaca ttcaaaccgg ctcttatgaa 660 aaagacggta agcaagtata cactacagac gtggtagttg atcgctttga atttatcgaa 720 gcagcaccta agaaagaaga atcaccattc taacgagaac agagatgaga gctttggctc 780 tctctcattt caattaaata tgacatatat gtcaaggagg gagtaagatg aaaacactta 840 aagctttagg ttattgtgtt atagcttttg tagtatttaa tgtatccatg tttacaacat 900 catttattat tgataagggg tatgaaaatg aacaagtcac acctagcaca acaattgaag 960 aaatacaaac aacagcaaca gaaactacac attcaaatga atcaactcaa gaagaagtaa 1020 caccaaaata tacgccagat aacacagaag aatacacagt aaatgagtat atgggttatg 1080 accctaatct aaacggcgac tcctactcta tcactcgtaa ttctgacggt atggaaggtg 1140 tgttaacaat cgataaccaa gctaatatgc aatggtacta ttatgatgct gaaggtattc 1200 acacaaacta ggaggaaaaa caaatgctaa aattcaaaga aggacaaact tatatctgta 1260 caaaatctaa taattcatgg tggactgtag gaagagagta tgtagtatca ctatacgatt 1320 atagagtacc ttatttgact gatgatgatg gtggtaagtg gactgcggaa cacttggata 1380 aacgtagtaa cagattcaaa ctcaaggagg aaacaacaat gacagactta attgacaacc 1440 aaccacacta caaaaaccaa gggattgaac ctattgtatt aatgaagact aacttctcta 1500 aagaagagtt tgctggattc ttacaaggta atgtgttaaa atacatgttg cgctacaaag 1560 ataagaatgg aattgaagat ttaaagaaag ctaagacgta cctaacatgg ttgattgagg 1620 agttagatga tgatggcgtt cgccgttcag taagttaagg aggaagaaaa atgataatgt 1680 atcttgtaat aattaaccta ttagctgcag ttggtgttgt agcaatcatc tatatcattg 1740 aggagattaa aacagaaaag aaaaagaaac gtaaagaaaa gtttgagcaa gatgtttttg 1800 aagtggtatt aaaagtgtta gaccagatta aggaggaaga aaatgccgaa gtataacgta 1860 actgataaat catacggtaa ctataatggt aggttaaact acagttatgc ttttgataca 1920 actaagttta acggtttagc tagggataca gaaagagttc taaaaggttt agagagagct 1980 tttaactatt taccaggcga acaccacttt ccattcaaac atcctatcgc ttatatcgat 2040 gcaaaagcat tagtaagcgg tgcttatgtt gcgcctgtta gagctatttt aaatcacttt 2100 aacgctaaat catccgaaca tagagcaaat aaacacaaag aagaaatagc tagagatgct 2160 atggaatcac aatcacctag agaactaata aaggaattag ataaactaaa caagttattt 2220 gaaaaatatg cagacgatga attcaaagga cgacgtaaat tcgctattgt tgcatacaat 2280 gagttaggtt ataacggata ggaggaaaac aatggcatta tttaagaaga cccaagaaga 2340 aaagttttta aaatttattg aaaaatttag aaaagaaaat atcccctcaa actttaacca 2400 agcgagaggg gttgaaaatt taatcaatcc ggataatgat tacacggtat ctatctcttc 2460 acgtacagac ggtaaatcat ttaactatat gggaatgtta atggctgttg cttttgagtt 2520 tgacttaggt attttatttt tagatcgtca ctacaccgta agacaggctt atatggaaaa 2580 tatttgggag atcatgcacg ttatgaccca cttcgatgag caagagctaa cttttgagcg 2640 tagtgatgag tacgtgacag tcatctataa agacaaaaca atcggagcca ttacagatat 2700 aaacaacagt tccgacttga agaaccactc gcatttccta gctaaatttc ctatcattgt 2760 atatgatgaa tttttaacaa ttgaaacaga ctacgtacct ttagaggata aacactttga 2820 acgtatttac cgttctgttg accgtaacga caacatccct ttaattggta ttcctaaggt 2880 gtttctctta ggtaatcctg agaacttcgc tagcccttta attgccatgt tagatttata 2940 caacaagtta gagaaacata caatcaacac aggtaaacac tatgataata tttatttaga 3000 aatgttccga aacgataacc agaatgcaaa gcgtaaccta agagcattta aatacgcgga 3060 taaaagttct atctctggag agtttcaaat caataacacg tatattgcta acgatgaaat 3120 aagagcgatg ataaaaagag gaggctttaa cacaacgatt attaagttag aggaaggttt 3180 tcttaaacta acctacaaca ttgaaacacg taagacatta atcagctacg tgtcaagtgt 3240 ttccgattat gatttctgta cccaagtatc tgataagaaa gagggagtgg tatttttagg 3300 agaaacttat tataaagaat cacatctaaa atatcacgaa agagggttct attattatga 3360 taacagttat accaaatcac ttatcacaag agatacaact ctcgttacat taaagctttg 3420 gaagtgtgta tcaaaacacg ctatagaaca tcaagcttca gaaccagata gaattgataa 3480 agaatatcaa gacacttact tagaaaacac attaaaagcg attcataaga aatttatgat 3540 agacaacttt taacatatat gttatagtat gtatgaggtg aggttatgga gtatgtaaaa 3600 tatttagatg gaacaacaaa agaggatatt atcaggtatt taagtaacaa cagagtacac 3660 caatttgata tcgatattga aacattaagc tataacatga atatgagtaa aaagaaacca 3720 acagctatga agagtcgcat gttcacattt acagcttcat ggtttgatga aacaggtcgc 3780 atttacacta ttgcaacacc atcagttaaa tatttcattg atgatttctt acttccttat 3840 gctaaaagaa cgaaaggcaa aagtaaacga agtgggtttg ctcgtgtagt attcaatgtt 3900 cacaatggga acaagtttga taatcatttc ttagctaaag agattcacga aacttatcac 3960 gctaaatata aaaattggga agatgataaa agtaaagtta aagttaactt aggtaaacac 4020 atgatgaaag gagaaaatac agaaaactat atcctagaga agagagtgaa gggtatgagc 4080 cacctctctt tctctgcggt tattggtgat ttacaaatcg atacagtcga taccgttatg 4140 aaaacaggat gtgccttatc aacagtagga caaatgcttt tatcagctgg atgtattaca 4200 gaagacaaac taaagacaac atttgattat gctaaatatc attatcaaga cgatatgaca 4260 gaaattgaag tcaatacaaa agttttagag atctttaatt ctctttcgga agaagagtgg 4320 atttatattg caaacgacac ttatatactt tcttcattaa gaagggattt cagtaaaata 4380 ttcccagggt tcgattatga caaacaaact aaaacccaaa acatcattaa tgcttacaaa 4440 agaaacaatg tggcagagtt tcaaatacta ggaaaaatca ccgttcaaga caagatatct 4500 ttaagacctg atatctacga ggtgaattat agtgactacg atgtacaagg tgagaaccta 4560 gctaagattg tacagaactt ttacaaagga ggattgaact tctataatca agaatacgtg 4620 ggtaaacttt taacaggaga gtttatcagt tttgatatta acagttctta tccctcaatc 4680 atgtatgaat ttccaatgcc ttggttaatc agaaatcatt atgaacacaa atgtattaaa 4740 aaggtatcga ctaatctaca cgaagaattt acgctttaca agataaccaa agaagtattt 4800 aatcgtttat tggatttaat tggttcccgc gtagtaaagc aaatgttaac aaaatacttt 4860 agaagagaag gcgaattcgt ttatctaacg acatggactt ttaaaatgtt aaaagaaaat 4920 ttccatttag aagttccaga aatagagtgt gaacaatggt ttgaatttag cgtgaagtac 4980 tttggtgcta gagacgctat tacagaattt tattttacta agacacaagg taaagctaag 5040 aatgttgttg agtttgttga taacaaccca atgaatatta ttgtacatga agacaaaaag 5100 aatgagaaag tttttacgaa acctatgatt gatattgcta aagtcaatct taacggtatt 5160 tacggagcac cagctttaaa accagcttac tcagtgggtt atcgtgatga agatggtttt 5220 ttacatgtag agcgtaacgg atatctaaac acggaacgta acgctatgac aagtgtcttc 5280 actacaggtg gtgcgctatg gcgtttaaca caacctctgc aatacttaac aaatgaagag 5340 attgacgatg cttttcttta ctgtgacaca gatagtcttt atatgaaacg ttacatctta 5400 cctaaattac ctaaagaaat gttccatcct atgaacttag gttcttggga cattgagcac 5460 aatttaatta cagaatttta cgtgttaaac cataaaaaat atgcttttta tgataatgga 5520 attgtctttc actgtggtgg tatcccagaa gaagctttcg atacagatat gacctttaaa 5580 gagtttattg atacacaatt tcataaaggg gttcaaatgc ctaacaacag aagcattatg 5640 acaacagaag gaacaatcgt tatctatgaa agcagcactg aattagatga aggtggttct 5700 taccctactc attacacaga tgaagatgaa ttaattgaac aactagtcaa aaaagaatta 5760 agaaaactgt tgacagaaca agatgacaat gatattatgt atatagaata tgagctaggt 5820 tcaatggctt tgaaagatgt ttatcctttc gagtatgaaa cagcaaaaga ttcattagct 5880 ggattgtatt atgattcggg agatattaga aataatattt ctcgataatt taaacaaaag 5940 agttgacata tatgtcaaat agtgatatac ttagttcata aagttaaagg gggaattaac 6000 atgttaaaag aatataaagt aatgaatgaa agagtgtaca aggtgattgc catagatgaa 6060 gatggatatg aaatggattt ctttattaag ggtgaagatt caaaccacgc acaacacaaa 6120 ttcatcaaat tggcgcaatt taatgagaat ggtttaatat acaaatcaat caaggagatt 6180 acaatcatat gaaaaactat aaaataactt tttatgatgc ttgggatggt acaacacaag 6240 aaacatttat ttttgctcaa acagctttag acgcggaaat tatcttctcc ttgtgtgaag 6300 acagttttgt tcaggtgctt gacgtaaagg aggaagaatg atgaagttat ttatcaaaag 6360 tttgtttaca ttatcctata tattatttat aggagcaaca atagtgtatg cttcaaacga 6420 aggttttcta actactgctg gaaatattct agtatctatg ttatttggag ctagtataaa 6480 aataatggtt gattggttaa ctaaggagga agaataatgc tagcaataat ctggattatc 6540 ccaatacttg cttttattgc tttagtacat tttatggagg gagataatga ttaacaaatt 6600 aaaagatttc ttaaaaagaa ttttaatcgc aggagggtta atgaccgcat gaaaaatgaa 6660 gaaagaaaag agttccttaa aagattttgg attgctatgg aaacaatcat ggaagaaaaa 6720 cgtctaccac attctaaggt ggtaggtaac aacacatcac ttgctaaaag tcgcgagtta 6780 aaccctacaa ttgtaagact taaatctttt gctgataact tagatatgga ctttgaacgt 6840 tttataacca aactaaatga agagatggag caaattagat gaacttaaac cctgaatttt 6900 tagaagatgc aatgaatccc gaaaagaact acaccgctaa catgctcgct agatgggaaa 6960 ttgttgctgt taaccctaac tatgaggtat cagacttagg ggaagtgaga aatatcaaaa 7020 caggacgtat tttaaaacaa aagcttcgta acggttatgc tgctgttaat ctatcaaatg 7080 aaggtaaagt gacaactcat aacgttcacc gtttagttgc tgatgctttc ttatataatt 7140 ctgacaacaa agaacaagtg aaccacaaag acgaaattaa aacaaacaac tctttaatta 7200 atttggaata tgttacacct aaagagaata ttaaatactc aagagcgaaa caagtcattg 7260 gtaaagatat taatggaaat gttgttatgg agtttgaatc tttaacagag gcagccaaag 7320 aaggttactt ctctaattat gtcaaacgat ctcacatgta tcatctacca tataagggta 7380 tatacttttc tatcttgcca cctaaagccc aagaatataa ggagagaaaa atatggcaat 7440 aacaaaacaa gatttcggaa atgcgaaaat caatgaatca ttcggcatat cagatgatgt 7500 gctggtgaaa tatgtacaaa ataatgtacc aggttctcgt tggtggcgtt attggaaaga 7560 tgacgaagca aaaataagag aaacttttga cattgtaaga caaaatggaa tgtctgctgc 7620 tctcttagtg gtaaaagaaa aagctgaagg tgtcggtttt acaaataatg acatcgatgg 7680 atggggtaat cattatgata aacctgaccc caatccgtta atcgatctag ctaattacgc 7740 taaagtaact gtaaaaactt ctaactctac cgcttataac ccagcttgga tagatgctgg 7800 ttatccggtt aattgtgtgc ctactgacgt tattcaatcc ggtaacgctg attttaaaaa 7860 cactcctgta ggaacagtca aacgtgctta cgtaccgatg accgctgcat ctacttgggc 7920 ttactattac ccaccagcat taaaagctag tgttaacggc aaacaaaatt atgggaaccc 7980 catccaacaa tgtacagatt atttgaatga aatgggagca aaaataacag ggtctggagg 8040 ttctggctct ggtggtacta cacctccagg agataataac gatgtaaaac ctggagtacc 8100 agaccaaaat aaacctttag agggtagtgg tggagctacc tctacaaacc caacttctat 8160 tcttgaggcg attaaaaaag caattcaaca gattttagat ttgttaacac agaactattc 8220 tcaagaaaaa ttatcgattt acggttctaa aacaggatta atgttcaccc aagtaaataa 8280 gaactatatt tttgcctatg atttcttcaa tgaaaatgtt aaatcaatca ctgatatctt 8340 aaatgatacg gtaaaagatg aattagcaga aagtgggaat gtccaagatc aattacctaa 8400 tggaatcaat caaacagtgg aagttctatt aactagagca ttaaaatatg ttggtagtga 8460 tgtaaaatat gatgaaaata aaaacgggaa tccagaagac ggagcgactg ataatgctcg 8520 tttcatgtct tggtgtgtgc aaaccattca tccttcttta aaaggttctt cacataaaga 8580 cttctttaat aaattcaacg ctgctggata cgtcagacat cgcggaacgt ggtcttccat 8640 tgctaaaaat ctacaaattg gggatattgt atgctgtgcg gataacttaa cagtggacac 8700 aggagggaag gaatacttta ttgctatagg aaatgatgag tgtgtggagg cttacccttg 8760 ggaaaacggt gaaagaagta gtaaaaacgg caaacctgta gctcgtgggg tcgctaaatt 8820 taaactatct gaaagagctg ctctaggttg tgctgatatt gcaattatca gaccttactc 8880 acctaaagaa gttgaaaatc ctaccccttc aaatcctaac ccaccaagta gtactgtaaa 8940 agctgcttta gctcatattg catcttcaat aggtaatagg attggttctg gacagtgtta 9000 tgctttatgt gctgaatacg caggatttct aggaggacct ggtcttgggg ctggtacaaa 9060 atatgggtta tcgggtttaa gcggtcaggg gtcaaccagc gctgccgcag atatcggtat 9120 ttgttacaaa tgggcttcat atggttggac tgtgattcaa aatcctcaat tatcacaaat 9180 cactgttggt tgtatcttaa atatcaatcg tagtgcaaac gttactgaga gtttaggagc 9240 taacacatcc tcttggttta attggtcagc agatcctact tacggacata cgttagtggt 9300 ggaaaaagtg gaaggtgata ttctaaccgt attagagcag tggggtggta atcgagaata 9360 taccatgcgg aacatgatga aatattctgg ttctggggca tcaggaatct caagtatttg 9420 tattcctcca agtgcataag gtcaaatgat cccgttttag aagaaaacga aaaacaagcc 9480 cgctctaggc gggcgttttt ctttgattaa aatttcttta atcctgaaaa gatatcgtgt 9540 gttggttgct tcacgtttcc tttttcgtct tgcgtgcgtc ggaagactac ttttactttt 9600 ttacctttta cttgagagta tttaaattca gcatgtaaac caaatgcgta tttctcactt 9660 acattcaagg ctttattcac atcttttctg acaattcctt ttgatttctt acggtcgatt 9720 tcccgtcctt tttcatctaa caagatcgcg tagttatacc cacttatttg aggtacataa 9780 acccaaccat cacaaatgat tttatctcct tgtttctcaa atttatcaat acttcctaca 9840 ggacttgggt gaggttttcc tgcaggtgca gaacctccca tatgcttctt gatttcgctg 9900 atgaagtaat cctgtgtttt agctccagca ccatgaattt cttgcgagcg gtgtggacat 9960 gctgttgcgc tgaatgaatt atgaagtcta acagttgatt tatttggttg taatccatga 10020 cgttttaata tttcagcagc taatttaatc gctttggctt cattagctaa aaactgattt 10080 ttatccccca ttgattgaca agcttcaata ccaataaagt tagcattccc atcagcgtta 10140 gctgtatgcc aagccatatt accctcatct tccatttgta gtgtaccatc actagcaata 10200 tagtaatgag caaaaccgtt cgcggcattg ttgtttgtta cgtatccttt ataaaaagag 10260 gtgtttgctg ccttagagcc agcgtcatta tgaatgacaa ccccttttac ttttccattt 10320 ctacgtcctg caattccttt tgtaatcaat taacatcttc ctctctcgta aattttaaac 10380 taatgttttc aatcattgac ttgataattt ctacgtctcc caacgcatca gtcagttttt 10440 ggattgtttc ttggtaacgc aattctcgac cgttgttaga tttcatcacc cacacgaaaa 10500 gtcctacaaa tagagctgca aaactgattt cactagggtt agttaacatg ttttccacaa 10560 attgttccat ctaatcacct cctctcattc tattataaca catttctaat caatttataa 10620 tactattatt cataattgat tgattaaatg gatttgttac gtcttgtctg ttgtgccaga 10680 atttcacacc aatctgaagt aaagctttga tttgttgcat gaattgtggc ggtactcctg 10740 gaatattgta taccccatca atttgaaggt agtttacaat tctcatcgaa tctagacgct 10800 ctgttctacc ttgctcatcg aattggaacc caaatgtctt gtaataacgt ctgatttttt 10860 ctttttctct tgcacttggg gcacttaatt taactgtaaa cccaaagata tcgttagcaa 10920 tagagaatgc ttcacctcca ctttgagaag atagagaggg tgaagataaa gccatatcag 10980 caaattctgc tctttgttgg cgataaaagt tttgctcgtc aataatctta ctaccaatgt 11040 ttaaagggtt taatgaacct cctaagatag tgtatgcatc tgtaaaaacg ttcgtcgctt 11100 tttctgttgt gttttgtgaa tcatcacttg cagtacgccc gatatttttc acgcgaccgg 11160 ttagcgtgtt cgcttcagct aacgctctct tatttgcatt catagcaaga cctaggttat 11220 agttatccac taatacaggc acttctgtcc agttagagaa gtttaaactg ttgtttaaga 11280 aagtaccagc tttaacattt ccttgattag tttctccatc agcagcccag tttaatggga 11340 agatagatat tttattagaa taaccgatac tagtgaccat atggaatttt aaccctgtag 11400 aaggtaagaa acctaagtct aaatcaagtg attcacctac ccagttatac acttctaacg 11460 taccgtattc gttacggagt aaatgttttt catctgctgg ataacctagg taatccataa 11520 ttgtttctgg tgaccagtta atatctgaaa tgtctttatt tgctgatact ttaccattaa 11580 caaatttttt caagtaagca tctgttttac cattgaattt acaagattct aaatcccctg 11640 tatcgattaa gtctttagga atcaacataa cagatttaat gttctgacca atccaagcga 11700 agtctgtcaa tgctgctgag aatgatttga aatgttctaa gtcaatcgca tacaatccta 11760 caggagaaac aattttatca tatttaaccc cttttgatag tttaatgttt ggtttgttta 11820 ccgtaccaaa ttcagcctct aaatctacac tacattggaa tacacaaatc atttcagtcc 11880 agataactga ttttgaatac acgtatttct tagtggtggt ttttaagaca tcgttattcg 11940 ttcttagttc tggtaatctc tcaccataag ttgttagagg taagtgttga cgtttaattg 12000 ttacgttctc aataccttct aacactctcc cttgagtgaa tgttgccatc acgtcaatca 12060 cgatttgaaa agctgtcact tggtcattca cgtattgtgt agccatcacg tagaagaagt 12120 aaggtttatt tgtaaaacca tcaacgaaac aaccatagtt caccccctca gtattttcat 12180 actgaatagg agcgttgatt gttagtctat ctcttaccat attaaattgt ttctgaaatt 12240 ctactttttg gaactttcta tctaaccaat tcttataagc tgatacagta tcgtaatgta 12300 cggtattgtt aaagtctgtc attggtgtgt tgtagtataa agtaattgta cttaatttca 12360 tggtttccct cctaagtgat actgaaaata attggttcta agaaaattgg tgttgtacct 12420 gttttagata cgatagagca taaaccgtta ggtcttacgt aaatttttgc tgcatctgtt 12480 tgagtggtgc ttcgacctgg tagtaaatag tgatattcgt atccatcgtt tggaggtctg 12540 aagccttccg gtagggtgaa acaatcacct gtaccatcag ataatacacc tctcatataa 12600 accactccgt tttgacgttt cacttgaatt tgctgtacag ctttaaaagg acttgttggg 12660 gtgacatttg tccaagctgt atctacaacc ttactttcta aggtgtttaa acggttgttt 12720 tgtgtagcat tgatagatac ttgcgtatcg atttctgttt gaatttttgt atctaaagca 12780 gaaatgttag ttgtttgtgc ttgaatatcg gatttattct tagcaatgtc agttgtattg 12840 gtagtgattt tatctgtgtt atttgcgata tctgtcgtgt tcttattcag ttgtgcttta 12900 acaagttcta catcacctaa ggcattaaga gctgtttcta aatcagataa acgaatatta 12960 ataccagtaa aagctccttc aatgtttgaa agtcgattaa cgatagctgg tagttgattt 13020 tgaattgttt caatgtttga tttatttaca tcaatttcta agagtttctt tttgatagtt 13080 tcaagttcta aatcgacaat ttcatcgaaa tctactaaat cccctatcat agtaatcatt 13140 cggtcgataa tttcagtgta atcatcaaac aacatttctt tgtagacacc tttaatcttg 13200 atataaatcc gaccttctcg gtcagctacc atatctaatt ctttactagt gttaagagga 13260 agagatttaa catctcttac ctctagtgtt tttcttacga aatcttccat tagtattcac 13320 cgtccactcc aacaatttta gctccttcat cttctgagaa tgagatactt ccatcaggtt 13380 tgtaagttac taaccggcat ttagcgtatt tcagcacaac gttatctcga accatcttta 13440 aattcattcg tacttgataa gcttcattga ttgtacctgc tacggcgttg aacatgattt 13500 gtgggttatc aactgctgga acttgaacac gaaacatcgt gccaataaat tcaatagcga 13560 aaatcaatga gccaaaacga cgagttgaaa cagggaagtt catattttgc cctacagaaa 13620 caggtgtacc ttctcctttt ctccatagtg gtgttctttg aggtatgttt ttccatacga 13680 gtgagttatt gttcccacca ccattaccag tgttttggga agtatttaaa taccagagcc 13740 ctccgttctt ttgtacagtc attaagtagc atttgcgccg gttgtcttca ccccacactt 13800 ttaaggtgat taaatctcca ggagtgtttg ttggaacatc aggcataggg atagtgtcat 13860 gccaagcata aacagtggca taagtaccaa atgggatatc ccaaaaagaa tatttatctc 13920 ctaaaaaatt gtctttatgc gcataaccgt gagcttggcg aaaagcatct gaactcatca 13980 atccattgtt taattcggtt gctaacatgt gggggttagt tccatcacca tctgagtgat 14040 ttccaaattc tttagctagc ttgtttaccg attgttttaa gtttaaaatt tcttcgtttg 14100 tattcatagt atcctcctta gtatgtgata atgtctgtca aagcagcatt tccgatgggt 14160 tttaatgttg ctgttcggtt agcaatgtct attgttacca cgtgaaaacc gacttgcacg 14220 ttcgtaccga ctttagcaga atcagcaggg aaagcttgtc gcatactaat gatattgaag 14280 tttgttttcg tcccttttaa acttacttca tctttatgca ggtgaccatg gaagtaacca 14340 acaaatttgg aagtttcacg tgcttcaaac tcttctaaca catcaatgag taactgaccg 14400 ttcttcataa cggttacgtc taagttatga tgacccataa caatgatatg gtagtttgct 14460 tcaagattgt ttagttgtcc ttttaaccaa ctcaattgag cttcaccgac accagaatca 14520 tatgttaaca tttcttgata agttaaatcc atcgtattta accagatgat agcaacttgt 14580 ttgttaggga ttttgtgaac gccaaaaggt gcattaccgt aaatcttagc taattcagaa 14640 ggtttaatga tttctttctc atgtctagga tttccatcac gtaaataagg aagaccgcca 14700 gggtcatggt taccaattaa catgaatgtg tcaacaggtt cattaagaac taaagcattc 14760 gctactttac gggcagctgt aaggtttgag tacttcacac gttctggagg aattacagca 14820 gcgtgctctg tgacctgacc attgataccg tccatattat cacccatgta gaacactgaa 14880 tcagtttcac gagccacatc ttgaatatta tagattgaat cataatactg acgagaacta 14940 aagccgttga tgtctgttct taaatgaata tctgtaacca ttgttaggtt cgtgatgtta 15000 gggtcgtttt taattaaact attcttgact tgttgttgat tgctttgtgt gactaagtca 15060 tgactgtatt ggttaaaacc tccaatcatt acagtggacc cgtctacatt gatttcgtcc 15120 acacgattaa ttaatgtgtt tacgtgttcg tctaacttat agactaaagc atataagtct 15180 tttacaaaca atccgtcttc tttagctact acagcatttt gtaattccat cactaatggt 15240 tttggtagta agctgtttaa ttgtccgtca cctgtagcag ctgagattaa tacatcagct 15300 ttgattttga tgatttcttc taggtctaag ttagggtcga gctggtctac aaaatcacca 15360 atcttagtta aattaacggt atttgtatcg ataacatcaa cgtcacgatt gattagtcta 15420 gcaactaatt tcataatact atctaactgt tcattgagtg ttccaaggta ttctaaataa 15480 ctttcagcgt tagtcgagaa gtcttgttta atgttatagt taggatttcc gaatctggat 15540 aagttataca tgtatttcgc tccttttacc aagtttgtaa gaaacaacga cggtcatatt 15600 cgttgaaata accttcccac aacccttgaa gttttttgaa tgtttcagca ttatagctgg 15660 tgtttgatgt ttctgaatga cctgtaacag ttgagttccc taagtcttcc ctcttcgatt 15720 tagaaacgga attatcatca gcataagcta aatcgttttt atcaaggttc aagttcactt 15780 ggtcttgagg tagggtagta tttgcacttc tgtcgctact gttagaacta ttgtttgatt 15840 gactcttatt gttattgtct gatactgatt tattctcgtt cgatagagct gattcaaaat 15900 tgtgatagat agcttcaatc tcttgctcgt gtattaaaga ggtgtaaaca acttgagaag 15960 agaagtcttc caatgtctgt ctaccaatct cacgatttaa gaatcttacg gtaaatgctt 16020 ttttaaaaaa ggtgtcaact tctggtgtag gaaaggtgta atcactgaag attacttctg 16080 tcactatctt cgcgatgtct tcatcataac gtaaagcttt ttgaataaat cgataatctt 16140 tgttgttacc agtgtattga ttcccattaa agaattcatt tttacccttc ttcactaatt 16200 cggttcgcaa gacttgcatt aaggatactg tcgtcgctcc cattgtcgtt acctccttgc 16260 tcgttcataa ctgtaagtgt actgattgca ttgtcatcaa atagtgggtg aatttcataa 16320 ccataacgct tatttaacaa acgacaagca ttgtttcttg aagttaaata gatattacca 16380 tttgctgtgg tgtatgattt agctgagtta ctttctcctt ctgatacacc actttcttta 16440 tctacaccta aggttgagaa acctagaata gcgtttaatt catttaacgt gttttggtac 16500 tcacgtttaa gctctgctag cataccacct acatttgagt tgttccactc tttaatgtgt 16560 tcgtctgggt caaagtgacc agtaaccttt gtaataggtg aaccgttgta aagactttca 16620 gcgatgttgt ttactgtttg gtcattaggt tctccaatta agaatgtgct aatcttagct 16680 tgcattttaa gtgagtaacg agataaaaca atttcagcta actcattcgt atagtgttga 16740 atgatttcat aatcactatt ataagctagc gtcttattac gaaaaacaac aaagttaccg 16800 ctttgtgcgt tgtccacatc cgtgatttct ttcatacgtg gtaatcttaa ttctttaggg 16860 ataacccatt gaatatcttt accccttaaa ggtttagctg ctaacacgtt agctgggtca 16920 gatggtgtca tacgttgctt agatactcct aacactctga tatttcctag tgacgtttct 16980 ccaataataa catcatagtt acttcttagc ataacttcca ctttcaacca gtcaactgta 17040 ggaatctgat taccattgta gtatttaatt gttgagggta atagttcaag ataacgagaa 17100 tagaaaatgc gggcaaatcg atttctatga tttactaccc gcacgtctat tttctcggaa 17160 agctcttgtt ctatacccat attcatgggc tcaaacatgt agtaacctcc tattctgatt 17220 taggacctgt aatgagaatt ttgttataga aaggtgagat agctttcatt gagtagtaat 17280 gaatccaatg agttacttca tcaaactctg ggttgtagaa aggttctttc aacattcctt 17340 tagtataacg gttgtattta atagcatcta ggtcaaagat gtaagcccac aattcagaag 17400 atggtttaat ttcttcgaac ttagctgcgt ctgcttctgg taagaactcg ttaacatcaa 17460 atgttacaac agaaccttta gggatgatgt cagttgctgt cgtttggtaa tcacctagtt 17520 cgcggaaagc tttaacagct tcagcagaaa gagtaatatc ttctttcgct cggaacgctc 17580 cacctaagtc ttcaaaagag atgatacgtt tagagatgtc gataccttca gatgcgaatg 17640 tgttcgctag ttttgtgtta agcaagtatg ctttcatagc atctgtagtt aagatagcga 17700 tttttgaaag ttttgtaaca gttgtgtagc gagcgatcgc gccgccagaa gctaagcgag 17760 cttcattgta tttgtctgag ttattttgta aattcattaa cgcttcagcg attttattga 17820 ataaatcttc ttctgatgta gcggtacgac gagccttagc aggagttacc ttctcagcat 17880 aatcaactaa catcgcacga atttgacgtt cttcgtcaac gttgatgtca gagattttct 17940 ttttgtaaac cccaatagca tattttactc catcagataa tttagcaaag ttcatacgtt 18000 ggtcattatt gttaagtgtg aatttcatct tacgcatgat gccaggtccg tataatttag 18060 tgatcatttg cggatagtta cgtttaagca ttaaagcttc atctttactt aagtccatag 18120 ttgttggaat tgtgtcacga ataacatact cttctgagta ttgtcctaca aagtcaactt 18180 ctttagctaa ccattcaaac gagttaccta gagctgattg aatcaacgct gtttcgttta 18240 atttagggaa taagaatttg ttgataaatg tttcaaattc agttcctacg cttgtccagt 18300 tcgttcctaa tgtccacgct cgtccttgtg tgtggttaaa gtcttgtaaa gcttgtgcaa 18360 tattcgttcc taatactccc atgttattct tgtcctccta cattgtgaat tttattttca 18420 tactcatctc gcgatgatgg atcgatactc atttctgggg ttaattgagg tgatttgtca 18480 aatttgtaat cttgaccgcc accttttgta aaactcttta attcgtgtga ttgtaaaatt 18540 tcgtccatat tctttctctc cttataagtc tagtagtttt tcgatgtcct tagggtcatc 18600 ttgttttggt tcttctggtg atccagactg tggtgcttct ggttctggtt ctggttggat 18660 agtagataga gcatcaactt gtgcttgtaa ctgtttaacc aaagccataa cttcttcaat 18720 actagctgca tcacctacat cctcaaccgg tggattagca atcttatctg gtgttggttc 18780 tggttcgatt ggtattggtt ctggttctgg ttcagttgtt tttggttttg gttcaccttc 18840 aactttctta gctttttctt tatcttccaa atcatcaacc aatttgtttg tttcttcatc 18900 tgccaaattc gttacacctc ctaaaaatat ttgagccttt tcacgtcgtc gctgtaagtg 18960 ctgttgctca ggacaggata tgtaaagcaa gagaggttaa tcaaggttta aggtagtcac 19020 taacaaggag tgcaagtatg tttaatgcct ctcttactaa tataagtata acatataatt 19080 ataacattgt gcaagctttt attcattaaa atgaataagt ttttaataag gatttaatga 19140 gaaatctagt tacctaaggt gggggtctag gttaacgggt ggttcacctc tattta 19196

Claims (9)

Having a killing ability specific to Enterococcus hirae, consisting of a nucleotide sequence represented by SEQ ID NO: 1, accession number KFCC11783P, bacteriophage EH11.
delete delete A composition for antibiotics comprising the bacteriophage EH11 of claim 1.
A composition for feed addition, comprising the bacteriophage EH11 of claim 1.
A feed comprising the composition for feed addition of claim 5.
Claim 1 containing the bacteriophage EH11, disinfectant.
A cleaning agent comprising the bacteriophage EH11 of claim 1.
delete

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