WO2021010595A1 - Novel enterobacter aerogenes bacteriophage ent-aep-1, and use thereof for inhibiting growth of enterobacter aerogenes and enterobacter cloacae bacteria - Google Patents

Abstract

The present invention relates to: siphoviridae bacteriophage Ent-AEP-1 (accession number KCTC 13569BP) which is isolated from nature and characterized by having the ability to kill enterobacter aerogenes bacteria and enterobacter cloacae bacteria, and having a genome represented by SEQ ID NO: 1; and a method for using a composition including same as an active ingredient to prevent and treat diseases caused by the enterobacter aerogenes bacteria or the enterobacter cloacae bacteria.

Description

Novel Enterobacter arogenes bacteriophage ENT-AEP-1 and its use to inhibit the growth of Enterobacter arogenes and Enterobacter cloaca bacteria

The present invention is to prevent infection of Enterobacter arogenes and Enterobacter cloaca using a bacteriophage isolated from nature capable of infecting Enterobacter arogenes bacteria and killing Enterobacter arogenes bacteria, and a composition containing the same as an active ingredient, and It relates to a method of treatment, and more particularly, has the ability to kill Enterobacter arogenes bacteria and has a genome represented by SEQ ID NO: 1, characterized in that it has a genome represented by SEQ ID NO: 1, Ent-AEP- 1 (Accession No. KCTC 13569BP), and a composition containing the bacteriophage as an active ingredient to prevent infection of Enterobacter arogenes and Enterobacter cloaca bacteria and a treatment method after infection.

Enterobacter arogenes ( Enterobacter aerogenes ) is a gram-negative rod, which is about 1-3 μm in size and has flagella related to motility. Enterobacter arogenes bacteria are known to be pathogens that cause diseases due to opportunistic infections, although they exist in the intestinal tract of humans and animals. Enterobacter arogenes infection could be treated by treatment with antibiotics commonly used in the hospital, but the risk has emerged as antibiotic-resistant strains with beta-lactamase have recently been reported.

Enterobacter arogenes bacteria are community-infected bacteria that cause various infections such as respiratory infections, urinary tract infections, bacteremia, and skin infections. In particular, patients with reduced immunity or long-term hospitalization in the intensive care unit are known to have high morbidity and mortality because they are particularly susceptible to this fungus. In the 2014 antibiotic resistance rate survey by the Korea Centers for Disease Control and Prevention, it was reported that the main isolates of Enterobacter arogenes bacteria were respiratory (26.9%) and urine (26.3%).

Enterobacter cloaca ( Enterobacter cloacae ) is a Gram-negative rod that belongs to the family Enterobacteriaceae and is widely distributed in natural environments where humans survive, such as water, soil, and feces. Enterobacter cloaca is one of the major causative bacteria of nosocomial infection, and the infection caused by Enterobacter cloaca is Staphylococcus. aureus ), Escherichia coli ) and the like, but are known to show more serious symptoms clinically. In Korea, cases of bacteremia, sepsis, and group infections due to endoscopy have been reported in Korea, and in foreign countries, it is reported as a major causative agent of the occurrence of sepsis in burn centers, dialysis centers, intensive care units, neonates and pediatric wards Has been done. Recently, a wide range of cephalosporins (extended-spectrum cephalosporins) resistant strains have emerged and are prevalent, leading to an increased clinical risk.

Various antibiotics have been used for the purpose of preventing or treating infection of Enterobacter arogenes or Enterobacter cloaca, and the appearance of resistant bacteria due to misuse of antibiotics is on the rise. Therefore, there is an urgent need to develop drugs that have properties different from those of existing antibiotics.

Recently, the use of bacteriophage as a countermeasure against bacterial infectious diseases is receiving great attention. In particular, it can be said that interest in bacteriophage is higher than ever due to the preference of a nature-friendly method. Bacteriophages are very small microorganisms that infect bacteria and are usually shortened to phage (Phage). Bacteriophages have the ability to kill bacteria by proliferating inside the bacterial cells after infection with bacteria, and destroying the cell wall of the 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 due to this, a specific bacteriophage kills only specific bacteria and does not affect other bacteria. Bacterial specificity of these bacteriophages provides antibacterial effects only against the target bacteria and does not affect the environment or flora in animals. Conventional antibiotics, which have been widely used for treating bacteria, have an effect on several types of bacteria at the same time. This caused problems such as environmental pollution or disturbance of normal bacterial flora of animals. Unlike this, bacteriophages work only against specific bacteria, so the use of bacteriophages does not cause disturbances in the body's normal flora. Therefore, it can be said that the use of bacteriophage is very safe compared to the use of antibiotics, and the possibility of causing side effects by using it is relatively low.

Bacteriophage is a British bacteriologist Twort 1915 became discovered while conducting research on Staphylococcus aureus (Micrococcus) melting the colonies are transparent by any developer. In addition, in 1917, d'Herelle, a French bacteriologist, discovered that there was an action to dissolve Shigella dysenteriae in the filtrate of a person with a disease. Through this study, he independently discovered bacteriophage and eats bacteria. It was named bacteriophage in the meaning of. Since then, bacteriophages against various pathogenic bacteria such as Shigella, typhoid, and cholera have been continuously discovered.

Due to its special ability to kill bacteria, bacteriophage has been expected to be an effective countermeasure against bacterial infection since its discovery, and there have been many related studies. However, after the discovery of penicillin by Fleming, as the spread of antibiotics became more common, research on bacteriophage was limited to some Eastern European countries and the former Soviet Union. However, after 2000, due to the increase in antibiotic-resistant bacteria, the limitations of existing antibiotics appeared, and the possibility of development as a substitute for the existing antibiotics emerged. As a result, bacteriophage is again attracting attention as an anti-bacterial agent. In particular, as government-level regulations on the use of antibiotics have been strengthened worldwide, interest in bacteriophage is increasing, and industrial use cases are gradually increasing.

As described above, bacteriophage has very high specificity for bacteria. Due to the high specificity of these bacteriophages against bacteria, bacteriophages often exert antibacterial effects only against some strains, even if they belong to the same species. In addition, the strength of the antibacterial activity itself of bacteriophage exerted according to the target bacterial strain may vary. For this reason, it is necessary to secure various kinds of useful bacteriophages in order to secure an effective control method for certain kinds of bacteria. In order to develop an effective bacteriophage utilization method in response to Enterobacter arogenes or Enterobacter cloaca, it is of course necessary to secure various types of bacteriophages that can provide antibacterial effects against Enterobacter arogenes and Enterobacter cloaca. It is necessary, and furthermore, it is necessary to select and utilize a bacteriophage that has a comparative advantage in terms of the strength of antibacterial activity or the antibacterial range among the various useful bacteriophages obtained.

Thus, the present inventors use a bacteriophage isolated from nature capable of killing Enterobacter arogenes and Enterobacter cloaca to prevent and treat infections of Enterobacter arogenes and Enterobacter cloaca. After trying to develop a composition that can be used and to develop a method for preventing or treating infection of Enterobacter arogenes and Enterobacter cloaca using this composition, the bacteriophage suitable for this was isolated from nature, and this separation After obtaining the gene sequence information of the genome so that the resulting bacteriophage can be differentiated from other bacteriophages, a composition using the bacteriophage as an active ingredient was developed, and the composition was then applied to Enterobacter arogenes and Enterobacter cloaca. The present invention was completed by confirming that it can be effectively used for prevention and treatment of infection.

Therefore, the object of the present invention is Siphoviridae isolated from nature, characterized in that it has the ability to kill Enterobacter arogenes bacteria or Enterobacter cloaca bacteria and has a genome represented by SEQ ID NO: 1 ( Siphoviridae ) bacteriophage Ent -To provide AEP-1 (accession number KCTC 13569BP).

Another object of the present invention is Enterobacter arogenes bacterium or Enterobacter cloa comprising a bacteriophage Ent-AEP-1 (accession number KCTC 13569BP) capable of killing Enterobacter arogenes bacteria or Enterobacter cloaca bacteria as an active ingredient. It is to provide a composition that can be used to prevent infection of Aca bacterium and a method for preventing infection of Enterobacter arogenes bacteria or Enterobacter cloaca bacterium using the composition.

Another object of the present invention is Enterobacter arogenes bacterium or Enterobacter cloa comprising a bacteriophage Ent-AEP-1 (accession number KCTC 13569BP) capable of killing Enterobacter arogenes bacteria or Enterobacter cloaca bacteria as an active ingredient. It is to provide a composition that can be used to treat a disease caused by Aca bacterium, and a method for treating a disease caused by Enterobacter arogenes bacteria or Enterobacter cloaca bacterium using the composition.

Another object of the present invention is Enterobacter arogenes bacteria or Enterobacter arogenes using compositions containing as an active ingredient the bacteriophage Ent-AEP-1 (accession number KCTC 13569BP) capable of killing Enterobacter cloaca bacteria or It is to provide a pharmaceutical composition for the purpose of preventing or treating Enterobacter cloaca infection.

Another object of the present invention is to provide a disinfectant comprising a bacteriophage Ent-AEP-1 (accession number KCTC 13569BP) capable of killing Enterobacter arogenes bacteria or Enterobacter cloaca bacteria as an active ingredient. This disinfectant can be used to prevent infection with Enterobacter arogenes or Enterobacter cloaca, and is particularly effective in preventing nosocomial infection.

Another object of the present invention is to provide an antibiotic comprising a bacteriophage Ent-AEP-1 (accession number KCTC 13569BP) capable of killing Enterobacter arogenes bacteria or Enterobacter cloaca bacteria as an active ingredient. This antibiotic is effective in preventing and treating Enterobacter arogenes or Enterobacter cloaca infections.

The present invention has the ability to kill Enterobacter arogenes bacteria or Enterobacter cloaca bacteria, and has a genome represented by SEQ ID NO: 1, characterized in that it has a genome represented by SEQ ID NO: 1 Sipovirida bacteriophage Ent-AEP-1 (consigned No. KCTC 13569BP), and a method for preventing and treating infection of Enterobacter arogenes bacteria or Enterobacter cloaca bacteria using a composition comprising the same as an active ingredient.

Bacteriophage Ent-AEP-1 was deposited by the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center on June 29, 2018 after being isolated by the inventors (accession number KCTC 13569BP).

In addition, the present invention provides a pharmaceutical composition comprising a bacteriophage Ent-AEP-1 that can be used to prevent or treat infection of Enterobacter arogenes bacteria or Enterobacter cloaca bacteria as an active ingredient. As an application example of the pharmaceutical composition, a disinfectant or antibiotic may be suggested, but it is obvious that the present invention is not limited thereto.

The bacteriophage Ent-AEP-1 contained in the composition of the present invention effectively kills Enterobacter arogenes bacteria or Enterobacter cloaca bacteria, so respiratory infections, urinary tract infections, and It is effective in the prevention (prevention of infection) and treatment (infection treatment) of diseases such as bacteremia and skin infections. Accordingly, the composition of the present invention can be used for the purpose of preventing and treating diseases caused by Enterobacter arogenes bacteria or Enterobacter cloaca bacteria. Diseases caused by Enterobacter arogenes bacteria or Enterobacter cloaca bacteria in the present specification include respiratory infections, urinary tract infections, bacteremia, skin infections, and the like.

Enterobacter arogenes bacteria or Enterobacter cloaca bacteria in the present specification may be sensitive to existing antibiotics or resistant to existing antibiotics. In other words, it does not matter whether or not resistance to existing antibiotics is acquired.

As used herein, the term "prevention" or "prevention" means (i) inhibiting the entry of Enterobacter arogenes bacteria or Enterobacter cloaca bacteria into the body, or Enterobacter arogenes bacteria or Enterobacter cloaca Preventing infection of Enterobacter arogenes or Enterobacter cloaca in a manner that inhibits the growth of Aca bacterium; Or (ii) It means inhibiting the development of a disease caused by Enterobacter arogenes bacteria or Enterobacter cloaca bacteria infection.

As used herein, the terms “treatment” or “treatment” include (i) inhibition of diseases caused by Enterobacter arogenes or Enterobacter cloaca; Or (ii) Any action that alleviates the pathological condition of a disease caused by Enterobacter arogenes bacteria or Enterobacter cloaca bacteria.

In the present specification, "separated", "separated" or "separated" refers to separating a bacteriophage from a natural state by using various experimental techniques and separating it from other bacteriophages to secure a characteristic capable of specifying the bacteriophage of the present invention Refers to, and in addition to this, it includes proliferating the bacteriophage of the present invention to be industrially utilized by biotechnology.

Pharmaceutically acceptable carriers included in the composition of the present invention are commonly used in formulation, and are lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate. , Microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc., but are limited thereto. no. The composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components.

The composition of the present invention may be used as a method of applying or spraying to a diseased area, and may be administered by oral administration or parenteral administration. In the case of parenteral administration, intravenous administration, intraperitoneal administration, intramuscular administration , Subcutaneous administration or topical administration may be used.

Suitable application, spray and dosage of the pharmaceutical composition of the present invention include the method of formulation, mode of administration, age, weight, sex, severity of disease symptoms, food, administration time, route of administration, excretion rate, and It varies depending on factors such as response sensitivity, and usually a skilled physician or veterinarian can easily determine and prescribe an effective dosage for the desired treatment.

The composition of the present invention contains bacteriophage Ent-AEP-1 as an active ingredient. The bacteriophage contained at this time Ent-AEP-1 is included in 1×10 ¹ pfu/ml to 1×10 ³⁰ pfu/ml, or 1×10 ¹ pfu/g to 1×10 ³⁰ pfu/g, preferably 1 × 10 ⁴ pfu/ml-1 × 10 ¹⁵ pfu/ml, or 1 × 10 ⁴ pfu/g-1 × 10 ¹⁵ pfu/g.

The composition of the present invention is prepared in a unit dosage form by being formulated using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person having ordinary knowledge in the technical field to which the present invention belongs, or It can also be manufactured by putting it inside a multi-volume container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or a stabilizer.

The composition of the present invention may be implemented with a disinfectant or antibiotic, but is not limited thereto, depending on the method of use. In the present specification, the term “antibiotic” refers to preservatives, fungicides and antibacterial agents.

Bacteriophages capable of providing antimicrobial activity against other bacterial species may be added to the composition of the present invention in order to increase the efficiency for this purpose of use. In addition, other types of bacteriophages having antibacterial activity against Enterobacter arogenes bacteria or Enterobacter cloaca bacteria may also be added. Even bacteriophages having antibacterial activity against Enterobacter arogenes bacteria or Enterobacter cloaca bacteria differ from each other in terms of the strength of the antibacterial activity and the antibacterial range, so an appropriate combination of them can maximize the effect.

The method of preventing or treating infection of Enterobacter arogenes or Enterobacter cloaca using a composition containing the bacteriophage Ent-AEP-1 of the present invention as an active ingredient is compared to a method based on conventional antibiotics, etc. It can provide an advantage that the specificity for bacteria and Enterobacter cloaca is very high. This means that it can be used for the purpose of preventing or treating infection of Enterobacter arogenes bacteria or Enterobacter cloaca bacteria without affecting other useful flora, and it means that there are very few side effects from its use. Normally, when antibiotics are used, common organisms are also damaged, resulting in various side effects. On the other hand, bacteriophage, even if it is a bacterial species capable of exerting antibacterial activity, in exerting antibacterial effect, the strength of the antibacterial activity or the antibacterial range [individual bacteria in terms of several strains belonging to Enterobacter arogenes or Enterobacter cloaca The range in which the antibacterial activity of bacteriophage is exhibited against the main. Typically, bacteriophage can exert antibacterial activity against some strains belonging to the same bacterial species. That is, even if they belong to the same bacterial species, there may be differences in susceptibility to bacteriophages depending on individual bacterial strains] Since there is a difference in terms of the present invention, other bacteriophages having antibacterial activity against Enterobacter arogenes or Enterobacter cloaca Compared to, it can provide a differential antibacterial effect. This provides a big difference in its effectiveness when used in industrial sites.

1 is an electron micrograph of the bacteriophage Ent-AEP-1.

2 is an experimental result showing the killing ability of the bacteriophage Ent-AEP-1 against Enterobacter arogenes bacteria and Enterobacter cloaca bacteria. The upper part is the dropping of only the buffer solution (Buffer) that does not contain the bacteriophage Ent-AEP-1, the lower part is the dropping of the liquid containing the bacteriophage Ent-AEP-1. The transparent part observed from the bottom is the lysis plaque formed as a result of lysis of the bacteria to be tested by the action of the bacteriophage Ent-AEP-1.

Hereinafter, the present invention will be described in more detail based on examples, but these examples are only examples of the present invention, and the scope of the present invention is not limited to these examples.

Example 1: Enterobacter Arrowjins Isolation of bacteriophage that can kill bacteria

Samples obtained from the natural environment were used to separate the bacteriophage that can kill Enterobacter arogenes bacteria. On the other hand, the Enterobacter arogenes bacteria used to separate the bacteriophage was pre-sale from the antibiotic-resistant strain bank and used (pre-sale number CCARM16001).

The bacteriophage separation process will be described in detail, TSB (Tryptic Soy Broth) medium (casein digest, 17 g/L; Soybean digest, 3 g/L; dextrose, 2.5) inoculated with Enterobacter arogenes bacteria at a ratio of 1/1,000. Samples collected in g/L; NaCl, 5 g/L; dipotassium phosphate, 2.5 g/L) were added together, followed by shaking culture at 37°C for 3-4 hours. After incubation, the supernatant was recovered by centrifugation at 8,000 rpm for 20 minutes. The collected supernatant was inoculated with Enterobacter arogenes bacteria at a ratio of 1/1,000, and then cultured with shaking again at 37°C for 3-4 hours. When the bacteriophage was included in the sample, this process was repeated a total of 5 times so that the number of bacteriophages (Titer) could be sufficiently increased. After repeating 5 times, the culture solution was centrifuged at 8,000 rpm for 20 minutes. After centrifugation, the recovered supernatant was filtered using a 0.45 μm filter. It was investigated whether there is a bacteriophage capable of killing Enterobacter arogenes bacteria through a usual spot assay using the filtrate thus obtained.

The instillation experiment was carried out as follows. Enterobacter arogenes bacteria were inoculated in TSB medium at a ratio of 1/1,000, and then cultured with shaking at 37°C for one night. 3 ml of the culture solution of Enterobacter arogenes bacteria prepared in this way (OD ₆₀₀ is 1.5) was added to TSA (Tryptic Soy Agar) plate medium (casein digest, 15 g/L; Soybean digest, 5 g/L; NaCl, 5 g/). L; agar, 15 g/L) was spreading. The smeared plate medium was left in a clean bench for about 30 minutes to allow the smear to dry. After drying, 10 μl of the previously prepared filtrate was dropped onto the plate medium plated with Enterobacter arogenes bacteria. It was left to stand for about 30 minutes and dried. After drying, the instilled flat medium was cultured at 37°C for one day, and then it was investigated whether a clear zone was formed at the location where the filtrate was removed. In the case of the filtrate in which the transparent ring is generated, it can be determined that it contains bacteriophage capable of killing Enterobacter arogenes bacteria. Through this investigation, it was possible to obtain a filtrate containing bacteriophage having a killing ability against Enterobacter arogenes bacteria.

Separation of pure bacteriophage was performed using a filtrate in which the presence of bacteriophage having a killing ability against Enterobacter arogenes bacteria was confirmed. For the separation of pure bacteriophage, a conventional plaque assay was used. To explain this in detail, one lysis plate formed in the lysis plate analysis was recovered using a sterilized tip, and then it was added to the culture medium of Enterobacter arogenes bacteria and incubated at 37° C. for 4-5 hours. After incubation, centrifugation at 8,000 rpm for 20 minutes to obtain a supernatant. To the obtained supernatant, a culture solution of Enterobacter arogenes was added in a volume of 1/50, and then cultured again at 37° C. for 4-5 hours. In order to increase the number of bacteriophages, this process was performed at least 5 times, and then finally centrifuged at 8,000 rpm for 20 minutes to obtain a supernatant. Using the obtained supernatant, lysis was performed again. Since the separation of pure bacteriophage is not usually achieved with only one of the above processes, the previous step was repeated entirely again using the lysing plate formed at this time. This process was repeated at least 5 times to obtain a solution containing pure bacteriophage. Typically, separation of pure bacteriophage was repeatedly performed until the size and shape of the formed lysis plate were all similar. And finally, it was confirmed whether the pure separation of the bacteriophage through electron microscopy analysis. The above-described process was repeated until pure separation was confirmed by electron microscopy analysis. Electron microscopy analysis was performed according to a conventional method. Briefly explaining this is as follows. A solution containing pure bacteriophage was buried on a copper grid, negative staining and drying were performed with 2% uranyl acetate, and the shape was observed through a transmission electron microscope. An electron microscope photograph of the purely isolated bacteriophage is shown in FIG. 1. When judging by the morphological characteristics, it was confirmed that the newly secured bacteriophage belonged to the Siphoviridae bacteriophage.

The solution containing pure bacteriophage confirmed in this way went through the following purification process. The solution containing pure bacteriophage was added with a culture solution of Enterobacter arogenes in a volume of 1/50 of the total volume of the solution, followed by incubation for 4-5 hours again. After incubation, centrifugation at 8,000 rpm for 20 minutes to obtain a supernatant. This process was repeated a total of 5 times to obtain a solution containing a sufficient number of bacteriophage. The supernatant obtained by the final centrifugation was filtered using a 0.45 μm filter, followed by a conventional polyethylene glycol (PEG) precipitation process. Specifically, PEG and NaCl were added to 100 ml of the filtrate to become 10% PEG 8000/0.5 M NaCl, and then allowed to stand at 4° C. for 2-3 hours, and then centrifuged at 8,000 rpm for 30 minutes to obtain a bacteriophage precipitate. . The thus obtained bacteriophage precipitate was suspended in 5 ml of a buffer solution (Buffer; 10 mM Tris-HCl, 10 mM MgSO ₄ , 0.1% Gelatin, pH 8.0). This is referred to as bacteriophage suspension or bacteriophage liquid.

Purified pure bacteriophage was obtained through the above process, and this bacteriophage was named bacteriophage Ent-AEP-1, and then deposited with the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center on June 29, 2018 (accession number KCTC 13569BP). ).

Example 2: Bacteriophage Ent - AEP -1 genome isolation and genome sequence analysis

The genome of the bacteriophage Ent-AEP-1 was isolated as follows. For dielectric separation, a suspension of bacteriophage obtained in the same manner as in Example 1 was used. First, in order to remove DNA and RNA of Enterobacter arogenes bacteria that may be contained in the suspension, 200 U of DNase I and RNase A were added to 10 ml of the bacteriophage suspension, and then left at 37°C for 30 minutes. To remove the activities of DNase I and RNase A after standing for 30 minutes, 500 μl of 0.5 M ethylene diamine tetraacetic acid (EDTA) was added, and then allowed to stand for 10 minutes. And after allowing it to stand at 65° C. for an additional 10 minutes, 100 μl of proteinase K (20 mg/ml) was added to break the outer wall of the bacteriophage, and then reacted at 37° C. for 20 minutes. Thereafter, 500 μl of 10% sodium dodecyl sulfate (SDS) was added, followed by reaction at 65° C. for 1 hour. After 1 hour reaction, 10 ml of a mixture of phenol: chloroform: isoamyl alcohol having a composition ratio of 25:24:1 was added to the reaction solution, and then mixed well. Then, this was centrifuged at 13,000 rpm for 15 minutes to separate the layers, and then the upper layer was taken from the separated layers, and 1.5 volume ratio of isopropyl alcohol was added thereto, followed by centrifugation at 13,000 rpm for 10 minutes. The dielectric was precipitated. After recovering the precipitate, 70% ethanol was added to the precipitate, followed by centrifugation at 13,000 rpm for 10 minutes to wash the precipitate. The washed precipitate was recovered, dried in vacuum, and then dissolved in 100 μl of water. The above process was repeated to secure a large amount of the genome of the bacteriophage Ent-AEP-1.

The genome obtained in this way is obtained by performing Next generation sequencing analysis using a Pac-bio instrument at the National Instrumentation Center for Environmental Management, Seoul National University, and the genome sequence information of the bacteriophage Ent-AEP-1. Secured. The finally analyzed bacteriophage Ent-AEP-1 genome has a size of 51,529 bp, and the entire genome sequence is shown in SEQ ID NO: 1.

Based on the genome sequence information of the secured bacteriophage Ent-AEP-1, using BLAST on the Web, we investigated the homology with the previously known bacteriophage genome sequence. As a result of BLAST investigation, it was confirmed that the genome sequence of the bacteriophage Ent-AEP-1 has relatively high homology with the sequence of Clapsiella bacteriophage KOX-1 (Genbank Accession No. KY780482.1) (query coverage: 88%, identity: 97%).

However, bacteriophage Ent-AEP-1 has a linear genome, whereas Clapsiella bacteriophage KOX-1 has a circular genome, and the number of open reading frames (ORFs) on the bacteriophage Ent-AEP-1 genome Clapsiella bacteriophage KOX-1 had 81 open reading frames, and the arrangement of open reading frames in the genome was different, whereas the two bacteriophages could be judged to have a clear genetic difference.

Based on these facts, it could be concluded that the bacteriophage Ent-AEP-1 is a novel bacteriophage different from the previously reported bacteriophages. Along with this fact, from the fact that the strength of the antibacterial activity and the range of antibacterial activity that can be provided are different if the types of bacteriophage are different in general, it can be judged that the bacteriophage Ent-AEP-1 can provide antibacterial effects different from other previously reported bacteriophages. there was.

Example 3: Bacteriophage Ent - AEP -1 Enterobacter Arrowjins , Enterobacter Cloaca Against bacteria Mortality Research

The ability of the isolated bacteriophage Ent-AEP-1 to kill Enterobacter arogenes bacteria was investigated. The killing ability investigation was carried out in a manner of examining whether or not a transparent ring was generated through the drop experiment presented in Example 1. The Enterobacter arogenes strains used in the killing ability investigation were either received in sale from the antibiotic-resistant strain bank in Korea or were isolated by the present inventors and were identified as Enterobacter arogenes bacteria, for a total of 4 weeks. The bacteriophage Ent-AEP-1 had a killing ability for all 4 weeks of Enterobacter arogenes, which were the subjects of the experiment. In addition, as a result of investigating the killing ability of the bacteriophage Ent-AEP-1 against Enterobacter cloaca through a drop experiment, it was confirmed that the bacteriophage Ent-AEP-1 has apoptosis for 1 out of 5 Enterobacter cloaca . Representative experimental results are presented in FIG. 2. On the other hand, the investigation of the antibacterial activity of the bacteriophage Ent-AEP-1 was carried out by Staphylococcus aureus ), Bordetella bronchiseptica , Enterococcus faecalis ), Enterococcus faecium , Streptococcus mitis ), Streptococcus uberis) and were carried out about the Pseudomonas Ke rugi labor (Pseudomonas aeruginosa), bacteriophage Ent-AEP-1 as a result did not have apoptotic function for these species.

As a result of the above, it was confirmed that the bacteriophage Ent-AEP-1 has excellent killing ability against Enterobacter arogenes bacteria and can exert an antibacterial effect against a number of Enterobacter arogenes strains. In addition, it was confirmed that the bacteriophage Ent-AEP-1 can exert an antibacterial effect against Enterobacter cloaca. This shows that the bacteriophage Ent-AEP-1 can be utilized as an active ingredient of a composition for the purpose of preventing or treating diseases caused by Enterobacter arogenes or Enterobacter cloaca.

Example 4: Bacteriophage Ent - AEP -1 Enterobacter Arrowjins Or Enterobacter Cloaca For the prevention of fungal infections Experimental example

In the present invention, "infection prevention" or "prevention" means that Enterobacter arogenes or Enterobacter cloaca is inhibited from entering the body, or the growth of Enterobacter arogenes or Enterobacter cloaca bacteria introduced into the body As previously explained, it means preventing the infection of Enterobacter arogenes or Enterobacter cloaca by inhibiting the infection, or inhibiting the development of a disease caused by infection of Enterobacter arogenes or Enterobacter cloaca. There is a bar. Therefore, in order to provide the "preventive" effect for the purpose of the present invention, it is possible to reduce the number by inhibiting the proliferation of Enterobacter arogenes bacteria or Enterobacter cloaca bacteria, or killing Enterobacter arogenes bacteria or Enterobacter cloaca bacteria. You must have the ability to do it. This example was carried out to investigate whether the bacteriophage Ent-AEP-1 of the present invention has the ability to meet such purpose.

Add 100 μl of 1×10 ⁸ pfu/ml level of bacteriophage Ent-AEP-1 solution to one tube containing 9 ml of TSB medium, and only the same amount of TSB medium in the other tube containing 9 ml of TSB medium. Further added. Then, a culture solution of Enterobacter arogenes or Enterobacter cloaca was added to each tube so that the absorbance at 600 nm was about 0.3. After adding Enterobacter arogenes or Enterobacter cloaca, the tubes were transferred to an incubator at 37° C. and cultured with shaking to observe the growth state of Enterobacter arogenes or Enterobacter cloaca. As shown in Tables 1 and 2, in the tube to which the bacteriophage Ent-AEP-1 solution was added, inhibition of the growth of Enterobacter arogenes or Enterobacter cloaca was observed, whereas in the tube to which the bacteriophage solution was not added No inhibition of growth of Enterobacter arogenes or Enterobacter cloaca was observed.

Inhibiting the growth of Enterobacter arogenes bacteria

division	OD 600 absorbance value
	0 minutes	30 minutes	1 hours	1.5 hours	2 hours
No bacteriophage liquid added	0.3	0.52	1.28	2.12	4.41
Addition of bacteriophage liquid	0.3	0.52	1.13	0.25	0.15

Inhibition of the growth of Enterobacter cloaca bacteria

division	OD 600 absorbance value
	0 minutes	30 minutes	1 hours	1.5 hours	2 hours
No bacteriophage liquid added	0.3	0.41	1.03	2	3.6
Addition of bacteriophage liquid	0.3	0.33	0.32	0.27	0.1

From these results, it was found that the bacteriophage Ent-AEP-1 of the present invention not only inhibits the growth of Enterobacter arogenes and Enterobacter cloaca, but also has the ability to kill Enterobacter arogenes and Enterobacter cloaca. From this, it could be concluded that the bacteriophage Ent-AEP-1 can be used as an active ingredient of a composition for the purpose of preventing infection of Enterobacter arogenes or Enterobacter cloaca.

Example 5: Bacteriophage Ent - AEP Enterobacter with -1 Arrowjins Fungus or Enterobacter Cloaca Fungal infection Prevention example

The effect of the bacteriophage Ent-AEP-1 on the prevention of infection caused by Enterobacter arogenes or Enterobacter cloaca was investigated as follows. Forty eight-week-old mice were divided into four groups of 10 mice per group, and then the mice of each group were separated and reared in mouse cages for each group for 14 days. The animals belonging to the control group 1 and 2 were fed freely with regular feed every day from the first day to the end of the test, and the animals belonging to the remaining two groups (group 3 and group 4) were fed first. From the day to the end of the day, a feed of the same composition containing the bacteriophage Ent-AEP-1 was fed freely at an amount of 10 ⁶ pfu/g daily. The feed containing bacteriophage was prepared by spraying the feed with a bacteriophage suspension (Suspension) of which the concentration (Titer) is known and drying it. The administration of bacteriophage can be administered by direct oral administration instead of feeding with feed, but in order to reduce bacteriophage damage caused by gastric juice, a method of feeding with feed was applied. When actually used in the field, the bacteriophage wrapped in an enteric capsule can be used or a method of taking it with a neutralizing agent can be applied. And on the 8th, 9th, and 10th days of the start of the test, for all test animals belonging to groups 1 and 3, a suspension of Enterobacter arogenes (10 ³ cfu/ml) was added to 20 ml per cage per day. It was sprayed once, and a suspension of Enterobacter cloaca (10 ³ cfu/ml) was sprayed twice a day at 20 ml per cage to all test animals belonging to groups 2 and 4. The suspension of Enterobacter arogenes bacteria was prepared as follows. Enterobacter arogenes bacteria were cultured at 37°C for 18 hours using TSB medium, and then only the cells were recovered. The recovered cells were suspended in physiological saline (pH 7.2) to 1×10 ³ cfu/ml. The suspension of Enterobacter cloaca was prepared as follows. Enterobacter cloaca was cultured at 37° C. for 18 hours using TSB medium, and then only the cells were recovered. The recovered cells were suspended in physiological saline (pH 7.2) to 1×10 ³ cfu/ml. From the 8th day of the test start to the end of the test (the 14th day of the start of the test), the number of dead individuals and the number of individuals with decreased activity were observed. The results were as follows.

Prevention effect investigation test result

group	Number of objects with reduced activity/number of dead objects
	D8	D9	D10	D11	D12	D13	D14
Group 1	0/0	1/0	3/0	5/0	6/1	6/1	5/1
Group 2	0/0	0/0	2/0	4/0	6/0	7/1	6/1
Group 3	0/0	0/0	0/0	0/0	0/0	0/0	0/0
Group 4	0/0	0/0	0/0	0/0	0/0	0/0	0/0

From this result, it was confirmed that the bacteriophage Ent-AEP-1 of the present invention is also very effective in preventing infectious diseases caused by Enterobacter arogenes bacteria or Enterobacter cloaca bacteria.

Example 6: Bacteriophage Ent - AEP Enterobacter with -1 Arrowjins Fungus or Enterobacter Cloaca Fungal infections Treatment example

The therapeutic effect of the bacteriophage Ent-AEP-1 on infectious diseases caused by Enterobacter arogenes or Enterobacter cloaca was investigated as follows. Forty eight-week-old mice were divided into four groups of 10 mice per group, and then the mice of each group were separated and reared in mouse cages for each group for 14 days. It went through the acclimatization process for 7 days, and at this time, normal feed was freely fed to all test animals. On the 8th day of the start of the test, all test animals belonging to groups 1 and 3 were administered orally (10 ⁹ cfu/mouse) of Enterobacter arogenes suspension, and all test animals belonging to groups 2 and 4 were administered orally. For this, a suspension of Enterobacter cloaca was administered by oral administration (10 ⁹ cfu/mouse). The suspension of Enterobacter arogenes bacteria was prepared as follows. Enterobacter arogenes bacteria were cultured at 37°C for 18 hours using TSB medium, and then only the cells were recovered. The recovered cells were suspended in physiological saline (pH 7.2) to 1×10 ¹⁰ cfu/ml. The suspension of Enterobacter cloaca was prepared as follows. Enterobacter cloaca was cultured at 37° C. for 18 hours using TSB medium, and then only the cells were recovered. The recovered cells were suspended in physiological saline (pH 7.2) to 1×10 ¹⁰ cfu/ml. From 2 hours after the administration of the bacteria to the end of the test, animals belonging to groups 1 and 2 were fed freely with normal feed, and animals belonging to the remaining two groups (group 3 and 4) as the experimental group To the bacteriophage at a content of 10 ⁶ pfu/g, a feed of the same composition containing the bacteriophage Ent-AEP-1 was freely fed from 2 hours to the end of the test. The feed containing bacteriophage was prepared by spraying the feed with a bacteriophage suspension (Suspension) of which the concentration (Titer) is known and drying it. The administration of bacteriophage can be administered by direct oral administration instead of feeding with feed, but in order to reduce bacteriophage damage caused by gastric juice, a method of feeding with feed was applied. When actually used in the field, the bacteriophage wrapped in an enteric capsule can be used or a method of taking it with a neutralizing agent can be applied. The number of dead individuals in each group was observed from the bacterial administration day (D8) to the end of the test (D14). The results were as follows.

Treatment effect investigation test result

group	The number of dead individuals
	D8	D9	D10	D11	D12	D13	D14
Group 1	0	0	One	2	2	One	0
Group 2	0	One	0	2	One	One	One
Group 3	0	0	0	0	0	0	0
Group 4	0	0	0	0	0	0	0

From this result, it was confirmed that the bacteriophage Ent-AEP-1 of the present invention is also very effective in the treatment of infectious diseases caused by Enterobacter arogenes or Enterobacter cloaca.

As a result of the above, a specific part of the present invention has been described in detail, and it is obvious that this specific technology is only a preferred embodiment for those of ordinary skill in the art, and the scope of the present invention is not limited thereto. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

[Accession number]

Depository name: KCTC

Accession number: KCTC 13569BP

Consignment Date: 20180629

Claims (4)

1. Sipoviridae bacteriophage Ent-AEP-1 isolated from nature, characterized by having the ability to kill Enterobacter arogenes and Enterobacter cloaca bacteria and having a genome represented by SEQ ID NO: 1 (accession number KCTC 13569BP).
2. Claim 1 bacteriophage Ent-AEP-1 (accession number KCTC 13569BP) containing as an active ingredient, Enterobacter arogenes bacteria or Enterobacter cloaca bacteria for preventing and treating diseases caused by a composition.
3. The composition containing the bacteriophage Ent-AEP-1 (accession number KCTC 13569BP) of claim 1 as an active ingredient is sprayed into the surrounding environment where Enterobacter arogenes or Enterobacter cloaca bacteria may exist, or infection concerns or infected persons A method for preventing and treating diseases caused by Enterobacter arogenes bacteria or Enterobacter cloaca bacteria, comprising the step of administering to a subject.
4. The method according to claim 3, wherein the composition is administered to animals other than humans in the form of a pharmaceutical composition such as a disinfectant or antibiotic, and prevents diseases caused by Enterobacter arogenes bacteria or Enterobacter cloaca bacteria, and How to treat.

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