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WO2016126009A1 - Nouveau bactériophage edw-tap-1 de l'edwardsiella tarda et utilisation associée pour inhiber la prolifération de l'edwardsiella tarda - Google Patents

Nouveau bactériophage edw-tap-1 de l'edwardsiella tarda et utilisation associée pour inhiber la prolifération de l'edwardsiella tarda Download PDF

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Publication number
WO2016126009A1
WO2016126009A1 PCT/KR2016/000031 KR2016000031W WO2016126009A1 WO 2016126009 A1 WO2016126009 A1 WO 2016126009A1 KR 2016000031 W KR2016000031 W KR 2016000031W WO 2016126009 A1 WO2016126009 A1 WO 2016126009A1
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bacteriophage
tarda
tap
edw
composition
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English (en)
Korean (ko)
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윤성준
강상현
전수연
권안성
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Intron Biotechnology Inc
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Intron Biotechnology Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/195—Antibiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/10—Washing or bathing preparations

Definitions

  • the present invention prevents the infection of Edward siela tarda using a composition containing the bacteriophage isolated from nature capable of killing Edward siela tarda and killing Edward siela tarda and an active ingredient. And it relates to a method of treatment, more specifically myobidae isolated from nature characterized in that it has a genome represented by SEQ ID NO: 1 having the ability to specifically kill Eddsiella tarda bacteria Bacteriophage Edw-TAP-1 (Accession No. KCTC 12740BP), and a method for preventing and post-infection treatment of Ed Ward siella tarda bacteria using the composition comprising the bacteriophage as an active ingredient.
  • Edwardsiella tarda is a Gram-negative bacillus belonging to Enterobacteriaceae, and has cell-like antigens (Somatic, O-antigens) and flagella antigens (Flagella, H-antigens) of lipopolysaccharides (LPS) on the cell wall. Combinations of these antigens can be divided into various serotypes.
  • Edward Siela tarda is a major causative agent of Edwardsiellosis and is widely known to infect marine and freshwater fish such as flounder, salmon, catfish, eel, and tilapia.
  • Fish infected with Edward disease are visually observed with blackening, abdominal bloating, and hernia, and anatomical findings include nodules in the liver and kidneys, and turbidity and odor in the presence of ascites.
  • a combination of streptococcal disease and the infection occurs, and in some cases muscle abscesses.
  • fish with Edward disease often die of gangrene and septicemia, causing serious economic losses in the aquaculture industry.
  • Edward disease is prevalent in high water temperatures, but recent developments suggest that it is not necessarily due to water temperature or seasonal effects. In addition, there are few cases of death in a short period of time, so once the onset of death is a long-term mortality is often economic damage. In most cases, bacterial infections alone or mixed infections are treated with antibiotics. However, these methods can cause problems such as resistant bacteria and environmental pollution due to the abuse of antibiotics. Therefore, it is necessary to develop a more natural way.
  • Bacteriophages are tiny microorganisms that infect bacteria, often called phage. Bacteriophages have the ability to infect bacteria and multiply inside bacterial cells, and kill off bacteria by destroying the cell wall of the host bacteria when the progeny bacteriophages come out of the bacteria. Bacteriophage bacterial infections are highly specific, so there are only a few types of bacteriophages that can infect certain bacteria. That is, certain bacteriophages can infect only a specific category of bacteria, so that certain bacteriophages kill only certain bacteria and do not affect other bacteria.
  • the bacteriophage was discovered in 1915 by a British bacteriologist Twort, who studied the phenomenon of colonization of the micrococcus colony transparently by something.
  • French bacteriologist d'Herelle discovered that some of the filtrates of foreign patients had a function of dissolving Shigella disenteriae , and through this study, they independently discovered bacteriophages and consumed them. In the sense, they named it bacteriophage. Since then, bacteriophages have been found for many pathogenic bacteria such as dysentery, typhoid, and cholera.
  • bacteriophages Because of its special ability to kill bacteria, bacteriophages have been expected to be an effective way to combat bacterial infections since their discovery and many studies have been done. However, after the discovery of penicillin by Flemming, the prevalence of antibiotics has led to studies of bacteriophages only limited to some Eastern European countries and the former Soviet Union. However, since 2000, due to the increase of antibiotic-resistant bacteria, the limit of existing antibiotics appears, and as the possibility of developing an alternative to the existing antibiotics is highlighted, bacteriophage is attracting attention as an anti-bacterial agent.
  • the present inventors have developed a composition that can be used to prevent or treat an infection of Edwardela tarta bacteria by using bacteriophage isolated from nature capable of selectively killing Edwardela tarta bacteria. After trying to develop a method for preventing or treating the infection of Ed C. tarda by using the composition, it is possible to isolate suitable bacteriophages from nature and to distinguish them from other bacteriophages. By securing a gene sequence of the genome so as to develop a composition comprising the bacteriophage as an active ingredient, and then confirming that the composition can be effectively used to prevent and treat the infection of Ed C. Was completed.
  • an object of the present invention is Myoviridae bacteriophage Edw- isolated from nature, characterized by having a genome represented by SEQ ID NO: 1 having the ability to specifically kill Eddsiella tarda. TAP-1 (Accession No. KCTC 12740BP).
  • the present invention provides a composition that can be used to treat an infection, and a method for treating infection of Eddsiella tarda using the composition.
  • Still another object of the present invention is to provide a medicament for the purpose of preventing and treating Eddsiella tarda infection using the compositions.
  • Another object of the present invention is to provide a feed additive for the purpose of providing a specification effect through the prevention and treatment of Ed ward siella tarda infection using the compositions.
  • the present invention is a Myobiridae bacteriophage Edw-TAP-1 (accession number), characterized in that it has a genome represented by SEQ ID NO: 1 having the ability to specifically kill the Edwadsiella tarda bacteria KCTC 12740BP), and a method for preventing and treating infection with Eddsiella tarda using the composition comprising the same as an active ingredient.
  • Bacteriophage Edw-TAP-1 was isolated by the inventors and deposited in the Korea Institute of Biotechnology and Biotechnology Center on January 8, 2015 (Accession No. KCTC 12740BP).
  • the present invention also provides a bath and feed additive comprising bacteriophage Edw-TAP-1 as an active ingredient that can be used to prevent or treat the infection of Ed C.
  • Bacteriophage Edw-TAP-1 included in the composition of the present invention effectively kills Eddsiella tarda bacteria, thus preventing (infecting) or treating Edward disease caused by Eddsiella tarda bacteria (infection treatment) ) To effect. Therefore, the composition of the present invention can be used for the purpose of prevention and treatment of Ed Ward disease caused by Ed Ward Siella tarda.
  • treatment refers to (i) suppression of Edward disease caused by Eddsiella tarda; And (ii) alleviation of Edward disease caused by the Eddwardella tarda bacterium.
  • isolated refers to the separation of bacteriophages from the natural state using various experimental techniques, and to securing specific characteristics that can be distinguished from other bacteriophages. This includes growing the bacteriophages for industrial use.
  • compositions of the present invention are those commonly used in the formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate , Microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but are not limited thereto. no.
  • the composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like in addition to the above components.
  • the composition of the present invention includes bacteriophage Edw-TAP-1 as an active ingredient.
  • the bacteriophage Edw-TAP-1 included at this time is included as 1 ⁇ 10 1 pfu / ml 1 ⁇ 10 30 pfu / ml or 1 ⁇ 10 1 pfu / g to 1 ⁇ 10 30 pfu / g, preferably 1 ⁇ 10 4 pfu / ml to 1 ⁇ 10 15 pfu / ml or 1 ⁇ 10 4 pfu / g to 1 ⁇ 10 15 pfu / g.
  • compositions of the present invention may be prepared in unit dosage form by being formulated with pharmaceutically acceptable carriers and / or excipients, according to methods which may be readily practiced by those skilled in the art. It may also be prepared by incorporation into a multi-dose container.
  • the formulations here may be in the form of solutions, suspensions or emulsions in oils or aqueous media or in the form of extracts, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.
  • composition of the present invention may be implemented as a bath and feed additives.
  • Infection prevention and treatment method of Ed Ward siella tarda using a composition comprising the bacteriophage Edw-TAP-1 of the present invention as an active ingredient Ed Wad Siela compared to conventional methods based on chemicals such as antibiotics It can provide the advantage of very high specificity for tar. This means that it can be used for the purpose of preventing or treating Edwadsiella tarda without influencing other useful flora, and the side effects of its use are very small. In general, the use of chemicals, such as antibiotics will also damage the common flora bacteria, resulting in a decrease in the immunity of animals, resulting in various side effects. On the other hand, the present invention can also provide the advantage of being very natural because it is used as an active ingredient of the composition to separate the bacteriophage already present in nature.
  • Figure 2 is an experimental result showing the killing ability of Ed Wd Siela Tarda bacteria of bacteriophage Edw-TAP-1.
  • the transparent part is the lysate plaque formed by lysis of bacteria.
  • Ella necessarily the collected sample and the Ed tar is inoculated with the bacteria in TSB 1/1000 ratio (T S oy ryptic roth B) medium (Casein Digest, 17 g / L; Soy bean Digest, 3 g / L; dextrose, 2.5 g / L; NaCl, 5 g / L; dipotassium phosphate, 2.5 g / L) together and shaken at 37 ° C. for 3-4 hours. After incubation, the supernatant was recovered by centrifugation at 8,000 rpm for 20 minutes.
  • T S oy ryptic roth B T S oy ryptic roth B
  • Casein Digest 17 g / L
  • Soy bean Digest 3 g / L
  • dextrose 2.5 g / L
  • NaCl 5 g / L
  • dipotassium phosphate 2.5 g / L
  • Edwad siella tarda was inoculated in the recovered supernatant at a rate of 1/1000 and then shaken again for 3-4 hours at 37 °C.
  • the process was repeated five times in order to increase the number of bacteriophages (Titer). After five repetitions, the culture was centrifuged at 8,000 rpm for 20 minutes. After centrifugation, the recovered supernatant was filtered using a 0.45 ⁇ m filter. It was examined whether there was a bacteriophage capable of killing Eddsiella tarda by the usual spot assay using the obtained filtrate.
  • the filtrate in which the transparent ring is formed contains bacteriophages capable of killing Eddsiella tarda. Through this investigation, it was possible to obtain a filtrate including bacteriophage having the ability to kill Edwardsiella tarda.
  • Separation of pure bacteriophages was carried out using a filtrate in which the presence of bacteriophages with killing ability against Edward siella tarda was confirmed. Separation of pure bacteriophage was carried out using a conventional Plaque assay. To explain this in detail, one of the lytic plaques formed in the lytic plaque assay was recovered using a sterile tip, and then added to the Eddsiella tarda bacterial culture and incubated together at 37 ° C. for 4-5 hours. After incubation, the supernatant was obtained by centrifugation at 8,000 rpm for 20 minutes.
  • the Edd Siella tarda bacteria culture solution was added at a volume of 50/50, and then incubated at 37 ° C for 4-5 hours. In order to increase the number of bacteriophages, this procedure was performed at least five times, and finally, the supernatant was obtained by centrifugation at 8,000 rpm for 20 minutes. Using the obtained supernatant, lysis plate analysis was performed again. Since the separation of the pure bacteriophage is not usually achieved in one step of the above process, the previous step was repeated again using the lysate formed. This procedure was carried out at least five times to obtain a solution containing pure bacteriophage.
  • the solution containing pure bacteriophage identified in this way was subjected to the following purification process.
  • Edwadsiella tarda culture medium was added to a volume of 1/50 of the total volume of the solution including the pure bacteriophage, and then incubated again at 37 ° C. for 4-5 hours. After incubation, the supernatant was obtained by centrifugation at 8,000 rpm for 20 minutes. This procedure was repeated a total of five times to obtain a solution containing a sufficient number of bacteriophages.
  • the supernatant obtained by the final centrifugation was filtered using a 0.45 ⁇ m filter followed by a conventional polyethylene glycol (PEG) precipitation process.
  • PEG polyethylene glycol
  • PEG and NaCl were added to 100 ml of the filtrate to be 10% PEG 8000 / 0.5 M NaCl, and then allowed to stand at 4 ° C. for 2-3 hours, followed by centrifugation at 8,000 rpm for 30 minutes to obtain a bacteriophage precipitate.
  • the bacteriophage precipitate thus obtained was suspended in 5 ml of buffer (Buffer; 10 mM Tris-HCl, 10 mM MgSO 4 , 0.1% Gelatin, pH 8.0). This is called bacteriophage suspension or bacteriophage solution.
  • the genome of bacteriophage Edw-TAP-1 was isolated as follows. Bacteriophage suspension obtained in the same manner as in Example 1 was used for dielectric separation. First, in order to remove DNA and RNA of Eddsiella tarda, which may be included in the suspension, 200 U of each of DNase I and RNase A was added to 10 ml of the bacteriophage suspension, followed by standing at 37 ° C for 30 minutes. In order to remove the activity of DNase I and RNase A after 30 minutes, 500 ⁇ l of 0.5 M ethylenediaminetetraacetic acid (EDTA) was added and allowed to stand for 10 minutes. The mixture was left at 65 ° C.
  • EDTA ethylenediaminetetraacetic acid
  • bacteriophage Edw-TAP-1 could be considered as a novel bacteriophage that has not been reported previously.
  • the killing ability of the isolated bacteriophage Edw-TAP-1 against Edward siella tarda was investigated.
  • the killing ability was investigated by the drop test in the same manner as in Example 1 to determine the production of transparent rings.
  • Ed ward siela tarda bacteria used for killing ability investigation were separated by the present inventors and 12 species were identified as ed ward siela tarda bacteria.
  • Bacteriophage Edw-TAP-1 had the ability to kill against 9 species of Edward sidella tarda. Representative experimental results are shown in FIG. 2.
  • the bacteriophage Edw-TAP-1 has a specific killing ability against Ed Ward siella tarda, and it can be confirmed that it can exert an antimicrobial effect against a number of Ed Ward siella tarda. This means that the bacteriophage Edw-TAP-1 can be used as an active ingredient of the composition for preventing and treating Eddsiella tarda.
  • Example 4 bacteriophage Edw Of TAP-1 Edwad Siela About prevention of infection of tar Experimental Example
  • the bacteriophage Edw-TAP-1 of the present invention has the ability to inhibit the growth and kill the Edwadsiella tarda, and from this, the bacteriophage Edw-TAP-1 is Edwad City. It was concluded that it can be used as an active ingredient of a composition for the purpose of preventing the infection of Ella tarda.
  • the treatment effect of bacteriophage Edw-TAP-1 in halibut induced by Edward sickle tarda bacteria was investigated.
  • Four-month-old halibut fry (6-9 cm) was divided into two groups, which were divided into two groups, and then separated and bred in a tank for 14 days.
  • the environment of the bath was controlled and the temperature of the laboratory containing the bath was kept constant.
  • the feed containing Edward sidella tarda bacteria was fed twice a day by the usual feed method at a level of 1 ⁇ 10 8 cfu / g for 3 days from the 5th day from the start of the experiment.
  • individuals with clinical symptoms of Edward disease were identified from the last day of feed containing the Edwardsiella tarda.
  • the flounder in the experimental group was 1 ⁇ 10 8 pfu / g of bacteriophage Edw-.
  • Feed containing TAP-1 was fed according to a conventional feed feeding method.
  • the flounders of the control group (not administered bacteriophage) were fed with the same composition without the bacteriophage Edw-TAP-1.
  • Ed Ward's disease status was investigated by measuring the body color blackening index.
  • the body color blackening index was measured by measuring the dark coloration (DC) score (normal: 0, light blackening: 1, dark blackening: 2) which is commonly used. The results were shown in Table 2.
  • Body color blackening index measurement results (average) date D8 D9 D10 D11 D12 D13 D14 Control group (not administered bacteriophage) 1.00 1.36 1.64 1.68 1.72 1.52 1.36 Experimental group (bacteriophage administration) 1.00 0.76 0.24 0.16 0.16 0.08 0.00
  • a feed additive was prepared using bacteriophage Edw-TAP-1 solution to include 1 ⁇ 10 8 pfu of bacteriophage Edw-TAP-1 per g of feed additive.
  • the method of preparing a feed additive was prepared by adding maltodextrin to the bacteriophage solution (40%, w / v) and adding trihalose to a total of 10%, followed by lyophilization. Finally, it was ground to a fine powder form.
  • the drying process in the manufacturing process may be replaced by reduced pressure drying, warming drying, room temperature drying.
  • the feed additive without bacteriophage also used the buffer used to prepare the bacteriophage solution (Buffer; 10 mM Tris-HCl, 10 mM MgSO 4 , 0.1% Gelatin, pH 8.0) instead of the bacteriophage solution. It was prepared by.
  • Each of the two feed additives thus prepared was mixed with 250 times the fish feed for fish in a weight ratio to prepare the final two feeds.
  • the bacteriophage Edw-TAP-1 solution was used to prepare a bath so that 1 ⁇ 10 8 pfu of bacteriophage Edw-TAP-1 per ml of the bath detergent was included.
  • the method of preparing the bath detergent is prepared by adding the bacteriophage Edw-TAP-1 solution so that 1 ⁇ 10 8 pfu of bacteriophage Edw-TAP-1 is included per 1 ml of the buffer used to prepare the bacteriophage solution. It was.
  • the buffer itself used in the preparation of the bacteriophage solution was used as it is.
  • the two types of baths thus prepared were diluted with 1,000 times water by volume and used as the final bath.
  • Example 6 and Example 7 Using the feed prepared in Example 6 and Example 7 and the bath was investigated whether the improvement of the specifications results when flounder breeding. In particular, the survey was conducted in terms of mortality.
  • a total of 500 halibuts were divided into two groups (group-A fed and group-B treated with a bath) for four weeks. Each group was divided into 125 subgroups, and each subgroup was divided into a small group (small group-1) to which the bacteriophage Edw-TAP-1 was applied and a small group (small group-2) to which the bacteriophage was not applied.
  • the flounder was a four-month-old flounder fry, and flounders from each subgroup were raised in separate tanks at regular intervals. Each subgroup is divided and referred to as Table 3 below.
  • the feed prepared according to the feed preparation method described in Example 6 was fed according to the conventional feed feeding method according to the classification of Table 3, and in the case of the treatment of the bathing agent, the bathing bath described in Example 7
  • the bath preparation prepared according to the preparation method was treated according to the conventional bath treatment method according to the classification of Table 3.
  • the test results are shown in Table 4.

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Abstract

La présente invention concerne le bactériophage Edw-TAP-1 Myoviridae (N° d'entrée KCTC 12740BP) isolé du milieu naturel, caractérisé par le maintien d'une capacité à tuer de manière spécifique l'Edwardsiella tarda et présentant le génome représenté par SEQ ID N° 1, et un procédé pour la prévention et le traitement de l'infection par l'Edwardsiella tarda à l'aide d'une composition contenant le bactériophage en tant que principe actif.
PCT/KR2016/000031 2015-02-03 2016-01-05 Nouveau bactériophage edw-tap-1 de l'edwardsiella tarda et utilisation associée pour inhiber la prolifération de l'edwardsiella tarda Ceased WO2016126009A1 (fr)

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KR1020150016533A KR101609111B1 (ko) 2015-02-03 2015-02-03 신규한 에드와드시엘라 타르다 박테리오파지 ＥｄＷ-ＴＡＰ-1 및 이의 에드와드시엘라 타르다 균 증식 억제 용도
KR10-2015-0016533 2015-02-03

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CN114958778A (zh) * 2022-01-28 2022-08-30 武汉格瑞农生物科技有限公司 一株副溶血弧菌裂解性噬菌体gvp-p20及其在中华绒螯蟹中副溶血弧菌的防治应用
CN115216453A (zh) * 2021-12-31 2022-10-21 华东理工大学 爱德华氏菌特异性噬菌体和其组合物及其应用
CN120173894A (zh) * 2025-05-22 2025-06-20 聊城大学 一种迟缓爱德华氏菌噬菌体及其应用

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KR101792522B1 (ko) * 2016-06-14 2017-11-02 주식회사 인트론바이오테크놀로지 신규한 비브리오 파라헤몰리티쿠스 박테리오파지 Vib-PAP-5 및 이의 비브리오 파라헤몰리티쿠스 균 증식 억제 용도
KR102368437B1 (ko) * 2016-07-25 2022-02-28 주식회사 인트론바이오테크놀로지 오일이 코팅된 박테리오파지를 포함한 양어용 사료 및 이의 제조 방법
KR20180011518A (ko) * 2016-07-25 2018-02-02 주식회사 인트론바이오테크놀로지 오일이 코팅된 박테리오파지를 포함한 양어용 사료 및 이의 제조 방법
CN114736877B (zh) * 2022-05-20 2023-07-07 青岛诺安百特生物技术有限公司 一株杀鱼爱德华氏菌噬菌体、其噬菌体组合物及其应用

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