KR20160044637A - Novel bacteriophage CAU130028 having killing activity specific to Salmonella Enteritidis - Google Patents

Abstract

The present invention relates to a novel bacteriophage which specifically kills Salmonella enteritidis. More specifically, provided is a pharmaceutical composition for treating and preventing disease induced by bacteria from the genus Salmonella, containing bacteriophage as an active ingredient. The novel bacteriophage of the present invention can specifically kill Salmonella enteritidis, thereby being effective. Thus, the bacteriophage can be useful for developing food additives after separation from food sources.

Description

A novel bacteriophage CAU130028 {Novel bacteriophage CAU130028 having killing activity specific to Salmonella Enteritidis), which specifically kills Salmonella enteritidis,

The present invention relates to a novel bacteriophage which specifically kills Salmonella Enteritidis , and more particularly to a pharmaceutical composition for preventing and treating a Salmonella enteric disease which comprises bacteriophage as an active ingredient .

Recently, the problem of the emergence and increase of antibiotic-resistant bacteria in livestock industry has become very important in many aspects and is one of the areas of greatest interest. As researches on antibiotic resistance have progressed, many studies focusing on the development of new preventive and therapeutic technologies that can replace antibiotics have been carried out. One such study is the use of bacteriophages or phage. A study on the treatment of infectious diseases using bacteriophage began in 1917 when Twort first discovered bacteriophage in 1915 and d'Herelle applied it to Salmonella Gallinarum, the cause of avian typhoid. In the 1930s and 1940s, not only animals but also humans have been applied in many countries. However, since the discovery of antibiotics has reduced the use of bacteriophages, many studies have been conducted recently. This is because the technique of using bacteriophage for many pathogenic bacteria resistant to antibiotics is very useful.

Bacteriophages exist extensively in nature and exist in common environments with bacteria that are their hosts. That is, these bacteriophages already exist in various environments related to human and animal ministers, food, soil, water, and wetlands. In order to use the bacteriophage for therapeutic purposes, it is necessary to show an appropriate infection rate between the host cell and the bacteriophage, and to show the specificity between the host cell and the bacteriophage. Thus, It is very important to develop a bacteriophage having specificity to lyse the bacteriophage.

Salmonella is an acute intestinal infectious agent in humans and animals. It is the causative agent of acquired infectious diseases that can be transmitted to humans through old food. It is a disease that causes huge economic damage to swine industry due to delayed growth due to death or chronic diarrhea caused by acute sepsis or enteritis when infected with pigs. In addition to this, salmonella which has been infected with pigs is contaminated with pork and has a very high risk of spreading to humans.

Salmonella is a gram-negative bacterium that does not form gram-negative apo and is a motile bacterium belonging to the intestinal microflora and is distributed throughout the natural world including humans and animals. It is caused by ingesting contaminated food that causes infection in the intestinal tract. Illnesses caused by Salmonella include food poisoning, enteritis, poultry typhus, and salmonellosis. Salmonella enteritidis and Salmonella typhi cause salmonellosis in pigs, cattle, and poultry. When Salmonella enteritidis and Salmonella typhi are infected with pigs, cattle, and poultry, Salmonella is resistant to various antibiotics, making it difficult to completely eliminate them as antibiotics. Therefore, a method for effectively removing and controlling Salmonella is required.

Until now, most antibiotics have been used to remove this salmonella. However, antibiotics resistant to antibiotic abuse and abuse in biota farms are emerging as a problem in the production of safe livestock products due to the appearance of various types of antibiotic resistant bacteria and residual antibiotics. Therefore, treatment with antibiotics is very difficult, And various attempts have been made.

Bacteriophages are bacterial-specific viruses that can be called phage and propagate when bacterial hosts are present. Bacteriophages are not interested because of their inefficiency in controlling bacteria due to their host-specific properties, but they are attracting attention again with the emergence of super bacteria resistant to antibiotics. In Europe, Listerix-P100 has been developed as a food additive to control listeria using bacteriophage, and has been approved by the US FDA.

Until now, the isolate of bacteriophages isolated is the feces of river water and livestock which are mostly salmonella. However, when commercialization is considered, there is a disadvantage that the separation source may cause a consumer's displeasure to treat the bacteriophages isolated from the feces or the rivers.

Accordingly, the present inventors have established effective treatment methods for swine diarrhea, which causes enormous economic damage to the swine industry, and have found that, when infected with a human body, for the purpose of treating Salmonella infection, the major pathogenic bacteria among bacteriophages isolated from domestic pig farms and domestic sewage The bacteriophage CAU130028 showing specificity to Salmonella enterica serovar Enteritidis was isolated and the ability of Salmonella enteritidis to kill was confirmed by the test, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a bacteriophage CAU130028 having a specific killing activity against Salmonella enteritidis isolated from a food source.

It is still another object of the present invention to provide a pharmaceutical composition for preventing and treating Salmonella entericidid diseases including Salmonella enteritidis including bacteriophage CAU130028 as an active ingredient.

In order to achieve the above object, the present invention provides a bacteriophage CAU130028 having a specific killing activity against Salmonella enteritidis isolated from a food source.

In order to accomplish still another object of the present invention, the present invention provides a pharmaceutical composition for preventing and treating Salmonella entericidis diseases including Salmonella enteritidis including bacteriophage CAU130028 as an active ingredient.

Hereinafter, the present invention will be described in detail.

Unless defined otherwise, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art and are consistent with those described in the following references (Singleton et al, J. Wiley & Sons, 1994; and Janeway, C., Travers, P., Walport, M., Shlomchik, Immunobiology, 5th Ed., Garland Publishing, 2001).

The present invention provides bacteriophage CAU130028 having specific killing activity against Salmonella enteritidis isolated from a food source.

In the present invention, the bacteriophage is a deposit number KCTC18321P. The present inventors selected new bacteriophages having specific killing ability against Salmonella enteritidis from the environment and deposited the isolated bacteriophage in the genetic bank of the Korea Biotechnology Research Institute.

The bacteriophage CAU130028 of the present invention can be applied to the medical industry, the food industry, the biotechnology industry, and the like to effectively kill the harmful Salmonella enteritidis in the target place or the target material without any problem about the antimicrobial resistance.

The present invention provides a pharmaceutical composition for the prevention and treatment of Salmonella entericidis diseases including Salmonella enteritidis including bacteriophage CAU130028 as an active ingredient.

The bacteriophage contained in the composition of the present invention is less harmful to the emergence of resistant bacteria than general antimicrobial agents, exhibits a lytic action only in bacteria, and is excellent in selective toxicity that does not act on eukaryotic cells, and thus is harmless to humans and animals. .

As used herein, the term " treatment " refers to the prevention of diseases caused by Salmonella spp .; Inhibition of diseases caused by Salmonella spp .; And mitigation of diseases caused by Salmonella spp.

The composition of the present invention may further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers to be included in the composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate , Microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, no.

The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc., in addition to the above components.

The pharmaceutical composition of the present invention can be used as a method of applying or spraying on a diseased site, or may be administered through oral administration or parenteral administration. In the case of parenteral administration, intravenous administration, intraperitoneal administration, Intravenous, subcutaneous, or topical administration.

Suitable application, spray, and dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, disease symptom, food, administration time, administration route, And the ordinarily skilled physician can readily determine and prescribe dosages effective for the desired treatment. Generally, the pharmaceutical composition of the present invention comprises 1 x 10 ⁶ to 1 x 10 ⁹ pfu / ml of bacteriophage.

The pharmaceutical composition of the present invention may be formulated into pharmaceutically acceptable carriers and / or excipients according to a method which can be easily carried out by those skilled in the art, Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

The present invention is characterized in that the Salmonella enterococcal disease including Salmonella enteritidis is food poisoning, enteritis, salmonellosis, poultry typhus or acute bacteremia.

Salmonella spp. Causes diseases in humans and animals by contaminated food, water, utensils and various infectious agents. Salmonella enteropathic diseases include food poisoning, gastroenteritis, enteritis, bacteremia, acute bacteremia, poultry typhus, salmonellosis, chronic infections and local infections.

Accordingly, the present invention relates to a novel bacteriophage that specifically kills Salmonella Enteritidis , and more particularly, to a pharmaceutical composition for preventing and treating Salmonella Enteritidis including bacteriophage as an active ingredient do. INDUSTRIAL APPLICABILITY The present invention is effective to specifically kill Salmonella enteritidis, and is easy to develop as a food additive by separating it from a food source.

Figure 1 is a photograph of a plaque assay result of bacteriophage CAU130028.
2 is a graph showing the killing effect of Salmonella on bacteriophage over time.

Hereinafter, the present invention will be described in detail.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

≪ Example 1 >

Isolation of single bacteriophage capable of specifically killing Salmonella enteritidis

In general, daily fermented foods such as kimchi and soy sauce are collected in a conventional market and then incubated with 1 ml of Salmonella enteritidis in 100 ml of LB medium per 20 ml of each sample for incubation with Salmonella enteritidis at 37 ° C and 180 rpm For 18 hours or more, and centrifuged at 8000 rpm for 15 minutes to recover the supernatant. The recovered supernatant was filtered using a filter of 0.2 탆 which could only be filtered with bacteriophage to recover the bacteriophage solution inactivated. The filtrate thus obtained was analyzed for the presence of the bacteriophage by means of a flotation analysis. A photograph of the result of the analysis of the flask was shown in Fig. The soft agar overlay method was performed by adding the filtrate and Salmonella culture solution to 5 ml of LB soft agar 0.6% agar (w / v) in LB agar at a ratio of 1: 1. The bacteriophage confirmed the plaque on which the bacteria were lysed, and the plaque selected with the sterilized tip was reacted with Salmonella enteritidis again to perform a pure culture. In order to obtain a large amount of isolated bacteriophages, 5 ml of SM solution was poured into plaque-rich agar and lysate was performed at 100 rpm at room temperature for 3 hours. Then, 1 ml of salmonella cultured in LB medium preheated at 60 ° C for 1 hour Lysate solution was added and incubated at 37 ° C for 5 hours. This was filtered with a 0.2 μm pore size filter and stored in a refrigerator.

≪ Example 2 >

Comparison of the effect of Salmonella enteritidis on the isolation of bacteriophages

CAU130028, CAU130051 and CAU130049 bacteriophage samples were obtained from kimchi through the method described above. Salmonella enteritidis was cultivated at 37 ° C for 18 hours and then separated into MOI (Ministry of Information) of 10, 100, 1000, 10000 to confirm the killing effect of Salmonella enteritidis-specific bacteriophage. Bacteriophage was treated. After incubation at 37 ° C, samples were taken at 0, 10, 20, 40, and 60 minutes for each time period, and cultured at 37 ° C for 18 hours. As shown in FIG. 2, CAU130028 showed the highest Salmonella enteritidis killing effect, and CAU130051 and CAU130049 showed the next highest enteritis effect.

≪ Example 3 >

Identification of the virulence factor of isolated bacteriophages

Genetic analysis was conducted to determine whether the bacteriophage isolated from salmonella contaminated with food contained Salmonella pathogenicity factors. First, we isolated the Salmonella gene and identified the pathogenic factors shown in Table 1. The nucleotide sequences of the pathogenic factors mentioned in the present invention are given in various references. The results are shown in Table 2. In CAU130028 bacteriophage of the present invention, avrA and sodC1, only two adult patients were confirmed. On the other hand, in the CAU130049 and CAU130051 bacteriophages, eight hospitalizers were identified. Among the bacteriophages isolated from the same kimchi, the isolates contained few pathogenic factors and had a high mortality of Salmonella enteritidis.

Identification of pathogenic factors of Salmonella and primer used in the present invention Target gene Gene function Primer Tm (° C) Product  size ( bp ) order
number avrA SPI 1 encoded protein, inhibits the key proinflammatory, antiapoptotic NF-kappa B pathway F5'-CCTGTATTGTTGAGCGTCTGG-3
R5'-AGAAGAGCTTCGTTGAATGTCC-3 ' 58 422 One 2 gipA Gifsy-1 peyer's patch-specific virulence factor GipA F5'-ACGACTGAGCAGGCTGAG-3 '
R5'-TTGGAAATGGTGACGGTAGAC-3 ' 422 3 4 mgtC SPI 3 encoded putative transcriptional regulator, required for intramacrophage survival and growth in low MG2 + F5'-TGACTATCAATGCTCCAGTGAAT-3 '
R5'-ATTTACTGGCCGCTATGCTGTTG-3 ' 656 5 6 siiD SPI 4 encodes with SiiC SiiD and SiiF membrane pore for SiiE, T1 SS F5'-GAATAGAAGACAAAGCGATCATC-3 '
R5'-GCTTTGTCCACGCCTTTCACT-3 ' 1243 7 8 sodC1 Gifsy-2 prophage encoded, copper / zinc superoxide dismutase F5'-CCAGTGGAGCAGGTTTATCG-3 '
R5'-GGTGCGCTCATCAGTTGTTC-3 ' 418 9 10 sopB SPI 5 encoded translocated effector protein F5'-TCAGAAGRCGTCTAACCACTC-3 '
R5'-TACCGTCCTCATGCACACTC-3 ' 518 11 12 sopE1 Translocated effector protein, encoded by P2-like cryptic bacteriophage F5'-CGGGCAGTGTTGACAAATAAAG-3 '
R5'-TGTTGGAATTGCTGTGGAGTC-3 ' 455 13 14 spvC Salmonella plasmid virulence: hydrophilicprotein, providesrapidgrowthandsurvivalwithinthehost F5'-ACTCCTTGCACAACCAAATGCGGA-3 '
R5'-TGTCTTCTGCATTTCGCCACC-3 ' 571 15 16 ssaQ SPI 2 encoded secretion system apparatus protein F5'-GAATAGCGAATGAAGAGCGTCC-3 '
R5'-CATCGTGTTATCCTCTGTCAGC-3 ' 644 17 18 bcfC  Bovine colonizaiton factor, fimrial usher F5'-ACCAGAGACATTGCCTTCC-3 '
R5'-TTCTGATCGCCGCTATTCG-3 ' 467 19 20

VIRULENCE FACTOR result of CAU130028 sample number origin virulence factor avrA ssaQ mgtC siiD sopB gipA sodC1 sopE1 spvC bcfC CAU130028 kimchi + - - - - - + - - - CAU130049 kimchi + + + + + - + + + - CAU130051 kimchi + + + + + - + + + -

As described above, the bacteriophage specific to Salmonella enteritidis isolated from the food source of the present invention is more suitable for application to food than the conventional bacteriophage because it has a high killing power and safety of Salmonella enteritidis, It is highly available.

Korea Biotechnology Research Institute KCTC18321P 20140911

<110> Chung-Ang University Industry-Academy Cooperation Foundation <120> Novel bacteriophage CAU130028 having killing activity specific to          Salmonella Enteritidis <130> NP14-0080 <160> 20 <170> Kopatentin 2.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> avrA forward primer <400> 1 cctgtattgt tgagcgtctg g 21 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> avrA reverse primer <400> 2 agaagagctt cgttgaatgt cc 22 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> gipA forward primer <400> 3 acgactgagc aggctgag 18 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> gipA reverse primer <400> 4 ttggaaatgg tgacggtaga c 21 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> mgtC forward primer <400> 5 tgactatcaa tgctccagtg aat 23 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> mgtC reverse primer <400> 6 atttactggc cgctatgctg ttg 23 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> siiD forward primer <400> 7 gaatagaaga caaagcgatc atc 23 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siiD reverse primer <400> 8 gctttgtcca cgcctttcac t 21 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sodC1 forward primer <400> 9 ccagtggagc aggtttatcg 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sodC1 reverse primer <400> 10 ggtgcgctca tcagttgttc 20 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> sopB forward primer <400> 11 tcagaagrcg tctaaccact c 21 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sopB reverse primer <400> 12 taccgtcctc atgcacactc 20 <210> 13 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> sopE1 forward primer <400> 13 cgggcagtgt tgacaaataa ag 22 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> sopE1 reverse primer <400> 14 tgttggaatt gctgtggagt c 21 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> spvC forward primer <400> 15 actccttgca caaccaaatg cgga 24 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> spvC reverse primer <400> 16 tgtcttctgc atttcgccac c 21 <210> 17 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ssaQ forward primer <400> 17 gaatagcgaa tgaagagcgt cc 22 <210> 18 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ssaQ reverse primer <400> 18 catcgtgtta tcctctgtca gc 22 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> bcfC forward primer <400> 19 accagagaca ttgccttcc 19 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> bcfC reverse primer <400> 20 ttctgatcgc cgctattcg 19

Claims (3)

Bacteriophage CAU130028 identified as Accession No. KCTC 18321P, which has a specific killing activity against Salmonella enteritidis isolated from a food source.
A pharmaceutical composition for the prevention and treatment of Salmonella entericidid diseases including Salmonella enteritidis comprising the bacteriophage of claim 1 as an active ingredient.
[Claim 3] The pharmaceutical composition according to claim 2, wherein the Salmonella enteritidis-inducing disease including Salmonella enteritidis is food poisoning, enteritis, salmonellosis, poultry typhus or Bacteremia.

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