阅读说明：本技术 新型猪链球菌噬菌体str-sup-2及其在抑制猪链球菌细菌增殖方面的用途 (Novel streptococcus suis bacteriophage STR-SUP-2 and application thereof in inhibiting streptococcus suis bacterial proliferation ) 是由 尹圣君 俊苏云 权安升 李恩吉 康桑贤 于 2019-05-31 设计创作，主要内容包括：本发明涉及一种从自然界分离出的长尾噬菌体科噬菌体Str-SUP-2(保藏号：KCTC 13515BP),其特征在于具有杀死猪链球菌的能力,并且具有由SEQ ID NO:1表示的基因组,本发明还涉及一种用于使用包含所述长尾噬菌体科噬菌体Str-SUP-2作为活性成分的组合物来预防和治疗由猪链球菌引起的疾病的方法。(The present invention relates to a bacteriophage Str-SUP-2 of Long-tailed bacteriophages (accession No: KCTC 13515BP) isolated from the natural world, characterized by having the ability to kill Streptococcus suis and having a genome represented by SEQ ID NO:1, and a method for preventing and treating diseases caused by Streptococcus suis using a composition comprising the bacteriophage Str-SUP-2 of Long-tailed bacteriophages as an active ingredient.)

1. A bacteriophage Str-SUP-2 (accession No: KCTC 13515BP) of the family Long-tailed bacteriophage, isolated from nature, having the ability to kill Streptococcus suis and having a genome represented by SEQ ID NO: 1.

2. A composition for preventing and treating diseases caused by Streptococcus suis, comprising the bacteriophage Str-SUP-2 (deposition No: KCTC 13515BP) according to claim 1 as an active ingredient.

3. The composition of claim 2, wherein the composition is used to prepare a feed supplement, a drinking water supplement, or a disinfectant.

4. A method for preventing and treating diseases caused by streptococcus suis, comprising:

the composition according to claim 2 comprising the bacteriophage Str-SUP-2 (deposition No: KCTC 13515BP) as an active ingredient is sprayed into the environment.

5. The method of claim 4, wherein the composition is in the form of a disinfectant.

6. A method for preventing and treating diseases caused by streptococcus suis, comprising:

the composition comprising the bacteriophage Str-SUP-2 (deposition number: KCTC 13515BP) as an active ingredient according to claim 2 is administered to an animal other than human.

7. The method of claim 6, wherein the composition is in the form of a drinking water supplement or a feed supplement.

Technical Field

The present invention relates to a bacteriophage isolated from the natural world, which infects streptococcus suis, thereby killing streptococcus suis, and a method for preventing and treating diseases caused by streptococcus suis using a composition comprising the aforementioned bacteriophage as an active ingredient. More particularly, the present invention relates to a bacteriophage Str-SUP-2 of the family Long-tailed bacteriophages isolated from the natural world (accession No: KCTC 13515BP) having the ability to kill Streptococcus suis and having the genome represented by SEQ ID NO:1, and a method for preventing or treating diseases caused by Streptococcus suis using a composition comprising the aforementioned bacteriophage as an active ingredient.

Background

Streptococcus suis is a peanut-shaped gram-positive bacterium, and streptococcus suis infection is known to be a significant animal-derived disease that occurs worldwide. Streptococcus suis bacteria are classified into 29 serotypes according to Capsular antigen (capsula, K). Based on the serotype report of streptococcus suis bacteria worldwide, serotypes 1 to 9 are widely distributed, accounting for about 75% of their total number, and serotype 2 is known to be the most common serotype isolated from pathogenic swine bodies in most countries.

Meanwhile, swine infected with streptococcus suis mainly show symptoms of anorexia, lethargy, eruption, fever and paralysis. In particular, respiratory tract infections such as pneumonia and the like may occur in fattening pigs, thereby causing serious economic loss to the pig breeding industry. In addition, streptococcus suis is a known major pathogen causing swine to develop meningitis, septicemia, arthritis, endocarditis, and vaginitis, and outbreaks of streptococcus suis have been reported worldwide (including korea, north america, europe, and the like). Therefore, there is an urgent need to develop methods for preventing and treating Streptococcus suis infection.

Although various antibiotics have been used for the prevention or treatment of diseases caused by streptococcus suis, currently, the resistance of bacteria to such antibiotics is increasing, and thus, there is an urgent need to develop methods other than antibiotics.

Recently, the use of bacteriophages as a countermeasure against infectious bacterial diseases has attracted much attention. In particular, these bacteriophages have received great attention due to their strong antibacterial activity against antibiotic-resistant bacteria. Bacteriophages are very small microorganisms that infect bacteria. Once a bacteriophage infects a bacterium, the bacteriophage will proliferate within the bacterial cell. After propagation, the progeny of the phage will break the bacterial cell wall and escape from the host bacteria, indicating that the phage has the ability to kill bacteria. The way in which the bacteriophage infects bacteria is characterized by its very high specificity, and therefore, the range of types of bacteriophage infecting a particular bacterium is limited. That is, a certain bacteriophage can only infect a certain specific bacterium, which means that a certain bacteriophage can only exert an antibacterial effect against a certain specific bacterium. Due to this bacterial specificity of bacteriophages, bacteriophages have an antibacterial effect only on the target bacteria, without affecting the symbiotic bacteria in the environment or in the animal body. Conventional antibiotics, which have been widely used for bacterial therapy, can affect many other kinds of bacteria, which causes problems such as environmental pollution and interference with normal flora in animals. In contrast, the use of bacteriophage does not interfere with the normal flora in the animal because the target bacteria are selectively killed by the use of bacteriophage. Thus, the phage can be safely utilized, thus greatly reducing the possibility of adverse effects from the use of phage compared to antibiotics.

The phage was originally discovered by the british bacteriologist Twort in 1915, when he noticed that micrococcus colonies were softened and became transparent by something unknown. In 1917, a french bacteriologist d' Herelle found that shigella dysenteriae in stool filtrate of a patient with dysentery was destroyed by something, and further studied the phenomenon. As a result he independently identified phages and named them "phages", meaning "bacterial phagocytes". Since then, phages acting on pathogenic bacteria such as Shigella, Streptococcus typhi and Vibrio cholerae have been continuously identified.

Because of the unique ability of bacteriophages to kill bacteria, bacteriophages have since been discovered and have received much attention as an effective strategy to address bacterial infections. However, since the discovery of penicillins by fleming, the study of bacteriophages has only continued in some eastern european countries and the former soviet union due to the widespread dissemination of antibiotics. Since 2000, the limitations of conventional antibiotics have become apparent due to the increase of antibiotic-resistant bacteria, the possibility of developing bacteriophages as alternatives to conventional antibiotics has been highlighted, and thus bacteriophages as antibacterial agents have attracted attention again.

As mentioned above, bacteriophages tend to be highly specific for the target bacteria. Because of the high specificity of bacteriophages for bacteria, bacteriophages usually exhibit an antibacterial effect only on certain bacterial strains, even within the same species. In addition, the antibacterial strength of the bacteriophage varies depending on the target strain. Therefore, in order to effectively control a specific bacterium, it is necessary to collect a variety of useful phages. Therefore, in order to develop an effective phage utilization method for controlling Streptococcus suis, it is necessary to obtain a plurality of phages that exhibit antibacterial effects against Streptococcus suis. In addition, it is necessary to screen out phages superior to other phages in terms of antibacterial intensity and spectrum from the obtained phages.

Disclosure of Invention

Technical problem

Accordingly, the present inventors have endeavored to develop a composition suitable for preventing and treating diseases caused by Streptococcus suis using bacteriophage that is isolated from the natural world and can kill Streptococcus suis, and further endeavored to establish a method for preventing and treating diseases caused by Streptococcus suis using the composition. As a result, the present inventors isolated a phage suitable for this purpose from nature, and determined a genomic sequence that distinguishes the isolated phage from other phages. Thereafter, the present inventors developed a composition comprising bacteriophage as an active ingredient, and determined that the composition can be effectively used for the prevention and treatment of diseases caused by streptococcus suis, thereby forming the present invention.

Accordingly, it is an object of the present invention to provide a bacteriophage Str-SUP-2 of the family Long-tailed bacteriophages isolated from nature (accession No: KCTC 13515BP) having the ability to kill Streptococcus suis and having the genome represented by SEQ ID NO: 1.

It is another object of the present invention to provide a composition suitable for preventing or treating diseases caused by Streptococcus suis, the composition comprising, as an active ingredient, an isolated bacteriophage Str-SUP-2 (accession No: KCTC 13515BP), the isolated bacteriophage Str-SUP-2 infecting Streptococcus suis, thereby killing the Streptococcus suis.

It is still another object of the present invention to provide a method for preventing and treating diseases caused by Streptococcus suis using a composition suitable for preventing and treating diseases caused by Streptococcus suis, the composition comprising, as an active ingredient, an isolated bacteriophage Str-SUP-2 (accession No: KCTC 13515BP), the isolated bacteriophage Str-SUP-2 infecting Streptococcus suis, thereby killing Streptococcus suis.

It is still another object of the present invention to provide a disinfectant for preventing and treating diseases caused by streptococcus suis using the composition.

It is still another object of the present invention to provide a drinking water additive that plays a role in cultivation management by preventing and treating diseases caused by streptococcus suis using the composition.

It is still another object of the present invention to provide a feed additive effective for cultivation management by preventing and treating diseases caused by streptococcus suis using the composition.

Technical solution

The present invention provides a bacteriophage Str-SUP-2 (accession No: KCTC 13515BP) of the family Long-tailed bacteriophages isolated from the nature, having the ability to specifically kill Streptococcus suis and having the genome represented by SEQ ID NO:1, and a method for preventing and treating diseases caused by Streptococcus suis using a composition comprising the bacteriophage as an active ingredient.

The present inventors isolated the bacteriophage Str-SUP-2 and then deposited it at the Korean type culture Collection of the Korean institute for bioscience and biotechnology (accession No: KCTC 13515BP) 24.4.2018.

In addition, the present invention provides a disinfectant, a drinking water additive and a feed additive suitable for the prevention and treatment of diseases caused by Streptococcus suis, which comprise the bacteriophage Str-SUP-2 as an active ingredient.

Since the bacteriophage Str-SUP-2 comprised by the composition of the present invention is effective in killing Streptococcus suis, it is effective in preventing (preventing infection) or treating (treating infection) diseases caused by Streptococcus suis. Therefore, the composition of the present invention can be used for the prevention and treatment of diseases caused by streptococcus suis.

The term "prevention" as used herein refers to (i) prevention of infection by Streptococcus suis and (ii) inhibition of the development of disease caused by Streptococcus suis infection.

The term "treatment" as used herein refers to (i) inhibition of a disease caused by streptococcus suis and (ii) alleviation of the pathological condition of a disease caused by streptococcus suis.

The term "isolation" as used herein refers to an act of isolating a bacteriophage from the natural world by using various experimental techniques and taking a characteristic that can distinguish the bacteriophage of the present invention from other bacteriophages, and further includes an act of propagating the bacteriophage of the present invention using a biotechnology to make the bacteriophage industrially applicable.

The pharmaceutically acceptable carrier included in the composition of the present invention is a carrier generally used for preparing pharmaceutical preparations, and examples thereof include: lactose, glucose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil, but are not limited thereto. In addition to the above ingredients, the compositions of the present invention may also include lubricating agents, wetting agents, sweetening agents, flavoring agents, emulsifying agents, suspending agents and preserving agents.

The composition of the present invention comprises the bacteriophage Str-SUP-2 as an active ingredient. At 1 × 10¹pfu/ml to 1X10³⁰pfu/ml or 1X10¹pfu/g to 1X10³⁰Concentration of pfu/g, preferably at 1X10⁴pfu/ml to 1X10¹⁵pfu/ml or 1X10⁴pfu/g to 1X10¹⁵The concentration of pfu/g comprises the bacteriophage Str-SUP-2.

The compositions of the present invention may be formulated according to methods that can be readily practiced by those skilled in the art using pharmaceutically acceptable carriers and/or excipients to prepare the compositions in unit dosage form or to add the compositions to multi-dose containers. Here, the formulation may be provided in the form of a solution, a suspension, or an emulsion in an oil or aqueous medium, or in the form of an extract, a powder, a granule, a tablet, or a capsule, and may further contain a dispersing agent or a stabilizer.

The composition of the present invention may be prepared as a disinfectant or a drinking water additive or a feed additive according to its purpose of use, but is not limited thereto. To enhance its effectiveness, phages having antibacterial activity against other bacterial species may also be included in the compositions of the invention. In addition, other types of bacteriophages having antibacterial activity against Streptococcus suis may also be included in the compositions of the invention. These phages may be combined appropriately to maximize the antibacterial effect of the composition, since the respective antibacterial activity of these phages against s.

Advantageous effects

According to the present invention, the method for preventing and treating diseases caused by Streptococcus suis using a composition comprising the bacteriophage Str-SUP-2 as an effective ingredient has the advantage of having very high specificity to Streptococcus suis, compared to conventional methods based on existing antibiotics. This means that the composition can be used for the prevention and treatment of diseases caused by streptococcus suis without affecting other useful commensal bacteria and with less side effects due to its use. Generally, when antibiotics are used, symbiotic bacteria are also damaged, eventually reducing the immunity of animals, and thus, the use of antibiotics causes various side effects. Meanwhile, for each bacteriophage showing antibacterial activity against the same bacterial species, the antibacterial activity of the bacteriophage differs depending on the bacterial strain even for the same bacterial species because of the antibacterial intensity or antibacterial spectrum [ i.e., the antibacterial activity spectrum of the bacteriophage applied to individual bacterial strains, in the case of each bacterial strain belonging to streptococcus suis, the bacteriophage generally acts only on some bacterial strains even within the same species. Therefore, the present invention can provide an antibacterial activity against Streptococcus suis that is different from the antibacterial activity of other phages acting on Streptococcus suis, which will make a great difference in effectiveness when it is applied to the industrial field.

Drawings

FIG. 1 is an electron micrograph showing the morphology of bacteriophage Str-SUP-2.

FIG. 2 is a schematic diagram showing the difference in gene characteristics by comparing the genomic sequence of the bacteriophage Str-SUP-2 with the genomic sequence of the streptococcal bacteriophage phi5218, which has a relatively high genomic sequence homology thereto.

FIG. 3 is a photograph showing the results of an experiment regarding the ability of the bacteriophage Str-SUP-2 to kill Streptococcus suis. Based on the centerline of the plate medium, only the buffer containing no phage Str-SUP-2 was spotted on the left side of the plate medium, while the solution containing phage Str-SUP-2 was spotted on the right side of the plate medium. The clean zone observed on the right side is a plaque formed by lysis of the target bacteria due to the action of bacteriophage Str-SUP-2.

Detailed Description

The present invention will be better understood by the following examples, which are intended to illustrate the invention and should not be construed as limiting the scope of the invention.

Example 1: isolation of phage capable of killing Streptococcus suis

A sample collected from nature is used to isolate phages capable of killing streptococcus suis. Here, the Streptococcus suis strain for phage isolation was obtained from the Korean type culture Collection (accession No: KCTC 3557).

The process of isolating the phage is described in detail below. The collected sample was added to THB (Todd Hewitt broth) medium (cardiac infusion, 3.1 g/L; protein, 20 g/L; glucose, 2 g/L; sodium chloride, 2 g/L; disodium phosphate, 0.4 g/L; sodium carbonate, 2.5g/L) inoculated with Streptococcus suis at a rate of 1/1,000, followed by shaking culture at 37 ℃ for 3 to 4 hours. After completion of the culture, centrifugation was performed at 8,000rpm for 20 minutes, and the supernatant was recovered. The recovered supernatant was inoculated with Streptococcus suis at a rate of 1/1000, and then cultured with shaking at 37 ℃ for 3 to 4 hours. When the phage was included in the sample, the above process was repeated 5 times in total to sufficiently increase the number of phage (titer). After repeating this process 5 times, the culture broth was centrifuged at 8,000rpm for 20 minutes. After centrifugation, the recovered supernatant was filtered using a 0.45 μm filter. The filtrate thus obtained was used in a typical spot assay to assess whether phages capable of killing streptococcus suis were included therein.

The spot measurement was performed as follows. THB medium inoculated with Streptococcus suis at a rate of 1/1,000 was then cultured overnight at 37 ℃ with shaking. 3ml (OD600 of 1.5) of the Streptococcus suis culture prepared as described above were spread on a THA (Todd Hewitt agar: cardiac infusion, 3.1 g/L; protein, 20 g/L; glucose, 2 g/L; sodium chloride, 2 g/L; disodium phosphate, 0.4 g/L; sodium carbonate, 2.5 g/L; agar, 15g/L) plate. The plate was placed on a clean bench for approximately 30 minutes to dry the dispersed solution. After drying, 10. mu.l of the filtrate prepared as described above was spotted on a plate on which Streptococcus suis was scattered, and then left to dry for about 30 minutes. After drying, the spotted plate was incubated at 37 ℃ for 1 day with standing, and then examined for the formation of a clear zone at the position where the filtrate was dropped. If the filtrate produced a clean zone, it was judged that phages capable of killing streptococcus suis were included. By the above examination, a filtrate containing phages having the ability to kill streptococcus suis can be obtained.

Pure phage were isolated from the filtrate that was confirmed to have phage capable of killing streptococcus suis. A typical plaque assay was used to isolate pure phage. Specifically, the plaque formed during plaque assay was recovered using a sterile pipette tip, added to the streptococcus suis culture broth, and then cultured at 37 ℃ for 4 to 5 hours. Thereafter, centrifugation was performed at 8,000rpm for 20 minutes to obtain a supernatant. To the obtained supernatant was added a culture broth of Streptococcus suis at a volume ratio of 1/50, and then, the culture was carried out at 37 ℃ for 4 to 5 hours. To increase the number of phages, the above procedure was repeated at least 5 times, after which centrifugation was carried out at 8,000rpm for 20 minutes to obtain the final supernatant. The final supernatant thus obtained was used to perform plaque assay again. Generally, when the above process is performed once, the isolation of pure phage is not completed, and thus the process is repeated using the plaque formed as described above. After at least 5 repetitions, a solution containing pure phage was obtained. The process of isolating pure phage is repeated until the plaques produced become approximately similar to each other in both size and morphology. Additionally, electron microscopy was used to confirm the final isolation of pure phage. The above procedure was repeated until the isolation of pure phage was confirmed by electron microscopy. Electron microscopy was performed in a typical manner. Briefly, a solution containing pure phage was loaded onto a copper mesh, then negatively stained with 2% uranyl acetate and dried. Then, the morphology thereof was observed using a transmission electron microscope. An electron micrograph of the isolated pure phage is shown in figure 1. Based on its morphological characteristics, it was confirmed that the novel phages isolated as above belong to phages of the family of Long-tailed bacteriophages.

The solution containing the pure phage confirmed as above was subjected to the following purification treatment. To the solution containing the pure phage, a streptococcus suis culture broth was added in a volume ratio of 1/50 based on the total volume of the phage solution, and then, cultured for an additional 4 to 5 hours. Thereafter, centrifugation was performed at 8,000rpm for 20 minutes to obtain a supernatant. This process was repeated 5 times in total to obtain a solution containing a sufficient number of phages. The supernatant from the last centrifugation was filtered using a 0.45 μm filter and then subjected to a typical polyethylene glycol (PEG) precipitation treatment. Specifically, to 100ml of the filtrate was added PEG and NaCl to reach 10% PEG 8000 and 0.5M NaCl, and then it was left to stand at 4 ℃ for 2 to 3 hours. Thereafter, centrifugation was performed at 8,000rpm for 30 minutes to obtain a phage precipitate. The resulting phage pellet was suspended in 5ml of buffer (10mM Tris-HCl, 10mM MgSO4, 0.1% gelatin, pH 8.0). The resulting material may be referred to as a phage suspension or phage solution.

The purified phages as described above were collected, designated as phage Str-SUP-2, and then deposited at the Korean type culture Collection of the Korean institute of bioscience and biotechnology (accession No: KCTC 13515BP) on 24/4.2018.

Example 2: isolation and sequence analysis of the genome of the bacteriophage Str-SUP-2

The genome of the phage Str-SUP-2 was isolated as follows. The genome was isolated from the phage suspension obtained using the same method as described in example 1. First, in order to remove DNA and RNA of Streptococcus suis included in the suspension, 200U of DNase I and 200U of RNase A were added to 10ml of phage suspension, which was then allowed to stand at 37 ℃ for 30 minutes. After leaving it for 30 minutes, 500. mu.l of 0.5M ethylenediaminetetraacetic acid (EDTA) was added thereto in order to inactivate the activities of DNase I and RNase A, and the resulting mixture was left to stand for 10 minutes. Further, the resulting mixture was allowed to stand at 65 ℃ for another 10 minutes, and then 100. mu.l of proteinase K (20mg/ml) was added thereto to destroy the outer wall of the phage, followed by reaction at 37 ℃ for 20 minutes. Thereafter, 500. mu.l of 10% Sodium Dodecyl Sulfate (SDS) was added thereto, followed by reaction at 65 ℃ for 1 hour. After the reaction was allowed to occur for 1 hour, 10ml of a mixed solution of phenol, chloroform and isoamyl alcohol at a composition ratio of 25: 24: 1 was added to the resulting reaction solution, which was then thoroughly mixed. The resulting mixture was then centrifuged at 13,000rpm for 15 minutes to separate the layers of the mixture. Among the separated layers, the upper layer was selected, to which isopropanol was added at a volume ratio of 1.5, and which was centrifuged at 13,000rpm for 10 minutes to precipitate the genome. After collecting the precipitate, 70% ethanol was added to the precipitate, and it was centrifuged at 13,000rpm for 10 minutes to wash the precipitate. The washed precipitate was recovered, dried under vacuum and then dissolved in 100. mu.l of water. This process is repeated to obtain a sufficient amount of genome of the bacteriophage Str-SUP-2.

The next generation sequencing analysis was performed using the Illumina Mi-Seq sequencer supplied by Macrogen to obtain sequence information on the thus obtained genome of the bacteriophage Str-SUP-2. The final analyzed genome size of the phage Str-SUP-2 was 32,673bp, and the entire genome sequence was represented by SEQ ID NO: 1.

The homology (similarity) of the phage Str-SUP-2 genomic sequence obtained as above with previously reported phage genomic sequences was investigated using BLAST on the web. Based on the results of the BLAST survey, the genomic sequence of bacteriophage Str-SUP-2 was found to have relatively high homology with the sequence of Streptococcus phage phi5218(GenBank accession No.: KC 348600.1). However, the bacteriophage Str-SUP-2 has the morphological characteristics of the long-tailed family of bacteriophages, while the streptococcal bacteriophage phi5218 has the morphological characteristics of the short-tailed family of bacteriophages, with obvious morphological differences between them. Furthermore, the number of Open Reading Frames (ORFs) of the phage Str-SUP-2 genome was 59, while we found that streptococcal phage phi5218 had 64 open reading frames, based on which these two phages were also assessed to be genetically different. The differences in morphological and genetic characteristics between the two phages may indicate that there are external and functional differences in the various characteristics expressed in various ways between the two phages. Moreover, the difference between these two phages also suggests a difference in the industrial applicability of the two phages. Meanwhile, the difference in gene characteristics observed by comparing the genome sequences of the two phages is schematically shown in fig. 2.

Therefore, the following conclusions can be drawn: the bacteriophage Str-SUP-2 is a novel bacteriophage different from previously reported bacteriophages. Further, since the antibacterial intensity and antibacterial spectrum of the bacteriophage generally vary according to the type of the bacteriophage, it is considered that the bacteriophage Str-SUP-2 may provide an antibacterial activity different from that of any other previously reported bacteriophage.

Example 3: evaluation of the killing Capacity of bacteriophage Str-SUP-2 against Streptococcus suis

The isolated phage Str-SUP-2 was evaluated for its ability to kill Streptococcus suis. To evaluate its killing power, the formation of denuded zones was observed using a spot assay in the same manner as described in example 1. A total of 10 strains of streptococcus suis, which were isolated by the inventors of the present invention and identified, or a total of 10 strains obtained from KCTC or the korean veterinary culture collection, were used as streptococcus suis strains for evaluation of killing ability. Phage Str-SUP-2 has the ability to kill 8 strains (including KCTC 3557) out of 10 Streptococcus suis strains in total, reaching experimental goals. Representative experimental results are shown in fig. 3. At the same time, the ability of the bacteriophage Str-SUP-2 to kill Bordetella bronchiseptica, enterococcus faecalis, enterococcus faecium, Streptococcus mitis, Streptococcus uberis and Pseudomonas aeruginosa was examined. As a result, the bacteriophage Str-SUP-2 does not have the ability to kill these microorganisms.

Therefore, the following conclusions can be drawn: the bacteriophage Str-SUP-2 has a strong ability to kill Streptococcus suis and exhibits antibacterial effects against many strains of Streptococcus suis, indicating that the bacteriophage Str-SUP-2 can be used as an active ingredient of a composition for preventing and treating diseases caused by Streptococcus suis.

Example 4: experiment for prevention of Streptococcus suis infection Using bacteriophage Str-SUP-2

To a test tube containing 9ml of THB medium was added 100. mu.l of a 1X10 concentration⁸pfu/ml phage Str-SUP-2 solution. To another tube containing 9ml of THB medium was added only the same amount of THB medium. Then, to each test tube, a culture broth of Streptococcus suis was added so that the absorbance at 600nm reached approximately 0.5. After addition of Streptococcus suis, the tubes were transferred to an incubator at 37 ℃ and then subjected to shaking culture, during which the growth state of Streptococcus suis was observed. As shown in table 1 below, it was observed: growth of Streptococcus suis was inhibited in tubes to which phage Str-SUP-2 solution was added, while growth of Streptococcus suis was not inhibited in tubes to which phage solution was not added.

[ TABLE 1 ]

Growth inhibition of Streptococcus suis

The above results show that: the bacteriophage Str-SUP-2 of the present invention not only inhibits the growth of Streptococcus suis, but also has the ability to kill Streptococcus suis. Therefore, the following conclusions are drawn: the bacteriophage Str-SUP-2 can be used as an active ingredient in a composition for preventing diseases caused by Streptococcus suis.

Example 5: animal test for preventing diseases caused by Streptococcus suis Using bacteriophage Str-SUP-2

The prophylactic effect of bacteriophage Str-SUP-2 on diseases caused by Streptococcus suis was evaluated using weaned piglets. Ten 25-day-old weaned piglets were divided into 2 groups (5 pigs per group) and fed separately in a laboratory pig house (1.1m × 1.0m) for 14-day experiments. The ambient environment was controlled using a heater and the temperature and humidity in the swine room were kept constant and the floor of the swine room was washed daily. Pigs in the experimental group (given the feed containing the phage) were provided with a feed containing 1X10 in a typical feeding manner from the start to the end of the experiment⁸pfu/g phage Str-SUP-2 feed. For comparison, pigs in the control group (given feed not containing the phage) were provided with feed having the same composition but not containing phage Str-SUP-2 in the same feeding manner from the start of the experiment to the end of the experiment. The feed was supplemented with 1X10 of a feed for 2 days from day 7 after the start of the experiment⁸cfu/g of Streptococcus suis, feed was provided twice daily to all pigs in the experimental group (given feed containing the phage) and the control group (given feed not containing the phage), resulting in infection of Streptococcus suis. From the date of feeding with the feed containing streptococcus suis (from day 7 after the start of the experiment), the levels of streptococcus suis detected in nasal secretions of all the test animals were examined daily.

Detection of streptococcus suis in nasal secretions (nasal swabs) was performed as follows. Nasal secretion samples were spread on blood agar plates and then incubated at 37 ℃ for 18 to 24 hours. Among the colonies obtained, a colony presumed to be Streptococcus suis was isolated. The thus selected colonies were used as samples, and subjected to a streptococcus suis-specific Polymerase Chain Reaction (PCR), thereby finally confirming whether the corresponding colonies were streptococcus suis. The results of the bacterial detection are shown in table 2 below.

[ TABLE 2 ]

Test results (average value) for Streptococcus suis

From the above results, it was confirmed that the bacteriophage Str-SUP-2 of the present invention is very effective in preventing diseases caused by Streptococcus suis.

Example 6: treatment of diseases caused by Streptococcus suis with bacteriophage Str-SUP-2

The therapeutic effect of the phage Str-SUP-2 on diseases caused by Streptococcus suis was evaluated as follows. Eight 25-day-old weaned piglets were divided into 2 groups in total and fed separately in a laboratory pig house (1.1m × 1.0m) for 14-day experiments. The ambient environment was controlled using a heater to keep the temperature and humidity in the swine room constant, and the floor of the swine room was washed daily. From day 4 after the start of the experiment, 5ml of a Streptococcus suis solution (10)⁹cfu/ml) were sprayed into the nasal cavity of all pigs. A solution of streptococcus suis for nasal administration was prepared as follows. After culturing Streptococcus suis bacteria at 37 ℃ for 18 hours using THB medium, the cells thereof were separated and then suspended in physiological saline (pH 7.2) to adjust the cell concentration to 10⁹cfu/ml. From the day after forced infection with Streptococcus suis, pigs in the experimental group (group given the phage solution) were administered twice a day 10 times in the same manner as the administration of the Streptococcus suis solution⁹Intranasal administration of pfu phage Str-SUP-2. The pigs in the control group (group not administered with the phage solution) were not treated at all. The control group and the experimental group were provided with feed and drinking water in the same manner. All the test animals were examined for the development of atrophic rhinitis caused by Streptococcus suis bacteria from day 3 after forced infection with Streptococcus suis (from day 7 after the start of the experiment). Regulation of atrophic rhinitis by Streptococcus suis bacteria by measuring the amount of nasal secretionsAnd (6) checking. According to the observation of the experimenter, the amount of nasal secretions was indicated by labeling the normal level as "0", the slightly higher level as "1" and the severe level as "2". The results are shown in table 3 below.

[ TABLE 3 ]

Results of nasal secretion investigation (mean)

As can be seen from the above results, it was confirmed that the bacteriophage Str-SUP-2 of the present invention is very effective in treating diseases caused by Streptococcus suis.

Example 7: preparation of feed additive and feed

Using bacteriophage Str-SUP-2 solution to prepare feed additive, each gram of feed additive contains 1 × 10 amount⁸pfu phage Str-SUP-2. The feed additive was prepared in the following manner: maltodextrin (50%, w/v) was added to the phage solution, which was then lyophilized and finally pulverized into a fine powder. In the above preparation process, the drying treatment may be replaced by drying under reduced pressure, drying by heating or drying at room temperature. To prepare a control for comparison, the phage solution was not used, but the buffer (10mM Tris-HCl, 10mM MgSO4, 0.1% gelatin, pH 8.0) used to prepare the phage solution was used to prepare a feed additive that did not contain the phage.

The thus prepared two feed additives were mixed with the pig feed at a weight ratio of 1,000, respectively, to finally prepare two feeds.

Example 8: additive and disinfectant for preparing drinking water

Drinking water additives and disinfectants are prepared in the same manner because they differ only in use but are identical in formulation. Using bacteriophage Str-SUP-2 solutionPreparing the drinking water additive (or disinfectant). In a method for preparing a drinking water additive (or disinfectant), a bacteriophage Str-SUP-2 solution is added such that the bacteriophage solution comprises an amount of 1 × 10 per ml of buffer used to prepare the bacteriophage solution⁹pfu phage Str-SUP-2, and thoroughly mixed. To prepare a control for comparison, the buffer used to prepare the phage solution was used as is as a drinking water additive (or disinfectant) that did not contain the phage.

The thus prepared two drinking water additives (or disinfectants) were diluted with water at a volume ratio of 1,000 to obtain final drinking water or disinfectants.

Example 9: confirmation of raising effect of pig raising

Whether the feed, drinking water and disinfectant prepared in examples 7 and 8 were effective for pig breeding was evaluated. In particular, the present assessment places emphasis on measuring the degree of weight gain. A total of sixty 25-day-old weaned piglets were divided into three groups, each comprising 20 pigs (group a: fed with feed, group B: fed with drinking water, group C: treated with disinfectant), and subjected to four-week experiment. Each group was subdivided into subgroups each including 10 pigs, and the subgroups were classified into a subgroup to which phage Str-SUP-2 was applied (subgroup-r) and a subgroup to which no phage was applied (subgroup-r). In this experiment, weaned piglets were fed separately in separate subgroups. The subgroups are sorted and named as shown in table 4 below.

[ TABLE 4 ]

Subgroup classification and expression in pig breeding experiments

In terms of providing the feed, as shown in table 4, the feed prepared in example 7 was provided in a typical feeding manner, and as shown in table 4, the drinking water prepared in example 8 was provided in a typical feeding manner. In terms of disinfection, disinfection is alternated with regular disinfection, 3 times per week. The sterilization using a typical disinfectant is not performed on the day of spraying the disinfectant of the present invention. Based on the experimental results, the degree of weight gain was significantly better in the group to which the bacteriophage Str-SUP-2 was added, compared to the group to which the bacteriophage Str-SUP-2 was not added (Table 5). For reference, the isolation rate of streptococcus suis bacteria in nasal secretions of test animals was also investigated as shown in example 5. Streptococcus suis bacteria were detected in nasal secretions of some animals in the group not administered bacteriophage Str-SUP-2. On the other hand, in all animals in the group to which the bacteriophage Str-SUP-2 was administered, no Streptococcus suis bacteria were detected during the experimental period.

[ TABLE 5 ]

Results of pig breeding experiments

The above results show that feeding with the feed and drinking water prepared according to the present invention and the use of the disinfectant according to the present invention are effective for pig breeding. Thus, it can be concluded that the composition of the invention is effective when used for feeding pigs.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those skilled in the art that the detailed description is of preferred embodiments only, and that the scope of the present invention is not limited thereto. Accordingly, the scope of the invention should be determined by the appended claims and their equivalents.

[ deposit No. ]

The name of the depository institution: KCTC

The preservation number is as follows: KCTC 13515BP

The preservation date is as follows: 20180424