Vibrio parahaemolyticus bacteriophage Vib-PAP-7 and use of same for inhibiting vibrio parahaemolyticus bacteria proliferation

Abstract

The present invention relates to a Myoviridae bacteriophage Vib-PAP-7 (Accession number: KCTC 13247BP) isolated from nature, which has the ability to kill Vibrio parahaemolyticus and has the genome represented by SEQ ID NO: 1, and a method for preventing or treating a disease caused by Vibrio parahaemolyticus using a composition containing the Myoviridae bacteriophage Vib-PAP-7 as an active ingredient.

Claims

1. A composition for blocking a Vibrio parahaemolyticus infection, inhibiting the development of diseases caused by a Vibrio parahaemolyticus infection, suppressing diseases caused by Vibrio parahaemolyticus, or alleviating the pathological condition of the diseases caused by Vibrio parahaemolyticus, comprising: maltodextrin and 1×10.sup.4 plaque-forming units per gram (pfu/g) to 1×10.sup.15 pfu/g of proliferated and purified Myoviridae bacteriophage Vib-PAP-7, which has an ability to specifically kill Vibrio parahaemolyticus and a genome represented by SEQ ID NO: 1, and is deposited in the Korean Collection for Type Cultures (KCTC) under accession number KCTC 13247BP, wherein the maltodextrin and Myoviridae bacteriophage Vib-PAP-7 are in a dried mixture.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 is an electron micrograph showing the morphology of the bacteriophage Vib-PAP-7.

(2) FIG. 2 is a photograph showing results of experiment of the ability of the bacteriophage Vib-PAP-7 to kill Vibrio parahaemolyticus. Based on the center line of the plate culture medium, only the buffer containing no bacteriophage Vib-PAP-7 is spotted on the left side thereof and a solution containing the bacteriophage Vib-PAP-7 is spotted on the right side thereof. The clear zone observed on the right side is a plaque formed by lysis of the target bacteria due to the action of the bacteriophage Vib-PAP-7.

MODE FOR INVENTION

(3) A better understanding of the present invention will be given through the following examples. These examples are merely set forth to illustrate the present invention but are not to be construed as limiting the scope of the present invention.

Example 1: Isolation of Bacteriophage Capable of Killing Vibrio parahaemolyticus

(4) Samples were collected from nature to isolate the bacteriophage capable of killing 5/7/i Vibrio parahaemolyticus. Here, the Vibrio parahaemolyticus strains used for the bacteriophage isolation are Vibrio parahaemolyticus that had been previously isolated and identified as Vibrio parahaemolyticus by the present inventors.

(5) The procedure for isolating the bacteriophage is described in detail herein below. The collected sample was added to LB (Luria-Bertani) culture medium (tryptone, 10 g/L; yeast extract, 5 g/L; sodium chloride, 10 g/L) inoculated with Vibrio parahaemolyticus at a ratio of 1/1,000, followed by shaking culture at 37° C. for 3 to 4 hr. Thereafter, centrifugation was performed at 8,000 rpm for 20 min and a supernatant was recovered. The recovered supernatant was inoculated with Vibrio parahaemolyticus at a ratio of 1/1,000 and then subjected to shaking culture at 37° C. for 3 to 4 hr. When the sample contained the bacteriophage, the above procedure was repeated a total of 5 times in order to sufficiently increase the number (titer) of bacteriophages. After repeating the procedure 5 times, the culture broth was subjected to centrifugation at 8,000 rpm for 20 min. Thereafter, the recovered supernatant was filtered using a 0.45 μm filter. The filtrate thus obtained was used in a typical spot assay for evaluating whether or not a bacteriophage capable of killing Vibrio parahaemolyticus was included therein.

(6) The spot assay was performed as follows: LB culture medium was inoculated with Vibrio parahaemolyticus at a ratio of 1/1,000 and then subjected to shaking culture at 37° C. overnight. 3 ml (OD.sub.600 of 1.5) of the culture broth of Vibrio parahaemolyticus prepared above was spread on LA (Luria-Bertani Agar: tryptone, 10 g/L; yeast extract, 5 g/L; sodium chloride, 10 g/L; agar, 15 g/L) plate. The plate was left on a clean bench for about 30 min to dry the spread solution. After drying, 10 μl of the prepared filtrate was spotted onto the plate which Vibrio parahaemolyticus was spread and then left for about 30 min to dry. Thereafter, the plate that was subjected to spotting was standing-cultured at 37° C. for one day, and then examined for the formation of clear zones at the positions where the filtrate was dropped. In the case in which the filtrate generated a clear zone, it was judged that a bacteriophage capable of killing Vibrio parahaemolyticus was included therein. Through the above examination, it was possible to obtain a filtrate containing a bacteriophage having the ability to kill Vibrio parahaemolyticus.

(7) The pure bacteriophage was isolated from the filtrate confirmed above to have the bacteriophage capable of killing Vibrio parahaemolyticus. A typical plaque assay was used to isolate the pure bacteriophage. In detail, a plaque formed in the course of the plaque assay was recovered using a sterilized tip, added to the culture broth of Vibrio parahaemolyticus, and then cultured at 37° C. for 4 to 5 hr. Thereafter, centrifugation was performed at 8,000 rpm for 20 min to obtain a supernatant. The culture broth of Vibrio parahaemolyticus was added to the obtained supernatant at a volume ratio of 1/50 and then cultured at 37° C. for 4 to 5 hr. In order to increase the number of bacteriophages, the above procedure was repeated at least 5 times. Then, centrifugation was performed at 8,000 rpm for 20 min in order to obtain the final supernatant. A plaque assay was further performed using the final supernatant thus obtained. In general, isolation of a pure bacteriophage is not completed through a single iteration of a procedure, so the above procedure was repeated using the resulting plaque formed above. After at least 5 repetitions of the procedure, the solution containing the pure bacteriophage was obtained. The procedure for isolating the pure bacteriophage was repeated in its entirety until the generated plaques became similar to each other with respect to size and morphology. In addition, final isolation of the pure bacteriophage was confirmed using electron microscopy. The above procedure was repeated until the isolation of the pure bacteriophage was confirmed using electron microscopy. The electron microscopy was performed through a typical method. Briefly, the solution containing the pure bacteriophage was loaded on a copper grid, followed by negative staining with 2% uranyl acetate and drying. The morphology thereof was then observed using a transmission electron microscope. The electron micrograph of the pure bacteriophage that was isolated is shown in FIG. 1. Based on the morphological characteristics thereof, the above bacteriophage was confirmed to belong to the Myoviridae bacteriophage.

(8) The solution containing the pure bacteriophage confirmed above was subjected to the following purification process. The culture broth of Vibrio parahaemolyticus was added to the solution containing the pure bacteriophage at a volume ratio of 1/50 based on the total volume of the bacteriophage solution, and then further cultured for 4 to 5 hr. Thereafter, centrifugation was performed at 8,000 rpm for 20 min to obtain a supernatant. This procedure was repeated a total of 5 times in order to obtain a solution containing a sufficient number of bacteriophages. The supernatant obtained from the final centrifugation was filtered using a 0.45 μm filter, followed by a typical polyethylene glycol (PEG) precipitation process. Specifically, PEG and NaCl were added to 100 ml of the filtrate reaching 10% PEG 8000/0.5 M NaCl, which was then allowed to stand at 4° C. for 2 to 3 hr. Thereafter, centrifugation was performed at 8,000 rpm for 30 min to obtain a bacteriophage precipitate. The bacteriophage precipitate thus obtained was suspended in 5 ml of a buffer (10 mM Tris-HCl, 10 mM MgSO.sub.4, 0.1% gelatin, pH 8.0). The resulting material may be referred to as a bacteriophage suspension or bacteriophage solution.

(9) As a result, the pure bacteriophage purified above was collected, was named Vib-PAP-7, and deposited at the Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology on Apr. 12, 2017 (Accession number: KCTC 13247BP).

Example 2: Separation and Sequence Analysis of Genome of Bacteriophage Vib-PAP-7

(10) The genome of the bacteriophage Vib-PAP-7 was separated as follows. The genome was separated from the bacteriophage suspension obtained using the same method as in Example 1. First, in order to eliminate DNA and RNA of Vibrio parahaemolyticus included in the suspension, 200 U of each of DNase I and RNase A was added to 10 ml of the bacteriophage suspension and then allowed to stand at 37° C. for 30 min. After being allowed to stand for 30 min, in order to inactivate the DNase I and RNase A activity, 500 μl of 0.5 M ethylenediaminetetraacetic acid (EDTA) was added thereto, and the resulting mixture was then allowed to stand for 10 min. In addition, the resulting mixture was further allowed to stand at 65° C. for 10 min, and 100 μl of proteinase K (20 mg/ml) was then added thereto so as to break the outer wall of the bacteriophage, followed by reacting at 37° C. for 20 min. Thereafter, 500 μl of 10% sodium dodecyl sulfate (SDS) was added thereto, followed by reacting at 65° C. for 1 hr. After reaction for 1 hr, the resulting reaction solution was added with 10 ml of the solution of phenol:chloroform:isoamyl alcohol, which were mixed at a component ratio of 25:24:1, followed by mixing thoroughly. In addition, the resulting mixture was subjected to centrifugation at 13,000 rpm for 15 min to separate layers. Among the separated layers, the upper layer was selected, and isopropyl alcohol was added thereto at a volume ratio of 1.5, followed by centrifugation at 13,000 rpm for 10 min to precipitate the genome. After collecting the precipitate, 70% ethanol was added to the precipitate, followed by centrifugation at 13,000 rpm for 10 min to wash the precipitate. The washed precipitate was recovered, vacuum-dried and then dissolved in 100 μl of water. This procedure was repeated to obtain a sufficient amount of the genome of the bacteriophage Vib-PAP-7.

(11) Information on the sequence of the genome of the bacteriophage Vib-PAP-7 thus obtained was secured by performing next-generation sequencing analysis using an Illumina Mi-Seq apparatus provided by the Macrogen. The finally analyzed genome of the bacteriophage Vib-PAP-7 had a size of 76,187 bp, and the whole genome sequence is represented by SEQ ID NO: 1.

(12) The homology (similarity) of the bacteriophage Vib-PAP-7 genomic sequence obtained above with conventionally reported bacteriophage genomic sequences was investigated using BLAST on the web. Based on the results of the BLAST investigation, the genomic sequence of the bacteriophage Vib-PAP-7 was found to have relatively high homology with the sequence of the Vibrio bacteriophage SSP002 (GenBank Accession No. JQ692107.1) and the sequence of vB_VpaS_MAR10 (GenBank Accession No. JX556418.1) (96%/97% and 67%/79%, respectively, in the order of query coverage/identity). However, the number of open reading frames (ORFs) on the bacteriophage Vib-PAP-7 genome is 101, whereas the bacteriophage SSP002 has 102 open reading frames and the bacteriophage vB-VpaS_MAR10, having slightly low homology therewith, has 104 open reading frames, from which these bacteriophages are also evaluated to be different.

(13) Therefore, it can be concluded that the bacteriophage Vib-PAP-7 is a novel bacteriophage different from existing reported bacteriophages. Moreover, since the antibacterial strength and spectrum of bacteriophages typically depend on the type of bacteriophage, it is considered that the bacteriophage Vib-PAP-7 can provide antibacterial activity different from that of any other bacteriophages reported conventionally.

Example 3: Investigation of Killing Ability of Bacteriophage Vib-PAP-7 for Vibrio parahaemolyticus

(14) The killing ability of the isolated bacteriophage Vib-PAP-7 for Vibrio parahaemolyticus was investigated. In order to evaluate the killing ability, the formation of clear zones was observed using a spot assay in the same manner as described in connection with Example 1. A total of 25 Vibrio parahaemolyticus strains were used for the investigation of killing ability, and were obtained from a strain bank or were isolated and identified as Vibrio parahaemolyticus by the present inventors. The bacteriophage Vib-PAP-7 had the ability to kill a total of 21 strains, among 25 strains of Vibrio parahaemolyticus, that is, the experimental target. The representative experimental results thereof are shown in FIG. 2. Meanwhile, the ability of the bacteriophage Vib-PAP-7 to kill Edwardsiella tarda, Vibrio anguillarum, Vibrio ichthyoenteri, Lactococcus garvieae, Streptococcus parauberis, Streptococcus iniae, and Aeromonas salmonicida was also measured. Consequently, the bacteriophage Vib-PAP-7 did not have the ability to kill these microorganisms.

(15) Therefore, it can be concluded that the bacteriophage Vib-PAP-7 has high ability to kill Vibrio parahaemolyticus and an antibacterial effect against many Vibrio parahaemolyticus strains, indicating that the bacteriophage Vib-PAP-7 can be used as an active ingredient of the composition for preventing or treating diseases caused by Vibrio parahaemolyticus.

Example 4: Experiment for Prevention of Vibrio Parahaemolyticus Infection Using Bacteriophage Vib-PAP-7

(16) 100 μl of a bacteriophage Vib-PAP-7 solution at a level of 1×10.sup.8 pfu/ml was added to a tube containing 9 ml of LB culture medium. To another tube containing 9 ml of LB culture medium, only the same amount of LB culture medium was further added. A culture broth of Vibrio parahaemolyticus was then added to each tube so that absorbance reached about 0.5 at 600 nm. After the addition of Vibrio parahaemolyticus, the tubes were transferred to an incubator at 37° C., followed by shaking culture, during which the growth of Vibrio parahaemolyticus was observed. As shown in Table 1 below, it was observed that the growth of Vibrio parahaemolyticus was inhibited in the tube to which the bacteriophage Vib-PAP-7 solution was added, whereas the growth of Vibrio parahaemolyticus was not inhibited in the tube to which the bacteriophage solution was not added.

(17) TABLE-US-00001 TABLE 1 Growth inhibition of Vibrio parahaemolyticus OD.sub.600 absorbance value 0 min after 60 min after 120 min after Classification culture culture culture Not added with 0.52 1.18 1.56 bacteriophage solution Added with 0.52 0.23 0.19 bacteriophage solution

(18) The above results show that the bacteriophage Vib-PAP-7 of the present invention is not only capable of inhibiting the growth of Vibrio parahaemolyticus but also capable of killing Vibrio parahaemolyticus. Therefore, it is concluded that the bacteriophage Vib-PAP-7 can be used as an active ingredient of the composition for preventing diseases caused by Vibrio parahaemolyticus.

Example 5: Animal Testing for Preventing Disease Caused by Vibrio parahaemolyticus Using Bacteriophage Vib-PAP-7

(19) A total of 2 groups of sixty juvenile sea bass per group (body weight: 5 to 7 g and body length: 8 to 10 cm) were prepared and farmed separately in water tanks, and an experiment was performed for 14 days. The environment surrounding the water tanks was controlled, and the temperature in the laboratory where the water tanks were located was maintained constant. Over the whole experimental period from the 1st day of the experiment, sea bass in an experimental group (the group to which the bacteriophage was administered) were fed with a feed containing the bacteriophage Vib-PAP-7 at 1×10.sup.8 pfu/g in a typical feeding manner. In contrast, sea bass in a control group (the group to which the bacteriophage was not administered) were fed with the same feed as the experimental group except that the bacteriophage Vib-PAP-7 was not contained in the same manner as in the experimental group. For 2 days from the 7th day after the experiment started, the provided feed was added with Vibrio parahaemolyticus at a level of 1×10.sup.8 cfu/g and then provided respectively twice a day so as to induce a Vibrio parahaemolyticus infection. From the 9th day after the experiment started (the 2.sup.nd day after the Vibrio parahaemolyticus infection was induced), vibriosis pathogenesis was examined in all test animals on a daily basis. The vibriosis pathogenesis was evaluated by measuring a body-darkening index. The measurement of the body-darkening index was performed using a typical process of measuring a dark coloration (DC) score (0: normal, 1: slight darkening, 2: strong darkening). The results are shown in Table 2 below.

(20) TABLE-US-00002 TABLE 2 Result of measurement of body-darkening index (mean) DC score (mean) Days D 9 D 10 D 11 D 12 D 13 D 14 Control group (not 0.68 0.72 0.84 0.88 1.00 1.12 administered with bacteriophage) Experimental group 0.32 0.12 0.04 0.04 0 0 (administered with bacteriophage)

(21) As is apparent from the above results, it can be concluded that the bacteriophage Vib-PAP-7 of the present invention is very effective in the prevention of diseases caused by Vibrio parahaemolyticus.

Example 6: Treatment of Disease Caused by Vibrio parahaemolyticus Using Bacteriophage Vib-PAP-7

(22) The therapeutic effect of the bacteriophage Vib-PAP-7 on diseases caused by Vibrio parahaemolyticus was evaluated as follows. A total of 2 groups of sixty juvenile sea bass per group (body weight: 5 to 7 g and body length: 8 to 10 cm) were prepared and farmed separately in water tanks, and an experiment was performed for 14 days. The environment surrounding the water tanks was controlled, and the temperature in the laboratory where the water tanks were located was maintained constant. For 3 days from the 5th day after the experiment started, the feed contaminated with Vibrio parahaemolyticus at a level of 1×10.sup.8 cfu/g was provided twice a day in a typical feeding manner. Sea bass subjects showing clinical symptoms of vibriosis were observed in both water tanks from the last day of the procedure in which the feed contaminated with Vibrio parahaemolyticus was provided. From the next day after the feed contaminated with Vibrio parahaemolyticus was provided for 3 days (the 8th day after the experiment started), sea bass in an experimental group (the group to which the bacteriophage was administered) were fed with a feed containing the bacteriophage Vib-PAP-7 (1×10.sup.8 pfu/g) in a typical feeding manner. In contrast, sea bass in a control group (the group to which the bacteriophage was not administered) were fed with the same feed as the experimental group except that the bacteriophage Vib-PAP-7 was not contained in the same manner as in the experimental group. From the 3/d day after the forced infection of Vibrio parahaemolyticus (the 8th day after the experiment started), vibriosis pathogenesis was examined in all test animals on a daily basis. The vibriosis pathogenesis caused by Vibrio parahaemolyticus was examined by measuring a body-darkening index as in Example 5. The results are shown in Table 3 below.

(23) TABLE-US-00003 TABLE 3 Result of measurement of body-darkening index (mean) DC score (mean) Days D 8 D 9 D 10 D 11 D 12 D 13 D 14 Control group (not 0.87 0.93 1.03 1.13 1.13 1.30 1.37 administered with bacteriophage) Experimental group 0.90 0.87 0.83 0.70 0.37 0.13 0.13 (administered with bacteriophage)

(24) As is apparent from the above results, it can be concluded that the bacteriophage Vib-PAP-7 of the present invention is very effective in the treatment of diseases caused by Vibrio parahaemolyticus.

Example 7: Preparation of Feed Additives and Feeds

(25) Feed additives were prepared using a bacteriophage Vib-PAP-7 solution so that a bacteriophage Vib-PAP-7 was contained in an amount of 1×10.sup.8 pfu for 1 g of the feed additives. The method of preparing the feed additive was as follows: Maltodextrin (50%, w/v) was added to the bacteriophage solution, and the resulting mixture was then freeze-dried. Finally, the dried mixture was ground into fine powder. In the above-described preparation procedure, the drying process may be replaced with drying under reduced pressure, drying with heat, or drying at room temperature. In order to prepare the control for comparison, a feed additive was prepared that did not contain the bacteriophage but contained the buffer (10 mM Tris-HCl, 10 mM MgSO.sub.4, 0.1% gelatin, pH 8.0) used to prepare the bacteriophage solution.

(26) The two kinds of feed additives thus prepared were each mixed with a raw fish-based moist pellet at a weight ratio of 250, thus ultimately preparing two kinds of final feeds.

Example 8: Preparation of Medicine Bath Agent

(27) The method of preparing a medicine bath agent was as follows: The medicine bath agent was prepared using a bacteriophage Vib-PAP-7 solution so that a bacteriophage Vib-PAP-7 was contained in an amount of 1×10.sup.8 pfu for 1 ml of the medicine bath agent. In the method of preparing the medicine bath agent, the bacteriophage Vib-PAP-7 solution was added so that the bacteriophage Vib-PAP-7 was contained in an amount of 1×10.sup.8 pfu for 1 ml of a buffer used to prepare the bacteriophage solution, and mixing was sufficiently performed. In order to prepare the control for comparison, the buffer used to prepare the bacteriophage solution was used as the medicine bath agent that did not contain the bacteriophage.

(28) The two kinds of medicine bath agents thus prepared were diluted with water at a volume ratio of 1,000, resulting in the final medicine bath agent.

Example 9: Confirmation of Feeding Effect on Sea Bass Farming

(29) The improvement in the feeding result upon sea bass farming was investigated using the feeds and the medicine bath agents prepared in Examples 7 and 8. In particular, the investigation was focused on mortality ratio. A total of 1,000 juvenile sea bass were divided into two groups, each including 500 sea bass (group A: fed with the feed; and group B: treated with the medicine bath agent), and an experiment was performed for 4 weeks. Each group was further divided into subgroups each including 250 sea bass, and the subgroups were classified into a subgroup to which the bacteriophage Vib-PAP-7 was applied (subgroup-{circle around (1)}) and a subgroup to which the bacteriophage was not applied (subgroup-{circle around (2)}). In the present experiment, the target sea bass was juvenile (body weight: 5 to 7 g and body length: 8 to 10 cm), and the juvenile sea bass of the experimental subgroups were farmed in separate water tanks spaced apart from each other at a certain interval. The subgroups were classified and named as shown in Table 4 below.

(30) TABLE-US-00004 TABLE 4 Sub-group classification and expression in sea bass feeding experiment Sub-group classification and expression Bacteriophage Vib- Bacteriophage Application PAP-7 is applied is not applied Group fed with feed A-{circle around (1)} A-{circle around (2)} Group treated with B-{circle around (1)} B-{circle around (2)} medicine bath agent

(31) In the case of provision of the feeds, the feeds prepared in Example 7 were provided according to conventional feeding method as classified in Table 4. The treatment using the medicine bath agent was performed according to a conventional treatment method using a medicine bath agent, in which fish bodies are immersed in a diluted solution of the medicine bath agent, as classified in Table 4 using the medicine bath agent prepared as described in Example 8. The results are shown in Table 5.

(32) TABLE-US-00005 TABLE 5 Mortality ratio of sea bass in feeding experiment Dead sea bass/total sea Mortality Group bass of experiment (No.) ratio (%) A-{circle around (1)}  7/250 2.8 A-{circle around (2)} 43/250 17.2 B-{circle around (1)} 11/250 4.4 B-{circle around (2)} 56/250 22.4

(33) The above results indicate that the provision of the feed prepared according to the present invention and the treatment using the medicine bath agent prepared according to the present invention were effective at reducing mortality ratio in the farming of sea bass. Therefore, it is concluded that the composition of the present invention could be efficiently applied to improving the results of feeding of sea bass.

(34) While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, those skilled in the art will appreciate that the specific description is only a preferred embodiment, and that the scope of the present invention is not limited thereto. It is therefore intended that the scope of the present invention be defined by the claims appended hereto and their equivalents.

(35) [Accession Number]

(36) Name of Depositary Authority: KCTC

(37) Accession number: KCTC 13247BP

(38) Accession date: 20170412