<i>Vibrio parahaemolyticus </i>bacteriophage Vib-PAP-5 and use thereof for suppressing proliferation of <i>Vibrio parahaemolyticus </i>bacteria

Abstract

The present invention relates to a Myoviridae bacteriophage Vib-PAP-5 (accession number KCTC 13029BP) isolated from nature characterized by having a capability for specifically killing Vibrio parahaemolyticus bacteria and having a genome expressed by the SEQ ID NO:1, and to a method for preventing and treating infections from Vibrio parahaemolyticus bacteria by means of a composition comprising the Myoviridae bacteriophage Vib-PAP-5 as an active ingredient.

Claims

1. A method for treating a Vibrio parahaemolyticus infection, the method comprising: administering to an animal other than a human a composition comprising an isolated Myoviridae bacteriophage Vib-PAP-5 (Accession number: KCTC 13029BP) that can kill Vibrio parahaemolyticus specifically as an active ingredient, wherein the Myovirdae bacteriophage Vib-PAP-5 is prepared by bacterial culture with inoculum of bacteriophage Vib-PAP-5 and comprises a genome encoded by the nucleotide sequence of SEQ ID NO:1, wherein the composition is administered as a feed additive or a medicine bath agent.

2. The method of claim 1, wherein said composition further comprises a pharmaceutically acceptable carrier.

3. The method of claim 2, wherein said composition for the medicine bath agent comprises the bacteriophage Vib-PAP-5 at a concentration of 110.sup.1 pfu/ml to 110.sup.30 pfu/ml.

4. The method of claim 2, wherein said composition for the food additive comprises the bacteriophage Vib-PAP-5 at a concentration of 110.sup.1 pfu/g to 110.sup.30 pfu/g.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 is an electron micrograph showing the morphology of the bacteriophage Vib-PAP-5.

(2) FIG. 2 is a photograph showing the results of an experiment on the ability of the bacteriophage Vib-PAP-5 to kill Vibrio parahaemolyticus. The clear zone is a plaque formed by lysis of the target bacteria.

MODE FOR INVENTION

(3) Hereinafter, the present invention will be described in more detail with reference to Examples. However, the Examples are merely examples of the present invention, and the scope of the present invention is not limited to the Examples.

Example 1: Isolation of Bacteriophage Capable of Killing Vibrio parahaemolyticus

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

(5) The isolation procedure of the bacteriophage is described in detail hereinafter. The collected sample was added to an 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 hours. Upon completion of the culture, centrifugation was performed at 8,000 rpm for 20 minutes and a supernatant was recovered. The recovered supernatant was inoculated with Vibrio parahaemolyticus at a ratio of 1/1,000, followed by shaking culture at 37 C. for 3 to 4 hours. 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 the bacteriophages. After repeating the procedure 5 times, the culture solution was subjected to centrifugation at 8,000 rpm for 20 minutes. After the centrifugation, the recovered supernatant was filtered using a 0.45 m filter. The obtained filtrate was used in a typical spot assay for examining 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, followed by shaking culture at 37 C. for overnight. 3 ml (OD.sub.600 of 1.5) of the culture solution 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 minutes to dry the spread solution. After drying, 10 l of the prepared filtrate was spotted onto the plate culture medium on which Vibrio parahaemolyticus was spread and then left for about 30 minutes to dry. After drying, the plate culture medium that was subjected to spotting was stationary-cultured at 37 C. for one day, and then examined for the formation of a clear zone at the position at which the filtrate was dropped. In the case of the filtrate generating the clear zone, it is judged that the bacteriophage capable of killing Vibrio parahaemolyticus is included therein. Through the above examination, the filtrate containing the bacteriophage having the ability to kill Vibrio parahaemolyticus could be obtained.

(7) The pure bacteriophage was isolated from the filtrate confirmed above to have the bacteriophage capable of killing Vibrio parahaemolyticus. A conventional plaque assay was used for the isolation of the pure bacteriophage. In detail, a plaque formed in the course of the plaque assay was recovered using a sterilized tip, which was then added to the culture solution of Vibrio parahaemolyticus, followed by culturing at 37 C. for 4 to 5 hours. After the culturing, centrifugation was performed at 8,000 rpm for 20 minutes to obtain a supernatant. The Vibrio parahaemolyticus culture solution was added to the obtained supernatant at a volume ratio of 1/50, followed by culturing at 37 C. for 4 to 5 hours. 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 minutes to obtain the final supernatant. A plaque assay was further performed using the resulting supernatant. In general, the 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 the isolation of the pure bacteriophage was generally repeated until the generated plaques became similar to each other in size and morphology. In addition, the final isolation of the pure bacteriophage was confirmed using electron microscopy. Until the isolation of the pure bacteriophage was confirmed using the electron microscopy, the above procedure was repeated. The electron microscopy was performed according to a conventional 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, the novel bacteriophage isolated above 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 Vibrio parahaemolyticus culture solution 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, followed by further culturing for 4 to 5 hours. After the culturing, centrifugation was performed at 8,000 rpm for 20 minutes to obtain a supernatant. This procedure was repeated a total of 5 times to obtain a solution containing sufficient numbers of the bacteriophage. The supernatant obtained from the final centrifugation was filtered using a 0.45 m filter, followed by a conventional polyethylene glycol (PEG) precipitation process. Specifically, PEG and NaCl were added to 100 ml of the filtrate until reaching 10% PEG 8000/0.5 M NaCl, and then left at 4 C. for 2 to 3 hours. Thereafter, centrifugation was performed at 8,000 rpm for 30 minutes to obtain the bacteriophage precipitate. The resulting bacteriophage precipitate 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 was referred to as a bacteriophage suspension or bacteriophage solution.

(9) As a result, the pure bacteriophage purified above was collected, was named the bacteriophage Vib-PAP-5, and then deposited at Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology on May 26, 2016 (Accession number: KCTC 13029BP).

Example 2: Separation and Sequence Analysis of Genome of Bacteriophage Vib-PAP-5

(10) The genome of the bacteriophage Vib-PAP-5 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 left at 37 C. for 30 minutes. After being left for 30 minutes, in order to remove the DNase I and RNase A activity, 500 l of 0.5 M ethylenediaminetetraacetic acid (EDTA) was added thereto and then left for 10 minutes. In addition, the resulting mixture was further left at 65 C. for 10 minutes, and 100 l of proteinase K (20 mg/ml) was then added thereto so as to break the outer wall of the bacteriophage, followed by reaction at 37 C. for 20 minutes. After that, 500 l of 10% sodium dodecyl sulfate (SDS) was added thereto, followed by reaction at 65 C. for 1 hour. After the reaction for 1 hour, 10 ml of the solution of phenol:chloroform:isoamyl alcohol mixed at a component ratio of 25:24:1 was added to the reaction solution, followed by mixing well. In addition, the resulting mixture was subjected to centrifugation at 13,000 rpm for 15 minutes 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 minutes to precipitate the genome. After collecting the precipitate, 70% ethanol was added to the precipitate, followed by centrifugation at 13,000 rpm for 10 minutes to wash the precipitate. The washed precipitate was recovered, vacuum-dried and then dissolved in 100 l of water. This procedure was repeated to obtain a sufficient amount of the genome of the bacteriophage Vib-PAP-5.

(11) Information on the sequence of the genome of the bacteriophage Vib-PAP-5 obtained above was secured by performing next-generation sequencing analysis using Illumina Mi-Seq equipment from Macrogen, Inc. The finally analyzed genome of the bacteriophage Vib-PAP-5 had a size of 58,970 bp and the sequence of the whole genome was expressed by SEQ. ID. NO: 1.

(12) The homology (similarity) of the bacteriophage Vib-PAP-5 genomic sequence obtained above with previously reported bacteriophage genomic sequences was investigated using BLAST (ncbi.nlm.nih.gov/BLAST/) on the web. As a result of the BLAST investigation, bacteriophage sequences with homology of 50% or more were not confirmed.

(13) Based upon this result, it is concluded that the bacteriophage Vib-PAP-5 must be a novel bacteriophage that has not been reported previously. Further, since the antibacterial strength and spectrum of bacteriophages typically depend on the type of bacteriophage, it is considered that the bacteriophage Vib-PAP-5 can provide antibacterial activity different from that of any other bacteriophages reported previously.

Example 3: Investigation of Ability of Bacteriophage Vib-PAP-5 to Kill Vibrio parahaemolyticus

(14) The ability of the isolated bacteriophage Vib-PAP-5 to kill Vibrio parahaemolyticus was investigated. In order to investigate the killing ability, the formation of clear zones was observed using the spot assay in the same manner as described in Example 1. A total of 18 strains which had been isolated and identified as Vibrio parahaemolyticus by the present inventors were used as Vibrio parahaemolyticus for the investigation of killing ability. The bacteriophage Vib-PAP-5 had the ability to kill a total of 17 strains among 18 strains of Vibrio parahaemolyticus as the experimental target. The representative experimental result is shown in FIG. 2. Meanwhile, the ability of the bacteriophage Vib-PAP-5 to kill Edwardsiella tarda, Vibrio anguillarum, Vibrio ichthyoenteri, Lactococcus garvieae, Streptococcus parauberis, Streptococcus iniae, and Aeromonas salmonicida was also investigated in a separate experiment. As a result, the bacteriophage Vib-PAP-5 did not have the ability to kill these microorganisms.

(15) Therefore, it is confirmed that the bacteriophage Vib-PAP-5 has the specific ability to kill Vibrio parahaemolyticus and a broad antibacterial spectrum against Vibrio parahaemolyticus, suggesting that the bacteriophage Vib-PAP-5 can be used as an active ingredient of the composition for preventing and treating Vibrio parahaemolyticus infection.

Example 4: Experimental Example Regarding Prevention of Vibrio parahaemolyticus Infection Using Bacteriophage Vib-PAP-5

(16) 100 l of a bacteriophage Vib-PAP-5 solution at a level of 110.sup.8 pfu/ml was added to a tube containing 9 ml of an LB culture medium. To another tube containing 9 ml of an LB culture medium, only the same amount of LB culture medium was further added. A Vibrio parahaemolyticus culture solution was then added to each tube so that absorbance reached about 0.5 at 600 nm. After Vibrio parahaemolyticus was added, the tubes were transferred to an incubator at 37 C., followed by shaking culture, during which the growth of Vibrio parahaemolyticus was observed. As presented in Table 1, it was observed that the growth of Vibrio parahaemolyticus was inhibited in the tube to which the bacteriophage Vib-PAP-solution was added, while 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 minutes after 60 minutes after 120 minutes after Classification culture culture culture Bacteriophage 0.501 0.966 1.681 solution is not added Bacteriophage 0.501 0.302 0.226 solution is added

(18) The above results indicate that the bacteriophage Vib-PAP-5 of the present invention not only inhibits the growth of Vibrio parahaemolyticus but also has the ability to kill Vibrio parahaemolyticus. Therefore, it is concluded that the bacteriophage Vib-PAP-5 can be used as an active ingredient of the composition for preventing a Vibrio parahaemolyticus infection.

Example 5: Animal Experiment on Prevention of Vibrio parahaemolyticus Infection Using Bacteriophage Vib-PAP-5

(19) The preventive effect of the bacteriophage Vib-PAP-5 on sea bass subjected to Vibrio parahaemolyticus infection was investigated. 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) was 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 1.sup.st day of the experiment, sea bass in an experimental group (the group to which the bacteriophage was administered) was fed with a feed containing the bacteriophage Vib-PAP-5 at 110.sup.8 pfu/g according to a conventional feeding method. In contrast, sea bass in a control group (the group to which the bacteriophage was not administered) was fed with the same feed as in the experimental group except that the bacteriophage Vib-PAP-5 was not contained according to the same method as in the experimental group. From the seventh day after the experiment started, the feed to be provided was contaminated with Vibrio parahaemolyticus at a level of 110.sup.8 cfu/g for two days and thereafter provided respectively twice a day so as to induce a Vibrio parahaemolyticus infection. From the ninth day after the experiment started (the second day after the Vibrio parahaemolyticus infection was induced), vibriosis pathogenesis was examined in all test animals on a daily basis. The vibriosis pathogenesis was examined by measuring a body darkening index. The measurement of the body darkening index was performed using a conventional method for measuring a dark coloration (DC) score (0: normal, 1: slight darkening, 2: strong darkening). The results are shown in Table 2.

(20) TABLE-US-00002 TABLE 2 Result of measurement of body darkening index (mean) DC score (mean) Days D9 D10 D11 D12 D13 D14 Control group 0.72 0.72 0.76 0.80 1.00 1.08 (bacteriophage is not administered) Experimental group 0.20 0.04 0 0 0 0 (bacteriophage is administered)

(21) From the above results, it is confirmed that the bacteriophage Vib-PAP-5 of the present invention could be very effective in inhibiting Vibrio parahaemolyticus infection.

Example 6: Example of Treatment of Infectious Diseases of Vibrio parahaemolyticus Using Bacteriophage Vib-PAP-5

(22) The treatment effect of the bacteriophage Vib-PAP-on sea bass suffering from vibriosis caused by Vibrio parahaemolyticus was investigated. 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) was 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 stayed was maintained. From the fifth day after the experiment started, the feed contaminated with Vibrio parahaemolyticus at a level of 110.sup.8 cfu/g was provided twice a day for three days according to a conventional feeding method. 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 three days (the eighth day after the experiment started), sea bass in an experimental group (the group to which the bacteriophage was administered) was fed with a feed containing the bacteriophage Vib-PAP-5 (lx 10.sup.8 pfu/g) according to a conventional feeding method. In contrast, sea bass in a control group (the group to which the bacteriophage was not administered) was fed with the same feed as in the experimental group except that the bacteriophage Vib-PAP-5 was not contained according to the same method as in the experimental group. From the third day after the forced infection of Vibrio parahaemolyticus (the eighth 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.

(23) TABLE-US-00003 TABLE 3 Result of measurement of body darkening index (mean) DC score (mean) Days D8 D9 D10 D11 D12 D13 D14 Control group 0.93 1.03 1.10 1.17 1.20 1.30 1.33 (bacteriophage is not administered) Experimental group 1.03 0.93 0.87 0.77 0.43 0.23 0.17 (bacteriophage is administered)

(24) From the above results, it is confirmed that the bacteriophage Vib-PAP-5 of the present invention could be very effective in the treatment of infectious diseases caused by Vibrio parahaemolyticus.

Example 7: Preparation of Feed Additives and Feeds

(25) Feed additives were prepared using a bacteriophage Vib-PAP-5 solution so that a bacteriophage Vib-PAP-5 was contained in an amount of 110.sup.8 pfu per 1 g of the feed additives. The method of preparing the feed additives 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 powders. In the above-described preparation procedure, the drying procedure can be replaced with drying under a reduced pressure, drying with heat, or drying at room temperature. In order to prepare the control for comparison, the feed additives that did not contain the bacteriophage but contained a buffer (10 mM Tris-HCl, 10 mM MgSO.sub.4, 0.1% Gelatin, pH 8.0) used to prepare the bacteriophage solution was prepared.

(26) The two kinds of feed additives that were prepared above were each mixed with a raw fish-based moist pellet at a weight ratio of 250, thus 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-5 solution so that a bacteriophage Vib-PAP-5 was contained in an amount of 110.sup.8 pfu per 1 ml of the medicine bath agent. In the method of preparing the medicine bath agent, the bacteriophage Vib-PAP-5 solution was added so that the bacteriophage Vib-PAP-5 was contained in an amount of 110.sup.8 pfu per 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 prepared kinds of medicine bath agents 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) Improvement in the feeding result upon sea bass farming was investigated using the feed and the medicine bath agents prepared in Examples 7 and 8. In particular, the investigation was focused on mortality. A total of 800 juvenile sea bass was divided into two groups, each including 400 sea bass (group A; fed with the feeds and group B; treated with the medicine bath agent), and an experiment was performed for four weeks. Each group was divided into sub-groups each including 200 sea bass, and the sub-groups were classified into a sub-group to which the bacteriophage Vib-PAP-5 was applied (sub-group-{circle around (1)}) and a sub-group to which the bacteriophage was not applied (sub-group-{circle around (2)}). In the present experiment, the target sea bass was the juvenile (body weight: 5 to 7 g and body length: 8 to 10 cm), and the juvenile sea bass of the experimental sub-groups was farmed in separate water tanks placed apart from each other at a certain space interval. The sub-groups were classified and named as shown in Table 4.

(30) TABLE-US-00004 TABLE 4 Sub-group classification and expression in sea bass feeding experiment Sub-group classification and expression Bacteriophage Vib-PAP-5 is Bacteriophage is not Application applied applied Group fed with feeds A-{circle around (1)} A-{circle around (2)} Group treated with medicine B-{circle around (1)} B-{circle around (2)} bath agent

(31) In the case of provision of the feeds, the feeds prepared in Example 7 were provided according to a 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 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 of sea bass in feeding experiment Dead sea bass/total sea bass Mortality Group of experiment (No.) (%) A-{circle around (1)} 7/200 3.5 A-{circle around (2)} 39/200 19.5 B-{circle around (1)} 9/200 4.5 B-{circle around (2)} 58/200 29.0

(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 in improving the feeding result 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 animal feeding.

(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) Name of Depositary Authority: KCTC

(36) Accession number: KCTC 13029BP

(37) Accession date: 20160526