<i>Lactococcus garvieae </i>bacteriophage Lac-GAP-3 and use thereof in inhibiting proliferation of <i>Lactococcus garvieae </i>bacteria

Abstract

The present invention relates to Siphoviridae bacteriophage Lac-GAP-3 (Accession Number KCTC 12816BP) having the ability to specifically kill Lactococcus garvieae bacteria and a genome represented by SEQ ID NO: 1 and isolated from nature, and a method for prevention and treatment of Lactococcus garvieae bacterial infection by using a composition containing the same bacteriophage as an effective ingredient.

Claims

1. A method for treating a Lactococcus garvieae infection, the method comprising: administering to an animal other than a human the composition including the Siphoviridae bacteriophage Lac-GAP-3 (Accession number: KCTC 12816BP) which has an ability to specifically kill Lactococcus garvieae and which includes a genome expressed by SEQ. ID. NO: 1 as an active ingredient.

2. The method for treating the Lactococcus garvieae infection of claim 1, wherein said composition is administered to the animal other than the human as a medicine bath agent or a feed additive.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 is an electron micrograph showing the morphology of the bacteriophage Lac-GAP-3.

(2) FIG. 2 is a photograph showing the results of an experiment on the ability of the bacteriophage Lac-GAP-3 to kill Lactococcus garvieae. Based on the center line of the plate culture medium, only the buffer containing no bacteriophage Lac-GAP-3 is spotted on the left side thereof, and the solution containing bacteriophage Lac-GAP-3 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 bacteriophage Lac-GAP-3.

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 Lactococcus garvieae

(4) Samples were collected from nature to isolate the bacteriophage capable of killing Lactococcus garvieae. Meanwhile, the Lactococcus garvieae used for the bacteriophage isolation was purchased from the Korea Environmental Microorganisms Bank (Accession number: KEMB 2221-072).

(5) The isolation procedure of the bacteriophage is described in detail hereinafter. The collected sample was added to a TSB (Tryptic Soy Broth) culture medium (casein digest, 17 g/L; soybean digest, 3 g/L; dextrose, 2.5 g/L; NaCl, 5 g/L; dipotassium phosphate, 2.5 g/L) inoculated with Lactococcus garvieae at a ratio of 1/1,000, followed by shaking culture at 30° 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 Lactococcus garvieae at a ratio of 1/1,000, followed by shaking culture at 30° 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 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 μm filter. The obtained filtrate was used in a typical spot assay for examining whether or not a bacteriophage capable of killing Lactococcus garvieae was included therein.

(6) The spot assay was performed as follows: TSB culture medium was inoculated with Lactococcus garvieae at a ratio of 1/1,000, followed by shaking culture at 30° C. overnight. 3 ml (OD600 of 1.5) of the culture solution of Lactococcus garvieae prepared as described above was spread on TSA (Tryptic Soy Agar: casein digest, 15 g/L; soybean digest, 5 g/L; NaCl, 5 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 μl of the prepared filtrate was spotted onto the plate which Lactococcus garvieae was spread and then left for about 30 minutes to dry. After drying, the plate that was subjected to spotting was standing-cultured at 30° 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 Lactococcus garvieae is included therein. Through the above examination, the filtrate containing the bacteriophage having the ability to kill Lactococcus garvieae could be obtained.

(7) The pure bacteriophage was isolated from the filtrate confirmed above to have the bacteriophage capable of killing Lactococcus garvieae. 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 Lactococcus garvieae, followed by culturing at 30° C. for 4 to 5 hours. After the culturing, centrifugation was performed at 8,000 rpm for 20 minutes to obtain a supernatant. The Lactococcus garvieae culture solution was added to the obtained supernatant at a volume ratio of 1/50, followed by culturing at 30° 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 above was confirmed to belong to the Siphoviridae bacteriophage.

(8) The solution containing the pure bacteriophage confirmed above was subjected to the following purification process. The Lactococcus garvieae 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 μm 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 Lac-GAP-3, and was then deposited at Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology on May 20, 2015 (Accession number: KCTC 12816BP).

Example 2: Separation and Sequence Analysis of Genome of Bacteriophage Lac-GAP-3

(10) The genome of the bacteriophage Lac-GAP-3 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 Lactococcus garvieae 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 inactivate the DNase I and RNase A activity, 500 μl 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 μl 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 μl 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 μl of water. This procedure was repeated to obtain a sufficient amount of the genome of the bacteriophage Lac-GAP-3.

(11) Next-generation sequencing analysis using Illumina Mi-Seq equipment from Macrogen, Inc. was performed, and information on the sequence of the genome of the bacteriophage Lac-GAP-3 obtained above was then secured. The finally analyzed genome of the bacteriophage Lac-GAP-3 had a size of 24,375 bp and the sequence of the whole genome was expressed by SEQ. ID. NO: 1.

(12) The homology (similarity) of the bacteriophage Lac-GAP-3 genomic sequence obtained above with previously reported bacteriophage genomic sequences was investigated using 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 Lac-GAP-3 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 Lac-GAP-3 can provide antibacterial activity different from that of any other bacteriophages reported previously.

Example 3: Investigation of Ability of Bacteriophage Lac-GAP-3 to Kill Lactococcus garvieae

(14) The ability of the isolated bacteriophage Lac-GAP-3 to kill Lactococcus garvieae 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 15 strains which had been isolated and identified as Lactococcus garvieae by the present inventors were used as Lactococcus garvieae for the investigation of killing ability. The bacteriophage Lac-GAP-3 had the ability to kill a total of 13 strains among 15 strains of Lactococcus garvieae as the experimental target. The representative experimental result is shown in FIG. 2. Meanwhile, the ability of the bacteriophage Lac-GAP-3 to kill Edwardsiella tarda, Vibrio parahaemolyticus, Vibrio anguillarum, Streptococcus iniae, Streptococcus parauberis, and Aeromonas salmonicida was also investigated in a separate experiment. As a result, the bacteriophage Lac-GAP-3 did not have the ability to kill these microorganisms.

(15) Therefore, it is confirmed that the bacteriophage Lac-GAP-3 has the specific ability to kill Lactococcus garvieae and a broad antibacterial spectrum against Lactococcus garvieae, suggesting that the bacteriophage Lac-GAP-3 can be used as an active ingredient of the composition for preventing and treating Lactococcus garvieae infection.

Example 4: Experimental Example Regarding Prevention of Lactococcus garvieae Infection Using Bacteriophage Lac-GAP-3

(16) 100 μl of a bacteriophage Lac-GAP-3 solution at a level of 1×10.sup.8 pfu/ml was added to a tube containing 9 ml of a TSB culture medium. To another tube containing 9 ml of a TSB culture medium, only the same amount of TSB culture medium was further added. A Lactococcus garvieae culture solution was then added to each tube so that absorbance reached about 0.5 at 600 nm. After Lactococcus garvieae was added, the tubes were transferred to an incubator at 30° C., followed by shaking culture, during which the growth of Lactococcus garvieae was observed. As presented in Table 1, it was observed that the growth of Lactococcus garvieae was inhibited in the tube to which the bacteriophage Lac-GAP-3 solution was added, while the growth of Lactococcus garvieae was not inhibited in the tube to which the bacteriophage solution was not added.

(17) TABLE-US-00001 TABLE 1 Growth inhibition of Lactococcus garvieae OD.sub.600 absorbance value 0 minutes 60 minutes 120 minutes Classification after culture after culture after culture Bacteriophage 0.498 0.992 1.364 solution is not added Bacteriophage 0.498 0.294 0.168 solution is added

(18) The above results indicate that the bacteriophage Lac-GAP-3 of the present invention not only inhibits the growth of Lactococcus garvieae but also has the ability to kill Lactococcus garvieae. Therefore, it is concluded that the bacteriophage Lac-GAP-3 can be used as an active ingredient of the composition for preventing a Lactococcus garvieae infection.

Example 5: Animal Experiment on Prevention of Lactococcus garvieae Infection Using Bacteriophage Lac-GAP-3

(19) The preventive effect of the bacteriophage Lac-GAP-3 on olive flounder subjected to Lactococcus garvieae infection was investigated. A total of 2 groups of fifty juvenile olive flounder 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, olive flounder in an experimental group (the group to which the bacteriophage was administered) was fed with a feed containing the bacteriophage Lac-GAP-3 at 1×10.sup.8 pfu/g according to a conventional feeding method. In contrast, olive flounder 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 Lac-GAP-3 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 Lactococcus garvieae at a level of 1×10.sup.8 cfu/g for two days and thereafter provided respectively twice a day so as to induce a Lactococcus garvieae infection. From the ninth day after the experiment started (the second day after the Lactococcus garvieae infection was induced), streptococcosis pathogenesis was examined in all test animals on a daily basis. The streptococcosis 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 D 9 D 10 D 11 D 12 D 13 D 14 Control group 0.80 0.80 0.76 1.04 1.08 1.12 (bacteriophage is not administered) Experimental group 0.24 0.12 0.04 0.04 0 0 (bacteriophage is administered)

(21) From the above results, it is confirmed that the bacteriophage Lac-GAP-3 of the present invention could be very effective in inhibiting Lactococcus garvieae infection.

Example 6: Example of Treatment of Infectious Diseases of Lactococcus garvieae Using Bacteriophage Lac-GAP-3

(22) The treatment effect of the bacteriophage Lac-GAP-3 on olive flounder suffering from streptococcosis caused by Lactococcus garvieae was investigated. A total of 2 groups of eighty juvenile olive flounder 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. From the fifth day after the experiment started, the feed contaminated with Lactococcus garvieae at a level of 1×10.sup.8 cfu/g was provided twice a day for three days according to a conventional feeding method. Olive flounder subjects showing clinical symptoms of streptococcosis were observed in both water tanks from the last day of the procedure in which the feed contaminated with Lactococcus garvieae was provided. From the next day after the feed contaminated with Lactococcus garvieae was provided for three days (the eighth day after the experiment started), olive flounder in an experimental group (the group to which the bacteriophage was administered) was fed with a feed containing the bacteriophage Lac-GAP-3 (1×10.sup.8 pfu/g) according to a conventional feeding method. In contrast, olive flounder 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 Lac-GAP-3 was not contained according to the same method as in the experimental group. From the third day after the forced infection of Lactococcus garvieae (the eighth day after the experiment started), streptococcosis pathogenesis was examined in all test animals on a daily basis. The streptococcosis pathogenesis caused by Lactococcus garvieae 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 D 8 D 9 D 10 D 11 D 12 D 13 D 14 Control group 0.85 0.93 1.03 1.13 1.23 1.20 1.30 (bacteriophage is not administered) Experimental group 0.90 0.78 0.75 0.73 0.43 0.20 0.13 (bacteriophage is administered)

(24) From the above results, it is confirmed that the bacteriophage Lac-GAP-3 of the present invention could be very effective in the treatment of infectious diseases caused by Lactococcus garvieae.

Example 7: Preparation of Feed Additives and Feeds

(25) Feed additives were prepared using a bacteriophage Lac-GAP-3 solution so that a bacteriophage Lac-GAP-3 was contained in an amount of 1×10.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 additive 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 Lac-GAP-3 solution so that a bacteriophage Lac-GAP-3 was contained in an amount of 1×10.sup.8 pfu per 1 ml of the medicine bath agent. In the method of preparing the medicine bath agent, the bacteriophage Lac-GAP-3 solution was added so that the bacteriophage Lac-GAP-3 was contained in an amount of 1×10.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 Olive Flounder Farming

(29) Improvement in the feeding result upon olive flounder 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 olive flounder was divided into two groups, each including 400 olive flounder (group A; fed with the feed 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 olive flounder, and the sub-groups were classified into a sub-group to which the bacteriophage Lac-GAP-3 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 olive flounder was the juvenile (body weight: 5 to 7 g and body length: 8 to 10 cm), and the juvenile olive flounder of the experimental sub-groups were 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 olive flounder feeding experiment Sub-group classification and expression Bacteriophage Bacteriophage Lac-GAP-3 is is not Application applied 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 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 olive flounder in feeding experiment Dead olive flounder/total olive Mortality Group flounder of experiment (No.) (%) A-{circle around (1)}  6/200 3.0 A-{circle around (2)} 41/200 20.5 B-{circle around (1)}  9/200 4.5 B-{circle around (2)} 53/200 26.5

(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 olive flounder. 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: Korea Research Institute of Bioscience and Biotechnology

(36) Accession number: KCTC 12816BP

(37) Accession date: 20150520