NOVEL LACTOCOCCUS GARVIEAE BACTERIOPHAGE LAC-GAP-1 AND USE THEREOF IN SUPPRESSING PROLIFERATION OF LACTOCOCCUS GARVIEAE BACTERIA

Abstract

The present invention relates to a Myoviridae bacteriophage Lac-GAP-1 that is isolated from the nature and can kill specifically Lactococcus garvieae cells, which has a genome represented by the nucleotide sequence of SEQ. ID. NO: 1 (Accession NO: KCTC 12686BP), and a method for preventing and treating the infections of Lactococcus garvieae using the composition comprising said bacteriophage as an active ingredient.

Claims

1. A Myoviridae bacteriophage Lac-GAP-1 that is isolated from the nature and can kill Lactococcus garvieae cells specifically, which has the genome represented by the nucleotide sequence of SEQ. ID. NO: 1.

2. A composition for preventing and treating the infections of Lactococcus garvieae, which comprises the bacteriophage Lac-GAP-1 of claim 1 as an active ingredient.

3. The composition for preventing and treating the infections of Lactococcus garvieae according to claim 2, wherein said composition is used to prepare an immersion agent or a feed additive.

4. A method for preventing or treating the infections of Lactococcus garvieae, which comprises a step of administering to a subject the composition of claim 2 comprising the bacteriophage Lac-GAP-1 as an active ingredient.

5. The method for preventing or treating the infections of Lactococcus garvieae according to claim 4, wherein said composition is administered to a subject in the form of an immersion agent or a feed additive.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:

[0027] FIG. 1 is an electron micrograph showing the morphology of the bacteriophage Lac-GAP-1.

[0028] FIG. 2 is a photograph illustrating the capability of the bacteriophage Lac-GAP-1 to kill Lactococcus garvieae cells. The clear zone on the dish is the formation of plaque by lysis of bacteria cells.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.

[0030] However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

Example 1: Isolation of Bacteriophage Capable of Killing Lactococcus Garvieae

[0031] Samples were collected from the nature to screen the bacteriophage capable of killing Lactococcus garvieae. In the meantime, the Lactococcus garvieae cells used for the bacteriophage isolation herein were obtained from Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology (Accession NO: KCTC 5619).

[0032] The isolation procedure of the bacteriophage is described in detail hereinafter. The collected sample was added to the TSB (Tryptic Soy Broth) medium (pancreatic digest of casein, 17 g/L; papaic digest of soybean, 3 g/L; dextrose, 2.5 g/L; sodium chloride, 5 g/L; dipotassium phosphate, 2.5 g/L) inoculated with Lactococcus garvieae at the ratio of 1/1000, followed by shaking culture at 30-C for 3˜4 hours. Upon completion of the culture, centrifugation was performed at 8,000 rpm for 20 minutes and supernatant was recovered. The recovered supernatant was inoculated with Lactococcus garvieae at the ratio of 1/1000, followed by shaking culture at 30° C. for 3˜4 hours. When the sample contained the bacteriophage, the above procedure was repeated total 5 times in order to increase the titer of the bacteriophage. After repeating the procedure 5 times, the culture solution proceeded to centrifugation at 8,000 rpm for minutes and the resulting supernatant was recovered. The recovered supernatant was filtrated by using a 0.45 μm filter. The obtained filtrate was used in spot assay for examining whether or not the bacteriophage capable of killing Lactococcus garvieae was included therein.

[0033] Spot assay was performed as follows; TSB medium was inoculated with Lactococcus garvieae at the ratio of 1/1000, followed by shaking culture at 30° C. for overnight. 3 ml (1.5 of OD.sub.600) of the culture broth of Lactococcus garvieae prepared above was spread on the TSA (Tryptic Soy Agar; pancreatic digest of casein, 15 g/L; papaic digest of soybean, 5 g/L; sodium chloride, 5 g/L; agar, 15 g/L) plate. The plate stood in a chamber for about 30 minutes to dry. After drying, 10 μl of the resulting filtrate was spotted directly onto the surface of the Lactococcus garvieae lawns and dried for about 30 minutes. Following drying, the plate was incubated at 30° C. for a day and then, examined for the formation of clear zone on the surface of the bacterial lawns. If a clear zone was generated where the filtrate was dropped, it could be judged that the bacteriophage capable of killing Lactococcus garvieae was included in the filtrate. Through the above procedure, the filtrate containing the bacteriophage having the killing ability of Lactococcus garvieae could be obtained.

[0034] After that, the bacteriophage was isolated from the filtrate confirmed above to have the bacteriophage capable of killing Lactococcus garvieae. The conventional plaque assay was used for the isolation of pure bacteriophages. In detail, a plaque formed in the course of the plaque assay was picked up by using a sterilized tip, which was then added to the culture solution of Lactococcus garvieae, followed by culturing at 30° C. for 4-5 hours. Upon completion of the culture, centrifugation was performed at 8,000 rpm for 20 minutes to obtain supernatant. The recovered supernatant was inoculated with Lactococcus garvieae culture at the ratio of 1/50, followed by culturing at 30° C. for 4 5 hours. To increase the titer of the bacteriophage, the above procedure was repeated at least 5 times. Then, centrifugation was performed at 8,000 rpm for 20 minutes to obtain supernatant. Plaque assay was performed with the obtained supernatant. In general, the pure bacteriophage isolation is not completed by one-time procedure, so the above procedure was repeated by using the plague formed above. After at least 5 times of repeated 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 in sizes and morphologies. And the final pure bacteriophage isolation was confirmed by the observation under electron microscope. Until the pure bacteriophage isolation was confirmed under electron microscope, the above procedure was repeated. The observation under electron microscope was performed by the conventional method. Briefly, the solution containing the pure bacteriophage was loaded on copper grid, followed by negative staining with 2% uranyl acetate. After drying thereof, the morphology was observed under transmission electron microscope. The electron micrograph of the bacteriophage isolated in the present invention is presented in FIG. 1. From the morphological observation, the bacteriophage isolated above was identified as belonging to the family Myoviridae.

[0035] The solution containing the pure bacteriophage confirmed above proceeded to purification. The culture broth of Lactococcus garvieae was added to the solution containing the pure bacteriophage at the volume of 1/50 of the total volume of the bacteriophage solution, followed by culturing again for 4˜5 hours. Upon completion of the culture, centrifugation was performed at 8,000 rpm for 20 minutes to obtain supernatant. This procedure was repeated 5 times to obtain a solution containing enough numbers of the bacteriophage. The supernatant obtained from the final centrifugation was filtered by a 0.45 μm filter, followed by the conventional polyethylene glycol (PEG) precipitation. Particularly, PEG and NaCl were added to 100 Mk of the filtrate until reaching 10% PEG 8000/0.5 M NaCl, which stood at 4° C. for 2˜3 hours. Then, centrifugation was performed at 8,000 rpm for 30 minutes to obtain the bacteriophage precipitate. The resulting bacteriophage precipitate was resuspended in 5 ml of buffer (10 mM Tris-HCl, 10 mM MgSO.sub.4, 0.1% Gelatin, pH 8.0). This solution was called as the bacteriophage suspension or bacteriophage solution.

[0036] As a result, the pure bacteriophage purified above was collected, which was named as the bacteriophage Lac-GAP-1 and then deposited at Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology in Oct. 1, 2014 (Accession NO: KCTC 12686BP).

Example 2: Separation and Sequence Analysis of the Bacteriophage Lac-GAP-1 Genome

[0037] The genome of the bacteriophage Lac-GAP-1 was separated as follows. The genome was separated from the bacteriophage suspension obtained in Example 1. First, in order to eliminate DNA and RNA of Lactococcus garvieae included in the suspension, DNase I and RNase A were added 200 U each to 10 ml of the bacteriophage suspension, which was incubated at 37° C. for 30 minutes. 30 minutes later, to remove the DNase I and RNase A activity, 500 μl of 0.5 μM ethylenediaminetetraacetic acid (EDTA) was added thereto, which was incubated for 10 minutes. The suspension was further incubated at 65° C. for 10 minutes and then added with 100 μl of proteinase K (20 mg/ml) to break the outer wall of the bacteriophage, followed by incubation at 37° C. for 20 minutes. After that, 500 μl of 10% sodium dodecyl sulfate (SDS) solution was added thereto, followed by incubation at 65° C. for 1 hour. 10 ml of the mixture of phenol:chloroform:isoamylalcohol in a ratio of 25:24:1 was added thereto, followed by mixing well. The mixture was centrifuged at 13,000 rpm for 15 minutes to separate each layer. The upper layer was obtained, to which isopropyl alcohol was added at the volume of 1.5 times the volume of the upper layer, followed by centrifugation at 13,000 rpm for 10 minutes to precipitate the genome of the bacteriophage. 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 bacteriophage Lac-GAP-1 genome.

[0038] The nucleotide sequence of the genome of the bacteriophage Lac-GAP-1 obtained above was analyzed by Next Generation Sequencing (NGS) using illumina Mi-Seq device at National Instrumentation Center for Environmental Management, Seoul National University. As a result, it is suggested that the final genome of bacteriophage Lac-GAP-1 have 34,587 bp of size and the nucleotide sequence of the whole genome has SEQ. ID. NO: 1.

[0039] Similarity of the genomic sequence of the bacteriophage Lac-GAP-1 obtained above with the previously reported bacteriophage genome sequences was investigated by using BLAST on Web (http://www.ncbi.nlm.nih.gov/BLAST/). From the BLAST result, it was difficult to find bacteriophage sequences having more than 50% of sequence homology with this bacteriophage sequence.

[0040] Based upon this result, it is concluded that the bacteriophage Lac-GAP-1 should be a novel bacteriophage not reported previously.

Example 3: Investigation of Killing Ability of the Bacteriophage Lac-GAP-1 Against Lactococcus Garvieae

[0041] The killing ability of the isolated bacteriophage Lac-GAP-1 against Lactococcus garvieae was investigated. To do so, the formation of clear zone was observed by the spot assay by the same manner as described in Example 1. The Lactococcus garvieae used for this investigation were total 9 strains which had been isolated and identified as Lactococcus garvieae previously by the present inventors. The bacteriophage Lac-GAP-1 demonstrated the killing ability against 7 strains of Lactococcus garvieae used in this experiment. The representative result of the killing ability test is shown in FIG. 2. In the meantime, the activity of the bacteriophage Lac-GAP-1 to kill Edwardsiella tarda, Vibrio anguillarum, Vibrio ichthyoenteri, Streptococcus parauberis and Streptococcus iniae was also investigated. As a result, it is decided that the bacteriophage Lac-GAP-1 did not have the killing activity against these microorganisms.

[0042] Therefore, it is confirmed that the bacteriophage Lac-GAP-1 has the specific ability to kill Lactococcus garvieae cells and a broad antibacterial spectrum against Lactococcus garvieae, suggesting that the bacteriophage Lac-GAP-1 of the present invention could be used as an active ingredient of the composition for preventing and treating the infections of Lactococcus garvieae.

Example 4: Preventive Effect of Bacteriophage Lac-GAP-1 on the Infections of Lactococcus Garvieae

[0043] 100 μl of the bacteriophage Lac-GAP-1 solution at 1×10.sup.8 pfu/mL was added to a tube containing 9 mL of TSB. To another tube containing 9 mL of TSB, the same amount of TSB was further added. Lactococcus garvieae culture solution was added to each tube until OD.sub.600 reached 0.5. Then, the tubes were transferred in a 30° C. incubator, followed by shaking-culture, during which the growth of Lactococcus garvieae was observed. As presented in Table 1, the growth of Lactococcus garvieae was inhibited in the tube adding the bacteriophage Lac-GAP-1 solution, while the growth of Lactococcus garvieae was not inhibited in the tube not adding the bacteriophage solution.

TABLE-US-00001 TABLE 1 Inhibition of growth of Lactococcus garvieae OD.sub.600 Treatment 0 min. 60 min. 120 min. −bacteriophage 0.50 1.01 1.60 solution +bacteriophage 0.50 0.38 0.21 solution

[0044] The above results indicate that the bacteriophage Lac-GAP-1 not only inhibited the growth of Lactococcus garvieae but also could kill the bacterial cells. Therefore, the bacteriophage Lac-GAP-1 can be used as an active ingredient of the composition in order to prevent the infections of Lactococcus garvieae.

Example 5: Therapeutic Effect of Bacteriophage Lac-GAP-1 on the Infections of Lactococcus Garvieae

[0045] Therapeutic effect of the bacteriophage Lac-GAP-1 on the flatfish suffered from streptococcosis by the infections of Lactococcus garvieae was investigated. Particularly, total 2 groups of juvenile flatfish (10 juvenile flatfish per group, body length 6˜9 cm) at 4 months old were prepared, which were cultured separately in different water tanks for 14 days. Surrounding environment of the water tanks was controlled. The temperature and humidity in the laboratory where the water tanks stayed were also controlled. From the 5.sup.th day of the experiment, feeds adding Lactococcus garvieae cells at 1×10.sup.8 CFU/g were provided twice a day for 3 days according to the conventional feed supply procedure. Flatfish subjects showing clinical symptoms of streptococosis from the last day of this procedure, were observed in both water tanks. From the next day of providing feeds adding Lactococcus garvieae cells for 3 days (the 8.sup.th day of the experiment), flatfish of the experimental groups (adding the bacteriophage) were fed with feeds adding the bacteriophage Lac-GAP-1 at 1×10.sup.8 PFU/g according to the conventional feed supply procedure, while flatfish of the control group (without the bacteriophage) were fed with the same feeds without the bacteriophage Lac-GAP-1 according to the conventional procedure. After the 8.sup.th day of the experiment, all the test animals were examined whether being suffered from streptococcosis or not. The outbreak of streptococcosis was detected by measuring body darkening index. The measurement of body darkening index was performed by the conventional method obtaining Dark Coloration (DC) score (0: normal, 1: light coloration, 2: dark coloration). The results are shown in Table 2.

TABLE-US-00002 TABLE 2 Dark coloration score (average values) Days D8 D9 D10 D11 D12 D13 D14 Control group 1.0 1.3 1.4 1.5 1.3 1.2 1.2 (−bacteriophage) Experimental group 1.0 0.7 0.3 0.2 0.2 0.1 0 (+bacteriophage)

[0046] From the above results, it is confirmed that the bacteriophage Lac-GAP-1 of the present invention could be very efficient to treat the infections of Lactococcus garvieae.

Example 6: Preparation of Feed Additives and Feeds

[0047] Feed additives were prepared by adding the bacteriophage Lac-GAP-1 solution at the concentration of 1×10.sup.8 pfu/g feed additives. The preparation method thereof was as follows: Maltodextrin (40%, w/v) was added to the bacteriophage solution and then trehalose was added to reach 10 weight %. After mixing well, the resulting mixture was freeze-dried. Lastly, the dried mixture was grinded into fine powders. The drying procedure above can be replaced with drying under a reduced pressure, drying at warm temperature, or drying at room temperature. To prepare the control for comparison, feed additives that did not contain the bacteriophage but contained only buffer (10 mM Tris-HCl, 10 mM MgSO.sub.4, 0.1% Gelatin, pH 8.0) were prepared.

[0048] The above two kinds of feed additives were mixed with raw fish-based moist pellet at the volume of 250 times the volume of additives, resulting in two kinds of final feed additives.

Example 7: Preparation of an Immersion Agent (Medicine Bath Agent)

[0049] An immersion agent comprising 1×10.sup.8 pfu/ml of bacteriophage Lac-GAP-1 was prepared. The preparation method was as follows: 1×10.sup.8 pfu of the bacteriophage Lac-GAP-1 was added to 1 mL of buffer, which was well mixed. To prepare the control, the buffer itself that is the same with the one used for the mixture of the bacteriophage solution was prepared.

[0050] The prepared two kinds of immersion agents were diluted with water at the ratio of 1:1,000, resulting in the final immersion agents for the experiment.

Example 8: Effect on Flatfish Aquafarming

[0051] The effect of the feeds and the immersion agents prepared in Example 6 and Example 7 on flatfish aquafarming was investigated. Particularly, the investigation was focused on the mortality. Total 400 flatfish were grouped into two, 200 flatfish for each group, which proceeded to the following experiment (group A; fed with feeds, group B; treated with immersion agent). Each group was divided to two sub-groups again, group of 100 flatfish each (sub-group-{circle around (1)}: treated with the bacteriophage Lac-GAP-1, sub-group-{circle around (2)}: not-treated with the bacteriophage Lac-GAP-1). The flatfish used for this experiment were the juvenile flatfish at 4 months old. Each sub-group flatfish were aquacultured in separate water tanks placed at a certain space interval. Each sub-group was distinguished and named as shown in Table 3.

TABLE-US-00003 TABLE 3 Sub-groups of aquafarming experiment of flatfish Sub-group Treated with the Not-treated bacteriophage with the Treatment Lac-GAP-1 bacteriophage Fed with feeds A-{circle around (1)} A-{circle around (2)} Treated with B-{circle around (1)} B-{circle around (2)} immersion agents

[0052] Feeds were provided according to the conventional feed supply procedure as presented in Table 3 with the feeds prepared as described in Example 6. The treatment of immersion agent was also performed by the conventional procedure as presented in Table 3 with the immersion agent prepared as described in Example 7. The test result is shown in Table 4.

TABLE-US-00004 TABLE 4 Mortality of flatfish in aquafarming Group Mortality (%) A-{circle around (1)} 5 A-{circle around (2)} 22 B-{circle around (1)} 9 B-{circle around (2)} 24

[0053] The above results indicate that the feeds prepared by the present invention and the immersion agent prepared according to the present invention are effective to reduce the mortality of the cultured flatfish. Therefore, it is concluded that the composition of the present invention could be efficiently applied to improve outcomes of flatfish aquaculture.

[0054] Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended Claims.