<i>Streptococcus iniae </i>bacteriophage Str-INP-1 and use of the same for inhibiting the proliferation of <i>Streptococcus iniae</i>

Abstract

The present invention relates to a Siphoviridae bacteriophage Str-INP-1 (Accession NO: KCTC 12687BP) that is isolated from the nature and can kill specifically Streptococcus iniae cells, which has a genome represented by the nucleotide sequence of SEQ. ID. NO: 1, and a method for preventing and treating the infections of Streptococcus iniae using the composition comprising said bacteriophage as an active ingredient.

Claims

1. A pharmaceutical composition for inhibiting or treating the infections of Streptococcus iniae comprising an effective amount of a Siphoviridae bacteriophage Str-INP-1 as an active ingredient, wherein said Siphoviridae bacteriophage is isolated from the nature and can kill Streptococcus iniae cells specifically, wherein the genome of said Siphoviridae bacteriophage comprises the nucleotide sequence of SEQ. ID. NO: 1, and wherein said pharmaceutical composition is formulated in the form of a bath treatment agent or a feed additive.

2. A method for inhibiting or treating the infections of Streptococcus iniae in a subject in need thereof, which comprises administering to the subject a therapeutically effective amount of the pharmaceutical composition as set forth in claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:

(2) FIG. 1 is an electron micrograph showing the morphology of the bacteriophage Str-INP-1.

(3) FIG. 2 is a photograph illustrating the capability of the bacteriophage Str-INP-1 to kill Streptococcus iniae cells. The clear zone on the dish is the formation of plaque by lysis of target bacteria cells.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

(4) Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.

(5) 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 Streptococcus iniae

(6) Samples were collected from the nature to screen the bacteriophage capable of killing Streptococcus iniae. In the meantime, the Streptococcus iniae cells used for the bacteriophage isolation herein were isolated by the present inventors previously and identified to Streptococcus iniae.

(7) The isolation procedure of the bacteriophage is described in detail hereinafter. The collected sample was added to the THB (Todd Hewitt Broth) medium (heart infusion, 3.1 g/L; peptone, 20 g/L; dextrose, 2 g/L; sodium chloride, 2 g/L; disodium phosphate, 0.4 g/L; sodium carbonate, 2.5 g/L) inoculated with Streptococcus iniae at the ratio of 1/1000, followed by shaking culture at 30 C. for 34 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 Streptococcus iniae at the ratio of 1/1000, followed by shaking culture at 30 C. for 34 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 20 minutes and the resulting supernatant was recovered. The recovered supernatant was filtrated by using a 0.45 m filter. The obtained filtrate was used in spot assay for examining whether or not the bacteriophage capable of killing Streptococcus iniae was included therein.

(8) Spot assay was performed as follows; THB medium was inoculated with Streptococcus iniae 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 Streptococcus iniae prepared above was spread on the THA (Todd Hewitt Agar; heart infusion, 3.1 g/L; peptone, 20 g/L; dextrose, 2 g/L; sodium chloride, 2 g/L; disodium phosphate, 0.4 g/L; sodium carbonate, 2.5 g/L; agar, 15 g/L) plate. The plate stood in a chamber for about 30 minutes to dry. After drying, 10 l of the resulting filtrate was spotted directly onto the surface of the Streptococcus iniae 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 is judged that the bacteriophage capable of killing Streptococcus iniae should be included in the filtrate. Through the above procedure, the filtrate containing the bacteriophage having the killing ability of Streptococcus iniae can be obtained.

(9) After that, the bacteriophage was isolated from the filtrate confirmed above to have the bacteriophage capable of killing Streptococcus iniae. The conventional plaque assay was used for the isolation of pure bacteriophage. 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 Streptococcus iniae, followed by culturing at 30 C. for 45 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 Streptococcus iniae culture at the ratio of 1/50, followed by culturing at 30 C. for 45 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 plaque 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 Siphoviridae.

(10) The solution containing the pure bacteriophage confirmed above proceeded to purification. The culture broth of Streptococcus iniae 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 45 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 m filter, followed by the conventional polyethylene glycol (PEG) precipitation. Particularly, PEG and NaCl were added to 100 ml of the filtrate until reaching 10% PEG 8000/0.5 M NaCl, which stood at 4 C. for 23 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.

(11) As a result, the pure bacteriophage purified above was collected, which was named as the bacteriophage Str-INP-1 and then deposited at Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology in Oct. 1, 2014 (Accession NO: KCTC 12687BP).

Example 2: Separation and Sequence Analysis of the Bacteriophage Str-INP-1 Genome

(12) The genome of the bacteriophage Str-INP-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 Streptococcus iniae cells 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 l of 0.5 M 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 l 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 l 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 l of water. This procedure was repeated to obtain a sufficient amount of the bacteriophage Str-INP-1 genome.

(13) The nucleotide sequence of the bacteriophage Str-INP-1 genome obtained above was determined by Next Generation Sequencing analysis using Roche 454 GS Junior device from Chun Lab. Ltd. As a result, it is suggested that the final genome of bacteriophage Str-INP-1 has 33,269 bp of size and the nucleotide sequence of the whole genome has SEQ. ID. NO: 1.

(14) Similarity of the genomic sequence of the bacteriophage Str-INP-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.

(15) Based upon this result, it is concluded that the bacteriophage Str-INP-1 should be a novel bacteriophage not reported previously. Either, it is referred that when bacteriophages are different in their kind, their antibacterial strength and spectrum become different typically. As a consequence, it is confirmed that the bacteriophage Str-INP-1 provides have more remarkable antibacterial activity than any other bacteriophages aforementioned.

Example 3: Investigation of Killing Ability of the Bacteriophage Str-INP-1 Against Streptococcus iniae

(16) The killing ability of the isolated bacteriophage Str-INP-1 against Streptococcus iniae 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 Streptococcus iniae used for this investigation were total 43 strains which had been isolated and identified as Streptococcus iniae previously by the present inventors. The bacteriophage Str-INP-1 demonstrated the killing ability against 36 strains of Streptococcus iniae among these 43 strains 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 Str-INP-1 to kill Edwardsiella tarda, Vibrio anguillarum, Vibrio ichthyoenteri, Lactococcus garvieae and Streptococcus parauberis was also investigated respectively. As a result, it is decided that the bacteriophage Str-INP-1 should not have the killing activity against these microorganisms.

(17) Therefore, it is confirmed that the bacteriophage Str-INP-1 has the specific ability to kill Streptococcus iniae cells and a broad antibacterial spectrum against Streptococcus iniae, suggesting that the bacteriophage Str-INP-1 of the present invention can be used as an active ingredient of the composition for preventing and treating the infections of Streptococcus iniae.

Example 4: Preventive Effect of Bacteriophage Str-INP-1 on the Infections of Streptococcus iniae

(18) 100 l of the bacteriophage Str-INP-1 solution at 110.sup.8 pfu/ml was added to a tube containing 9 ml of THB. To another tube containing 9 ml of THB, the same amount of THB was further added. Streptococcus iniae culture solution was added to each tube until OD.sub.600 reached about 0.5. Then, the tubes were transferred to a 30 C. incubator, followed by shaking-culture, during which the growth of Streptococcus iniae was observed. As presented in Table 1, the growth of Streptococcus iniae was inhibited in the tube adding the bacteriophage Str-INP-1 solution, while the growth of Streptococcus iniae was not inhibited in the tube without adding the bacteriophage solution.

(19) TABLE-US-00001 TABLE 1 Inhibition of growth of Streptococcus iniae OD.sub.600 Treatment 0 min. 60 min. 120 min. bacteriophage 0.498 0.982 1.564 solution +bacteriophage 0.498 0.295 0.142 solution

(20) The above results indicate that the bacteriophage Str-INP-1 should not only inhibit the growth of Streptococcus iniae but also can kill the bacterial cells. Therefore, it is concluded that the bacteriophage Str-INP-1 can be used as an active ingredient of the composition in order to prevent the infections of Streptococcus iniae.

Example 5: Therapeutic Effect of Bacteriophage Str-INP-1 on the Infections of Streptococcus iniae

(21) Therapeutic effect of the bacteriophage Str-INP-1 on the olive flounder suffered from streptococcosis by the infections of Streptococcus iniae was investigated. Particularly, total 2 groups of juvenile olive flounder (50 juvenile olive flounder per group, body length 69 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 Streptococcus iniae cells at 110.sup.8 cfu/g were provided twice a day for 3 days according to the conventional feed supply procedure. Olive flounder 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 Streptococcus iniae cells for 3 days (the 8.sup.th day of the experiment), olive flounder of the experimental groups (adding the bacteriophage) were fed with feeds adding the bacteriophage Str-INP-1 at 110.sup.8 pfu/g according to the conventional feed supply procedure, while olive flounder of the control group (without the bacteriophage) were fed with the same feeds without adding the bacteriophage Str-INP-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.

(22) TABLE-US-00002 TABLE 2 Dark coloration score (average values) Days D 8 D 9 D 10 D 11 D 12 D 13 D 14 Control group 1.04 1.40 1.64 1.72 1.68 1.36 1.16 (bacteriophage) Experimental group 1.00 0.84 0.32 0.20 0.12 0.08 0.04 (+bacteriophage)

(23) From the above results, it is confirmed that the bacteriophage Str-INP-1 of the present invention could be very effective to treat the infection of Streptococcus iniae.

Example 6: Preparation of Feed Additives and Feeds

(24) Feed additives were prepared by adding the bacteriophage Str-INP-1 solution at the concentration of 110.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.

(25) 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)

(26) An immersion agent comprising 110.sup.8 pfu/ml of bacteriophage Str-INP-1 was prepared. The preparation method was as follows: 110.sup.8 pfu of the bacteriophage Str-INP-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.

(27) 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 Olive Flounder Aquafarming

(28) The effect of the feeds and the immersion agents prepared in Example 6 and Example 7 on olive flounder aquafarming was investigated. Particularly, the investigation was focused on the mortality. Total 600 olive flounder were grouped into two, 300 olive flounder for each group, which proceeded to the following experiment (group A; fed with feed, group B; treated with immersion agent). Each group was divided to two sub-groups again, group of 150 olive flounder each (sub-group-{circle around (1)}: treated with the bacteriophage Str-INP-1, sub-group-{circle around (2)}: not-treated with the bacteriophage Str-INP-1). The olive flounder used for this experiment were the juvenile olive flounder at 4 months old. Each sub-group olive flounder were aquacultured in separate water tanks placed at a certain space interval. Each sub-group was distinguished and named as shown in Table 3.

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

(30) 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.

(31) TABLE-US-00004 TABLE 4 Mortality of olive flounder in aquafarming Dead fish/total test Group fish (No.) Mortality (%) A-{circle around (1)} 6/150 4.0 A-{circle around (2)} 37/150 24.7 B-{circle around (1)} 8/150 5.3 B-{circle around (2)} 43/150 28.7

(32) 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 olive flounder. Therefore, it is concluded that the composition of the present invention could be efficiently applied to improve outcomes of olive flounder aquaculture.

(33) 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.