Vibrio parahaemolyticus bacteriophage Vib-PAP-2 and use thereof for inhibiting proliferation of <i>Vibrio parahaemolyticus</i>

Abstract

The present invention relates to: Podoviridae bacteriophage Vib-PAP-2 (accession number KCTC 12910BP) which has the capability to specifically destroy Vibrio parahaemolyticus, is characterized by having a genome represented by SEQ ID NO: 1, and is isolated from nature; and a method for preventing and treating Vibrio parahaemolyticus infections, using a composition containing bacteriophage Vib-PAP-2 as an active ingredient.

Claims

1. A method for treating or decreasing the probability for developing an infection of Vibrio parahaemolyticus, which comprises a step of administering to a subject a composition comprising the Podoviridae bacteriophage Vib-PAP-2 (Accession NO: KCTC 12910BP) that can kill Vibrio parahaemolyticus cells specifically, wherein the Podoviridae bacteriophage Vib-PAP-2 comprises a genome represented by the nucleotide sequence of SEQ. ID. NO: 1, as an active ingredient.

2. The method for treating or decreasing the probability for developing an infection of Vibrio parahaemolyticus according to claim 1, 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

(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 Vib-PAP-2.

(3) FIG. 2 is a photograph illustrating the capability of the bacteriophage Vib-PAP-2 to kill Vibrio parahaemolyticus 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 Vibrio Parahaemolyticus

(6) Samples were collected from the nature to screen the bacteriophage capable of killing Vibrio parahaemolyticus. In the meantime, the Vibrio parahaemolyticus strain used for the bacteriophage isolation herein was obtained from Korean Collection of Type Cultures, Korea Research Institute of Bioscience and Biotechnology (Accession NO: KCTC 2729).

(7) The isolation procedure of the bacteriophage is described in detail hereinafter. The collected sample was added to LB (Luria-Bertani; tryptone, 10 g/L; yeast extract, 5 g/L; sodium chloride, 10 g/L) broth inoculated with Vibrio parahaemolyticus at the ratio of 1/1,000, followed by shaking culture at 37° 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 Vibrio parahaemolyticus at the ratio of 1/1,000, followed by shaking culture at 37° 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 20 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 Vibrio parahaemolyticus was included therein.

(8) Spot assay was performed as follows; LB broth was inoculated with Vibrio parahaemolyticus at the ratio of 1/1,000, followed by shaking culture at 37° C. for overnight. 3 ml (1.5 of OD.sub.600) 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 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 Vibrio parahaemolyticus lawns and dried for about 30 minutes. Following drying, the plate was incubated at 37° 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 Vibrio parahaemolyticus should be included in the filtrate. Through the above procedure, the filtrate containing the bacteriophage having the killing ability of Vibrio parahaemolyticus can be obtained.

(9) After that, the bacteriophage was isolated from the filtrate confirmed above to have the bacteriophage capable of killing Vibrio parahaemolyticus. 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 Vibrio parahaemolyticus, followed by culturing at 37° 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 Vibrio parahaemolyticus culture at the ratio of 1/50, followed by culturing at 37° 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 by using the resulting supernatant. In general, the pure bacteriophage isolation is not completed by one-time procedure, so the above procedure was repeated by using the resulting 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 electron microscopy. Until the pure bacteriophage isolation was confirmed by electron microscopy, the above procedure was repeated. The electron microscopy 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 using a transmission electron microscope. The electron micrograph of the bacteriophage isolated in the present invention is presented in FIG. 1. Based on the morphological characteristics, the bacteriophage isolated above was confirmed as belonging to the family Podoviridae.

(10) The solution containing the pure bacteriophage confirmed above proceeded to purification. The culture broth of Vibrio parahaemolyticus 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 ml 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 suspended 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 Vib-PAP-2 and then deposited at Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology in Sep. 22, 2015 (Accession NO: KCTC 12910BP).

Example 2

Separation and Sequence Analysis of the Bacteriophage Vib-PAP-2 Genome

(12) The genome of the bacteriophage Vib-PAP-2 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 Vibrio parahaemolyticus 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 μl 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 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 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 Vib-PAP-2 genome.

(13) The nucleotide sequence of the bacteriophage Vib-PAP-2 genome obtained above was determined by Next Generation Sequencing analysis using Roche 454 GS Junior device. As a result, it is suggested that the final genome of bacteriophage Vib-PAP-2 has 43,221 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 Vib-PAP-2 obtained above with the previously reported bacteriophage genome sequences was investigated by using BLAST. From the BLAST result, it is demonstrated that the genomic sequence of bacteriophage Vib-PAP-2 has a relatively high homology with the genomic sequence of Vibrio bacteriophage VP93 (Genbank Accession NO: FJ896200.1) (Query coverage/identity: 95%/94%). Nevertheless, the bacteriophage Vib-PAP-2 has a circular genome while the Vibrio bacteriophage VP93 has a linear genome. Thus, it is determined that they should be different kinds of bacteriophages. In addition, the genomic sequence of bacteriophage Vib-PAP-2 was compared to that of Vibrio bacteriophage VP93 by using NEBcutter V2.0 Web program. As a result, it is illustrated that the bacteriophage Vib-PAP-2 genome can be digested in a single cut by 8 kinds of restriction enzymes (AhdI, BglI, BsaI, BseYI, BssHII, EarI, MscI, PsiI), while the Vibrio bacteriophage VP93 can be singly cut by 10 kinds (AcuI, AfeI, BmtI, BseRI, BssHII, EarI, MscI, NheI, NsiI, PflMI). Therefore, it is clarified again that they should be different kinds of bacteriophages.

(15) Based upon this result, it is concluded that the bacteriophage Vib-PAP-2 should be a novel bacteriophage not reported previously. Either, it is referred that when bacteriophages are different in their kinds, their antibacterial strength and spectrum become different typically. As a consequence, it is confirmed that the bacteriophage Vib-PAP-2 provides have more remarkable antibacterial activity than any other bacteriophages aforementioned.

Example 3

Investigation of Killing Ability of the Bacteriophage Vib-PAP-2 Against Vibrio Parahaemolyticus

(16) The killing ability of the isolated bacteriophage Vib-PAP-2 against Vibrio parahaemolyticus 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 Vibrio parahaemolyticus used for this investigation were total 14 strains which had been isolated and identified as Vibrio parahaemolyticus previously by the present inventors. The bacteriophage Vib-PAP-2 demonstrated the killing ability against 13 strains of Vibrio parahaemolyticus 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 Vib-PAP-2 to kill Edwardsiella tarda, Vibrio anguillarum, Vibrio ichthyoenteri, Lactococcus garvieae, Streptococcus parauberis, Streptococcus iniae and Aeromonas salmonicida was also investigated respectively. As a result, it is concluded that the bacteriophage Vib-PAP-2 does not have the killing activity against these microorganisms.

(17) Therefore, it is confirmed that the bacteriophage Vib-PAP-2 has the specific ability to kill Vibrio parahaemolyticus cells and a broad antibacterial spectrum against Vibrio parahaemolyticus, suggesting that the bacteriophage Vib-PAP-2 of the present invention can be used as an active ingredient of the composition for preventing and treating the infections of Vibrio parahaemolyticus.

Example 4

Preventive Effect of Bacteriophage Vib-PAP-2 on the Infections of Vibrio Parahaemolyticus

(18) 100 μl of the bacteriophage Vib-PAP-2 solution at 1×10.sup.8 pfu/ml was added to a tube containing 9 ml of LB broth. To another tube containing 9 ml of LB broth, the same amount of LB broth was further added. Vibrio parahaemolyticus culture solution was added to each tube until OD.sub.600 reached about 0.5. Then, the tubes were transferred to a 37° C. incubator, followed by shaking-culture, during which the growth of Vibrio parahaemolyticus was observed. As presented in Table 1, the growth of Vibrio parahaemolyticus was inhibited in the tube adding the bacteriophage Vib-PAP-2 solution, while the growth of Vibrio parahaemolyticus was not inhibited in the tube without adding the bacteriophage solution.

(19) TABLE-US-00001 TABLE 1 Growth inhibition of Vibrio parahaemolyticus OD.sub.600 Treatment 0 min. 60 min. 120 min. −bacteriophage 0.52 0.92 1.84 solution +bacteriophage 0.53 0.31 0.15 solution

(20) The above results indicate that the bacteriophage Vib-PAP-2 should not only inhibit the growth of Vibrio parahaemolyticus but also can kill Vibrio parahaemolyticus. Therefore, it is concluded that the bacteriophage Vib-PAP-2 can be used as an active ingredient of the composition in order to prevent the infections of Vibrio parahaemolyticus.

Example 5

Preventive Effect of Bacteriophage Vib-PAP-2 on the Infections of Vibrio Parahaemolyticus in Animal Model

(21) Preventive effect of the bacteriophage Vib-PAP-2 on sea basses suffered from Vibrio parahaemolyticus infection was investigated. Particularly, total 2 groups of juvenile sea bass (50 juvenile sea basses per group; body weight 5˜7 g, body length 8˜10 cm) 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 1.sup.st day of the experiment, sea basses of the experimental groups (adding the bacteriophage) were fed with feeds adding the bacteriophage Vib-PAP-2 at 1×10.sup.8 pfu/g according to the conventional feed supply procedure, while sea basses of the control group (without adding the bacteriophage) were fed with the same feed without adding the bacteriophage according to the conventional procedure. From the 7.sup.th day of the experiment, the feeds of both groups were contaminated with Vibrio parahaemolyticus at 1×10.sup.8 pfu/g for 2 days and thereafter provided respectively twice a day so as to bring about the infections of Vibrio parahaemolyticus. From the next day of inducing such an infection for 2 days (the 9.sup.th day of the experiment), the feeds without contaminated Vibrio parahaemolyticus were provided again respectively for both the groups. Then, all the test animals were examined whether being suffered from Vibrio parahaemolyticus infection or not. The outbreak of infectious disease caused by Vibrio parahaemolyticus was detected by measuring a 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 9 D 10 D 11 D 12 D 13 D 14 Control group 0.68 0.72 0.76 0.92 1.08 1.20 (−bacteriophage) Experimental group 0.16 0 0 0 0 0. (+bacteriophage)

(23) From the above results, it is confirmed that the bacteriophage Vib-PAP-2 of the present invention could be very effective to prevent infectious diseases caused by Vibrio parahaemolyticus.

Example 6

Therapeutic Effect of Bacteriophage Vib-PAP-2 on the Infections of Vibrio Parahaemolyticus

(24) Therapeutic effect of the bacteriophage Vib-PAP-2 on sea basses suffered from Vibrio parahaemolyticus infection was investigated. Particularly, total 2 groups of juvenile sea bass (60 juvenile sea basses per group; body weight 5˜7 g, body length 8˜10 cm) 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 Vibrio parahaemolyticus cells at 1×10.sup.8 cfu/g were provided twice a day for 3 days according to the conventional feed supply procedure. Sea bass subjects showing clinical symptoms of infectious disease caused by Vibrio parahaemolyticus from the last day of this procedure, were observed in both water tanks. From the next day of providing feeds adding Vibrio parahaemolyticus cells for 3 days (the 8.sup.th day of the experiment), sea basses of the experimental groups (adding the bacteriophage) were fed with feeds adding the bacteriophage Vib-PAP-2 at 1×10.sup.8 pfu/g according to the conventional feed supply procedure, while sea basses of the control group (without the bacteriophage) were fed with the same feeds without adding the bacteriophage Vib-PAP-2 according to the conventional procedure. After the 8.sup.th day of the experiment, all the test animals were examined whether being suffered from infectious disease caused by Vibrio parahaemolyticus or not. The outbreak of infectious disease caused by Vibrio parahaemolyticus 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 3.

(25) TABLE-US-00003 TABLE 3 Dark coloration score (average values) Days D 8 D 9 D 10 D 11 D 12 D 13 D 14 Control group 0.93 1.00 1.07 1.13 1.23 1.37 1.40 (−bacteriophage) Experimental group 0.87 0.80 0.77 0.63 0.40 0.23 0.20 (+bacteriophage)

(26) From the above results, it is confirmed that the bacteriophage Vib-PAP-2 of the present invention could be very effective to treat the infectious disease caused by Vibrio parahaemolyticus.

Example 7

Preparation of Feed Additives and Feeds

(27) Feed additives were prepared by adding the bacteriophage Vib-PAP-2 solution at the concentration of 1×10.sup.8 pfu/g feed additives. The preparation method thereof was as follows: Maltodextrin (50%, w/v) was added to the bacteriophage solution, mixed and then 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.

(28) 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 8

Preparation of an Immersion Agent (Medicine Bath Agent)

(29) An immersion agent comprising 1×10.sup.8 pfu/ml of bacteriophage Vib-PAP-2 was prepared. The preparation method was as follows: 1×10.sup.8 pfu of the bacteriophage Vib-PAP-2 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.

(30) 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 9

Effect on Sea Bass Aquafarming

(31) The effect of the feeds and the immersion agents prepared in Example 7 and Example 8 on sea bass aquafarming was investigated. Particularly, the investigation was focused on the mortality. Total 500 juvenile sea basses (body weight 5˜7 g, body length 8˜10 cm) were grouped into two, 250 sea basses 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 125 sea basses each (sub-group—{circle around (1)}: treated with the bacteriophage Vib-PAP-2, sub-group—{circle around (2)}: not-treated with the bacteriophage Vib-PAP-2). Each sub-group sea bass were aquacultured in separate water tanks placed at a certain space interval. Each sub-group was distinguished and named as shown in Table 4.

(32) TABLE-US-00004 TABLE 4 Sub-groups of sea bass in aquafarming experiment Sub-group Treated with the bacteriophage Vib- Not-treated with Treatment PAP-2 the bacteriophage Fed with feed A-{circle around (1)} A-{circle around (2)} Treated with B-{circle around (1)} B-{circle around (2)} immersion agent

(33) Feeds were provided according to the conventional feed supply procedure as presented in Table 4 with the feeds prepared as described in Example 7. The treatment of immersion agent was also performed by the conventional procedure as presented in Table 4 with the immersion agent prepared as described in Example 8. The test result is shown in Table 5.

(34) TABLE-US-00005 TABLE 5 Mortality of sea bass in aquafarming Dead fish/total test Group fish (No.) Mortality (%) A-{circle around (1)}  3/125 2.4 A-{circle around (2)} 25/125 20.0 B-{circle around (1)}  8/125 6.4 B-{circle around (2)} 32/125 25.6

(35) 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 sea basses. Therefore, it is concluded that the composition of the present invention could be efficiently applied to improve outcomes of sea bass aquaculture.

(36) 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.