NOVEL ENTEROPATHOGENIC E. COLI BACTERIOPHAGE ESC-CHP-2 AND USE THEREOF FOR INHIBITING PROLIFERATION OF ENTEROPATHOGENIC E. COLI

Abstract

The present invention relates to a Myoviridae bacteriophage Esc-CHP-2 that is isolated from the nature and can kill specifically enteropathogenic E. coli strains, which has a genome represented by the nucleotide sequence of SEQ. ID. NO: 1 (Accession NO: KCTC 12661BP), and a method for preventing and treating the infections of enteropathogenic E. coli using the composition comprising said bacteriophage as an active ingredient.

Claims

1. A Myoviridae bacteriophage Esc-CHP-2 that is isolated from the nature and can kill enteropathogenic E. coli 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 enteropathogenic E. coli, which comprises the bacteriophage Esc-CHP-2 of claim 1 as an active ingredient.

3. A composition for preventing and treating the infections of enteropathogenic E. coli according to claim 2, wherein said composition is used to prepare a feed additive, a drinking water additive, or a disinfectant.

4. A method for preventing and treating the infections of enteropathogenic E. coli, which comprises a step of administering to a subject the composition of claim 2 comprising the bacteriophage Esc-CHP-2 as an active ingredient.

5. The method for preventing and treating the infections of enteropathogenic E. coli according to claim 4, wherein said composition is administered to a subject in the form of a feed additive, a drinking water additive, or a disinfectant.

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 Esc-CHP-2.

[0028] FIG. 2 is a photograph illustrating the capability of the bacteriophage Esc-CHP-2 to kill enteropathogenic E. coli. 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 Enteropathogenic E. coli

[0031] Samples were collected from the nature to screen the bacteriophage capable of killing enteropathogenic E. coli. The enteropathogenic E. coli used for the bacteriophage isolation herein were the one that had been isolated by the present inventors and identified as enteropathogenic E. coli previously.

[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 enteropathogenic E. coli at the ratio of 1/1000, 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 enteropathogenic E. coli at the ratio of 1/1000, 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 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 enteropathogenic E. coli was included therein.

[0033] Spot assay was performed as follows; TSB medium was inoculated with enteropathogenic E. coli at the ratio of 1/1000, followed by shaking culture at 37° C. for overnight. 3 ml (1.5 of OD.sub.600) of the culture broth of enteropathogenic E. coli prepared above was spread on the TSA (Tryptic Soy Agar; pancreatic digest of casein, 17 g/L; papaic digest of soybean, 3 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 enteropathogenic E. coli 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 zones 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 enteropathogenic E. coli was included in the filtrate. Through the above procedure, the filtrate containing the bacteriophage having the killing ability of enteropathogenic E. coli could be obtained.

[0034] After that, the bacteriophage was isolated from the filtrate confirmed above to have the bacteriophage capable of killing enteropathogenic E. coli. 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 enteropathogenic E. coli, 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 enteropathogenic E. coli 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 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 enteropathogenic E. coli 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 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 Esc-CHP-2 and then deposited at Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology in Aug. 21, 2014 (Accession NO: KCTC 12661BP).

Example 2: Separation and Sequence Analysis of the Bacteriophage Esc-CHP-2 Genome

[0037] The genome of the bacteriophage Esc-CHP-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 enteropathogenic E. coli 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 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 Esc-CHP-2 genome.

[0038] The nucleotide sequence of the genome of the bacteriophage Esc-CHP-2 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 Esc-CHP-2 have 53,363 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 Esc-CHP-2 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 is confirmed that the genomic sequence of the bacteriophage Esc-CHP-2 has a relatively high homology (91%) with the sequence of Enterobacteria bacteriophage phiEcoM-GJ1 (Genbank Accession NO: EF460875.1). However, the number of ORFs (Open Reading Frame) within the genome of bacteriophage Esc-CHP-2 was determined to 83 ORFs, while that of Enterobacteria bacteriophage phiEcoM-GJ1 was 75 ORFs.

[0040] Based upon this result, it is concluded that the bacteriophage Esc-CHP-2 should be a novel bacteriophage not reported previously.

Example 3: Investigation of Killing Ability of the Bacteriophage Esc-CHP-2 Against Enteropathogenic E. Coli

[0041] The killing ability of the isolated bacteriophage Esc-CHP-2 against enteropathogenic E. coli 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 enteropathogenic E. coli used for this investigation were total 12 strains which had been isolated and identified as enteropathogenic E. coli previously by the present inventors. The bacteriophage Esc-CHP-2 demonstrated the killing ability against 9 strains of the enteropathogenic E. coli 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 Esc-CHP-2 to kill Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Lactobacillus plantarum, Streptococcus uberis and Pseudomonas aeruginosa was also investigated. As a result, it is decided that the bacteriophage Esc-CHP-2 did not have the killing activity against these microorganisms.

[0042] Therefore, it was confirmed that the bacteriophage Esc-CHP-2 has the specific ability to kill enteropathogenic E. coli and a broad antibacterial spectrum against enteropathogenic E. coli, suggesting that the bacteriophage Esc-CHP-2 of the present invention could be used as an active ingredient of the composition for preventing and treating the infections of enteropathogenic E. coli.

Example 4: Preventive Effect of Bacteriophage Esc-CHP-2 on the Infections of Enteropathogenic E. coli

[0043] 100 μl of the bacteriophage Esc-CHP-2 solution at 1×10.sup.8 pfu/was added to a tube containing 9 ml of TSB. To another tube containing 9 ml of TSB, only the same volume of TSB was added. Then, the enteropathogenic E. coli culture was added to each tube to prepare bacterial suspension in 0.5 of OD.sub.600. After that, the tubes were transferred to an incubator at 37° C., followed by shaking culture, during which the growth of enteropathogenic E. coli was observed. As presented in Table 1, the growth of enteropathogenic E. coli was inhibited in the tube added with the bacteriophage Esc-CHP-2 solution, while the growth of enteropathogenic E. coli was not inhibited in the tube without the bacteriophage Esc-CHP-2 solution.

TABLE-US-00001 TABLE 1 Inhibition of growth of enteropathogenic E. coli OD.sub.600 Culturing Culturing Culturing Item 0 min. 60 min. 120 min. (−) bacteriophage 0.5 1.3 1.9 solution (+) bacteriophage 0.5 0.3 0.2 solution

[0044] The above results indicate that the bacteriophage Esc-CHP-2 not only inhibited the growth of enteropathogenic E. coli but also could kill them. Therefore, the bacteriophage Esc-CHP-2 can be used as an active ingredient of the composition for preventing the infections of enteropathogenic E. coli.

Example 5: Therapeutic Effect of Bacteriophage Esc-CHP-2 on the Infections of Enteropathogenic E. coli

[0045] Therapeutic effect of the bacteriophage Esc-CHP-2 on animals affected by enteropathogenic E. coli was investigated. 4 weaning pigs at 25 days of age were grouped together; total 2 groups of pigs were raised in a pig pen (1.1 m×1.0 m) for 14 days. Heating system was furnished and the surrounding environment was controlled. The temperature and the humidity of the pig pen were controlled and the floor was cleaned every day. On the 7.sup.th day of the experiment, all the animals were orally administered with 1 mL of enteropathogenic E. coli suspension using an oral injection tube. The enteropathogenic E. coli suspension administered above was prepared as follows: enteropathogenic E. coli was cultured in TSB medium at 37° C. for 18 hours and the bacterial cells were collected by centrifugation. Saline (pH 7.2) was added to the bacterial cell pellet to make cell suspension at a concentration of 10.sup.9 CFU/ml. From the next day of the enteropathogenic E. coli challenge, the experimental group (bacteriophage solution treated pigs) were orally administered with the bacteriophage Esc-CHP-2 (10.sup.9 PFU/head) via the same way as used for the above administration twice a day. The control group (bacteriophage solution non-treated pigs) was treated with nothing. Feeds and drinking water were equally provided to both groups. After the challenge of enteropathogenic E. coli, all the animals were observed every day whether or not they experienced diarrhea. The observation was performed by measuring the diarrhea index. The diarrhea index was set as follows according to Fecal Consistency (FC) score (normal: 0, loose stool: 1, moderate diarrhea: 2, and severe diarrhea: 3). The results are shown in Table 2.

TABLE-US-00002 TABLE 2 Fecal Consistency score Days after enteropathogenic E. coli challenge 0 1 2 3 4 5 6 7 Control group 2.25 2.75 2.5 2.5 2 2 1.5 1.5 (−bacteriophage solution) Experimental group 2.25 2 1 0.5 0.5 0.25 0 0 (+bacteriophage solution)

[0046] From the above results, it is confirmed that the bacteriophage Esc-CHP-2 of the present invention could be very effective to treat the infections of enteropathogenic E. coli.

Example 6: Preparation of Feed Additives and Feeds

[0047] Feed additive containing bacteriophage Esc-CHP-2 at a concentration of 1×10.sup.8 pfu/g was prepared using the bacteriophage Esc-CHP-2 solution. 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% of final concentration. After mixing well, the mixture was freeze-dried. Lastly, the dried mixture was grinded into fine powders. The drying process above can be replaced with vacuum-drying, drying at warm temperature, or drying at room temperature. To prepare the control feed additive for comparison, feed additive that did not contain the bacteriophage but contained buffer (10 mM Tris-HCl, 10 mM MgSO.sub.4, 0.1% Gelatin, pH 8.0) only was prepared.

[0048] The above two kinds of feed additives were mixed with the 1,000 times volume of feed for pig farming respectively, resulting in two kinds of final feeds.

Example 7: Preparation of Drinking Water Additives and Disinfectants

[0049] Drinking water additive and disinfectant are different in intended use but same in the composition, so they have been prepared by the same manner. Drinking water additive (or disinfectant) containing bacteriophage Esc-CHP-2 at a concentration of ix 10.sup.8 pfu/ml was prepared using the bacteriophage Esc-CHP-2 solution. Particularly, to prepare drinking water additive (or disinfectant), the bacteriophage ESC-CHP-2 solution was added to buffer solution to reach 1×10.sup.8 pfu/ml, which was mixed well. For the comparison, the above buffer solution itself was used as the drinking water additive (or disinfectant) that did not contain the bacteriophage.

[0050] The prepared two kinds of drinking water additives (or disinfectants) were diluted in water at the ratio of 1:1000, and then used as drinking water or disinfectant.

Example 8: Effect on Pig Farming

[0051] The effect of the feeds, drinking water, and disinfectant prepared in Example 6 and Example 7 on pig farming was investigated. Particularly, the investigation was focused on diarrhea conditions by fecal consistency score used in Example 5. Total 30 piglets were grouped into three groups, and each group was composed of 10 piglets (group A: feed test group, group B: drinking water test group; and group C: disinfectant test group). The experiment was continued for 2 weeks. Each group was divided by two sub-groups comprising 5 piglets each. The sub-groups were divided according to the treatment of the bacteriophage Esc-CHP-2 or not (sub-group-{circle around (1)}: treated with the bacteriophage Esc-CHP-2; and sub-group-{circle around (2)}: not-treated with the bacteriophage). The piglets used in this experiment were weaning pigs at 20 days of age and raised in a separated room placed at a sufficient distance from each other. Each sub-group was divided and named as shown in Table 3.

TABLE-US-00003 TABLE 3 Sub-groups of pig farming experiment Sub-group Treated with the bacteriophage Not-treated with Item Esc-CHP-2 the bacteriophage Fed with feeds A-{circle around (1)} A-{circle around (2)} Provided with B-{circle around (1)} B-{circle around (2)} drinking water Treated with C-{circle around (1)} C-{circle around (2)} disinfectant

[0052] Feeds were provided according to the conventional feed supply method as presented in Table 3 with the feeds prepared in Example 6. Drinking water was provided according to the conventional water supply method as presented in Table 3 with the drinking water prepared in Example 7. Disinfectant was treated three times a week with taking turns with the conventional disinfectant. That is, on the day when the disinfectant of the present invention was sprayed, the conventional disinfectant was not treated. The results are shown in Table 4.

TABLE-US-00004 TABLE 4 Fecal consistency score of pig farming experiment Group Fecal consistency score d1 d2 d3 d4 d5 d6 d7 d8 d9 d10 d11 d12 d13 d14 A-{circle around (1)} 0.2 0 0 0.2 0 0 0 0 0.2 0 0.2 0 0 0 A-{circle around (2)} 0 0 0.4 0.4 0.4 0.2 0.4 0.4 0.2 0.4 0.4 0.2 0.4 0.2 B-{circle around (1)} 0 0.2 0 0 0.2 0 0.2 0 0 0 0.2 0 0 0 B-{circle around (2)} 0.2 0 0.4 0.4 0.2 0.4 0.2 0.4 0.4 0.2 0.2 0.4 0.2 0.2 C-{circle around (1)} 0 0.2 0 0 0.2 0 0 0.2 0 0 0 0.2 0 0 C-{circle around (2)} 0.2 0 0.2 0.4 0.4 0.4 0.4 0.2 0.4 0.4 0.2 0.4 0.2 0.4

[0053] From the above results, it is confirmed that the feeds, drinking water, and the disinfectant prepared according to the present invention were effective in reducing the animal diarrhea. Therefore, it is concluded that the composition of the present invention could be efficiently applied for the improvement of productivity of animal farming.

[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.