<i>Escherichia coli </i>bacteriophage Esc-COP-7, and use thereof for suppressing proliferation of pathogenic <i>Escherichia coli</i>

Abstract

The present invention relates to a Myoviridae bacteriophage ESC-COP-7 (accession number KCTC 13130BP) isolated from nature, and a method for preventing and treating infections from pathogenic Escherichia coli by means of a composition containing the Myoviridae bacteriophage ESC-COP-7 as an active ingredient, the Myoviridae bacteriophage ESC-COP-7 being characterized by having the capability to specifically kill Escherichia coli, and genome expressed by the SEQ ID 1.

Claims

1. A method for treating a pathogenic Escherichia coli infection, the method comprising: administering to an animal other than a human a composition, wherein the composition comprises a Myoviridae bacteriophage Esc-COP-7 which has an ability to specifically kill Escherichia coli, the genome represented by the nucleotide sequence of SEQ ID NO: 1, and is deposited as accession number: KCTC 13130BP.

2. The method for treating the pathogenic Escherichia coli infection of claim 1, wherein said composition is administered to the animal other than the human as a feed additive, a drinking-water additive, or a disinfectant.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 is an electron micrograph showing the morphology of the bacteriophage Esc-COP-7.

(2) FIG. 2 is a photograph showing the results of an experiment on the ability of the bacteriophage Esc-COP-7 to kill Escherichia coli. The clear zone is a plaque formed by lysis of the target bacteria.

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 Escherichia coli

(4) Samples were collected from nature to isolate the bacteriophage capable of killing Escherichia coli. Here, the Escherichia coli strains used for the bacteriophage isolation had been previously isolated and identified as pathogenic Escherichia coli by the present inventors.

(5) The procedure for isolating 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 Escherichia coli at a ratio of 1/1000, followed by shaking culture at 37° 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 Escherichia coli at a ratio of 1/1000, followed by shaking culture at 37° 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 the bacteriophage. 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 Escherichia coli was included therein.

(6) The spot assay was performed as follows: TSB culture medium was inoculated with Escherichia coli at a ratio of 1/1000, followed by shaking culture at 37° C. overnight. 3 ml (OD.sub.600 of 1.5) of the culture solution of Escherichia coli prepared above was spread on TSA (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 culture medium on which Escherichia coli was spread and then left to dry for about 30 minutes. After drying, the plate culture medium that was subjected to spotting was stationary-cultured at 37° C. for one day, and then examined for the formation of clear zones at the positions where the filtrate was dropped. In the case of the filtrate generated a clear zone, it is judged that the bacteriophage capable of killing Escherichia coli is included therein. Through the above examination, the filtrate containing the bacteriophage having the ability to kill Escherichia coli could be obtained.

(7) The pure bacteriophage was isolated from the filtrate confirmed above to have the bacteriophage capable of killing Escherichia coli. A conventional plaque assay was used to isolate 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 Escherichia coli, followed by culturing at 37° C. for 4 to 5 hours. After the culturing, centrifugation was performed at 8,000 rpm for 20 minutes to obtain a supernatant. The Escherichia coli culture solution was added to the obtained supernatant at a volume ratio of 1/50, followed by culturing at 37° 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 in order 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, a solution containing the pure bacteriophage was obtained. The procedure for isolating the pure bacteriophage was generally repeated until the generated plaques became similar to each other in size and morphology. In addition, final isolation of the pure bacteriophage was confirmed using electron microscopy. The above procedure was repeated until the isolation of the pure bacteriophage was confirmed using electron microscopy. 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 isolated above was confirmed to belong to the Myoviridae bacteriophage.

(8) The solution containing the pure bacteriophage confirmed above was subjected to the following purification process. The Escherichia coli 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.

(9) 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 in order 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.

(10) As a result, the pure bacteriophage purified above was collected, was named the bacteriophage Esc-COP-7, and then deposited under the Budapest Treaty on the International Procedure at Korea Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daijeon 305-806, Republic of Korea; the deposit was made on Oct. 17, 2016 (Accession number: KCTC 13130BP).

Example 2: Separation and Sequence Analysis of Genome of Bacteriophage Esc-COP-7

(11) The genome of the bacteriophage Esc-COP-7 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 Escherichia coli 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 remove 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 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 thoroughly. 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 in order 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 Esc-COP-7.

(12) Information on the sequence of the genome of the bacteriophage Esc-COP-7 obtained above was secured by performing next-generation sequencing analysis using Illumina Mi-Seq equipment from the National Instrumentation Center for

(13) Environmental Management, Seoul National University. The finally analyzed genome of the bacteriophage Esc-COP-7 had a size of 54,200 bp, and the sequence of whole genome was expressed by SEQ. ID. NO: 1.

(14) The homology (similarity) of the bacteriophage Esc-COP-7 genomic sequence obtained above with previously reported bacteriophage genomic sequences was investigated using BLAST (www.ncbi.nlm.nih.gov/BLAST/) on the web. As a result of the BLAST investigation, the genomic sequence of the bacteriophage Esc-COP-7 was found to have a relatively high homology with the sequence of the Escherichia coli bacteriophage phiEcoM-GJ1 (Genbank Accession No. EF460875.1) (identity: 89%). However, the number of open reading frames (ORFs) on the bacteriophage Esc-COP-7 genome is 77, whereas Escherichia coli bacteriophage phiEcoM-GJ1 has 75 open reading frames, unlike the bacteriophage Esc-COP-7.

(15) Based upon this result, it is concluded that the bacteriophage Esc-COP-7 must be a novel bacteriophage different from conventionally reported bacteriophages. Further, since the antibacterial strength and spectrum of bacteriophages typically depend on the type of bacteriophage, it is considered that the bacteriophage Esc-COP-7 can provide antibacterial activity different from that of any other bacteriophages reported previously.

Example 3: Investigation of Ability of Bacteriophage Esc-COP-7 to Kill Pathogenic Escherichia coli

(16) The ability of the isolated bacteriophage Esc-COP-7 to kill pathogenic Escherichia coli 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 10 strains that had been isolated and identified as pathogenic Escherichia coli by the present inventors were used as pathogenic Escherichia coli for the investigation of killing ability. The bacteriophage Esc-COP-7 had the ability to kill a total of 9 strains among 10 strains of pathogenic Escherichia coli as the experimental target. The experimental result thereof is shown in FIG. 2. Meanwhile, the ability of the bacteriophage Esc-COP-7 to kill Bordetella bronchiseptica, Enterococcus faecalis, Enterococcus faecium, Streptococcus mitis, Streptococcus uberis and Pseudomonas aeruginosa was also investigated in a separate experiment. As a result, the bacteriophage Esc-COP-7 did not have the ability to kill these microorganisms.

(17) Therefore, it is confirmed that the bacteriophage Esc-COP-7 has strong ability to kill pathogenic Escherichia coli and a broad antibacterial spectrum against pathogenic Escherichia coli, suggesting that the bacteriophage Esc-COP-7 can be used as an active ingredient of the composition for preventing and treating pathogenic Escherichia coli infection.

Example 4: Experimental Example Regarding Prevention of Pathogenic Escherichia coli Infection Using Bacteriophage Esc-COP-7

(18) 100 μl of a bacteriophage Esc-COP-7 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 pathogenic Escherichia coli culture solution was then added to each tube so that absorbance reached about 0.5 at 600 nm. After pathogenic Escherichia coli was added, the tubes were transferred to an incubator at 37° C., followed by shaking culture, during which the growth of pathogenic Escherichia coli was observed. As presented in Table 1, it was observed that the growth of pathogenic Escherichia coli was inhibited in the tube to which the bacteriophage Esc-COP-7 solution was added, while the growth of pathogenic Escherichia coli was not inhibited in the tube to which the bacteriophage solution was not added.

(19) TABLE-US-00001 TABLE 1 Growth inhibition of pathogenic Escherichia coli OD.sub.600 absorbance value 0 minutes 30 minutes 60 minutes after after after Classification culture culture culture Bacteriophage solution is 0.5 0.8 1.7 not added Bacteriophage solution is 0.5 0.2 0.1 added

(20) The above results indicate that the bacteriophage

(21) Esc-COP-7 of the present invention not only inhibits the growth of pathogenic Escherichia coli but also has the ability to kill pathogenic Escherichia coli. Therefore, it is concluded that the bacteriophage Esc-COP-7 can be used as an active ingredient of the composition for preventing a pathogenic Escherichia coli infection.

Example 5: Example of Treatment of Infectious Diseases of Pathogenic Escherichia coli Using Bacteriophage Esc-COP-7

(22) The therapeutic effect of the bacteriophage Esc-COP-7 on pigs afflicted with pathogenic Escherichia coli was investigated. A total of 2 groups of four 25-day-old weaning pigs per group were prepared and reared separately in experimental farming pig pens (1.1 m×1.0 m), and the experiment was performed for 14 days. The environment surrounding the pens under the warming facility was controlled, and the temperature and humidity in the pig pens were maintained constant, and the floor of the pig pen was cleaned every day. On the 7.sup.th day after the start of the experiment, all pigs were orally administered with a pathogenic Escherichia coli solution using an oral injection tube. The administered pathogenic Escherichia coli solution was prepared as follows. Pathogenic Escherichia coli was cultured at 37° C. for 18 hours using a TSB culture medium, after which the bacteria were isolated and adjusted to 10.sup.9 CFU/ml using physiological saline (pH 7.2). From the day following administration of the pathogenic Escherichia coli, the bacteriophage Esc-COP-7 of 10.sup.9 PFU was orally administered to the pigs in the experimental group (bacteriophage solution-administered group) twice a day in the same manner as the administration of the pathogenic Escherichia coli solution. The pigs in the control group (the group not administered with bacteriophage solution) were not subjected to any treatment. Feed and drinking water were provided to both the control and experimental groups. Diarrhea was examined in all test animals on a daily basis after administration of the pathogenic Escherichia coli. The extent of diarrhea was determined by measuring according to a diarrhea index. The diarrhea index was measured using a commonly used Fecal Consistency (FC) score (normal: 0, soft stool: 1, loose diarrhea: 2, severe diarrhea: 3). The results are shown in Table 2.

(23) TABLE-US-00002 TABLE 2 Result of measurement of diarrhea index Days after administration with pathogenic E. coli 0 1 2 3 4 5 6 Control group 1.0 1.5 1.5 1.25 1.0 1.0 0.75 (bacteriophage solution not administered) Experimental group 0.5 0.5 0.25 0.25 0 0 0 (administered with bacteriophage solution)

(24) From the above results, it is confirmed that the bacteriophage Esc-COP-7 of the present invention could be potentially very effective in the treatment of infectious diseases caused by pathogenic Escherichia coli.

Example 6: Preparation of Feed Additives and Feed

(25) Feed additives were prepared using a bacteriophage Esc-COP-7 solution so that a bacteriophage Esc-COP-7 was contained in an amount of 1×10.sup.9 pfu for 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 powder. In the above-described preparation procedure, the drying procedure can be replaced with drying under reduced pressure, drying with heat, or drying at room temperature. In order to prepare the control for comparison, the feed additives 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 thus prepared were each mixed with a pig-based feed at a weight ratio of 1,000, thus ultimately preparing two kinds of feed.

Example 7: Preparation of Drinking-Water Additives and Disinfectants

(27) Drinking-water additives and disinfectants were prepared in the same manner because they differ only in utilization and are the same in dosage form. The drinking-water additives (or disinfectants) were prepared using a bacteriophage Esc-COP-7 solution so that a bacteriophage Esc-COP-7 was contained in an amount of 1×10.sup.9 pfu for 1 ml of the drinking-water additives (or disinfectants). In the method of preparing the drinking-water additives (or disinfectants), the bacteriophage Esc-COP-7 solution was added so that the bacteriophage Esc-COP-7 was contained in an amount of 1×10.sup.9 pfu for 1 ml of the 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 itself was used as the drinking-water additive (or disinfectant) that did not contain the bacteriophage.

(28) The prepared two kinds of drinking-water additives (or disinfectants) were diluted with water at a volume ratio of 1,000, thus ultimately preparing drinking-water additives (or disinfectants).

Example 8: Confirmation of Feeding Effect on Pig Farming

(29) Improvement in pig farming as the result of feeding was investigated using the feed, drinking water or disinfectant prepared in Examples 6 and 7. In particular, the investigation was focused on mortality. A total of 30 piglets were divided into three groups, each including 10 piglets (group A: fed with the feed, group B: fed with the drinking water, and group C: treated with the disinfectant), and an experiment was performed for four weeks. Each group was divided into sub-groups each including 5 piglets, and the sub-groups were classified into a sub-group to which the bacteriophage Esc-COP-7 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 piglets were 20-day-old weaning piglets, and the piglets of the experimental sub-groups were farmed in separate pens placed apart from each other at a certain space interval. The sub-groups were classified and named as shown in Table 3.

(30) TABLE-US-00003 TABLE 3 Sub-group classification and expression in pig feeding experiment Sub-group classification and expression Bacteriophage Esc-COP-7 Bacteriophage Application is applied is not applied Group fed with feed A-{circle around (1)} A-{circle around (2)} Group fed with drinking B-{circle around (1)} B-{circle around (2)} water Group treated with C-{circle around (1)} C-{circle around (2)} disinfectant

(31) In the case of provision of the feed, the feed prepared in Example 6 was provided according to a conventional feeding method as classified in Table 3, and the drinking water prepared in Example 7 was provided according to a conventional drinking-water feeding method as classified in Table 3. In the case of disinfection, the disinfection was carried out alternately with the existing disinfection 3 times a week. Disinfection using a typical disinfectant was not performed on the day at which the disinfectant of the present invention was sprayed. The experimental results are shown in Table 4.

(32) TABLE-US-00004 TABLE 4 Group Mortality (%) A-{circle around (1)} 0 A-{circle around (2)} 60 B-{circle around (1)} 0 B-{circle around (2)} 40 C-{circle around (1)} 0 C-{circle around (2)} 80

(33) The above results indicate that the provision of the feed and the drinking water prepared according to the present invention and the disinfection according to the present invention were effective in reducing mortality upon pig farming. Therefore, it is concluded that the composition of the present invention is capable of being effectively applied to improving the results of pig 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) [Accession Number]

(36) Name of Depositary Authority: KCTC

(37) Accession number: KCTC 13130BP

(38) Accession date: 20161017