<i>Clostridium perfringens </i>bacteriophage Clo-PEP-2 and use for inhibiting <i>Clostridium perfringens </i>proliferation of same

Abstract

The present invention relates to Siphoviridae bacteriophage Clo-PEP-2 (accession number KCTC 13185BP), separated from nature, which is capable of killing Clostridium perfringens and has a genome expressed by sequence number 1 and a method for preventing or treating diseases, induced by Clostridium perfringens, by means of a composition comprising the Siphoviridae bacteriophage Clo-PEP-2 as an active ingredient.

Claims

1. A method of preventing or treating a disease caused by Clostridium perfringens, the method comprising: administering to an animal other than a human a composition comprising an effective amount of Siphoviridae bacteriophage Clo-PEP-2 having a genome set forth in SEQ ID NO: 1 and deposited under the accession number of KCTC 13185BP, which has an ability to kill Clostridium perfringens.

Description

DESCRIPTION OF DRAWINGS

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

(2) FIG. 2 is a photograph showing the results of an experiment on the ability of the bacteriophage Clo-PEP-2 to kill Clostridium perfringens, in which the clear zone is a plaque formed by lysis of the 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 Clostridium Perfringens

(4) Samples were collected from nature to isolate the bacteriophage capable of killing Clostridium perfringens. Here, the Clostridium perfringens strains used for the bacteriophage isolation had been previously isolated and identified as Clostridium perfringens 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 Clostridium perfringens at a ratio of 1/1000, and then cultured at 37° C. for 3 to 4 hr under anaerobic conditions. After completion of the culture, centrifugation was performed at 8,000 rpm for 20 min and a supernatant was recovered. The recovered supernatant was inoculated with Clostridium perfringens at a ratio of 1/1000, and then cultured at 37° C. for 3 to 4 hr under anaerobic conditions. 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 bacteriophages. After repeating the procedure 5 times, the culture broth was subjected to centrifugation at 8,000 rpm for 20 min. After 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 Clostridium perfringens was included therein.

(6) The spot assay was performed as follows: TSB culture medium was inoculated with Clostridium perfringens at a ratio of 1/1000, and then cultured at 37° C. overnight under anaerobic conditions. 3 ml (OD.sub.600 of 1.5) of the culture broth of Clostridium perfringens prepared above was spread on a TSA (Tryptic Soy Agar: casein digest, 15 g/L; soybean digest, 5 g/L; NaCl, 5 g/L; agar, 15 g/L) plate in an anaerobic incubator. The spread plate culture medium was left in the anaerobic incubator for about 30 min to thus dry the spread solution. After drying, 10 μl of the prepared filtrate was spotted onto the plate culture medium on which Clostridium perfringens was spread and then left to dry for about 30 min in the anaerobic incubator. After drying, the plate culture medium that was subjected to spotting was cultured without shaking at 37° C. for one day under anaerobic conditions, and then examined for the formation of clear zones at the positions where the filtrate was dropped. In the case in which the filtrate generated a clear zone, it was judged that the bacteriophage capable of killing Clostridium perfringens was included therein. Through the above examination, it was possible to obtain a filtrate containing the bacteriophage having the ability to kill Clostridium perfringens.

(7) The pure bacteriophage was isolated from the filtrate confirmed above to have the bacteriophage capable of killing Clostridium perfringens. 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 broth of Clostridium perfringens, followed by culturing at 37° C. for 4 to 5 hr under anaerobic conditions. After the culturing, centrifugation was performed at 8,000 rpm for 20 min to obtain a supernatant. The Clostridium perfringens culture broth was added to the obtained supernatant at a volume ratio of 1/50, followed by culturing at 37° C. for 4 to 5 hr under anaerobic conditions. 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 min 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, the solution containing the pure bacteriophage was obtained. The procedure for isolating the pure bacteriophage was repeated in its entirety until the generated plaques became similar to each other with respect to 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 thereof, the novel bacteriophage that was isolated above was confirmed to be a Siphoviridae bacteriophage.

(8) The solution containing the pure bacteriophage confirmed above was subjected to the following purification process. The Clostridium perfringens culture broth 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 hr under anaerobic conditions. After the culturing, centrifugation was performed at 8,000 rpm for 20 min to obtain a supernatant. This procedure was repeated a total of 5 times in order to obtain a solution containing a sufficient number of bacteriophages. 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 reaching 10% PEG 8000/0.5 M NaCl, which was then left at 4° C. for 2 to 3 hr. Thereafter, centrifugation was performed at 8,000 rpm for 30 min 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 may be referred to as a bacteriophage suspension or bacteriophage solution.

(9) As a result, the pure bacteriophage purified above was collected, was named the bacteriophage Clo-PEP-2, and deposited under the Budapest Treaty on the International Procedure at the Korean 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 Jan. 11, 2017 (Accession number: KCTC 13185BP).

Example 2

Separation and Sequence Analysis of Genome of Bacteriophage Clo-PEP-2

(10) The genome of the bacteriophage Clo-PEP-2 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 Clostridium perfringens 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 min. After being left for 30 min, in order to stop the DNase I and RNase A activity, 500 μl of 0.5 M ethylenediaminetetraacetic acid (EDTA) was added thereto, and the resulting mixture was then left for 10 min. In addition, the resulting mixture was further left at 65° C. for 10 min, 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 reacting at 37° C. for 20 min. Thereafter, 500 μl of 10% sodium dodecyl sulfate (SDS) was added thereto, followed by reacting at 65° C. for 1 hr. After reaction for 1 hr, 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 min to thus 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 min in order to precipitate the genome. After the precipitate was recovered, 70% ethanol was added to the precipitate, followed by centrifugation at 13,000 rpm for 10 min to wash the precipitate. The washed precipitate was recovered, vacuum-dried and then dissolved in 100 μl of water. This procedure was repeated to thus obtain a large amount of the genome of the bacteriophage Clo-PEP-2.

(11) Information on the sequence of the genome of the bacteriophage Clo-PEP-2 obtained above was secured by performing next-generation sequencing analysis using an Illumina Mi-Seq apparatus provided by the National Instrumentation Center for Environmental Management, Seoul National University. The finally analyzed genome of the bacteriophage Clo-PEP-2 had a size of 39,456 bp, and the whole genome sequence is represented by SEQ ID NO: 1.

(12) The homology (similarity) of the bacteriophage Clo-PEP-2 genomic sequence obtained above with previously reported bacteriophage genomic sequences was investigated using BLAST on the web. Based on the results of the BLAST investigation, the genomic sequence of the bacteriophage Clo-PEP-2 was found to have homology with the sequence of the Clostridium perfringens bacteriophage phi130 (GenBank Accession number: JF767208.1), but the identity thereof was low, namely about 69%. Also, the bacteriophage Clo-PEP-2 has an annular genome and Clostridium perfringens bacteriophage phi130 has a linear genome, and thus there is a significant difference in the genomic shape in addition to the sequence between the genomes of these two bacteriophages. Furthermore, the number of open reading frames (ORFs) on the bacteriophage Clo-PEP-2 genome is 64, whereas the Clostridium perfringens bacteriophage phi130 has 55 open reading frames, from which these two bacteriophages are also evaluated to be different.

(13) Therefore, it can be concluded that the bacteriophage Clo-PEP-2 is a novel bacteriophage different from conventionally reported bacteriophages. Moreover, since the antibacterial strength and spectrum of bacteriophages typically depend on the type of bacteriophage, it is considered that the bacteriophage Clo-PEP-2 can provide antibacterial activity different from that of any other bacteriophages reported previously.

Example 3

Investigation of Killing Ability of Bacteriophage Clo-PEP-2 for Clostridium Perfringens

(14) The killing ability of the isolated bacteriophage Clo-PEP-2 for Clostridium perfringens was investigated. In order to evaluate the killing ability, the formation of clear zones was observed using a spot assay in the same manner as described in connection with Example 1. A total of 10 strains that had been isolated and identified as Clostridium perfringens by the present inventors were used as Clostridium perfringens for the investigation of killing ability. The bacteriophage Clo-PEP-2 had the ability to kill a total of 9 strains, among 10 strains of Clostridium perfringens, that is, the experimental target. The representative experimental results thereof are shown in FIG. 2. Meanwhile, the ability of the bacteriophage Clo-PEP-2 to kill Bordetella bronchiseptica, Enterococcus faecalis, Enterococcus faecium, Streptococcus mitis, Streptococcus uberis and Pseudomonas aeruginosa was also measured. Consequently, the bacteriophage Clo-PEP-2 was found not to have the ability to kill these microorganisms.

(15) Therefore, it can be concluded that the bacteriophage Clo-PEP-2 has high ability to kill Clostridium perfringens and an antibacterial effect against many Clostridium perfringens strains, indicating that the bacteriophage Clo-PEP-2 can be used as an active ingredient of the composition for preventing or treating diseases caused by Clostridium perfringens.

Example 4

Experiment for Prevention of Clostridium Perfringens Infection Using Bacteriophage Clo-PEP-2

(16) 100 μl of a bacteriophage Clo-PEP-2 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 Clostridium perfringens culture broth was then added to each tube under anaerobic conditions so that absorbance reached about 0.5 at 600 nm. After addition of Clostridium perfringens, the tubes were cultured at 37° C. under anaerobic conditions, during which the growth of Clostridium perfringens was observed. As shown in Table 1 below, it was observed that the growth of Clostridium perfringens was inhibited in the tube to which the bacteriophage Clo-PEP-2 solution was added, whereas the growth of Clostridium perfringens was not inhibited in the tube to which the bacteriophage solution was not added.

(17) TABLE-US-00001 TABLE 1 Growth inhibition of Clostridium perfringens OD.sub.600 absorbance value 0 min after 60 min after 120 min after Classification culture culture culture Not added with 0.5 0.7 1.3 bacteriophage solution Added with bacteriophage 0.5 0.3 0.2 solution

(18) The above results show that the bacteriophage Clo-PEP-2 of the present invention not only inhibits the growth of Clostridium perfringens but also has the ability to kill Clostridium perfringens. Therefore, it is concluded that the bacteriophage Clo-PEP-2 can be used as an active ingredient of the composition for preventing diseases caused by Clostridium perfringens.

Example 5

Animal Testing for Preventing Disease Caused by Clostridium Perfringens Using Bacteriophage Clo-PEP-2

(19) The preventive effect of the bacteriophage Clo-PEP-2 on diseases caused by Clostridium perfringens was evaluated using weaning pigs. 20 of 25-day-old weaning pigs were divided into a total of 2 groups of 10 pigs per group and reared separately in experimental pig-rearing rooms (1.1 m×1.0 m), and the test was performed for 14 days. The surrounding environment was controlled using a heater, and the temperature and humidity in the pig rooms were maintained constant, and the pig room floors were washed every day. A feed containing 1×10.sup.8 pfu/g of bacteriophage Clo-PEP-2 was supplied to pigs in the experimental group (administered with feed containing the bacteriophage) in a typical feeding manner starting from the test until the end of the test. In contrast, a feed having the same composition but excluding bacteriophage Clo-PEP-2 was supplied to pigs in the control group (administered with feed not containing the bacteriophage) in the same feeding manner starting from the test until the end of the test. For 2 days from the seventh day after the start of the test, the feed was further added with 1×10.sup.8 pfu/g of Clostridium perfringens and then supplied to all of the pigs in the experimental group (administered with feed containing the bacteriophage) and the control group (administered with feed not containing the bacteriophage) twice a day, thereby inducing infection with Clostridium perfringens. The detected level of Clostridium perfringens in the feces of all test animals was examined daily from the date of the feeding with the feed containing Clostridium perfringens (from the seventh day after the start of the test), and the extent of diarrhea of the pigs was also examined.

(20) The detection of Clostridium perfringens in feces was carried out as follows. The fecal sample was spread on a Clostridium-perfringens-selective medium (a TSC agar plate; OXOID) and then cultured at 37° C. for 18 to 24 hr under anaerobic conditions. Among the resulting colonies, colonies presumed to be Clostridium perfringens were isolated. The colonies thus obtained were used as samples and subjected to polymerase chain reaction (PCR) specific to Clostridium perfringens, and thus whether or not the corresponding colonies were Clostridium perfringens was finally confirmed.

(21) 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).

(22) The results are shown in Tables 2 and 3 below.

(23) TABLE-US-00002 TABLE 2 Results of detection of Clostridium perfringens (mean) Number of colonies of Clostridium perfringens bacteria detected per plate medium dish Classification D7 D8 D9 D10 D11 D12 D13 D14 Control group 20 18 15 14 16 15 14 13 (administered with feed not containing bacteriophage) Experimental 10 7 4 3 1 0 0 0 group (administered with feed containing bacteriophage)

(24) TABLE-US-00003 TABLE 3 Diarrhea index Classification D7 D8 D9 D10 D11 D12 D13 D14 Control group 1.1 1.4 2.0 1.5 1.4 1.5 1.4 1.4 (administered with feed not containing bacteriophage) Experimental 0.5 0.3 0.2 0 0 0 0 0 group (administered with feed containing bacteriophage)

(25) As is apparent from the above results, it can be concluded that the bacteriophage Clo-PEP-2 of the present invention is very effective in the prevention of diseases caused by Clostridium perfringens.

Example 6

Treatment of Disease Caused by Clostridium Perfringens Using Bacteriophage Clo-PEP-2

(26) The therapeutic effect of the bacteriophage Clo-PEP-2 on diseases caused by Clostridium perfringens was evaluated as follows. 2 groups of forty 2-day-old chicks per group were prepared and reared separately, and the test was performed for 14 days. For 3 days from the fifth day after the start of the test, a feed containing 1×10.sup.7 cfu/g of Clostridium perfringens was supplied in a typical feeding manner. From the last day of feeding with feed containing Clostridium perfringens, Clostridium perfringens was found in the feces of both groups. From the next day (the eighth day after the start of the test) after the feeding with the feed containing Clostridium perfringens for 3 days, a feed containing 1×10.sup.8 pfu/g of bacteriophage Clo-PEP-2 was supplied to chicks in the experimental group (administered with feed containing the bacteriophage) in a typical feeding manner. In contrast, a feed having the same composition but excluding bacteriophage Clo-PEP-2 was supplied to chicks in the control group (administered with feed not containing the bacteriophage) in the same manner. From the ninth day after the start of the test, the number of Clostridium perfringens bacteria in the feces of the test animals was measured. A Clostridium-perfringens-selective medium (a TSC agar plate) was used to prevent interference with other contaminating bacteria in the measurement of the number of Clostridium perfringens bacteria in this example. The sample was spread on the selective medium under anaerobic conditions and then cultured at 37° C. for 18 to 24 hr under anaerobic conditions. Colonies presumed to be Clostridium perfringens were isolated from the selective medium, after which Clostridium perfringens was identified through polymerase chain reaction (PCR) (the case where the number of colonies identified as Clostridium perfringens through PCR is 10.sup.2 cfu/ml or more=2, the case where the number of colonies identified as Clostridium perfringens through PCR is 10.sup.1˜10.sup.2 cfu/ml=1, and the case where the number of colonies identified as Clostridium perfringens through PCR is 10°˜10.sup.1 cfu/ml=0). The results are shown in Table 4 below.

(27) TABLE-US-00004 TABLE 4 Results of measurement of the number of Clostridium perfringens bacteria (mean) Day D9 D10 D11 D12 D13 D14 Control group (administered 1.0 1.1 1.0 1.1 1.1 1.2 with feed not containing bacteriophage) Experimental group 0.2 0.1 0 0 0 0 (administered with feed containing bacteriophage)

(28) As is apparent from the above results, it can be concluded that the bacteriophage Clo-PEP-2 of the present invention is very effective in the treatment of diseases caused by Clostridium perfringens.

Example 7

Preparation of Feed Additive and Feed

(29) A feed additive was prepared using a bacteriophage Clo-PEP-2 solution so that a bacteriophage Clo-PEP-2 was contained in an amount of 1×10.sup.9 pfu for 1 g of the feed additive. The method of preparing the feed additive 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 process may 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 additive 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.

(30) The two kinds of feed additives thus prepared were each mixed with a poultry-based feed at a weight ratio of 1,000, thus ultimately preparing two kinds of feed.

Example 8

Preparation of Drinking-Water Additive and Disinfectant

(31) A drinking-water additive and a disinfectant were prepared in the same manner because they differ only in utilization and are the same in dosage form. The drinking-water additive (or disinfectant) was prepared using a bacteriophage Clo-PEP-2 solution so that a bacteriophage Clo-PEP-2 was contained in an amount of 1×10.sup.9 pfu for 1 ml of the drinking-water additive (or disinfectant). In the method of preparing the drinking-water additive (or disinfectant), the bacteriophage Clo-PEP-2 solution was added so that the bacteriophage Clo-PEP-2 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 was used without change as the drinking-water additive (or disinfectant) that did not contain the bacteriophage.

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

Example 9

Confirmation of Feeding Effect on Chicken Farming

(33) An improvement in chicken farming as the result of feeding was investigated using the feed, drinking water or disinfectant prepared in Examples 7 and 8. In particular, the investigation was focused on mortality. 120 of 2-day-old chicks were divided into three groups, each including 40 chicks (group A: fed with the feed, group B: fed with the drinking water, and group C: treated with the disinfectant), and the test was performed for four weeks. Each group was divided into subgroups each including 20 chicks, and the subgroups were classified into a subgroup to which the bacteriophage Clo-PEP-2 was applied (subgroup-{circle around (1)}) and a subgroup to which the bacteriophage was not applied (subgroup-{circle around (2)}). In the present test, the chicks were raised separately in individual subgroups. The subgroups were classified and named as shown in Table 5 below.

(34) TABLE-US-00005 TABLE 5 Subgroup classification and expression in chicken feeding test Subgroup classification and expression Bacteriophage Clo-PEP-2 is Bacteriophage Application 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

(35) In the case of provision of the feed, the feed prepared in Example 7 was provided according to a typical feeding method as classified in Table 5, and the drinking water prepared in Example 8 was provided according to a typical drinking-water provision method as classified in Table 5. In the case of disinfection, the disinfection was carried out alternately with existing disinfection 3 times a week. Disinfection using a typical disinfectant was not performed on the day on which the disinfectant of the present invention was sprayed. The test results are shown in Table 6 below.

(36) TABLE-US-00006 TABLE 6 Mortality in chicken feeding test Group Mortality (%) A-{circle around (1)} 0 A-{circle around (2)} 40 B-{circle around (1)} 5 B-{circle around (2)} 40 C-{circle around (1)} 0 C-{circle around (2)} 40

(37) 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 chicken farming. Therefore, it is concluded that the composition of the present invention is capable of being effectively applied to improving the results of chicken feeding.

(38) 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.

(39) [Accession Number]

(40) Name of Depositary Authority: KCTC

(41) Accession number: KCTC 13185BP

(42) Accession date: 20170111