NOVEL SALMONELLA TYPHIMURIUM BACTERIOPHAGE STP-2 AND USE THEREOF FOR INHIBITING PROLIFERATION OF SALMONELLA TYPHIMURIUM

Abstract

The present invention relates to a Siphoviridae bacteriophage STP-2 (Accession number: KCTC 12853BP) isolated from nature and characterized by having the ability to destroy Salmonella Typhimurium and having a genome represented by SEQ ID NO:1; and to a method for preventing and treating diseases caused by Salmonella Typhimurium using Siphoviridae bacteriophage STP-2 containing the same as an active ingredient.

Claims

1. A Siphoviridae bacteriophage STP-2 (Accession number: KCTC 12853BP) isolated from nature, which has an ability to kill Salmonella Typhimurium and has a genome represented by SEQ ID NO: 1.

2. A composition for preventing and treating a disease caused by Salmonella Typhimurium, comprising the bacteriophage STP-2 (Accession number: KCTC 12853BP) of claim 1 as an active ingredient.

3. The composition of claim 2, wherein the composition is used in a form of a feed additive, a drinking-water additive or a disinfectant.

4. A method of preventing and treating a disease caused by Salmonella Typhimurium, comprising: administering to an animal other than a human the composition comprising, as the active ingredient, the bacteriophage STP-2 (Accession number: KCTC 12853BP) of claim 2.

5. The method of claim 4, wherein the composition is administered to the animal other than the human in a form of a feed additive, a drinking-water additive or a disinfectant.

Description

DESCRIPTION OF DRAWINGS

[0027] FIG. 1 shows an electron micrograph of the bacteriophage STP-2; and

[0028] FIG. 2 shows the results of an experiment on the ability of the bacteriophage STP-2 to kill Salmonella Typhimurium, in which, based on the center line of the plate medium, the left side shows the results obtained using only a buffer not containing bacteriophage STP-2, and the right side shows the results obtained using a solution containing bacteriophage STP-2, in which the clear zone, which is observed at the right side, is a plaque formed due to lysis of the target bacteria by the action of bacteriophage STP-2.

MODE FOR INVENTION

[0029] A better understanding of the present invention will be given through the following examples, which are merely set forth to illustrate the present invention, and are not to be construed as limiting the scope of the present invention.

Example 1: Isolation of Bacteriophage Capable of Killing Salmonella Typhimurium

[0030] Samples collected from nature were used to isolate a bacteriophage capable of killing Salmonella Typhimurium. Here, the Salmonella Typhimurium bacteria used for the bacteriophage isolation were obtained from the American Type Culture Collection (ATCC) (ATCC14028).

[0031] The procedure for isolating the bacteriophage is specified below. 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 Salmonella Typhimurium at a ratio of 1/1,000, followed by shaking culture at 37 C. for 3 to 4 hr. After completion of the culture, centrifugation was performed at 8,000 rpm for 20 min and the supernatant was recovered. The recovered supernatant was inoculated with Salmonella Typhimurium at a ratio of 1/1000, followed by shaking culture at 37 C. for 3 to 4 hr. When the bacteriophage was included in the sample, the above procedure was repeated a total of 5 times in order to sufficiently increase the number (titer) of bacteriophages. After the procedure was repeated 5 times, the culture solution was centrifuged at 8,000 rpm for 20 min. After centrifugation, the recovered supernatant was filtered using a 0.45 m filter. The filtrate thus obtained was used in a typical spot assay for examining whether or not a bacteriophage capable of killing Salmonella Typhimurium was included therein.

[0032] The spot assay was performed as follows. A TSB culture medium was inoculated with Salmonella Typhimurium at a ratio of 1/1,000, followed by shaking culture at 37 C. overnight. 3 ml (OD.sub.600 of 1.5) of the Salmonella Typhimurium culture solution prepared as described above was spread on a TSA (Tryptic Soy Agar) plate medium (casein digest, 15 g/L; soybean digest, 5 g/L; NaCl, 5 g/L; agar, 15 g/L). The plate was allowed to stand on a clean bench for about 30 min to dry the spread solution. After drying, 10 l of the filtrate prepared as described above was spotted onto the plate medium on which Salmonella Typhimurium was spread, and was then allowed to stand for about 30 min to dry. After drying, the plate medium that was subjected to spotting was subjected to stationary culture at 37 C. for one day, and then examined for the formation of a clear zone at the position at which the filtrate was dropped. In the case in which the filtrate generated a clear zone, it was judged that a bacteriophage capable of killing Salmonella Typhimurium was included therein. Through the above examination, it was possible to obtain a filtrate containing a bacteriophage having the ability to kill Salmonella Typhimurium.

[0033] The pure bacteriophage was isolated from the filtrate confirmed to have the bacteriophage capable of killing Salmonella Typhimurium. A typical plaque assay was used to isolate the pure bacteriophage. Specifically, a plaque formed in the course of the plaque assay was recovered using a sterilized tip, added to the Salmonella Typhimurium culture solution, and then cultured at 37 C. for 4 to 5 hr. Thereafter, centrifugation was performed at 8,000 rpm for 20 min to obtain a supernatant. The supernatant thus obtained was added with the Salmonella Typhimurium culture solution at a volume ratio of 1/50 and then cultured at 37 C. for 4 to 5 hr. In order to increase the number of bacteriophages, the above procedure was repeated at least 5 times, after which centrifugation was performed at 8,000 rpm for 20 min to obtain a final supernatant. A plaque assay was performed again using the supernatant thus obtained. In general, isolation of a pure bacteriophage is not completed through a single iteration of a procedure, so the above procedure was repeated using the plaque formed above. After at least 5 repetitions of the procedure, the solution containing the pure bacteriophage was obtained. The procedure for isolation of the pure bacteriophage was repeated until the generated plaques became generally similar to each other with regard to size and morphology. Additionally, final isolation of the pure bacteriophage was confirmed using electron microscopy. The above procedure was repeated until isolation of the pure bacteriophage was confirmed using electron microscopy. The electron microscopy was performed according to a typical 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.

[0034] The solution containing the pure bacteriophage confirmed above was subjected to the following purification process. The solution containing the pure bacteriophage was added with the Salmonella Typhimurium culture solution at a volume ratio of 1/50, based on the total volume of the solution, and then cultured for 4 to 5 hr. Thereafter, 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 m filter, followed by a typical polyethylene glycol (PEG) precipitation process. Specifically, 100 ml of the filtrate was added with 10% PEG 8000 and 0.5 M NaCl, allowed to stand at 4 C. for 2 to 3 hr, and then centrifuged at 8,000 rpm for 30 min to obtain a 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.

[0035] The bacteriophage purified as described above was collected, was named bacteriophage STP-2, and was then deposited at the Korea Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology on Jun. 23, 2015 (Accession number: KCTC 12853BP).

[0036]

Example 2: Separation and Sequence Analysis of Genome of Bacteriophage STP-2

[0037] The genome of the bacteriophage STP-2 was separated as follows. The genome was separated from a bacteriophage suspension obtained using the same method as described in Example 1. First, in order to eliminate DNA and RNA of Salmonella Typhimurium included in the suspension, 10 ml of the bacteriophage suspension was added with 200 U of each of DNase I and RNase A and then allowed to stand at 37 C. for 30 min. After being allowed to stand for 30 min, in order to stop the DNase I and RNase A activity, 500 l of 0.5 M ethylenediaminetetraacetic acid (EDTA) was added thereto, and the resulting mixture was then allowed to stand for 10 min. In addition, the resulting mixture was again allowed to stand at 65 C. for 10 min, and 100 pl 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 min. Thereafter, 500 l of 10% sodium dodecyl sulfate (SDS) was added thereto, followed by reaction at 65 C. for 1 hr. After the reaction for 1 hr, the resulting reaction solution was added with 10 ml of a mixed solution of phenol, chloroform and isoamyl alcohol at a component ratio of 25:24:1 and then mixed thoroughly. Then, the resulting mixture was centrifuged at 13,000 rpm for 15 min to thus separate layers. Among the separated layers, the upper layer was selected, added with isopropyl alcohol at a volume ratio of 1.5, and centrifuged at 13,000 rpm for 10 min in order to precipitate the genome. The precipitate was recovered and washed by addition with 70% ethanol, then centrifuged at 13,000 rpm for 10 min. The washed precipitate was recovered, dried in a vacuum and then dissolved in 100 l of water. This procedure was repeated to thus obtain a large amount of the genome of the bacteriophage STP-2.

[0038] The genome thus obtained was subjected to next-generation sequencing analysis using a Pac-bio sequencer from the National Instrumentation Center for Environmental Management, Seoul National University, and then information on the sequence of the genome of bacteriophage STP-2 was obtained. The finally analyzed genome of the bacteriophage STP-2 had a size of 59,043 bp, and the sequence of the whole genome is represented by SEQ ID NO: 1.

[0039] The homology (similarity) of the bacteriophage STP-2 genomic sequence obtained above with previously reported bacteriophage genomic sequences was investigated using BLAST on the web. Based on the results of BLAST investigation, the genomic sequence of the bacteriophage STP-2 was identified to have relatively high homology with the sequence of the Salmonella bacteriophage iEPS5 (GenBank Accession number: KC677662.1) (identity: 99%). However, the bacteriophage STP-2 has a circular genome and the Salmonella bacteriophage iEPS5 has a linear genome. Furthermore, the number of open reading frames (ORFs) on the bacteriophage STP-2 genome was 102, whereas the Salmonella bacteriophage iEPS5 was found to have 73 open reading frames, based on which these two bacteriophages were evaluated to be genetically different. The difference in genetic characteristics between these two bacteriophages can indicate that there are external and functional differences in various characteristics expressed in various ways between the two bacteriophages. Furthermore, the difference between these two bacteriophages also indicates that there is a difference in industrial use of the two bacteriophages.

[0040] Therefore, it can be concluded that the bacteriophage STP-2 is a novel bacteriophage different from previously reported bacteriophages. Moreover, since the antibacterial strength and spectrum of bacteriophages typically vary depending on the type of bacteriophage, it is considered that the bacteriophage STP-2 can provide antibacterial activity different from that of any other previously reported bacteriophage.

[0041]

Example 3: Evaluation of Ability of Bacteriophage STP-2 to Kill Salmonella Typhimurium

[0042] The ability of the isolated bacteriophage STP-2 to kill Salmonella Typhimurium was evaluated. 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 Example 1. A total of 10 strains that had been isolated and identified as Salmonella Typhimurium by the present inventors or obtained from ATCC were used as Salmonella Typhimurium strains for evaluation of killing ability. The bacteriophage STP-2 had the ability to kill a total of 9 strains, including ATCC14028, among 10 strains of Salmonella Typhimurium, which was the experimental target. The representative experimental result is shown in FIG. 2. Meanwhile, the ability of the bacteriophage STP-2 to kill Bordetella bronchiseptica, Enterococcus faecalis, Enterococcus faecium, Streptococcus mitis, Streptococcus uberis and Pseudomonas aeruginosa was also measured. Consequently, the bacteriophage STP-2 was found not to have the ability to kill these microorganisms.

[0043] Therefore, it can be concluded that the bacteriophage STP-2 has strong ability to kill Salmonella Typhimurium and can exhibit antibacterial effects against many Salmonella Typhimurium strains, indicating that the bacteriophage STP-2 can be used as an active ingredient of a composition for preventing and treating diseases caused by Salmonella Typhimurium.

[0044]

Example 4: Experiment for Prevention of Salmonella Typhimurium Infection Using Bacteriophage STP-2

[0045] 100 l of a bacteriophage STP-2 solution at a concentration of 110.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 Salmonella Typhimurium culture solution was then added to each tube so that absorbance reached about 0.5 at 600 nm. After the addition of Salmonella Typhimurium, the tubes were transferred to an incubator at 37 C., followed by shaking culture, during which the growth of Salmonella Typhimurium was observed. As shown in Table 1 below, it was observed that the growth of Salmonella Typhimurium was inhibited in the tube to which the bacteriophage STP-2 solution was added, whereas the growth of Salmonella Typhimurium was not inhibited in the tube to which the bacteriophage solution was not added.

[0046]

TABLE-US-00001 TABLE 1 Growth inhibition of Salmonella Typhimurium OD.sub.600 absorbance value 0 min after 60 min after 120 min after Classification culture culture culture Not added with bacteriophage 0.5 0.9 1.6 solution Added with bacteriophage 0.5 0.2 0.1 solution

[0047] The above results show that the bacteriophage STP-2 of the present invention not only inhibits the growth of Salmonella Typhimurium but also has the ability to kill Salmonella Typhimurium. Therefore, it is concluded that the bacteriophage STP-2 can be used as an active ingredient in a composition for preventing diseases caused by Salmonella Typhimurium.

Example 5: Animal Testing for Prevention of Disease Caused by Salmonella Typhimurium Using Bacteriophage STP-2

[0048] The preventive effect of the bacteriophage STP-2 on diseases caused by Salmonella Typhimurium was evaluated using weaning pigs. Twenty 25-day-old weaning pigs were divided into a total of 2 groups (10 pigs per group) and reared separately in experimental pig-rearing rooms (1.1 m1.0 m), and an experiment 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 110.sup.8 pfu/g of bacteriophage STP-2 was provided to pigs in an experimental group (administered with feed containing the bacteriophage) in a typical feeding manner starting from the beginning of the experiment until the end of the experiment. For comparison therewith, a feed having the same composition but not containing bacteriophage STP-2 was provided to pigs in a control group (administered with feed not containing the bacteriophage) in the same feeding manner starting from the beginning of the experiment until the end of the experiment. For 2 days from the seventh day after the start of the experiment, the feed was further added with 110.sup.8 cfu/g of Salmonella Typhimurium and then provided 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 Salmonella Typhimurium. The detected level of Salmonella Typhimurium in the feces of all test animals was examined daily from the date of feeding with the feed containing Salmonella Typhimurium (from the seventh day after the start of the experiment), and the severity of diarrhea of the pigs was also examined.

[0049] The detection of Salmonella Typhimurium in feces was carried out as follows. The fecal sample was spread on a Salmonella-Typhimurium-selective medium (RAMBACH agar; Merck) and then cultured at 37 C. for 18 to 24 hr. Among the resulting colonies, colonies presumed to be Salmonella Typhimurium were isolated. The colonies thus obtained were used as samples and subjected to polymerase chain reaction (PCR) specific to Salmonella Typhimurium, and thus whether or not the corresponding colonies were Salmonella Typhimurium was finally confirmed.

[0050] The occurrence of diarrhea was determined through comparison with a diarrhea index. The diarrhea index was measured according to a commonly used Fecal Consistency (FC) score (normal: 0, soft stool: 1, loose diarrhea: 2, severe diarrhea: 3).

[0051] The results thereof are shown in Tables 2 and 3 below.

[0052]

TABLE-US-00002 TABLE 2 Results of detection of Salmonella Typhimurium (mean) Number of colonies of Salmonella Typhimurium detected per plate Classification D7 D8 D9 D10 D11 D12 D13 D14 Control group 24 21 18 17 16 17 14 14 (administered with feed not containing bacteriophage) Experimental group 12 8 7 3 1 0 0 0 (administered with feed containing bacteriophage)

TABLE-US-00003 TABLE 3 Diarrhea index Classification D7 D8 D9 D10 D11 D12 D13 D14 Control group 1.6 1.8 2.0 1.6 1.4 1.5 1.2 1.2 (administered with feed not containing bacteriophage) Experimental group 0.6 0.4 0.2 0 0 0 0 0 (administered with feed containing bacteriophage)

[0053] As is apparent from the above results, it can be confirmed that the bacteriophage STP-2 of the present invention was very effective in the prevention of diseases caused by Salmonella Typhimurium.

Example 6: Treatment of Disease Caused by Salmonella Typhimurium Using Bacteriophage STP-2

[0054] The therapeutic effect of the bacteriophage STP-2 on diseases caused by Salmonella Typhimurium was evaluated as follows. 2 groups of forty 2-day-old chicks per group were prepared and reared separately, and an experiment was performed for 14 days. For 3 days from the fifth day after the start of the experiment, a feed containing 110.sup.7 cfu/g of Salmonella Typhimurium was provided in a typical feeding manner. From the next day after the feeding with the feed containing Salmonella Typhimurium for 3 days (from the eighth day after the start of the experiment), a feed containing lx 10.sup.8 pfu/g of bacteriophage STP-2 was provided to chicks in an experimental group (administered with feed containing the bacteriophage) in a typical feeding manner. For comparison, a feed having the same composition but not containing bacteriophage STP-2 was provided to chicks in a control group (administered with feed not containing the bacteriophage) in the same feeding manner. From the eighth day after the start of the experiment, the number of Salmonella Typhimurium bacteria in the feces of the test animals was measured. A Salmonella-Typhimurium-selective medium (RAMBACH agar,

[0055] Merck) was used to prevent interference due to contamination with other bacteria in the measurement of the number of Salmonella Typhimurium bacteria in this example. The sample was spread on the selective medium and then cultured at 37 C. for 18 to 24 hr. The colonies presumed to be Salmonella Typhimurium were isolated from the cultured selective medium, after which Salmonella Typhimurium was identified through polymerase chain reaction (the case where the number of colonies identified as Salmonella Typhimurium through polymerase chain reaction is 10.sup.2 cfu/ml or more=2, the case where the number of colonies identified as Salmonella Typhimurium through polymerase chain reaction is 10.sup.1 to 10.sup.2 cfu/ml=1, and the case where the number of colonies identified as Salmonella Typhimurium through polymerase chain reaction is 10.sup.0 to 10.sup.1 cfu/ml=0). For reference, Salmonella Typhimurium bacteria were observed in the feces of both groups from the day after the end of administration of the feed containing Salmonella Typhimurium bacteria (from the eighth day after the start of the experiment), indicating that forced infection was well induced. The results of measurement of the number of Salmonella Typhimurium bacteria are shown in Table 4 below.

[0056]

TABLE-US-00004 TABLE 4 Results of measurement of number of Salmonella Typhimurium bacteria (mean) Days D8 D9 D10 D11 D12 D13 D14 Control group 1.2 1.2 1.0 1.1 1.0 1.1 1.0 (administered with feed not containing bacteriophage) Experimental group 0.3 0.2 0.2 0 0 0 0 (administered with feed containing bacteriophage)

[0057] As is apparent from the above results, it can be confirmed that the bacteriophage STP-2 of the present invention was very effective in the treatment of diseases caused by Salmonella Typhimurium.

Example 7: Preparation of Feed Additive and Feed

[0058] Feed additives were prepared using a bacteriophage STP-2 solution so that bacteriophage STP-2 was contained in an amount of 110.sup.8 pfu per gram of the feed additive. The feed additives were prepared in a manner in which the bacteriophage solution was added with maltodextrin (50%, w/v) and then freeze-dried, followed by final pulverization into a fine powder. In the above preparation procedure, the drying process may be embodied as drying under reduced pressure, drying with heat, or drying at room temperature. In order to prepare the control for comparison, the feed additives not containing the bacteriophage were prepared using the buffer (10 mM Tris-HCl, 10 mM MgSO.sub.4, 0.1% gelatin, pH 8.0) used in the preparation of the bacteriophage solution, in lieu of the bacteriophage solution.

[0059] Each of the two kinds of feed additives thus prepared was mixed with a poultry feed at a weight ratio of 1,000, thus finally obtaining two kinds of feed.

[0060]

Example 8: Preparation of Drinking-Water Additive and Disinfectant

[0061] 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 STP-2 solution so that the bacteriophage STP-2 was contained in an amount of 110.sup.9 pfu per ml of the drinking-water additive (or disinfectant). In the method of preparing the drinking-water additive (or disinfectant), the bacteriophage STP-2 solution was added so that the bacteriophage STP-2 was contained in an amount of 110.sup.9 pfu per ml of the buffer used in the preparation of the bacteriophage solution, and mixing was sufficiently performed. In order to prepare the control for comparison, the buffer used in the preparation of the bacteriophage solution was used without change as a drinking-water additive (or disinfectant) not containing the bacteriophage.

[0062] The two kinds of drinking-water additives (or disinfectants) thus prepared were diluted with water at a volume ratio of 1,000, thus obtaining final drinking water or disinfectants.

[0063]

Example 9: Confirmation of Feeding Effect on Chicken Farming

[0064] The improvement in chicken farming as the result of feeding was evaluated using the feed, drinking water and disinfectant prepared in Examples 7 and 8. In particular, the present evaluation was focused on mortality ratio. A total of 120 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 an experiment 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 STP-2 was applied (subgroup-{circle around (1)}) and a subgroup to which the bacteriophage was not applied (subgroup-{circle around (2)}). In the present experiment, the chicks were raised separately in individual subgroups. The subgroups were classified and named as shown in Table 5 below.

[0065]

TABLE-US-00005 TABLE 5 Subgroup classification and expression in chicken-feeding experiment Subgroup classification and expression Bacteriophage STP-2 is Bacteriophage is not Application applied 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

[0066] In the case of provision of the feed, the feed prepared in Example 7 was provided in a typical feeding manner, as shown in Table 5, and the drinking water prepared in Example 8 was provided in a typical feeding manner, as shown in Table 5. In the case of disinfection, the disinfection was carried out alternately with conventional 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 experimental results thereof are shown in Table 6 below.

[0067]

TABLE-US-00006 TABLE 6 Mortality in chicken-feeding experiment Group Mortality (%) A-{circle around (1)} 0 A-{circle around (2)} 45 B-{circle around (1)} 5 B-{circle around (2)} 45 C-{circle around (1)} 5 C-{circle around (2)} 45

[0068] 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 at reducing mortality ratio upon chicken farming. Therefore, it is concluded that the composition of the present invention is effective when used to feed chickens.

[0069]

[0070] 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.

[0071]

[0072] [Accession number]

[0073] Name of Depositary Authority: KCTC

[0074] Accession number: KCTC 12853BP

[0075] Accession date: 20150623

[0076]