Clostridium perfringens bacteriophage as well as composition, kit and application thereof

Abstract

The invention relates to a clostridium perfringens phage and application thereof. The bacteriophage is a clostridium perfringens bacteriophage P2ACQJS03 (wherein the clostridium perfringens bacteriophage P2ACQJS03), and the preservation number of the clostridium perfringens bacteriophage P2ACQJS03 in the China Center for Type Culture Collection (CCTCC) is CCTCC NO: M 20242430. The bacteriophage provided by the invention is a virulent bacteriophage separated from the nature, tests prove that the bacteriophage has no toxic effect on normal microbial flora, and the DNA of the bacteriophage cannot encode virulence genes, so that the bacteriophage is high in stability; the bacteriophage disclosed by the invention is wide in host range, and large-scale industrial production can be realized. The bacteriophage provides an excellent strain resource for developing a novel antibacterial preparation, and has a good application and development prospect.

Description

Technical Field

[0001] The present application belongs to the field of bacteriophage technology, and in particular relates to a Clostridium perfringens bacteriophage and a composition, a kit and an application thereof. Background Art

[0002] Clostridium perfringens, also known as Clostridium perfringens, is a spore-forming Gram-positive anaerobic bacterium widely distributed in soil, feed, sewage, animal digestive tract and feces. The bacterium can produce six major toxins (α, β, ε, ι, β2 and enterotoxins), which are foodborne pathogens that can cause food poisoning in humans and necrotic enteritis in animals. It causes diseases in the production process of animal products and spreads through the processing links, threatening human health and causing huge economic losses to the breeding industry. Clostridium perfringens is the main pathogen that causes necrotic enteritis (NE) in animals, causing great economic losses.

[0003] The long-term use and abuse of antibiotics have led to an increasingly serious problem of drug resistance in Clostridium perfringens. The resistance rates of Clostridium perfringens from chicken, pig and sheep were 66%, 82% and 100% respectively, and the resistance rates to erythromycin, lincomycin, neomycin, tetracycline and ciprofloxacin were very high. In addition, resistance genes and virulence genes can be transmitted and spread in Clostridium perfringens through plasmids, making the prevention and control of Clostridium perfringens more difficult.

[0004] Bacteriophages are a type of virus that specializes in lysing bacteria. Their main chemical components are proteins and nucleic acids. They are widely found in soil, air, water and organisms, and have strong specificity. Due to their high efficiency in self-replication, specificity against pathogens, no impact on normal flora and no resistance, there have been many reports of successful applications abroad, and they have great potential in replacing antibiotics to prevent and treat Clostridium perfringens infections.

[0005] The related art discloses a Myoviridae phage Clo-PEP-1 that can specifically kill Clostridium perfringens cells, as well as a method for preventing Clostridium perfringens infection and then treating using a composition containing the phage as an active ingredient.

[0006] The related technology discloses a phage composition comprising 4 new strains of Clostridium perfringens phages, and the composition is used to prepare a lysed Clostridium perfringens product for the prevention and treatment of Clostridium perfringens infection in breeding enterprises, thereby reducing the use of antibiotics in breeding enterprises and improving the economic benefits of the enterprises.

[0007] The above-mentioned prior art solutions have the following defects: the lysis ability of the above-mentioned Clostridium perfringens phage or the tolerance to high temperature environment above 50° C. is limited, and it is urgent to provide a new Clostridium perfringens phage. Summary of the invention

[0008] In view of the deficiencies in the prior art, the present application provides a new Clostridium perfringens phage, which has a strong lysis ability and can withstand a high temperature environment of 55 to 70°C or above, and can be well applied in the prevention and treatment of Clostridium perfringens related diseases.

[0009] To this end, the first aspect of the present application provides a Clostridium perfringens phage, wherein the phage is Clostridium perfringens phage P2ACQJS03 (Clostridium perfringens phage P2ACQJS03), and the deposit number of the phage in the China Center for Type Culture Collection is CCTCC NO: M 20242430.

[0010] In some embodiments, the bacteriophage has a polyhedral three-dimensional symmetrical head and a short contracted tail, the head has a diameter of 40 to 50 nm, the tail has a length of 10 to 13 nm, and the tail has a diameter of 4 to 6 nm.

[0011] In some embodiments, the phage was cultured for 4 h under MOI=0.001 and its titer was as high as 2.2 x 10 13 PFU / mL.

[0012] In this application, the multiplicity of infection (MOI) is the ratio of the number of phages to the number of bacteria, and is an important basis for studying the relationship between the amount of phage-infected bacteria and the output of phage progeny. The Clostridium perfringens phage described in this application only needs to be added in an appropriate amount to infect Clostridium perfringens to proliferate and obtain a large number of progeny phages. Therefore, the Clostridium perfringens phage described in this application can provide a source of high-quality phage strains for industrial production of biocides.

[0013] In some embodiments, the bacteriophage is resistant to temperatures between 55°C and 70°C.

[0014] In the present application, the "tolerance" means that the titer of the Clostridium perfringens phage when cultured at a temperature of 55-70°C is only slightly lower than that when cultured at the optimal temperature, but the order of magnitude does not change. The Clostridium perfringens phage described in the present application can tolerate a high temperature environment, thereby meeting the specificity requirements.

[0015] In the present application, the Clostridium perfringens phage has good stability under the condition of pH=4-11, and the phage has good tolerance under acidic conditions and has a certain tolerance to alkaline conditions.

[0016] The second aspect of the present application provides a phage composition, which comprises the Clostridium perfringens phage as described in the first aspect of the present application.

[0017] In some embodiments, the composition further comprises at least one chemical sterilant.

[0018] The present application does not clearly limit the specific type of the chemical bactericide in the composition, and those skilled in the art can make a conventional selection. For example, the chemical bactericide can be any one of didecyldimethylammonium chloride with a concentration of 0.05% and chlorhexidine with a concentration of 0.01%. As an exemplary illustration, the ratio of the Clostridium perfringens phage to the chemical bactericide can be determined by those skilled in the art in combination with the present invention, the actual application field, and the common sense in the art.

[0019] The third aspect of the present application provides a biofungicide, which is suitable for lysing Clostridium perfringens. The preparation of the biofungicide uses the Clostridium perfringens phage or the phage composition for lysing Clostridium perfringens; the dosage form of the biofungicide is one of a solution, a powder, a gel, a granule or a lyophilized agent.

[0020] The Clostridium perfringens phage or phage composition described in the present application can specifically kill Clostridium perfringens in the environment, and thus can be used in the preparation of a biocide for killing Clostridium perfringens. The biocide can kill Clostridium perfringens, thereby improving the distribution of microorganisms in the environment; at the same time, the biocide can also be sprayed in a food production workshop to prevent and control contamination by Clostridium perfringens and other bacteria during food processing.

[0021] The fourth aspect of the present application provides a medicine, which is suitable for treating or preventing diseases caused by Clostridium perfringens infection, and the preparation of the medicine uses the Clostridium perfringens phage.

[0022] The Clostridium perfringens phage described in the present application can kill Clostridium perfringens efficiently and specifically, and thus can be used in the preparation of a drug for treating or preventing a disease caused by Clostridium perfringens infection. When an animal eats the drug, the Clostridium perfringens phage in the drug can specifically kill Clostridium perfringens in the animal, thereby treating or preventing a disease caused by Clostridium perfringens infection.

[0023] As used herein, the term "treating" means (i) inhibiting a disease caused by Clostridium perfringens cells; and (ii) ameliorating a disease caused by Clostridium perfringens cells.

[0024] The fifth aspect of the present application provides a feed additive, wherein the preparation of the feed additive uses the Clostridium perfringens phage.

[0025] Since the Clostridium perfringens phage described in the present application can kill Clostridium perfringens efficiently and specifically, adding the feed additive containing the Clostridium perfringens phage to the feed can specifically and continuously prevent and treat the contamination of Clostridium perfringens in feed production, storage and animal breeding. In addition, it can also inhibit the infection of Clostridium perfringens in the intestinal tract of animals and enhance the resistance of animals.

[0026] This application has the following beneficial effects:

[0027] (1) The Clostridium perfringens phage described in the present application has a strong ability to withstand high temperatures, can specifically partially or completely inactivate Clostridium perfringens, and provides a source of phage strains for the industrial production of phage fungicides;

[0028] (2) The Clostridium perfringens phage described in this application is a virulent phage isolated from nature, and toxicological experiments have proven that it is safe and has no side effects; the phage DNA used for testing cannot encode proteins that may cause potential health risks, and does not contain virulence genes or adverse genes; this application has not made any genetic modification to the phage used for testing;

[0029] (3) The Clostridium perfringens phage described in the present application proliferates well in the Clostridium perfringens host, and thus can be fermented and cultured on a large scale;

[0030] (4) The interaction between the Clostridium perfringens phage and non-host bacteria described in the present application cannot identify any strain of the non-host bacteria tested, and has good specificity;

[0031] (5) The Clostridium perfringens phage described in the present application has no antagonistic effect on other combined substances and can be used in combination with chemical bactericides; the Clostridium perfringens phage described in the present application can be used as an active ingredient in various products used for environmental disinfection, such as, but not limited to, disinfection and decontamination of water distribution systems, irrigation facilities, aquaculture facilities, public and private facilities or other environmental surfaces in the form of liquid immersion, spraying, and combined use with aqueous carriers, which can effectively control the growth and activity of target bacteria; the liquid immersion and spraying forms include, but are not limited to, detergents, disinfectants, detergents, etc.; the aqueous carriers include, but are not limited to, phosphate buffer, TSB medium, LB medium, chlorine-free water, etc.;

[0032] (6) The Clostridium perfringens phage described in the present application can be used to prepare a composition, a reagent or a kit for rapid detection of Clostridium perfringens, including but not limited to detecting Clostridium perfringens in a target sample in the form of a test strip, a kit, etc., or screening target pathogens in clinical samples, to effectively ensure the sensitivity of the detection;

[0033] (7) The Clostridium perfringens phage described in the present application can effectively kill Clostridium perfringens on the surface of meat food and prevent Clostridium perfringens contamination of meat food during storage, and can be used as an effective ingredient in various food protection products. The present application includes but is not limited to the prevention of food spoilage caused by Clostridium perfringens infection by liquid immersion, spraying, and combined use with synthetic components, and is particularly suitable for cooked food or food that is not suitable for sterilization. The liquid immersion and spraying forms include but are not limited to food sterilizers, food disinfectants, food preservatives, etc.; the synthetic components in the present invention include but are not limited to benzoic acid, sodium benzoate, sorbic acid, potassium sorbate, calcium propionate, etc.;

[0034] (8) The Clostridium perfringens phage described in the present application can be used alone or in combination, and can be prepared into a biocide, feed additive and therapeutic drug by a person skilled in the art based on the disclosure of the present invention and the common knowledge in the field, and applied to treat or prevent infectious diseases caused by Clostridium perfringens. Hosts that can be infected by Clostridium perfringens include humans, livestock (pigs, cattle, sheep, etc.), poultry (chickens, ducks, geese, etc.), and various mammals, fish, rodents, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a photo of the plaque morphology of the Clostridium perfringens phage described in this application.

[0036] Figure 2 This is a schematic diagram of the morphological structure of the Clostridium perfringens phage described in the present application under a transmission electron microscope.

[0037] Figure 3 The figure is a predicted map of the gene structure of the Clostridium perfringens phage.

[0038] Figure 4 It is a restriction enzyme cleavage site distribution map of the Clostridium perfringens phage genome.

[0039] Figure 5 It is a genetic evolutionary tree analysis diagram of the Clostridium perfringens phage.

[0040] Figure 6 It is the lysis ability of the Clostridium perfringens phage to Clostridium perfringens.

[0041] Figure 7The lysis result of the Clostridium perfringens phage on non-host pathogenic bacteria; Figure 7 In the figure, the numbers 1-16 of the bacterial species represent: 1. Clostridium difficile, 2. Clostridium equi, 3. Clostridium haemolyticum, 4. Clostridium novyi, 5. Clostridium botulinum, 6. Clostridium tetani, 7. Streptococcus agalactiae, 8. Streptococcus dysgalactiae, 9. Salmonella, 10. Staphylococcus aureus, 11. Pseudomonas aeruginosa, 12. Klebsiella pneumoniae, 13. Acinetobacter baumannii, 14. Escherichia coli, 15. Listeria monocytogenes, 16. Shigella.

[0042] Figure 8 The lysis result of the Clostridium perfringens phage on non-pathogenic beneficial bacteria; Figure 8 In the figure, the bacterial species numbers 17-22 represent: 17. Clostridium butyricum, 18. Clostridium sporogenes, 19. Non-pathogenic rhizobium, 20. Non-pathogenic Bacillus subtilis, 21. Non-pathogenic Bacillus megaterium, 22. Non-pathogenic Bacillus licheniformis. DETAILED DESCRIPTION

[0043] In order to make the present application easier to understand, the present application will be further described in detail below in conjunction with the examples, which are merely illustrative and are not intended to limit the scope of application of the present application. The raw materials or components used in the present application can be obtained through commercial routes or conventional methods unless otherwise specified.

[0044] Biological deposit information

[0045] Classification and nomenclature: Clostridium perfringens bacteriophage;

[0046] Collection institution: China Center for Type Culture Collection

[0047] Abbreviation of depository institution: CCTCC

[0048] Address: School of Life Sciences, Wuhan University

[0049] Date of deposit: November 4, 2024

[0050] Registration number of the Collection Center: CCTCC NO: M 20242430.

[0051] The trypsin-sulfite-cycloserine agar base (TSC) used in the examples was purchased from Qingdao Haibo Biological Company.

[0052] In the following examples, the strain codes involved are all the naming methods of our company.

[0053] The formula of the culture medium adopted in the following examples is as follows:

[0054] The formula of TSB liquid medium is: 15g tryptone, 5g soy peptone, 5g sodium chloride, 1000mL distilled water, 121℃, 20min high pressure steam sterilization;

[0055] The formula of TSA solid medium is: 15g tryptone, 5g soy peptone, 5g sodium chloride, 15g agar, 1000mL distilled water, 121℃, 20min high pressure steam sterilization;

[0056] The formula of TSB semi-solid agar medium is: 15g tryptone, 5g soy peptone, 5g sodium chloride, 7g agar, 1000mL distilled water, 121℃, 20min high pressure steam sterilization;

[0057] The formula of SM solution is: sodium chloride 5.8g, magnesium sulfate 2g, 1mol / LTris-HCl 50mL, gelatin 0.25g, distilled water 1000mL, 121℃, high pressure steam sterilization for 20min;

[0058] The formula of porcine artificial gastric juice is as follows: dissolve 0.32g pepsin in 100mL 0.03M NaCl solution prepared with sterile water, adjust the pH of the mixed solution to about 1.2 with hydrochloric acid, and use it immediately after preparation;

[0059] The formula of artificial small intestinal fluid is as follows: dissolve 1g pancreatin in 100mL 0.05M KHPO4 solution prepared with sterile water, adjust the pH to 7.5, and use it immediately after preparation;

[0060] The formula of 2% bile salt solution is: 2g porcine bile salt, 98mL distilled water, dissolved and filtered for sterilization.

[0061] Example 1: Isolation, preparation and purification of Clostridium perfringens phage

[0062] The source sample of the Clostridium perfringens phage described in the present application was collected from sewage from a farm in Suqian City, Jiangsu Province, filtered through double-layer filter paper, centrifuged at low speed at room temperature, and then the supernatant was filtered through a 0.22 μm filter membrane.

[0063] Isolation of bacteriophage: Take 10mL of the filtered supernatant, add it to 10mL of 2x TSB liquid medium, and add 1mL of the bacteriophage host bacteria-Clostridium perfringens ACQJS03 logarithmic phase bacterial liquid, place it at 37℃ and culture it under anaerobic conditions for 8h, then take the above culture, centrifuge it at 8000rpm for 10min, filter it with a 0.22μm filter membrane to obtain the supernatant, and set it aside. Take 0.5mL of the bacteriophage host bacteria logarithmic phase bacterial liquid, add it to 5mL, 48℃ semi-solid TSB medium, mix it, pour it on the TSA plate, and prepare a double-layer plate containing the host bacteria. Take 10μl of the above supernatant after filtration, drip it on the solidified double-layer plate, air-dry it under sterile conditions, and place it at 37℃ and culture it under anaerobic conditions for 8h to form bacteriophage droplet plaques.

[0064] Purification of phage: Pick up phage plaques and place them in 1 mL SM buffer, shake for 1 min, perform 10-fold gradient dilution, and take 10 2 , 10 4 and 10 6 Add 0.5 mL of logarithmic phase host bacteria to the diluent, mix well and let stand for 15 minutes, then add 5 mL of 48°C semi-solid TSB medium, immediately pour on the TSA plate, shake well and let stand for 5 minutes, wait for it to solidify, place it in a 37°C incubator for anaerobic culture for 8 hours, and observe to obtain a double-layer plate containing a single plaque. Pick up a single plaque and add it to 1 mL of SM buffer, purify it at least 3 times as described above, and finally obtain a single plaque with consistent morphology and size on the double-layer plate ( Figure 1 ), which is the purified phage, named as Clostridium perfringens phage P2ACQJS03. A single plaque of phage P2ACQJS03 was picked and placed in 50 mL TSB medium containing 1 mL of logarithmic phase host bacteria solution, and cultured for 8 hours at 37°C under anaerobic conditions. The culture was centrifuged at 8000 rpm for 10 minutes, and the supernatant was filtered with a 0.22 μm filter membrane to obtain the pure culture solution of Clostridium perfringens phage P2ACQJS03.

[0065] The morphology of the Clostridium perfringens phage under transmission electron microscopy is as follows Figure 2 As shown, it has a polyhedral symmetrical head and a short contracted tail. The head has a diameter of 40 to 50 nm, the tail is 10 to 13 nm long, and the tail has a diameter of 4 to 6 nm.

[0066] The deposit number of Clostridium perfringens phage P2ACQJS03 is CCTCC NO: M 20242430.

[0067] Example 2: Preparation of Clostridium perfringens phage particles and extraction and sequencing of genome

[0068] 1. Preparation of Clostridium perfringens phage particles and extraction and sequencing of genome

[0069] Take 100mL of the pure culture fluid of Clostridium perfringens phage P2ACQJS03 prepared in Example 1, add DNaseI and RNaseA at a final concentration of 1μg / mL, incubate at 37°C for 60min, then add 5.84g NaCl (final concentration 1mol / L), and place in an ice bath for 1h after dissolving. Centrifuge at 11000rpm for 10min at 4°C, and transfer the supernatant to a new centrifuge tube. Add solid polyethylene glycol (PEG8000) to a final concentration of 10% (w / v), wait for it to completely dissolve, and place in an ice bath for at least 1h. Centrifuge at 11000rpm for 20min at 4°C, resuspend the precipitate with a small amount of SM solution, and obtain a phage particle concentrate, which is stored at 4°C for use.

[0070] The phage nucleic acid was extracted and sequenced using a lambda phage genomic DNA kit. After nucleotide sequencing, the Clostridium perfringens phage P2ACQJS03 had a nucleotide sequence shown in SEQ ID No.1.

[0071] Example 3: Whole genome analysis of Clostridium perfringens phage

[0072] 1. Genome Sequence Analysis of Clostridium perfringens Phage

[0073] Sequencing of the Clostridium perfringens phage P2ACQJS03 showed that the phage P2ACQJS03 genome was a linear double-stranded DNA with a total genome length of 23352 bp, the genome A, T, G, and C base contents were 35%, 35.4%, 14.3%, and 15.2%, respectively, and the G+C content was 29.5%.

[0074] The complete genome of bacteriophage P2ACQJS03 was initially predicted online using PHASTEST software (https: / / phastest.ca / ), which showed 46 phage gene sequences. The structural details are shown in Figure 3 .

[0075] 2. Analysis of biological characteristics of genomic restriction sites of Clostridium perfringens phage

[0076] The whole genome sequence of the Clostridium perfringens phage P2ACQJS03 was analyzed for restriction sites of 209 conventional endonucleases using SnapGene software. The distribution of restriction enzyme cutting sites of the genome is shown in the following figure. Figure 4 shown.

[0077] 3 Genetic evolution analysis of Clostridium perfringens phages

[0078] The phage P2ACQJS03 genome was used for genetic tree analysis in NCBI. Figure 5 The results showed that the phage closest to P2ACQJS03 was Clostridium phage phi24R (NC_019523.1).

[0079] Based on the unique genome information and electron microscopic morphology of the phage P2ACQJS03, according to the definition of the International Committee on Taxonomy of Viruses (ICTV), the phage P2ACQJS03 is a Clostridium perfringens phage belonging to the Gregsiragusavirus genus of the Denniswatsonvirinae subfamily of the Guelinviridae family of the Caudoviricetes class.

[0080] Example 4: Determination of the titer of Clostridium perfringens phage

[0081] Using SM solution as diluent, the pure culture solution of Clostridium perfringens phage P2ACQJS03 (prepared in Example 1) was diluted 10 times in a gradient stepwise manner to 10 8 times. Take l0 5 , l0 6 , l0 7 and l0 8 1000 μL of the diluted phage culture solution was evenly mixed with 300 μL of its host bacterial solution, and allowed to stand for 15 minutes to allow it to fully bind to the receptors on the bacterial surface. The above mixture was added to 4 mL of TSB semi-solid agar medium cooled to 50°C, mixed and immediately spread on a solidified solid agar plate, and after the agar solidified, inverted and cultured at 37°C under anaerobic conditions for 8 hours. Three parallel samples were required for each dilution, and the average number of plaques of the three parallel samples of this dilution was taken for counting. Among them, the phage titer (PFU / mL) = average number of plaques × dilution factor, and the results are shown in Table 1.

[0082] Table 1 Titer of Clostridium perfringens phage

[0083]

[0084] It can be concluded from Table 1 that the Clostridium perfringens phage P2ACQJS03 has a 10 13 PFU / mL or above.

[0085] Example 5: Determination of the optimal multiplicity of infection (MOI) of Clostridium perfringens phage against Clostridium perfringens

[0086] Pick a single colony of Clostridium perfringens, inoculate it into a test tube containing 3mL TSB liquid culture medium, and culture it for 4h at 37°C under anaerobic conditions to obtain a host bacterial suspension. The bacterial suspension is transferred to 10mL TSB culture medium at a ratio of 1:100, and cultured to the early logarithmic stage at 37°C under anaerobic conditions. Add the pure culture solution of Clostridium perfringens phage (prepared by Example 1) and host bacteria (MOI=phage number / bacterial number) according to the ratio of infection multiplicity of 1000, 100, 10, 1, 0.1, 0.01 and 0.001, add TSB liquid culture medium to make the total volume of each tube the same, and culture it for 8h at 37°C under anaerobic conditions. After the culture is completed, centrifuge at 10000g for 10min and collect the supernatant, and use the double-layer plate method to determine the titer of the phage filtrate after filtering through a 0.22μm filter membrane. Each point was cultured in duplicate tubes and the average value was taken, and the MOI that produced the highest phage titer was taken as the optimal multiplicity of infection. The experiment was repeated 3 times.

[0087] The phage filtrate obtained by the method can be used as a pure culture solution of Clostridium perfringens phage P2ACQJS03 and stored at 4°C for future use.

[0088] The results are shown in Table 2. The titer of Clostridium perfringens phage reached the highest (2.2x10 13 PFU / mL), its MOI=0.001.

[0089] Table 2 Titers of Clostridium perfringens phages at different infection multiplicity

[0090]

[0091] Example 6: Detection of virulence gene or adverse gene deletion in Clostridium perfringens phage

[0092] The present invention selected 103 virulence genes identified as lysogenic phages from pathogenic bacteria (Table 3), and determined whether the phage P2ACQJS03 contained the above virulence genes by measuring the whole genome of the phage P2ACQJS03 of Clostridium perfringens and performing bioinformatics analysis on it. The results showed that the phages tested in the present invention did not contain the following virulence genes.

[0093] Table 3 Major known toxic genes of lysogenic phages in pathogenic bacteria

[0094]

[0095]

[0096]

[0097] Example 7: Toxicology Experiment

[0098] Forty experimental mice of similar age, half male and half female, were randomly divided into four groups (phage group 1, phage group 2, phage group 3, and control group) after three days of adaptive feeding, with 10 mice in each group (5 males and 5 females). Phage group 1, phage group 2, and phage group 3 were given 0.2 mL / mouse, 0.5 mL / mouse, and 1 mL / mouse of the titer of 6.7x10 9 PFU / mL of pure culture fluid of Clostridium perfringens phage (prepared in Example 5), and the control group was given an equal amount of normal saline. The administration was continued for 15 days, and the experimental mice were killed by cervical dislocation to examine the internal organs.

[0099] Forty one-day-old chicks were randomly divided into four groups (phage group 1, phage group 2, phage group 3, and control group), with 10 chicks in each group. Phage group 1, phage group 2, and phage group 3 were given 0.4 mL / chicken, 1 mL / chicken, and 2 mL / chicken, respectively, with a titer of 6.7x10 9 PFU / mL of pure culture fluid of Clostridium perfringens phage (prepared in Example 5), and the control group was given an equal amount of normal saline. The administration was continued for 15 days, and the experimental chicks were killed and the internal organs were examined.

[0100] Thirty newborn piglets were randomly divided into five groups (phage group 1, phage group 2, phage group 3, phage group 4, and control group), with 6 piglets in each group. Phage group 1, phage group 2, phage group 3, and phage group 4 were given 2 mL / pig, 6 mL / pig, 10 mL / pig, and 20 mL / pig, respectively, with a titer of 6.7x10 9 PFU / mL of pure culture fluid of Clostridium perfringens phage (prepared in Example 5), and the control group was given an equal amount of normal saline, and the administration was continued for 15 days.

[0101] The experimental results showed that in the above treatment groups, different doses of Clostridium perfringens phage had no effect on the daily behavior of the test animals, and no symptoms such as depression, fever, vomiting and diarrhea appeared. The viscera of mice and chicks were examined and no abnormalities were found. This shows that the Clostridium perfringens phage has biosafety and can be used as a feed additive.

[0102] Example 8: Thermal stability test of Clostridium perfringens phage

[0103] Take 1000 μL of each and the titer is 6.7x109 PFU / mL of pure culture fluid of Clostridium perfringens phage (prepared in Example 5) was placed in a sterile EP tube and exposed to water baths at 25°C (CK), 55°C, 65°C, and 75°C for 2h, 24h, and 48h, respectively. After the exposure time, the sample tube was taken out and immediately placed in an ice bath for cooling. After appropriate dilution, the phage titer was determined by the double-layer plate method. The experiment was repeated 3 times.

[0104] Table 4 The titer of Clostridium perfringens phage P2ACQJS03 at different temperatures

[0105]

[0106] The results are shown in Table 4. In the experimental group, the Clostridium perfringens phage P2ACQJS03 was more likely to survive at 55°C. The Clostridium perfringens phage P2ACQJS03 had good high temperature resistance. Compared with the control, it still had an 82% survival rate after being bathed in water at 65°C for 2 hours, and a 10% survival rate after being bathed in water at 75°C for 2 hours. 7 PFU / mL titer.

[0107] Example 9: pH stability test of Clostridium perfringens phage

[0108] Take sterile EP tubes and add 900 μL of TSB medium with different pH (1-14) respectively. Then place the EP tubes in a constant temperature water bath at 25°C. After the temperature is balanced, add 100 μL of pure culture solution of Clostridium perfringens phage (prepared in Example 5) to make its initial titer 3.5×10 9 PFU / mL, and stand at room temperature for 1 hour. After the reaction time is over, the sample is appropriately diluted and the phage titer is determined by the double-layer plate method. The above process is then repeated after 4h, 8h, 24h and 96h, and the experiment is repeated 3 times. The results are shown in Table 5.

[0109] Table 5 pH stability of Clostridium perfringens phage (initial titer 3.5x10 9 PFU / mL)

[0110]

[0111] As shown in Table 5, the potency of the Clostridium perfringens phage described in the present application did not change significantly between pH 4 and pH 11, indicating that it has good stability under neutral, slightly acidic and alkaline conditions; the Clostridium perfringens phage described in the present application still has 10 7 The PFU / mL titer indicated that the phage had a certain tolerance to alkaline conditions.

[0112] Example 10: Survival stability test of Clostridium perfringens phage

[0113] 5 mL of pure culture solution of Clostridium perfringens phage (prepared in Example 5) was dispensed into sterile test tubes and placed at 4°C, 25°C, and 30°C, respectively. The phage titer was determined by the double-layer plate method after appropriate dilution at regular intervals. The results are shown in Table 6.

[0114] Table 6 Survival stability of Clostridium perfringens phage at different storage temperatures

[0115]

[0116]

[0117] As shown in Table 6, the titer of Clostridium perfringens phage stored at 4°C for 50 weeks only decreased by about half, still with a titer of 1.82x10 10 PFU / mL; at 25°C, the titer of Clostridium perfringens phage did not decrease by more than one order of magnitude within 10 weeks, and the titer could still be detected after 39 weeks; at 30°C, the titer of Clostridium perfringens phage could still be detected after 14 weeks. 8 PFU / mL titer, the titer can still be detected after 30 weeks; Clostridium perfringens phage has a strong lysis ability on host bacteria at all storage temperatures. Clostridium perfringens phage is suitable for storage at 4℃.

[0118] Example 11: Lysis experiment of Clostridium perfringens phage on Clostridium perfringens

[0119] The double-layer plate spot method was used to determine the lysis spectrum of bacteriophages. Single colonies of 5 toxin types and different regional origins of Clostridium perfringens were selected, totaling 145 strains, and they were inoculated into test tubes containing 3mL TSB liquid culture medium and cultured at 37℃ under anaerobic conditions for 4h to obtain bacterial suspensions of each strain. 300μL of bacterial suspension was mixed with TSB semi-solid culture medium and spread on ordinary agar plates, and 5μL of the titer of 1x10 8 PFU / mL of Clostridium perfringens phage (prepared in Example 5) were dropped onto each plate, and after natural air drying, they were cultured at 37°C under anaerobic conditions for 8 hours, and the results were observed. Figure 6 shown.

[0120] from Figure 6 It can be seen that the Clostridium perfringens phage described in the present application has a wide host range, can identify various toxin types of Clostridium perfringens, and has a lysis rate of more than 92%, and can be well used in the preparation of a biofungicide for lysing Clostridium perfringens.

[0121] Example 12: Lysis test of Clostridium perfringens phage against non-host pathogenic bacteria

[0122] The lysis spectrum of phage was determined by double-layer plate spot method.

[0123] 284 single colonies of non-host pathogenic bacteria were picked, including 31 strains of Clostridium difficile, 13 strains of Clostridium equi, 16 strains of Clostridium hemolyticum, 23 strains of Clostridium novyi, 33 strains of Clostridium botulinum, 19 strains of Clostridium tetani, 5 strains of Streptococcus agalactiae, 6 strains of Streptococcus dysgalactiae, 22 strains of Salmonella, 17 strains of Staphylococcus aureus, 20 strains of Pseudomonas aeruginosa, 15 strains of Klebsiella pneumoniae, 23 strains of Acinetobacter baumannii, 24 strains of Escherichia coli, 7 strains of Listeria, and 10 strains of Shigella. They were inoculated in test tubes containing 3 mL of TSB, and the bacterial suspension was cultured to the logarithmic phase under the optimal culture conditions for each test bacteria to obtain the bacterial suspension of each strain. 300 μL of bacterial suspension was mixed with semi-solid culture medium and spread on ordinary agar plates, and 5 μL of titer of 1x10 8 PFU / mL of Clostridium perfringens phage stock solution (prepared in Example 5) was dropped onto each plate. After being naturally air-dried, the plate was cultured overnight under the optimal culture conditions for each test bacterium and the results were observed. Figure 7 shown.

[0124] Depend on Figure 7 It can be seen that the Clostridium perfringens phage described in the present application has certain recognition for the tested Clostridium difficile, Clostridium equi, Clostridium haemolyticum, Clostridium novyi and Clostridium tetani, but cannot recognize Clostridium botulinum and other tested bacteria. This shows that the tested phage has extremely strong intra-genus specificity and has certain application potential for controlling other pathogenic bacteria of the genus Clostridium.

[0125] Example 13: Lysis test of Clostridium perfringens phage on non-pathogenic beneficial bacteria

[0126] 122 strains of non-pathogenic beneficial bacteria were selected, including 43 strains of Clostridium butyricum, 35 strains of Clostridium sporogenes, 18 strains of non-pathogenic rhizobia, 11 strains of non-pathogenic Bacillus subtilis, 9 strains of non-pathogenic Bacillus megaterium, and 16 strains of non-pathogenic Bacillus licheniformis. They were inoculated in test tubes containing 3 mL of TSB, and the bacterial suspension was cultured to the logarithmic phase under the optimal culture conditions for each test bacteria to obtain bacterial suspension of each strain. 300 μL of bacterial suspension was mixed with semi-solid culture medium and spread on ordinary agar plates, and 5 μL of the titer of 1x10 8 PFU / mL of Clostridium perfringens phage (prepared in Example 5) were dropped onto each plate. The cells were cultured overnight under the optimal culture conditions for each test bacterium and the results were observed. Figure 8 shown.

[0127] Depend on Figure 8 It can be seen that the Clostridium perfringens phages described in the present application cannot identify any of the 122 strains of non-pathogenic beneficial bacteria tested, indicating that the tested phages have no damaging effect on the beneficial bacterial microbial community.

[0128] Example 14: Biological Activity Test of Clostridium perfringens Phage Spray Powder and Lyophilized Powder

[0129] 1. Biological activity test of Clostridium perfringens phage spray powder

[0130] 2% starch (W / V) and 4% sorbitol (W / V) were dissolved in a solution with a titer of 3.1x10 9 PFU / mL Clostridium perfringens phage P2ACQJS03 pure culture solution (prepared in Example 5), incubate with phage at 37°C and 180r / min for 2h for phage coating. Spray the coated phage solution on the carrier defatted rice bran at a ratio of 1:4 (W / W), stir to mix evenly, dry at 50°C for 2h, humidity <10%, and pass through a 40-mesh sieve. After the preparation is completed, take a sample and use the double-layer plate method to determine the phage titer, and repeat the experiment 3 times. The phage spray powder is stored at room temperature.

[0131] 2. Biological activity test of lyophilized powder of Clostridium perfringens phage

[0132] 2% starch (W / V) and 4% sorbitol (W / V) were dissolved in a solution with a titer of 3.1x10 9 PFU / mL Clostridium perfringens phage P2ACQJS03 pure culture solution (prepared in Example 5), incubate with phage at 37°C and 180r / min for 2h for phage coating. Add the coated phage solution to the material tray, vacuum freeze-dry for 50±2h, crush through a 40-mesh sieve, and the moisture content of the freeze-dried powder is <10%. After the preparation is completed, samples are taken and the phage titer is determined by the double-layer plate method. The experiment is repeated 3 times. The phage freeze-dried powder is stored at room temperature.

[0133] The results are shown in Table 7. There was no significant difference in the potency of the Clostridium perfringens phage P2ACQJS03 before and after the preparation of spray powder and freeze-dried powder, respectively, indicating that the phage has good tolerance and stability to the powder preparation conditions of spray powder and freeze-dried powder.

[0134] Table 7 Titer of Clostridium perfringens phage powder before and after preparation

[0135]

[0136] Example 15: Stability test of Clostridium perfringens phage in gastric juice, small intestinal juice and bile salt solution

[0137] Take a sterile EP tube and add porcine artificial gastric juice, artificial small intestinal juice, 2% bile salt solution (W / V) and 900 μL of SM solution respectively. Then place the EP tube in a constant temperature water bath at 25°C. After the temperature is balanced, add 100 μL of pure culture solution of Clostridium perfringens phage (prepared in Example 5) to make its initial titer 5.4×10 9 PFU / mL, and stand at room temperature for 1 hour, with SM solution treatment as the control. After the reaction time is over, the sample is appropriately diluted and the phage titer is determined by the double-layer plate method, and the experiment is repeated 3 times.

[0138] The results are shown in Table 8. There is no significant change in the potency of the tested phage in pig artificial gastric juice, artificial small intestinal juice and 2% bile salt solution, indicating that the phage can be used for animal gavage.

[0139] Table 8 Stability of Clostridium perfringens phage in gastric juice, small intestinal juice and bile salt solution

[0140]

[0141] Example 16: Preparation of a Composition of Clostridium perfringens Phage

[0142] The titer is 1x10 9 PFU / mL pure culture solution of Clostridium perfringens phage P2ACQJS03 (prepared in Example 5) and didecyldimethylammonium chloride were uniformly mixed in equal volumes to prepare a composition, and the final concentration of didecyldimethylammonium chloride in the composition was 0.05% (Composition 1).

[0143] The titer is 1x10 9 PFU / mL pure culture solution of Clostridium perfringens phage P2ACQJS03 (prepared in Example 5) and chlorhexidine were uniformly mixed in equal volumes to prepare a composition, and the final concentration of chlorhexidine in the composition was 0.01% (composition 2).

[0144] The titer is 1x10 9 PFU / mL pure culture solution of Clostridium perfringens phage P2ACQJS03 (prepared in Example 5), didecyldimethylammonium chloride and chlorhexidine were uniformly mixed in equal volumes to prepare a composition, and the final concentrations of didecyldimethylammonium chloride and chlorhexidine in the composition were 0.05% and 0.01%, respectively (Composition 3).

[0145] Example 17: Prevention and treatment of Clostridium perfringens contamination during storage of chicken samples using Clostridium perfringens phage and its composition

[0146] 1. Preparation method of composition 1 to composition 3: the same as the method described in Example 16.

[0147] 2. Pure culture solution of Clostridium perfringens phage P2ACQJS03: prepared in Example 5.

[0148] 3. Cut the high-pressure steam sterilized chicken into 90 small cubes of 1 cm square, divide them into 6 groups (single phage P2ACQJS03 group, composition 1, composition 2, composition 3, control group and blank group), 15 cubes in each group, and place them in sterile plates. Each cube of the experimental group was inoculated with 3.5x10 3 cfu / mL Clostridium perfringens and the dose is 10 6 PFU / kg of the tested phage; the control group was inoculated with 3.5x10 3 cfu / mL of Clostridium perfringens and an equal amount of sterile water; the blank group was given an equal amount of sterile saline. Each treatment was placed in an incubator at 37°C for anaerobically culture. Every 2 hours, a piece of chicken was placed in 10 mL of sterile water, shaken thoroughly, and the Clostridium perfringens content of the liquid was determined using a TSC plate. The experiment was repeated 3 times. The results are shown in Table 9.

[0149] Table 9 The results of the prevention and treatment of Clostridium perfringens phage P2ACQJS03 and its combination on Clostridium perfringens contamination during the storage of chicken samples

[0150]

[0151] The results in Table 9 show that after 8 hours, a large amount of Clostridium perfringens grew on the surface of the chicken in the control group; while in each experimental group, due to the addition of Clostridium perfringens phage P2ACQJS03 and its composition 1, composition 2, and composition 3, the Clostridium perfringens on the surface of the chicken was always controlled at an extremely low level. Clostridium perfringens phage P2ACQJS03 and its composition 1, composition 2, and composition 3 can be used as a biocide to effectively kill Clostridium perfringens on the surface of chicken and prevent the contamination of Clostridium perfringens during the storage of chicken. Of course, Clostridium perfringens phage P2ACQJS03 can also be prepared into health products or medicines for application.

[0152] Example 18: Therapeutic effect of Clostridium perfringens phage on chickens infected with Clostridium perfringens

[0153] 150 one-day-old healthy chicks were randomly divided into three groups (bacteriophage P2ACQJS03 group, positive control group, negative control group), with 50 chicks in each group. The pure culture solution of Clostridium perfringens bacteriophage P2ACQJS03 was prepared as in Example 5.

[0154] This experiment adopts the method of online flat breeding. Before the experiment, the chicken house, chicken pen, feed trough, waterer, etc. were thoroughly cleaned and disinfected, and the chicken house was fumigated and disinfected as a whole. The chicks were free to eat and drink, with continuous light, regular cleaning and disinfection, and vaccination according to the normal immunization program. The temperature of the chicken house was controlled at 32-34℃ in the first week of the experiment, 30-32℃ in the second week, and about 25℃ from the third week to the end of the experiment. The humidity of the chicken house was 50%-60%.

[0155] During the experiment, all groups were fed with a basic diet. Except for the negative control group, the other two groups of chicks were continuously fed with a Clostridium perfringens suspension (1x10 8 CFU / mL), and each chick was gavaged 1 mL daily for 8 consecutive days. The chicks in the phage P2ACQJS03 group were orally gavaged with the test phage P2ACQJS03 (1x10 8 PFU / mL, prepared by Example 5) pure culture solution, each chick was gavaged with 1 mL per day for 8 consecutive days. At the same time, the chicks in the negative control group were gavaged with the same dose of TSB medium. The mortality of the experimental chicks was counted on the 21st day and the 30th day, and the jejunal intestinal damage was scored. The results are shown in Table 10.

[0156] Mortality rate = (number of dead chicks / total number of chicks) × 100%.

[0157] Scoring criteria for intestinal injury: 0 = no visible injury; 1 = thin or fragile intestinal wall (congestion); 2 = small focal necrosis or ulceration (1 to 5 foci); 3 = focal necrosis or ulceration.

[0158] Table 10 Effect of Clostridium perfringens phage on growth performance of chicks

[0159]

[0160] As can be seen from Table 10, compared with the control group without adding Clostridium perfringens phage, the mortality rate of chicks in the experimental group fed with Clostridium perfringens phage was significantly reduced, indicating that Clostridium perfringens phage has a good therapeutic effect on chicks challenged with Clostridium perfringens. Therefore, Clostridium perfringens phage can be preferably used in the preparation of drugs for treating or preventing diseases caused by Clostridium perfringens infection.

[0161] Example 19: Preparation and use of a kit for Clostridium perfringens phage

[0162] The kit contains 5 to 10 mL of a 3×10 9The pure culture solution of Clostridium perfringens phage P2ACQJS03 (Clostridium perfringens phage P2ACQJS03), 1LTSB liquid culture medium, 1LTSB semi-solid culture medium, and 1LTSA solid culture medium; wherein the Clostridium perfringens phage P2ACQJS03 (Clostridium perfringens phage P2ACQJS03) is the pure culture solution of phage P2ACQJS03 prepared in Example 1, or the pure culture solution of phage P2ACQJS03 prepared in Example 5.

[0163] The method of using the kit is: take a titer of 3×10 9 PFU / mL Clostridium perfringens phage P2ACQJS03 (Clostridium perfringens phage P2ACQJS03) phage liquid, the double-layer plate spot method is used to determine the lysis spectrum of the test phage. Pick a single colony to be tested, inoculate it in the target liquid culture medium, and shake culture it at the target temperature in combination with the growth characteristics of the strain to be tested to obtain the strain liquid to be tested. Take 300μL of the strain suspension to be tested and mix it with 5mL TSB semi-solid culture medium and spread it on a TSA solid plate, take 10μL of Clostridium perfringens phage P2ACQJS03 (Clostridium perfringens phage P2ACQJS03) liquid and spot it on the plate. After natural air drying, culture it at the target temperature according to the growth characteristics of the strain to be tested, and observe the results.

[0164] The Clostridium perfringens phage P2ACQJS03 of the present invention can be used to prepare a composition, a reagent or a kit, and is applied to the rapid detection of Clostridium perfringens, including but not limited to detecting Clostridium perfringens in a target sample in the form of a reagent, a kit, etc., or screening target pathogens in clinical samples, effectively ensuring the sensitivity of the detection.

[0165] It should be noted that the embodiments described above are only used to explain the present application and do not constitute any limitation to the present application. The present application is described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present application may be modified as specified within the scope of the claims of the present application, and the present invention may be revised without departing from the scope and spirit of the present application. Although the present application described therein relates to specific methods, materials and embodiments, it does not mean that the present application is limited to the specific examples disclosed therein. On the contrary, the present application can be extended to all other methods and applications with the same functions.

Claims

1. A Clostridium perfringens phage, characterized in that: The phage is Clostridium perfringens phage P2ACQJS03 ( Clostridium perfringens phage P2ACQJS03), and the deposit number of the Chinese Center for Type Culture Collection is CCTCC NO:M 20242430.

2. The Clostridium perfringens phage according to claim 1, characterized in that The bacteriophage has a polyhedral three-dimensional symmetrical head and a short contracted tail, the head has a diameter of 40 to 50 nm, the tail has a length of 10 to 13 nm, and the tail has a diameter of 4 to 6 nm.

3. The Clostridium perfringens phage according to claim 2, characterized in that The phage was cultured for 4 h under MOI = 0.001, and its titer was as high as 2.2 x 10 13 PFU / mL.

4. The Clostridium perfringens phage according to claim 3, characterized in that The bacteriophage is tolerant under the condition of temperature of 55-70°C.

5. A bacteriophage composition, characterized in that: The composition comprises the Clostridium perfringens phage according to any one of claims 1 to 4.

6. The bacteriophage composition according to claim 5, characterized in that The composition also includes a chemical germicide.

7. A biocide, characterized in that: The biofungicide is suitable for lysing Clostridium perfringens. The preparation of the biofungicide uses the Clostridium perfringens phage described in any one of claims 1 to 4 or the phage composition for lysing Clostridium perfringens described in any one of claims 5 to 6; the dosage form of the biofungicide is one of a solution, a powder, a gel, a granule or a lyophilized agent.

8. A drug, characterized in that The medicine is suitable for treating or preventing diseases caused by Clostridium perfringens infection, and the preparation of the medicine uses the Clostridium perfringens phage according to any one of claims 1 to 4.

9. A feed additive, characterized in that: The feed additive is prepared by using the Clostridium perfringens phage as described in any one of claims 1 to 4.

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