Method for reducing dosage of antibiotics for treating clostridium perfringens infection, bactericidal composition, bacteriophage and application
By combining a specific bacteriophage RDP-CP-2310008 with multiple antibiotics, the problems of antibiotic resistance and excessive dosage have been solved, resulting in a significant reduction in antibiotic usage, a delay in the development of resistance, and a guarantee of food safety.
Patent Information
- Application Number
- CN202512040313.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-31
- Publication Date
- 2026-04-07
AI Technical Summary
Existing technologies lack clear methods and directions for screening specific bacteriophages to significantly reduce antibiotic use, and antibiotic resistance is spreading rapidly, leading to increased chicken farming costs and economic losses.
The use of a specific Clostridium perfringens phage, RDP-CP-2310008, in combination with multiple antibiotics demonstrated a remarkable synergistic bactericidal effect, significantly reducing the minimum inhibitory concentration of antibiotics and thus reducing the amount of antibiotics used.
Without completely abandoning antibiotics, we should significantly reduce antibiotic usage, delay the development of drug resistance, reduce drug residues, and ensure food safety and public health security.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial technology, and more specifically to a method for reducing the dosage of antibiotics used to treat Clostridium perfringens infections, a bactericidal composition, a bacteriophage, and its application. Background Technology
[0002] With the rapid large-scale development of the poultry industry, bacterial diseases are becoming increasingly prominent, hindering the sustainable and healthy development of China's poultry industry. *Clostridium perfringens* (Cp) is a Gram-positive, spore-forming anaerobic bacterium widely distributed in soil, dust, animal production environments, and feed. It primarily causes necrotic enteritis in poultry. Infected chickens have fragile intestines filled with gas, and necrosis occurs in the jejunum and ileum, forming pseudomembranes. Studies have shown that *Clostridium perfringens* colonizes up to 95% of the intestines, causing clinical or subclinical necrotic enteritis, increasing chicken farming costs by $0.05 per bird and resulting in significant economic losses. Growth-promoting antibiotics, such as bacitracin, lincomycin, tylosin, and penicillin, are typically added to feed to prevent *Clostridium perfringens* infection in chickens. However, with the rapid spread of antibiotic resistance, AGPs have been gradually phased out. The ban on AGP may increase the rapid proliferation of Clostridium perfringens in poultry environments, leading to reduced feed digestibility and absorption in chickens, slow weight gain, increased farming costs, and ultimately increased burden on consumers.
[0003] Bacteriophages, as viruses capable of specifically lysing bacteria, are ideal alternatives to antibiotics. Although there is a general idea in academia that bacteriophages can be used in combination with antibiotics, current technologies offer no specific and reliable technical insights or experimental evidence regarding the core objective of achieving this combination, which types of antibiotics can be used in combination, and whether and how to quantify the reduction in antibiotic dosage after combination. Those skilled in the art lack a clear motivation and direction to screen for a specific bacteriophage that can achieve significant antibiotic reduction.
[0004] Therefore, how to minimize antibiotic usage while ensuring efficacy, thereby delaying the emergence of drug-resistant bacteria and reducing drug residues, has become a crucial but unresolved technical problem in this field.
[0005] The information disclosed in this background section is only intended to enhance the understanding of the background technology of this application and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention
[0006] This invention aims to overcome the shortcomings of existing technologies and provide a specific Clostridium perfringens bacteriophage. The key feature of this bacteriophage is its ability to produce unexpected and significant synergistic effects with a variety of antibiotics, thereby reducing antibiotic usage to 1 / 256 to 1 / 2 of the conventional concentration. This provides a novel, efficient, and safe technical solution for achieving antibiotic reduction goals in aquaculture.
[0007] The technical solution of this invention is as follows: In a first aspect, the present invention provides a method for reducing the dosage of antibiotics used to treat Clostridium perfringens infection, wherein Clostridium perfringens bacteriophage (… Clostridium perfringens bactrriophage RDP-CP-2310008 can be used in combination with one or more antibiotics. The Clostridium perfringens phage RDP-CP-2310008 was deposited on November 29, 2023, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO.45778. The inventors unexpectedly discovered that the Clostridium perfringens bacteriophage RDP-CP-2310008 exhibits a remarkable synergistic bactericidal effect when used in combination with various antibiotics, including β-lactams, aminoglycosides, and tetracyclines. Experiments confirmed that in the presence of the bacteriophage, the minimum inhibitory concentration (MIC) of these antibiotics against Clostridium perfringens can be significantly reduced to 1 / 64 to 1 / 2 of that when used alone. This unexpected technical effect makes the bacteriophage of outstanding industrial value in the preparation of compositions that reduce antibiotic dosage.
[0008] In a second aspect, the present invention provides a bactericidal composition comprising the aforementioned Clostridium perfringens phage RDP-CP-2310008 and one or more antibiotics.
[0009] Thirdly, the present invention provides the use of the Clostridium perfringens bacteriophage or bactericidal composition in the preparation of medicaments and disinfectants for the prevention and / or treatment of Clostridium perfringens infection.
[0010] The beneficial effects achieved by this invention are as follows: 1. This invention provides and verifies for the first time a specific technical solution that can significantly reduce antibiotic usage (up to 64 times) by combining a specific bacteriophage with antibiotics, providing a new approach to solving the problem of antibiotic abuse in the aquaculture industry; 2. The Clostridium perfringens phage RDP-CP-2310008 in this invention can produce synergistic effects with various antibiotics with different mechanisms of action, making it widely applicable and highly practical. This method significantly reduces the amount of antibiotics used without completely eliminating them, which helps delay the development of drug resistance, reduces drug residues, and ensures food safety and public health safety. 3. The Clostridium perfringens phage RDP-CP-2310008 in this invention also has the advantages of high potency, genetic stability and high lysis rate, making it suitable for industrial production. Attached Figure Description
[0011] Figure 1 This is an electron micrograph of the bacteriophage isolated in this invention.
[0012] Figure 2 This is an experimental diagram of the one-step growth curve of the bacteriophage of this invention.
[0013] Figure 3 This is a diagram of the thermal stability experiment of the bacteriophage of this invention.
[0014] Figure 4 This is a diagram of the pH stability experiment of the bacteriophage of this invention.
[0015] Figure 5 This is a diagram of the phage stability genetic experiment of this invention.
[0016] Figure 6 This is the MIC diagram of the antibiotic used in this invention. Detailed Implementation
[0017] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0018] In this invention, unless otherwise specified, the equipment and raw materials used are commercially available or commonly used in the field. The methods in the following embodiments, unless otherwise specified, are conventional methods in the field. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In this invention, *Clostridium perfringens* CP-9.6 is referred to as "host CP-9.6" or "CP-9.6", and *Clostridium perfringens* bacteriophage RDP-CP-2310008 is referred to as "bacteriophage RDP-CP-2310008" or "RDP-CP-2310008".
[0019] Among them: culture medium formulation Components of 2×TY liquid culture medium (g / L): Casein peptone: 16 Yeast extract: 10 Sodium chloride: 5.0 Magnesium chloride: 0.01 Calcium chloride: 0.01 pH: 7.2-7.6 Upper culture medium composition (g / L): Casein peptone: 16 Yeast extract: 10 Sodium chloride: 5.0 Magnesium chloride: 0.01 Calcium chloride: 0.01 Agar: 4.0 pH: 7.2-7.6 Example 1: Isolation and identification of Clostridium perfringens CP-9.6 1.1 Pathological sample collection and processing Liver tissue and cecal contents were collected from dead chickens under aseptic conditions as samples for pathogen and bacteriophage isolation. Cecal contents and fecal samples were suspended in physiological saline at a ratio of 1:5 (w / v), thoroughly shaken, and allowed to stand for 4 hours. Subsequently, the samples were centrifuged at 6000 rpm for 20 min, and the supernatant was filtered through a 0.45 μm filter membrane. The filtrate was stored at 4℃ for later use.
[0020] 1.2 Isolation and purification of Clostridium perfringens 100 μL of the above filtrate was spread onto TSC (tryptone-sulfite-cycloserine) selective solid medium plates and incubated at 42°C in an anaerobic environment for 24 h. Clostridium perfringens produces hydrogen sulfide and θ toxin, resulting in typical black colonies on TSC medium.
[0021] A single black colony was selected and streaked onto an agar plate containing 5% sheep defibrinated blood, and incubated anaerobicly at 42°C for 24 hours. Clostridium perfringens forms a double hemolytic zone on the blood agar plate (the inner zone is completely hemolyzed, and the outer zone is partially hemolyzed). Colonies exhibiting the typical double hemolytic zone on the blood agar plate were streaked three times consecutively on TSC and blood agar plates to obtain a pure culture.
[0022] 1.3 Preliminary identification of Clostridium perfringens The purified strains were identified as follows: Morphological identification: Purified colonies were picked from blood agar plates, stained with Gram stain, and examined under a microscope. Gram-positive, short, thick bacilli were observed, consistent with the typical morphological characteristics of Clostridium perfringens. Biochemical identification: The purified strain was inoculated into milk culture medium and cultured at 37°C for 24 hours. The "violent fermentation" phenomenon (acid production caused casein to coagulate, and gas production caused the coagulation to break) was observed, which further confirmed Clostridium perfringens. Hemolytic characteristics identification: Observation of blood agar culture results showed that the strain produced a double hemolytic ring (the inner layer was complete hemolysis caused by α toxin, and the outer layer was incomplete hemolysis caused by θ toxin), which is a characteristic hemolytic manifestation of Clostridium perfringens. Serological identification: The slide agglutination test was used, and the strain was reacted with Clostridium perfringens type A specific antiserum. The strain showed obvious agglutination, and the isolate was finally identified as Clostridium perfringens type A and named CP-9.6.
[0023] 1.4 Strain activation The CP-9.6 strain, stored at -80℃, was streaked onto TSC plates and anaerobically cultured at 42℃ for 24 h. Typical black colonies were picked and inoculated onto 5% sheep blood agar plates and cultured under the same conditions for 16–24 h. Single colonies were then inoculated into 2×TY liquid medium and anaerobically cultured at 42℃ with shaking until the logarithmic growth phase (approximately 6–8 h, bacterial concentration approximately 1×10⁻⁶). 8 (CFU / mL), used for subsequent phage isolation experiments.
[0024] Example 2: Isolation and identification of Clostridium perfringens phage RDP-CP-2310008 2.1 Phage co-culture enrichment Take 100 μL of the host bacterium CP-9.6 in the logarithmic growth phase (approximately 1 × 10⁻⁶). 8 The phage was enriched by mixing 200 μL of the pretreated filtrate from Example 1.1 with 2×TY liquid medium (CFU / mL) and incubating overnight in an anaerobic environment at 42°C. 2.2 Phage Isolation and Purification Phages were isolated using the double-layer plate method: Centrifuge the co-culture at 12,000 rpm for 10 minutes, collect the supernatant and filter it through a 0.22 μm filter membrane to remove bacteria, and collect the crude phage extract; Take 100 μL of crude phage extract and mix it with 100 μL of host bacterial culture. Add the melted and cooled upper culture medium to about 45°C, mix quickly, and pour it onto the bottom of a sterile plate (pre-laid solid culture medium) to make a double-layer plate. Anaerobic culture at 42℃ for 24 hours, observe and select larger, clear single phage plaques; The selected phage plaques were inoculated onto a new double-layer plate, and the above separation steps were repeated 4 times until the phage plaques on the plate were uniform in size and morphology, and the purified phage was obtained and named RDP-CP-2310008. 2.3 Phage amplification culture and preservation The purified phage plaques were picked, soaked in sterile physiological saline for 30 minutes, and then filtered through a 0.22 μm filter membrane to remove bacteria, thus obtaining a phage suspension. The phage suspension was serially diluted 10-fold. 100 μL of the appropriate concentration dilution was mixed with 200 μL of the host bacterial suspension, and the mixture was spread into double-layer plates. The plates were then anaerobically incubated at 42°C for 24 hours. Plates with cloud-like phage plaques were selected. Add sterile physiological saline to the above plate, soak, scrape the upper culture medium into a 50 mL centrifuge tube, shake for 10 minutes, centrifuge at 5000 rpm for 10 minutes, collect the supernatant and filter it through a 0.22 μm filter membrane to obtain a high concentration of bacteriophage stock solution; The phage stock solution and the freeze-drying protectant (16% skim milk powder + 2% mannitol + 0.5% vitamin C) were mixed evenly at a volume ratio of 1:1 and dispensed into sterile ampoules. Ampoules were pre-frozen in a freeze dryer for 4 hours, then vacuum freeze-dried for 48 hours. After freeze-drying, they were vacuum sealed and stored at -80°C for long-term preservation.
[0025] 2.4 Electron microscopic observation of bacteriophages Take a value higher than 10 5 50 μL of PFU / mL phage sample was dropped onto a microporous copper grid and allowed to precipitate for 1 min. Excess liquid was then absorbed with filter paper. 50 μL of 1% phosphotungstic acid (PTA) was added to the copper grid and stained for 5 min. Excess stain was absorbed with filter paper, and the sample was dried and observed and photographed using a transmission electron microscope.
[0026] As shown in Figure 1, electron microscopy revealed that this bacteriophage has a polyhedral, three-dimensionally symmetrical head encapsulating nucleic acid, with a diameter of approximately 42 nm, and a tail approximately 30 nm long. According to the definition of the International Committee on Taxonomy of Viruses (ICTV), the morphology of this bacteriophage conforms to the characteristics of the Brachypophagidae family, and it is named RDP-CP-2310008. Sequencing and gene analysis were performed by Qingdao Fubes Biotechnology Co., Ltd., and the results showed that its gene sequence is shown in SEQ ID NO.1, with a sequence length of 18241 bp.
[0027] 2.5 Determination of the optimal multiple of infection for bacteriophages Phage proliferation solution and host were added to liquid culture medium at multiplicity of infection ratios of 100:1, 10:1, 1:1, 1:10, 1:100, and 1:1000, ensuring that the total volume of the culture system was the same. The medium was incubated at 42℃ under anaerobic conditions for 12 h, centrifuged at 12000 rpm for 5 min at room temperature, and the supernatant was plated on double plates to determine its titer. The medium was then incubated at 42℃ under anaerobic conditions for 24 h.
[0028] Table 1 Results of the Optimal Multiplicity of Infection Experiment
[0029] The experimental results are shown in Table 1. When the multiplicity of infection is 10:1, the titer is highest after 8 hours of culture, indicating that the optimal multiplicity of infection for RDP-CP-2310008 is 0.1.
[0030] 2.6 One-step growth curve of Clostridium perfringens bacteriophage The host bacteria and phages were mixed at the optimal MOI ratio, and the mixture was incubated for 30 min. The mixture was then centrifuged at 11,000 r / min for 10 min at 4 °C, and the supernatant was collected. The precipitate was resuspended in 2×TY medium and anaerobic cultured at 42 °C. Samples were taken every 5 min for the first 30 min, counted every 10 min from 30 to 60 min, and sampled every 30 min from 60 to 420 min. The samples were centrifuged at 11,000 r / min for 10 min at 4 °C, filtered, and the phage titer was determined using the double-layer plate method.
[0031] A growth curve was plotted with time on the x-axis and the logarithm of phage titer on the y-axis to obtain the phage latency and lysis period, and to calculate the average lysis rate.
[0032] Average lysis rate = phage titer at the end of the outbreak / host bacterial concentration at the beginning of infection.
[0033] The results are as follows Figure 2 As shown, the titer of the bacteriophage did not change significantly within 20 minutes after infecting the host bacteria, indicating a latency period of approximately 20 minutes. The titer increased significantly within 30-120 minutes post-infection, then stabilized, indicating a phage lysis period of approximately 90 minutes. The phage lysis rate was approximately 115 PFU / infected cell, demonstrating extremely strong phage lysis and replication capabilities.
[0034] 2.7 Determination of the thermal stability of bacteriophages The phage stock solution was aliquoted into 50 mL sterile centrifuge tubes and incubated for 30 min at 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 80℃ and 90℃ respectively. Then, it was serially diluted 10 times with physiological saline and plated on double plates to determine its potency.
[0035] As shown in Figure 3, the phage has a relatively high titer at temperatures below 50℃. As the temperature increases, the titer of the phage decreases rapidly, indicating that RDP-CP-2310008 is relatively sensitive to temperature.
[0036] 2.8 Determination of pH stability of bacteriophages The pH of the physiological saline was adjusted using dilute hydrochloric acid and dilute NaOH solution to prepare buffer solutions with pH values of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. Then, the bacteriophage RDP-CP-2310008 was diluted to a concentration of 1×10⁻⁶ using the prepared buffer solutions. 8 The pfu / mL concentration was determined by incubating the diluted solution in a water bath at 42°C for 1 hour, then diluting it 10-fold with physiological saline and plating it onto double plates for titer determination. The solution was then incubated at 42°C under anaerobic conditions for 24 hours.
[0037] The experimental results are shown in Figure 4. RDP-CP-2310008 at pH 5.0 The phage maintained good activity within a pH range of 10.0, with no significant change in phage titer. Furthermore, the highest phage titer, reaching 2.13 × 10⁻⁶, was observed at pH 7.0. 8 pfu / mL. The phage titer decreases rapidly with increasing or decreasing pH; therefore, phage RDP-CP-2310008 can tolerate weak acids and moderately strong alkalis, exhibiting good acid and alkali tolerance.
[0038] 2.9 Phage Stability Genetic Experiment RDP-CP-2310008 was passaged 30 times consecutively. In each generation, equal amounts of RDP-CP-2310008 and CP-9.6 were added to the culture medium. Each generation was cultured for 24 hours. Then, double plates were laid and the potency was determined.
[0039] Depend on Figure 5 It can be seen that the titer gradually decreases from generation 1 to generation 7, reaching its lowest point in generation 7. Then, it fluctuates significantly in generations 8 and 9, gradually stabilizing between generations 10 and 30, indicating that RDP-CP-2310008 has good genetic stability. To improve its titer, phages from generation 10 and later are selected for production.
[0040] Example 3: Phage lysis rate experiment 3.1 Experimental Methods Under aseptic conditions, take 1 mL of the sample and 1 mL of the host bacterial culture (1×10⁻⁶) respectively. 7 (CFU / mL), incubate at 42℃ for 15 minutes, mix well, and dilute with physiological saline to 10. 7 10 6 10 5For each gradient, 100 μL was spread onto a solid plate and incubated at 42°C under anaerobic conditions for 48 h. Each gradient was repeated twice. Simultaneously, 1 mL of physiological saline and 1 mL of host bacterial culture were used as blank controls. The above steps were repeated. Plates with 30-300 colonies were selected for colony counting. This experiment was repeated three times, and the average value was used.
[0041] Phage lysis rate = (1 - number of colonies in the treatment group / number of colonies in the control group) × 100%.
[0042] 3.2 Experimental Results and Analysis Table 2 Experimental data on the lysis rate of Clostridium perfringens bacteriophages
[0043] The experimental results are shown in Table 2. The lysis rate of RDP-CP-2310008 reached 98.35%, which has a good lysis effect on the host and is suitable for use in the breeding process.
[0044] Example 4: Synergistic Experiment of Bacteriophage and Antibiotic 4.1 Experimental Methods Antibiotic solutions were prepared using 2×TY medium, and the antibiotic concentrations are shown in Table 3.
[0045] Generally, the initial concentration of antibiotics is 1.024%. Following a doubling dilution method, the antibacterial effect of antibiotics is significantly concentration-dependent; as the dilution increases, the antibacterial effect gradually diminishes. In a 96-well plate, this manifests as the culture medium changing from clear to turbid (i.e., the antibiotic has become ineffective, and bacteria can grow normally). 100 μL of sterile TY medium was added to each well of a 96-well plate. 100 μL of different prepared antibiotics were added to each well in the first column, and the mixture was serially diluted up to the 11th column and then discarded. The last column served as a positive control. 100 μL of purified bacterial suspension (diluted 100 times with proliferation medium) was then added to each well and the plate was incubated anaerobically at 42 °C for 24 h.
[0046] Table 3 Antibiotic MIC
[0047] Antibiotic solutions were prepared using TY medium, with concentrations shown in Table 3. 100 μL of sterile TY medium was added to each well of a 96-well plate. Then, 100 μL of the prepared antibiotic solution was added to each well in the first column, and this was serially diluted up to the 11th column, which was then discarded. The last column served as a positive control. Next, 100 μL of purified bacterial suspension (diluted 100-fold with proliferation broth) and phage proliferation broth (diluted 10000-fold) were added, and the plates were incubated at 42°C under oxygen for 24 hours.
[0048] The combined use of bacteriophages and antibiotics can enhance bactericidal effects and reduce the development of drug resistance. Its mechanism of action involves multiple interrelated mechanisms, including alterations in bacterial physiological state, biofilm disruption, resistance suppression, and the involvement of the immune system.
[0049] The results are as follows Figure 6 In the presence of bacteriophages, the minimum susceptibility concentration (MSC) of antibiotics is significantly reduced. Furthermore, in the presence of bacteriophages, antibiotics exhibit synergistic effects on bacterial inhibition / killing through different mechanisms. With the same antibacterial effect, the dosage of antibiotics can be reduced to 1 / 64 to 1 / 2 of the original dosage. In poultry farming, this can significantly reduce antibiotic usage, decrease the emergence of drug-resistant bacteria, improve farming efficiency, and ensure food safety.
[0050] The embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.
Claims
1. A method for reducing the dosage of antibiotics used to treat Clostridium perfringens infections, characterized in that: Clostridium perfringens bacterium phage RDP-CP-2310008 was used in combination with one or more antibiotics; said Clostridium perfringens bacterium phage RDP-CP-2310008 was deposited on November 29, 2023, at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.45778.
2. The method according to claim 1, characterized in that: The combined use reduces the minimum inhibitory concentration of the antibiotic against Clostridium perfringens to 1 / 64 to 1 / 2 of that when the antibiotic is used alone.
3. The method according to claim 1 or 2, characterized in that: The antibiotic is selected from one or more of the following: β-lactam antibiotics, aminoglycoside antibiotics, tetracycline antibiotics, macrolide antibiotics, lincosamide antibiotics, polypeptide antibiotics, and polyphosphate antibiotics.
4. The method according to claim 3, characterized in that: The antibiotic is selected from one or more of the following: amoxicillin, apramycin, amikacin, neomycin, kanamycin monosulfate, doxycycline, florfenicol, chlortetracycline, oxytetracycline, tylosin, tilmicosin, lincomycin, colistin, and fosfomycin sodium.
5. A bactericidal composition, characterized in that: The active ingredients of the composition include: a) Clostridium perfringens bacterium phage RDP-CP-2310008, with accession number CGMCC NO.45778; and b) One or more antibiotics used against Clostridium perfringens.
6. The bactericidal composition according to claim 5, characterized in that: The antibiotic is selected from one or more of the following: β-lactam antibiotics, aminoglycoside antibiotics, tetracycline antibiotics, macrolide antibiotics, lincosamide antibiotics, polypeptide antibiotics, and polyphosphate antibiotics.
7. The bactericidal composition according to claim 6, characterized in that: The antibiotic is selected from one or more of the following: amoxicillin, apramycin, amikacin, neomycin, kanamycin monosulfate, doxycycline, florfenicol, chlortetracycline, oxytetracycline, tylosin, tilmicosin, lincomycin, colistin, and fosfomycin sodium.
8. A Clostridium perfringens phage RDP-CP-2310008, characterized in that: The Clostridium perfringens phage RDP-CP-2310008 was deposited on November 29, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.45778.
9. The use of the Clostridium perfringens phage RDP-CP-2310008 according to claim 8 in the preparation of a medicament for the prevention and / or treatment of Clostridium perfringens infection.
10. A disinfectant, characterized in that: Its active ingredient comprises Clostridium perfringens phage RDP-CP-2310008 as described in claim 8 or the bactericidal composition as described in any one of claims 5 to 7.