Salmonella phage cocktail and application thereof

Abstract

The invention discloses a salmonella bacteriophage cocktail and application thereof, and belongs to the technical field of bioengineering, the salmonella bacteriophage cocktail is characterized by comprising a salmonella bacteriophage vBSenMP9, a salmonella bacteriophage vBSalSPS87 and a salmonella bacteriophage vBSalSPMY153, and the salmonella bacteriophage vBSalSPMY153 is prepared from salmonella bacteriophage vBSenMP9, salmonella bacteriophage The salmonella bacteriophage vBSenMP9 is preserved in the China Center for Type Culture Collection, and the preservation number of the salmonella bacteriophage vBSenMP9 is CCTCC M 20231900; the salmonella bacteriophage vBSalSPS87 is preserved in the China Center for Type Culture Collection, and the preservation number of the salmonella bacteriophage vBSalSPS87 is CCTCC M 20242948; the salmonella bacteriophage vBSalSPMY153 is preserved in the China Center for Type Culture Collection, and the preservation number of the salmonella bacteriophage vBSalSPMY153 is CCTCC M 20242949. The salmonella bacteriophage cocktail has a wide splitting spectrum for salmonella, and genes related to lyogen, drug resistance and toxicity are not found in each salmonella bacteriophage genome, so that the salmonella bacteriophage cocktail is safe when being applied to research and development of medicines and sterilization products.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a Salmonella phage cocktail and its application. Background Art

[0002] Salmonella belongs to Gram-negative enterobacteria and is a common foodborne pathogen and a typical zoonotic pathogen. After infection with Salmonella, it mostly presents as intestinal diseases such as diarrhea, vomiting, gastroenteritis, etc., and the symptoms are relatively mild. However, it is easy to spread from the intestine to other parts of the body, causing invasive diseases such as sepsis, meningitis, osteomyelitis, and even causing death. Due to the extensive use of antibiotics globally, especially in the medical and aquaculture industries, resistant strains of Salmonella have emerged continuously. There is an urgent need to develop new antibiotic alternative drugs to control the infections caused by drug-resistant Salmonella.

[0003] A phage is a type of bacteriophage virus that completes the lysis or its own lysogenic cycle through specific adsorption to bacteria. Phages are divided into virulent phages and temperate phages. Virulent phages achieve the purpose of reproducing offspring by lysing bacteria. After a virulent phage infects a host, the tail fiber protein specifically binds to the receptor on the surface of the host, and then injects its own genetic material into the host cell. It uses the metabolic system of the host cell to replicate nucleic acids, synthesize proteins and various required enzymes. Subsequently, the host cell lyses from the inside under the action of the lytic proteins encoded by the genes, releasing progeny phages and simultaneously achieving the effect of lysing the cells. With the continuous emergence of resistant strains, the antibiotic therapy and application are restricted. Therefore, in the "post-antibiotic era", phage therapy has once again attracted the attention of many researchers.

[0004] Phages have the characteristics of high specificity, high efficiency, and easy availability compared with traditional antibiotic treatments. They can quickly kill specific pathogens without affecting the normal flora of the body, and also have a good killing effect on drug-resistant strains. Research at home and abroad has shown that phages have great potential in preventing and controlling bacterial infections. At present, personalized phage therapy has not been widely promoted. Compared with traditional broad-spectrum antibiotics, phage therapy also faces problems such as strong specificity, narrow bactericidal spectrum, and easy bacterial tolerance. Compared with a single phage, a combination of several different phages, namely a phage cocktail, has more advantages, such as resisting bacterial mutations, broadening the host spectrum, and inhibiting the formation of biofilms. Therefore, the development of a Salmonella phage cocktail is of great significance for preventing and controlling Salmonella infections. Summary of the Invention

[0005] The purpose of the present invention is to provide a Salmonella phage cocktail to solve the problems raised in the above background art.

[0006] To achieve the above object, the present invention provides the following technical solutions: A Salmonella phage cocktail, comprising: Salmonella phage vB_SenM_P9, Salmonella phage vB_SalS_PS87, and Salmonella phage vB_SalS_PMY153; the Salmonella phage vB_SenM_P9 is deposited in the China Center for Type Culture Collection, with the deposit number CCTCC M 20231900, and the deposit date is October 16, 2023; the Salmonella phage vB_SalS_PS87 is deposited in the China Center for Type Culture Collection, with the deposit number CCTCC M20242948, and the deposit date is December 30, 2024; the Salmonella phage vB_SalS_PMY153 is deposited in the China Center for Type Culture Collection, with the deposit number CCTCC M 20242949, and the deposit date is December 30, 2024; the above deposit addresses are all at Wuhan University, Wuhan, Hubei Province, China.

[0007] Preferably, the titer ratio of the Salmonella phage vB_SenM_P9, Salmonella phage vB_SalS_PS87, and Salmonella phage vB_SalS_PMY153 is 1:1:1.

[0008] Another object of the present invention is to provide an application of the above Salmonella phage cocktail in the preparation of drugs for preventing and / or treating infectious diseases caused by Salmonella.

[0009] Another object of the present invention is to provide an application of the above Salmonella phage cocktail in killing Salmonella in the spatial environment.

[0010] Preferably, the above Salmonella phage cocktail can be used to kill Salmonella in humans and animals; humans include those infected or suspected of being infected with Salmonella; animals include all animals infected or suspected of being infected with Salmonella; by using the above Salmonella phage cocktail, emergency prevention and treatment can be carried out.

[0011] Preferably, the spatial environment includes medical environments, natural environments, and animal breeding environments.

[0012] Preferably, the animal breeding environment includes feeding troughs, floors, walls, feces, and bedding; the medical environment includes, for example, wards and treatment rooms; the natural environment includes forests, soil, and rivers; by spraying the above Salmonella phage cocktail to reduce Salmonella in the environment, the purpose of purifying the environment can be achieved.

[0013] Another object of the present invention is to provide a broad-spectrum drug or bactericide for killing Salmonella, which comprises the above Salmonella phage cocktail as an effective active ingredient.

[0014] Preferably, the Salmonella phage cocktail is one of a liquid preparation, a freeze-dried preparation or an oral solid preparation.

[0015] A Salmonella phage cocktail provided by the present invention contains three Salmonella phages, has a broad lysis spectrum against Salmonella, and no genes related to lysogeny, drug resistance and virulence are found in the genomes of each Salmonella phage, indicating its safety for the research and development of drugs and bactericidal products; among them, the Salmonella phage cocktail has bactericidal activity against Salmonella of serotypes O4, O7, O8, O9, O10, and O19, and also has bactericidal activity against Salmonella from different regions; it can be used alone or in combination with other substances, providing a safe and non-toxic phage therapeutic drug and disinfection product for the treatment of Salmonella infection in vivo and in vitro and the disinfection and purification of Salmonella in the environment. Description of the Drawings

[0016] Figure 1 For the plaque morphology and electron microscopy images of phage vB_SalS_PMY153 and phage vB_SalS_PS87; Figure 2 For the results of the determination of the optimal multiplicity of infection of phage vB_SenM_P9, phage vB_SalS_PMY153 and phage vB_SalS_PS87; Figure 3 For the results of the one-step growth curve determination of phage vB_SenM_P9, phage vB_SalS_PMY153 and phage vB_SalS_PS87; Figure 4 For the results of the temperature stability determination of phage vB_SenM_P9, phage vB_SalS_PMY153 and phage vB_SalS_PS87; Figure 5 For the results of the pH stability determination of phage vB_SenM_P9, phage vB_SalS_PMY153 and phage vB_SalS_PS87.

[0017] Figure 6 For the results of the antibacterial activity determination of single phages and Salmonella phage cocktails.

[0018] Figure 7 For the results of the lysis spectrum analysis of single phages and Salmonella phage cocktails. Detailed Embodiments

[0019] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0020] The following embodiments are implementation cases of the technical solutions of the present invention in actual applications, but are not limited thereto. The reagents and experimental equipment involved are all commercially available products.

[0021] The three Salmonella phages vB_SenM_P9, phage vB_SalS_PMY153, and phage vB_SalS_PS87 in the embodiments of the present invention are phages isolated from sewage.

[0022] Among them, phage vB_SenM_P9 has an icosahedral head and a contractile tail, and can be directly obtained from the China Center for Type Culture Collection, with the preservation number CCTCC M 20231900.

[0023] Phage vB_SalS_PMY153 has an icosahedral head. The phage can form clear plaques on LB agar medium, without a halo around, with clear and regular edges, and a diameter of 0.5 - 1 mm. It has been preserved in the China Center for Type Culture Collection on December 30, 2024, with the preservation number CCTCC M 20242949, and the preservation address is: Wuhan University, Wuhan, China.

[0024] Phage vB_SalS_PS87 has an icosahedral head. The phage can form clear plaques on LB agar medium, without a halo around, with clear and regular edges, and a diameter of 0.5 - 3 mm. It has been preserved in the China Center for Type Culture Collection on December 30, 2024, with the preservation number CCTCC M 20242948, and the preservation address is: Wuhan University, Wuhan, China.

[0025] Example 1: This example provides a method for isolating and preparing phage vB_SalS_PMY153 and phage vB_SalS_PS87, which is specifically as follows: The isolation process of the phage is described in detail below. The sewage samples in the examples of the present invention were collected from Changchun Park in Changchun City, and the host bacteria were Salmonella MY153 and S87. The sewage was collected, filtered through gauze, centrifuged at 6000 rpm for 10 min, the supernatant was taken, and the treated sewage was used to replace ddH2O to prepare LB medium (100 mL); 1 mL of the host bacteria cultured overnight was added to the medium, and cultured at 37°C for 10-12 h; 1 mL of the culture was taken, centrifuged at 12,000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm filter to form the phage stock solution and stored. The obtained filtrate was used for plaque testing to check whether it contained phages capable of lysing the host bacteria.

[0026] Plaque testing: Inoculate Salmonella in 5 mL of LB liquid medium at a ratio of 2%, and culture it overnight with shaking at 37°C. Take 0.1 mL of the above-prepared bacterial culture solution and drop it in the center of the plate, and spread the bacterial solution evenly with a spreading rod; after it dries, take 20 μL of the above phage stock solution and drop it in one area; after it dries naturally, place it in an incubator at 37°C for 10 h, and observe whether there are plaques formed in the area where the phage was dropped.

[0027] If plaques are formed, it proves the existence of phages. Take 0.1 mL of the phage stock solution and perform a series of 10-fold dilutions. Take 10 -2 、10 -4 and 10 -6 dilution solutions, each 0.1 mL, are mixed with 0.1 mL of the host bacteria culture. After reacting at room temperature for 15 min, add about 7 mL of 45°C LB semi-solid medium, mix well and quickly pour it onto the upper layer of the 1.5% LB agar medium plate, shake well and leave it flat for 10 min. Wait for it to solidify, place it in an incubator at 37°C for 8 h and observe to obtain a double-layer plate with single plaques formed.

[0028] Example 2: This example provides a method for amplifying and purifying phages, which is as follows: On the double-layer plate with plaques formed, use a sterile pipette tip to pick a single plaque with a large diameter, rounder and translucent shape, inoculate it into 5 mL of LB liquid medium, add 200 μL of the phage host bacteria solution, mix well, react at room temperature for 15 min, culture at 37°C for 10-14 h, centrifuge at 12000 rpm at 4°C for 1 min, and take the supernatant; repeat the double-layer plate experiment, and pick single plaques 4-5 times in this way to purify the phages into plaques of the same size.

[0029] Take 1 mL of freshly cultured host bacteria and add 300 μL of phage lysate (in the ratio of a single phage culture to host bacteria at 1:1, 1:10, and 1:100 respectively). Incubate at 37°C for 20 min to allow phage particles to adsorb to the host bacteria; add 800 mL of LB liquid medium, shake and culture at 37°C for 6 - 8 h, centrifuge at 12,000 rpm at 4°C for 10 min, and take the supernatant, which is the phage lysate.

[0030] PEG purification: Add RNaseA and DNaseⅠ to the phage lysate to a final concentration of 1 μg / mL each, and let it stand at room temperature for 30 min; add NaCl to a final concentration of 1 mol / L, mix well, and then incubate on ice for 1 - 2 h; centrifuge at 8,000 rpm at 4°C for 15 - 20 min and collect the supernatant; add PEG - 8000 at a rate of 10 g per 100 mL, gently stir to dissolve it, and incubate on ice for more than 2 h to allow the phage to form a precipitate under the action of PEG - 8000; centrifuge at 12,000 rpm at 4°C for 10 - 20 min, recover the precipitated phage particles, add 2 mL of SM solution, wash the precipitate thoroughly, and let it act at room temperature for 1 h; add an equal volume of chloroform for extraction, gently shake for 30 s; centrifuge at 5,000 rpm at 4°C for 10 min to separate the organic phase and the hydrophilic phase, and recover the hydrophilic phase containing phage particles to obtain purified phage.

[0031] The double - layer plate method was used to detect the phage titer: Dilute the above - purified phage liquid by 10 - fold gradients. Take 0.1 mL of the phage dilution solutions of several corresponding gradients and mix them well with 0.1 mL of the host bacteria solution, spread them on a double - layer agar plate, and incubate at 37°C for about 10 h. Count the number of plaques on each agar plate. Select the plate with about 100 - 200 plaques, and calculate the initial concentration of the phage according to the dilution factor to obtain the phage titer. As for the purified phage Figure 1 shown, phage vB_SalS_PMY153 can form clear and transparent plaques in 1.5% LB agar medium, without a halo around, with clear and regular edges, and a diameter of 0.5 - 1 mm; phage vB_SalS_PS87 can form clear and transparent plaques in 1.5% LB agar medium, without a halo around, with clear and regular edges, and a diameter of 0.5 - 3 mm.

[0032] The purified phages, named vB_SenM_P9, vB_SalS_PMY153, and vB_SalS_PS87 respectively, are deposited at the China Center for Type Culture Collection. The address of the depository is Wuhan University, Wuhan, China, 430072. The deposit numbers are CCTCC M 20231900, CCTCC M 20242949, and CCTCC M 20242948 respectively. Their taxonomic names are Salmonella phage P9, Salmonella phage vB_SalS_PMY153, and Salmonella phage vB_SalS_PS87 respectively. The deposit dates are both December 30, 2024.

[0033] Example 3: Transmission electron microscopy (TEM) observation of the phages was carried out. Specifically, the phages purified by PEG in Example 2 were used for TEM observation. The specific operation steps were as follows: 10 μL of the sample was dropped onto a copper grid, allowed to precipitate for 15 min, the excess liquid was blotted with filter paper, stained with 2% phosphotungstic acid (PTA) for 1 - 2 min, and then observed using a transmission electron microscope (Hitachi H-7650) after drying. The observation results are as Figure 1 shown. The head of phage vB_SalS_PMY153 is an icosahedron, with a head diameter of approximately 78.4 ± 4.7 nm and a tail length of approximately 140.5 ± 4.3 nm. The head of phage vB_SalS_PS87 is an icosahedron, with a head diameter of approximately 49.9 ± 2.6 nm. According to the "Virus Taxonomy - Eighth Report of the International Committee on Taxonomy of Viruses" published by the International Committee on Taxonomy of Viruses (ICTV) in 2005, phages vB_SalS_PMY153 and vB_SalS_PS87 belong to the family Siphoviridae.

[0034] Example 4: This example provides a method for determining the optimal multiplicity of infection (MOI) of three Salmonella phages, namely vB_SenM_P9, vB_SalS_PMY153, and vB_SalS_PS87, as follows: The bacterial liquid cultured to the logarithmic phase was adjusted to a concentration of 10 8CFU / mL, and then the phages were mixed with the bacterial solution at ratios of phage / bacteria of 0.001, 0.01, 0.1, 1, 10, and 100, transferred to LB liquid medium, and cultured with shaking at 37 °C for 4 h. The culture solution was centrifuged at 10,000 rpm for 15 min at 4 °C, and the supernatant was filtered through a disposable filter with a pore size of 0.22 μm to obtain the phage propagation solution. The titer of the propagation solution was measured using the double-layer plate method, and the phage / bacteria ratio with the highest titer was the optimal MOI. The results are as Figure 2 shown, and the MOI of phage vB_SenM_P9 is 10 -1 when the phage titer is the highest, reaching 1×10 9 PFU / mL; the MOI of phage vB_SalS_PMY153 is 10 -4 -10 -5 when the phage titer is the highest, reaching 1.4×10 8 PFU / mL; the MOI of phage vB_SalS_PS87 is between 1 and 10 -6 when the phage titer can be relatively high, reaching 9.2×10 8 PFU / mL. Therefore, the optimal MOI of phage vB_SenM_P9 is 10 -1 and the optimal MOI of phage vB_SalS_PMY153 is 10 -4 -10 -5 and the optimal MOI of phage vB_SalS_PS87 is between 1 and 10 -5 .

[0035] Example 5: This example provides a method for measuring the one-step growth curve of three Salmonella phages, namely phage vB_SenM_P9, phage vB_SalS_PMY153, and phage vB_SalS_PS87, which is as follows: The host bacteria cultured to the logarithmic phase were mixed with the phages at a ratio of MOI = 0.1, placed at 4 °C for 15 min, the precipitate was suspended with fresh LB liquid medium, and the suspension was cultured with shaking at 37 °C. Samples were taken every 10 min from 0 min to 100 min to measure the phage titer, thereby plotting the one-step growth curve of phage-infected bacteria. The results are as Figure 3 shown. The latent period of phage vB_SenM_P9 is 15 min, and the entire lysis cycle lasts for 70 min; the latent period of phage vB_SalS_PMY153 is 10 min, and the entire lysis cycle lasts for 80 min; the latent period of phage vB_SalS_PS87 is 15 min, and the entire lysis cycle lasts for 90 min.

[0036] Example 6: This example provides a method for measuring the temperature stability of three Salmonella phages, namely phage vB_SenM_P9, phage vB_SalS_PMY153, and phage vB_SalS_PS87, which is as follows: Adjust the titers of phages vB_SenM_P9, vB_SalS_PMY153, and vB_SalS_PS87 to 10 7 PFU / mL, and place them in water baths at 28°C, 37°C, 50°C, 60°C, and 70°C respectively. Take samples every 20 minutes until 80 minutes, and measure the phage titers in different samples. The results are as Figure 4 shown. Phages vB_SenM_P9 and vB_SalS_PS87 can maintain good activity between 28°C and 60°C; when the temperature is greater than 70°C, the activity gradually weakens; phage vB_SalS_PMY153 can maintain good activity between 28°C and 50°C; when the temperature is greater than 60°C, the activity gradually weakens; once the temperature reaches 70°C and above, the phages will quickly inactivate and die.

[0037] Example 7: This example provides a method for measuring the pH stability of phages, which is as follows: Mix phages vB_SenM_P9, vB_SalS_PMY153, and vB_SalS_PS87 with SM buffer solutions of different pH values (pH ranging from 1 to 12) respectively, and then incubate them at 37°C for 1 h, and measure the phage titers in different samples. The results are as Figure 5 shown. Phages vB_SenM_P9, vB_SalS_PMY153, and vB_SalS_PS87 can all maintain good activity at pH values from 4 to 11; in acidic conditions, when the pH value is less than 4, their activities gradually weaken until inactivation; in alkaline conditions, when the pH value is greater than 11, their activities significantly weaken until inactivation.

[0038] Example 8: This example provides a method for measuring the in vitro bactericidal effect of a Salmonella phage cocktail (phage composition), which is as follows: Mix phages vB_SenM_P9, vB_SalS_PMY153, and vB_SalS_PS87 in a ratio of 1:1:1 to form a phage composition (Pc group), and the titer of each phage is 10 8 PFU / mL. Measure the in vitro bactericidal effects of single phages and the phage composition on Salmonella FHC01, and analyze the bactericidal abilities of single phages and the phage composition in vitro. Culture Salmonella FHCO1 in LB liquid medium until the logarithmic phase OD 600mm= 0.8. The phages and phage compositions were added to the logarithmic-phase bacterial solution at an MOI of 0.1 and cultured with shaking in a constant-temperature shaker at 37 °C for 5 h. Under the same culture conditions, but the bacteria without added phages were used as the negative control (Negative group). 200 μL of the culture solution was collected every 1 h, and the phage titer in the culture solution was measured using a double-layer plate. The experiment was repeated three times. The results are as Figure 6 shown. The number of colonies in the phage composition Pc treatment group was basically the same as that in the single phage treatment group in the first 3 h. However, after 3 h, the number of Salmonella in the single phage treatment group began to increase, while the number of Salmonella in the phage composition Pc treatment group remained unchanged. This result indicates that, compared with single phages, the phage composition inhibits the generation of resistant bacteria and has a better antibacterial effect.

[0039] Example 9: This example provides a method for analyzing the host spectrum of a Salmonella phage cocktail, which is as follows: The titers of the phages vB_SenM_P9, vB_SalS_PMY153, and vB_SalS_PS87 were adjusted to 10 9 PFU / mL and then mixed and reserved for use. In the experiment, 108 strains of Salmonella were selected as the objects, and the plaque assay was used to determine the host spectra of the phages vB_SenM_P9, vB_SalS_PMY153, vB_SalS_PS87, and the phage cocktail composed of the three phages. The specific operations are as follows: 0.1 mL of the overnight culture of the test strain to be measured was taken respectively, and they were spread into a uniform bacterial lawn on an LB agar plate using a spreading rod. 10 μL of the phage was added dropwise on the surface of the bacterial lawn. After the liquid drop dried, it was inverted and cultured in an incubator at 37 °C for 12 - 16 h. The results were observed. If plaques were produced, it was recorded as "+", otherwise as "-". The results are as Figure 7 shown. The phage vB_SenM_P9 could produce plaques on the plates of 102 strains of Salmonella, and the positive rate was 94.44% (102 / 108); the phage vB_SalS_PMY153 could produce plaques on the plates of 45 strains of Salmonella, and the positive rate was 41.67% (45 / 108); the phage vB_SalS_PS87 could produce plaques on the plates of 69 strains of Salmonella, and the positive rate was 63.89% (69 / 108). And the host lysis spectra of these three phages were different. The phage cocktail composed of the three phages could produce plaques on the plates of 106 strains of Salmonella (serotypes including O4, O7, O8, O9, O10, O19), and the lysis rate was 98.1% (106 / 108), indicating that the phage cocktail broadened the lysis spectrum against Salmonella.

[0040] In summary, a broad-spectrum Salmonella phage cocktail provided by an embodiment of the present invention has a broad lysis spectrum against Salmonella, and no genes related to lysogeny, drug resistance, and virulence are found in the genomes of each phage, indicating its safety for use in the research and development of drugs and bactericidal products; it has bactericidal activity against Salmonella of serotypes O4, O7, O8, O9, O10, and O19; it has bactericidal activity against Salmonella from different regions; it can be used alone or in combination with other substances, providing a safe and non-toxic phage therapeutic drug and disinfection product for the treatment of Salmonella infections in vivo and in vitro and the disinfection and purification of Salmonella in the environment.

[0041] Inspired by the above ideal embodiments of the present invention, through the above description, relevant staff can completely make various changes and modifications without departing from the technical idea of the present invention. The technical scope of the present invention is not limited to the content in the specification.

Claims

1. A Salmonella phage cocktail, characterized in that: include: Salmonella phage vB_SenM_P9, Salmonella phage vB_SalS_PS87 and Salmonella phage vB_SalS_PMY153; the Salmonella phage vB_SenM_P9 is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC M 20231900; the Salmonella phage vB_SalS_PS87 is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC M 20242948; the Salmonella phage vB_SalS_PMY153 is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC M20242949.

2. The Salmonella phage cocktail according to claim 1, characterized in that The titer ratio of the Salmonella phage vB_SenM_P9, the Salmonella phage vB_SalS_PS87 and the Salmonella phage vB_SalS_PMY153 is 1:1:

1.

3. Use of the Salmonella phage cocktail as claimed in claim 1 or 2 in the preparation of a drug for preventing and / or treating infectious diseases caused by Salmonella.

4. Use of the Salmonella phage cocktail as claimed in claim 1 or 2 in killing Salmonella in a space environment.

5. The use according to claim 4, characterized in that: The space environment includes a medical environment, a natural environment and an animal breeding environment.

6. The use according to claim 5, characterized in that: The animal breeding environment includes feed troughs, floors, walls, feces and bedding; the medical environment includes wards and treatment rooms; the natural environment includes forests, soil and rivers; and the purpose of purifying the environment is achieved by spraying the Salmonella phage cocktail to eliminate the Salmonella in the environment.

7. A broad-spectrum drug or bactericide for killing Salmonella, characterized in that: Comprising the Salmonella phage cocktail according to claim 1 or 2.

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