Salmonella phage targeting salmonella lipopolysaccharide o antigen and applications thereof

Abstract

The application belongs to the technical field of bacteriophages, and particularly relates to a Salmonella bacteriophage targeting Salmonella lipopolysaccharide O antigen and application thereof. The Salmonella bacteriophage vB_SalM-ps4 disclosed by the application does not contain virulence genes, has high safety, good stability within a certain temperature and acid-base range, a wide tolerance range, can effectively inhibit and remove biofilm formed by Salmonella, has a removal and inhibition effect on various Salmonella, has a wide lysis spectrum, has a good prevention and removal effect on Salmonella in food and feed infected by Salmonella; meanwhile, the Salmonella bacteriophage vB_SalM-ps4 can also cross-species lyse Escherichia coli O157:H7, and has a wide application field.

Description

Technical Field

[0001] This invention belongs to the field of phage technology, specifically relating to a Salmonella phage that targets the Salmonella lipopolysaccharide O antigen and its application. Background Technology

[0002] Salmonella ( Salmonella Salmonella (Salmonella) is a Gram-negative bacterium and one of the most important zoonotic foodborne pathogens worldwide, causing a variety of diseases. According to statistics, over 2,500 serotypes of Salmonella have been identified, some of which, particularly *Salmonella enteritidis* and *Salmonella typhimurium*, are highly susceptible to microbial contamination leading to illness in humans and animals. Salmonella is typically found in the intestines of warm-blooded animals, making it easily transferred from the environment to meat during slaughter and processing. Poultry and other foods such as fresh vegetables and fruits are all vectors for human exposure to Salmonella, and infections transmitted through these routes underscore the urgent need to control and prevent Salmonella contamination.

[0003] Currently, antibiotics remain the primary treatment for Salmonella infections in livestock production. However, repeated and inappropriate use of antibiotics has led to drug resistance in pathogens, posing a threat to global health. Therefore, a stable, effective, and environmentally friendly alternative antibiotic therapy is urgently needed for the prevention and treatment of Salmonella infections.

[0004] Bacteriophages are viruses that can infect bacteria. They possess high host specificity and are the most abundant biological entities on Earth. Compared to traditional antibiotics, they can specifically infect target bacteria without affecting the normal microbial environment. They utilize the ability to replicate themselves using bacteria and pose no harm to animals or humans. After nearly a century of development, bacteriophages have become environmentally friendly biocontrol agents for eliminating foodborne pathogens. Currently, commercially recognized bacteriophage products are available in my country, such as bacteriophage modifiers for improving poultry farming environments. However, current Salmonella bacteriophage strains suffer from a narrow lytic spectrum, significantly limiting their application in livestock and poultry farming. Summary of the Invention

[0005] The purpose of this invention is to provide a Salmonella phage that targets the Salmonella lipopolysaccharide O antigen and its application. The Salmonella phage can recognize a variety of Salmonella, has a broad lysis spectrum, and can also lyse O157 serotype Escherichia coli across species.

[0006] This invention provides a Salmonella bacteriophage ( SalmonellaThe Salmonella phage vB_SalM-ps4 has the accession number CCTCC NO: M 20241333.

[0007] This invention provides the application of the Salmonella bacteriophage vB_SalM-ps4 described in the above technical solution in one or both of the following: 1) Prepare formulations to inhibit and / or eliminate Salmonella; 2) Prepare formulations that inhibit and / or eliminate Escherichia coli.

[0008] Preferably, the formulation in 1) includes a formulation that targets Salmonella polysaccharides and / or removes Salmonella biofilms.

[0009] Preferably, the Escherichia coli includes Escherichia coli serotype O157.

[0010] Preferably, the Salmonella includes Salmonella enteritidis (Salmonella enteritidis). S. Enteritidis, Salmonella pullorum ( S. Pullorum, Salmonella Stanley ( S. Stanley), Salmonella Chester S. Chester, Salmonella infantis ( S. Infantis and Salmonella typhimurium ( S. One or more of the following: Typhimurium.

[0011] Preferably, the formulation comprises one or more of the following four items: 1) Medications for the prevention and / or treatment of diseases caused by Escherichia coli infection; 2) Medications for the prevention and / or treatment of diseases caused by Salmonella infection; 3) Disinfectant.

[0012] The present invention also provides a formulation comprising excipients and the Salmonella phage vB_SalM-ps4 described in the above technical solution.

[0013] Preferably, the content of Salmonella phage vB_SalM-ps4 in the formulation is 10. 6 PFU / mL ~10 9 PFU / mL.

[0014] Beneficial effects: This invention provides a Salmonella bacteriophage ( SalmonellaThe Salmonella phage vB_SalM-ps4, with accession number CCTCC NO: M 20241333, is described in this invention. This Salmonella phage vB_SalM-ps4 does not contain virulence genes, exhibits high safety, good stability within a certain temperature and pH range, and a wide tolerance range. It can effectively inhibit and remove biofilms formed by Salmonella, thus possessing a clearing and inhibitory effect on various Salmonella species. It also has a broad lysis spectrum, providing excellent prevention and removal of Salmonella in food and feed infected with Salmonella. Furthermore, the Salmonella phage vB_SalM-ps4 can also lyse Escherichia coli O157:H7 across species, making it suitable for a wide range of applications.

[0015] Biological Preservation Information Salmonella phage ( Salmonella phage vB_SalM-ps4 was deposited on June 21, 2024 at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China. The accession number is CCTCC NO: M 20241333. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 This is a morphological image of the plaques of Salmonella phage vB_SalM-ps4 purified using Salmonella enteritidis SE006 as the host bacterium in Example 1. Figure 2 This is a transmission electron microscope (TEM) image of Salmonella bacteriophage vB_SalM-ps4 from Example 1. Figure 3 This is a circled diagram of the genome annotation of Salmonella phage vB_SalM-ps4 in Example 2; Figure 4 This is a diagram showing the identification of the target receptor for Salmonella bacteriophage vB_SalM-ps4 in Example 4; Figure 5 This is a diagram showing the optimal MOI experimental results for Salmonella phage vB_SalM-ps4 in Example 5; Figure 6 This is a graph showing the one-step growth curve of Salmonella phage vB_SalM-ps4 in Example 6; Figure 7 This is a graph showing the pH stability results of Salmonella bacteriophage vB_SalM-ps4 in Example 7; Figure 8 This is a graph showing the thermal stability results of Salmonella bacteriophage vB_SalM-ps4 in Example 8; Figure 9 The inhibition of biofilm by Salmonella bacteriophage vB_SalM-ps4 in Example 9; Figure 10 This refers to the scavenging effect of Salmonella bacteriophage vB_SalM-ps4 on mature biofilms in Example 10; Figure 11 The determination of Salmonella contamination in chicken feed controlled by Salmonella bacteriophage vB_SalM-ps4 in Example 11; Figure 12 This refers to the inhibitory effect of Salmonella phage vB_SalM-ps4 on Salmonella in chicken meat in Example 12. Detailed Implementation

[0018] This invention provides a Salmonella bacteriophage ( Salmonella The Salmonella phage vB_SalM-ps4 has the accession number CCTCC NO: M 20241333.

[0019] The Salmonella phage vB_SalM-ps4 described in this invention was isolated and screened from urban sewage. It forms clear plaques on LB double-layer plates. Transmission electron microscopy revealed that the head of Salmonella phage vB_SalM-ps4 has a regular polyhedral structure, and the tail is extensible. Its head diameter is 85±5 nm, and its tail length is 90±5 nm, classifying it as belonging to the Myocerviridae family. The Salmonella phage vB_SalM-ps4 described in this invention exhibits stable activity after 1 hour at a pH of 3-11. It remains stable at temperatures between 4℃ and 50℃, with the titer remaining essentially unchanged. Even at 60℃, it maintains good stability, and the phage titer can still be maintained at 1×10⁻⁶. 7 PFU / mL. The genome size of the Salmonella phage vB_SalM-ps4 described in this invention is 158583 bp, and the genome sequence has been submitted to GeneBank, accession number PP533474; whole genome sequencing results show that the Salmonella phage vB_SalM-ps4 does not contain virulence genes or drug resistance genes. Experimental verification revealed that the Salmonella phage vB_SalM-ps4 described in this invention, using the Salmonella lipopolysaccharide O antigen as a target receptor, can lyse a wide range of Salmonella species, including Salmonella enteritidis, Salmonella pullorum, Salmonella Stanley, Salmonella Chester, Salmonella infantis, and Salmonella typhimurium, exhibiting a broad lysis spectrum; simultaneously, it can lyse Escherichia coli O157:H7 across species, making it suitable for a wide range of applications.

[0020] Based on the above advantages, the present invention provides the application of the Salmonella phage vB_SalM-ps4 described in the above technical solution in one or both of the following: 1) preparing a preparation for inhibiting and / or eliminating Salmonella; 2) preparing a preparation for inhibiting and / or eliminating Escherichia coli.

[0021] The formulations of this invention preferably include those targeting Salmonella polysaccharides and / or those scavenging Salmonella biofilms. The *Escherichia coli* of this invention preferably includes serotype O157 *Escherichia coli*; the *Salmonella* preferably includes one or more of *Salmonella enteritidis*, *Salmonella pullorum*, *Salmonella Stanley*, *Salmonella Chesteri*, *Salmonella infantis*, and *Salmonella typhimurium*, more preferably *Salmonella enteritidis* and / or *Salmonella typhimurium*.

[0022] The formulations of this invention preferably include one or more of the following four items: 1) drugs for preventing and / or treating diseases caused by Escherichia coli infection; 2) drugs for preventing and / or treating diseases caused by Salmonella infection; and 3) disinfectants. The Escherichia coli of this invention preferably includes serotype O157 Escherichia coli; the Salmonella preferably includes one or more of Salmonella enteritidis, Salmonella pullorum, Salmonella Stanley, Salmonella Chester, Salmonella infantis, and Salmonella typhimurium, more preferably Salmonella enteritidis and / or Salmonella typhimurium.

[0023] This invention also provides a formulation comprising excipients and the Salmonella phage vB_SalM-ps4 described in the above-described technical solution. Preferably, the content of Salmonella phage vB_SalM-ps4 in the formulation of this invention is 10. 6 PFU / mL ~10 9 PFU / mL, further preferably 10 7 PFU / mL ~10 8 PFU / mL.

[0024] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0025] Example 1 Isolation, purification, proliferation and morphological analysis of bacteriophages (I) Isolation and purification of bacteriophages The collected urban sewage samples were centrifuged at 6000 g for 5 min to remove impurities. The supernatant was filtered through a 0.22 µm filter and set aside. 2 mL of a logarithmic growth phase Salmonella enterica SE006 suspension (disclosed in Gao D, Ji H, Wang L, Li X, Hu D, Zhao J, Wang S, Tao P, Li X, Qian P. Fitness Trade-Offs in Phage Cocktail-Resistant Salmonella enterica Serovar Enteritidis Results in Increased Antibiotic Susceptibility and Reduced Virulence. Microbiol Spectr. 2022 Oct26;10(5):e0291422. doi: 10.1128 / spectrum.02914-22. Epub 2022 Sep 27. PMID:36165776; PMCID: PMC9603643.) was added to 3 Mix 2 mL of filtered wastewater with 1 mL of LB liquid medium and incubate at 37 °C for 6 h in a shaker. Centrifuge the enriched mixture at 12000 g for 5 min, filter the supernatant through a 0.22 µm filter, and perform serial dilutions. Take 100 µL of each dilution and mix it with 100 µL of host bacteria SE006. Add the mixture to 5 mL of melted LB semi-solid medium (containing 0.7% agar), mix well, and then cover the mixture with LB solid agar (containing 1.5% agar). Incubate at 37 °C. Select a clear single plaque, pick it up with a pipette tip, and resuspend it in SM buffer to obtain phage extract. Filter the extract through a 0.22 µm filter and continue to detect phage single plaques using the double-layer plate method. Amplify and culture the phage single plaques after at least 3 rounds of purification, detect their titers, store them at 4 °C, and name them vB_SalM-ps4.

[0026] (ii) Proliferation of bacteriophages The purified bacteriophage vB_SalM-ps4 extract was added to Salmonella enterica SE006 cultured in the logarithmic growth phase and incubated at 37°C in a shaker for 4 h. After the bacteriophages completed lysis and multiplied from the host bacteria, the lysate was centrifuged at 8000 g for 5 min, and the supernatant was filtered through a 0.22 µm filter to remove residual bacteria, yielding the multiplied bacteriophage extract. After measuring its titer, it was stored at 4°C.

[0027] (III) Observe the morphology of bacteriophage plaques. The purified bacteriophage vB_SalM-ps4 was serially diluted, and 100 µL of each dilution was mixed with 100 µL of host bacteria SE006. This mixture was then added to 5 mL of melted LB semi-solid medium, mixed thoroughly, and evenly spread onto LB solid agar. After cooling, the mixture was incubated upside down in a 37°C incubator. The morphology of the plaques was observed after 12 h. The results are as follows: Figure 1 As shown, the formation of a transparent circle on the double-layer plate proves that the bacteriophage was successfully isolated and is real.

[0028] (iv) Observation of bacteriophage morphology using transmission electron microscopy The copper mesh was immersed in a drop of purified high-titer phage extract for 10 min using phosphotungstic acid negative staining. The residual suspension was then blotted off with filter paper, and the mesh was negatively stained with 2% phosphotungstic acid (PTA, 2% w / v) for 2 min, followed by air drying. The prepared copper mesh was then observed under a transmission electron microscope. The results are as follows: Figure 2 As shown. Transmission electron microscopy revealed that this bacteriophage belongs to the order Caudovirales and the family Myoviridae in morphological classification.

[0029] Example 2 Analysis of the genome of bacteriophage vB_SalM-ps4 The complete genome of bacteriophage vB_SalM-ps4 was assembled, and the resulting genome sequence was submitted to GenBank (accession number PP533474). The genome was annotated using the RAST online database (http: / / rast.nmpdr.org / ), and manually validated using NCBI's BLST tool (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The presence of antibiotic resistance and virulence genes in the bacteriophage genome was analyzed using the CARD online database (https: / / card.mcmaster.ca / analyze / rgi), and the presence of lysogen-associated proteins was detected.

[0030] The results are as follows Figure 3 As shown in the functional annotation diagram of the bacteriophage genome, there are no antibiotic resistance and virulence genes in the bacteriophage genome, nor are there any lysogen-related proteins, indicating that the bacteriophage does not contain any components that could pose a threat to humans and can be used clinically.

[0031] Example 3 The detection method for the lysis spectrum of bacteriophage vB_SalM-ps4 is as follows: The bacteria used to determine the lysis profile of bacteriophage vB_SalM-ps4 were all from laboratory-preserved sources. In this embodiment, the double-layer plate method was used to determine the lysis profile of the bacteriophage. Bacterial strains were added to LB liquid medium and incubated overnight at 37°C. 100 µL of the bacterial solution was added to 5 mL of melted LB semi-solid medium, mixed well, and poured into double-layer plates. After cooling and solidification, bacteriophage vB_SalM-ps4 was serially diluted 10-fold. 10 µL of each dilution was dripped onto the double-layer plates. After air-drying until non-flowing, the plates were inverted and incubated at 37°C for 6–12 h. The lysis of the tested strains by the bacteriophage was observed, and the phage plaque formation efficiency (EOP) was calculated according to Formula I.

[0032] EOP = Test bacterial phage titer / Host bacterial phage titer (Formula I).

[0033] The test results are shown in Table 1. This phage can lyse a variety of different serotypes of Salmonella and can also lyse Escherichia coli O157:H7. It has cross-species lysis capability and a relatively broad spectrum of Salmonella lysis.

[0034] Table 1. Phage lysis spectrum

[0035] Note: "+" indicates cleavage, "-" indicates no cleavage.

[0036] Example 4 The identification of bacteriophage vB_SalM-ps4 targeting and recognizing the host bacterial LPS receptor was performed using the following method: There are many ways to identify phage host receptors, such as sequencing phage-resistant mutant strains and comparing gene mutation sites, screening transposon random mutant libraries, and constructing libraries of gene knockout strains of commonly used phage receptors. This study identified receptors by constructing gene knockout strains of commonly used phage receptors. *Salmonella enterica* SE006 was used as a wild-type control, and the previously constructed gene-deleted strains regulating the lipopolysaccharide O antigen, SE006-ΔrfbP and SE006-ΔrfaL, were used as receptor identification strains (Gao et al., 2022, Fitness Trade-Offs in Phage Cocktail-Resistant *Salmonella enterica* Serovar Enteritidis Results in Increased Antibiotic Susceptibility and Reduced Virulence).

[0037] The ability of bacteriophages to lyse SE006-Δ was determined using the double-layer agar speckle method. rfbP and SE006-Δ rfaL If the bacteriophage cannot lyse the gene-deleted strain, it indicates that the gene is its target receptor. The results are as follows... Figure 4 As shown, bacteriophage vB_SalM-ps4 cannot lyse the regulator of the O antigen. rfbP and rfaL The gene deletion indicates that the receptor targeted by this bacteriophage is lipopolysaccharide O antigen.

[0038] Example 5 Determination of the optimal multiple of infection (MOI) of bacteriophage vB_SalM-ps4 The concentration of host bacteriophage SE006 was determined using the plate spread method. Bacteriophage vB_SalM-ps4 (titer 10) stored at 4℃ was used. 8 PFU / mL) extract, and Salmonella enteritidis cultured to the logarithmic growth phase (10 PFU / mL) extract. 7 The bacterial culture was mixed with bacteriophage at concentrations of 100, 10, 1, 0.1, 0.01, 0.001, and 0.0001 (CFU / mL). The mixture was then incubated at 37 °C with shaking for 4 h, followed by centrifugation at 12000 g for 1 min at 4 °C. Immediately after centrifugation, the supernatant was serially diluted 10-fold. The bacteriophage titer at different multiplicity of infection (MOI) was determined using the double-layer plate method. The MOI at the highest MOI was determined by comparing plaque counts.

[0039] The results are as follows Figure 5 As shown, the phage exhibits the best bacterial lysis efficiency and highest titer when the multiplicity of infection (MOI) is 1. Therefore, the optimal MOI for this phage is 1. Figure 4 It can also be observed that the phage titer does not fluctuate much under different MOIs, indicating that the phage can reproduce well under different MOIs.

[0040] Example 6 Determination of one-step growth curve of bacteriophage vB_SalM-ps4 Based on the MOI=1 for bacteriophage vB_SalM-ps4, bacteriophage vB_SalM-ps4 was mixed with Salmonella enterica SE006 cultured to the logarithmic growth phase at an MOI of 1. After mixing, the mixture was incubated at 37°C with shaking for 10 min. Then, it was centrifuged at 8000 g for 10 min at 4°C, the supernatant was discarded, and the bacterial pellet was washed with preheated LB broth at 37°C. This washing was repeated three times. The pellet was then resuspended in an equal volume of preheated LB broth and incubated at 37°C with shaking. Samples were taken every 10 min until 150 min. After centrifugation at 10000 g for 2 min, the supernatant was immediately aspirated and serially diluted 10-fold. The bacteriophage titer at each point was determined using the double-layer plate method. This process was repeated three times. A one-step growth curve was plotted with infection time on the x-axis and bacteriophage titer on the y-axis.

[0041] The results are as follows Figure 6 As shown, the incubation period of the phage was determined by sampling every 10 minutes. After 20 minutes, the curve showed an exponential increase, which was the lysis period of the phage. The titer increased significantly. After 70 minutes, the logarithmic value of the phage titer remained basically unchanged, and it entered the stationary period.

[0042] Example 7 Method for determining the temperature stability of bacteriophage vB_SalM-ps4 The stock solution of bacteriophage vB_SalM-ps4, stored at 4℃, was placed into 1.5mL EP tubes after autoclaving. Seven temperature gradients were set up, and the bacteriophage was placed in a water bath at 4℃, 25℃, 37℃, 50℃, 60℃, 70℃, and 80℃ for 60 min. After serial dilution with SM buffer, the bacteriophage titer at different temperatures was determined using the double-layer plate method. The above operation was repeated three times.

[0043] The results are as follows Figure 7 As shown, the titer of this bacteriophage remained essentially unchanged for 60 minutes at temperatures below 50°C, maintaining its original activity. Even after 60 minutes of exposure at 60°C, the phage titer remained at 1×10⁻⁶. 7 The concentration is approximately PFU / mL. This indicates that within a certain temperature range, temperature has a relatively low impact on this bacteriophage, demonstrating good thermal stability.

[0044] Example 8 Method for determining the pH stability of bacteriophage vB_SalM-ps4 The pH of the buffer solution was adjusted using HCl and NaOH solutions to prepare 12 buffer solutions with different pH gradients (2-13). Bacteriophage vB_SalM-ps4 cells stored at 4℃ were transferred to autoclaved 1.5 mL EP tubes. 100 µL of each phage was added to the buffer solution at each pH value, and the tubes were incubated in a 37℃ water bath for 60 min. After 10-fold serial dilutions with SM buffer, the phage titers at different pH values ​​were determined using the bilayer plate method. This process was repeated three times.

[0045] The results are as follows Figure 8 As shown, the titer of this bacteriophage remained at 1×10⁻⁶ after being placed in a pH range of 3–10 for 60 minutes. 7 With a concentration above PFU / mL, the titer did not decrease significantly after 60 min at pH 11, remaining at 1×10⁻⁶. 6 The concentration of PFU / mL indicates that the phage can survive stably under different pH conditions, demonstrating good pH stability.

[0046] Example 9 Determination of biofilm inhibition by bacteriophage vB_SalM-ps4 Before the assay, prepare a 96-well polyvinyl chloride plate, place it in a clean bench and irradiate it with ultraviolet light for 20 min, and then serially dilute the Salmonella enteritidis SE006 bacterial suspension to 1×10⁻⁶. 5 After CFU / mL concentration, 200 µL of bacterial culture was added to a 96-well plate. The positive control was bacterial culture, and the negative control was PBS buffer. Treatment groups were mixed with phage at MOIs of 1000 and 10000, respectively, and 200 µL of the mixture was added to each well. The plates were incubated at 37°C for 48 h. After incubation, the liquid was aspirated, and the wells were washed three times with PBS buffer, removing any remaining liquid in the third wash. 200 µL of methanol was added, and the plates were allowed to stand for 15 min, then the methanol was aspirated and allowed to air dry. 200 µL of 0.1% crystal violet was added, and the plates were stained for 15 min, then rinsed three times with running water. The liquid in the wells was aspirated, and the plates were allowed to air dry. 200 µL of 30% acetic acid was added, and the plates were measured at 595 nm (OD500) using a microplate reader. 595 The absorbance was measured at ( ).

[0047] The results are as follows Figure 9 As shown, the phage mixed with the bacterial solution can significantly inhibit the formation of biofilms of Salmonella enteritidis SE006.

[0048] Example 10 Determination of the effect of bacteriophage vB_SalM-ps4 on mature biofilms Before the assay, prepare a 96-well polyvinyl chloride plate, place it in a clean bench and irradiate with ultraviolet light for 20 min, then serially dilute Salmonella SE006 to 1×10⁻⁶. 5 After obtaining CFU / mL, 200 µL of bacterial culture was added to a 96-well plate and incubated at 37°C for 48 h. After incubation, the liquid in the wells was aspirated, and the plates were washed three times with PBS and allowed to air dry. Then, 200 µL of phage was added to each well at MOIs of 1000 and 10000, and the plates were incubated at 37°C for 24 h. After incubation, the liquid was aspirated, and the wells were washed three times with PBS buffer, removing any remaining liquid on the third wash. 200 µL of methanol was added, and the plates were allowed to stand for 15 min, then the methanol was washed off and the plates were allowed to air dry. 200 µL of 0.1% crystal violet was added, and the plates were stained for 15 min, then rinsed three times with running water. The liquid in the wells was aspirated, and the plates were allowed to air dry. 200 µL of 30% acetic acid was added, and the plates were measured at 595 nm (OD500) using a microplate reader. 595 The absorbance was measured at ( ).

[0049] The results are as follows Figure 10 The bacteriophage shown can effectively remove mature biofilms.

[0050] Example 11 Determination method for effective control of Salmonella contamination in chicken feed using bacteriophage vB_SalM-ps4 Prepare three sets of pelleted chicken feed, 10g each. Autoclave and dry the chicken feed before use. Dilute the overnight cultured host bacteria SE006 to a bacterial concentration of 10. 6 CFU / mL, using a sterile sprayer, 1 mL of diluted SE006 was evenly sprayed onto the spread feed surface. The control group was sprayed with an equal volume of PBS buffer. The experimental groups were sprayed with 1 mL of phage at MOI=100, MOI=10000, and MOI=10000, respectively, and then incubated at 25℃. Samples were taken at 2 h, 4 h, 6 h, 12 h, and 24 h, 1 g each time, dissolved in PBS buffer, serially diluted, and the SE006 content of the host bacteria was determined.

[0051] The results are as follows Figure 11 As shown, the number of bacteria in the feed can be significantly reduced at different times, indicating that the bacteriophage can effectively reduce the number of Salmonella enteritidis in the feed, reduce Salmonella contamination in the feed, and block the spread of Salmonella in the feed.

[0052] Example 12 Determination of the inhibitory effect of bacteriophages on Salmonella in chicken meat The temperature was set at 25℃. The chicken was purchased from a local supermarket on the day of the test. The chicken was first pre-treated. Using sterile scissors, the chicken was cut evenly into 1 cm pieces. 3After cutting the chicken into small pieces, evenly coat the surface of the chicken with 75% alcohol (by volume) and irradiate it with ultraviolet light in a clean bench for 30 minutes to prepare sterile chicken samples. (The text then repeats the process of preparing 1×10...) 5 CFU / mL of host bacteria SE006 was uniformly sprayed onto the surface of sterile chicken samples to obtain artificially infected chicken samples with SE006. To ensure sufficient adsorption of host bacteria SE006 on the chicken surface, the chicken samples were air-dried in a laminar flow hood. Phages were uniformly sprayed onto the treated chicken surface at MOI ratios of 1000 and 10000. The control group consisted of the same volume of PBS buffer. Samples from both groups at different temperatures were placed in a 4℃ refrigerator and a 25℃ incubator, respectively. Samples were taken at 0, 2, 4, 6, 12, and 24 h, respectively. After mixing in PBS buffer, the samples were centrifuged at 8000g for 5 min, the supernatant was discarded, and the precipitate was serially diluted 10-fold. The antibacterial effect of the phage was then calculated using the plate spread method. The bactericidal efficiency was calculated using the formula: (number of bacteria in the control group - number of bacteria in the experimental group) ÷ number of bacteria in the control group × 100%, Formula II.

[0053] The results are as follows Figure 12 As shown, after treating chicken samples with bacteriophage at 25℃ with MOI=1000 for 6 hours, the bacterial count decreased by 2.41 lg (CFU / mL) compared to the control group, with a sterilization efficiency of 99.5%. At 25℃, after treating chicken samples with bacteriophage at MOI=10000 for 6 hours, the bacterial count decreased by 2.30 lg (CFU / mL) compared to the control group, with a sterilization efficiency of 99.6%, and the antibacterial effect was even more pronounced at 24 hours.

[0054] The antibacterial experiment on chicken meat described in Example 10 demonstrated that the phage had a significant inhibitory effect on Salmonella enteritidis in food.

[0055] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A Salmonella phage targeting Salmonella lipopolysaccharide O antigen as its receptor ( Salmonella The Salmonella phage vB_SalM-ps4 has the accession number CCTCC NO: M 20241333.

2. The use of the Salmonella phage vB_SalM-ps4 according to claim 1 in one or both of the following: 1) Prepare formulations to inhibit and / or eliminate Salmonella; 2) Prepare formulations to inhibit and / or eliminate serotype O157 Escherichia coli; The Salmonella mentioned is Salmonella enteritidis (Salmonella enteritidis). S.Enteritidis Salmonella pullorum ( ), S. pullorum Salmonella Stanley ( S. Stanley Salmonella Chesteria S.Chester Salmonella infantis ( S.Infantis ) and Salmonella typhimurium ( S.Typhimurium ).

3. The application according to claim 2, characterized in that, The formulation in 1) is a formulation that targets Salmonella polysaccharide O antigen and / or removes Salmonella biofilm.

4. The application according to claim 2, characterized in that, The formulation is one or more of the following three: 1) Medications for treating diseases caused by Escherichia coli infection; 2) Medications for treating diseases caused by Salmonella infection; 3) Disinfectant.

5. A formulation, characterized in that, The formulation includes excipients and the Salmonella phage vB_SalM-ps4 as described in claim 1.

6. The formulation according to claim 5, characterized in that, The preparation contains 10g of Salmonella phage vB_SalM-ps4. 6 PFU / mL ~10 9 PFU / mL.

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