Salmonella virulent phage vBSalPNW15 and application thereof

Abstract

The invention discloses a salmonella virulent bacteriophage vBSalPNW15 and application thereof, the bacteriophage is separated from farm sewage, belongs to a long-tail bacteriophage family, has a regular icosahedron head and a long-tail structure, and keeps stable under the conditions that the pH is 4-13 and the temperature is 40-60 DEG C. Genomic analysis shows that the kit does not contain virulence genes and drug-resistant genes and is good in safety. The bacteriophage and cinnamyl aldehyde are combined to be used for preventing and treating salmonella infection, both the bacteriophage and cinnamyl aldehyde show a remarkable synergistic bacteriostatic effect (FICI = 0.5) in vitro and in vivo, and the bacteriophage and cinnamyl aldehyde can effectively inhibit growth of multi-drug-resistant salmonella, remove biological membranes, relieve tissue pathological damage and remarkably improve the survival rate of chicks. The invention provides a safe and efficient antibiotic replacement scheme for preventing and treating salmonella infection, and is suitable for the fields of livestock and poultry breeding and food safety.

Description

Technical Field

[0001] This invention relates to the fields of microbiology and veterinary medicine, and more particularly to a novel virulent Salmonella bacteriophage vB_SalP_NW15, comprising a composition of the bacteriophage and cinnamaldehyde, and its use in the prevention and treatment of Salmonella infections. Background Technology

[0002] Salmonella is a Gram-negative bacterium belonging to the Enterobacteriaceae family and is an important zoonotic pathogen. In poultry farming, Salmonella infection can cause diseases such as pullorum disease and fowl typhoid, leading to high mortality rates, stunted growth, and reduced egg production in chicks, resulting in severe economic losses. Simultaneously, Salmonella can be transmitted to humans through contaminated poultry products, causing food poisoning and threatening public health. Currently, the control of salmonellosis mainly relies on antibiotics. However, the long-term and excessive use of antibiotics has led to increasingly serious problems of Salmonella resistance, with multidrug-resistant strains constantly emerging, reducing the effectiveness of traditional antibiotic treatments and even rendering them ineffective. Furthermore, antibiotic residues also pose a threat to food safety and the ecological environment. Therefore, developing new, safe, and efficient Salmonella control strategies has become an urgent need in the current poultry industry and public health sectors.

[0003] Bacteriophages are viruses that infect bacteria. They possess host specificity, capable of lysing pathogens without affecting host cells or other normal flora. Phage therapy, as a potential antibiotic alternative, offers advantages such as high specificity, self-replication, and low residue levels. However, single-phage therapy has limitations including a narrow host spectrum, susceptibility to bacterial immune system clearance, and the potential to induce phage resistance in bacteria.

[0004] Plant essential oils and their active ingredients, such as cinnamaldehyde, possess broad-spectrum antibacterial activity and are unlikely to induce bacterial resistance. Studies have shown that cinnamaldehyde exerts its antibacterial effects through multiple mechanisms, including disrupting bacterial cell membrane structure and inhibiting energy metabolism. Combining bacteriophages with plant-derived antibacterial agents may enhance antibacterial efficacy through multi-target synergistic effects, reduce the dosage of single ingredients, and lower the risk of bacterial resistance.

[0005] Currently, although there are research reports on the use of bacteriophages or plant essential oils alone for Salmonella control, there are no reports on systematic studies of the combined use of Salmonella bacteriophages and cinnamaldehyde, especially on efficacy evaluation in animal models. Therefore, isolating a new, highly effective Salmonella bacteriophage and exploring its synergistic antibacterial effect with cinnamaldehyde is of great significance for developing novel Salmonella control products. Summary of the Invention

[0006] The purpose of this invention is to provide a novel virulent Salmonella phage to overcome the shortcomings of the prior art.

[0007] Another object of the present invention is to provide a composition of the bacteriophage and cinnamaldehyde.

[0008] Another object of the present invention is to provide the use of the bacteriophage or the bacteriophage and cinnamaldehyde composition.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides a Salmonella bacteriophage vB_SalP_NW15, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 47008 and deposit date of December 17, 2025.

[0011] Furthermore, the bacteriophage vB_SalP_NW15 has the following morphological characteristics: the head is an icosahedron with a diameter of approximately 58 nm; the tail is a slender cylindrical shape with a length of approximately 96 nm and a diameter of approximately 10 nm; fibrous structures are visible at the end of the tail. According to the classification criteria of the International Committee on Taxonomy of Viruses (ICTV), this bacteriophage belongs to the order Caudata and the family Longtailophageidae.

[0012] Furthermore, the bacteriophage vB_SalP_NW15 possesses at least one of the following biological characteristics:

[0013] (1) Good stability: The phage titer remained stable and the activity did not decrease significantly after being treated for 1 hour in the pH range of 4-13 or at 40-60℃ for 1 hour.

[0014] (2) High lysis efficiency: short latency period, about 10 minutes; large lysis volume, about 96 PFU / cell;

[0015] (3) Safety: No virulence genes or antibiotic resistance genes were found in whole-genome sequencing analysis.

[0016] In a second aspect, the present invention provides an antibacterial composition comprising the Salmonella virulent bacteriophage vB_SalP_NW15 as described above and cinnamaldehyde.

[0017] Furthermore, the concentration of bacteriophage vB_SalP_NW15 in the antibacterial composition is 10. 4 PFU / mL up to 10 10 PFU / mL, preferably 10 6 PFU / mL up to 10 8 PFU / mL; the concentration of cinnamaldehyde is 0.1 times the minimum inhibitory concentration (MIC) to 2 times the MIC, preferably 0.5 times the MIC to 1 times the MIC.

[0018] Furthermore, the concentration ratio of bacteriophage vB_SalP_NW15 to cinnamaldehyde in the antibacterial composition is 1:0.1 to 1:10, preferably 1:1 to 1:5.

[0019] Thirdly, the present invention provides the use of the Salmonella virulent bacteriophage vB_SalP_NW15 as described above or the antimicrobial composition as described above in the prevention or treatment of Salmonella infection.

[0020] Furthermore, the Salmonella mentioned is multidrug-resistant Salmonella enteritidis.

[0021] Furthermore, the drug is used to prevent or treat Salmonella enteritidis infection in poultry.

[0022] Furthermore, the drug is administered orally.

[0023] The beneficial effects of this invention are as follows:

[0024] 1. A novel, highly efficient Salmonella phage is provided: This invention isolates a novel virulent Salmonella phage, vB_SalP_NW15, from farm wastewater. This phage exhibits good pH and temperature stability, high lytic efficiency, and genomic analysis shows that it does not contain virulence or drug resistance genes, demonstrating high safety and excellent application potential.

[0025] 2. A highly effective antibacterial composition was developed: This invention is the first to combine bacteriophage vB_SalP_NW15 with the plant-derived active ingredient cinnamaldehyde. In vitro experiments confirmed that the two have a significant synergistic antibacterial effect (FICI=0.5), effectively inhibiting the growth of planktonic Salmonella and significantly enhancing the removal of biofilms.

[0026] 3. The good in vivo therapeutic effect was verified: In the Salmonella infection model of chicks, the combined treatment of bacteriophage vB_SalP_NW15 and cinnamaldehyde can significantly increase the survival rate of infected chicks to 100%, effectively reduce the bacterial load in the blood and liver, and significantly alleviate the pathological damage to tissues such as the heart and liver. The therapeutic effect is significantly better than the use of bacteriophage or cinnamaldehyde alone.

[0027] 4. Provides a new antibiotic alternative: The phage-cinnamaldehyde composition of the present invention provides a safe, efficient, and non-resistant new antibiotic alternative for the prevention and control of Salmonella infection, especially multidrug-resistant Salmonella infection. It helps to reduce the use of antibiotics in the livestock industry, protect the health of livestock and poultry and food safety, and has important economic and social value. Attached Figure Description

[0028] Figure 1Plaque morphology of bacteriophage vB_SalP_NW15 (A, B) and transmission electron microscopy morphology (C).

[0029] Figure 2 Biological characteristics of bacteriophage vB_SalP_NW15. (A) Optimal multiplicity of infection assay; (B) One-step growth curve; (C) Temperature stability; (D) pH stability.

[0030] Figure 3 Genome loop diagram (A) and phylogenetic tree based on terminal large subunit protein of bacteriophage vB_SalP_NW15 (B).

[0031] Figure 4 Synergistic antibacterial effect of bacteriophage vB_SalP_NW15 and cinnamaldehyde in vitro. (A) Time-inhibition curves of different concentrations of cinnamaldehyde combined with fixed bacteriophage concentration; (B) Time-inhibition curves of different concentrations of bacteriophage combined with fixed 1 / 2 MIC cinnamaldehyde; (C) Scavenging effect on Salmonella biofilm.

[0032] Figure 5 Symptoms (AC) of Salmonella enteritis infection model in chicks and pathological changes (D) of dead chicks after necropsy.

[0033] Figure 6 In vivo effects of combined treatment with bacteriophage vB_SalP_NW15 and cinnamaldehyde. (A) Survival rate of chicks in each group; (B) Weight changes; (C) Blood bacterial load; (D) Liver bacterial load; (E) Histopathological observation of heart, liver and other tissues. Detailed Implementation

[0034] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0035] In this embodiment of the invention, the Salmonella virulent bacteriophage vB_SalP_NW15 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 47008 and deposit date of December 17, 2025.

[0036] The bacteriophage vB_SalP_NW15 has the following morphological characteristics: the head is an icosahedron with a diameter of approximately 58 nm; the tail is a slender cylindrical shape with a length of approximately 96 nm and a diameter of approximately 10 nm; fibrous structures are visible at the end of the tail. According to the classification criteria of the International Committee on Taxonomy of Viruses (ICTV), this bacteriophage belongs to the order Caudata and the family Longtailophageidae.

[0037] The bacteriophage vB_SalP_NW15 has at least one of the following biological characteristics:

[0038] (1) Good stability: The phage titer remained stable and the activity did not decrease significantly after being treated for 1 hour in the pH range of 4-13 or at 40-60℃ for 1 hour.

[0039] (2) High lysis efficiency: short latency period, about 10 minutes; large lysis volume, about 96 PFU / cell;

[0040] (3) Safety: No virulence genes or antibiotic resistance genes were found in whole-genome sequencing analysis.

[0041] An antibacterial composition comprising the virulent Salmonella bacteriophage vB_SalP_NW15 as described above and cinnamaldehyde.

[0042] In the antibacterial composition, the concentration of bacteriophage vB_SalP_NW15 is 10. 4 PFU / mL up to 10 10 PFU / mL, preferably 10 6 PFU / mL up to 10 8 PFU / mL; the concentration of cinnamaldehyde is 0.1 times the minimum inhibitory concentration (MIC) to 2 times the MIC, preferably 0.5 times the MIC to 1 times the MIC.

[0043] The concentration ratio of bacteriophage vB_SalP_NW15 to cinnamaldehyde in the antibacterial composition is 1:0.1 to 1:10, preferably 1:1 to 1:5.

[0044] To make the present invention fully disclosed, more specific embodiments are described below.

[0045] Example 1: Isolation and purification of bacteriophage vB_SalP_NW15

[0046] Sample collection and processing: Wastewater and fecal samples were collected from five different live poultry markets or farms in Nanning City, Guangxi Province. 10 mL of each sample was mixed with an equal volume of physiological saline, homogenized thoroughly, and centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant was filtered through a 0.22 μm microporous membrane, and the resulting filtrate was used as the phage stock solution, which was stored at 4°C for later use.

[0047] Phage isolation and preliminary validation: Multidrug-resistant Salmonella enteritidis S55 isolated from the liver of diseased chickens was used as the indicator bacterium. A double-layer agar plate method was employed, with 10 μL of the phage mother droplet added to a semi-solid agar plate containing the host bacterium, and incubated upside down at 37°C for 12-24 hours. The presence of clear plaques was observed to preliminarily validate the phage's existence.

[0048] Plaque purification: Using a sterile pipette tip, pick a single, clearly shaped plaque and place it in a centrifuge tube containing 0.5 mL LB broth. Shake well to elute. Serially dilute the eluent 10-fold and incubate using the double-layer plate method. Pick single plaques again. Repeat this purification step 6-10 times until the obtained plaques are uniform in size and morphology.

[0049] Phage amplification and preservation: The purified single phage plaque eluent was mixed with the logarithmic-phase host bacterial culture (S55) at a certain ratio and incubated at 37°C and 180 rpm with shaking for 4-6 hours until the culture medium became clear. The culture medium was centrifuged at 4°C and 12,000 rpm for 10 minutes, and the supernatant was filtered through a 0.22 μm filter membrane to obtain purified phage lysate. The lysate was mixed with an equal volume of 50% glycerol, aliquoted, and stored at -80°C.

[0050] Result: As Figure 1 As shown in A and B, bacteriophage vB_SalP_NW15 can form single plaques of uniform size and consistent characteristics on a double-layer plate.

[0051] Example 2: Morphological observation of bacteriophage vB_SalP_NW15

[0052] Add 10 μL of purified phage lysis buffer to a copper mesh support membrane, let stand at room temperature for 5 minutes, and blot away excess liquid with filter paper. Add 2% phosphotungstic acid (pH 7.0) for negative staining for 1 minute, aspirate the stain, and allow to dry at room temperature. Observe and photograph using a JEM-1200EXII transmission electron microscope at an accelerating voltage of 80 kV.

[0053] Result: As Figure 1 As shown in Figure C, bacteriophage vB_SalP_NW15 exhibits typical tailed phage morphology. Its head is icosahedral with a diameter of approximately 58±3 nm; the tail is slender, non-contractable, cylindrical, with a length of approximately 96±3 nm and a diameter of approximately 10±3 nm. Fibrous synaptic structures are visible at the tail tip. Based on its morphological characteristics, this phage is classified into the family Longtailed Phagesidae.

[0054] Example 3: Biological characteristics analysis of bacteriophage vB_SalP_NW15

[0055] Optimal multiple of infection (MOI) determination: Adjust the host bacterial S55 concentration to 10. 8CFU / mL. Phage lysates were diluted to different concentrations and mixed with an equal volume of host bacterial culture to obtain MOIs of 10, 1, 0.1, 0.01, and 0.001. After incubation at 37°C with shaking for 3 hours, the lysates were obtained by centrifugation and filtration. The phage titer (PFU / mL) of the lysates at each MOI was determined using the double-layer plate method. The MOI with the highest titer was the optimal MOI.

[0056] Result: As Figure 2 As shown in Figure A, the phage titer was highest when the MOI was 0.0001, reaching 6.625 × 10⁻⁶. 11 PFU / mL, therefore the optimal MOI for determining phage vB_SalP_NW15 is 0.0001.

[0057] One-step growth curve: The phage was mixed with the host bacteria at the optimal MOI. After adsorption for 5 minutes, unadsorbed phage was removed by centrifugation, and the precipitate was resuspended in a large amount of fresh LB medium. The mixture was incubated at 37°C with shaking, and samples were taken every 10 minutes to determine the phage titer for 120 minutes.

[0058] Result: As Figure 2 As shown in B, the incubation period of bacteriophage vB_SalP_NW15 is approximately 10 minutes, the outbreak period is approximately 110 minutes, and the average lysis rate is approximately 96 PFU / cell.

[0059] Temperature stability: The phage lysate was placed in water baths at 40℃, 50℃, 60℃, 70℃ and 80℃, and samples were taken at 20, 40 and 60 minutes. The samples were immediately cooled in an ice bath before the titer was determined.

[0060] Result: As Figure 2 As shown in Figure C, treatment at 40-60℃ for 60 minutes did not significantly change the titer of bacteriophages. Treatment at 70℃ for 60 minutes significantly decreased the titer (p<0.001). Treatment at 80℃ for 20 minutes completely inactivated the bacteriophages.

[0061] pH stability: Adjust the pH of LB liquid medium to 1-13 with HCl or NaOH. Mix the phage lysate with mediums of different pH values, incubate at 37°C for 1 hour, then immediately dilute with neutral pH medium and determine the titer.

[0062] Result: As Figure 2 As shown in D, the titer of bacteriophages remained stable after treatment within the pH range of 4-13 for 1 hour. Activity decreased significantly at pH < 4 and was completely inactivated at pH = 1.

[0063] Example 4: Whole genome sequencing and analysis of bacteriophage vB_SalP_NW15

[0064] Genomic DNA extraction and sequencing: High-purity phage DNA was extracted using the EZNA® Bacteria DNA Kit. Paired-end sequencing (2×150 bp) was performed using the Illumina NovaSeq 6000 platform.

[0065] Sequence assembly and annotation: FastP was used for data quality control, and ABySS and GapCloser were used for sequence assembly to obtain the complete genome. Gene function annotation was performed using databases such as NCBI NR, SwissProt, KEGG, and COG. Virulence genes and drug resistance genes were screened using VFDB and CARD databases, respectively.

[0066] Genomic analysis results:

[0067] Key characteristics: The genome of bacteriophage vB_SalP_NW15 is double-stranded DNA, 42,821 bp in length, with a GC content of 49.06%. The genome diagram is shown below. Figure 3 As shown in Figure A.

[0068] Gene annotation: A total of 57 open reading frames (ORFs) were predicted, of which 35 were annotated with their functions, mainly including structural protein genes (capsid proteins, tail proteins), DNA replication and metabolism-related genes, lyase and depolymerase genes, etc. No tRNA genes were found.

[0069] Safety assessment: Comparison with the Virulence Factor Database (VFDB) and the Antibiotic Resistance Gene Database (CARD) revealed no known virulence genes or antibiotic resistance genes, indicating that this bacteriophage has high biosafety.

[0070] Phylogenetic analysis: Construction of a phylogenetic tree based on terminal large subunit enzyme sequences ( Figure 3 B). The results showed that bacteriophage vB_SalP_NW15 is most closely related to bacteriophages of the Jerseyvirus genus, belonging to the class Caudoviricetes and subfamily Guernseyvirinae.

[0071] Example 5: Determination of the minimum inhibitory concentration (MIC) of cinnamaldehyde against Salmonella

[0072] The MIC of cinnamaldehyde against indicator bacterium S55 was determined using the microbroth dilution method. In 96-well plates, cinnamaldehyde was serially diluted twofold with MHB broth to concentrations ranging from 20 µL / mL to 0.15625 µL / mL. An equal volume of diluted S55 bacterial suspension (final concentration approximately 5 × 10⁻⁶) was added to each well. 5(CFU / mL). Set up control wells for bacterial growth without the drug and control wells for culture medium without the drug. Incubate the 96-well plate at 37°C for 24 hours. The minimum drug concentration at which no bacterial growth is observed visually is defined as the MIC.

[0073] Results: The MIC of cinnamaldehyde against Salmonella enteritidis S55 was 0.625 µL / mL.

[0074] Example 6: Evaluation of the in vitro synergistic effect of bacteriophage vB_SalP_NW15 and cinnamaldehyde

[0075] The checkerboard method was used to determine the synergistic index (FICI): In a 96-well plate, cinnamaldehyde (0-16×MIC) and bacteriophage (10...) were added... 3 -10 10 Eight serial dilutions of drug A (PFU / mL) were performed, arranged in a checkerboard pattern. 50 µL of drug A, 50 µL of drug B, and 100 µL of S55 bacterial suspension (final concentration 10) were added to each well. 6 CFU / mL). After incubation at 37℃ for 24-48 hours, read the results. Calculate the partial inhibitory concentration index (FICI): FICI = (MIC of A when combined with other drugs / MIC of A when alone) + (MIC of B when combined with other drugs / MIC of B when alone).

[0076] Results: The FICI value of bacteriophage vB_SalP_NW15 combined with cinnamaldehyde was 0.5. According to the judgment criteria (FICI ≤ 0.5 indicates synergistic effect), this indicates that the two have a significant synergistic antibacterial effect.

[0077] Time-inhibition curves: Dynamic inhibition experiments were conducted in 96-well plates. Different groups were set up: (A) Fixed phage concentration (10 8 (A) Combined with different concentrations of cinnamaldehyde (2, 1, 1 / 2, 1 / 4 MIC); (B) Fixed cinnamaldehyde concentration (1 / 2 MIC), combined with different concentrations of bacteriophage (10 PFU / mL), 7 10 8 10 9 10 10 PFU / mL). Single-drug groups, positive controls (bacterial suspension), and negative controls (culture medium) were also set up. The total volume per well was 200 µL, and the final bacterial suspension concentration was 10. 6 CFU / mL. Incubate at 37℃ with shaking, and measure OD600 values ​​at 0, 2, 4, 8, 12, 18, and 24 hours.

[0078] Result: As Figure 4As shown in A and 4B, using phage alone or at sub-inhibitory concentrations (1 / 2, 1 / 4 MIC) of cinnamaldehyde, bacteria resumed growth in the later stages of culture (12–24 hours). However, when phage was used in combination with cinnamaldehyde, even at lower concentrations (e.g., 1 / 2 MIC CA + 10), bacterial growth was inhibited. 8 PFU / mL Phage can also continuously and completely inhibit bacterial growth for 24 hours, showing a significant synergistic antibacterial effect.

[0079] Biofilm removal assay: S55 biofilms were cultured in 96-well plates for 48 hours. The supernatant was discarded, and the cells were gently washed three times with PBS. Culture media containing different treatment drugs were added: phage alone (10... 8 The study included tests for cinnamaldehyde (1 / 2 and 1 / 4 MIC), phage combined with cinnamaldehyde, and a PBS control. After treatment at 37°C for 24 hours, the cells were washed with PBS, stained with crystal violet, and the OD595 value was measured to calculate the biofilm clearance rate.

[0080] Result: As Figure 4 As shown in Figure C, the biofilm clearance rate of bacteriophage alone or cinnamaldehyde alone was only 20.63%-29.66%. However, when the two were used in combination, the biofilm clearance rate was significantly increased to 45.73%-51.60%, demonstrating that the combined regimen has synergistic anti-biofilm activity.

[0081] Example 7: In vivo efficacy evaluation of bacteriophage vB_SalP_NW15 and cinnamaldehyde (chick infection model)

[0082] Infection model establishment and LD50 determination: Sixty 5-day-old SPF broiler chickens were randomly divided into 6 groups of 10 each. Different doses of S55 bacterial suspension (10 mg / L) were administered intraperitoneally. 6 Up to 10 10 CFU / animal, while the control group was injected with an equal volume of physiological saline. Observations were conducted for 7 consecutive days, mortality was recorded, and the median lethal dose (LD50) was calculated.

[0083] Result: The LD50 of S55 for chicks was 10. 7 CFU / mL. At this dose, an acute systemic infection model was successfully established in chicks, who exhibited symptoms such as lethargy, diarrhea, and unsteady gait. Figure 5 AC), with a mortality rate of approximately 50%. Autopsy of dead chicks revealed significant lesions in organs such as the heart, liver, and spleen. Figure 5 D).

[0084] Grouping and treatment: 70 five-day-old SPF white-feathered broilers were randomly divided into 7 groups of 10 each.

[0085] Group 1 (NC): Blank control, injected and orally administered normal saline.

[0086] Group 2 (PN): Phage control, injected with physiological saline + orally administered high-dose phage (10 8 PFU / mL, 1 mL / animal).

[0087] Group 3 (PC): Bacterial model group, injected with S55 bacterial solution (10 8 (CFU / mL, 100 µL / animal) + oral saline.

[0088] Group 4 (P): Phage treatment group, infection + oral phage (10 8 PFU / mL, 1 mL / animal).

[0089] Group 5 (C): Cinnamaldehyde treatment group, infection + oral cinnamaldehyde (200 mg / kg).

[0090] Group 6 (CPH): Combined high-dose group, infection + oral phage (10 4 PFU / mL (1 mL / animal) + cinnamaldehyde (200 mg / kg).

[0091] Group 7 (CPL): Combined low-dose group, infection + oral phage (10 4 PFU / mL, 1 mL / animal) + cinnamaldehyde (100 mg / kg).

[0092] The first dose was administered 2 hours after infection, once daily for 3 consecutive days.

[0093] Treatment observation and sample collection: Chicks were observed continuously for 7 days, and their mental state, clinical symptoms, and mortality were recorded. On day 7, all surviving chicks were weighed and then euthanized. Blood and liver tissue were collected for bacterial load determination. Heart, liver, spleen, and ileum tissue were collected, fixed in 10% formalin, paraffin-embedded, sectioned, and stained with H&E for histopathological analysis.

[0094] Treatment results:

[0095] Survival rate: such as Figure 6 As shown in Figure A, the survival rate of chicks in both the combined high- and low-dose treatment groups (CPH, CPL) reached 100%, significantly higher than that in the model group (PC, 50%), the phage monotherapy group (P, 70%), and the cinnamaldehyde monotherapy group (C, 80%). The survival rate of the phage control group (PN) was 100%, indicating that the high-dose phage itself was non-toxic to chicks.

[0096] Weight changes: such as Figure 6As shown in Figure B, the model group experienced a significant decrease in body weight compared to the control group. The combined high-dose therapy (CPH) group showed no significant difference in body weight compared to the control group, indicating the best recovery outcome.

[0097] Bacterial load: such as Figure 6 As shown in C and 6D, the bacterial load in the blood and liver of each treatment group was significantly lower than that of the model group (p<0.05). Among them, the combined treatment group showed the most significant bacterial clearance effect, with the bacterial load in the liver decreasing by several orders of magnitude compared with the model group (p<0.001).

[0098] Histopathology: such as Figure 6 As shown in Figure E, severe inflammation and necrosis were observed in the heart, liver, and ileum of chicks in the model group. The lesions were alleviated in the single-drug treatment group, but edema and inflammatory cell infiltration were still visible. The lesions were further improved in the combined low-drug group. No significant pathological changes were observed in any of the examined tissues in the combined high-drug group and the blank control group, indicating that the combined treatment effectively alleviated tissue damage caused by Salmonella infection.

[0099] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Various modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope defined by the claims should be included within the scope of protection of the present invention.

Claims

1. Salmonella virulent bacteriophage vB_SalP_NW15, characterized in that, The bacteriophage is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 47008 and deposit date of December 17, 2025.

2. The Salmonella virulent bacteriophage vB_SalP_NW15 according to claim 1, characterized in that, The bacteriophage has the following morphological characteristics: The head is an icosahedron with a diameter of 55-61 nm; the tail is cylindrical with a length of 93-99 nm and a diameter of 8-12 nm.

3. The Salmonella virulent bacteriophage vB_SalP_NW15 according to claim 1, characterized in that, The bacteriophages were treated at pH 4-13 for 1 hour, and the titer remained above 90% of the initial titer.

4. The Salmonella virulent bacteriophage vB_SalP_NW15 according to claim 1, characterized in that, The bacteriophage, when treated at 40-60℃ for 1 hour, retains more than 90% of its initial titer.

5. The Salmonella virulent bacteriophage vB_SalP_NW15 according to claim 1, characterized in that, The phage has a latency period of 8-12 minutes and a lysis rate of 90-100 PFU / cell.

6. The Salmonella virulent bacteriophage vB_SalP_NW15 according to claim 1, characterized in that, The full-length genome of the bacteriophage is 42,821 bp, with a GC content of 49.06%, and it does not contain virulence genes or drug resistance genes.

7. An antibacterial composition, characterized in that, It contains the Salmonella virulent bacteriophage vB_SalP_NW15 as described in any one of claims 1-6 and cinnamaldehyde.

8. The antibacterial composition according to claim 7, characterized in that, The concentration of the bacteriophage was 10. 4 -10 10 PFU / mL, wherein the concentration of cinnamaldehyde is 0.1-2 MIC.

9. The antibacterial composition according to claim 8, characterized in that, The concentration ratio of bacteriophage to cinnamaldehyde in the antibacterial composition is 1:0.1-10.

10. The use of the antimicrobial composition according to any one of claims 7-9 in the prevention and treatment of Salmonella infection.

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