Salmonella enteritidis resistant strain phage, phage preparation and application thereof

Abstract

The application belongs to the technical field of biology and relates to a bacteriophage, in particular to a Salmonella enteritidis resistant strain bacteriophage, a bacteriophage preparation and application thereof. The bacteriophage has a preservation number of CCTCC No. M 20251313 and is preserved in the China Center for Type Culture Collection on June 9, 2025. The bacteriophage sBR4p4 is isolated from a host bacterium of the bacteriophage BS3 resistant strain BR4, and the bacteriophage is easy to isolate. The biological property determination result shows that the sBR4p4 has a wide lysis spectrum and temperature tolerance range, good stability and long in-vitro lysis duration. The whole genome sequencing analysis shows that the bacteriophage sBR4p4 does not contain virulence genes, drug resistance genes and lysogeny genes; and the application provides a basis for further large-scale production of a drug for preventing and treating Salmonella enteritidis and Escherichia coli infection.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a bacteriophage, specifically to a bacteriophage of a Salmonella enteritidis resistant strain, a bacteriophage preparation, and its application. Background Technology

[0002] Salmonella enteritidis is a common pathogenic serotype of Salmonella, widely distributed in nature. It can contaminate meat, eggs, and dairy products. Infection in humans and animals primarily causes gastrointestinal symptoms, and in severe cases, can lead to sepsis. With the widespread use of antibiotics, Salmonella resistance is increasing. To address this issue, the development of novel antimicrobial agents has received considerable attention.

[0003] Bacteriophages, as naturally occurring viruses, can specifically recognize and lyse target bacteria without affecting the normal flora. Therefore, phage therapy is considered a promising treatment option. However, a major challenge of phage therapy is the development of resistant strains. Resistant strains typically develop gradually through genetic variation or adaptive changes during long-term exposure to phages. Despite the high specificity and targeting of phages, the development of resistant strains may limit their clinical efficacy. Therefore, improving the long-term efficacy of phage therapy has become an urgent problem to be solved. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a phage strain of Salmonella enteritidis resistant to phage. This invention isolates a phage from a Salmonella enteritidis strain that has developed phage resistance, determines the biological characteristics of the isolated phage, and further analyzes its whole genome sequence, identifying it as a novel phage strain, providing important evidence for its application.

[0005] The present invention further provides a formulation containing the above-mentioned bacteriophage.

[0006] Another object of the present invention is to provide the use of the above-mentioned bacteriophage or bacteriophage preparation in the preparation of a medicament for treating Salmonella enteritidis.

[0007] The technical solution adopted by the present invention to achieve the above objectives is as follows: This invention provides a bacteriophage of a Salmonella enteritidis resistant strain, wherein the bacteriophage Salmonella The accession number for phagesBR4p4 is CCTCC No.M 20251313, and it was deposited at the China Center for Type Culture Collection on June 9, 2025.

[0008] The present invention also provides the application of the above-mentioned bacteriophage in the preparation of drugs for treating infections caused by Salmonella enteritidis and Escherichia coli.

[0009] The present invention further provides a phage preparation comprising the above-mentioned phage.

[0010] Preferably, antibiotics are also included.

[0011] Preferably, the antibiotic is amoxicillin.

[0012] This invention also provides the application of the above-mentioned phage preparation in the preparation of drugs for treating infections caused by Salmonella enteritidis and Escherichia coli.

[0013] The beneficial effects of this invention are as follows: (1) This invention uses the BS3-resistant bacteriophage strain BR4 as the host bacteriophage to isolate bacteriophage sBR4p4, which is easy to isolate. Biological characteristic assays show that sBR4p4 has a wide lysis spectrum and temperature tolerance range, good stability, and a long in vitro lysis duration. Whole-genome sequencing analysis shows that bacteriophage sBR4p4 does not contain virulence genes, drug resistance genes, or lysogenic genes; this provides a foundation for further large-scale production of drugs for the prevention or treatment of Salmonella enteritidis and Escherichia coli infections.

[0014] (2) The phage sBR4p4 obtained by screening in this invention, when used in combination with amoxicillin, can reduce the dosage of amoxicillin and delay the emergence of drug-resistant strains; in vivo synergistic test showed that it had an inhibitory effect on Salmonella enteritidis BR4 infection. Based on a comprehensive evaluation of clinical manifestations, autopsy scores, liver and spleen bacterial load, and cytokines, all experimental groups showed different degrees of antibacterial effect, among which the intervention effect of the prevention group was the most significant.

[0015] Preservation Information Biomaterials: Salmonella phage sBR4p4 Preservation period: June 9, 2025 Preservation Institution: China Center for Type Culture Collection Accession number: CCTCC NO: M 20251313 Address of the repository: Wuhan University Collection Center, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. Attached Figure Description

[0016] Figure 1 Plaques of bacteriophage sBR4p4 (A) and electron microscopic morphology (B); Figure 2 One-step growth curve of bacteriophage sBR4p4; Figure 3 Temperature stability of bacteriophage sBR4p4; Figure 4 pH stability of bacteriophage sBR4p4; Figure 5 Stability of bacteriophage sBR4p4 to ultraviolet light; Figure 6 The sensitivity of bacteriophage sBR4p4 to chloroform. : P≤ 0.01; Figure 7 In vitro bactericidal activity of bacteriophage sBR4p4; Figure 8 The sensitivity of bacteriophage sBR4p4 to room temperature and refrigeration. : P≤ 0.01; Figure 9 A map of the whole genome of bacteriophage sBR4p4; in the map, clockwise represents forward transcription and counterclockwise represents reverse transcription; Figure 10 Phylogenetic tree based on the whole genome sequence of bacteriophage sBR4p4; Figure 11 Results of in vitro synergistic antibacterial assay of bacteriophage sBR4p4 and amoxicillin; in the figure, different lowercase letters indicate significant differences between treatment groups. P ≤0.05; Figure 12 Clinical observation and autopsy tissue lesion scoring; in the figure, different lowercase letters indicate significant differences between treatment groups. P ≤0.05; Figure 13 Liver bacterial load in different groups; in the figure, different lowercase letters indicate significant differences between treatment groups. P ≤0.05; Figure 14 Spleen bacterial load in different groups; in the figure, different lowercase letters indicate significant differences between treatment groups. P ≤0.05; Figure 15 TNF-α expression levels at different time points in each group; in the figure, different lowercase letters indicate significant differences between treatment groups. P ≤0.05; Figure 16 Levels of IL-1β in each group at different time points; Figure 17 IL-10 levels at different time points in each group; in the figure, different lowercase letters indicate significant differences between treatment groups. P≤ 0.05. Detailed Implementation

[0017] The technical solution of the present invention will be further explained and described below through specific embodiments.

[0018] Example 1: Isolation and determination of biological characteristics of Salmonella resistant enteritis phage 1. Method 1.1 Isolation, purification, and titer determination of resistant bacteriophage strains 1.1.1 Resuscitation of the strain The BS3-resistant strain of Salmonella enteritidis, which was frozen at -80℃, was revived.

[0019] 1.1.2 Isolation of resistant bacteriophage strains Wastewater and feces collected from a chicken farm in Weifang, Shandong Province, were soaked for 12 hours and then allowed to stand to remove sediment and impurities. Bacteriophages were isolated using the BS3-resistant strain BR4 as the host bacteriophage. The filtrate was then mixed with 10... 9 Mix 200 μL each of CFU / mL fresh BR4 bacterial culture and add to 5 mL of LB liquid medium. Incubate at 37°C on a shaker until bacterial fragments appear in the test tube, approximately 6 hours later. Isolate bacteriophages using the double-layer agar method and observe for the formation of transparent plaques.

[0020] 1.1.3 Purification of resistant strain bacteriophages Different sized phage plaques were picked up with a pipette tip and added to 1 mL of LB liquid medium. Fresh BR4 bacterial culture was added, and the mixture was incubated at 37°C in a shaker for 6 h. The plaques were centrifuged, filtered, and plated multiple times to form double-layer plates. Single plaques were then purified until the plaques were of uniform size.

[0021] 1.2 Morphological observation of bacteriophages 10 10 20 μL of PFU / mL phage proliferation solution was placed on a copper grid for adsorption for 15 min, then stained with 1% phosphotungstic acid for 2 min, dried at 70℃ for 20 min, and the phage morphology was observed using transmission electron microscopy.

[0022] 1.3 Determination of phage lysis profile The sBR4p4 lysis profiles of 50 Salmonella and Escherichia coli strains from different host sources were determined using the spot method. Freshly cultured suspensions of the aforementioned host bacteria (10 μL / mL) were used for analysis. 9 7 μL of each of the CFU / mL phage culture solution was spotted on NA plates. After solidification, 3 μL of phage proliferation solution was spotted on the surface of the solidified plaque. After complete absorption, the plates were incubated at 37°C for 6 h. At the same time, a positive control of bacterial culture and a negative control of physiological saline were prepared to observe whether transparent phage plaques were formed.

[0023] 1.4 Determination of the optimal multiple of infection (MOI) for bacteriophages Dilute the phage proliferation solution 10-fold and mix it with 1.6 × 10⁻⁶ ppm. 9BR4 bacterial suspensions at CFU / mL were mixed and incubated at ratios of 10, 1, 0.1, 0.01, and 0.001. After 4 h of shaking culture, the mixtures were centrifuged at 12000 r / min for 10 min. Phage titer was determined using the double-layer plate method, with three replicates per group. The MOI with the highest phage titer was selected as the optimal MOI.

[0024] 1.5 Determination of one-step growth curve of bacteriophage Mix 400 μL each of phage proliferation broth and BR4 bacterial culture at an MOI ratio of 10, incubate at 37°C for 5 min, centrifuge at 12000 r / min for 1 min, wash twice with LB medium to remove unadsorbed phage, and finally resuspend in 1 mL of LB medium. Place the resuspended solution in 9 mL of LB medium. Take 200 μL of sample at each time point (5 min intervals for the first h, 20 min intervals for the second h, and 30 min intervals for the third h), centrifuge, filter, and dilute the supernatant. Determine the phage titer using the double-layer plate method, with three replicates each time.

[0025] 1.6 Determination of the thermal stability of bacteriophages The phage proliferation solution was incubated at 40℃, 50℃, 60℃, 70℃ and 80℃ for 1 h, and samples were taken every 20 min. The phage titer was determined by the droplet method, with three replicates per group.

[0026] 1.7 pH stability assay of bacteriophages Add 100 μL of phage proliferation solution to LB liquid medium with pH adjusted to 2-13, and incubate at 37°C for 1 h, 2 h, and 3 h, respectively. Determine the phage titer using the droplet method, with three replicates per group.

[0027] 1.8 Stability of bacteriophages to ultraviolet light Take 2 mL of phage proliferation solution and place it under a UV lamp at a distance of 40 cm. Take 20 μL every 20 min and determine the titer using the droplet method. Each group has 3 replicates. The measurement is carried out continuously for 120 min. Plot the phage change curve over time based on the titer.

[0028] 1.9 Phage susceptibility to chloroform Add 40 μL of chloroform to 1 mL of phage proliferation medium with known titer, and incubate at 37°C with shaking for 30 min. At the same time, set up a blank control without chloroform and determine the phage titer using the droplet method.

[0029] 1.10 In vitro bactericidal activity of bacteriophages Bacteriophages and host bacteria BR4 were mixed in 96-well plates and incubated at 37°C with different MOIs (1000, 100, 10, 1, 0.1, 0.01, 0.001). For the first 14 hours, 200 μL samples were taken every hour to measure the OD. 600 Absorbance values. Positive and negative controls were included, and three replicates were performed at each time point.

[0030] 1.11 Sensitivity of bacteriophages to ambient temperature and refrigeration environments Three samples of bacteriophages with known titers were stored at room temperature and 4°C for three months, and the changes in the titers of the bacteriophages were measured.

[0031] 1.12 Isolation, whole-genome sequencing, and analysis of bacteriophages Phage proliferation broth was sent to Guangdong Megagene Technology Co., Ltd. for sequencing. The sequencing results were used to annotate the complete phage sequence using RAST and GeneMark. Predicted ORFs were validated using BLASTp. tRNAscan-SE was used to search for tRNA-encoding genes. Lysogenic genes within the genome were detected using the online tool PHASTER, and drug resistance and virulence genes of the phage were analyzed using CGE server. A complete genome map was constructed using SnapGene.

[0032] 2. Results and Analysis 2.1 Phage plaques and electron microscopic morphology A resistant bacteriophage strain, named sBR4p4, was isolated from wastewater from a chicken farm. Figure 1 It was found that purified bacteriophage formed transparent plaques with a diameter of about 3 mm on a double-layer plate after 24 hours, and the titer of the host bacterium BR4 could reach ~10. 10 PFU / mL. Under transmission electron microscopy, the head of bacteriophage sBR4p4 has an icosahedral symmetry structure with a diameter of about 60 nm, and the tail is a long-tailed bacteriophage with a wavy and curved shape of about 120 nm.

[0033] 2.2 Results of phage lysis profile determination As shown in Table 1, the lysis rate of the bacteriophage on the tested host bacteria was 30% (15 / 50). It is noteworthy that the bacteriophage sBR4p4 not only lysed the parent strain S3 and the tolerant strain BR4, but also lysed the two avian Escherichia coli strains tested.

[0034] Table 1. Lysis spectrum of bacteriophage sBR4p4 Note: +: can be broken down; -: cannot be broken down. 2.3 Optimal MOI determination results for bacteriophages As shown in Table 2, the titer of phage sBR4p4 was highest when the MOI was 1, so the optimal MOI was determined to be 1.

[0035] Table 2. Optimal MOI determination results for bacteriophage sBR4p4 2.4 One-step growth curve of bacteriophage The one-step growth curve of phage sBR4p4, measured with an MOI of 10, is shown in the figure. Figure 2 The calculated sBR4p4 valence is 10. 9 The concentration of PFU / mL was 20 min, the flare-up period was 100 min, and the flare-up dose was 100 PFU / cell. 2.5 Results of phage thermal stability test Depend on Figure 3 The results show that the titer of bacteriophage sBR4p4 remained essentially unchanged after incubation at 40℃ for 60 min; after incubation at 50℃ and 60℃ for 60 min, the titer decreased by approximately one order of magnitude; after incubation at 70℃ for 60 min, the titer decreased by approximately four orders of magnitude; and after incubation at 80℃ for 20 min, the phage was completely inactivated. These results indicate that bacteriophage sBR4p4 has good stability below 60℃.

[0036] 2.6 Results of pH stability assay for bacteriophages Depend on Figure 4 It can be seen that the activity of bacteriophage sBR4p4 is stable within 3 hours at pH 5-11, and its titer drops sharply and it is rapidly inactivated at pH <5 or >11. 2.7 Results of phage sensitivity testing to ultraviolet light Depend on Figure 5 It can be seen that the titer of bacteriophage sBR4p4 continuously decreases with prolonged ultraviolet irradiation time, and the bacteriophage is completely inactivated after 110 minutes of ultraviolet irradiation. This shows that bacteriophage sBR4p4 is sensitive to ultraviolet light.

[0037] 2.8 Phage Sensitivity to Chloroform Depend on Figure 6 It can be seen that after 30 minutes of contact with chloroform, the titer of bacteriophage sBR4p4 decreased by approximately 0.5 orders of magnitude, which was highly significant compared to the control group without chloroform. P≤ (0.01), indicating that bacteriophage sBR4p4 is sensitive to chloroform.

[0038] 2.9 In vitro bactericidal curve of phage sBR4p4 Depend on Figure 7It can be seen that when bacteriophage sBR4p4 was co-cultured with the host bacterium BR4 for 4-6 h, all groups showed a trend of growth followed by decline, with MOI ranging from 0.001 to 1000. The bactericidal curves almost overlapped with those of the negative control group, indicating that bacteriophage sBR4p4 has a good bactericidal effect.

[0039] 2.10 Stability of bacteriophages to room temperature and refrigerated environments Depend on Figure 8 It was found that the potency of bacteriophage sBR4p4 decreased by approximately five orders of magnitude after 3 months of storage at room temperature. At 4°C, the potency decreased by approximately 1.5 orders of magnitude. The difference in potency between room temperature and 4°C storage was extremely significant. P≤ (0.01), indicating that phage sBR4p4 is more suitable for cold storage.

[0040] 2.11 Phage whole genome sequencing results Whole-genome sequencing of bacteriophage sBR4p4 revealed that the genome is a double-stranded DNA, 44,208 bp in length, with a GC content of 47.81%, and 68 open reading frames (ORFs) were identified. Of these, 26 were forward transcribed and 42 were reverse transcribed. Analysis showed that the sBR4p4 genome does not contain tRNA. Online CGE server and PHASTER analyses confirmed that the sBR4p4 genome does not contain virulence genes, drug resistance genes, or lysogenic genes. Alignment of the 68 ORFs of sBR4p4 using the online BLASTp tool is shown in Table 3. Figure 9 As shown, 41 ORFs have known functions, mainly including two categories: phage structural proteins and lysis-related proteins. The remaining ORFs are hypothetical proteins. By comparing the whole genome of phage sBR4p4 with BLASTn, phage sBR4p4 belongs to... Caudoviricetes division, Dhillonvirus Through phylogenetic tree construction, bacteriophage sBR4p4 was found to be related to bacteriophage sBR4p4. Escherichia .phage B2 (GenBank accession number: NC_073069.1) has a coverage of 92% and a homology of 93.77%.

[0041] Table 3. Results of ORF prediction analysis of the whole genome of bacteriophage sBR4p4 2.12 Phage whole genome phylogenetic tree results A phylogenetic tree was constructed based on the sBR4p4 whole genome. Figure 10 It can be seen that sBR4p4 and bacteriophage Escherichia.phage B2 (GenBank login number: NC_073069.1) is the most closely related.

[0042] Example 2: Synergistic antibacterial effect of bacteriophage sBR4p4 and amoxicillin 1. Method 1.1 In vitro lysis assay of bacteriophage sBR4p4 with amoxicillin The sBR4p4 phage proliferation medium was centrifuged at 12000 r / min, and the supernatant was filtered through a 0.22 μm filter. The sBR4p4 and amoxicillin were then subjected to in vitro lysis experiments according to the groupings in Table 4. The final concentration of 1 / 2 MIC amoxicillin was 128 μg / mL, and the final concentrations of BS3 phage and sBR4p4 phage were 1×10⁻⁶. 6 PFU / mL, the final concentration of BR4 bacterial culture was 1×10⁻⁶. 6 CFU / mL, final volume 200 μL, OD measured at 6 h, 12 h, and 24 h time points. 600 The absorbance value.

[0043] Table 4. In vitro lysis assay of sBR4p4 with amoxicillin (AMX) 1.2 Phage safety and blood entry test 1.2.1 Incubation of hatching eggs Place the purchased SPF hatching eggs in a sterilized incubator, set the temperature to 38℃ and the humidity to 60%, and incubate until hatching. Add water regularly in the morning and evening, and observe the operation of the incubator regularly to ensure normal incubation.

[0044] 1.2.2 Safety and Blood Entry Tests of Bacteriophages SPF chicks were raised to 7 days of age, and 12 chicks were randomly divided into two groups. The experimental group received oral administration of phage sBR4p4 (10 mg / kg). 10 Chickens were administered 0.5 mL of phage sBR4p4 (PFU / mL) orally, while the control group received 0.5 mL of LB. Changes in the flock were observed. Blood was collected from the wing vein at 1 h, 2 h, 4 h, 6 h, and 8 h. The undiluted blood and a 10-fold dilution were mixed with the BR4 bacterial culture using a pour plate method, then mixed with heated and cooled 0.7% LB supernatant, shaken well, and incubated at 37°C for 12 h. The number of bacteriophage plaques on the plates was calculated, and the phage entry into the bloodstream was evaluated based on the plaque formation at each time point.

[0045] 1.3 Determination of the median infectious dose of Salmonella SPF chicks were raised to 7 days of age, and 50 chicks were randomly selected, with 10 chicks per group. Each group of chicks was orally administered 0.5 mL of a different concentration (1×10⁻⁶).10 CFU / mL, 1×10 9 CFU / mL, 1×10 8 CFU / mL, 1×10 7 CFU / mL, 1×10 6 The chicks were given BR4 bacterial suspension (CFU / mL) daily, and their mental state, respiratory rate, and feces were observed. Scores were assigned based on their condition. The specific scoring criteria are shown in Table 5.

[0046] Table 5 Scoring criteria for chick challenge tests 1.4 Synergistic effect of bacteriophage and amoxicillin in preventing Salmonella enteritis in chicks 1.4.1 Preparation of antibiotic-phage mixture Amoxicillin is administered at a dose of 30 mg / kg body weight. The amoxicillin and phage sBR4p4 suspension are mixed at a 1:1 volume ratio. After sterility testing of the phage, it is mixed with amoxicillin to form a total volume of 0.5 mL. It should be prepared and used immediately.

[0047] 1.4.2 Synergistic effect of bacteriophage and amoxicillin in preventing Salmonella enteritis in chicks Ninety-six SPF chicks were raised to 7 days old and then randomly divided into 6 groups of 16 chicks each. Different experimental groups were set up as shown in Table 6. The concentration of BR4 bacterial solution was 10⁸ CFU / mL and the titer of phage sBR4p4 was 10⁸ PFU / mL.

[0048] Table 6. Experimental protocol for the synergistic prevention and treatment of Salmonella enteritis in chicks by bacteriophage sBR4p4 and amoxicillin (AMX). After the above experiment was completed, the chicks were fed normally. The chicks' mental state was observed regularly every day, and they were scored according to the scoring criteria in Table 5. The differences between the different groups were statistically analyzed.

[0049] 1.4.3 Determination of bacterial load in liver and spleen SPF chicks raised to 14 days old were euthanized by neck dislocation. 0.5 g of liver and spleen were taken and 0.5 mL of physiological saline was added. After thorough grinding, the supernatant was collected and diluted 10-fold. 20 μL of the original solution and supernatant of different dilutions were spotted onto SS plates in triplicate. The bacterial load of the liver and spleen was calculated by colony counting.

[0050] 1.4.4 Serum Cytokine Detection At 24 h, 48 h, and 72 h after infection, blood was collected from the jugular vein of 3 chickens in each group. The blood samples were centrifuged at 5000 r / min for 10 min at low temperature to obtain serum. The serum levels of cytokines IL-1β, IL-10, and TNF-α were measured according to the instructions of the ELISA kit (eBioscience, California, USA).

[0051] 1.4.5 Statistical Analysis and Graphing Graphpad 6.02 and IBM SPSS Statistics 26 were used for graphing and analysis.

[0052] 2 Results and Analysis 2.1 Results of in vitro synergistic assay of bacteriophage sBR4p4 and AMX Depend on Figure 11 It can be seen that, after 6 hours of treatment, there was no significant difference between the 1 / 2MIC AMX group, the 1 / 2MIC AMX + sBR4p4 group, and the BR4 challenge group. P >0.05), while the BR4B4 group was significantly lower than the other groups ( P ≤0.05); after 12 h of treatment, there was no significant difference between the 1 / 2MIC AMX+sBR4p4 group and the sBR4p4 group ( P >0.05), all significantly lower than the 1 / 2 MIC AMX group and the BR4 challenge group ( P ≤0.05); at 24 h of action, the 1 / 2MIC + sBR4p4 group was significantly lower than the 1 / 2MIC AMX group and the sBR4p4 group (P≤0.05), while there was no significant difference between the 1 / 2MIC AMX group and the sBR4p4 group. OD in all groups 600 All were significantly lower than those in the BR4 challenge group. This indicates that the combined use of bacteriophage and amoxicillin can reduce the amount of antibiotics used.

[0053] 2.2 Phage safety and blood entry test results After 7 days of clinical observation, there was no significant difference in the mental state of chicks in the oral phage group and the LB group, indicating that oral administration of 10... 10 The PFU / mL phage is safe. Blood transfusion assays showed that phage sBR4p4 (35 PFU / mL) was detectable in blood 2 hours after oral administration; the phage titer in blood reached its peak (4.1 × 10⁻⁶) at 4 hours. 4 (PFU / mL). Bacteriophages were not detected at 6 h and other time points.

[0054] 2.3 Results of the median infection dose of Salmonella enteritidis During the 7-day observation period, no chicks in any of the experimental groups died. Scores for each group were calculated based on the scoring criteria (Table 7). Using the Reed-Muench formula, the optimal dosage for oral administration of 0.5 mL of 7.5 × 10⁻⁶ ppm was determined. 8 CFU containing Salmonella enteritidis can cause clinical symptoms in 50% of chicks.

[0055] Table 7 Results of Median Infection Level in Chicks 2.4 Results of the experiment on the synergistic prevention and control of Salmonella enteritis in chicks by bacteriophage and amoxicillin 2.4.1 Clinical observation and autopsy tissue lesion scoring results Depend on Figure 12 It can be seen that there are significant differences between the prevention group, the single treatment group, the triple treatment group, the amoxicillin group, and the challenge group. P (≤0.05), with the prevention group showing the best intervention effect.

[0056] 2.4.2 Results of Liver and Spleen Bacterial Load Measurement Depend on Figure 13 , 14 It was found that at 24 h of treatment, BR4 bacteria were detected in the liver and spleen only in the challenge group. At 48 h of treatment, BR4 bacteria were detected in the liver and spleen of the single treatment group, the triple treatment group, and the amoxicillin group. There was no significant difference in bacterial load in liver tissue among the groups. p >0.05), while the spleen levels in other groups were significantly lower than those in the challenge group ( P ≤0.05), there was no significant difference between one and three phage treatments within 48 hours. p >0.05), all significantly lower than the amoxicillin group ( P ≤0.05). At 72 h of treatment, the bacterial load in the liver and spleen was consistent, with no significant difference between the single-treatment group, the three-treatment group, and the amoxicillin group. p >0.05), but showed a significant difference from the challenge group ( P (≤0.05). The challenge group maintained a high bacterial load in the liver and spleen throughout the treatment. The prevention group showed no detectable bacterial load from 24 to 72 hours, indicating that each treatment group had varying degrees of inhibitory effect on BR4, with the prevention group showing the best control over bacterial spread in the liver and spleen in the early stages.

[0057] 2.4.3 Serum cytokine detection results Depend on Figure 15 It can be seen that there was no significant difference in TNF-α expression level at 24 h in the prevention group, the group that received one dose of phage, and the group that received three doses of phage. p >0.05), significantly lower than the challenge group, but significantly higher than the amoxicillin group and the blank control group ( P≤0.05); TNF-α expression levels at 48 h showed no significant difference between the prevention group and the group treated with phage once. p >0.05), but significantly lower than the three-times group, antibiotic group, and challenge group, and significantly higher than the blank control group ( P ≤0.05); TNF-α expression levels at 72 h showed no significant difference among the phage application groups (one application, three applications, and antibiotic group). p >0.05), significantly lower than the challenge group, but significantly higher than the prevention group ( P (≤0.05). This indicates that each intervention group can reduce inflammation caused by bacterial infection. Depend on Figure 16 It can be seen that at 24 h of the experiment, the IL-1β expression level in the prevention group was significantly lower than that in other groups. P ≤0.05), there was no significant difference among the phage application group (one application), the three application groups, and the antibiotic application group ( p >0.05), and there were significant differences between other groups and the challenge group ( P ≤0.05); at 48 h of the experiment, the IL-1β expression level in the prevention group was not significantly different from that in the group that received only one phage application ( p >0.05), but significantly lower than the challenge group, the three-treatment group, and the amoxicillin group ( P ≤0.05), significantly higher than the blank control group ( P ≤0.05), there were no significant differences between the single-use and triple-use phage groups and the antibiotic group. p >0.05); at 72 h of the experiment, the IL-1β expression level in the prevention group was significantly lower than that in the phage application group (one-time application), the three-time application, the antibiotic application group, and the challenge group ( P ≤0.05), with no significant difference from the blank control group ( p >0.05), there was no significant difference among the phage application group (one-time application), the three-time application group, and the antibiotic application group ( p >0.05), but all were significantly lower than the challenge group and higher than the blank control group ( P (≤0.05). This indicates that each intervention group can reduce the occurrence of inflammatory responses.

[0058] Depend on Figure 17 It can be seen that the IL-10 expression level at 24 h was not significantly different between the prevention group, the phage application three times group, and the challenge group. p >0.05), significantly lower than the phage administration group and the antibiotic group, but significantly higher than the blank control group ( P ≤0.05), the antibiotic group had the highest IL-10 expression level, significantly higher than other groups ( P≤0.05); IL-10 expression levels at 48 h were significantly lower in the prevention group than in the phage administration group (one-time application, three-time application, and antibiotic group). P ≤0.05), which was significantly higher than that of the challenge group and the blank control group ( P ≤0.05), there were no significant differences among the phage application groups (one-time application, three-time application, and antibiotic application). p >0.05); IL-10 expression levels at 72 h showed no significant difference between the prevention and treatment groups. p >0.05), all significantly higher than the challenge group and the blank control group ( P ≤0.05), the expression level in the challenge group was significantly higher than that in the blank control group ( P (≤0.05). This indicates that the other intervention groups modulate immune function and reduce the occurrence of inflammatory responses by upregulating IL-10 expression levels.

Claims

1. A phage strain resistant to Salmonella enteritidis, characterized in that, The bacteriophage Salmonella The accession number for phagesBR4p4 is CCTCC No.M 20251313, and it was deposited at the China Center for Type Culture Collection on June 9, 2025.

2. A phage preparation, characterized in that, It includes the bacteriophage as described in claim 1.

3. The phage preparation according to claim 2, characterized in that, It also includes antibiotics.

4. The phage preparation according to claim 3, characterized in that, The antibiotic in question is amoxicillin.

Images