Broad-spectrum salmonella bacteriophage SD40-16 and application thereof

Abstract

The invention belongs to the technical field of microbial sterilization preparations, and particularly discloses a broad-spectrum salmonella bacteriophage SD40-16 and application of the broad-spectrum salmonella bacteriophage SD40-16. The bacteriophage SD40-16 can be used for cracking six serotypes of salmonella and escherichia coli including salmonella enteritidis and salmonella typhimurium, is wide in host range and strong in killing capability, and can be used for inhibiting the growth of bacteria. And meanwhile, the strain has good thermal stability and acid-base tolerance, and is easy to proliferate and enrich. The bacteriophage can enhance the antibacterial ability of kanamycin, provides a new drug combination strategy, has an obvious synergistic effect on salmonella resistance when being combined with kanamycin, and has a good application prospect in the aspect of preventing and treating salmonella infection.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial antibacterial preparations, and in particular relates to a broad-spectrum Salmonella phage SD40-16 and an application thereof. Background Art

[0002] Salmonella, a Gram-negative bacillus belonging to the Enterobacteriaceae family, is recognized as the second most common zoonotic pathogen in the European Union. It not only causes salmonellosis in livestock and poultry, seriously threatening the healthy development of the livestock industry, but can also contaminate livestock and poultry products through multiple channels, causing food safety issues and endangering human health.

[0003] Bacteriophages are viruses that can infect and utilize bacterial metabolism to reproduce, and are widely present in nature and in the human body. Bacteriophages are small in size, diverse in morphology, and simple in structure. They are antimicrobial substances found in large quantities in nature, primarily killing bacteria by causing a lytic effect. They are promising biocontrol agents and a reasonable alternative to antibiotics. Therefore, in current practical applications, due to the large number of Salmonella serotypes, the bactericidal range of a single phage is limited by its lytic specificity. Therefore, isolating a broad-spectrum Salmonella phage, broadening the bactericidal range of phages, and increasing their bactericidal activity have always been technical challenges that need to be addressed in this field. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention provides a broad-spectrum Salmonella bacteriophage SD40-16 and its use in the preparation of a Salmonella antibacterial agent. The bacteriophage SD40-16 has a broad host range, strong lytic activity against Salmonella, strong temperature and pH stability, and is easily proliferated and enriched. It has the potential for commercial development as a Salmonella infection preventive and control agent.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] In the first aspect, the present invention provides a broad-spectrum Salmonella phage SD40-16, which was isolated from sewage in a hospital in Guangdong Province. The phage SD40-16 has a regular hexahedral head with a transverse diameter of approximately 67 nm and a long diameter of approximately 60 nm, and an elongated cylindrical tail with a length of approximately 109 nm and a diameter of approximately 11 nm. According to the 9th report of the International Committee on Taxonomy of Viruses, this phage belongs to the order Caudata, family Myoceraceae.

[0007] In a second aspect, the present invention provides a use of the Salmonella phage SD40-16 in the preparation of an antibacterial agent. The use refers to the fact that the phage SD40-16 can lyse Salmonella and Escherichia coli, has a strong killing ability, and can inhibit bacterial growth.

[0008] Furthermore, phage SD410-16 has good environmental tolerance, with good tolerance under acidic and alkaline conditions and an optimal pH value of 4 to 12. It also has good thermal stability, with an optimal temperature of 4 - 50 °C. It is easy to proliferate and enrich, with a latency period of 15 min, explosive growth at 15 - 40 min, and then leveling off, reaching up to 1010.5 PFU / mL at most, and a burst size of 164 PFU / cell. The optimal multiplicity of infection MOI is 10, and the highest titer is 13.34 Lg(PFU / mL).

[0009] Furthermore, the Salmonella includes 6 serotypes of Salmonella, including Salmonella enteritidis, Salmonella typhimurium, Salmonella typhimurium variant, Salmonella indiana, Salmonella derby, and Salmonella st. paul.

[0010] Furthermore, the Escherichia coli includes Escherichia coli ST410 and Escherichia coli ST1011.

[0011] In the third aspect, the present invention provides the application of phage SD40-16 in the preparation of an antibiotic antibacterial synergist, where the antibiotic is kanamycin and the bacterium is Salmonella. It can effectively reduce the dosage of the antibiotic in the antibacterial agent and improve the antibacterial effect.

[0012] In the fourth aspect, the present invention provides an antibacterial composition, including the phage SD40-16 and kanamycin described above. Experiments have proved that when phage SD410-16 and kanamycin act together, they show a synergistic effect, which can reduce the effective MIC of kanamycin by 4 times.

[0013] Furthermore, the concentrations of phage SD40-16 and kanamycin in the composition are 10 3 -10 9 PFU / mL and 0.25 - 258 μg / mL, respectively.

[0014] In the fifth aspect, the present invention provides the application of the above antibacterial composition in the preparation of a drug for treating Salmonella infection.

[0015] Furthermore, the Salmonella includes Salmonella enteritidis SD410, which is multi-drug resistant to tetracycline, ampicillin, spectinomycin, amikacin, and nalidixic acid (MIC > 128 μg / mL).

[0016] Furthermore, the drug contains phage SD40-16 and kanamycin, as well as a pharmaceutically acceptable carrier or excipient.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] (1) A broad-spectrum Salmonella phage SD410-16 was isolated in the present invention. It can lyse Salmonella of 6 serotypes including Salmonella enteritidis and Salmonella typhimurium, as well as Escherichia coli. It has a wide host range and strong killing ability, and can inhibit bacterial growth. It can be used as a potential therapeutic drug for bacterial infections caused by Salmonella and Escherichia coli, for preventing or treating bacterial infections caused by Salmonella. It can also be added to feed as a feed additive, which can specifically and continuously prevent the survival and reproduction of Salmonella in feed, and prevent the contamination of Salmonella in feed storage and animal breeding.

[0019] (2) Phage SD410-16 has good environmental tolerance, good tolerance under acidic and alkaline conditions, and the optimal pH value is 4-12. It also has good thermal stability, and the optimal temperature is 4-50 °C. It is easy to proliferate and enrich, with a latent period of 15 min, explosive growth at 15-40 min, and then tending to be flat, up to 10 10.5 PFU / mL, and the burst size is 164 PFU / cell. The optimal multiplicity of infection MOI is 10, and the highest titer is 13.34 Log(PFU / mL).

[0020] (3) When phage SD410-16 is combined with kanamycin for the bactericidal composition for preventing and treating Salmonella, it shows a synergistic effect, which can reduce the effective MIC of kanamycin by 4 times. It is confirmed by animal experiments that the combination of phage and kanamycin can effectively inhibit bacterial colonization in the liver, spleen, kidney, cecum, etc. caused by Salmonella infection. The phage of the present invention can also be further prepared into a composition with other phages for a broader antibacterial or bacteriostatic application. Description of the Drawings

[0021] Figure 1 It is a plaque picture formed by phage SD410-16.

[0022] Figure 2 It is an electron micrograph of phage SD410-16.

[0023] Figure 3 It is the experimental result of the optimal MOI of phage SD410-16.

[0024] Figure 4 It is the experimental result of the temperature stability of phage SD4410-16.

[0025] Figure 5 It is the experimental result of the acid-base stability of phage SD410-16.

[0026] Figure 6 It is the one-step growth curve of phage SD4410-16.

[0027] Figure 7 This is the antibacterial curve of phage SD410-16.

[0028] Figure 8 This is the experimental result of the combined application of phage SD410-16 and antibiotics.

[0029] Figure 9 This is the in vivo therapeutic effect of the combined use of phage SD410-16 and antibiotics. Specific implementation manners

[0030] Next, in combination with the embodiments of the present invention, the technical solutions of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0031] In the embodiments of the present invention, the test methods used are all conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0032] Example 1 Isolation, purification and preservation of phage

[0033] 1.1 Isolation of Salmonella phage SD410-16

[0034] The double-layer plate spotting method was used to isolate the phage. Take about 5 mL of sewage collected from a hospital in Guangdong, add it to a test tube containing 5 mL of LB liquid medium, culture it in a constant temperature shaker at 37 °C for 4 hours, centrifuge at 10000 r / min for 5 min, and filter and sterilize the supernatant with a 0.22-μm filter membrane. Take 100 μL of Salmonella preserved in the laboratory and mix it with 5-7 mL of LB semi-solid medium cooled to about 55 °C in a centrifuge tube, pour it onto the LB solid plate medium, and let it stand until it solidifies to make a double-layer plate. Take 10 μL of the above-filtered and sterilized supernatant and spot it on the double-layer plate, and culture it at 37 °C for 6 hours, and observe the generation of plaques.

[0035] 1.2 Purification of Salmonella phage SD410-16

[0036] On the double-layer plate with plaques, use a sterile pipette tip to pick out a large and translucent single plaque, place it in SM buffer and let it stand for 12 hours, filter it with a 0.22-μm filter membrane, take 10 μL of the filtrate and spot it on the double-layer plate prepared with its host bacteria, culture it at 37 °C for 6 hours, repeat picking out a single plaque for purification 4-6 times, and form plaques with the same morphology and size on the double-layer plate. As Figure 1 shown, the purified phage SD410-16 was obtained.

[0037] 1.3 Preservation of phages

[0038] Mix the phage proliferation solution with 50% glycerol at a ratio of 6:4, dispense it into 2-ml cryotubes, store it in a -80°C refrigerator, or make it into freeze-dried powder and store it in a 4°C refrigerator.

[0039] Example 2 Identification of phages

[0040] 2.1 Electron microscopy morphological identification of phages

[0041] Prepare a phage high-titer lysate by the plate amplification method. Take 100 μL of the host bacteria liquid grown to the logarithmic phase, mix it with the phage lysate at the optimal multiplicity of infection ratio, prepare a double-layer plate, incubate it at 37°C for 12 hours, add 10 mL of SM buffer to the culture dish, shake it on a shaker at 100 rpm for 4 hours, collect the eluate, centrifuge it at 12000 r / min for 5 min, and filter it through a 0.22-μm filter membrane to obtain the phage high-titer lysate. As determined by the double-layer plate method, the titer of this phage lysate is 2.6×1010 pfu / mL. Stain it negatively with 2% phosphotungstic acid (w / v, pH 7.0), observe the morphology of the phages under a transmission electron microscope, take pictures at an accelerating voltage of 100 kV, and the electron micrograph is as Figure 2 shown.

[0042] As Figure 2 can be seen, the head of phage SD410-16 is a regular hexahedron, with a transverse diameter of about 67 nm and a longitudinal diameter of about 60 nm. The tail is a slender cylindrical shape, about 109 nm long and about 11 nm in diameter. According to the 9th report of the International Committee on Taxonomy of Viruses, it can be judged that this phage belongs to the order Caudovirales and the family Myoviridae.

[0043] Example 3 Determination of the host spectrum of phage SD410-16

[0044] Twenty-eight Salmonella strains (including 7 S. Enteritidis strains, 7 S. Typhimurium strains, 6 S. Indiana strains, 4 S. Kentucky strains, 3 I,4,[5],12:i:- strains, and 1 S. Saintpaul strain) and 4 Escherichia coli strains identified and preserved in the laboratory were selected. The double-layer plate spotting method was used to determine the host range of phage SD410-16. Pour about 5-7 mL of LB solid medium into a sterile petri dish and let it solidify at room temperature as the bottom medium. Place the pre-sterilized LB semi-solid medium in a 50 °C constant temperature water bath. Take 5-7 mL of the semi-solid medium in the 50 °C water bath and add it to a 10 mL sterile centrifuge tube. Cool it to about 46 °C and add 100 μL of Salmonella bacterial solution, then quickly mix well and pour it onto the solid plate. Let it stand for at least 15 min until it solidifies. Make double-layer plates for each strain. Take 10 μL of phage SD410-16 and spot it on each of the above double-layer plates. Incubate overnight at 37 °C and observe whether there are plaques, as well as the size and transparency of the plaques. Classify and record according to the state of the spotted plaques: +2, large and transparent plaques, complete lysis of bacteria; +1, incomplete lysis, greater turbidity in the spotting area; 0, no plaques. The results are shown in Table 1.

[0045] Table 1

[0046]

[0047]

[0048] As can be seen from Table 1, among the 28 Salmonella strains tested, phage SD410-16 could form plaques on 27 strains, accounting for about 96.4%, including 7 S. Enteritidis strains (7 / 7, 100%), 7 S. Typhimurium strains (6 / 7, 85.7%), and 6 S. Indiana strains (6 / 6, 100%), indicating a broad host range. Among them, 10 strains had transparent plaques (+2), accounting for about 35.7%. The proportion of strains that could form transparent plaques on the double-layer plates of S. Enteritidis was 7 / 7 (100%).

[0049] Example 4 Determination of the titer of phage SD410-16

[0050] The titer of phage was determined by the double-layer plate method. Pour about 5-7 mL of LB solid medium into a sterile petri dish and let it solidify at room temperature as the bottom medium. Place the pre-sterilized LB semi-solid medium in a 50 °C constant temperature water bath. Take 9 sterile 1.5 mL centrifuge tubes and number them according to the dilution factor. Add 900 μL of sterile physiological saline to each tube, and add 100 μL of the purified bacterial filtrate to the first centrifuge tube and mix well. Replace the pipette tip and take 100 μL to the second tube, and sequentially perform 10-fold serial dilutions of the bacterial filtrate. Take 100 μL of the bacterial dilutions at different dilution factors and mix with 100 μL of the bacterial suspension of Salmonella SD410-16 in a 10 mL sterile centrifuge tube, incubate at 37 °C for 5 min, add 5-7 mL of 50 °C LB semi-solid medium to the mixture, and then immediately pour it into a petri dish with LB solid medium at the bottom. Let it stand for at least 15 min to prepare double-layer plates, and culture at 37 °C for 18-24 hours, and count the plaques. Select the plates with 20-300 plaques for counting. The titer (PFU / mL) = average number of plaques × 10 × dilution factor.

[0051] The titer of phage SD410-16 measured by the double-layer plate method was 6.1×10 10 PFU / mL.

[0052] Example 5 Determination of the Optimal Multiplicity of Infection and One-Step Growth Curve of Phage SD410-16

[0053] 5.1 Determination of the Optimal Multiplicity of Infection of Phage SD410-16

[0054] The multiplicity of infection (MOI) refers to the ratio of the number of phages that can adsorb to the number of host bacteria within a specific time. In practical applications, the ratio of phages to host bacteria when the highest phage titer is obtained under certain culture conditions is usually taken as the optimal MOI. Adjust the concentration of the host bacterium Salmonella enterica serovar Typhimurium SD410 to 10 7 CFU / mL, and perform 10-fold serial dilutions of the phage SD410-16 lysate with sterile LB liquid medium according to the ratios of MOI being 100, 10, 1, 0.1, 0.01, and 0.001 respectively. Take 100 μL each of the phage dilution and the host bacterium culture solution, add them to 5 mL of pre-warmed LB liquid medium, and culture with shaking at 37 °C and 180 r / min for 4 hours. Centrifuge the mixed culture at 12000 r / min for 2 min and filter through a 0.22 μm filter membrane to obtain the lysate. The titer of the phage lysate was determined by the double-layer plate method, repeated three times, and the multiplicity of infection with the highest lysate titer was the optimal multiplicity of infection of this phage. The results are as Figure 3 shown.

[0055] AsFigure 3 It can be seen that when the MOI is 10, the progeny production rate of phage SD410-16 is the highest, the multiplication factor of the phage reaches the highest value, and the titer is 13.34 Lg(PFU / mL), indicating that the optimal MOI of phage SD410-16 is 10.

[0056] 5.2 Determination of the one-step growth curve of phage SD410-16

[0057] Mix the phage SD410-16 lysate and the host bacterium SD410 bacterial solution according to the optimal multiplicity of infection ratio, place it in a 37°C water bath for 5 min, centrifuge at 12,000 r / min for 5 min, discard the supernatant, wash it twice with pre-warmed LB liquid medium at 37°C, add 1 mL of pre-warmed LB liquid medium at 37°C, resuspend the precipitate, add it to 100 mL of pre-warmed LB liquid medium at 37°C, culture it in a constant temperature shaker at 37°C, sample every 10 min, and measure the phage titer by the double-layer plate method for 180 min, repeating three times. Using time as the horizontal axis and phage titer as the vertical axis, draw the one-step growth curve of the phage, as specifically shown in Figure 6 shown. Calculate the burst size according to the following formula: burst size = final phage titer / initial number of host bacteria.

[0058] From Figure 6 it can be seen that the latent period of phage SD410-16 is 15 min, it undergoes explosive growth from 15 to 40 min, then flattens out, reaching a maximum of 1010.5 PFU / mL, and the burst size is 164 PFU / cell.

[0059] Example 6 Determination of the temperature and acid-base stability of phage SD410-16

[0060] 6.1 Determination of the temperature stability of phage SD410-16

[0061] Put 5 tubes of phage lysate (500 μL / tube) into water baths at 40°C, 50°C, 60°C, and 70°C respectively, sample once at 30 min and 60 min, and measure the phage titer by the double-layer plate method, repeating three times. Take 15 sterilized 1.5 mL centrifuge tubes, add 500 uL of SD410-16 lysate to each tube, with 3 tubes in a group, and place them in water baths at 40°C, 50°C, 60°C, and 70°C respectively. Take out one tube from each water bath at different temperatures at 30 min and 60 min, and use Salmonella enteritidis SD410 as the host bacterium to measure the phage titer by the double-layer plate method, repeating three times. The experimental results are as shown in Figure 4 shown.

[0062] From Figure 4It can be seen that phages have a certain temperature stability. Under the conditions of 4 - 50 °C, the titer of phage SD410 - 16 tends to be stable within 60 min, that is, the optimal temperature is 4 - 50 °C; when the temperature exceeds 60 °C, with the prolongation of time, the activity of the phage gradually decreases, but still maintains partial activity.

[0063] 6.2 Determination of the acid - base stability of phage SD410 - 16

[0064] Use 1 mol / L HC1 and NaOH to adjust the pH value of LB liquid medium to 1 - 13 respectively. Take 900 μL of LB liquid medium with different pH values and add 1 mL of phage, mix evenly, and incubate in a water bath at 37 °C for 1 hour. Use the double - layer plate method with Salmonella enteritidis SD410 as the host bacterium to determine the phage titer under different pH treatments. The experimental results are as Figure 5 shown.

[0065] It can be Figure 5 seen that the optimal pH of phage SD410 - 16 is 4 - 12; when pH ≤ 3 or ≥ 12, the phage activity gradually weakens; when pH is 2 and 13, the phage is completely inactivated. This shows that phage SD410 - 16 has a certain tolerance to acids and bases.

[0066] Example 7 Determination of the antibacterial experiment of phage SD410 - 16

[0067] Cultivate the host bacterium Salmonella enteritidis SD410 to the logarithmic phase, and adjust the absorbance value (OD 600 value) of the bacterial liquid to 0.3 and then dilute it appropriately. Mix the phage and the bacterial liquid in equal amounts at ratios of MOI = 10, 1, 0.1, 0.01, and 0.001, add them to a 96 - well plate, and place them in a constant - temperature incubator at 37 °C and let them stand. Design an equal amount of LB broth as a negative control in the control group, and mix the bacterial liquid and LB broth in equal amounts as a positive control. Use an enzyme - linked immunosorbent assay (ELISA) reader to measure the OD600 value every 30 min, record the results, and draw a growth curve. The results are as Figure 7 shown.

[0068] It can be Figure 7 seen that in the control group, OD 600It continued to rise and reached a maximum of about 1.2 at 10 h, and then decreased at 12 h. For phage SD410-16 with different MOIs, the absorbance values of all phage groups with different multiplicities of infection decreased at 4 h, and then had a slight upward trend after 5 h, but all remained at a relatively low absorbance level, indicating that within 5 h, phage SD410-16 could significantly inhibit the growth of bacteria in the logarithmic growth phase. However, after 5 h, the absorbance value increased significantly, probably due to the large reproduction of phage-resistant bacteria, and the absorbance was less than that of the control group within 12 h. Moreover, it can be seen from the figure that the absorbance values of phage groups with different MOIs were all about 1.2 at 12 h, indicating that there was no significant difference in their antibacterial effects (P>0.05).

[0069] Example 8 Combined application of phage SD410-16 and antibiotic (PAS)

[0070] Salmonella enteritidis SD410 was inoculated into LB broth after overnight culture and cultured for 4 h, and the bacterial suspension concentration was adjusted to 10 6 CFU / mL. Then 100 μL of the bacterial suspension was added to a 96-well plate. Subsequently, different concentrations of phage (50 μL, final titer 10 3 -10 9 PFU / mL) and the antibiotic kanamycin (KAN) (50 μL, concentration 0.5 - 256 μg / mL) were added to the 96-well plate. The positive control group was only the bacterial suspension, and the negative control was only phage or antibiotic. Finally, MH broth was added to make the volume of each well 200 μL. It was cultured in a constant temperature incubator at 37 °C for 24 h. After the culture was completed, the OD600 value of each well was measured and the results were recorded. The results are as Figure 8 shown.

[0071] As Figure 8 can be seen, when treated with 8 μg / mL kanamycin, the graph showed almost complete inhibition of bacterial growth (bacterial reduction rate ≥ 90%). When treated with phage SD410-16 alone, it gradually decreased from 10 3 to 10 6 PFU / mL. When in combination treatment, when the kanamycin concentration dropped to 2 μg / mL, the bacteria were reduced by more than 90%, indicating that the combination of kanamycin and 10 7 、10 8 PFU / mL phage SD410-16 reduced the effective MIC of kanamycin by 4-fold, indicating that phage SD410-16 showed an additive or synergistic effect when combined with kanamycin.

[0072] Example 9 Therapeutic test of in vivo infection with the combination of phage SD410-16 and antibiotic

[0073] 9.1 Sterility test of phage

[0074] Take 5 μL of the phage SD410-16 lysate and spot it on an LB solid plate. After absorption, invert the plate and incubate it in a constant temperature incubator at 37 °C for 24 hours, and observe whether there is colony growth.

[0075] The lysate was spotted on an LB solid plate and incubated at 37 °C for 24 hours. No colony growth was found on the plate, indicating that the phage lysate is sterile and can be used for treatment experiments.

[0076] 9.2 Experimental animals and ethics

[0077] SPF-grade female BALB / c mice (5-7 weeks old, weighing 18-22 g) were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. [License number: SCXK(Xiang)2021-0002] and adaptively raised in a barrier environment animal house for 5 days. All experimental operations have been approved by the Experimental Animal Ethics Committee of South China Agricultural University (Approval number: 2024c094) and strictly follow the 3R principle.

[0078] 9.3 Establishment of infection model

[0079] 72 h before the experiment, chloramphenicol was intermittently added to the sterilized drinking water to a final concentration of 20 μg / mL, and the fresh medicinal solution was replaced daily for 3 days. Salmonella enteritidis SD410 stored at -80 °C was inoculated into LB broth and cultured with shaking at 37 °C until the logarithmic growth phase. After gradient dilution, the concentration of the bacterial suspension was calibrated by the plate counting method to prepare an infectious bacterial solution of 2×10 6 CFU / mL. Each mouse was intraperitoneally injected with 100 μL of the bacterial solution to establish a systemic Salmonella infection model.

[0080] 9.4 In vivo treatment effect of the combination of phage SD410-16 and antibiotics

[0081] 1 h after infection, the mice were divided into 4 groups (n = 5) by the stratified random method, namely the control group, the phage group, the antibiotic group, and the combination group of phage and antibiotic. The control group was injected with 100 μL of PBS, the phage group was injected with 100 μL of 10 9 PFU / mL phage SD410-16, the antibiotic group was injected with 100 μL of 4 μg / mL kanamycin, and the combination group of phage and antibiotic was injected with 100 μL of 10 9PFU / mL phage SD410-16 and 100 μL of 4 μg / mL kanamycin. All administration methods were completed by intraperitoneal injection. 48 h after administration, the mice were euthanized, and the kidney, spleen, liver, and cecum tissues were aseptically removed and weighed and recorded using a precision electronic balance. After homogenizing each tissue, 100 μL of the homogenate was taken for 10-fold serial dilution, and the bacterial load was quantified by the colony-forming unit (CFU) counting method. The bacterial load was expressed as Lg CFU / g tissue. The results are as Figure 9 shown.

[0082] As Figure 9 can be seen, compared with the groups treated with kanamycin alone, phage SD410-16, and PBS (control group), PAS treatment showed better therapeutic effects, reducing the bacterial density by an average of 3.97 log CFU / g (Mann-Whitney t test, all P = 0.0286). Specifically, in the liver, the bacterial load decreased by 1.67 log CFU / g when SD410-16 was used alone, 1.86 log10 units when kanamycin was used alone, and 2.97 log10 units when PAS was used. The PAS treatment reduced the bacterial density by 1.3, 1.11, and 2.97 log CFU / g compared with the groups treated with SD410-16 alone, kanamycin alone, and the control group, respectively. Similar trends were also observed in other organs (spleen, kidney, and cecum). In the spleen, the PAS treatment reduced the bacterial density by 1, 1.08, and 2.44 log CFU / g compared with the groups treated with SD410-16 alone, kanamycin alone, and the control group, respectively. In the kidney, the PAS treatment reduced the bacterial density by 1.81, 0.97, and 3.87 log CFU / g compared with the groups treated with SD410-16 alone, kanamycin alone, and the control group, respectively. In the cecum, the PAS treatment reduced the bacterial density by 1.24, 1.76, and 3.31 log CFU / g compared with the groups treated with SD410-16 alone, kanamycin alone, and the control group, respectively. In summary, the combined treatment of phage SD410-16 and kanamycin is more effective than single phage or antibiotic treatment for Salmonella infection, significantly inhibiting the colonization of pathogenic bacteria.

[0083] Obviously, the specific implementation schemes described above only further elaborate on the purpose, technical solutions, and beneficial effects of the present invention. It should be understood that the above are only specific examples of the present invention and are not used to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of a broad-spectrum Salmonella phage SD40-16 in the preparation of an antibacterial agent, characterized in that, The phage SD40-16 can lyse and inhibit bacterial growth.

2. The application according to claim 1, characterized in that, The bacteria refer to Salmonella and Escherichia coli.

3. The application according to claim 2, wherein The Salmonella is any one of Salmonella enteritidis, Salmonella typhimurium, Salmonella typhimurium variant, Salmonella indiana, Salmonella derby, and Salmonella st. paul.

4. The application according to claim 2, wherein The Escherichia coli is one of Escherichia coli ST410 and Escherichia coli ST1011.

5. Use of bacteriophage SD40-16 in preparing an antibiotic bacteriostatic synergist, characterized in that, The antibiotic is kanamycin, and the bacteria targeted by the bacteriostatic agent are Salmonella.

6. Use of phage SD40-16 combined with kanamycin in the preparation of a reagent for anti-Salmonella infection.

7. The application according to claim 6, characterized in that, The Salmonella is selected from any one of Salmonella enteritidis, Salmonella typhimurium, Salmonella typhimurium variant, Salmonella indiana, and Salmonella derby.

8. An antibacterial composition, characterized in that, It includes phage SD40-16 and kanamycin.

9. The antibacterial composition according to claim 8, characterized in that, The concentrations of phage SD40-16 and kanamycin in the composition are 10 3 -10 9 PFU / mL and 0.25 - 258 μg / mL, respectively.

10. Use of the antibacterial composition according to claim 8 or 9 in the preparation of a medicament for preventing Salmonella infection, characterized in that, The drug includes phage SD40-16 and kanamycin, as well as pharmaceutically acceptable carriers or excipients.

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