Cocktail preparation based on duck-origin salmonella adaptive evolution bacteriophage and application of cocktail preparation

Abstract

The invention discloses a cocktail preparation based on duck-origin salmonella adaptive evolution bacteriophage and application of the cocktail preparation, and belongs to the technical field of biological medicine. The cocktail preparation consists of a phage phi ST 705 with the preservation number of CCTCC (China Center for Type Culture Collection) NO: M 2025836, a phage phi R2 with the preservation number of CCTCC NO: M 2025837 and a phage phi R1-1-1 with the preservation number of CCTCC NO: M 2025838. The bacteriophage phi ST 705 is separated and purified from sewage of a farm, and the host bacterium is salmonella ST 705; the bacteriophage phi R2 and the bacteriophage phi R1-1-1 correspond to bacteriophages separated from the salmonella ST 705 in an adaptive evolution strain under the continuous selection pressure of the bacteriophage phi ST 705. The cracking capacity and the host spectrum of the cocktail preparation are remarkably expanded, and the resistant mutation rate of salmonella ST 705 can be effectively reduced.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to a duck-derived Salmonella adaptive evolution phage cocktail formulation and its application. Background Technology

[0002] Salmonella, a significant zoonotic pathogen, is characterized by its highly concealed, multi-stage, and high-risk transmission. Its contamination has permeated the entire supply chain of farming, processing, and distribution, posing a substantial challenge to public health prevention and control systems. Antibiotics are the primary means of combating Salmonella infection, but the increasingly serious problem of bacterial resistance severely limits their effectiveness, leading to greater caution in their use. Studies show that the resistance rate of Salmonella to commonly used antibiotics is increasing year by year, with most strains exhibiting multidrug resistance. More seriously, resistance genes in drug-resistant strains can spread rapidly between different strains through mobile genetic elements such as plasmids and integrons, resulting in a continuously expanding resistance spectrum of Salmonella and severely weakening the effectiveness of antibiotics. Therefore, it is necessary to develop new antibacterial methods to replace antibiotics.

[0003] Phage therapy, as a highly promising innovative strategy, offers a new direction for solving the problem of drug-resistant bacterial infections. Unlike traditional antimicrobial drugs, which have the limitation of broad-spectrum killing and easily causing dysbiosis, phages can achieve precise infection by recognizing specific receptors on the bacterial surface (such as lipopolysaccharides and capsular polysaccharides), effectively eliminating pathogens while minimizing the impact on beneficial bacteria in the host's gut. Currently, phages have made great progress in the prevention and treatment of Salmonella infections. However, there are numerous Salmonella serotypes, and the differences in surface receptors among different serotypes result in significant differences in the clearance ability of a single phage against different Salmonella serotypes. Furthermore, Salmonella can develop resistance to single phages through adaptive evolution and evade infection through multiple mechanisms. This continuous evolutionary game between phages and bacteria makes single or fixed combinations of phage preparations highly susceptible to ineffectiveness.

[0004] Phage cocktail therapy is an emerging approach to enhance the antibacterial efficacy of phages. Combining multiple phages with different host spectra and lytic properties can effectively broaden the bactericidal spectrum, enhance efficacy, and maximally inhibit the development of bacterial resistance. An ideal cocktail formulation should contain phages that can target different genotypes or species of the same bacteria to address polymorphic or mixed infections. Screening for suitable phage combinations, enabling each phage to target different bacterial surface receptors, can not only significantly enhance therapeutic efficacy but also increase the bacterial evolutionary cost, thereby delaying the development of resistance.

[0005] In the long-term process of co-evolution, bacteriophages and bacteria have driven each other's evolution through selective pressure. To cope with the multiple defense mechanisms of bacteria, bacteriophages have continuously evolved diverse countermeasures to maintain their infectious adaptability. For example, they can adapt to new receptors by modifying receptor-binding proteins; encode variable-specific RBPs; and expand their host range through gene mutations. Through these continuously evolving countermeasures, bacteriophages constantly break through bacterial defense barriers, maintaining a dynamic balance and co-evolution with their host bacteria. Utilizing the co-evolution of the host and bacteriophage can continuously improve the infectious adaptability of bacteriophages. Based on the theory of bacterial-bacteriophage co-evolution, strategies for rationally designing cocktail formulations by simulating natural evolution have been developed in this field. The typical path is as follows: first, screen for bacteriophages that can lyse the original target bacteria (first-line bacteriophages); then, use these bacteriophages to induce resistant mutant strains; then, use these mutant strains as hosts to screen for new bacteriophages (second-line bacteriophages); finally, combine the first-line and second-line bacteriophages to obtain formulations with a broader lysis spectrum and a lower risk of resistance induction.

[0006] However, such general strategies relying on directed evolutionary screening inherently possess unpredictable randomness in the antibacterial efficacy of the resulting phage combinations. While this method addresses the procedural problem of "how to obtain a combination," it cannot guarantee that any particular combination will produce a strong synergistic effect beyond the conventional additive effect; nor can it predict whether the combination will produce an extreme and quantifiable suppressive effect on the resistance evolution of target bacteria. Therefore, the core challenge currently facing the field of phage cocktail therapy has shifted from "how to screen" to "how to identify and obtain specific phage combinations with a definite, powerful, and verifiable synergistic ability to inhibit bacterial resistance evolution." Summary of the Invention

[0007] In order to overcome the technical defects of existing phage cocktail formulation screening methods, such as uncertain combination efficacy and difficulty in obtaining specific products with extreme resistance inhibition capabilities, this invention aims to provide a phage cocktail formulation composed of specific phages with a definite and excellent resistance inhibition effect and its application.

[0008] This invention addresses the aforementioned core challenges by successfully identifying and obtaining a cocktail formulation composed of specific bacteriophages through adaptive evolution strategies and systematic validation. This specific combination not only solves the problem of "how to obtain" but, more importantly, provides an experimentally proven solution with a defined and powerful synergistic inhibitory resistance capability.

[0009] The technical solution adopted in this invention is as follows: In a first aspect of the invention, a phage cocktail formulation is provided, comprising phage ΦST 705, phage ΦR2 and phage ΦR1-1-1; The bacteriophage ΦST 705 was deposited on April 21, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan, China, with accession number CCTCC NO: M 2025836, and classified as Salmonella Typhimurium bacteriophage ΦST 705. Salmonella enterica subsp. enterica serovar Typhimurium phage ΦST705; The bacteriophage ΦR2 was deposited on April 21, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan, China, with accession number CCTCC NO: M 2025837, and classified as Salmonella Typhimurium bacteriophage ΦR2. Salmonella enterica subsp. enterica serovar Typhimurium phage ΦR2; The bacteriophage ΦR1-1-1 was deposited on April 21, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan, China, with accession number CCTCC NO: M 2025838, and classified as Salmonella Typhimurium bacteriophage ΦR1-1-1. Salmonella enterica subsp. enterica serovar Typhimurium phage ΦR1-1-1.

[0010] Preferably, the liquid matrix used in the phage cocktail formulation is SM buffer solution or LB liquid medium.

[0011] In a second aspect of the invention, the use of the phage cocktail formulation in the preparation of a medicament for reducing the antiphage mutation rate of Salmonella, the formulation being able to reduce the antiphage mutation rate of Salmonella to 10. -2 Magnitude.

[0012] The phage cocktail formulation described in this invention exhibits an unexpected synergistic effect in inhibiting resistance. Experiments have shown that this cocktail formulation can reduce the phage-resistant mutation rate of Salmonella ST 705 from 10% under single phage treatment with ΦST 705. -1 (i.e., 10%) level, drastically reduced to 10 -2 (i.e., 0.1% to 1%). This reduction of 1 to 2 orders of magnitude significantly exceeds the general expectation in the field that phage cocktail formulations can only delay the development of resistance, demonstrating an unexpected technical effect.

[0013] Preferably, the formulation is a liquid dosage form, and is a colorless or yellow transparent liquid.

[0014] Preferably, in the phage cocktail formulation, the stock solutions of phage ΦST 705, phage ΦR2, and phage ΦR1-1-1 are mixed to ensure that the final concentration of each phage in the resulting cocktail formulation is not less than 1×10⁻⁶. 8 PFU / mL.

[0015] In a third aspect of the invention, the use of the aforementioned phage cocktail formulation in the preparation of a medicament for the prevention or treatment of Salmonella infection is provided.

[0016] Preferably, the Salmonella includes duck-derived Salmonella typhimurium ST 705.

[0017] Preferably, the drug is administered orally.

[0018] Preferably, the drug is a feed additive or a drinking water additive.

[0019] Preferably, the animals used in the application include, but are not limited to, poultry (chickens, ducks, etc.), livestock (pigs, cattle, sheep, etc.) and other Salmonella hosts.

[0020] In a fourth aspect of the invention, the phage cocktail formulation is used in the preparation of disinfectants or antibacterial agents for in vitro lysis of Salmonella.

[0021] Preferably, the Salmonella includes, but is not limited to, duck-derived Salmonella typhimurium ST 705.

[0022] Preferably, the in vitro scenarios in the application include, but are not limited to, in vitro antibacterial experiments in the laboratory and the killing of Salmonella in livestock and poultry farming environments.

[0023] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects: This invention is based on bacterial-bacteriophage co-evolution and develops a cocktail formulation composed of a primitive host bacteriophage and an adaptively evolved host bacteriophage.

[0024] This particular combination exhibited unexpected resistance-inhibiting capabilities, reducing the bacterial resistance mutation rate from 10% to 10% with a single phage. -1 Significantly reduced to 10 -2 The magnitude of the reduction was more than one order of magnitude, effectively overcoming the defect that a single phage is prone to inducing resistance.

[0025] This formulation effectively overcomes the shortcomings of single phages, such as narrow lysis spectrum and easy induction of resistance, through the functional complementarity and synergistic effect of multiple phages, and achieves broad-spectrum, high-efficiency and long-lasting antibacterial effect. At the same time, it indirectly weakens the pathogenicity of pathogens by utilizing the adaptive cost of bacteria in the evolution of antiphages.

[0026] This phage cocktail preparation can effectively reduce the mortality rate of ducklings and can be used as an antibiotic alternative to prevent and treat Salmonella infections in livestock and poultry farming. Attached Figure Description

[0027] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0028] Figure 1 The adsorption kinetics curves of bacteriophage ΦST 705, bacteriophage ΦR2, and bacteriophage ΦR1-1-1 on Salmonella ST 705 are shown.

[0029] Figure 2 The figures are: A) In vitro lysis curves of bacteriophage ΦST 705, bacteriophage ΦR2, and bacteriophage ΦR1-1-1 under different MOI conditions; B) In vitro lysis curves of bacteriophage ΦST 705 under different MOI conditions; C) In vitro lysis curves of bacteriophage ΦR2 under different MOI conditions; D) In ​​vitro lysis curves of bacteriophage ΦR1-1-1 under different MOI conditions.

[0030] Figure 3 The survival rates of bacteriophages ΦST 705, ΦR2, and ΦR1-1-1 against larvae of the large wax moth at different doses are as follows: A represents the survival rate of bacteriophage ΦST 705 against larvae of the large wax moth at different doses; B represents the survival rate of bacteriophage ΦR2 against larvae of the large wax moth at different doses; and C represents the survival rate of bacteriophage ΦR1-1-1 against larvae of the large wax moth at different doses.

[0031] Figure 4 In vitro lysis curves of phage cocktail formulations with different formulations.

[0032] Figure 5 This is a diagram showing the changes in the host spectrum between a single bacteriophage and a bacteriophage cocktail formulation.

[0033] Figure 6 Comparison of anti-phage mutation rates between phage ΦST 705 and phage cocktail formulations; Note: Data in the figure are expressed in orders of magnitude.

[0034] Figure 7 The survival curves of a Salmonella-infected duckling model after 7 days of different treatments.

[0035] Figure 8 This is the genome map of bacteriophage ΦST 705.

[0036] Figure 9 This is the genome map of bacteriophage ΦR2.

[0037] Figure 10 This is the genome map of bacteriophage ΦR1-1-1. Detailed Implementation

[0038] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0039] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0040] The host bacteria and methods for their availability involved in this invention: This invention provides a standard method for obtaining the initial host bacteriology for phage cocktail formulations. The initial host bacteriology specifically refers to a duck-derived *Salmonella typhimurium* strain capable of being efficiently lysed by phage ΦST 705 and subsequently producing adaptively evolving strains. Obtaining this host bacteriology does not depend on any unpreserved specific strain, but is achieved through the following method, which includes key screening steps: Methods for isolation, identification, and screening of initial host bacteria: Step 1: Sample Pretreatment After recording and numbering the dead embryo samples, pre-enrichment treatment was performed: In a sterile laminar flow hood, sterile BPW enrichment broth was dispensed into shakers, 4.5 mL per tube. Approximately 0.5 g of sample (from the yolk sac of the liver or intestine) was transferred to each shaker using sterile forceps. The tubes were capped and numbered, and then incubated at 37°C and 220 rpm for 8-12 h.

[0041] Step 2: Selective Enrichment Transfer Transfer the BPW enrichment broth to three different selective enrichment media (SC, TTB, and RVS media; TTB media requires the addition of iodine solution and brilliant green before use, and thorough mixing). Take 500 μL from each BPW enrichment broth and transfer them sequentially to 4.5 mL SC, TTB, and RVS shakers. Cap the tubes, label them, and incubate at 37°C and 220 rpm for 18–20 h.

[0042] Step 3: Streak plate separation and pure culture Gently shake the SC, TTB, and RVS enrichment broths to mix thoroughly. Using a sterile inoculation loop, take a small amount of the bacterial suspension and inoculate it onto XLD agar plates using the three-zone streak method. After labeling, invert the plates and incubate at 37°C for 24-48 h. Observe and select typical Salmonella colonies (e.g., Salmonella typhimurium appears as smooth colonies with a black center on XLD plates). Pick three suspected colonies from each plate and inoculate them into LB liquid medium, incubating at 37°C for 6-8 h using a shaker. Finally, using the bacterial suspension as a template, Salmonella standard strains as a positive control, and ddH2O as a negative control, perform PCR identification using Salmonella-specific primers fimW. Perform 1% agarose gel electrophoresis on 5 μL of the PCR amplification product. Under conditions where the positive and negative controls are valid, if a specific amplification band appears at the 477 bp position, the result is considered positive for Salmonella. Primer sequences are shown in Table 1.

[0043] Table 1. Salmonella-specific fimW primer sequences Primer sequences (5′–3′) Product size (bp) fimW-F 5'-AACAGTCACTTTGAGCATGGGTT-3' 477 fimW-R 5'-GAGTGACTTTGTCTGCTCTTCA-3' 477

[0044] Prepare a 25 μL PCR reaction system for PCR amplification. The PCR reaction system is shown in Table 2.

[0045] Table 2 PCR reaction system Element Volume (μL) 2×PCR Master Mix 12.5 upstream primer 1 Downstream primer 1 <![CDATA[ddH2O]]> 9.5 Bacterial template 1

[0046] The PCR procedure is shown in Table 3.

[0047] Table 3 PCR reaction procedure reaction process Reaction conditions Pre-variation 94℃ for 4 minutes transsexual 94℃ for 1 minute annealing 50℃ for 1 minute extend 72℃ for 1 minute Further extension 72℃ for 10 minutes

[0048] Step 4: Purification, culture, verification, and preservation of the strain Salmonella strains that tested positive by PCR were purified and cultured. They were then inoculated onto XLD plates using the three-zone streak method and incubated upside down at 37°C for 48 hours. Single colonies were picked and inoculated onto LB broth, and cultured in a shaker at 37°C for 6-8 hours. Using the bacterial culture as a template, the target strain was then re-verified by PCR. After successful verification, the strain was preserved: 1 mL of bacterial culture was added to a 2 mL centrifuge tube, along with an equal volume of sterile glycerol. The mixture was thoroughly mixed and then stored at -20°C for long-term cryopreservation.

[0049] Step 5: Functional screening using phage ΦST 705 This step is a decisive method for determining the initial host bacteria suitable for this invention from identified Salmonella strains: Obtaining screening tools: Using the phage ΦST 705 (accession number CCTCC NO: M2025836) disclosed in this invention, a titer ≥1×10⁻⁶ was prepared.8 PFU / mL lysis buffer.

[0050] Sensitivity testing was performed: phage lysis activity was determined using the droplet method. Salmonella strains identified as positive by PCR were shaken to prepare double-layer agar plates (10 mL of LB solid agar cooled to 50°C was poured into a sterile petri dish as the bottom layer; after solidification, 20 μL was taken to achieve a final concentration of 10). 7 Mix the activated bacterial culture (CFU / mL) with 5 ml of LB semi-solid agar cooled to 40°C until homogeneous. Pour the mixture onto the bottom plate and spread it evenly. After the semi-solid agar has solidified, place the plate in a clean bench to dry the surface moisture. Add 10 μL of phage ΦST 705 lysis buffer to the center of the plate and allow it to stand before incubation and observation.

[0051] Determination and Acquisition: The bacteria were incubated at 37°C for 3-4 hours. Strains that formed clear, fused lysis zones around the dropper point were identified as the initial host bacteria meeting the requirements of this invention. This result is intuitive and objective.

[0052] Regarding the host bacterium ST 705 and reproducibility: All data in the embodiments of this invention were obtained using a specific host bacterium isolated and identified from a duck farm in Shandong Province using the above method. This bacterium was numbered as duck-derived Salmonella typhimurium ST 705 in the experiment.

[0053] Unless otherwise specified, in all the embodiments described below, "duck-derived Salmonella typhimurium ST 705" refers to the host bacterium obtained by screening using the above method and possessing the aforementioned functions.

[0054] It should be clearly stated that the core of protection of this invention is the cocktail formulation product itself, and its application, composed of specific preserved bacteriophages ΦST 705 (preservation number CCTCC NO: M 2025836), phage ΦR2 (preservation number CCTCC NO: M 2025837), and phage ΦR1-1-1 (preservation number CCTCC NO: M 2025838). The host bacterium ST 705 used in the examples is only a test model and the source host of phages ΦR2 and ΦR1-1-1, selected solely to verify the superior effects of this specific product (such as extremely low resistance mutation rate, broad-spectrum lytic ability, and in vivo efficacy).

[0055] Any person skilled in the art, upon obtaining the three preserved bacteriophages, can directly mix them to prepare the cocktail formulation of this invention, and utilize their respective susceptible Salmonella strains (including but not limited to the host bacterium ST705 in the examples) to verify their synergistic effects in reducing resistance mutation rates, expanding the host spectrum, and preventing infection. Therefore, the implementation and efficacy verification of this invention rely entirely on the specific preserved bacteriophages, and not on any unpreserved specific host strains. The isolation and screening methods for the host bacterium ST705 are merely illustrative of the original acquisition background of bacteriophages ΦR2 and ΦR1-1-1, and are not necessary prerequisites for implementing this invention.

[0056] Example 1: Preparation of host bacteria ST 705 Bacterial suspensions stored at -20 ℃ were streaked onto XLD agar plates and incubated at 37 ℃ for 18–24 h. Single colonies were picked and inoculated into shaker tubes containing 3 mL of LB liquid medium and activated by shaking at 37 ℃ and 220 rpm for 6–8 h. Simultaneously, double-layer agar plates were prepared: 10 mL of LB solid agar cooled to 50 ℃ was poured into a sterile petri dish, gently shaken to spread, and allowed to solidify to form the bottom layer; 5 mL of LB solid agar was added to the bottom layer. 7 Add 5 mL of cooled semi-solid agar to a centrifuge tube containing CFU / mL activated bacterial solution, mix well, and pour onto a bottom plate. After the semi-solid agar has solidified, place the plate in a clean bench to dry the surface moisture.

[0057] Example 2: Isolation and purification of bacteriophage ΦST 705 Phage isolation: Take 5 mL of farm wastewater into a centrifuge tube, add 1 mol / L CaCl2 solution to achieve a final concentration of 1 mmol / L, mix thoroughly, centrifuge at 8000 g for 10 min, and filter the supernatant through a 0.22 μm filter membrane for sterilization. Add 3 mL of LB liquid culture medium and 3 mL of filtered wastewater to a shaker, and inoculate to a final concentration of 10 mmol / L. 7 The activated bacterial culture at CFU / mL was cultured at 37 ℃ and 220 rpm for 6 h on a shaker. After centrifuging the culture at 8000 g for 10 min, the supernatant was filtered through a 0.22 μm filter membrane. The filtrate was serially diluted 10-fold with SM buffer, and 10 μL of each dilution was spotted onto dried double-layer agar plates and incubated at 37 ℃ for 3-4 h.

[0058] Phage purification: A single plaque was picked up with a sterile pipette and added to 1 mL of SM buffer. The mixture was vortexed for 1 min to detach the phage. The suspension was serially diluted 10-fold, and 10 μL was spotted onto a double-layer agar plate and incubated at 37 ℃ for 3–4 h. This process was repeated 2–3 times until the plaques were uniform in morphology and size. The purified phage was named phage ΦST705. This phage was deposited on April 21, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan, China, with accession number CCTCC NO: M 2025836, and classified as Salmonella Typhimurium phage ΦST705. Salmonella enterica subsp. enterica serovar Typhimurium phage ΦST 705.

[0059] Example 3: Analysis of basic characteristics of bacteriophage genomes Whole-genome sequencing and bioinformatics annotation of the three bacteriophages involved in this invention were performed, and the results are as follows: (1) The genome annotation of phage ΦST 705 identified 73 coding sequences (CDS), of which 49 were located on the sense strand and 24 on the antisense strand. Functional prediction showed that 34 CDS were annotated as known functional genes, mainly involving connexins, DNA / RNA and nucleotide metabolism, head and packaging, integration and excision, cleavage, helper metabolic genes and host regulation, tail and transcriptional regulation, etc. For details, see [link to relevant documentation]. Figure 8 .

[0060] (2) Phage ΦR2 genome annotation identified 57 coding sequences (CDS), of which 6 were located on the sense strand and 51 on the antisense strand. Functional prediction showed that 37 CDS were annotated as known functional genes, mainly involving connective proteins, DNA / RNA and nucleotide metabolism, head and packaging, integration and excision, cleavage, helper metabolic genes and host regulation, tail and transcriptional regulation, etc. For details, see [link to relevant documentation]. Figure 9 .

[0061] (3) The phage ΦR1-1-1 genome annotation identified 73 coding sequences (CDS), of which 50 were located on the sense strand and 23 on the antisense strand. Functional prediction showed that 35 CDS were annotated as known functional genes, mainly involving connective proteins, DNA / RNA and nucleotide metabolism, head and packaging, integration and excision, cleavage, helper metabolic genes and host regulation, tail and transcriptional regulation, etc. For details, see [link to relevant documentation]. Figure 10 .

[0062] The above genomic feature analysis shows that the three bacteriophages described in this invention all possess typical virulent bacteriophage genetic structures, encoding genes necessary for complete viral replication and bacterial lysis, providing a molecular biological basis for their efficient in vitro lysis ability and in vivo therapeutic potential.

[0063] Example 4: Screening and validation of adaptively evolved Salmonella strains R2 and R1-1-1 Duck-derived Salmonella ST 705 was transferred at a concentration of 1% to 1 mL of LB liquid medium, and the bacterial concentration was diluted to OD200. 600 =0.1, at the optimal multiple of infection (MOI) of 0.01, phage ΦST 705 was mixed with the bacterial suspension and incubated at 37 ℃ and 220 rpm for 3-4 h in a shaker. The bacterial suspension was then streaked onto XLD agar plates and incubated overnight at 37 ℃. Verification was performed using the spotting method: a single colony was picked and streaked onto an LB solid agar plate, and 10 μL of a high concentration (10 μL) was spotted at the colony site. 8 Phage ΦST705 (PFU / mL) was incubated at 37°C, and the growth of the strains was observed. Colonies that could still grow normally at the phage spotting site were selected as candidate strains resistant to phage ΦST705. These candidate strains were purified through three consecutive subcultures, and the above-mentioned "spotting method" was repeated after each subculture to ensure the stability of their resistance phenotype. Through this process, this invention obtained several stable adaptive evolutionary strains. Two representative strains were selected and named adaptive evolutionary Salmonella strain R2 and adaptive evolutionary Salmonella strain R1-1-1. These strains can be stably and reproducibly obtained by those skilled in the art using the disclosed induction and screening methods described above.

[0064] Example 5: Isolation and purification of bacteriophage ΦR2 and bacteriophage ΦR1-1-1 The isolation and purification of bacteriophage ΦR2 and bacteriophage ΦR1-1-1 were carried out in accordance with Example 2, and the host bacteria used were Salmonella strain R2 and Salmonella strain R1-1-1, respectively.

[0065] The bacteriophage ΦR2 was deposited on April 21, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan, China, with accession number CCTCC NO: M 2025837, and classified as Salmonella Typhimurium bacteriophage ΦR2. Salmonella enterica subsp. enterica serovar Typhimurium phage ΦR2.

[0066] The bacteriophage ΦR1-1-1 was deposited on April 21, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan, China, with accession number CCTCC NO: M 2025838, and classified as Salmonella Typhimurium bacteriophage ΦR1-1-1. Salmonella enterica subsp. enterica serovar Typhimurium phage ΦR1-1-1.

[0067] Example 6: Adsorption experiment of bacteriophage ΦST 705, bacteriophage ΦR2, and bacteriophage ΦR1-1-1 on host bacterium ST 705 Example 6: Adsorption experiment of bacteriophage ΦST 705, bacteriophage ΦR2, and bacteriophage ΦR1-1-1 on host bacterium ST 705 Take 2 mL of the stationary host bacterium ST 705, centrifuge at 10000 rpm for 2 min, resuspend in fresh LB liquid medium, and adjust to OD200. 600 For a multiplicity of infection (MCI) of 2.0, phage and bacterial suspension were mixed at a ratio of 0.1, and LB liquid medium was added to make a volume of 10 mL. The mixture was shaken and mixed, and then dispensed into 1.5 mL centrifuge tubes. Three tubes were centrifuged every 3 min to collect the supernatant, which was then filtered through a 0.22 μm disposable sterile water filter. The filtrate was serially diluted 10-fold with SM buffer and spotted onto a double-layer agar plate containing the host bacteria. The plate was incubated overnight at 37 °C, and the phage titer was calculated.

[0068] Adsorption rate calculation: The phage titer was calculated by counting plaques on the double-layer agar plate after cultivation, and the adsorption rate was calculated by counting the number of unadsorbed phages. The formula is: Adsorption rate = (Initial phage count - Number of unadsorbed phages) / Initial phage count × 100%. The adsorption rate results are shown below. Figure 1 As shown, all three bacteriophages exhibited rapid initial adsorption capacity to the host bacterium ST 705. In the early stages of infection (0–6 min), the adsorption rates of bacteriophages ΦST 705 and ΦR1-1-1 increased rapidly, significantly higher than that of bacteriophage ΦR2. At 6 min, the adsorption rate of bacteriophage ΦST 705 reached approximately 97%, and subsequently stabilized (final adsorption rate >98%), indicating that its receptor-binding protein has a high affinity for the receptor on the surface of the host bacterium ST 705.

[0069] In contrast, the adsorption kinetics curves of phage ΦR2 and phage ΦR1-1-1 showed a more gradual increase, with final adsorption rates of approximately 90% and 58%, respectively. This result suggests that the host recognition mechanism of these two phages, isolated from adaptively evolved strains, may have undergone adaptive changes, leading to a decrease in their binding efficiency to the host bacterium ST 705.

[0070] Adsorption is a crucial first step in phage infection. Phage ΦST 705 exhibits optimal adsorption performance, consistent with its evolutionary background as the original host phage. The moderately reduced adsorption capacity of phages ΦR2 and ΦR1-1-1 may reflect receptor-binding protein (RBP) variations that occurred during their co-evolution to infect new hosts (adaptively evolved Salmonella strains R2 and R1-1-1). This differentiated adsorption characteristic suggests that the three phages may mediate infection through not entirely identical surface receptors, providing a theoretical basis for constructing phage cocktail formulations that can inhibit phage resistance development at multiple targets.

[0071] Example 7: In vitro lysis experiments of bacteriophages ΦST 705, ΦR2, and ΦR1-1-1 under different MOI conditions The host bacterium ST 705 was inoculated into LB liquid medium and cultured to the logarithmic growth phase. The bacterial suspension was mixed with different concentrations of phage suspension at MOIs of 10, 1, 0.1, 0.01, and 0.001, with a control of bacterial suspension without phage. The mixture was aliquoted into 96-well plates, 200 μL per well. The microplate reader was programmed and incubated at 37 °C. The OD of the bacterial suspension was measured every 30 min. 600 Value. By comparing the changes in bacterial concentration between the experimental group and the control group, the value of bacteriophage ΦST 705 was evaluated. Figure 2 A), bacteriophage ΦR2 ( Figure 2 B in the text), bacteriophage ΦR1-1-1 ( Figure 2 The lysis effect of strain C on the strain was investigated and lysis curves were plotted.

[0072] The lysis effect of bacteriophage ΦST 705 is as follows: Figure 2 As shown in Figure A, phage ΦST 705 exhibits rapid and complete lysis under high MOI conditions (MOI=10 and 1), reducing the OD of the bacterial culture by 2-3 hours. 600 The value decreased to near baseline. As the MOI decreased, the lysis initiation time was delayed and the lysis rate slowed down. At MOI=0.001, a significant antibacterial effect was still observed, indicating that phage ΦST 705 has a highly efficient infection and lysis capability against the original host bacterium ST 705.

[0073] Lysis effect of bacteriophage ΦR2 Figure 2 As shown in B, significant antibacterial effects were observed under different multiples of infection (MOI) treatments. During the experimental period, the OD values ​​for MOIs of 10, 1, and 0.1 were... 600The value remained stable at around 0.1, effectively inhibiting bacterial proliferation; even under low MOI (0.01) conditions, the bacterial density only showed a brief and slight increase in the early stage, and was subsequently stabilized and inhibited.

[0074] The lysis effect of bacteriophage ΦR1-1-1 is as follows: Figure 2 As shown in C, bacteriophages effectively inhibited bacterial proliferation under different multiples of infection (MOI) treatments, with the experimental group at MOI=0.1 showing the most significant inhibitory effect. 600 The values ​​were consistently significantly lower than those in the control group; the other experimental groups also maintained stable antibacterial effects, with mid-term OD values ​​significantly lower than those in the control group. 600 A slight increase followed by a decrease; even at MOI=0.001, bacteriophages were still able to reduce the bacterial proliferation rate, resulting in a bacterial concentration lower than that of the control group.

[0075] All three phage strains exhibited obvious MOI-dependent lysis characteristics: the higher the MOI, the faster the lysis initiation and the more thorough the effect.

[0076] This result provides an important basis for the construction of subsequent cocktail formulations: by combining bacteriophages with different lysis kinetics, they can exert synergistic effects at different stages of infection, improve the overall control of bacterial populations, and reduce the risk of treatment failure due to MOI fluctuations or changes in bacterial count.

[0077] Example 8: Safety evaluation of bacteriophage ΦST 705, bacteriophage ΦR2, and bacteriophage ΦR1-1-1 Healthy wax moth larvae were selected and randomly divided into 4 groups of 10 larvae each. Each group was injected with 10 mg of ... 5 10 6 10 7 10 8 or / and 10 9 10 μL of phage suspension with PFU / mL was injected, with an equal volume of SM buffer as a negative control. Larvae were cultured at 37 °C after injection, and their status and survival were observed every 12 h for a total of 48 h. The mortality rate of larvae in each group was statistically analyzed to determine the phage ΦST 705 (…). Figure 3 A), bacteriophage ΦR2 ( Figure 3 B in the text), bacteriophage ΦR1-1-1 ( Figure 3 C) Biosafety in animal models.

[0078] The results are as follows Figure 3 The above describes an injection dose as high as 10 9Within the PFU / larval range, there was no significant difference in larval survival rates between the three phage treatment groups and the SM buffer control group (p>0.05). Survival rates in all groups remained above 90% within 48 hours, and the time distribution of deaths was consistent with the control group, mostly occurring in the later stages of the experiment (36-48 hours). This suggests that the deaths may be related to experimental procedures or the larval physiological state itself, rather than direct phage toxicity.

[0079] In summary, bacteriophages ΦST 705, ΦR2, and ΦR1-1-1 demonstrated good biosafety against the larvae of the giant wax moth at the experimental doses, providing preliminary safety evidence for their subsequent application in animal models such as poultry.

[0080] Example 9: Preparation of a bacteriophage cocktail formulation Phage culture stored at -20 °C was inoculated onto XLD agar plates and incubated at 37 °C for 18–24 h. Single colonies were picked and inoculated into shaker tubes containing 3 mL of LB liquid medium and activated at 37 °C and 220 rpm for 6–8 h. Subsequently, 10% LB liquid medium was added to 5 mL of LB liquid medium to a final concentration of 10%. 8 CFU / mL of host bacterial culture and a final concentration of 10 6 Phages at PFU / mL were cultured at 37 ℃ and 220 rpm for 3–4 h on a shaker. The state of the culture was observed, and after the culture medium changed from turbid to clear, it was filtered through a 0.22 μm filter membrane to obtain a single-strain phage suspension. Phage suspensions of ΦST 705, ΦR2, and ΦR1-1-1 were combined to obtain phage cocktail formulations. For example, the three can be mixed in equal volume ratios to prepare a complete cocktail formulation containing all three phage strains; alternatively, phage ΦST 705 can be mixed separately with phage ΦR2 or phage ΦR1-1-1 in equal volume ratios to prepare a two-component cocktail formulation for comparative studies.

[0081] Example 10: In vitro lysis experiment of bacteriophage cocktail formulation The host bacterium ST 705 was inoculated into LB liquid medium and cultured to the logarithmic growth phase. The bacterial suspension was mixed with different formulations of phage cocktails at an MOI of 0.1, with a bacterial suspension without phage added serving as a control. The mixture was aliquoted into 96-well plates, 200 μL per well. The microplate reader was programmed and incubated at 37 °C. The OD of the bacterial suspension was measured at 30 min every 30 min. 600 Value. By comparing the changes in bacterial concentration in the experimental and control groups, the lysis effect of the phage cocktail formulation on the bacterial strain was evaluated. Figure 4 ).

[0082] The results are as follows Figure 4As shown, all phage treatment groups can inhibit bacterial growth to varying degrees, but the lysis efficiency is positively correlated with the combinatorial complexity.

[0083] Although a single phage group (phage ΦST 705, phage ΦR2, phage ΦR1-1-1) can induce OD 600 The bacterial count decreased, but varying degrees of bacterial regeneration were observed in the later stages of infection (>12 h), suggesting that resistant subpopulations are easily induced under single selection pressure.

[0084] The dual-phage combination exhibited superior lysis kinetics, OD 600 The descent was deeper and regeneration was delayed, with the combination of phage ΦST 705 + phage ΦR2 showing the most significant effect.

[0085] The triple phage cocktail formulation (phage ΦST 705 + phage ΦR2 + phage ΦR1-1-1) exhibited the strongest synergistic lysis effect: its OD 600 The levels rapidly dropped below baseline within 4 hours and remained stable throughout the 24-hour monitoring period without any rebound. This indicates that the three bacteriophage strains, through multi-target attack, completely blocked the bacteria's escape routes, achieving sustained and complete bacterial clearance.

[0086] It is evident that the multiphage cocktail formulation constructed based on the adaptive evolution strategy of this invention can significantly enhance the breadth, depth, and persistence of antibacterial effects through functional complementarity and synergistic effects.

[0087] Example 11: Host spectrum test of phage cocktail formulation The host bacteria to be tested were activated and cultured to the logarithmic growth phase. Each bacterial suspension was then spread onto LB agar plates using a disposable sterile swab, and 10... 7 PFU / mL phages and phage cocktails were spotted onto a plate. The presence and clarity of plaques were used to determine the phage's infectivity against each host bacterium. Finally, based on the degree of lysis, phages were categorized as completely lysed, incompletely lysed, or non-lysed. Figure 5 ).

[0088] A total of 27 Salmonella strains were used for host spectrum testing. Information on 10 known serotypes of Salmonella is shown in Table 4, including duck, chicken, and environmental isolates.

[0089] Table 4 strain number serotype Separation source ST1328 Salmonella typhimurium Duck source ST14028 Salmonella typhimurium Chicken source 4392 Salmonella Indiana Duck source 714 Salmonella ceresodes Duck source 1687 Salmonella typhimurium Duck source 1063 Salmonella typhimurium Duck source 20FJ03 Salmonella enteritidis Chicken source F118 Salmonella enteritidis Chicken source 18Y23 Kentucky Salmonella Duck source ST699 Salmonella typhimurium Chicken source

[0090] The results are as follows Figure 5As shown, each bacteriophage exhibits a relatively narrow and complementary host range: bacteriophage ΦST 705 can efficiently lyse 13 strains, including 4 with known serotypes; bacteriophage ΦR2 can lyse 10 strains, including 2 with known serotypes; and bacteriophage ΦR1-1-1 can lyse 5 strains, including 1 with known serotype. The host ranges of the three only partially overlap.

[0091] The three-phage cocktail formulation exhibited a significant synergistic amplification effect: the number of lysable strains increased to 24, including 7 known serotypes, and the clarity of lysis (plaque size and transparency) for most strains was superior to any single phage. Of particular note was the complete or incomplete lysis of strains such as ST14028, which are insensitive to a single phage, demonstrating that the multiphage combination can overcome the host's receptor barrier through receptor complementarity or synergistic adsorption.

[0092] It is evident that the cocktail formulation, by integrating the host recognition characteristics of different bacteriophages, significantly expands the lysis spectrum, providing a more effective solution for addressing Salmonella serotype diversity and mixed infections.

[0093] Example 12: Host Bacterium Antiphage Mutation Rate Experiment The following groups were set up: a control group, a ΦST 705 treatment group, a ΦR2 treatment group, a ΦR1-1-1 treatment group, a ΦST 705+ΦR2 treatment group, a ΦR2+ΦR1-1-1 treatment group, a ΦST 705+ΦR1-1-1 treatment group, and a three-phage cocktail treatment group, with two replicates for each group. Fresh host bacterium ST 705 culture was adjusted to OD... 600 =0.2, add 270 mL of diluted bacterial solution to each tube. Except for the control group, add 30 μL of 2×10 to each of the other groups. 10 For multi-phage combinations, the suspension used is prepared by mixing equal volumes of the original phage solutions from each component to ensure a consistent total phage titer across all experimental groups. Incubate at 37°C with shaking for 90 min. Then, use 2×10⁻⁶ PFU / mL phage solution. 9 Serial dilutions of the same phage in SM buffer (PFU / mL) were applied to XLD agar plates and incubated at 37°C for 24 h. Colony counts were observed and recorded. Using the control group concentration as a reference, the phage resistance mutation rate for each group was calculated. Phage resistance mutation rate = (Number of bacteria in the treatment group / Number of bacteria in the original solution) × 100% Figure 6 ).

[0094] The results are as follows Figure 6 As shown, the results reveal the high specificity of the combined effect. The resistance mutation rate of single phage ΦST 705 treatment is as high as 1×10⁻⁶. -1The results, or above, confirm that single-drug therapy readily identifies resistant strains. It is noteworthy that the effectiveness of dual-phage combinations is not always superior to that of single-phage treatments. In particular, the mutation rate of the combination of phage ΦR2 and phage ΦR1-1-1 was even higher than that of phage ΦR1-1-1 alone, suggesting that these two evolved phages may have induced a common susceptible escape pathway.

[0095] However, the two-strain combinations containing the original phage ΦST 705 (phage ΦST 705 + phage ΦR2 and phage ΦST 705 + phage ΦR1-1-1) showed a clear synergistic advantage, reducing the mutation rate to 1×10⁻⁶. -2 The magnitude is not specified. However, the complete cocktail formulation containing all three phage strains (phage ΦST 705 + phage ΦR2 + phage ΦR1-1-1) achieved the best synergistic effect, significantly and stably suppressing the resistance mutation rate to 1×10⁻⁶. -2 The following is the lowest among all processing groups.

[0096] These results demonstrate that the design of cocktail formulations exhibits strict combination specificity. Simple pairwise combinations may not effectively reduce the risk of resistance and could even be counterproductive. Only through carefully screened and validated specific three-strain combinations can the evolutionary threshold of bacteria be maximized through multi-target, high-cost selection pressure, thereby achieving the goal of persistently inhibiting the development of resistance.

[0097] Example 13: Experiment on the prevention and treatment of Salmonella infection in ducklings using a phage cocktail formulation Two-day-old Cherry Valley ducks were randomly divided into three experimental groups based on their body weight, ensuring no significant difference in mean body weight among the groups. Each group consisted of 27 ducks: Group A (Salmonella challenge group), Group B (phage cocktail treatment group), and Group D (control group). Groups A and B received an intramuscular injection of 300 μL of a final concentration of 10... 7 CFU / mL ST 705 bacterial suspension; Group D was injected with an equal volume of physiological saline; Group B was orally administered 300 μL of a phage cocktail containing 0.3% sodium bicarbonate and SM buffer; Groups A and D were orally administered an equal volume of SM buffer containing 0.3% sodium bicarbonate. Monitoring was conducted continuously for 7 days, and duckling mortality was observed and recorded. Figure 7 ).

[0098] The results are as follows Figure 7 As shown, the survival rate of the blank control group (Group D) remained at 100% throughout the experiment, indicating that the experimental operation itself had no adverse effect on the ducklings.

[0099] The challenge control group (Group A) began to die on the second day after infection, and the survival rate dropped rapidly. By the seventh day, the cumulative survival rate was only 70%, which confirmed that the host bacterium ST 705 had a strong pathogenicity to ducklings and successfully established an infection model.

[0100] The cocktail therapy group (Group B) showed significant protective effects. Compared with Group A, the mortality process was significantly delayed and moderated, with a final 7-day survival rate as high as 95%, which was highly significant compared with Group A (p<0.01). This indicates that oral gavage with a phage cocktail preparation can effectively clear Salmonella infection in the body and significantly reduce infection-related mortality.

[0101] It is evident that the phage cocktail formulation developed in this study demonstrated excellent therapeutic efficacy in animal models of Salmonella infection, effectively improving the survival rate of infected ducklings and possessing the potential to replace antibiotics for clinical prevention and control.

[0102] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A phage cocktail formulation, characterized in that, It consists of bacteriophage ΦST 705, bacteriophage ΦR2, and bacteriophage ΦR1-1-1; The bacteriophage ΦST 705 was deposited on April 21, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan, China, with accession number CCTCC NO: M 2025836, and classified as Salmonella Typhimurium bacteriophage ΦST 705. Salmonella enterica subsp. enterica serovar Typhimurium PhageΦST 705; The bacteriophage ΦR2 was deposited on April 21, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan, China, with accession number CCTCC NO: M 2025837, and classified as Salmonella Typhimurium bacteriophage ΦR2. Salmonella enterica subsp. enterica serovar Typhimurium phageΦR2; The bacteriophage ΦR1-1-1 was deposited on April 21, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan, China, with accession number CCTCC NO: M 2025838, and classified as Salmonella Typhimurium bacteriophage ΦR1-1-1. Salmonella enterica subsp. enterica serovar Typhimurium phageΦR1-1-1.

2. The phage cocktail formulation according to claim 1, characterized in that, The formulation is a colorless or yellow transparent liquid, and its liquid matrix is ​​SM buffer solution or LB liquid culture medium.

3. The use of the phage cocktail formulation according to claim 1 or 2 in the preparation of a drug for reducing the antiphage mutation rate of Salmonella, characterized in that, The formulation can reduce the antiphage mutation rate of Salmonella to 10%. -2 Magnitude.

4. The application according to claim 3, characterized in that, The Salmonella strain in question is duck-derived Salmonella typhimurium ST705.

5. The use of the phage cocktail formulation according to claim 1 or 2 in the preparation of a medicament for the prevention or treatment of Salmonella infection in animals.

6. The application according to claim 5, characterized in that, The Salmonella mentioned includes duck-derived Salmonella typhimurium ST705.

7. The application according to claim 5 or 6, characterized in that, The drug is administered orally.

8. The application according to claim 7, characterized in that, The drug is in the form of a feed additive or a drinking water additive.

9. The application according to claim 5, characterized in that, The animals mentioned include poultry or livestock.

10. The use of the phage cocktail formulation according to claim 1 or 2 in the preparation of a disinfectant or antibacterial agent for in vitro lysis of Salmonella.

Images