Phage composition and application thereof, biological preparation and application thereof

Abstract

The invention discloses a bacteriophage composition and application thereof, and a biological preparation and application thereof, and relates to the technical field of microorganisms, Escherichia coli bacteriophage 81P-58 and Escherichia coli bacteriophage 87P-16 can well inhibit clinically separated Escherichia coli, and the two bacteriophages have the characteristics of wide lysis spectrum, high proliferation performance, good physicochemical factor tolerance and the like. The composition of normal flora is not damaged, the influence of bacterial drug resistance is avoided, the problem of drug residue is avoided, and the use safety is high. The bacteriophage cocktail formed by combining the bacteriophage 87P-16 and the bacteriophage 81P-58 has the splitting rate of 92% (46 / 50) on clinically separated multi-drug-resistant escherichia coli, and has good prevention and treatment effects on an escherichia coli infection model of greater wax moths; and the combined use of the phage cocktail and a low-dose antibiotic has a remarkable effect of synergistically inhibiting the multi-drug-resistant escherichia coli, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a bacteriophage composition and its application, and biological agents and their applications. Background Technology

[0002] Escherichia coli is an opportunistic pathogenic Gram-negative bacterium widely found in animal intestines and the farming environment. Under current large-scale farming models, pathogenic E. coli can cause various diseases in animals, such as gastrointestinal infections, peritonitis, and septicemia, significantly increasing mortality rates and causing huge economic losses to the livestock industry.

[0003] Currently, livestock farms mainly rely on antibiotics to control E. coli infections. However, the extensive use of antibiotics has accelerated the emergence and spread of severely drug-resistant and multidrug-resistant E. coli, making antibiotic resistance a growing problem. In the latest list of drug-resistant bacteria published by the World Health Organization, E. coli is listed as a critical priority pathogen. The emergence of drug-resistant strains not only increases the difficulty of treatment but also leads to higher treatment costs and mortality rates, seriously threatening the healthy development of the livestock industry. Therefore, developing new antibiotic alternatives to control the increasingly serious drug-resistant E. coli infections is urgently needed.

[0004] Bacteriophages are widely distributed in the natural environment and can specifically recognize bacterial surface receptors to infect and lyse bacteria. They possess numerous advantages, including abundant resources, low production costs, high specificity, immunity to bacterial drug resistance, and no residue. However, the lytic spectrum of a single bacteriophage is typically narrow, limiting its effectiveness. Formulating bacteriophages with different lytic spectra into cocktail formulations can effectively broaden the antibacterial spectrum without disrupting the normal flora structure, making it an effective means of combating drug-resistant bacterial infections. Notably, the fewer bacteriophage strains in a phage cocktail, the lower its production cost. Because the lytic spectrum of *E. coli* bacteriophages is generally narrow, current clinical *E. coli* cocktails require a large number of bacteriophage strains to meet clinical needs. This not only significantly increases production costs but also leads to unstable antibacterial effects due to complex interactions between bacteriophage strains. Therefore, developing phage cocktail formulations with fewer components and a broad lytic spectrum will be a crucial breakthrough in controlling clinically resistant *E. coli* infections. Summary of the Invention

[0005] The main objective of this invention is to provide a phage composition and its application, as well as a biological agent and its application, with the aim of providing a phage composition that can inhibit pathogenic Escherichia coli.

[0006] To achieve the above objectives, the present invention provides a phage composition comprising at least one of Escherichia coli phage 81P-58 and Escherichia coli phage 87P-16, wherein:

[0007] The accession number of the Escherichia coli phage 81P-58 is CCTCC NO: M 2025835, the accession date is April 21, 2025, and the accession address is China Center for Type Culture Collection, Wuhan University, Wuhan, China.

[0008] The accession number of the Escherichia coli phage 87P-16 is CCTCC NO: M 2025834, the accession date is April 21, 2025, and the accession address is China Center for Type Culture Collection, Wuhan University, Wuhan, China.

[0009] In one embodiment, the phage composition comprises Escherichia coli phage 81P-58 and Escherichia coli phage 87P-16:

[0010] The ratio of Escherichia coli phage 81P-58 to Escherichia coli phage 87P-16 is 1:1.

[0011] The present invention also provides the application of the aforementioned phage composition in the preparation of an inhibitor of chicken-derived multidrug-resistant pathogenic Escherichia coli.

[0012] In one embodiment, the chicken-derived multidrug-resistant pathogenic Escherichia coli includes at least one of the following types: O8, O15, O55, O78, O86, O111, O114, O125, and O127.

[0013] The present invention also provides the application of the aforementioned phage composition in the preparation of biological agents for the prevention and treatment of diseases or contamination caused by chicken-derived multidrug-resistant pathogenic Escherichia coli.

[0014] The present invention also provides a biological agent comprising the aforementioned bacteriophage composition.

[0015] In one embodiment, the biological agent comprises a bacteriophage composition and an antibiotic, wherein the antibiotic comprises at least one of amoxicillin and ceftiofur sodium.

[0016] In one embodiment, the antibiotic comprises amoxicillin, and the concentration of the bacteriophage composition in the biological agent is ≥10. 6 pfu / mL, wherein the concentration of amoxicillin in the biological agent is ≤2 μg / mL; or,

[0017] The antibiotic includes ceftiofur sodium, and the concentration of the bacteriophage composition in the biological agent is ≥10. 6 pfu / mL, wherein the concentration of ceftiofur sodium is ≤16 μg / mL.

[0018] In one embodiment, the antibiotic comprises amoxicillin, and the concentration of the bacteriophage composition in the biological agent is ≥10. 7 pfu / mL, wherein the concentration of amoxicillin in the biological agent is ≤1 μg / mL; or,

[0019] The antibiotic includes ceftiofur sodium, and the concentration of the bacteriophage composition in the biological agent is ≥10. 8 pfu / mL, wherein the concentration of ceftiofur sodium is ≤1 μg / mL.

[0020] In one embodiment, the biological agent includes feed additives, drinking water additives, meat cleaning agents, or drugs.

[0021] In the technical solution of this invention, both Escherichia coli phage 81P-58 and Escherichia coli phage 87P-16 can effectively inhibit clinically isolated Escherichia coli. The optimal multiplicity of infection (MOI) of phage 81P-58 is 0.001, and that of phage 87P-16 is 0.0001. The titers of both phages remain stable at pH 4–11 and temperatures below 50°C. Both phages have a broad lysis spectrum, high proliferation performance, and good tolerance to physicochemical factors. They do not disrupt the normal flora composition, are not affected by bacterial drug resistance, and have no drug residue issues, thus exhibiting high safety in use. Furthermore, the phage cocktail composed of phage 87P-16 and phage 81P-58 achieved a lysis rate of 92% (46 / 50) against clinically isolated multidrug-resistant Escherichia coli. This means that the phage cocktail could significantly inhibit the proliferation of a mixture of 46 multidrug-resistant Escherichia coli strains within 24 hours. It also showed good preventive and therapeutic effects on the Escherichia coli infection model of Escherichia coli in the giant wax moth. Moreover, the phage cocktail combined with low-dose antibiotics had a significant synergistic inhibitory effect on multidrug-resistant Escherichia coli, showing promising application prospects. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0023] Figure 1 Image (A) shows the plaque morphology of Escherichia coli phage 81P-58 provided by this invention; Figure 1 Image (B) shows the plaque morphology of Escherichia coli phage 87P-16 provided by this invention;

[0024] Figure 2 Image A is a transmission electron microscope image of Escherichia coli phage 81P-58 provided by the present invention; Figure 2 Image B is a transmission electron microscope image of Escherichia coli bacteriophage 87P-16 provided by the present invention;

[0025] Figure 3 Image (A) shows the results of the thermal stability test of Escherichia coli phage 87P-16 provided by this invention; Figure 3 (B) is a graph showing the results of the thermal stability test of Escherichia coli phage 81P-58 provided by the present invention;

[0026] Figure 4 (A) is a graph showing the pH stability test results of Escherichia coli phage 81P-58 provided by the present invention; Figure 4 (B) is a graph showing the pH stability test results of Escherichia coli phage 87P-16 provided by the present invention;

[0027] Figure 5 Image (A) shows the results of ultraviolet sensitivity testing of Escherichia coli phage 81P-58 provided by this invention; Figure 5 (B) is a graph showing the results of ultraviolet sensitivity testing of Escherichia coli phage 87P-16 provided by the present invention;

[0028] Figure 6 (A) is a graph showing the results of the one-step growth curve determination of Escherichia coli phage 81P-58 provided by the present invention; Figure 6 (B) is a graph showing the results of the one-step growth curve determination of Escherichia coli phage 87P-16 provided by the present invention;

[0029] Figure 7 Image (A) shows the 8-hour in vitro antibacterial effect of the cocktail XD1 composed of Escherichia coli phage 81P-58 and Escherichia coli phage 87P-16 provided by the present invention. Figure 7 (B) is a diagram showing the 16-hour in vitro antibacterial effect of the cocktail XD1 composed of Escherichia coli phage 81P-58 and Escherichia coli phage 87P-16 provided by the present invention. Figure 7 (C) is a 24-hour in vitro antibacterial effect diagram of the cocktail XD1 composed of Escherichia coli phage 81P-58 and Escherichia coli phage 87P-16 provided by the present invention;

[0030] Figure 8(A) is a graph showing the results of the in vitro synergistic antibacterial effect of the cocktail XD1 composed of Escherichia coli phage 81P-58 and Escherichia coli phage 87P-16 provided by the present invention with the antibiotic amoxicillin for 0 h. Figure 8 (B) is a graph showing the results of the in vitro synergistic antibacterial effect of the cocktail XD1 composed of Escherichia coli phage 81P-58 and Escherichia coli phage 87P-16 provided by the present invention and the antibiotic ceftiofur sodium for 0 h. Figure 8 (C) is a graph showing the results of the in vitro synergistic antibacterial effect of the cocktail XD1 composed of Escherichia coli phage 81P-58 and Escherichia coli phage 87P-16 provided by the present invention and the antibiotic amoxicillin for 16 hours. Figure 8 (D) is a graph showing the results of the in vitro synergistic antibacterial effect of the cocktail XD1, composed of Escherichia coli phage 81P-58 and Escherichia coli phage 87P-16 provided by the present invention, and the antibiotic ceftiofur sodium for 16 hours. Figure 8 In (E), the cocktail XD1, composed of Escherichia coli phage 81P-58 and Escherichia coli phage 87P-16 provided by the present invention, is prepared at 10... 7 Figure showing the results of synergistic in vitro antibacterial activity of pfu / mL concentration and 1μg / mL antibiotic amoxicillin for 16h; Figure 8 (F) represents a cocktail XD1 composed of Escherichia coli phage 81P-58 and Escherichia coli phage 87P-16 provided for this invention, in 10... 8 Figure showing the results of synergistic in vitro antibacterial activity of pfu / mL and 1μg / mL ceftiofur sodium for 16h;

[0031] Figure 9 Figure (A) shows the results of in vivo treatment of the *E. coli* infection model of the giant wax moth using the cocktail XD1 composed of *E. coli* phage 81P-58 and *E. coli* phage 87P-16 provided by this invention. Figure 9 Figure (B) shows the in vivo efficacy of the cocktail XD1, composed of Escherichia coli phage 81P-58 and Escherichia coli phage 87P-16 provided by the present invention, in preventing Escherichia coli.

[0032] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially. Furthermore, the meaning of "and / or" throughout the text includes three parallel solutions; for example, "A and / or B" includes solution A, or solution B, or a solution where both A and B are satisfied simultaneously. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Escherichia coli is an opportunistic pathogenic Gram-negative bacterium widely found in animal intestines and the farming environment. Under current large-scale farming models, pathogenic E. coli can cause various diseases in animals, such as gastrointestinal infections, peritonitis, and septicemia, significantly increasing mortality rates and causing huge economic losses to the livestock industry.

[0035] Currently, livestock farms mainly rely on antibiotics to control E. coli infections. However, the extensive use of antibiotics has accelerated the emergence and spread of severely drug-resistant and multidrug-resistant E. coli, making antibiotic resistance a growing problem. In the latest list of drug-resistant bacteria published by the World Health Organization, E. coli is listed as a critical priority pathogen. The emergence of drug-resistant strains not only increases the difficulty of treatment but also leads to higher treatment costs and mortality rates, seriously threatening the healthy development of the livestock industry. Therefore, developing new antibiotic alternatives to control the increasingly serious drug-resistant E. coli infections is urgently needed.

[0036] Bacteriophages are widely distributed in the natural environment and can specifically recognize bacterial surface receptors to infect and lyse bacteria. They possess numerous advantages, including abundant resources, low production costs, high specificity, immunity to bacterial drug resistance, and no residue. However, the lytic spectrum of a single bacteriophage is typically narrow, limiting its effectiveness. Formulating bacteriophages with different lytic spectra into cocktail formulations can effectively broaden the antibacterial spectrum without disrupting the normal flora structure, making it an effective means of combating drug-resistant bacterial infections. Notably, the fewer bacteriophage strains in a phage cocktail, the lower its production cost. Because the lytic spectrum of *E. coli* bacteriophages is generally narrow, current clinical *E. coli* cocktails require a large number of bacteriophage strains to meet clinical needs. This not only significantly increases production costs but also leads to unstable antibacterial effects due to complex interactions between bacteriophage strains. Therefore, developing phage cocktail formulations with fewer components and a broad lytic spectrum will be a crucial breakthrough in controlling clinically resistant *E. coli* infections.

[0037] In view of this, the present invention provides a phage composition comprising at least one of Escherichia coliphage 81P-58 and Escherichia coliphage 87P-16, wherein: the accession number of Escherichia coliphage 81P-58 is CCTCC NO: M2025835, the accession date is April 21, 2025, and the accession address is China Center for Type Culture Collection, Wuhan University, Wuhan, China; the accession number of Escherichia coliphage 87P-16 is CCTCC NO: M 2025834, the accession date is April 21, 2025, and the accession address is China Center for Type Culture Collection, Wuhan University, Wuhan, China.

[0038] In the technical solution of this invention, both Escherichia coli phage 81P-58 and Escherichia coli phage 87P-16, isolated from nature, can effectively inhibit clinically isolated Escherichia coli. The optimal multiplicity of infection (MMO) of phage 81P-58 is 0.001, and that of phage 87P-16 is 0.0001. The titers of both phages remain stable at pH 4–11 and temperatures below 50°C. Both phages exhibit a wide lysis spectrum, high proliferation capacity, and good tolerance to physicochemical factors. The phage genomes do not carry harmful genes such as virulence genes or drug resistance genes. As a nemesis of bacteria, phages can specifically lyse pathogenic Escherichia coli without disrupting the normal flora of the body. They are not affected by bacterial drug resistance and have no drug residue issues, thus ensuring high safety in use. Furthermore, the phage cocktail composed of phage 87P-16 and phage 81P-58 achieved a lysis rate of 92% (46 / 50) against clinically isolated Escherichia coli, meaning that the phage cocktail could significantly inhibit the proliferation of a mixture of 46 multidrug-resistant Escherichia coli strains within 24 hours. It also showed good preventive and therapeutic effects on the Escherichia coli infection model of Escherichia coli in the giant wax moth. Moreover, the phage cocktail combined with low-dose antibiotics had a significant synergistic inhibitory effect on multidrug-resistant Escherichia coli, showing promising application prospects.

[0039] Specifically, the bacteriophages 81P-58 and 87P-16 isolated in this invention both possess a distinct regular polyhedral head structure and a retractable tail structure, classifying them as myotail bacteriophages. The head of bacteriophage 81P-58 is hexagonal with icosahedral symmetry, an aspect ratio of approximately 1.3, a diameter of approximately 108 nm, and a tail of approximately 98 nm. The head of bacteriophage 87P-16 is also hexagonal with icosahedral symmetry, an aspect ratio of approximately 1.2, a diameter of approximately 115 nm, and a tail of approximately 80 nm. The titer of bacteriophage 81P-58 is 1.6 × 10⁻⁶. 9 pfu / mL, the titer of phage 87P-16 is 2.4 × 10⁻⁶. 9 pfu / mL; the optimal multiplicity of infection (MMO) for both E. coli phage 81P-58 and E. coli phage 81P-58 was 0.001. The lysis rate of phage 81P-58 was 72% (36 / 50), and that of phage 87P-16 was 50% (25 / 50). The combination of these two phages to form cocktail XD1 achieved a lysis rate of 92% (46 / 50), indicating that cocktail XD1 has a synergistic inhibitory effect on E. coli. It should be noted that cocktail XD1 was prepared by directly mixing the two phages.

[0040] In some embodiments, the phage composition comprises *E. coli* phage 81P-58 and *E. coli* phage 87P-16 in a 1:1 ratio. It is understood that the 1:1 ratio of *E. coli* phage 81P-58 to *E. coli* phage 87P-16 indicates that the resulting phage cocktail XD1 exhibits good in vitro inhibitory effects against *E. coli* and can synergistically inhibit *E. coli* with lower doses of antibiotics.

[0041] This invention also provides the use of the aforementioned phage composition in the preparation of inhibitors for chicken-derived multidrug-resistant pathogenic Escherichia coli. Chicken-derived multidrug-resistant pathogenic Escherichia coli refers to a specific type of Escherichia coli that can cause disease in chickens and is resistant to multiple drugs. In some embodiments, the chicken-derived multidrug-resistant pathogenic Escherichia coli includes at least one of Escherichia coli types O8, O15, O55, O78, O86, O111, O114, O125, and O127. The above types refer to the serotypes of Escherichia coli.

[0042] This invention also provides the application of the aforementioned phage composition in the preparation of biological agents for the prevention and treatment of diseases or contamination caused by multidrug-resistant pathogenic Escherichia coli from chickens. In some embodiments, the biological agent includes feed additives, drinking water additives, meat cleaners, or drugs. That is, the phage composition of this invention can be compounded with other solutions or carriers to form feed additives, drinking water additives, or meat cleaners for eliminating Escherichia coli present in the environment, or for preventing and treating Escherichia coli infections in chickens or humans, or the phage composition can be combined with a drug carrier to prepare a drug for treating diseases caused by Escherichia coli.

[0043] The present invention also provides a biological agent comprising the aforementioned bacteriophage composition. Therefore, the biological agent possesses all the beneficial effects of the aforementioned bacteriophage composition, which will not be elaborated further here.

[0044] In some embodiments, the biological agent comprises a bacteriophage composition and an antibiotic, wherein the antibiotic comprises at least one of amoxicillin and ceftiofur sodium. It is understood that both the bacteriophage composition and the aforementioned two antibiotics have a synergistic inhibitory effect on *Escherichia coli*.

[0045] In some embodiments, the antibiotic includes amoxicillin, and the concentration of the phage composition in the biological agent is ≥10. 6 pfu / mL, wherein the concentration of amoxicillin in the biological agent is ≤2 μg / mL; or, wherein the antibiotic comprises ceftiofur sodium, and the concentration of the phage composition in the biological agent is ≥10 pfu / mL. 6The concentration of ceftiofur sodium is ≤16 μg / mL. It is understood that amoxicillin alone typically inhibits *E. coli* at a concentration of 8 μg / mL, while when used with a phage combination, a significant synergistic inhibitory effect on *E. coli* can be achieved at sub-MIC concentrations of amoxicillin. It is also understood that increasing the amount of phage can further reduce the amount of antibiotic used, while still achieving a good inhibitory effect on *E. coli*.

[0046] More preferably, in some embodiments, the antibiotic includes amoxicillin, and the concentration of the phage composition in the biological agent is ≥10. 7 pfu / mL, wherein the concentration of amoxicillin in the biological agent is ≤1 μg / mL; or, wherein the antibiotic comprises ceftiofur sodium, and the concentration of the phage composition in the biological agent is ≥10 pfu / mL. 8 The concentration of ceftiofur sodium is ≤1 μg / mL. Increasing the concentration of the phage composition can further reduce the amount of antibiotic used.

[0047] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0048] Example 1: Isolation and purification of Escherichia coli phage 81P-58 and Escherichia coli phage 87P-16

[0049] (1) Experimental materials:

[0050] Environmental samples were collected from chicken farms in Zhucheng City, Shandong Province. The host bacteria used to isolate bacteriophages were 50 clinical isolates of pathogenic Escherichia coli (Table 1), which were isolated from tissues and organs of diseased chickens from various provinces across the country and preserved in the laboratory.

[0051] (2) Experimental methods:

[0052] Using 50 pathogenic Escherichia coli isolates as host bacteria (Table 1), bacteriophages were isolated from multiple environmental samples. 20g of the collected environmental samples were added to 30mL of LB liquid medium and soaked for 4 hours. After centrifugation at 4000rpm for 30 minutes, 20mL of the supernatant was transferred to a new centrifuge tube, and a mixed bacterial culture of 50 overnight-cultured Escherichia coli strains (each strain having a concentration of 10) was added. 9 Mix 30 mL of CFU / mL solution thoroughly and incubate at 37°C in a shaking incubator for 4 hours. Centrifuge at 4000 rpm for 30 minutes. Take 5 mL of the supernatant and filter it using a 0.45 μm aqueous filter. Store the filtrate at 4°C for later use.

[0053] From each E. coli strain bacterial suspension (1×10)9 Take 7 μL of each of the following solutions (CFU / mL): mix with 3 μL of filtrate, spot onto agar plates, and incubate at 37°C for 2.5 h. Observe the plaques. Record the bacterial suspension and filtrate corresponding to the plaques. Then, using the recorded bacterial suspension as the host bacteria, purify the corresponding filtrate using the double-layer plate method. For each plaque, add physiological saline, incubate at 37°C for 2 h, centrifuge at 12000 rpm for 5 min, collect the supernatant, filter through a 0.22 μL filter, and add 100 μL of the filtrate to an equal volume of host bacterial suspension. Incubate using the double-layer plate method. Repeat the above process three times to obtain a total of 90 phage strains with uniform plaque morphology, i.e., 90 purified phage stock solutions. Add 30% glycerol to the stock solution and store at -80°C for later use.

[0054] Table 1. Host bacterial background information and phage lysis spectrum

[0055]

[0056]

[0057] Example 2: Determination of the lysis spectra of Escherichia coli phage 81P-58 and Escherichia coli phage 87P-16

[0058] The lysis profile of isolated bacteriophages was determined using the double-layer plate method. Fifty *E. coli* isolates were propagated to the logarithmic growth phase to obtain freshly cultured host bacteria. 1 mL of the purified bacteriophage stock solution from Example 1 and 1 mL of freshly cultured host bacteria were added to 10 mL of LB broth and incubated at 37°C with shaking for 2 h. 5% chloroform (v / v) was added, and the culture was continued with shaking for 30 min. The mixture was then centrifuged at 12000 rpm for 2 min, and the supernatant was collected to obtain the bacteriophage propagation solution. 100 μL of the freshly propagated host bacteria solution was mixed with an equal volume of the bacteriophage propagation solution, and the mixture was incubated using the double-layer plate method. Bacteriophage plaques were observed, and the lysis profile was statistically analyzed. Bacteriophages with broad lysis profiles were screened, resulting in bacteriophages 81P-58 and 87P-16. As shown in Table 1, the lysis rate of phage 81P-58 was 72% (36 / 50), and the lysis rate of phage 87P-16 was 50% (25 / 50). The combination of these two phages to form cocktail XD1 achieved a lysis rate of 92% (46 / 50). In Table 1, AMX represents amoxicillin, ENR represents enrofloxacin, FLR represents florfenicol, ARM represents azithromycin, / represents unknown serotype, - represents no lysis, + represents lysis, and R indicates drug resistance.

[0059] Example 3: Morphological observation of Escherichia coli phage 81P-58 and Escherichia coli phage 87P-16

[0060] Take 100 μL of the proliferation solution of phage 81P-58 obtained in Example 2 and 100 μL of the proliferation solution of phage 87P-16 obtained in Example 2 (10 4 pfu / mL), and 100 μL of host bacteria E28 culture (1×10⁻⁶ pfu / mL) were respectively mixed with pfu / mL. 9 Mix well with CFU / mL, incubate using the double-layer plate method for 3 hours, and observe the morphology of the phage plaques. The results are as follows: Figure 1 As shown. Bacteriophage 81P-58 ( Figure 1 (A) and phage 87P-16 ( Figure 1 Both B and C can form translucent phagocytic plaques on the fungal moss, with clear and regular edges and a diameter of 1–1.5 mm.

[0061] 10 μL of the phage proliferation solution from Example 2 was dropped onto a copper grid, dried, and then stained with 2% phosphotungstic acid. After drying, the phage morphology was observed using a transmission electron microscope. Under the transmission electron microscope, phage 81P-58 ( Figure 2 (A) and phage 87P-16 ( Figure 2 Both B and C exhibit a distinct regular polyhedral head structure and a retractable tail structure, classifying them as myotail phages. Figure 2 As shown in (A), the head of bacteriophage 81P-58 is hexagonal with icosahedral symmetry, an aspect ratio of approximately 1.3, and a diameter of approximately 108 nm, while the tail is approximately 98 nm. From... Figure 2 As shown in (B), the head of bacteriophage 87P-16 is hexagonal with icosahedral symmetry, an aspect ratio of approximately 1.2, a diameter of approximately 115 nm, and a tail of approximately 80 nm.

[0062] Example 4: Titer determination of Escherichia coli phage 81P-58 and Escherichia coli phage 87P-16

[0063] The proliferation media of phage 81P-58 and phage 87P-16 were serially diluted 10-fold, respectively, with a dilution gradient of 10. -1 ~10 -7 Take 100 μL of each dilution of the proliferation solution and an equal volume of host bacterial culture (1×10⁻⁶). 9 Mix the CFU / mL solution thoroughly and count plaques using the double-layer agar method, performing triplicate for each dilution. Calculate the phage titer based on the number of plaques; the titer of phage 81P-58 was determined to be 1.6 × 10⁻⁶. 9 pfu / mL, the titer of phage 87P-16 is 2.4 × 10⁻⁶. 9 pfu / mL.

[0064] Example 5: Determination of the optimal multiple of infection (MOI) for Escherichia coli phage 81P-58 and Escherichia coli phage 87P-16

[0065] Fresh host bacterial culture E28, proliferated to the logarithmic growth phase, was collected. The proliferation media of phages 81P-58 and 87P-16 from Example 2 were mixed with their corresponding host bacterial cultures E28 at MOI ratios of 0.001, 0.01, 0.1, 1, and 10, respectively. The mixtures were incubated at 37°C with shaking for 4 hours, centrifuged at 12000 rpm for 20 minutes, and filtered through a 0.22 μm filter to remove bacteria, yielding fresh phage proliferation media. The titer of the phages in the fresh phage proliferation media was determined using the double-layer plate method, with three replicates per group. The MOI corresponding to the highest phage titer was considered the optimal multiplicity of infection. The results are shown in Table 2.

[0066] Table 2 Optimal Multiplicity of Infection for Bacteriophages

[0067]

[0068] As shown in Table 2, when the multiplicity of infection (MOF) is 0.001, the titer of Escherichia coli phage 81P-58 reaches a maximum of 3.75 × 10⁻⁶. 9 pfu / mL. Therefore, the optimal multiplicity of infection (MOI) for E. coli phage 81P-58 is 0.001. When the MOI is 0.0001, the titer of E. coli phage 87P-16 reaches a maximum of 8.15 × 10⁻⁶ pfu / mL. 9 pfu / mL. Therefore, the optimal multiplicity of infection for Escherichia coli phage 81P-58 is 0.0001.

[0069] Example 6: Determination of the thermal stability of Escherichia coli phage 81P-58 and Escherichia coli phage 87P-16

[0070] Take 1 mL of the phage proliferation medium of 81P-58 and 87P-16 from Example 2 respectively (10 9 The phage (pfu / mL) was placed in a metal bath at 40, 50, 60, 70, and 80℃ for 1 hour. 100 μL samples were taken at 20, 40, and 60 minutes respectively. The titer of the phage at different temperatures was determined using the double-layer plate method. Each temperature treatment was performed in triplicate. The results are shown in the figure. Figure 3 .

[0071] from Figure 3 (A) and Figure 3 As shown in (B), the titers of bacteriophages 81P-58 and 87P-16 remain stable below 50℃; when the temperature is 60℃, the titer of the bacteriophages decreases by 1 to 2 orders of magnitude within 60 minutes; when the temperature exceeds 70℃, bacteriophage 81P-58 is rapidly inactivated; bacteriophage 87P-16 is gradually inactivated within 40 minutes at 70℃ and is rapidly inactivated at 80℃.

[0072] Example 7: pH stability determination of Escherichia coli phage 81P-58 and Escherichia coli phage 87P-16

[0073] Adjust the pH of LB broth to 3–12, add 200 μL of phages 81P-58 and 87P-16 respectively, and incubate at 37°C. Take samples at 1 h, 2 h, and 3 h, and determine the phage titer using the double-layer plate method, with three replicates at each time point. Plot the phage pH stability curves, as shown below. Figure 4 As shown.

[0074] from Figure 4 (A) and Figure 4 As shown in (B), bacteriophages 81P-58 and 87-16P maintain stable titers within the pH range of 4–10. At pH 11, bacteriophage 87-16P still retains 10 titers after 1 hour. 5 The survival rate of pfu / mL indicates that phage 87-16P has good alkali resistance. Among them, 81P-58 still showed detectable levels of 10 pfu / mL at pH 3. 4 The PFU / mL phage concentration indicates that phage 81P-58 is sensitive to strong alkalis and has good acid resistance.

[0075] Example 8: Determination of UV sensitivity of Escherichia coli phage 81P-58 and Escherichia coli phage 87P-16

[0076] Take 2 mL of the proliferation solution of phages 81P-58 and 87P-16 from Example 2 (10 9 The phage proliferation solution (pfu / mL) was placed in a petri dish and continuously irradiated under a 30W UV lamp (approximately 40 cm above the plate). Every 10 minutes, 100 μL of the phage proliferation solution was collected, and the phage titer was determined using the double-layer plate method. Measurements were performed continuously for 60 minutes, with three replicates at each time point. A UV sensitivity curve for the phage was plotted, as shown below. Figure 5 As shown.

[0077] from Figure 5 (A) and Figure 5 As shown in Figure (B), the titers of bacteriophages 81P-58 and 87P-16 decreased within 20 minutes of UV irradiation. The titers of both bacteriophages gradually decreased with prolonged UV irradiation, but they still exhibited a certain degree of resistance.

[0078] Example 9: One-step growth curve determination of Escherichia coli phage 81P-58 and Escherichia coli phage 87P-16

[0079] Take 100 μL of the proliferation solution of phages 81P-58 and 87P-16 from Example 2 respectively (10 7 pfu / mL) and 100 μL of host bacterium E28 culture from Table 1 (10 pfu / mL)8 Mix (CFU / mL) thoroughly, add 7 mL LB broth, and incubate at 37°C with shaking at 220 rpm. Take 200 μL of the proliferation solution periodically (starting from 0 min, with 10 min intervals for the first hour, 20 min intervals for the second hour, and 30 min intervals for the third hour), and filter through a 0.22 μm filter. Perform a 10-fold serial dilution of the filtrate, determine the phage titer using the double-layer plate method (3 replicates), and plot a one-step growth curve of the phage. Figure 6 .

[0080] from Figure 6 (A) and Figure 6 As shown in Figure (B), bacteriophages 81P-58 and 87P-16 possess strong infectivity and proliferative capacity against bacteria. The incubation period for 81P-58 is 20 minutes, with an outbreak time ranging from 20 to 100 minutes, and a titer reaching 8.4 × 10⁻⁶. 9 The pfu / mL concentration was high, with a burst dose of 112 pfu / cell; the latency of 87P-16 was 30 min, the burst time ranged from 30 to 150 min, and the titer reached 1.05 × 10⁻⁶. 10 pfu / mL, burst rate 1235 pfu / cell.

[0081] Example 10: Determination of the in vitro antibacterial effect of phage cocktail XD1

[0082] Using 50 clinical isolates of *Escherichia coli* listed in Table 1 as target bacteria, the in vitro antibacterial effect of cocktail XD1 was evaluated using the micro-broth method. The proliferation broth of bacteriophages 81P-58 and 87P-16 from Example 1 (2 × 10⁻⁶) was used. 9 Mix equal volumes of pfu / mL to obtain phage cocktail XD1, with a proliferation medium concentration of 1×10⁻⁶. 9 pfu / mL.

[0083] In a 96-well microplate, 20 μL of each of the major *Escherichia coli* strains listed in Table 1 was taken as the target bacteria at an MOI of 1 and mixed with 100 μL of phage cocktail XD1 proliferation solution. This mixture was then added to 80 μL of LB broth medium. A control group without phages was also included. All three experimental groups were incubated at 37°C for 24 h, and the OD values ​​of each well were measured at 8 h, 16 h, and 24 h. 600 Values, results are shown below Figure 7 .

[0084] Depend on Figure 7 (A) Figure 7 (B) and Figure 7 As shown in Figure (C), the inhibitory effect of phage cocktail XD1 varied among different bacterial strains. At 8 h, 87.5% of the target strains in the XD1 cocktail treatment group showed a lower OD rate compared to the control group.600 The value decreased; at 16 h, 89.5% of the target strains in the cocktail XD1 treatment group had lower OD values ​​than the control group. 600 Value; by 24h, 68.7% of the OD of the cocktail XD1 treatment group 600 The value was still lower than that of the control group. Therefore, the phage cocktail XD1 had a good in vitro antibacterial effect against Escherichia coli at an MOI of 1.

[0085] Example 11: Determination of the synergistic effect of phage cocktail XD1 with antibiotics

[0086] Amoxicillin and ceftiofur sodium solutions with a concentration of 512 μg / mL were serially diluted 2-fold. Amoxicillin and ceftiofur sodium solutions of different concentrations (1 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL, 128 μg / mL, 256 μg / mL, 512 μg / mL) were then diluted with different concentrations (10 pfu / mL, 10... 2 pfu / mL, 10 3 pfu / mL, 10 4 pfu / mL, 10 5 pfu / mL, 10 6 pfu / mL, 10 7 pfu / mL, 10 8 Add 100 μL of E. coli phage XD1 proliferation medium (pfu / mL) to each well of a 96-well plate, mix well, and then add 100 μL of a 10 pfu / mL solution. 8 100 μL of E. coli 28 bacterial suspension (cfu / mL) was used as controls. Wells containing bacterial suspension + antibiotic-free phage, bacterial suspension + antibiotic-free phage, and bacterial suspension alone were incubated at 37°C for 16 hours. OD was measured. 600 Values ​​were used to create a heatmap of the combined antibacterial effect of bacteriophage and antibiotics, based on the measurement results. The results are as follows: Figure 8 As shown.

[0087] Depend on Figure 8 (A) Figure 8 As shown in Figure (B), the initial concentrations of the bacterial cultures at 0 h were relatively uniform, with no significant differences; after 16 h of treatment, the concentrations of the cultures decreased. Figure 8 (C) and Figure 8 As shown in Figure (D), compared with the bacterial control wells, the addition of any concentration of phage cocktail XD1 effectively inhibited bacterial growth. When the XD1 concentration reached 10... 6At pfu / mL and above, it showed a significant synergistic antibacterial effect with amoxicillin at a sub-MIC concentration (2 μg / mL), and also showed a significant synergistic antibacterial effect when used in combination with ceftiofur sodium at a sub-MIC concentration (16 μg / mL). When the XD1 concentration reached 10... 7 At pfu / mL, it showed a significant synergistic antibacterial effect with amoxicillin at a sub-MIC concentration (1 μg / mL). Figure 8 (E); when the XD1 concentration reaches 10 8 At pfu / mL, it showed a significant synergistic antibacterial effect with sub-MIC concentration (1 μg / mL) of ceftiofur sodium. Figure 8 (F).

[0088] Example 12: In vivo antibacterial effect determination of phage cocktail XD1

[0089] A *Escherichia coli* E28 strain was selected as the pathogenic strain to establish an infection model in the large wax moth. Eighty large wax moth larvae of similar body length were selected and divided into four groups of 20 larvae each. Freshly proliferated *E. coli* E28 was washed three times with MgSO4 solution. The bacterial suspension was then serially diluted 10-fold to 10⁻⁶. 6 CFU / mL, 10 7 CFU / mL and 10 8 CFU / mL. 5 μL of diluted bacterial solution was injected into the larvae of the large wax moth. MgSO4 solution was used as a control group. The large wax moth larvae were cultured in the dark, and mortality was observed at any time. The results are shown in Table 3.

[0090] Table 3. Determination of the median lethal dose (LD50) of *Escherichia coli* infection in *Gnaphalium affine*.

[0091]

[0092]

[0093] Based on the results in Table 3, and according to the formula LD50 = X k The median lethal dose (LD50) of E. coli E28, calculated using the formula -i(∑p-0.5), is 2.7 × 10⁻⁶. 7 CFU / mL. Where X k ∑p is the logarithmic dose corresponding to the highest dose group, i is the interval between dose groups, and ∑p is the sum of mortality rates (p) of each group.

[0094] The control effect of phage cocktail XD1 was determined using an established Escherichia coli infection model in *Escherichia coli*. Healthy *Escherichia coli* borers of similar body length were selected and divided into 9 groups: bacterial E28 infection group, MgSO4 control group, and XD1 (10... 8 PFU / mL) control group, XD1 (10 8 PFU / mL) high-dose treatment group, XD1 (107 PFU / mL) medium-dose treatment group, XD1 (10 6 PFU / mL) low-dose treatment group, XD1 (10 8 PFU / mL) high-dose prevention group, XD1 (10 7 PFU / mL) medium-dose prevention group, XD1 (10 6 Low-dose prevention group (PFU / mL). Except for the MgSO4 control group, all other groups were injected with 5 μL of Escherichia coli E28 bacterial solution (3 × 10⁻⁶ PFU / mL). 7 (CFU / mL). Two hours before and two hours after infection, phage groups were injected with 5 μL of different concentrations of XD1 cocktail. The larvae of the large wax moth were cultured in a 30℃ incubator in the dark, and survival was recorded every 12 hours. Survival curves were plotted based on the mortality rate of the large wax moths. Results are shown below. Figure 9 .

[0095] Depend on Figure 9 The results in (A) show that when XD1 was administered 2 hours after infection with E. coli E28, no deaths occurred in the control group of large wax moths, indicating that the diluent and phage have high safety and no obvious toxic side effects on large wax moths. The survival rates of the three XD1 treatment groups were 90%, 40%, and 50%, respectively, while the survival rate in the E28 infection group was only 30%, indicating that 10 6 Phage cocktail XD1 with PFU / mL or higher showed significant therapeutic effects against *E. coli* infection in *E. coli* moth. Figure 8 The results in (B) show that when XD1 was administered 2 hours before infection with E. coli E28, the survival rates in the prevention groups were 70%, 60%, and 50%, respectively, indicating that 10 6 Phage cocktail XD1 with a concentration of PFU / mL or higher can provide significant protection against Escherichia coli infection in the giant wax moth.

[0096] In conclusion, 10 8 PFU / mL phage cocktail XD1 can effectively prevent and treat infections caused by E. coli E28, a type of worm.

[0097] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. A bacteriophage composition, characterized in that, Including at least one of Escherichia coli phage 81P-58 and Escherichia coli phage 87P-16, wherein: The accession number of the Escherichia coli phage 81P-58 is CCTCC NO: M 2025835, the accession date is April 21, 2025, and the accession address is China Center for Type Culture Collection, Wuhan University, Wuhan, China. The accession number of the Escherichia coli phage 87P-16 is CCTCC NO: M 2025834, the accession date is April 21, 2025, and the accession address is China Center for Type Culture Collection, Wuhan University, Wuhan, China.

2. The phage composition according to claim 1, characterized in that, The phage composition comprises Escherichia coli phage 81P-58 and Escherichia coli phage 87P-16: The ratio of Escherichia coli phage 81P-58 to Escherichia coli phage 87P-16 is 1:

1.

3. The use of a phage composition as described in claim 1 or 2 in the preparation of an inhibitor of chicken-derived multidrug-resistant pathogenic Escherichia coli.

4. The use of the phage composition as described in claim 3 in the preparation of an inhibitor of chicken-derived multidrug-resistant pathogenic Escherichia coli, characterized in that, The chicken-derived multidrug-resistant pathogenic Escherichia coli includes at least one of the following types: O8, O15, O55, O78, O86, O111, O114, O125, and O127.

5. The use of a phage composition as described in claim 1 or 2 in the preparation of a biological agent for the prevention and treatment of diseases or contamination caused by chicken-derived multidrug-resistant pathogenic Escherichia coli.

6. A biological agent, characterized in that, Includes the bacteriophage composition as described in claim 1 or 2.

7. The biological agent as described in claim 6, characterized in that, The biological agent includes a bacteriophage composition and an antibiotic, wherein the antibiotic includes at least one of amoxicillin and ceftiofur sodium.

8. The biological agent as described in claim 7, characterized in that, The antibiotics include amoxicillin, and the concentration of the bacteriophage composition in the biological agent is ≥10. 6 pfu / mL, wherein the concentration of amoxicillin in the biological agent is ≤2 μg / mL; or, The antibiotic includes ceftiofur sodium, and the concentration of the bacteriophage composition in the biological agent is ≥10. 6 pfu / mL, wherein the concentration of ceftiofur sodium is ≤16 μg / mL.

9. The biological agent as described in claim 7, characterized in that, The antibiotics include amoxicillin, and the concentration of the bacteriophage composition in the biological agent is ≥10. 7 pfu / mL, wherein the concentration of amoxicillin in the biological agent is ≤1 μg / mL; or, The antibiotic includes ceftiofur sodium, and the concentration of the bacteriophage composition in the biological agent is ≥10. 8 pfu / mL, wherein the concentration of ceftiofur sodium is ≤1 μg / mL.

10. The biological agent according to claim 6, characterized in that, The biological agents include feed additives, drinking water additives, meat product cleaners, or drugs.

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