Broad-spectrum bacteriophage vB_EcoM_GXW16 of drug-resistant escherichia coli and application thereof

Abstract

The application discloses an avian drug-resistant Escherichia coli broad-spectrum bacteriophage vB_EcoM_GXW16 and application thereof. The bacteriophage is separated from a sewage sample of a chicken farm in Guangxi, and is identified as belonging to the Caudoviricetes class, Myoviridae family and Darcivirus genus, and has a icosahedral capsid and a stretchable tail. Whole genome sequencing proves that the bacteriophage does not contain virulence genes, drug resistance genes and lysogeny-related genes, and has high biological safety. The bacteriophage has a wide host range, can lyse 68.42% (39 / 57) of Escherichia coli isolates, the optimal multiplicity of infection is 0.001, the latent period is 10 min, and the bacteriophage has good stability within the range of 40-60 DEG C and pH 4-12. The bacteriophage can efficiently inhibit the growth of drug-resistant avian pathogenic Escherichia coli, has important application value in the preparation of medicine, feed additive and other products for preventing and treating avian Escherichia coli disease, and provides a new solution for antibiotic replacement therapy.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and veterinary medicine, specifically relating to a broad-spectrum bacteriophage vB_EcoM_GXW16 targeting avian drug-resistant Escherichia coli, its isolation, identification methods and applications, and is particularly suitable for the prevention and control of avian pathogenic Escherichia coli (APEC) infection. Background Technology

[0002] Avian pathogenic Escherichia coli (APEC) is an important group of extraintestinal pathogenic Escherichia coli (ExPEC). It can cause a variety of diseases in poultry such as chickens, turkeys, and ducks, including perihepatitis, air sacculitis, and pericarditis. These diseases are collectively known as avian colibacillosis and are among the most common and economically destructive bacterial diseases in the global poultry industry. APEC infection can lead to a mortality rate of up to 53.5% in chicks, causing huge economic losses to the poultry industry.

[0003] Currently, clinical prevention and control of APEC infections mainly relies on antibiotics. However, with the long-term overuse of antibiotics, antimicrobial resistance (AMR) has become an increasingly serious problem, posing a major threat to global public health and veterinary health. The widespread prevalence of drug-resistant strains not only leads to treatment failure and increased economic losses, but the drug-resistant genes they carry can also spread horizontally, posing a potential risk to human health. Studies have shown that 76.5% of APEC strains exhibit multidrug resistance, and existing vaccines only target specific serogroups, offering limited protection and failing to effectively combat complex drug-resistant strain infections. Therefore, the development of novel antimicrobial agents to replace antibiotics is urgently needed.

[0004] Bacteriophages, viruses capable of specifically infecting and lysing bacteria, are the most abundant and ubiquitous organisms on Earth, possessing natural antibacterial properties. Compared to traditional antibiotics, phage therapy offers advantages such as strong host specificity, low screening costs, and low likelihood of inducing drug resistance, making it an important research direction in addressing the antibiotic resistance crisis. Numerous clinical trials have confirmed the safety and efficacy of phage therapy in treating severe bacterial infections, demonstrating broad application prospects. However, different phages exhibit significant differences in host range, biological characteristics, and therapeutic effects; therefore, screening for phages with broad-spectrum lytic activity, environmental stability, and good in vivo therapeutic efficacy is crucial for their clinical application.

[0005] Based on this, this study isolated and screened broad-spectrum lytic phages against avian drug-resistant Escherichia coli from the natural environment, systematically analyzed their biological characteristics and genomic features, and evaluated their in vitro antibacterial activity and in vivo therapeutic potential, aiming to provide a safe and effective antibiotic alternative and offer a new strategy for the prevention and control of avian drug-resistant Escherichia coli infection. Summary of the Invention

[0006] To address the problems of increasing antibiotic resistance and limited vaccine protection in the prevention and control of avian drug-resistant Escherichia coli infection, this invention provides a broad-spectrum, highly effective, and safe broad-spectrum phage vB_EcoM_GXW16 for avian drug-resistant Escherichia coli. This phage has a wide host range, strong environmental adaptability, and high biosafety, and can effectively prevent and treat avian coli infections.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a broad-spectrum avian pathogenic Escherichia coli bacteriophage vB_EcoM_GXW16, named Escherichia coli phage vB_EcoM_GXW16, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 47011 and deposit date of January 7, 2026.

[0009] Furthermore, the bacteriophage vB_EcoM_GXW16 possesses the following morphological characteristics: a clearly defined icosahedral head and a retractable tail; the head diameter is 86±3 nm, the tail length is 87±3 nm, and it has relatively long tail fibers. According to the classification criteria of the International Committee on Taxonomy of Viruses (ICTV), this bacteriophage belongs to the family Myocaudidae in the order Caudophages.

[0010] Furthermore, the avian drug-resistant Escherichia coli broad-spectrum phage vB_EcoM_GXW16 has a full-length genome of 170605 bp, a GC content of 39.51%, and 267 open reading frames annotated. It does not carry virulence genes, antibiotic resistance genes, or lysogen-related genes.

[0011] Furthermore, the bacteriophage vB_EcoM_GXW16 possesses at least one of the following biological characteristics:

[0012] (1) Wide host range: It can effectively lyse 39 of the 57 Escherichia coli isolates, with a lysis rate of 68.42%, and has a good targeted lysis effect on multidrug-resistant avian pathogenic Escherichia coli.

[0013] (2) Good stability: After treatment for 1 hour in the pH range of 4-12, the titer remains above 90% of the initial titer, or after treatment for 1 hour at 40-60℃, the phage titer remains stable and the activity does not decrease significantly.

[0014] (3) High lysis efficiency: The optimal multiplicity of infection is only 0.001, the incubation period is short, about 10 minutes; the lysis volume is large, the outbreak period lasts for 110 minutes, about 81 PFU / cell;

[0015] Escherichia coli isolates with 68.42% (39 / 57) lysis rate were found, including the multidrug-resistant strain Escherichia coli_O117:H25_E5.

[0016] (4) High adsorption rate: The adsorption rate of host bacteria can reach 81.43% within 10 minutes, which can quickly start the infection process.

[0017] (5) Safety: No lysogen-related genes, virulence genes and antibiotic resistance genes were found in the whole genome sequencing analysis.

[0018] (6) Strong in vitro antibacterial activity: At three concentrations of MOI=1, 0.1 and 0.01, it can significantly inhibit the growth of drug-resistant pathogenic Escherichia coli in birds, inhibit the proliferation of host bacteria within 2 hours, and significantly reduce the number of bacteria compared with the control group within 12 hours (P<0.001).

[0019] Secondly, the present invention provides the use of the above-described Escherichia coli bacteriophage vB_EcoM_GXW16 in the prevention or treatment of Escherichia coli infection.

[0020] Furthermore, the Escherichia coli is a drug-resistant avian Escherichia coli.

[0021] Thirdly, the present invention provides a preparation for preventing and controlling avian drug-resistant Escherichia coli infection, wherein the active ingredient comprises the aforementioned avian drug-resistant Escherichia coli broad-spectrum bacteriophage vB_EcoM_GXW16.

[0022] Furthermore, the preparation is used for the prevention or treatment of avian Escherichia coli infection.

[0023] Furthermore, the preparation is administered orally.

[0024] The beneficial effects of this invention are as follows:

[0025] 1. Wide host range and high lysis efficiency: Phage vB_EcoM_GXW16 can lyse 68.42% of E. coli isolates, and is especially effective against multidrug-resistant avian pathogenic E. coli, solving the problem of narrow host range of existing phages.

[0026] 2. Excellent biological characteristics: low optimal multiplicity of infection (0.001), short incubation period (10 min), fast adsorption rate, strong environmental adaptability, wide range of temperature and acid / alkali resistance, and easy to produce, process, store, transport and apply.

[0027] 3. Significant prevention and control effects: It can rapidly inhibit bacterial proliferation in vitro and effectively improve the survival rate of infected chicks, reduce bacterial load, and alleviate organ damage in vivo, providing an effective means of prevention and control for avian colibacillosis.

[0028] 4. Broad application prospects: It can be made into a variety of products such as medicines, feed additives, and environmental disinfectants. It is applicable to the prevention and control of E. coli diseases in poultry farming, providing an ideal alternative to antibiotics and having important economic and social value. Attached Figure Description

[0029] Figure 1 Morphological image of bacteriophage vB_EcoM_GXW16. (A) Plaque morphology; (B) Transmission electron microscope morphology image.

[0030] Figure 2 Biological characteristics of bacteriophage vB_EcoM_GXW16. (A) Optimal multiplicity of infection; (B) Adsorption rate; (C) One-step growth curve; (D) Temperature stability; (E) Storage experiment; (F) pH stability.

[0031] Figure 3 : Antibacterial effect of bacteriophage vB_EcoM_GXW16 in vitro. (A) OD of host bacteria 600 Detection; (B) Bacterial count of the host bacteria.

[0032] Figure 4 Genome analysis of bacteriophage vB_EcoM_GXW16. (A) Circular genome map; (B) A phylogenetic tree constructed based on the gene sequence of the large subunit of the termination enzyme.

[0033] Figure 5 Symptoms of E. coli infection in chicks (A) Full-body image of an infected chicken; (B) Changes observed during abdominal necropsy in the infected group; (C) Comparison of heart, liver, and spleen tissues from infected and normal chickens.

[0034] Figure 6 : The therapeutic effect of bacteriophage vB_EcoM_GXW16 on a chick infection model. (A) Survival rate of chicks in each group; (B) Average weight of surviving chicks in each group; (C) Liver bacterial load; (D) Histopathological sections of heart, liver, and spleen tissues (HE, 200x). Detailed Implementation

[0035] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the following embodiments are merely illustrative of the invention and are not intended to limit the scope of the invention in any way. Unless otherwise specified, the experimental methods used in this invention are conventional methods. Unless otherwise specified, the materials, reagents, etc., used in this invention are commercially available. Furthermore, other terms used in this invention, unless otherwise stated, generally have the meanings commonly understood by those skilled in the art.

[0036] In this embodiment of the invention, the broad-spectrum phage vB_EcoM_GXW16 of avian drug-resistant Escherichia coli is deposited at the China General Microbiological Culture Collection Center (CGMCC), at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 47011 and deposit date of January 7, 2026.

[0037] The following describes the process through more specific examples.

[0038] Example 1: Isolation and purification of bacteriophage vB_EcoM_GXW16

[0039] Sample Collection and Processing: Wastewater and fecal samples were collected from various live poultry markets and farms in Guigang, Nanning, Chongzuo, and Beihai, Guangxi. 2 g (or 2 mL) of each sample was added to 5 mL of LB liquid medium and incubated at 37℃ and 180 r / min for 4 h on a shaker. The mixture was then centrifuged at 10000 r / min for 10 min, and the supernatant was collected. The supernatant was filtered through a 0.22 μm sterile membrane to obtain the phage stock solution, which was stored at 4℃ for later use.

[0040] Phage isolation and preliminary validation: *Escherichia coli* avian subtype O117:H25_E5, isolated from the liver of diseased chickens, was used as the indicator bacterium. A double-layer agar plate method was employed, with 5 μL of the phage mother bacteria added dropwise to a semi-solid agar plate containing the host bacteria. The plates were incubated upside down at 37°C for 6–12 hours. The formation of transparent phage plaques was observed.

[0041] Plaque purification: Using a sterile pipette tip, pick up a single, well-defined, and transparent plaque and place it in a centrifuge tube containing 0.5 mL LB broth. Shake well to elute. Serially dilute the eluent 10-fold and incubate using the double-layer plate method. Pick single plaques again. Repeat this purification step 4-6 times until uniformly shaped and sized plaques appear on the plates, indicating that the purified phage has been obtained.

[0042] Phage amplification and preservation: The purified single phage plaque eluent was mixed with the logarithmic-phase host bacterial culture at a certain ratio and incubated at 37°C and 180 rpm with shaking for 4-6 hours. After removal, the culture was centrifuged at 4°C and 12,000 rpm for 10 minutes. The supernatant was filtered through a 0.22 μm filter to obtain purified phage lysis buffer. The lysis buffer was mixed with an equal volume of 50% glycerol, aliquoted, and stored at -80°C.

[0043] Result: As Figure 1 As shown in Figure A, phage vB_EcoM_GXW16 can form single phage plaques of uniform size and consistent characteristics on a double-layer plate.

[0044] Example 2: Morphological observation of bacteriophage vB_EcoM_GXW16

[0045] Add 10 μL of purified phage lysis buffer to a copper mesh support membrane, let stand at room temperature for 5 min, and blot away excess liquid with filter paper. Add 2% phosphotungstic acid (pH 7.0) for negative staining for 1 min, aspirate the stain, and allow to dry at room temperature. Observe and photograph using a JEM-1200EXII transmission electron microscope at an accelerating voltage of 80 kV.

[0046] Result: As Figure 1 As shown in Figure B, bacteriophage vB_EcoM_GXW16 has a typical myotail bacteriophage structure, with an icosahedral head approximately 86±3 nm in diameter and a retractable tail approximately 87±3 nm in length, bearing a relatively long tail fiber. According to the International Committee on Taxonomy of Viruses (ICTV) guidelines, this bacteriophage belongs to the class Caudoviricetes, genus Myotail Virus, genus Kodakavirus.

[0047] Example 3: Biological characteristics analysis of bacteriophage vB_EcoM_GXW16

[0048] Optimal multiple of infection (MOI) determination: The concentration of host bacterium Escherichia coli_O117:H25_E5 was adjusted to 10. 8 CFU / mL. Phage lysates were diluted to different concentrations and mixed with an equal volume of host bacterial culture to obtain MOIs of 10, 1, 0.1, 0.01, and 0.001. After incubation at 37°C with shaking for 4 hours, the lysates were obtained by centrifugation and filtration. The phage titer (PFU / mL) at each MOI was determined using the double-layer plate method, with three replicates for each treatment. The MOI with the highest titer was considered the optimal MOI.

[0049] Result: As Figure 2 As shown in Figure A, the phage titer was highest at an MOI of 0.001, reaching 3.47 × 10⁻⁶. 9PFU / mL, therefore the optimal MOI for determining phage vB_EcoM_GXW16 is 0.001.

[0050] Adsorption rate determination: The phage and host bacteria were mixed at the optimal MOI and incubated at 37°C for 12 min. Samples were taken every 2 min, filtered through a 0.22 µm filter membrane, and after serial dilution, the titer of unabsorbed phage in the supernatant was measured. The percentage of phage adsorption rate at different time points was calculated as follows: [(initial phage titer - phage titer in supernatant) / initial phage titer] ×100%.

[0051] Result: As Figure 2 As shown in B, the adsorption rate of bacteriophage was 25.52% within 2 min, 56.92% within 6 min, and 81.43% within 10 min.

[0052] One-step growth curve: Phages and host bacteria were mixed at the optimal MOI and incubated in a 37°C water bath for 5 min to allow for full adsorption of the host bacteria. Subsequently, unadsorbed phages were removed by centrifugation, and the precipitate was washed twice with preheated LB broth at 37°C to remove unadsorbed free phages. The washed precipitate was resuspended in a large volume of fresh LB broth. The medium was incubated at 37°C with shaking, sampling every 10 min for the first 120 min, and every 20 min thereafter, for a total of 180 min. Phage titer was determined using the double-layer plate method.

[0053] Result: As Figure 2 As shown in Figure C, the latency period of bacteriophage vB_EcoM_GXW16 is approximately 10 min, the outbreak period is approximately 110 min, and the average lysis rate is approximately 81 PFU / cell.

[0054] Temperature stability: The phage lysate was placed in water baths at 40℃, 50℃, 60℃, 70℃ and 80℃, and samples were taken at 20, 40 and 60 min. After being immediately cooled in an ice bath, the phage titer was determined using the double-layer plate method.

[0055] Result: As Figure 2 As shown in D, the titer of bacteriophages treated at 40-60℃ for 60 min did not change significantly. However, at 70℃, the phage titer gradually decreased with increasing time, and when the temperature was raised to 80℃, it was completely inactivated after 20 min.

[0056] Storage experiment: The phage suspension was stored at room temperature and 4°C in a refrigerator, and samples were taken once a week for 4 consecutive weeks. The phage titer was determined to evaluate its storage stability.

[0057] Result: As Figure 2As shown in E, the phages did not show a significant decrease in titer after being stored at room temperature and 4°C for 4 weeks, and still maintained high activity.

[0058] pH stability: Adjust the pH of LB liquid medium to 1-13 with HCl or NaOH. Mix the phage lysate with mediums of different pH values, incubate at 37°C for 1 hour, then immediately dilute with neutral pH medium and determine the titer.

[0059] Result: As Figure 2 As shown in F, the bacteriophages were completely inactivated at pH=1 and pH=13; their activity decreased at pH=2-3; and their activity was stable in the pH=4-12 range, with the best activity at pH=7-9.

[0060] Example 4: In vitro antibacterial experiment of bacteriophage vB_EcoM_GXW16

[0061] The bacteriophage was used to infect the host bacteria at three different MOIs (1, 0.1, 0.01). The host strain was then adjusted to OD. 600 nm is 0.2 (approximately 10 8 The phage was added to the host bacteria at MOIs of 0.01, 0.1, and 1 (CFU / ml), and the mixture was incubated at 37°C for 12 h. Measurements were taken every 2 h. 96-well plate 600 nm Bacterial growth was determined by measuring absorbance at a specific temperature. Simultaneously, the number of viable *E. coli* was determined by MacConkey plate counting. Phage-free bacterial cultures were used as positive controls, and LB broth as negative controls.

[0062] Result: As Figure 3 As shown in Figure A, within 4 h of treatment with vB_EcoM_GXW16, the growth of Escherichia coli_O117:H25_E5 was completely inhibited under all MOIs, and the OD within 12 h was [missing data]. 600 The values ​​were still significantly different from the positive control (p). < 0.001). For example... Figure 3 As shown in B, compared with the control group, the number of viable cells in the three phage-treated groups (MOI=1, 0.1, and 0.01) decreased rapidly within 2 hours, and then gradually increased, although the number of bacteria in all treatment groups exceeded 10 at 12 hours. 8 CFU / ml, but there was still a significant difference compared with the positive control (p). < 0.001).

[0063] Example 5: Whole genome sequencing and analysis of bacteriophage vB_EcoM_GXW16

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

[0065] Sequence assembly and annotation: FastP was used for data quality control, and ABySS and GapCloser were used for sequence assembly to obtain the complete genome. Gene function annotation was performed using databases such as NCBI NR, SwissProt, KEGG, and COG. Virulence genes and drug resistance genes were screened using VFDB and CARD databases, respectively. Based on the large subunit gene sequence of the terminase, multiple sequence alignment was performed using MEGA12 software, a phylogenetic tree was constructed using the neighbor-joining method, and visualized using the iTOL online tool.

[0066] Genomic analysis results:

[0067] Basic characteristics: bacteriophage vB_EcoM_GXW16 is a double-stranded DNA bacteriophage with a full-length genome sequence of 170,605 bp, a (G+C) content of 39.51%, and a gene length accounting for 95.2% of the entire genome. 。 Genome circle diagram as follows Figure 4 As shown in Figure A.

[0068] Gene annotation: A total of 267 open reading frames (ORFs) were annotated across the entire genome, with 41 located on the positive strand and the remaining 226 on the negative strand. 127 ORFs (47.57%) were annotated as genes with known functions, including DNA replication and metabolism-related proteins, structural / packaging proteins, and host cleavage proteins. The remaining 140 ORFs (52.43%) were annotated as hypothetical proteins.

[0069] Safety assessment: The absence of tRNA in the genome indicates that the phage... vB_EcoM_GXW16 relies entirely on the host for protein synthesis. Comparison with the Virulence Factor Database (VFDB) and the Antibiotic Resistance Gene Database (CARD) revealed no known virulence genes or antibiotic resistance genes, suggesting the safety of bacteriophage vB_EcoM_GXW16 in clinical applications.

[0070] Phylogenetic analysis: such as Figure 4 As shown in Figure B, a phylogenetic tree was constructed based on the gene sequence of the large subunit of the termination enzyme. The results showed that phage vB_EcoM_GXW16 is closely related to other E. coli phages and belongs to the genus Codacardivirus of the class Caudoviricetes.

[0071] Example 6: In vivo efficacy evaluation of bacteriophage vB_EcoM_GXW16 (chick infection model)

[0072] Infection model establishment and LD50 determination: Sixty healthy broiler chickens were raised to 7 days of age and randomly divided into 6 groups of 10 chickens each. Each group of broilers was intraperitoneally injected with different doses of Escherichia coli O117:H25_E5 (10... 10 10 9 , 10 8 , 10 7 , 10 6 CFU (0.1 ml) bacterial suspension was administered, while the control group received the same volume of sterile saline. Chick survival was recorded for 7 days post-challenge, and the median lethal dose (LD50) was calculated using SPSS software. 50 ).

[0073] result: Escherichia coli_O117:H25_E5 of LD 50 for 4.6×10 8 CFU / mL. Infected chickens exhibit obvious symptoms such as lethargy, standing listlessly with eyes closed, and drooping wings. Figure 5 A. Upon necropsy, the abdominal organs were covered with a layer of fibrous exudate; the heart was adhered to the sternum / liver, as shown in the image. Figure 5 B. Autopsy of the dead chicken showed varying degrees of hepatomegaly, with a layer of fibrinous exudate covering the liver surface; the capsule was thick and easily peeled off; the pericardium was significantly thickened and covered with a large amount of yellowish-white or purulent exudate; and the spleen was enlarged. The pathological changes are shown in the figure. 5C.

[0074] Treatment trial grouping: 96 Only 1 Healthy white-feathered broiler chickens of [age] were raised under laboratory conditions. 6 On the seventh day, they were randomly divided into 8 groups (phage prevention group A, phage prevention group B, phage treatment group C, phage treatment group D, E. coli control group, antibiotic treatment group, blank control group, and phage control group), with 12 birds in each group. The broilers in groups A-D, the E. coli control group, and the antibiotic treatment group were injected intraperitoneally with 50% of the lethal dose (LD50). 50Escherichia coli_O117:H25_E5 was used as the control group, while the phage control group was replaced with physiological saline. Phage prevention groups A and B received oral administration of 10 mg of the drug 3 hours before infection. 5 PFU and 10 8 PFU vB_EcoM_GXW16 once. Phage treatment groups C and D received 10 mg orally 3 hours after infection. 5 PFU and 10 8 PFU vB_EcoM_GXW16 was administered for 3 consecutive days. Florfenicol in the antibiotic treatment group was purchased from Hubei Yuanhao Biotechnology Co., Ltd., and administered according to the instructions. The phage control group received 10 mg of PFU orally for three consecutive days. 8 PFU vB_EcoM_GXW16. A blank control group and an E. coli control group were replaced with physiological saline. All groups were fed under identical conditions, with chickens having free access to feed and water daily.

[0075] Treatment observation and sample collection: Chicks were observed continuously for 7 days, and their mental state, clinical symptoms, and mortality were recorded. On day 7, all surviving chicks were weighed and then euthanized. Liver tissue was collected for bacterial load determination. Heart, liver, and spleen were collected, fixed in 10% formalin, paraffin-embedded, sectioned, and stained with H&E for histopathological analysis.

[0076] Treatment results:

[0077] Survival rate: such as Figure 6 As shown in Figure A, survival rates improved to varying degrees in all groups after adopting different treatment regimens. (Post-infection) 3 h using 10 8 The highest survival rate was observed in chicks treated with phage at a concentration of PFU / ml for 3 days. 83.33%. 3 days post-infection h gives 10 5 phage at PFU / ml was administered 3 days and 3 hours before infection. 8 The survival rate of the phage group treated once with PFU / ml was 75%. Furthermore, the survival rate of the phage control group was 100%, indicating that vB_EcoM_GXW16 (10 8 PFU has no side effects on broiler chickens.

[0078] Average weight of each group: e.g. Figure 6 As shown in B, compared with the blank control group, the chicks in the E. coli treatment group had a significantly lower body weight (p < 0.001). However, compared with the group that received only E. coli, the phage treatment groups B, C, and D showed higher body weight (p < 0.01).

[0079] Bacterial load: such as Figure 6As shown in C, the number of viable liver bacteria in the phage treatment groups B, C, and D was significantly lower than that in the bacterial control group (p < 0.001).

[0080] Histopathology: such as Figure 6 As shown in D, in the bacterial control group, the chicks' heart tissue showed extensive necrosis of the epicardium and underlying myocardium, with abundant proliferation of fibrous connective tissue, accompanied by infiltration of numerous lymphocytes, macrophages, and heterophilic granulocytes. The liver tissue showed localized thickening of the capsule, proliferation of fibrous connective tissue, and significant lymphocyte infiltration, with widespread hepatocyte edema and swelling, and numerous focal lymphocyte infiltrations. The spleen showed significant necrosis of both red and white pulp cells. In contrast, in the phage treatment groups C and D, the heart tissue showed clear epicardial and endocardial structures without obvious abnormalities, with a small number of lymphocytes visible in the myocardium; a small amount of hepatocyte edema and lymphocyte infiltration; and no obvious abnormalities were observed in the spleen tissue.

[0081] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Various modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope defined by the claims should be included within the scope of protection of the present invention.

Claims

1. Avian drug-resistant Escherichia coli broad-spectrum bacteriophage vB_EcoM_GXW16, characterized in that, This bacteriophage is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 47011 and deposit date of January 7, 2026.

2. The avian drug-resistant Escherichia coli broad-spectrum bacteriophage vB_EcoM_GXW16 according to claim 1, characterized in that, This bacteriophage is a tailed bacteriophage with an icosahedral head of 86±3 nm in diameter and a retractable tail of 87±3 nm in length.

3. The avian drug-resistant Escherichia coli broad-spectrum bacteriophage vB_EcoM_GXW16 according to claim 1, characterized in that, The genome is 170,605 bp in length, with a GC content of 39.51%, and 267 open reading frames are annotated. It does not carry virulence genes, antibiotic resistance genes, or lysogen-related genes.

4. The avian drug-resistant Escherichia coli broad-spectrum bacteriophage vB_EcoM_GXW16 according to claim 1, characterized in that, The bacteriophage, when treated at pH 4-12 for 1 hour, maintained a titer of over 90% of its initial titer.

5. The avian drug-resistant Escherichia coli broad-spectrum bacteriophage vB_EcoM_GXW16 according to claim 1, characterized in that, The phage remained stable in potency after being treated at 40-60℃ for 1 hour.

6. The avian drug-resistant Escherichia coli broad-spectrum bacteriophage vB_EcoM_GXW16 according to claim 1, characterized in that, The optimal multiplicity of infection was 0.001, the incubation period was 10 min, the outbreak period lasted 110 min, and the outbreak dose was 81 PFU / cell.

7. The avian drug-resistant Escherichia coli broad-spectrum bacteriophage vB_EcoM_GXW16 according to claim 1, characterized in that, It can lyse 68.42% of Escherichia coli isolates, including the multidrug-resistant strain Escherichia coli_O117:H25_E5.

8. The use of the broad-spectrum bacteriophage vB_EcoM_GXW16 of avian drug-resistant Escherichia coli according to any one of claims 1-7 in the preparation of a formulation for the prevention and control of avian drug-resistant Escherichia coli infection.

9. A preparation for preventing and controlling avian drug-resistant Escherichia coli infection, characterized in that, The active ingredient comprises the broad-spectrum phage vB_EcoM_GXW16 of avian drug-resistant Escherichia coli as described in any one of claims 1-7.

10. The preparation for preventing and controlling avian drug-resistant Escherichia coli infection according to claim 9, characterized in that, The preparation is administered orally.

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