Escherichia coli phage XHX-E-025P for lysing escherichia coli and salmonella and application thereof

Abstract

The application discloses an Escherichia coli phage XHX-E-025P for lysing Escherichia coli and Salmonella and an application thereof, and relates to the technical field of microorganisms. The phage screened by the application has a wide lysis spectrum, can not only efficiently lyse Escherichia coli, but also can cross-species lyse Salmonella, and has a high lysis rate on various clinical isolates, thereby effectively coping with mixed infections. In particular, the lysis activity of the phage on multiple drug-resistant strains is better than that of commercially available phages, thereby providing a new solution for solving drug-resistant infections. In addition, in piglet diarrhea treatment tests, the cure rate of the phage is significantly higher than that of a control group, and the application prospect is good. Whole genome sequencing shows that the phage carries various tail fiber proteins and tail structure proteins, thereby providing a structural basis for cross-species lysis. Meanwhile, phylogenetic analysis shows that the phage is a novel Escherichia coli phage that has not been reported.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a bacteriophage, Escherichia coli phage XHX-E-025P, that lyses Escherichia coli and Salmonella, and its applications. Background Technology

[0002] Escherichia coli and Salmonella are two of the most prevalent pathogens in livestock and poultry farming, infecting various farmed animals such as pigs, chickens, and ducks. This leads to diarrhea in piglets, septicemia in poultry, stunted growth, and increased mortality, causing significant economic losses. Simultaneously, Salmonella can also infect humans through contaminated meat, eggs, and dairy products, causing food poisoning, gastroenteritis, and even septicemia, making it a significant zoonotic pathogen. In recent years, bacterial diseases caused by E. coli and Salmonella have become increasingly serious, posing a major obstacle to the healthy development of the livestock and poultry farming industry.

[0003] Currently, aquaculture production relies heavily on antibiotics to control E. coli and Salmonella infections. However, long-term use has led to the emergence of drug-resistant strains, increasing the difficulty of treatment. Furthermore, drug-resistant genes may spread through the food chain, threatening human health. National bacterial resistance monitoring reports show that E. coli has high resistance rates to third-generation cephalosporins and quinolones; Salmonella also exhibits a continuously rising trend in resistance rates to multiple commonly used antibiotics. Simultaneously, conventional disinfection measures are insufficient to completely eliminate pathogens in the aquaculture environment, as these bacteria can form biofilms on the surfaces of aquaculture facilities, leading to recurrent disease outbreaks. Therefore, there is an urgent need to develop safe and highly effective new antibacterial agents.

[0004] Bacteriophages are a class of viruses that specifically infect and lyse bacteria. They possess advantages such as high species specificity, environmental friendliness, and the ability to self-replicate, showing promising application prospects in the control of pathogenic bacteria in livestock and poultry. However, most existing Escherichia coli bacteriophages and Salmonella bacteriophages generally suffer from narrow host ranges and poor environmental stability. Most bacteriophages can only lyse a single bacterial species or are only effective against a few strains, making it difficult to cope with the complex situation of mixed infections of multiple pathogens in the breeding environment. Furthermore, they are easily inactivated under acidic, alkaline, or high-temperature conditions, making stable application in actual breeding environments difficult. Large-scale production and formulation development also present challenges.

[0005] In practical aquaculture applications, bacteriophages may face severe challenges from multidrug-resistant strains. The widespread prevalence of clinical isolates of drug-resistant Escherichia coli and multidrug-resistant Salmonella makes it difficult for traditional antibiotic treatments to effectively control outbreaks. Existing bacteriophages often lack inhibitory effects on drug-resistant strains due to their narrow host range. If bacteriophages cannot effectively lyse multidrug-resistant strains, they will be unable to play a role in replacing or supplementing antibiotics in practical applications.

[0006] Therefore, discovering a broad-spectrum bacteriophage that can simultaneously lyse Escherichia coli and Salmonella and has good lytic activity against multidrug-resistant strains, and developing corresponding drug formulations, feed additives and environmental disinfection products, is of great significance for green prevention and control in livestock and poultry farming and for ensuring public health and safety.

[0007] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides a bacteriophage, Escherichia coli phage XHX-E-025P, that lyses Escherichia coli and Salmonella, and its applications, in order to solve the problems mentioned in the background art.

[0009] A bacteriophage of *Escherichia coli* that lyses *Escherichia coli* and *Salmonella*, named *Escherichia coli phage* XHX-E-025P, was deposited on March 17, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China 430072, China, with accession number CCTCC NO: M 2025497, and classified as *Escherichia coli phage*. Escherichia coli phage ).

[0010] Preferably, its tail protein has an amino acid sequence as shown in SEQ ID NO.1.

[0011] The use of the above-described Escherichia coli phage in the preparation of a medicament for the prevention or treatment of Escherichia coli or Salmonella infection.

[0012] An application of the above-described Escherichia coli bacteriophage in the preparation of feed or feed additives.

[0013] An application of the above-described Escherichia coli bacteriophage in the preparation of environmental disinfectants.

[0014] A feed additive comprising the aforementioned Escherichia coli bacteriophage.

[0015] An antimicrobial preparation comprising the above-described Escherichia coli bacteriophage and pharmaceutically or veterinarily acceptable excipients.

[0016] According to the above-described antibacterial agent, the dosage form of the antibacterial agent is an oral solution, a spray solution, or an injection solution.

[0017] An environmental disinfectant comprising the aforementioned Escherichia coli bacteriophage.

[0018] A feed composition comprising a base feed and the feed additives described above.

[0019] The present invention provides a bacteriophage, Escherichia coliphage XHX-E-025P, for lysing Escherichia coli and Salmonella, and its application, which have the following beneficial effects: (1) This phage can not only efficiently lyse Escherichia coli, but also lyse Salmonella across genera. Host spectrum analysis results show that this phage has a high lysis rate for clinical isolates of Escherichia coli and Salmonella from different regions, indicating that this phage has a wide lysis spectrum and can effectively cope with the complex situation of mixed infection of Escherichia coli and Salmonella in the breeding environment.

[0020] (2) This phage maintains high lytic activity against clinical isolates of multidrug-resistant Escherichia coli and multidrug-resistant Salmonella. Compared with commercially available Escherichia coli phages, the phage of this invention has a significantly better lytic effect on multidrug-resistant strains than existing products, which can effectively solve the problem of having no drugs available after antibiotic treatment failure, and provide a new treatment method for multidrug-resistant bacterial infections.

[0021] (3) The phage can maintain stable lytic activity over a wide range of temperature and pH. pH tolerance test shows that after treatment for a certain period of time over a wide range of pH, the phage titer does not change significantly, and it can maintain activity in different acidic and alkaline environments of the gastrointestinal tract of farmed animals; temperature stability test shows that the phage still maintains high lytic activity after treatment for a certain period of time over a wide range of temperature, and has good thermal stability, which is convenient for large-scale production and storage.

[0022] (4) The phage has a short incubation period after infecting the host bacteria, and can rapidly proliferate and lyse the host bacteria in a short time, releasing a large number of progeny phages to achieve efficient sterilization. The phage can achieve efficient proliferation and lysis at extremely low infection doses, and has good amplification efficiency, which is beneficial to reducing production costs.

[0023] (5) The results of the piglet diarrhea treatment experiment showed that after continuous oral administration of the phage suspension, the cure rate of diarrheal piglets was significantly higher than that of the control group, indicating that the phage has a significant therapeutic effect on piglet diarrhea caused by Escherichia coli and Salmonella and has good clinical application prospects.

[0024] (6) Whole-genome sequencing and functional annotation results showed that the phage genome contained multiple gene modules related to structural proteins, DNA replication and regulation, host lysis and packaging, and annotated various tail fibrin proteins and tail-related structural proteins, providing a structural basis for the phage to recognize and adsorb host bacterial surface receptors. Phylogenetic analysis showed that this phage was located in an independent branch position in the Escherichia coli phage population, and was distantly related to known Escherichia coli phages, belonging to a novel phage strain. Attached Figure Description

[0025] Figure 1 This image shows the plaque morphology of the *Escherichia coli* phage XHX-E-025P purified in Example 1 of this invention on a plate.

[0026] Figure 2 Transmission electron microscopy image of Escherichia coli phage XHX-E-025P.

[0027] Figure 3 The figure shows the pH tolerance results of Escherichia coli phage XHX-E-025P.

[0028] Figure 4 Figure showing the temperature tolerance results of Escherichia coli phage XHX-E-025P.

[0029] Figure 5 This is a one-step growth curve of Escherichia coli phage XHX-E-025P.

[0030] Figure 6 The figure shows the optimal multiplicity of infection determination results for Escherichia coli phage XHX-E-025P.

[0031] Figure 7 Phylogenetic tree of Escherichia coli phage XHX-E-025P. Detailed Implementation

[0032] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. 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.

[0033] I. Experimental Materials and Reagents 1×PBS buffer (1L): Weigh 99g of PBS solid powder into 1L of distilled water, heat to boiling until completely dissolved to prepare 10×PBS buffer. Take 100 mL of 10×PBS solution and add it to 900 mL of distilled water. Autoclave at 121 °C for 15 min and cool for later use.

[0034] 0.6% LB agar medium / plate (200 mL): Weigh 2 g tryptone, 2 g sodium chloride, 1 g yeast extract and 1.2 g agar into 200 mL distilled water, heat to boiling until completely dissolved, autoclave at 121 °C for 15 min, and cool for later use.

[0035] LB Broth: Weigh 10.0 g of tryptone, 5.0 g of yeast extract, and 10.0 g of sodium chloride, dissolve in 1000 ml of distilled water, and autoclave at 121 °C for 15 min.

[0036] LB nutrient agar (1000 ml): Weigh 10.0 g tryptone, 5.0 g yeast extract, 10.0 g sodium chloride, and 15.0 g agar, dissolve in 1000 ml distilled water, and autoclave at 121 °C for 15 min. II. Specific Implementation Methods Example 1: Isolation, preparation, purification, and culture of Escherichia coli phage XHX-E-025P.

[0038] 1. Sample pretreatment (1) In May 2024, manure was collected from a sow farm in Lüliang City, Shanxi Province, China, and transported back to the laboratory after being stored at low temperature. (2) Larger particulate impurities are removed by gauze filtration; (3) Centrifuge the sample at 8000 rpm for 10 min; (4) The supernatant was sterilized by filtration using a 0.22 μm microporous membrane; 2. Isolation of bacteriophages (1) Take 10 ml of sterile double LB concentrated broth and add 10 mL of filtered wastewater.

[0039] (2) Inoculate with 0.1 mL of host bacterial culture that has been cultured overnight and culture with shaking at 37 °C.

[0040] (3) After culturing for 24 h, centrifuge at 8000 rpm for 10 min.

[0041] (4) The supernatant was sterilized by using a 0.22 μm microporous membrane.

[0042] 3. Bacteriophage spot test (1) Mix 100 μl of overnight cultured bacterial solution with 5 mL of melted and incubated semi-solid culture medium at 50 °C, and pour the mixture onto the bottom agar plate to prepare bacterial moss for each strain.

[0043] (2) Add a few drops of 10 μl of the sample filtrate to the bacterial moss until the droplets are completely dry.

[0044] (3) Incubate the plate in a 37 °C constant temperature incubator overnight. Check for any clear or dull lysis areas.

[0045] 4. Plate method for isolating bacteriophages from samples (1) Prepare solid agar plates and number the plates according to the dilution of the sample. Prepare enough semi-solid agar medium, and place it in a water bath or oven at 45-48 °C after completely melting the semi-solid agar.

[0046] (2) Prepare sufficient sterile capped test tubes or microcentrifuge tubes. Add 0.9 mL of PBS diluent to the sterile test tubes or microcentrifuge tubes according to the experimental requirements. Number the test tubes or microcentrifuge tubes according to the dilution.

[0047] (3) Add 0.1 mL of phage lysis buffer to the first tube, mix well, and then transfer 0.1 mL to the second tube. Repeat this process for 10-fold dilution.

[0048] (4) Transfer 0.1 mL of phage dilution to warm semi-solid culture medium test tubes, immediately add 0.1 mL of overnight host culture, mix evenly and pour onto the surface of solid culture medium.

[0049] (5) After the semi-solid layer solidifies in 15-30 min, it is inverted and cultured at 37 °C. After continuous culture for 18-24 h, plates with a single plaque are selected for phage purification.

[0050] 5. Purification of bacteriophages (1) Pick up a single phage plaque with an autoclaved pipette tip, suspend it in 1 mL of PBS buffer, and vortex thoroughly to dissolve the phage completely in the PBS buffer; (2) Use a filter membrane with a pore size of 0.22 μm to remove contaminants; (3) Dilute the PBS buffer mixed with the phage plaques appropriately, and passage the phages continuously using the double-layer plate culture method. When the observed phage plaques are basically consistent in size and morphology, the purified phage individuals can be obtained. The morphology of the purified phages in this experiment is as follows: Figure 1 Medium-sized plaques, transparent, round, with smooth edges.

[0051] 6. Preservation of bacteriophages Mix 0.5 ml of phage stock solution with 0.5 ml of sterile glycerol in a 2 ml cryovial to a final concentration of 50%, and store at -80 °C.

[0052] Example 2: Electron microscopic observation of Escherichia coli phage XHX-E-025P Figure 2 shows a negative-stained transmission electron microscope image of Escherichia coli phage XHX-E-025P, magnified 60,000 times, with a scale bar of 100 nm. It can be seen that the phage has a complete ellipsoidal icosahedral head and a characteristic short and thick tail, and its morphology is consistent with the typical structure of the Short-tailed Phagesidae family.

[0053] Example 3: Acid-base tolerance test of Escherichia coli phage XHX-E-025P (1) Using LB broth, a series of solutions with different pH values ​​were prepared by adding appropriate amounts of concentrated hydrochloric acid or sodium hydroxide, ranging from pH 2.0 to 12.0.

[0054] (2) 100 μl of phage suspension was added to 900 μl of solutions with different pH values, and the phage was incubated under the set pH conditions. The incubation temperature was 37 °C and the incubation time was 2 h.

[0055] (3) After incubation, the titer of bacteriophages under different pH conditions was determined by double-layer agar plate method. The survival rate of bacteriophages was determined by calculating the number of plaques.

[0056] (4) Based on the measured titer data, plot the relationship curve between phage titer and pH value, and analyze the stability of phage under different pH conditions.

[0057] The results are as follows Figure 3 As shown, the titer of bacteriophage Escherichia coli phage XHX-E-025P did not change significantly at pH 3.0-11.0, and it could maintain a relatively stable level.

[0058] Example 4: Temperature stability experiment of Escherichia coli phage XHX-E-025P (1) Take 500 μl of phage suspension and dispense it into multiple test tubes.

[0059] (2) Set a series of different temperatures: 0 °C, 10 °C, 20 °C, 30 °C, 40 °C, 50 °C, 60 °C, 70 °C, 80 °C, and 90 °C. Temperatures of 10 °C, 20 °C, 30 °C, 40 °C, 50 °C, 60 °C, 70 °C, 80 °C, and 90 °C were controlled using a constant temperature incubator, while 0 °C was controlled using an ice-water bath. The incubation time was 30 min.

[0060] (3) After the incubation is completed, the test tube should be removed immediately to terminate the temperature treatment process.

[0061] (4) The titer of phages treated at different temperatures was determined using the double-layer agar plate method. The formation of phage plaques was observed. The number of phage plaques was calculated, and the titer of the phages was determined.

[0062] The results are as follows Figure 4 As shown, the titer of bacteriophage *Escherichia coli* phage XHX-E-025P remained stable within the temperature range of 10-50 °C, with no significant change. At 60 °C, the titer decreased slightly, but the magnitude was small. This indicates that *Escherichia coli* phage XHX-E-025P exhibits good temperature stability.

[0063] Example 5: One-step growth curve of Escherichia coli phage XHX-E-025P (1) Take an appropriate amount of host bacterial culture in the logarithmic growth phase and adjust its concentration to 10. 8 Approximately CFU / mL. Aliquot the host bacterial culture into multiple 2 ml test tubes, 500 μl per tube.

[0064] (2) Take 500 μl of phage suspension as 10 4 PFU / mL was added to the host bacterial culture to make the ratio of bacteriophage to host bacteria 1:10000. The mixture was incubated at 37 °C for 15 min to allow the bacteriophage to fully adsorb onto the host bacteria.

[0065] (3) Centrifuge the mixed bacterial culture at 4 °C and 8000 rpm for 5 min to remove unadsorbed phages. Wash the precipitated host bacteria with PBS buffer, and repeat the centrifugation and washing steps twice to ensure that all unadsorbed phages are removed.

[0066] (4) Resuspend the washed host bacteria precipitate in 10 ml of fresh LB medium and incubate the resuspended bacterial solution in a 37 °C water bath. Starting from 0 min, take a 0.5 mL sample every 10 min until the host bacteria are completely lysed. After taking the sample, immediately place the sample in an ice bath to cool it and terminate the reaction.

[0067] (5) The phage titer at each time point was determined using the double-layer agar plate method, and the formation of phage plaques was observed. The number of phage plaques at each time point was calculated to determine the phage titer.

[0068] (6) Based on the measured titer data, plot the curve of phage titer change over time. Analyze the latency, lysis period, and stationary period of the phage using the curve.

[0069] The results are as follows Figure 5 As shown, within 0-10 min of phage infection of the host, the phage titer is low and remains essentially unchanged; within 10-80 min, the phage titer gradually increases. Subsequently, within 80-120 min, the phage titer remains at a high level and almost unchanged. This indicates that the latency period of phage Escherichia coli phage XHX-E-025P is in the first 10 min, the lysis period is from 10 to 80 min, and the stationary period is after 80 min.

[0070] Example 6: Optimal Multiple of Infection for Escherichia coli phage XHX-E-025P (1) Take an appropriate amount of host bacterial culture in the logarithmic growth phase and adjust its concentration to 10. 6 Approximately CFU / mL. The host bacterial culture was aliquoted into multiple test tubes containing 5ml LB broth, 100μl per tube.

[0071] (2) Take an appropriate amount of phage suspension and dilute it with LB broth to different concentrations, corresponding to different MOI values. The MOI values ​​are 0.000001, 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100.

[0072] (3) Add phage suspensions of different concentrations to the host bacterial culture, so that the ratio of phage to host bacteria in each test tube is the set MOI value.

[0073] (4) Incubate at 37 °C with shaking for 4 h.

[0074] (5) The titer of phage in each tube was determined by the double-layer agar plate method.

[0075] (6) Based on the measured titer data, plot the curves of phage titer change over time under different MOI conditions. Analyze the phage proliferation under different MOI conditions and determine the optimal MOI value.

[0076] The results are as follows Figure 6 As shown, under the condition of MOI=0.000001, the titer of bacteriophage Escherichia coli phage XHX-E-025P reached its highest level (5.0 × 10⁻⁶). 9 PFU / ml).

[0077] Example 7: Host profile analysis of Escherichia coli phage XHX-E-025P (1) In this experiment, a total of 30 strains of Escherichia coli from different regions were selected and cultured to the logarithmic growth phase. 100 μl of bacterial solution was mixed with 5 mL of semi-solid solution, poured onto the surface of solid culture medium, shaken thoroughly, and cooled.

[0078] (2) Take 5 μl of the titer, which is 1X10. 8 A drop of PFU / mL phage solution was placed in the center of a semi-solid culture. After the liquid was completely absorbed, the culture was incubated overnight at 37 °C. Bacteria that could form plaques were marked as "+", indicating a positive result, and those that could not were marked as "-", indicating a negative result. The results are summarized in Tables 1 and 2.

[0079] Table 1 Host spectrum analysis of Escherichia coli phage XHX-E-025P 2 Hubei + 17 Shandong + 3 Hubei + 18 Shanxi + 4 Hubei + 19 Guangxi + 5 Hubei + 20 Guangxi + 6 Henan + 21 Guangxi + 7 Jiangsu + 22 Jiangxi + 8 Jiangsu + 23 Shanxi - 9 Shandong + 24 Fujian + 10 Shandong + 25 Liaoning - 11 Shandong + 26 Sichuan - 12 Shanxi + 27 Liaoning + 13 Fujian + 28 Guizhou + 14 Fujian - 29 Liaoning + 15 Fujian + 30 Guangdong + Table 2. Analysis of Salmonella lysates from Escherichia coli phage XHX-E-025P. 2 Sichuan + 11 Anhui - 3 Sichuan - 12 Sichuan + 4 Sichuan + 13 Sichuan + 5 Sichuan + 14 Sichuan + 6 Sichuan + 15 Anhui + 7 Sichuan + 16 Shandong + 8 Sichuan + 17 Shandong + 9 Anhui + 18 Shandong + Table 1 shows that the Escherichia coli phage XHX-E-025P lyses 26 strains of Escherichia coli. The strains in this experiment were all collected from diseased materials from different farms, and the lysis rate reached 86%, indicating that the lysis spectrum of the phage Escherichia coli phage XHX-E-025P is relatively broad.

[0080] Table 2 shows that the Escherichia coli phage XHX-E-025P has a lytic effect on 16 Salmonella strains. The strains in this experiment were all collected from diseased materials from different farms, and the lysis rate reached 88%, indicating that the phage Escherichia coli phage XHX-E-025P has a wide lysis spectrum.

[0081] Example 8: The therapeutic effect of Escherichia coli phage XHX-E-025P on diarrhea in piglets. Forty-two 4-day-old piglets with diarrhea from a pig farm in Yancheng, Jiangsu Province, belonging to a certain group company, were randomly divided into a control group and an Escherichia coli phage oral administration test group. The oral administration test group was given Escherichia coli phage XHX-E-025P orally daily, 10ml per piglet (1×10⁻⁶ ml). 6 The control group received the same dose of normal saline orally for three consecutive days (PFU / ml) and the cure rate was calculated. The results are summarized in Table 3.

[0082] Table 3. The therapeutic effects of Escherichia coli bacteriophage on piglet diarrhea.

[0083] Table 3 shows that the phage Escherichia coli phage XHX-E-025P achieved a cure rate of 76.2% in piglets with diarrhea, demonstrating significant efficacy.

[0084] Example 9: Lytic activity experiment of Escherichia coli phage XHX-E-025P against multidrug-resistant strains This experiment selected 30 multidrug-resistant Escherichia coli strains and 18 multidrug-resistant Salmonella strains from different regions and conducted lysis experiments. The drug-resistant strains were resistant to most of 13 drugs, including cefotaxime, ciprofloxacin, gentamicin, and tetracycline. Comparison with the lysis results of this bacteriophage showed that it maintained high lysis activity against both multidrug-resistant Escherichia coli and multidrug-resistant Salmonella strains. The results are summarized in Tables 4-5.

[0085] Table 4. Analysis of drug resistance lysis results of 30 strains of Escherichia coli

[0086] Table 5. Analysis of drug resistance lysis results of 18 Salmonella strains

[0087] Example 10: Whole genome sequencing and evolutionary analysis of Escherichia coli phage XHX-E-025P Genomic DNA was extracted from *E. coli* bacteriophages using a phage genomic DNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd.). Genome sequencing was performed by Qingdao Qingke Co., Ltd. (Qingdao, China) using an Illumina HiSeq 2500 sequencer. Sequencing was then performed, and the genome was assembled using SPAdes genome assembly software (v3.11.1) and A5-miseq (v20160825). The d1011321 phage read length was 42291 bp; the average sequencing depth was 54.8-fold. Phage characteristics were predicted using Phabox (v2.1.13).

[0088] Possible protein-coding sequences (CDS) were predicted using bakta_v1.11.4 software. CDS prediction was performed using Prodigal (v2.6.3); tRNA and rRNA genes were identified using tRNAscan-SE (v2.0.11) and Infernal (v1.1.5) based on the Rfam (v14.9) database, respectively. Functional annotation was performed by alignment with the UniProtKB (release 2024_01), NCBI RefSeq, and Pfam (v36.0) databases.

[0089] To identify homologous sequences for phylogenetic analysis, target nucleotide sequences were used as query sequences. BLASTn (v2.13.0) was used to search the NCBI non-redundant nucleotide (nr / nt) database, with an E-value threshold of 1e-5 and a minimum query coverage of 70%. Sequences with nucleotide identity ≥ 80% were selected for subsequent analysis. All statistical analyses and phylogenetic reconstructions were performed in R software (v4.3.1), using the following R packages: ape (v5.7), phangorn (v2.11), seqinr (v4.2), ggtree (v3.10), ggplot2 (v3.4), reshape2 (v1.4), and rentrez (v1.2).

[0090] Homologous nucleotide sequences obtained from the NCBI nr / nt database were searched using BLASTn and imported and processed using the seqinr package. Multiple sequence alignment was performed using the msa or DECIPHER package. The phylogenetic tree was constructed using maximum likelihood (ML) with the phangorn package, and the optimal nucleotide substitution model was automatically selected based on the Akaike Information Criterion (AIC) using the phangorn package. Node support was evaluated using 1,000 bootstrap replicates. The final phylogenetic tree was visualized, annotated, and beautified using the ggtree package, and further graphically adjusted using ggplot2. The reshape2 package was used for data reconstruction before plotting related heatmaps or multiple combination plots.

[0091] Based on second-generation whole-genome sequencing analysis, we extracted the complete genome information of the *Escherichia coli* phage *XHX-E-025P*. By assembling the contig sequences, we determined the *Escherichia coli* phage *XHX-E-025P* genome to be 42291 bp in length. Phanbox analysis identified it as a lytic phage. Protein annotation tools revealed the presence of multiple Tail proteins with strong binding capabilities to bacterial membrane proteins or lipopolysaccharides. The phage genome is 42291 bp in length, with a GC content of 48.09%, and 319 ORFs were extracted.

[0092] To determine its specificity, we compared it with databases such as NCBI and then constructed its phylogenetic tree using meta-analysis software and the online function of itol. The results are as follows: Figure 7 As shown, the *Escherichia coli* phage XHX-E-025P occupies an independent branch position within the *E. coli* phage population, indicating a distant phylogenetic relationship with known *E. coli* phages. This suggests that the phage is unique within the *E. coli* phage population and belongs to a novel phage strain. This result further supports the uniqueness of the phage's host recognition mechanism, consistent with its ability to lyse *Salmonella* across genera.

[0093] It should be noted that, in order to facilitate an accurate understanding of the known meanings of the various English abbreviations in this invention document, the following explanations are provided in this invention document, specifically referring to the English abbreviations and their Chinese meanings comparison table shown in Table 6.

[0094] Table 6: Comparison of English Abbreviations and Chinese Meanings 1 Escherichia coli phage XHX-E-025P Escherichia coli bacteriophage XHX-E-25P 2 CCTCC China Center for Type Culture Collection 3 NO. serial number 4 M Microbial preservation classification code (CCTCC preservation number prefix) 5 SEQ ID NO.1 Sequence number 1 (amino acid sequence) 7 PBS Phosphate buffer 8 1×PBS 1x working concentration phosphate buffer 9 10×PBS 10x concentrated phosphate buffer 10 LB LB medium (Luria-Bertani medium) 11 LB agar medium LB agar medium (solid) 12 LB Broth LB liquid culture medium 13 LB nutrient agar LB nutrient agar (solid) 14 0.6% LB agar 0.6% LB semi-solid agar medium 40 ORFs ORF refers to a continuous sequence of nucleotides in DNA or RNA that potentially encodes a protein, starting from the start codon and ending at the stop codon. 41 CDS The specific nucleotide sequence of the amino acid sequence in a protein 42 tRNA transfer RNA 43 rRNA Ribosomal RNA 44 E value In a purely random scenario, what is the expected number of times that the sequence you are currently comparing will produce a match "no worse than the current score" with sequences in the target database? 45 ML ML is a statistical algorithm used to find, among many possible phylogenetic trees, the tree that maximizes the probability (likelihood) of the currently observed sequence data (such as the DNA or protein sequence you have). 46 AIC AIC = -2 × (logarithm of maximum likelihood) + 2 × (number of model parameters) 47 read This refers to the number of bases contained in a single read. 48 contigs Assembly Fragments 49 genome Genome 50 Tail Protein Tail fibroin 51 Illumina HiSeq 2500 Whole genome sequencing instruments 52 SPAdes (v3.11.1) Virus / bacterial assembly tools 53 A5-miseq (v20160825) Virus / bacterial assembly tools 54 Phabox (v2.1.13) Life history prediction tools 55 bakta (v1.11.4) Comment tools 56 Prodigal (v2.6.3) Comment tools 57 tRNAscan-SE (v2.0.11) tRNAscan-SE is a bioinformatics software specifically designed for identifying and annotating transfer RNA (tRNA) genes in genomic sequences. 58 Infernal (v1.1.5) Unlike conventional sequence alignment tools (such as BLAST), Infernal is based on a model called covariance. 59 Rfam (v14.9) Rfam is a comprehensive database dedicated to storing information on non-coding RNA (ncRNA) families, and is one of the most essential public resources in this field. 60 UniProtKB (release 2024_01) Protein Database 61 NCBI RefSeq American Bioinformatics Database 62 Pfam (v36.0) Rfam is a comprehensive database dedicated to storing information on non-coding RNA (ncRNA) families, and is one of the most essential public resources in this field. 63 NCBI nr / nt Sequence alignment database 64 BLASTn (v2.13.0) Sequence alignment tools 65 R (v4.3.1) R language 66 ape (v5.7) Phylogenetic and Evolutionary Analysis R Package 67 phangorn (v2.11) Phylogenetic and Evolutionary Analysis R Package 68 seqinr (v4.2) Phylogenetic and Evolutionary Analysis R Package 69 ggtree (v3.10) Phylogenetic and Evolutionary Analysis R Package 70 ggplot2 (v3.4) Plotting R package 71 reshape2 (v1.4) Data processing R packages 72 rentrez (v1.2) Data processing R packages 73 msa Data processing R packages 74 DECIPHER Data processing R packages 75 modelTest() Data processing R packages 76 meta Data processing R packages 77 itol Evolutionary Tree Drawing Tool The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An Escherichia coli bacteriophage that lyses Escherichia coli and Salmonella, characterized in that, Named *Escherichia coli* bacteriophage XHX-E-025P, it is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2025497, and classified as *Escherichia coli* bacteriophage (…). Escherichia coli phage ).

2. The Escherichia coli bacteriophage lysing Escherichia coli and Salmonella according to claim 1, characterized in that, Its tail protein has the amino acid sequence shown in SEQ ID NO.

1.

3. The use of an Escherichia coli bacteriophage according to claim 1 or 2 in the preparation of a medicament for the prevention or treatment of Escherichia coli or Salmonella infection.

4. The use of an Escherichia coli bacteriophage according to claim 1 or 2 in the preparation of feed or feed additives.

5. The application of an Escherichia coli bacteriophage according to claim 1 or 2 in the preparation of an environmental disinfectant.

6. A feed additive, characterized in that, Includes the Escherichia coli bacteriophage as described in claim 1 or 2.

7. An antibacterial preparation, characterized in that, Includes the Escherichia coli bacteriophage as described in claim 1 or 2 and pharmaceutically or veterinarily acceptable excipients.

8. The antibacterial agent according to claim 7, characterized in that, The antibacterial preparation is available in the form of oral administration solution, spray solution, or injection solution.

9. An environmental disinfectant, characterized in that, Includes the Escherichia coli bacteriophage as described in claim 1 or 2.

10. A feed composition, characterized in that, Includes basic feed and the feed additives as described in claim 6.

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