An Efficient Escherichia coli Bacteriophage Lysis and Its Application

Abstract

The present invention relates to an efficient Escherichia coli phage and its application. The phage is named LYE-01, and its taxonomic name is Escherichia coliphage. The preservation number is CGMCC No. 46095, the preservation date is August 7, 2024, and the preservation institution is the General Microbiology Center of the China Committee for Culture Collection of Microorganisms. The present invention provides a phage LYE-01 of Escherichia coli. The optimal multiplicity of infection of this phage is 1. It has strong acid resistance and thermal stability. Under the conditions of pH value 4-11, temperature 10°C-50°C, and ultraviolet irradiation within 20 minutes, this phage can maintain a high titer. The one-step growth curve experiment shows that the latent period of this phage infecting Escherichia coli is about 10 minutes, the burst period is about 40 minutes thereafter, and the stable plateau period is after 50 minutes of phage infection.

Description

Technical Field

[0001] The present invention belongs to the field of microorganism screening, in particular to a highly efficient lysis Escherichia coli phage, a preparation and an anti-Escherichia coli application. Background Art

[0002] Manure is an important fertilizer resource. After processing, it can be used as a soil conditioner and humus supplement, which can be applied to the soil to increase crop yields. However, in addition to being rich in nutrients such as organic matter, nitrogen, and phosphorus, manure also contains a large number of pathogens, making it a significant source of environmental pollution and public health problems. For example, the COVID-19 virus that has broken out in recent years can be transmitted through the fecal-oral route, posing a potential risk of infection to humans.

[0003] Escherichia coli (E. coli), a typical opportunistic pathogen in feces, can, under certain conditions, cause digestive tract infections, urinary tract infections, and sepsis in humans and animals. It is a major zoonotic pathogen, posing a significant threat to human health and the livestock industry. Furthermore, E. coli is a significant cause of diarrhea in infants and young children in developing countries and travelers' diarrhea in developed countries, and is one of the leading causes of death in children under five years of age in developing countries. Therefore, the control and treatment of E. coli remains a crucial public health issue.

[0004] In 2019, antibiotic resistance in six pathogens (Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa) caused over 1.27 million deaths, with E. coli causing the most deaths. Antibiotics are increasingly limiting the treatment of E. coli, and the increasing prevalence of resistance is an urgent issue that demands additional treatment options to curb the spread of resistance. Consequently, increasing interest in alternative antibiotic therapies is being pursued, with a focus on discovering green, safe, and efficient treatment technologies to mitigate the development of bacterial resistance and thus reduce harm to the environment and human health.

[0005] In this context, phage therapy provides a new countermeasure. Bacteriophages (phages) are a class of viruses that specifically infect and lyse bacteria and are considered a potential alternative to antibiotics. Unlike antibiotics, phages are highly host-specific, meaning they can precisely target and kill specific bacterial strains with minimal impact on the body's normal flora or other beneficial microorganisms. With the frequent emergence of drug-resistant pathogens, the use of phages to treat bacterial infections has gradually attracted people's attention.

[0006] At present, although there have been reports of using phage therapy to clinically treat Escherichia coli infections, in the fields of environmental governance and waste resource utilization, phages, as an ancient yet new force, have become a research hotspot for specifically killing drug-resistant pathogenic bacteria. Summary of the Invention

[0007] In view of this, the purpose of the present invention is to provide a highly efficient Escherichia coli-lysing phage and its application. The phage is a lytic phage that specifically infects Escherichia coli. Through the study of the biological characteristics, morphology, and whole-genome analysis of this phage, it is found that as a new potential therapeutic agent, this phage has broad application prospects in the field of fecal sewage harmless treatment.

[0008] To achieve the above purpose, the present invention provides the following technical solutions:

[0009] A highly efficient Escherichia coli-lysing phage, named LYE-01, classified as Escherichia coliphage, with a preservation number of CGMCC No. 46095, preservation date: August 7, 2024, preservation institution: General Microbiology Center, China Microbial Culture Collection Center, preservation address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0010] A microbial preparation, including the phage named LYE-01.

[0011] A bactericide or disinfectant, including the phage named LYE-01 or its microbial preparation.

[0012] Application of the phage preparation named LYE-01 in the preparation of products for inhibiting or killing Escherichia coli. The phage preparation against Escherichia coli is used for treating Escherichia coli infections in fecal sewage environments, including applications in treating toilet fecal sewage or livestock and poultry fecal sewage.

[0013] The titer of LYE-01 in the phage preparation is above 1×10 6 PFU / mL.

[0014] The applicable pH value of the phage is 4 - 11, and the applicable temperature of the phage is 10℃ - 70℃.

[0015] Application of the phage named LYE-01 in the preparation of an agent against Escherichia coli.

[0016] The agent against Escherichia coli is a drug for treating Escherichia coli infections, or the agent against Escherichia coli is used for treating toilet fecal sewage or livestock and poultry fecal sewage preparations.

[0017] An Escherichia coli-resistant preparation, characterized in that the active ingredient includes a phage named LYE-01.

[0018] The present invention has the following beneficial effects:

[0019] The present invention provides a phage LYE-01 of Escherichia coli. The optimal multiplicity of infection of this phage is 1. It has strong acid resistance and thermal stability. Under the conditions of pH value 4 - 11, temperature 10°C - 50°C, and ultraviolet irradiation within 20 min, this phage can maintain a high titer. The one-step growth curve test shows that the latent period of this phage infecting Escherichia coli is about 10 min, the burst period is about 40 min thereafter, and the stable plateau period is after 50 min of phage infection.

[0020] According to the classification of the International Committee on Taxonomy of Viruses (ICTV) and the morphology of phage LYE-01, phage LYE-01 is officially classified as a member of the family Straboviridae. The discovery of this phage fills the gap in the species resources of Escherichia coli phages. The whole-genome sequencing results show that the full genome length of phage LYE-01 is 165,614 bp. The base contents of genomic A, C, G, and T are 32.61%, 16.73%, 18.8%, and 31.86% respectively, and the overall GC content is 35.52%.

[0021] According to the phage genome map and genome annotation, LYE-01 has 264 coding sequences (CDSs). 119 ORFs are predicted to be hypothetical proteins, and 145 ORFs are predicted to be functional proteins, mainly involved in phage structure, host cell lysis, progeny DNA replication, and phage metabolism and other functions. In addition, the phage LYE-01 genome does not contain virulence genes and antibiotic resistance genes, and has good biosafety. Description of the Drawings

[0022] Figure 1 For the morphological characteristics of phage LYE-01; among them, Figure A is a plate picture of the isolation of phage LYE-01 by the spot method; Figure B is a plate picture of the purification of phage LYE-01 by the double-layer plate method; Figure C is a transmission electron microscope photograph of phage LYE-01;

[0023] Figure 2Biological characteristics of phage LYE-01; among them, Figure A is a bar chart of the titer of phage LYE-01 at different multiplicity of infection; Figure B is the one-step growth curve of phage LYE-01; Figure C is a bar chart of the titer of phage LYE-01 after incubation at different pH values; Figure D is a bar chart of the titer of phage LYE-01 in an ultraviolet irradiation environment at 254 nm; Figure E is a bar chart of the titer of phage LYE-01 after incubation at different temperatures;

[0024] Figure 3 Genomic map of phage LYE-01;

[0025] Figure 4 Proteomic relationship between phage LYE-01 and other related phages;

[0026] Figure 5 In vitro antibacterial plate picture of phage LYE-01. Detailed implementation mode

[0027] The following further illustrates the present invention in conjunction with embodiments. The following embodiments are narrative and not restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0028] The present invention provides a highly efficient Escherichia coli phage named LYE-01, which is classified as Escherichia coliphage. It was deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee on August 7, 2024. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC No. 46095.

[0029] The present invention isolated and purified LYE-01 from pig manure samples from a small pig farm in Dongli District, Tianjin, China. The optimal multiplicity of infection of this phage is 1, and it has strong acid resistance and thermal stability. Under the conditions of pH value 4 - 11, temperature 10°C - 50°C, and ultraviolet irradiation within 20 minutes, this phage can maintain a high titer. The one-step growth curve experiment shows that the latent period of this phage infecting Escherichia coli is about 10 minutes, the burst period is about 40 minutes thereafter, and the stable plateau period is after 50 minutes of phage infection. It shows that the time from the specific adsorption of phage to bacteria until the synthesis of the next generation in bacteria is short, and the replication efficiency of this phage is high. Generally speaking, compared with other types of phages reported in the current literature, this phage has a relatively short lysis cycle and can be better applied to the harmless treatment environment of manure.

[0030] The results of whole-genome sequencing showed that the whole genome of phage LYE-01 was 165,614 bp in length. The base contents of genomic A, C, G, and T were 32.61%, 16.73%, 18.8%, and 31.86%, respectively, and the overall GC content was 35.52%. According to the phage genome map and genome annotation, LYE-01 had 264 coding sequences (CDSs), 119 ORFs were predicted to be hypothetical proteins, and 145 ORFs were predicted to be functional proteins, mainly involved in phage structure, host cell lysis, progeny DNA replication, and phage metabolism and other functions. In addition, the genome of phage LYE-01 did not contain virulence genes and antibiotic resistance genes, showing good biosafety. In the NCBI database, BLAST was used to compare and analyze the similarity between LYE-01 and other phages. The results showed that phage MLF4 (GenBank accession number: NC_055780.1) had the highest similarity with LYE-01, with a homology of 95.06%. Compared with the closest phage MLF4, LYE-01 could be identified as an existing species of the genera Duplodnaviria, Heungongvirae, Uroviricota, Caudoviricetes, Straboviridae, Tevenvirinae, and Tequatrovirus. Using the VipTree platform for whole-sequence analysis of the proteome sequence of Escherichia coli phage LYE-01, 1090 prokaryotic dsDNA virus proteome sequences were identified, of which 234 (21.5%) belonged to Straboviridae ( Figure 4 ). Further analysis of 194 Escherichia coli phages in the database found only 86 Straboviridae phages (including Escherichia coli phage LYE-01). Therefore, according to the classification of the International Committee on Taxonomy of Viruses (ICTV) and the morphology of phage LYE-01, phage LYE-01 was officially classified as a member of the family Straboviridae. The discovery of the phage filled the gap in the species resources of Escherichia coli phages.

[0031] The culturing method of LYE-01 of the present invention includes:

[0032] The phages lysing Escherichia coli in the samples were preliminarily screened by the drop method. The clear spots were selected and purified multiple times by the double-layer plate method to ensure that the isolated phages had high specificity and high activity. The purified phages were proliferated in a liquid medium rich in Escherichia coli by the liquid proliferation method. The phage enrichment solution was stored in SM buffer (5.8 g / L sodium chloride, 2.0 g / L magnesium sulfate, 50 mL / L 1M Tris-Cl buffer, pH = 7.5) and refrigerated at 4 °C.

[0033] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments.

[0034] In the following embodiments, the preparation method of the LB liquid medium is as follows: accurately weigh 10 g of tryptone, 10 g of yeast extract, and 5 g of NaCl, dissolve them in 1000 mL of ultrapure water, accurately adjust the pH to 7.5, autoclave at 121 °C for 25 min, and store at 4 °C for later use.

[0035] The preparation method of the upper layer medium (0.75% agar) is as follows: accurately weigh 10 g of tryptone, 10 g of yeast extract, 5 g of NaCl, and 7.5 g of agar powder, dissolve them in 1000 mL of ultrapure water, and autoclave at 121 °C for 25 min.

[0036] The preparation method of the lower layer medium (1.5% agar) is as follows: accurately weigh 10 g of tryptone, 10 g of yeast extract, 5 g of NaCl, and 15 g of agar powder, dissolve them in 1000 mL of ultrapure water, autoclave at 121 °C for 25 min, and pour it into a 90 mm sterile petri dish after cooling to about 50 °C to make the bottom plate.

[0037] The preparation method of the SM buffer is as follows: accurately weigh 5.8 g of NaCl, 2 g of MgSO4·7H2O, and 0.1 g of gelatin.

[0038] 50 mL of 1 mol / L Tris-HCl with pH = 7.5, make up the volume to 1 L with ultrapure water, shake well until fully dissolved, autoclave at 121 °C for 25 min, and store at 4 °C for later use.

[0039] Example 1

[0040] Isolation and purification of phage LYE-01

[0041] The sewage sample was collected from the pig manure sample of a small pig farm in Dongli District, Tianjin, China. Take 50 mL of the pig manure sample, filter it with gauze, add CaCl2 to make its concentration 1 mmol / L, dispense it into 50 mL centrifuge tubes, centrifuge at 4500 r / min for 10 min, pass the supernatant through a 0.22 μm bacterial filter membrane, discard the precipitate, and transfer it to a 300 mL conical flask.

[0042] Isolation of phages: Use the spot method to screen phages against Escherichia coli from the sewage sample. The purification result is as shown in Figure 1 Figure A in it, where circular transparent plaques can be clearly observed.

[0043] Purification of phages: The double-layer plate method was used for the isolation and purification of phages. The isolated phages were purified 6 - 8 times to form phage plaques with uniform size and morphology on the plate. Use a 20 μL pipette tip to pick one phage plaque with obvious differences in size and morphology each, dissolve it into a centrifuge tube containing 1 mL of sterilized SM buffer, gently shake it, and place it at 4°C overnight to allow the phages to be fully released into SM; centrifuge at 10000 rpm at 4°C for 5 min, collect the supernatant, pass it through a 0.22 μm bacterial filter membrane, and perform gradient dilution of the phage filtrate to 10 -9 , double-layer culture, repeat 6 - 8 times. When the morphology and size of the phage plaques that appear are relatively uniform, the purified phage LYE-01 is obtained. As shown in Figure 1 Figure B in it, where phage plaques with uniform size and morphology can be clearly observed.

[0044] Proliferation of phages: The liquid proliferation method was used for the proliferation of phages. The phage stock solution was added to the host bacterial solution cultured for 6 - 7 h at a ratio of 1:10, and then cultured in a shaker at 37°C for 3 - 4 h. The mixed solution was centrifuged at 8000 rpm at 4°C for 5 min, and the supernatant was collected, which is the phage proliferation solution.

[0045] Storage of phages: The phage enrichment solution was stored in SM buffer (5.8 g / L sodium chloride, 2.0 g / L magnesium sulfate, 50 mL / L 1M Tris-Cl buffer, pH = 7.5) and refrigerated at 4°C.

[0046] Example 2

[0047] Biological characteristics of phages

[0048] (1) Morphological observation of phages

[0049] The morphology of the phage was observed by transmission electron microscopy. 10 μL of the above phage preservation solution was dropped onto a copper mesh and allowed to precipitate for 1 min. After standing at room temperature for 10 min, the excess liquid was blotted with filter paper. It was stained with 2% phosphotungstic acid staining solution for 2 min, and the excess staining solution was blotted with filter paper and air-dried at room temperature for 20 min. The morphology of the phage was observed at an accelerating voltage of 80 - 120 kV in the HT7800 transmission electron microscope. The Nano measurer 1.2 software was used to statistically analyze the dimensions such as the length and diameter of the phage particles.

[0050] The electron micrograph of the phage is as shown in Figure 1 Figure C in []. The phage LYE-01 is in a "tadpole" shape, and its head (diameter 88 ± 4 nm) is icosahedral. It has a contractile long tail with a length of 109 ± 4 nm, showing helical symmetry, and also has tail fibers and tail pins. Based on the classification of the International Committee on Taxonomy of Viruses (ICTV), the phage is tentatively classified as a member of the family Myoviridae.

[0051] (2) Identification of the host range of the phage

[0052] Eleven potential host bacteria were selected for the determination of the phage host spectrum, among which 3 were Escherichia coli. After inoculating the potential host bacteria in LB medium and culturing overnight, the phage host spectrum was tested by the spot method: 50 μL of the above bacterial solution was spread on a double-layer LB agar plate in sequence, and allowed to stand at room temperature for 15 min. Then, 1.5 μL of the phage preservation solution was pipetted onto the double-layer LB agar plate containing the bacterial solution, and 5 drops were evenly dropped. It was cultured at 37 °C for 20 min. After the phage filtrate was completely absorbed, it was inverted. The experiment was repeated three times, and whether plaques appeared on the medium after culturing overnight was observed, using the double-layer agar plate without dropping the phage filtrate as a blank control.

[0053] In the experiments of infecting common drug-resistant pathogenic bacteria in fecal pollution such as Escherichia coli, Salmonella, and Enterococcus faecalis, it was found that the phage only had a lysis effect on Escherichia coli and had no killing effect on other bacterial strains (Table 1).

[0054] Table 1 Determination of the host spectrum of phage LYE-01

[0055] Bacterial name Lysis ability Escherichia coli (Migula) Castellani and Chalmers BNCC332831 + Escherichia coli (Migula) Castellani and Chalmers BNCC305635 + Escherichia coli - Enterococcus faecalis BNCC102668 - Halomonas ST1 - Halomonas ST5 - Halovibrio ST17 - Salmonella - Halomonas ST9 - Halomonas ST13 - Halomonas Y32 -

[0056] + indicates having a lysis effect on it; - indicates having no lysis effect on it

[0057] (3) Detection of the optimal multiplicity of infection of the phage

[0058] Determination of the concentration of host bacteria: Take a single colony of Escherichia coli and inoculate it into 50 mL of LB liquid medium. Incubate it at 37 °C with shaking at 160 r / min until the early logarithmic growth phase. Using the LB liquid medium as the blank control group, measure the OD of the host bacteria at a wavelength of 600 nm using an ultraviolet spectrophotometer. 600 When the value ≈ 0.2, serially dilute the bacterial solution by a factor of 10. Take 0.1 mL of the diluted solution at different dilution factors and evenly spread it on the LB solid plate. Incubate it at 37 °C overnight. Count the colonies on the plate the next day. Bacterial concentration = number of colonies on the plate × dilution factor × 10 (CFU / mL).

[0059] Preparation of phage solutions with different concentrations: Take a single plaque and inoculate it into 50 mL of the host bacterial solution in the logarithmic growth phase. Incubate it at 37 °C with shaking until the liquid becomes clear. Centrifuge at 10000 r / min for 10 min, take the supernatant, and serially dilute it 10-fold with LB liquid medium. Take 100 μL from each dilution and measure the phage titer by the double-layer plate method. Store the supernatant at 4 °C for later use. Observe and count the results the next day. Phage titer = number of plaques × dilution factor × 10 (PFU / mL).

[0060] Mix the phage solution and the host bacterial solution according to the multiplicity of infection (MOI) ratios of 0.001, 0.01, 0.1, 1, 10, and 100 respectively, and then add LB liquid medium to make the total volume of different proportion culture systems the same. Incubate at 37 °C with shaking at 160 r / min overnight. Take 1 mL and centrifuge at 10000 r / min for 10 min. Take the supernatant and serially dilute it 10-fold. Spread the double-layer plate and incubate it at 37 °C overnight to measure the phage titer. At the same time, set up a control group with phage without adding host bacteria and host bacteria without adding phage.

[0061] The results are as shown in Figure 2 Figure A in. After measurement, the optimal multiplicity of infection of the phage is 1 PFU / CFU. Therefore, we use MOI = 1 as the standard dose in subsequent experiments.

[0062] (4) One-step growth curve of the phage

[0063] Add the phage filtrate and the fresh cultured Escherichia coli bacterial solution with OD 600 = 0.6 to a sterile test tube according to the optimal MOI. Incubate at 37 °C with shaking at 160 r / min. Take out 500 μL at 0, 10, 20, 30, 40, 50, 60, 70, 80, and 90 min respectively, centrifuge at 10000 r / min for 5 min, take 100 μL of the supernatant, make serial 10-fold dilutions, and measure the phage titer.

[0064] The one-step growth curve of phage LYE-01 is as shown in Figure 2As shown in Figure B, the results show that the latency period is about 10 minutes, and the burst period is about 40 minutes thereafter. The stable plateau period occurs 50 minutes after phage infection. Phages have a relatively short lysis cycle, and this excellent biological characteristic is of great value for the rapid removal of pathogenic bacteria in fecal sewage.

[0065] (5) Determination of the tolerance of phages to pH, ultraviolet light, and temperature

[0066] Using 1 mol / L NaOH and HCl solutions, adjust the pH value of the SM buffer to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 respectively. Take 100 μL of the phage filtrate and add it to 900 μL of the SM buffer, mix well in a 1 mL centrifuge tube, set up two additional parallel groups for each set, after water bath at 37 °C for 1 h, perform 10-fold serial dilutions, and determine the titer by the double-layer agar plate method.

[0067] Add 2 ml of the phage filtrate (titer 1×10 7 PFU / mL) to a sterile petri dish, expose it to ultraviolet light in a biosafety cabinet, take samples at different treatment times (5, 10, 15, 25, 30 min) respectively, and determine the titer of the phage by the double-layer plate method.

[0068] Take 500 μL of the phage filtrate in a sterile centrifuge tube, set up multiple groups, and place them in a constant temperature water bath at 10, 30, 50, 70, and 90 °C respectively. Set up two additional parallel groups for each set and incubate for 1 h. Using Escherichia coli (OD 600 = 0.6 - 0.8) as the host, determine the titer of the phage by the double-layer plate method.

[0069] The pH stability test results of phage LYE-01 are as shown in Figure 2 Figure C. Within the pH range of 4 - 11, the phage can maintain a relatively high titer. As the pH approaches the extreme values of decrease and increase, the phage will be completely inactivated.

[0070] The ultraviolet stability test results of phage LYE-01 are as shown in Figure 2 Figure D. In the ultraviolet irradiation environment of 254 nm, the phage shows a gradually decreasing trend and is completely inactivated after 25 minutes of irradiation.

[0071] The temperature stability test results of phage LYE-01 are as shown in Figure 2 Figure E. When the phage is incubated in the environment of 10 - 50 °C for 1 h, the titer of the phage can be stably maintained at about 10 10 PFU / mL and is basically not affected. However, under the treatment at temperatures above 70 °C, the titer of the phage drops sharply until it is completely inactivated at 90 °C.

[0072] Example 3

[0073] Whole-genome analysis of phage

[0074] Genomic DNA was extracted from phage LYE-01 using a λ phage genomic DNA extraction kit (PEG precipitation method). Take 12 mL of freshly lysed cell suspension treated with 0.5% chloroform, and centrifuge at 8000 g for 5 min to remove host bacterial debris. Filter the supernatant containing most of the virus particles through a 0.22 μm syringe filter to remove other impurities. Add RNaseA and DNaseI to 1 mL of TE buffer respectively, and gently pipette to mix evenly. After complete dissolution, aliquot and store at -20 °C according to the usage amount each time. Add 0.5 μL of RNaseA and 1 μL of DNase I to 1 mL of phage concentrate, mix well, and incubate at 37 °C for 30 min. Add 400 μL of phage precipitation solution to the supernatant, shake well until completely dissolved, and incubate overnight at 4 °C. Centrifuge at 10000 g in a refrigerated centrifuge at 4 °C for 20 min, and discard the supernatant. Add 1 mL of SM buffer, thoroughly wash the attachment on the tube wall and the precipitate at the bottom of the tube, then transfer to a new centrifuge tube, add 4 μL of phage lysate, and incubate at 68 °C for 15 min. Add an equal volume of protein scavenging solution, gently mix, centrifuge at 12000 g for 5 min, and take the supernatant. Add an appropriate amount of 70% ethanol solution, mix well, centrifuge at 8000 g at 4 °C for 8 min, and discard the supernatant. After drying the phage DNA at room temperature, add an appropriate amount of TE buffer and store at -20 °C.

[0075] The phage genomic DNA was sequenced on the Illumina HiSeq novaseq 6000 sequencing platform. Through processes such as sequencing quality control (fastq), genomic sequence splicing (Spades), and assembled sequence correction (PhageTerm), an accurate and complete phage genomic sequence was obtained. The obtained sequence was aligned with viral homologous sequences in the NCBI database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The complete annotated sequence of the phage genome was stored in the GenBank viral nucleotide database (accession number: PQ303785). The phage virus proteomics tree was drawn using the ViPTree platform (https: / / www.genome.jp / viptree). The circular map of the phage whole genome was drawn using CGView.

[0076] The full genome length of LYE-01 is 165,614 bp. The contents of bases A, C, G, and T in the genome are 32.61%, 16.73%, 18.8%, and 31.86% respectively, and the total GC content is 35.52%. According to the phage genome map and genome annotation, LYE-01 has 264 coding sequences (CDSs). The gene annotation is shown in Table 2, among which 119 ORFs are predicted to be hypothetical proteins, and 145 ORFs are predicted to be functional proteins, mainly involved in phage structure, host cell lysis, progeny DNA replication, and phage metabolism and other functions. In addition, the genome of phage LYE-01 does not contain virulence genes and antibiotic resistance genes, showing good biosafety. In the NCBI database, BLAST was used to compare and analyze the similarity between LYE-01 and other phages. The results showed that phage MLF4 (GenBank accession number: NC_055780.1) had the highest similarity with LYE-01, with a homology of 95.06%. Compared with the closest phage MLF4, LYE-01 can be identified as an existing species of the genera Duplodnaviria, Heungongvirae, Uroviricota, Caudoviricetes, Straboviridae, Tevenvirinae, and Tequatrovirus. Using the VipTree platform to perform a full-sequence analysis of the proteome sequence of Escherichia coli phage LYE-01, 1090 prokaryotic dsDNA virus proteome sequences were identified, of which 234 (21.5%) belonged to Straboviridae( Figure 4 ). Further analysis of 194 Escherichia coli phages in the database found only 86 Straboviridae phages (including Escherichia coli phage LYE-01). Therefore, according to the classification of the International Committee on Taxonomy of Viruses (ICTV) and the morphology of phage LYE-01, phage LYE-01 was officially classified as a member of the family Straboviridae. The discovery of this phage fills the gap in the species resources of Escherichia coli phages.

[0077] Table 2 Genome annotation of phage LYE-01

[0078]

[0079]

[0080]

[0081]

[0082] Example 4

[0083] Detection of bacteriostatic effect of phage LYE-01

[0084] Uniformly coat the host bacterium Escherichia coli on an LB agar plate, then take 5 μL of the phage LYE-01 solution and drop it on the LB agar plate coated with Escherichia coli, and incubate it overnight in an incubator at 37 °C. Observe whether there are plaques on the LB agar plate the next day.

[0085] The results are as Figure 5 shown. The circular transparent plaques indicate the plaques on Escherichia coli. The results show that the bacteriostatic effect of the single phage LYE-01 is good and no resistant strains appear.

Claims

1. An efficient Escherichia coli phage lysate, characterized in that: Named LYE-01, classified as Escherichia coli phage ( Escherichia phage ), with the deposit number CGMCC No. 46095, deposit date: August 7, 2024, deposit institution: China General Microbiological Culture Collection Center, deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

2. A microbial preparation, characterized in that: The bacteriophage according to claim 1.

3. A fungicide or disinfectant, characterized in that: The method comprises the LYE-01 according to claim 1 or the microbial preparation according to claim 2.

4. Use of the highly efficient Escherichia coli phage lysis as claimed in claim 1 in the preparation of a product for inhibiting or killing Escherichia coli ( Escherichia coli ).

5. Use of the highly efficient Escherichia coli lysing phage according to claim 1 in treating Escherichia coli infection in fecal sewage environment.

6. The application according to claim 5, characterized in that: The concentration of the phage is 1×10 6 PFU / mL or higher.

7. Use of the phage according to claim 1 in the preparation of a preparation against Escherichia coli ( Escherichia coli ).

8. An Escherichia coli-resistant preparation, characterized in that, The active ingredient comprises the bacteriophage LYE-01 according to claim 1.

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