Efficient splitting coliphage and application thereof

Abstract

The invention relates to an efficient splitting escherichia coli bacteriophage and application thereof, the bacteriophage is named as LYE-01 and classified as Escherichia coli bacteriophage, the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.46095, the preservation date is August 7, 2024, and the preservation institution is China General Microbiological Culture Collection Center. The invention provides a bacteriophage LYE-01 of escherichia coli, the optimal infection complex number of the bacteriophage is 1, the bacteriophage has very strong acid resistance and thermal stability, and the bacteriophage can keep high titer under the conditions that the pH value is 4-11, the temperature is 10-50 DEG C and the ultraviolet irradiation is within 20 minutes. A one-step growth curve test shows that the incubation period of escherichia coli infected by the bacteriophage is about 10 minutes, the outbreak period is about 40 minutes later, and the stable plateau period of the bacteriophage is obtained after the bacteriophage is infected for 50 minutes.

Description

Technical Field

[0001] The invention belongs to the field of microorganism screening, in particular to a highly efficient lysis of 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 and applied to the soil to increase crop yields. However, in addition to rich organic matter, nitrogen, phosphorus and other nutrients, manure also contains a large number of pathogens, becoming an important source of environmental pollution and public health problems. For example, the COVID-2019 virus that broke 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) is a typical conditional pathogen in feces. It can cause digestive tract infection, urinary tract infection and sepsis in humans or animals under certain conditions. It is an important zoonosis pathogen, which poses a great threat to human safety and animal husbandry. In addition, E. coli is an important pathogen of diarrhea in infants and young children in developing countries and travelers in developed countries, and is one of the main pathogens causing death in children under 5 years old in developing countries. Therefore, the control and treatment of E. coli has always been an important issue in the field of public health.

[0004] In 2019, antibiotic resistance caused by six pathogens (Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa) caused more than 1.27 million deaths, of which antibiotic resistance caused by Escherichia coli caused the most deaths. Antibiotics have gradually become limited in the treatment of Escherichia coli, and its increasing resistance is an urgent problem that needs to be addressed. Other treatment methods are needed to curb the spread of resistance. Therefore, more and more people are beginning to pay attention to alternative antibiotic therapies and are committed to discovering green, safe, and efficient treatment technologies to reduce the occurrence of bacterial resistance, thereby reducing harm to the environment and human health.

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

[0006] At present, although there have been reports of phage therapy being used in the clinical treatment of 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 pathogens. Summary of the invention

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

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

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

[0010] A microbial preparation comprising a bacteriophage named LYE-01.

[0011] A bactericide or disinfectant comprising a bacteriophage named LYE-01 or a microbial preparation thereof.

[0012] The invention relates to the use of a phage preparation named LYE-01 in the preparation of a product for inhibiting or killing Escherichia coli. The anti-Escherichia coli phage preparation is used for treating Escherichia coli infection in feces and sewage environments, including treating toilet feces or livestock and poultry feces.

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

[0014] The applicable pH value of the bacteriophage is 4-11, and the applicable temperature of the bacteriophage is 10°C to 70°C.

[0015] Use of a bacteriophage named LYE-01 in the preparation of an anti-Escherichia coli preparation.

[0016] The anti-Escherichia coli preparation is a medicine for treating Escherichia coli infection, or the anti-Escherichia coli preparation is used as a preparation for treating toilet waste or livestock and poultry waste.

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

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

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

[0020] According to the classification of the International Committee on Taxonomy of Viruses (ICTV) and the morphology of bacteriophage LYE-01, bacteriophage LYE-01 is officially classified as a member of the Straboviridae family. The discovery of this phage fills the gap in Escherichia coli phage species resources. The results of whole genome sequencing showed that the whole genome length of bacteriophage LYE-01 is 165,614bp. The A, C, G and T base contents of the genome 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 (CDS), 119 ORFs are predicted to be hypothetical proteins, and 145 ORFs are predicted to be functional proteins, which are mainly involved in phage structure, host cell lysis, offspring DNA replication, 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The morphological characteristics of bacteriophage LYE-01; Figure A is a plate picture of bacteriophage LYE-01 separated by the droplet method; Figure B is a plate picture of bacteriophage LYE-01 purified by the double-layer plate method; Figure C is a transmission electron microscope picture of bacteriophage LYE-01;

[0023] Figure 2The biological characteristics of bacteriophage LYE-01; Figure A is a titer bar graph of bacteriophage LYE-01 at different infection multiplicities; Figure B is a one-step growth curve of bacteriophage LYE-01; Figure C is a titer bar graph of bacteriophage LYE-01 after incubation at different pH values; Figure D is a titer bar graph of bacteriophage LYE-01 under 254nm ultraviolet irradiation; Figure E is a titer bar graph of bacteriophage LYE-01 after incubation at different temperatures;

[0024] Figure 3 is the genome map of bacteriophage LYE-01;

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

[0026] Figure 5 This is a picture of the in vitro antibacterial plate of bacteriophage LYE-01. DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0028] The present invention provides a highly efficient lysing Escherichia coli phage, the Escherichia coli phage is named LYE-01, classified as Escherichia coli phage (Escherichia coliphage), and is deposited in the General Microbiology Center of China National Committee for the Preservation of Microbiological Cultures on August 7, 2024, with a preservation address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and a preservation number of CGMCC No. 46095.

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

[0030] The results of whole genome sequencing showed that the whole genome of phage LYE-01 was 165,614bp in length. The A, C, G and T base contents of the genome 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 has 264 coding sequences (CDS), 119 ORFs are predicted to be hypothetical proteins, and 145 ORFs are predicted to be functional proteins, which are mainly involved in phage structure, host cell lysis, progeny DNA replication, phage metabolism and other functions. In addition, the genome of phage LYE-01 does not contain virulence genes and antibiotic resistance genes, and has good biosafety. In the NCBI database, BLAST was used to compare the similarity of LYE-01 with other phages. The results showed that bacteriophage MLF4 (GenBank ID: NC_055780.1) had the highest similarity with LYE-01, with a homology of 95.06%. Compared with the nearest bacteriophage MLF4, LYE-01 can be identified as a species of the existing genera Duplodnaviria, heungongvirae, Uroviricota, Caudoviricetes, Straboviridae, Tevenvirinae, and Tequatrovirus. The full sequence analysis of the Escherichia coli phage LYE-01 proteome sequence using the VipTree platform identified 1090 prokaryotic dsDNA virus proteome sequences, of which 234 (21.5%) belonged to the Straboviridae ( Figure 4 ). Further analysis of the 194 E. coli phages in the database revealed only 86 Straboviridae phages (including E. coli phage LYE-01). Therefore, based on the classification of the International Committee on Taxonomy of Viruses (ICTV) and the morphology of phage LYE-01, phage LYE-01 was formally classified as a member of the Straboviridae family. The discovery of the phage fills the gap in the resource of E. coli phage species.

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

[0032] The samples were preliminarily screened for phages capable of lysing E. coli by the spot method. Transparent 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 culture medium rich in E. 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 stored in a refrigerator at 4°C.

[0033] The technical solution provided by the present invention is described in detail below in conjunction with embodiments.

[0034] In the following examples, the LB liquid culture medium was prepared by accurately weighing 10 g of tryptone, 10 g of yeast powder and 5 g of NaCl, dissolving them in 1000 mL of ultrapure water, accurately adjusting the pH to 7.5, sterilizing by high pressure at 121° C. for 25 min, and storing at 4° C. for later use.

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

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

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

[0038] 1mol / L Tris-HCl (pH=7.5) 50mL, add ultrapure water to make up to 1L, shake until fully dissolved, sterilize at 121℃ high pressure for 25min, and store at 4℃ for later use.

[0039] Example 1

[0040] Isolation and purification of bacteriophage LYE-01

[0041] The wastewater samples were collected from pig manure samples from a small pig farm in Dongli District, Tianjin, China. 50 mL of pig manure sample was taken, filtered with gauze, CaCl2 was added to make the concentration 1 mmol / L, and divided into 50 mL centrifuge tubes, centrifuged at 4500 r / min for 10 min, the supernatant was filtered through a 0.22 μm bacterial filter, the precipitate was discarded, and the supernatant was placed in a 300 mL conical flask.

[0042] Isolation of bacteriophages: Phages targeting Escherichia coli were screened from sewage samples using the droplet method. The purification results are shown in Figure 2. Figure 1 As shown in Figure A, a circular transparent plaque can be clearly observed in the figure.

[0043] Purification of phages: The double-layer plate method was used to separate and purify phages. The separated phages were purified 6 to 8 times to form plaques of uniform size and shape on the plate. One plaque with obvious differences in shape and size was picked up with a 20μL pipette tip, and dissolved in a centrifuge tube containing 1mL of sterilized SM buffer. The tube was gently shaken and placed at 4°C overnight to allow the phages to be fully released into the SM. The supernatant was collected and filtered through a 0.22μm bacterial filter. The phage filtrate was gradiently diluted to 10 -9 , double-layer culture, repeated 6 to 8 times, when the plaques appear with uniform morphology and size, the purified phage LYE-01 is obtained. Figure 1 As shown in Figure B, plaques of uniform size and shape can be clearly observed in the figure.

[0044] Phage proliferation: The liquid proliferation method is used to proliferate phages. The phage stock solution is added to the host bacterial solution cultured for 6 to 7 hours at a ratio of 1:10, and then cultured in a 37°C shaker for 3 to 4 hours. The mixed solution is centrifuged at 4°C and 8000 rpm for 5 minutes, and the supernatant is 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 1 M Tris-Cl buffer, pH = 7.5) and stored in a refrigerator at 4°C.

[0046] Example 2

[0047] Biological characteristics of bacteriophages

[0048] (1) Morphological observation of bacteriophage

[0049] Observe the morphology of bacteriophages by transmission electron microscopy. Take 10 μL of the above phage preservation solution and drop it on the copper grid to precipitate for 1 minute. After standing at room temperature for 10 minutes, use filter paper to absorb the excess liquid. Stain with 2% phosphotungstic acid stain for 2 minutes, use filter paper to absorb the excess stain, and dry at room temperature for 20 minutes. Observe the morphology of bacteriophages at an accelerating voltage of 80-120 kV on a HT7800 transmission electron microscope. Use Nano measurer 1.2 software to perform statistical analysis on the length, diameter and other dimensions of phage particles.

[0050] Electron micrograph of bacteriophage Figure 1 As shown in Figure C, the phage LYE-01 is tadpole-shaped, with an icosahedral head (diameter 88±4nm). It has a long contractile tail with a length of 109±4nm, spiral symmetry, and a tail filament and a tail spike. Based on the classification of the International Committee on Taxonomy of Viruses (ICTV), the phage is temporarily classified as a member of the Myoviridae family.

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

[0052] Eleven strains of potential host bacteria were selected for determination of the host spectrum of the phage, including three strains of Escherichia coli. After the potential host bacteria were inoculated into LB medium and cultured overnight, the phage host spectrum was tested by the drop method: 50 μL of the above bacterial solution was spread on double-layer LB agar plates in turn, and allowed to stand at room temperature for 15 minutes, and then 1.5 μL of phage preservation solution was aspirated and dropped on the double-layer LB agar plate containing the bacterial solution, and 5 drops were evenly dropped, and cultured at 37°C for 20 minutes. After the phage filtrate was completely absorbed, it was inverted, and the experiment was repeated three times to observe whether plaques appeared in the culture medium after overnight culture, and the double-layer agar plate without phage filtrate was used as a blank control.

[0053] In experiments on infection with common drug-resistant pathogens in feces such as Escherichia coli, Salmonella and Enterococcus faecalis, it was found that bacteriophages only had a lysis effect on Escherichia coli and had no killing effect on other bacterial strains (Table 1).

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

[0055] Bacteria name Cracking capacity Escherichiacoli(Migula)CastellaniandChalmersBNCC332831 + Escherichiacoli(Migula)CastellaniandChalmersBNCC305635 + Escherichiacoli - EnterococcusfaecalisBNCC102668 - HalomonasST1 - HalomonasST5 - HalovibrioST17 - Salmonella - HalomonasST9 - HalomonasST13 - HalomonasY32 -

[0056] +: has a cracking effect on it; -: has no cracking effect on it

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

[0058] Determination of host bacteria concentration: Take a single colony of E. coli and inoculate it into 50 mL LB liquid culture medium, and culture it at 37°C and 160 r / min with shaking until the initial logarithmic growth. Use LB liquid culture medium as the blank control group, and measure the host bacteria OD at a wavelength of 600 nm using an ultraviolet spectrophotometer. 600 When the value is ≈0.2, dilute the bacterial solution 10 times, take 0.1mL of the dilutions of different dilutions and evenly spread them on the LB solid plate, and culture them at 37℃ 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 solution of different concentrations: Take a single plaque and inoculate it into 50mL of host bacterial solution in the logarithmic growth phase, shake and culture at 37℃ until the liquid is clear, centrifuge at 10000r / min for 10min, take the supernatant, and make 10-fold serial dilutions with LB liquid culture medium. Take 100μL from each dilution, determine the phage titer by double-layer plate method, and store the supernatant at 4℃ for later use. Observe the results and count the next day. Phage titer = number of plaques × dilution factor × 10 (PFU / mL).

[0060] The phage fluid and host bacterial fluid were mixed at a ratio of 0.001, 0.01, 0.1, 1, 10, and 100 respectively, and then LB liquid medium was added to make the total volume of the culture system of different ratios consistent. Cultured overnight at 37℃160r / min with shaking, 1mL was taken and centrifuged at 10000r / min for 10min, and the supernatant was diluted 10 times. Double-layer plates were spread, cultured overnight at 37℃, and the phage titer was determined. At the same time, phage without host bacteria and host bacteria without phage were set up as control groups.

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

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

[0063] According to the optimal MOI, add phage filtrate and fresh culture OD into a sterile test tube. 600 =0.6 E. coli liquid, 37°C, 160r / min shaking culture. Take out 500μL at 0, 10, 20, 30, 40, 50, 60, 70, 80 and 90min, centrifuge at 10000r / min for 5min, take 100μL of supernatant, make 10-fold serial dilution, and determine the phage titer.

[0064] The one-step growth curve of bacteriophage LYE-01 is as follows Figure 2As shown in Figure B, the results show that the incubation period is about 10 minutes, and the outbreak period is about 40 minutes thereafter. After 50 minutes of phage infection, it is a stable plateau period. Bacteriophages have a relatively short lysis cycle, and this excellent biological characteristic is of great value for achieving rapid removal of pathogenic bacteria in feces.

[0065] (5) Determination of phage tolerance to pH, UV and temperature

[0066] Use 1 mol / L NaOH and HCl solution to adjust the pH value of SM buffer to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. Take 100 μL of phage filtrate and add it to 900 μL SM buffer, mix it in a 1 mL centrifuge tube, set up two parallel groups for each group, and after 1 hour in a 37°C water bath, dilute it 10 times in a gradient, and determine the titer by double-layer agar plate method.

[0067] 2 ml of phage filtrate (titer 1×10 7 PFU / mL) was added into a sterile culture dish and exposed to ultraviolet light in a biosafety cabinet. Samples were taken at different treatment times (5, 10, 15, 25, and 30 min) and the phage titer was determined by the double-layer plate method.

[0068] Take 500 μL of phage filtrate in a sterile centrifuge tube and set up multiple groups, which were placed in a constant temperature water bath at 10, 30, 50, 70, and 90°C, respectively. Each group was set up in parallel with two other groups and incubated for 1 hour. 600 =0.6~0.8) was used as the host, and the phage titer was determined by the double-layer plate method.

[0069] The pH stability test results of bacteriophage LYE-01 are as follows Figure 2 As shown in Figure C, within the pH range of 4-11, the phage can maintain a high titer. As the pH limit decreases and increases, the phage will be completely inactivated.

[0070] The UV stability test results of bacteriophage LYE-01 are as follows: Figure 2 As shown in Figure D, under the 254nm ultraviolet irradiation environment, the phage showed a gradual downward trend and was completely inactivated after 25 minutes of irradiation.

[0071] The temperature stability test results of bacteriophage LYE-01 are as follows Figure 2 As shown in Figure E, the phage titer can be stably maintained at 10 when incubated at 10-50°C for 1 hour. 10 PFU / mL is about 100%, which is basically unaffected. However, when the temperature is above 70℃, the titer of the phage drops sharply until it is completely inactivated at 90℃.

[0072] Example 3

[0073] Whole genome analysis of bacteriophages

[0074] Extract genomic DNA from phage LYE-01 using the lambda phage genomic DNA extraction kit (PEG precipitation method). Take 12mL of fresh bacterial lysate treated with 0.5% chloroform and centrifuge at 8000g for 5min to remove host bacterial debris. Filter the supernatant containing most of the virus particles through a 0.22μm injection filter to remove other impurities. Add RNaseA and DNaseI to 1mL of TE buffer respectively and mix gently by pipetting. After fully dissolved, divide and freeze at -20℃ according to the amount used each time. Add 0.5μL RNaseA and 1μLDNase I to 1mL of phage concentrate, mix thoroughly, and incubate at 37℃ for 30min. Add 400μL phage precipitation solution to the supernatant, shake until fully dissolved, and incubate at 4℃ overnight. Centrifuge at 10000g in a refrigerated centrifuge at 4℃ for 20min and discard the supernatant. Add 1mL SM buffer, thoroughly clean the tube wall attachments and the bottom sediment, then transfer to a new centrifuge tube, add 4μL phage lysis solution, and incubate at 68℃ for 15min. Add an equal volume of protein removal solution, gently mix, centrifuge at 12000g for 5min, and take the supernatant. Add an appropriate amount of 70% ethanol solution, mix, centrifuge at 8000g for 8min at 4℃, and discard the supernatant. After drying the phage DNA at room temperature, add an appropriate amount of TE buffer and store at -20℃.

[0075] The phage genomic DNA was sequenced on the Illumina HiSeq novaseq 6000 sequencing platform. After sequencing quality control (fastq), genome sequence splicing (Spades), assembly sequence correction (PhageTerm), etc., an accurate and complete phage genome sequence was obtained. The obtained sequence was compared with the viral homology sequence in the NCBI database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The complete annotated sequence of the phage genome is stored in the GenBank Virus Nucleotide Database (Accession No. PQ303785). The ViPTree platform (https: / / www.genome.jp / viptree) was used to draw the phage virus proteomics tree. CGView was used to draw the circle map of the phage genome.

[0076] The total genome length of LYE-01 is 165,614 bp, and the contents of A, C, G and T bases 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 (CDS). The gene annotations are shown in Table 2, of which 119 ORFs are predicted to be hypothetical proteins and 145 ORFs are predicted to be functional proteins, which are 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, and has good biosafety. In the NCBI database, BLAST was used to compare the similarity of LYE-01 with other phages. The results showed that bacteriophage MLF4 (GenBank ID: NC_055780.1) had the highest similarity with LYE-01, with a homology of 95.06%. Compared with the nearest bacteriophage MLF4, LYE-01 can be identified as a species of the existing genera Duplodnaviria, heungongvirae, Uroviricota, Caudoviricetes, Straboviridae, Tevenvirinae, and Tequatrovirus. The full sequence analysis of the Escherichia coli phage LYE-01 proteome sequence using the VipTree platform identified 1090 prokaryotic dsDNA virus proteome sequences, of which 234 (21.5%) belonged to the Straboviridae ( Figure 4 ). Further analysis of the 194 E. coli phages in the database revealed only 86 Straboviridae phages (including E. 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 formally classified as a member of the Straboviridae family. The discovery of this phage fills the gap in the resource of E. coli phage species.

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

[0078]

[0079]

[0080]

[0081]

[0082] Example 4

[0083] Antibacterial effect detection of bacteriophage LYE-01

[0084] The host bacteria Escherichia coli was evenly spread on the LB agar plate, and then 5 μL of the phage LYE-01 solution was dropped on the LB agar plate coated with Escherichia coli, and incubated in a 37°C incubator overnight. The LB agar plate was observed the next day for the presence of plaques.

[0085] The results are as follows Figure 5 As shown, the round transparent plaques represent plaques on Escherichia coli. The results show that the single phage LYE-01 has a good antibacterial effect and no resistant strains appear.

Claims

1. A highly efficient method for lysing Escherichia coli phage, characterized in that: The name is LYE-01, the classification name is Escherichia coliphage, the preservation number is CGMCC No.46095, the preservation date is August 7, 2024, the preservation institution is the General Microbiology Center of China Microbiological Culture Collection Administration, and the preservation address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

2. A microbial preparation, characterized in that: Comprising the bacteriophage described in claim 1.

3. A bactericide or disinfectant, characterized in that: Including LYE-01 according to claim 1 or the microbial preparation according to claim 2.

4. Use of the bacteriophage preparation according to any one of claims 1 to 3 in the preparation of a product for inhibiting or killing Escherichia coli.

5. The use according to claim 4, characterized in that: The anti-Escherichia coli phage preparation is used for treating Escherichia coli infection in fecal sewage environment.

6. The bacteriophage preparation according to claim 5, characterized in that: The titer of LYE-01 in the phage preparation was 1×10 6 PFU / mL or above.

7. The highly efficient Escherichia coli phage lysis according to claim 1, characterized in that: The applicable pH value of the bacteriophage is 4-11, and the applicable temperature of the bacteriophage is 10°C to 70°C.

8. Use of the bacteriophage according to claim 1 in the preparation of an anti-Escherichia coli preparation.

9. The use according to claim 8, characterized in that: The anti-Escherichia coli preparation is a medicine for treating Escherichia coli infection, or the anti-Escherichia coli preparation is used for treating toilet sewage or livestock and poultry sewage.

10. An anti-Escherichia coli preparation, characterized in that: The active ingredient comprises the bacteriophage LYE-01 described in claim 1.

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