A bacteriophage capable of lysing high-risk clones of Escherichia coli and targeting multiple serotypes and its application

Abstract

This invention relates to the field of microbiology, and particularly to a high-risk clonal strain of *Escherichia coli* that targets multiple serotypes and its applications. This application discloses a high-risk clonal strain of *Escherichia coli* that targets multiple serotypes, named *Escherichia phage vB_EcoM_32M3Y*, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 45969. The phage described in this application has a low MOI, good stability, excellent lytic activity, and no drug resistance or virulence genes in its genome sequence, thus possessing good clinical application value.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a high-risk clone of Escherichia coli that targets multiple serotypes and its applications. Background Technology

[0002] Antimicrobial resistance has become one of the major public health threats of the 21st century. According to the UK's Antimicrobial Resistance Assessment, by 2050, the number of people dying annually from drug-resistant bacterial infections will reach 10 million (O'Neill, 2016). The problem of resistance in Gram-negative bacteria is particularly prominent, sometimes even leading to a situation where no effective drugs are available. In 2017, the World Health Organization (WHO) listed carbapenem-resistant Acinetobacter baumannii, carbapenem-resistant Pseudomonas aeruginosa, carbapenem-resistant and extended-spectrum β-lactamase-producing Enterobacteriaceae as the highest priority in its global resistance priority list, all of which are Gram-negative bacteria (Tacconelli et al., 2018). Highest priority drug-resistant bacteria are often vertically transmitted through clonal strains. Some highly resistant and virulent strains become high-risk clonal strains that spread widely in global ecological niches, posing a serious challenge to clinical anti-infective treatment (Nicolas-Chanoine et al., 2014; Mathers et al., 2015; Mendes et al., 2023).

[0003] Among Enterobacteriaceae, high-risk clones of species such as *Escherichia coli*, *Klebsiella pneumoniae*, and *Salmonella* have attracted researchers' attention, as they are often important causes of urinary tract infections, bloodstream infections, pneumonia, and gastroenteritis (Paterson, 2006). *Escherichia coli* ST410, a multidrug-resistant extraintestinal pathogenic *E. coli*, has been recognized as a new high-risk clone (Roer et al., 2018). *Enterobacteria coli* resistant to multiple carbapenems and producing extended-spectrum β-lactamases (ECLA) are also a significant risk factor globally. Enterobacteriae , CRE; Extended-spectrum Beta-lactamase producing Enterobacteriae In ESBL resistance monitoring, ST410 was frequently detected and often carried by other drugs. bla OXA-181 , bla CTX-M-15 , bla NDM-5Drug resistance genes (Pitout et al., 2019). In addition to clinical applications, carbapenem-resistant Escherichia coli ST410 has also been detected in large numbers in the environment and animals (Kyung et al., 2022; He et al., 2023), becoming a major threat to global public health.

[0004] The impending crisis of finding effective treatments for bacteriophages has caused widespread concern, highlighting the urgent need to find alternative antibiotic therapies. Bacteriophages (phages) are a type of virus that parasitizes prokaryotic microorganisms such as bacteria and archaea. They are figuratively called "bacterial viruses" or "bacterial predators" and are among the most diverse biological entities on Earth. It is estimated that there are 10 million bacteriophages on Earth. 31 In some ecosystems, bacteriophages outnumber bacteria by up to 10 times, playing a crucial role in maintaining the balance of the Earth's biosphere and ecosystems. As viruses of bacteria, bacteriophages can be used to treat bacterial infections, especially those caused by multidrug-resistant bacteria. However, phage therapy also has limitations, such as the high host specificity of phages, which limits its therapeutic scope and has hindered its widespread application for a long time. Nevertheless, compared to other treatment methods, phage therapy offers advantages such as high specificity, minimal disruption of bacterial community structure, and continuous self-replication and expansion during bacterial lysis, making it a popular alternative to antibiotics. Therefore, it is possible to mine specific phages from high-risk clones and establish phage libraries for clinical use. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies and address the challenges in treating infections caused by multidrug-resistant clones and the limitations of narrow phage lysis range, this application provides a phage strain that lyses high-risk Escherichia coli clones and targets multiple serotypes, along with its applications. The phage has a low MOI, good stability, and excellent lysis activity. Its genome sequence contains no drug resistance or virulence genes, making it valuable for clinical applications.

[0006] The technical solution adopted by this invention to solve its technical problem is:

[0007] A high-risk clone of lysing Escherichia coli targeting multiple serotypes, named Escherichia phage vB_EcoM_32M3Y, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 45969.

[0008] Preferably, the bacteriophage is sourced from wastewater from a livestock farm.

[0009] Preferably, the bacteriophage belongs to the family Ackermannviridae, subfamily Aglimvirinae, and genus Agtrevirus.

[0010] Preferably, the genome of the bacteriophage is a linear double-stranded DNA with a size of 152,897 bp and a G+C content of 49%. The genome of the bacteriophage does not contain virulence factors or drug resistance genes.

[0011] Preferably, the optimal multiplicity of infection of the bacteriophage against Escherichia coli is 0.00001:1, the incubation period is 40 min, the outbreak period is 70 min, and the outbreak amount is 108±10 PFU / cell.

[0012] Preferably, the phage contains four different tail spike proteins, which can lyse four serotypes of Escherichia coli.

[0013] Preferably, the bacteriophage maintains good activity at 4–50°C and / or at a pH of 3–12.

[0014] The present invention also provides a bactericidal composition comprising the bacteriophage described above.

[0015] The present invention also provides the use of bacteriophages in the preparation of medicaments or formulations for the treatment or prevention of bacterial infections in mice caused by Escherichia coli.

[0016] Preferably, the phage targets the B5 / H24RxC subtype of high-risk clone Escherichia coli ST410 and / or targets specific serotypes of Escherichia coli and / or targets the O antigen.

[0017] The beneficial effects of this invention are:

[0018] 1. The bacteriophage provided in this application Escherichia Phage vB_EcoM_32M3Y is a subtype of B5 / H24RxC that can lyse high-risk clones of ST410 Escherichia coli. This subtype is characterized by carbapenem resistance and high toxicity, and is widely prevalent and spread in human clinical medicine and animal husbandry.

[0019] 2. The phage provided in this application has a low MOI, good stability, good lysis activity, and no drug resistance or virulence genes in its genome sequence, thus having good clinical application value.

[0020] 3. The phage provided in this application has four tail spike proteins and an O antigen as its receptor. It can lyse four serotypes of Escherichia coli, including ST410 Escherichia coli carrying Onovel1 and ST9388 Escherichia coli carrying another untyped O antigen. Compared with phages with a single tail spike protein, it has a broader phage spectrum.

[0021] Preservation Information:

[0022] A high-risk clonal strain of *Escherichia coli* that targets multiple serotypes was named [name of the phage]. Escherichia Phage vB_EcoM_32M3Y was deposited on April 24, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 45969. Attached Figure Description

[0023] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0024] Figure 1 For morphological observation of phage plaques during the phage screening process described in this application;

[0025] Where a: morphology of a single phage spot; b: halo on the surface of the phage;

[0026] Figure 2 Transmission electron micrograph of the bacteriophage described in this application (46000×).

[0027] Figure 3 This represents the optimal multiplicity of infection for the bacteriophage described in this application;

[0028] Figure 4 The stability evaluation results of the bacteriophage described in this application

[0029] Where a: temperature stability; b: pH stability;

[0030] Figure 5 This is a one-step growth curve of the bacteriophage described in this application;

[0031] Figure 6 The in vitro therapeutic effect (bactericidal curve) of the bacteriophage described in this application;

[0032] Figure 7 The therapeutic effect of the bacteriophage described in this application on mice;

[0033] Figure 8 This refers to the complete genome information of the bacteriophage described in this application;

[0034] Figure 9 For the TSP sequence alignment of the bacteriophage described in this application;

[0035] Where a: TSP sequence of P32M-3-Y; b: alignment of different TSP sequences. Detailed Implementation

[0036] To facilitate understanding by those skilled in the art, the present invention will be further described below with reference to embodiments. The content mentioned in the embodiments is not intended to limit the present invention.

[0037] For reagents or instruments used in the following text where specific technical or conditional specifications are not given, standard experimental conditions shall apply. If no reagent company instructions are explicitly provided, the conditions recommended in those instructions shall be followed. Reagents or instruments whose manufacturers are not specified are all commercially available standard products.

[0038] Terminology Explanation:

[0039] Drug resistance: refers to the ability of bacteria and other microorganisms to resist drugs such as antibiotics, making the originally effective drugs unable to inhibit or kill these microorganisms.

[0040] High-risk clones: These are bacterial strains with high drug resistance and virulence that can spread globally through cloning, posing a threat to public health.

[0041] Carbapenems: a class of broad-spectrum antibiotics used to treat a variety of bacterial infections, especially those resistant to other antibiotics.

[0042] ESBL (Extended-Spectrum Beta-Lactamases): These are enzymes that can hydrolyze a variety of β-lactam antibiotics, leading to bacterial resistance to these antibiotics.

[0043] MOI (Multiplicity of Infection): In a phage infection experiment, the ratio of phage particles to host bacterial cells is considered the multiplicity of infection.

[0044] PFU (Plaque-Forming Units): These are units used to measure phage concentration. One PFU represents one phage particle capable of producing a visible plaque.

[0045] The latency period refers to the time from when a bacteriophage infects a bacteriophage until the bacteria lyse and begin releasing new bacteriophages. This is the process of the bacteriophage replicating and assembling within the bacteria.

[0046] The burst period refers to the phase in which a large number of newly released bacteriophages rapidly increase after bacteria are lysed by bacteriophages. This is a crucial stage in the bacteriophage reproduction cycle.

[0047] An open reading frame (ORF) is a segment of a DNA sequence that encodes a protein and runs from the start codon to the stop codon without being interrupted by other non-coding sequences.

[0048] ST type (Sequence Type): A bacterial typing method based on the polymorphism of certain gene sequences in the bacterial genome.

[0049] O antigen: The polysaccharide portion of the outer membrane of the Escherichia coli cell wall, which is associated with bacterial serotype classification.

[0050] Tail Spike Protein (TSP): A protein in the tail of a bacteriophage that is responsible for recognizing and binding to receptors on the surface of host bacteria.

[0051] Agtrevirus A classification of bacteriophages, belonging to Ackermannviridae Virology.

[0052] VirulenceFinder and ResFinder: Two bioinformatics tools used to predict virulence factors and drug resistance genes in bacterial genomes.

[0053] NCBI-blastn: An algorithm for aligning nucleic acid sequences, which can identify the similarity between sequences.

[0054] FastANI: A software used to calculate the average nucleotide identity between bacterial or bacteriophage genomes.

[0055] Semisolid medium is a medium made by adding a small amount of solidifying agent (such as agar) to a liquid medium. Its solidification degree is between that of liquid and solid mediums. It is often used to observe the growth and spread of microorganisms such as bacteriophages.

[0056] This invention provides a high-risk phage strain of lysing Escherichia coli that targets multiple serotypes, classified and named Escherichia coli phage. Escherichia Phage vB_EcoM_32M3Y (abbreviated as P32M-3-Y), with accession number CGMCC No.45969, deposited on April 24, 2024, is deposited at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences.

[0057] We have currently discovered that the bacteriophage described in this application belongs to... Ackermannviridae Virology ， Aglimvirinae Subfamily, Agtrevirus The genome of bacteriophage P32M-3-Y is a linear double-stranded DNA, 152,897 bp in size, with a G+C content of 49%, and was successfully annotated to 187 open reading frames (ORFs). VirulenceFinder and ResFinder predictions indicate that the P32M-3-Y genome does not contain virulence factors or drug resistance genes.

[0058] Furthermore, the optimal multiplicity of infection for bacteriophage against Escherichia coli was 0.00001:1, with an incubation period of 40 min, an outbreak period of 70 min, and an outbreak dose of 108±10 PFU / cell.

[0059] Furthermore, the bacteriophage maintains good activity at 4–50°C and at pH 3–12, demonstrating good temperature stability and excellent acid and alkali tolerance.

[0060] Furthermore, bacteriophages have shown good therapeutic effects in mouse bacterial infection models, significantly delaying and reducing mortality rates in mice.

[0061] Furthermore, the phage targets the B5 / H24RxC subtype of the high-risk clone ST410 Escherichia coli, which is characterized by carbapenem resistance and high toxicity.

[0062] Furthermore, the phage can target four serotypes of Escherichia coli, including ST410 Escherichia coli carrying Onovel1 and ST9388 Escherichia coli with an untyped O antigen. Both are popular clones that persist and spread in the aquaculture field and have a broad phage spectrum.

[0063] Furthermore, the phage specifically targets the O antigen, exhibiting serotype specificity. Phage genome analysis revealed that the phage contains four distinct tail spike proteins (TSPs), named TSP1, TSP2, TSP3, and TSP4 sequentially according to transcriptional direction. No sequence similarity exists among the four TSPs. NCBI-blastn screening identified five sequences most similar to the phage's TSP sequences. The TSP sequences from different phages are conserved only at the N-terminus, while significant differences exist in the C-terminal domain, which is responsible for receptor recognition and degradation. This indicates that the host range of this phage differs significantly from other phages in the same genus, thus supplementing existing limitations on phage host range.

[0064] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0065] Prepare materials:

[0066] The formula for LB nutrient agar is: 10.0g tryptone, 5.0g yeast extract, 10.0g sodium chloride, 15.0g agar, and 1000mL distilled water.

[0067] The recipe for LB broth is as follows: 10.0g tryptone, 5.0g yeast extract, 10.0g sodium chloride, and 1000mL deionized water.

[0068] The SM solution formula is as follows: 8.5g sodium chloride, 2g magnesium sulfate, 50 mL 1mol / L Tris-HCl, 0.25g gelatin, and 1000mL deionized water.

[0069] The formula for LB semi-solid agar is: 7.2g tryptone, 3.6g yeast extract, 7.2g sodium chloride, 7.0g agar, and 1000mL deionized water.

[0070] Aseptic double-concentrated broth with 2mM CaCl2: 20g LB broth powder, 88.78mg CaCl2, 400mL deionized water.

[0071] Example 1

[0072] Isolation, preparation, purification, and culture of bacteriophages.

[0073] 1. Sample pretreatment

[0074] (1) Wastewater was collected from a slaughterhouse in Guangzhou and stored at low temperature before being transported back to the laboratory;

[0075] (2) Larger particulate impurities are removed by gauze filtration;

[0076] (3) Centrifuge the sample at 4000g for 20 minutes to remove bacteria and other impurities;

[0077] (4) Bacterial sterilization filtration was performed using microporous membranes of 0.45μm and 0.22μm in sequence;

[0078] (5) Use a small tangential flow to ultrafilter and concentrate the filtered sewage sample to about 50 mL of liquid to obtain the phage stock solution, to which a small amount of chloroform may be added.

[0079] 2. Isolation of bacteriophages:

[0080] (1) Take 10 mL of sterile double-concentrated broth containing 2 mmol / L CaCl2 and add 10 mL of clarified (or filtered) wastewater;

[0081] (2) Inoculate with 0.1 mL of overnight cultured host bacteria and culture gently (50 rpm / min) with shaking at an appropriate growth temperature (usually 37°C);

[0082] (3) After culturing for 24–48 h, centrifuge at 10000 g for 10 min. Pour the supernatant into a small vial with a screw cap or a stoppered test tube. Add 0.5 mL of chloroform to the clear crude lysis buffer, shake gently, and store at 4 °C.

[0083] 3. Phage spot test

[0084] (1) Mix 100 μL of overnight cultured cells 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.

[0085] (2) Add a few drops of 5 μL of the sample enrichment solution to the bacterial moss until the droplets are completely dry.

[0086] (3) Incubate the plates overnight in a 37°C constant temperature incubator. Check for any clear or cloudy lysis zones.

[0087] 4. Isolation of bacteriophages

[0088] (1) Add 0.9 mL of SM Buffer to a sterile test tube or centrifuge tube, and number the centrifuge tubes according to the dilution;

[0089] (2) Add 0.1 mL of phage enrichment solution to the first tube, mix well, and then transfer 0.1 mL to the second tube. Repeat this process for 10-fold dilutions.

[0090] (3) Transfer 0.1 mL of phage dilution to warm semi-solid culture medium test tubes, immediately add 0.1 mL of host bacteria cultured to the logarithmic growth phase, mix well, and then introduce it onto the surface of solid culture medium; after the semi-solid layer solidifies, invert the tubes for culture at the optimal culture temperature. Culture for 8–10 h, and select plates with a single plaque for phage purification.

[0091] 5. Purification of bacteriophages

[0092] (1) Pick up a single phage plaque with the autoclaved tip, suspend it in 1 mL of SM Buffer, and mix thoroughly by vortexing to dissolve the phage completely in the SM Buffer;

[0093] (2) Use a filter membrane with a pore size of 0.22 μm to remove bacteria;

[0094] (3) Dilute the SM 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, purified phage individuals can be obtained, such as... Figure 1 As shown, Escherichia phage P32M-3-Y (Escherichia phage vB_EcoM_32M3Y) was obtained, with the accession number CGMCC No.45969.

[0095] 6. Preservation of bacteriophages

[0096] In a 2 mL preservation tube, mix 0.5 mL of phage stock solution with 0.5 mL of sterile glycerol to a final concentration of 50%. Store at -80 °C.

[0097] Example 2: Electron microscopic observation of bacteriophages

[0098] (1) Take the phage culture prepared in Example 1 and concentrate 50 mL of phage solution to 2 mL using a 100 kDa Amicon Ultra centrifuge ultrafiltration tube;

[0099] (2) Take 15 μL of the concentrated phage solution and drop it onto a copper grid, and let it precipitate for 15 min;

[0100] (3) Use filter paper to gently absorb the excess liquid on the copper grid, then add 2% phosphotungstic acid (PTA, pH=7.0) to the copper grid and stain for 10 min. After the copper grid dries, observe and photograph it under an electron microscope.

[0101] The results are as follows Figure 2 As shown, the morphology of Escherichia coli phage P32M-3-Y observed under a transmission microscope revealed that the head structure of the phage was about 85±4 nm in diameter and had a tail structure with a length of about 126±3 nm.

[0102] Example 3: Extraction and sequencing of bacteriophage genomes

[0103] (1) Take the phage culture prepared in Example 1 and concentrate 50 mL of phage solution to 2 mL using a 100 kDa Amicon Ultra centrifuge ultrafiltration tube;

[0104] (2) Take 2 mL of phage sample that has passed through a 0.22 µm microporous membrane and put it into a 15 mL centrifuge tube that has been autoclaved and has a smooth surface;

[0105] (3) Add 2 µL of DNase I and RNase (1 mg / mL final concentration 1 µg / mL), gently invert to mix, and incubate at 37 °C for 1 h;

[0106] (4) In a 15 mL centrifuge tube, add 2 mL of formamide, 20 µL of EDTA, and 200 µL of 2 M Tris HCl / 0.2 M EDTA (TE), gently invert and mix, and let stand at room temperature for 30 min.

[0107] (5) Use the phage genome extraction kit (M13 Isolation Kit D6900) to complete the extraction of genomic DNA according to the instructions.

[0108] The extracted phage product was sent to Annoroad for sequencing, yielding the nucleotide sequence of Escherichia coli phage P32M-3-Y. The genome annotation results are as follows: Figure 8 As shown, the genome of phage P32M-3-Y is a linear double-stranded DNA, 152,897 bp in size, with a G+C content of 49%, and successfully annotated to 187 open reading frames (ORFs). VirulenceFinder and ResFinder predictions indicate that the P32M-3-Y genome does not contain virulence factors or drug resistance genes. Using blastn to compare the P32M-3-Y phage genome, phage sequences with a query coverage >85% were selected. The average nucleotide identity (ANI) was calculated using FastANI software. P32M-3-Y was most closely related to Escherichia phage 2307YX22 (GenBank: OR776998.1), with an identity of 98.0957%, both belonging to the Ackermannviridae family, Aglimvirinae subfamily, and Agtrevirus genus.

[0109] Example 4: Confirmation of the optimal multiplicity of infection for bacteriophages

[0110] (1) The host bacteria were cultured to the logarithmic growth phase and then plated for counting by serial dilution of tenfold;

[0111] (2) Determine the phage titer by performing a double-layer plate test on the phage fluid;

[0112] (3) Adjust the concentration of the bacterial solution and the titer of the phage solution to appropriate amounts, and mix the phage solution and bacterial solution at a ratio of 1:1 with MOI=10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, and 0.000001 respectively, and let stand for 10 min;

[0113] (4) Add fresh LB broth to bring the volume to 10 mL, and incubate at 37 °C and 180 rpm for 4 h with shaking.

[0114] (5) Centrifuge the mixture at 10000 g for 2 min, take the supernatant and filter it through a 0.22 μm filter membrane;

[0115] (6) Perform a double-layer plate test on the phage fluid of each group to determine the phage titer, and perform three replicates for each experimental group.

[0116] The results are as follows Figure 3 As shown, under the condition of MOI=0.00001, the titer of phage P32M-3-Y reached the highest level (5.73×10¹⁰ PFU / mL).

[0117] Example 5: Determination of the stability of bacteriophage P32M-3-Y

[0118] (1) Temperature stability: The host bacteria were cultured to the logarithmic growth phase. The metal bath was pre-set to 4 ℃, 25 ℃, 37 ℃, 50 ℃, 60 ℃, and 70 ℃. After temperature stabilization, the phage fluid was incubated in each temperature for 1 h. After incubation, a double-layer plate test was performed to determine the phage titer. Each experimental group was repeated in triplicate; for example... Figure 4 The results showed that after incubation at 4 ℃, 25 ℃, 37 ℃ and 50 ℃ for 1 h, the phage titer did not change significantly, and the phage maintained good activity, indicating that P32M-3-Y has good temperature stability.

[0119] (2) pH stability: The host bacteria were cultured to the logarithmic growth phase. The pH of the LB broth was adjusted to 3–12 beforehand. Bacteriophages were added to LB broths at different pH values ​​at a ratio of 1:10 and incubated at room temperature for 1 h. After incubation, a double-layer plate test was performed to determine the phage titer. Each experimental group was repeated in triplicate. Figure 4 b. When phage P32M-3-Y was incubated at pH 6 to 12 for 1 h, the phage titer did not change significantly and could maintain a relatively stable level. When incubated at pH 3 to 5 for 1 h, the phage titer decreased slightly, but the change did not exceed one order of magnitude, indicating that P32M-3-Y has good pH tolerance.

[0120] Example 6: Determination of the lysis spectrum of bacteriophage P32M-3-Y

[0121] In this experiment, a total of 86 strains of ST-type Escherichia coli, including ST410 and ST9388, were selected and cultured to the logarithmic growth phase. 200 μL of the bacterial culture was added to LB agar plates, and 4.8 mL of semi-solid was added. The plates were shaken thoroughly and cooled.

[0122] (2) Take 5 μL of phage solution with a titer of 1×10⁸ PFU / mL and drop it into the center of the semi-solid. After the liquid is completely absorbed, incubate overnight at 37 ℃. Mark bacteria that can form plaques as "+" for positive results and "-" for negative results. The lysis effect is summarized in Tables 1 and 2.

[0123] Table 1. Lytic effect of bacteriophage P32M-3-Y on ST410 strain

[0124]

[0125] Table 2. Lytic effect of phage P32M-3-Y on various prevalent ST serotypes of Escherichia coli.

[0126]

[0127] Tables 1 and 2 show that phage P32M-3-Y lysed 26 ST410 *E. coli* strains, with a lysis rate of 52% (26 / 50). According to the phylogenetic classification of ST410 strains by Roer et al. (2018) and Ba et al. (2024), the ST410 strains preserved in our laboratory belong to three clusters: B3 / H24Rx, B4 / H24RxC, and B5 / H24RxC. P32M-3-Y lysed some strains within the B3 / H24Rx and B5 / H24RxC clusters, and serotype analysis revealed that the O antigen of these strains was the novel Onovel. In addition to the ST410 strains, P32M-3-Y also lysed *E. coli* type ST9388, which encodes a serotype different from Onovel1.

[0128] for Ackermannviridae For bacteriophages of the Viridae family, their receptor recognition protein is generally the tail spike protein (TSP), and it can encode up to four different tail spike proteins (Sørensen et al., 2021). Compared to bacteriophages containing only a single TSP, they have a wider host range. Bacteriophage P32-3-Y contains four different tail spike proteins, which are named TSP1, TSP2, TSP3, and TSP4 in order of transcription direction, such as... Figure 9 As shown in Figure a. Five sequences most similar to the P32M-3-Y TSP sequence were selected using NCBI-blastn, and the sequence alignment results are shown below. Figure 9As shown in b, the TSP sequences of different bacteriophages are conserved only at the N-terminus, while their C-terminal domains, which recognize and degrade receptors, show significant differences. These catalytic domains are highly diverse and have been demonstrated by multiple studies to bind to polysaccharide receptors (such as O antigens or K antigens) (Hsu et al., 2013; Greenfield et al., 2019; Plattner et al., 2019). TSP4 is highly similar to E. coli bacteriophage AV101 (GenBank: WJJ54142.1) (100% coverage, 78.34% identity), suggesting that they likely recognize similar receptors. TSP1–4 of AV101 bacteriophage have been shown to recognize and degrade O8, O82, O153, and O159 antigens (Sorensen et al., 2024), and it is speculated that P32M-3-Y may also recognize four different O antigens.

[0129] Example 7: In vitro therapeutic effects of Escherichia coli bacteriophage

[0130] (1) such as Figure 5 As shown, the host bacteria were cultured to the logarithmic growth phase, and the phage fluid and bacterial suspension were mixed 1:1 at MOI ratios of 10, 1, 0.1 and 0.01. The blank group was a 1:1 mixture of LB broth and bacterial suspension, and incubated at 37 °C.

[0131] (2) OD was measured every 1 hour using an enzyme-linked immunosorbent assay (ELISA) reader. 600 Values. Each experimental group was repeated 3 times.

[0132] The results are as follows Figure 6 As shown, when the phage MOI=10, the bacterial count can be reduced to OD within 3 hours. 600 =0.1, which can rapidly reduce the number of bacteria to the minimum within 5 hours, indicating that bacteriophage P32M-3-Y has a good in vitro bactericidal effect.

[0133] Example 8: In vivo therapeutic effect of Escherichia coli bacteriophage

[0134] Eighteen C57BL / 6 mice aged 29-35 days were randomly divided into two groups: a control group of 8 mice and a phage:bacterial ratio (MOI) of 10 mice (10 mice). All mice in both groups were intraperitoneally injected with 10... 8 CFU E. coli culture, such as Figure 7As shown, mice began to exhibit lethargy after 2 hours. The MOI=10:1 group received an intraperitoneal injection of 10⁹ PFU of phage fluid, while the Control group received an intraperitoneal injection of the same volume of PBS. Results showed that after 24 hours, the survival rate of mice in the Control group was only 25%, while the MOI=10:1 treatment group had 100% survival. Even after 48 hours, the survival rate in the Control group remained at 30%, indicating that the use of phage P32M-3-Y significantly delayed and reduced mouse mortality.

[0135] The above embodiments are preferred implementations of the present invention. In addition, the present invention can be implemented in other ways. Any obvious structural, process or formula substitutions without departing from the concept of the present invention are within the protection scope of the present invention.

Claims

1. A high-risk phage strain of lysing *Escherichia coli* that targets multiple serotypes, characterized in that... The bacteriophage was named Escherichia phage vB_EcoM_32M3Y and is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 45969.

2. A bactericidal composition, characterized in that, Includes the bacteriophage as described in claim 1.

3. Use of the bacteriophage of claim 1 in the preparation of a medicament for treating or preventing bacterial infections in mice caused by Escherichia coli; The phage targets the B5 / H24RxC subtype of the high-risk clone ST410 Escherichia coli.

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