Phage for splitting escherichia coli high-risk clone strain and targeting multiple serotypes and application

Abstract

The invention relates to the technical field of microorganisms, in particular to a bacteriophage capable of splitting a high-risk escherichia coli clone strain and targeting multiple serotypes and application of the bacteriophage. The invention discloses a bacteriophage capable of splitting a high-risk escherichia coli clone strain and targeting multiple serotypes, the bacteriophage is named as Escherichia phage vBEcoM32M3Y, the bacteriophage is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.45969. The invention further discloses a preparation method of the bacteriophage. The bacteriophage disclosed by the invention is low in MOI, good in stability, good in cracking activity, free of drug-resistant and virulence genes in a genome sequence, and relatively good in clinical application value.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a bacteriophage capable of lysing a high-risk clone of Escherichia coli and targeting multiple serotypes and its application. Background Art

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

[0003] Among the Enterobacteriaceae, high-risk clones of Escherichia coli, Klebsiella pneumoniae, Salmonella and other species have attracted the attention of researchers. They are often important causes of diseases such as urinary tract infections, bloodstream infections, pneumonia, and gastroenteritis (Paterson, 2006). Escherichia coli ST410 is an extraintestinal pathogenic Escherichia coli associated with multidrug resistance and has been recognized as a new high-risk clone (Roer et al., 2018). In many carbapenem-resistant, extended-spectrum β-lactamase-producing Enterobacteriaceae (Carbapenem-resistant Enterobacteriae , CRE; Extended-spectrum Beta-lactamase producing Enterobacteriae In the monitoring of ESBL resistance, ST410 is frequently detected and often carries bla OXA-181 , bla CTX-M-15 , bla NDM-5In addition to clinical practice, 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), and has become a major threat to global public health.

[0004] The crisis of phages being unavailable for treatment in the future has caused widespread concern, and there is an urgent need to find alternative treatments to antibiotics. Bacteriophages (phages) are a type of virus that parasitizes prokaryotic microorganisms such as bacteria and archaea. They are vividly called "bacterial viruses" or "bacterial predators" and are the most abundant and diverse biological entities on Earth. It is estimated that the number of bacteriophages on Earth is 10 31 In some ecosystems, the number of bacteriophages is even 10 times that of bacteria, and their existence plays a very important role in maintaining the balance of the earth's biosphere and ecosystem. Phages, as viruses of bacteria, can be used to treat bacterial infections, especially infections of multidrug-resistant bacteria. However, phage therapy also has limitations, such as the high host specificity of phages, which leads to a limited range of phage treatment, and phages have not been widely used for a long time. But compared with other treatment methods, phage therapy has the characteristics of strong specificity, no destruction of bacterial structure, and continuous self-replication and amplification during bacterial lysis, making it one of the hot spots of antibiotic alternative therapy. Therefore, specific phages can be mined for high-risk clones to establish a phage library that can be used for clinical treatment. Summary of the invention

[0005] In order to overcome the shortcomings of the prior art, aiming at the treatment difficulties of multi-drug resistant clone infections and the limitation of the narrow phage lysis range, the present application provides a phage that lyses high-risk clones of Escherichia coli and targets multiple serotypes and its application. The phage has low MOI, good stability, good lysis activity, no resistance and virulence genes in the genome sequence, and has good clinical application value.

[0006] The technical solution adopted by the present invention to solve its technical problem is: A bacteriophage that lyses high-risk clones of Escherichia coli and targets multiple serotypes, the bacteriophage is named Escherichia phage vB_EcoM_32M3Y, and is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration with a deposit number of CGMCC No.45969.

[0007] Preferably, the source of the bacteriophage is farm wastewater.

[0008] Preferably, the bacteriophage belongs to the Ackermannviridae family, the Aglimvirinae subfamily, the Agtrevirus genus.

[0009] 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%, and the genome of the bacteriophage does not contain virulence factors and drug resistance genes.

[0010] Preferably, the optimal infection multiplicity of the bacteriophage to Escherichia coli is 0.00001:1, the incubation period is 40 min, the burst period is 70 min, and the burst volume is 108±10 PFU / cell.

[0011] Preferably, the bacteriophage contains four different tail spike proteins and can lyse four serotypes of Escherichia coli.

[0012] Preferably, the bacteriophage maintains good activity at 4-50° C. and / or maintains good activity in an environment with a pH of 3-12.

[0013] The present invention also provides a bactericidal composition, comprising the above-mentioned bacteriophage.

[0014] The present invention also provides the use of bacteriophage in preparing medicine or preparation for treating or preventing bacterial infection in mice caused by Escherichia coli.

[0015] Preferably, the phage targets the B5 / H24RxC subtype of the high-risk clone strain ST410 of Escherichia coli and / or targets a specific serotype of Escherichia coli and / or targets the O antigen.

[0016] The beneficial effects of the present invention are: 1. Bacteriophage provided by this application Escherichia phage vB_EcoM_32M3Y can lyse the B5 / H24RxC subtype of the high-risk clone strain of ST410 Escherichia coli, which is characterized by carbapenem resistance and high virulence and is widely prevalent and spread in human clinical medicine and animal husbandry.

[0017] 2. The phage provided in this application has low MOI, good stability, good lytic activity, no drug resistance and virulence genes in the genome sequence, and has good clinical application value.

[0018] 3. The bacteriophage provided in the present application has four tail spike proteins, and its receptor is O antigen. It can lyse four serotypes of Escherichia coli, including ST410 Escherichia coli carrying Onovel1 and ST9388 Escherichia coli carrying another untyped O antigen. It has a wider phage spectrum than bacteriophages with a single tail spike protein.

[0019] Storage information description: A bacteriophage that lyses high-risk clones of Escherichia coli and targets multiple serotypes is named Escherichia phage vB_EcoM_32M3Y was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on April 24, 2024, with the deposit number CGMCC No.45969. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0021] Figure 1 The morphological observation of phage plaques during the phage screening process described in this application; Where a: single plaque morphology of bacteriophage; b: halo on the surface of bacteriophage; Figure 2 This is a transmission electron micrograph (46000×) of the bacteriophage described in this application; Figure 3 is the optimal infection multiplicity of the bacteriophage described in this application; Figure 4 The stability evaluation results of the bacteriophage described in this application Where a: temperature stability b: pH stability; Figure 5 is a one-step growth curve of the bacteriophage described in this application; Figure 6 The in vitro therapeutic effect (bactericidal curve) of the bacteriophage described in this application; Figure 7 The therapeutic effect of the phage described in this application on mice; Figure 8 The complete genome information of the bacteriophage described in this application; Fig. 9 Alignment of TSP sequences of bacteriophages described in this application; Wherein a: TSP sequence of P32M-3-Y; b: alignment of different TSP sequences. DETAILED DESCRIPTION

[0022] In order to facilitate the understanding of those skilled in the art, the present invention is further described below in conjunction with embodiments, and the contents mentioned in the implementation modes are not intended to limit the present invention.

[0023] If the reagents or instruments used in the following text do not specify specific techniques or conditions, then the experiments shall be carried out according to conventional experimental conditions. If there is no clear instruction manual provided by the reagent company, then the conditions recommended in the instruction manual shall be followed. If the reagents or instruments used do not specify the manufacturer, they are all conventional products that can be purchased commercially.

[0024] Terminology explanation: Drug resistance: refers to the ability of bacteria and other microorganisms to resist drugs such as antibiotics, making it impossible for originally effective drugs to inhibit or kill these microorganisms.

[0025] High-risk clones: refers to bacterial strains with high drug resistance and high virulence. They can be spread through cloning and spread globally, posing a threat to public health.

[0026] Carbapenems: A class of broad-spectrum antibiotics used to treat a wide range of bacterial infections, particularly those that are resistant to other antibiotics.

[0027] ESBL (Extended-Spectrum Beta-Lactamases): Extended-spectrum β-lactamases are a class of enzymes that can hydrolyze a variety of β-lactam antibiotics, causing bacteria to become resistant to these antibiotics.

[0028] MOI (Multiplicity of Infection): Multiplicity of infection refers to the ratio of phage particles to host bacterial cells in a phage infection experiment.

[0029] PFU (Plaque-Forming Units): Plaque-forming units, used to measure the concentration of bacteriophages. One PFU represents one phage particle that can produce visible plaques.

[0030] Latency period: refers to the time period from when a bacteriophage infects a bacterium to when the bacterium lyses and begins to release new bacteriophages. This is the process by which bacteriophages replicate and assemble in bacteria.

[0031] Burst period: refers to the stage when the newly released phages increase in large numbers in a short period of time after the bacteria are lysed by phages. This is a key stage in the phage reproduction cycle.

[0032] Open Reading Frame (ORF): It is a segment in the DNA sequence that can encode proteins and starts from the start codon and ends with the stop codon without being interrupted by other non-coding sequences.

[0033] ST type (Sequence Type): Sequence type, a bacterial typing method based on the polymorphism of certain gene sequences in the bacterial genome.

[0034] O Antigen: The polysaccharide part of the outer membrane of the Escherichia coli cell wall, which is related to the serotype classification of bacteria.

[0035] 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.

[0036] Agtrevirus : A classification of bacteriophages belonging to Ackermannviridae Virology.

[0037] VirulenceFinder and ResFinder: two bioinformatics tools for predicting virulence factors and drug resistance genes in bacterial genomes.

[0038] NCBI-blastn: An algorithm for nucleic acid sequence alignment that can find similarities between sequences.

[0039] FastANI: A software for calculating average nucleotide identity between bacterial or bacteriophage genomes.

[0040] Semisolid Medium: A medium made by adding a small amount of coagulant (such as agar) to a liquid medium. Its degree of coagulation is between that of liquid and solid medium. It is often used to observe the growth and spread of microorganisms such as bacteriophages.

[0041] The present invention provides a bacteriophage that lyses high-risk clones of Escherichia coli and targets multiple serotypes, which is classified and named as Escherichia coli phage Escherichia phage vB_EcoM_32M3Y (abbreviated as P32M-3-Y), the deposit number is CGMCC No.45969, the deposit date is April 24, 2024, the depositor is the General Microbiology Center of China Microbiological Culture Collection Administration, and the address is the Institute of Microbiology, Chinese Academy of Sciences.

[0042] We have now discovered that the bacteriophage described in this application belongs to Ackermannviridae Virus ， Aglimvirinae Subfamily, Agtrevirus The genome of bacteriophage P32M-3-Y is a linear double-stranded DNA with a size of 152,897 bp and a G+C content of 49%. 187 open reading frames (ORFs) were successfully annotated. VirulenceFinder and ResFinder prediction results showed that the P32M-3-Y genome did not contain virulence factors and drug resistance genes.

[0043] Furthermore, the optimal infection multiplicity of the phage against E. coli was 0.00001:1, the incubation period was 40 min, the burst period was 70 min, and the burst volume was 108 ± 10 PFU / cell.

[0044] Furthermore, the phage maintains good activity at 4 to 50°C and in an environment of pH 3 to 12. The phage has good temperature stability and excellent acid-base tolerance.

[0045] Furthermore, bacteriophages have a good therapeutic effect in a mouse bacterial infection model and can significantly delay and reduce the mortality rate of mice.

[0046] Furthermore, the phage targets the B5 / H24RxC subtype of the high-risk clone strain ST410 of Escherichia coli, which is characterized by carbapenem resistance and high virulence.

[0047] Furthermore, the phage can target four serotypes of E. coli, including ST410 E. coli carrying Onovel1 and another untyped O-antigen ST9388 E. coli, both of which are prevalent clones that persist and spread in the aquaculture field and have a broad phage spectrum.

[0048] Furthermore, the phage specifically targets the antigen O antigen and has serotype specificity. Phage genome analysis showed that the phage contained four different tail spike proteins (TSP), which were named TSP1, TSP2, TSP3 and TSP4 in order according to the transcription direction, and there was no sequence similarity between the four TSPs. Five sequences most similar to the TSP sequence of the phage were screened by NCBI-blastn. The TSP sequences of different phages were conserved only at the N-terminus, while there were obvious differences in the C-terminal domain that recognized and degraded the receptor, indicating that the host range of the phage was significantly different from that of other phages of the same genus, and could supplement the limitations of the host range of existing phages.

[0049] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0050] Prepare the materials: The formula of LB nutrient agar is: 10.0 g tryptone, 5.0 g yeast extract powder, 10.0 g sodium chloride, 15.0 g agar, and 1000 mL distilled water.

[0051] The formula of LB broth is: 10.0 g of tryptone, 5.0 g of yeast extract powder, 10.0 g of sodium chloride, and 1000 mL of deionized water.

[0052] The formula of SM solution is: 8.5 g sodium chloride, 2 g magnesium sulfate, 50 mL 1 mol / L Tris-HCl, 0.25 g gelatin, and 1000 mL deionized water.

[0053] The formula of LB semisolid agar is: 7.2 g of tryptone, 3.6 g of yeast extract powder, 7.2 g of sodium chloride, 7.0 g of agar, and 1000 mL of deionized water.

[0054] Sterile double concentrated broth with 2mM CaCl2: LB broth powder 20g, CaCl2 88.78mg, deionized water 400mL.

[0055] Example 1 Isolation, preparation and purification of bacteriophages.

[0056] 1. Sample Pretreatment (1) Collect wastewater from a slaughterhouse in Guangzhou, store it at low temperatures, and then transport it back to the laboratory; (2) Remove larger particles through gauze filtering; (3) Centrifuge the sample at 4000 g for 20 min to remove bacteria and other impurities; (4) Use 0.45 μm and 0.22 μm microporous filter membranes to sterilize and filter bacteria; (5) Using a small tangential flow, the filtered sewage sample is ultrafiltered and concentrated into about 50 mL of liquid to obtain a phage stock solution, to which a small amount of chloroform may be added.

[0057] 2. Isolation of bacteriophage: (1) Take 10 mL of sterile double concentrated broth containing 2 mmol / L CaCl2 and add 10 mL of clarified (or filtered) sewage; (2) Inoculate 0.1 mL of overnight host bacterial culture and culture at an appropriate growth temperature (usually 37°C) with gentle shaking (50 rpm / min); (3) After 24 to 48 hours of incubation, centrifuge at 10,000 g for 10 minutes. Pour the supernatant into a vial with a screw cap or a stoppered test tube. Add 0.5 mL of chloroform to the clarified crude lysate, shake gently and store at 4°C.

[0058] 3. Phage spot test (1) 100 μL of overnight cultured bacteria was mixed with 5 mL of semi-solid medium melted and kept at 50°C and poured onto the bottom agar plate to prepare a bacterial lawn of each strain.

[0059] (2) Add several drops of 5 μL of the sample enrichment solution to the bacterial lawn until the droplets are completely dry.

[0060] (3) Place the plate in a 37°C incubator and incubate overnight. Check for clear or turbid lysis zones.

[0061] 4. Isolation of bacteriophage (1) Add 0.9 mL of SM Buffer to a sterile test tube or centrifuge tube and number the tube according to the dilution. (2) Add 0.1 mL of phage enrichment solution to the first tube, mix well, and transfer 0.1 mL to the second tube. Repeat this method to perform 10-fold dilutions; (3) Transfer 0.1 mL of phage dilution to a warm semi-solid culture medium test tube, immediately add 0.1 mL of host bacteria cultured to the logarithmic growth phase, mix well, and introduce to the surface of the solid culture medium; after the semi-solid layer solidifies, invert and culture at the optimal culture temperature. Culture for 8 to 10 hours, and select a plate with a single plaque for phage purification.

[0062] 5. Purification of phage (1) Pick up a single phage plaque with a sterilized pipette tip, suspend it in 1 mL of SM Buffer, and vortex thoroughly to fully dissolve the phage in SM Buffer. (2) Filter using a 0.22 μm pore size filter to remove bacteria; (3) Dilute the SM Buffer mixed with the plaques appropriately and use the double-layer plate culture method to continuously propagate the phages. When the observed plaques are basically consistent in size and morphology, purified phage individuals can be obtained, such as Figure 1 As shown, the Escherichia coli phage P32M-3-Y (Escherichia phage vB_EcoM_32M3Y) was obtained, with the deposit number being CGMCC No.45969.

[0063] 6. Preservation of bacteriophage In a 2 mL storage tube, mix 0.5 mL of phage stock solution with 0.5 mL of sterile glycerol to a final concentration of 50%. Store in a -80 ℃ refrigerator.

[0064] Example 2: Electron microscopic observation of bacteriophage (1) Take the phage culture prepared in Example 1 and use a 100 kDa Amicon Ultra centrifugal ultrafiltration tube to concentrate 50 mL of the phage liquid to 2 mL; (2) Take 15 μL of concentrated phage solution and drop it on the copper grid and let it settle for 15 min; (3) Use filter paper to gently absorb excess liquid on the copper mesh, then add 2% phosphotungstic acid (PTA, pH = 7.0) to the copper mesh and stain it for 10 min. After the copper mesh is dry, perform electron microscopy observation and photography.

[0065] The results are as follows Figure 2 As shown, the morphology of Escherichia coli phage P32M-3-Y was observed under a transmission microscope and it was found 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.

[0066] Example 3: Extraction and sequencing of bacteriophage genome (1) Take the phage culture prepared in Example 1 and use a 100 kDa Amicon Ultra centrifugal ultrafiltration tube to concentrate 50 mL of the phage liquid to 2 mL; (2) Take 2 mL of phage sample that has been filtered through a 0.22 µm microporous filter and place it in a 15 mL centrifuge tube with a smooth surface that has been sterilized by high pressure; (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. (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 to mix, and let stand at room temperature for 30 min. (5) Use the bacteriophage genome extraction kit (M13 Isolation Kit D6900) to extract genomic DNA according to the instructions.

[0067] The extracted phage product was sent to Annoroad for sequencing, and the nucleotide sequence of Escherichia coli phage P32M-3-Y was obtained. The genome annotation results are as follows Figure 8As shown in the figure, the genome of bacteriophage P32M-3-Y is a linear double-stranded DNA with a size of 152,897 bp and a G+C content of 49%. 187 open reading frames (ORFs) were successfully annotated. VirulenceFinder and ResFinder prediction results showed that the P32M-3-Y genome did not contain virulence factors and drug resistance genes. The phage genome of phage P32M-3-Y was compared using blastn, and phage sequences with a query coverage of >85% were selected. The average nucleotide identity (ANI) was calculated using FastANI software. P32M-3-Y was closest to Escherichia phage 2307YX22 (GenBank: OR776998.1), with a consistency of 98.0957%. They both belonged to the Ackermannviridae family, Aglimvirinae subfamily, and Agtrevirus genus.

[0068] Example 4: Confirmation of the optimal infection multiplicity of phage (1) Cultivate the host bacteria to the logarithmic growth phase, dilute them tenfold, and then plate them for counting; (2) Perform a double-layer plate test on the phage fluid to determine the phage titer; (3) Adjust the concentration of bacterial solution and the titer of phage solution to appropriate amounts, mix the phage solution and bacterial solution in a ratio of 1:1 at MOI = 10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, 0.000001, and 0.000001, respectively, and let stand for 10 min; (4) Add fresh LB broth to make up to 10 mL and incubate at 37 °C, 180 rpm, and shake for 4 h. (5) Centrifuge the mixture at 10,000 g for 2 min, take the supernatant and filter it through a 0.22 μm filter membrane; (6) The phage titer of each group was determined by double-layer plate test, with three replicates for each experimental group.

[0069] 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 (5.73×1010 PFU / mL).

[0070] Example 5: Determination of the stability of bacteriophage P32M-3-Y (1) Temperature stability: Cultivate the host bacteria to the logarithmic growth phase. Set the metal bath to 4 ℃, 25 ℃, 37 ℃, 50 ℃, 60 ℃ and 70 ℃ in advance. After the temperature stabilizes, place the phage solution in each temperature for 1 h. After the incubation is completed, perform a double-layer plate test to determine the phage titer. Each experimental group is repeated three times; if Figure 4 a The results showed that the phage titer did not change significantly when incubated at 4 ℃, 25 ℃, 37 ℃ and 50 ℃ for 1 h, and the phage maintained good activity, indicating that P32M-3-Y has good temperature stability.

[0071] (2) pH stability: Cultivate the host bacteria to the logarithmic growth phase. Adjust the pH value of the LB broth to 3-12 in advance, add phages to the LB broths with different pH values ​​at a ratio of 1:10 and incubate at room temperature for 1 h. After the incubation is completed, perform a double-layer plate test to determine the phage titer. Perform three replicates for each experimental group. Figure 4 b. When the phage P32M-3-Y was incubated at a pH of 6 to 12 for 1 h, the phage titer did not change significantly and was able to maintain a relatively stable level. When it was incubated at a pH of 3 to 5 for 1 h, the phage titer decreased slightly, but the change did not exceed 1 order of magnitude, indicating that P32M-3-Y has good tolerance to pH.

[0072] Example 6: Determination of the lysis spectrum of bacteriophage P32M-3-Y 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 bacterial solution was added to the LB agar plate, and 4.8 mL of semi-solid was added, shaken thoroughly, and cooled; (2) Take 5 μL of phage liquid with a titer of 1×108 PFU / mL and drop it in the center of the semi-solid. After the liquid is completely absorbed, place it in a 37 ℃ incubator for overnight culture. Bacteria that can form plaques are recorded as "+" for positive results, and vice versa as "-" for negative results. The lysis effects are summarized in Tables 1 and 2.

[0073] Table 1 Lysis effect of bacteriophage P32M-3-Y on ST410 strain

[0074] Table 2 Lysis effect of bacteriophage P32M-3-Y on various popular ST types of Escherichia coli

[0075] From Tables 1 and 2, it can be found that phage P32M-3-Y has a lytic effect on 26 strains of ST410 Escherichia coli, with a lysis rate of 52% (26 / 50). According to the evolutionary lineage classification of ST410 strains by Roer et al. (2018) and Ba et al. (2024), the ST410 strains preserved in this laboratory belong to three clusters: B3 / H24Rx, B4 / H24RxC, and B5 / H24RxC. P32M-3-Y has a lytic effect on some strains in B3 / H24Rx and B5 / H24RxC. After serotype analysis, the O antigens of these strains are all new Onovel. In addition to the ST410 strain, P32M-3-Y has a lytic effect on ST9388 Escherichia coli, which encodes a serotype different from Onovel1.

[0076] for Ackermannviridae For phages of the Viridae family, their receptor recognition proteins are generally tail spike proteins (TSPs), and they can encode up to four different tail spike proteins (Sørensen et al., 2021). Compared with phages containing only a single TSP, they have a wider host range. Phage P32-3-Y contains four different tail spike proteins, which are named TSP1, TSP2, TSP3, and TSP4 according to the transcription direction. Fig. 9 As shown in a. Five sequences most similar to the TSP sequence of P32M-3-Y were screened by NCBI-blastn. The sequence alignment results are shown in Fig. 9 As shown in Figure 2, the TSP sequences of different phages are only conserved at the N-terminus, while there are obvious differences in the C-terminal domains that recognize and degrade receptors. These catalytic domains are highly diverse and have been shown in many studies to bind to polysaccharide receptors (such as O antigen or K antigen) (Hsu et al., 2013; Greenfield et al., 2019; Plattner et al., 2019). TSP4 is highly similar to Escherichia coli phage AV101 (GenBank: WJJ54142.1) (coverage 100%, consistency 78.34%), suggesting that the two are likely to recognize similar receptors. TSP1-4 of AV101 phage have been shown to recognize and degrade O8, O82, O153 and O159 antigens (Sorensen et al., 2024). It is speculated that P32M-3-Y can also recognize four different O antigens.

[0077] Example 7: In vitro therapeutic effect of Escherichia coli phage (1) If Figure 5As shown, the host bacteria were cultured to the logarithmic growth phase, and the phage solution and bacterial solution were mixed in a ratio of 1:1 at MOI = 10, 1, 0.1 and 0.01. The blank group selected LB broth and bacterial solution mixed in a ratio of 1:1 and cultured at 37 °C; (2) Measure OD with an ELISA reader every 1 h 600 Each experimental group was repeated 3 times.

[0078] The results are as follows Figure 6 When the phage MOI = 10, the bacterial count can be reduced to OD within 3 hours. 600 =0.1, and the number of bacteria can be quickly reduced to the lowest within 5 h, indicating that bacteriophage P32M-3-Y has a good in vitro bactericidal effect.

[0079] Example 8: In vivo therapeutic effect of Escherichia coli phage Eighteen C57BL / 6 mice aged 29-35 days were divided into two groups: a blank group (i.e., control group) with eight mice and a group with a phage:bacteria ratio (MOI) of 10:1 with ten mice injected intraperitoneally. 8 CFU of Escherichia coli, such as Figure 7 As shown, 2 hours later, the mice began to show symptoms of mental depression. The MOI=10:1 group was intraperitoneally injected with 109 PFU of phage solution, and the Control group was intraperitoneally injected with the same volume of PBS. The results showed that after 24 hours, the survival rate of mice in the Control group was only 25%, while the mice in the MOI=10:1 treatment group survived 100%, and after 48 hours, the survival rate of mice in this group was still 30%, indicating that the use of phage P32M-3-Y can significantly delay and reduce the mortality rate of mice.

[0080] The above embodiments are preferred implementation schemes of the present invention. In addition, the present invention may also be implemented in other ways. Any obvious structural, process or formula replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. A bacteriophage that lyses high-risk clones of Escherichia coli and targets multiple serotypes, characterized in that: The bacteriophage is named as Escherichia phage vB_EcoM_32M3Y and is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration with a deposit number of CGMCC No.45969.

2. The bacteriophage according to claim 1, characterized in that The source of the bacteriophage is farm wastewater.

3. The bacteriophage according to claim 1, characterized in that The bacteriophage belongs to the Ackermannviridae virus family, the Aglimvirinae subfamily, and the Agtrevirus genus.

4. The bacteriophage according to claim 1, characterized in that 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 and drug-resistant genes.

5. The bacteriophage according to claim 1, characterized in that The optimal infection multiplicity of the bacteriophage to Escherichia coli is 0.00001:1, the incubation period is 40 min, the burst period is 70 min, and the burst volume is 108±10 PFU / cell.

6. The bacteriophage according to claim 1, characterized in that The bacteriophage contains four different tail spike proteins and can lyse four serotypes of Escherichia coli.

7. The bacteriophage according to claim 1, characterized in that The bacteriophage maintains good activity at 4-50° C. and / or maintains good activity in an environment with a pH of 3-12.

8. A bactericidal composition, characterized in that: The bacteriophage comprising any one of claims 1 to 7.

9. Use of the bacteriophage according to any one of claims 1 to 7 in the preparation of a medicament or preparation for treating or preventing bacterial infection in mice caused by Escherichia coli.

10. The use according to claim 9, characterized in that The bacteriophage targets the B5 / H24RxC subtype of the high-risk clone strain ST410 type Escherichia coli and / or targets a specific serotype of Escherichia coli and / or targets the O antigen.

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