Plasmid-dependent bacteriophage and uses thereof

Abstract

The application discloses a plasmid-dependent bacteriophage and application thereof. The plasmid-dependent bacteriophage has a preservation number of CGMCC No.45976, is preserved in the China General Microbiological Culture Collection Center on May 9, 2024, and has a preservation name of Escherichia coli Escherichia phage vB_EcoP_LHP, and / or the plasmid-dependent bacteriophage has a preservation number of CGMCC No.45977, is preserved in the China General Microbiological Culture Collection Center on May 9, 2024, and has a preservation name of Escherichia coli Escherichia phage vB_EcoP_IUE. The plasmid-dependent bacteriophage can be used for preventing and treating soil pollution.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microorganisms, in particular to a plasmid-dependent bacteriophage and uses thereof. BACKGROUND

[0002] Bacteriophages are viruses that infect prokaryotes, and are the most abundant biological entities on earth so far, which can specifically infect bacterial hosts in lysogeny or lysis or both. The overuse of antibiotics accelerates the emergence of multi-drug resistant (MDR) bacteria. According to the United Nations, by 2050, 10 million people will die each year due to MDR bacterial infections, which will pose a serious threat to society and economy 1 . Bacteriophages are attracting attention as an alternative to antibiotics due to their good efficacy and safety in in vitro models and human studies 2 . Unlike antibiotics, bacteriophages have lower development costs, are sustainable and renewable, and can ensure the biosafety of bacteriophages by screening the bacteriophage genome to exclude antibiotic resistance genes (ARGs), virulence factors and lysogeny-related genes 3 . In addition, bacteriophages can specifically lyse susceptible bacteria without affecting non-host bacteria, and the bacteriophages will also decrease after the pathogenic host bacteria are inactivated, thereby maintaining the stability and diversity of microorganisms 4 . Therefore, bacteriophage therapy can be used to treat diseases caused by bacterial infections, including by MDR pathogenic bacteria 5 .

[0003] In addition to disease treatment, bacteriophage therapy also has great potential in food safety, such as food detection and food pathogen prevention. Studies have shown that the addition of bacteriophages can reduce the pathogen density in tomato planting systems, increase soil bacterial diversity, and reduce the disease incidence by 80% 6 . Considering that strong lytic bacteriophages have the ability to infect and quickly kill bacteria, the isolation and screening of lytic bacteriophages and the evaluation of the ability of bacteriophages to control soil antibiotic resistant bacteria (ARB) pollution as a biological control technology can provide theoretical support and technical guarantee for the environmental application of bacteriophages. SUMMARY

[0004] The present application utilizes a strong lytic bacteriophage isolated from the environment by multi-drug resistant bacteria E. coli K12 (RP4), and evaluates the lytic ability and biosafety of the bacteriophage by analyzing the biological characteristics, genome and phylogenetic relationship of the bacteriophage. In addition, by establishing a pure bacteriophage-soil microcosm model, the effect of single species bacteriophage technology in preventing and controlling soil ARB pollution is explored.

[0005] The present application aims to provide a plasmid-dependent strong lytic bacteriophage. It has a biological control effect on E. coli K12 (RP4) contaminated soil.

[0006] To achieve the above-mentioned purpose, the present application provides a plasmid-dependent bacteriophage, characterized in that the plasmid-dependent bacteriophage has a preservation number of CGMCC No. 45976, is preserved in the China General Microbiological Culture Collection Center on May 9, 2024, and has a preservation name of Escherichia phage vB_EcoP_LHP, and / or

[0007] The preservation number of the plasmid-dependent bacteriophage is CGMCC No. 45977, which is preserved in the China General Microbiological Culture Collection Center on May 9, 2024, and the preservation name is Escherichia phage vB_EcoP_IUE.

[0008] The present application also provides the use of the plasmid-dependent bacteriophage for preventing and controlling soil pollution.

[0009] Further, the soil pollution is that the soil is contaminated by E. coli K12 (RP4).

[0010] The present application also provides a biological control method for contaminated soil, characterized in that the plasmid-dependent bacteriophage vB_EcoP_LHP and / or the plasmid-dependent bacteriophage vB_EcoP_IUE are used.

[0011] Further, the plasmid-dependent bacteriophage is added to the contaminated soil and mixed evenly, then sterile water is added to make the humidity 70±5% of the field water holding capacity, then it is placed in a constant temperature incubator at 30±5℃ for 2-10 days, and carbon source is supplemented on the 0th, 1st, 2nd, 3rd, 5th and 7th days, and the amount of carbon source added is 2-3 mol / L of glucose.

[0012] Further, the amount of carbon source added is 2.8 mol / L of glucose.

[0013] Further, the ratio of the amount of the plasmid-dependent bacteriophage added to the content of drug-resistant bacteria in the contaminated soil is 1 PFU:1 CFU.

[0014] Further, the time for the culture is 2-7 days. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is a morphological and transmission electron microscopy of the phage.

[0016] Figure 2 is a biological property chart of the phage.

[0017] Figure 3 is a gene map and phylogenetic analysis chart of the phage vB_EcoP_LHP.

[0018] Figure 4 is a gene map and phylogenetic analysis chart of the phage vB_EcoP_IUE.

[0019] Figure 5 is a chart of the effect of phage treatment / non-treatment on E. coli K12 (RP4) contaminated soil. DETAILED DESCRIPTION

[0020] Embodiments of the present application are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar components are denoted by the same or similar reference numerals throughout. The embodiments described below are exemplary, and are intended to explain the present application, and are not to be understood as limiting the present application. In the embodiments, unless a specific technique or condition is mentioned, the technique or condition described in the literature or according to the product manual is used. In the embodiments, unless the manufacturer is mentioned, the reagent or instrument used is a general product that can be commercially available.

[0021] In the following examples, the farmland soil and river water samples used for phage screening and ARB-contaminated soil model construction were collected from Xiamen City, Fujian Province (118°05’E, 24°64’N) at a depth of 0-20 cm. The soil used for the ARB-contaminated model was sieved through a 2 mm sieve to remove dead branches, leaves, and large stone particles, and the pH of the soil was 7.25. When measuring the pH, a 1:2.5 (sample: deionized water, w / v) solution was prepared, 220 rpm, 1 h, and the pH was measured after standing using an Orion Star™ A211 pH meter (ThermoFisher Scientific, USA).

[0022] Strain E. coli K12 (RP4) was used for isolation and screening of phages, and some strains were also cultured for determination of host spectrum of phages (Table 1). Plasmid RP4 carries kanamycin resistance gene (Kan, 50 mg / mL), ampicillin resistance gene (Amp, 100 mg / mL) and tetracycline resistance gene (Tet, 50 mg / mL). Strain E. coli K12 itself is resistant to rifampicin (Rif, 100 mg / mL). LB liquid medium, LB solid medium and SM buffer (SM buffer preparation method: 5.8 g NaCl, 2 g MgS04·7H20, 50 mL Tris-Cl (1 M, pH 7.5), add ultrapure water to 1 L, autoclave) were used for culture of bacteria and isolation and purification of phages, and the culture temperature of liquid medium was 37 °C, and the culture temperature of phage culture plate was 30 °C.

[0023] Table 1 Host spectrum table of phages

[0024]

[0025]

[0026] Wherein, “+” represents that the bacteria are gram-positive bacteria, and “-” represents that the bacteria are gram-negative bacteria. In other columns, “+++” represents that the lysis area is completely transparent, “++” represents that the lysis area is relatively transparent, but there is a small amount of bacterial growth, “+” represents that the lysis area is relatively turbid, and “-” represents that the bacteria are not sensitive to the phage. The E. coli K12 used in the experiment is induced by standard strain E. coli MG1655 (ATCC 47076) by antibiotics, and is resistant to 100 mg / L of rifampicin; and the E. coli MG1655 in the table is E. coli MG1655::lacIq-pLpp-mCherry-KmR, and is resistant to 50 mg / L of kanamycin.

[0027] Example 1 Isolation and purification of phages

[0028] (1) Preparation of host bacteria liquid and pretreatment of soil samples

[0029] The frozen E. coli K12 (RP4) was inoculated on an LB plate and cultured overnight at 37 °C; a single colony was picked and inoculated in LB liquid medium, and cultured overnight at 37 °C and 120 rpm; 1 conical flask containing 50 mL of LB was taken, 5 g of soil or 10 mL of river water was added, and after overnight culture at 37 °C and 120 rpm, the precipitate was taken out and allowed to stand.

[0030] (2) Propagation and isolation of phage

[0031] Take 10 mL of supernatant after standing in step (1), centrifuge at 5000 rpm for 10 min, take 2 mL of supernatant and filter through a 0.22 μm filter; take 500 μL of filtrate through the 0.22 μm filter, add 5 mL of LB, add 100 μL of E. coli K12 (RP4) overnight culture, incubate at 37°C, 120 rpm, overnight; then, centrifuge the mixed culture of the overnight culture of phage and host bacteria at 5000 rpm for 10 min, filter through a 0.22 μm filter, and use the spot plating method 7 to spot 5 μL of filtrate on a double-layer medium containing E. coli K12 (RP4) and incubate at 30°C overnight.

[0032] (3) Purification and enrichment of phage

[0033] When plaques appear in step (2), pick them out and add to LB together with 100 μL of E. coli K12 (RP4) overnight culture, incubate at 37°C, 120 rpm, overnight; then, centrifuge the mixed culture of the overnight culture of phage and host bacteria at 5000 rpm for 10 min, filter through a 0.22 μm filter, and perform 10 1 -10 8 gradient dilution, and further purify the phage using the double-plate method 7 After purification, pick the phage from the plate and elute into SM buffer, and perform 10 1 -10 8 gradient dilution, and further purify the phage using the double-plate method, which is repeated 4-6 times until the plaques on the plate have consistent morphology; then, perform small-scale expansion, i.e., pick 1 plaque from the purified plate and add to 100 μL of fresh culture of the host in the logarithmic growth phase, add 5 mL of LB, incubate at 30°C, 120 rpm, overnight; centrifuge the mixed culture of the small-scale expanded phage and host bacteria at 5000 rpm for 10 min, filter through a 0.22 μm filter, take 4 mL of filtrate, add 500 μL of fresh culture of the host in the logarithmic growth phase, add 50 mL of LB to a conical flask, incubate at 30°C, 120 rpm, overnight (the system is expanded as needed); then, centrifuge at 5000 rpm for 10 min, filter through a 0.22 μm filter, and use the filtrate for phage preservation, phage morphology observation, phage biological property determination, genome sequencing, and establishment of a phage-controlled soil ARB infection model.

[0034] The present embodiment collects samples from river water and vegetable field soil in Xiamen City, Fujian Province, using E. coli K12 (RP4) as the host bacteria to isolate bacteriophages. The morphology, transparency and reproductive capacity of the plaques are used as evaluation criteria to screen two strains of bacteriophages with strong lytic ability and high efficiency in infecting host bacteria, named vB_EcoP_LHP and vB_EcoP_IUE Figure 1 The morphology of the plaques is shown in (a) and (b), and the morphology of the bacteriophages under a transmission electron microscope is shown in (c) and (d). Each plaque of each bacteriophage is continuously subcultured for more than 6 generations, and still forms stable plaques on the double-layer solid plate, indicating that the bacteriophages can effectively infect the host E. coli K12 (RP4).

[0035] Example 2: Observation of bacteriophage morphology and determination of biological characteristics

[0036] (1) Electron microscope morphology of bacteriophages

[0037] Phosphotungstic acid counterstaining method 8 The samples are prepared into a film, and the morphology and structure of the isolated bacteriophages are characterized by transmission electron microscopy (Hitachi H-7650 TEM). The specific process is as follows: first, 8 μL of the sample is dropped on a copper mesh and left for 5 min; the excess water is absorbed with filter paper, and 8 μL of phosphotungstic acid is dropped on the sealing film; the copper mesh carrying the sample is covered on the phosphotungstic acid droplet, and left for 10 s for staining; the excess water is absorbed with filter paper, and dried at room temperature for 15 min; the morphology of the bacteriophages is observed by transmission electron microscopy.

[0038] The morphology of the bacteriophages is observed by transmission electron microscopy, and the size of the bacteriophages is determined Figure 1 ), and the results show that the bacteriophages all have tails, belonging to the Caudoviricetes class. The head layer of the bacteriophages mainly shows a polyhedral shape, with a diameter of about 60 nm for LHP and about 71 nm for IUE.

[0039] (2) Bacteriophage lysis spectrum

[0040] The range of the isolated bacteriophages is determined by the spot plaque method, and a total of 29 bacterial strains are cultured (Table 1). Briefly, after each test strain is streaked for activation, a single colony is picked into LB, and cultured at 37°C, 120 rpm, overnight. 100 μL of the logarithmic phase strain is added to 4.5 mL of 0.7% LB semi-solid with a temperature lower than 60°C, mixed uniformly, and then poured into a 1.5% LB solid plate prepared in advance, and used after the upper semi-solid medium is solidified. The bacteriophage culture is diluted to a titer of 10 9PFU / mL, 5 μL of the phage working solution was dropped on the surface of the plate, and after the plate was dried, it was transferred to a 30°C incubator for overnight culture, and then the phage plaques were observed.

[0041] The lysis spectrum of plasmid-dependent phages LHP and IUE was explored by spot test and double plate method. A total of 29 laboratory strains were used (Table 1), including 12 strains of E. coli. The results showed that the phages mainly lysed E. coli. LHP and IUE showed different phage plaque infectivity on E. coli MG1655 containing different plasmids, i.e. LHP and IUE could effectively lyse E. coli MG1655 (RP4), but the lysis effect on E. coli MG1655 (pKJK5) was poor. In addition, LHP and IUE could efficiently lyse E. coli K12 (RP4) and E. coli K12 (pKJK5). In addition, LHP and IUE also had lysis ability on strains E. coli K12 and M. yixingense ACCC 19709.

[0042] (3) Optimal multiplicity of infection of phage

[0043] Freshly cultured phage and E. coli K12 (RP4) in the logarithmic growth phase were mixed at a multiplicity of infection (MOI) of 0.01, 0.1, 1, 10 and 100, and then 5 mL of LB was added. The mixture was incubated at 37°C, 120 rpm for 12 h, and then centrifuged at 12,000 g for 2 min at room temperature to remove residual bacterial cells. Then, the supernatant was filtered through a 0.22 μm filter. The filtered solution was gradiently diluted, and the phage titer at each MOI was determined by double plate method, in which the MOI corresponding to the highest titer of phage was the optimal MOI of the phage. Each MOI determination included three biological replicates.

[0044] E. coli K12 (RP4) was used as the host strain of plasmid-dependent phages LHP and IUE, and the host strain was mixed with different proportions of phage for culture. The results of MOI determination are shown in (a) of FIG. 8. The optimal MOI of plasmid-dependent phages LHP and IUE was 100. Figure 2

[0045] (4) One-step growth curve

[0046] Fresh phage (titer 10 8 PFU / mL) was mixed with host bacteria in the logarithmic growth phase (concentration 10 9 ​CFU / mL) were mixed homogeneously at MOI = 0.1, and incubated at 37°C for 10 min; then, the mixed culture was centrifuged at 7,000 g for 5 min, and the precipitate was eluted with LB (2 times); then, the mixed system was resuspended with LB, and incubated at 37°C at 200 rpm, and sampled according to time, and the sampling time was: 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150 min, and 0.5 mL sample was collected each time; finally, each sample was centrifuged at 16,000 g for 2 min, and the titer of the phage was determined by the double-plate method. The burst size was calculated as the ratio of the final count of released phage particles to the initial count of phage particles 9 Each sampling point included three biological replicates.

[0047] The one-step growth curve of the phage is shown in (b) of Figure 2 The latent period of LHP was 10-20 min, and then the virus particles were rapidly released, the burst period was 80 min, the burst size was 40 PFU / cell, and the final titer reached 8 x 10 8 PFU / mL; the IUE had a shorter latent period (less than 10 min), and then the virus particles were rapidly released, the burst period was 140 min, the burst size was 600 PFU / cell, and the final titer reached 6 x 10 9 PFU / mL.

[0048] (5) Thermal stability experiment

[0049] Take 0.5 mL of the phage working solution in a 1.5 mL centrifuge tube, and incubate in a constant-temperature water bath at 40, 50, 60, 70, 80 and 90°C for 1 h, and then determine the titer of the phage by the double-plate method. Each temperature included three biological replicates.

[0050] The stability of the phage was tested by incubating the phage at different temperatures for 1 h, as shown in (c) of Figure 2 The thermal stability of LHP was poor, and it lost activity after incubation at 60°C for 1 h; the IUE did not have significant changes in phage titer (P > 0.05) after incubation at 50°C or below for 1 h, and then the activity of the phage decreased significantly (P < 0.05) as the temperature increased, and it lost activity after incubation at 90°C for 1 h.

[0051] Example 3 Sequencing of the phage genome and data analysis

[0052] The phage culture was concentrated by 100-kDa ultrafiltration tubes (Millipore, USA), and the free exogenous DNA fragments in the phage concentrate were removed by DNase I (RNase-free, TransGen Biotech, China) according to the manufacturer's instructions. Subsequently, the phage DNA was extracted using the TIANamp Virus DNA / RNA Kit (Tiangen, China), and the concentration of the DNA was determined using the Qubit dsDNA HS Assay Kit (ThermoFisher Scientific, USA). The DNA samples that passed the quality control were sent to Guangzhou Meigene Scientific Biotechnology Co., Ltd. for sequencing. The DNA that passed the quality control was randomly broken into DNA fragments, and the concentration of the DNA was determined using the Qubit dsDNA HS Assay Kit (ThermoFisher Scientific, USA). The DNA fragments were constructed into a library using the NEBNext®Ultra II DNA Library Prep Kit for Illumina®(New England Biolabs, USA) according to the standard protocol. The constructed amplicon library was subjected to PE150 sequencing using the Illumina NovaSeq 6000 platform, and the sequencing throughput was 2 GB. TM 4.0 fluorometer (ThermoFisher Scientific, USA) to determine the concentration of the DNA. The DNA samples that passed the quality control were sent to Guangzhou Meigene Scientific Biotechnology Co., Ltd. for sequencing. The DNA that passed the quality control was randomly broken into DNA fragments, and the concentration of the DNA was determined using the Qubit dsDNA HS Assay Kit (ThermoFisher Scientific, USA). The DNA fragments were constructed into a library using the NEBNext®Ultra II DNA Library Prep Kit for Illumina®(New England Biolabs, USA) according to the standard protocol. The constructed amplicon library was subjected to PE150 sequencing using the Illumina NovaSeq 6000 platform, and the sequencing throughput was 2 GB. Ultra TM IIDNA Library Prep Kit for (NewEngland Biolabs,USA) standard protocol. The constructed amplicon library was subjected to PE150 sequencing using the Illumina NovaSeq 6000 platform, and the sequencing throughput was 2 GB.

[0053] After obtaining the sequencing data, the present study first used Trimmomatic (v0.36) 10 to remove the adapters and low-quality fragments of the sequences according to the default parameters, and used FastQC (v0.11.5) to perform quality control on the sequences; used SPAdes (v3.13.0) 11 to perform sequence assembly on the high-quality Reads; used RAST to predict potential genes encoding proteins; used BLASTN to search for phages with high similarity to the isolated phage in the Non-Redundant Database (NR) of the National Center for Biotechnology Information (NCBI); used the Protein Basic Local Alignment Search Tool (BLASTp) 12 to perform protein function annotation on the isolated phage; used tRNAscan-SE (v2.0) 13 to find potential tRNA encoding genes; used the online prediction platform Virulence Finder 14 and ResFinder 14Detection of virulence factors and antibiotic resistance genes in the phage genome; use of geNomad 15 Detection of lysogeny genes in the phage genome; use of GCview 16 Draw the genetic map of the phage; finally, align the protein sequences of the major coat protein and large subunit of the terminase using the ClustalW algorithm and construct and display the phylogenetic tree of the phage using the Neighbor-Joining method in MEGA11 17 with 1000 replicates. The GenBank sequence accession numbers of the relevant phages used to construct the phylogenetic tree are shown in Table 2.

[0054] Table 2 GenBank accession numbers of sequences used to construct the phylogenetic tree

[0055]

[0056]

[0057] (1) Whole genome analysis and phylogenetic analysis of vB_EcoP_LHP

[0058] By assembling and annotating the sequencing data, the genome map of E. coli phage vB_EcoP_LHP is shown in Fig. Figure 3 The genome of LHP consists of a circular double-stranded DNA with a length of 42,055 bp, with a GC content of 54.56% (Table 3). A total of 48 open reading frames (ORFs) were identified from the phage genome, of which 47 ORFs on the positive strand and 1 ORF on the negative strand. A total of 26 ORFs were predicted to be functional, mainly including five functional modules, i.e., DNA replication and regulation, metabolism, structure and packaging, and lysis, and the rest were hypothetical proteins (Table 4). The DNA replication and regulation module of LHP mainly includes DNA primase, DNA polymerase, RNA polymerase, ATP-dependent DNA helicase, etc., DNA metabolism mainly includes cytosine-specific methyltransferase and endonuclease, etc., the proteins related to the structure and packaging function of the phage mainly include major tail protein, large subunit of terminase and domain protein, etc., and the proteins related to the lysis function of the phage mainly include holin protein and lysozyme protein. Using the online software tRNAscan-SE, no tRNA genes were found in the genome of LHP, indicating that the replication of LHP depends on the translation mechanism of the host. Through the online software Virulence Finder, ResFinder and geNomad, no virulence factors, drug resistance genes or lysogeny-related genes were found in the genome of LHP, indicating that the phage has biological safety for subsequent laboratory bacterial inactivation tests and practical applications.

[0059] According to the International Committee on Taxonomy of Viruses (ICTV), species identification requires less than 95% similarity to the genome sequence, while genus identification requires less than 70%. Comparison of the LHP genome with the NCBI gene database revealed that LHP shares the highest nucleotide identity with Cronobacter phage EspYZU05 (MW882933), which belongs to the genus Cronosvirus within the subfamily Melnykvirinae of the family Autographiviridae. The sequence identity between LHP and EspYZU05 is 44.10% (coverage (59.00%) x homology (74.75%) = 44.10%). Therefore, LHP was preliminarily identified as a novel bacteriophage and deposited with the strain. The deposit information is as follows.

[0060] Strain name: Escherichia coli phage vB_EcoP_LHP;

[0061] Deposit date: May 9, 2024;

[0062] Depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China General Microbiology Center (CGMCC);

[0063] Deposit number: CGMCC No.45976.

[0064] The major capsid protein sequence of phages is highly conserved and suitable for phylogenetic analysis. In addition, the large subunit of the terminase, which assists viral assembly by pushing viral DNA into the capsid, is not only a key component of the DNA packaging machinery but is also generally well conserved among tailed phages. Therefore, to illustrate the phylogenetic relationship between LHP and other homologous phages, this study was based on the major capsid protein ( Figure 3 (b)) and the large subunit of the terminase ( Figure 3 A phylogenetic tree was constructed based on the amino acid sequences of LHP and other phages belonging to the Autographiviridae family. The results showed that LHP was highly homologous to Cronobacter phage Dev-CD-23823, a member of the genus Cronosvirus within the subfamily Melnykvirinae. This suggests that LHP and Cronosvirus phages may share similar DNA packaging mechanisms, consistent with the BLASTN alignment results. Therefore, LHP is a member of the genus Cronosvirus within the subfamily Melnykvirinae.

[0065] (2) Whole genome analysis and phylogenetic analysis of vB_EcoP_IUE

[0066] By assembling and annotating the sequencing data, the genome map of Escherichia coli phage vB_EcoP_IUE was Figure 4 (a) shows the IUE genome. The IUE genome consists of a circular double-stranded DNA of 41,174 bp in length with a GC content of 58.21% (Table 3). A total of 47 ORFs were identified from the phage genome, including 22 ORFs on the plus strand and 25 ORFs on the minus strand. A total of 16 ORFs were predicted to be functional, mainly including five functional modules, namely DNA replication and regulation, metabolism, structure and packaging, and lysis, while the rest were hypothetical proteins (Table 5). The DNA replication and regulation modules of IUE mainly include DNA primase, RNA polymerase, and DNA helicase, while DNA metabolism mainly includes cytosine-specific methyltransferase and endonuclease. Proteins related to the phage structure and packaging function mainly include the main tail protein, terminase large subunit, and domain protein, while proteins related to the phage lysis function mainly include lytic transglycokinase, holin protein, and lysozyme protein. Using tRNAscan-SE online software, no tRNA genes were found in IUE, indicating that IUE replication depends on the host's translational machinery. Virulence Finder, ResFinder, and geNomad online software also detected no virulence factors, drug resistance genes, or lysogeny-related genes in the IUE genome, demonstrating the biosafety of this phage for subsequent laboratory bacterial inactivation testing and practical applications.

[0067] Comparison of the IUE genome with the NCBI gene database revealed that IUE shared the highest nucleotide identity with Kosakonia phage Kc259 (MW258712), which belongs to the class Caudoviricetes and is not specifically classified. The sequence identity between IUE and Kc259 was 97.12% (coverage (99.00%) x homology (98.10%) = 97.12%). To clarify the phylogenetic relationships between IUE and other homologous phages, this study used the major capsid protein ( Figure 4 (b)) and the large subunit of the terminase ( Figure 4The amino acid sequence of (c)) was used to construct a phylogenetic tree of IUE and other phages with higher genome similarity. The results showed that IUE was highly homologous to Kosakonia phage Kc166B belonging to the Caudoviricetes class, indicating that IUE and phages of the Caudoviricetes class may have similar DNA packaging mechanisms, which is consistent with the BLASTN alignment results. Therefore, IUE belongs to the members of the Caudoviricetes class, and has not been classified in detail. The LHP was deposited as a bacterial strain, and the deposit information is as follows.

[0068] Strain name: Escherichia phage vB_EcoP_IUE;

[0069] Date of deposit: May 09, 2024;

[0070] Depositary: Institute of Microbiology, Chinese Academy of Sciences, No. 1, Yabian West Road, Chaoyang District, Beijing, China General Microbiological Center of China Microorganism Culture Collection Committee (CGMCC);

[0071] Accession number: CGMCC No. 45977.

[0072] Table 3 Characteristic table of phage genome

[0073]

[0074] Table 4 OFRs function prediction table of phage LHP

[0075]

[0076]

[0077]

[0078]

[0079] Table 5 OFRs function prediction table of phage IUE

[0080]

[0081]

[0082]

[0083]

[0084] Example 4 Phage control ARB pollution model

[0085] The resistant bacteria in the soil were analyzed using double-resistant plates (Rif 100 mg / L and Tet 50 mg / L). The specific operation process was as follows: 1 g of soil was taken, 5 mL of sterile SM buffer was added, and after incubation at 220 rpm for 30 min, it was ultrasonicated at 40 KHz for 10 min, and then coated on double-resistant plates and cultured at 37°C and 30°C for 24 h, respectively. After ensuring that there were no bacteria resistant to Rif and Tet in the soil, the soil was used to establish the soil ARB pollution model.

[0086] 400 g of soil was taken and fresh cultured E. coli K12 (RP4) in logarithmic growth was added (washed twice with SM buffer, and the final concentration of E. coli K12 (RP4) in the soil was 10 8 cells / g dry soil), followed by the addition of 2.8 mol / L glucose (0.6% of the amount of carbon added) (calculated based on the soil, 2.8 mol of glucose was added per 1 L of soil) 18 , mixed well, and then incubated at 30°C for 6 h. After incubation, 15 portions of soil, each weighing 20 g, were weighed into 50 mL glass beakers. Phages were added according to the experimental design.

[0087] According to the MOI of the phage (MOI LHP = 1; MOI IUE = 10), the phage and the above soil were mixed well, sterile water was added to make the humidity 70% of the field water holding capacity, and then incubated in a constant temperature incubator at 30°C. Sample collection and carbon source replenishment were performed at 0, 1, 2, 3, 5, and 7 days. Part of the collected soil samples were used for DNA extraction, and part were used for determination of the residual amount of E. coli K12 (RP4) in the soil. The specific experimental design was as follows: CK, soil; LHP, soil + LHP; IUE, soil + IUE, each treatment included three biological replicates. Through the pure phage-soil microcosm experiment, the biological control effect of the phage on the E. coli K12 (RP4) contaminated soil was evaluated Figure 5 , where (a) is the effect of phage treatment / non-treatment on the abundance of soil E. coli K12 (RP4) (a), (b) is the effect of phage treatment / non-treatment on soil bacteria, (c) is the effect of phage treatment / non-treatment on the relative abundance of gfp gene, and (d) is the effect of phage treatment / non-treatment on the absolute abundance of gfp gene).

[0088] The use of Soil DNA was extracted using the Spin Kit (MP Biomedical, USA) according to the manufacturer's instructions. The concentration of RP4 was calculated by quantifying the gfp gene, using the 16S rRNA gene as an internal reference gene. Table 6 lists the primer sequences of the target genes. Briefly, PCR was first used to obtain PCR products for all target genes. The PCR amplification system was 50 μL, i.e., 25 μL 2× LightCycler 480 Green I Master Mix (Roche Inc., USA), 1 μL of each front and back primer (10 μM), 21 μL of sterile ultrapure water, and 2 μL of soil DNA. The PCR amplification conditions were as follows: pre-denaturation at 95°C for 5 minutes, 30 cycles of amplification, each cycle including denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 30 seconds, and extension at 72°C for 10 minutes. The size of the PCR product was observed by 1% agarose gel electrophoresis at a voltage of 110 V. After ensuring the accuracy of the product size, the PCR product was purified using AMPure XP Beads (Beckman Coulter, USA); secondly, the purified target fragment was inserted into the Amp-resistant -T vector (A3600, Promega, China) and transformed into E. coli DH5α (Takara, Japan); finally, blue-white screening and resistance plate screening methods were used to obtain a strain carrying a high-copy plasmid of the target gene. The constructed strain was cultured on a large scale, and the plasmid carrying the target gene was extracted. Subsequently, the plasmid was serially diluted to obtain a standard sample of the corresponding gene, which was used to construct a standard curve for the target gene in subsequent PCR. The qPCR amplification system for each sample was 20 μL, namely 10 μL 2× LightCycler 480 SYBR Green I Master (Roche Inc., USA), 0.2 μL bovine serum albumin (BSA, 20 mg / L), 0.8 μL of each front and back primer (10 μM), 6.2 μL sterile ultrapure water, and 2 μL DNA. qPCR amplification conditions were: pre-denaturation at 95°C for 5 minutes, followed by 40 cycles of amplification, each consisting of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds (signal collection at 31 seconds). Subsequently, a melting curve analysis was performed using the following protocol: 95°C for 15 seconds, 60°C for 1 minute, and 95°C for 15 seconds. The expression level of the target gene in the sample was calculated using the qPCR standard curve.

[0089] Table 6 Primer sequences for genes gfp and 16S rRNA

[0090]

[0091]

[0092] Overall, the concentration of E. coli K12(RP4) and the abundance of RP4 in all treatments showed a downward trend with the extension of the incubation time. IUE also gradually showed strong lytic ability to E. coli K12(RP4) from the second day, and the concentration of E. coli K12(RP4) in the soil treated with IUE significantly decreased (P < 0.05; Table 7) with the extension of the incubation time, from 4.83 x 10 6 CFU / mL to 2.23 x 10 5 CFU / mL. The addition of carbon source significantly improved the absolute abundance of soil bacteria (P < 0.05; Figure 5 (b)), while the addition of carbon source did not improve the concentration of E. coli K12(RP4) in the soil (a). Figure 5 The absolute abundance and relative abundance of gfp in the soil system showed the same downward trend, and the abundance of gfp in the IUE treatment was the lowest on the 7th day of the system incubation (P < 0.05; Figure 5 (c), Figure 5 (d) and Table 7).

[0093] Table 7 Difference table of bacteriostatic activity of single phage and phage cocktail

[0094]

[0095] where CK represents no treatment, E. coli K12(RP4) represents the detected number of E. coli K12(RP4) in the soil, 16S rRNA-ABS represents the absolute abundance of 16S rRNA gene, RP4-RA represents the relative abundance of gfp gene, and RP4-ABS represents the absolute abundance of gfp gene.

[0096] 1. Usman, S. S.; Uba, A. I.; Christina, E., Bacteriophage genome engineering for phage therapy to combat bacterial antimicrobial resistance as an alternative to antibiotics. Mol. Biol. Rep. 2023, 50, (8), 7055-7067.

[0097] 2. Luong, T.; Salabarria, A. C.; Roach, D. R., Phage therapy in the resistance era: where do we stand and where are we going? Clin. Ther. 2020, 42, (9), 1659-1680.

[0098] 3. Abdelsattar, A.; Dawoud, A.; Makky, S.; Nofal, R.; Aziz, R.; El-Shibiny, A., Bacteriophages: from isolation to application. Curr. Pharm. Biotechnol. 2022, 23, (3), 337-360.

[0099] 4. Ye, M.; Sun, M.-M.; Huang, D.; Zhang, Z.-Y.; Zhang, H.; Zhang, S.-T.; Hu, F.; Jiang, X.; Jiao, W.-T., A review of bacteriophage therapy for pathogenic bacteria inactivation in the soil environment. Environ. Int. 2019, 129, 488-496.

[0100] 5. Wittebole, X.; De Roock, S.; Opal, S. M., A historical overview of bacteriophage therapy as an alternative to antibiotics for the treatment of bacterial pathogens. Virulence 2014, 5, (1), 226-235.

[0101] 6. Wang, X.-F.; Wei, Z.; Yang, K.-M.; Wang, J.-N.; Jousset, A.; Xu, Y.-C.; Shen, Q.-R.; Friman, V.-P., Phage combination therapies for bacterial wilt disease in tomato. Nature Biotechnology 2019, 37, (12), 1513-1520.

[0102] 7. Luong, T.; Salabarria, A. C.; Edwards, R. A.; Roach, D. R., Standardized bacteriophage purification for personalized phage therapy. Nat. Protoc. 2020, 15, (9), 2867-2890.

[0103] 8. Klimenko, S. M.; Tikchonenko, T. I.; Andreev, V. M., Packing of DNA in head of bacteriophage T2. Journal of Molecular Biology 1967, 23, (3), 523-533.

[0104] 9. Sun, W.-J.; Liu, C.-F.; Yu, L.; Cui, F.-J.; Zhou, Q.; Yu, S.-L.; Sun, L., A novel bacteriophage KSL-1 of 2-Keto-gluconic acid producer Pseudomonas fluorescens K1005: isolation, characterization and its remedial action. BMC Microbiol. 2012, 12, 127.

[0105] 10. Bolger, A. M.; Lohse, M.; Usadel, B., Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 2014, 30, (15), 2114-2120.

[0106] 11. Nurk, S.; Meleshko, D.; Korobeynikov, A.; Pevzner, P. A., metaSPAdes: a new versatile metagenomic assembler. Genome Res. 2017, 27, (5), 824-834.

[0107] 12. Altschul, S. F.; Madden, T. L.; Schaffer, A. A.; Zhang, J. H.; Zhang, Z.; Miller, W.; Lipman, D. J., Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997, 25, (17), 3389-3402.

[0108] 13. Schattner, P.; Brooks, A. N.; Lowe, T. M., The tRNAscan-SE, snoscan and snoGPS web servers for the detection of tRNAs and snoRNAs. Nucleic Acids Res. 2005, 33, 686-689.

[0109] 14. Kleinheinz, K. A.; Joensen, K. G.; Larsen, M. V., Applying the ResFinder and VirulenceFinder web-services for easy identification of acquired antibiotic resistance and E. coli virulence genes in bacteriophage and prophage nucleotide sequences. Bacteriophage 2014, 4, (1), 27943.

[0110] 15. Camargo, A. P.; Roux, S.; Schulz, F.; Babinski, M.; Xu, Y.; Hu, B.; Chain, P. S. G.; Nayfach, S.; Kyrpides, N. C., Identification of mobile genetic elements with geNomad. Nature Biotechnology 2023.

[0111] 16. Stothard, P.; Wishart, D. S., Circular genome visualization and exploration using CGView. Bioinformatics 2005, 21, (4), 537-539.

[0112] 17. Tamura, K.; Stecher, G.; Kumar, S., MEGA11 molecular evolutionary genetics analysis version 11. Mol. Biol. Evol. 2021, 38, (7), 3022-3027.

[0113] 18. Reischke, S.; Kumar, M. G. K.; E., Threshold concentration of glucose for bacterial growth in soil. Soil Biol. Biochem. 2015, 80, 218-223.

[0114] 19. Hale, L.; Luth, M.; Crowley, D., Biochar characteristics relate to its utility as an alternative soil inoculum carrier to peat and vermiculite. Soil Biol. Biochem. 2015, 81, 228-235.

[0115] 20. Yang, K.; Chen, Q.-L.; Chen, M.-L.; Li, H.-Z.; Liao, H.; Pu, Q.; Zhu, Y.-G.; Cui, L., Temporal dynamics of antibiotic resistome in the plastisphere during microbial colonization. Environ. Sci. Technol. 2020, 54, (18), 11322-11332.

[0116] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and purpose of the present invention.

Claims

1. A plasmid-dependent bacteriophage, characterized in that, The plasmid-dependent phage is an Escherichia coli phage ( Escherichia phage ) vB_EcoP_LHP, whose deposit number is CGMCC No.45976, or The plasmid-dependent phage is an Escherichia coli phage ( Escherichia phage ) vB_EcoP_IUE, its deposit number is CGMCC No.45977.

2. Use of the plasmid-dependent bacteriophage according to claim 1 for preventing soil pollution; the soil is contaminated with antibiotic-resistant Escherichia coli, the antibiotic being kanamycin, ampicillin, tetracycline and rifampicin.

3. Use according to claim 2, characterized in that, The soil pollution is soil contaminated with kanamycin-, ampicillin-, tetracycline- and rifampicin-resistant Escherichia coli K12.

4. A method for biological control of contaminated soil, characterized in that, The plasmid-dependent bacteriophage according to claim 1 is used for preventing soil contaminated with antibiotic-resistant Escherichia coli, the antibiotic being kanamycin, ampicillin, tetracycline and rifampicin.

5. The method for biological control of contaminated soil according to claim 4, wherein The plasmid-dependent bacteriophage according to claim 1 is added to the contaminated soil, mixed evenly, and then sterile water is added to make the humidity 70 ± 5% of the field water holding capacity, followed by incubation in a constant temperature incubator at 30 ± 5℃ for 2-10 days, and carbon source supplement is carried out on the 0th, 1st, 2nd, 3rd, 5th and 7th days, the carbon source and its addition amount being 2-3 mol / L glucose.

6. The method for biological control of contaminated soil according to claim 5, wherein The carbon source and its addition amount are both 2.8 mol / L glucose.

7. The method for biological control of contaminated soil according to claim 5, wherein The addition amount of the plasmid-dependent bacteriophage is 1 PFU: 1 CFU of the resistant bacteria content of the contaminated soil.

8. The method for biological control of contaminated soil according to claim 5, wherein The incubation time is 2-7 days.

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