Escherichia coli phage and application thereof

Abstract

The invention discloses an escherichia coli bacteriophage and application thereof, and belongs to the technical field of microorganisms. The invention relates to an Escherichia coli phage vBSflSz7, which is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC NO.45768, the preservation date is January 2, 2024, the preservation address is Institute of Microbiology, Chinese Academy of Sciences, 3 #, No.1 yard, West Beichen Road, Chaoyang District, Beijing, the postcode is 100101, and the Escherichia coli phage is classified and named as Escherichia coli phage; the phage is named as the phage vBSflSz7. The novel Escherichia coli bacteriophage obtained by separation and purification can specifically and effectively kill Escherichia coli, and has good in-vivo and in-vitro antibacterial effects on drug-resistant Escherichia coli.

Description

Technical Field

[0001] The invention relates to the technical field of microbial medicine, and in particular to an Escherichia coli phage and application thereof. Background Art

[0002] Escherichia coli ( Escherichia coli , E. coli ) is also known as Escherichia coli, which is a type of Gram-negative bacteria in the Enterobacteriaceae family. Non-pathogenic Escherichia coli is part of the healthy human intestinal flora, but pathogenic Escherichia coli is still one of the common pathogens that cause community-acquired infections and nosocomial infections, showing a global epidemic trend. Due to the irrational use of antibiotics over the years, the drug resistance of Escherichia coli has become increasingly severe, and the effect of antibiotic treatment has been poor. Even carbapenem-resistant Escherichia coli has become increasingly widespread. Antibiotic-resistant Escherichia coli has been detected many times in patients with ventilator-associated pneumonia and other types of infections in intensive care units. This type of infection is difficult to control and has a high mortality rate. It has become an important risk factor for death of patients in intensive care units.

[0003] Bacteriophages are widely distributed in nature. They are viruses that can specifically infect bacteria, causing metabolic disorders or lysis and death of host bacteria. Phage therapy is an emerging approach that uses phages to specifically inhibit infectious pathogens. In the face of increasingly severe drug resistance, phage therapy has once again attracted the attention of researchers around the world. Phages can fight pathogens including Staphylococcus, Streptococcus, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, etc., and can treat diseases including purulent wound infections, dermatitis, pneumonia, empyema, gastroenteritis, sepsis, osteomyelitis, etc. Phage therapy has the advantages of exponential proliferation, strong specificity, easy real-time update, few side effects, no residue, short cycle, and low cost, so it has broad development prospects. At present, cases of successful clinical use of phages to treat multidrug-resistant bacterial infections have been widely reported, and a large number of studies have also provided a solid theoretical basis for the transformation of phage therapy into clinical practice.

[0004] Therefore, a method is provided to specifically recognize and cleave E. coli The bacteriophages are of great significance for the treatment of infections caused by E. coli. Summary of the invention

[0005] The purpose of the present invention is to provide an Escherichia coli phage and provide a specific application of the phage to make up for the deficiencies of the prior art.

[0006] In order to achieve the above object, the specific technical solution adopted by the present invention is: An Escherichia coli phage vB_SflS_z7, the phage is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration (CGMCC), the deposit number is: CGMCC NO.45768, the deposit date is: January 2, 2024, the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, and the classification name is Escherichia coli phage Escherichia coli phage ; Named as phage vB_SflS_z7.

[0007] The application of the Escherichia coli phage vB_SflS_z7 in the preparation of anti-Escherichia coli drugs.

[0008] The application of the Escherichia coli phage vB_SflS_z7 in the preparation of anti-Escherichia coli pneumonia drugs.

[0009] Compared with the prior art, the present invention has the following beneficial effects: The novel Escherichia coli phage vB_SflS_z7 isolated and purified by the present invention can specifically and effectively eliminate Escherichia coli and has good in vivo and in vitro antibacterial effects on drug-resistant Escherichia coli. The present invention provides an experimental basis for the clinical development of preparations for preventing and treating drug-resistant Escherichia coli infections and has great clinical application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The morphology of bacteriophage vB_SflS_z7 on a double-layer agar plate.

[0011] Figure 2 Transmission electron microscope photos of bacteriophage vB_SflS_z7; a is a 20,000-fold magnified photo, and b is a 40,000-fold magnified photo.

[0012] Figure 3 The effect of different temperatures on the titer of bacteriophage vB_SflS_z7.

[0013] Figure 4 The effect of pH on the lysis ability of bacteriophage vB_SflS_z7.

[0014] Figure 5 This is the one-step growth curve of bacteriophage vB_SflS_z7.

[0015] Figure 6 This is the genome map of bacteriophage vB_SflS_z7.

[0016] Figure 7 Virus phylogenetic tree of the bacteriophage vB_SflS_z7 genome.

[0017] Figure 8Phylogeny of bacteriophage vB_SflS_z7 and the most recent 25 viral genomes.

[0018] Fig. 9 Phylogenetic and comparative genomic analysis of bacteriophage vB_SflS_z7.

[0019] Fig.10 For bacteriophage vB_SflS_z7 and virus clusters from the NCBI RefSeq database.

[0020] Fig.11 This is a heat map of shared genes between bacteriophage vB_SflS_z7 and viruses under the same VC as vB_SflS_z7.

[0021] Fig.12 To construct an evolutionary tree based on the terminal large subunit of bacteriophage vB_SflS_z7, different colors represent different bacteriophages, and * specifically represents vB_SflS_z7.

[0022] Fig.13 Figure 2 shows the bacterial load and phage load in the lungs of mice after administration of bacteria. Figure a shows the bacterial load in the lungs of mice in different treatment groups. Figure b shows the phage load in the lungs of mice in different treatment groups. # indicates p<0.05 compared with the control.

[0023] Fig.14 The lung pathological conditions of mice in different groups. The Con group was only added with saline, the Phage group was only added with vB_SflS_z7 phage, the E.coli group was only added with E. coli, and the E.coli+Phage group was added with vB_SflS_z7 phage 2 hours after the addition of E. coli.

[0024] Fig.15 is the content of TNF-α and IL-6 cytokines in different groups of mice 24 hours later; a is the content of TNF-α cytokines in different treatment groups of mice 24 hours later, and b is the content of IL-6 cytokines in different treatment groups of mice 24 hours later. # and * represent p<0.05 compared with the control. DETAILED DESCRIPTION

[0025] The present invention is further explained and illustrated below through specific embodiments in conjunction with the accompanying drawings.

[0026] Example 1: Isolation and purification of bacteriophage vB_SflS_z7 Escherichia coli is commercially available and used for screening and identification of bacteriophages isolated from sewage.

[0027] The method for isolating bacteriophage vB_SflS_z7 comprises the following steps: (1) Use a 0.22 μm microporous membrane filter to filter hospital sewage to remove bacteria and other impurities; (2) Escherichia coli was used as the host bacteria to screen and identify bacteriophages isolated from sewage; the presence of bacteriophages was detected by double-layer plate method: Solid LB medium: 1% (wt / vol) peptone, 1% (wt / vol) agar.

[0028] Semisolid LB medium: 1% (wt / vol) fish meal peptone, 0.75% (wt / vol) agar.

[0029] Liquid LB medium: 1% (wt / vol) fish meal peptone.

[0030] (3) Mix E. coli with filtered sewage, spread on plates, and obtain plaques, such as Figure 1 As indicated, the cells were incubated overnight at a constant 37°C.

[0031] (4) Repeat the double-layer plate test, such as Figure 1 As shown, pick 3 to 5 single plaques and purify the phages until the plaques have a uniform size.

[0032] (5) Suspend the purified phage in 3-5 mL of sterile SM buffer with the following formula: 100mM NaCl 81.2mM MgSO4·7H2O 50 mM Tris-HCl (pH 7.5) 0.01% gelatin (6) Store the purified phage vB_SflS_z7 at 4°C.

[0033] Example 2: Characteristic Experiment of Bacteriophage vB_SflS_z7 1. Electron microscopy test and titer determination: (1) Observation of phage morphology using transmission electron microscopy The morphology of phage vB_SflS_z7 was observed using a transmission electron microscope (TEM). 20 μL of concentrated purified phage solution (109 PFU / mL) was dropped onto a carbon-coated copper grid and air-dried after 1 minute of exposure. Subsequently, the sample was negatively stained with 2% phosphotungstic acid (pH 7.5) for 5 minutes. TEM (JEOL JEM-1200EX, Japan) was used for observation at an accelerating voltage of 100 kV.

[0034] (2) Phage titer determination Dilute the purified phage solution tenfold to 10^-8. Take 200μL of each gradient dilution and mix it with 200μL of host bacteria in the logarithmic growth phase in a 5mL cryotube and let it stand for 30 minutes. Then, add 3.5mL of melted LB semi-solid medium, vortex to mix, pour into LB plate, and culture at room temperature overnight. Select a plate with a moderate number of transparent plaques with clear edges for counting and calculating the phage titer.

[0035] like Figure 2 As shown, the plaques formed by phage vB_SflS_z7 are large, transparent, and have clear edges, with a diameter of about 3 mm. The phage titer was determined by the double-layer plate method, and the number of plaques diluted to 10^-7 was 37, and the phage titer was 1.85×10^9 PFU / mL.

[0036] 2. Phage thermal stability determination The purified phage solution was diluted to 10^-7, and 500 μL of the dilution was added to the cryopreservation tubes, and then placed in a -20℃ refrigerator, a 4℃ refrigerator, a 15℃, 25℃, 35℃, 45℃, 55℃, 65℃, 75℃ and 85℃ metal bath for 2 hours. After the treatment time, the phage activity was immediately determined by the double-layer agar plate method. Three parallel samples were set for each temperature condition. By counting the number of plaques formed after treatment at different temperatures, the titer of the phage under different temperature conditions was calculated to evaluate its thermal stability.

[0037] like Figure 3 As shown in the figure, phage vB_SflS_z7 maintains high activity in the range of -20℃ to 55℃, with a titer of up to 10^9 PFU / mL; when the temperature is higher than 55℃, the phage activity drops sharply and is completely inactivated at 65℃. This shows that phage vB_SflS_z7 has a high tolerance to low and normal temperatures, but is intolerant to high temperatures.

[0038] 3. Phage pH stability determination Dilute the purified phage solution to 10^-6. Take 180μL of SM buffer with different pH values ​​(pH = 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) and 20μL of diluted phage solution and add them to 5mL cryovials and let stand for 1 hour. Then add 200μL of host bacteria in the logarithmic growth phase and let stand for 30 minutes to ensure that the phage completely infects the host. Then, add 3mL of melted LB semi-solid medium, mix well, pour into LB solid plate, and culture at room temperature. The pH stability of the phage is evaluated by counting the number of plaques after treatment with different pH values ​​and calculating the phage titer.

[0039] like Figure 4As shown, after being treated under different pH conditions for the same time, the phage titer was measured to evaluate its pH stability. The results showed that the phage vB_SflS_z7 exhibited a higher titer at pH values ​​of 3 to 11, which could reach 10^9 PFU / mL; while at pH values ​​of 2 and 12, the phage was completely inactivated. This indicates that the phage vB_SflS_z7 has a large acid-base tolerance range and maintains a high activity between pH values ​​of 3 and 11.

[0040] 4. One-step growth curve determination of bacteriophage The host bacteria were inoculated into 3 mL of LB liquid medium and cultured at 28°C and 140 RPM with shaking until the logarithmic growth phase, with a bacterial concentration of about 1×10^8 CFU / mL. 500 μL of phage stock solution was mixed with 500 μL of host bacteria in the logarithmic growth phase in a 1.5 mL centrifuge tube and allowed to stand for 20 minutes to ensure complete adsorption of the phage. Then centrifuge at 8000×g for 1 minute, wash the precipitate three times with LB liquid medium, suspend it in 50 mL of LB liquid medium, vortex and mix thoroughly before starting the timer.

[0041] From 0 to 30 minutes, samples were taken every 5 minutes; from 30 to 90 minutes, samples were taken every 10 minutes; from 90 to 120 minutes, samples were taken every 30 minutes. After gradient dilution of each sample, the phage titer was determined using the double-layer agar plate method. Three parallel samples were set up for the above experiment. With the sampling time as the horizontal axis and the logarithm of the phage titer as the vertical axis, a one-step growth curve was drawn to determine the incubation period, outbreak period, and lysis amount of the phage.

[0042] like Figure 5 As shown, the one-step growth curve of phage vB_SflS_z7 shows that within 0 to 10 minutes of phage infection of the host, the phage titer is low and remains basically unchanged; within 10 to 60 minutes, the phage titer gradually increases, exceeding 10^8 PFU / mL at 60 minutes. Subsequently, within 60 to 180 minutes, the phage titer remains at a high level and remains almost unchanged. It shows that the incubation period of phage vB_SflS_z7 infection of Escherichia coli is about 10 minutes, the outbreak period is about 50 minutes, and it enters the plateau phase at the 60th minute after infection. The lysis amount of phage vB_SflS_z7 is about 65 PFU / cell.

[0043] 5. Genomic DNA extraction and bioinformatics analysis of bacteriophage vB_SflS_z7 In order to further understand the genomic structure and function of bacteriophage vB_SflS_z7 and develop efficient therapeutic strategies against Escherichia coli, genomic DNA extraction and bioinformatics analysis were performed.

[0044] Phage genomic DNA was extracted using the OMEGA viral DNA kit, and its purity and integrity were tested by electrophoresis. The extracted phage genomic DNA was sent to Tianjin Novogene for Illumina Miseq 2×300 paired-end sequencing. The raw data were filtered and assembled using SOAP de novo v2.04. The generated reads were further filtered using Trimmomatic v0.36 to remove adapter sequences and low-quality regions, ensuring that the length was greater than 36 bp and the average phred value was not less than 15.

[0045] PhageTerm v1.0.11 was used to identify the ends of the phages, and the reads with the highest coverage were considered to be the ends of the phages. RAST v2.0 was used to predict the coding DNA sequences of the phages. All open reading frames (ORFs) were annotated in the NCBI non-redundant protein (NR) database by BLASTp, with an e-value of <1e-5. At the same time, the Pfam database and the KEGG database were searched, and the online server HHpred was used for function prediction. The schematic diagram of the phage genome was generated using the CLC Main Workbench software.

[0046] like Figure 6 As shown in Figure 1, the whole genome of bacteriophage vB_SflS_z7 was sequenced, and the results showed that its genome was a linear double-stranded DNA (dsDNA) with a total length of 76,537 bp and a GC content of 42.05%. Some of the sequences are shown in Sequence Table 1. Through BLASTp, HHpred, KEGG and Pfam search analysis, a total of 128 ORFs were predicted, of which 65 ORFs had unknown functions, accounting for about 51%, and were hypothetical proteins; another 63 ORFs matched known functions, including 13 auxiliary metabolic genes (AMGs) (such as orf2, 56, 59, 65, 66, 68, 70, 79, 82, 85, 86, 88, 108), 7 structural proteins (such as orf7, 9, 11, 12, 16, 18, 117), 3 packaging proteins (such as orf7, 9, 11, 12, 16, 18, 117), and 3 packaging proteins (such as orf7, 9, 11, 12, 16, 18, 117). orf4, 5, 119), 28 proteins related to DNA replication and regulation (such as orf10, 21, 22, 27, 29, 30, 31, 32, 39, 40, 45, 46, 49, 52, 58, 60, 62, 64, 67, 75, 76, 77, 78, 91, 98, 111, 115, 122), 8 lytic proteins (such as orf13, 14, 15, 20, 34, 35, 110, 112) and 4 other functional proteins (such as orf17, 26, 33, 102). In addition, the genome also contains a tRNA gene (Orf103_rna).

[0047] 6. Phylogenetic analysis and comparative genomic analysis Based on the whole-genome amino acid sequences of phage vB_SflS_z7 and Escherichia coli phages, a proteome tree was generated using ViPTreev3 and genome comparisons were performed. vConTACT 2.0 used the ICTV classification dataset for clustering, and BLASTp queried the coding sequence of vB_SflS_z7 in the IMG / VR database to find related homologous phages. VIRIDIC calculated the ANI value of vB_SflS_z7, phatmap visualized the results, screened closely related phage sequences, and constructed a phylogenetic tree using IQ-TREE and visualized it using iTOL.

[0048] Figure 7 Displays a circular proteome tree based on the whole genome amino acid sequence, Figure 8 This is the rectangular proteome tree of the 25 most similar viruses. Fig. 9 tBLASTx genome comparison shows that vB_SflS_z7 and Escherichia phage (NC_018835, NC_028903, NC_027395, NC_010324) have high similarity.

[0049] vConTACT 2.0 clustered all viruses, the vB_SflS_z7 reference sequence, and four similar UViGs, and Gephi visualized the viral genome classification ( Fig.10 ), the results showed that vB_SflS_z7 and Escherichia phage (NC_018835, NC_028903, NC_010324, NC_027395, NC_018859, NC_023593) were grouped into the same VC (VC_1) and belonged to the genus Kuravirus.

[0050] Fig.11 Nucleotide-based genome-wide analysis showed that vB_SflS_z7 had high similarity with the shared genes of these phages. Phylogenetic analysis showed that vB_SflS_z7 had high similarity with Escherichia phage Bacteriophages should be classified in the same virus family. Fig.12 The phylogenetic tree based on the terminal large subunit sequence is shown, and the results show that vB_SflS_z7 Escherichia phage In the same branch.

[0051] In summary, vB_SflS_z7 has a close evolutionary relationship with the discovered Escherichia coli phages and should be classified as the Kuravirus genus.

[0052] Example 3: Evaluation of the effect of bacteriophage vB_SflS_z7 in treating Escherichia coli pneumonia in mice 1. Experimental Animals All experimental animals were clean-grade 6-7-week-old male BALB / c mice provided by the Experimental Animal Center of the Affiliated Hospital of Qingdao University. The mice were kept in a normal temperature environment with free access to water and food.

[0053] 2. Establishment of Escherichia coli pneumonia model The mice were intubated under direct vision under isoflurane gas anesthesia and given 50 μL of 1×10^8 CFU / mL Escherichia coli suspension. After anesthesia, the mice were fixed on a tilted surgical board with their heads facing up for endotracheal intubation. After intubation, the mice were restored to their normal posture and returned to their cages.

[0054] 3. Treatment with bacteriophage vB_SflS_z7 One hour after inoculation with E. coli, the mice were intubated again and given 30 μL of phage vB_SflS_z7 solution (MOI = 10). After 24 hours, the alveolar lavage fluid of the mice was collected to detect changes in cytokines. The mice were then dissected to determine the load of bacteria and phages in the lungs, and the lung tissues were fixed in paraformaldehyde for pathological section analysis.

[0055] 4. Results Analysis Depend on Fig.13 (# represents p<0.05 compared with the control) E. coli The bacterial load in the lungs of mice in the E. coli + 1000 times of Phage group, E. coli The phage load in the +Phage group was about 10 times that in the Phage group. E. coli Phages in the +Phage group were able to proliferate in vivo and effectively lyse bacteria.

[0056] Depend on Fig.14 The results showed that the alveolar structure of the E.coli group was abnormal, and most of the alveolar space was infiltrated by inflammatory exudates and immune cells with hemorrhage and integration. The E.coli+Phage group had significant relief, local lesions, and most of the lung tissue was normal. Both control groups showed normal lung tissue morphology.

[0057] Depend on Fig.15 It can be seen that the levels of TNF-α and IL-6 in the E. coli group were about twice that of the E. coli+Phage group, indicating that bacteriophage can effectively reduce the content of cytokines and reduce inflammatory responses.

[0058] From the above, it can be concluded that the bacteriophage vB_SflS_z7 provided by the present invention can effectively lyse bacteria and alleviate inflammatory response.

[0059] Although the present invention has been described above with reference to the embodiments, various modifications may be made thereto and parts thereof may be replaced by equivalents without departing from the scope of the present invention. In particular, as long as there is no structural conflict, the various features in the embodiments disclosed in the present invention may be used in combination with each other in any manner, and the fact that these combinations are not exhaustively described in this specification is only for the sake of omitting space and saving resources. Therefore, the present invention is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. An Escherichia coli phage vB_SflS_z7, which is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration (CGMCC), with a deposit number of CGMCC NO.45768, a deposit date of January 2, 2024, and a deposit address of: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, and is classified and named Escherichia coli phage Escherichia coli phage ; Named as phage vB_SflS_z7.

2. Use of the Escherichia coli phage vB_SflS_z7 according to claim 1 in the preparation of anti-Escherichia coli drugs.

3. Use of the Escherichia coli phage vB_SflS_z7 according to claim 1 in the preparation of anti-Escherichia coli pneumonia drugs.

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