Carbon penicillin-resistant escherichia coli bacteriophage and application thereof

Abstract

The present application relates to the field of biotechnology, in particular to a carbapenem-resistant escherichia coli phage and application thereof.A new escherichia coli phage vB_EOP_Se009 is isolated and purified, and the phage has good sterilization effect on carbapenem-resistant escherichia coli.The phage has strong tolerance to temperature and pH, is high in specificity and stability, can effectively solve the problem that carbapenem-resistant escherichia coli is resistant to multiple antibiotics, and provides an experimental basis for developing a preparation for preventing or treating carbapenem-resistant escherichia coli infection in clinic, and has great clinical application potential.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a carbapenem-resistant Escherichia coli bacteriophage and its application. Background Technology

[0002] Escherichia coli is a common commensal and pathogenic bacterium, a major cause of intestinal and urinary tract infections and sepsis, placing a significant burden on global healthcare systems. In recent years, the overuse of antibiotics in clinical practice has led to the emergence of carbapenem-resistant Escherichia coli, posing a serious challenge to clinical treatment. A Lancet report indicates that in 2019, 1.27 million people died directly from drug-resistant bacterial infections, and 4.95 million deaths were related to drug-resistant bacterial infections. According to the 2023 China Antimicrobial Resistance Surveillance Report, Escherichia coli is one of the most frequently isolated Gram-negative bacteria in clinical settings, and its resistance rates to meropenem and imipenem are increasing year by year. Carbapenem-resistant Escherichia coli primarily resist carbapenem antibiotics by producing carbapenemases (such as KPC, NDM, VIM, and OXA-48 enzymes). These enzymes can break down drugs, leading to antibiotic resistance in the bacteria. Their resistance genes are often located on transferable plasmids, which can spread between different bacteria, increasing the risk of resistance proliferation. Carbapenem-resistant Escherichia coli exhibit resistance to multiple antibiotics, resulting in very limited treatment options. This poses a significant threat to public health and presents a major challenge to clinical treatment. To effectively address this issue, it is necessary to strengthen surveillance and prevention efforts, while simultaneously promoting the research and application of novel therapeutic drugs and strategies.

[0003] Bacteriophages are viruses capable of infecting and replicating within bacterial cells. They are among the most abundant and ubiquitous organisms on Earth, playing a crucial role in microbial physiology, population dynamics, evolution, and therapeutics. After infecting bacteria, bacteriophages multiply within the cell, disrupting its structure and releasing progeny to continue infecting. They exhibit high specificity, efficiency, and safety. However, with the discovery of antibiotics, the antibacterial applications of bacteriophages were once overlooked.

[0004] Currently, antibiotic overuse has led to a serious problem of drug-resistant bacteria, posing a major challenge to global public health. Phage therapy, utilizing bacteriophages to target and eliminate bacterial infections, can effectively kill drug-resistant bacteria as an alternative or supplement to traditional antibiotic treatment. Therefore, the application of bacteriophages to treat infections caused by clinical superbugs has once again become a focus of attention. However, the biological characteristics of bacteriophages still require further research. The diversity of bacteriophage types and the lack of strong evidence to support the efficacy and tolerability assessment of their mixtures add uncertainty to the clinical application of bacteriophages. It is believed that with continued research, the mechanisms of action of bacteriophages will be increasingly revealed, the regulation of bacteriophage therapy will be increasingly determined, and the application prospects of bacteriophages are very promising. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a carbapenem-resistant Escherichia coli bacteriophage and its applications. This invention offers a biological method capable of efficiently killing carbapenem-resistant Escherichia coli, and can also be developed into an antibacterial product, possessing significant market application value.

[0006] The present invention adopts the following technical solution: A type of Escherichia coli bacteriophage ( Escherichia coli Phage vB_EOP_Se009 was deposited on April 27, 2025, at the China General Microbiological Culture Collection Center (CGMCC), address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, accession number: CGMCC No. 46435.

[0007] Furthermore, the Escherichia coli bacteriophage vB_EOP_Se009 is a long-tailed bacteriophage with a polyhedral head structure, a head diameter of 75.05 nm, and a tail length of 120.80 nm.

[0008] Furthermore, the *Escherichia coli* bacteriophage vB_EOP_Se009 exhibits tolerance under conditions of pH 4–10.0 and temperature -20°C–70°C. After culturing for 3 hours at an MOI of 0.1, the titer reaches 4.5 × 10⁻⁶. 12 PFU / mL.

[0009] The application of the above-mentioned Escherichia coli bacteriophage vB_EOP_Se009 in the preparation of products for treating diseases caused by Escherichia coli.

[0010] Furthermore, the products include pharmaceuticals, health products, food preservatives, feed additives, cleaning agents, or disinfectants.

[0011] A drug for the prevention and / or treatment of diseases caused by Escherichia coli, wherein the active ingredient of the drug comprises the aforementioned Escherichia coli bacteriophage vB_EOP_Se009.

[0012] An antibacterial or bactericidal drug comprising the aforementioned Escherichia coli bacteriophage vB_EOP_Se009.

[0013] A disinfectant or cleaning agent comprising the aforementioned Escherichia coli bacteriophage vB_EOP_Se009.

[0014] A bactericidal composition for preventing and treating Escherichia coli infection, comprising the aforementioned Escherichia coli bacteriophage vB_EOP_Se009.

[0015] Furthermore, the bactericidal composition further includes other antibacterial or bactericidal active ingredients; more preferably, the other antibacterial or bactericidal active ingredients include other bacteriophages and / or antibiotics.

[0016] Beneficial effects: This invention isolates and purifies a novel Escherichia coli bacteriophage, vB_EOP_Se009, which exhibits excellent bactericidal activity against carbapenem-resistant Escherichia coli. This bacteriophage demonstrates strong tolerance to temperature and pH, high specificity, and stability, effectively addressing the problem of carbapenem-resistant Escherichia coli developing resistance to multiple antibiotics. This invention provides an experimental basis for the clinical development of formulations for the prevention or treatment of carbapenem-resistant Escherichia coli infections and possesses significant clinical application potential. Attached Figure Description

[0017] Figure 1 Image of a plaque formed by the lysis of host bacteria by bacteriophage vB_EOP_Se009.

[0018] Figure 2 Transmission electron microscopy image of bacteriophage vB_EOP_Se009.

[0019] Figure 3 The bar chart shows the phage titer under different concentration ratios of host bacteria and phage vB_EOP_Se009.

[0020] Figure 4 This is a one-step growth curve of bacteriophage vB_EOP_Se009.

[0021] Figure 5 This is a statistical chart showing the number of surviving bacteriophage vB_EOP_Se009 under different pH conditions.

[0022] Figure 6 This is a statistical chart showing the number of surviving bacteriophage vB_EOP_Se009 cells under different temperature conditions.

[0023] Figure 7 The adsorption rate curve of bacteriophage vB_EOP_Se009 is shown.

[0024] Figure 8 The inhibition curve of bacteriophage vB_EOP_Se009.

[0025] Figure 9 Genetic analysis map of bacteriophage vB_EOP_Se009. Detailed Implementation

[0026] The specific implementation methods of the present invention will be further explained and described below with reference to the embodiments.

[0027] The formulations of the reagents and culture media used in the following examples are as follows: LB liquid medium: Weigh 10g of LB medium powder, dissolve it in 400mL of deionized water, mix thoroughly, autoclave at 121℃, cool and store at 4℃ for later use.

[0028] LB solid medium: Weigh 10g of LB medium powder and 4g of agar powder, dissolve them in 400mL of deionized water, mix thoroughly, autoclave at 121℃, cool to 50-60℃, mix well and pour into sterile Petri dishes. After the medium solidifies, invert and store at 4℃ for later use.

[0029] LB semi-solid medium: Weigh 10g of LB medium powder and 3g of agar powder, dissolve them in 400mL of deionized water, mix thoroughly, sterilize at 121℃ under high temperature and pressure, and store at 4℃ for later use after the medium has solidified. It can be used after heating.

[0030] Host bacteria used in the examples E. coli _009 is a carbapenem-resistant Escherichia coli isolate from a clinical sputum specimen (provided by the Department of Laboratory Medicine, Shanxi Academy of Traditional Chinese Medicine). It was streaked onto MacConkey agar and incubated at 37°C (5% CO2, 95% O2) for 48 hours. A single isolate was then picked. E. coli _009 was cultured in 5 mL of LB liquid medium at 37°C (5% CO2, 95% O2) for 24 h to obtain a fresh host bacterial culture for phage isolation. E. coli _009 is resistant to imipenem (IPM), ampicillin / sulbactam (SAM), ampicillin (AMP), aztreonam (ATM), ceftriaxone (CRO), cefotaxime (CTX), cefepime (FEP), piperacillin (PIP), cefuroxime axetil (CXA), cefuroxime (CXM), ceftazidime (CAZ), cefotetan (CTT), amikacin (AK), ciprofloxacin (CIP), levofloxacin (LVE), cefazolin (CFZ), and trimethoprim / sulfamethoxazole (SMZ / TMP), and sensitive to meropenem (MEM), cefoxitin (FOX), tobramycin (TOP), and gentamicin (GM), and moderately sensitive to cefoperazone / sulbactam (CST). For the purpose of replicating the relevant experiments of this invention, the above-mentioned host bacteria can be obtained from the Shanxi Academy of Traditional Chinese Medicine. E. coli _009 is only for repeating the relevant experiments of this invention and should not be used for other purposes.

[0031] Example 1: Isolation, enrichment and purification of Escherichia coli bacteriophages: (1) Wastewater samples were collected from Shanxi Provincial Hospital of Traditional Chinese Medicine. The samples were centrifuged at 5000g for 10 minutes to remove large particulate impurities. The supernatant was filtered through a 0.45 μm needle filter to remove residual bacteria.

[0032] (2) Phage enrichment: Take 3 mL of the wastewater sample filtered in step (1) and 3 mL of host bacteria in the logarithmic growth phase and mix with 2 mL of 3×LB liquid medium. After shaking and culturing at 37℃ and 160 rpm for 24 hours, centrifuge at 8000g for 5 minutes. Filter the supernatant twice through a 0.22 μm needle filter and collect the filtrate, which is the phage enrichment solution.

[0033] (3) Phage purification: Spread 100 μL of host bacterial suspension evenly on the surface of LB solid medium. After the surface dries, drop 10 μL of phage enrichment solution into the center of the plate and incubate upside down at 37°C for 24 hours. Pick phage plaques with clear edges and suspend them in 1 mL of SM buffer, vortex to mix, and incubate overnight at 4°C. The next day, centrifuge at 8000 g for 3 minutes, and take the supernatant for serial dilution (10 μL to 10 μL). -1 Up to 10 -10 Repeat the purification process 3-5 times until uniform plaques are obtained.

[0034] The results are as follows Figure 1 As shown, the present invention isolated a bacteriophage (named vB_EOP_Se009), which can form 1 mm circular plaques in LB solid medium with no halo around it and clear and regular edges, and is a typical lytic bacteriophage.

[0035] The above-mentioned Escherichia coli bacteriophage ( Escherichia coli Phage vB_EOP_Se009 was deposited on April 27, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46435.

[0036] Example 2: Phage morphology observation Phage morphology was observed using uranium acetate negative staining. A carbon-coated support membrane copper mesh was placed on a sealing film, and a drop of purified phage suspension was placed on the mesh. After 5 minutes, excess phage suspension was blotted off from the edge of the mesh with filter paper. The mesh was dried for 1 minute, and then 10 μL of 1% uranium acetate solution was added for staining for 1 minute. Excess stain was blotted off with filter paper, and the mesh was allowed to dry at room temperature before being observed and images acquired under a transmission electron microscope.

[0037] Transmission electron microscopy observation results as follows Figure 2As shown, bacteriophage vB_EOP_Se009 is a long-tailed bacteriophage with a polyhedral head structure, a head diameter of 75.05 nm, and a tail length of 120.80 nm.

[0038] Example 3: Optimal Multiple of Infection (MOI) determination Prepare host bacterial culture in logarithmic growth phase (cell concentration 1.5 × 10⁻⁶). 8 Take equal volumes of phage suspension and host bacterial suspension, and mix them at phage number (PFU) / host bacterial number (CFU) ratios of 100:1, 10:1, 1:1, 1:10, 1:100, 1:1000, and 1:10000. Add 5 mL of LB liquid medium and incubate at 37°C with shaking for 3 h. After incubation, centrifuge at 8000 g and 4°C for 5 min. Filter the supernatant through a 0.22 μm needle filter, recover the filtrate, and determine the phage titer in the filtrate using the double-layer plate method. The multiplicity of infection (MLI) that produces the highest titer is the optimal MII of infection.

[0039] The results are as follows Figure 3 As shown, the optimal MOI for phage vB_EOP_Se009 is 0.1, at which point the phage titer reaches its peak of 4.5 × 10⁻⁶. 12 PFU / mL.

[0040] Example 4: Phage one-step growth curve determination The phage was mixed with the host bacteria at the optimal MOI, adsorbed at 37°C for 10 minutes, centrifuged to remove unadsorbed phage, and resuspended in 10 mL of fresh LB liquid medium. The mixture was then cultured at 37°C with shaking. Samples were taken every 10 minutes to determine the phage titer and plot a one-step growth curve. The latency period and lysis rate were calculated.

[0041] The results are as follows Figure 4 As shown, the latency period of bacteriophage vB_EOP_Se009 is approximately 10 minutes, and it enters a plateau phase after lysis lasts for 50 minutes. The highest bacteriophage titer can reach 3.8 × 10⁻⁶. 14 PFU / mL.

[0042] Example 5: Phage pH tolerance assay Adjust the pH of LB liquid medium to 2.0–12.0 using HCl and NaOH solutions, and mix with phage suspension (1.2 × 10⁻⁶). 8 Phage titers were determined by mixing PFU / mL at a volume ratio of 9:1 and incubating at 37°C for 1 hour. The survival rate of phages under different pH conditions was evaluated by using the double-layer plate method.

[0043] The results are as follows Figure 5 As shown, bacteriophage vB_EOP_Se009 maintains a survival rate of over 90% at pH 4.0 to pH 10.0, while extreme pH ≤ 2.0 or pH ≥ 12.0 will lead to complete inactivation.

[0044] Example 6: Phage temperature tolerance assay Bacteriophage suspension (1.2×10) 8 Phages (PFU / mL) were treated at -20℃, 4℃, 37℃, 50℃, 70℃, 80℃, 90℃, and 100℃ for 1 hour, respectively. The phage titer was determined using the double-layer plate method to evaluate the phage survival rate under different temperature conditions.

[0045] The results are as follows Figure 6 As shown, bacteriophage vB_EOP_Se009 is stable in activity within the range of -20℃ to 70℃, with a survival rate of over 80%, and is completely inactivated above 80℃.

[0046] Example 7: Phage adsorption experiment Bacteriophage suspension (1.5×10) 7 PFU / mL) and host bacterial culture (1.5×10) 8 The phages (CFU / mL) were mixed at the optimal MOI ratio and incubated with shaking at 37°C. At 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 min, 100 μL of the mixture was taken and mixed with 900 μL of SM buffer. The mixture was then immediately centrifuged (13000×g, 1 min). The titer of the free phages was measured using the double-layer plate method. The percentage of the adsorbed phage titer relative to the total phage titer at each time point was calculated, and the adsorption rate curve was plotted.

[0047] The results are as follows Figure 7 As shown, the adsorption rate of bacteriophage vB_EOP_Se009 reached over 90% at 6 min, and then tended to stabilize.

[0048] Example 8: bacteriophage inhibition experiment Different MOIs (10) -2 10 -1 Phage suspensions and host bacterial suspensions (1, 10) were mixed at a volume ratio of 1:1. The control group was mixed with host bacterial suspension and LB liquid medium at a ratio of 1:1. The mixtures were incubated at 37°C with shaking at 160 rpm, and OD was measured every 30 minutes. 600 Values ​​were used to plot the antibacterial curve.

[0049] The results are as follows Figure 8As shown, phages with different MOIs can inhibit the growth of host bacteria, and the higher the MOI, the better the antibacterial effect.

[0050] Example 9: Phage plaque formation efficiency (EOP) assay Phages exhibiting the broadest bactericidal host range in the droplet assay were selected to more thoroughly assess productive infection as defined by plating efficiency (EOP). The host bacteria used in the experiment... E. coli _001~ E. coli _010、 E. coli _015~ E. coli All strains _018 are Escherichia coli, isolated from clinical specimens (provided by the Department of Laboratory Medicine, Shanxi Academy of Traditional Chinese Medicine). E. coli ATCC25922 E. coli ATCC8739 E. coli ATCC35218 was the standard strain, purchased from the American Type Culture Collection. Different host bacterial suspensions (100 μL) were evenly spread on the surface of LB solid medium. After the surface dried, 10 μL of phage suspension was spotted onto different bacterial plates. After drying, the plates were incubated overnight at 37°C, and the appearance of phage plaques was observed. 100 μL of the host bacterial suspension showing phage plaques was combined with 100 μL of appropriately diluted phage suspension for the double-layer plate method to determine the number of plaques. The plates were incubated overnight at 37°C, and the number of plaques formed (PFU) for each combination was calculated. The EOP (average PFU of the tested host bacteria / host bacteria) was calculated. E. coli (Average PFU of _009).

[0051] The results are shown in Table 1. Among the 18 Escherichia coli isolates, bacteriophage vB_EOP_Se009 was sensitive to 4 Escherichia coli isolates, and the bacteriophage produced obvious lysis zones on them.

[0052] Table 1. Results of plating efficiency (EOP) determination of bacteriophage vB_EOP_Se009

[0053] Note: EOP of 0.5~1.0 indicates high efficiency; EOP of 0.1~0.5 indicates medium efficiency; EOP of 0.001~<0.1 indicates low efficiency; EOP<0.001 indicates ineffective; "-" indicates no plaques are produced.

[0054] Example 10: Phage genome extraction and sequencing Phage DNA was extracted using magnetic beads, and DNA quality was assessed by agarose gel electrophoresis, Nanodrop (A260 / A280 detection), and Qubit quantification. Library construction and sequencing: Libraries were constructed using the SQK-LSK109 kit. After end repair, adapter ligation, and magnetic bead purification, whole-genome sequencing was performed using the Oxford Nanopore platform. Bioinformatics analysis: Gene function was annotated using tools such as Prokka, BLAST, and PHACTS. Genes related to host recognition and cleavage were screened, and virulence and drug resistance genes were excluded.

[0055] Analysis results as follows Figure 9 As shown, the results indicate that the full-length genome of bacteriophage vB_EOP_Se009 is 68010 bp, with a GC content of 46.20%. 101 functional genes were annotated in the COG, GO, KEGG, KOG, Swissprot, TrEMBL, and nr databases for bacteriophage vB_EOP_Se009, representing an annotation rate of 93.07%. BLAST alignment in the NCBI database confirmed that bacteriophage vB_EOP_Se009 belongs to the class Caudoviricetes, genus Wifcevirus. Using BLASTp, virulence and drug resistance genes were queried in the VFDB and CARD databases, respectively; no virulence or drug resistance genes were found in bacteriophage vB_EOP_Se009.

Claims

1. A type of Escherichia coli bacteriophage ( Escherichia coli Phage vB_EOP_Se009 was deposited on April 27, 2025, at the China General Microbiological Culture Collection Center (CGMCC), address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, accession number: CGMCC No. 46435.

2. The *Escherichia coli* bacteriophage vB_EOP_Se009 as described in claim 1, characterized in that, The Escherichia coli bacteriophage vB_EOP_Se009 is a long-tailed bacteriophage with a polyhedral head structure, a head diameter of 75.05 nm, and a tail length of 120.80 nm.

3. The use of the Escherichia coli bacteriophage vB_EOP_Se009 of claim 1 in the preparation of products for treating diseases caused by Escherichia coli, said products comprising cleaning agents or disinfectants.

4. A disinfectant or cleaning agent, characterized in that, Includes the Escherichia coli bacteriophage vB_EOP_Se009 as described in claim 1.

5. A bactericidal composition for preventing and treating Escherichia coli infection, characterized in that, Includes the Escherichia coli bacteriophage vB_EOP_Se009 as described in claim 1.

6. The bactericidal composition according to claim 5, characterized in that, The bactericidal composition also includes other antibacterial or bactericidal active ingredients.

7. The bactericidal composition according to claim 6, characterized in that, Other antibacterial or bactericidal active ingredients include other bacteriophages and / or antibiotics.

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