Multi-drug-resistant Escherichia coli bacteriophage and application thereof

Abstract

The invention relates to the technical field of biomedicine, in particular to a multi-drug-resistant Escherichia coli bacteriophage and application thereof. The Escherichia coli bacteriophage vBEOPSe004 is obtained through screening, the bacteriophage has a good killing effect on multi-drug-resistant Escherichia coli obtained through clinical separation, the tolerance to temperature and pH is high, and the stable survival rate is kept when the pH value ranges from 4 to 10 and the temperature ranges from-20 DEG C to 70 DEG C. The bacteriophage can effectively solve the problem of drug resistance of multi-drug-resistant Escherichia coli to various antibiotics in a targeted manner, and has great advantages in the aspect of clinical treatment of infection.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a multidrug-resistant Escherichia coli bacteriophage and its applications. Background Technology

[0002] Escherichia coli is a Gram-negative, rod-shaped bacterium and one of the most common opportunistic pathogens in clinical practice. It is a major cause of urinary tract infections, abdominal infections, and diarrhea, and can also cause cholecystitis, neonatal meningitis, bacteremia, and pneumonia. In recent years, with the overuse of antibiotics, the problem of drug resistance in Escherichia coli has become increasingly serious. Escherichia coli carries drug resistance genes through mobile genetic elements such as plasmids and integrons, exhibiting widespread resistance to β-lactams, quinolones, and carbapenems. The spread of multidrug-resistant strains not only leads to increased infection mortality but also exacerbates the healthcare burden, necessitating the development of novel antimicrobial strategies.

[0003] With the widespread use and overuse of antibiotics, more and more bacteria are developing resistance, and even "superbugs" resistant to multiple antibiotics have emerged. The development of new antibacterial drugs is far outpaced by the rate at which drug-resistant bacteria are emerging, rendering antibiotic therapy ineffective against these superbugs, creating an urgent need for new treatment methods. Bacteriophages, with their unique advantages and potential, have re-entered the scientists' field of vision.

[0004] Bacteriophages are viruses that infect microorganisms such as bacteria, fungi, actinomycetes, or spirochetes. They are obligate intracellular parasites that can only replicate and multiply within living microbial cells. They possess the basic characteristics of viruses: they are tiny, can pass through cell filters, lack cellular structure, and are mainly composed of a protein capsid and nucleic acid contained within it. The name bacteriophage comes from the Greek word "phagos," meaning "to devour," precisely because of their ability to lyse host bacteria. Bacteriophages are widely distributed in the natural environment, and their numbers are enormous, reaching approximately 10^6 kilophates. 31 Animals are extremely diverse biological entities on Earth, numbering approximately 10 times more than bacteria.

[0005] Phage therapy utilizes the specific recognition and lysis capabilities of bacteriophages to precisely target and eliminate specific superbugs without disrupting the normal flora. Furthermore, bacteriophages possess the ability to self-reproduce, multiplying exponentially in infected areas to rapidly eradicate pathogens. In conclusion, with the overuse of antibiotics and the increasing resistance of bacteria, phage therapy has made significant progress in treating bacterial infections in humans and animals, and even in the biocontrol of foodborne illnesses. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a multidrug-resistant Escherichia coli bacteriophage and its applications. The bacteriophage provided by this invention can efficiently kill multidrug-resistant Escherichia coli, overcoming the problems of existing antibiotic therapies being ineffective against multidrug-resistant bacteria and broad-spectrum antibiotics disrupting the microecological balance. It provides a highly efficient, safe, and self-amplifying biological antibacterial solution, which can be used to develop antibacterial products and has significant market application value.

[0007] The present invention adopts the following technical solution: A type of Escherichia coli bacteriophage ( Escherichia coli Phage vB_EOP_Se004 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. 46434.

[0008] According to a preferred embodiment of the present invention, the Escherichia coli bacteriophage vB_EOP_Se004 is a tailed bacteriophage with a head diameter of 82.99 nm and a tail length of 145.20 nm.

[0009] According to a preferred embodiment of the present invention, the *Escherichia coli* bacteriophage vB_EOP_Se004 exhibits tolerance at pH 4.0–10.0 and temperatures ranging from -20°C to 70°C. When cultured for 3 hours at an MOI of 0.1, its titer reaches 1.69 × 10⁻⁶. 13 PFU / mL.

[0010] The above-mentioned Escherichia coli bacteriophage vB_EOP_Se004 is used in the preparation of products for the prevention and / or treatment of diseases caused by Escherichia coli.

[0011] According to a preferred embodiment of the present invention, the product includes pharmaceuticals, health products, food preservatives, feed additives, cleaning agents, or disinfectants.

[0012] A bactericidal composition for preventing and treating Escherichia coli infection, comprising an effective amount of Escherichia coli bacteriophage vB_EOP_Se004.

[0013] According to a preferred embodiment of the present invention, 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.

[0014] A drug for the prevention and treatment of Escherichia coli infection, comprising an effective amount of Escherichia coli bacteriophage vB_EOP_Se004.

[0015] A food preservative or feed additive for preventing Escherichia coli infection, comprising an effective amount of Escherichia coli bacteriophage vB_EOP_Se004.

[0016] A cleaning agent or disinfectant for preventing and treating Escherichia coli infection, comprising an effective amount of Escherichia coli bacteriophage vB_EOP_Se004.

[0017] Beneficial effects: The bacteriophage vB_EOP_Se004 of this invention has a good killing effect on multidrug-resistant Escherichia coli isolated clinically, and has strong tolerance to temperature and pH, maintaining a stable survival rate between pH 4 and 10 and temperature -20℃ to 70℃. This bacteriophage can effectively address the drug resistance problem of multidrug-resistant Escherichia coli to multiple antibiotics, and has great advantages in the clinical treatment of infections. The advantages are as follows: (1) High specificity, the bacteriophage specifically targets multidrug-resistant Escherichia coli and will not affect other beneficial bacteria; (2) It will not cause bacterial drug resistance and resistance; (3) It can rely on the proliferation of host bacteria, and a single dose can achieve the effect of multiple doses of other antibacterial drugs; (4) Low side effects, bacteriophage therapy has a low risk of side effects because they only attack specific bacteria; (5) Combination therapy potential, the bacteriophage can be combined with other bacteriophages to form a cocktail preparation, or it can be used in combination with antibiotics to enhance the therapeutic effect and overcome the limitations of single therapy. The bacteriophage vB_EOP_Se004 provided by this invention is a biological method that can effectively kill multidrug-resistant Escherichia coli. It has the characteristics of high efficiency, specificity, and few side effects. It can kill pathogens without disrupting the balance of the in vivo flora, which is unmatched by antibiotics and other antibacterial drugs. Attached Figure Description

[0018] Figure 1 Image of a plaque formed by the lysis of host bacteria by bacteriophage vB_EOP_Se004.

[0019] Figure 2 Transmission electron microscopy image of bacteriophage vB_EOP_Se004.

[0020] Figure 3 The bar chart shows the phage titer under different concentration ratios of host bacteria and phage vB_EOP_Se004.

[0021] Figure 4 This is a one-step growth curve of bacteriophage vB_EOP_Se004.

[0022] Figure 5 This is a statistical chart showing the survival rate of bacteriophage vB_EOP_Se004 under different pH conditions.

[0023] Figure 6 This is a statistical chart showing the number of surviving bacteriophage vB_EOP_Se004 under different temperature conditions.

[0024] Figure 7 The adsorption rate curve of bacteriophage vB_EOP_Se004 is shown.

[0025] Figure 8 The inhibition curve of bacteriophage vB_EOP_Se004.

[0026] Figure 9 Genetic analysis map of bacteriophage vB_EOP_Se004. Detailed Implementation

[0027] The specific embodiments of the present invention will be described in detail below with reference to the examples. Obviously, the described embodiments are only some, not all, of the embodiments described in this application. All other embodiments obtained by those skilled in the art based on the embodiments in this application are within the scope of protection of this application.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] Host bacteria used in the examples E. coli _004 is a multidrug-resistant Escherichia coli isolate from a clinical sputum specimen (provided by the Department of Laboratory Medicine, Shanxi Provincial 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 _004 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_004 is resistant to ampicillin (AMP), aztreonam (ATM), ceftriaxone (CRO), cefotaxime (CTX), cefepime (FEP), cefoxitin (FOX), piperacillin (PIP), cefuroxime axetil (CXA), cefuroxime (CXM), ceftazidime (CAZ), cefazolin (CFZ), and trimethoprim / sulfamethoxazole (SMZ / TMP). It is sensitive to cefotetan (CTT), amikacin (AK), tobramycin (TOP), gentamicin (GM), ciprofloxacin (CIP), levofloxacin (LEV), nitrofurantoin (NIT), piperacillin / tazobactam (TZP), cefoperazone / sulbactam (CST), meropenem (MEM), and imipenem (IPM), and moderately sensitive to ampicillin / sulbactam (SAM). 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 _004 is only for repeating the relevant experiments of this invention and should not be used for other purposes.

[0032] Example 1: Isolation, enrichment and purification of Escherichia coli bacteriophages: (1) Phage isolation: Wastewater samples were collected from Shanxi Provincial Hospital of Traditional Chinese Medicine. Large particles were removed by centrifugation at 5000g for 10 minutes. The supernatant was filtered through a 0.45μm needle filter to remove residual bacteria.

[0033] (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 culture medium. After shaking and culturing at 37℃ and 160 rpm for 24 hours, centrifuge at 8000 g for 5 minutes. Filter the supernatant twice through a 0.22 μm needle filter and collect the filtrate, which is the phage enrichment solution.

[0034] (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 at 37°C upside down 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 / 10000 μL / 10000 μL). -1 Up to 10 -10 Repeat the purification process 3-5 times until uniform plaques are obtained, such as... Figure 1 As shown.

[0035] This invention isolates an Escherichia coli phage, named vB_EOP_Se004, 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 phage.

[0036] The above-mentioned Escherichia coli bacteriophage ( Escherichia coli Phage vB_EOP_Se004 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. 46434.

[0037] Example 2: Phage morphology observation: Phage morphology was observed using uranium acetate negative staining. A carbon-coated support film copper mesh was placed on a sealing film, and a drop of purified phage suspension was placed on the copper mesh. After 5 min, excess phage suspension was absorbed from the edge of the copper mesh with filter paper. After drying for 1 min, 10 μL of 1% uranium acetate solution was added, and staining was performed for 1 min. Excess staining solution was absorbed with filter paper, and after drying at room temperature, the phage was observed under a transmission electron microscope, and images were acquired.

[0038] Transmission electron microscopy observation results as follows Figure 2 As shown, bacteriophage vB_EOP_Se004 is a tailed bacteriophage with a head diameter of approximately 82.99 nm and a tail length of 145.20 nm, which can enhance the ability to penetrate the biofilm of drug-resistant bacteria.

[0039] Example 3: Optimal multiple of infection (MOI) determination: Preparation of host bacterial suspension in logarithmic growth phase (cell concentration of 1×10⁻⁶) 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.

[0040] The results are as follows Figure 3 As shown, the optimal MOI for phage vB_EOP_Se004 is 0.1, at which point the phage titer reaches its peak of 1.69 × 10⁻⁶. 13 The short latency and high lysis rate of this bacteriophage (PFU / mL) allow it to rapidly reach therapeutic concentrations at the site of infection.

[0041] 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 the one-step growth curve. The latency period and lysis rate were calculated.

[0042] The results are as follows Figure 4 As shown, the latency period of bacteriophage vB_EOP_Se004 is approximately 30 minutes, and it enters the plateau phase after lysis lasts for 40 minutes. The highest bacteriophage titer can reach 1.58 × 10⁻⁶. 11 PFU / mL.

[0043] Example 5: Phage pH tolerance assay: LB liquid medium was adjusted to pH 2.0–12.0 using HCl and NaOH solutions, and phage suspension (1.2 × 10⁻⁶) was mixed with the phage suspension. 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.

[0044] The results are as follows Figure 5 As shown, the survival rate of bacteriophage vB_EOP_Se004 remains above 80% in the pH range of 4.0 to 10.0, while extreme pH ≤ 2.0 or pH ≥ 12.0 will lead to complete inactivation.

[0045] Example 6: Phage temperature tolerance test: Phage suspension (1.2 × 10⁻⁶) 8 Phages (PFU / mL) were treated at -20℃, 4℃, 37℃, 50℃, 60℃, 70℃, 80℃, 90℃, and 100℃ for 1 hour. The phage titer was determined using the double-layer plate method to evaluate the phage survival rate under different temperature conditions.

[0046] The results are as follows Figure 6 As shown, bacteriophage vB_EOP_Se004 exhibits stable activity within a temperature range of -20℃ to 70℃, maintaining a survival rate of over 80%, and is completely inactivated above 80℃. This bacteriophage demonstrates broad pH and temperature tolerance, facilitating the processing and storage of pharmaceutical formulations.

[0047] Example 7: Phage adsorption experiment: Phage suspension (1.5 × 10⁻⁶) 8 PFU / mL) and host bacterial culture (1.5×10) 9The 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 (13000g, 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.

[0048] The results are as follows Figure 7 As shown, only 3% of the bacteriophage vB_EOP_Se004 remained in a free state within 4 minutes, and the adsorption rate of the bacteriophage to the host bacteria reached 97%, after which it tended to stabilize.

[0049] Example 8: Phage 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.

[0050] The results are as follows Figure 8 As shown, phages with different MOIs can inhibit the growth of host bacteria, and the higher the MOI, the better the antibacterial effect.

[0051] Example 9: Phage plaque formation efficiency assay: Phages exhibiting the broadest bactericidal host range in the drop method were selected to more thoroughly assess productive infection as defined by plaque formation efficiency (EOP). 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. coliATCC35218 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 _004).

[0052] The results are shown in Table 1. Among the 18 Escherichia coli isolates, bacteriophage vB_EOP_Se004 was sensitive to 5 Escherichia coli isolates, and the bacteriophage produced obvious lysis zones on them.

[0053] Table 1. Results of plating efficiency (EOP) of bacteriophage vB_EOP_Se004

[0054] 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.

[0055] 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 lysis were screened, and virulence and drug resistance genes were excluded.

[0056] Analysis results as follows Figure 9As shown, the results indicate that the full-length genome of bacteriophage vB_EOP_Se004 is 87672 bp, with a GC content of 38.99%. 135 functional genes of bacteriophage vB_EOP_Se004 were annotated in the COG, GO, KEGG, KOG, Swissprot, TrEMBL, and nr databases, representing an annotation rate of 94.07%. BLAST alignment in the NCBI database confirmed that bacteriophage vB_EOP_Se004 is a new species or subgroup (unclassified Mooglevirus) of the class Caudoviricetes. 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_Se004.

Claims

1. A type of Escherichia coli bacteriophage ( Escherichia coli Phage vB_EOP_Se004 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. 46434.

2. The *Escherichia coli* bacteriophage vB_EOP_Se004 as described in claim 1, characterized in that, The Escherichia coli bacteriophage vB_EOP_Se004 is a tailed bacteriophage with a head diameter of 82.99 nm and a tail length of 145.20 nm.

3. The use of the Escherichia coli bacteriophage vB_EOP_Se004 according to claim 1 in the preparation of products for the prevention and / or treatment of diseases caused by Escherichia coli.

4. The application as described in claim 3, characterized in that, The products include pharmaceuticals, health products, food preservatives, feed additives, cleaning agents, or disinfectants.

5. A bactericidal composition for preventing and treating Escherichia coli infection, characterized in that, Including the effective content of Escherichia coli bacteriophage vB_EOP_Se004.

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.

8. A medicine for preventing and treating Escherichia coli infection, characterized in that, Including the effective content of Escherichia coli bacteriophage vB_EOP_Se004.

9. A food preservative or feed additive for preventing and treating Escherichia coli infection, characterized in that, Including the effective content of Escherichia coli bacteriophage vB_EOP_Se004.

10. A cleaning agent or disinfectant for preventing and treating Escherichia coli infections, characterized in that, Including the effective content of Escherichia coli bacteriophage vB_EOP_Se004.

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