Escherichia coli ML001, specific bacteriophage thereof and application of escherichia coli ML001 in prevention of alcoholic liver diseases

Abstract

The invention relates to Escherichia coli ML001, a specific bacteriophage thereof and an application of the Escherichia coli ML001 in preventing alcoholic liver diseases. The Escherichia coli strain ML001 and the specific bacteriophage thereof are preserved in China General Microbiological Culture Collection Center (CGMCC). Animal experiments prove that the bacteriophage can effectively reduce the level of glutamic-pyruvic transaminase and glutamic oxalacetic transaminase in the plasma of an alcoholic liver disease model mouse, which indicates that the bacteriophage has a definite liver protection function. The histopathologic examination further shows that after the phage is administered, the number of lipid droplets in the liver of the mouse is obviously reduced, and the liver lipid accumulation is effectively relieved. On the molecular mechanism level, the bacteriophage can down-regulate expression of fatty acid synthesis genes Srebf1 and Acly at the same time and inhibit a lipid synthesis pathway; meanwhile, the expression of a chemotactic factor Ccl6 is reduced, and the inflammatory response of the liver is relieved.

Description

Technical Field

[0001] This invention belongs to the field of bacteriophage technology, specifically relating to a strain of Escherichia coli ML001, its specific bacteriophage, and its application in the prevention of alcoholic liver disease. Background Technology

[0002] Alcohol-associated liver disease (ALD) is a series of liver damage caused by long-term excessive alcohol consumption. Its spectrum includes alcoholic fatty liver disease, alcoholic hepatitis, cirrhosis, liver necrosis, and liver cancer. According to the latest statistics from the World Health Organization, alcohol consumption causes approximately 3 million deaths annually, accounting for 5.3% of all deaths worldwide, placing a heavy burden on public health and the socioeconomic system. However, to this day, the pathogenesis of alcoholic liver disease is not fully understood, and preventative drugs and strategies for alcoholic liver disease remain very limited.

[0003] Clinical prevention of alcoholic liver disease (ALD) fundamentally relies on abstinence from alcohol. For early-stage patients, abstinence combined with nutritional support can reverse the disease; however, for critically ill patients with alcoholic hepatitis (AH), in addition to abstinence, drug therapy is still necessary. Drugs are mainly used as adjuncts to abstinence and anti-inflammatory treatment (such as glucocorticoids), but they generally have limitations such as high hepatotoxicity risks, significant individual differences in efficacy, and the inability to reverse disease progression. Therefore, developing novel ALD prevention methods that can precisely intervene in disease progression and have high safety has become an urgent need in this field.

[0004] In recent years, the role of the gut-liver axis in the pathogenesis of ALD has become increasingly prominent. Studies have shown that long-term alcohol consumption leads to gut microbiota dysbiosis and impaired intestinal barrier function, allowing gut microbes and their metabolites (such as endotoxins) to translocate into the portal circulation, thereby activating hepatic immune cells and triggering and sustaining inflammatory responses and liver damage. Therefore, regulating the gut microbiota is considered a novel strategy for preventing ALD.

[0005] Bacteriophages, as key members of the enterovirome, are natural predators of bacteria and exhibit high strain specificity. Recent metagenomic studies have revealed significant changes in the intestinal phage composition of liver disease patients, suggesting that the phage-bacteria interaction network may be closely related to the progression of liver disease. Theoretically, using phages to precisely target and eliminate specific pathogenic bacteria in the gut can directly reshape the gut microbiota structure. While the above research provides a theoretical basis, translating this theory into practical applications remains a technical challenge in this field. Specifically, there is currently a lack of specific phages that can specifically target key pathogenic strains closely related to the occurrence and development of ALD, and have been validated for effective prevention of ALD, along with their defined clinical application protocols. In particular, for strains isolated from the livers of ALD mice that are associated with the occurrence and development of ALD (such as *Escherichia coli* ML001 involved in this invention), there are currently no publicly reported isolation, preparation, or application in ALD prevention of specific phages.

[0006] Therefore, isolating bacteriophages that can specifically target ALD-associated pathogens and developing their preparation methods and pharmaceutical applications is of great scientific significance and clinical value for breaking through existing bottlenecks in ALD prevention and providing a new and precise prevention strategy. Summary of the Invention

[0007] Technical problem to be solved: This invention addresses the lack of strategies for preventing alcoholic liver disease by providing Escherichia coli ML001, its specific bacteriophage, and its application in the prevention of alcoholic liver disease.

[0008] Technical solution: An Escherichia coli strain, E. coli ML001, has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 11, 2025, with accession number CGMCC No. 36154, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0009] A bacteriophage capable of specifically lysing the aforementioned Escherichia coli strain E. coli ML001, named Escherichia coli phage EcP001, has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 11, 2025, with accession number CGMCCNo.46681, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] The application of the above-mentioned Escherichia coli strain E. coli ML001 in screening bacteriophages for the prevention of alcoholic liver disease.

[0011] The application of the above-mentioned Escherichia coli strain E. coli ML001 in the preparation of reagents or kits for screening bacteriophages for the prevention of alcoholic liver disease.

[0012] The above-mentioned bacteriophages are used in the preparation of drugs for the prevention of alcoholic liver disease.

[0013] A pharmaceutical composition for the prevention of alcoholic liver disease comprises the above-mentioned bacteriophage and a pharmaceutically acceptable carrier.

[0014] Preferably, the dosage form of the above-mentioned drug is an oral preparation.

[0015] Preferably, the above-mentioned oral preparation is an enteric-coated preparation.

[0016] The above-mentioned pharmaceutical composition further comprises at least one drug for the prevention of alcoholic liver disease.

[0017] Beneficial Effects: Animal experiments have demonstrated that this phage can effectively reduce the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the plasma of mice with alcoholic liver disease, indicating its clear hepatoprotective function. Histopathological examination further showed that after administration of this phage, the number of lipid droplets in the liver of mice was significantly reduced, and hepatic lipid accumulation was effectively alleviated. At the molecular level, this phage can simultaneously downregulate the expression of fatty acid synthesis genes Srebf1 and Acly, inhibiting the lipid synthesis pathway; it also reduces the expression of the chemokine Ccl6, alleviating liver inflammation. This dual regulatory effect on lipid metabolism and inflammatory response reflects the multi-target synergistic effect of this phage in improving alcoholic liver disease. More importantly, this invention reveals for the first time the key role of Escherichia coli ML001 in the development and progression of alcoholic liver disease, and uses this specific strain as a target to achieve precise intervention in the disease using its specific phage. This prevention strategy based on specific pathogen-phage interactions not only provides new ideas for the prevention of alcoholic liver disease, but also demonstrates a more targeted therapeutic advantage than traditional broad-spectrum antibiotics or anti-inflammatory drugs. Attached Figure Description

[0018] Figure 1 This report compares the relative abundance of *Escherichia coli* ML001 in the liver and cecal contents of mice in the control diet group and the alcohol diet group, obtained from metagenomic sequencing analysis. The results showed that the abundance of *E. coli* ML001 in the cecum and liver of mice in the alcohol diet group was significantly higher than that in the control group (*P < 0.05), indicating that *E. coli* ML001 is associated with the development and progression of alcoholic liver disease.

[0019] Figure 2This invention relates to the effects of Escherichia coli ML001 on plasma liver function indicators in a mouse model of alcoholic liver disease: the left figure is a bar chart of ALT levels, and the right figure is a bar chart of AST levels. The results showed that the ALT and AST levels in the E. coli ML001 gavage group were significantly higher than those in the solvent control group (*P < 0.05), indicating that E. coli ML001 can worsen alcohol-induced liver injury.

[0020] Figure 3 The images show the morphological characteristics of the Escherichia coli ML001 bacteriophage isolated in this invention (Escherichia coli phage EcP001); the left image shows the morphology of the phage plaques, and the right image is a transmission electron microscope (TEM) image of the phage. The results show that after purification, the phage plaques on the plate are uniform in morphology and size, indicating that a purified phage clone has been obtained. TEM results show that this phage belongs to the order Tailed Phages, with an icosahedral symmetric capsid structure, a diameter of approximately 90 nm, a tail length of approximately 140 nm, and a tail diameter of approximately 25 nm.

[0021] Figure 4 The effects of the Escherichia coli ML001 bacteriophage (Escherichia coli phage EcP001) of this invention on plasma liver function indicators in a mouse model of alcoholic liver disease are shown in the following figures: the left figure is a bar chart of ALT levels, and the right figure is a bar chart of AST levels. The results show that the ALT and AST levels in the phage-treated group were significantly lower than those in the adjuvant control group (*P < 0.05), indicating that this phage can effectively inhibit alcohol-induced liver damage.

[0022] Figure 5 The images show the histopathological changes in mouse livers after treatment with the Escherichia coli ML001 phage (Escherichia coli phage EcP001) of this invention. The top image shows the hematoxylin-eosin (H&E) staining results, and the bottom image shows the Oil Red O staining results. The results show that the number of lipid droplets in the liver of mice treated with the phage was significantly reduced, and lipid accumulation in the liver was alleviated.

[0023] Figure 6This is a comparison of real-time quantitative PCR results showing the mRNA expression levels of lipid metabolism-related genes and inflammation-related genes in mouse liver tissue after treatment with the Escherichia coli ML001 phage (Escherichia coli phage EcP001) of this invention. The results showed that the expression of fatty acid synthesis genes (Srebf1, Acly) and chemokine gene (Ccl6) was significantly downregulated in the phage-treated group (*P < 0.05), indicating that this phage exerts a protective effect by inhibiting lipid synthesis and inflammatory responses. Detailed Implementation

[0024] The following embodiments are provided to enable those skilled in the art to more fully understand the present invention, but do not limit the invention in any way.

[0025] Example 1: Isolation and Identification of Escherichia coli ML001

[0026] 1.1 Laboratory Animals and Grouping

[0027] Twelve SPF-grade female mice (20 ± 2 g) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd., with a strain of C57BL / 6N. They were randomly divided into two groups with the same average weight, with six mice in each group: A) control group; B) alcohol group.

[0028] The disease model used was a mouse model of alcoholic liver disease characterized by chronic alcohol feeding followed by a single acute alcohol gavage, also known as the NIAAA model. For the first 5 days, the mice were fed an adaptive liquid diet. On day 6, the alcohol group was switched to a Lieber-DeCarli liquid diet containing 5% (vol / vol) ethanol, which was continued for 10 days. The control group continued to be fed the Lieber-DeCarli control liquid diet. On the morning of day 11, the mice were weighed. Subsequently, the control group was gavaged with 45% (wt / vol) maltodextrin (g = 20 μL), while the alcohol group was gavaged with 31.5% (vol / vol) ethanol (g = 20 μL). The mice were euthanized 9 hours later.

[0029] 1.2 Strains Isolation

[0030] Under aseptic conditions, euthanized mice underwent tissue dissection. The liver was completely removed using sterile surgical instruments and cut into small pieces with sterile scissors. 1 mL of sterile PBS and 1 mm sterile magnetic beads were added to each piece. All samples were homogenized in a tissue homogenizer at 0 °C and 60 Hz for 1 min, repeated 2-3 times to obtain a homogenate. One-tenth (120 μL) of the liver homogenate volume was spread onto mGAM agar plates and incubated in an anaerobic environment (85% N2, 5% CO2, 10% H2) at 37 °C. Colony growth was observed after 24 h; if colonies were small, incubation was extended to 2-4 days. A plate without colonies after 5 days was considered devoid of revivable bacteria.

[0031] 1.3 Strain Identification

[0032] Colonies were randomly picked from each plate using a disposable inoculation loop and inoculated into 96-well plates containing 200 μL of mGAM. After amplification, 2 μL of bacterial suspension was used as a template for PCR-based 16S rRNA gene sequence amplification. The PCR mixture contained 2×PrimeSTAR® HS buffer and 250 nM primers (27F (SEQ ID NO.1): 5'-AGAGTTTGATCMTGGCTCAG-3'; 1492R (SEQ ID NO.2): 5'-TACGGYTACCTTGTTACGACTT-3'). The reaction program was as follows: pre-denaturation at 95 °C for 3 minutes, followed by 35 cycles (denaturation at 95 °C for 15 seconds, annealing at 52 °C for 30 seconds, extension at 72 °C for 60 seconds). Finally, extension at 72 °C for 5 minutes. PCR products were sent to a third-party sequencing institution for sequencing. The sequencing results were compared with the 16S rRNA sequence database using NCBI BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). After preliminary identification, whole-genome sequencing was performed on the isolate to further clarify its taxonomic position. The analysis results showed that the average nucleotide identity of this isolate with the Escherichia coli model strain E. coli O83:H1 was 98.8%, which is higher than the 96% species threshold, thus confirming it as Escherichia coli at the genomic level. Based on the above results and the unique isolation source of this strain (isolated from the liver of a mouse model of alcoholic liver disease), we named this newly screened strain E. coli ML001. This strain has been deposited at the China General Microbiological Culture Collection Center.

[0033] Example 2: Abundance and pathogenicity verification of Escherichia coli ML001 in the liver of mice with alcoholic liver disease

[0034] 2.1 Abundance Validation

[0035] Total bacterial genomic DNA was extracted from mouse liver and cecal contents samples collected in Section 1.1 of Example 1, and metagenomic sequencing was performed to analyze the relative abundance of *Escherichia coli* ML001. The results are as follows: Figure 1 As shown, compared with the control group, the abundance of Escherichia coli ML001 in the cecum and liver of mice in the alcohol group was significantly increased (*P < 0.05). This result indicates that Escherichia coli ML001 is closely related to the occurrence and development of alcoholic liver disease.

[0036] 2.2 Laboratory Animals and Grouping

[0037] Twelve SPF-grade female mice (20 ± 2 g) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd., strain C57BL / 6N, and randomly divided into two groups with the same average weight, with 6 mice in each group: A) bacterial solution group, which was administered Escherichia coli ML001 by gavage; B) solvent group, which was administered an equal volume of sterile PBS by gavage.

[0038] The disease model used was a mouse model of alcoholic liver disease induced by chronic alcohol feeding followed by one acute alcohol gavage. For the first 5 days, the mice were fed an adaptive liquid diet, and on day 6, they were switched to a Lieber-DeCarli liquid diet containing 5% (vol / vol) ethanol, which was continued for 10 days. The bacterial culture group received 6.7 × 10⁶ ml of the diet via gavage daily. 9 CFU / mL Escherichia coli ML001 was administered to mice in the solvent group, with an equal volume of sterile solvent. On the morning of day 11, the mice were weighed and administered 31.5% (vol / vol) alcohol by gavage at a volume of (mouse weight (g) × 20) μL. The mice were euthanized 9 h later.

[0039] 2.3 Liver function index measurement

[0040] After euthanasia, blood was collected via the inferior vena cava into centrifuge tubes containing EDTA anticoagulant. The tubes were centrifuged at 3000 g for 10 min to separate the plasma, and the activities of ALT and AST in the plasma were measured. Results are as follows: Figure 2 As shown: Compared with the solvent control group, the levels of ALT and AST in the E. coli ML001 gavage group were significantly increased (*P < 0.05), suggesting that the Escherichia coli E. coli ML001 of the present invention can aggravate alcohol-induced liver injury.

[0041] Example 3: Screening for Escherichia coli ML001 phage

[0042] Wastewater samples were collected from natural water bodies (such as lakes and rivers), centrifuged at 5000 g for 1 minute to remove mud and impurities, and then filtered through a 0.22 µm filter membrane to obtain clarified wastewater. 100 µL of clarified wastewater was mixed with 100 µL of overnight cultured *Escherichia coli* ML001, and then LB semi-solid medium was added to a final volume of 4 mL. After gentle mixing, the mixture was poured onto LB agar plates (double-layer plate method) and incubated overnight at 37 °C. Once circular, transparent plaques appeared on the semi-solid plates, individual plaques were picked and dissolved in SM buffer (1 L: 5.8 g sodium chloride, 2 g anhydrous magnesium sulfate, 50 mL pH=7.5 Tris base-hydrochloric acid), and 5 mL OD245 was added. 600nm Escherichia coli ML001 bacterial suspension with a concentration of 0.6 was incubated at 37 °C for 6 hours, then centrifuged at 12900 g for 10 minutes at 4 °C. The supernatant phage lysate was serially diluted. The double-layer plate method was repeated for purification, iterating 3-5 times until uniform phage plaques were obtained. RNase and DNase were added to the purified high-concentration phage lysate to a final concentration of 10 μg / mL, and the mixture was reacted at 37 °C for 1 hour. PEG8000 was added to a final concentration of 10% (w / v, g / mL), and the mixture was incubated overnight at 4 °C to precipitate. The phage precipitate was obtained by centrifugation at 12000 g for 10 minutes at 4 °C. Transmission electron microscopy results showed that the phage belonged to the order Caudataphages, with an icosahedral symmetric capsid structure, a diameter of approximately 90 nm, a tail length of approximately 140 nm, and a tail diameter of approximately 25 nm.

[0043] Example 4: Validation of the protective effect of E. coli ML001 phage against alcoholic liver disease in mice.

[0044] 4.1 Laboratory Animals and Grouping

[0045] Twelve SPF-grade female mice (20 ± 2 g) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd., with a strain of C57BL / 6N. They were randomly divided into two groups with the same average weight, with six mice in each group: A) phage administration group; B) adjuvant control group.

[0046] The disease model used was a mouse model of alcoholic liver disease induced by chronic alcohol feeding followed by a single acute alcohol gavage. For the first 5 days, the mice were fed an adaptive liquid diet. On day 6, the diet was switched to Lieber-DeCarli liquid diet containing 5% (vol / vol) ethanol, and this feeding continued for 10 days. The phage administration group received 1×10⁻⁶ ethanol via gavage on day 1 of the experiment. 11A suspension of PFU / mL phage targeting Escherichia coli ML001 (Escherichia coli phage EcP001) was administered once, and the same concentration of phage was administered by gavage every other day after changing the alcohol diet. The adjuvant group was administered the same volume of SMBuffer by gavage at the same time. On the morning of day 11, the mice were weighed and administered 31.5% (vol / vol) alcohol by gavage at a volume of (mouse body weight (g) × 20) μL. The mice were euthanized 9 h later, and various indicators were measured.

[0047] 4.2 Liver function index measurement

[0048] At the end of the experiment, mice in each group were euthanized, and blood was collected via the inferior vena cava into centrifuge tubes containing EDTA anticoagulant. The tubes were centrifuged at 3000 g for 10 min to separate the plasma, and the activities of ALT and AST in the plasma were measured. Results are as follows: Figure 4 As shown, compared with the adjuvant control group, the ALT and AST levels in the phage administration group were significantly reduced (*P < 0.05), suggesting that the phage of the present invention can effectively inhibit alcohol-induced liver injury.

[0049] 4.3 Histopathological examination

[0050] Left and right middle lobes of the liver were fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, and then sectioned to a thickness of 5 µm. Hematoxylin and eosin (H&E) staining was performed to observe the tissue morphology and structure. The caudate lobe was immediately embedded in OCT embedding medium, flash-frozen in liquid nitrogen, and then sectioned to a thickness of 5 µm. The results are as follows: Figure 5 As shown, compared with the adjuvant control group, the number of lipid droplets in the liver of mice in the phage administration group was significantly reduced, indicating that the phage provided by the present invention can effectively alleviate alcohol-induced liver lipid accumulation.

[0051] 4.4 Real-time quantitative PCR (qPCR)

[0052] Approximately 30 mg of liver tissue was collected, and total RNA was extracted using the TRIzol method. After reverse transcription, the expression of lipid metabolism-related genes was detected by SYBR Green qPCR. 18S rRNA was used as an internal control, and the primer sequences are shown in Sequence Listing 1.

[0053] Table 1 List of qPCR primers

[0054]

[0055] The results are as follows Figure 6As shown, compared with the adjuvant control group, the expression of fatty acid synthesis genes (Srebf1, Acly) was significantly downregulated in the phage-administered group (*P < 0.05), indicating that the lipid synthesis pathway was inhibited; the expression of chemokine gene (Ccl6) was significantly decreased in the phage-administered group (*P < 0.05), indicating that the pro-inflammatory pathway was inhibited. Therefore, the phage of this invention can exert a protective effect against alcohol-induced fatty liver by inhibiting lipid synthesis and inflammatory response.

[0056] 4.5 Statistical Methods

[0057] All data are expressed as mean ± SEM, with sample sizes n = 6–12 per group. Two-tailed unpaired Mann-Whitney U tests were performed using GraphPad Prism 9.0 software to compare differences between groups. P < 0.05 was considered statistically significant, and a star (*) was used in the figures to indicate significant differences (*P < 0.05).

Claims

1. An Escherichia coli strain, E. coli ML001, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36154.

2. A bacteriophage, characterized in that, It is capable of specifically lysing the Escherichia coli strain E. coli ML001 as described in claim 1.

3. The bacteriophage according to claim 2, named Escherichia coliphage EcP001, is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 46681.

4. The use of the Escherichia coli strain E. coli ML001 of claim 1 in screening bacteriophages for the prevention of alcoholic liver disease.

5. The use of the Escherichia coli strain E. coli ML001 according to claim 1 in the preparation of reagents or kits for screening bacteriophages for the prevention of alcoholic liver disease.

6. The use of the bacteriophage according to claim 2 or 3 in the preparation of a medicament for the prevention of alcoholic liver disease.

7. A pharmaceutical composition for the prevention of alcoholic liver disease, characterized in that, It includes the bacteriophage as described in claim 2 or 3 and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, wherein the dosage form is an oral formulation.

9. The pharmaceutical composition according to claim 8, wherein the oral formulation is an enteric-coated formulation.

10. The pharmaceutical composition according to claim 7, further comprising at least one medicament for preventing alcoholic liver disease.

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