Preparation method for bacteriophage targeting expec and use thereof

Abstract

Provided are a preparation method for a bacteriophage targeting ExPEC and the use thereof. The bacteriophage targeting ExPEC is obtained by means of uniformly mixing, in proportion, a logarithmic phase bacterial liquid of swine-derived parenteral pathogenic Escherichia coli as a host strain with a bacteriophage filtrate and a culture medium, and then carrying out multiplication culture, separation and purification. The bacteriophage targeting ExPEC is capable of accurately targeting ExPEC in the intestinal tract without affecting other intestinal floras, thereby mitigating the effect thereof on exacerbating hepatic steatosis and inflammatory injury caused by alcohol. The bacteriophage targeting ExPEC therapy is expected to become one of new effective treatment methods for alcohol-related liver diseases.

Description

Preparation method and application of phage targeting ExPEC Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to a preparation method and application of bacteriophages targeting ExPEC. Background Art

[0002] Alcohol-related liver disease (ALLD) refers to a range of liver lesions caused by excessive or chronic alcohol consumption, including alcoholic fatty liver disease, alcoholic hepatitis, alcoholic cirrhosis, and alcoholic liver cancer. Surveys show that both the proportion of alcohol users and the prevalence of ALD are increasing worldwide. In 2019, approximately 25% of deaths from cirrhosis worldwide were alcohol-related, and the annual incidence of hepatocellular carcinoma (HCC) in patients with alcohol-related cirrhosis ranged from 0.9% to 5.6%. Clearly, ALD imposes a significant socioeconomic and global health burden. However, to date, there are no approved effective treatments for ALD other than alcohol abstinence and liver transplantation. These treatments are difficult to maintain and lack donors, making them clinically feasible and widely implemented. Therefore, the search for new treatments for ALD is urgent.

[0003] 1. Etiology and Pathogenesis of Alcohol-Related Liver Disease

[0004] The etiology and pathogenesis of alcohol-related liver disease are complex. Current research, both domestically and internationally, has identified high-risk factors for alcohol-related liver disease, including the type of alcoholic beverage consumed, the amount consumed, the duration of drinking, nutritional status, genetic susceptibility, and ethnicity. To date, the pathological mechanisms of alcohol-related liver disease have not been fully elucidated, but are generally believed to be related to the following three factors: First, alcohol and its active metabolite, acetaldehyde, can directly cause toxic liver damage; second, liver damage can further induce the release of damage-associated molecular patterns, which in turn recruit innate and adaptive immune cells, triggering a series of immune inflammatory responses and exacerbating liver damage; finally, alcohol intake can affect the abundance and composition of the gut microbiota, disrupting the intestinal barrier, and allowing the gut microbiota and their metabolites to translocate to the liver, triggering an immune response and liver damage.

[0005] Treatment of alcohol-related liver disease

[0006] The principles of treatment for alcohol-related liver disease include abstinence and nutritional support, reducing the severity of alcoholic liver disease, improving existing secondary malnutrition, and symptomatic treatment of alcoholic cirrhosis and its complications. Abstinence is the most important measure for treating alcohol-related liver disease, and withdrawal syndrome should be prevented and treated during the abstinence process. It is worth noting that patients with alcohol-related liver disease may still progress to alcoholic liver fibrosis and cirrhosis after abstinence. For patients with advanced alcoholic cirrhosis, simple abstinence cannot effectively reverse liver damage, and liver transplantation is required. Of course, liver transplantation can also be considered for patients with alcoholic hepatitis who do not respond to drug treatment. Liver transplantation is difficult to become a widely implemented treatment method in clinical practice due to its high cost and lack of donors. Therefore, there is an urgent need to develop more effective and universal treatments and regimens for alcohol-related liver disease. In fact, many clinical studies have focused on this and have developed some potential treatments for alcohol-related liver disease. Based on the existing literature, the following drugs with potential therapeutic potential are summarized:

[0007] (1) Alleviate liver cell damage

[0008] Accumulating evidence suggests that hepatocellular injury, at least in part caused by ethanol-induced oxidative stress and innate immune responses, plays a crucial role in the progression of alcohol-related liver disease. Therefore, protecting hepatocellular cells from damage is considered a potential therapeutic strategy. S-adenosylmethionine can restore mitochondrial glutathione and improve steatosis in rodents, and thus may be a potential treatment option for alcohol-related liver disease. Granulocyte colony-stimulating factor (G-CSF) is a potent growth factor that has been proposed to promote hepatocellular regeneration in severe alcoholic hepatitis. A meta-analysis showed that G-CSF was associated with a greater than 70% reduction in 90-day mortality in patients with alcoholic hepatitis. In addition, F-652, an agonist of the anti-inflammatory cytokine IL-22, has been shown in a phase 2 clinical trial to reduce inflammatory markers and promote liver regeneration in patients with alcoholic hepatitis.

[0009] (2) Alleviate inflammatory response

[0010] Chronic inflammation is a key factor in the development of alcohol-related liver disease, suggesting that modulating the inflammatory response is a promising therapeutic strategy for improving alcohol-related liver disease. Glucocorticoids (such as prednisolone) are currently commonly used as first-line anti-inflammatory drugs for patients with severe alcoholic hepatitis; however, prednisolone is ineffective in most patients and increases the risk of bacterial and fungal infections. In a clinical study in the UK, liver biopsies of patients with alcoholic hepatitis who were treated with an IL-1β antibody for 28 days showed histological improvement in their livers.

[0011] Targeted microbial therapy

[0012] In recent years, with increasing understanding of the impact of ethanol on intestinal pathophysiology, the gut microbiome has become a major target for developing treatments for alcohol-related liver disease. In animal studies, probiotics have helped restore gut microbial diversity and ameliorated liver damage and intestinal barrier impairment in mice. In a multicenter randomized controlled trial, probiotics significantly reduced TNF-α and LPS levels in patients with alcoholic hepatitis compared with placebo. Furthermore, clinical studies have shown that even in patients with severe alcoholic hepatitis, microbiota can be altered and liver damage can be ameliorated without complications after receiving microbiota from healthy donors. Furthermore, in a recent study, Duan et al. identified cytolytic Enterobacter faecalis strains as a significant factor in exacerbating hepatocellular damage and mortality in patients with severe alcoholism. Using humanized mice colonized with bacteria collected from the feces of patients with alcoholic hepatitis (AH), the researchers found that certain phages specifically targeted cytolytic E. faecalis, reduced cytolytic activity in the liver, and abrogated ethanol-induced liver disease. This phage therapy offers a method for precisely editing the gut microbiome, but larger human clinical trials are needed to validate these results.

[0013] 3. Bacteriophage

[0014] Bacteriophages are viruses that invade bacterial cells. This invasion causes bacterial cell lysis, disrupting bacterial metabolism and leading to bacterial self-destruction. Phage therapy is a treatment method that uses phages to lyse bacteria to treat pathogenic infections. In recent years, scientists have made progress in using phage therapy to treat bacteria such as Mycobacterium, Klebsiella pneumoniae, and Escherichia coli, as well as influenza viruses, and diseases such as alcoholic liver disease and inflammatory bowel disease associated with intestinal bacteria.

[0015] Phage therapy has the following advantages: (1) Bacterial host specificity: Each phage attacks only a very limited number of bacteria and is almost specific to one type of bacteria. Therefore, they can target specific pathogenic bacteria without affecting the normal bacterial flora in the host. (2) Easy to grow and purify: Similar to antibiotics, bacteria can also develop resistance to phages, but developing new phages is much simpler than developing new antibiotics. It only takes a few weeks to obtain new phages, while it takes many years to obtain new antibiotics. (3) Strong penetration: They can reach all sites of bacterial infection, while the concentration of antibiotics will quickly decrease below the infected surface, so local use of phages has special advantages. (4) Reproducibility: The number of phages will increase as the target bacterial population spreads, so small doses can be administered. (5) Non-toxicity: phages only invade bacteria and not human cells. (6) Self-limiting: Once the target bacterial population is eliminated, the number of phages will drop sharply. In general, phages have become an effective choice for combating bacterial infections due to their many advantages. Summary of the Invention

[0016] The purpose of the present invention is to provide a method for preparing a phage targeting ExPEC, so as to obtain a phage targeting ExPEC for use in preparing a drug for treating or preventing alcohol-related liver disease, thereby opening up a new treatment method for alcohol-related liver disease.

[0017] In view of this, the scheme of the present invention is as follows:

[0018] In one aspect of the present invention, a method for preparing a bacteriophage targeting ExPEC is proposed, which is obtained by mixing a host bacterium, a bacteriophage filtrate, and a culture medium in proportion, and then proliferating, culturing, isolating, and purifying the bacterium; the host bacterium is a logarithmic phase bacterial solution of extraintestinal pathogenic Escherichia coli from porcine.

[0019] Furthermore, the phage filtrate is obtained by centrifuging environmental wastewater, removing the supernatant, and sterilizing it by microfiltration. The environmental wastewater includes, but is not limited to, aquaculture wastewater, domestic wastewater, food processing wastewater, and river water, which are wastewater conducive to phage reproduction; aquaculture wastewater includes poultry farm wastewater and livestock farm wastewater; and domestic wastewater includes market wastewater and residential wastewater.

[0020] Furthermore, the porcine extraintestinal pathogenic Escherichia coli is obtained by inoculating and culturing Escherichia coli PCN033 strain.

[0021] Furthermore, the volume ratio of the host bacteria, phage filtrate, and culture medium is 1:1:2; and / or the culture medium is TSB culture medium.

[0022] Furthermore, the separation step adopts a double-layer plate method to separate the phage; and / or the purification step adopts a dot-blot method to purify the phage.

[0023] The second aspect of the present invention provides a bacteriophage obtained by the preparation method described in the first aspect.

[0024] The third aspect of the present invention provides the use of the bacteriophage described in the second aspect in the preparation of a drug for treating or preventing alcohol-related liver disease.

[0025] Furthermore, the drug further comprises one or more pharmaceutically or physiologically acceptable carriers, and / or excipients, and / or diluents.

[0026] Furthermore, the drug reduces the content of liver enzymes in serum, and / or reduces the expression of genes related to liver inflammation, and / or alleviates lipid deposition and inflammatory cell infiltration in the liver.

[0027] Preferably, the liver enzymes include ALT and AST; and the liver inflammation-related genes include IL-1β, Cxcl1 and Cxcl2.

[0028] Compared with the prior art, the beneficial effects of the present invention include but are not limited to:

[0029] 1. The present invention provides a method for preparing the ExPEC-targeting phage, which has readily available materials, a simple and convenient process, and is suitable for promotion.

[0030] 2. The ExPEC-targeted phages obtained in this invention can precisely target ExPECs in the intestine without affecting other intestinal flora, thereby mitigating their exacerbating effects on alcohol-induced liver steatosis and inflammatory damage. Therefore, phage therapy has the potential to become a new and effective treatment for alcohol-related liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0032] FIG1 is a schematic diagram of phage lysis plaques in Example 1.

[0033] Figure 2 is a TEM electron microscope image of the bacteriophage in Example 2.

[0034] FIG3 shows the effects of different treatments in Example 3 on liver enzyme levels in mouse serum.

[0035] FIG4 is a graph showing the effects of different treatments on the expression of genes related to liver inflammation in mice in Example 3.

[0036] FIG5 is a graph showing the effects of different treatments on fatty degeneration and inflammation in mouse liver tissue in Example 3. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solution and beneficial technical effects of the present invention clearer, the technical solution of the present invention is further described in detail in conjunction with the embodiments. It should be understood that the specific implementation methods described in this specification are only for explaining the present invention and are not intended to limit the present invention.

[0038] Example 1 Preparation of bacteriophage

[0039] A method for preparing a bacteriophage targeting intestinal ExPEC comprises the following steps:

[0040] (1) Sample processing: The collected environmental sewage was centrifuged at 8000 rpm for 15 min, and the supernatant was sterilized by filtering through a 0.22 μm microporous filter membrane to obtain the filtrate containing bacteriophages.

[0041] (2) Preparation of host bacteria: A single colony of Escherichia coli PCN033 was selected and inoculated into 1 ml of LB broth. The culture was carried out at 220 rpm and 37°C for 24 h to obtain a host bacteria suspension for later use.

[0042] (3) Phage proliferation: The host bacteria PCN033 culture liquid was transferred to TSB medium at a ratio of 1:100 and cultured at 220 rpm and 37°C for 1.5-3 h to the logarithmic phase. The host bacteria, phage filtrate, and TSB medium were added to a sterilized conical flask at a ratio of 1:1:2 (volume ratio), mixed, and cultured at 220 rpm and 37°C for 2-3 h to enrich the phage.

[0043] (4) Phage isolation: After the above phage valerization solution was allowed to stand at 4°C for 2 h, 20 mL was taken and centrifuged at 7000 rpm at 4°C for 10 min. The supernatant was sterilized by filtering through a 0.22 μm microporous filter to obtain the phage stock solution for phage isolation.

[0044] (5) Use the double-layer plate method to isolate phages. Take 300 μL of the host bacterial suspension and place it in a sterile 10 mL EP tube. Add 1 mL of phage stock solution and then add melted semi-solid to 8 mL. Quickly pour the suspension onto a prepared plate with TSA culture medium as the bottom layer. Tilt and rotate the plate to evenly distribute the suspension. After the agar solidifies, incubate the suspension at 37°C overnight and observe the results. If phages are present in the stock solution, they will form eroded, transparent spots on the upper agar plate, forming a sharp contrast with the yellow-white, misty bacterial lawn.

[0045] (6) Phage purification: Phage purification was performed using the dot plaque method. Use a sterile 200μL pipette tip to pick up a plaque with a relatively uniform shape and size, inoculate it into 3ml of PBS, shake it and let it stand at 4℃ for 2h, take the supernatant and sterilize it through a 0.22μm microporous filter. Dilute the filtrate with PBS for later use. Take 300μL of the host bacterial suspension into a sterile 10mL EP tube, add the melted semi-solid agar medium to 8mL, and quickly pour it into the prepared plate with TSA medium as the bottom layer. Tilt and rotate the plate to make it evenly distributed. After the agar solidifies, take 10μL of the phage filtrate according to the dilution and dot it on the plate. After drying, invert and culture it at a constant temperature of 37℃ overnight. Use a sterile 10μL pipette tip to pick up a single plaque and inoculate it into 1mL of PBS, shake it and let it stand at 4℃ for 2h, take the supernatant and sterilize it through a 0.22μm microporous filter, and mark it as F1. Repeat this step 3-5 times, and the size and morphology of the plaques observed each time remain uniform, as shown in Figure 1, to obtain purified phage.

[0046] Example 2 Characterization of phage targeting ExPEC

[0047] Take 10 μL of phage proliferation solution (10 9A 10 μL (0.175 μg / mL) solution (100 μL) of 2% phosphotungstic acid (PTFA) was dripped onto a copper grid and allowed to stand for 15 minutes. Excess liquid was then removed with filter paper. The cells were then stained with 10 μL of 2% phosphotungstic acid for 5-10 minutes, air-dried, and the morphological characteristics of the phage were observed under a transmission electron microscope. Transmission electron microscopy (TEM) images are shown in Figure 2. These images reveal that the head of ph033 is approximately 92 nm in diameter and the tail is approximately 107 nm long. Phylogenetic analysis based on the nucleotide sequence of the phage's major capsid protein indicates that ph033 is a member of the genus Tequatrovirus in the family Myoviridae.

[0048] Example 3 Effects of phage targeting ExPEC

[0049] 1. Objective: To establish an internationally recognized alcoholic liver disease model (NIAAA model) and administer different treatments (oral administration of PBS, ExPEC, and ExPEC plus phage targeting ExPEC) to examine liver steatosis and inflammation-related markers. This study aimed to determine whether ExPEC exacerbates alcohol-induced liver damage and, if so, to further determine whether phage targeting ExPEC can reverse the exacerbating effect of ExPEC on the pathological progression of alcohol-related liver disease.

[0050] 2. Experimental methods: C57 mice (8 weeks old) were used. After one week of adaptation, they were first given a Lieber-DeCarli control liquid diet for 5 days. Then, the "alcohol model group" was given a 5% (vol / vol) Lieber-DeCarli alcohol liquid diet for 10 days. The "control group" was given a control diet with the same calorie content as the alcohol group. According to the different treatments of each group from the 6th to the 15th day, the "control group" and the "alcohol model group" were further divided into the "PBS group" (each mouse was gavaged with 100ul PBS on the 6th, 9th, 12th and 15th day of modeling), the "ExPEC group" (each mouse was gavaged with 100ul ExPEC on the 6th, 9th, 12th and 15th day of modeling, and the number of ExPEC was 10^9 CFU / mouse) and the "ExPEC+phage group" (each mouse was gavaged with 100ul ExPEC on the 6th, 9th, 12th and 15th day of modeling, and the number of ExPEC was 10^9 CFU / mouse, and on the 15th day, 1 hour after the mice were gavaged with ExPEC, each mouse was gavaged with 100ul phage, and the number was 10^11 PFU / mouse). Finally, on day 16, mice in the alcohol model group were gavaged with a large dose of alcohol (40% vol / vol, 5g / kg). Three hours later, each mouse in the "PBS group" was gavaged with 100µl of PBS, and each mouse in the "ExPEC" and "ExPEC + phage" groups was gavaged with 100µl of ExPEC, at a concentration of 109 CFU / mouse. One hour later, each mouse in the "PBS" and "ExPEC" groups was gavaged with 100µl of PBS, and each mouse in the "ExPEC + phage" group was gavaged with 100µl of phage, at a concentration of 1011 PFU / mouse. Nine hours after the alcohol gavage, the mice were anesthetized, and blood and liver samples were collected for analysis.

[0051] 3. Experimental results:

[0052] 1) ExPEC can further aggravate the alcohol-induced increase in liver enzymes (alanine aminotransferase ALT and aspartate aminotransferase AST, which are positively correlated with the degree of liver damage), while bacteriophage can reverse this aggravating effect.

[0053] Figure 3 shows the serum liver enzyme levels in each group. Panel A: ALT levels in serum of mice in each group; Panel B: AST levels in serum of mice in each group. Differences between the three groups were analyzed using the One-Way Anova test. If data did not conform to normality or homogeneity of variance, the Kruskal-Wallis test was used. *: p < 0.05, **p < 0.01. As can be seen in Figure 3, liver enzyme levels in the alcohol model group were significantly higher than those in the control group, indicating successful establishment of the alcoholic liver disease model. Furthermore, within the alcohol model group, liver enzyme levels in the ExPEC group were significantly elevated compared to the PBS group; whereas, liver enzyme levels in the ExPEC + phage group decreased compared to the ExPEC group. This biochemical analysis demonstrates that ExPEC exacerbates alcohol-induced liver damage, while phage reverses this exacerbating effect of ExPEC.

[0054] 2) ExPEC can further aggravate the upregulation of inflammatory-related genes (IL-1β, Cxcl1, Cxcl2) induced by alcohol, while phage can reverse this aggravating effect.

[0055] Figure 4 shows the expression levels of inflammation-related genes (IL-1β, Cxcl1, and Cxcl2) in the livers of each group. These are common tissue inflammatory factors; higher expression levels indicate more severe inflammatory damage. Panel A: Liver IL-1β expression; Panel B: Liver Cxcl1 expression; Panel C: Liver Cxcl2 expression. Differences between the three groups were analyzed using the One-Way Anova test. If the data did not meet normality and homogeneity of variance, the Kruskal-Wallis test was used. *: p < 0.05, **p < 0.01. As can be seen in Figure 4, the levels of IL-1β, Cxcl1, and Cxcl2 in the "alcohol model group" were significantly higher than those in the "control group," indicating that the alcoholic liver disease model was successfully established. Furthermore, in the alcohol model group, compared with the PBS group, the ExPEC group showed significantly increased levels of IL-1β, Cxcl1, and Cxcl2. However, compared with the ExPEC group, the ExPEC+phage group showed decreased levels of IL-1β, Cxcl1, and Cxcl2. This transcriptomic analysis suggests that ExPEC can aggravate alcohol-induced liver inflammation, while phage can reverse this exacerbating effect of ExPEC.

[0056] 3) ExPECs can further aggravate alcohol-induced liver steatosis and inflammation, while phages can reverse this aggravating effect.

[0057] Figure 5 shows the pathological findings of liver tissue from each group, magnified 200x. HE staining of the liver identifies hepatic steatosis and hepatitis by observing the presence of vacuoles of varying sizes within the hepatocyte cytoplasm and the distribution and composition of inflammatory cells. Compared with the control group, the alcohol model group showed a significant increase in fat vacuoles and the presence of inflammatory cell infiltration, primarily lymphocytes, demonstrating successful alcoholic liver disease modeling. Furthermore, within the alcohol model group, the ExPEC group showed significantly larger and more numerous fat vacuoles, with more pronounced inflammatory cell infiltration, compared with the PBS group. In contrast, the ExPEC + phage group showed significantly smaller and fewer fat vacuoles, and a less pronounced inflammatory cell infiltration, compared with the ExPEC group. This histological analysis demonstrates that ExPEC exacerbates alcohol-induced hepatic steatosis and inflammation, while phage reverses these exacerbating effects of ExPEC.

[0058] 4. Experimental Conclusions: Phage targeting ExPEC can significantly reverse the aggravating effect of ExPEC on alcohol-related liver disease at multiple levels: at the biochemical level, the phage of the present invention can significantly reduce the levels of serum biochemical indicators reflecting liver damage (ALT and AST); at the transcriptional level, it can significantly reduce the expression of liver inflammation-related genes (IL-1β, Cxcl1 and Cxcl2); at the tissue level, it can reduce lipid deposition and inflammatory cell infiltration in the liver.

[0059] Although the present invention is disclosed as above, the scope of protection disclosed by the present invention is not limited thereto. Those skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention, and these changes and modifications will fall within the scope of protection of the present invention.

Claims

1. A method for preparing a phage targeting ExPEC, characterized in that: The host bacteria is obtained by mixing host bacteria, phage filtrate and culture medium in proportion, then multiplying, culturing, separating and purifying; the host bacteria is a logarithmic phase bacterial solution of extraintestinal pathogenic Escherichia coli from pigs.

2. The preparation method according to claim 1, characterized in that The bacteriophage filtrate is obtained by centrifuging environmental sewage to obtain the supernatant and performing microfiltration for sterilization.

3. The preparation method according to claim 1, characterized in that The porcine extraintestinal pathogenic Escherichia coli is obtained by inoculating and culturing the Escherichia coli PCN033 strain.

4. The preparation method according to claim 1, characterized in that The volume ratio of the host bacteria, phage filtrate and culture medium is 1:1:2; and / or the culture medium is TSB culture medium.

5. The preparation method according to claim 1, characterized in that The separation step adopts a double-layer plate method to separate the phage; and / or the purification step adopts a dot-blot method to purify the phage.

6. The bacteriophage obtained by the preparation method according to any one of claims 1 to 5.

7. Use of the bacteriophage according to claim 6 in the preparation of a drug for treating or preventing alcohol-related liver disease.

8. The use according to claim 7, characterized in that The medicament further comprises one or more pharmaceutically or physiologically acceptable carriers, and / or excipients, and / or diluents.

9. The use according to claim 7, characterized in that The drug reduces the content of liver enzymes in serum, and / or reduces the expression of genes related to liver inflammation, and / or alleviates lipid deposition and inflammatory cell infiltration in the liver.

10. The use according to claim 9, characterized in that The liver enzymes include ALT and AST; the liver inflammation-related genes include IL-1β, Cxcl1 and Cxcl2.

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