Preparation method of phage targeting extra-intestinal pathogenic escherichia coli (ExPEC) and use

Abstract

A method for preparing a bacteriophage targeting extra-intestinal pathogenic Escherichia coli (ExPEC) comprising mixing pig (porcine) extraintestinal E. coli host cells at logarithmic phase with phage

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the technical field of biomedicine, in particular to a preparation method of a phage targeting extra-intestinal pathogenic Escherichia coli (ExPEC) and use. BACKGROUND

[0002] Alcohol-related liver disease (ALD) refers to a series of hepatic lesions caused by excessive or long-term drinking, including alcoholic fatty liver, alcoholic hepatitis, alcoholic cirrhosis and alcoholic liver cancer. According to the survey, both the proportion of drinkers and the prevalence of ALD are on the rise worldwide. In 2019, about 25% of cirrhosis-induced deaths worldwide were related to alcohol, and the annual incidence rate of hepatocellular carcinoma in patients with ALD ranged from 0.9% to 5.6%. Obviously, ALD imposes a huge burden on the social economy and global health. Nevertheless, so far, there are no approved effective therapies for ALD other than alcohol abstinence and liver transplantation. However, alcohol abstinence and liver transplantation cannot be widely and effectively implemented as treatment options in clinical practice due to their difficulty in long-term persistence and lack of donors. Therefore, it is urgent to find new treatment options for ALD.

[0003] I. The etiology and pathogenesis of ALD

[0004] The etiology and pathogenesis of ALD are complex. The high-risk factors for ALD identified in current domestic and international research include: type of alcoholic beverage, alcohol consumption, duration of alcohol consumption, nutritional status, genetic susceptibility, and ethnicity. So far, the pathological mechanism of ALD has not been fully elucidated, but it is generally believed to be related to the following three points. Firstly, alcohol and its active metabolite acetaldehyde can directly cause hepatotoxicity and liver injury; secondly, liver injury can further induce the release of damage-associated molecular patterns, thereby recruiting innate and adaptive immune cells, causing a series of immune inflammatory reactions, and exacerbating liver injury; finally, alcohol intake can affect the abundance and composition of intestinal flora, disrupt the intestinal barrier, and cause the translocation of intestinal flora and its metabolites to the liver, leading to immune responses and liver injury.

[0005] II. Treatment of ALD

[0006] The treatment principles of ALD include alcohol abstinence and nutritional support, reducing the severity of alcoholic liver disease, improving existing secondary malnutrition, and symptomatic treatment of alcoholic cirrhosis and its complications. Alcohol abstinence is the most important measure for the treatment of ALD. Abstinence syndrome should be prevented and treated during alcohol abstinence. It is worth noting that patients with ALD may still progress to alcoholic liver fibrosis and cirrhosis after alcohol abstinence. For patients with advanced alcoholic cirrhosis, alcohol abstinence alone cannot effectively reverse liver injury and liver transplantation is needed. Of course, patients with alcoholic hepatitis who do not respond to drug therapy may also consider undergoing liver transplantation. Liver transplantation is difficult to become a widely implemented treatment option in clinical practice due to its high cost and shortage of donors. Therefore, there is an urgent need to develop more effective and universal medications and therapeutic regimens for ALD. In fact, a plurality of clinical studies have focused on this and developed some promising medicaments for the treatment of ALD. Based on the existing literature, the medicaments that may have therapeutic potential are summarized as follows:

[0007] (i) Alleviation of liver cell injury

[0008] More and more evidence suggests that liver cell injury caused at least in part by ethanol-induced oxidative stress and innate immune response plays a crucial role in the progression of ALD. Therefore, protecting hepatocytes from injury is considered as a potential treatment strategy. S-Adenosylmethionine can restore glutathione in mitochondria and improve steatosis in rodents, so it may be a potential treatment option for ALD. Granulocyte colony-stimulating factor (G-CSF) is an effective growth factor that has been proposed to promote hepatocyte regeneration in severe alcoholic hepatitis. A meta-analysis showed that G-CSF was associated with a reduction of the 90-day mortality by more than 70% in patients with alcoholic hepatitis. In addition, the agonist F-652 of anti-inflammatory cytokine interleukin-22 (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] (ii) Alleviation of inflammatory response

[0010] Chronic inflammation is a key factor in the development of ALD, suggesting that regulating the inflammatory response is a promising treatment strategy for improving ALD. Glucocorticoids (such as prednisolone) are currently used as first-line anti-inflammatory drugs for patients with severe alcoholic hepatitis; however, prednisolone is ineffective for most patients and increases the risk of bacterial and fungal infections. In a clinical study in the UK, 28 days after treatment with anti-interleukin-1 beta (IL-ip) antibody in patients with alcoholic hepatitis, the results of hepatic biopsy in these patients showed a histological improvement in their livers.

[0011] (iii) Targeted microbial therapy

[0012] In recent years, with the increasing understanding of the effect of ethanol on intestinal pathophysiology, intestinal microflora has become one of the main targets for the development of therapies for ALD. In animal experiments, probiotics can help restore intestinal microbial diversity in mice, and further improve liver injury and intestinal barrier damage in mice. In a multicenter randomized controlled trial (RCT), tumor necrosis factor alpha (TNF-a) and lipopolysaccharide (LPS) in patients with alcoholic hepatitis were significantly reduced in the probiotics group compared to the placebo group. In addition, clinical studies have shown that even patients with severe alcoholic hepatitis can experience changes in their intestinal microflora and improvement in liver injury after receiving microbiota from healthy donors, without the occurrence of complications. In addition, in a recent study, Duan et al. found that the cytolysin-secreting Enterococcus faecalis strain was an important factor for aggravating liver cell injury and death in patients with severe alcoholism. Using humanized mice colonized with bacteria collected from the feces of patients with alcoholic hepatitis (AH), researchers found that certain phages specifically targeted Enterococcus faecalis with cytolysis, reduced cytolysin in the liver, and eliminated ethanol-induced liver disease. This phage therapy provides a method for precisely editing the intestinal microflora, but more people are required to participate in clinical trials to verify these results.

[0013] III. Phage

[0014] Phages are viruses that invade bacterial cells. The invasion of these viruses can lead to the lysis of bacterial cells, destroy the metabolism of bacteria, and cause bacterial self-destruction. Phage therapy is a treatment method that uses phages to lyse bacteria and treat pathogenic infections. In recent years, scientists have made progress in using phage therapy to treat bacteria such as Mycobacterium sp., Klebsiella pneumoniae, and Escherichia cole influenza virus, as well as diseases such as alcoholic liver disease and inflammatory bowel diseases associated with intestinal bacteria.

[0015] Phage therapy has the following advantages: (1) Bacterial host specificity: Each phage attacks only a very limited number of bacterial species and is almost specific to one bacterial species. Therefore, they can target specific pathogenic bacteria without affecting the normal flora in the host. (2) Proneness to growth and purification: Similar to antibiotics, bacteria can also develop resistance to phages, but developing new phages is much simpler than developing new antibiotics. It takes only a few weeks to obtain new phages, while it takes a plurality of years to obtain new antibiotics. (3) Strong penetrativity: Phages can reach all sites of bacterial infection, and the concentration of antibiotics will decrease rapidly below the infected surface, so the topical use of phages has special advantages. (4) Multiplicability: The number of phages will increase when the target flora diffuses, so drugs can be administered in small doses. (5) Non-toxicity: Phages only invade bacteria but not human cells. (6) Self-limiting: Once the target flora is eliminated, the number of phages will sharply decrease. In general, phages have become an effective choice for anti-bacterial infection due to a plurality of advantages thereof. SUMMARY

[0016] An objective of the present disclosure is to provide a preparation method of a phage targeting ExPEC. The phage targeting ExPEC obtained is used in preparing a medicament for treatment or prophylaxis of ALD, opening up a new treatment option for ALD.

[0017] In view of this, the technical solutions of the present disclosure are as follows:

[0018] A first aspect of the present disclosure is to provide a preparation method of a phage targeting ExPEC, where the phage targeting ExPEC is obtained by mixing a bacterial host, a phage filtrate, and a culture medium in proportion, followed by proliferation, culture, isolation, and purification; and the bacterial host is a suspension of porcine extra-intestinal pathogenic Escherichia coli at logarithmic phase.

[0019] Further, the phage filtrate is obtained by centrifugation of environmental sewage to collect a supernatant and microfiltration to remove cells. The environmental sewage includes but is not limited to livestock sewage, domestic sewage, food processing sewage, river water, and other sewage conducive to phage multiplication. The livestock sewage is selected from the group consisting of livestock and poultry breeding sewage and livestock farm sewage, and the domestic sewage is selected from the group consisting of vegetable market sewage and living area sewage.

[0020] Further, the porcine extra-intestinal pathogenic Escherichia coli is obtained by inoculating and culturing an Escherichia coli PCN033 strain.

[0021] Further, the bacterial host, the phage filtrate, and the culture medium have a volume ratio of 1:1:2; and / or the culture medium is Tryptic Soy Broth (TSB).

[0022] Further, the isolation is achieved by a double layer agar plate method for phage isolation; and / or the purification is achieved by dot blotting for phage purification.

[0023] A second aspect of the present disclosure provides a phage obtained by the preparation method in the first aspect.

[0024] A third aspect of the present disclosure provides use of the phage in the second aspect in preparing a medicament for treatment or prophylaxis of ALD.

[0025] Further, the medicament further includes one or more pharmaceutically or physiologically acceptable carriers, and / or a vehicle, and / or a diluent.

[0026] Further, the medicament lowers content of liver enzymes in serum, and / or down-regulates expression of liver inflammation-associated genes, and / or alleviates lipid deposition and inflammatory cell infiltration in the liver.

[0027] Preferably, the liver enzymes include alanine transaminase (ALT) and aspartate aminotransferase (AST); and the liver inflammation-associated genes include interleukin-1 beta (IL-1 ft), C-X-C motif chemokine ligand 1 (Cxcllf and C-X-C motif chemokine ligand 2 (Cxcl2).

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

[0029] 1. The preparation method of a phage targeting ExPEC provided by the present disclosure is characterized by easy availability of materials, simple and convenient process, which is suitable for promotion.

[0030] 2. The phage targeting ExPEC obtained by the present disclosure can precisely target the ExPEC in the intestine without affecting other intestinal flora, thereby alleviating exacerbating effects of the ExPEC on alcohol-induced hepatic steatosis and inflammatory injury. Therefore, phage therapy is expected to become one of the new effective therapies for ALD. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to describe the technical solutions in the examples of the present disclosure more clearly, the accompanying drawings required for describing the examples will be briefly described below. Obviously, the accompanying drawings in the following description show merely some examples of the present disclosure, and a person of ordinary skill in the art can further derive other accompanying drawings from these accompanying drawings without creative efforts.

[0032] FIG. 1 is a schematic diagram of lytic phage plaques in Example 1;

[0033] FIG. 2 is a transmission electron microscopy (TEM) image of phages in Example 2;

[0034] FIG. 3 illustrates effects of different treatments on levels of liver enzymes in mouse serum in Example 3;

[0035] FIG. 4 illustrates results of effects of different treatments on expression of mouse liver inflammation-associated genes in Example 3; and

[0036] FIG. 5 illustrates results of effects of different treatments on hepatic steatosis and inflammation in mice in Example 3. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0037] To make the objectives, technical solutions, beneficial effects of the present disclosure clearer, the technical solutions of the present disclosure will be further described below with reference to the examples. It should be understood that the specific implementations described herein are merely intended to explain the present disclosure, rather than to limit the present disclosure.

[0038] Example 1 Preparation of phage

[0039] A preparation method of a phage targeting ExPEC was provided, including the following steps:

[0040] (1) Sample treatment: The collected environmental sewage was centrifuged at 8,000 rpm for 15 min, and the supernatant was collected and filtered through a 0.22 pm millipore filter to remove cells and obtain a phage filtrate.

[0041] (2) Preparation of bacterial host: A single colony of Escherichia coli PCN033 was inoculated in 1 ml of Luria-Bertani (LB) broth at 220 rpm for 24 h at 37°C to obtain a bacterial host suspension for later use.

[0042] (3) Phage multiplication: The bacterial host PCN033 suspension was transferred to TSB at a ratio of 1:100 and cultured at 220 rpm for 1.5-3 h at 37°C to logarithmic phase. The bacterial host, phage filtrate, and TSB were added to a sterilized Erlenmeyer flask at a ratio of 1:1:2 (volume ratio), mixed well, and cultured at 220 rpm for 2-3 h at 37°C to enrich phages.

[0043] (4) Phage isolation: After the above phage multiplication suspension was left to stand at 4°C for 2 h, 20 mL of the suspension was centrifuged at 7,000 rpm for 10 min at 4°C. The supernatant was then filtered through a 0.22 pm millipore filter to obtain a phage stock solution, which was used for phage isolation.

[0044] (5) The phages were isolated by double layer agar plate method; 300 pL of the bacterial host suspension was charged into a sterile 10 mL EP tube and supplemented with 1 mL of the phage stock solution; then, the melted semi-solid was added to 8 mL and quickly poured into a well-prepared Petri dish with TSA at the bottom, and the Petri dish was tilted and rotated to make it evenly distributed; after the agar was solidified, it was inverted overnight at a constant temperature of 37°C to observe the result. If there are phages in the stock solution, a silkworm-eaten transparent plaque can be formed on the upper agar plate, which is in sharp contrast to the yellowish white misty lawn.

[0045] (6) Phage purification: The phages were purified by dot blotting. A relatively uniform sized plaque was picked up with a sterile 200 pL pipette tip, inoculated into 3 mL of phosphate-buffered saline (PBS), shaken, and left to stand for 2 h at 4°C. The supernatant was filtered through a 0.22 pm millipore filter to remove cells. The filtrate was diluted with PBS, respectively. Then 300 pL of the bacterial host suspension was charged into a sterile 10 mL EP tube; the melted semi-solid agar medium was added to 8 mL and quickly poured into a well-prepared Petri dish with TSA at the bottom, and the Petri dish was tilted and rotated to make it evenly distributed; after the agar was solidified, 10 pL each of phage filtrate was, by dilution, applied on a Petri dish, dried, inverted and cultured at a constant temperature of 37°C overnight. A single plaque was picked up with a sterile 10 pL pipette tip, inoculated into 1 mL of PBS, shaken, and left to stand for 2 h at 4°C. The supernatant was filtered through a 0.22 pm millipore filter to remove cells, and labeled as Fl. This step was repeated 3-5 times. The size and morphology of the plaque observed each time remained uniform, as shown in FIG. 1, and the purified phages were obtained.

[0046] Example 2 Characterization of phage targeting ExPEC

[0047] Ten microlitre (10 pL) of phage multiplication suspension (109 PFU / mL) was added dropwise to a copper mesh, and left to stand for 15 min; the excess liquid was sucked by filter paper; 10 pL of 2% phosphotungstic acid was added dropwise to stain for 5-10 min, and then dried naturally. The morphological characteristics of phage were observed under a transmission electron microscope (TEM). The TEM image is shown in FIG. 2. The TEM image reveals that the head of ph033 is about 92 nm in length, and the tail is about 107 nm in length. The phylogenetic analysis based on the nucleotide sequences of the major capsid proteins of phages showed that ph033 was a member of the genus Tequatrovirus in the family Myoviridae.

[0048] Example 3 The effect of the phage targeting ExPEC

[0049] 1. Purpose: Different treatments (PBS, ExPEC, ExPEC + phage targeting ExPEC were given by gavage, respectively) were given by constructing an internationally recognized alcoholic liver model (NIAAA model). Indices related to hepatic steatosis and inflammation were detected to determine whether ExPEC aggravated alcohol-induced liver injury. If so, it was further clarified whether the phage targeting ExPEC reversed the exacerbating effect of ExPEC on the pathological progression of ALD.

[0050] 2. Methods: C57 mice (8 weeks old) were acclimatised for one week, Lieber-DeCarli control liquid feed was given for 5 days, and then the "alcohol model group" was given 5% (vol / vol) Lieber-DeCarli alcohol liquid feed for 10 days. The "control group" was given a control diet with the same calorie as the alcohol group. According to the different treatments of each group from days 6 to 15, the "control group" and "alcohol model group" were further divided into "PBS group" (each mouse was given 100 pL of PBS by gavage at 6, 9, 12, and 15 days after modeling), "ExPEC group" (each mouse was given 100 pL of ExPEC by gavage at 6, 9, 12, and 15 days after modeling, with an ExPEC count of 10A9 CFU / mouse), and "ExPEC + phage group" (each mouse was given 100 pL of ExPEC by gavage at 6, 9, 12, and 15 days after modeling, with an ExPEC count of 10A9 CFU / mouse; on day 15, at 1 h after administration of ExPEC by gavage, each mouse was given 100 pL of phages by gavage, with a phage count of 10Al 1 PFU / mouse), respectively. Finally, on day 16, the mice in the alcohol model group were given a large volume of alcohol (40% vol / vol, 5 g / kg) by gavage; after 3 h, each mouse in the "PBS group" was given 100 pL of PBS by gavage, while each mouse in the "ExPEC group" and "ExPEC + phage group" was given 100 pL of ExPEC by gavage, with an ExPEC count of 10A9 CFU / mouse. After another 1 h, each mouse in the "PBS group" and "ExPEC group" was given 100 pL of PBS, while each mouse in the "ExPEC + phage group" was given 100 pL of phages, with a phage count of 10Al 1 PFU / mouse. At 9 h after administration of alcohol by gavage, the mice were anaesthetised, and blood, liver and other specimens were sampled for related tests.

[0051] 3. Results:

[0052] 1) ExPEC further exacerbated the elevation of liver enzymes (ALT and AST, positively correlated with the severity of liver injury) induced by alcohol, and the phage reversed this exacerbating effect.

[0053] FIG. 3 illustrates the levels of liver enzymes in the serum of each group. FIG. 3A illustrates mouse serum ALT levels of each group; FIG. 3B illustrates mouse serum AST levels of each group. The difference among the three groups was tested by one-way ANOVA. If the data do not conform to normality or homogeneity of variance, Kruskal-Wallis test is used. *: P <0.05, ** P <0.01. It can be seen from FIG. 3 that the "alcohol model group" shows significantly higher levels of liver enzymes than the "control group", indicating that the alcoholic liver model is successfully established. In addition, in the "alcohol model group", the levels of liver enzymes were significantly elevated in the "ExPEC group" compared with the "PBS group"; the levels of liver enzymes fell back in the "ExPEC + phage group" compared with the "ExPEC group". This indicated at the biochemical level that ExPEC could exacerbate alcohol-induced liver injury, and the phage could reverse this exacerbating effect of ExPEC.

[0054] 2) ExPEC further exacerbated the up-regulation of the expression of alcohol-induced inflammation-associated genes (11-1 / ), Cxcll, and Cxcl2\ and the phage reversed this exacerbating effect.

[0055] FIG. 4 illustrates the expression levels of liver inflammation-associated genes of each group (11-1 / ), Cxcll and Cxcl2 are common tissue inflammatory factors, and the higher the expression level is, the more severe the inflammatory injury is). FIG. 4A illustrates the expression of IL-1 ft in the liver; FIG. 4B illustrates the expression of Cxcll in the liver; FIG. 4C illustrates the expression of Cxcl2 in the liver. The difference among the three groups was tested by one-way ANOVA. If the data do not conform to normality or homogeneity of variance, Kruskal-Wallis test is used. *: P <0.05, ** P <0.01. It can be seen from FIG. 4 that the "alcohol model group" shows significantly higher levels of 11-1 / ), Cxcll and Cxcl2 than the "control group", indicating that the alcoholic liver model is successfully established. In addition, in the "alcohol model group", the levels of IL-ip, Cxcll and Cxcl2 were significantly elevated in the "ExPEC group" compared with the "PBS group"; the levels of IL-ip, Cxcll and Cxcl2 fell back in the "ExPEC + phage group" compared with the "ExPEC group". This indicated at the transcriptome level that ExPEC could exacerbate alcohol-induced liver inflammation, and the phage could reverse this exacerbating effect of ExPEC.

[0056] 3) ExPEC further exacerbated the alcohol-induced hepatic steatosis and inflammation, and the phage reversed this exacerbating effect.

[0057] FIG. 5 illustrates the hematoxylin-eosin (HE) staining results of the pathological conditions of the liver tissues in each group at 200* magnification. Liver HE staining determined hepatic steatosis and hepatitis by means of the distribution and composition of vacuoles of different sizes in the cytoplasms of hepatocytes and inflammatory cells. Compared with the "control group", the "alcohol model group" showed a significant increase in fat cavitations, and infiltration of inflammatory cells, predominantly lymphocytes, was observed, indicating that the alcoholic liver model was successfully established. In addition, in the "alcohol model group", the volume and quantity of fat cavitations were significantly increased and the inflammatory cell infiltration was more significant in the "ExPEC group" compared with the "PBS group"; compared with the "ExPEC group", the "ExPEC + phage group" showed a significant reduction in the volume and quantity of fat cavitations, as well as a reduction in inflammatory cell infiltration. This indicated at the histological level that ExPEC could exacerbate alcohol-induced hepatic steatosis and inflammation, and the phage could reverse this exacerbating effect of ExPEC.

[0058] 4. Conclusion: The phage targeting ExPEC can significantly reverse the exacerbating effect of ExPEC on ALD at a plurality of levels: at the biochemical level, the phage provided by the present disclosure can significantly reduce the levels of serum biochemical indices (ALT and AST) that reflect liver injury; at the transcriptional level, it can significantly down-regulate the expression of liver inflammation-associated genes (IL-1 / 3, Cxcll, and Cxcl2\ at the tissue level, it can alleviate lipid deposition and inflammatory cell infiltration in the liver.

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

Claims

CLAIMED IS:

1. A phage targeting extra-intestinal pathogenic Escherichia coli (ExPEC) for use in the treatment or prophylaxis of alcohol-related liver disease (ALD), wherein the phage targeting ExPEC is obtained by mixing a bacterial host, a phage filtrate, and a culture medium in proportion, followed by proliferation, culture, isolation, and purification; and the bacterial host is a suspension of porcine extra-intestinal pathogenic Escherichia coli at logarithmic phase.

2. The phage targeting ExPEC according to claim 1, wherein the phage filtrate is obtained by centrifugation of environmental sewage to collect a supernatant and microfiltration to remove cells.

3. The phage targeting ExPEC according to claim 1, wherein the porcine extra-intestinal pathogenic Escherichia coli is obtained by inoculating and culturing an Escherichia coli PCN033 strain.

4. The phage targeting ExPEC according to claim 1, wherein the bacterial host, the phage filtrate, and the culture medium have a volume ratio of 1:1:2; and / or the culture medium is Tryptic Soy Broth (TSB).

5. The phage targeting ExPEC according to claim 1, wherein the isolation is achieved by a double layer agar plate method for phage isolation; and / or the purification is achieved by dot blotting for phage purification.

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