A swarming type Enterobacter Ludwigii and its use

Abstract

The present invention belongs to the field of new microbial application technology, specifically a Ludwig's Enterobacter and its application in improving inflammatory bowel disease. The strain is named Ludwig's Enterobacter (Enterobacter ludwigii) LS-57, which is deposited in the China General Microbiological Culture Collection Management Center on July 22, 2024, and the deposit number is CGMCC NO. 31424. The strain has the ability of cluster movement, has a strong adaptability to the intestinal environment, tolerates the acidity of gastric juice well, is sensitive to a variety of commonly used antibiotics, and has a good improvement effect on colitis.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and in particular relates to a swarming motile Enterobacter Ludwigii and a use thereof. Background Art

[0002] Inflammatory bowel disease (IBD) is a group of chronic, recurrent inflammatory diseases of the intestine that primarily affect the mucosal layer of the intestine, including two major subtypes: Crohn's disease (UC) and ulcerative colitis (CD). Symptoms of IBD may vary from person to person, but common symptoms include abdominal pain and diarrhea, bloody stools, weight loss, fatigue, malnutrition, etc., which affect the patient's quality of life. In addition, the chronic nature and unpredictable course of IBD impose long-term psychological and economic burdens on patients and their families. The exact cause of IBD is not fully understood, but it is generally believed to be the result of a combination of factors, including host genetic susceptibility, intestinal flora disorders, intestinal mucosal barrier damage, and intestinal mucosal immune abnormalities. IBD itself is not fatal, but it may increase the risk of other diseases such as colon cancer, blood clots, and liver disease.

[0003] The treatment of IBD is mainly drug therapy, which is mainly used to relieve symptoms. There is no complete cure yet. The main clinical treatment drugs are salicylates, glucocorticoids and immunosuppressants. Salicylates can play an anti-inflammatory role by reducing the release of pro-inflammatory factors in the intestine, but salicylates have serious gastrointestinal adverse reactions and even cause drug-induced kidney injury; glucocorticoids can relieve inflammation by inhibiting the release of inflammatory substances. They are commonly used in patients with moderate to severe symptoms of enteritis, but long-term or large-scale use can easily lead to drug resistance and multiple adverse reactions; immunosuppressants can inhibit the proliferation of inflammatory cells, but patients are prone to adverse reactions such as nausea, vomiting and diarrhea after taking them. In short, although salicylates, hormone drugs or immunosuppressants can relieve the clinical symptoms of IBD, they all have shortcomings such as easy relapse after discontinuation of medication and low compliance of patients with long-term use.

[0004] Compared with healthy individuals, the intestinal flora of patients with active IBD showed changes in bacterial diversity, composition and / or abundance, while similar microbial composition patterns were found between IBD patients, such as reduced microbial diversity, reduced abundance of Firmicutes, and increased abundance of Proteobacteria. Intestinal commensal bacteria showed significant positive effects in the prevention and treatment of IBD, and the protective effects of these microorganisms, such as regulation of intestinal microbial repair, immunomodulation, enhancement of anti-inflammatory effects, and intestinal barrier repair, appeared to be strain-specific. Therefore, screening strains that have an ameliorative effect on inflammatory bowel disease is of great significance for the development of new therapies for inflammatory bowel disease.

[0005] The patent of this invention adopts a method for isolating and purifying symbiotic clustering bacteria from the feces of patients with colon cancer based on the clustering motility of the strains, and screened out a new strain of Ludwig's Enterobacter, which came from a fecal sample of a 78-year-old male patient with colon cancer and had the ability to cluster. Summary of the invention

[0006] Based on the above existing technology,

[0007] The first aspect of the present invention provides a novel strain of Enterobacter ludwigii LS-57, which is deposited in the China General Microbiological Culture Collection Center with a deposit date of July 22, 2024 and a deposit number of CGMCC NO. 31424.

[0008] The strain has the ability of swarming, specifically, it can grow monoclonal colonies in a film-like diffusion pattern.

[0009] The strain has a short rod-like morphology; further, it is a white dot-like clone.

[0010] This strain is a Gram-negative bacterium.

[0011] The 16S rDNA gene sequence of the strain is shown in SEQ ID NO.3.

[0012] The second aspect of the present invention provides a composition comprising the Enterobacter Ludwigii strain LS-57, or provides a bacterial agent comprising the Enterobacter Ludwigii strain LS-57.

[0013] The present invention further provides a microbial preparation product comprising the Ludwig's Enterobacter strain LS-57, which can be a medicine.

[0014] The viable bacteria count is not less than 1.5E+09 CFU / g.

[0015] The preferred route of administration is oral;

[0016] The dosage form can be tablets, capsules, granules, pills or oral liquids, etc.;

[0017] In some specific embodiments, the drug further includes a pharmaceutical carrier and / or a pharmaceutical excipient. The pharmaceutical carrier includes microcapsules, microspheres, nanoparticles, and liposomes; the pharmaceutical excipient includes one or more of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, excipients, additives, and release retardants.

[0018] The type of the bacterial agent may be an inactivated bacterial agent or a live bacterial agent, but in order to retain the bacterial activity to the greatest extent, the bacterial agent is preferably a live bacterial agent.

[0019] The bacterial agent further comprises fermentation products and non-fermentation products of Enterobacter Ludwigii, wherein the non-fermentation products mainly include metabolites.

[0020] The pharmaceutical composition contains the above-mentioned Enterobacter Ludwigii strain LS-57, a drug carrier and / or a pharmaceutical excipient.

[0021] In a specific embodiment, the present invention provides a pharmaceutical composition comprising Enterobacter ludwigii strain LS-57.

[0022] The third aspect of the present invention provides the use of Ludwig's Enterobacter strain LS-57

[0023] Specifically, it is the use of the Ludwig's Enterobacter strain LS-57 in the preparation of medicines for treating, relieving colitis and / or improving intestinal health;

[0024] The application includes at least one of the following functions

[0025] (1) Stabilize the weight of the treated subjects and, further, significantly improve the weight loss of the treated subjects;

[0026] (2) Improve the subject’s symptoms of sticky stool;

[0027] (3) Improve the patient's bloody stool symptoms;

[0028] Furthermore, the E. ludwigii strain LS-57 can reduce the disease activity index (Disease activity index DAI);

[0029] (4) Improved the pathological changes in colon tissue of subjects with acute colitis;

[0030] (5) Repair colon inflammation.

[0031] The fourth aspect of the present invention provides a method for separating bacteria

[0032] The invention provides a method for separating bacteria, wherein the separation method is to screen strains based on clustering movement ability.

[0033] Furthermore, in the separation method, the standard for screening strains is to dilute the separated sample and apply it to a solid culture medium, and then observe whether there are monoclonal colonies that diffuse like a thin film, so as to determine the target strains with clustering ability. Furthermore, in the separation method, the strains screened are derived from fecal samples, and the fecal samples are derived from patients with colon cancer.

[0034] Furthermore, the isolation method includes homogenizing the stool samples of patients with colon cancer, inoculating them into solid culture medium after dilution, and culturing them at 37°C and 20% humidity. The colonies show wavy diffusion and multi-layer diffusion, and thin film diffusion-like colonies are observed to grow. The edges of the colonies are further inoculated and streaked for purification until the purified single strain grows thin film diffusion-like monoclonal colonies, indicating that the strain is a clustered bacterium.

[0035] The inoculation medium is a 0.8%-1.5% LB agar plate; the homogenization treatment refers to homogenizing the feces sample to a concentration of 200 mg / mL.

[0036] Swarming motility refers to a form of bacterial swarming motion, in which bacteria move collectively and rapidly on a surface by driving their flagella. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Culture of swarming bacteria from homogenized fecal samples

[0038] Figure 2 The swarming movement of LS-57 bacteria

[0039] Figure 3 Gram staining of Enterobacter ludwigii LS-57

[0040] Figure 4 The total number of viable bacteria in the original bacterial solution treated with PBS at different pH

[0041] Figure 5 Body weight changes in mice with DSS-induced colitis

[0042] Figure 6 Body weight changes of different groups after treatment (The results are expressed as "mean ± standard error", and the experimental data were analyzed by one-way analysis of variance (one-ANOVA) using GraphPad Prism 10.1.2, * represents P < 0.05.)

[0043] Figure 7 Evaluation of disease activity index (DAI) in different groups (The results are expressed as "mean ± standard error". The experimental data were analyzed by T test (Tukey's test) using GraphPad Prism 10.1.2. ** represents P < 0.01, **** represents P < 0.0001.)

[0044] Figure 8 Spleen index of different groups (The results are expressed as "mean ± standard error", and the experimental data were analyzed by one-way analysis of variance (one-ANOVA) using GraphPad Prism 10.1.2. ** represents P < 0.01.)

[0045] Fig. 9 Comparison of tissue section staining of mice treated in different groups

[0046] Fig.10 Changes in MPO in mice treated with different methods (The results are expressed as "mean ± standard error". The experimental data were analyzed by T test (Tukey's test) using GraphPad Prism 10.1.2. ** represents P < 0.01, **** represents P < 0.0001.)

[0047] Fig.11 Changes of inflammatory factors in mice treated with different methods (The results are expressed as "mean ± standard error", and the experimental data were analyzed by one-way analysis of variance (one-ANOVA) using GraphPad Prism 10.1.2.) DETAILED DESCRIPTION

[0048] 1. Isolation, Screening and Identification of Enterobacter Ludwigii

[0049] 1. Sample processing

[0050] Fecal samples from patients with colon cancer were thawed step by step as follows: the samples were removed from a -80°C freezer, placed in a -25°C freezer for 1 hour, and then thawed on ice for 1 hour. On a scale, weigh a sterile microcentrifuge tube and set to peel. In a sterile operating table, remove the sample with a sterile pipette tip and place it in a microcentrifuge tube, and weigh the stool sample using the same scale. Add enough sterile phosphate buffered saline (PBS: pH=7.4, room temperature) to the microcentrifuge tube to make the final stool concentration reach 200 mg / mL. Rotate the sterile pestle in the microcentrifuge tube about twenty times to completely homogenize the stool pellet.

[0051] 2. Plate culture of bacteria

[0052] Vortex the microcentrifuge tube containing the stool sample for about 10 seconds, and then inoculate 5 μL of the homogenized stool solution on the center surface of a 0.8% LB agar plate. The plate was placed in an incubator at 37°C and 20% humidity. After 16 hours of incubation (i.e., swarming condition incubation), film-like diffuse colonies were observed, such as Figure 1 As shown, the bacterial film has grown all over the culture dish, and is a wave-like layered diffusion growth.

[0053] 3. Screening of monoclonal strains based on swarming motility

[0054] For plates with swarm-expanded colonies, use a sterile inoculation loop to gently scrape the edges of the colonies at four different points, and then streak them on 1.5% LB agar plates. The streaked plates were incubated overnight in a 37°C incubator.

[0055] After the streaked plates have grown sufficiently, take two single colonies from each plate and place them in 5 ml LB broth. Incubate the samples on a shaker at 200 rpm and 37°C for 16 hours. Inoculate 5 μL of the bacterial suspension in the center of the surface of a 0.8% LB agar plate and incubate at 37°C and 20% humidity for 16 hours to confirm again whether the bacteria have the ability to colonize. Figure 2 As shown, the inoculated bacterial solution grew monoclonal colonies in the form of thin film diffusion, indicating that the strain was a swarming bacterium (i.e., "swarming culture verification").

[0056] At the same time, the bacterial suspension was streaked on a 1.5% LB agar plate and cultured at 37°C to observe whether it was a pure culture.

[0057] Two tubes of bacteria were randomly selected for each sample for PCR. The bacterial solution after shaking for 16 hours was diluted 50 times and PCR amplification reaction was performed according to Table 1. Prepare 1% agarose gel (1g agarose plus 100mL deionized water), heat and boil 2-3 times, add 10μL 4S Green buffer when the temperature drops to about 55℃, mix well, and pour into the gel tank. After solidification, add 1μL Loading buffer and 5 μL PCR product mixture into the gel well, and perform gel electrophoresis at 220V and 30min. If a band appears at the target band (1500 bp), send the sample for 16S rRNA sequencing. Compare the sequencing results on the NCBI website.

[0058] Table 1 PCR amplification reaction system

[0059]

[0060] 4. Purification and identification of strains

[0061] Morphological identification:

[0062] Use Gram staining to stain LS-57 bacteria, and observe its morphology under a 100x oil microscope. The specific steps are as follows. Inoculate LS-57 bacteria into 5 mL of LB broth medium and shake the bacteria at 37 °C and 200 rpm for 16 h. Streak the bacterial liquid on a 1.5% LB agar plate and culture it overnight at 37 °C and 40% humidity. Take a small amount of LS-57 single colony, dip it in 2 µL of saline, and spread it clockwise to about 1 cm 2 Evenly thin and round, place at room temperature until the bacterial solution is dry. Fix the slide by passing it through the flame of an alcohol lamp 1-2 times. Be careful not to overheat it. The slide should not be too hot. Take crystal violet staining solution and add it to the bacteria, stain for 1 minute, and wash with water. Add iodine solution and stain for 1 minute, wash with water. Add decolorizing solution, shake the slide, decolorize for 30 seconds, wash with water, and absorb the water. Add safranin staining solution, stain for 1 minute, and wash with water. Use filter paper to absorb excess water and examine under a 100x oil microscope.

[0063] Gram stain microscopy results, gram-negative bacteria (see Figure 3 ), in the shape of a short rod.

[0064] Molecular Biology Identification:

[0065] 16S rDNA amplification universal primer sequence:

[0066] 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO: 1)

[0067] 1492R: 5'-TACGGYTACCTTGTTACGACTT-3' (SEQ ID NO: 2)

[0068] PCR product sequencing results:

[0069]

[0070] 2. Evaluation of acid tolerance of Enterobacter ludwigii

[0071] The acid tolerance of strain LS-57 was expressed as the ratio of the total number of viable bacteria in the bacterial solution treated with PBS of different pH values ​​to the initial total number of viable bacteria. The pH values ​​were determined based on the pH values ​​of fasting simulated gastric fluid, standard simulated gastric fluid, and full simulated gastric fluid, which were 2.0, 3.0, and 4.0, respectively.

[0072] Inoculate LS-57 strain into 5mL LB broth and culture in a constant temperature shaker at 37℃ and 200 rpm for 16h. Take 30mL PBS (pH = 7.4) buffer in a centrifuge tube and slowly add 1 mol / L HCl, measuring with a pH meter while adding until pH = 2.0, pH = 3.0 and pH = 4.0 are just reached. Take 5ml of PBS (pH = 7.4) and PBS of different pH values ​​and mix with the bacterial solution in equal volumes, and culture according to Table 1. Both PBS buffer and bacterial solution must be prepared and used immediately.

[0073] After the culture was completed, the mixture was diluted 10 3 , 10 5 and 10 7 Pipette 100 µL of the dilution into a sterile dish, and make two sterile dishes for each dilution. At the same time, pipette 100 µL of PBS (pH = 2.0, pH = 3.0, and pH = 4.0) into a sterile dish as a blank control. Pour about 15 mL of 1.5% LB agar medium cooled to 50°C into a sterile dish, rotate the sterile dish clockwise or counterclockwise at least 20 times to mix it evenly, and after the agar solidifies, turn the plate over and culture it in a 37°C, 40% humidity incubator overnight. If the colony has no growth or grows small, you can choose to culture it for 48 to 72 hours. After culture, count all the colonies on the plate. From sample dilution to plate pouring, it is required to be completed within 15 minutes.

[0074] The total number of viable bacteria in the sample bacterial solution treated with PBS (pH = 7.4) is recorded as N1, and the total number of viable bacteria in the sample bacterial solution treated with PBS at pH = 2.0, pH = 3.0 or pH = 4.0 is recorded as N1'. Repeat the above steps to complete two acid tolerance tests, in which the total number of viable bacteria in the sample bacterial solution treated with PBS (pH = 7.4) is recorded as N2 and N3, and the total number of viable bacteria in the sample bacterial solution treated with PBS at pH = 2.0, pH = 3.0 or pH = 4.0 is recorded as N2` and N3`. Calculate the relative average deviation of the total number of viable bacteria determination results of N1, N2, N3 and N1`, N2`, N3`. The relative average deviation of the two sets of data should not be greater than 15%, otherwise the acid tolerance test needs to be completed again. See the results for details. Figure 2 The pH shown in indicates the color change.

[0075] Table 2 Main parameters of the acid tolerance test method for LS-57 bacteria

[0076]

[0077] Acid tolerance is calculated according to the following formula:

[0078]

[0079] Where:

[0080] A -- Acid tolerance, expressed as a percentage (%)

[0081] N1--The total number of initial viable bacteria in the first acid tolerance test, in CFU / mL;

[0082] N2--The total number of viable bacteria at the beginning of the second acid tolerance test, in CFU / mL;

[0083] N3--The total number of initial viable bacteria in the third acid tolerance test, in CFU / mL;

[0084] N1'--The total number of viable bacteria after the first acid tolerance test, in CFU / mL;

[0085] N2'--The total number of viable bacteria after the second acid tolerance test, in CFU / mL;

[0086] N3'--The total number of viable bacteria after the third acid tolerance test, in CFU / mL;

[0087] 1 / 3--average of three parallel data;

[0088] The calculation result is expressed as an integer.

[0089] according to Figure 4The results of "Total number of viable bacteria in original bacterial solution treated with PBS at different pH values" shown in the figure show that the tolerance of LS-57 to pH = 2.0 is 70%, the tolerance of LS-57 to pH = 3.0 is 105%, and the tolerance of LS-57 to pH = 4.0 is 107%.

[0090] This indicates that the Ludwig Enterobacter LS-57 can tolerate the acidity of gastric juice well and is suitable for making oral live bacteria products.

[0091] 3. Antibiotic Susceptibility Testing of Enterobacter Ludwigii

[0092] Antibiotic susceptibility testing is used to determine whether bacteria can be inhibited by the concentrations achieved by conventional doses of antimicrobial drugs to demonstrate their safety.

[0093] Experimental method: LS-57 bacteria were cultured overnight in TSA broth at 37°C and 200 rpm, and then streaked onto 1.5% TSA agar plates. Three to four pure colonies cultured on the TSA plates for 18 to 24 hours were aseptically picked and inoculated into 5 mL of tryptic soy broth (TSB). The culture was shaken at 37°C and 200 rpm for 2 hours, and the OD value was calibrated to 0.5 McFarland units (OD) using a McFarland standard turbidimetric tube. 625 = 0.08~0.10), which is equivalent to a bacterial concentration of 1.5×10 8 CFU / mL.

[0094] The prepared bacterial solution should be used within 15 minutes. Add 300 µL of bacterial solution to the surface of 1.5% MHA agar and spread it evenly with a sterile L-stick. Leave it at room temperature for 3 to 5 minutes until the water on the plate is completely absorbed by the agar.

[0095] Use sterile tweezers to stick standard antimicrobial drug paper sheets one by one as needed, and gently press them to make them close to the agar surface. Once the paper sheets touch the plate, they cannot be moved. The distance between the centers of each paper sheet should be greater than 24 mm, and the distance between the paper sheet and the inner edge of the plate should be greater than 15 mm. Generally, no more than 5 paper sheets should be placed on a plate with a diameter of 90 mm. Turn the agar plate over within 15 minutes after sticking the paper sheets, and incubate at 37℃ and 40% for 17 h±1 h. During the incubation process, the plates should be placed separately, and no more than 2 should be stacked together.

[0096] Take out the plate and measure the diameter of the antibacterial ring of each paper piece (including the diameter of the paper piece) on the back of the plate with a vernier caliper under transmitted light. The edge of the antibacterial ring is limited to the point where no obvious bacterial growth can be seen with the naked eye. The growth of tiny colonies at the edge of the antibacterial ring that can only be observed under a magnifying glass can be ignored.

[0097] Table 3 Interpretation of the antibacterial diameter of LS-57 bacteria

[0098]

[0099] The results are shown in Table 2. LS-57 is sensitive to multiple antibiotics, including ticarcillin (75µg / tablet), tetracycline (30µg / tablet) and polymyxin B (300 µg / tablet). (Bacterial resistance refers to the gradual weakening of bacterial sensitivity to antimicrobial drugs after repeated contact with antimicrobial drugs, and even the ability to resist rather than be inhibited and killed. This characteristic of bacteria is called resistance. It is usually measured by antimicrobial susceptibility testing (AST), and the results can be expressed as sensitive (S), intermediate (I) and resistant (R). Resistant (R) When conventional doses are used, the growth and reproduction of bacteria are inhibited by the achievable concentration level of antimicrobial drugs. Intermediate (I) Conventional doses of antimicrobial drugs are less effective against bacteria than sensitive strains, and the drugs are effective at physiological concentration sites or can be treated with drugs at higher than normal doses.)

[0100] The results of the antibacterial experiment showed that the diameter of the antibacterial ring fell within the range of certain bacterial resistance mechanisms, and the bacteria could not be inhibited by the concentrations reached by conventional doses of antibacterial drugs. It can be seen that LS-57 bacteria are sensitive to most antibiotics, indicating that the strain is relatively safe and can be used in biological preparations.

[0101] Effects of Enterobacter ludwigii on growth performance and organ indices in mice with DSS-induced colitis

[0102] Mouse source and treatment method

[0103] The experiment used 8-week-old, female C57BL / 6 mice, SPF grade, from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd. After arrival, the animals were first adapted to the institution for 1 week, with approximately 12 h / 12 ​​h alternating light / dark lighting per day, and the ambient temperature and relative humidity of the animal room were controlled at 22-26 ºC and 40-70%, respectively.

[0104] Mouse grouping

[0105] Description: Thirty healthy C57BL / 6 mice were randomly divided into three groups according to their body weight, with 10 mice in each group, namely No DSS group / blank control group, DSS model group and DSS+LS-57 group.

[0106] Table 4 Grouping and treatment of experimental mice

[0107]

[0108] DSS-induced colitis model mice

[0109] Each group of mice (except the No DSS group) was given drinking water containing 3% DSS (Dextran Sulfate Sodium Salt) (free drinking). The mice were gavaged every day at the beginning of the experiment. The mice were weighed before gavage until the weight loss rate of the DSS group mice was > 20% (indicating successful modeling). The formula for the weight loss rate of mice is as follows:

[0110] Mouse weight loss rate (%) = (initial weight - current weight) / initial weight * 100%

[0111] Results Figure 5 shown.

[0112] Treatment of mice in the control and experimental groups

[0113] The mice in the blank control group did not have DSS added to their drinking water, and were gavaged with 200 μL of LB liquid culture medium every day during the experiment; the mice in the experimental group had 3% DSS added to their drinking water, and were gavaged with 200 μL of LS-57 fresh culture medium every day during the experiment. Figure 5 The body weight changes of different groups after treatment are shown. On the 9th day of the experiment, the body weight of mice in the DSS+LS-57 group was significantly higher than that in the DSS group, indicating that LS-57 can significantly improve the weight loss of mice induced by DSS.

[0114] Disease Activity Index (DAI) Assessment

[0115] During the experiment, the clinical progression of colitis was evaluated every day. DAI is a comprehensive score based on the animal's weight loss rate, stool viscosity, and blood in the stool. DAI = (weight change score + stool consistency score + blood in the stool score) / 3. On the 5th, 6th, and 7th days of the experiment, the DAI score of mice in the DSS+LS-57 group was significantly lower than that in the DSS group, indicating that oral administration of LS-57 significantly improved the decline in the disease index caused by DSS induction.

[0116] Mouse fecal occult blood detection method: Use fecal occult blood qualitative detection kit (o-tolidine method, Regen Biotech) for detection (see the instructions for specific operations), and refer to the fecal occult blood interpretation table for result interpretation. If there is visible blood in the stool, it will be directly rated as 4 points.

[0117] Table 5 Scoring criteria for the disease activity index (DAI) of mice

[0118]

[0119] *Normal stool is formed; loose stool is loose or soft stool that does not stick around the anus; loose stool is unformed stool that sticks around the anus.

[0120] **The degree of blood in stool is assessed according to Table 4. If there is visible blood in the stool, it is directly scored as 4 points.

[0121] Table 6 Fecal occult blood interpretation table

[0122]

[0123] Spleen index

[0124] Methods: After euthanasia, the spleens of mice were taken out and weighed for the determination of spleen index.

[0125] Spleen index = spleen weight (mg) / mouse body weight (g)

[0126] like Figure 7 As shown, the spleen index of mice in the DSS+LS-57 group was significantly lower than that in the DSS model group, indicating that the degree of inflammation in mice gavaged with LS-57 was significantly reduced.

[0127] 5. Effects of Enterobacter Ludwigii on biochemical parameters of DSS-induced colitis mice

[0128] Tissue section staining

[0129] After measuring the length of the colon, the cecum was cut off, and the colon was wound into a spiral on a horizontal plane and fixed in 4% tissue fixative. The intestinal tissue hematoxylin-eosin staining (HE staining) was sent to Wuhan Saiweier Biotechnology Co., Ltd.

[0130] like Fig. 9 As shown in the figure, the colon tissue in the DSS group had large-area ulcers, the mucosal epithelium was lost at the ulcer site, the intestinal gland structure disappeared and was replaced by proliferating connective tissue, accompanied by a large number of lymphocytes and granulocytes infiltration, and the intestinal glands around the ulcer were irregularly arranged; the submucosal layer was slightly edematous, the connective tissue was loosely arranged, and there was a small amount of lymphocytes and granulocytes infiltration; the muscular layer was severely thickened. The surface of the colon tissue in the DSS+LS-57 group was a single-layer columnar epithelium with normal morphology and structure, abundant intestinal glands in the lamina propria, goblet cells were distributed between epithelial cells, and the mucosal muscular layer composed of double-layer smooth muscle cells separated the intestinal crypts from the submucosal layer. The submucosal layer was composed of connective tissue, and the remaining part of the intestinal wall included the muscular layer and serosa composed of smooth muscle cells. This shows that oral administration of LS-57 significantly improved the pathological changes in the colon tissue of mice with acute colitis caused by DSS.

[0131] MPO detection

[0132] Myeloperoxidase (MPO) is a marker of neutrophil activation, and its level and activity represent the function and state of neutrophils. Under physiological conditions, MPO is part of the innate immune system and can fight against the invasion of pathogens such as bacteria and fungi. Under certain conditions, MPO can catalyze reactions to generate excessive oxidants. When it exceeds the body's antioxidant defense response, it will cause oxidative stress and oxidative tissue damage, and participate in the occurrence of various diseases, such as inflammation, small vessel vasculitis, tumors, nephritis, and atherosclerosis.

[0133] Four colon samples were randomly selected from each experimental group, and each sample was divided into the proximal segment (proximal, close to the cecum), the middle segment (middle) and the distal segment (distal, close to the rectum) for MPO detection. The MPO in colon tissue was determined using a kit purchased from Nanjing Jiancheng Bioengineering Institute. The MPO in the colon was quantitatively analyzed using an ultraviolet spectrophotometer. The specific operation steps were carried out according to the instructions of the kit.

[0134] like Fig.10 As shown, the MPO of the middle and distal segments of the colon of mice in the DSS + LS-57 group were significantly lower than those in the DSS group, and the MPO of the distal segment of the colon of mice in the DSS + LS-57 group was extremely significantly lower than that in the DSS group, indicating that the Ludwig Escherichia coli LS-57 significantly repaired inflammation in the colon of mice.

[0135] Pro-inflammatory / anti-inflammatory factor expression

[0136] The expression levels of pro-inflammatory factors TNF-α, IL-6 and TNFR-2 genes in the colon were analyzed by qPCR.

[0137] Colon tissue stored at -80°C was lysed with TriZol lysis buffer, and then total RNA was extracted under chloroform, isopropanol, and 75% ethanol treatment. The absorbance values ​​at A260 / A280 and A260 / A230 were measured using an ultra-micro spectrophotometer to detect the concentration and purity of RNA. The qualified RNA was reverse transcribed into cDNA using the TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix Kit from Beijing Quanshijin Biotechnology Co., Ltd. The specific operation steps were carried out according to the instructions of the kit. The reaction system was 20 μl, containing 2×TS Reaction Mix 10 μl, Random Primer 1 μl, TransScript® RT / RI Enzyme Mix 1 μl, gDNA Remover 1 μl, RNase-free Water to 20 μl, and total RNA 500 ng. Reaction conditions: incubate at 25°C for 10 min, incubate at 42°C for 15 min, and heat at 85°C for 5 s to inactivate TransScript® RT / RI and gDNA Remover.

[0138] The primer sequences for real-time fluorescence quantitative PCR (qPCR) reaction are detailed in Table 6, 7 μL ddH2O. PCR amplification conditions: 95°C pre-denaturation for 3 min; 95°C denaturation for 10 s, 60 °C annealing for 30 s, for a total of 39 cycles. TBP was used as the internal reference, and the experimental results were analyzed using the 2−ΔΔCt calculation method. The gene expression calculation formula was: 2Cttarget (control) – Ct target (treatment) / 2Ct TBP (control) – Ct TBP (treatment).

[0139] like Fig.11 The results showed that the expression levels of pro-inflammatory factors TNF-α, IL-6 and TNFR-2 genes in the colon tissue of mice in the DSS+LS-57 group were lower than those in the DSS model group, showing a downward trend, indicating that LS-57 has a good therapeutic effect on DSS-induced acute colitis in mice.

Claims

1. A strain of Enterobacter ludwigii LS-57, deposited in China General Microbiological Culture Collection Center, with a deposit date of July 22, 2024 and a deposit number of CGMCC NO. 31424.

2. The Enterobacter Ludwigii according to claim 1, characterized in that: The 16S rDNA gene sequence thereof comprises the sequence shown in SEQ ID NO.

3.

3. The Enterobacter Ludwigii according to claim 1, wherein the bacterium has the ability of swarming and can grow into a monoclonal colony in a film-diffusion-like manner. 4 . The Enterobacter Ludwigii according to claim 1 , wherein the bacterium is a Gram-negative bacterium in a short rod-shaped form and is a white-like colony.

5. A composition comprising the Enterobacter ludwigii according to any one of claims 1 to 4.

6. Use of the Enterobacter Ludwigii described in claims 1 to 4 or the composition described in claim 5 in the preparation of a medicament for treating or alleviating colitis. The composition according to claim 5 , which is an oral bacterial preparation.

8. The composition according to claim 7, wherein the oral bacterial agent is a live bacterial agent, and the number of live bacteria is not less than 1.5E+09 CFU / g.

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