Enterobacter agglomerans cluster movement and uses thereof

Abstract

The application belongs to the technical field of new application of microorganisms, and particularly relates to an enterobacter and application of the enterobacter in improving inflammatory bowel disease. The strain is named LS-68, is preserved in the China General Microbiological Culture Collection Center, has a preservation date of October 28, 2024, and has a preservation number of CGMCC NO.32368. The strain has the ability of group movement, has strong adaptability to the intestinal environment, well tolerates the acidity of gastric juice, is sensitive to a plurality of commonly used antibiotics, and has a good improvement effect on colitis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microorganisms, and particularly relates to a swarm-typed enterobacter and uses thereof. BACKGROUND

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

[0003] The main treatment for IBD is drug therapy, which is mainly used to relieve symptoms, and there is currently no complete cure. Clinical treatment drugs mainly include salicylic acid, glucocorticoids, and immunosuppressants. Salicylic acid drugs can exert anti-inflammatory effects by reducing the release of pro-inflammatory factors in the intestine, but salicylic acid drugs have serious adverse reactions such as gastrointestinal reactions, and even cause drug-induced kidney damage; glucocorticoid drugs can relieve inflammation by inhibiting the release of inflammatory substances, and are often used in patients with moderate to severe symptoms of intestinal inflammation, but long-term or large-dose use can easily produce drug resistance and various adverse reactions; immunosuppressants can inhibit the proliferation of inflammatory cells, but patients taking them are prone to adverse reactions such as nausea, vomiting, and diarrhea. In summary, although salicylic acid drugs, hormone drugs, or immunosuppressant drugs can relieve the clinical symptoms of IBD, they all have the disadvantages of easy recurrence after stopping medication, low patient compliance for long-term use, etc.

[0004] Compared with healthy individuals, the intestinal flora of patients in the active phase of IBD shows changes in bacterial diversity, composition, and / or abundance, and there are similar microbial composition patterns among IBD patients, such as reduced microbial diversity, reduced abundance of Firmicutes, and increased abundance of Proteobacteria. Intestinal symbiotic bacteria show significant positive effects in the prevention and treatment of IBD, and the protective effects of these microorganisms, such as regulation of intestinal microbial repair, immune regulation, enhancement of anti-inflammatory effects, and repair of intestinal barrier, etc., seem to be strain-specific. Therefore, screening for strains that have an improving effect on inflammatory bowel disease is of great significance for the development of new therapies for inflammatory bowel disease.

[0005] This invention patent employs a method for isolating and purifying symbiotic cluster bacteria from the feces of colon cancer patients based on the cluster motility of bacterial strains. A novel Enterobacter strain was screened from a preoperative fecal sample of an 82-year-old male colon cancer patient, which exhibits cluster motility. Summary of the Invention

[0006] Based on the above-mentioned existing technology situation,

[0007] The first aspect of this invention provides an Enterobacter strain LS-68, which is deposited at the China General Microbiological Culture Collection Center on October 28, 2024, with accession number CGMCC NO. 32368.

[0008] This strain has the ability to move in clusters; specifically, it can grow monoclonal colonies that spread like a thin film.

[0009] The strain is short rod-shaped; furthermore, it is a white, dot-like clone.

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

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

[0012] A second aspect of the present invention provides a composition comprising Enterobacter LS-68, or a bacterial agent comprising Enterobacter LS-68.

[0013] The present invention further provides a microbial preparation product containing Enterobacter strain LS-68, said product being a pharmaceutical product.

[0014] The number of live bacteria is not less than 1.5E+09 CFU / g.

[0015] Oral administration is preferred.

[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 pharmaceutical excipients. The pharmaceutical carrier comprises microcapsules, microspheres, nanoparticles, and liposomes; the pharmaceutical excipients comprise one or more of the following: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and anti-flocculators, filter aids, excipients, additives, and release inhibitors.

[0018] The bacterial agent can be either an inactivated bacterial agent or a live bacterial agent, but to maximize the preservation of bacterial activity, a live bacterial agent is preferred.

[0019] The microbial agent also contains fermentation products of Enterobacteriaceae and non-fermentation products, the non-fermentation products mainly including metabolites.

[0020] The pharmaceutical composition contains the aforementioned Enterobacter LS-68 strain, a drug carrier, and / or pharmaceutical excipients.

[0021] In one specific embodiment, the present invention provides a pharmaceutical composition comprising Enterobacter strain LS-68.

[0022] A third aspect of this invention provides the use of Enterobacter strain LS-68.

[0023] Specifically, the use of Enterobacter LS-68 in the preparation of medicines for treating, alleviating colitis and / or improving gut health;

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

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

[0026] (2) Improves the symptoms of sticky stool in the treated patients;

[0027] (3) Improves the symptoms of rectal bleeding in the treated patients;

[0028] Furthermore, Enterobacter strain LS-68 can reduce the Disease Activity Index (DAI).

[0029] (4) It improved the pathological changes of colon tissue in patients with acute colitis.

[0030] (5) Repair colon inflammation.

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

[0032] This invention provides a method for isolating bacteria, which is based on screening strains for swarming ability.

[0033] Furthermore, in the isolation method, the criterion for screening strains is to dilute the isolated sample and spread it on a solid culture medium, then observe whether there are monoclonal colonies exhibiting a thin-film diffusion pattern, thereby determining whether it is a target strain with clustering motility. Furthermore, in the isolation method, the screened strains are derived from fecal samples, which are obtained from colon cancer patients.

[0034] Furthermore, the separation method includes homogenizing fecal samples from colorectal cancer patients, diluting them, inoculating them into a solid culture medium, and culturing them at 37°C and 20% humidity. The colonies exhibit wavy diffusion and multi-layer diffusion patterns, and a thin film diffusion-like colony is observed to grow. The colony edges are further inoculated and streaked for purification until the purified single strain grows a thin film diffusion-like monoclonal colony, indicating that the strain is a cluster of bacteria.

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

[0036] Swarming refers to a form of bacterial swarming movement, in which bacteria collectively and rapidly move across a surface by driving their flagella. Attached Figure Description

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

[0038] Figure 2 Cluster movement of Enterobacter LS-68

[0039] Figure 3 Gram staining of Enterobacter LS-68

[0040] Figure 4 Growth curve of Enterobacter LS-68

[0041] Figure 5 Total viable count of the original bacterial culture treated with PBS at different pH values

[0042] Figure 6 DSS-induced weight changes in mice with colitis

[0043] Figure 7 Weight changes after different treatment groups (Results are expressed as mean ± standard error. Experimental data were analyzed using one-way ANOVA with GraphPad Prism 10.1.2. * indicates P < 0.05.)

[0044] Figure 8 Disease Activity Index (DAI) assessment for different groups (results are expressed as mean ± standard error; experimental data were analyzed using GraphPad Prism 10.1.2 with a t-test (Tukey's test), * represents P < 0.01, **** represents P < 0.0001).

[0045] Figure 9Spleen indices of different groups (Results are expressed as mean ± standard error. Experimental data were analyzed using GraphPad Prism 10.1.2 using one-way ANOVA. ** indicates P < 0.01.)

[0046] Figure 10 Comparison of staining of tissue sections from mice treated with different groups

[0047] Figure 11 Changes in MPO in mice under different treatments (Results are expressed as mean ± standard error. Experimental data were analyzed using a t-test (Tukey's test) with GraphPad Prism 10.1.2. * represents P < 0.01, **** represents P < 0.0001.)

[0048] Figure 12 Changes in inflammatory factors in mice under different treatments (results are expressed as mean ± standard error; experimental data were analyzed using one-way ANOVA with GraphPad Prism 10.1.2). Detailed Implementation

[0049] I. Isolation, Screening and Identification of Enterobacter LS-68

[0050] 1. Sample processing

[0051] The stool samples were obtained from a colon cancer patient. The samples were thawed stepwise 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. A sterile microcentrifuge tube was weighed on a balance and the tare setting was applied. In a sterile operating room, the sample was removed using a sterile pipette tip and placed into the microcentrifuge tube, and the stool sample was weighed again using the same scale. Sufficient sterile phosphate-buffered saline (PBS: pH 7.4, room temperature) was added to the microcentrifuge tube to achieve a final stool concentration of 200 mg / mL. The sterile vibrator was rotated approximately twenty times in the microcentrifuge tube to completely homogenize the stool particles.

[0052] 2. Bacterial plate culture

[0053] Vortex the microcentrifuge tube containing the fecal sample for approximately 10 seconds, then inoculate the center of a 0.8% LB agar plate with 5 μL of homogenized fecal solution. The plate is placed in an incubator at 37 ℃ and 20% humidity and incubated for 16 hours (i.e., swarming conditions). Thin-film diffusion-like colonies are observed, such as... Figure 1 As shown, the bacterial film has covered the entire culture dish, exhibiting a wave-like, layered diffusion growth pattern.

[0054] 3. Screening of monoclonal strains based on swarming ability

[0055] For plates showing Swarm expanded colonies, gently scrape the edges of four different colonies using a sterile inoculation loop and streak them onto 1.5% LB agar plates. Incubate the streaked plates overnight at 37 °C.

[0056] After the streaked plates have grown sufficiently, two single colonies from each plate are placed in 5 ml of LB broth. The samples are then incubated on a shaker at 200 rpm and 37°C for 16 hours. A 5 μL bacterial suspension is then inoculated into the center of a 0.8% LB agar plate and incubated at 37°C and 20% humidity for 16 hours to further confirm the bacteria's ability to aggregate. Figure 2 As shown, the inoculated bacterial solution grows monoclonal colonies that spread like a thin film, indicating that the strain is a cluster of bacteria (i.e., "swarming culture verification").

[0057] Meanwhile, the bacterial suspension was streaked on 1.5% LB agar plates and incubated at 37 °C to observe whether it was a pure culture.

[0058] Two tubes of bacterial strain were randomly selected from each sample for PCR. The bacterial culture, after 16 hours of shaking, was diluted 50-fold and PCR amplification was performed according to Table 1. A 1% agarose gel was prepared (1g agarose in 100mL deionized water), heated to boiling 2-3 times, and then cooled to approximately 55℃. 10μL of 4S Green buffer was added and mixed thoroughly before pouring into the gel casting tank. After solidification, 1μL of loading buffer and 5μL of PCR product mixture were added to the gel wells, and gel electrophoresis was performed at 220V for 30min. If a band appeared at the target position (1500 bp), the sample was sent for 16S rRNA sequencing. The sequencing results were compared with those on the NCBI website.

[0059] Table 1 PCR amplification reaction system

[0060]

[0061] 4. Purification and Identification of Strains

[0062] Morphological identification:

[0063] LS-68 was stained using Gram staining and its morphology was observed under a 100x oil immersion microscope. The specific procedures are as follows: LS-68 was inoculated into 5 mL of LB broth and incubated at 37 ℃ and 200 rpm for 16 h. The bacterial culture was then streaked onto 1.5% LB agar plates and incubated overnight at 37 ℃ and 40% humidity. A small amount of a single LS-68 colony was taken, dipped into 2 µL of physiological saline, and spread clockwise to form a colony approximately 1 cm in diameter. 2 Prepare a uniform, thin, round slide and place it at room temperature until the bacterial culture dries. Fix the slide by passing it through an alcohol lamp flame 1-2 times, being careful not to overheat it; the slide should be warm to the touch. Add crystal violet staining solution to the bacteria and stain for 1 minute, then rinse with water. Add iodine solution and stain for 1 minute, then rinse with water. Add destaining solution, shake the slide, destain for 30 seconds, rinse with water, and blot dry. Add safranin staining solution and stain for 1 minute, then rinse with water. Blot dry with filter paper and examine under a 100x oil immersion microscope.

[0064] Gram staining microscopy results indicate Gram-negative bacteria (see...) Figure 3 It has a short rod-like shape.

[0065] Molecular biological identification:

[0066] Universal primer sequences for 16S rDNA amplification:

[0067] 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO: 1)

[0068] 1492R: 5'-TACGGYTACCTTGTTACGACTT-3' (SEQ ID NO: 2)

[0069] (Y represents any base of A, T, C, or G)

[0070] PCR product sequencing results:

[0071]

[0072] (SEQ ID NO:3)

[0073] 5. Determination of the growth curve of the strain

[0074] The growth curve of the bacteria was determined using a turbidimetric method. The OD600 value was indirectly measured using a UV-Vis spectrophotometer to infer the growth trend of the bacteria in the sample. Inoculation and culture of the bacteria: A suitable amount of glycerol bacteria or a single clone of bacteria from agar was inoculated into 5 mL of culture medium and cultured until the plateau phase. A sample was then streaked to verify the purity of the bacterial solution. The 5 mL bacterial solution was expanded to a volume of 25-30 mL, and after reaching the plateau phase, a sample was streaked to verify the purity of the bacterial solution. The OD600 value of the bacterial solution was measured, and an appropriate dilution was performed to achieve an OD600 value between 0.02 and 0.1. The seed culture was inoculated into an appropriate amount of liquid culture medium according to the dilution factor. The number of test groups was set according to the characteristics of the bacteria. After mixing the bacterial solution, it was aliquoted into 15 mL centrifuge tubes, with 4-5 mL per tube. Each group was performed in triplicate, and the culture medium was reserved as a blank control. The initial OD600 value of the mixed bacterial solution was measured. The blank control and the aliquoted bacterial solution were placed together in a shaker for incubation. OD600 value determination: Set the UV-Vis spectrophotometer to 600 nm, and use a blank culture medium as a control to determine the OD600 value of the bacterial culture at different incubation times, and record the incubation time and OD600 value. When setting sampling points, the time interval should be shortened accordingly during the exponential growth phase of the bacterial strain (the time interval can be shortened to 15-30 min per measurement). Data processing: Use Origin or Prism software for graphing and data analysis (the nonlinear fitting equation is Logistic).

[0075] Growth curve assay of Enterobacter LS-68 (participating in...) Figure 4 After approximately 0.5-1 hour of cultivation, it enters the logarithmic growth phase, and after 4 hours of cultivation, its growth enters a plateau phase.

[0076] II. Acid tolerance evaluation of Enterobacter LS-68

[0077] The acid tolerance of Enterobacter LS-68 was expressed as the ratio of the total number of viable bacteria in the bacterial culture treated with PBS at 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 juice, standard simulated gastric juice, and satiated simulated gastric juice, which were 2.0, 3.0, and 4.0, respectively.

[0078] The LS-68 strain was inoculated into 5 mL of LB broth and cultured in a shaker at 37°C and 200 rpm for 16 h. 30 mL of PBS (pH = 7.4) buffer was placed in a centrifuge tube, and 1 mol / L HCl was slowly added dropwise, with pH measured continuously until the pH reached 2.0, 3.0, and 4.0. 5 mL of PBS (pH = 7.4) and PBS at different pH values ​​were then mixed with an equal volume of the bacterial culture and cultured according to Table 1. Both the PBS buffer and the bacterial culture should be prepared fresh before use.

[0079] After incubation, the mixture was diluted 10 μL with sterile LB broth. 3 10 5 and 10 7 For each dilution, pipette 100 µL of the diluted solution into sterile petri dishes, making two sterile plates for each dilution. Simultaneously, pipette 100 µL of PBS (pH = 2.0, pH = 3.0, and pH = 4.0) into sterile petri dishes as blank controls. Pour approximately 15 mL of 1.5% LB agar medium cooled to 50°C into the sterile petri dishes, rotating the dishes clockwise or counterclockwise at least 20 times to mix thoroughly. After the agar solidifies, invert the plates and incubate overnight at 37°C and 40% humidity. If there is no growth or the growth is small, incubation can be extended to 48–72 h. After incubation, count all colonies on the plates. The entire process from sample dilution to plate pouring should be completed within 15 minutes.

[0080] The total viable count of bacterial suspensions treated with PBS (pH = 7.4) is recorded as N1, and the total viable count of bacterial suspensions 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. The total viable count of bacterial suspensions treated with PBS (pH = 7.4) is recorded as N2 and N3, and the total viable count of bacterial suspensions treated with PBS at pH = 2.0, pH = 3.0, or pH = 4.0 is recorded as N2' and N3'. Calculate the relative mean deviation between the results of the total viable count measurements of N1, N2, N3 and N1', N2', N3'. The relative mean deviation between the two sets of data should not exceed 15%; otherwise, the acid tolerance test must be repeated.

[0081] Table 2. Main parameters of the acid tolerance test method for Enterobacter LS-68

[0082]

[0083] Acid tolerance is calculated using the following formula:

[0084]

[0085] In the formula:

[0086] A -- Acid tolerance, expressed as a percentage (%)

[0087] N1 -- The initial total number of viable bacteria in the first acid tolerance test, in CFU / mL;

[0088] N2 -- The initial total viable count of the second acid tolerance test, in CFU / mL;

[0089] N3 -- The initial total viable count of the third acid tolerance test, in CFU / mL;

[0090] N1' -- Total viable bacterial count after the first acid tolerance test, in CFU / mL;

[0091] N2' -- Total viable bacteria count after the second acid tolerance test, in CFU / mL;

[0092] N3' -- Total viable bacterial count after the third acid tolerance test, in CFU / mL;

[0093] 1 / 3 -- Take the average of three parallel data points;

[0094] The calculation result is expressed as an integer.

[0095] according to Figure 5 The results of "total viable bacteria count in PBS treatment at different pH values" show that the Enterobacter LS-68 exhibits a tolerance of 122% to pH=4.0, 101% to pH=3.0, and 114% to pH=2.0, indicating that LS68 has good acid tolerance. This suggests that Enterobacter LS-68 can well tolerate the acidity of gastric juice and is suitable for making oral live bacterial products.

[0096] III. Antibiotic susceptibility testing for Enterobacter LS-68

[0097] Antibiotic susceptibility testing determines whether bacteria can be inhibited by concentrations achieved with conventional doses of antibacterial drugs, thus demonstrating their safety.

[0098] Experimental Methods: LS-68 strain was cultured overnight in TSA broth at 37 °C and 200 rpm, and then streaked onto 1.5% TSA agar plates. Aseptically, 3-4 pure colonies cultured for 18-24 h on the TSA plates were picked and inoculated into 5 mL of tryptone soy broth (TSB). The plates were incubated at 37 °C and 200 rpm with shaking for 2 h. The culture was then corrected to 0.5 McFarland standard turbidimetric units (OD) using a McFarland standard turbidimetric tube. 625 = 0.08~0.10), equivalent to a bacterial concentration of 1.5×10.8 CFU / mL.

[0099] The prepared bacterial suspension should be used within 15 minutes. Add 300 µL of bacterial suspension to the surface of 1.5% MHA agar and spread it evenly using a sterile L-shaped rod. Incubate at room temperature for 3–5 minutes to allow the agar to completely absorb the moisture from the plate.

[0100] Using sterile forceps, apply standard antibiotic strips one by one as needed, pressing gently to ensure they adhere firmly to the agar surface. Once a strip touches the plate, it should not be moved. The center-to-center distance between each strip should be greater than 24 mm, and the strip should be more than 15 mm from the inner edge of the plate. Generally, no more than 5 strips should be placed on a 90 mm diameter plate. Within 15 minutes of applying the strips, invert the agar plate and incubate at 37°C and 40% concentration for 17 h ± 1 h. During incubation, the plates should be kept separate and no more than two should be stacked together.

[0101] Remove the plate and measure the diameter of the inhibition zone (including the diameter of the paper) of each paper piece on the back of the plate under transmitted light using a vernier caliper. The edge of the inhibition zone should be limited to a point where no obvious bacterial growth is visible to the naked eye. The growth of tiny colonies at the edge of the inhibition zone that can only be observed under a magnifying glass can be ignored.

[0102] Table 3. Interpretation of the diameter of the inhibition zone of Enterobacter LS-68

[0103]

[0104] The results are shown in Table 3. LS-68 was sensitive to multiple antibiotics, including gentamicin (10 µg / tablet), tetracycline (30 µg / tablet), ciprofloxacin (5 µg / tablet), streptomycin (10 µg / tablet), polymyxin B (300 IU), and kanamycin (30 µg / tablet). (Antimicrobial resistance refers to the gradual decrease in bacterial sensitivity to antimicrobial drugs after repeated exposure, eventually leading to resistance without inhibition or eradication. This characteristic of bacteria is called drug resistance. It is usually determined by antimicrobial susceptibility testing (AST), and the results can be expressed as sensitive (S), intermediate (I), and resistant (R). Resistant (R): Bacterial growth and reproduction are inhibited by achievable antimicrobial concentrations at conventional doses. Intermediate (I): Conventional doses of antimicrobial drugs are less effective against bacteria than sensitive strains; the drug is effective at physiological concentration sites or higher doses can be used for treatment.)

[0105] The antibacterial experiment results showed that the diameter of the inhibition zone fell within the range of certain bacterial resistance mechanisms, and the bacteria could not be inhibited by the concentrations achieved by conventional doses of antibacterial drugs. This indicates that LS-68 showed sensitivity to most antibiotics, suggesting that this strain is relatively safe and can be used in biological agents or dietary supplements.

[0106] IV. Effects of Enterobacter LS-68 on growth performance and organ index in DSS-induced colitis mice

[0107] 1. Mouse source and processing methods

[0108] Eight-week-old female C57BL / 6 mice, SPF grade, were used in the experiment and were obtained from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. After arrival, the animals were acclimatized in the facility for one week. Approximately 12 hours / 12 hours of alternating light and dark lighting were provided daily. The ambient temperature and relative humidity of the animal room were controlled at 22-26 ºC and 40-70%, respectively.

[0109] 2. Mouse grouping

[0110] Description: Fifteen healthy C57BL / 6 mice were randomly divided into three groups of five mice each according to their body weight: No DSS group / blank control, DSS model group, and DSS+LS-68 group.

[0111] Table 4. Grouping and treatment of experimental mice

[0112]

[0113] 3. DSS-induced colitis model mice

[0114] Mice in each group (except the No DSS group) were given drinking water containing 3% DSS (Dextran Sulfate Sodium Salt) (free access to drinking water). At the beginning of the experiment, mice were administered the solution via gavage daily, with mouse weight measured before each administration, until the DSS group showed a weight loss rate > 20% (indicating successful model establishment). The formula for the mouse weight loss rate is as follows:

[0115] Mouse weight loss rate (%) = (initial weight - current weight) / initial weight * 100%

[0116] See results Figure 6 As shown.

[0117] 4. Treatment of mice in the control and experimental groups

[0118] The experimental group mice had 3% DSS added to their drinking water, while the control group mice were administered 200 μL of fresh LS-68 culture medium by gavage daily. Figure 6The changes in body weight after different treatment groups are shown. On day 10 of the experiment, the body weight of mice in the DSS+LS-68 group was significantly higher than that in the DSS group, indicating that LS-68 can significantly improve the decrease in body weight of mice induced by DSS.

[0119] 5. Disease Activity Index (DAI) Assessment

[0120] During the experiment, the clinical progression of colitis was assessed daily. The Disease Index (DAI) was a comprehensive score combining the animal's weight loss rate, stool consistency, and degree of rectal bleeding. DAI = (weight change score + stool consistency score + rectal bleeding score) / 3. On day 9 of the experiment, the DAI score of mice in the DSS+LS-68 group was significantly lower than that in the DSS group, indicating that gavage administration of LS-68 significantly improved the DSS-induced decrease in the disease index.

[0121] Method for detecting fecal occult blood in mice: The fecal occult blood qualitative detection kit (o-toluidine method, Regen Biosciences) was used for detection (see the instruction manual for detailed operation). The results are interpreted according to the fecal occult blood interpretation table. If there is visible blood in the stool, it is directly rated as 4 points.

[0122] Table 5 Disease Activity Index (DAI) Scoring Criteria for Mice

[0123]

[0124] *Normal stool is formed; loose stool is loose or soft stool that does not stick to the anus; watery stool is unformed stool that sticks to the anus.

[0125] The degree of rectal bleeding is assessed according to Table 4. If there is visible bleeding in the stool, it is directly rated as 4 points.

[0126] Table 6. Fecal Occult Blood Interpretation Table

[0127]

[0128] 6. Spleen Index

[0129] Methods: The spleens of mice were harvested and weighed after euthanasia for the determination of spleen index.

[0130] Spleen index = spleen weight (mg) / mouse body weight (g)

[0131] like Figure 8 As shown, the spleen index of mice in the DSS+LS-68 group was significantly lower than that in the DSS model group, indicating that the degree of inflammation in mice administered LS-68 by gavage was significantly reduced.

[0132] V. Effects of Enterobacter LS-68 on Biochemical Indicators in DSS-Induced Colitis Mice

[0133] 1. Staining of tissue sections

[0134] After measuring the length of the colon, the cecum was removed, and the colon was coiled into a spiral on a horizontal plane and fixed in 4% tissue fixative. Hematoxylin-eosin staining (HE staining) of the intestinal tissue was prepared by Wuhan Saiweier Biotechnology Co., Ltd.

[0135] like Figure 10 As shown, the DSS group exhibited extensive ulceration of the colonic tissue, with loss of mucosal epithelium at the ulceration sites, disappearance of intestinal gland structure replaced by proliferating connective tissue, accompanied by abundant infiltration of lymphocytes and granulocytes, and irregular arrangement of intestinal glands around the ulceration sites; mild edema of the submucosa, loose arrangement of connective tissue, and a small amount of infiltration of lymphocytes and granulocytes; and severe thickening of the muscular layer. In the DSS+LS-68 group, the colonic tissue surface consisted of a single layer of columnar epithelium with normal morphology and structure, abundant intestinal glands in the lamina propria, goblet cells distributed between epithelial cells, and a double layer of smooth muscle cells separating the intestinal crypts from the submucosa. The submucosa was composed of connective tissue, and the remaining part of the intestinal wall included a muscular layer composed of smooth muscle cells and a serosa. This indicates that gavage administration of LS-68 significantly improved the pathological changes in the colonic tissue of mice with acute colitis induced by DSS.

[0136] 2. MPO detection

[0137] 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, fighting against the invasion of pathogens such as bacteria and fungi. Under certain conditions, MPO can catalyze reactions that produce excess oxidants. When this exceeds the body's antioxidant defense response, it leads to oxidative stress and oxidative tissue damage, contributing to the development of various diseases such as inflammation, small vessel vasculitis, tumors, nephritis, and atherosclerosis.

[0138] Four colon samples were randomly selected from each experimental group. Each sample was further divided into proximal (near the cecum), middle, and distal segments (near the rectum) for MPO detection. Colonic MPO was measured using a kit purchased from Nanjing Jiancheng Bioengineering Institute. Quantitative analysis of MPO in the colon was performed using a UV spectrophotometer, with specific operating procedures following the kit's instructions.

[0139] like Figure 11 As shown, the MPO in the colon of mice in the DSS +LS -68 group was significantly lower than that in the DSS group, indicating that the Enterobacter LS-68 significantly repaired inflammation in the colon of mice.

[0140] 3. Expression levels of pro-inflammatory / anti-inflammatory factors

[0141] The expression levels of pro-inflammatory genes TNF-α, IL-6, and TNFR-2 in the colon were analyzed by qPCR.

[0142] Colon tissue stored at -80°C was lysed with TriZol lysis buffer, and total RNA was extracted after treatment with chloroform, isopropanol, and 75% ethanol, respectively. The absorbance at A260 / A280 and A260 / A230 was measured using a micro spectrophotometer to determine 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 TransGen Biotech Co., Ltd. The specific procedures were performed according to the kit instructions. The reaction volume was 20 μl, containing 10 μl of 2×TSReaction Mix, 1 μl of Random Primer, 1 μl of TransScript® RT / RI Enzyme Mix, 1 μl of gDNARemover, and RNase-free water to a final volume of 20 μl, with a total RNA content of 500 ng. Reaction conditions: Incubate at 25°C for 10 min, incubate at 42°C for 15 min, heat at 85°C for 5 s to inactivate TransScript®RT / RI and gDNA Remover.

[0143] The primer sequences for quantitative real-time PCR (qPCR) are detailed in Table 6. 7 μL ddH2O was used. 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 an internal control. The experimental results were analyzed using the 2−ΔΔCt calculation method. The gene expression level was calculated using the formula: 2Cttarget (control) – Cttarget (treatment) / 2CtTBP (control) – CtTBP (treatment).

[0144] like Figure 12 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-68 group were lower than those in the DSS model group, and showed a decreasing trend, indicating that LS-68 has a good therapeutic effect on DSS-induced acute colitis in mice.

Claims

1. An Enterobacter LS-68 strain, deposited at the China General Microbiological Culture Collection Center on October 28, 2024, with accession number CGMCC NO. 32368; its 16S rDNA gene sequence contains the sequence shown in SEQ ID NO.

3.

2. A composition comprising the Enterobacter as described in claim 1, wherein the composition is a pharmaceutical product.

3. The use of the Enterobacterium of claim 1 and the composition of claim 2 in the preparation of a medicament for treating and relieving colitis.

4. The composition according to claim 2, wherein the composition is an oral probiotic.

5. The composition according to claim 4, wherein the oral probiotic is a live probiotic with a live count of not less than 1.5E+09 CFU / g.

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