Swarming-motility enterobacter ludwigii and use thereof

Abstract

Provided are an Enterobacter ludwigii strain and use thereof in the amelioration of inflammatory bowel disease. The present invention belongs to the technical field of new applications of microorganisms. The strain is named Enterobacter ludwigii LS-57 and is deposited in China General Microbiological Culture Collection Center under the accession number CGMCC NO. 31424, with a deposit date of July 22, 2024. The strain exhibits swarming motility, has relatively strong adaptability to the intestinal environment, tolerates gastric juice acidity well, is sensitive to various commonly used antibiotics, and has a good ameliorative effect on colitis.

Description

A swarming, motile Enterobacter ludwigii bacterium and its uses Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to a swarming, motile Enterobacter ludwigii and its uses. Background Technology

[0002] Inflammatory bowel disease (IBD) is a group of chronic, relapsing inflammatory bowel diseases that primarily affect the intestinal mucosa, including two main subtypes: Crohn's disease (UC) and ulcerative colitis (CD). Symptoms of IBD can vary from person to person, but common symptoms include abdominal pain and diarrhea, bloody stools, weight loss, fatigue, and malnutrition, impacting patients' quality of life. Furthermore, the chronic nature and unpredictable course of IBD impose a long-term psychological and economic burden on patients and their families. The exact cause of IBD is not fully understood, but it is generally believed to result from a combination of factors, including host genetic susceptibility, gut microbiota dysbiosis, disruption of the intestinal mucosal barrier, and abnormal intestinal mucosal immunity. IBD itself is not fatal, but it may increase the risk of other diseases such as colon cancer, blood clots, and liver disease.

[0003] Treatment for IBD primarily involves medication to relieve symptoms; currently, there is no complete cure. Clinically used medications mainly include salicylates, glucocorticoids, and immunosuppressants. Salicylates exert their anti-inflammatory effect by reducing the release of pro-inflammatory factors in the intestines; however, they have serious gastrointestinal side effects and can even cause drug-induced kidney damage. Glucocorticoids relieve inflammation by inhibiting the release of inflammatory substances and are commonly used for patients with moderate to severe enteritis; however, long-term or high-dose use can easily lead to drug resistance and various adverse reactions. Immunosuppressants inhibit the proliferation of inflammatory cells, but patients are prone to adverse reactions such as nausea, vomiting, and diarrhea after taking them. In summary, although salicylates, hormones, and immunosuppressants can relieve the clinical symptoms of IBD, they all have drawbacks such as easy relapse upon discontinuation and low patient compliance with long-term use.

[0004] Compared to healthy individuals, patients with active IBD exhibit altered gut microbiota diversity, composition, and / or abundance. Similar patterns of microbial composition exist among IBD patients, such as decreased microbial diversity, reduced Firmicutes abundance, and increased Proteobacteria abundance. Gut symbiotic bacteria have shown significant positive effects in the prevention and treatment of IBD. The protective functions of these microorganisms, such as regulating gut microbiota repair, immune modulation, enhanced anti-inflammatory effects, and intestinal barrier repair, appear to be strain-specific. Therefore, screening for strains that ameliorate inflammatory bowel disease is crucial for developing new therapies for IBD.

[0005] Several Ludwig's Enterobacter species have been screened and their applications provided in the prior art. CN119220429A discloses Ludwig's Enterobacter XL0404, which produces IAA at a content of 18.94 mg / L and ACC deaminase at a content of 484.48 U / L. It also possesses multiple functions such as phosphorus solubilization, potassium solubilization, nitrogen fixation, and ferophile production. Furthermore, it exhibits inhibitory effects on major root and stem disease pathogens such as *Phytophthora indicum*, *Rhizoctonia solani*, *Ustilago maydis*, *Fusarium oxysporum*, and *Fusarium solani*, and is primarily used for the biological control of tobacco root and stem diseases. CN118909823A discloses Ludwig's Enterobacter ES2 and its application in the degradation of nicosulfuron, mainly for the remediation of nicosulfuron-contaminated soil. CN118126897A discloses a heavy metal immobilizing endophytic proliferator, *Enterobacter ludwigii*, its screening and application, which is tolerant to and adsorbs cadmium, can colonize tobacco and promote tobacco growth in cadmium-polluted environments, reduces cadmium content in tobacco and cadmium cytotoxicity to tobacco, and increases soil cadmium stability. CN117946933A discloses a strain of *Enterobacter ludwigii* ZTB-A30, capable of removing formaldehyde from industrial wastewater and indoor air. CN114806952B discloses a *Enterobacter ludwigii* and its application in the conversion of 5-hydroxymethylfurfural. CN105861352B discloses a *Enterobacter ludwigii* that, under conditions providing carbon, nitrogen, and inorganic salts, can survive and grow in a culture environment containing dichloroquinoline acid, exhibiting tolerance and degradation ability to dichloroquinoline acid, thus solving the problems of phytotoxicity and environmental pollution caused by this chemical residue.

[0006] However, the screening methods used in existing technologies are generally based on conventional dilution and isolation methods. Furthermore, the Ludwig C. Ludwig strains screened by existing technologies are mainly used for treating heavy metals in petroleum and soil. Existing technologies have not found any use for strain isolation through swarming, nor have they reported any medicinal value for treating or improving intestinal diseases.

[0007] 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 ludwigia strain was screened from a fecal sample of a 78-year-old male colon cancer patient, which exhibits cluster motility. Summary of the Invention

[0008] Based on the above-mentioned existing technology situation,

[0009] The first aspect of this invention provides a novel Enterobacter ludwig strain LS-57, which is deposited at the China General Microbiological Culture Collection Center on July 22, 2024, with accession number CGMCC NO. 31424.

[0010] This strain has the ability to move in clusters; specifically, it can grow monoclonal colonies that spread like a thin film.

[0011] The strain is short rod-shaped; furthermore, it is a white, dot-like clone.

[0012] This strain is a Gram-negative bacterium.

[0013] The 16S rDNA gene sequence of this strain is shown in SEQ ID NO.3.

[0014] Swarming motility refers to the collective migration of bacteria on a low-hardness agar surface (0.3-1.5%) through the coordinated action of flagella. It is characterized by the radial or wavy diffusion of the colony edge. The bacteria move rapidly and collectively on the surface by driving their flagella, which is different from swimming or planktonic movement.

[0015] A second aspect of the present invention provides a composition comprising Ludwig C. LS-57, or a bacterial agent comprising Ludwig C. LS-57.

[0016] The present invention further provides a microbial preparation product containing Ludwig's Enterobacter LS-57 strain, which can be used as food, pharmaceuticals, or health products.

[0017] The number of live bacteria is not less than 1.5E+09 CFU / g.

[0018] Oral administration is preferred.

[0019] The dosage form can be tablets, capsules, granules, pills, or oral liquids, etc.

[0020] 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, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculators, filter aids, excipients, additives, and release inhibitors. Microcapsules refer to microcapsules containing natural (e.g., chitosan) or synthetic polymers (e.g., PLGA) encapsulating bacterial cells, with a diameter of 1-1000 μm, capable of sustained-release of active ingredients. Microspheres are spherical particles with a diameter of 10-500 μm formed by adsorbing bacterial cells onto porous carrier materials (such as calcium alginate), and are commonly used for oral delivery. Enteric coating technology, specifically for oral formulations, uses hydroxypropyl methylcellulose phthalate (HPMCP) as the enteric coating material, ensuring a bacterial survival rate of >80% in the acidic environment of the stomach.

[0021] In one specific embodiment, the health food further includes food additives, which are selected from: acidity regulators, anti-caking agents, defoamers, antioxidants, bleaching agents, leavening agents, coloring agents, color-protecting agents, enzyme preparations, flavor enhancers, nutritional fortifiers, preservatives, sweeteners, thickeners, and flavorings.

[0022] 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.

[0023] The bacterial agent also contains fermentation products and non-fermentation products of Ludwig C., wherein the non-fermentation products mainly include metabolites.

[0024] The pharmaceutical composition contains the aforementioned Ludwig's Enterobacter LS-57 strain, a drug carrier, and / or pharmaceutical excipients.

[0025] In one specific embodiment, the present invention provides a food composition capable of improving gut health, the composition comprising Ludwig's Enterobacter LS-57. The food composition may be a functional food and may contain commonly used additives in food compositions. The food composition may be a dietary supplement.

[0026] In one specific embodiment, the present invention provides a health supplement composition capable of improving gut health, the health supplement composition comprising Ludwig Cronobacter LS-57.

[0027] In one specific embodiment, the present invention provides a pharmaceutical composition comprising Ludwig's Enterobacter LS-57.

[0028] Furthermore, Enterobacter ludwigii LS-57 can be co-cultured with other synergistic strains to enhance its efficacy in improving gut health, reducing inflammation, or regulating metabolism. No specific limitations are required, but if needed, these can be synergistic strains for anti-inflammatory and intestinal barrier repair, and / or strains that enhance metabolic function, and / or strains that inhibit pathogens and compete for niches. Examples include Clostridium plasminogen lysate, Akkermansia, Bifidobacterium infantis, EcN probiotics, Lactobacillus plantarum, and Enterobacter rotagenes.

[0029] A third aspect of this invention provides the use of Ludwig's Enterobacter LS-57 strain.

[0030] Specifically, the use of Ludwig's Enterobacter LS-57 in treatment and pharmaceuticals; specifically, in the treatment of intestinal-related diseases; more specifically, in the treatment of inflammatory bowel disease; and more specifically, in the treatment, relief of colitis, and / or improvement of gut health.

[0031] Use in the preparation of medicines for the treatment, relief of colitis and / or improvement of gut health.

[0032] Application of Ludwig's Enterobacter LS-57 in the preparation of food compositions that improve gut health.

[0033] The application includes at least one of the following functions.

[0034] (1) Stabilize the weight of the patient, and further, significantly improve the weight loss of the patient;

[0035] (2) Improves the symptoms of sticky stool in the treated patients;

[0036] (3) Improves the symptoms of rectal bleeding in the treated patients;

[0037] Furthermore, Ludwig's Enterobacter strain LS-57 can reduce the Disease Activity Index (DAI).

[0038] (4) It improved the pathological changes of colon tissue in patients with acute colitis.

[0039] (5) Repair colon inflammation.

[0040] The fourth aspect of the present invention provides a method for isolating bacteria.

[0041] This invention provides a method for isolating bacteria, wherein the isolation method is based on screening strains for swarming ability.

[0042] 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.

[0043] 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.

[0044] 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. Attached Figure Description

[0045] Figure 1. Culture of clustered motile bacteria in homogenized fecal samples.

[0046] Figure 2. Cluster movement of bacteria LS-57.

[0047] Figure 3. Gram staining of Ludwig's Enterobacter LS-57.

[0048] Figure 4. Total viable bacteria count of the original bacterial culture treated with PBS at different pH values.

[0049] Figure 5. Weight changes in mice with colitis induced by DSS.

[0050] Figure 6. Weight changes after different treatment groups (Results are expressed as mean ± standard error. Experimental data were analyzed using GraphPad Prism 10.1.2 using one-way ANOVA. * indicates P < 0.05).

[0051] Figure 7. Assessment of Disease Activity Index (DAI) in different groups (Results are expressed as mean ± standard error. Experimental data were tested using GraphPad Prism 10.1.2 with a t-test (Tukey's test). * represents P < 0.01, **** represents P < 0.0001.)

[0052] Figure 8. Spleen index of different groups (Results are expressed as mean ± standard error. Experimental data were analyzed by one-way ANOVA using GraphPad Prism 10.1.2. ** indicates P < 0.01).

[0053] Figure 9. Comparison of staining of tissue sections from mice treated with different groups.

[0054] Figure 10. Changes in MPO in mice under different treatments (Results are expressed as mean ± standard error. Experimental data were tested using GraphPad Prism 10.1.2 using Tukey's test. * represents P < 0.01, **** represents P < 0.0001).

[0055] Figure 11. 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

[0056] I. Isolation, Screening and Identification of Enterobacter ludwig's bacillus

[0057] 1. Sample processing

[0058] 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.

[0059] 2. Bacterial plate culture

[0060] Vortex the microcentrifuge tube containing the fecal sample for approximately 10 seconds, then inoculate 5 μL of homogenized fecal solution onto the center surface of a 0.8% LB agar plate. The plate was placed in an incubator at 37 ℃ and 20% humidity and incubated for 16 hours (i.e., swarming conditions). Thin-film diffusion-like colonies were observed, as shown in Figure 1. The bacterial film had completely covered the entire culture dish, exhibiting a wave-like, layered diffusion growth pattern.

[0061] 3. Screening of monoclonal strains based on swarming ability

[0062] For plates showing Swarm expansion 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.

[0063] After the streaked plates had grown sufficiently, two single colonies from each plate were placed in 5 ml of LB broth. The samples were then incubated on a shaker at 200 rpm and 37°C for 16 hours. A 5 μL bacterial suspension was 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 swarm. As shown in Figure 2, the inoculated bacterial solution grew thin-film-diffusion-like single-clonal colonies, indicating that the strain was a swarming bacterium (i.e., "swarming culture verification").

[0064] 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.

[0065] 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 sequencing. The sequencing results were compared with those on the NCBI website.

[0066] Table 1 PCR amplification reaction system

[0067]

[0068] 4. Purification and Identification of Strains

[0069] Morphological identification:

[0070] Gram staining was used to stain LS-57 bacteria, and its morphology was observed under a 100x oil immersion microscope. The specific procedures are as follows: LS-57 bacteria were 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-57 colony was taken, dipped into 2 µL of physiological saline, and spread clockwise to form a colony approximately 1 cm in diameter. 2Prepare 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.

[0071] The Gram staining microscopy results showed that it was a Gram-negative bacterium (see Figure 3), with a short rod-like morphology.

[0072] Molecular biological identification:

[0073] Universal primer sequences for 16S rDNA amplification:

[0074] 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO: 1)

[0075] 1492R: 5'-TACGGYTACCTTGTTACGACTT-3' (SEQ ID NO: 2)

[0076] PCR product sequencing results:

[0077]

[0078] II. Acid tolerance evaluation of Enterobacter ludwig's bacterium

[0079] The acid tolerance of strain LS-57 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.

[0080] The LS-57 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.

[0081] 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.

[0082] The total viable count of the bacterial culture in the PBS-treated sample (pH = 7.4) is recorded as N1, and the total viable count of the bacterial culture in the PBS-treated sample (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 the bacterial culture in the PBS-treated sample (pH = 7.4) is recorded as N2 and N3, and the total viable count of the bacterial culture in the PBS-treated sample (pH = 2.0, pH = 3.0, or pH = 4.0) is recorded as N2' and N3'. Calculate the relative average deviation between the results of the N1, N2, N3 and N1', N2', N3' viable count measurements. The relative average deviation between the two sets of data should not exceed 15%; otherwise, the acid tolerance test must be repeated. See Figure 2 for the pH indicator color change results.

[0083] Table 2. Main parameters of the acid tolerance test method for bacteria LS-57

[0084]

[0085] Acid tolerance is calculated using the following formula:

[0086] ;

[0087] In the formula:

[0088] A -- Acid tolerance, expressed as a percentage (%)

[0089] N1 -- The initial total number of viable bacteria in the first acid tolerance test, in CFU / mL;

[0090] N2 -- The initial total viable count of the second acid tolerance test, in CFU / mL;

[0091] N3 -- The initial total viable count of the third acid tolerance test, in CFU / mL;

[0092] N1' -- Total viable bacterial count after the first acid tolerance test, in CFU / mL;

[0093] N2' -- Total viable bacteria count after the second acid tolerance test, in CFU / mL;

[0094] N3' -- Total viable bacterial count after the third acid tolerance test, in CFU / mL;

[0095] 1 / 3 -- Take the average of three parallel data points;

[0096] The calculation result is expressed as an integer.

[0097] Based on the results of "total viable bacteria count of original bacterial solutions treated with PBS at different pH values" shown in Figure 4, it can be calculated that LS-57 bacteria have a tolerance of 70% to pH = 2.0, a tolerance of 105% to pH = 3.0, and a tolerance of 107% to pH = 4.0.

[0098] This indicates that the Ludwig C. LS-57 strain can well tolerate the acidity of gastric juice, making it suitable for oral live bacteria products.

[0099] III. Antibiotic susceptibility testing of Enterobacter ludwig's bacillus

[0100] Antibiotic susceptibility testing determines whether bacteria can be inhibited by concentrations achieved with conventional doses of antibacterial drugs, thus demonstrating their safety.

[0101] Experimental Methods: LS-57 bacteria were 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.

[0102] 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 any moisture from the plate.

[0103] 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 ℃ 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.

[0104] 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.

[0105] Table 3. Interpretation of the inhibition rate of bacteria LS-57

[0106]

[0107] The results are shown in Table 2. LS-57 bacteria were sensitive to multiple antibiotics, including ticarcillin (75 µg / tablet), tetracycline (30 µg / tablet), and polymyxin B (300 µg / tablet). (Antimicrobial resistance refers to the gradual decrease in bacterial sensitivity to antibiotics after repeated exposure, eventually leading to resistance without inhibition or eradication. This characteristic of bacteria is called resistance. It is usually determined through 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 antibiotic concentrations at conventional doses. Intermediate (I): Conventional doses of antibiotics are less effective against bacteria than sensitive strains; the drug is effective at physiological concentration sites or higher doses may be used for treatment.)

[0108] 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-57 bacteria showed sensitivity to most antibiotics, suggesting that this strain is relatively safe and can be used in biological agents or dietary supplements.

[0109] IV. Effects of Enterobacter ludwig's bacterium on growth performance and organ index in DSS-induced colitis mice

[0110] 1. Mouse source and processing methods

[0111] 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.

[0112] 2. Mouse grouping

[0113] Description: Thirty healthy C57BL / 6 mice were randomly divided into three groups of 10 mice each according to their body weight: No DSS group / blank control, DSS model group, and DSS+LS-57 group.

[0114] Table 4. Grouping and treatment of experimental mice

[0115]

[0116] 3. DSS-induced colitis model mice

[0117] 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:

[0118] Mouse weight loss rate (%) = (initial weight - current weight) / initial weight * 100%

[0119] The results are shown in Figure 5.

[0120] 4. Treatment of mice in the control and experimental groups

[0121] The control group mice received no DSS in their drinking water and were administered 200 μL of LB liquid culture medium by gavage daily during the experiment. The experimental group mice received 3% DSS in their drinking water and were administered 200 μL of fresh LS-57 culture medium by gavage daily during the experiment. Figure 5 shows the changes in body weight after treatment in different groups. On day 9 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 DSS-induced decrease in body weight in mice.

[0122] 5. Disease Activity Index (DAI) Assessment

[0123] 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 days 5, 6, and 7 of the experiment, the DAI scores of mice in the DSS+LS-57 group were significantly lower than those in the DSS group, indicating that gavage administration of LS-57 significantly improved the DSS-induced decrease in the disease index.

[0124] 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.

[0125] Table 5 Disease Activity Index (DAI) Scoring Criteria for Mice

[0126]

[0127] *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.

[0128] 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.

[0129] Table 6. Fecal Occult Blood Interpretation Table

[0130]

[0131] 6. Spleen Index

[0132] Methods: The spleens of mice were harvested and weighed after euthanasia for the determination of spleen index.

[0133] Spleen index = spleen weight (mg) / mouse body weight (g)

[0134] As shown in Figure 7, 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 administered LS-57 by gavage was significantly reduced.

[0135] V. Effects of Enterobacter ludwig's bacterium on biochemical indicators in DSS-induced colitis mice

[0136] Tissue section staining

[0137] 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.

[0138] As shown in Figure 9, 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; slight edema in 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-57 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-57 significantly improved the pathological changes in the colonic tissue of mice with acute colitis induced by DSS.

[0139] MPO detection

[0140] 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.

[0141] 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.

[0142] As shown in Figure 10, the MPO of the middle and distal segments of the colon in the DSS +LS -57 group was significantly lower than that in the DSS group, and the MPO of the distal segment of the colon in the DSS +LS -57 group was extremely significantly lower than that in the DSS group, indicating that the Ludwig C. LS-57 significantly repaired inflammation in the colon of mice.

[0143] Expression levels of pro-inflammatory / anti-inflammatory factors

[0144] The expression levels of pro-inflammatory genes TNF-α, IL-6, and TNFR-2 in the colon were analyzed by qPCR.

[0145] 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×TS Reaction Mix, 1 μl of Random Primer, 1 μl of TransScript® RT / RI Enzyme Mix, 1 μl of gDNA Remover, 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.

[0146] 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: 2Ct target (control) – Ct target (treatment) / 2Ct TBP (control) – Ct TBP (treatment).

[0147] As shown in Figure 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, and showed a decreasing 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 at the China General Microbiological Culture Collection Center on July 22, 2024, with accession number CGMCC NO. 31424.

2. The Ludwig's Enterobacter according to claim 1, characterized in that: Its 16S rDNA gene sequence contains the sequence shown in SEQ ID NO.

3.

3. The Ludwig's Enterobacter according to claim 1, wherein the bacteria have the ability to move in clusters; further, the bacteria are capable of growing monoclonal colonies that spread like a thin film.

4. The Ludwig's Enterobacter according to claim 1, wherein the bacterium is a short rod-shaped Gram-negative bacterium, and further, is a white-like clone.

5. A composition comprising *Enterobacter ludwigii* as described in any one of claims 1-4, wherein the *Enterobacter ludwigii* composition may be a food, pharmaceutical, or health product.

6. Use of the Ludwig's Enterobacterium of claims 1-4 and the compositions of claims 5-6 in treatment and pharmaceutical manufacturing; specifically, in the treatment of intestinal-related diseases; more specifically, in the treatment of inflammatory bowel disease; more specifically, in the treatment, relief of colitis and / or improvement of intestinal health; and in the preparation of medicines or foods for the treatment, relief of colitis and / or improvement of intestinal health.

7. A food composition capable of improving gut health, said composition comprising Ludwig Cronobacter LS-57.

8. The composition according to claim 5, wherein the composition is an oral probiotic; preferably, the oral probiotic is a live probiotic, and the live count is not less than 1.5E+09 CFU / g.

9. A method for isolating bacteria, wherein the isolation method is based on screening strains for swarming motility.

10. In the isolation method according to claim 9, the criterion for screening strains is to observe whether there are film-diffusion-like monoclonal colonies after diluting and spreading the isolated sample on a solid culture medium, thereby determining whether it is a target strain with cluster motility; further, in the isolation method, the screened strains are derived from fecal samples, and the fecal samples are derived from colon cancer patients; further, the isolation method includes homogenizing the fecal samples from colon cancer patients, diluting them, and inoculating them into a solid culture medium. After the appearance of swarm expansion bacteria, single colonies are further inoculated until film-diffusion-like monoclonal colonies grow, indicating that the strain is a clustering bacterium; further, the inoculation culture medium is 0.5%-1.5% LB agar plates; the homogenization treatment refers to homogenizing the fecal sample to a concentration of 100 mg / mL.

Images