Bacteroides fragilis and application thereof

Abstract

The present application relates to a strain of Bacteroides fragilis and its application, the strain is preserved in China General Microbiological Culture Collection Center (CGMCC) on April 14, 2026, and the preservation number is: CGMCC No. 38257.The strain can regulate STING-dependent training immunity, effectively resist Salmonella typhimurium and H9N2 avian influenza virus infection, maintain the integrity of the intestinal barrier of the host, and reduce inflammatory damage.The Bacteroides fragilis of the present application has good biological safety, and the strain can be used as a training immunity inducer, and the strain is expected to be a candidate probiotic or vaccine adjuvant for preventing / treating bacterial and viral infections, suitable for human health and poultry farming, and provides a new solution to resist antibiotic resistance.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering and immunology technology, specifically relating to a strain of Bacteroides fragilis and its applications. Background Technology

[0002] The global overuse of antibiotics has triggered a dual crisis: the incidence of antibiotic resistance continues to rise, and the balance of the host's gut microbiota is disrupted. This not only makes the body more susceptible to pathogen invasion but also limits effective treatment options. This serious dilemma highlights the urgent need to develop alternative strategies that can enhance the host's inherent resistance without exacerbating dysbiosis.

[0003] Training immunity, as an innovative anti-infection strategy, has emerged and become a research hotspot. Unlike adaptive immunity, which relies on the clonal expansion of T and B cells, training immunity refers to the reprogramming of innate immune cells and their bone marrow progenitor cells after their first exposure to microorganisms or endogenous stimuli, thereby producing a non-specific protective effect. When these trained cells return to homeostasis, they will initiate a faster and stronger pro-inflammatory response when they encounter homologous or heterologous pathogens again.

[0004] The gut microbiota is a key regulator of immune training, and commensal bacteria and their metabolites can serve as natural training agents. Bacteroides fragilis, an obligate anaerobic bacterium that colonizes the mammalian gut, is an ideal candidate for next-generation probiotics. Studies have confirmed its ability to regulate innate immunity and strengthen the intestinal epithelial barrier. However, current research has not clarified whether Bacteroides fragilis induces broad-spectrum anti-infective protection through immune training, nor has it elucidated the molecular mechanisms and applications of this immune training mechanism.

[0005] Salmonella typhimurium is a significant zoonotic pathogen, posing a serious threat to human health and poultry farming. Similarly, H9N2 avian influenza virus is a major viral pathogen, also endangering poultry farming and public health. Current control strategies against these pathogens heavily rely on antibiotics or traditional vaccines, but issues such as increasing antibiotic resistance, strong vaccine specificity, and susceptibility to viral mutations limit their effectiveness. Furthermore, while there are reports of using BCG and β-glucan to induce training immunity, these inducers have drawbacks such as pathogenicity of the source, potential to induce pathological inflammation, or unsuitability for animal consumption. Therefore, developing a safe, efficient, symbiotic-derived training immune inducer with broad-spectrum cross-protective activity has significant translational value for reducing and replacing antibiotics in human health and livestock farming. Summary of the Invention

[0006] Purpose of the invention: The primary objective of this invention is to provide a strain of Bacteroides fragilis with good biocompatibility that can be induced to train immunity.

[0007] A second objective of the present invention is to provide a composition or compound formulation containing the aforementioned Bacteroides fragilis.

[0008] A third objective of the present invention is to provide the use of the aforementioned Bacteroides fragilis in the preparation of drugs, feed additives or vaccines that protect against infection by pathogens such as Salmonella typhimurium and / or H9N2 avian influenza virus.

[0009] Technical solution: To solve the above-mentioned technical problems, the present invention provides a strain of Bacteroides fragilis, which is classified as Bacteroides fragilis and was deposited on April 14, 2026 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38257 and deposit address in Beijing, China.

[0010] The strain described in this invention has a survival rate in an acidic environment of pH 3.0 and a bile salt concentration of 0.1%, is non-hemolytic, has no obvious systemic toxicity in the host, does not disrupt immune organ homeostasis, does not cause intestinal structural abnormalities, and has good biosafety.

[0011] The present invention also provides a composition or compound formulation comprising any one or more of the following: live Bacteroides fragilis, inactivated Bacteroides fragilis, its culture, its lysate, its extract, its culture supernatant, its outer membrane vesicles, its fermentation product, or its metabolites; preferably, it further comprises a pharmaceutically, food-grade, health food, or cosmetically acceptable combination of excipients.

[0012] The composition or compound formulation of the present invention comprises at least one of the following components:

[0013] (1) Whole Bacteroides fragilis;

[0014] (2) Outer membrane vesicles secreted by Bacteroides fragilis;

[0015] (3) Cyclic diadenosine monophosphate (cDMA), a metabolite of Bacteroides fragilis.

[0016] The present invention also provides an outer membrane vesicle of Bacteroides fragilis, which is secreted by the above-mentioned Bacteroides fragilis strain, has a particle size of 150-160 nm, a biconcave disc-shaped structure, carries cyclic diadenosine monophosphate, and can activate the cGAS-STING signaling pathway.

[0017] The present invention also provides a method for preparing the outer membrane vesicles of the above-mentioned Bacteroides fragilis, comprising the following steps:

[0018] 1) The above-mentioned Bacteroides fragilis strain was inoculated into a special anaerobic culture medium and cultured in an anaerobic incubator to obtain bacterial solution;

[0019] 2) The bacterial culture was separated and purified, the supernatant was collected, and the culture was centrifuged and filtered to obtain crude outer membrane vesicles of Bacteroides fragilis;

[0020] 3) The crude outer membrane vesicles were further purified and identified by nanoparticle tracking analysis and scanning electron microscopy to obtain intact Bacteroides fragilis outer membrane vesicles.

[0021] The present invention also provides the use of the aforementioned Bacteroides fragilis or the aforementioned composition or compound preparation in the preparation of medicaments for the prevention or treatment of Salmonella typhimurium infection and / or H9N2 avian influenza virus infection.

[0022] The present invention also provides the use of the aforementioned Bacteroides fragilis or the aforementioned composition or compound preparation in the preparation of a medicament for inhibiting or killing Salmonella typhimurium and / or H9N2 avian influenza virus.

[0023] The drug includes oral preparations, vaccines, topical preparations, or inactivated preparations thereof for combined oral and topical use. Preferably, the oral preparation includes gavage or feed additives.

[0024] The drug is one that inhibits the colonization and spread of pathogens in the host, maintains the integrity of the intestinal barrier, reverses the downregulation of tight junction protein expression, alleviates inflammatory response, and / or inhibits the overactivation of the STING signaling pathway.

[0025] The drug is applied to mammals and / or poultry, preferably, the mammals include mice and humans, and the poultry include chickens.

[0026] The present invention also provides an antipathogenic drug, immune inducer, feed additive, or vaccine, wherein the drug, immune inducer, feed additive, or vaccine comprises the aforementioned Bacteroides fragilis or the aforementioned composition or compound formulation.

[0027] The antipathogenic drug, immune training inducer, feed additive, or vaccine includes at least one of the outer membrane vesicles of Bacteroides fragilis as an active ingredient.

[0028] The antipathogenic drugs, immune training inducers, feed additives, or vaccines also contain pharmaceutically acceptable carriers or excipients.

[0029] The training immune inducer activates macrophages to polarize to the M1 type, regulates the cGAS-STING-IRF3 signaling pathway, induces a training immune response, and achieves broad-spectrum protection against pathogens.

[0030] The mechanism of the application is as follows: inhibiting the colonization and spread of pathogens in the host, maintaining the integrity of the intestinal barrier, reversing the downregulation of tight junction proteins Occludin and Claudin, alleviating the inflammatory response, and inhibiting the overactivation of the STING signaling pathway.

[0031] Beneficial Effects: Compared with existing technologies, this invention has the following advantages: This invention is the first to isolate and identify three strains of *Bacteroides fragilis* with good biocompatibility from infant feces. These strains exhibit moderate acid / bile salt tolerance, are non-hemolytic, and do not affect host growth, immune organ function, or intestinal structure, providing a solid safety foundation for their subsequent applications. This invention clarifies that *Bacteroides fragilis* can induce STING-dependent training immunity through the secretion of outer membrane vesicles. These outer membrane vesicles, as key effector components, carry cyclic diadenosine monophosphate (cGAS) and can specifically activate the cGAS-STING signaling pathway, regulating macrophage M1 polarization. This elucidates a novel mechanism of symbiotic bacteria-mediated training immunity and expands the sources of training immunity inducers. The *Bacteroides fragilis* strains and their outer membrane vesicles of this invention have broad-spectrum antipathogenic activity, simultaneously resisting infection by *Salmonella typhimurium* (bacteria) and H9N2 avian influenza virus (virus), overcoming the limitations of traditional vaccines' strong specificity and antibiotics' effectiveness only against bacteria, providing a new candidate for broad-spectrum anti-infection strategies. The protective mechanism of the *Bacteroides fragilis* strain and its outer membrane vesicles of this invention is highly conserved in mice and chickens, making it suitable for both mammals and poultry. It can be applied simultaneously to human health protection and disease control in poultry farming, aligning with the concept of a unified health approach and possessing broad application prospects. The outer membrane vesicles of *Bacteroides fragilis* of this invention can be purified and stabilized, and can be formulated into various dosage forms such as feed additives and mucosal vaccines, circumventing the challenges of live probiotic delivery and eliminating the risk of antibiotic residues. The *Bacteroides fragilis* strain of this invention exhibits good biocompatibility and holds promise as a candidate probiotic or vaccine adjuvant for the prevention / treatment of bacterial and viral infections, suitable for human health and poultry farming, providing a new solution to combat antibiotic resistance. Attached Figure Description

[0032] Figure 1 Figure showing the results of the isolation and identification of Bacteroides fragilis, safety evaluation, and correlation analysis with diarrhea.

[0033] Figure 2 Figure showing the results of training mice to resist Salmonella Typhimurium infection by inducing immunity with Bacteroides fragilis.

[0034] Figure 3 The image shows the results of Bacteroides fragilis enhancing the body's immunity to resist Salmonella typhimurium infection by training macrophages.

[0035] Figure 4The results show that Bacteroides fragilis protects mice from Salmonella typhimurium infection by regulating the STING signaling innate immune pathway.

[0036] Figure 5 Figure showing the identification of Bacteroides fragilis outer membrane vesicles (OMVs) and their activation of M1 macrophages via the cGAS-STING pathway.

[0037] Figure 6 Figure showing the results of the application validation of Bacteroides fragilis-induced trained immunity in chickens to resist Salmonella typhimurium infection.

[0038] Figure 7 The figure shows the results of the broad-spectrum protection against H9N2 avian influenza virus by the trained immunity induced by Bacteroides fragilis. Detailed Implementation

[0039] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims. Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in the art.

[0040] The present invention will be further described below with reference to specific embodiments and accompanying drawings. Unless otherwise specified, the methods used in the following embodiments are conventional methods. The specific materials and reagents involved are as follows:

[0041] 1. Materials:

[0042] (1) This invention was approved by the Animal Experiment Ethics Committee of Nanjing Agricultural University, and all experimental procedures complied with relevant Chinese animal experiment regulations. Male C57BL / 6 mice aged 6-8 weeks were purchased from the Experimental Animal Center of Yangzhou University, and STING knockout heterozygous mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. The experimental mice were randomly divided into groups of 6 mice each and fed with standard feed. Seven-day-old Arbor Acres broiler chickens were purchased from local poultry farms and fed according to the nutritional standards for broiler chickens. The experimental chickens were randomly divided into groups of 6 mice each.

[0043] (2) Mouse macrophage cell line RAW264.7 was purchased from Beijing Solarbio Science & Technology Co., Ltd. After rapid thawing of frozen cells in a 37°C water bath, they were seeded into high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics and cultured in a 37°C, 5% CO2 incubator. Chicken macrophage cell line HD11 was purchased from Wuxi Newgen Biotech Co., Ltd. and cultured in high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics. The medium was changed every 3 days, and the cells were passaged according to conventional methods after confluence.

[0044] (3) Salmonella Typhimurium SL1344 was a strain preserved in our laboratory (SL1344 is from the article Trainedimmunity using probiotics and inactivated pathogens enhances resistance to Salmonella enterica serovar Typhimurium infection by activating the cGAS-STING signal pathway in mice and chickens, DOI:10.1016 / j.jare.2025.03.011), and was cultured overnight in LB liquid medium containing 50 μg / mL streptomycin; Bacteroides fragilis was cultured in a dedicated anaerobic medium (BHI medium, Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd., catalog number HB8297-5) in an anaerobic incubator.

[0045] (4) Isolation of primary peritoneal macrophages and dendritic cells: Primary peritoneal macrophages were isolated from mice using a classic method: The peritoneal cavity of mice was flushed with 5 mL of sterile Hans' balanced salt solution, the flushing fluid was collected and centrifuged to obtain cell pellet, and the pellet was resuspended in RPMI v1640 complete medium containing antibiotics and antifungal agents to obtain primary peritoneal macrophages. The isolation of dendritic cells was performed according to relevant classic methods.

[0046] 2. Reagents:

[0047] DMEM culture medium (Nanjing Senbeijia Biotechnology Co., Ltd.); fetal bovine serum (Gibco); 0.05% trypsin (Gibco); 0.25% trypsin (Nanjing Senbeijia Biotechnology Co., Ltd.); RIPA strong lysis buffer (Guangzhou Shuopu Biotechnology Co., Ltd.); Trizol (Nanjing Novizan Biotechnology Co., Ltd.); chloroform (Shenzhen Sanpin Technology Co., Ltd.); DEPC water (Wuhan Gaiyuntian Biotechnology Co., Ltd.); anhydrous ethanol (Shenzhen Sanpin Technology Co., Ltd.); reverse transcription kit (Nanjing Novizan Biotechnology Co., Ltd., catalog number R323-01); real-time fluorescence kit (Nanjing Novizan Biotechnology Co., Ltd.). Vizan Biotechnology Co., Ltd. (Catalog No. Q312-02); 10% protein precast gel (Shanghai Yamei Biomedical Technology Co., Ltd.); 5×SDS denaturing protein loading buffer (Shanghai Yamei Biomedical Technology Co., Ltd.); HPR-labeled GAPDH (Shanghai Ebibio Biotechnology Co., Ltd., Catalog No. AB0037); Occludin monoclonal antibody (Wuhan Sanying Co., Ltd., Catalog No. 27260-1-AP); Claudind monoclonal antibody (Wuhan Sanying Co., Ltd., Catalog No. 13050-1-AP); secondary antibody goat anti-rabbit (Nanjing Baode Biotechnology Co., Ltd., Catalog No. BS13278).

[0048] 3. General evaluation indicators and test procedures:

[0049] (1) Assessment of colonic injury: Distal colonic tissue from mice was taken, and a 0.5 μm segment was cut, fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with H&E. The tissue damage was observed under a microscope. The histopathological scoring of colonic tissue was based on seven indicators: inflammatory response, crypt injury, crypt abscess, inflammatory cell infiltration, submucosal edema, goblet cell reduction, and epithelial hyperplasia. The inflammatory response was scored as 0.4 points based on the extent of involvement of the mucosa, submucosa, muscularis propria, and serosa; crypt injury was scored as 0.4 points based on the degree of crypt structure destruction from normal to complete destruction with ulceration; crypt abscess was scored as 0.2 points based on whether it was unifocal or multifocal; and epithelial hyperplasia was scored as 0.3 points based on the extent of epithelial proliferation from unifocal to diffuse. This scoring system can comprehensively assess the severity and type of colonic injury in each group of mice. The specific steps are as follows:

[0050] Tissue fixation: Freshly removed tissue was fixed in centrifuge tubes containing 4% paraformaldehyde.

[0051] Trimming and dehydration: Place the fixed tissue on filter paper, cut two smooth planes with a blade, and then transfer it to a new 10 mL centrifuge tube. Soak the trimmed tissue block in anhydrous ethanol at concentrations of 75% (overnight, about 12 h), 85% (1 h), 95% (1 h), and 100% (1 h) in sequence, repeating the anhydrous ethanol treatment twice.

[0052] Tissue clearing: Place the dehydrated tissue in a fume hood and immerse it in xylene, adjusting the clearing time according to the size of the tissue.

[0053] Tissue paraffin infiltration: After the tissue has been cleared, it is quickly transferred to a 60°C paraffin bath and placed in a beaker containing paraffin solution for 2 hours.

[0054] Tissue embedding: Place one plane of the above tissue vertically in a rectangular cardboard box containing wax liquid, and then wait for the wax liquid to completely solidify.

[0055] Tissue sectioning: Place the embedded paraffin block in a paraffin microtome, cut out tissue sections with a thickness of 5 μm, attach them to an adhesive slide, and dry them at 37°C for later use.

[0056] (2) Macrophage depletion in vivo: Macrophages in mice were depleted by intraperitoneal injection of clophosphonate-loaded liposomes. 2 mg of liposomes (purchased from Shanghai Yisheng Biotechnology Co., Ltd., catalog number 40337ES08) was injected per 20 g of mouse body weight. Control mice were injected intraperitoneally with an equal volume of blank liposomes (purchased from Shanghai Yisheng Biotechnology Co., Ltd., catalog number 40338ES08). To verify the macrophage depletion efficiency, mononuclear cells were isolated from the lungs and spleen of mice and stained with CD11b and F4 / 80 antibodies (purchased from Hangzhou Lianke Biotechnology Co., Ltd., catalog number of CD11b antibody: F41011b03, catalog number of F4 / 80 antibody: F21480A02). The proportion of macrophages was detected by flow cytometry. Meanwhile, mouse fecal homogenate was serially diluted 10-fold, and 10 μL of the diluted solution was spread on LB agar plates and incubated at 37°C under aerobic and anaerobic conditions, respectively. Colony-forming units were counted after 24 h and 48 h to monitor the depletion of intestinal flora.

[0057] (3) Real-time quantitative PCR and Western blotting: Total RNA was extracted from cells after treatment with TRIzol reagent, reverse transcribed into cDNA, and then subjected to real-time quantitative PCR. The specific steps of reverse transcription are as follows: Using the extracted total RNA as a template, reverse transcription was performed using the Novizan RT SuperMix for qPCR kit. First, a reaction system to remove genomic DNA was prepared (Table 1), and incubated at 42℃ for 2 min to remove interference from genomic DNA in subsequent experiments. Then, 4 μL of 5×HiScript Ⅲ qRT SuperMix was added to the reaction tube of the first step, and the mixture was gently pipetted again; the reverse transcription was completed at 37℃ for 15 min, and then the enzyme was inactivated at 85℃ for 5 s. After the reaction, the cDNA product was stored at -20℃ for subsequent real-time quantitative PCR.

[0058] Table 1 Reaction system for removing genomic DNA

[0059]

[0060] PCR reactions were performed using the SYBR Green quantitative PCR kit on a LightCycler 480 II quantitative PCR instrument. The PCR reaction system is shown in Table 2, and the reaction conditions are shown in Table 3. Using β-actin as an internal reference gene, the expression levels of genes such as ZO-1, Occludin, Claudin, cGAS, STING, TANK-binding kinase 1 (TBK1), IRF3, TNF-α, IL-1β, and IL-6 were quantitatively analyzed using the 2-ΔΔCT method. Primers used in the experiments were synthesized by Shanghai Sangon Biotech Co., Ltd., and their sequences are detailed in Table 4.

[0061] Table 2 PCR reaction system

[0062]

[0063] Table 3 PCR Reaction Conditions

[0064]

[0065] Table 4 Primer sequences used in PCR reactions

[0066]

[0067] (4) In the Western blot experiment, the following antibodies were used: cGAS antibody (Nanjing Bayode Biotechnology Co., Ltd., catalog number BS80526), ​​STING antibody (Nanjing Bayode Biotechnology Co., Ltd., catalog number BS79500), TBK1 antibody (Nanjing Bayode Biotechnology Co., Ltd., catalog number BS60714), IRF3 antibody (Nanjing Bayode Biotechnology Co., Ltd., catalog number BZ16696), Occludin monoclonal antibody (Wuhan Sanying Co., Ltd., catalog number 27260-1-AP), and Claudind monoclonal antibody. The primary antibodies (Wuhan Sanying Co., Ltd., catalog number 13050-1-AP) and β-actin antibody (Nanjing Bayode Biotechnology Co., Ltd., catalog number BS00241-T) were used as primary antibodies for incubation. After the primary antibody incubation, horseradish peroxidase-labeled secondary antibody goat anti-rabbit (Nanjing Bayode Biotechnology Co., Ltd., catalog number BS13278) was added for binding. Finally, the protein bands were developed using an enhanced chemiluminescence reagent (Shandong Saikexide Technology Co., Ltd., catalog number ED0015-C), and the protein bands were quantitatively analyzed using ImageJ software.

[0068] (5) Flow cytometry and fluorescence-activated cell sorting: Mice were euthanized by cervical dislocation, fixed in a supine position, and 5 mL of PBS buffer (pH 7.2-7.4, 0.01M, Beijing Solarbio Science & Technology Co., Ltd., catalog number P1020) was slowly injected into the right lower abdomen. The abdomen was gently massaged to allow for full diffusion, and the mixture was allowed to stand for 5 min. Peritoneal lavage fluid was then collected, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the precipitate was collected and counted using a cell counter. 1×10⁻⁶ cells were collected. 8 For each cell, add 5 μL of PE-labeled F4 / 80 antibody (Hangzhou Duoning Biotechnology Co., Ltd., catalog number: F21480A02) and APC-labeled CD11b antibody (Hangzhou Duoning Biotechnology Co., Ltd., catalog number: F41011b03) and incubate for 30 minutes in the dark. After incubation, add 2 mL of flow cytometry staining buffer to each tube, centrifuge at 1000 rpm for 10 min, and discard the supernatant. Finally, resuspend the precipitate in 500 μL of flow cytometry staining buffer, filter, and perform flow cytometry analysis.

[0069] (6) Immunization training and Salmonella challenge experiments in mice and chickens: Immunization training experiment in mice: C57BL / 6J mice were randomly divided into different training groups, and the training stimulus was 1×10⁻⁶ mg / L. 8 CFU / mL Bacteroides fragilis bacterial suspension. Mice in each group were administered 200 μL of the bacterial suspension via gavage three times a week to induce immune training. In the challenge experiment, mice were administered 200 μL of a 1×10⁻⁶ concentration via gavage. 8 Mice were administered CFU / mL Salmonella Typhimurium bacterial suspension, while the control group received an equal volume of sterile PBS via gavage. Body weight, survival rate, and clinical symptoms were monitored in mice for 7 consecutive days after challenge. Broiler immunization training experiment: Seven-day-old Albert Illegible white-feathered broilers were selected and raised in a standard environment according to broiler nutritional standards, and randomly divided into different experimental groups. To induce immunization training, each group of chickens was gavaged with 400 μL of the corresponding treatment solution three times within one week; after immunization, 1 mL of a 1×10⁻⁶ concentration was administered via gavage. 8 Chickens were challenged with a CFU / mL Salmonella Typhimurium bacterial suspension, while the control group received an equal volume of sterile PBS via gavage. Weight changes, survival rate, and clinical symptoms were monitored in chickens for 7 consecutive days after challenge.

[0070] Example 1: Isolation, identification, and safety evaluation of Bacteroides fragilis strains

[0071] 1. Strains Isolation

[0072] Infant stool samples were collected and inoculated onto Bacteroides bile esculin agar (BBE) medium (Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd., catalog number HB7028) and anaerobically cultured at 37°C for 48 h. Results are as follows: Figure 1Morphological identification of strain A showed that after Gram staining and microscopic observation, the purified strain was a Gram-negative anaerobic bacillus with typical characteristics of Bacteroides fragilis. Figure 1 Results B in the study showed that it can produce melanin and hydrolyze esculin, and its 16S rRNA gene sequence is that of the *Bacteroides fragilis* reference strain ATCC W03014 (gene bank accession number X83953). The three *Bacteroides fragilis* strains were identified by 16S rRNA and their phylogenetic relationships were analyzed, with the following results: Figure 1 The D in the data shows that Bf-10 has 98% homology with Bf-18, while Bf-19 has 79% homology with the other two strains.

[0073] 2. Molecular identification

[0074] Genomic DNA was extracted from the strain, the 16S rRNA gene was amplified, and sequencing analysis was performed. The results are as follows: Figure 1 The C-value in the data shows that the isolated strain has 98%~99% sequence homology with the Bacteroides fragilis reference strain ATCC W03014 (gene bank accession number X83953), confirming its taxonomic status as Bacteroides fragilis. The three strains were named F10-414-A-10 (Bf-10), F08-010-A-18 (Bf-18), and F10-440-A-19 (Bf-19), respectively.

[0075] 3. Safety evaluation of Bacteroides fragilis strains

[0076] (1) Grouping of experimental animals

[0077] Male C57BL / 6 mice aged 6-8 weeks were randomly divided into 4 groups of 6 mice each: control group, Bf-10 group, Bf-18 group, and Bf-19 treatment group.

[0078] (2) Treatment method

[0079] Mice in each bacterial strain treatment group were administered 200 μL of a 1×10⁻⁶ concentration via gavage each time. 8 Bacteroides fragilis culture at CFU / mL was administered via gavage three times within one week; the control group was administered an equal volume of sterile PBS via gavage for 7 consecutive days. Body weight, spleen index, and colon length were monitored; colon tissue was collected for H&E staining to observe pathological damage; and the expression of tight junction proteins Occludin and Claudin was detected by Western blotting.

[0080] (3) Safety testing indicators

[0081] Weight monitoring: Mouse weight was monitored daily. The average weight of each group of mice was recorded at the end of the experiment. Results are shown in [link to relevant documentation]. Figure 1In the E group, the body weight of mice in each strain treatment group was not significantly different from that in the control group, indicating that the strain had no obvious systemic toxicity.

[0082] Immune organ testing: Mice were dissected, spleens were harvested, spleen weight was measured, and spleen index was calculated. Figure 1 The results showed that there were no significant differences in spleen weight and spleen index between the treatment groups and the control group, and no splenomegaly or splenic atrophy, indicating that the strains did not disrupt the homeostasis of immune organs.

[0083] Intestinal structure examination: The length of the mouse intestine was measured, and the results are shown in [reference needed]. Figure 1 In the F strain, the intestinal length of mice in each treatment group was 6.5–7.0 cm, which was not significantly different from the 6.6 cm in the control group. Colon tissue was taken for H&E staining to observe intestinal morphology, and the results were as follows: Figure 1 The G value indicates that the intestinal mucosa, villus structure, and crypt depth in each treatment group were consistent with those in the control group, with no tissue damage. Further analysis using Western blot revealed the expression levels of tight junction proteins in the colonic tissue. Figure 1 The H results showed that, compared with the blank control group, Bf-18 and Bf-19 treatments upregulated the expression levels of Occludin and Claudin proteins, while Bf-10 mainly promoted the expression of Occludin protein.

[0084] Hemolytic activity test: Each strain was inoculated onto blood agar plates (Changde Bickman Biotechnology Co., Ltd., BKMAMLAB) and anaerobic incubated for 48 hours. The formation of hemolytic rings was observed, and the results were as follows: Figure 1 The D-values ​​in the data show that none of the strains formed a hemolytic zone and were not hemolytic.

[0085] Acid / Bile Salt Tolerance Test: Each strain was inoculated into acidic medium (pH 3.0) and 0.1% bile salt medium, respectively. Survival rates were then assessed after incubation. Results are shown below. Figure 1 The I-values ​​show that strain Bf-10 had the highest survival rate, and all strains exhibited moderate acid / bile salt tolerance. This Bf-10 strain is identified as *Bacteroides fragilis*, abbreviated as dipro-X37, and classified as *Bacteroides fragilis*. It was deposited on April 14, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38257.

[0086] Example 2: Bacteroides fragilis induces and trains mice to resist Salmonella typhimurium infection (mouse model)

[0087] (1) Construction of training immune model and challenge experiment

[0088] Animal grouping: Male C57BL / 6 mice aged 6-8 weeks were randomly divided into 4 groups of 6 mice each: control group, *Bacteroides fragilis* treatment group, *Salmonella typhimurium* infection group, and *Bacteroides fragilis* pretreatment + infection group. Immunotherapy training: Mice in the *Bacteroides fragilis* treatment group and the *Bacteroides fragilis* pretreatment + infection group were administered 200 μL of a 1×10⁻⁶ concentration via gavage each time. 8 CFU / mL Bf-10 bacterial suspension was administered by gavage three times within one week; the control and infected groups were administered an equal volume of sterile PBS by gavage. Challenge experiment: After training and immunization induction, mice in the infected group and the Bacteroides fragilis pretreatment + infection group were administered 200 μL of 1×10⁻⁶ CFU / mL Bf-10 bacterial suspension by gavage. 8 CFU / mL Salmonella Typhimurium SL1344 bacterial suspension; control group and Bacteroides fragilis treatment group were administered an equal volume of sterile PBS by gavage. Mouse body weight and survival rate were monitored for 7 consecutive days post-challenge. Monitoring indicators included: body weight change, fecal / liver bacterial load (CFU plate count), colon length, colonic H&E pathological score, and tight junction protein expression.

[0089] (2) Results of detection indicators

[0090] See weight and survival results. Figure 2 C and Figure 2 In group B, the infected mice experienced a significant 7% decrease in body weight and a reduced survival rate; mice pretreated with *Bacteroides fragilis* and infected mice maintained stable body weight, and their survival rate was not significantly different from the control group. This indicates that *Bacteroides fragilis* can alleviate the weight loss caused by *Salmonella typhimurium* infection and improve survival rate. Bacterial load detection: Colony forming units (CFU) were counted from mouse feces and liver. The results are as follows: Figure 2 The F-values ​​in the study showed that the bacterial load in the Bacteroides fragilis pretreatment + infection group was significantly lower than that in the infection group, indicating that Bacteroides fragilis can inhibit the colonization and spread of Salmonella typhimurium.

[0091] Intestinal barrier detection: Measuring mouse intestinal length, see [link to relevant documentation]. Figure 2 In groups D and E, the intestinal length of the infected group was significantly shortened to 5 cm, while the intestinal length of the Bacteroides fragilis pretreatment + infection group remained at 6 cm, close to the 7 cm of the control group; the colon HE staining results are as follows. Figure 2 H and Figure 2 The results in the G series show that pretreatment with *Bacteroides fragilis* can reverse the damage to the colonic epithelial barrier in the infected group. Western blot results are as follows: Figure 2 The results showed that pretreatment with Bacteroides fragilis reversed the downregulation of Occludin and Claudin proteins in the infected group, thus maintaining the integrity of the intestinal barrier.

[0092] Inflammation and signaling pathway detection: Results of Western blot analysis and real-time quantitative PCR are as follows: Figure 2 J and Figure 2 K in the study showed that pretreatment with Bacteroides fragilis could inhibit the overactivation of STING, cGAS, and IRF3 in the infected group, reduce the expression of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α, and alleviate the inflammatory response.

[0093] Example 3: Macrophages and the STING pathway are the core of the protective effect induced by Bacteroides fragilis training.

[0094] (1) Macrophage exhaustion experiment

[0095] Six-week-old C57BL / 6 wild-type mice were randomly divided into four groups, with six mice in each group. The groups included a macrophage-cleared blank control group (Clo-Control), a macrophage-cleared *Bacteroides fragilis* treatment group (Clo-Bf), a macrophage-cleared *Salmonella typhimurium* group (Clo-St), and a macrophage-cleared *Bacteroides fragilis* pretreatment group (Clo-B.f+St). A macrophage-cleared mouse model was established by tail vein injection of clophosphonate liposomes (Shanghai Yisheng Biotechnology Co., Ltd., catalog number 40337ES08) 7 days and 4 days before gavage with *Salmonella typhimurium*. Six days and 3 days before gavage with *Salmonella typhimurium*, mice in the Clo-Bf and Clo-B.f+St groups were administered 1×10⁻⁶ *Bacteroides fragilis* via gavage. 8 CFU / mL, 200 μL / mouse; the remaining mice were administered 200 μL PBS by gavage; on day 0, Clo-St and Clo-B.f+St were infected with Salmonella Typhimurium 1×10⁻⁶. 8 CFU / mL, 200 μL / mouse; the remaining mice were administered 200 μL of sterile PBS by gavage. Mice were sacrificed and samples were collected 5 days after infection with Salmonella Typhimurium.

[0096] The detection was performed using the aforementioned flow cytometry method.

[0097] (2) Test results

[0098] like Figure 3 Flow cytometry analysis, shown in Figure A, confirmed successful macrophage depletion. Figure 3 The C-results showed that no deaths occurred in either the blank control group or the Bacteroides fragilis group after macrophage removal; the mortality rate of mice in the Salmonella typhimurium infection group was 50%, while the mortality rate of mice pretreated with Bacteroides fragilis was 60%; body weight monitoring showed... Figure 3 As shown in D, after macrophage depletion, the body weight of the Bacteroides fragilis pretreatment + infection group decreased by 13.5%, which was not significantly different from the infection group after depletion, indicating that the protective effect of Bacteroides fragilis completely disappeared; intestinal damage ( Figure 3 E and G in the sample), bacterial load test results ( Figure 3The F in the study also confirmed that after macrophages were depleted, Bacteroides fragilis could not play a protective role, indicating that macrophages are the core training cells. Figure 3 The H values ​​in the study showed that Salmonella typhimurium infection significantly upregulated the expression levels of various inflammatory factors; however, even with the removal of macrophages, Bacteroides fragilis pretreatment failed to inhibit the infection-induced high expression of inflammatory factors, and the body remained in an inflammatory state.

[0099] (3) STING gene deletion model

[0100] Experimental animals: Wild-type (WT) C57BL / 6 mice and STING heterozygotes (STING) were selected. + / - Mice were randomly divided into eight groups of six each, with each group consisting of either a WT-Control or STING control group. + / - -Control), Bacteroides fragilis treatment group (WT-Bf or STING) + / - -Bf), infection group (WT-St group or STING) + / - -St), Bacteroides fragilis pretreatment + infection group (WT-B.f+St group or STING) + / - -B.f+St). Training and challenge were performed according to the protocol of Example 2, and body weight, bacterial load, colon length, and expression of STING pathway proteins (cGAS, STING, IRF3) were measured.

[0101] (4) Test results Figure 4 The B in the diagram shows that the WT-St group is related to the STING group. + / - All patients in the -St group died after infection, with a mortality rate of 66.67%; while no deaths occurred in the WT-B.f+St group, but under STING knockdown conditions, STING... + / - Deaths still occurred in the -B.f+St group, with a mortality rate of 33.35%; in wild-type mice, pretreatment with Bacteroides fragilis significantly alleviated infection-induced weight loss. Figure 4 C) Intestinal damage (colon length and staining results as shown) Figure 4 As shown in D and E), inhibiting bacterial load ( Figure 4 (G in the middle); while in STING + / - In mice, the protective effect of Bacteroides fragilis was completely lost; the pretreatment + infection group showed no significant differences in body weight loss, bacterial load, and intestinal damage compared to the infection group; STING pathway protein detection showed... Figure 4 The F-value in the data indicates that Bacteroides fragilis cannot regulate STING. + / - STING pathway expression in mice, and the results of inflammatory factor detection are as follows: Figure 4 As shown in H, the results indicate that the STING signaling pathway is an essential pathway for the protective effect mediated by Bacteroides fragilis.

[0102] Example 4: Extraction and functional identification of outer membrane vesicles (OMVs) of Bacteroides fragilis

[0103] (1) Extraction of Bacteroides fragilis OMVs: Bf-10 strain was selected and inoculated into a special anaerobic medium (BHI medium, Qingdao Haibo Biotechnology HB8297-5). The culture was anaerobic until the logarithmic growth phase to obtain the bacterial suspension. The bacterial suspension was centrifuged at 8000 r / min for 10 min at 4℃, and the supernatant was collected. The supernatant was filtered through a 0.22 μm filter membrane to remove impurities and bacterial debris. The filtered supernatant was ultracentrifuged at 100000 r / min for 2 h at 4℃, and the precipitate was collected and resuspended in PBS to obtain crude outer membrane vesicles. The crude outer membrane vesicles were further purified by ultracentrifugation (4℃, 120000×g, 80 min) to obtain Bacteroides fragilis OMVs, such as... Figure 5 A in the middle.

[0104] (2) The target components were collected, resuspended in PBS, and their morphology was identified by NTA particle size analysis and transmission electron microscopy (TEM).

[0105] (3) Identification of OMVs extracted from Bacteroides fragilis: The size of outer membrane vesicles was detected by nanoparticle tracking analysis. The results are shown in [reference needed]. Figure 5 The C-type particles, with a diameter of 150–160 nm, are consistent with typical bacterial outer membrane vesicle characteristics; observed using scanning electron microscopy (see [reference]). Figure 5 B in the image shows that the outer membrane vesicles have a biconcave disc-like structure, confirming that complete Bacteroides fragilis outer membrane vesicles were obtained.

[0106] (4) Cell training model: RAW264.7 macrophages were co-incubated with 20 ng of OMVs extracted from Bacteroides fragilis for 24 h, the stimulants were washed off, and the cells rested for 3 days. Lipopolysaccharide (LPS) (100 ng / mL) was then added for stimulation. Protein samples were collected to detect the STING pathway and M1 / M2 polarization markers. RAW264.7 macrophages were seeded in 6-well plates and divided into control group, outer membrane vesicle group, LPS group, outer membrane vesicle + LPS group, whole Bacteroides fragilis + LPS group, and cyclic diadenosine monophosphate + LPS group.

[0107] (5) Cell training model detection results are as follows Figure 5 As shown: External membrane vesicle (OMV) treatment upregulated the expression of STING, cGAS, and iNOS in a time-dependent manner (3h, 6h, 12h, 24h). Figure 5 D and Figure 5The expression of the M2 marker Arg1 was not significantly different, indicating that outer membrane vesicles can activate macrophage M1 polarization. Treatment of outer membrane vesicles with lipopolysaccharide (LPS) significantly upregulated STING and IRF3 expression, with effects comparable to cyclic diadenosine monophosphate (cDDP) treatment with LPS, while whole Bacteroides fragilis culture with LPS did not have this effect, confirming that outer membrane vesicles are a key component of Bacteroides fragilis-mediated immune training. ELISA confirmed the detection of c-di-AMP in Bacteroides fragilis cultures.

[0108] Example 5: Bacteroides fragilis induces and trains immunity against Salmonella typhimurium infection (chicken model)

[0109] (1) Experimental design: Animal grouping: Seven-day-old Arbor Acqua broiler chickens (Nanjing Tegeli Planting Professional Cooperative) were randomly divided into four groups of six each: control group, Bacteroides fragilis treatment group, Salmonella typhimurium infection group, and Bacteroides fragilis pretreatment + infection group. Immunization training: Chickens in each strain treatment group and pretreatment + infection group were administered 400 μL of 1×10⁻⁶ protein solution via gavage each time. 8 Bf-10 bacterial suspension at CFU / mL was administered via gavage three times within one week; the control and infected groups were administered an equal volume of sterile PBS via gavage. Challenge experiment: After training and immunization induction, chickens in the infected group and the pre-treated + infected group were administered 1 mL of 1×10⁻⁶ CFU / mL Bf-10 bacterial suspension via gavage. 8 CFU / mL Salmonella Typhimurium bacterial suspension; control group and Bacteroides fragilis treatment group were administered an equal volume of sterile PBS by gavage. Chicken body weight was monitored for 7 consecutive days after challenge. Monitoring indicators: body weight change, fecal / liver bacterial load (CFU plate count), colon length, colonic H&E pathological score, and tight junction protein expression.

[0110] Test results as follows Figure 6 As shown, the weight monitoring results indicate ( Figure 6 In group B), *Salmonella typhimurium* infection caused a significant decrease in body weight in chickens in the simple infection group, with a weight reduction of 15.2 ± 1.8% on day 5 post-infection compared to the control group; while chickens in the *Bacteroides fragilis* pretreatment group only showed a slight weight loss (3.8 ± 0.9%), significantly lower than the simple infection group, and no significant difference compared to the control group or the *Bacteroides fragilis*-only treatment group. Quantitative detection results of *Salmonella typhimurium* colony-forming units (…). Figure 6 The study (H) confirmed that pretreatment with *Bacteroides fragilis* significantly inhibited its colonization and spread in chicken feces and liver. Furthermore, quantitative real-time fluorescence assays were used to detect the 16S rRNA expression levels of *Salmonella typhimurium* in the cecum and liver tissues. Figure 6 The bacterial load of *Salmonella typhimurium* in the cecum and liver tissues was significantly increased, while pretreatment with *Bacteroides fragilis* significantly reduced the bacterial load of *Salmonella typhimurium* in the cecum and liver. The H&E staining results of chicken ileum tissue are shown below. Figure 6As shown in C, the intestinal damage in the group infected with Salmonella typhimurium alone was severe, manifested as shortened villi, destroyed crypt structures and extensive infiltration of inflammatory cells; while the intestinal structure of the chickens in the Bacteroides fragilis pretreated infection group was intact, and the villi height and pathological score were similar to those of the control group and significantly better than those of the simple infection group. Figure 6 The results showed that the length of intestinal villi in chicks infected with typhus was significantly shortened, and Bacteroides fragilis could reverse the villi shortening.

[0111] Western blot analysis showed that Salmonella typhimurium infection significantly downregulated the expression levels of tight junction proteins Claudin and Occludin. Figure 6 The expression levels of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α were significantly increased in the ileum and liver tissues of chickens infected with Salmonella typhimurium alone. Pretreatment with Bacteroides fragilis reversed this downregulation trend. Figure 6 (I) and pretreatment with Bacteroides fragilis significantly alleviated this inflammatory response. Consistent with mouse experiments, pretreatment with Bacteroides fragilis also restored STING signaling activated by Salmonella typhimurium to normal levels by regulating the STING signaling pathway in chickens. Figure 6 (G in the text). This confirms that the protective effect of Bacteroides fragilis is also effective in chickens, demonstrating that this mechanism is highly conserved between mammals and birds.

[0112] Example 6: Broad-spectrum protection against H9N2 avian influenza virus by Bacteroides fragilis-induced trained immunity.

[0113] (1) Experimental design: Animal grouping: Male C57BL / 6 mice aged 6-8 weeks were randomly divided into 4 groups of 6 mice each: control group, Bacteroides fragilis treatment group, H9N2 infection group, and Bacteroides fragilis pretreatment + infection group. Immunization training: In the treatment group and the pretreatment + infection group, 400 μL of 1×10⁻⁶ bacteria were administered by gavage each time. 8 Bf-10 bacterial suspension at CFU / mL was administered via gavage three times within one week; the control and infected groups were administered an equal volume of sterile PBS via gavage. Challenge experiment: After training and immunization induction, mice in the infected group and the pre-treated + infected group were infected with H9N2 avian influenza virus (10 CFU / mL). 6 PFU); the control group and the Bacteroides fragilis treatment group were given an equal volume of sterile PBS. Samples were taken for testing 5 days after infection. Monitoring indicators: weight change, colon length, lung and colonic H&E pathological scores, and tight junction protein expression.

[0114] (2) Test results are as follows Figure 7 As shown, Figure 7Results B showed that Bacteroides fragilis significantly improved the weight loss in mice after H9N2 virus infection, and the colon length in the H9N2-infected group was significantly shortened. Figure 7 In the H9N2-infected group, mice showed severe lung damage, alveolar wall thickening, alveolar atrophy with inflammatory cell infiltration, colonic epithelial damage, and intestinal gland atrophy; while pretreatment with Bacteroides fragilis improved lung and colonic damage (e.g., Figure 7 In mice with D, E, and F in the *Bacteroides fragilis* pretreatment + infection group, colon length was restored, lung and colon inflammation was reduced, HA protein expression was significantly decreased, and viral replication was inhibited. Tight junction protein detection results are shown in Figure G; pretreatment reversed the downregulation of Occludin and Claudin expression. STING pathway detection results are shown in Figure G. Figure 7 As shown in G, pretreatment can suppress H9N2-induced overactivation of the STING signaling pathway; Figure 7 The H in the data shows that, using quantitative real-time PCR to detect TNF-α validation factor expression, *Bacteroides fragilis* reduced the high expression of inflammatory factors induced by H9N2 infection. ELISA detection confirmed this. Figure 7 In the middle stage, *Bacteroides fragilis* produces cyclic diadenosine monophosphate (cDMA), which participates in mediating the training effect, indicating that *Bacteroides fragilis* also has a protective effect against H9N2 avian influenza virus. This result suggests that training immunity induced by *Bacteroides fragilis* has a dual broad-spectrum protective effect against both bacteria and viruses.

Claims

1. A strain of Bacteroides fragilis, characterized in that, The Bacteroides fragilis species was classified as Bacteroides fragilis and was deposited on April 14, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38257.

2. A composition or compound formulation, characterized in that, The composition comprises any one or more of the following: live Bacteroides fragilis, inactivated Bacteroides fragilis, its culture, its lysate, its extract, its culture supernatant, its outer membrane vesicles, its fermentation product, or its metabolites as described in claim 1; preferably, it also includes a pharmaceutically, food-grade, health food, or cosmetically acceptable combination of excipients.

3. The use of the Bacteroides fragilis of claim 1 or the composition or compound of claim 2 in the preparation of a medicament for the prevention or treatment of Salmonella typhimurium infection and / or H9N2 avian influenza virus infection.

4. The use of Bacteroides fragilis according to claim 1 or the composition or compound formulation according to claim 2 in the preparation of a medicament for inhibiting or killing Salmonella typhimurium and / or H9N2 avian influenza virus.

5. The application according to claim 3, characterized in that, The drug includes oral preparations, vaccines, topical preparations, or inactivated preparations thereof for combined oral and topical use. Preferably, the oral preparation includes gavage or feed additives.

6. The application according to claim 3 or 4, characterized in that, The drug is an agent that inhibits the colonization and spread of pathogens in the host, maintains the integrity of the intestinal barrier, reverses the downregulation of tight junction protein expression, alleviates inflammatory response, and / or inhibits the overactivation of the STING signaling pathway.

7. The application according to claim 3 or 4, characterized in that, The drug is applied to mammals and / or poultry, preferably, the mammals include mice and humans, and the poultry include chickens.

8. An antipathogenic drug, an immune training inducer, a feed additive, or a vaccine, characterized in that, The drug-induced immune inducer, feed additive, or vaccine comprises Bacteroides fragilis as described in claim 1 or the composition or compound formulation as described in claim 2.

9. The antipathogenic drug, immune training inducer, feed additive, or vaccine according to claim 6, characterized in that, The drug or vaccine includes at least one of the outer membrane vesicles of Bacteroides fragilis as described in claim 1 as an active ingredient.

10. The antipathogenic drug or vaccine according to claim 7, characterized in that, The drug or vaccine also contains pharmaceutically acceptable carriers or excipients.

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