A next-generation probiotic strain for gut health: Bacteroides cellulosilyticus LYH2 variant with anti-inflammatory and metabolic advantages

Cong Lan; Xiaofeng Deng; Shen Jin; Hua Li; Yang Liu; Aimin Wu; Yanjiao Liu; Yifan Zhang; Jun He; Zhaolai Dai; Daiwen Chen; Bing Yu; Zhiqing Huang; Xiangbing Mao; Ping Zheng; Jie Yu; Junqiu Luo; Hui Yan; Yuheng Luo

doi:10.1016/j.ebiom.2026.106232

. 2026 Mar 25;126:106232. doi: 10.1016/j.ebiom.2026.106232

A next-generation probiotic strain for gut health: Bacteroides cellulosilyticus LYH2 variant with anti-inflammatory and metabolic advantages

Cong Lan ^a,^c, Xiaofeng Deng ^a,^c, Shen Jin ^a,^c, Hua Li ^a,^c, Yang Liu ^a,^c, Aimin Wu ^a, Yanjiao Liu ^a, Yifan Zhang ^a, Jun He ^a, Zhaolai Dai ^b, Daiwen Chen ^a, Bing Yu ^a, Zhiqing Huang ^a, Xiangbing Mao ^a, Ping Zheng ^a, Jie Yu ^a, Junqiu Luo ^a, Hui Yan ^a, Yuheng Luo ^a,^∗

PMCID: PMC13051721 PMID: 41886971

Summary

Background

The rising global incidence of inflammatory bowel disease (IBD) underscores the pressing demand for effective therapeutic approaches. Among promising next-generation probiotics (NGPs), certain Bacteroides species, notably Bacteroides cellulosilyticus, have attracted increasing interests. However, their mechanisms of action remain incompletely elucidated.

Methods

Six Bacteroides strains were isolated from porcine intestine, among which B. cellulosilyticus LYH2 was selected for detailed functional characterisation. Genomic and metabolic profiling were conducted to evaluate its polysaccharide-degrading capacity, short-chain fatty acid (SCFA) production, and antimicrobial metabolite synthesis. In vitro and in vivo studies assessed its dose-dependent effects on pathogen inhibition, macrophage infection, immune modulation, and metabolic safety. A dextran sulphate sodium (DSS)-induced colitis model (male C57BL/6J mice), along with Ffar3-deficient mice, was employed to investigate anti-inflammatory efficacy and underlying mechanisms.

Findings

B. cellulosilyticus LYH2 demonstrated broad-spectrum antimicrobial activity, effectively suppressed macrophage infection, and reduced pro-inflammatory gene expression in vitro. Long-term oral administration proved safe in mice and improved metabolic parameters. In DSS-induced colitis, B. cellulosilyticus LYH2 outperformed a reference Bacteroides strain, mesalamine, and Lactobacillus reuteri in mitigating inflammation and restoring colonic goblet cell numbers. Mechanistically, B. cellulosilyticus LYH2 enhanced colonic propionate production, modulated gut microbiota composition, and activated Ffar3 signalling, consequently attenuating M1 macrophage polarisation. Genetic ablation of Ffar3 largely abolished these protective effects.

Interpretation

B. cellulosilyticus LYH2 represents a safe and functionally potent NGP that alleviates experimental colitis via propionate–Ffar3 signalling, supporting its therapeutic potential for intestinal inflammatory disorders.

Funding

This research was supported by grants from National Natural Science Foundation of China (32372900) and Natural Science Foundation of Sichuan Province (2023NSFSC0237).

Keywords: Bacteroides cellulosilyticus, Next-generation probiotics, Colitis, Propionate, Ffar3 signalling

Research in context.

Evidence before this study

Inflammatory bowel disease (IBD) has become increasingly prevalent worldwide, with its incidence rising in both developed and developing countries. The growing burden of IBD, which includes conditions such as ulcerative colitis and Crohn's disease, has led to an urgent need for effective therapeutic approaches. Traditional treatments, while beneficial, often come with side effects and limited long-term efficacy, highlighting the necessity for alternative strategies. In recent years, microbial-based therapies, including probiotics, have gained significant attention as a promising approach to modulating gut health and managing IBD. The role of gut microbiota in maintaining intestinal homoeostasis has been well-established, and dysbiosis is now recognised as a key factor in the pathogenesis of IBD. Among the numerous microbial candidates being explored, Bacteroides species, particularly Bacteroides cellulosilyticus, have emerged as promising next-generation probiotics (NGPs) due to their unique ability to degrade complex carbohydrates and produce beneficial metabolites such as short-chain fatty acids (SCFAs). Previous studies have shown that Bacteroides species, including B. cellulosilyticus, exhibit anti-inflammatory properties and can restore gut microbiota balance, offering potential benefits for patients with IBD. Despite these promising findings, the precise mechanisms by which B. cellulosilyticus contributes to gut health and inflammation control remain underexplored, underscoring the need for further investigation. A comprehensive review of the literature, including studies from PubMed, Scopus, and Web of Science, reveals a gap in knowledge regarding the full probiotic potential of B. cellulosilyticus and its impact on gut inflammation in IBD models. This study aims to fill that gap by investigating B. cellulosilyticus LYH2 as a potential probiotic strain for the management of intestinal inflammation.

Added value of this study

This study advances current understanding by providing a systematic functional characterisation of B. cellulosilyticus LYH2, a porcine gut-derived strain, as a versatile NGP. Through integrated genomic, metabolic, and experimental analyses, we demonstrate that B. cellulosilyticus LYH2 possesses broad antimicrobial activity, exerts beneficial metabolic effects, and displays potent anti-inflammatory functions. Moving beyond previous correlative observations linking Bacteroides to gut health, this work offers comprehensive functional validation, including multi-dose safety profiling, metabolic improvements, and direct comparative assessment against a reference strain and a clinically used anti-inflammatory drug. Notably, by employing Ffar3-deficient mice, we establish a clear mechanistic connection between B. cellulosilyticus LYH2-derived metabolites, particularly propionate, and host immune regulation. Collectively, these findings elevate B. cellulosilyticus from a gut commensal with associative health benefits to a mechanistically defined next-generation probiotic with translational relevance for inflammatory bowel disease.

Implications of all the available evidence

The rising global burden of IBD, alongside growing interest in microbiota-targeted therapies, underscores the clinical significance of these results. Accumulating evidence positions B. cellulosilyticus LYH2 as a promising NGP capable of modulating host immunity, reshaping gut microbiota, and alleviating intestinal inflammation. By confirming both safety and functional efficacy in preclinical models, this study supports the potential of B. cellulosilyticus LYH2 as a complementary or alternative intervention for IBD management. Importantly, the delineation of a propionate-Ffar3-dependent pathway offers mechanistic insight that could inform patient stratification and rational combination with existing anti-inflammatory regimens. Further clinical investigations are warranted to evaluate the long-term efficacy, optimal dosing, and translational potential of B. cellulosilyticus LYH2 in human IBD and related inflammatory conditions.

Introduction

Probiotics are live microorganisms that, upon administration in adequate amounts, impart numerous health advantages to the host. This encompasses the first-generation probiotics (FGPs), a group that notably includes Lactobacillus, Bacillus, Bifidobacterium, and yeast species, primarily functioning to uphold overall health status.¹ Conversely, next-generation probiotics (NGPs) represent a distinct class of microorganisms isolated from the gut microbiota of animals, specifically designed to address and treat specific health ailments within the host.² These NGPs, predominantly strict anaerobes, transcend the realm of mere health promotion and are utilised for the targeted prevention and treatment of specific conditions.

The Bacteroides genus, a dominant category of symbiotic bacteria residing in the guts of humans and pigs,³ harbours several promising NGP candidates that have demonstrated efficacy in various studies, notably those related to inflammatory bowel disease (IBD), depression, obesity, and colorectal cancer (CRC). Many Bacteroides species excel at carbohydrate metabolism, playing a pivotal role in nutrient digestion and gut health maintenance.⁴ Among these, Bacteroides cellulosilyticus (B. cellulosilyticus) stands out in the human gut microbiome for its exceptional capacity, boasting the highest number of carbohydrate-active enzymes (CAZymes) compared to its sequenced counterparts within the genus.⁵ These enzymes empower it to degrade intricate molecules such as cellulose, fostering a mutually beneficial relationship with humans. Intriguingly, a recent study observed a decrease in the abundance of B. cellulosilyticus in mice infected with Schistosoma mansoni, hinting at its potential anti-inflammatory function in mitigating parasite-induced gut inflammation.⁶ Furthermore, the administration of specifically designed polysaccharides, like arabinogalactan (AG), has been shown to enhance the prevalence of B. cellulosilyticus, thereby ameliorating DSS-induced colitis in experimental models.⁷ Nevertheless, functional investigations into the role of B. cellulosilyticus within the gut ecosystem remain limited and incomplete, highlighting the need for further research in this promising area.

In this study, a B. cellulosilyticus strain LYH2 with potential probiotic properties was isolated and screened from the gut microbiome of healthy weaned piglets. A comprehensive characterisation of its biological features, including genomic and metabolomic analyses, was first performed to evaluate its functional potential. Subsequently, an in vitro infection model using Salmonella Typhimurium-induced intracellular infection in RAW264.7 macrophages was established to assess the immunomodulatory capacity of B. cellulosilyticus LYH2 and its metabolites. To further investigate its in vivo effects, two independent in vivo experiments were conducted. Mice received oral administration of B. cellulosilyticus LYH2 at different doses to evaluate its long-term safety, potential metabolic benefits, and protective efficacy against DSS-induced colitis. Furthermore, using Ffar3-deficient mice, direct genetic evidence was provided demonstrating that the protective effects of B. cellulosilyticus LYH2 against colitis are mediated through a strain-propionate-Ffar3 signalling axis.

By integrating these in vitro and in vivo approaches, this study aimed to delineate the molecular and immunological mechanisms through which B. cellulosilyticus LYH2 alleviates intestinal inflammation, thereby providing a comprehensive foundation for its development as a promising next-generation probiotic.

Methods

Bacterial isolation, Bacteroides cultivation and preliminary screening with IPEC-J2 cells

Fresh faecal samples were aseptically and anaerobically collected from healthy suckling piglets and processed to prepare faecal suspensions. The bacterial isolation procedure is detailed in Supplementary Information. The preparation of the enrichment medium adhered to the described methodology.⁸ Six Bacteroides species (B. cellulosilyticus, Bacteroides ovatus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus, and Bacteroides fragilis) were inoculated at a 1% (v/v) concentration into anaerobic BHI medium and incubated for 72 h. Following centrifugation at 4000 × g for 5 min, the resultant supernatants were carefully collected and subsequently utilised in further experiments. To assess the impact of these supernatants on IPEC-J2 cell viability, experiments were conducted according to the established protocols of our group.⁹ IPEC-J2 cells (RRID: CVCL_2246) were obtained from DSMZ and cultured in DMEM (Gibco, C11995500BT) supplemented with 10% foetal bovine serum (FBS; Gibco, A5669701), 100 U/mL penicillin and 100 μg/mL streptomycin at 37 °C in a humidified incubator with 5% CO₂. The materials and methods for cell viability screening, bacterial anti-inflammatory activity, cell migration assays, phenotypic identification, antibiotic resistance testing of B. cellulosilyticus LYH2, and anaerobic medium preparation are detailed in the Supplementary Information.

Strains

This investigation also encompassed three prevalent pathogenic strains: Escherichia coli ATCC 25922 (E. coli), Staphylococcus aureus ATCC 25923 (S. aureus), and Salmonella Typhimurium (laboratory‑maintained strain, S.Typhimurium). Notably, these strains were previously curated and maintained by the esteemed Animal Nutrition Institute of Sichuan Agricultural University. B. cellulosilyticus JCM 15632 (BNCC353576) and Lactobacillus reuteri ATCC BAA-2837 were purchased from the BeNa Culture Collection (Beijing, China).

Identification of strains with 16S rDNA sequencing

The purified strains were inoculated into anaerobic BHI medium and incubated at 37 °C for 48 h. Following this, DNA extraction was performed using a boiling method described previously,¹⁰ and the extracted DNA was stored at −20 °C for subsequent use. The PCR amplification, Sanger sequencing, and NCBI database alignment methods are detailed in the Supplementary Information. Strains with >98.65% similarity were confirmed as the same species.¹¹

Whole genome sequencing and pangenome analysis of B. cellulosilyticus LYH2

We isolated a strain closely related to B. cellulosilyticus JCM 15632, designated as B. cellulosilyticus LYH2. The strain was sent to Shanghai Majorbio Bio-Pharm Technology Co., Ltd. for whole-genome sequencing using Illumina HiSeq platforms. Bioinformatic analysis was performed on the Majorbio Cloud Platform (http://cloud.majorbio.com). Detailed procedures for whole genome sequencing and pangenome analysis are provided in the Supplementary Information.

Analysis of metabolites in the culture supernatant of B. cellulosilyticus LYH2

Upon activation, B. cellulosilyticus LYH2 was inoculated into BHI medium at a 10% volumetric concentration, followed by anaerobic incubation at 37 °C for 72 h. The cultures were then subjected to centrifugation, with the resulting supernatant being carefully harvested, culminating in a total collection of six samples: three derived from a blank culture and three from the B. cellulosilyticus LYH2 strain culture. These samples underwent non-targeted metabolomic analysis, facilitated by APTBIO (Shanghai, China). Detailed methods for metabolomics are provided in the Supplementary Information. The intricate procedures for sample processing and analysis adhered strictly to the methodologies outlined in our previous study.¹²

Determining antibacterial efficacy via co-cultivation techniques

After 48 h of incubation, the supernatant of B. cellulosilyticus LYH2 was collected by centrifugation at 8000 × g for 10 min. Its metabolites were extracted using ethyl acetate.¹³ Antibacterial activity against E. coli ATCC 25922, S. aureus ATCC 25923, and S. Typhimurium ATCC 14028 was assessed by co-culturing, with detailed methods provided in the Supplementary Information.

Animal trial

To evaluate the safety and protective effects of B. cellulosilyticus LYH2, a total of five independent animal experiments were conducted using male C57BL/6J mice (RRID: IMSR_JAX:000664): Two independent experiments were performed to assess the safety of B. cellulosilyticus LYH2. In the first experiment, thirty-six 4-week-old male C57BL/6J mice were individually housed and randomised into four groups (n = 9 per group): control (CON, PBS), low-dose (B. cellulosilyticus LYH2 at 3 × 10⁸ CFU/200 μL), medium-dose (3 × 10⁹ CFU/200 μL), and high-dose (3 × 10¹⁰ CFU/200 μL). Bacterial suspension or PBS was administered by oral gavage every other day for 64 days. Detailed experimental timelines and animal welfare considerations are provided in the Supplementary Information. In the second experiment, twenty 3-week-old male C57BL/6J mice were individually housed and randomised into two groups (n = 10 per group): CON (PBS, 200 μL) and Ce (B. cellulosilyticus LYH2 at 3 × 10⁹ CFU/200 μL). Treatments were administered on alternate days throughout the 64-day experimental period. At the end of both safety experiments, mice were anaesthetised prior to sample collection.

Three independent experiments were conducted to evaluate the protective effects of B. cellulosilyticus LYH2 in DSS-induced colitis models. In the first experiment, forty-two 6-week-old male C57BL/6J mice were individually housed and randomised into to six groups (n = 7 per group): CON, DSS (3% DSS), Ce (3% DSS and B. cellulosilyticus LYH2 at 10⁹ CFU/200 μL), JCM (3% DSS and B. cellulosilyticus JCM 15632 at 10⁹ CFU/200 μL), 5-ASA (3% DSS and 5-aminosalicylic acid at 100 mg/kg),¹⁴ and LR (3% DSS and L. reuteri ATCC BAA-2837 at 10⁹ CFU/200 μL). All bacterial strains and 5-ASA (Cat# HY-15027, MedChemExpress, USA) were administered by oral gavage on alternate days for 21 days, while control mice received PBS. Colitis was subsequently induced by providing 3% DSS in drinking water for 8 days. Bacterial administration was discontinued during this period, whereas 5-ASA treatment was maintained. In the second experiment, sixty 6-week-old male C57BL/6J mice were individually housed and randomised into four groups (n = 10 per group): CON, DSS (3% DSS), Ce (3% DSS and B. cellulosilyticus LYH2 at 10⁹ CFU/200 μL), and Ce-M (3% DSS and B. cellulosilyticus LYH2-derived metabolites). Treatments were administered on alternate days for 21 days prior to DSS challenge, and discontinued during the 7-day DSS exposure. In the third experiment, forty 10-week-old male mice, including twenty wild-type (WT) and twenty Ffar3-deficient (Ffar3^−/−) C57BL/6J mice, were individually housed and randomised into eight groups (n = 5 per group): WT, WT-DSS, WT-Ce (B. cellulosilyticus LYH2 at 10⁹ CFU/200 μL), WT-NaP (150 mM sodium propionate), Ffar3^−/−, Ffar3^−/−-DSS, Ffar3^−/−-Ce, and Ffar3^−/−-NaP. Mice underwent a 21-day pretreatment with B. cellulosilyticus LYH2 (alternate-day gavage) or sodium propionate (continuous in drinking water), followed by colitis induction with 3% DSS for 9 days. Bacterial administration was discontinued during DSS challenge, while propionate treatment was maintained. Disease Activity Index (DAI) scores are summarised in Table 1. At the end of all experiments, mice were anaesthetised prior to sample collection.

Table 1.

Evaluation of disease activity index (DAI^a).

Decrease of body weight (%)	Stool characteristics	Stool occult blood	Score
0	Normal stools	Normal stools	0
1–5	Loose stools	Positive for occult blood	1
5–10	Loose stools	Positive for occult blood	2
10–15	Watery stools	Stools with visible blood	3
>15	Watery stools	Stools with visible blood	4

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DAI = (weight loss rate + fecal characteristics + bleeding situation)/3.

All mice were housed at the Institute of Animal Nutrition, Sichuan Agricultural University, under controlled conditions (temperature 22 ± 2 °C, 12 h light/dark cycle, 50–60% humidity) with free access to water and standard chow. Environmental enrichment (nesting material) was provided.

Measurement of organ, muscle, and fat indices

After the removal of any adhering fat, the weights of the heart, thymus, lungs, spleen, liver, and kidneys were measured. The calculation formulae for organ and fat indices are provided in the Supplementary Information.

Bacterial translocation detection

DNA was extracted from 0.2 g of liver and spleen samples using the CTAB method. Real-time quantitative PCR was performed with SYBR Green and specific primers targeting total bacterial and Bacteroides spp. 16S rRNA genes (see Table S1). The qPCR protocol followed the procedures outlined in our previous studies.¹⁵^,¹⁶

Analysis of haematological parameters, serum biochemical parameters and flow cytometry-based assessment of T lymphocyte subpopulations

A 50 μL aliquot of anticoagulated whole blood was analysed using a haematology analyser (Mindray MC-80, China). Frozen serum samples were gently thawed at 4 °C and subsequently analysed using an automated biochemical analyser (Hitachi 7020, Japan). Serum biochemical markers are detailed in the Supplementary Information. For flow cytometry, 100 μL of blood was stained with antibodies against CD3⁺, CD3⁺CD4⁺, and CD3⁺CD8⁺ to differentiate T lymphocytes, helper T cells, and cytotoxic T cells, following the method described previously by our group.¹⁷

Histological staining, mucus layer staining, and immunofluorescence analysis

Intestinal tissues were fixed in paraformaldehyde and Carnoy's fixative (60% methanol, 30% chloroform, 10% acetic acid). Paraffin sections were prepared for H&E staining, Alcian Blue (AB) staining, and immunofluorescence to analyse intestinal morphology, histopathological scoring, mucus layer thickness, and the expression and quantification of CD86, ZO-1, Occludin, and Claudin-1. Detailed methods are provided in the Supplementary Information.

Measurement of SCFA concentration

The analysis was performed on colonic digesta samples obtained from mice and on the supernatant of anaerobic cultures of B. cellulosilyticus LYH2 grown in BHI broth for 72 h. The specific methodology employed in this study was adapted from a previously published work by our research group.¹⁶

16s rRNA amplicon sequencing

The sequencing procedure encompassed six replicate samples. Following the manufacturer's instructions, genomic DNA was extracted from each frozen stool sample using the E.Z.N.A Stool DNA Kit (Omega Bio-Tek, Georgia, USA). High-throughput sequencing was then performed by Novogene Co., Ltd. (Beijing, China). Specific parameters are provided in the Supplementary Information.

RNA extraction and real-time PCR analysis

RNA from cells and tissues was extracted using the Trizol method, followed by reverse transcription and qPCR analysis. The primer sequences and other details for this process are outlined in Table S1. As internal controls, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin were employed to normalise the data. The relative mRNA expression levels of the target genes were quantified using the 2^−ΔΔCt method.¹⁸ The real-time PCR quantification and all reaction procedures were conducted according to the protocols established by our research group.⁹

Protein extraction and Western blotting

Proteins were extracted from mouse colon tissues using RIPA lysis buffer (Cat# G2002, Servicebio, Wuhan, China). The lysates were denatured at 37 °C for 30 min and then subjected to Western blot analysis according to standard protocols. The following primary antibodies were used: polyclonal anti-GPR41 (Cat# PA5-75521, RRID: AB_2719249, Thermo Fisher Scientific) and recombinant anti-β-actin (mouse monoclonal; Cat# GB15001, RRID: AB_3083704, Servicebio, Wuhan, China). Western blotting was performed in three independent biological replicates. Band intensities were quantified by densitometric analysis.

Statistical analysis

Data analysis and visualisation were conducted using Microsoft Excel 2019 and GraphPad Prism 9.4.1. Normality of data distribution was assessed using the Shapiro–Wilk test. For comparisons between two groups, an unpaired two-tailed Student's t-test was applied when the data met the normality assumption; otherwise, the non-parametric Mann–Whitney U test was used. For experiments involving multiple groups, one-way analysis of variance (ANOVA) followed by Tukey's honestly significant difference (HSD) post hoc test, or Dunnett's test using the DSS group as a reference control, was performed for normally distributed data, whereas the Kruskal–Wallis test followed by Dunn's multiple comparisons test was used for non-normally distributed data. To evaluate the effects of treatment, genotype, and their interaction in studies involving Ffar3 knockout mice, two-way ANOVA was employed, with Dunnett's test for post hoc multiple comparisons using the WT-DSS and Ffar3^−/− DSS groups as reference controls. Additionally, ordinary linear models (value ∼ genotype) were fitted separately for each treatment group using R to assess genotype effects, and the resulting P values were adjusted for multiple comparisons using the Bonferroni correction. For high-dimensional untargeted analyses (e.g., metabolomics), the false discovery rate (FDR) correction was applied to account for multiple testing. Sample sizes were determined based on prior studies using similar DSS-induced colitis models and outcome measures, which have demonstrated adequate statistical power to detect biologically relevant effects. Data are presented as mean ± standard error of the mean (SEM) unless otherwise indicated in the figure legends. Statistical significance was set at P<0.05.

Ethics

All animal procedures used in this study were approved by the Animal Care and Use Committee of Sichuan Agricultural University (licence number: CD-SYXK-2017-015).

Role of funders

The funding bodies had no involvement in study design, data collection, statistical analysis, data interpretation, or manuscript preparation.

Results

Isolation, identification, and probiotic potential assessment of Bacteroides strains

From healthy piglets, six Bacteroides strains: B. cellulosilyticus, B. ovatus, B. thetaiotaomicron, B. uniformis, B. vulgatus, and B. fragilis were isolated (Fig. 1A). The supernatants of these strains were co-cultured with IPEC-J2 cells for preliminary screening based on cell viability. At dilutions of 1:20 and 1:10, B. cellulosilyticus, B. ovatus, B. thetaiotaomicron, and B. uniformis significantly enhanced cell viability (P < 0.05, one-way ANOVA with Tukey's post-hoc test, Fig. 1B), with B. cellulosilyticus, B. ovatus, and B. uniformis demonstrating the most pronounced effects, and were therefore selected for further analysis. Based on these results, dilutions of 1:20 and 1:10 were chosen for subsequent experiments. We further evaluated IL8 expression in IPEC-J2 cells after incubation with supernatants from the selected strains and ETEC challenge. ETEC significantly upregulated IL8 mRNA expression (P < 0.05, one-way ANOVA with Tukey's post-hoc test, Fig. 1C), while B. cellulosilyticus at a 1:20 and 1:10 dilution substantially downregulated IL8 expression (P < 0.05, one-way ANOVA with Tukey's post-hoc test, Fig. 1C). Cell migration assays (Fig. 1D and E) revealed that B. cellulosilyticus at a 1:20 dilution had no significant effect on cell migration rates (P > 0.05, one-way ANOVA with Tukey’s post-hoc test, Fig. 1F), whereas a 1:10 dilution significantly enhanced migration rates compared to the other groups (P < 0.05, one-way ANOVA with Tukey’s post-hoc test, Fig. 1G).

Fig. 1 — **Comprehensive assessment of *Bacteroides* strains on cell viability, IL8 expression, and wound healing migration assays.** (A) Schematic representation illustrating the isolation and identification processes of *Bacteroides* strains. (B) Evaluation of IPEC-J2 cells viability following treatment with varying concentrations (1:20, 1:10, and 1:5) of bacterial culture supernatants diluted with cell culture medium (n = 3). Data are presented as mean ± SEM, with significant differences denoted by asterisks (∗*P <* 0.05; ∗∗*P <* 0.01). (C) Comparative analysis of relative mRNA expression levels of the pro-inflammatory cytokine IL8 in IPEC-J2 cells. Cells were preconditioned with distinct bacterial culture supernatants for 24 h prior to a 2-h challenge with ETEC. Distinct alphabetical letters signify statistically significant differences between treatment groups (*P <* 0.05). (D) Wound healing migration assays of IPEC-J2 cells exposed to bacterial supernatants at a 1:20 dilution over 24 h. Representative images are depicted for the control (CON), BHI medium, and supernatants from *B. ovatus*, *B. cellulosilyticus*, and *B. uniformis* at 4× migration. Scale bar = 100 μm. (E) Quantitative analysis of migration area percentages of IPEC-J2 cells exposed to bacterial supernatants at a 1:20 dilution over 24 h (n = 3). Boxes show IQR with median (line); whiskers, min–max. n = 3. (F) Wound healing migration assays of IPEC-J2 cells treated with bacterial supernatants at a 1:10 dilution over 24 h. Representative images are depicted for the control (CON), BHI medium, and supernatants from *B. ovatus*, *B. cellulosilyticus*, and *B. uniformis* at 4× migration. Scale bar = 100 μm. (G) Quantitative analysis of migration area percentages of IPEC-J2 cells exposed to bacterial supernatants at a 1:10 dilution over 24 h (n = 3). Boxes show IQR with median (line); whiskers, min–max. n = 3. ∗∗*P <* 0.01; ∗∗∗*P <* 0.001.

Investigation and characterisation of biological features of B. cellulosilyticus LYH2 derived from porcine sources

This B. cellulosilyticus strain was closely related to B. cellulosilyticus JCM 15632 (Similarity 98.99%, NCBI alignment), indicating they are different strains within the same genus (Fig. 2A). Hence, the strain was named B. cellulosilyticus LYH2. B. cellulosilyticus LYH2 is a Gram-negative, non-motile bacterium, negative for catalase and oxidase activities, and does not form spores. Scanning electron microscopy revealed smooth, semi-transparent, circular colonies and short rod-shaped cells (Fig. 2B). Haemolytic assays were negative (Fig. 2C). B. cellulosilyticus LYH2 exhibits specific resistance to several antibiotics (Table 2).

Fig. 2 — **Comprehensive analysis of *B. cellulosilyticus* LYH2 encompassing morphological characterisation, haemolytic activity, whole-genome sequencing, and pangenome assessment.** (A) A phylogenetic tree constructed using 16S rRNA gene sequences from *B. cellulosilyticus* LYH2 and 33 closely related strains sourced from the NCBI database. The tree was built employing the neighbour-joining method in MEGA 7 and rendered using iTol (https://itol.embl.de). (B) High-resolution electron micrograph showcasing *B. cellulosilyticus* LYH2 at a magnification of 20,000×. (C) Growth pattern of *B. cellulosilyticus* LYH2 on blood agar plates. (D) A Circular Genome Map of *B. cellulosilyticus* LYH2 and (E) GO (Gene Ontology, http://www.geneontology.org) Annotation of *B. cellulosilyticus* LYH2. (F) A pangenome curve illustrating the dynamic variation in the number of gene clusters among six strains of *B. cellulosilyticus* analysed with OrthoFinder. (G) Core genome curve corresponding to the same six strains. (H) Phylogenetic analysis rooted in single-copy genes. (I) Average Nucleotide Identity (ANI) analysis of the six strains, visually represented through a colour-coded matrix, where each colour corresponds to the ANI value between pairs of genomes, facilitating quick comparisons of genetic similarity. (J) Pangenome profile of all six *B. cellulosilyticus* strains analysed. (K) Upset plot displaying the unique genes present in each strain as well as the genes shared between any two strains.

Table 2.

Diameter of the inhibitory zone of different antibiotics on B. cellulosilyticus LYH2.^a

Antibiotics	Titre (μg)	B. cellulosilyticus LYH2
Antibiotics	Titre (μg)	Inhibitory Zone Diameter IZD (mm)	Sensitivity
Amoxicillin	10	46	+
Enrofloxacin	5	15	+
Sulfadiazine	300	0	–
Streptomycin	10	0	–
Rifampicin	5	38	+
Polymyxin	300	13	+
Tetracycline	30	16	+
Clindamycin	2	0	–
Ampicillin	20	32	+
Linezolid	30	48	+
Cefepime	30	35	+
Imipenem	10	31	+
Clarithromycin	15	0	–
Vancomycin	30	25	+
Penicillin G	10	0	–
Gentamicin	10	0	–

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The antimicrobial susceptibility assay was performed in triplicate, and representative results are shown, as all independent experiments yielded consistent outcomes. Sensitivity: +indicates susceptible, –indicates resistant.

Whole-genome sequencing of B. cellulosilyticus LYH2 generated its genomic circular map (Fig. 2D), with detailed genomic features provided in the Supplementary Information. Gene ontology (GO, Fig. 2E), KEGG pathway (Fig. S1A), and COG analysis (Fig. S1B) highlighted the significance of B. cellulosilyticus LYH2 in carbohydrate metabolism. The B. cellulosilyticus LYH2 genome harbours 83 antibiotic resistance genes, primarily linked to macrolides, fluoroquinolones, phenicols, tetracyclines, and aminoglycosides (Table S2). Virulence factor analysis using the VFDB database identified 29 potential virulence factors (Table S2). Additionally, it encodes 476 CAZyme genes, including enzymes like α-l-fucosidase and α-l-galactosidase, crucial for glycoside hydrolase activity (Table S2). Pangenome analysis of six B. cellulosilyticus strains, including LYH2, revealed an open pangenome with gene clusters increasing from 5200 to 9800 (Fig. 2F), while the core genome remained stable (5400–5900, Fig. 2G). This indicates that LYH2 contributes unique genes to the species, reflecting high genetic diversity. Phylogenetic and ANI analysis confirmed a high similarity between B. cellulosilyticus LYH2 and BFG-250 (Fig. 2H and I). Pangenome analysis of six B. cellulosilyticus strains revealed 3128 core gene clusters, with B. cellulosilyticus LYH2 contributing 1227 novel clusters and sharing 576 clusters with B. cellulosilyticus BFG-250 (Fig. 2J and K).

Metabolic profiling and antibacterial and anti-inflammatory activity of B. cellulosilyticus LYH2

B. cellulosilyticus LYH2 enhanced the production of acetate (AA) and propionate (PA) compared with the CON group (Fig. 3A, P < 0.05, Student's t-test). Additionally, isovalerate was detected only in the Ce group and was absent in the BHI control, demonstrating that B. cellulosilyticus LYH2 specifically promotes isovalerate production. Non-targeted metabolomics analysis of the supernatant from BHI medium inoculated with B. cellulosilyticus LYH2 revealed an increase in metabolite identification compared to the blank medium, with 354 metabolites upregulated and 520 downregulated in positive ion mode, and 591 upregulated and 813 downregulated in negative ion mode (Fig. 3B and C). A total of 258 unique metabolites were identified, including six antibiotic substances, with 2-hydroxy-6-methylquinoline-3-carbaldehyde and Mollicellin I showing the highest concentrations (Table S3). Principal Component Analysis (PCA) highlighted distinct metabolite profiles between the control and Ce groups (Fig. 3D and E). KEGG pathway enrichment revealed significant pathways, such as protein digestion and absorption, GABAergic synapse, and the citrate cycle (Table S3). Metabolic shifts in the Ce group were visualised through bar charts of pathway difference abundance (Fig. 3F). Metabolites extracted with ethyl acetate were solidified using a nitrogen evaporator (Fig. 3G). In vitro co-culture experiments showed that the supernatant of B. cellulosilyticus LYH2 inhibited the growth of E. coli, S. aureus, and S. Typhimurium (Fig. 3H–J). In the in vitro inflammation model of S. Typhimurium infection in RAW264.7 macrophages, B. cellulosilyticus LYH2 metabolites reduced Salmonella infection and inflammation (Fig. S2, detailed results in Supplementary Information).

Prolonged administration of porcine-derived B. cellulosilyticus LYH2 is well-tolerated and improves host metabolic parameters

Following 64 days of B. cellulosilyticus LYH2 administration, mice in the low- and medium-dose groups displayed a significant increase in body weight by day 64 compared to controls (Fig. 4A and B, P < 0.05, one-way ANOVA with Tukey's post-hoc test). Serum biochemical analyses showed that aspartate aminotransferase (AST) levels were lower in the low- and medium-doses groups (Fig. 4C, P < 0.05, one-way ANOVA with Tukey's post-hoc test). In contrast, serum albumin (Alb) and non-esterified fatty acids (NEFA) levels remained comparable across all groups (Fig. 4D and E, P > 0.05, one-way ANOVA with Tukey's post-hoc test). Notably, serum complement component 3 (C3) was elevated specifically in the high-dose group relative to others (Fig. 4F, P < 0.05, one-way ANOVA with Tukey's post-hoc test).

Small intestinal length was greater in B. cellulosilyticus LYH2-treated mice than in controls (Fig. 4G, P < 0.05, one-way ANOVA with Tukey's post-hoc test), whereas colon length did not differ among groups (Fig. 4H, P > 0.05, one-way ANOVA with Tukey's post-hoc test). Organ indices showed no variation (Fig. 4I, P > 0.05, one-way ANOVA with Tukey's post-hoc test). However, the brown adipose tissue (BAT) index was significantly higher in the medium-dose group (Fig. 4J, P < 0.05, one-way ANOVA with Tukey's post-hoc test). Histological examination revealed no marked differences in intestinal morphology or goblet cell counts. Consistently, the villus height-to-crypt depth ratios in the small intestine and goblet cell numbers in the colon were similar across groups (Fig. 4K–N and Fig. S3A–D, P > 0.05, one-way ANOVA with Tukey's post-hoc test). Moreover, expression of tight junction proteins and inflammation-related genes in the colonic mucosa remained unchanged (Fig. 4O and P, P > 0.05, one-way ANOVA with Tukey's post-hoc test).

To further assess the long-term safety and experimental consistency of B. cellulosilyticus LYH2, a follow-up extended feeding study was performed using the medium dose. Although body weight showed an upward trend in the Ce group (Fig. S4A, P = 0.055, Student's t-test), growth performance parameters did not differ from controls (Fig. S4B–D, P > 0.05, Student's t-test). The Ce group exhibited a significantly increased cardiac index (Fig. S4E, P < 0.05, Student's t-test). No significant differences were found in gastrocnemius muscle weight or indices (Fig. S4F–G, P > 0.05, Student's t-test), but BAT weight and index were higher in the Ce group, with no changes in subcutaneous white adipose tissue (sWAT, Fig. S4H–I, P < 0.05, Student's t-test). Intestinal lengths were unaffected (Fig. S4J, P > 0.05, Student's t-test), and histological analysis showed no liver damage (Fig. S4K–L).

Serum analyses (Table 3) revealed lower total protein (TP), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) in the Ce group (P < 0.05, Student's t-test). The Ce group also showed a significant decrease in white blood cell (WBC) count, lymphocyte count (Lym#), and eosinophil percentage (Eos%) (Table 4, P < 0.05, Student's t-test). Long-term gavage of B. cellulosilyticus LYH2 did not significantly impact the total bacterial copy number or Bacteroides abundance in the liver (Fig. S4M−N, P > 0.05, Student's t-test) or spleen (Fig. S4O, P > 0.05). In the hindgut, Tgfb1 expression tended to be downregulated in Ce group colonic tissue (Fig. S4P, P = 0.089, Student's t-test), with significant downregulation of Cldn2 (Fig. S4Q, P < 0.05, Student's t-test).

Table 3.

Effects of long-term oral administration of B. cellulosilyticus LYH2 on serum biochemical parameters in mice.^a

Item	CON	Ce	P-value
ALT, U/L	21.23 ± 5.67	22.42 ± 9.73	0.741
AST, U/L	49.86 ± 19.61	58.99 ± 19.75	0.314
TP, g/L	47.75 ± 2.61	45.15 ± 1.7	0.017
Alb, g/L	17.35 ± 1.78	16.19 ± 0.93	0.084
TC, mmol/L	2.61 ± 0.29	2.33 ± 0.29	0.049
LDL-C, mmol/L	0.5 ± 0.07	0.42 ± 0.04	0.005
HDL-C, mmol/L	1.66 ± 0.25	1.55 ± 0.23	0.342
C3, g/L	0.17 ± 0.06	0.15 ± 0.02	0.370
NEFA, mmol/L	2506.64 ± 365.98	2376.73 ± 347.95	0.427
TG, mmol/L	1.51 ± 0.22	1.32 ± 0.25	0.084

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Data are presented as mean ± SEM. n = 10. Statistical significance was determined using an unpaired two-tailed Student's t-test.

Table 4.

Effects of long-term oral administration of B. cellulosilyticus LYH2 on complete blood count in mice.^a

Item	CON	Ce	P-value
WBC, 10⁹/L	3.43 ± 0.68	2.7 ± 0.69	0.028
Neu#, 10⁹/L	1.06 ± 0.40	0.73 ± 0.35	0.065
Lym#, 10⁹/L	1.81 ± 0.51	1.26 ± 0.36	0.013
Mon#, 10⁹/L	0.12 ± 0.08	0.13 ± 0.08	0.665
Eos#, 10⁹/L	0.45 ± 0.35	0.58 ± 0.095	0.267
Neu%, %	31.05 ± 10.05	25.86 ± 5.34	0.166
Lym%, %	52.02 ± 7.61	46.66 ± 5.62	0.090
Mon%, %	3.56 ± 2.01	4.9 ± 1.94	0.147
Eos%, %	13.33 ± 10.32	22.55 ± 6.14	0.026
RBC, %	9.04 ± 0.39	8.66 ± 0.48	0.069
HGB, %	143.9 ± 5.95	137.1 ± 8.6	0.055
HCT, 10¹²/L	39.59 ± 1.75	37.62 ± 2.49	0.056
MCV, g/L	43.82 ± 1.00	43.42 ± 0.50	0.272
MCH, %	15.94 ± 0.32	15.82 ± 0.15	0.296
MCHC, fL	363.8 ± 8.34	364.3 ± 2.21	0.857
RDWCV, pg	13.84 ± 0.60	14.06 ± 0.47	0.376
RDWSD, g/L	27.63 ± 1.05	27.92 ± 1.29	0.588
PLT, %	328.5 ± 162.65	275.4 ± 121.91	0.42
MPV, fL	5.58 ± 0.31	5.58 ± 0.32	1.000
PDW, 10⁹/L	16.39 ± 0.34	16.45 ± 0.38	0.714
PCT, fL	0.18 ± 0.08	0.15 ± 0.07	0.390

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Data are presented as mean ± SEM. n = 10. Statistical significance was determined using an unpaired two-tailed Student's t-test.

Porcine-derived B. cellulosilyticus LYH2 attenuates DSS-induced colitis with superior efficacy to reference strains and 5-ASA

Administration of DSS successfully induced experimental colitis, resulting in a significant reduction in body weight relative to controls (Fig. 5A and B, P < 0.05, one-way ANOVA with Dunnett's post-hoc test). At the study endpoint, mice treated with either B. cellulosilyticus LYH2 (Ce group) or JCM 15632 (JCM group) exhibited higher body weights than the DSS group (Fig. 5B, P < 0.05, one-way ANOVA with Dunnett's post-hoc test). While DAI scores were elevated in DSS-treated mice (Fig. 5C, P < 0.05, one-way ANOVA with Dunnett's post-hoc test), no significant differences were observed between the DSS group and any treatment group. Colon length was shorter in the DSS group compared to both the CON and Ce groups (Fig. 5D and E, P < 0.05, one-way ANOVA with Dunnett's post-hoc test). Serum cytokine analysis by ELISA showed that DSS challenge significantly increased levels of MCP-1, IL-1β, and TNF-α, while reducing IL-10 (Fig. 5F–I, P < 0.05, one-way ANOVA with Dunnett's post-hoc test). Ce treatment effectively reversed these changes. The other treatments modulated cytokine levels to varying degrees. Histopathological scoring of colon tissue confirmed severe damage in DSS mice, which was significantly ameliorated by all treatments (Fig. 5J and K, P < 0.05, one-way ANOVA with Dunnett's post-hoc test). AB-PAS staining revealed a pronounced loss of goblet cells in the DSS group, an effect that was partially restored in both the Ce and 5-ASA groups (Fig. 5L and M, P < 0.05, one-way ANOVA with Dunnett's post-hoc test). Consistent with these findings, colonic mucosal mRNA expression of pro-inflammatory genes (Tnf, Il1b, Mcp1, and Cxcl10) was upregulated by DSS (Fig. 5N, P < 0.05, Kruskal–Wallis test with Dunn's post-hoc test). All treatments significantly downregulated Tnf expression (Fig. 5N, P < 0.05, Kruskal–Wallis test with Dunn's post-hoc test).

Fig. 5 — **Effects of B. *cellulosilyticus* LYH2 and comparator treatments on disease severity, inflammation, and goblet cell number in a DSS-induced colitis mouse model.** Six-week-old male C57BL/6J mice were randomly assigned to six groups (n = 7): CON (normal drinking water), DSS (3% DSS in drinking water), Ce (3% DSS and B. *cellulosilyticus* LYH2 at 1 × 10⁹ CFU/mL), JCM (3% DSS and B. *cellulosilyticus* JCM 15632 at 1 × 10⁹ CFU/mL), 5-ASA (3% DSS and 5-aminosalicylic acid at 100 mg/kg), and LR (3% DSS and *Lactobacillus reuteri* ATCC BAA-2837 at 1 × 10⁹ CFU/mL). Bacterial strains and 5-ASA were given by oral gavage every other day for 21 days before DSS exposure. Colitis was then induced with 3% DSS for 8 days, during which bacterial treatments were paused while 5-ASA continued. (A) Body weight ratio during the experiment. (B) Final body weight. (C) DAI scores during DSS treatment. (D) Representative images of colons from each group. (E) Colon length. (F) Serum monocyte chemoattractant protein-1 (MCP-1) levels. (G) Serum interleukin-1β (IL-1β) levels. (H) Serum tumour necrosis factor-α (TNF-α) levels. (I) Serum interleukin-10 (IL-10) levels. (J) Representative H&E-stained colon sections. Scale bars = 200 μm, 10× objective. (K) Histological scores of colonic tissues. (L) Representative AB-PAS-stained colon sections. Scale bars = 200 μm, 10× objective. (M) Goblet cell number per crypt. (N) Relative mRNA expression of inflammatory and chemokine-related genes (*Tnf*, *Il1b*, *Mcp1*, and *Cxcl10*) in colon. Data are mean ± SEM (n = 7). Statistical significance was evaluated using either one-way ANOVA followed by Dunnett's post-hoc test (when normality and homogeneity of variance were met) or the Kruskal–Wallis test followed by Dunn's post-hoc test with Bonferroni correction (when these assumptions were violated), for comparisons of each group versus the DSS group. ∗*P <* 0.05, ∗∗*P <* 0.01, ∗∗∗*P <* 0.001.

B. cellulosilyticus LYH2 enhances intestinal integrity and barrier function, alleviating DSS-induced colitis in mice

In a DSS-induced colitis mouse model, DSS treatment led to significant body weight loss and higher DAI scores compared to controls (Fig. 6A and B, P < 0.05, one-way ANOVA with Tukey's post-hoc test). It also shortened colon length (Fig. 6C and D, P < 0.05, one-way ANOVA with Tukey's post-hoc test), which was restored by live B. cellulosilyticus LYH2 (Fig. 6D, P < 0.05, one-way ANOVA with Tukey's post-hoc test). Histological analysis showed intact mucosa and thicker mucus in controls (Fig. 6E and F), whereas DSS caused mucosal ulceration, epithelium loss, goblet cell depletion, and inflammation. The DSS group had higher pathological scores (Fig. 6G, P < 0.05, one-way ANOVA with Tukey's post-hoc test), which were alleviated by live B. cellulosilyticus LYH2 and its metabolites (Fig. 6G, P < 0.05, one-way ANOVA with Tukey's post-hoc test). Only live B. cellulosilyticus LYH2 restored colonic mucus thickness (Fig. 6H, P < 0.05, one-way ANOVA with Tukey's post-hoc test).

The spleen index was elevated in all DSS-treated groups (Fig. 6I, P < 0.05, one-way ANOVA with Tukey's post-hoc test). Muc2 expression was lower in the DSS group (Fig. 6J, P < 0.05, one-way ANOVA with Tukey's post-hoc test), while the Ce group showed a trend towards higher expression (Fig. 6J, P = 0.065, one-way ANOVA with Tukey's post-hoc test). No significant changes were found in Ocln and Tjp1 (Fig. 6K and L, P > 0.05, one-way ANOVA with Tukey's post-hoc test). Cldn1 expression was lower in both DSS and Ce-M groups (Fig. 6M, P < 0.05, one-way ANOVA with Tukey's post-hoc test), with the Ce group showing a trend towards higher levels than the DSS group (Fig. 6M, P = 0.062). The live bacteria group had significantly higher Cldn1 expression than the metabolite-treated group (Fig. 6M, P < 0.05, one-way ANOVA with Tukey's post-hoc test). Immunofluorescence confirmed reduced ZO-1, Occludin, and Claudin-1 in the DSS group (Fig. 6N–Q, Fig. S5, P < 0.05, one-way ANOVA with Tukey's post-hoc test), with the Ce group showing higher levels of ZO-1 and Claudin-1 (Fig. 6O and Q, P < 0.05, one-way ANOVA with Tukey's post-hoc test).

In serum analyses, the DSS group had reduced TG (P < 0.05, one-way ANOVA with Tukey's post-hoc test, Table 5) and elevated C3, TBA, and TP (P < 0.05, one-way ANOVA with Tukey's post-hoc test, Table 5). Compared to DSS, the Ce group showed decreased TBA and TP (P < 0.05, one-way ANOVA with Tukey's post-hoc test, Table 5) and increased TG (P < 0.05, one-way ANOVA with Tukey's post-hoc test, Table 5). The Ce-M group also exhibited lower TBA and TP (P < 0.05, one-way ANOVA with Tukey's post-hoc test, Table 5).

Table 5.

Effect of oral administration of B. cellulosilyticus LYH2 on serum biochemical indices in DSS-induced colitis mice.^a

Item	CON	DSS	Ce	Ce-M	P-value
Alb, g/L	16.16 ± 4.82	12.26 ± 0.96	12.25 ± 1.87	12.81 ± 3.56	0.152
C3, g/L	0.13 ± 0.03^a	0.21 ± 0.05^b	0.19 ± 0.05^ab	0.18 ± 0.03^ab	0.010
CRP, mg/L	2.39 ± 0.58	2.32 ± 0.36	2.49 ± 0.3	2.48 ± 0.33	0.781
IgG, g/L	1.17 ± 0.34	1.23 ± 0.50	1.42 ± 0.61	1.25 ± 0.48	0.811
TBA, μmol/L	1.36 ± 0.49^b	27.42 ± 7.95^a	2.59 ± 2.80^b	10.66 ± 14.16^b	0.000
LDH, U/L	680.08 ± 588.51	257.47 ± 149.93	377.97 ± 233.03	354.73 ± 206.09	0.126
TC, mmol/L	2.47 ± 0.35	2.73 ± 0.41	2.63 ± 0.21	2.69 ± 0.29	0.584
TG, mmol/L	1.94 ± 0.39^a	0.67 ± 0.32^c	1.44 ± 0.44^ab	1.12 ± 0.39^bc	0.000
TP, g/L	2.47 ± 0.35^b	2.73 ± 0.41^a	2.63 ± 0.21^b	2.69 ± 0.29^b	0.000

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Data are presented as mean ± SEM. n = 10. Statistical significance was determined using one-way ANOVA with Tukey's post-hoc test. Different superscript letters in the same column indicate significant differences between groups (P < 0.05).

Impact of live B. cellulosilyticus LYH2 and its metabolites on colon immune function and gut microbiota composition in mice with DSS-induced colitis

Flow cytometric analysis of T lymphocyte subsets revealed significant differences in the proportions of CD3⁺, CD3⁺CD4⁺, and CD3⁺CD8⁺ T cells in DSS-treated mice compared to controls (Fig. 7A and B, P < 0.05, one-way ANOVA with Dunnett's post-hoc test). The Ce group showed reduced proportions of these subsets (Fig. 7B, P < 0.05, one-way ANOVA with Dunnett's post-hoc test), and the Ce-M group had a notable decrease in CD3⁺CD4⁺ T cells (Fig. 7B, P < 0.05, one-way ANOVA with Dunnett's post-hoc test). SCFA concentrations in colonic contents were higher in the Ce group, with significant increases in PA and isovalerate (IVA) compared to the DSS group (Fig. 7C, P < 0.05, one-way ANOVA with Tukey's post-hoc test). mRNA expression of Ffar3 was elevated in the Ce group (Fig. 7D, P < 0.05, one-way ANOVA with Tukey's post-hoc test), whereas Ffar2 showed no significant differences (Fig. 7E, P > 0.05, one-way ANOVA with Tukey's post-hoc test). Western blot analysis showed that Ffar3 protein expression was increased in the Ce and Ce-M groups compared to the DSS group (Fig. S6A and B, P < 0.05, one-way ANOVA with Tukey's post-hoc test). 16S rRNA sequencing showed no significant differences in α-diversity between the CON and DSS groups (Fig. 7F, P > 0.05, Kruskal–Wallis test with Dunn's post-hoc test), but the Ce group exhibited improved diversity indices (Chao1, Faith's PD, observed features) compared to both CON and DSS (Fig. 7F, P < 0.05, Kruskal–Wallis test with Dunn's post-hoc test). The Ce-M group also significantly increased Faith's PD values (Fig. 7F, P < 0.05, Kruskal–Wallis test with Dunn's post-hoc test). Administration of live B. cellulosilyticus LYH2 and its metabolites increased the relative abundance of B. cellulosilyticus (Fig. 7G, P < 0.05, Kruskal–Wallis test with Dunn's post-hoc test).

PCoA revealed distinct microbial community shifts, with DSS separated from controls and Ce and Ce-M treatments inducing significant changes (Fig. 7H). Genus-level analysis showed a decrease in Bacteroides in the DSS group, reversed by B. cellulosilyticus LYH2 (Fig. 7I and J). Redundancy analysis (RDA) confirmed significant microbial distribution differences, validated by a permutation test (P = 0.001, Fig. 7K). The Ce and Ce-M groups were strongly associated with higher SCFA levels, particularly PA and butyrate, while the DSS group showed weaker associations with SCFAs and beneficial taxa, indicating dysbiosis (Fig. 7K).

Analysis of SCFA-associated genera revealed that Parabacteroides_B_862066 was lower in the Ce and Ce-M groups compared to CON (Fig. 7L, P < 0.05, Kruskal–Wallis test with Dunn's post-hoc test). The abundance of Bacteroides_H was significantly increased in the Ce-M group compared with the DSS group (Fig. 7L, P < 0.05, Kruskal–Wallis test with Dunn's post-hoc test). Additionally, Kineothrix was enriched in both DSS and Ce groups compared to CON (Fig. 7L, P < 0.05, Kruskal–Wallis test with Dunn's post-hoc test).

Live B. cellulosilyticus LYH2 and its metabolites attenuate intestinal inflammation via downregulation of M1 macrophages in the colon of DSS-treated mice

Treatment with DSS significantly upregulated the mRNA expression of inflammatory and immune markers related to M1 macrophages (Cd86, Nos2, Cxcl10, Mcp1, Tnf, Il6, Il1b) in colonic tissue compared to the CON group (P < 0.05, one-way ANOVA with Tukey's post-hoc test, Fig. 8A). Oral administration of live B. cellulosilyticus LYH2 and its metabolites downregulated these markers (P < 0.05, one-way ANOVA with Tukey's post-hoc test, Fig. 8A). For M2 macrophage-associated genes, B. cellulosilyticus LYH2 upregulated Ym1, Mgl2, and Il4 (P < 0.05, one-way ANOVA with Tukey's post-hoc test, Fig. 8B). However, Fizz1 expression was lower in all DSS-treated groups (P < 0.05, one-way ANOVA with Tukey's post-hoc test, Fig. 8B), and Il10 expression was higher in the DSS group (P < 0.05, one-way ANOVA with Tukey's post-hoc test, Fig. 8B). Immunofluorescence staining for CD86 confirmed that M1 macrophages were significantly increased in the DSS group (P < 0.05, one-way ANOVA with Tukey's post-hoc test, Fig. 8C and D). B. cellulosilyticus LYH2 and its metabolites reduced CD86-positive cells.

Fig. 8 — *B. cellulosilyticus* LYH2 and its metabolites primarily downregulate the number of M1 macrophages. (A) mRNA expression levels of genes associated with M1 macrophages (n = 10). (B) mRNA expression levels of genes associated with M2 macrophages. (C) Immunofluorescence staining of CD86, a marker for M1 macrophages, in mouse colon tissue. In the immunofluorescence images, red indicates CD86, and blue represents DAPI-stained nuclei. Scale bars = 50 μm, n = 3. (D) Quantification of CD86-positive area fraction in colon (n = 3). Statistical significance was evaluated by one-way ANOVA with Tukey's post-hoc multiple-comparison tests. Groups sharing identical lowercase letters indicate no statistically significant difference, whereas different letters denote significant differences. ∗*P <* 0.05, ∗∗*P <* 0.01, ∗∗∗*P <* 0.001.

Ffar3 is required for propionate-mediated protection against colitis

To determine whether the protective effects of B. cellulosilyticus LYH2 (Ce) against DSS-induced colitis depend on PA and Ffar3 signalling, we generated Ffar3^−/− mice via sgRNA-induced frameshift mutations (Fig. S7A–D). DSS challenge reduced body weight in both wild-type (WT) and Ffar3^−/− mice (Fig. 9A and B, P < 0.05, two-way ANOVA with Dunnett's post-hoc test). In WT mice, NaP significantly restored body weight, whereas this effect was attenuated in Ffar3^−/− mice (Fig. 9C, P < 0.05, ordinary linear models with Bonferroni correction for genotype effects). DSS elevated DAI scores in both genotypes, and NaP reduced DAI scores in WT but not in Ffar3^−/− mice, with significant treatment × genotype interactions (Fig. S8, P < 0.001, two-way ANOVA with Dunnett's post-hoc test).

Colon shortening induced by DSS was ameliorated by Ce and NaP in WT mice, but not in Ffar3^−/− mice (Fig. 9D and E, P < 0.05, two-way ANOVA with Dunnett's post-hoc test). In WT mice, NaP significantly restored colon length, whereas this effect was attenuated in Ffar3^−/− mice (Fig. 9F, P < 0.05, ordinary linear models with Bonferroni correction for genotype effects). Similarly, DSS-induced leucocytosis was suppressed by Ce and NaP in WT mice, with only modest reduction in Ffar3^−/− mice (Fig. 9G, two-way ANOVA with Dunnett's post-hoc test). Ce increased faecal PA in both genotypes, whereas NaP elevated PA selectively in Ffar3^−/− mice (Fig. 9H, P < 0.05, two-way ANOVA with Dunnett's post-hoc test). Histologically, Ce and NaP markedly attenuated epithelial damage and inflammatory infiltration in WT colon, but these improvements were diminished in Ffar3^−/− mice (Fig. 9I and J, P < 0.05, two-way ANOVA with Dunnett's post-hoc test). AB-PAS staining further showed that goblet cell loss was reversed by Ce and NaP in WT mice, but not in Ffar3^−/− mice (Fig. 9K and L, P < 0.05, two-way ANOVA with Dunnett's post-hoc test).

Ffar3 mediates T cell and macrophage regulation by B. cellulosilyticus LYH2 and propionate

Flow cytometry revealed that DSS disrupted peripheral T cell homoeostasis in WT mice, increasing CD8⁺ T cells and lowering the CD3⁺CD4⁺/CD3⁺CD8⁺ ratio. NaP restored T cell balance in WT mice, but not in Ffar3^−/− mice (Fig. 10A and B, P < 0.05, two-way ANOVA with Dunnett's post-hoc test). Serum levels of MCP-1, TNF-α, and IL-1β were elevated by DSS in both genotypes. Ce and NaP reduced these cytokines in WT mice, but not in Ffar3^−/− mice, with significant genotype × treatment interactions (Fig. 10C, P < 0.05, two-way ANOVA with Dunnett's post-hoc test).

In colonic mucosa, DSS downregulated Ffar3 expression, which was restored by Ce and NaP (Fig. 10D, P < 0.05, two-way ANOVA with Dunnett's post-hoc test). DSS also upregulated M1‑associated genes (Il1b, Nos2, Tnf, and Cd86) in WT mice, as well as Tnf and Cd86 in Ffar3^−/− mice (Fig. 10E, P < 0.05, two-way ANOVA with Dunnett's post-hoc test). Ce and NaP suppressed this induction in WT mice but had no effect in Ffar3^−/− mice. Immunofluorescence confirmed that DSS increased CD86⁺ cells in colon, an effect attenuated by Ce and NaP only in WT mice (Fig. 10F and G, P < 0.05, two-way ANOVA with Dunnett's post-hoc test).

Discussion

Bacteroides, a preeminent bacterial genus abundant within the human gut ecosystem,¹⁹^,²⁰ possesses remarkable metabolic prowess. Bacteroides plays a vital role in preserving gut homoeostasis,⁴^,²¹ significantly influencing immune function, intestinal barrier integrity, and metabolic activities of the host.22, 23, 24 However, it is imperative to acknowledge that under specific conditions, Bacteroides may contribute to gut injury.²⁵ Consequently, while investigating its potential as a probiotic, a rigorous safety evaluation becomes paramount to ensure its safe and beneficial application.

We isolated B. cellulosilyticus LYH2 from the gastrointestinal tracts of healthy pigs and screened it for probiotic traits. Phenotypic testing revealed that B. cellulosilyticus LYH2 lacked haemolytic activity and motility, indicating low toxicity and supporting its potential safe use. Genomic analysis of B. cellulosilyticus LYH2 revealed the presence of certain virulence factors, though not the typical Bacteroides virulence markers, such as B. fragilis enterotoxin gene (bft).²⁶ Probiotics can harbour such factors without adverse health effects.²⁷^,²⁸ B. cellulosilyticus LYH2 also contains antibiotic resistance genes but remains sensitive to certain antibiotics, similar to other probiotics like Lactobacillus, which show multi-drug resistance.²⁹ These resistance and virulence factors may help LYH2 adapt and thrive in the gut environment.³⁰^,³¹ Pan-genome analysis categorised LYH2 as part of an open pan-genome, revealing its phylogenetic ties with other B. cellulosilyticus strains. B. cellulosilyticus LYH2 possesses a rich array of CAZymes, predominantly glycoside hydrolases (GHs) across six major classes, enabling it to efficiently degrade cellulose and hemicellulose, key polysaccharides in plant-based feedstocks.³² Like other Bacteroides species, it contains polysaccharide utilisation loci (PULs) for synthesising CAZymes that break down complex glycans.³³ This metabolic capability drives the production of SCFAs under simple culture conditions.³⁴

Metabolomic profiling of B. cellulosilyticus LYH2 reveals its metabolic flexibility, particularly in pathways like the tricarboxylic acid (TCA) cycle and amino acid metabolism, highlighting its adaptation to different substrates.³⁵ In addition, B. cellulosilyticus LYH2 produces antibiotic-like compounds and these compounds allow B. cellulosilyticus LYH2 to inhibit the growth of pathogens such as E. coli, S. aureus, and S. Typhimurium, supporting its potential as a biological control agent or probiotic with antimicrobial properties. While supernatants from other gut microbes (e.g., E. coli) may also exert antimicrobial effects, our study was limited to comparisons with blank BHI medium. The potential contributions of additional microbial supernatants therefore remain unassessed. Nevertheless, the robust pathogen-inhibitory activity of B. cellulosilyticus LYH2 remains clearly demonstrated. Future studies incorporating a broader panel of microbial supernatants would help to better situate the antimicrobial profile of B. cellulosilyticus LYH2 within the complex gut ecosystem.

To investigate the anti-inflammatory effects of B. cellulosilyticus LYH2 and its metabolites in vitro, we used an S. Typhimurium infection model in macrophages, a common zoonotic pathogen.³⁶ The ability of Salmonella to survive and proliferate in host cells, particularly phagocytes, is key to its virulence.³⁷ In our experiment, S. Typhimurium induced a strong inflammatory response, consistent with previous studies.³⁸ We also observed upregulation of anti-inflammatory cytokines IL-10 and TGF-β, suggesting immune evasion by Salmonella.³⁹^,⁴⁰ Both viable B. cellulosilyticus LYH2 and its metabolites reduced macrophage infection and inflammation, with metabolites showing a more pronounced effect. This supports previous reports that Bacteroides species, particularly those producing propionate, inhibit Salmonella growth by disrupting intracellular pH balance.¹³

A long-term gavage study was conducted to evaluate the In vivo effects of B. cellulosilyticus LYH2 in mice. The results demonstrated that oral administration of 3 × 10⁸, 3 × 10⁹ and 3 × 10¹⁰ CFU of viable cells was non-pathogenic, confirming its safety. These dosages is suitable for commercial use, maintaining bacterial viability and integrity after lyophilisation and reconstitution.⁴¹ Notably, prolonged administration showed a trend toward increased body weight, consistent with previous human studies.⁴²

B. cellulosilyticus LYH2 enhanced BAT weight and index, important for thermogenesis and energy balance, while reducing serum total protein, TC, LDL-C, and TG. BAT activation is linked to increased energy expenditure and reduced obesity risk, whereas elevated TC, LDL-C, and TG are associated with higher cardiovascular disease risk.43, 44, 45, 46 These findings suggest that B. cellulosilyticus LYH2, administered at a dose of 3 × 10⁹ CFU, may improve cardiovascular and metabolic health. However, the role of BAT activation in these effects requires further investigation. Blood analysis post-gavage revealed a decrease in white blood cells and lymphocytes, alongside an increase in eosinophils, which are associated with nutrient metabolism, particularly proteins and lipids.⁴⁷ The rise in cardiopulmonary indices may reflect a compensatory response to slight changes in red blood cell counts. As no inflammatory damage to organs was observed, immune cell changes likely stem from metabolic shifts. Notably, eosinophil counts are inversely related to LDL-C levels,⁴⁸ suggesting additional complexity. Further studies are underway to explore the role of B. cellulosilyticus LYH2 in inflammation.

A reduction in gut commensals, including Bacteroides, is commonly observed during active IBD.49, 50, 51 Supplementation with certain Bacteroides strains has shown promise in alleviating colitis symptoms.⁵²^,⁵³ DSS is widely used to induce experimental colitis, in which disruption of the intestinal mucosal barrier is a central pathogenic event. In strain-comparison experiments, DSS markedly reduced colonic goblet cell numbers. By contrast, intervention with B. cellulosilyticus LYH2 significantly restored body weight, goblet cell numbers, and both systemic and colonic local inflammatory responses in DSS-treated mice, with efficacy comparable to the clinical drug 5-ASA. These results demonstrate that B. cellulosilyticus LYH2 robustly protects the intestinal mucosal barrier.

Although the B. cellulosilyticus JCM reference strain and Lactobacillus partially attenuated DSS-induced intestinal injury, their capacity to restore goblet cell numbers was limited relative to B. cellulosilyticus LYH2. This indicates substantial functional divergence among probiotic strains in maintaining mucus-layer homoeostasis. The strong goblet-cell-protective effect of B. cellulosilyticus LYH2 suggests that this strain may more effectively promote mucus secretion or sustain goblet cell differentiation, thereby enhancing barrier integrity and host defence against inflammatory damage.

Following confirmation of its anti-inflammatory activity and goblet-cell-restorative capacity, we further assessed the impact of B. cellulosilyticus LYH2 on intestinal barrier integrity. In the DSS colitis model, while B. cellulosilyticus LYH2 did not significantly improve body weight loss or DAI scores, it markedly attenuated DSS-induced colon shortening. Moreover, treatment with B. cellulosilyticus LYH2 significantly elevated colonic expression of tight-junction proteins, including ZO-1, occludin, and claudin-1. Notably, a reduction in MUC-2, crucial for gut barrier integrity, is linked to greater colitis susceptibility.⁵⁴^,⁵⁵ In contrast, tight junction proteins help preserve barrier function,⁵⁶ suggesting B. cellulosilyticus LYH2 may support gut barrier integrity and alleviate colitis symptoms. DSS treatment increased lymphocyte counts in mouse blood, consistent with previous reports,⁵⁷ and reduced red blood cell counts, indicating acute anaemia due to intestinal bleeding.⁵⁸ B. cellulosilyticus LYH2 treatment significantly decreased lymphocyte counts while elevating monocyte and eosinophil levels. These findings suggest B. cellulosilyticus LYH2 reduces inflammation and limits immune cell infiltration in colitis. Blood biochemical markers showed systemic inflammation in DSS-treated mice, which was alleviated by B. cellulosilyticus LYH2. Flow cytometry revealed DSS treatment increased T lymphocyte subpopulations, indicating immune activation.⁵⁹ In contrast, B. cellulosilyticus LYH2 modulated T lymphocyte profiles, attenuating the inflammatory response.

B. cellulosilyticus, a strict anaerobe in the hindgut, can ferment complex carbohydrates, as confirmed by genomic analysis. In the murine model of colitis, administration of live B. cellulosilyticus LYH2 markedly elevated SCFA levels in the colon, specifically propionate and isovalerate, and upregulated the expression of the intestinal receptor Ffar3. Acting via SCFA receptors, including Ffar2 and Ffar3, these SCFAs enhance intestinal barrier integrity by promoting MUC2 expression, strengthening tight junction proteins, and modulating protein profiles in the gut, collectively supporting gastrointestinal homoeostasis.60, 61, 62, 63, 64 Our 16S rRNA sequencing further revealed a substantial increase in B. cellulosilyticus LYH2 abundance in the gut post-administration, accompanied by notable shifts in overall microbiota composition. Together, these results indicate that the protective effects of B. cellulosilyticus LYH2 are likely mediated through both partial colonisation and microbiota-driven modulation of intestinal metabolism and immune responses. Meanwhile, 16S rRNA sequencing revealed a strong correlation between microbiota structure and SCFA concentrations. These findings highlight the potential of B. cellulosilyticus LYH2 and its metabolites in modulating the microbiota and promoting SCFA production, supporting intestinal health. Macrophages are essential for intestinal immune homoeostasis, distinguishing harmless antigens from pathogens to maintain immune tolerance. M1 macrophage polarisation plays a key role in the progression of colitis in mice.⁶⁵ In the DSS-induced colitis model, we observed an increase in colonic M1 macrophages and pro-inflammatory M1-related genes,⁶⁶ indicating strong inflammation.

Several limitations warrant consideration. Although the dextran sulphate sodium–induced colitis model is widely used to evaluate anti-inflammatory efficacy, it primarily reflects chemically induced epithelial injury rather than the complex, immune-mediated pathogenesis characteristic of human IBD. Therefore, the translational relevance of B. cellulosilyticus LYH2 should be further validated in complementary models, such as chronic colitis or genetically susceptible models that more closely recapitulate immune dysregulation. Moreover, while our findings support a propionate–Ffar3-dependent mechanism underlying the protective effects of B. cellulosilyticus LYH2, SCFAs signal through multiple receptors and pathways, including Ffar2 and histone deacetylase inhibition, which were not systematically examined in this study. Although Ffar3 deficiency largely abrogated the protective phenotype, residual responses suggest that additional microbial metabolites or host signalling pathways may also contribute. Finally, despite the apparent metabolic safety observed following long-term oral administration in mice, comprehensive toxicological evaluation—including extended histopathological and systemic immune assessments—remains necessary prior to clinical translation. Given that this strain was originally isolated from porcine intestine, its host specificity, colonisation efficiency, and functional stability in humans require careful investigation.

To elucidate the underlying mechanisms, we utilised Ffar3 knockout mice and demonstrated that B. cellulosilyticus LYH2 alleviates DSS-induced colitis by enhancing intestinal propionate production in a Ffar3-dependent manner, thereby co-ordinately reinforcing epithelial barrier integrity and immune homoeostasis. Loss of Ffar3 substantially attenuated the protective effects of both B. cellulosilyticus LYH2 and exogenous propionate against inflammatory phenotypes, goblet cell depletion, M1 macrophage activation, and T-cell imbalance, establishing a “B. cellulosilyticus LYH2-propionate-Ffar3” axis as a critical functional pathway. These findings not only provide experimental insights into host–microbe interactions mediated by Bacteroides probiotics, but also offer mechanistic support for microbiota-derived metabolite-based interventions targeting Ffar3 in the treatment of inflammatory bowel diseases (Fig. 11).

Fig. 11 — Proposed model summarising the mechanistic insights.

Contributors

Cong Lan, Xiaofeng Deng, Shen Jin, Hua Li, and Yang Liu completed strain isolation, animal experiments, sample testing, and the initial draft of the paper. Aimin Wu, Yanjiao Liu, and Yifan Zhang completed cell culture experiments. Jun He, Zhaolai Dai, Daiwen Chen, and Bing Yu assisted in designing animal experiments and cell experiments, helped revise the initial draft of the paper, and completed some sample testing work. Zhiqing Huang and Xiangbing Mao assisted in strain culture and recovery. Ping Zheng, Jie Yu, and Junqiu Luo assisted in animal experiments and sample collection. Hui Yan helped complete cell experiments and some sample testing. Yuheng Luo designed the technical route for this study, provided financial support for this research, completed bioinformatics analysis and manuscript revision. All authors have read and approved the final version of the manuscript. Cong Lan and Shen Jin have accessed and verified the underlying data.

Data sharing statement

The raw data reported have been deposited in the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2021) in National Genomics Data Center (Nucleic Acids Res 2022), China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (PRJCA033624) that are publicly accessible at https://ngdc.cncb.ac.cn.⁶⁷^,⁶⁸ All the clean genome sequencing data were deposited in the Genome Sequence Archive (GSA) of NGDC under the accession number CRA037838 and CRA037820. The metabolome data of the single-strain culture and faecal metabolome data have been deposited in the Open Archive for Miscellaneous Data (OMIX) at the National Genomics Data Center (NGDC) under accession numbers OMIX008276.

Declaration of interests

The author declares that there are no competing interests.

Acknowledgements

This research was supported by grants from National Natural Science Foundation of China (32372900) and Natural Science Foundation of Sichuan Province (2023NSFSC0237).

Footnotes

^{Appendix A}

Supplementary data related to this article can be found at https://doi.org/10.1016/j.ebiom.2026.106232.

Appendix A. Supplementary data

Supplementary Materials

mmc1.docx^{(22.9MB, docx)}

Supplementary Table S2

mmc2.xlsx^{(110.4KB, xlsx)}

Supplementary Table S3

mmc3.xlsx^{(5.5MB, xlsx)}

WB blots

mmc4.docx^{(268KB, docx)}

RAW264.7 cell STR profiling report

mmc5.pdf^{(392.7KB, pdf)}

RAW264.7 cell quality control report

mmc6.pdf^{(2.2MB, pdf)}

IPEC-J2 cell line authentication report

mmc7.pdf^{(817KB, pdf)}

IPEC-J2 Mycoplasma testing report

mmc8.pdf^{(284.5KB, pdf)}

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

mmc1.docx^{(22.9MB, docx)}

Supplementary Table S2

mmc2.xlsx^{(110.4KB, xlsx)}

Supplementary Table S3

mmc3.xlsx^{(5.5MB, xlsx)}

WB blots

mmc4.docx^{(268KB, docx)}

RAW264.7 cell STR profiling report

mmc5.pdf^{(392.7KB, pdf)}

RAW264.7 cell quality control report

mmc6.pdf^{(2.2MB, pdf)}

IPEC-J2 cell line authentication report

mmc7.pdf^{(817KB, pdf)}

IPEC-J2 Mycoplasma testing report

mmc8.pdf^{(284.5KB, pdf)}

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PERMALINK

A next-generation probiotic strain for gut health: Bacteroides cellulosilyticus LYH2 variant with anti-inflammatory and metabolic advantages

Cong Lan

Xiaofeng Deng

Shen Jin

Hua Li

Yang Liu

Aimin Wu

Yanjiao Liu

Yifan Zhang

Jun He

Zhaolai Dai

Daiwen Chen

Bing Yu

Zhiqing Huang

Xiangbing Mao

Ping Zheng

Jie Yu

Junqiu Luo

Hui Yan

Yuheng Luo

Summary

Background

Methods

Findings

Interpretation

Funding

Research in context.

Evidence before this study

Added value of this study

Implications of all the available evidence

Introduction

Methods

Bacterial isolation, Bacteroides cultivation and preliminary screening with IPEC-J2 cells

Strains

Identification of strains with 16S rDNA sequencing

Whole genome sequencing and pangenome analysis of B. cellulosilyticus LYH2

Analysis of metabolites in the culture supernatant of B. cellulosilyticus LYH2

Determining antibacterial efficacy via co-cultivation techniques

Animal trial

Table 1.

Measurement of organ, muscle, and fat indices

Bacterial translocation detection

Analysis of haematological parameters, serum biochemical parameters and flow cytometry-based assessment of T lymphocyte subpopulations

Histological staining, mucus layer staining, and immunofluorescence analysis

Measurement of SCFA concentration

16s rRNA amplicon sequencing

RNA extraction and real-time PCR analysis

Protein extraction and Western blotting

Statistical analysis

Ethics

Role of funders

Results

Isolation, identification, and probiotic potential assessment of Bacteroides strains

Fig. 1.

Investigation and characterisation of biological features of B. cellulosilyticus LYH2 derived from porcine sources

Fig. 2.

Table 2.

Metabolic profiling and antibacterial and anti-inflammatory activity of B. cellulosilyticus LYH2

Fig. 3.

Prolonged administration of porcine-derived B. cellulosilyticus LYH2 is well-tolerated and improves host metabolic parameters

Fig. 4.

Table 3.

Table 4.

Porcine-derived B. cellulosilyticus LYH2 attenuates DSS-induced colitis with superior efficacy to reference strains and 5-ASA

Fig. 5.

B. cellulosilyticus LYH2 enhances intestinal integrity and barrier function, alleviating DSS-induced colitis in mice

Fig. 6.

Table 5.

Impact of live B. cellulosilyticus LYH2 and its metabolites on colon immune function and gut microbiota composition in mice with DSS-induced colitis

Fig. 7.

Live B. cellulosilyticus LYH2 and its metabolites attenuate intestinal inflammation via downregulation of M1 macrophages in the colon of DSS-treated mice

Fig. 8.

Ffar3 is required for propionate-mediated protection against colitis

Fig. 9.

Ffar3 mediates T cell and macrophage regulation by B. cellulosilyticus LYH2 and propionate

Fig. 10.

Discussion

Fig. 11.

Contributors