The strains Blautia wexlerae MW-022, Dorea longicatena MW-023, and Roseburia faecis MW-024 were originally isolated from fecal samples of healthy adults and deposited at the State Key Laboratory of Food Science and Resources. Coprococcus eutactus DSM107541 was purchased from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Germany. All microorganisms were cultured in yeast extract-casitone-fatty acids (YCFA) medium (Hopebio, China) under anaerobic conditions of 5% H₂, 5% CO₂, and 90% N₂ at 37 °C^[22].

A total of 42 six-week-old male C57BL/6J mice with an initial body weight of approximately 21 g were obtained from Nanjing GemPharmatech. They were maintained under specific pathogen-free (SPF) conditions with a 12-h light/dark cycle and provided with ad libitum access to standard chow and water. After seven days of acclimation, the mice were randomized into seven groups (n = 6 per group): a normal control group (B), a model group (M), and five intervention groups.

All mice except those in the B group underwent gut microbiota depletion via a 7-day oral antibiotic cocktail (ampicillin 100 mg/kg, metronidazole 100 mg/kg, vancomycin 50 mg/kg, and neomycin 100 mg/kg)^[23]. Beginning on day 3 of antibiotic treatment, IBS was induced by daily oral gavage of senna leaf decoction (0.5 g/mL, 10 mL/kg BW) combined with restraint stress for 15 days^[24-26]

Following a one-day washout of antibiotics, the gut microbiota of these mice was reconstructed by oral gavage of a synthetic community (SynCom-23) for 3 consecutive days^[27-29]. SynCom-23 was composed of 23 strains detailed in Supplementary Table 1. Individual strains were grown anaerobically in YCFA medium at 37 °C and adjusted to the concentrations listed in Supplementary Table 1. Equal volumes of each culture were then combined to produce a final suspension at 2.1 × 10⁹ colony forming unit (CFU)/mL; 0.2 mL of this suspension was administered to each mouse.

Immediately following the SynCom-23 reconstruction, the 10-day bacterial intervention phase began. Mice in the R.f, D.l, B.w, and C.e groups received oral gavage of 10⁸ CFU live cells of R. faecis MW-024, D. longicatena MW-023, B. wexlerae MW-022, and C. eutactus DSM107541, respectively, each suspended in 0.2 mL phosphate-buffered saline (PBS) (pH 7.2). The mix group was given a mixture of the four bacteria cells (10⁸ CFU in total) by gavage. Meanwhile, the mice in the B and M groups received equivalent volumes of PBS as a vehicle control.

Throughout the experiment, the body weight and fecal status of the mice were monitored daily. The time to first loose stool was defined as the interval from the end of senna leaf gavage to the first observation of loose stool (unformed, watery feces with perianal soiling). This parameter was recorded independently by two blinded researchers. Fecal consistency was assessed semi-quantitatively using the Bristol Stool Form Scale^[30]. Upon completion of the study, the mice were anesthetized and sacrificed by cervical dislocation. Serum samples, fecal samples, colon tissues including contents, and prefrontal cortex were carefully collected and appropriately preserved for subsequent analysis.

All animal experiments in this study complied with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. They were approved by the Experimental Animal Care and Use Committee of Nanchang University (approval No. NCULAE-20250916003).

The Y-maze test^[31] was employed to assess spatial working memory, as originally described by Dellu et al. The apparatus consisted of a custom-built maze with three identical arms (50 cm × 18 cm × 35 cm) joined at a 120° angle. Distinct geometric shapes were affixed to the inner surface of each arm for visual differentiation. For each trial, a mouse was initially placed in the central junction, facing a randomly selected arm, and allowed to explore freely for 5 min. Its behavior was recorded by a video tracking system. The following parameters were documented: Entries into an arm were counted whenever all four paws crossed into the arm (total arm entries). Spontaneous alternation was recorded when a mouse entered three different arms consecutively. The percentage of spontaneous alternation was calculated as [Number of Alternations / (Total Arm Entries - 2)] × 100%. After each trial, the maze was wiped down with 75% ethanol to remove any odor cues.

The open field test^[32] was conducted to evaluate general locomotor activity and anxiety-like behavior. Prior to testing, all animals were acclimatized to the behavioral testing room for 2 h under quiet, sound-attenuated conditions. Each mouse was gently placed in the center of a white, square open-field arena (40 cm × 40 cm × 60 cm) and allowed to explore freely for 5 min. An overhead camera, fixed 1.5 m above the arena, recorded the movements. Video tracking software was used to quantify total distance traveled as a measure of locomotor activity. Anxiety-like behavior was inferred from the time spent and the number of entries into the central zone. Reduced activity in this zone was taken as an indicator of higher anxiety. The arena was cleaned with 75% ethanol between each trial. Each subsequent test began only after the ethanol had fully evaporated.

Colon tissue samples were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at a thickness of 4 μm. Sections were then stained with hematoxylin and eosin (H&E)^[33]. Whole-slide imaging was performed using a digital pathology scanning system (Leica Aperio LV1).

Fresh fecal pellets were collected from each animal, placed in sterile tubes, and stored at -80 °C. Total genomic DNA was isolated with the FastPure Stool DNA Isolation Kit (MJYH, Shanghai, China) following the manufacturer’s protocol. DNA concentration and purity were measured on a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). The V3-V4 hypervariable region of the 16S rRNA gene was amplified using the barcoded primer pair 338F/806R^[34] on an ABI GeneAmp® 9700 thermal cycler. The amplicons were cleaned up with a DNA Gel Extraction Kit (PCR Clean-Up Kit, YuHua, China) and quantified on a Qubit 4.0 fluorometer (Thermo Fisher Scientific, USA). Equimolar quantities of the purified products were then combined and sequenced in paired-end mode on an Illumina NextSeq 2000 platform (Illumina, San Diego, USA). Sequencing was performed by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China).

Raw sequencing data were demultiplexed, quality-filtered with fastp (version 0.19.6)^[35], and merged with FLASH (version 1.2.11)^[36]. Subsequently, the DADA2^[37] plugin within the QIIME2 (version 2020.2)^[38] pipeline was employed to denoise the sequences and resolve them into amplicon sequence variants (ASVs). To standardize for sequencing depth, the ASV table was rarefied to 20,000 sequences per sample for downstream alpha and beta diversity analyses. This rarefaction depth achieved an average Good's coverage of 97.90%. Taxonomic classification of ASVs was performed using the Naive Bayes consensus taxonomy classifier in QIIME2 against the SILVA 16S rRNA database (version 138).

Bioinformatic and statistical data analyses were conducted on the Majorbio Cloud Platform. Alpha diversity indices (Chao1 and Sobs) were calculated using mothur^[39], and differences between groups were assessed using the Wilcoxon rank-sum test. For beta diversity, Bray-Curtis dissimilarities were calculated and visualized using Principal Coordinate Analysis (PCoA). Permutational multivariate analysis of variance (PERMANOVA) was applied to test for significant differences in microbial community structure across groups. Linear discriminant analysis effect size (LEfSe) was employed to identify bacterial taxa with significantly different abundances between groups. Taxa detected from phylum to genus level were retained with an linear discriminant analysis (LDA) score > 2 and P < 0.05. Distance-based redundancy analysis (db-RDA) was used to investigate the influence of clinical indicators on gut bacterial community structure. Subsequently, linear regression analysis was applied to assess the impact of these key clinical indicators on alpha diversity indices. Finally, a Spearman correlation network^[41] was constructed to visualize microbial co-occurrence patterns (|r| > 0.6, P < 0.05).

Total RNA was reverse transcribed into cDNA using the PrimeScript RT Reagent Kit (Takara). Subsequently, quantitative real-time PCR (RT-qPCR) was carried out using TB Green Premix Ex Taq II (Takara). The reactions were run on a QuantStudio 7 Flex Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). This assay was used to quantify the mRNA expression of genes related to visceral hypersensitivity, tight junction integrity, and neurotransmitter systems. Primer sequences are listed in Supplementary Table 2. β-actin served as the endogenous reference gene for normalization, and relative gene expression levels were calculated using the 2^-ΔΔCt method^[33].

Distal colon specimens (30 mg) were homogenized in ice-cold PBS, and the homogenates were centrifuged at 10,000 × g for 10 min at 4 °C^[42]. The supernatants were then harvested for subsequent assays. Levels of myeloperoxidase (MPO), malondialdehyde (MDA), superoxide dismutase (SOD), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in the colon were determined using commercial enzyme-linked immunosorbent assay (ELISA) kits (Fcmacs, Nanjing, China) according to the manufacturer’s instructions. All measurements were normalized to the total protein content of each sample, as quantified with a BCA protein assay kit (Beyotime, Shanghai, China).

All statistical analyses were performed using GraphPad Prism (version 10.1.0) and SPSS (version 26.0). Data are presented as mean ± standard error of the mean (SEM). For two-group comparisons, Student’s t-test was used. For multiple-group comparisons, one-way analysis of variance (ANOVA) followed by Tukey's post hoc test was applied to control the family-wise error rate. For correlation analyses involving multiple variables, Spearman's rank correlation coefficients were computed using the R packages psych, pheatmap, and reshape2. Raw P-values from these analyses were corrected using the Benjamini-Hochberg false discovery rate (FDR) procedure, with statistical significance set at adjusted q < 0.05. A two-tailed P-value (or adjusted q-value) of less than 0.05 was considered statistically significant for all tests unless otherwise stated.

We established a standardized IBS model by first depleting the endogenous gut microbiota of mice with an antibiotic cocktail, followed by colonization with SynCom-23 [Figure 1A]. Following model establishment, mice exhibited significant weight loss; however, this effect was substantially alleviated by intervention with the four selected Lachnospiraceae strains [Figure 1B]. Regarding diarrheal symptoms, treatments with B. wexlerae MW-022, R. faecis MW-024, or the four-strain combination significantly delayed the onset of diarrhea. This was indicated by the prolonged time to first loose stool [Figure 1C]. Notably, all interventions improved stool consistency [Figure 1D]. At the molecular level, all treatments significantly modulated the colonic expression of visceral hypersensitivity markers, calcitonin gene-related peptide (CGRP) and transient receptor potential vanilloid 1 (TRPV1) [Figure 1E and F]. Consistent with the core pathology of IBS, which is characterized by the absence of overt organic lesions^[5], histological analysis revealed no significant structural damage to the colonic mucosa in any group [Supplementary Figure 1]. Collectively, these data confirmed the successful establishment of an IBS mouse model and demonstrated that the selected Lachnospiraceae strains effectively ameliorated core IBS phenotypes and regulated visceral sensitivity.

All interventions significantly reduced the levels of pro-inflammatory cytokines TNF-α and IL-6, while increasing anti-inflammatory cytokine IL-10 [Figure 1G-I]. Furthermore, all Lachnospiraceae strains effectively modulated key oxidative stress indicators: compared with the M group, all interventions elevated SOD content, albeit not to normal levels, and reduced MPO activity and MDA content to near-normal levels [Figure 1J-L], indicating a role in mitigating intestinal low-grade inflammation and oxidative damage. To assess intestinal barrier integrity, we measured the expression of tight junction proteins. Among all strains, R. faecis MW-024 significantly upregulated the expression of ZO-1, Claudin-1, and Occludin. The other three single-strain interventions also effectively upregulated the expression of Claudin-1 and Occludin, but showed strain-specific differences in the regulation of zonula occludens-1 (ZO-1): B. wexlerae MW-022, D. longicatena MW-023, and C. eutactus DSM107541 did not significantly alter the expression level of ZO-1 [Figure 1M-O]. Notably, R. faecis MW-024 exhibited the most pronounced upregulation effect on these three tight junction proteins, underscoring its potent capacity to enhance the intestinal barrier function.

To assess the therapeutic potential of Lachnospiraceae strains on IBS-related behavioral abnormalities, we first characterized the deficits in our mouse model using the open field and Y-maze tests. These tests are commonly used to capture anxiety-like behavior and spatial working memory mediated through the gut-brain axis. Compared to the B group, IBS mice exhibited significant anxiety-like behavior and diminished exploratory motivation, evidenced by reduced time in the central zone and decreased total distance traveled in the open field test [Figure 2A and B]. In the Y-maze test, they showed a specific impairment in spatial working memory, indicated by a significantly reduced spontaneous alternation rate, while general locomotion was unaffected [Figure 2C and D].

Following intervention with the Lachnospiraceae strains, these anxiety-like behaviors and diminished exploratory motivation were effectively reversed. All treatments also alleviated the cognitive impairment observed in the Y-maze test, though with varying degrees of efficacy. Notably, the D.l group achieved the highest spontaneous alternation rate. The R.f, C.e and Mix groups restored cognitive function to a level equivalent to the B group. While B. wexlerae MW-022 also significantly increased the spontaneous alternation rate, its performance remained significantly lower than that of the B group. To elucidate the underlying neural mechanisms, we further examined the expression of key factors in neurotransmitter systems within the prefrontal cortex^[47].

IBS mice exhibited significant dysregulation in both the serotonin (5-HT) system^[48] [Figure 2E-G] and the GABA/glutamate (GABA/Glu) system^[49] [Figure 2H-K]. Each strain exerted distinct, strain-specific neuromodulatory effects on these pathways. Within the 5-HT system, C. eutactus DSM107541 reversed the expression of Slc6a4 and Htr1a; D. longicatena MW-023 reversed the expression of Tph2 and Htr1a; both reversed two out of the three key indicators (Tph2, Slc6a4 and Htr1a) respectively, thus achieving a more prominent effect. In contrast, R. faecis MW-024 solely downregulated Htr1a, while B. wexlerae MW-022 and the mixed strains specifically upregulated Tph2. Regarding the GABA/Glu system, B. wexlerae MW-022 demonstrated the most potent capacity, significantly regulating all three markers (Gad1, Slc1a2, and Gabbr1). R. faecis MW-024 notably regulated two of the three markers (Gad1 and Slc1a2), whereas D. longicatena MW-023 solely reversed abnormalities in Gad1. The mixed strains upregulated Gad1 and Gabbr1. Additionally, all four strains effectively downregulated the astrocytic marker glial fibrillary acidic protein (GFAP), with the exception of the mixed formulation.

To elucidate the potential impact of Lachnospiraceae on host metabolism, we first analyzed the intrinsic metabolic capabilities of the individual strains and their combination. The analysis focused on the production of SCFAs and neuroactive metabolites. Analysis of SCFA production revealed distinct functional specialization among strains [Figure 3A]. R. faecis MW-024 and C. eutactus DSM107541 were identified as efficient butyrate producers, whereas B. wexlerae MW-022 and D. longicatena MW-023 primarily generated acetate. Additionally, B. wexlerae MW-022 and C. eutactus DSM107541 produced modest amounts of propionate. The mixed-strain combination exhibited metabolic complementarity, simultaneously producing multiple SCFAs. Furthermore, in vitro cultures confirmed that all four Lachnospiraceae strains synthesized GABA at comparable levels [Supplementary Figure 2A].

In vivo experiments revealed that the four strains significantly increased the colonic concentrations of acetic, propionic, and butyric acid in IBS mice. This effect was observed both when strains were administered individually and in combination [Figure 3B].

At the neuromodulatory level, these interventions partially reversed the neurological abnormalities induced by the IBS-D model, albeit through strain-specific pathways. Analysis of the tryptophan-serotonin pathway revealed that D. longicatena MW-023 and B. wexlerae MW-022 were the most effective modulators. These strains elevated tryptophan (Trp) levels while reducing serotonin (5-HT) and kynurenine (KYN) concentrations, thereby normalizing the KYN/Trp ratio [Figure 3C-F]. In contrast, R. faecis MW-024, C. eutactus DSM107541, and the mixed strains did not effectively recover KYN levels. Additionally, all four strains notably reversed the expression of quinolinic acid (QUIN) [Figure 3G]. Regarding the GABA/Glu pathway, all treatments, with the exception of the B.w group, reduced GABA concentrations [Figure 3H]. Conversely, both D.l and B.w groups demonstrated restoration of glutamate levels [Figure 3I]. A common effect observed across all treatments was the downregulation of trimethylamine N-oxide (TMAO) [Figure 3J].

Bile acid profiles, which are shaped by gut microbiota and influence intestinal homeostasis and neuromodulation^[50], were quantified in ileal content [Supplementary Figure 2B]. Bacterial interventions consistently drove a shift in BA composition toward a neuroprotective state. Specifically, they significantly reduced neurotoxic deoxycholic acid (DCA) and lithocholic acid (LCA) [Figure 3K-L], with DCA in the B. wexlerae MW-022 group being the sole exception. At the same time, they increased the neuroprotective bile acids ursodeoxycholic acid (UDCA) and tauroursodeoxycholic acid (TUDCA)^[51] [Figure 3M and N]. This rebalancing was reflected in the functional BA index, (UDCA + TUDCA)/(LCA + DCA). This metric was significantly elevated across all intervention groups, indicating a shift from toxic to a protective BA pool [Figure 3O, Supplementary Figure 2C and D]. This beneficial shift was further supported by marked reductions in the DCA/cholic acid (CA) and LCA/chenodeoxycholic acid (CDCA) ratios, both indicators of primary-to-secondary BA conversion, across all treated groups relative to the model [Figure 3P and Q, Supplementary Figure 2E and F]. Finally, all treatments also reversed the abnormal increase in histamine [Figure 3R].

Lachnospiraceae interventions effectively reversed the disease-induced decline in gut microbiota α-diversity (Chao1 and Sobs indexes, Figure 4A and B) and regulated the overall microbial structure (PCoA, Figure 4C). Taxonomic analysis revealed that the IBS model group exhibited significant gut microbiota dysbiosis, characterized by a marked increase in Pseudomonadota and a significant decrease in Actinomycetota, alongside a notable reduction in beneficial genera such as Adlercreutzia, Clostridia_UCG-014, Lachnospiraceae_NK4A136_group, and Roseburia [Supplementary Figure 3].

LEfSe analysis further clarified potential microbial biomarkers specifically induced by individual strains [Figure 4D]: the R.f group enriched Bacilli, Staphylococcus, Eubacterium_coprostanoligenes_group, Blautia, Lachnospiraceae_UCG-006, and RF39. The D.l group specifically enriched Anaerovoracaceae, RF39, Lachnospiraceae, Muribaculum, Butyribacter, and Colidextribacter. The B.w group significantly enriched core members of the order Lactobacillales, including Lactobacillaceae, Ligilactobacillus, and Thomasclavelia. The C.e group showed enrichment focused on the Lachnospirales and Oscillospirales orders, including the key butyrate-producing genus Lachnospiraceae_NK4A136_group. The mix group primarily enriched Parabacteroides, Staphylococcaceae, Mucispirillum, and Deferribacteraceae.

To link these microbial shifts to functional outcomes, we performed correlation analyses between the microbiota, metabolites, and disease phenotypes. Ligilactobacillus and Acutalibacter positively correlated with all SCFAs. Oscillospiraceae was associated with the reversal of BA profiles (DCA, LCA, UDCA and TUDCA). In parallel, Lachnospiraceae_NK4A136 correlated with improvements in LCA, UDCA and TUDCA. Adlercreutzia, Candidatus_Saccharimonas, Oscillospiraceae, Desulfovibrionaceae, Dubosiella, RF39, and Faecalibaculum showed positive correlations with the regulation of the GABA/Glu system. In contrast, Monoglobus and Turicibacter were positively linked exclusively to improvements of glutamine and TMAO [Figure 4E]. Finally, Adlercreutzia correlated with improvements in cytokines, oxidative-stress markers, and tight junction proteins; conversely, Clostridia_UCG-014, Candidatus_Saccharimonas, and Dubosiella correlated only with improvements in cytokines and tight junction proteins [Figure 4F].

To investigate the gut-brain axis, we conducted multi-level correlation analyses to map the potential pathways of how Lachnospiraceae treatments alleviate IBS. A global analysis revealed significant associations among BAs, SCFAs, and neurological factors [Supplementary Figure 4A], establishing an across-system foundation.

Further dissection uncovered a pivotal regulatory cascade. Butyrate, a key SCFA produced by Lachnospiraceae, was significantly negatively correlated with toxic BAs (DCA and LCA, Figure 5A and B). It also showed positive correlations with neuroprotective BAs (UDCA and TUDCA, Figure 5C and D). Acetate showed similar trends [Supplementary Figure 4B-E], suggesting that its regulatory pathways concerning BAs may partially overlap with those of butyrate. This rebalancing profoundly influenced the nervous system [Figure 5E]. All three SCFAs positively correlated with Gad1 and Tph2, but negatively correlated with GFAP, QUIN, histamine, TMAO, and GABA. Acetic acid was positively related to Slc1a2, Gabbr1, and Trp, and negatively related to Htr1a and Slc6a4. Conversely, propionic acid positively correlated with Trp, while butyric acid negatively correlated with Htr1a. Furthermore, shifts in BA profiles, specifically the downregulation of toxic BAs (DCA and LCA) and upregulation of neuroprotective BAs (UDCA and TUDCA), displayed close connections with Gad1, Trp and Gabbr1. Additionally, inflammatory cytokines (TNF-α and IL-6) demonstrated significant correlations with various neurological factors [Figure 5F-K, Supplementary Figure 4F-K]. This finding links peripheral inflammation to central outcomes.

This study has several methodological limitations that warrant careful consideration. First, the colonization efficiency of individual SynCom-23 members was not monitored using qPCR or strain-specific primers. Additionally, no baseline microbiota sample was collected in the window between SynCom-23 colonization and the Lachnospiraceae intervention. Without direct colonization tracking and pre-intervention profiling, it is challenging to fully disentangle ecological remodeling driven by the target strains from the natural temporal drift of the synthetic community. Second, the inherent gas-producing property of D. longicatena may pose a risk for IBS patients with impaired intestinal gas handling. In addition, metabolic cross-interference among consortium members and the potential horizontal transfer of antibiotic resistance genes in the mixed-strain formulation should not be overlooked. Therefore, the long-term safety of these strains and their combination requires more comprehensive evaluation. Third, the gut-brain axis causal inferences drawn in this study rest primarily on correlation analyses and gene expression data. They lack direct functional validation through receptor-knockout models, pathway inhibition, or single-strain colonization experiments in germ-free mice. Consequently, the current conclusions should be regarded as a biologically plausible mechanistic hypothesis grounded in multi-dimensional evidence rather than definitive proof of causality. Future studies incorporating pre-intervention baseline sampling, strain-specific tracking of SynCom-23 members, and rigorous functional validation using gene-knockout or pathway-inhibition approaches will be essential to address these limitations. Such efforts will further consolidate the scientific foundation of this work.

This study systematically reveals that the four representative strains effectively alleviate IBS symptoms in mice with potentially differential pathways: B. wexlerae MW-022 generates considerable acetic acid, stimulates Lactobacillaceae proliferation, and targets the GABA/Glu system. R. faecis MW-024 specializes in butyrate production and affects the GABA/Glu system. D. longicatena MW-023 enhances the 5-HT system at synthesis and reception levels, whereas C. eutactus DSM107541 modulates the 5-HT system via the “clearance-reception” mode. Furthermore, these strains in combination achieve more comprehensive effects on gut micro-ecology.