SPF grade BALB/c mice (4–6 weeks old) were purchased from Hangzhou Charles River Co., Ltd. (Hangzhou, China). Mice were fed a standard rodent chow diet (provided by Hangzhou Charles River Co., Ltd.) ad libitum. Feed and water were refreshed every two days. All animal procedures were conducted in compliance with the guidelines for the care and use of laboratory animals approved by the Experimental Animal Ethics Committee of Zhejiang Z&F University (Approval number is ZAFUAC202458). L. rhamnosus CIQ249, Lactobacillus reuteri (L. reuteri), Enterococcus faecium (E. faecium), Escherichia coli (E. coli) K99, E. coli O157, Listeria monocytogenes (L. monocytogenes), Salmonella typhimurium (S. typhimurium), Shigella spp., Vibrio mimicus (V. mimicus), V. parahaemolyticus, Yersinia enterocolitica (Y. enterocolitica) were maintained in our laboratory. Porcine intestinal epithelial cells (PIEC) kept in our laboratory were also used in this study.

We used 5(6)-carboxyfluorescein diacetate succinimidyl ester (cFDA-SE) to evaluate the digestive tract colonization ability of L. rhamnosus CIQ249, according to the assay previously described (Xu and Li, 2007). Briefly, L. rhamnosus CIQ249 was cultured and adjusted to 5 × 10⁸ CFU/mL for oral administration. A 10 mM cFDA-SE (ThermoFisher, USA) stock solution was diluted 1:800 and mixed with an equal volume of the CIQ249. After incubation at 37°C for 30 min, the cells were washed and resuspended to 10¹⁰ CFU/mL. Samples were fixed with 0.75% formaldehyde, and the labeling efficiency was determined by flow cytometry. Mice were randomly divided into two groups (n = 20 per group): normal control group (sterile PBS group) and cFDA-SE-labeled CIQ249 group (2 × 10⁹ CFU/mouse). At 1, 7, 15 and 20 days post-oral administration, five mice per group were euthanized to collect jejunum, ileum, and colon segments from each mouse. Individual section was cut longitudinally, and any visible residual food particles or fecal material was removed from the intestine before being examined for the presence of adhering cFDA-SE-labeled CIQ249. This examination was performed by adding 150 μL of PBS to every 1.0 cm of tissue and dislodging microbes from the mucosal surfaces of the tissues. Cell extracts were fixed with formaldehyde (0.75%, vol/vol) prior to flow cytometry analysis with three technical replicates.

To assess growth-promoting effects, mice were randomly divided into two groups (n = 10 per group). One group received the strain CIQ249 (2 × 10⁹ CFU per mouse) orally, while the control group received sterile PBS, for 28 consecutive days. Daily feed intake and body weight gain were recorded.

BALB/c mice were randomly divided into two groups (n = 5 per group): experimental group (oral CIQ249, 2 × 10⁹ CFU/mouse) and control group (sterile PBS). After 28 days oral administration, three mice per group were euthanized to collect duodenum, jejunum, and ileum. Tissues were fixed in 10% neutral formaldehyde, and processed for Hematoxylin and eosin (H&E) staining. Villus height, crypt depth, and the villus-to-crypt ratio were analyzed.

Strain CIQ249 was activated, inoculated (1%) into 10 mL sterile MRS broth, and incubated at 37°C for 24 h. The culture supernatant was filter-sterilized and adjusted to pH 7.0. Indicator strains (E. coli K99, E. coli O157, L. monocytogenes, Shigella spp., S. typhimurium, L. reuteri, E. faecium, V. parahaemolyticus, Y. enterocolitica, and V. mimicus) were grown to OD₆₀₀ ≈ 1.0. Aliquots (200 µL) of each bacterial culture were spread on appropriate agar media, and 6 mm wells were punched. Wells were filled with CIQ249 supernatant, sterile MRS broth (negative control), or penicillin/streptomycin (10000 U/mL, positive control). Plates were incubated at 37°C for 24 h, and the diameter of inhibition zones was measured. All experiments were performed with three independent biological replicates, each consisting of three technical replicates.

L. rhamnosus CIQ249 was grown in 100 mL MRS broth (1% inoculum) at 37°C for 24 h. S. typhimurium and E. coli K99 were activated and separately added (1%) to the filter-sterilized CIQ249 supernatant (pH 7.0), followed by incubation at 37°C for 24 h. Controls were incubated in sterile MRS broth. Bacterial morphology was examined by scanning electron microscopy. The experiment was performed with three independent biological replicates, each consisting of three technical replicates.

Mice were randomly divided into eight groups (n = 10 per group): (1) normal control (sterile PBS) group; (2) CIQ249 alone (2 × 10⁹ CFU/mouse); (3) CIQ249 for 7 days, then infection with S. typhimurium (2 × 10⁷ CFU/mouse) group; (4) CIQ249 for 7 days, then infection with E. coli K99 (2 × 10⁷ CFU/mouse) group; (5) S. typhimurium alone (2 × 10⁷ CFU/mouse group); (6) E. coli K99 alone (2 × 10⁷ CFU/mouse) group; (7) co-administration of CIQ249 and S. typhimurium (1:1, v/v); (8) co-administration of CIQ249 and E. coli K99 (1:1, v/v). Mortality was recorded daily for 8 days. Body weight changes and clinical symptoms (scored according to Supplementary Table S1) were monitored. Duodenum, jejunum, and ileum were collected for histopathological examination.

PIEC was used for assessment in vitro. Briefly, PIEC was seeded in 24-well plates. CIQ249, S. typhimurium and E. coli K99 were grown to OD₆₀₀ ≈ 1.0, washed, and resuspended in DMEM at 1 × 10⁸ CFU/mL. Cells were treated as follows: (1) Control (untreated PIEC); (2) PIEC + CIQ249 (1 × 10⁷ CFU/mL); (3) PIEC + typhimurium (1 × 10⁷ CFU/mL); (4) PIEC + E. coli K99 (1 × 10⁷ CFU/mL); (5) PIEC pre-incubated with CIQ249 (1 × 10⁷ CFU/mL, 2 h, removed) then challenged with S. typhimurium (1 × 10⁷ CFU/mL); (6) PIEC pre-incubated with CIQ249 then challenged with E. coli K99; (7) PIEC co-cultured with CIQ249 (5×10⁶ CFU/mL) and S. typhimurium (5 × 10⁶ CFU/mL); (8) PIEC co-cultured with CIQ249 (5 × 10⁶ CFU/mL) and E. coli K99 (5 × 10⁶ CFU/mL). All treatments lasted 6 h at 37°C. For immunofluorescence assay (IFA), post-treatment, cells were washed, fixed with 4% paraformaldehyde, and blocked with 0.3% BSA. The cells were then incubated with rabbit anti-pig ZO-1 (1:100) or Claudin-1 (1:100) primary antibodies (Proteintech, China), followed by FITC-labeled goat anti-rabbit IgG (1:400, Abcam, UK). Tight junction integrity was observed by fluorescence microscopy. For quantitative RT-PCR analysis (qRT-PCR) of ZO-1 and Claudin-1 expression, total RNA was extracted for each treatment group, reverse transcribed, and amplified using SYBR Green (Takara, China) on a 7500 real-time PCR system (ABI, USA) with the primers listed in Supplementary Table S2. Gene expression fold changes were calculated using the 2^-ΔΔCt method, using β-actin as the internal control. For Western blot analysis of ZO-1 and Claudin-1 expression, cells were lysed, and proteins were separated by 12% SDS-PAGE, transferred to PVDF membranes (Bio-Rad, USA), and blocked with 5% skim milk. Membranes were probed with rabbit anti-pig ZO-1 or Claudin-1 (1:2000, Proteintech) and HRP-labeled goat anti-rabbit IgG (1:5000). Signals were detected using a chemiluminescence kit (Applygen, China). All experiments were performed with three independent biological replicates, each consisting of three technical replicates.

Mice were used for assessment in vivo. Briefly, mice were divided into five groups:(1) Control (sterile PBS) group; (2) CIQ249 alone (2 × 10⁹ CFU/mouse for 5 days); (3) S. typhimurium or E. coli K99 alone (2 × 10⁷ CFU/mouse); (4) CIQ249 for 5 days, then infection with S. typhimurium or E. coli K99. Intestinal samples were collected for qRT-qPCR and Western blot analysis of ZO-1 and Claudin-1, using rabbit anti-mouse ZO-1 of Claudin-1 primary antibodies (Absin, China) and HRP-labeled goat anti-rabbit IgG secondary antibody. All experiments were performed with three independent biological replicates, each consisting of three technical replicates.

Mice (n = 15 per group) received oral L. rhamnosus CIQ249 (200 μL of 1 × 10¹⁰ CFU/mL) or sterile PBS (control). At 0, 7, 14, 21 and 28 days post- administration, three mice per group were euthanized to collect serum and intestinal mucus. Levels of IFN-γ, IL-2, IL-4, IL-17, and IL-27, as well as IgA in serum and sIgA in intestinal mucus were measured using commercial ELISA kit (TransGen Biotech, China) with three technical replicates.

These experiments were carried out in accordance with previously described protocols (Jia et al., 2020). To detect dendritic cells (DCs) activation in Peyer’s patches (PPs), the PPs were isolated from the intestines of mice in the CIQ249 and PBS control groups on day 7 post oral administration. The tissues were grinded homogenized in 5 mL of pre-cooled Hank’s Balanced Salt Solution (HBSS). The resulting cell suspension was filtered through a 300-mesh stainless steel sieve and centrifuged at 500 × g for 10 min. After centrifugation, the cell pellet was resuspended in 8 mL HBSS, carefully layered onto 5 mL of 70% Per-coll (Sigma, USA) solution, and centrifuged at 500 × g for 20 min. Cells on the interface were collected, resuspended in HBSS at a concentration of 10⁶/mL, and incubated with anti-CD11c antibody (Abcam, USA). After washing, the cells were stained with FITC-conjugated anti-CD40 or anti-CD86 antibodies (Abcam, USA), and analyzed by flow cytometry. The expression of Bcl-6 in T lymphocytes within the PPs was detected by immunohistochemistry (IHC). Briefly, PPs were isolated from mice in the CIQ249 and PBS control groups, fixed in 10% formaldehyde, and embedded in paraffin. The tissue sections were then prepared and incubated with a mouse anti-Bcl-6 antibody (Abcam, USA) at 37°C for 2 h, followed by incubation with an HRP-conjugated goat anti-mouse IgG (Sigma, USA) or FITC-conjugated goat anti-mouse IgG (ThermoFisher, USA). Signal development was followed by observation under inverted microscope.

To analyze the CIQ249-induced differentiation of IgA-secreting cells in PPs, a combination of flow cytometry and IHC was employed. Detailed methodologies for intestinal PPs isolation, single-cell preparation, and flow cytometry were as follows: for collection and processing of PPs, fresh intestinal segments containing visible PPs were aseptically excised from euthanized mice, fat tissue and mesentery were carefully removed under a stereomicroscope, and individual PPs were gently separated using fine forceps and immediately placed in cold, sterile PBS. For preparation of single-cell suspension, isolated PPs were transferred onto a sterile 300-mesh stainless-steel cell strainer and gently pressed with the plunger of a syringe while rinsing with cold PBS to release lymphocytes. The resulting cell suspension was collected and centrifuged at 500 × g for 5 min at 4°C. The pellet was resuspended in 5 mL of digestion medium consisting of RPMI-1640 supplemented with 1 mg/mL collagenase IV (Sigma) and 0.1 mg/mL DNase I (Sigma), followed by incubation at 37°C for 30 min with gentle shaking. After digestion, the cell suspension was passed through a 400-mesh cell strainer to remove debris and undigested tissue, washed twice with cold PBS and centrifuged at 500 × g for 5 min. The final pellet was resuspended in FACS buffer (PBS containing 2% fetal bovine serum and 1 mM EDTA) and adjusted to a concentration of 2 × 10⁶ cells/mL. For surface marker staining, 100 μL of cell suspension (2 × 10⁵ cells) was aliquoted into FACS tubes and incubated with the following antibody cocktails for 30 min at 4 °C in the dark: T-cell panel (for Tfh identification): experimental group: APC-conjugated anti-mouse CXCR5 (3 μL) + PE-conjugated anti-mouse CD4 (3 μL) (BD pharmingen, USA); single-color controls: APC-CXCR5 alone, and PE-CD4 alone; unstained control: cells + PBS only. B-cell/plasma-cell panel (for IgA-secreting lineage analysis): experimental group: FITC-conjugated anti-mouse IgA (4 μL) + PerCP-Cy5.5-conjugated anti-mouse B220 (2 μL) + PE-Cy7-conjugated anti-mouse IgM (2 μL) (BD pharmingen, USA); single-color controls: FITC-IgA alone, PerCP-B220 alone, and PE-Cy7-IgM alone; Unstained control: cells + PBS only. After incubation, cells were washed twice with cold FACS buffer, centrifuged at 500 × g for 5 min, and finally resuspended in 500 μL of PBS for immediate acquisition. Subsequently, the percentage of CXCR5⁺CD4⁺ T follicular helper (Tfh) cells, B220⁺IgM⁺ B lymphocytes, B220⁺IgA⁺ B lymphocytes, and B220⁻IgA⁺ plasma cells were analyzed on a flow cytometer (BD Biosciences), and the gating strategy was as follows—live cell gate: based on forward scatter (FSC-A) vs. side scatter (SSC-A) to exclude debris and dead cells; single-cell gate: FSC-H vs. FSC-A to exclude doublets; T-cell subset: from single cells, CD4⁺ cells were gated, and subsequent analysis of CXCR5 expression identified CD4⁺CXCR5⁺ Tfh cells; B-cell/plasma-cell subset: from single cells, B220⁺IgM⁺ conventional B cells, B220⁺IgA⁺ IgA-committed B cells, and B220⁻IgA⁺ IgA-secreting plasma cells were identified based on co-expression of B220, IgM, and IgA. Additionally, IgA-secreting cells within the PPs were visualized by IHC, as described in the previous section, using an anti-mouse IgA antibody (Abcam, USA). All experiments were performed with three independent biological replicates.

PIEC were subjected to treatments as follows:(1) Control (sterile PBS) group; (2) CIQ249 alone (2 × 10⁹ CFU/mL for 3 h); (3) S. typhimurium alone (2 × 10⁷ CFU/mL for 3 h); (4) strain CIQ249 for 3 h, then infection with S. typhimurium for 3 h. Subsequently, cells from each group were collected and sent for transcriptomic analysis by Guangzhou Genedenovo Co., Ltd. (Guangzhou, China). All experiments were performed with three independent biological replicates per condition (control, strain CIQ249, S. typhimurium, and CIQ249+S. typhimurium).

Data are presented as mean ± standard deviation (SD). Differences among groups were analyzed using one-way ANOVA followed by Tukey’s multiple-comparison test in GraphPad Prism V8.0. Significant differences are indicated with asterisks (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). All experiments were performed in at least triplicate.

Flow cytometry confirmed approximately 99.3% positive labeling rate for cFDA-SE-labeled CIQ249 (Figure 1A). The strain demonstrated significant intestinal colonization, with detection rates of 75.03%, 87.82%, and 82.75% in the jejunum, ileum, and colon, respectively, on day 1 post-administration, and remained detectable (~20%) up to day 20 (Figure 1A). In mice, the strain CIQ249 markedly increased average daily feed intake and average daily gain while reducing the feed-to-gain ratio (Figure 1B). Furthermore, it promoted villus growth in the duodenum, jejunum, and ileum, resulting in shallower crypts and a significantly increased V/C ratio in the duodenum and ileum (Figure 1C).

The filtered supernatant (pH 7.0) of the CIQ249 culture exhibited inhibitory activity against various pathogenic bacteria but had not effect on the probiotics L. reuteri and E. faecium (Figure 2A). Scanning electron microscopy (SEM) analysis revealed distinct shrinkage and damage to the cell walls of E. coli K99 and S. typhimurium following incubation with the CIQ249 supernatant (Figure 2B). In contrast, the morphology of the probiotics L. reuteri remained intact, with no signs of shrinkage or damage. These observations suggest that the damage to pathogens is not a nonspecific result of nutrient depletion but rather reflects the specific action of antagonistic substances present in the CIQ249 supernatant. In the mouse infection model, no mortality occurred in groups receiving CIQ249 alone or as a pre-treatment (Figure 3A). In contrast, mice infected with S. typhimurium or E. coli K99 alone began dying on days 1-2, with 100% mortality by day 7. Co-administration groups showed > 50% survival. Body weight loss was severe in pathogen-only groups but significantly attenuated in groups receiving the CIQ249 (Figure 3B). Clinical symptom scores were markedly lower in groups receiving the CIQ249 (either as pre-treatment or co-administration) compared to pathogen-only groups (Figure 3C). Histopathology confirmed severe villus breakage, autolysis, and crypt damage in S. typhimurium and E. coli K99-infected mice, while intestinal architecture remained relatively intact in CIQ249-treated groups (Figure 3D).

Indirect immunofluorescence showed diffuse and discontinuous staining of ZO-1 (Figure 4A) and Claudin-1 (Figure 4B) with intercellular breaks in PIEC treated with S. typhimurium or E. coli K99 alone. In contrast, the CIQ249 treatment maintained or restored continuous, intact tight junction protein localization, even upon pathogenic challenge. At the gene level, the CIQ249 significantly upregulated ZO-1 and Claudin-1 mRNA expression in both PIEC (Figure 5A) and mouse intestinal cells (Figure 5B) compared to controls, whereas pathogens downregulated their expression. Pre-treatment with the CIQ249 prevented the pathogen-induced downregulation. Consistent with the transcriptional regulation, Western blot revealed corresponding changes at the protein level. Densitometric quantification of bands, normalized to β-actin, was performed across biological replicates. In PIEC (Figure 5C), treatment with CIQ249 significantly upregulated the expression of ZO-1 and Claudin-1. In contrast, challenge with S. typhimurium or E. coli K99 alone markedly reduced the levels of both proteins. Pre-incubation with CIQ249 prior to pathogen challenge effectively prevented this downregulation. A similar protective pattern was observed in vivo in mouse intestinal tissues (Figure 5D). Oral administration of CIQ249 alone for 5 days increased both proteins expression compared to the PBS-treated control. Infection with S. typhimurium or E. coli K99 alone led to a significant reduction. Importantly, pre-treatment with CIQ249 completely abolished the pathogen-induced decrease, restoring the expression of both tight junction proteins.

Oral administration of the strain CIQ249 for 28 days gradually increased the levels of cytokines IFN-γ, IL-2, IL-4, IL-17, and IL-27 in both intestinal mucus (Figure 6A) and serum (Figure 6B). Serum IgA and intestinal sIgA levels were significantly elevated from day 14 onwards in the CIQ249 group compared to controls (Figure 7A). Flow cytometry revealed a significant increase in activated intestinal dendritic cells, evidenced by higher numbers of CD11c⁺CD40⁺ and CD11c⁺CD86⁺ cells (Figure 7B). Immunohistochemistry (Figure 7C) and fluorescence IHC (Figures 7D) showed increased Bcl-6 expression in T cells within the PPs of CIQ249-treated mice. Subsequently, flow cytometry confirmed a significant increase in CXCR5⁺CD4⁺ T follicular helper (Tfh) cells in PPs (Figure 7E). Furthermore, L. rhamnosus CIQ249 administration increased the percentages of B220⁺IgM⁺, B220⁺IgA⁺, and B220⁻IgA⁺ cells in PPs (Figure 8A), thereby promoting the differentiation of IgA-secreting cells, as corroborated by immunohistochemical analysis (Figures 8B, C).

In this work, the relationships between samples were visualized using correlation heatmaps. As shown in Supplementary Figure S1, the R² values between samples within each group were all greater than 0.8, indicating good reproducibility of the samples and supporting the feasibility of subsequent analyses. RNA-seq analysis yielded high-quality data (clean reads coverage > 95.57% alignment rate > 63.39%). Differential gene expression analysis (cutoff: P < 0.05 and |log2FC| > 2) identified 211 upregulated and 406 downregulated unique genes in the L. rhamnosus group vs. control; 30 upregulated and 84 downregulated genes in the Salmonella group vs. control; and 3987 upregulated and 1263 downregulated genes in the L. rhamnosus + Salmonella group vs. the Salmonella group (Figures 9A, B). To gain deeper mechanistic insight, a more informative KEGG pathway enrichment analysis was conducted for the following comparisons: control vs. L. rhamnosus, control vs. Salmonella, and Salmonella vs. L. rhamnosus + Salmonella (Figure 9C), and these data indicated that the top enriched pathways were predominantly associated with broad metabolic and immune response processes. Additionally, to confirm the RNA-seq trends, eight genes—Rap1gap, Anpep, Igf2, IL-10, Galnt6, IL-22ra1, Bdkrb1, and Masp2—were selected for validation by RT-qPCR. These genes were among the most significantly differentially expressed in the RNA-seq dataset and were functionally implicated in key biological processes relevant to this study: immune regulation (IL-10, IL-22ra1), inflammation (Bdkrb1), complement activation (Masp2), growth signaling (Igf2), epithelial function/metabolism (Anpep, Galnt6), and cell signaling (Rap1gap). As shown in Figure 9D, the RT-qPCR results corroborated the sequencing data, supporting the reliability of the transcriptomic findings.