Engineered Bacillus subtilis WB600/ZD reduces post-weaning diarrhea in piglets by modulating gut microbiota and aryl hydrocarbon receptor (AHR) signaling

Abstract

Post-weaning diarrhea (PWD) causes significant economic losses to the pig industry. A previous study demonstrated that engineered Bacillus subtilis WB600 expressing Zophobas atratus defensin (ZD), termed WB600/ZD, alleviates intestinal inflammation, modulates gut microbiota, and maintains redox homeostasis in Salmonella-challenged mice; however, the precise mechanisms remain unclear. In this study, a total of 50 weaned Landrace × Large White piglets at 21 d of age were assigned to four groups: healthy piglets fed standard diet (H group; 6.60 ± 0.48 kg, n = 15) or 2 × 10⁹ CFU/mL WB600/ZD (H + WB600/ZD group; 6.00 ± 0.68 kg, n = 15), and diarrheic piglets fed standard diet (PWD group; 6.51 ± 1.16 kg, n = 10) or 2 × 10⁹ CFU/mL WB600/ZD (PWD + WB600/ZD group; 6.91 ± 0.57 kg, n = 10). All groups received 7 d of treatment followed by 3 d of post-treatment monitoring. During the 10-d trial period, the body weight, feed intake per group, and diarrhea incidence were recorded. Results demonstrated that WB600/ZD reduced diarrhea incidence in both healthy (P < 0.001) and diarrheic piglets (P = 0.040). Additionally, WB600/ZD improved the growth performance, including final body weight (P = 0.017) and average daily gain (ADG; P = 0.007), without affecting average daily feed intake (ADFI; P = 0.907). Mechanistically, WB600/ZD increased the levels of serum glutathione peroxidase (GSH-Px; P = 0.014) and reduced myeloperoxidase (MPO; P < 0.001) and malondialdehyde (MDA; P < 0.001). Integrated fecal microbiota and metabolites showed that this protective effect of WB600/ZD was associated with gut microbiota-dependent tryptophan metabolism (P < 0.001). Furthermore, antibiotic-treated (pseudo-germ-free) mice receiving fecal microbiota transplantation (FMT) from WB600/ZD-treated piglets or administered the aryl hydrocarbon receptor (AHR) agonist 6-formylindolo[3,2-b]carbazole (FICZ) before Salmonella enterica subsp. enterica serovar Infantis (S. Infantis) challenge exhibited activation of the AHR/cytochrome P450 family 1 subfamily A member 1 (CYP1A1) signaling pathway (P = 0.022) and increased interleukin (IL)-22 secretion (P < 0.001), thereby alleviating S. Infantis infection. Overall, this study provides strong evidence that WB600/ZD is a promising antibiotic alternative for preventing PWD in newly weaned piglets.

1. Introduction

Weaning is one of the most important periods in a pig's life cycle (Pluske, 2016). The practice of early weaning in commercial operations, coupled with post-weaning dietary modifications, induces substantial perturbations in gut microbiota composition. These shifts increase host susceptibility to enteric pathogens such as Salmonella (Huang et al., 2024). Previous work has demonstrated that probiotic supplementation is a viable strategy to reduce Salmonella infections by modifying the porcine intestinal microbiota (Garvey et al., 2022; Yang et al., 2016, 2017; 2020; Zhang et al., 2018). However, the therapeutic efficacy of conventional probiotics is often limited, as exogenous probiotics struggle to compete with commensal bacteria and establish durable intestinal colonization (Stavropoulou and Bezirtzoglou, 2020).

Engineered probiotics are emerging as the next generation of live bio-therapeutic agents. These genetically modified microorganisms can be tailored to express specific proteins or modulate particular metabolic pathways, thereby exerting beneficial effects on host health (Aggarwal et al., 2020; Ma et al., 2022). A recent study has identified the Zophobas atratus defensin (ZD) as a potent natural antimicrobial peptide with significant bactericidal effects (Du et al., 2020). This peptide was expressed in both Escherichia coli and Bacillus subtilis (Wang et al., 2022). To enhance antibacterial activity, the peptide's amino acid sequence and structural properties were optimized via substitution and truncation. This optimized peptide was then validated in mouse models of E. coli-induced mastitis and skin burn wounds (Wang et al., 2022; Yang et al., 2024). A previous study discovered that the engineered probiotic WB600/ZD (an engineered B. subtilis strain, derived from the protease-deficient parental strain WB600, expressing the antimicrobial defensin peptide from Z. atratus, where “ZD” denotes the Z. atratus defensin) alleviates intestinal inflammation in mice caused by Salmonella enterica subsp. enterica serovar Infantis (S. Infantis) infection and significantly alters the colonic microbiota composition (Li et al., 2025).

The gut microbiota plays a crucial role in tryptophan metabolism, an essential amino acid pathway (Agus et al., 2018). This metabolic process generates aryl hydrocarbon receptor (AHR) agonists that support the development and maintenance of group 3 innate lymphoid cells (ILC3s) in the gut (Zhou and Sonnenberg, 2020). ILC3s function as counterparts to CD⁴⁺ T cells (cluster of differentiation 4 positive T lymphocyte) in adaptive immunity, and primarily produce interleukin (IL)-17 and IL-22. These cytokines are crucial for mucosal wound healing and antimicrobial peptides production (Song et al., 2020; Seillet and Jacquelot, 2019). The AHR/cytochrome P450 family 1 subfamily A member 1 (CYP1A1) signaling pathway, activated by gut microbiota tryptophan metabolism, is essential for maintaining intestinal homeostasis. This pathway regulates immune cell function, controls AHR signaling feedback, and maintains intestinal barrier integrity (Stockinger et al., 2021; Zhang et al., 2021).

In this study, a pseudo-germ-free mouse model was established using a quadruple antibiotic cocktail. To investigate whether WB600/ZD confers protection against S. Infantis infection by modulating gut microbiota and activating the AHR/CYP1A1 pathway, fecal microbiota transplantation (FMT) was conducted using feces from WB600/ZD-treated piglets. This hypothesis highlights the potential dual role of WB600/ZD in regulating microbes and enhancing host defense. This study aims to elucidate the health-promoting potential of WB600/ZD in regulating gut microbiota and enhancing host defense mechanisms against enteric pathogens.

2. Materials and methods

2.1. Animal ethics statement

All animal experiments adhered strictly to the Guidelines for Laboratory Animal Use and Care established by the Chinese Center for Disease Control and Prevention, as well as the Rules for Medical Laboratory Animals (1998) issued by the Chinese Ministry of Health. The animal experiments were conducted under a protocol approved by the Animal Ethics Committee of China Agricultural University (AW60904202-2-1).

2.2. Strain and bacterial cultures

S. Infantis CAU1508 was isolated from the intestinal contents of piglets with diarrhea (Yang et al., 2017). WB600/ZD was constructed and stored under standardized conditions in the laboratory (Li et al., 2025). Xylose Lysine Tergitol-4 Agar (XLT4, Qingdao Hope Bio-Technology Co., Ltd, Qingdao, Shangdong, China) was used for bacterial cultivation and isolation.

2.3. Animal experimental design

2.3.1. Piglet experimental design

A total of 50 newly weaned piglets at 21 d of age were randomized into two groups: 30 healthy piglets and 20 PWD piglets. Diarrheic piglets and healthy piglets were selected based on criteria from previous studies (Yang et al., 2016, 2017; 2020), in which diarrheic piglets were defined as those exhibiting watery diarrhea for at least two consecutive days, i.e., 2–3 d post-weaning at 21 d of age. Healthy piglets were defined as those that had never experienced diarrhea or other diseases. The housing conditions for piglets were maintained at 24–26 °C with 60% to 70% humidity and controlled ventilation to ensure optimal environmental conditions. For the administration of WB600/ZD to piglets via oral gavage, the procedure was performed once daily at 09:00 for 7 consecutive days. The 30 healthy piglets were further equally divided into two groups: 1) the healthy group fed a basal diet (H group, n = 15); and 2) the healthy + WB600/ZD group (H + WB600/ZD group) received oral gavage of 2 × 10⁹ CFU/mL WB600/ZD suspension while being fed the same basal diet (n = 15). The 20 diarrheal piglets were equally divided into two groups: 1) the post-weaning diarrhea (PWD) group fed a basal diet (n = 10); 2) the PWD + WB600/ZD group received oral gavage of 2 × 10⁹ CFU/mL WB600/ZD suspension while being fed the same basal diet (n = 10). These piglets did not receive any antibiotic treatment.

During the experiment, the body weight of all piglets was recorded on d 0, 2, 4, 6, 8, and 10. The ADG of the piglets was then calculated. The incidence and duration of diarrhea were documented. The formula for diarrhea rate is expressed as:

$$\text{Diarrhea}\text{rate}(\%)=\left[\text{Number}\text{of}\text{diarrhea}\text{incidents}\text{during}\text{the}\text{trial}\text{period}/\left(\text{Total}\text{number}\text{of}\text{piglets}\times \text{Trial}\text{duration}\text{in}\text{days}\right)\right]\times 100\text{.}$$

The feed intake of the piglets was measured daily. Blood samples of piglets were collected on d 0 and 8 for serum preparation and serum malondialdehyde (MDA), glutathione peroxidase (GSH-Px), and myeloperoxidase (MPO) detection. Fresh fecal samples were collected, divided into equal parts, immediately frozen, and stored at −80 °C for subsequent FMT, 16S rRNA sequencing, and metabolomics. In the FMT experiments, fecal samples were sourced from the PWD + WB600/ZD group and placed in physiological saline containing 10% sterile glycerol. The FMT experiment was performed as previously described (Zheng et al., 2019). Fresh donor piglet feces were suspended in physiological saline containing 10% sterile glycerol at a 1:10 (w/v) ratio. The suspension was homogenized and filtered through sterile mesh to remove large particulate matter. The mice did not undergo fasting prior to FMT administration.

2.3.2. 16S rRNA gene sequencing

The total genomic DNA was extracted from fecal samples of all piglets using the E.Z.N.A. Bacterial DNA Kit (Omega Biotek, Norcross, GA, USA) according to the manufacturer's instructions. The V3–V4 region of the 16S rRNA gene was amplified by PCR using universal primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). Subsequently, the PCR products were purified and used to construct sequencing libraries, followed by high-throughput sequencing on the Illumina MiSeq platform to generate paired-end sequencing data. Next, quality control, operational taxonomic units (OTU) clustering, and taxonomic annotation of the raw data were performed using QIIME2. Using default parameters, the quality-filtered and assembled sequences were processed for denoising using the DADA2 plugin in QIIME 2 version 2022.2 (Bolyen et al., 2019).

2.3.3. Untargeted metabolomics

Fecal metabolites were analyzed using a Thermo UHPLC-Q Exactive HF-X system equipped with an ACQUITY HSS T3 column (100 mm × 2.1 mm i.d, 1.8 μm; Waters Corp., Milford, MA, USA) at Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China), with mass spectrometric data acquisition performed using an electrospray ionization (ESI) source operating in both positive and negative ion modes. Partial least squares-discriminant analysis (PLS-DA) was performed using the R package “ropls" (Version 1.6.2). Metabolites with a variable importance in projection (VIP > 1) and P < 0.05 were considered different. Enrichment and pathway analysis of the differential metabolites were conducted based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/) using the Python package (SciPy. Stats, https://docs.scipy.org/doc/scipy/).

2.4. Diet formulation and nutritional analysis

The composition and nutritional components of the basal diet are provided in Table 1. The nutritional composition of the basal diet was analyzed according to the methods described by Hu et al. (2024). Gross energy (GE) content was determined using an oxygen bomb calorimeter (1108, Parr Instrument Co., Moline, Illinois, USA) following the standard method 983.23 (AOAC, 2007). The contents of crude protein (CP) were determined using a Kjeldahl automated apparatus (K9805, Shanghai Analytical Instrument Co., Ltd., Shanghai, China) following the method 976.06 (AOAC, 2007). Crude fiber (CF) level was analyzed using a fiber analyzer (Ankom Technology Corp., Macedon, NY, USA) according to Van Soest et al. (1991). Mineral contents, including phosphorus (P) and calcium (Ca), were analyzed using the 5110 Inductively coupled plasma optical emission spectrometer (ICP-OES, Agilent Technologies Australia (M) Pty. Ltd., Melbourne, Australia) following the methods 995.11 and 927.02 (AOAC, 2007). The contents of total lysine, methionine, and cysteine were determined using a high-performance liquid chromatography (HPLC) system (Waters Corp., Milford, MA, USA) following the method 982.30 (AOAC, 2007).

Table 1.

Formula and nutritional composition of weaned piglet feed in this study (air-dried basis, %).

Items	Content
Ingredients
Corn	19.22
Extruded corn	17.50
Wheat flour	25.00
Whey powder	7.50
Soybean oil	2.00
Extruded soybean	4.00
Soybean enzymolysis protein	5.00
Fermented concentrated feed	2.00
Fish meal	2.50
CaHPO4·2H2O	0.34
Acidifier	0.50
L-Lysine HCl (98.5%)	0.32
Feed-grade zinc oxide	0.12
Soy protein concentrate	5.00
Rice protein	5.00
Vitamin-mineral premix 1	4.00
Total	100.00
Nutrient composition2
Gross energy, MJ/kg	17.31
Crude protein	22.14
Crude fiber	2.62
Calcium	0.53
Phosphorus	0.67
Lysine	1.48
Methionine	0.59
Cystine	0.29

¹Vitamin-mineral premix feed provided the following per kilogram of diets: vitamin A 10,000 IU; vitamin D₃ 2000 IU; vitamin E 35 mg; vitamin K₃ 1.6 mg; vitamin B₁ 1.5 mg; vitamin B₂ 3.0 mg; vitamin B₆ 1.5 mg; vitamin B₁₂ 0.015 mg; niacin 25 mg; folic acid 2 mg; biotin 0.1 mg; Fe 120 mg; Cu 7 mg; Zn 75 mg; Mn 35 mg; I 0.5 mg; Se 0.3 mg.

²The nutritional components of the diet are all obtained from actual measurements.

2.5. Murine experimental design

Thirty C57BL/6 mice, aged 6 to 8 weeks and weighing 18 to 20 g, were randomly assigned to 5 groups (n = 6 per group). All downstream assays were performed in triplicate unless otherwise noted. To investigate the role of AHR in WB600/ZD-mediated protection in mice, AHR agonist 6-formylindolo[3,2-b] carbazole (FICZ) was used to activate AHR as a positive control and CH223191 (an AHR antagonist) during FMT to determine if the protective effects were AHR-dependent. To elucidate the mechanism by which WB600/ZD confers protection via microbiota-mediated activation of the AHR pathway, mice were randomly allocated into five groups: 1) CON group (Control): pseudo-germ-free mice receiving no further treatment; 2) S. Infantis infection group (SI group): pseudo-germ-free mice infected with S. Infantis to establish the disease model; 3) FMT + SI group: mice receiving fecal microbiota transplantation (FMT) from WB600/ZD-treated piglets prior to infection, to test the protective role of the modulated microbiota; 4) FICZ + SI group: mice treated with the AHR agonist FICZ before infection, serving as a positive control for AHR pathway activation; and 5) FMT + CH + SI group: mice receiving the AHR antagonist CH223191 concurrently with FMT to determine if the protection afforded by FMT is AHR-dependent. All mice were housed at the animal housing unit of China Agricultural University, Beijing, China. From d 1 to 7 of the experiment, all mice were administered 200 μL of antibiotic mixture (0.5 g/L vancomycin, 1 g/L ampicillin, 1 g/L neomycin, and 1 g/L metronidazole) twice daily via gavage to deplete the gut microbiota (Tirelle et al., 2020). After 3 d of gavage, fecal samples from mice were collected daily for colony plate counting. A reduction in colony counts of more than 90% compared to the CON group confirmed the successful establishment of the pseudo-germ-free model. From d 8 to 14, the CON group and SI group were daily gavaged with 200 μL of sterile phosphate buffer saline (PBS), the FMT + SI group received FMT treatment, the FICZ + SI group was injected with 1 μg of FICZ solution, and the FMT + CH + SI group underwent FMT and intraperitoneal injection of 10 mg/kg of CH223191. On d 15, the SI group, FMT + SI group, FICZ + SI group, and FMT + CH + SI group were gavaged with an S. Infantis suspension at a concentration of 1 × 10⁷ CFU/mL. On d 18, the mice were euthanized, and samples (blood, the colon, liver, and spleen) were collected. The tissue weight and colon length were measured. The body weight, fecal score, and overall behavioral conditions of each animal were recorded throughout the experimental period.

2.5.1. Fecal scoring and bacterial load

Approximately 0.5 g of fecal samples were collected, dried at 60 °C for 24 h, and weighed. The dry matter ratio of feces was calculated by comparing the dried weight to the initial weight. At 8, 24, 48, and 72 h post-infection, 0.5 g of fecal samples was collected, fecal scores were classified as follows: 0 to 1 (normal), 2 to 3 (soft or shapeless), and 4 to 5 (loose or watery stools). A 10% (w/v) homogenate was prepared using sterile PBS. Subsequently, the original homogenate and samples were diluted by 10-, 100-, and 1000-fold, and 100 μL of each dilution was spread onto XLT4 agar. After overnight incubation at 37 °C, Salmonella colonies were enumerated. The limit of detection via plating was 10 CFU/g feces.

2.5.2. Molecular and histological analyses

2.5.2.1. Real-time quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted from the ileal tissues of mice using TRIzol reagent according to the manufacturer's protocol (9109, Takara Biomedical Technology (Beijing) Co., Ltd., Beijing, China). Subsequently, complementary DNA (cDNA) was synthesized from the extracted RNA samples using a reverse transcription reagent kit (AE101–02, Beijing TransGen Biotech Co., Ltd., Beijing, China). RT-qPCR was performed in a 20 μL reaction volume using TransScript Green One-Step RT-qPCR SuperMix (AQ211, Beijing TransGen Biotech Co., Ltd., Beijing, China). and specific primers on an Applied Biosystems 7500 Fast Real-Time PCR System. The quantification of gene expression levels was performed utilizing the 2^−ΔΔCt method, which involved normalization to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primer sequences (Table S1) were designed via the National Center for Biotechnology Information (NCBI) Primer-Blast and synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). RT-qPCR was performed under the following conditions: an initial holding stage (94°C for 30 s), 40 amplification cycles (94 °C for 5 s, 60 °C for 15 s, 72 °C for 10 s), and a final melt curve analysis (95 °C for 15 s, 60 °C for 60 s, and 95 °C for 30 s).

2.5.2.2. Western blotting

The following antibodies were used for Western blot: AHR polyclonal antibody (AF6278, 1:500; Affinity Biosciences Ltd., Nanjing, China), CYP1A1 polyclonal antibody (AF5312, 1:500; Affinity Biosciences Ltd.), β-actin monoclonal antibody (66009-1-Ig, 1:8000; Proteintech Group, Inc., Wuhan, Hubei, China), claudin-1 polyclonal antibody (28674-1-AP, 1:5000; Proteintech Group, Inc.), occludin polyclonal antibody (27260-1-AP, 1:5000; Proteintech Group, Inc.).

The ileal tissues were homogenized in ice-cold RIPA lysis buffer containing 1 mmol/L phenylmethylsulfonyl fluoride (PMSF). Subsequently, the supernatant was obtained by centrifugation (12,000 × g, 4 °C, 15 min), and protein concentration was quantified using a Bicinchoninic Acid Assay (BCA) Protein Assay Kit (Beyotime Biotechnology, Inc., Shanghai, China). All samples were loaded with equal amounts of protein and separated by electrophoresis in sodium dodecyl sulfate (SDS) polyacrylamide gels and then transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with skim milk for 1.5 h, incubated with primary antibody overnight at 4 °C, and subsequently with a secondary peroxidase-labeled antibody for 1 h. Protein bands were visualized using a Tanon 5200 Chemiluminescent Imaging System (Shanghai, China). Band intensity was quantified with ImageJ 1.54f (National Institutes of Health, Bethesda, MD, USA).

2.5.2.3. Serum indicators detection

Blood samples were collected from mice and centrifuged at 4000 × g for 10 min to obtain the serum. Serum MDA (BC0025, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), MPO (BC5175, Beijing Solarbio Science & Technology Co., Ltd.,), GSH-Px (BC1195, Solarbio), and IL-22 (JL20258, Jonlnbio Industrial Co., Ltd., Shanghai, China) were measured using commercial kits following the manufacturer's instructions. Absorbance was measured using a Multiskan FC microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

2.5.2.4. Immunohistochemical staining

The immunohistochemistry staining (IHC) protocol was consistent with a previous study (Li et al., 2025). 3,3′-Diaminobenzidine (DAB), Horseradish Peroxidase Color Development Kit and enzyme-labeled goat anti-rabbit IgG polymer were obtained from Beijing Zhongsui Jinqiao Biotechnology Co., Ltd. (PV6001, Beijing, China). The colon and ileum tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 3 μm-thick slices. After dewaxing and rehydrating, tissue sections underwent antigen retrieval in citrate buffer (pH = 6) at 100 °C for 20 min in a steaming device. After blocking with 5% bovine serum albumin (BSA) for 30 min at room temperature, tissue sections were incubated overnight at 4 °C with rabbit polyclonal antibodies against mucin 2 (MUC2). Subsequently, a histochemical secondary antibody kit (PV6001; Beijing Zhongsui Jinqiao Biotechnology Co., Ltd., Beijing, China) was used for 1 h at room temperature. Finally, coverslips were applied by mounting with neutral resin. Microscopic observation was performed using an Olympus CX31 optical microscope (Evident Scientific, Tokyo, Japan), and images were captured with OVT view 3.7 software (OVT Software Technology Co., Ltd., Jiangsu, China).

2.5.2.5. Immunofluorescence staining

Zonula occludens-1 (ZO-1) expression in the ileum was detected by immunofluorescence staining. Staining was performed with rabbit polyclonal antibodies against ZO-1 (21773-1-AP, 1:500; Proteintech Group, Inc., Wuhan, Hubei, China), with tissue sections incubated overnight at 4 °C. The secondary antibody was detected using Alexa Fluor 555-labeled donkey anti-rabbit IgG (H + L) (A0453; Beyotime Biotechnology Inc., Shanghai, China), incubated for 1 h at room temperature. 4′,6-Diamidino-2′-phenylindole (DAPI) was incubated at room temperature for 10 min to stain the nuclei. Coverslips were mounted with an anti-fade mounting medium. Images were acquired using a TCS SP8 laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany).

2.5.2.6. Pathological assessment

Tissue sections were blindly scored based on the severity of lesions from 0 to 4. The scoring criteria were as follows: 0 = no visible damage; 1 = minor lesions with mild edema and epithelial cell detachment; 2 = mild lesions with inflammation and some inflammatory cell infiltration around blood vessels; 3 = moderate lesions with more inflammatory cell infiltration and substantial structural damage around blood vessels; and 4 = severe lesions with intense inflammatory cell infiltration and severe structural damage around blood vessels. Mice without S. Infantis infection were used as negative controls.

2.6. Statistical analysis

All microbial analysis methods were conducted on the Majorbio Cloud Platform (https://cloud.majorbio.com). Specifically, alpha diversity indexes were calculated using mother (Schloss et al., 2009), with Wilcoxon rank-sum tests employed to assess intergroup differences in alpha diversity. Beta diversity analysis was performed using Principal Coordinates Analysis (PCoA) based on Bray–Curtis distance metrics to evaluate microbial community structural similarities among samples. Additionally, Linear discriminant analysis effect size (LEfSe) analysis (Segata et al., 2011) was applied to identify bacterial taxa showing significant abundance differences (LDA score > 2, P < 0.05).

Statistical analysis and figure generation were conducted using GraphPad Prism 9.2.0. Differences were evaluated using Tukey's multiple comparisons test. Data are presented as means ± standard deviation (SD). The results are considered statistically significant at P < 0.05. For the data from both piglet and mouse experiments (including growth performance, serum biochemical parameters, cytokine levels, and gene/protein expression), statistical analysis was performed using a one-way analysis of variance (ANOVA). The statistical model is expressed as:

$$Y_{ij}=\mu +{\tau }_i+{\epsilon }_{ij}\text{,}$$

where Y_ij represents the observed value for the dependent variable; μ is the overall mean; τ_i is the fixed effect of the i-th treatment group (The treatments included Healthy, H + WB600/ZD, PWD, and PWD + WB600/ZD in the analysis of pig data; CON, SI, FMT + SI, FICZ + SI, and FMT + CH + SI in analysis of mice data); and ε_ij is the random error term.

Fecal dry/wet weight ratio and Salmonella load were subjected to two-way analysis of variance (ANOVA). To satisfy parametric assumptions, Salmonella loads reported below the limit of detection (1.0, log_₁₀ 10 CFU/g) were assigned the value of the detection limit for statistical analysis.The statistical model is as follows:

$$Y_{ijk}=\mu +{\alpha }_i+{\beta }_j+{\left(\alpha \beta \right)}_{ij}+{\epsilon }_{ijk}\text{,}$$

where Y_ijk represents the observed value for the k-th mouse in the i-th treatment group at the j-th time point; μ is the overall mean, α_i is the fixed effect of the i-th treatment group, including CON, SI, FMT + SI, FICZ + SI, and FMT + CH + SI; β_j is the fixed effect of the j-th time point; (αβ)_ij is the interaction effect between the treatment and time; and ε_ijk is the random error term.

3. Results

3.1. WB600/ZD alleviates weaning stress in newly weaned piglets

This study first validated the efficacy of WB600/ZD for preventing diarrhea and improving growth performance in post-weaning piglets. WB600/ZD significantly reduced the incidence of diarrhea in both healthy (P < 0.001) and diarrheic groups (P = 0.040) (Table 2). By d 10, the body weight of the PWD + WB600/ZD piglets was significantly higher than that of the PWD piglets (P = 0.017, Table 3). WB600/ZD increased the ADG (P = 0.007), but had no significant effect on the ADFI (P = 0.907). Notably, WB600/ZD enhanced anti-inflammatory and antioxidant capability, as evidenced by reduced levels of MDA (P < 0.001) and MPO (P < 0.001), and elevated GSH-Px (P = 0.014) activity in diarrheic piglets (Table 3). In summary, WB600/ZD ameliorated post-weaning diarrhea, improved growth performance, and modulated oxidative/inflammatory biomarkers in post-weaning piglets.

Table 2.

The impact of WB600/ZD on the incidence of diarrhea in piglets.

Groups1	At risk (n) 2	With diarrhea		Significance
		(n) 3	Diarrhea index,%
PWD	100	37	37	P = 0.040
PWD + WB600/ZD	100	23	23
H	150	45	30	P < 0.001
H + WB600/ZD	150	18	12

H = healthy; PWD = post-weaning diarrhea.

WB600/ZD, is an engineered Bacillus subtilis strain (derived from the protease-deficient parental strain WB600) expressing the antimicrobial defensin peptide from Zophobas atratus, where “ZD” denotes the Zophobas atratus defensin.

¹The healthy and PWD, groups were fed a standard diet; the other two groups were fed a trial diet supplemented with 2 × 10⁹ colony-forming units (CFU)/mL of WB600/ZD, bacterial suspension in their standard feed for 7 d.

²The number at risk indicates the total number of piglets per day for a period of time.

³The sum of the number of piglets with diarrhea during this period is considered to have diarrhea.

Table 3.

Effects of WB600/ZD on growth performance and antioxidant capacity in piglets ¹.

Items	H	H + WB600/ZD	PWD	PWD + WB600/ZD	P-value
Initial body weight, kg	6.60 ± 0.48	6.00 ± 0.68	6.51 ± 1.16	6.91 ± 0.57	0.173
Final body weight, kg	8.09 ± 0.60b	7.85 ± 0.49b	7.93 ± 1.06b	8.78 ± 0.63a	0.017
ADG, g	173.85 ± 74.37a	186.92 ± 67.87a	101.40 ± 37.55b	184.00 ± 31.05a	0.007
ADFI, g	172.13 ± 25.61	167 ± 29.99	159.38 ± 34.96	161.75 ± 33.50	0.907
MDA, nmol/mL	0.86 ± 0.07b	0.83 ± 0.05b	2.46 ± 0.19a	0.95 ± 0.09b	<0.001
MPO, U/mL	0.42 ± 0.04b	0.49 ± 0.02b	0.76 ± 0.03a	0.56 ± 0.03c	<0.001
GSH-Px, U/mL	148.96 ± 1.24a	124.35 ± 7.07a	83.51 ± 10.45c	111.41 ± 4.58b	<0.001

H = healthy; PWD = post-weaning diarrhea; MDA = malondialdehyde; MPO = myeloperoxidase; GSH-Px = glutathione peroxidase.

The healthy and PWD groups were fed a standard diet; the other two groups were fed a trial diet supplemented with 2 × 10⁹ CFU/mL of WB600/ZD, bacterial suspension in their standard feed for 7 d.

WB600/ZD, is an engineered Bacillus subtilis strain (derived from the protease-deficient parental strain WB600) expressing the antimicrobial defensin peptide from Zophobas atratus, where “ZD” denotes the Zophobas atratus defensin.

Values with different superscript lower-case letters within the same row are significantly different (P < 0.05).

¹The data are presented as means ± SD (n = 15 for groups H and H + WB600/ZD; n = 10 for groups PWD, and PWD + WB600/ZD).

3.2. WB600/ZD modulated the fecal microbiota composition in newly weaned piglets

WB600/ZD significantly modulated the fecal microbiota composition in newly weaned piglets, including both healthy and diarrheal piglets. The PCoA results indicated that supplementation with WB600/ZD altered the fecal microbiota structure in both healthy and diarrheal piglets (Fig. 1A). Compared with the PWD group, WB600/ZD treatment notably increased α-diversity, as evidenced by increases in the Chao1 (P = 0.017), Shannon (P = 0.008), and Abundance-based Coverage Estimator (ACE) (P = 0.020) indexes, reflecting increased microbial richness and diversity (Fig. 1B–D). At the phylum level, diarrheal piglets exhibited a decrease in the abundance of Bacteroidetes and an increase in the abundance of Firmicutes compared to healthy piglets (Fig. 1E). This imbalance was alleviated after WB600/ZD supplementation in diarrheic piglets. At the genus level, the abundance of Ruminococcus (P = 0.013), UCG-002 (P = 0.020), phascolarctobacterium (P = 0.031), and Lachnoclostridium (P = 0.045) in diarrheic piglets was significantly reduced compared to healthy piglets (Fig. 1F and G). WB600/ZD supplementation reduced the abundance of pathogenic Streptococcus and Escherichia-Shigella in diarrheic pigs (Fig. 1H and I). Overall, WB600/ZD has a significant regulatory effect on the fecal microbiota of both healthy and diarrheic piglets, improving the composition and function of the intestinal microbiota and providing beneficial effects on host health.

Fig. 1.

The effects of WB600/ZD on the fecal microbiota of healthy and post-weaning diarrhea (PWD) piglets. (A) Principal coordinates analysis (PCoA) based on weighted UniFrac analysis. (B-D) Alpha diversity indexes: Chao1, Shannon, and abundance-based coverage estimator (ACE). (E) Relative abundance of fecal microbiota at the phylum level. (F) Community heatmap. (G) Wilcoxon rank-sum test bar plot at the genus level between the healthy and the diarrheic piglets. (H) Wilcoxon rank-sum test bar plot at the genus level between the PWD and the PWD + WB600/ZD piglets. (I) Linear discriminant analysis effect size (LEfSe) at the genus level. WB600/ZD is an engineered Bacillus subtilis strain (derived from the protease-deficient parental strain WB600) expressing the antimicrobial defensin peptide from Zophobas atratus, where “ZD” denotes the Zophobas atratus defensin. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

3.3. WB600/ZD supplementation altered the gut microbiota metabolome

Microorganisms in the gut catabolize, transform, and synthesize nutrients ingested by the host through metabolic activity, producing a wide range of metabolites that influence host health (Flint et al., 2012; Li et al., 2024). The PLS-DA analysis indicated that WB600/ZD supplementation altered the composition of fecal metabolites in both healthy and diarrheal piglets (Fig. 2A). Diarrheal piglets exhibited an increase of 164 metabolites and a decrease of 316 metabolites compared to healthy piglets, and reduced metabolites are involved in tryptophan metabolism (P = 0.006) and primary bile acid biosynthesis (P < 0.001, Fig. 2B–E). After supplementation with WB600/ZD, diarrheal piglets experienced an increase of 820 metabolites and a decrease of 99 metabolites, and decreased metabolites were involved in tryptophan metabolism (P < 0.001) and primary bile acid biosynthesis (P = 0.029, Fig. 2D and E). Variable importance in projection (VIP) analysis and KEGG enrichment also confirmed that the WB600/ZD modulated tryptophan metabolism and primary bile acid biosynthesis (Fig. 2F–I). These results suggest that the anti-diarrheal mechanism of WB600/ZD involves microbiota-metabolite crosstalk.

Fig. 2.

The effects of WB600/ZD on the fecal metabolites of newly weaned piglets. (A) Partial least squares discriminant analysis (PLS-DA). (B) Altered metabolites between diarrheic pigs and healthy piglets. (C) Altered metabolites between diarrheic pigs with and without WB600/ZD treatment. The x-axis represents the logarithmic fold change of the metabolites (log₂FC), and the y-axis represents the negative logarithm of the P-value. (D, E) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis between diarrhea piglets versus healthy, diarrhea piglets versus diarrhea piglets treated with WB600/ZD. (F, G) KEGG topology analysis and variable importance in the projection (VIP) values of fecal metabolites in diarrheic and healthy piglets. (H, I) KEGG topology analysis and VIP values of fecal metabolites in diarrhea and diarrhea piglets treated with WB600/ZD. The P-value shown denote statistical significance for all pairwise comparisons between groups. WB600/ZD is an engineered Bacillus subtilis strain (derived from the protease-deficient parental strain WB600) expressing the antimicrobial defensin peptide from Zophobas atratus, where “ZD” denotes the Zophobas atratus defensin. PWD = post-weaning diarrhea; FC = fold change. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

3.4. Integrated microbiota-metabolite correlation analysis

Next, this study explored the relationship between fecal microbiota and metabolites by correlation analysis. Procrustes analysis revealed a significant correlation between microbiota and metabolites (P = 0.001, Fig. 3A). Compared to diarrheic piglets, the dominant fecal bacteria in healthy piglets including Ruminococcus, UCG-002, and Lachnoclostridium, showed significant positive correlations with most tryptophan metabolites, such as 2-amino-3-methoxybenzoic acid, 5-hydroxy-L-tryptophan, and 8-methoxyuric acid (Fig. 3B). After supplementation with WB600/ZD, the dominant gut microbiota in diarrheic piglets was found to elevate a variety of tryptophan metabolites, including kynurenic acid, 5-hydroxyindole-3-acetic acid, indole-3-acetic acid, and 2-aminomuconic acid (P = 0.046, Fig. 3C). These findings suggest the health-promoting effects of WB600/ZD on piglet diarrhea may be mediated through the modulation of host intestinal tryptophan metabolism.

Fig. 3.

Correlation analysis of tryptophan-involved metabolites and dominated gut microbiota from piglet feces. (A) Procrustes analysis was used to examine the correlation between the overall microbial community and metabolites. Pearson correlation coefficient analysis was conducted to examine the associations between dominant bacterial taxa and metabolites. (B) Correlation analysis between differential metabolites and bacteria at the genus level in newly weaned piglets without treatment (PWD vs. Healthy). (C) Correlation analysis between differential metabolites and bacteria at the genus level in newly weaned piglets treated with WB600/ZD (PWD + WB600/ZD vs. PWD). WB600/ZD is an engineered Bacillus subtilis strain (derived from the protease-deficient parental strain WB600) expressing the antimicrobial defensin peptide from Zophobas atratus, where “ZD” denotes the Zophobas atratus defensin. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

3.5. WB600/ZD-modulated gut microbiota prevented the suppression of the AHR/CYP1A1 pathway caused by S. Infantis

Fecal microbiota transplantation experiments in pseudo-germ-free mice demonstrated that the alleviation of S. Infantis-induced intestinal inflammation and the activation of the AHR/CYP1A1 pathway by WB600/ZD were dependent on the gut microbiota it modulated. The murine experimental design is shown in Fig. 4A. Results showed that S. Infantis infection downregulated AHR and CYP1A1 expression at both the protein (P < 0.001, P = 0.008) and mRNA (P = 0.048, P = 0.013) levels in the mouse ileum (Fig. 4B–D). This suppression was reversed in mice that received FMT prior to S. Infantis infection, which was consistent with the effects of the AHR agonist FICZ. As expected, the AHR inhibitor (CH223191) pretreatment reduced FMT-mediated activation of the pathway (P < 0.001). These findings indicate that WB600/ZD exerts its effects through the microbiota-AHR pathway.

Fig. 4.

WB600/ZD-modulated gut microbiota reactivated the AHR/CYP1A1 pathway in response to Salmonella enterica subsp. enterica serovar Infantis (S. Infantis). (A) The experimental design of a mouse model. (B) Ileal AHR and CYP1A1 protein expression by Western blot. (C, D) The mRNA expression of AHR and CYP1A1 in the ileum. The P-values shown denote statistical significance for all pairwise comparisons between groups. WB600/ZD is an engineered Bacillus subtilis strain (derived from the protease-deficient parental strain WB600) expressing the antimicrobial defensin peptide from Zophobas atratus, where “ZD” denotes the Zophobas atratus defensin. FMT = fecal microbiota transplantation; SI = S. Infantis infection ; CH CH223191. ABX = antibiotic mixture; FICZ = 6-formylindolo[3,2-b]carbazole; PBS = phosphate buffer saline; PWD = post-weaning diarrhea; AHR = aryl hydrocarbon receptor; CYP1A1 = cytochrome P450 family 1 subfamily A member 1; CFU = colony-forming unit.

3.6. WB600/ZD-modulated gut microbiota protected against S. Infantis infection via AHR

Fecal microbiota transplantation prior to S. Infantis infection improved fecal consistency and decreased fecal water content and S. Infantis shedding (P < 0.001, Table 4). While FICZ alleviated the clinical symptoms induced by S. Infantis infection, it did not reduce the fecal shedding of S. Infantis (Table 4). S. Infantis infection significantly elevated the liver (P = 0.008) and spleen (P < 0.001) indexes in mice. However, FMT following the administration of CH223191 did not alleviate the clinical manifestations induced by S. Infantis infection (Table 5). The effect of FMT pretreatment on the oxidative stress and inflammatory response induced by S. Infantis infection was further investigated. Pretreatment with FMT or FICZ alleviated Salmonella infection in mice. However, the protective effect of FMT on mice was inhibited by the prior injection of CH223191. These results indicate that WB600/ZD activated the AHR/CYP1A1 pathway in a gut microbiota-dependent manner, thereby improving the clinical manifestations of S. Infantis infection in mice.

Table 4.

The effects of fecal microbiota transplantation (FMT) on fecal scores and Salmonella load in mouse model ¹.

Items	CON	SI	FMT + SI	FICZ + SI	FMT + CH + SI	P-value
Fecal dry/wet weight ratio, %
0 h	0.45 ± 0.01	0.46 ± 0.01	0.46 ± 0.02	0.44 ± 0.01	0.45 ± 0.01	0.318
8 h	0.47 ± 0.02a	0.28 ± 0.01c	0.37 ± 0.01b	0.38 ± 0.01b	0.34 ± 0.02 bc	<0.001
24 h	0.47 ± 0.02a	0.34 ± 0.01c	0.40 ± 0.01b	0.44 ± 0.01a	0.39 ± 0.01 bc	<0.001
48 h	0.49 ± 0.01a	0.28 ± 0.01c	0.38 ± 0.02b	0.46 ± 0.01a	0.25 ± 0.01c	<0.001
72 h	0.42 ± 0.01a	0.26 ± 0.02b	0.41 ± 0.01a	0.38 ± 0.01a	0.31 ± 0.04b	<0.001
P-value (Two-way ANOVA)
Treatment 2, <0.001; Time, <0.001; interaction (Treatment × Time), <0.001.
Fecal Salmonella load3, log10(CFU/g)
8 h	<1.00c	7.81 ± 0.09a	5.49 ± 0.16b	6.26 ± 0.57b	6.20 ± 0.42b	<0.001
24 h	<1.00d	7.08 ± 0.17a	4.26 ± 0.37c	5.37 ± 0.10b	7.24 ± 0.11a	<0.001
48 h	<1.00d	7.88 ± 0.19b	4.36 ± 0.25c	4.75 ± 0.07c	8.08 ± 0.04a	<0.001
72 h	<1.00c	6.79 ± 0.07a	0c	5.52 ± 0.39b	6.74 ± 0.03a	<0.001
P-value (Two-way ANOVA)
Treatment 2, <0.001; Time, <0.001; interaction (Treatment × Time), <0.001.

Values with different superscript lower-case letters within the same row are significantly different (P < 0.05).

¹Data are presented as means ± SD, from n = 6 biologically independent mice per group. CON, control; SI, S. Infantis infection; FMT, fecal microbiota transplantation; FICZ, 6-formylindolo[3,2-b]carbazole; CH, CH223191 (AHR, antagonist).

²The treatments included CON, SI, FMT + SI, FICZ + SI, and FMT + CH + SI, in analysis of mice data.

³Limit of detection (LOD): Values below the LOD are denoted as “< 1.00” and were assigned the LOD value for statistical analysis.

Table 5.

Fecal microbiota transplantation (FMT) and aryl hydrocarbon receptor (AHR) activation protect against S. Infantis infection ¹.

Items	CON	SI	FMT + SI	FICZ + SI	FMT + CH + SI	P-value
GSH–Px, U/mL	249.52 ± 6.12a	184.83 ± 9.15b	269.82 ± 23.32a	239.38 ± 9.67a	210.96 ± 17.11b	0.001
MDA, nmol/mL	1.72 ± 0.13b	2.71 ± 0.11a	1.79 ± 0.11b	1.78 ± 0.08b	2.29 ± 0.28a	<0.001
MPO, U/mL	0.55 ± 0.02b	0.80 ± 0.06a	0.52 ± 0.03b	0.57 ± 0.02b	0.78 ± 0.03a	<0.001
IL-22, pg/mL	133.11 ± 9.39a	57.34 ± 14.83b	100.30 ± 9.58a	140.26 ± 17.43a	29.30 ± 7.35b	<0.001
Liver index, %	3.76 ± 0.05c	4.31 ± 0.11b	3.95 ± 0.02c	3.88 ± 0.35c	4.51 ± 0.19a	<0.001
Spleen index, mg/g	0.029 ± 0.002b	0.034 ± 0.001a	0.030 ± 0.001b	0.032 ± 0.001b	0.037 ± 0.002a	<0.001

GSH-Px = glutathione peroxidase; MDA = malonaldehyde; MPO = myeloperoxidase; IL-22 = interleukin-22.

Statistical analysis was performed by one-way ANOVA, followed by Tukey's multiple comparisons test.

Values with different superscript lower-case letters within the same row are significantly different (P < 0.05).

¹The data are presented as means ± SD, from n = 6 biologically independent mice per group. CON, control; SI, S. Infantis infection; FMT, fecal microbiota transplantation; FICZ, 6-formylindolo[3,2-b]carbazole; CH, CH223191 (AHR, antagonist).

Compared to the S. Infantis group, pretreatment with FMT resulted in decreased levels of serum MDA (P < 0.001) and MPO (P < 0.001), and increased levels of GSH-Px (P = 0.008, Table 5). Additionally, pre-treatment with FMT suppressed the expression of pro-inflammatory cytokine genes tumor necrosis factor-α (TNF-α; P = 0.004), IL-6 (P = 0.017), and IL-1β (P < 0.001) in the ileum (Fig. 5A–C). These findings are consistent with the results observed after FICZ treatment. However, the protective effects of FMT were abolished when CH223191 was administered in advance. Moreover, histopathological scores demonstrated that FMT alleviated S. Infantis-induced inflammatory cell infiltration in the ileum and liver, disruption of ileal villi, and disorganization of hepatic cord structure (P < 0.001, Fig. 5D and E). In summary, WB600/ZD-remodeled gut microbiota can inhibit the inflammatory response induced by Salmonella and help restore the body's redox balance.

Fig. 5.

WB600/ZD-modulated gut microbiota protected against Salmonella enterica subsp. enterica serovar Infantis (S. Infantis) infection via AHR in mice. (A-C) RT-qPCR detection of the mRNA expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1β in the ileum. (D) Histopathological examination of the liver and ileum. (E) Histopathological scores in the ileum and liver. All data are presented as means ± SD from at least three independent experiments. The P-values shown denote statistical significance for all pairwise comparisons between groups. WB600/ZD is an engineered Bacillus subtilis strain (derived from the protease-deficient parental strain WB600) expressing the antimicrobial defensin peptide from Zophobas atratus, where “ZD” denotes the Zophobas atratus defensin. FMT = fecal microbiota transplantation; SI = S. Infantis infection; CH CH223191. ABX = antibiotic mixture; FICZ = 6-formylindolo[3,2-b]carbazole; PWD = post-weaning diarrhea; AHR = aryl hydrocarbon receptor; CYP1A1 = cytochrome P450 family 1 subfamily A member 1.

3.7. WB600/ZD-modulated gut microbiota protected the intestinal barrier integrity from S. Infantis infection dependent on AHR

In the ileum, immunohistochemistry results demonstrated that S. Infantis infection reduced the expression of MUC2, whereas treatment with FMT and FICZ alleviated this phenomenon (P < 0.001). However, FMT following the administration of CH223191 did not increase the expression of MUC2 (P < 0.001, Fig. 6A, Table 6). Immunofluorescence results indicated that FMT could enhance the expression of tight junction protein ZO-1, which is consistent with the effects of FICZ treatment (Fig. 6B). Similarly, Western blot analysis showed that occludin and claudin-1 in the S. Infantis group were significantly decreased compared with the CON group (P < 0.001 and P = 0.004, Fig. 6C). Pretreatment with FMT significantly elevated the expression of tight junction proteins in the ileum (P = 0.014 and P = 0.030), consistent with the effects of FICZ (P = 0.044 and P = 0.047). Additionally, FMT significantly preserved the expression of MUC2 (P < 0.001), trefoil factor 3 (TFF3; P = 0.026), and galactose-3-O-sulfotransferase 2 (GAL3ST2; P < 0.001), which were reduced by S. Infantis infection (P < 0.001, Fig. 6D–F). Collectively, FMT protected the intestinal barrier integrity during S. Infantis infection in an AHR-dependent manner, as CH223191 abrogated FMT-mediated protection.

Fig. 6.

WB600/ZD-FMT and AHR activation alleviate ileal barrier damage caused by Salmonella entericasubsp. entericaserovar Infantis(S.Infantis) infection. (A) Immunohistochemical detection of mucins2 (MUC2) expression in the ileum (the black arrow). (B) Immunofluorescence detection of zonula occludens-1 (ZO-1) expression in the ileum. (C) Claudin-1 and occludin expression in the ileum by Western blot assays. (D-F) RT-qPCR detection of the expression of MUC2 and mucin-related genes trefoil factor 3 (TFF3) and Galactose-3-O-sulfotransferase 2 (GAL3ST2) in the ileum. All data are presented as means ± SD from at least three independent experiments. The P-values shown denote statistical significance for all pairwise comparisons between groups. WB600/ZD is an engineered Bacillus subtilis strain (derived from the protease-deficient parental strain WB600) expressing the antimicrobial defensin peptide from Zophobas atratus, where “ZD” denotes the Zophobas atratus defensin. FMT = fecal microbiota transplantation; SI = S. Infantis infection; CH CH223191. ABX = antibiotic mixture; FICZ = 6-formylindolo[3,2-b]carbazole; PWD = post-weaning diarrhea; AHR = aryl hydrocarbon receptor; CYP1A1 = cytochrome P450 family 1 subfamily A member 1.

Table 6.

Statistical analysis of mucin 2 (MUC2) immunohistochemistry in mouse ileum ¹.

Item	CON	SI	FMT + SI	FICZ + SI	FMT + CH + SI	P-value
IOD/Area 2	17.06 ± 0.19a	2.34 ± 0.91b	15.58 ± 0.95a	15.31 ± 1.60a	1.73 ± 0.47b	<0.001

Values with different superscript lower-case letters within the same row are significantly different (P < 0.05).

¹The data are presented as means ± SD, from n = 6 biologically independent mice per group. CON, control; SI, Salmonella enterica subsp. enterica serovar Infantis infection; FMT, fecal microbiota transplantation; FICZ, 6-formylindolo[3,2-b]carbazole; CH, CH223191 (AHR, antagonist).

²Integrated optical density (IOD)/Area represents relative optical density values, indicating staining intensity per unit area.

4. Discussion

This study demonstrates that the engineered probiotic WB600/ZD effectively improves gut health and growth performance in weaned piglets by remodeling gut microbiota and activating the tryptophan-AHR/CYP1A1 metabolic pathway, while also exhibiting anti-inflammatory effects in an S. Infantis-induced ileitis mouse model.

The weaning period represents a critical stage in pig production, during which piglets experience gut microbiota dysbiosis and underdeveloped intestinal barrier function, increasing susceptibility to diseases like diarrhea that compromise breeding efficiency (Luo et al., 2022; Zhu et al., 2022). Conventional probiotics often demonstrate limited efficacy due to poor colonization capacity (Mathipa and Thantsha, 2017; Huang et al., 2022). As a novel engineered probiotic, WB600/ZD addresses these limitations through enhanced functionality.

This study showed that the engineered probiotic WB600/ZD significantly increased the ADG of healthy weaned piglets, demonstrating its growth-promoting effects. Mechanistically, WB600/ZD exerted antioxidant activity by elevating GSH-Px activity and decreasing MPO and MDA levels. WB600/ZD combated oxidative stress through three synergistic mechanisms: boosting GSH-Px activity to scavenge free radicals, reducing MPO to suppress inflammatory oxidative bursts, and lowering MDA to preserve membrane integrity. Compared to the PWD group, WB600/ZD supplementation increased the α-diversity of fecal microbiota in diarrheic piglets, suggesting its potential to enhance disease resistance by restoring gut microecological balance. Microbial composition analysis showed that WB600/ZD effectively normalized the PWD-induced elevation in Firmicutes abundance. This normalization may contribute to reduced intestinal inflammation and improved barrier function. Notably, WB600/ZD significantly decreased the relative abundance of potential pathogens Streptococcus and Escherichia-Shigella, directly reducing infection risk. These findings indicate that WB600/ZD alleviates PWD and restores intestinal homeostasis through multi-target regulation of gut microbiota, including enhancing diversity, correcting dysbiosis, and suppressing opportunistic pathogens. These findings provide a theoretical foundation for developing microbiome-based PWD control strategies.

Integrated fecal microbiota and metabolites analysis identified tryptophan metabolism as an important mediator of WB600/ZD in enhancing the intestinal barrier function. Tryptophan, an essential amino acid, is metabolized by the gut microbiota into various bioactive compounds influencing host immunity, intestinal barrier function, and oxidative stress (Lin et al., 2022; Tan et al., 2022). Using a pseudo-germ-free murine model, we further verified the critical involvement of the AHR/CYP1A1 pathway. FMT, as well as treatment with AHR agonist FICZ, activated the AHR/CYP1A1 signaling pathway and promoted the secretion of IL-22. Previous studies have demonstrated that microbiota-derived tryptophan metabolites engage the AHR signaling pathway in ILC3s, driving IL-22 production through the AHR/IL-22 axis (Meynier et al., 2022; Stockinger et al., 2021). As a pivotal mediator of intestinal barrier function, IL-22 not only enhances the expression of tight junction proteins in intestinal epithelial cells but also stimulates goblet cells to secrete MUC2, thereby establishing a biochemical-physical barrier against pathogenic invasion (Zhang et al., 2024; Metidji et al., 2018; Stockinger et al., 2014). Concurrently, FMT and FICZ treatment also elevated serum GSH-Px, reduced MDA and MPO levels, and inhibited ileal pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α). Notably, these effects were abolished by AHR inhibition by CH223191, confirming that WB600/ZD alleviated S. Infantis-induced ileitis via gut microbiota-dependent activation of the AHR/CYP1A1 signaling pathway. In particular, activation of the AHR by tryptophan metabolites has been shown to regulate intestinal immune responses and maintain epithelial integrity (Sun et al., 2020; Pernomian et al., 2020).

Beyond tryptophan metabolism, fecal microbiota metabolomics also revealed that supplementation with WB600/ZD upregulated the primary bile acid biosynthesis pathway in diarrheic piglets, suggesting an additional mechanism for PWD amelioration, a finding consistent with prior research (Ren et al., 2022). However, the current study focused on tryptophan metabolism. Future research should investigate the bile acid metabolic pathway to fully elucidate the regulatory mechanisms of WB600/ZD on PWD.

Although zinc oxide (ZnO) supplementation has beneficial effects in improving growth performance, alleviating post-weaning diarrhea, and enhancing intestinal health in piglets (Kociova et al., 2020; Pluske, 2013), potential synergistic or antagonistic interactions between WB600/ZD and ZnO remain unexplored and warrant future study. Additionally, excessive ZnO use may cause environmental pollution, antibiotic resistance gene transmission, and toxicity (Markowicz et al., 2023; Shi et al., 2023). Therefore, reducing ZnO usage while maintaining efficacy will also be an important research focus in future studies.

This study demonstrated WB600/ZD's beneficial effects in ameliorating PWD, limitations of the study are as follows: 1) Validation across diverse PWD models and longer experimental durations is needed, as this study utilized only a natural infection model over a short period; 2) Although WB600/ZD upregulated tryptophan metabolites and beneficial bacterial genera, the specific functional strains involved remain unconfirmed. Future studies employing metagenomic sequencing and mono-colonization assays are needed to elucidate the precise mechanisms. These investigations will contribute to translating these preliminary findings into practical applications.

Compared to conventional probiotics, WB600/ZD synergistically combines inherent probiotic properties with the expression of Z. atratus defensin, resulting in enhanced antimicrobial activity. Although the defensin expressed by WB600/ZD demonstrates significant antimicrobial activity in vitro (Li et al., 2025), the current findings primarily highlight its ecological regulatory effects in vivo, including competitive exclusion of pathogens through enrichment of beneficial microbiota and modulation of microbial tryptophan metabolism. These results align with the emerging paradigm that engineered probiotics can exert protective effects by remodeling the gut microbiome. Nevertheless, the potential synergistic interactions between the direct bactericidal activity of defensin and microbiota-mediated protection require systematic validation in future studies. The application of WB600/ZD could reduce the reliance on antibiotics, aligning with the goals of sustainable livestock production.

5. Conclusion

In summary, the engineered probiotic WB600/ZD significantly enhanced the growth performance of newly weaned piglets and improved intestinal health. Potential mechanisms include regulating the gut microbiota, promoting nutrient absorption, improving antioxidant capacity, and activating the tryptophan metabolic pathway (AHR/CYP1A1). These findings highlight the translational potential of engineered probiotics in advancing sustainable practices within the pig industry.

Credit Author Statement

Wei Li: Writing – original draft, Software, Methodology. Xixi Wang: Supervision, Software, Data curation. Yu Zhang: Supervision, Software, Methodology. Hao Yang: Formal analysis, Data curation. Xue Wang: Formal analysis, Data curation. Wenwen Meng: Methodology, Investigation. Ting Hu: Methodology, Investigation. Wenqian Zhang: Methodology, Investigation. Yaohong Zhu: Writing – review & editing, Supervision, Resources, Project administration, Funding acquisition, Conceptualization. Jiufeng Wang: Writing – review & editing, Supervision, Resources, Project administration, Funding acquisition, Conceptualization. Guiyan Yang: Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization.

Availability of data and materials

All data generated or analyzed during this study are included in this article. The 16S rRNA sequencing data have been deposited in the NCBI Sequence Read Archive (SRA) under accession number (PRJNA1304681), and the untargeted metabolomics data are available in the METABOLIGHTS database under accession number (MTBLS12862).

Declaration of conflict of interests

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the content of this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article: