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

Post-weaning diarrhea (PWD) causes significant economic losses to the pig industry. A previous study demonstrated that engineered <i>Bacillus subtilis</i> WB600 expressing <i>Zophobas atratus</i> defensin (ZD), termed WB600/ZD, alleviates intestinal inflammation, modulates gut microbiota, and maintains redox homeostasis in <i>Salmonella</i>-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, <i>n</i> = 15) or 2 × 10⁹ CFU/mL WB600/ZD (H + WB600/ZD group; 6.00 ± 0.68 kg, <i>n</i> = 15), and diarrheic piglets fed standard diet (PWD group; 6.51 ± 1.16 kg, <i>n</i> = 10) or 2 × 10⁹ CFU/mL WB600/ZD (PWD + WB600/ZD group; 6.91 ± 0.57 kg, <i>n</i> = 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 (<i>P</i> < 0.001) and diarrheic piglets (<i>P</i> = 0.040). Additionally, WB600/ZD improved the growth performance, including final body weight (<i>P</i> = 0.017) and average daily gain (ADG; <i>P</i> = 0.007), without affecting average daily feed intake (ADFI; <i>P</i> = 0.907). Mechanistically, WB600/ZD increased the levels of serum glutathione peroxidase (GSH-Px; <i>P</i> = 0.014) and reduced myeloperoxidase (MPO; <i>P</i> < 0.001) and malondialdehyde (MDA; <i>P</i> < 0.001). Integrated fecal microbiota and metabolites showed that this protective effect of WB600/ZD was associated with gut microbiota-dependent tryptophan metabolism (<i>P</i> < 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 <i>Salmonella enterica</i> subsp. <i>enterica</i> serovar Infantis (<i>S.</i> Infantis) challenge exhibited activation of the AHR/cytochrome P450 family 1 subfamily A member 1 (CYP1A1) signaling pathway (<i>P</i> = 0.022) and increased interleukin (IL)-22 secretion (<i>P</i> < 0.001), thereby alleviating <i>S</i>. Infantis infection. Overall, this study provides strong evidence that WB600/ZD is a promising antibiotic alternative for preventing PWD in newly weaned piglets.