Novel integrated approach modeling proanthocyanidins and bacteriophages to combat multidrug Salmonella Typhimurium in challenged broilers

The emergence of multidrug bacterial isolates, including <i>Salmonella</i> (<i>S</i>.) Typhimurium, which primarily spreads to humans through chicken products, is correlated with a rising prevalence of antimicrobial therapy failure. Thus, we performed a comprehensive analysis of the combined impact of <i>Salmonella</i> bacteriophage (BP) and grape seed oligomeric pro-anthocyanidins (GSOPs) on growth performance, immune functions, antioxidant capacity, cecal microbiota, gut integrity, and <i>S</i>. Typhimurium resistance in challenged broilers. A total of 250 Ross-308 male broiler chicks were offered either a control diet or a diet supplemented with <i>Salmonella</i> BP alone at concentrations of 10⁹ PFU/0.1 ml, GSOPs alone at a level of 400 mg/kg diet, and a combination of both <i>Salmonella</i> BP and GSOPs, and experimentally infected with multidrug-resistant (MDR) <i>S</i>. Typhimurium strain at 14 days of age. Broilers administered BP, GSOPs, and their combination, particularly BP+GSOPs, had enhanced growth performance attributes even following a challenge with <i>S</i>. Typhimurium, alongside decreased mortality percentage, which was evidenced by increased expression of <i>MUC-2</i>, β-defensin-1, cathelicidins-2, <i>JAM</i>-2, occludin, and <i>CLDN-1</i> genes, reduced <i>S</i>. Typhimurium abundance, and downregulating its virulence-associated genes (<i>sopE</i> and <i>spvC</i>), alongside restored intestinal histological features. GSOPs+BP fortified group exhibited higher cecal beneficial bacteria counts (<i>Bacteroides, Firmicutes, Lactobacillus</i>, and <i>Bifidobacterium</i> species), lower cecal harmful bacteria loads (<i>Escherichia, Enterobacteriaceae</i>, and <i>Clostridium</i> clusters I and IV), decreased serum oxidative markers [H₂O₂, reactive oxygen species (ROS), and malondialdehyde (MDA)], and increased serum antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px)]. The incorporation of dietary BP, and GSOPs combination significantly downregulated the intestinal inflammatory regulated genes (<i>IL-1</i>β<i>, IL-6, CCL4, CCL20, COX-2</i>, and <i>iNOS</i>), and <i>mTOR</i> gene, and reduced the serum concentrations of LYZ, NO, CRP, and complement C3, alongside increased serum IgG, and IgM levels, and upregulation of autophagy-related genes (<i>atg5, atg7, atg12, BCLN-1</i>, and <i>LC3-II</i>). The aforementioned beneficial effects of the BP and GSOPs combination support their prospective use in avian nutrition to enhance performance and stimulate immune defense against gastrointestinal illnesses, including <i>S</i>. Typhimurium infection.