Prophylactic potential of a novel Gregsiragusavirus phage SLAM_phiCP2S1 combined with Pediococcus pentosaceus against Clostridium perfringens infection in broiler chicks

Necrotic enteritis caused by Clostridium perfringens in poultry is a widespread disease that incurs significant economic losses. With the global restriction of antibiotic growth promoters, there is an urgent need for effective alternative strategies to control bacterial disease. In this study, we isolated and characterized a novel virulent phage, SLAM_phiCP2S1, belonging to the Gregsiragusavirus genus, and evaluated its combined prophylactic potential with the probiotic Pediococcus pentosaceus SLAM_PEP01 to prevent C. perfringens infection in broiler chickens. In Caenorhabditis elegans, both the phage and probiotic individually improved host survival, whereas their co-administration produced the strongest protective effect. In broiler chicks, an experimental challenge with C. perfringens caused growth suppression and intestinal damage in infected controls. Phage-only treatment provided partial improvement, but the phage–probiotic combination most effectively preserved growth and intestinal health. Cecal bacterial counts and 16 S rRNA profiling showed that the phage–probiotic combination most effectively reduced C. perfringens burden, restoring levels close to those of uninfected controls. Metagenomic analysis further suggested enrichment of beneficial taxa and reduction of pathogen-associated genera in the combination group. These findings indicate that co-administration of SLAM_phiCP2S1 and SLAM_PEP01 more effectively curtailed C. perfringens infection and preserved growth and intestinal integrity by maintaining gut microbial homeostasis. This phage–probiotic combination highlights the potential and implications of an antibiotic-free phage-probiotics combination as sustainable strategies for intestinal health and performance in poultry.