Cross-resistance-guided phage cocktail design for effective mitigation of necrotic enteritis in poultry.

The withdrawal of antibiotic growth promoters from poultry production has led to the resurgence of Clostridium perfringens-induced necrotic enteritis (NE), resulting in annual global economic losses exceeding $6 billion. With conventional antibiotics becoming less accessible for disease prevention, there is an urgent need for effective non-antibiotic alternatives. Bacteriophage therapy represents a promising approach, although its utility is often constrained by narrow host range and rapid emergence of phage resistance. Here, we present a cross-resistance-guided strategy for rational phage cocktail design that combines phages with complementary and non-overlapping resistance profiles. Through systematic screening of eight C. perfringens-specific phages, we identified two host-range groups exhibiting complementary host ranges and reciprocal cross-resistance. A cocktail comprising CPD4 (siphovirus-like, 52,658 bp) and CPD7 (podovirus-like, 18,958 bp) was formulated based on their potency and divergence. Genomic and morphological analyses confirmed their strictly lytic lifecycle, absence of lysogenic or virulence genes, and high physicochemical stability. Notably, the cocktail demonstrated reciprocal cross-resistance, where resistance to one phage did not confer resistance to the other, thereby ensuring sustained lytic activity and mitigating the emergence of resistant strains. Furthermore, in vivo evaluation demonstrated that the cocktail significantly improved survival, reduced intestinal lesions, and suppressed C. perfringens colonization in NE-challenged chickens. Together, these results establish this cross-resistance-guided phage cocktail design as a practical and cost-effective strategy for NE mitigation in poultry.