A regrowth culture-guided screening strategy for isolating phages with prolonged inhibitory activity against Salmonella Enteritidis

Efficient screening methods are crucial for developing phage cocktails that can sustainably suppress bacterial growth. We developed a regrowth culture-guided phage screening strategy using Salmonella Enteritidis as a model. The strategy comprises four steps: (1) isolating an initial phage through conventional random screening, (2) generating a regrowth culture from bacteria that survive phage-induced inhibition, (3) enriching a phage pool by coincubating the regrowth culture with environmental samples, and (4) isolating phages that infect the regrowth-adapted bacterial population. Compared to cocktails derived from conventional random or mutant-based screening approaches, those prepared via this strategy demonstrated prolonged inhibitory activity, effectively suppressing S. Enteritidis regrowth for over 24 h in liquid culture. This approach provides a simple, reproducible, and ecologically relevant method for identifying phages with enhanced efficacy against phage-evasive bacterial populations. It may also serve as a valuable screening framework for developing robust phage cocktails against other pathogens.