Design of Lytic Phage Cocktails Targeting Salmonella: Synergistic Effects Based on In Vitro Lysis, In Vivo Protection, and Biofilm Intervention

<i>Salmonella</i> is a major zoonotic pathogen and phage cocktails offer a novel strategy against its infections. This study aimed to characterize <i>Salmonella</i> phages and assess the efficacy of various phage combinations, both in vitro and in vivo. Three phages (PJN012, PJN042, PJN065) were isolated, showing stability across a broad range of temperatures and pH values, and lacking genes associated with lysogenicity, virulence, and antibiotic resistance. Combined with two known phages (PJN025, vB_SalS_JNS02), they formed cocktails tested for lytic activity against <i>S</i>. Enteritidis and <i>S</i>. Typhimurium. Phage cocktails (comprising 2-5 phages) that demonstrated efficacy in vitro were validated using <i>Galleria mellonella</i> models. For <i>S</i>. Enteritidis strain 015, prophylactic cocktail C18 increased larval survival to 90% at 48 h (vs. 3% control). For <i>S</i>. Typhimurium strain 024, phage cocktail 26 showed the best therapeutic effect when co-injected with the bacterium, with a survival rate of up to 85% at 96 h, compared to 30% in the positive control group. Biofilm assays showed cocktails inhibited formation more effectively (e.g., at 24 h, C14 and C17 reduced biofilm formation by 93.74% and 94.21%, respectively) than removed established ones. The cocktails depended on bacterial type, phage genera, combinations, and incubation time. Robust in vitro screening remains crucial for optimizing phage formulations despite potential in vivo discrepancies.