Phage receptor specificity drives cross-resistance patterns and governs fitness trade-offs during sequential resistance acquisition in Salmonella.

Phages infect bacteria by binding to specific surface receptors, driving co-evolution in microbial communities and offering therapeutic potential. However, how receptor specificity shapes the cross-resistance patterns and evolutionary trade-offs during phage-bacteria co-evolution remains unclear. Here, we investigated the genetic basis and fitness trade-offs of phage resistance in Salmonella to phages targeting O-antigen, core oligosaccharide, and BtuB (TonB-dependent receptor for vitamin B12) under individual or combinatorial pressures. The interaction matrices between phage-resistant strains and phages targeting three different receptors showed that bacterial cross-resistance to phages depends on the receptor type. Lipopolysaccharide (LPS) truncation conferred cross-resistance to phages targeting either the O-antigen or core oligosaccharide; whereas resistance to phages targeting BtuB occurred exclusively through mutations in the btuB gene. For LPS receptors whose biosynthesis involves multiple genes, the fitness cost associated with phage resistance is gene-specific. Among mutations conferring resistance to both O-antigen-targeting and core-targeting phages, those in the rfaJ gene exhibited the lowest fitness cost. The three-phage combination targeting three receptors exhibited potent antibacterial effects. Under this selective pressure, Salmonella developed resistance through receptor modification. Resistance to O-antigen-targeting and core-targeting phages emerged first through mutations in LPS biosynthesis genes, with mutations in the rfaJ gene dominating. Subsequently, mutations in the btuB gene accumulated to resist BtuB-targeting phages, ultimately evading predation by all three phages. Our results reveal receptor-driven evolutionary trade-offs and sequential resistance acquisition in Salmonella under multiple phages pressure, enhancing understanding of microbial interactions and informing phage therapy strategies.