WaaL-WaaP receptor cross-talk drives phage-induced collateral sensitivity to colistin in Salmonella Pullorum

Phage therapy typically fails when bacteria rapidly evolve resistance, yet the inevitable fitness costs of escape mutants remain underexploited. In this study, an iterative and targeted phage-cocktail formulation strategy (ITPFS) was presented and developed. A potent lytic phage, PHZ055, was firstly isolated against a colistin-resistant Salmonella Pullorum strain. Co-cultured until resistant mutants emerged, a second lytic phage, PYW047_3, was screened for the resistant variant. The two phages were then combined as a cocktail. Results showed that bacterial growth was strongly suppressed and the emergence of resistance was markedly delayed. Receptor identification revealed that both phages use lipopolysaccharide (LPS) as a receptor, but at distinct sites: PHZ055 targets WaaL, and PYW047_3 targets WaaP. Mutation of WaaL was recently shown to be responsible for the loss of polymerized O-antigen units and thereby exposed the PYW047_3 binding region, indicating that phage cocktails targeting distinct receptor sites can be directionally screened. Moreover, mutants that escaped the two phages not only incurred a growth cost but also exhibited a 32-fold increase in colistin sensitivity. Combining the two phages together with 1/4 MIC colistin almost eradicated all bacteria within 24 h. In all, our study successfully provides a repeatable strategy that incorporates adaptive trade-offs to boost therapeutic efficacy.