Phage-driven coevolution reveals trade-off between antibiotic and phage resistance in Salmonella anatum

Abstract Phage therapy faces challenges against multidrug-resistant (MDR) Salmonella due to rapid phage-resistant mutant emergence. Understanding the intricate interplay between antibiotics and phages is essential for shaping Salmonella evolution and advancing phage therapy. In this study, MDR Salmonella anatum (S. anatum) 2089b coevolved with phage JNwz02 for 30 passages (60 days), then the effect of coevolution on the trade-off between phage and antibiotic resistance in bacteria was investigated. Our results demonstrated antagonistic coevolution between bacteria and phages, transitioning from arms race dynamics (ARD) to fluctuating selection dynamics (FSD). The fitness cost of phage resistance, manifested as reduced competitiveness, was observed. Bacteria evolved phage resistance while simultaneously regaining sensitivity to amoxicillin, ampicillin, and gentamicin, influenced by phage selection pressure and bacterial competitiveness. Moreover, the impact of phage selection pressure on the trade-off between antibiotic and phage resistance was more pronounced in the ARD stage than in the FSD stage. Whole genome analysis revealed mutations in the btuB gene in evolved S. anatum strains, with a notably higher mutation frequency in the ARD stage compared to the FSD stage. Subsequent knockout experiments confirmed BtuB as a receptor for phage JNwz02, and the deletion of btuB resulted in reduced bacterial competitiveness. Additionally, the mutations identified in the phage-resistant strains were linked to multiple single nucleotide polymorphisms (SNPs) associated with membrane components. This correlation implies a potential role of these SNPs in reinstating antibiotic susceptibility. These findings significantly advance our understanding of phage-host interactions and the impact of bacterial adaptations on antibiotic resistance.