Fitness costs of phage-driven resistance mutations in <i>Salmonella</i> Enteritidis populations

In the post-antibiotic era, bacteriophages have emerged as viable alternatives for combating antibiotic-resistant infections. Analogous to the emergence of antibiotic resistance, bacteria can also develop resistance to phages, a process often accompanied by a reduction in bacterial fitness. In this study, we investigated the mechanisms behind the development of phage resistance in <i>Salmonella enterica</i> serovar Enteritidis strain WJ48 during laboratory coevolution with phage GRNsp8, as well as the associated fitness costs and community dynamics. Three types of phage-resistant mutants, VP81, VP82, and VP84, were isolated. VP81 exhibited a frameshift mutation in the gene encoding glycosyltransferase and displayed partial resistance to GRNsp8. Both VP82 and VP84 carried frameshift mutations in <i>btuB</i>, conferring complete resistance to GRNsp8. Deletion of <i>btuB</i> abolished phage adsorption and conferred complete resistance. Meanwhile, complementation with <i>btuB</i> restored susceptibility. <i>In vitro</i> competition assays showed that the <i>btuB</i>-deletion strain was competitively disadvantaged relative to the wild-type strain within 24 h, exhibiting a relative fitness value of <1. Consistently, <i>in vivo</i> experiments in mice showed a marked attenuation of virulence in the mutant strain. Specifically, the LD₅₀ of the <i>btuB</i>-deficient strain in mice was 6.8 × 10⁸ CFU, 121 times higher than the wild-type strain. During the 9-day coevolution experiment, a single-point mutation in <i>btuB</i> consistently dominated and represented the primary mode of resistance. These findings shed light on the adaptive trade-offs that bacteria undergo to evade phage infection and provide valuable insights for designing more rational and effective phage therapy strategies that exploit these trade-offs.IMPORTANCEAs emerging antibacterial agents in the post-antibiotic era, Bacteriophages also face the challenge of bacterial resistance. However, phage resistance development by bacteria is frequently accompanied by a reduction in bacterial fitness. To elucidate the adaptive trade-offs associated with resistance, in this study, we used <i>Salmonella enterica</i> serovar Enteritidis strain WJ48 and the broad-host-range bacteriophage GRNsp8 as a model system. We found that the acquisition of phage resistance by bacteria was significantly associated with a reduction in virulence. These findings deepen our understanding of bacteria-phage coevolution but also offer key insights into leveraging the resistance-fitness trade-off to inform the strategic design of more effective phage therapies. The results highlight the potential for improving the application of phages in agriculture and animal husbandry, supporting the sustainable development of phage-based antimicrobial strategies.