A Gifsy prophage-encoded protein confers broad phage resistance in <i>Salmonella enterica</i> and is widely distributed across <i>Enterobacteriaceae</i>

Phage Felix O1 (FO1) is one of the most widely used bacteriophages for targeting <i>Salmonella enterica</i> and is applied in both research and food safety settings. Despite its broad host range, we observed significantly reduced efficiency of plating (EOP) on a subset of <i>Salmonella enterica</i> strains, including serovar Typhimurium 4/74 (<i>S</i>. 4/74). We found that O-antigen-mediated steric hindrance contributes to this resistance, which can be partially overcome by supplementation with the SP6 phage tailspike protein (TSP). However, full restoration of the EOP required deletion of the Gifsy-1 prophage, leading to the identification of a single prophage-encoded defense gene, <i>gipd474</i> (Gifsy-encoded phage defense protein from strain <i>S</i>. 4/74). Heterologous expression of this gene in <i>Escherichia coli</i> increased resistance to multiple tailed phages. Bioinformatic analysis revealed that this gene is widely spread across the <i>Enterobacteriaceae</i>, suggesting a conserved role in phage resistance. Together, these findings uncover a dual barrier to FO1 infectivity in <i>S</i>. 4/74 and highlight <i>gipd474</i> as a novel, prophage-encoded phage defense gene.IMPORTANCEPhage therapy and biocontrol are increasingly explored as alternatives to antibiotics, particularly against drug-resistant pathogens such as <i>Salmonella</i>. FO1 is a widely used lytic phage with broad activity across <i>Salmonella</i> serotypes. However, variability in phage infectivity limits its reliability against certain strains. Here, we report that <i>S</i>. 4/74 restricts FO1 through a combination of O-antigen-associated interference and a novel prophage-encoded resistance gene. This gene also impairs the infectivity of other tailed phages and is conserved across multiple <i>Enterobacteriaceae</i> genera. Our findings highlight the importance of prophage-encoded defense systems in shaping phage susceptibility and highlight the need to account for such elements when developing phage-based applications.