Prophage ΦESI promotes competitive dominance in the emergent Salmonella serovar Infantis lineage

The global emergence of multidrug-resistant Salmonella enterica serovar Infantis has been largely attributed to the pESI megaplasmid; however, additional factors contributing to the ecological success of this lineage remain poorly understood. Here, we characterize a phage of emergent Salmonella Infantis (ΦESI), a temperate bacteriophage discovered during coculture of an emergent pESI-positive strain (PM57) and a non-emergent strain (DR006). Phage ΦESI shows a siphovirus morphotype with a long tail and an elongated head, with a genome of 46,490 bp, which is integrated as a prophage at the 3'-end of an Arg-tRNA gene in PM57, but is absent in DR006, which is susceptible to ΦESI-mediated lysis. Screening of ΦESI genes across 20,429 global Salmonella Infantis genomes revealed a near-exclusive association of ΦESI and ΦESI-like prophages with the emergent pESI-positive lineage. Further bioinformatic analyses of complete chromosomes revealed diverse ΦESI insertion profiles showing geographic clustering and the presence of large-scale chromosomal inversions flanked by ΦESI genes. In vitro competition assays demonstrated that PM57 outcompeted DR006 in coculture, coinciding with high viral loads that selectively lysed DR006. Consistently, susceptibility assays showed that strains lacking ΦESI/ΦESI-like prophages were susceptible to ΦESI infection. Together, these findings identify ΦESI as a lineage-associated prophage that mediates competitive exclusion of susceptible strains. Our results support a role for prophage-driven competition in the ecological success of the multidrug-resistant Salmonella Infantis lineage.IMPORTANCEEmergent multidrug-resistant Salmonella enterica serovar Infantis strains carrying pESI megaplasmids have spread worldwide, posing a global public health threat and a significant economic burden. Nevertheless, the factors contributing to the success of this foodborne pathogen, beyond pESI, remain poorly understood. Here, we describe ΦESI, a novel temperate bacteriophage carried by the emergent lineage as a chromosomally integrated prophage. We show that ΦESI provides a competitive advantage to emergent strains by selectively killing non-emergent competitors while protecting lysogens from reinfection. Our findings uncover the contribution of ΦESI/ΦESI-like bacteriophages to the success of emergent Salmonella Infantis, highlighting how lineage-associated prophages can shape the ecological success of pathogenic bacteria.