The fitness of <i>Salmonella</i> Enteritidis in poultry is strongly shaped by interactions between bacteriophages, plasmids, and the microbial community

ABSTRACT Salmonella enterica serovar Enteritidis ( S . Enteritidis) is a major cause of foodborne illness linked to poultry across the globe, and its decreased susceptibility to quinolone antibiotics such as ciprofloxacin poses a significant threat to public health. Yet, the ecological and genetic pressures that drive the fitness of this pathogen in poultry environments remain poorly defined. In this study, we combined broiler chicken litter microcosm experiments, whole-genome sequencing, and machine learning to investigate factors that shape the fitness and antimicrobial resistance development of S . Enteritidis. We analyzed more than 10,000 genomes to assess the role bacteriophages, plasmids, and chromosomal mutations play in driving S . Enteritidis fitness. Our findings reveal that an underreported bacteriophage named ESSI-2 and IncX1 plasmids strongly influenced whether strains carried the gyrA mutations associated with decreased ciprofloxacin susceptibility. Strains of S . Enteritidis lacking both ESSI-2 phage and IncX1 plasmid harbored parallel mutations in genes linked to DNA topology, antimicrobial peptide resistance, and virulence. We found that ESSI-2 phage persistence in litter was shaped by distinct ecological community structures rather than random occurrence and that reused litter reduced S . Enteritidis survival compared to fresh pine wood shavings, suggesting that microbiome-based interventions could be leveraged to limit the spread of antibiotic-resistant Enteritidis strains. IMPORTANCE Foodborne illnesses linked to Salmonella enterica serovar Enteritidis strains with decreased susceptibility to quinolones, including ciprofloxacin, are on the rise in the United States, Canada, and Europe. In this study, we report for the first time that the gyrA mutation responsible for this phenotype is associated with a bacteriophage and a particular plasmid group. We demonstrated that when either of the two mobile genetic elements is carried by a S. Enteritidis strain, the likelihood of having gyrA mutation was negligible. Using broiler chicken litter microcosms, we provided insights into how the microbiome could be harnessed to limit the continued spread of these fitter lineages of S. Enteritidis strains. Our study shows that the survival and evolution of S. Enteritidis in poultry environments are strongly shaped by interactions among bacteriophages, plasmids, and the microbial community.