Salmonella enterica subsp. enterica serovar Heidelberg (S. Heidelberg) is a clinically-important serovar linked to food-borne illness, and commonly isolated from poultry. Investigations of a large, multistate outbreak in the USA in 2013 identified poultry litter (PL) as an important extra-intestinal environment that may have selected for specific S. Heidelberg strains. Poultry litter is a mixture of bedding materials and chicken excreta that contains chicken gastrointestinal (GI) bacteria, undigested feed, feathers, and other materials of chicken origin. In this study, we performed a series of controlled laboratory experiments which assessed the microevolution of two S. Heidelberg strains (SH-2813 and SH-116) in PL previously used to raise 3 flocks of broiler chickens. The strains are closely related at the chromosome level, differing from the reference genome by 109 and 89 single nucleotide polymorphisms/InDels, respectively. Whole genome sequencing was performed on 86 isolates recovered after 0, 1, 7 and 14 days of microevolution in PL. Only strains carrying an IncX1 (37kb), 2 ColE1 (4 and 6kb) and 1 ColpVC (2kb) plasmids survived more than 7 days in PL. Competition experiments showed that carriage of these plasmids was associated with increased fitness. This increased fitness was associated with an increased copy number of IncX1 and ColE1 plasmids. Further, all Col plasmid-bearing strains had hotspot mutations in 37 loci on the chromosome and in 3 loci on the IncX1 plasmid. Additionally, we observed a decrease in susceptibility to tobramycin, kanamycin, gentamicin, neomycin and fosfomycin for Col plasmid-bearing strains. Our study demonstrates how positive selection from poultry litter can change the evolutionary path of S. Heidelberg
