ColE1 plasmids potentiate the fitness of Salmonella Heidelberg in poultry litter

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 as an important extra-intestinal environment that may have selected for specific S. Heidelberg strains. Poultry litter (PL) is a mixture of bedding materials and chicken excreta that contains chicken gastrointestinal (GI) bacteria, undigested feed, feathers, and other materials of host origin. In this study, we performed a series of controlled laboratory experiments which assessed the evolution 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 by only 69 single-nucleotide variants. Whole genome sequencing was performed on 86 isolates recovered after 0, 1, 7 and 14 days of evolution 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 overall fitness. This increased fitness was associated with an increased copy number of IncX1 and ColE1 plasmids. Further, all Col plasmid-bearing strains had similar mutations in 102 genes encoded on the chromosome and in 3 genes encoded 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 acquisition of multiple Col-like plasmids can change the evolutionary path of S. Heidelberg in poultry litter.