Broiler litter moisture and trace metals contribute to the persistence of <i>Salmonella</i> strains that harbor large plasmids carrying siderophores

Broiler litter sampling has proven to be an effective method for determining the <i>Salmonella</i> status of a broiler chicken flock and understanding the ecology of <i>Salmonella</i> prior to harvest. In this study, we investigated the ecology of <i>Salmonella</i> within the litter (<i>n</i> = 224) from two commercial broiler houses in the United States. We employed culture enrichment methods and quantitative polymerase chain reaction to determine the prevalence and load of <i>Salmonella</i> and utilized antimicrobial susceptibility testing and whole-genome sequencing (WGS) to characterize select isolates. Additionally, we applied machine learning algorithms and <i>in vitro</i> experiments to identify environmental selective pressures that may contribute to the persistence of <i>Salmonella</i> in litter. Our findings indicate that the prevalence and abundance of <i>Salmonella</i> in broiler litter are influenced by the downtime between flocks as well as by the flock raised on the litter. A Decision Tree Classifier model developed demonstrated that the moisture in the caked part of litter was the most influential environmental parameter for predicting the prevalence of viable <i>Salmonella</i>. WGS analysis revealed that Typhimurium, Infantis, and Kentucky strains that harbored large self-conjugative plasmids encoding fitness factors for iron siderophore production were the dominant <i>Salmonella</i> population found in litter, and exposure to iron-limiting and copper-enriched culture media affected <i>Salmonella</i> growth. Our results suggest that trace metals may select for siderophores harbored on plasmids, and interventions that reduce litter moisture can potentially curtail the persistence of <i>Salmonella</i> in pre-harvest environments.IMPORTANCEBroiler chicken meat is the most consumed protein worldwide, and global poultry imports are projected to reach 17.5 million tons by 2031. To raise billions of chickens, litter is reused multiple times by the top global producers and exporters of chicken (Brazil and the United States). Chickens are in continuous contact with litter and depend on it for warmth and coprophagy. Consequently, litter serves as a major route for pathogens such as <i>Salmonella</i> to infect chickens, making it crucial to understand the environmental and genetic selective pressures that might explain why certain <i>Salmonella</i> strains persist on broiler farms more than others. In this study, we demonstrated that <i>Salmonella</i> strains that harbored siderophores on large conjugative plasmids persisted in litter and suggested that reducing litter moisture would significantly control <i>Salmonella</i> prevalence. However, a complete eradication of persisting <i>Salmonella</i> strains will require novel, innovative, and multifaceted approaches.