Identification of genes promoting fitness of a plant-associated Salmonella Choleraesuis strain on alfalfa sprouts during cold storage

Abstract Consumption of sprouted seeds, such as alfalfa sprouts, has increased in recent years due to their perceived health benefits. However, these food products have repeatedly been associated with outbreaks of foodborne pathogens, including Salmonella enterica serovars. An S. enterica serovar Choleraesuis strain previously isolated from melon fruit internal tissues was selected as a model to explore plant-pathogen interactions on alfalfa sprouts. Using this strain, we generated a barcoded transposon mutant library comprising approximately 33,000 unique insertions. This library and a collection of individual insertion mutants derived from it were used to identify genetic mechanisms contributing to the fitness of this S. Choleraesuis strain on alfalfa sprouts. The library was screened on sprouts during cold storage at 8°C. Negative selection for mutants with insertions in eda, fabF, lpp1_2, pnp, stpA, SCHChr_03621 and two intergenic regions were identified. Competition experiments between individual insertion mutants and the wild type confirmed the phenotype of three genes: eda, coding for a keto-hydroxyglutarate-aldolase/keto-deoxy-phosphogluconate aldolase involved in the Entner-Doudoroff pathway, mnmG, encoding the glucose-inhibited division protein, and fabF, involved in fatty acid biogenesis. This study offers a genome-wide perspective on the genes enabling a plant-associated Salmonella strain to persist on alfalfa sprouts. We highlight factors that are critical not only for persistence throughout the entire cold-storage period under conditions that closely simulate real shelf-life conditions in this high-risk food matrix. Footnotes Funding. This work was supported by a grant awarded to Irene Esteban Cuesta by the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG). Grant No. 494875429.