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

Consumption of sprouted seeds, such as alfalfa sprouts, has increased in recent years due to their nutritional value and antioxidant content. However, these products have repeatedly been implicated in outbreaks of foodborne pathogens, including Salmonella enterica . Although host-adapted Salmonella serovars are less frequently associated with foodborne illness, infections caused by these serovars often result in invasive and severe outcomes, highlighting the importance of understanding their persistence in food production systems. Moreover, the variability among Salmonella serovars requires characterization beyond the most prevalent types to support the development of precision food safety strategies effective across the diversity of serovars capable of contaminating fresh produce. Here, a plant-internalized Salmonella Choleraesuis strain was used as a model to investigate persistence mechanisms on alfalfa sprouts. A bar-coded transposon mutant library comprising approximately 33,000 unique insertions was generated, along with a collection of individual insertion mutants. These resources were used to identify genetic determinants contributing to strain fitness on sprouts under abusive cold storage (8°C) simulating commercial shelf-life environments. Genome-wide analyses identified negative selection for mutants with insertions in eda , fabF , lpp1_2 , pnp , stpA , SCHChr_03621 , and two intergenic regions. Competition assays confirmed fitness defects associated with eda , encoding a key enzyme of the Entner-Doudoroff pathway; mnmG , encoding a tRNA modification enzyme involved in translational fidelity; and fabF , involved in fatty acid biogenesis. These findings provide a genome-wide perspective on mechanisms enabling persistence on sprouts of a plant-associated, host-adapted Salmonella strain during cold storage and inform risk assessment and intervention design within precision food safety frameworks.