Genome-wide screening reveals opposing roles for central metabolism in planktonic and biofilm survival of Salmonella under combined limonene and heat treatment

Salmonella enterica serovar Typhimurium is a major cause of foodborne illness and remains a persistent challenge for food safety due to its ability to survive in both planktonic and biofilm-associated states along the farm-to-fork chain. While conventional control strategies rely on chemical preservatives and thermal treatments, increasing consumer demand for clean-label foods has driven interest in natural antimicrobials such as essential oils, of which limonene is a major citrus-derived component. However, the cellular mechanisms underlying Salmonella 's response to these strategies, and whether it differs between free and nanoemulsified formulations, or between planktonic and biofilm lifestyles, has not been defined. In this study, we applied an unbiased, transposon-directed insertion-site sequencing (TraDIS- Xpress ) to systematically identify every gene and pathway contributing to the survival of S. Typhimurium when exposed to limonene in both free and nanoemulsion, across different inactivation temperatures and in both planktonic culture and established biofilms. We show that inactivation of genes involved in central metabolism and oxidative stress responses improves planktonic survival of heat treatment, but that this protective effect is lost under nanoemulsified limonene at 54 °C, a condition under which survival was not detected. Interestingly, these same genes and pathways were essential for the survival of established biofilms irrespective of heat or oil treatment, revealing that the genetic determinants of susceptibility invert between the two lifestyles. Together, these findings provide a genome-wide view of the genetic architecture underpinning Salmonella survival in response to limonene and heat and reveal that planktonic and biofilm cells have opposite vulnerabilities, meaning that effective decontamination strategies must be tailored to the physiological state in which Salmonella persists..