Internalization of Salmonella in Leafy Greens and Impact on Acid Tolerance

Salmonella colonizes the surface or the inner part of leafy greens, while the ability of internalized bacteria to evade common disinfection practices may pose a considerable risk. Hereby, we aimed to assess how the colonization and internalization of Salmonella spp. (i) vary with the type of leafy green, the storage conditions (temperature, time), and Salmonella serovar at phenotypic and gene transcriptional level (regarding stress- and virulence- or type III secretion system [T3SS]-associated genes) and (ii) potentially impact the survival of the pathogen against subsequent exposure at lethal pH (2.7), mimicking the gastric acidity. Internalized Salmonella reached 3.0 to 5.0 log CFU/g depending on storage conditions and vegetable, with spinach and chicory allowing the highest (P < 0.05) internalization. Prolonged storage (48 h) at 20°C increased the recovery of internalized Salmonella in spinach and green amaranth by 1.0 to 1.5 log units. Colonization of Salmonella on/in leafy vegetables induced the transcription (maximum fold change [FCmax], ∼2,000) of T3SS-related genes. Interserovar variation regarding the internalization ability of Salmonella was observed only in lettuce and green amaranth in a time- and temperature-dependent manner. Attached cells exhibited higher survival rates against low pH than the internalized subpopulation; however, habituation at 20°C in lettuce and amaranth induced acid tolerance to internalized cells, manifested by the 1.5 to 2.0 log CFU/g survivors after 75 min at pH 2.7. Habituation of Salmonella in vegetable extracts sensitized it toward acid, while indigenous microbiota had limited impact on acid resistance of the organism. These findings reveal physiological aspects of Salmonella colonizing leafy vegetables that could be useful in fresh produce microbial risk assessment. IMPORTANCE Consumption of leafy greens has been increasingly associated with foodborne illnesses, and their contamination could occur at pre- and/or postharvest level. Human pathogens may become passively or actively internalized in plant tissues, thereby escaping decontamination procedures. Plant colonization may impact bacterial physiology such as stress resistance and virulence. In this study, it was demonstrated that internalization of Salmonella spp., at the postharvest level, varied with type of vegetable, serovar, and storage conditions. Attached and internalized subpopulations of Salmonella on/in leafy greens showed distinct physiological responses regarding transcriptional changes of stress- and virulence-associated genes, as well as survival capacity against subsequent exposure to lethal pH (2.7). These findings could contribute to a better understanding and potential (re)definition of the risk of enteric pathogens colonizing leafy greens, as well as to the design of intervention strategies aiming to improve the microbiological safety of fresh produce.