Impact of population density and stress adaptation on the internalization of Salmonella in leafy greens

Abstract

Salmonella enterica is capable of entering the interior of leafy greens and establishing in the apoplastic area, a phenomenon known as internalization. The ability of internalized bacteria to evade common disinfection practices poses a well-established risk. Our aim was to study the effect of: i) inoculum size and ii) prior adaptation of Salmonella to sublethal stresses, on the internalization of the pathogen in four leafy vegetables. Spinach, lettuce, arugula and chicory were inoculated, by immersion for 2 min at room temperature with: i) Salmonella Enteritidis at 3.0, 4.0, 5.0, 6.0, 7.0 log CFU/mL and ii) non-adapted or adapted S. Enteritidis to acid (in TSB with 1% glucose, incubated for 24 h at 37 °C), cold (in TSB for 7 days at 4 °C), starvation (0.85% NaCl of pH 6.6, 48 h at 37 °C) or desiccation (1.5 h at 42 °C, 4 days at 21 °C) stress at appx 3.5 log CFU/mL). Inoculated leafy greens were subsequently stored at 5 °C and 20 °C for 2 h and 48 h (n = 2 × 2). Population of internalized Salmonella, after surface decontamination with 1% w/v AgNO3, was assessed on selective media. Even the lowest initial bacterial inoculum was adequate for internalization of Salmonella to occur in leafy vegetables. Non-adapted Salmonella inoculum of 7.0 (maximum) and 3.0 log CFU/mL (lowest inoculation level tested) after short storage (2 h) resulted in 3.7–4.3 and 1.3–1.5 log CFU/g internalized bacterial population, respectively. Colonization (including both attachment and internalization processes), as well as internalization process, were positively correlated to initial inoculum level. These processes reached a different plateau beyond which, no further increase in internalization was observed. Adaptation of the pathogen to mild stresses enhanced internalization (P < 0.05), with desiccation- and acid-adapted Salmonella demonstrating the highest internalization capacity, regardless of the vegetable and storage temperature. These findings could contribute to further elucidation of colonization capacity of Salmonella in leafy vegetables and assist in selecting the proper conditions that contribute to the prevention of fresh produce contamination with Salmonella.