Internalization of Salmonella in lettuce leaves after irrigation using recycled wastewater

Fresh produce irrigated with recycled wastewater is prone to contamination by human pathogens. Compared to those attached to the surfaces, human pathogens reaching the interiors of plant tissues (internalization) pose a greater threat to food safety because they are often protected from surface disinfection. This study explored multiple questions about the internalization of Salmonella, a common human pathogen, into lettuce, one of the most consumed leafy green vegetables that are eaten raw. First, the effects of abiotic and biotic factors (i.e., soil texture, drought stress, and the presence of plant pathogens) on the internalization of Salmonella in lettuce were determined. Then, the structure of the indigenous microbial communities in the rhizosphere and the root of lettuce were elucidated using Illunima sequencing and the impacts of irrigation with Salmonella -laden water on the indigenous microbial communities were evaluated. Finally, modulation of protein expression in both Salmonella and lettuce upon the internalization of Salmonella in lettuce was revealed using quantitative proteomic analysis. Under optimal conditions for plant growth, the incidence of Salmonella internalization into lettuce leaves through root uptake was rare. However, it was noted that soil with a high level of sand content and exposure of lettuce to drought stress could increase the internalization frequency and the internalization concentration of Salmonella into lettuce leaves. In addition, Salmonella internalized in the leaves of Arabidopsis, a model plant, could benefit from the presence of a plant pathogen Pseudomonas syringae pv. tomato DC3000, because the latter suppressed the immune system of the plant. The bacterial community in the rhizosphere was slightly altered by the use of irrigation water containing Salmonella. In contrast, the bacterial community in the root of lettuce was not affected by the presence of Salmonella in irrigation water, suggesting the resilience of the bacterial community in the root. The relative abundances of several bacterial taxa known to be beneficial to plants, such as Oxalobacteraceae and Rhizobium, increased in the rhizosphere as a result of irrigation with water containing Salmonella. The modulation in protein expression suggested that internalized Salmonella cells up-regulated multiple stress response proteins to cope with stresses encountered in lettuce. On the other hand, lettuce responded to Salmonella internalization by up-regulating proteins involved in defense response to plant pathogens and induced R proteins, suggesting that the human pathogen triggered the defense mechanisms of lettuce