Visualizing pathogen internalization pathways in fresh tomatoes using MicroCT and confocal laser scanning microscopy

Pathogen contamination of fresh produce significantly impacts public health and consumer confidence in eating fresh fruits and vegetables. In tomato fruits, studies have shown that the stem scar plays an important role in pathogen infiltration. However, the exact mechanisms and pathways for pathogen movement inside tomatoes are not well documented. This study examined in detail the pathway of pathogen internalization in tomato fruit. Tomatoes were infused with potassium iodide and examined with Xradia Bio MicroCT at 0.5×∼4×. Micro CT images revealed that infiltrated potassium iodide moved along the vascular bundles inside the fruit. In separate trials, tomato fruits were further infiltrated with red fluorescent microspheres and E. coli O157:H7 -pGFP. Stem scars and core tissues were subsequently excised after fruit surface disinfection and observed using a Zeiss 710 laser confocal microscope, or stomached, cultured, and enumerated for infiltrated E. coli O157:H7. Populations of internalized E. coli O157:H7 were confined in xylem vessels and concentrated immediately beneath the stem-scar, with a sharp decline in population with vertical distance from the stem-scar. Our observations suggest that 1) vascular bundles, especially the xylem vessels in tomato fruit, play a critical role in pathogen internalization, and 2) pathogen internalization is a passive, rather than active process, as the infiltration and movement of both microspheres (non-living) and E. coli O157:H7 cells (living) behaved similarly inside tomatoes. This study presents visual evidence of the critical role of vascular bundles in pathogen internalization in tomato fruit using Micro CT and Confocal Laser Microscopy. The finding is important for developing science-based food safety practices, interventions in controlling pathogen internalization, and new tools for probing pathogen-plant interactions.