How colonizing alfalfa sprouts modulates the virulence of Shiga toxin-producing Escherichia coli

Shiga toxin-producing Escherichia coli (STEC), a significant cause of foodborne illnesses, is often associated with the consumption of fresh produce, including alfalfa sprouts. This study was executed to determine how quickly STEC grows, adapts, and colonizes alfalfa sprouts during production and storage, and whether the pathogen's virulence and infectious doses are affected by physiological adaptation to sprouts as an environment. A reporter STEC O157:H7 EDL933 strain was developed to track the transcription of eae, a virulence gene involved in colonizing human intestinal enterocytes. When the seeds were inoculated with 2.1 × 10³ CFU/g of the reporter strain, the pathogen's population increased to 1.5 × 10⁶ CFU/g sprouts within 1.38 days and then remained stable during the remainder of the 5-day sprouting, indicating physiological adaptation to this environment. Seeds were inoculated with ∼10⁸ CFU/g and subsequently treated with 2000 ppm calcium hypochlorite solution, followed by a water-rinse (treated seeds), or just rinsed with water (untreated seeds). After 5 days of sprouting, the resulting fresh sprouts were refrigerated for three days at 4 °C. Sprout samples were collected and treated with 2000 ppm calcium hypochlorite solution and rinsed thoroughly with water before counting internalized STEC, or just water-washed before measuring total STEC. The transcription of eae (normalized to cell count) was the highest on the second day of sprouting, but the transcription of other virulence and stress-related genes varied, with sodA being upregulated in STEC cells. Lethal dose 50 (LD₅₀) to Galleria mellonella, a STEC infection animal model, was lower (i.e., virulence was higher) in total STEC collected from fresh sprouts produced from treated seeds, compared to that from untreated seeds (1.9 × 10⁰ and 6.0 × 10¹ CFU/larva, respectively). Compared to refrigerated sprouts, the LD₅₀ of STEC from freshly produced sprouts was lower. Based on these findings, it can be concluded that (a) STEC quickly adapts physiologically to sprouts as an environment, (b) transcription of STEC virulence genes changed during sprouts production but generally decreased during refrigeration, and (c) STEC from fresh sprouts grown from sanitizer-treated seeds were more virulent in the animal model, but STEC from refrigerated sprouts were less virulent.