Transcriptomic analysis of Shiga toxin-producing Escherichia coli O157:H7 on alfalfa sprouts: Broad adaptive...

Shiga toxin-producing Escherichia coli (STEC) is a major foodborne pathogen associated with raw produce such as sprouts, where it encounters variable nutrient, temperature, and oxidative stress. This study elucidated the transcriptional adaptations of E. coli O157:H7 EDL933 during colonization and persistence on alfalfa sprouts under simulated commercial sprouting and refrigerated storage. RNA-sequencing was used to compare STEC grown in tryptic soy broth (TSB) (D₀), colonized sprouts for 5 days (D₅), and held for 3 days under refrigeration (D₈). Among 5002 expressed genes, 782 and 826 were differentially expressed (DEGs; |log₂FC| ≥ 2, adjusted p < 0.05) at D₅ vs. D₀, and D₈ vs. D₀, respectively, whereas 11 genes differed in the D₈ vs. D₅ comparison, indicating minimal shifts during refrigeration. Transition of STEC from TSB to sprouts tissues up-regulated de novo amino acid biosynthesis (hisA-H, leuABCD, and ilv operons), to compensate for nitrogen limitation within sprouts tissues. In sprouts environments, STEC induced high-affinity uptake systems for potassium (kdpA/B/C/F), phosphorus (pstSCAB), and sulfur (tauABCD), alongside enterobactin-mediated iron acquisition. To survive in the sprouts' microenvironment, STEC elevated the transcription of acid resistance (gadA/C, cadA/B), oxidative stress defense (sodA, katE/G), and envelope protection (degP, pspA-D, baeS) genes. Functional enrichment at D₅ and D₈, compared to D₀, indicated coordinated stress regulation and translation. Major transcriptional trends observed in RNA-seq were confirmed using RT-qPCR. Collectively, these findings highlight a broad transcriptional reprogramming, primarily during alfalfa sprout colonization, with the transcriptome stabilized by D₅ of sprouting whereas refrigeration (D₅-D₈) induced only minimal cold-induced changes. These findings provide insights that could be translated into STEC risk mitigation during sprouts production.