## 5 Conclusions  
Our study aimed to understand the microbial dynamics that occur when pathogenic cells such as STEC O157:H7 are exposed to temperature shifts during harvesting and storage. These findings can inform the assessment of risks posed by STEC O157:H7 on lettuce. We found that both low harvest temperatures and prolonged cold storage increased the transition of STEC O157:H7 into dormant states. This transition poses a risk of underestimating pathogen survival when microbial concentrations in lettuce are evaluated, which is important for more accurate risk modeling. Our study highlights the need to account for conditions that promote dormancy when using such data in QMRA. Furthermore, we demonstrated that the efficacy of sanitization processes, such as chlorine washing to minimize post-harvest cross-contamination, is influenced by the exposure of pathogens to temperatures at harvest or during storage. We also established that increasing the duration of cold storage did not result in evidence of increased strain virulence. Future risk assessments should therefore not only consider the effects of harvesting and cold storage temperatures on STEC O157:H7 die-off or growth but also account for physiological changes that could otherwise lead to an underestimation of risks. Entry into dormant states also poses a risk, as in routine culturing, only viable cells are considered. We concluded that colder harvest temperature followed by cold storage led to a higher transformation of STEC O157:H7 into dormant states. Length of cold storage led to greater reduction of STEC O157:H7 due to acid treatment, but not chlorine treatment. These results demonstrate that forward processing of lettuce does not lead to increased risk of STEC O157:H7 survival or stress tolerance. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/applmicrobiol5020045/s1](https://www.mdpi.com/article/10.3390/applmicrobiol5020045/s1)]([https://www.mdpi.com/article/10.3390/applmicrobiol5020045/s1](https://www.mdpi.com/article/10.3390/applmicrobiol5020045/s1)), Figure S1: Post-hoc tests for pairwise comparison of the interactive effect of strain and harvesting temperature on persistent percentage of STEC O157:H7 on Romaine lettuce. Figure S2: Post-hoc tests for pairwise comparison of the interactive effect of strain and harvesting temperature on persister percentage of STEC O157:H7 on Romaine lettuce. Figure S3: Pairwise comparison of the effect of strain on the on the injured and culturable cells of STEC O157:H7 during incubation of Romaine lettuce at harvesting temperature. Figure S4: Pairwise comparison of the predicted log reduction values of the culturable cells for different strains in lettuce harvested at different temperatures. Figure S5: Most important factors explaining the variance in (A) culturable and (B) VBNC cells and (C) acid and (D) chlorine tolerance. Figure S6: Pairwise comparison of the predicted increase in VBNC cells for different strains in lettuce harvested at different temperatures. Figure S7: Pairwise comparison of the predicted increase in log reduction due to acid treatment. Figure S8: Pairwise comparison of the predicted increase in log reduction due to chlorine treatment. Figure S9: Odd ratios for the factors affecting the survival of \(G\). _mellonella_ after inoculation with STEC cells from cold stored lettuce. Conceptualization, T.M.B. and J.M. ; methodology, D.S. and J.O.O. ; formal analysis, D.S. and J.O.O. ; investigation, D.S., J.O.O., C.C., A.E., C.J.K. and E.S.R. ; writing--original draft preparation D.S. and J.O.O. ; writing--review and editing, T.M.B., J.M., D.S. and J.O.O. ; visualization, D.S. and J.O.O. ; supervision, T.M.B. ; funding acquisition--T.M.B. and J.M. All authors have read and agreed to the published version of the manuscript. The funding for this study was made possible by The Center for Produce Safety and the U.S. Department of Agriculture's (USDA) Agricultural Marketing Service through grant AM21 SCBPCA1002. Data are available on request to the authors.