## 3 Results
### Harvest Temperatures and Cut-to-Cool Times of Romaine Lettuce from Salinas and Yuma Growing Regions
#### 3.2.1 Transitions in Physiological States of STEC O157:H7 on Inoculated Lettuce  
To assess the effects of harvest temperature on STEC O157:H7 physiology, we selected the harvest temperatures (9 \({}^{\circ}\)C or 17 \({}^{\circ}\)C) representing the 25 th and 75 th percentiles from the Yuma and Salinas growing regions, respectively. For the experiment, the cut-to-cool time of 240 min was selected, which was similar to the median cut-to-cool time for Romaine lettuce from the Yuma growing region. The greenhouse grown lettuce was inoculated with the different STEC O157:H7 strains and lettuce was incubated at the selected harvest temperatures for 240 min before cooling.  
The geometric means of culturable counts of STEC O157:H7 on harvested Romaine lettuce were 4.25-6.57 and 4.16-5.02 log CFU/g for the three strains at 9 and 17 \({}^{\circ}\)C, respectively. The average percentages of injured cells were 26.5 (sd = 12.4) to 81.1% (sd = 25.5) and 58.6 (sd = 24.3) to 72.7% (sd = 22.6), the average percentages of cells in the persister state were 2.4 (sd = 2.47) to 20.0 (sd = 7.95) and 6.7 (sd = 9.64) to 18.9 (sd = 20.1), and the average VBNC cell counts were \(-\)0.47 (sd = 0.7) to 0.54 (sd = 1.00) and \(-\)0.37 (sd = 0.56) to 0.99 (sd = 0.54) log CFU/g after harvest at 9 and 17 \({}^{\circ}\)C, respectively. The effect of harvest temperature on the vest temperature and strain on the physiological state was statistically evaluated using factorial ANOVA tests, accounting for the interactions. For changes in culturable and injured cells, significant differences (\(p<0.05\), ANOVA) were observed among the strains (Figure 3A,B). The change in persister percentages and VBNC counts were dependent on strain and harvest temperature, as evidenced by a significant interaction (\(p<0.05\), ANOVA; Figures S1 and S2). The Yuma outbreak strain had greater changes in culturable and injured cells compared to the Central Coast outbreak strain (Figure S3). The Salinas 2019 outbreak strain had a significantly higher persister percentage and amount of VBNC cells compared to that of the Central Coast 2018 outbreak strain on lettuce harvested at 9 \({}^{\circ}\)C (Figure 3C,D). For lettuce harvested at 17 \({}^{\circ}\)C, the Yuma outbreak strain had a higher amount of VBNC cells compared to the Central Coast 2019 outbreak strain.  
2.2 Effect of Harvest Temperature on Acid and Chlorine Tolerance of STEC O157:H7 on Inoculated Lettuce  
To quantify changes in acid and chlorine tolerance, the change in log values as Nt/N0 was over the 4 h period was calculated. The change in log reduction was computed as the  
Figure 3: Boxplots representing (**A**) change in culturable cells, (**B**) injured cell percentage, (**C**) persister cell percentage, and (**D**) VBNC cells at 4 h after harvest. X-axis shows harvest temperature. Culturable cells and VBNC cells are shown as change in log CFU/g, whereas persister cells, and injured cells are shown as change in percentages on \(Y\)-axis. Solid boxplots represent 25 th and 75 th percentile of the data. Outliers are shown by solid dots. Each boxplot represents one strain with at least three biological replicates and two technical replicates.  
difference of the LRV at harvest (0 h) and after 4 h of incubation at harvest temperatures (9 and 17 \({}^{\circ}\)C). Median log reduction values due to chlorine and acid treatments were reported for the Central Coast outbreak strain (0.17 and 0.30), the Salinas 2019 outbreak strain (\(-\)1.50 and \(-\)0.18), and the Yuma 2018 outbreak strain (0.05 and 0.04) at 9 \({}^{\circ}\)C and 17 \({}^{\circ}\)C harvesting temperatures, respectively (Figure 4). Despite the variations, strain and harvest temperature did not significantly (\(p\) > 0.05, ANOVA) affect the change in log reduction due to acid and chlorine treatments. The interaction of harvest temperature and strain did not significantly (\(p\) > 0.05, ANOVA) affect the change in log reduction due to acid nor chlorine treatments.