## 4 Discussion
### Increasing Cold Storage Time Led to Decreased Virulence in G. mellonella  
Even as sudden temperature fluctuations lead to production of heat and cold shock proteins to protect bacterial functions, shifts in temperature can also lead to the production of bacterial virulence factors [77]. Stress response and virulence genes such as _rpoS_, _stx1_, and _osmY_ have been shown to be highly expressed during cold shock transition from 37 \({}^{\circ}\)C to 4 \({}^{\circ}\)C or 7.5 \({}^{\circ}\)C [46; 78]. At longer cold storage temperatures, for cells inoculated on leafy greens, virulence genes such as _ila_, _stx2A_, and _ae_ have increased expression [49; 50]. In lettuce lysate, a cold shock of 10 min or 1 h led to increased expression of _stx1A_[78]. Shiga toxins have a cytotoxic effect, and cytotoxicity is an aspect of virulence that can be quantified in laboratory studies. A study in tissue culture cells showed that stressed STEC O157 cells had higher cytotoxicity than non-stressed controls [79]. This study also showed that chlorine-, starvation-, acid-, or oxidative-stressed STEC O157 cells have higher survival during cold storage of 4 wk at 8, 12, and 16 \({}^{\circ}\)C compared to non-stressed cells [79].  
We assessed relative virulence in our study with the insect model _Galeria mellonella_. Mammalian models of infection can raise ethical issues, generally are limited to small samples sizes, and are costly, and insect models provide an alternative representation of the innate immune response to pathogens. _G. mellonella_ is a common insect model that is used to investigate bacterial pathogenesis, including STEC O157:H7 [55; 57]. Our data shows that, in the Central Coast 2018 outbreak strain, there was a positive relationship between survival of larvae and cold storage, meaning that the virulence decreased with cold storage. This means that cold transportation of lettuce is likely to decrease virulence of STEC O157:H7 if it happened to contaminate lettuce. Our results are different from those reported in the previous study on cytotoxicity for stressed STEC O157 cells, although it is important to note that the previous research was conducted in cell culture rather than a more complex model host [79]. Previous studies have shown that cold temperature leads to increased virulence of gene expression, but only the effect of short-term shock has been evaluated on virulence phenotypes. Similar to our findings, a recent study showed decreased virulence of STEC O157:H7 in _G. mellonella_ following refrigerated storage of sprouts [80].  
Strains Affected the Variability in the Physiological, Tolerance, and Virulence Response of the Strains  
The strains that we used belong to different phylogenetic categories. The Central Coast 2018 and Salinas 2019 outbreak strains are more closely related to each other than to the Yuma outbreak strain, which belongs to the REPEXH01 category [3]. The Central Coast 2018 outbreak strain belongs to Clade 1, and the Salinas 2019 strain belongs to Clade 2 of REPEXH02 [6]. A recent study showed that a REPEXH01 strain had a lower persister cell formation and a higher VBNC cell formation compared to a REPEXH02 strain [81]. A similar trend was seen with our data, where the Yuma outbreak strain had a lower persister cell formation, and a higher VBNC cell formation compared to the Salinas strain. The Central Coast 2018 outbreak strain had the least increase in LRV during chlorine treatment, meaning that it was able to tolerate chlorine stress. Another study has shown that these REP strains, including Central Coast, CA 2018, and Salinas, CA 2019 outbreak strains, can form stronger biofilms, and exhibit a higher tolerance to gastric acid stress compared to other non-REP strains [82; 83]. Taken together, these data suggest differences in stress tolerance and entry into dormant states among REPEXH01 and REPEXH02 strains.