## 1 Introduction
When STEC O157:H7 was exposed to 8 \({}^{\circ}\)C in the phyllosphere of pre-harvest lettuce, transformation into VBNC cells occurred 8-9 days post-inoculation [36]. In post-harvest conditions, STEC in spinach wash water transformed into persister cells at 15 \({}^{\circ}\)C [35]. STEC inoculated into the process water transformed into VBNC cells when process water was treated with chlorine [37]. These studies suggest that STEC can alter its physiological state by entering dormant states when presented with stress, but whether the length of cold storage impacts the entry into dormant states on lettuce is still unknown. When bacteria experiences cold stress, translation efficiency is reduced due to RNA folding, with other changes such as reduced membrane permeability, lower substrate diffusion rates, and disrupted ion equilibrium [38; 39; 40]. Activation of the general stress response occurs when temperatures are shifted from 37-35 \({}^{\circ}\)C to 25-14 \({}^{\circ}\)C [41; 42; 43]. Various cold shock proteins (CSPs) are involved in adaptation to cold temperatures. A class of CSPs, called the RNA chaperones, keep the RNA single-stranded during initial stages of cold stress [40; 44]. At temperatures of 4 \({}^{\circ}\)C and 18 \({}^{\circ}\)C, _cspA_ and _cspG_, respectively, are reported to be upregulated in STEC O157:H7 in lettuce lysates [45; 46]. Expression of _cspH_ and survival of persisters is known to be associated [46]. CspD is regulated by (p)ppGpp independently of RpoS [47] and is also known to play a role in persister cell formation [48]. This could be one of the pathways that STEC O157:H7 utilizes to enter into the persister state in response to cold stress. Stresses similar to those imposed on lettuce, as well as entry into dormant states, are known to impact the virulence potential of STEC. STEC O157:H7 on lettuce stored at 4 \({}^{\circ}\)C for 9-10 days shows higher expression levels of virulence-related genes _stx1_, _eee_, _elnxA_, and _fliC_[49; 50]. For STEC O157:H7, which entered the VBNC state on lettuce at 8 \({}^{\circ}\)C, Shiga toxin was still detected, even when culturable cells were not present, indicating that the STEC retained the potential for virulence [36]. Together, these data demonstrate that cold temperatures can impact STEC O157:H7 VBNC formation and virulence, although it is unclear if the length of time in cold storage on lettuce may influence virulence potential. Lettuce transported from the West Coast to the East Coast may be held for days at refrigeration temperature during transport prior to processing, likely impacting pathogen physiology [16; 17]. Physiological changes such as entering a dormant state can protect cells from environmental stress [23]. While STEC O157:H7 can enter the persister or VBNC state on lettuce [23; 34; 35; 36], the extent to which this occurs during harvest, cooling, and refrigerated transport is unknown. Changes in the physiological state of STEC O157:H7 have the potential to impact risk of illness associated with contaminated lettuce, due to alterations in tolerance to sanitizers in wash water as well as virulence properties. The objectives of this study were to investigate the effect of the length of time in cold storage on (a) the physiological state of STEC O157:H7 on Romaine lettuce; (b) subsequent acid tolerance and chlorine tolerance; and (c) virulence of STEC O157:H7.