Escherichia coli O157:H7 stress and virulence gene expression on Romaine lettuce using comparative real-time PCR

Foodborne outbreaks attributed to the contamination of fresh produce with Escherichia coli O157:H7 are a growing concern. In particular, leafy-green vegetables, including lettuce and spinach, are susceptible to contamination by irrigation water, manure, and food processing and storage practices. The survival of E. coli O157:H7 and natural microflora on Romaine lettuce stored at 4 degrees C and 15 degrees C over a 9-day period was evaluated by plate counts. A two-step reverse-transcription comparative quantitative real-time PCR assay was employed to evaluate expression of genes coding for the A subunit of Shiga-toxin 1 and 2 (stx1A and stx2A), intimin (eaeA), flagellin (fliC), sigmaS--general stress sigma factor (rpoS) and iron superoxide dismutase (sodB) in E. coli O157:H7. Results indicate that reducing the storage temperature from 15 degrees C to 4 degrees C significantly (P<0.05) reduced the growth of Escherichia coli O157:H7 on Romaine lettuce, however, viable populations remained after the end of both storage periods. At end of the storage period, a 0.430 and 0.180 log decrease in E. coli O157:H7 was observed at 4 degrees C and 15 degrees C, respectively. Under both storage temperatures, total aerobic plate counts increased over the duration of the experiment. An increase in E. coli O157:H7 fold expression was observed with stx2A. Although stx1A exhibited upregulation for all storage conditions, variable gene expression was observed throughout the storage period. In addition, fliC was up-regulated during storage at 15 degrees C, while transcription at 4 degrees C storage changed only slightly. Expression of eaeA was variable at 15 degrees C with a tendency towards down-regulation, however, this gene was slightly up-regulated when stored at 4 degrees C. A slight upregulation of rpoS and sodB was also observed at 4 degrees C. In conclusion, our results suggest E. coli O157:H7 may become more virulent with prolonged storage of Romaine lettuce, particularly when stored at refrigerated temperatures.