Global gene expression of Escherichia coli O157 during survival on growing butterhead lettuce

Introduction
Numerous outbreaks with enteric pathogens such as Escherichia coli O157:H7 have been linked to consumption of fresh leafy vegetables (1). While the environmental factors which may influence the survival and proliferation of E. coli O157:H7 on growing plants are already intensively studied, little is known about the underlying genetic mechanisms.

Materials and Methods
Whole genome transcriptional profiles were generated from E. coli O157:H7 cells (isolate Sakai, stx-) inoculated on the leaves of growing butterhead lettuce. Four-week old plants were inoculated through spray inoculation and placed in a growth chamber at 18°C and 75% relative humidity. One hour and 2 days after inoculation, RNA was extracted from the E. coli O157:H7 cells recuperated from the leaves, and as a control, from the suspension used as inoculum. The reverse-transcribed DNA was then analyzed on E. coli microarray slides (Agilent).

Results
A total of 273 E. coli O157:H7 Sakai genes (5.04% of the whole genome) was significantly up or down regulated by at least twofold (P<0.01) when the pathogen was associated with the lettuce leaves. Almost 40% of these genes were poorly characterized or had an unknown function.
One hour after inoculation, the majority of the genes with a known function were upregulated (71%). These genes were mainly associated with metabolism (e.g. transport and metabolism of amino acids) and information storage and processing (transcription, translation, repair). At day 2, the known E. coli O157:H7 genes were mainly downregulated (65%) and were involved in among others carbohydrate transport, cell wall biogenesis and transcription.
Upregulation of numerous E. coli O157:H7 genes associated with oxidative stress and antimicrobial resistance, including the iron-sulfur cluster and the multiple antibiotic resistance (mar) operon, could be observed, whereas the Shiga toxin virulence genes were downregulated.

Discussion
Our findings reveal that the pathogen actively interacts with the plant environment by adapting its metabolism and responding to oxidative stress. Consequently, the pathogen may have acquired enhanced resistance against oxidative stress and cross-protection against various other stresses. On the other hand, a decrease in the expression of the Shiga toxin virulence genes was observed. Further research is needed to investigate how these adaptations may affect the pathogen’s subsequent survival during processing and consumption.

Acknowledgments
This study was funded by the Federal Public Service of Health, Food Chain Safety and Environment (contract RF 6202). The authors would like to thank Louise Birse, Dr. Pete Hedley and Dr. Jenny Morris for their help and processing of the microarrays.

References.
1.Olaimat, A. N., and R. A. Holley. 2012. Factors influencing the microbial safety of fresh produce: A review. Food Microbiology. 33:1-19.