The long-term survival of Escherichia coli O157:H7 and Salmonella on lettuce seeds and their subsequent survival and growth on germinating sprouts

Introduction
In recent years, numerous outbreaks with enteric pathogens have been associated with contaminated sprouts (2,3). Moreover, evidence suggests that the recent outbreaks in Germany and France with Escherichia coli O104:H4 could be attributed to contaminated seeds, namely fenugreek seeds (1). These seeds had possibly been contaminated for up to two years. But until now, bacteriological evidence for this hypothesis is lacking. This raises the question whether enteric pathogens can survive for such a long period on seeds and resuscitate and even proliferate on the germinating sprouts.

Purpose
The present study aims to investigate the long-term survival of enteric pathogens on lettuce seeds and their subsequent survival and growth on germinating sprouts.

Methods
Lettuce seeds were inoculated at a high level with two Salmonella and two E. coli O157:H7 strains (±8 log cfu/g). The survival of the pathogens was followed for at least one year (up to two years) using standard plating techniques on selective medium. After these two years, three methods were tested to recover the pathogens from the seeds. Last, the seeds were germinated and examined for the presence of the pathogens.

Results:
Both Salmonella strains survived significantly better than the E. coli O157:H7 strains. Two years post inoculation, Salmonella was still present on every seed while E. coli O157:H7 could be recovered from only from 4% to 14% of the seeds, depending on the recovery method used. Both pathogens were able to proliferate on the positive germinating sprouts. Densities of up to 5.92 log cfu per positive sprout were observed for Salmonella; for E. coli O157:H7 this rate was 4.41 log cfu per positive sprout.

Conclusions
Our study not only confirms the long-time survival of enteric pathogens on seeds but also shows that, even after long storage of the contaminated seeds, the pathogens are able to resuscitate and proliferate on the germinating sprouts.

Acknowledgments
This study was funded by the Federal Public Service of Health, Food Chain Safety and Environment (contract RF 6202).