Submitted to: Applied and Environmental Microbiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 2/14/2008
Publication Date: 4/1/2008
Citation: Brandl, M., Amundson, R. 2008. Leaf age as a risk factor in the contamination of lettuce with Escherichia coli O157:H7 and Salmonella enterica. Applied and Environmental Microbiology.74:2298-2306 Interpretive Summary: Outbreaks of Escherichia coli O157:H7 illness have been linked increasingly to leafy greens, particularly to lettuce. We present here the first evidence that this enteric pathogen can multiply on the leaves of Romaine lettuce plants and that its growth on pre-harvest and post-harvest lettuce leaves is dependent on leaf age. Its population size was consistently ca. 10-fold higher on the young (inner) leaves compared to the middle leaves. Similar observations were made with Salmonella enterica. Elemental analysis of the nutrients present on lettuce leaves of different ages showed that young leaves harbor higher amounts of both total nitrogen and carbon than middle leaves. Also, growth of E. coli O157:H7 was enhanced on middle leaves by supplementation with nitrogen, but not with glucose. This indicated that colonization of the middle lettuce leaves by the pathogen was limited by nitrogen. The results of our study suggest that young leaves may constitute a higher risk factor in contamination of lettuce than middle leaves. This should be taken into account in sampling procedures for detection of enteric pathogens on lettuce and in scientific studies on control and sanitization strategies.
Technical Abstract: Outbreaks of Escherichia coli O157:H7 illness have been linked increasingly to leafy greens, particularly to lettuce. We present here the first evidence that this enteric pathogen can multiply on the leaves of Romaine lettuce plants. The increase in population size of E. coli O157:H7 in the phyllosphere of young lettuce plants ranged from 16- to 100-fold under conditions of warm temperature and the presence of free water on the leaves, and varied significantly with leaf age. Its population size was consistently ca. 10-fold higher on the young (inner) leaves compared to the middle leaves. The growth rates of Salmonella enterica and of the natural bacterial microflora were similarly leaf age-dependent. Both enteric pathogens also achieved higher population sizes on young leaves than on middle leaves harvested from mature lettuce heads, suggesting that leaf age affects their pre-harvest as well as post-harvest colonization. Elemental analysis of the exudates collected from the surface of leaves of different ages revealed that young-leaf exudates were 2.9 and 1.5 times richer in total nitrogen and carbon, respectively, than middle-leaf exudates. This trend mirrored the nitrogen and carbon content of the leaf tissue. Application of ammonium nitrate, but not glucose, to middle leaves enhanced the growth of E. coli O157:H7 significantly, suggesting that low nitrogen limits its growth on these leaves. Our results indicate that leaf age and nitrogen content contribute to shaping the bacterial communities of pre-harvest and post-harvest lettuce and that young lettuce leaves may be associated with a greater risk of contamination with E. coli O157:H7.