Leaf age as a risk factor in the contamination of lettuce with Escherichia coli O157:H7 and Salmonella enterica

Produce Safety and Microbiology Research Unit, Agricultural Research Service, U.S.; Department of Agriculture, Albany, California 94710; and Division of Ecosystem Sciences, University of California, Berkeley, California 94720

^* To whom correspondence should be addressed. Email: mbrandl{at}pw.usda.gov.

	Abstract

Outbreaks of Escherichia coli O157:H7 infections 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.