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United States Department of Agriculture

Agricultural Research Service

Research Project: MICROBIAL ECOLOGY AND SAFETY OF FRESH ON-FARM ORGANICALLY GROWN PRODUCE Title: A Novel Approach to Investigate Internalization of Escherichia coli O157:H7 in Lettuce and Spinach

Author
item Sharma, Manan

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: August 22, 2008
Publication Date: September 11, 2008
Citation: Sharma, M. 2008. A Novel Approach to Investigate Internalization of Escherichia coli O157:H7 in Lettuce and Spinach. Meeting Abstract., Fresh Produce Safety Research Conference Research Synopses, p. 4-5.

Technical Abstract: The chromosomal integration of the green fluorescent protein (gfp) gene was successfully accomplished into four nalidixic acid resistant E. coli strains: two O157:H7 strains from produce outbreaks, 4407 and 5279, one O157:H7 strain from a beef-associated outbreak, 86-24h11, and a non-pathogenic commensal E. coli isolate (HS). An rpoS-deficient strain of E. coli O157:H7 was not able to be transformed with gfp because rpoS-functionality was required for this transformation. No E. coli cells were recovered by spiral plating from the internal tissues of baby spinach plants in sterile soils. Fluorescent E. coli cells were visualized in root tissue by microscopy. Three separate gfp-labeled inocula, E. coli O157:H7 strains 4407 and 5279 (Inoculum 1), E. coli O157:H7 strain 86-24h11 (Inoculum 2), and E. coli HS (Inoculum 3), consisting of gfp-labeled E. coli were applied to soils at two different inoculum levels (ca 7 log CFU/g sterile soil or ca. 3 log CFU/g sterile soil) in which baby spinach varieties were planted. In the 28-day study, no E. coli strains were recoverable by spiral plating from surface-sanitized internal tissues of spinach plants. However, Inoculum 1 survived at significantly higher populations in soils surrounding the rhizospheric soil than Inocula 3 after 21 and 28 d, indicating that produce outbreak strains of E. coli O157:H7 may be more viable in soils than non-pathogenic E. coli isolates. Cells of E. coli O157:H7 (Inoculum 2) were recovered by spiral plating from internal tissue spinach plants growing in hydroponic media for up to 14 days, and when these plants were transplanted into sterile soil, cells were still recovered from these tissues after 7 days. Internalized cells in spinach plants growing in hydroponic media were visualized microscopically. The virulence factors rfbE (O157-antigen) and ehx (hemolysin) were expressed at statistically significant higher levels in ground beef than on lettuce or in growth medium when grown at 37 deg C. However, no differences in the expression of eae (outer membrane adhesin intimin), espA (type III secretion filament), ihaA (adherence factor), and stxII (shiga toxin 2) were observed when E. coli O157:H7 were grown on ground beef, lettuce, or in laboratory growth medium. These data indicate that cut lettuce supported statistically similar levels of expression of these virulence factors of E. coli O157:H7 as ground beef.

Last Modified: 10/30/2014
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