Submitted to: Journal of Food Protection
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/2/2009
Publication Date: 7/1/2009
Publication URL: hdl.handle.net/10113/32522
Citation: Sharma, M., Ingram, D.T., Patel, J.R., Millner, P.D., Hull, A., Donnenberg, M. 2009. A novel approach to investigate the uptake and internalization of Escherichia coli O157:H7 in spinach cultivated in soil and hydroponic media. Journal of Food Protection. 72(7):1513-1520. Interpretive Summary: Uptake of E. coli O157:H7 through root tissues of leafy green commodities is a potential route of contamination. Several fluorescent strains of E. coli were modified in a simple and unique manner to carry the green fluorescent protein on the chromosome. E. coli cells were then inoculated at low and high populations into pasteurized soils or into hydroponic media in which baby spinach plants were cultivated. No bacterial populations were recovered from the surface-sterilized internal tissues of spinach plants on day 0, 7, 14, 21, or 28 when plants were grown in soils. When plants were grown hydroponically in the presence of high populations of E. coli, internalized cells of E. coli O157:H7 were recovered consistently on day 14 and day 21. Produce outbreak strains of E. coli O157:H7 survived at higher populations in soils than a commensal strain of E. coli HS. Fluorescent microscopy revealed that E. coli cells are mainly associated with root tissue. The physiological stress placed on E. coli in inoculated soils and the barriers that intact plant tissue present limit the uptake and internalization of E. coli O157:H7 from soils to spinach plants.
Technical Abstract: Internalization of E. coli O157:H7 into spinach plants through root uptake is a potential route of contamination. A Tn7-based plasmid vector was used to insert the green fluorescent protein (gfp) gene into the attTn7 site in the E. coli chromosome. Three gfp-labeled E. coli inocula, O157:H7 strains 4407 and 5279 (Inoculum 1), O157:H7 strain 86-24h11 (Inoculum 2), and HS (Inoculum 3), were cultivated in fecal slurries and applied to pasteurized soils at ca. 3 log CFU/g or 7 log CFU/g in which baby spinach was planted. In the 28-day study, no E. coli inocula were recovered by spiral plating from surface-sanitized internal tissues of spinach plants on d 0, 7, 14, 21, and 28. Inoculum 1 survived at significantly (P<0.05) higher populations in the Inoculum 3 after 14, 21 and 28 d, indicating that produce outbreak strains of E. coli O157:H7 may be less physiologically stressed in soils than non-pathogenic E. coli isolates. Inoculum 2 applied to hydroponic medium at a ca. 7 log CFU/ml were consistently recovered by spiral plating from the shoot tissues of spinach plants after 14 (3.73 log CFU/shoot) and 21 days (4.35 log CFU/shoot). Fluorescent E. coli cells primarily located in root tissues in 72 sections of shoot and root tissues of spinach plants, grown in inoculated soils, were observed by fluorescence microscopy. The stress encountered in the rhizosphere and intact plant tissue appears to prevent the uptake of E. coli O157:H7 from soils to spinach plant shoot tissue.