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A molecular method based on chromosomal integration of the green fluorescent protein (gfp) gene into E. coli O157:H7 is used in this project to provide an alternative, and potentially more accurate, approach to the measurement of E. coli O157:H7 internalization in fresh produce. Use of the gfp marker in previous studies typically involved integration of the gfp gene into plasmid DNA along with an antibiotic resistance gene marker, which may lead to detection of fewer E. coli O157:H7 cells than actually present under conditions of physiological, nutritional, or antimicrobial stress. The chromosomal integration method used in this study will help to determine whether and to what extent internalization of E. coli O157:H7 occurs in fresh produce.
The project will apply this novel approach to evaluate the following hypotheses: <ul>
<li>that E. coli O157:H7 strains isolated from produce-associated outbreaks are better able to be internalized into plants than are strains isolated from beef-associated outbreaks or non-pathogenic E. coli strains; <li>that E. coli O157:H7 requires an intact stress response to survive in plants; and <li>that established E. coli O157:H7 virulence factors are heightened, or up-regulated, during growth on leafy greens compared with growth in ground beef. </ul>
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Specific objectives of the project include: <ul>
<li>Development of strains of E. coli O157:H7 and non-pathogenic E. coli that contain the gfp gene inserted into the bacterial chromosome, using newly developed transformation methods based on transposons as vectors. These strains will have improved bacterial fitness over plasmid gfp strains and will not require a selective agent for detection, enabling accurate assessment of E. coli O157:H7 survival and growth.
<li>Determination of the survival and growth of gfp-labeled wild type versus stress response gene (rpoS)¬-deficient E. coli O157:H7 populations in internalized plant tissues in different spinach and lettuce varieties. Enumeration and microscopic analysis of E. coli O157:H7 from internalized tissues will reveal the possibility and extent of internalization in plant tissues at specific points during the growing process.
<li>Determination of differences in the expression of virulence factors in E. coli O157:H7 grown on leafy greens compared with E. coli O157:H7 grown in ground beef. Virulence factors such as ler (positive regulator of type III secretion), eae (outer membrane adhesin intimin), espA (type III secretion filament), ehxA (enterohemolysin), ihaA (adherence factor), rfbE (O157 antigen), and stxII (shiga toxin 2) will be evaluated by real time reverse transcriptase (RT) polymerase chain reaction (PCR) methods. A ratio between virulence factors and housekeeping genes will be determined, and the ratio for E. coli O157:H7 grown on lettuce will be compared to the ratio for E. coli O157:H7 grown on ground beef. This comparison will yield insights into the potential of E. coli O157:H7 to cause human illness through the consumption of contaminated produce.</ul>