FATE OF ESCHERICHIA COLI O157:H7 IN IRRIGATION WATER ON SOILS AND PLANTS AS VALIDATED BY CULTURE METHOD AND REAL-TIME PCR

Authors: Ibekwe, Abasiofiok - Mark; Watt, Pamela; Shouse, Peter; Grieve, Catherine

Submitted to: Canadian Journal of Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/20/2004
Publication Date: 2/11/2005
Citation: Ibekwe, A.M., Watt, P.M., Shouse, P.J., Grieve, C.M. 2005. Fate of Escherichia coli O157:H7 in irrigation water on soils and plants as validated by culture method and real-time PCR. Canadian Journal of Microbiology. 50:1007-1014.

Interpretive Summary: Reclaimed water is being increasingly used for the irrigation of crops, and there is the danger of inadequate water treatment resulting in incomplete removal of bacterial pathogens. One of the most common vehicles by which E. coli O157:H7 may be introduced into crops is by flood irrigation with water contaminated with cattle feces or by contaminated surface water. In this experiment, we used bacteria tagged with green fluorescent protein in flooded irrigation for easy identification of bacteria in soil, roots and leaf surfaces. After plating and PCR analysis, E. coli O157:H7 concentrations were higher on the soils containing root tissue than on either the leaf surfaces or soils without roots. Our results showed that PCR analysis was faster and more reliable in enumerating the pathogen than plate counts.

Technical Abstract: A real-time PCR method was developed to detect and quantify Escherichia coli 0157:H7/pGFP (E. coli) in phyllosphere, rhizosphere, and nonrhizosphere soils irrigated with contaminated water. Probes were designed to hybridize with the eae gene of E. coli O157:H7. The probes were incorporated into real-time PCR containing DNA extracted from the phyllosphere, rhizosphere, and nonrhizosphere soils irrigated with water artificially contaminated with E.coli 0157:H7. Quantification was done with samples containing 10 to 1000000000 CFU of E.coli 0157:H7 ml-1. Linearity was determined with DNA template ranging from 1000 to 1000000000 CFU ml-1. The detection limit for E.coli 0157:H7 assay was 1400 in rhizoshere and phyllosphere samples. The concentrations of E. coli O157:H7 obtained by real-time PCR were comparable to the concentrations obtained by traditional culture methods on mTSA during the first week of inoculation, and thereafter, the concentrations by real-time PCR were higher. The real-time PCR assay may be a useful tool for the rapid quantification and monitoring of E.coli 0157:H7 in irrigation water and contaminated fresh produce.