Analysis of E. coli DNA from selected source(s) using pulsed-field gel electrophoresis

The presence of fecal coliforms in surface waters is a public health issue throughout the United States and Canada. The presence of Escherichia coli (E. coli) indicates recent fecal contamination from either animal or human sources. To solve this growing problem one must identify the source(s) of such contamination by creating a database of DNA profiles of£. coli isolates. Hence, tracking the source(s) of E. coli can help link water contamination to animal source(s) of contamination. In this study, pulsed field gel electrophoresis (PFGE) was used to analyze DNA from E. coli isolates from known animals. For PFGE, the bacterial DNA was embedded in agarose plugs (to prevent shearing of DNA) and then cut with a restriction endonuclease (XbaI). The digested DNA was resolved on an agarose matrix to yield PFGE profiles. Unique DNA isolates that had been previously cut and resolved with were then cut with a second restriction endonuclease, Notl. Different DNA profiles of chromosomal fragments were generated for different animal sources. The following aspects, which are fundamentals of this study, were investigated to identify possible sources of contamination. Similar DNA profiles of E. coli were statistically analyzed to determine good identifier regions for rapid analysis. Four of the six established regions of a DNA profile were considered to be good identifier regions. It was determined that of the two restriction enzymes used, XbaI was a better enzyme to use to establish a database, since it allowed for better differentiation of strains within animals. Thus, it was possible to differentiate between animal types and individuals of the same animal in this database