Use of DNA fingerprinting and novel molecular methods to identify sources of Escherichia coli in the environment.

摘要

Fecal pollution affects the quality and safety of many water systems and can originate from both human and non-human sources including farm runoff, wildlife impact, agricultural waste, inadequate wastewater treatment, improper waste disposal, and septic failure. Understanding the origin of fecal pollution is paramount in assessing the proper risk and remedial action necessary after the problem has been identified. Feces from humans and animals contain a variety of pathogenic microorganisms, and many of these pathogens are not readily detectable in the environment by conventional methods as they are often present in very low numbers. In addition, different pathogens are harbored by different animal species, making identifying the type of pollution necessary for proper risk assessments to be performed. The prediction of the presence of pathogens is typically performed by the detection of established microbial indicators; however, these indicators are not adequate in identifying sources of pollution. Consequently, when they are detected in the environment using conventional tests, the source and the full extent of potential human health risks cannot be determined.; The first half of this study extended previous research using ribotyping to differentiate E. coli (a well-established fecal indicator) isolated from various animal species by including a greater number of isolates collected from a larger geographic region. As a result, it was determined that this method was not sufficient for differentiating sources of E. coli at the host species level outside of a confined watershed. For this reason and because ribotyping is time consuming and expensive, the second half of this study sought a more rapid molecular bacterial source tracking method by investigating the possibility that specific genetic differences exist between Escherichia coli isolated from animals and those isolated from humans. Several methods, including AFLP, PCR ribotyping, and rep-PCR were used to analyze the entire genome of E. coli from various sources for significant or subtle genetic differences. Single genes were also sequenced and analyzed for differences that could be useful in discriminating E. coli isolates based on host origin. Specifically, sequence analyses were performed on genes that code for fimbrial adhesins in E. coli. Once unique sequences were found, specific PCR primers were developed that were capable of amplifying these sequences. The result was a rapid, molecular tool that can aid in differentiating Escherichia coli derived from human and animal sources.