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DNA BASED MOLECULAR METHODS FOR BACTERIAL SOURCE TRACKING IN WATERSHEDS
Citation:
Simpson*, J M. DNA BASED MOLECULAR METHODS FOR BACTERIAL SOURCE TRACKING IN WATERSHEDS. Presented at EPA Science Forum 2003, Washington, DC, 5/5-7/2003.
Description:
Point and non-point pollution sources of fecal pollution on a watershed adversely impact the quality of drinking source waters and recreational waters. States are required to develop total maximum daily loads (TMDLs) and devise best management practices (BMPs) to reduce the pollutant inputs from the sources. Fecal indicator bacteria and pathogens from a variety of sources are among the most common biological contaminants found in source and recreational waters. Sensitive and reliable methods for tracking fecal microorganisms to their source(s) are urgently needed to assist in development of TMDLs and BMPs.
The presence of fecal bacteria does not always indicate human fecal input. Therefore it is necessary to distinguish among fecal sources (i.e. livestock, wildlife, pets, wastewater treatment). However, it is very difficult to determine the origin of biological water contamination using standard physiologic and biochemical characteristics of indicator bacteria. To avoid these limitations, we are applying genetics based techniques using 16S rRNA or rDNA genes for detecting and identifying fecal indicator or pathogenic microorganisms in water without the need for cultivation. Signature molecules such as the 16S rDNA gene may also be used to differentiate among types of contaminant sources to help implement new regulation standards and to evaluate the effectiveness of remedial actions.
This ORD in-house research project was initiated to evaluate potential target organisms and to determine appropriate molecular fingerprinting methods. Fingerprinting methods are based upon the generation of a unique DNA based pattern for each sample. The fingerprint generated is then compared to other fingerprints in a database to determine if a source can be identified. Potential DNA fingerprinting techniques include ribotyping, amplified fragment length polymorphism, repetitive PCR, denaturing gradient gel electrophoresis, and terminal fragment length polymorphism.
The goals of this project are to identify and characterize watershed bacterial community members of fecal origin using several molecular techniques, and to use this information to develop an integrated system for screening fecal bacteria to differentiate among animal sources of contamination. Thus far, the microbial community of a stream contaminated by horse manure has been examined in depth and a potential target (Bacteroides spp.) has been selected for further evaluation. Additionally, fecal samples from animals of agricultural significance have been evaluated for similarities in bacterial populations and a DNA fingerprint database has been initiated.
The selection of the best molecular method(s) along with the most suitable target bacterial population(s) would allow for rapid screening and identification of fecal contamination sources on a watershed. This, in turn, could facilitate development of better TMDL plans and lead to BMPs for effective remediation of fecal contaminant sources.