Molecular Detection of Anaerobic Bacteria as Indicator Species for Fecal Pollution in Water

EPA Grant Number: R827639
Title: Molecular Detection of Anaerobic Bacteria as Indicator Species for Fecal Pollution in Water
Investigators: Field, Katharine G.
Institution: Oregon State University
EPA Project Officer: Packard, Benjamin H
Project Period: November 1, 1999 through October 31, 2002
Project Amount: $223,829
RFA: Ecological Indicators (1999) RFA Text |  Recipients Lists
Research Category: Ecological Indicators/Assessment/Restoration , Aquatic Ecosystems


Fecal contamination of aquatic environments is a continuing problem, afflicting many regions of the U.S. Although the health risks to humans are well known, much remains to be learned about concomitant effects on microbial communities. Often the source of fecal contamination cannot be determined with certainty. For example, runoff from non-point sources such as manure from dairy pastures, failing septic systems, and overloads at sewage treatment facilities may all be candidates. The standard indicators for fecal pollution are fecal coliforms, which do not distinguish between human and animal sources. We have developed a novel indicator system based on the anaerobic gut bacterial group Bacteroides/Prevotella.. We do not grow the indicator bacteria, but instead measure molecular markers amplified from bacteria filtered from the water. With this method we can already distinguish human from cow fecal pollution in both estuarine and river waters. We propose to study a small, nutrient-rich, fecally-polluted estuary, Tillamook Bay, Oregon, and its tributary rivers. The objectives of this proposal are, first, to develop additional markers from other biologically-important polluting species, such as waterfowl. Second, we will identify the indicator strains or species that are host-specific. Finally, we will make the indicator system quantitative, to allow estimation of both the amount of total pollution in the water, and the proportions of different sources of fecal pollution.

Resulting Improvement in Risk Management: This research will result in an indicator system for fecal pollution which can be used in wetlands, estuaries, streams, and lakes. It will allow managers to distinguish the sources of fecal pollution in water, leading to better management practices to maintain ecosystem integrity and reduce the risk to human health.


We will amplify fecal genetic markers by the PCR with primers specific for I6S rDNA gene fragments from BacteroidesJPrevotella. We will digest amplicons with selected restriction enzymes, and rapidly screen them by separation on a DNA automated sequencer in GeneScan? mode (T-RFLP), which estimates the relative proportions of each fragment based on relative fluorescence. Fluorescence data are converted into an electropherogram that is highly diagnostic for specif~c bacterial constituents. With this method, we have identified peaks diagnostic for human and cow feces, and propose to do the same for waterfowl and other species that contribute fecal pollution. Each host-specific Bacteroides/Prevotella. marker will be identified with 16S rDNA clone libraries. The amount of fecal bacteria in samples, and the proportional contribution from cattle, human and other sources, will be quantified by means of real time quantitative PCR with a Roche Boehringer Mannheim Light Cycler. The method will be tested, and correlated to standard fecal coliform measurements, using water samples collected along a transect from the Tillamook River to the mouth of the bay.

Expected Results:

This proposed research develops a novel molecular-based indicator system to specifically address the effects of an anthropogenic threat to ecosystem integrity, fecal pollution in water. We will develop markers for waterfowl and any other species important to our system. We will identify the distinctive indicator species that differentiate cow from human and other fecal pollution, and create a database of the Bacteroides/Prevotella species present in feces from cow, human and other species. Using real-time quantitative PCR, we will make the technique quantitative, so that the total amount and relative proportions of different kinds of feces, not just their presence and absence, can be measured.

Publications and Presentations:

Publications have been submitted on this project: View all 38 publications for this project

Journal Articles:

Journal Articles have been submitted on this project: View all 8 journal articles for this project

Supplemental Keywords:

RFA, Scientific Discipline, Water, Ecosystem Protection/Environmental Exposure & Risk, Nutrients, Ecology, Wastewater, Environmental Chemistry, Ecosystem/Assessment/Indicators, Ecosystem Protection, Ecological Effects - Environmental Exposure & Risk, Ecological Indicators, risk assessment, ecological exposure, aquatic, health indicator, aquatic ecosystem, environmental monitoring, microbial indicators, nutrient transport, sewage treatment, bacteria, anaerobic bacteria, coliform, waterfowl, ecosystem indicators, estuarine ecosystems, water quality, coliforms, sewage treratment, water treatment, fecal pollution

Progress and Final Reports:

  • 2000 Progress Report
  • 2001 Progress Report
  • Final Report