Grantee Research Project Results
2000 Progress Report: Molecular Detection of Anaerobic Bacteria as Indicator Species for Fecal Pollution in Water
EPA Grant Number: R827639Title: 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 Period Covered by this Report: November 1, 1999 through October 31, 2000
Project Amount: $223,829
RFA: Ecological Indicators (1999) RFA Text | Recipients Lists
Research Category: Ecological Indicators/Assessment/Restoration , Aquatic Ecosystems
Objective:
Fecal pollution in water is a threat to ecosystem integrity that also poses health risks to humans. Often the problem is not mitigated because the sources of the pollution cannot be determined. For example, runoff from nonpoint sources such as farm manure and failing septic systems may be implicated. The standard indicator for fecal pollution, fecal coliforms, do not distinguish between human and animal sources. The central aim of this proposal is to continue the development of a novel indicator system, based on the fecal anaerobic group Bacteroides-Prevotella, for detecting, quantifying, and distinguishing the sources of fecal pollution in water. Individual objectives are to:
- Develop additional Bacteroides-Prevotella indicators from waterfowl and any other species that are believed to make a significant contribution to fecal pollution in the waters of Tillamook Bay and its feeder rivers.
- Analyze clone libraries of Bacteroides-Prevotella 16S rDNAs amplified from cow, human and waterfowl feces (and other species, identified in Objective 1). The goal is to identify the strains unique to different host species, which provide the species-specific indicator peaks detected by LH-PCR and T-RFLP. Sequences will be used to design oligonucleotide probes.
- Using real-time quantitative PCR, quantify the amount of
Bacteroides-Prevotella fecal bacteria in samples, and the proportional
contribution from cattle, human and other sources.
Progress Summary:
Objective 2: Using Bacteroides-specific PCR primers of our own design, we constructed a clone library of small-subunit 16S rDNA genes from fecal Bacteroides from both cow and human feces. We sorted the clones by restriction analysis, and chose approximately 50 from each species to sequence. Phylogenetic analysis of the sequences showed that there was no overlap between the two host species. Human fecal Bacteroides sequences clustered with known Bacteroides species such as B. vulgatus, whereas cow fecal Bacteroides sequences were not related to any known species and formed their own clusters. This made it easy to design unique PCR primers to selectively amplify either the human or the cow sequences. We tested the new primers with both fecal dilutions and natural water samples, and were able to recover sequences related to our human and cow clones. We also established the sensitivity of our primers in relation to the amount of feces in the water. Upon testing our primers for specificity, using fecal dilutions from a number of different animal species as targets, we found that although our human primers were very specific and only amplified Bacteroides from human feces, our cow primers amplified Bacteroides from other ruminant species, such as goat, sheep, deer, and elk. This is a potential problem, because in certain situations it could be important to distinguish the fecal pollution from wildlife from the fecal pollution of domestic animals.
Objective 1: We are in the process of designing new PCR primers to distinguish feces from more species. We have prepared Bacteroides small-subunit rDNA clone libraries from elk, and from domestic cats and dogs. These are in the process of being analyzed. We were unable to amplify Bacteroides from seagull feces, and await fecal samples from other waterfowl, such as ducks and geese, to be provided by collaborators. We are also planning to analyze beaver and harbor seal, potentially important species at our field sites.
Future Activities:
We will continue to design primers for different animal species. We are undertaking an experiment to quantify the sensitivity of detection of both general fecal pollution, and species-specific pollution, is relation to the standard coliforms-based methods. Finally, we will undertake Objective 3, using real-time PCR to make our method quantitative, during the second and third years of the project.Journal Articles on this Report : 2 Displayed | Download in RIS Format
Other project views: | All 38 publications | 10 publications in selected types | All 8 journal articles |
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Bernhard AE, Field KG. A PCR assay to discriminate human and ruminant feces based on host differences in Bacteroides-Prevotella genes encoding16S rRNA. Applied Environmental Microbiology 2000;66(10):4571-4574. |
R827639 (2000) |
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Bernhard AE, Field KG. Identification of nonpoint sources of fecal pollution in coastal waters by using host-specific 16S ribosomal DNA genetic markers from fecal anaerobes. Applied and Environmental Microbiology 2000;66(4):1587-1594. |
R827639 (2000) |
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Supplemental Keywords:
watersheds, marine, estuary, risk assessment, health effects, ecological effects, human health, pathogens, viruses, bacteria, effluent, innovative technology, remediation, decision making, monitoring, northwest, Oregon, OR, agriculture., RFA, Scientific Discipline, Water, Ecosystem Protection/Environmental Exposure & Risk, Nutrients, Ecology, Wastewater, Ecosystem/Assessment/Indicators, Ecosystem Protection, Environmental Chemistry, 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 pollutionRelevant Websites:
http://osu.orst.edu/dept/microbiology/fac/field2.html
Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.