2001 Progress Report: Molecular Detection of Anaerobic Bacteria as Indicator Species for Fecal Pollution in WaterEPA 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 Period Covered by this Report: November 1, 2000 through October 31, 2001
Project Amount: $223,829
RFA: Ecological Indicators (1999) RFA Text | Recipients Lists
Research Category: Ecological Indicators/Assessment/Restoration , Ecosystems
This project involves the extension and validation of a new method to rapidly detect fecal pollution in water, and discriminate its source, based on amplification of genetic markers from the Bacteroides-Prevotella group of fecal bacteria. The objectives of the project are to develop additional species-specific Bacteroides-Prevotella indicators, to identify unique Bacteroides-Prevotella 16S rDNA sequences in clone libraries from multiple animal species, and to develop a real-time quantitative polymerase chain reaction (PCR) assay to quantify the contributions of different species to fecal contamination in water.
We have constructed Bacteroides-Prevotella 16S rDNA clone libraries from feces of the following species: elk, pig, dog, cat, and horse. Clones were screened by terminal restriction fragment length analysis (T-RFLP). Multiple representatives from each restriction fragment pattern type were chosen for sequencing. Phylogenetic analysis found that ruminant sequences (elk and cow) comprise two novel gene clusters. Most elk sequences are very closely related to cow sequences. However, there are at least three small subclusters of elk-specific sequences. Most sequences from pig fell into the genus Prevotella, either as unknown lineages, or related to P. buccaformis and P. ruminicola. In most groups, fecal Bacteroides-Prevotella appear to have coevolved with host species. Sequences from dog and cat, however, were closely related to human sequences, suggesting that humans and their pets acquire fecal bacteria horizontally.
The sequence analyses were used to design general and species-specific PCR primers. Out of four tested, one general Bacteroides-Prevotella-specific primer worked very well, allowing us to exploit a variable region of the gene. Although there was overlap among human, dog, and cat sequences, both our original PCR primers designed to be human-specific did not amplify any sequences from dog and cat. We designed, optimized, and tested 12 new primers, only to find that many of them amplified targets from additional species. We concluded that it is not practical (because of the large cost) to construct clone libraries large enough to sample the entire range of sequence diversity found within each species. Nevertheless, the observed patterns of diversity indicate that there is substantial endemism within the Bacteroides-Prevotella group. Therefore, we have decided to try another approach to design Bacteroides-Prevotella primers. We will use our Bacteroides-Prevotella general primers to amplify sequences to use in subtractive hybridization.
We have designed real-time quantitative PCR nuclease (TaqMan) assays for both general Bacteroides-Prevotella (to measure total fecal pollution) and from human fecal pollution.
We will develop a rapid method of water quality assessment, and a method of quantifying the contribution of different sources to fecal pollution in water. We currently are testing these primer sets in real-time assays.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
|Other project views:||All 38 publications||10 publications in selected types||All 8 journal articles|
||Bernhard AE, Goyard T, Simonich MT, Field KG. Application of a rapid method for identifying fecal pollution sources in a multi-use estuary. Water Research 2003;37(4):909-913.||