In our BST projects we use antibiotic resistance analysis (ARA), carbon source profiles (the Biolog system), and pulsed-field gel electrophoresis (PFGE) to determine sources of fecal pollution. Enterococci are identified to genus and species by Biolog and E. coli isolates are identified by the API System. ARA is used primarily to differentiate between human, livestock, and wildlife sources, but can be used to identify specific sources (e.g. deer, cow, goose, etc.) if needed. Carbon source profiles is a new method under development in our lab. A manuscript has been submitted for publication; please contact Dr. Hagedorn for more information. PFGE is based on the method developed by Dr. George Simmons, Professor of Biology at VT. We use PFGE to cross-validate the souce classifications obtained with phenotypic methods. Our current projects are:

Bacterial Source Tracking Methods to Identify Nonpoint Fecal Pollution in Agricultural Watersheds. Funded by USDA's National Research Initiative (NRI). Co-Investigators are V. J. Harwood, Univ. of South Florida, and B. Wiggins, James Madison Univ.

BST Method Comparison Study (supplemental grant to the USDA-NRI project). Funded by USGS. Additional Co-Investigators are: D. Stoeckel (Ohio State Univ.), K. Strickler (WVa Dept. of Agriculture), and G. Lukasik Biological Consulting Services of North Florida, Inc.

Identifying Sources of Fecal Pollution in Shellfish Waters in Four Pilot Areas: Coan River, Little Wicomico River, LynnHaven Bay, and the Nansemond River. Funded by the Va Dept. of Environmental Quality. Co-Investigators are B. Wiggins, James Madison University, and H. Kator, Va Institute of Marine Sciences.

Identifying Sources of Fecal Pollution in Water as a Function of Sampling Frequency Under Low and High Stream Flow Conditions. Funded by Va Dept. of Conservation and Recreation. Co-Investigator is B. Wiggins, James Madison University.

TMDL Development in the Blackwater River, Franklin County, Va. Funded by Va Dept. of Conservation and Recreation. In cooperation with Map Tech, Inc.

TMDL Development in Naked Creek, Rockingham County, Va. Funded by Va Dept. of Conservation and Recreation.

Summary of BST Presentations at the American Society for Microbiology (ASM) Annual Meetings.

100th Annual ASM Meetings, May 21-25, 2000, Los Angeles, CA

Twelve posters were presented on BST. The methodologies in the posters used included 4 on antibiotic resistance analysis (ARA), 2 on randomly amplified polymorphic DNA (RAPD), and one each on ribotyping, source specific coliphages, biomarkers, sterols, multiple antibiotic resistance (MAR), and sorbitol-fermenting bifidobacteria. Most of the posters dealt with methodology development and building known source libraries.

101st Annual ASM Meeting, May 20-24, 2001, Orlando, FL

Twenty-three posters and two papers were presented on BST. The methodologies in the posters and papers included 6 on ARA, 5 on rep-PCR, 4 on ribotyping, 4 on source specific coliphages, 3 on multiple antibiotic resistance, two on pulsed-field gel electrophoresis, and one on source specific biotypes. Roughly half of the posters dealt with methodology development and library construction, while the other half involved using a BST method with a completed library to present source tracking results from a watershed, usually associated with a TMDL project.

BST Presentations by Students from the Hagedorn Lab at the 2001 ASM Meetings

Paper Q-318. Determining Sources of Fecal Pollution for a Rural Unsewered Community. Graves, A. K., A. M. Bowman, M. Mahal, and C. Hagedorn.

Bacterial Source Tracking (BST) methodology was used to determine sources of fecal pollution in Millwood, VA. Millwood consists of 71 homes, all served by individual septic systems of indeterminate age and performance, and a stream (Spout Run) passes through the center of the community. Stream and well samples were collected monthly starting in 5/99 and ending in 5/00, and analyzed for fecal coliforms and enterococci. Twelve percent of the well samples and 92% of the stream samples were positive for fecal coliforms, and 26% of the stream samples exceeded the recreational water standard (1000 fecal coliforms/100ml). Antibiotic Resistance Analysis (ARA) was preformed on enterococci recovered from Spout Run in upper, middle, and lower Millwood. Isolates of human origin appeared in upper, middle, and lower Millwood and ranged from 2.0% to 42% of the sourced isolates over sampling sites and seasons. The percentage of human origin isolates was highest in upper and middle Millwood, and declined downstream from that point. These results were obtained by comparing the antibiotic resistance profiles of stream isolates against a library of 1,174 known source isolates with correct classification rates for the known sources of 94.6% for human isolates, 93.7% for livestock isolates, and 87.8% for wildlife isolates. The main impairment in water samples was fecal coliforms, as no other problems were identified based on water chemistry analyses for both well and stream water samples. The sporadic instances where well samples were positive for fecal coliforms appeared mainly during the very dry fall sampling period.

Paper Q-372. Determining Sources of Fecal Pollution in the Blackwater River Watershed. Bowman, A. M., A. K. Graves, K. Hix, and C. Hagedorn.

Antibiotic resistance analysis (ARA) was used to determine sources of fecal pollution in the Blackwater River in south central Virginia. The Virginia Department of Environmental Quality designated seven segments as impaired due to fecal coliforms with non-point source (NPS) agriculture the suspected source of impairment. The Blackwater River watershed encompasses over 72,000 ha of dairy, beef, and intensive production agriculture, abundant wildlife populations in forested areas, and hundreds of homes with onsite septic systems. A library of antibiotic resistance profiles based on 30 drug/concentration combinations was developed for 1,451 enterococcus isolates from human, cattle, chicken, horse, goat, sheep, deer, raccoon, muskrat, goose, duck, coyote, and wild turkey. Each isolate was classified as human, wildlife or livestock. Correct classification rates were 82.3% for human, 86.2% for livestock and 87.4% for wildlife isolates. Profiles were then determined for 48 isolates from each stream sample, collected periodically from September 1999 to August 2000, and compared to the known sources using discriminant analysis. A human signature was found at each of 9 sites at least once during the sampling period, ranging from 2.1 to 56.2% of the profiled isolates. The livestock signature varied from 12.5% to 91.7% over sites and months, and the wildlife signature varied from 4.2% to 70.8%. The results indicate that both humans and wildlife contribute to fecal pollution in addition to the suspected source, livestock, and reducing fecal pollution in the Blackwater River watershed will require consideration of all three sources.