1. An Overview of Bacterial Source Tracking - Methods and Applications
Douglas Moyer & Kenneth Hyer
U.S. Geological Survey
Richmond, Virginia
October 17, 2003
An Overview of Bacterial Source Tracking - Methods and Applications
Douglas Moyer & Kenneth Hyer
U.S. Geological Survey
Richmond, Virginia
October 17, 2003

2. 303(D) List of Impaired Waters
More than 600 stream/rivers
listed as impaired
More than 50% listed for
violations of the fecal
coliform bacteria standard
According to the D water-quality report, prepared by the Department of Environmental Quality and Department of Conservation and Recreation, there are 240 streams and rivers that are listed as impaired due to violations of water-quality standards. Of these 240, 163 are listed due to violation of the fecal coliform bacteria standard which is 1000 colonies per 100 mL.
In order to clean up the streams, a major hurdle that has to be dealt with is determine the sources of fecal coliform bacteria causing the violations.
To address this problem the USGS has formed a partnership with DEQ, DCR, and Fairfax county to test ribotyping as bacteria source tracking method in VA and Demonstrate the utility of incorporating bacteria source tracking information into watershed models for better definition of waste-load allocations.
Determine the sources of
fecal coliform bacteria
causing the violations

3. Bacterial Source Tracking
UNKNOWN
KNOWN
Water
Samples
Source
Indicator
Organism
pattern C B A
Dog
Human
Goose
>The technique that we are incorporating to determine the dominant sources of fecal coliforms is Ribotyping. We have enlisted the help of Dr. Samadpour at the University of Washington to perform the genetic analysis associated this technique. Dr. Samadpour is at the forefront of developing the ribotyping technique and has successfully used this method to identify sources of fecal coliforms in several impaired streams in the state of Washington.
>How this technique works is as follows: we start off with the water samples and source samples. From both of these samples fecal coliform bacteria have been isolated. These samples are then sent to the Univ. of Washington for ribotyping.
>At the University of Washington, these samples are further processed to isolated E. coli. From the isolated E.coli, their respective DNA is isolated. Within this DNA Dr. Samadpour focuses on the section that is coded for the production of ribosomal RNA.
>Now it is this section that is used to distinguish among the E. coli that are derived from the different warm-blooded animal species.
>So we will obtain a genetic fingreprint from our water samples but the sources are unknown. These will be compared and matched to the unique genetic fingerprint obtained from our known source within the watershed.
>This information will be incorporated into our watershed model to ensure that we are accurately representing fecal coliform bacteria within the Christians Creek Watershed.

4. BST Methods Comparison Study
Study involves a direct comparison of 7 BST methods.
Each researcher is provided a collection of known library isolates.
Evaluation is based on the utility of each method to identify 200 blind isolates (which were from known sources).
Five Genotypic Methods (and investigators)
Ribotyping using two different enzyme sets
(George Lukasik, Mansour Samadpour)
Pulsed-field Gel Electrophoresis
(West Virginia Department of Agriculture)
rep-PCR using two different primer sets
(Howard Kator, Don Stoeckel)
Two Phenotypic Methods (and investigators)
Antibiotic Resistance Analysis (Bruce Wiggins)
Carbon Substrate Utilization (Chuck Hagedorn)
The approach that the USGS will take with respect to sample collection and processing is as follows.
In addition to the monthly samples collected by DEQ, we will be collecting water samples under three regimens
First, we will collect baseflow samples every six weeks. Second, we will collect samples from five independent storms. Finally, we will have 3 continuum events where we sample 6 locations along each stream reach.
The sample analyses are as follows: we will enumerate actual fecal coliform bacteria concentration from all water samples. This information will be incorporated into the watershed model
The Fecal coliforms obtained during baseflow and stormflow will be sent to the molecular genetics lab at the University of Washington for the ribotyping genetic analysis .
Finally, we will collect source samples from a variety of sources throughout the watershed. That means that we will be collecting feces from warm blooded animals such as people, pets, livestock, and wildlife.

5. Methods Comparison - Conclusions
In a general sense, we found that:
-In this study, under these conditions… These results may not be universal!
-All methods did not produce comparable results; some methods were more successful than others.
-Results will be published this winter, likely in Environmental Science and Technology. A draft manuscript is currently in the review process.
This is one of a few comparison studies being done. Need to see what some of these other comparison studies determine...
Without stepping through the results, we can offer these conclusions/recommendations:
-Perform considerable QA/QC in your BST work! This may include:
(1) analyzing blind collections of known isolates,
(2) use of multiple BST methods, and
(3) the use of other tracers to support the BST work.
-Source tracking has tremendous potential, just be cautious in your application of this new technology.
The approach that the USGS will take with respect to sample collection and processing is as follows.
In addition to the monthly samples collected by DEQ, we will be collecting water samples under three regimens
First, we will collect baseflow samples every six weeks. Second, we will collect samples from five independent storms. Finally, we will have 3 continuum events where we sample 6 locations along each stream reach.
The sample analyses are as follows: we will enumerate actual fecal coliform bacteria concentration from all water samples. This information will be incorporated into the watershed model
The Fecal coliforms obtained during baseflow and stormflow will be sent to the molecular genetics lab at the University of Washington for the ribotyping genetic analysis .
Finally, we will collect source samples from a variety of sources throughout the watershed. That means that we will be collecting feces from warm blooded animals such as people, pets, livestock, and wildlife.

6. Field Application of BST
Three Streams included on the DEQ 303(d) list:
Accotink Creek, Christians Creek, and Blacks Run
Field Data Collection
Water-sample collection
Baseflow - 8 samples every 6 weeks
Stormflow - 10 samples during 5 events
Source Samples
Bacteria Source Tracking Analysis – Ribotyping
TMDL Development Using HSPF
The approach that the USGS will take with respect to sample collection and processing is as follows.
In addition to the monthly samples collected by DEQ, we will be collecting water samples under three regimens
First, we will collect baseflow samples every six weeks. Second, we will collect samples from five independent storms. Finally, we will have 3 continuum events where we sample 6 locations along each stream reach.
The sample analyses are as follows: we will enumerate actual fecal coliform bacteria concentration from all water samples. This information will be incorporated into the watershed model
The Fecal coliforms obtained during baseflow and stormflow will be sent to the molecular genetics lab at the University of Washington for the ribotyping genetic analysis .
Finally, we will collect source samples from a variety of sources throughout the watershed. That means that we will be collecting feces from warm blooded animals such as people, pets, livestock, and wildlife.

7. BST Results: By Individual Contributor5 10 15 20 25 30 35
Goose
Human
Dog
Duck
Cat
Sea Gull
Raccoon
Cattle
Deer
Horse
Poultry
Percent of Known
Accotink Creek
Christians Creek
Blacks Run

8. Validation of BST: Human Signature
Caffeine
0.05
0.1
0.15
0.2
0.25
Caffeine (µg/L)
Cotinine
0.01
0.02
0.03
0.04
Cotinine (µg/L)
Accotink Creek
Christians Creek
Blacks Run

9. Results of BST: Comparison of Accotink Creek and Four Mile Run
Accotink Creek, BST Results
Four Mile Run, BST Results
(N=279)
(N=278)
Other
21.1%
12.0%
Dog
13.3%
9.0%
Waterfowl
38.7%
37.0%
Deer
1.4%
10.0%
Raccoon
5.4%
15.0%
Human
20.1%
17.0%

10. Blacks Run Fecal Coliform Modeling Process
Fecal Coliform Concentration
& BST Data
Fecal Coliform/Manure
Application/Deposition
Land
Surface
Wash-off
by Overland
Flow
Die-off
Point
Source
Release of
Bacteria with
Interflow
Shallow
Sub-Surface
Storage
Release of
Bacteria with
Baseflow
Deeper
Sub-Surface
Storage

11. Fecal Coliform Calibration
Observed BST
Initial Model Calibration
Final Model Calibration 5 10 15 20 25 30 35 40
Percent Signature
GOOSE
HUMAN
DOG
DUCK
CAT
RACCOON
DEER
MUSKRAT

12. USGS Contact Information
Watershed Model
Doug Moyer
1730 E. Parham Rd Phone:
Richmond, VA
Bacterial Source Tracking
Ken Hyer
1730 E. Parham Rd Phone:
Richmond, VA
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