Ken Hyer, U.S. Geological Survey Richmond, VA

1. Bacterial Source Tracking: Methods Comparison and Field Application Ken Hyer, U.S. Geological Survey Richmond, VA

2. Cooperators Cooperators VA Department of Environmental Quality VA Department of Environmental Quality Berkeley County, WV Berkeley County, WV VA Dept of Conservation and Recreation VA Dept of Conservation and Recreation WV Department of Environmental Protection WV Department of Environmental Protection WV Department of Agriculture WV Department of Agriculture Fairfax County, VA

3. Objectives for Talk • Describe a methods comparison study that evaluated 7 source tracking methods. • Describe a field application of BST and the associated quality control activities.

4. UNKNOWN KNOWN Isolated Water Source Samples E. coli Samples Isolated E. coli rRNA rRNA rRNA rRNA rRNA rRNA A C Dog Goose B Human Bacteria Source Tracking

5. BST Methods Comparison Study • Sampling in Berkeley County, West Virginia. • Involves researchers from across the nation and 3 different USGS district offices (Melvin Mathes of WV and Don Stoeckel of Ohio). • 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)

6. BST Methods Comparison Study • Prominent sources of fecal pollution being considered (based on NRCS input for Berkeley County): • Humans • Cattle (beef and dairy) • Chickens • Swine • Horses • Dogs • Canada Geese • Deer

7. Methods Comparison - Study Design • Collect feces from at least 20 individuals per source. • Isolate and confirm a library of known E. coli from the fecal samples: • Total of 70-100 confirmed E. coli per source • Total known library size of 900 isolates • Prepare a blind sample set comprised of 200 isolates that included three subsets: • 26 Replicates from the known library • 150 Fresh isolates from the 9 prominent sources • 24 Fresh isolates from sources that were not in the original known library (mice, cats, raccoons, etc.)

8. Methods Comparison - Study Design • Each researcher identified the source of each blind isolate. • Results were scored and the following were considered for each method: • Accuracy of isolate identification • Precision (reproducibility of replicate isolate analyses) • Robustness (isolates from sources not in the library are identified as unknown)

9. Methods Comparison - Results • In a general sense, we found that: -In this study, under these conditions… -Most methods did not perform as well as we expected, based on published literature. -Detailed study manuscript is in press at Environmental Science and Technology.

10. Results - Replicates • The first 3 methods used discriminant analysis (DA), the other 4 used direct matching techniques. • In scoring the replicates, the response “unknown” was considered incorrect (all isolates were in the known library). • For each method, considering an 8-way source classification: -ARA: 6 of 26 (23% correct) -CUP: 6 of 25 (24% correct) -RT-HindIII: 3 of 23 (13% correct) -RT-EcoR1: 14 of 26 (54% correct) -PFGE: 24 of 24 (100% correct) -BOX-PCR: 17 of 26 (65% correct) -REP-PCR: 10 of 23 (43% correct)

11. Results - Accuracy • For the accuracy subset, the response “unknown” was considered neutral (neither correct nor incorrect) – thus the number of isolates attempted is very important. • For each method, considering an 8-way source classification: -ARA: 36 of 150 (24% correct) -CUP: 20 of 143 (14% correct) -RT-HindIII: 19 of 147 (13% correct) -RT-EcoR1: 7 of 8 (88% correct) -PFGE: 15 of 40 (38% correct) -BOX-PCR: 32 of 149 (21% correct) -REP-PCR: 23 of 93 (25% correct)

12. Results - Ringers • In scoring the ringers, the response “unknown” was considered the only correct response because none of these isolates were in the library. • None of the DA methods attempted to identify and reject ringers. • For each method, considering an 8-way source classification: -ARA: 0 of 24 (0% correct) -CUP: 0 of 24 (0% correct) -RT-HindIII: 0 of 24 (0% correct) -RT-EcoR1: 24 of 24 (100% correct) -PFGE: 16 of 24 (67% correct) -BOX-PCR: 0 of 24 (0% correct) -REP-PCR: 8 of 24 (33% correct)

13. Reasons For Method Underperformance • Inadequate library size or structure • Temporal component in the source-library collection • Presence of many repetitive subtypes (transient strains) • Different statistical analyses may be needed • Regardless of these possible reasons, the study clearly demonstrates the need for QA/QC and proofing of methods.

14. Methods Comparison - Conclusions • This is only one of several ongoing comparison studies. It demonstrates that under these study conditions, none of these methods are performing at the levels we anticipated. • We can offer these 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. -Perform your QA/QC in such a way that you can detect if your method is working or failing.

15. 1. Accotink Creek – Urban 2. Blacks Run – Mixed Urban/Agricultural 3. Christians Creek - Agricultural 1 2 3 Example of an Applied Study

16. Study Design • Field Data Collection • Water-sample collection • Baseflow • Stormflow • Continuum • Source sample collection • Bacteria Source Tracking Analysis (Ribotyping)

17. 35 Accotink Creek 30 Christians Creek 25 Blacks Run 20 Percent of Known 15 10 5 0 Cat Dog Deer Duck Horse Cattle Goose Human Poultry Sea Gull Raccoon Results of MST:By Individual Contributor

18. 40 Warm (May-Sept) 30 Cool (Oct-March) Percent of Contribution 20 10 0 Cat Dog Deer Cattle Horse Poultry Human Waterfowl Seasonal Patterns in MST Data:Comparison of Warm and Cool Seasons

19. 0.25 0.04 Caffeine Cotinine 0.2 0.03 0.15 Cotinine (µg/L) Caffeine (µg/L) 0.02 0.1 0.01 0.05 0 0 Accotink Creek Christians Creek Blacks Run Validation of MST:Human Signature

20. 500 0.4 Discharge Arsenic 400 0.3 300 Total Arsenic (µg/L) Discharge (cfs) 0.2 200 0.1 100 0 0 10 20 30 40 50 60 Time (Hours) Validation of MST:Poultry Signature

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

22. Take Home Messages • Perform considerable QA/QC to ensure that you have confidence in your results. • Many different tools that can be applied to quality assure your BST data. • Under appropriate conditions, it appears BST can be used to successfully identify bacterial sources.

23. USGS Contact Information Ken Hyer 1730 E. Parham Rd Richmond, VA 23228 Email: kenhyer@usgs.gov Phone: 804-261-2636 On the web: http://va.water.usgs.gov/