Microbial Source Tracking as a Component of TMDL Development

1. Microbial Source Tracking as a Component of TMDL Development Y. Feng, R.U. Wijesinghe, and C.W. Wood Department of Agronomy & Soils Auburn University

2. Background • Fecal contamination in surface waters • Fecal indicator organisms: fecal coliforms, E. coli, and entercocci • Section 303(d) of the Clean Water Act requires each state to establish a list of waters that do not currently support designated uses and to establish the total maximum daily loads (TMDL) that will meet water quality standards for each listed waterbody.

3. TMDL Process • Identify quality limited waters: 303(d) Use Impairment Lists • Establish priority waters/watersheds • Develop TMDL: • Problem statement - cause of impairment • Numeric target(s) - water quality standards • Source analysis - relative contributions of pollutant sources • Loading capacity estimate • Allocations - wasteload allocations to point sources and nonpoint sources

4. What is Microbial Source Tracking (MST)? • MST (BST) is new technology being developed to determine the origins of enteric microorganisms from environmental samples. • MST can also be used to evaluate the outcomes of best management practices (BMP). • Potential sources: agriculture, wildlife, leaky septic tanks, sewer overflow, etc. • Various approaches have been used to identify fecal sources in waters.

5. Overview of MST • Phenotypic vs. genotypic methods • Phenotypic: physiological and biochemical characteristics expressed by organisms • Antibiotic resistance analysis (ARA) • Carbon utilization profile • Genotypic: genetic characteristics • Repetitive sequence-based polymerase chain reaction (rep-PCR) • Ribotyping • Pulsed field gel electrophoresis (PFGE) • Host specific genetic markers • Library dependent vs. independent methods • Library dependent methods: ARA, rep-PCR, ribotyping, PFGE • Library independent methods: viruses, host specific genetic markers (Bacteroides)

6. Which method is the best? • Method comparison studies • SCCWRP (Southern California Coastal Water Research Project) study (2003) • USGS study (2004)

7. Which method to use?

8. MST Decision Tree (EPA, 2005) • Is the fecal pollution problem defined? • Has an adequate sanitary survey been conducted? • How many sources were identified in the sanitary survey? • Proceed if multiple sources • Is the watershed/study area of manageable size? • What is the desired level of discrimination? • Human vs nonhuman • Group separation • Species specific • Specific individual host

9. MST in Alabama *

10. Repetitive sequence-based PCR (rep-PCR) • E. coli genome contains many copies of repetitive DNA sequences. During the polymerase chain reaction, primers bind to these specific repetitive sequences and multiple DNA fragments with various lengths are generated (www.bacbarcodes.com). • Advantages: simple, rapid, cost-effective, differentiation to the strain level

11. Fecal samples for the known-source library

12. MacConkey Agar MacConkey ChromAgar methyl red MRVP ------------------------------------> Red color 37°C Kovac’s reagent 1% tryptone ----------------> Red ring 37°C Citrate agar ----------------------------------> No color change 37°C EC mug ---------------------------> Gas production & 44.5°C fluorescent under UV

13. Rep-PCR DNA fingerprinting E. coli cells mixed with PCR reagents PCR with Box A1R primer Electrophoresis Data analysis Gel imaging

14. rep-PCR fingerprints generated using the BOX A1R primer 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 Lanes 1, 10, 19, 18, 28, and 35 contain 1-kb Plus DNA ladder. Lanes 2, and 33 contain ATCC 25922, and lane 34 is the negative control. Lanes 3-5 contain dog isolates, lanes 6-9, 11 and 12 chicken, lanes 13-18 horse, lanes 20-25 cattle, and lanes 26, 27, and 29 -32 wild turkey.

15. Known source library

16. MANOVA plot of E. coli rep-PCR DNA fingerprints from different source groups: eight vs. three source groups Human Domestic Human Cattle Chicken Dog Horse Deer Waterfowl Wild turkey Wildlife

17. Water samples

18. Source distribution of E. coli in a Minnesota watershed 16% unknown 14% geese 9% turkey 12% pigs 9% sheep 12% cats 9% deer 10% cows 9% human Microbial source tracking guide document, EPA 2005

19. Summary • “A TMDL is a quantitative assessment of water quality problems, contributing sources, and load reductions or control actions needed to restore and protect individual waterbodies.” (US EPA) • MST is a valuable tool for TMDL development. • MST techniques are still under development. • Needs for further research: • Library size • Geographic variability • Temporal variability • Search for better library-independent methods • Host specificity of microorganisms • Genetic change associated with habitat change

20. Acknowledgements • AU: Brenda Wood, Michele Owsley, John Owen, Tony Dawkins, Joey Shaw, Ludovic Capo Chichi • NRCS: April Jones • Soil & Water Conservation District: Wil Smith • MWWSSB: Buddy Morgan, Jerald Conway, Mark Causey • CH2M Hill: Sabra Sutton, Malene Watson • USGS: Brian Atkins, Amy Gill, Don Stoeckel • Alabama Water Resource Research Institute • Auburn University Environmental Institute • Montgomery County Soil and Water Conservation District