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Improving Life through Science and Technology.

Bacterial Source Tracking Leona River Emily Martin and Terry Gentry Soil & Aquatic Microbiology Laboratory Department of Soil & Crop Sciences Texas A&M University June 27, 2013 Stakeholder Meeting. Improving Life through Science and Technology.

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Improving Life through Science and Technology.

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  1. Bacterial Source Tracking Leona River Emily Martin and Terry Gentry Soil & Aquatic Microbiology Laboratory Department of Soil & Crop Sciences Texas A&M University June 27, 2013 Stakeholder Meeting Improving Life through Science and Technology.

  2. Where did the Bacteria (E. coli) Come From? • Potential sources • Humans • Domesticated animals • Wildlife • Methods for determining sources • Source survey • Modeling • Bacterial source tracking 2

  3. Sources of bacteria What is Bacterial Source Tracking (BST)? • Data collection and analysis to determine the sources of fecal contamination in a waterbody • Based on uniqueness of bacteria from individual sources • A variety of different methods are used • Differs from modeling in that it is not a predictive tool and does not require calibration and validation of input variables 3

  4. Library-Dependent BST Methods • Methods: • DNA fingerprinting • Enterobacterial repetitive intergenic consensus sequence-polymerase chain reaction (ERIC-PCR) • RiboPrinting® (RP) • Advantages/Disadvantages: • More discriminating • Allows ranking of sources • More expensive 4

  5. Sources Development of TexasE. coli BST Library Isolate E. coli DNA Fingerprint Add to Library 5

  6. Texas E. coli BST Library (v. 6-13) • Contains 1,524 E. coli isolates from 1,358 different human and animal samples • Collected from across Texas for BST studies including: • Waco / Belton Lake • San Antonio • Lake Granbury • Oyster Creek / Trinity River • Buck Creek • Little Brazos River Tributaries • Attoyac Bayou • Additional isolates being added from ongoing and future BST projects in other areas of Texas 6

  7. Use of Texas E. coli BST Library for Identifying Water Isolates Isolate E. coli DNA Fingerprint Compare to Library Source ID 7

  8. Comparison to Texas E. coli BST Library • Best match approach with 80% minimum similarity cutoff based on laboratory QC data • Water isolate must match library isolate ≥ 80% similarity or is considered unidentified • Identification to single library isolate with highest similarity – max similarity approach Similarity: 96.94% ERIC-PCR Similarity: 95.82% RP 8

  9. Three-way v. Seven-way Split of Results • Using the results • Is it from human sources? • Is it from livestock? • Is it from wildlife? • Biology • Large variety of wildlife • Cosmopolitan strains • Geographical and temporal differences • Statistics • Number of isolates collected • May only use three-way split for limited studies (1) Human (2) Livestock & Pets (3) Wildlife vs. Human (1) Pets (2) Cattle (3) Livestock, avian (4) Livestock, non-avian (5) Wildlife, avian (6) Wildlife, non-avian (7) 9

  10. Library-Independent BST Approach: • Genotypic detection of microorganisms based on marker genes (DNA) • Does not require known-source library • Most common approach targets Bacteroidales - - - + + + + 10

  11. What are Bacteroidales? • More abundant in feces than E. coli • Obligate anaerobes – less likely to multiply in environment • Subgroups appear to be host specific • Markers available for humans, ruminants, horse, hog • Not pathogens http://www.sourcemolecular.com/newsite/_images/bacteroidetes.jpg 11

  12. BST for Leona River • Library-independent • Target ~250 water samples from across the study area using Bacteroidales PCR for human, ruminant, horse, and hog markers • Severe drought, only analyzed ~100 samples • Limited library-dependent • Analyze E. coli from ~75 water samples from across the study area using both ERIC-PCR and RP fingerprinting • Supplement Texas E. coli BST Library with known-source fecal samples from the watershed 12

  13. Monitoring Stations 13

  14. Leona BST Samples 14

  15. Bacteroidales BST Results Stream Samples (n=71) 15

  16. Bacteroidales BST Results Fish Hatchery Samples (n=29) 16

  17. Known-Source Fecal Additions from Leona River • DNA fingerprinted 201 total isolates (ERIC-RP) from 201 individual fecal samples • Ultimately, 77 isolates were validated and added to the Texas E. coli BST Library • Domesticated animals and livestock (36 total) • Beef cattle (15), horses (5), chickens (4), dogs (3), goats (4), sheep (3), and ducks (2) • Wildlife (41 total) • Feral hog (17), deer (9), coyote (4), fox (4), various birds (4), raccoon (2), and road runner (1) 17

  18. E. coli BST Results Stream Samples (n=77) – 3-Way Split 18

  19. E. coli BST Results Stream Samples (n=77) – 7-Way Split 19

  20. BST Summary • Library-Independent Analysis • Limited detection of source-specific markers • Human marker only detected at wastewater plants • Limited Library-Dependent Analysis • Major E. coli sources in watershed appear to be wildlife (feral hogs, coyote, and deer) as well as domesticated animals (cattle) • Texas E. coli BST Library additions from Leona • Significant effort toward to include numerous samples from major potential sources in the watershed 20

  21. Questions? Emily Martin and Terry Gentry 2474 TAMU Texas A&M University College Station, TX 77843 Office: (979) 845-5323 Lab: (979) 845-5604 Email: emartin@ag.tamu.edu tgentry@ag.tamu.edu 21

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