Use of Bacterial Source Tracking to Characterize Texas Watersheds

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Randall Allen
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Emily Martin 3 1 Texas Water Resources Institute; 2 University of Texas

1 Use of Bacterial Source Tracking to Characterize Texas Watersheds Kevin Wagner 1, George Di Giovanni 2, Terry Gentry 3, Elizabeth Casarez 2, Emily Martin 3 1 Texas Water Resources Institute; 2 University of Texas School of Public Health, Houston, El Paso Branch Campus; 3 Texas A&M University

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

3 What is Bacterial Source Used to determine the sources of fecal contamination Tracking (BST)? Based on uniqueness of bacteria from individual sources A variety of different methods are used Often works best as part of a toolbox approach

4 BST Target Organisms Bacterial v. Microbial Source Tracking Different targets: E. coli Bacteroidales Bacteriophage Human viruses Chemicals

5 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

6 Development of Texas E. coli BST Library Sources Isolate E. coli DNA Fingerprint Add to Library

7 Texas E. coli BST Library (v. 6-13) Contains 1,524 E. coli isolates from 1,358 different human and animal samples Developed by collecting over 3,500 domestic sewage, wildlife, livestock, and pet fecal samples and screening over 6,000 isolates for clones and host specificity Samples from 13 watersheds across Texas for BST 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

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

9 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% Similarity: 95.82% RP ERIC-PCR

10 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) Other livestock, avian (4) Other livestock, non-avian (5) Wildlife, avian (6) Wildlife, non-avian (7)

11 Texas E. coli BST Library Composition & Rates of Correct Classification (RCC)

12 Library-Independent BST Approach: Genotypic detection of microorganisms based on marker genes (DNA) Does not require known-source library Most common approach targets Bacteroidales Presence/absence Relative abundance

13 What are Bacteroidales? More abundant in feces than E. coli Not pathogens Obligate anaerobes less likely to multiply in environment Subgroups appear to be host specific Markers available for humans, ruminants, horse, hog Others being tested (e.g., poultry) site/_images/bacteroidetes.jpg Limited wildlife markers

14 History of BST Use in Texas Lake Waco/Belton Project initiated Sep Funded by TSSWCB Evaluated utility & methods Completed Feb. 2006

15 History of BST Use in Texas Lake Waco/Belton Project Findings 4-method composite performed better than individual methods 2-method composites appeared promising ERIC-ARA = lower cost but more sample & data processing ERIC-RP = higher cost but automated TMDL Task Force Report 2007 Confirmed ERIC-RP as recommended method

16 Texas E. coli BST Library Contains 1,524 E. coli isolates From 1,358 different fecal samples Representing >50 animal subclasses Collected from 13 watersheds (& growing) across Texas Human 25% Domestic Animals 34% Wildlife 41%

17 Findings To Date

18 E. coli BST Results - Attoyac Base + Storm Samples 3-Way Split Unidentified (n=13) 13% Livestock and Domesticated Animals (n=21.5) 21% Humans (n=6) 6% Wildlife (n=63.5) 61%

19 E. coli BST Results - Attoyac Base + Storm Samples (7-Way Split) Humans (n=6) 6% Unidentified (n=13) 13% Cattle (n=10.5) 10% Other livestock, avian (n=3) 3% Other livestock, non-avian (n=3) 3% Wildlife, non-avian (n=47.5) 46% Pets (n=5) 5% Wildlife, avian (n=16) 15%

20 E. coli BST Results - Attoyac Base Flow vs Storm Flow (3-Way Split) Unidentified (n=1) 6% Base Flow Livestock and Domesticated Animals (n=6) 33% Storm Flow Unidentified (n=2) 11% Livestock and Domesticated Animals (n=6) 33% Wildlife (n=10) 56% Humans (n=1) 6% Wildlife (n=10) 56%

21 E. coli BST Results - Lampasas (Monthly 4-way Split All Sites Combined)

22 Summary of BST Study Findings (n=11) 3-Way Split Unidentified 12% Domesticated Animals 27% Wildlife 51% Human 10%

23 Summary of BST Study Findings (n=10) 5-Way Split Unidentified 11% Human 10% Non-Avian Wildlife 32% All Livestock 24% Pets 5% Avian Wildlife 18%

24 Summary of BST Study Findings (n=7) 7-Way Split Unidentified 12% Human 10% Non-Avian Wildlife 32% Cattle 13% Avian Livestock 5% Other Non-Avian Livestock 5% Pets 5% Avian Wildlife 18%

25 Future Methods & Approaches 1. Identify the Unidentified Continue expansion of BST library Evaluating naturalized E. coli 2. Improve Library-Independent BST Rapid and less expensive Limited markers, but new markers being developed Geographic stability of markers Quantification?

26 Questions? Kevin Wagner TWRI Assoc. Director George Di Giovanni Professor, UT School of Public Health El Paso Terry Gentry Assoc. Professor, Texas A&M AgriLife Research

Research Project Summary

Division of Science, Research and Technology Research Project Summary June, 25 Microbial Source Tracking in the Manasquan River Estuary Michael A. Palladino, Ph.D. and John A. Tiedemann Thomas B. Atherholt,