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.
Where did the Bacteria (E. coli) Come From?
• Potential sources
• Humans
• Domesticated animals
• Wildlife
• Methods for determining sources
• Source survey
• Modeling
• Bacterial source tracking
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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
Sources
of bacteria
• 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
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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
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Development of Texas
E. coli BST Library
Sources
Isolate
DNA
Add to
E. coli
Fingerprint
Library
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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
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Use of Texas E. coli BST Library for
Identifying Water Isolates
Isolate
DNA
Compare
E. coli
Fingerprint
to Library
Source
ID
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Comparison to Texas E. coli BST Library
• Best match approach with
80% minimum similarity
Similarity: 96.94%
cutoff based on laboratory
ERIC-PCR
QC data
• Water isolate must match
library isolate ≥ 80%
similarity or is considered
unidentified
Similarity: 95.82%
RP
• Identification to single
library isolate with
highest similarity – max
similarity approach
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Three-way v. Seven-way Split of Results
• Using the results
• Is it from human sources?
• Is it from livestock?
(1) Human
(2) Livestock & Pets
(3) Wildlife
• Is it from wildlife?
• Biology
• Large variety of wildlife
• Cosmopolitan strains
• Geographical and
temporal differences
• Statistics
• Number of isolates
collected
vs.
Human (1)
Pets (2)
Cattle (3)
Livestock, avian (4)
Livestock, non-avian (5)
Wildlife, avian (6)
Wildlife, non-avian (7)
• May only use three-way
split for limited studies
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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
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
http://www.sourcemolecular.com/new
site/_images/bacteroidetes.jpg
• Not pathogens
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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 knownsource fecal samples from the watershed
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Monitoring Stations
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Leona BST Samples
2011
2012
2013
Sept- Dec
Jan - Dec
Jan - May
Stream (13)
18
37
16
71
WWTFs (2)
8
20
8
36
Bacteroidales Total
26
57
24
107
24
39
14
77
Parameter (# sites)
Total
Collected
Bacteroidales
E. coli (ERIC-RP)
Stream (13)
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Bacteroidales BST Results
Stream Samples (n=71)
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Bacteroidales BST Results
Fish Hatchery Samples (n=29)
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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)
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E. coli BST Results
Stream Samples (n=77) – 3-Way Split
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E. coli BST Results
Stream Samples (n=77) – 7-Way Split
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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
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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
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