Improving Life through Science and Technology.
Bacterial Source TrackingLeona River Emily Martin and Terry GentrySoil & Aquatic Microbiology LaboratoryDepartment of Soil & Crop SciencesTexas A&M University
June 27, 2013Stakeholder Meeting
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
• 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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Sourcesof
bacteria
Library-Dependent BST Methods
Methods: • DNA fingerprinting
• Enterobacterial repetitive intergenic
consensus sequence-polymerase
chain reaction (ERIC-PCR)• RiboPrinting® (RP)
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Advantages/Disadvantages:• More discriminating• Allows ranking of
sources• More expensive
Development of TexasE. coli BST Library
Sources
Isolate
E. coli
DNA
Fingerprint
Add to
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
E. coli
DNA
Fingerprint
Compare
to Library
Source ID
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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%
ERIC-PCR
RP
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(1) Human(2) Livestock & Pets(3) Wildlife
Human (1)Pets (2)
Cattle (3)Livestock, avian (4)
Livestock, non-avian (5) Wildlife, avian (6)
Wildlife, non-avian (7)
vs.
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 9
Library-Independent BSTApproach:• 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• Not pathogens
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http://www.sourcemolecular.com/newsite/_images/bacteroidetes.jpg
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
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Monitoring Stations
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2011 2012 2013 Total CollectedParameter (# sites) Sept- Dec Jan - Dec Jan - May
Bacteroidales
Stream (13) 18 37 16 71WWTFs (2) 8 20 8 36
Bacteroidales Total 26 57 24 107
E. coli (ERIC-RP)
Stream (13) 24 39 14 77
Leona BST Samples
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Bacteroidales BST ResultsStream Samples (n=71)
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Bacteroidales BST ResultsFish 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 ResultsStream Samples (n=77) – 3-Way Split
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E. coli BST ResultsStream 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 TAMUTexas A&M UniversityCollege Station, TX 77843Office: (979) 845-5323Lab: (979) 845-5604Email: [email protected]
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