Improving Life through Science and Technology.
Bacterial Source TrackingBig Cypress CreekBacteria Assessment ProjectEmily Martin and Terry GentrySoil & Aquatic Microbiology LaboratoryDepartment of Soil & Crop SciencesTexas A&M University
August 26, 2010
Stakeholder Meeting
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
Genotypic BST Methods(Library-Dependent)
Methods:
• DNA fingerprinting
• Enterobacterial repetitive intergenic
consensus sequence-polymerase chain
reaction (ERIC-PCR)
• RiboPrinting® (RP)
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Advantages/Disadvantages:• More expensive• More discriminating
Library-Independent BSTApproach:
• Genotypic detection of microorganisms based on marker genes (DNA)
• Does not require known-source library
• Most common approach targets Bacteroidales
4+ + +- +- -
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 Big Cypress Creek
• Limited library-dependent• Analyze E. coli from ~100 water samples from across the
study area using both ERIC-PCR and RP fingerprinting
• Library will be supplemented with known-source fecal samples from the watershed
• Library-independent– Analyze ~250 water samples from across the study area
using Bacteroidales PCR for human, ruminant, horse, and hog markers
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Monitoring Stations
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2009 2010
Parameter Sept Oct Nov Dec Jan Feb Mar Apr May June July Aug Oct DecTotal
Collected To-Date
Target Number
To Collect
Bacteroidales
Stream (14) 14 14 14 14 14 14 14 14 14 20 X X X 146 188
WWTFs (2) 2 2 2 2 2 2 2 2 3 X X X 19 25
Storm - Stream (14) 14 14 14 X 42 56
Storm - WWTFs (2) 2 2 2 X 6 8
Bacteroidales Total 32 16 16 14 16 16 16 16 32 23 16 16 16 32 213 277
E. coli (ERIC-RP)
Stream (14) 14 12 14 X X X 40 82
WWTFs (2) 1 1 2 1 X X X 5 11
Storm - Stream (14) 11 X 11 25
Storm - WWTFs (2) 2 X 2 4
E. coli Total 14 1 13 2 13 15 16 14 32 58 122
Big Cypress BST Samples
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Status of BST Analyses
• E. coli (ERIC-PCR + RP)− 58 samples analyzed (58/122= 48% complete)
− Base flow = 44
− Storm flow = 14
• Bacteroidales PCR– 213 samples analyzed (213/277 = 77% complete)
– Base flow = 165
– Storm flow = 48
– Human, ruminant, hog, and horse markers
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Interim Bacteroidales BST ResultsBase v. Storm Samples
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0%10%20%30%40%50%60%70%80%90%
100%
Base Flow (n=165) Storm Flow (n=48)
Posi
tive H
its General
Human
Ruminant
Hog
Horse
Interim Bacteroidales BST ResultsSub-Watershed Stream Samples
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0%10%20%30%40%50%60%70%80%90%
100%
Hart Creek (n=70)
Tankersley Creek (n=52)
Big Cypress Creek (n=66)
Post
ive H
its General
Human
Ruminant
Hog
Horse
Interim Bacteroidales BST ResultsWastewater Treatment Plants
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0%10%20%30%40%50%60%70%80%90%
100%
Mt. Pleasant WWTP (n=13)
Pilgrim's Pride WWTP (n=12)
Posi
tive H
its General
Human
Ruminant
Hog
Horse
Interim E. coli BST ResultsBase + Storm Samples – 3-Way Split
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Human(n=3)
5%
Livestock and Pets
(n=23)40%
Wildlife(n=21)
36%
Unidentified(n=11)
19%
BST Summary• Limited Library-Dependent Analysis
• Existing Texas E.coli BST Library appears to be working relatively well (81% of isolates identified)
• Major sources in watershed appear to be domestic animals (livestock and pets) and wildlife (feral hogs, deer, birds, and small mammals)
• Known-Source Library additions from Big Cypress
• Library Independent Analysis • Hog/feral hog and ruminant markers most common
• Big spike in ruminant and hog hits during storm events
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Future BST • Additional data will be collected and analyzed through
January 2011
• 64 E. coli isolates
• 44 Bacteroidales samples
• ~50 Known source isolates into State Library
• Results will change as more data is collected
• Final results will be presented in 2011
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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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