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

7

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

13

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: emartin@ag.tamu.edu

tgentry@ag.tamu.edu

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Slide Number 1What is Bacterial Source Tracking (BST)?Genotypic BST Methods�(Library-Dependent)Library-Independent BSTWhat are Bacteroidales?Slide Number 6Slide Number 7Slide Number 8Slide Number 9Slide Number 10Slide Number 11Slide Number 12Slide Number 13Slide Number 14Slide Number 15Questions?