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: emartin@ag.tamu.edu

tgentry@ag.tamu.edu

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