FECAL SOURCE TRACKING FOR WATER QUALITY

C.A. CARSON

Food and Agriculture Policy Research InstituteColleges of Agriculture and Veterinary Medicine

University Of Missouri

GENERAL APPROACH TO FST/BST

• Routine water sampling shows unacceptable levels of sentinel indicator(s) bacteria indicative of fecal pollution

• FST test(s) are chosen to provide evidence of host source(s)

• A remediation plan can be developed to decrease pollution for compliance with water quality standards

EPA STANDARDS FOR RECREATIONAL WATERS

• Fresh waterNMT 200 Fecal coliforms / 100 ml.NMT 126 E. coli / 100 ml.

• Salt waterNMT 33 Enterococci / 100 ml.

EXAMPLES OF POTENTIAL SOURCES OF FECAL POLLUTION

• Human sewage treatment systems - private, collective (aging urban utilities)

• CAFO s

• Pastured animals

• Pet animals

• Migratory birds

• Wild animals

TARGETING FECAL POLLUTION

• Non-pathogenic bacteria-large numbers of harmless bacteria usually present for normal intestinal function

• Pathogenic (disease-producing) bacteria-normally absent or in low numbers

• Looking for pathogens (the real concern) in water samples resembles “looking for a needle in a haystack”

• Finding the haystack is easier• Fecal coliforms; E. coli; Enterococcus are

common/plentiful; useful as “indicators”

TIERED CONCERNS

• Human vs. nonhuman sources (public health risk)

• Identification of human and various nonhuman sources via E. coli or other common indicator organisms

Basis of BST Methods

• Particular strains of enteric bacteria (eg. E. coli) inhabit intestinal tracks of humans, animals and birds

• These various “host-specific” strains can be distinguished by their different biochemistry (function/phenotype) or different genetic/DNA structure (genotype)

• BST can be performed using either of these qualities

FIRST EXAMPLE METHODBacterial Fingerprinting / rep-PCR

Library-Based Genotyping Procedure

• Multiple copies of target repeat elements per E. coli genome

• Repeat numbers and locations vary per bacterial strain

• Primers amplify segments of DNA between repeats/signature of strain

Ribosomes Cell wall

Cytoplasmic membraneFlagellum

Bacterial chromosomes Cytoplasm

From: Principles of Microbiology by Atlas. W.C. Brown Co. 2nd Ed. 1997.From: Principles of Microbiology by Atlas. W.C. Brown Co. 2nd Ed. 1997.

PROKARYOTIC BACTERIAL CELLPROKARYOTIC BACTERIAL CELL

rep PCR test – based on location of target gene in rep PCR test – based on location of target gene in E. coliE. coli

E. coli

rep genes

DNA chromosome

1

2

3

1

2 31

23

Human Cow Dog

PCRMultiply 1,2,3

Different DNAFingerprint patterns

PCRMultiply 1,2,3

PCRMultiply 1,2,3

Bionumerics software Similarity coefficients of patterns calculated

by dice method with fuzzy logic option. Discriminant analysis via cross validation of

database

Select/Grow pure fecal E. coli

isolates

Lyse cellsPCR BOX A1R primer

Image capture

Pattern analysis by computer program

Electrophoresis of Rx mixture with Eth Br

+Larger Smaller

+++-

--

-

Marker Lane

MATERIALS AND METHODS MATERIALS AND METHODS rep PCR rep PCR

FECAL E. coli ISOLATES FROM TWO INDIVIDUAL HUMAN SAMPLES

(Bp)

400

600

800

1000

1500

2000

3000

500010000

400

600

800

1000

1500

2000

3000

500010000

(Bp)

200

FECAL E. coli ISOLATES FROM A LITTLE SAC RIVER WATER SAMPLE

(Bp)

(Bp)

400

600

800

1000

1500

2000

3000

5000 10000

400

600

800

1000

1500

2000

3000

500010000

200 200

PATTERN ANALYSIS

• DNA fingerprint patterns of fecal E. coli isolates are compiled in known-host database/library (human and non-human hosts)

• Environmental (water) E. coli isolates host-associated by comparison with database isolates; maximum similarity with a particular library pattern

• Arbitrary cutoff for “unknown” patterns – at least 80% similarity with library pattern; A-C quality factor

SECOND EXAMPLE METHODHost Specific/Gene Specific Targeting

Non library-based Procedure

• Bacteroides are most numerous human intestinal bacteria

• Different hosts have different species and strains• Bacteroides thetaiotaomicron (B. tim) is a

human-associated species• PCR test for presence of a B. tim gene in water

is used as an indicator of human fecal pollution

Bacteroides timTarget DNA

Bacteroides thetaiotaomicron Bacteroides thetaiotaomicron TestTest

PCRMultiply Target

Electrophoresis

542 size

HumanFecal pollution

No HumanFeces

Field Application of Bacterial Source Tracking Methods

UPPER SHOAL CREEK WATERSHED

• 3 county area in extreme SW part of MO; Newton, McDonald, Barry Counties

• One of most agriculturally productive areas in MO

• 91,000 acres in the watershed; 90% is pasture land grazed by over 300,000 head of cattle and fertilized by spreading poultry litter

• 50-80 million poultry produced here yearly• 13 miles of Shoal Creek are designated as

impaired due to high fecal coliform (FC) levels

Shoal Creek BST Data

Date FC/100 ml#

Patterns CattleDomestic Animals Human Poultry Wildlife

1/29/02 1,470 14 6 0 7 1 0

7/23/02 637 18 3 5 1 9 0

8/2/02 870 19 10 3 2 2 2

5/7/03 300 16 4 7 1 3 1

6/12/03 125 9 6 1 1 1 0

Seasonal Fecal E.Coli Sources (Average % Contribution)SUMMER WINTER

6%

11%

45%

19%

19%Cattle

Wildlife Human

Poultry

DomesticAnimals

25% 27%

21%17%

11%

CattleWildlife

HumanPoultry

DomesticAnimals

Fecal E.Coli Sources and Flow

SUMMER STORM FLOWS

SUMMER BASE FLOWS

Wildlife5%

8%

43%

15%

29% Cattle

Human

Poultry

Domestic Animals

7%

48%13%9%

23%Cattle

DomesticAnimals

Poultry

Human

Wildlife

STUDY CONCLUSIONS

• Cattle (particularly in streams) contribute substantially to water pollution

• Waste from pastured animals and spread poultry litter also contribute via runoff to streams

• There are multiple host sources of feces that combine for the total contribution

• Studies usually reveal multiple host sources, rather than a single host source

• Results from routine water quality monitoring, fecal source tracking and visual inspection can all combine to analyze problems and suggest solutions

BOTTOM LINE

• BST methods are powerful tools to resolve questions of host sources of fecal pollution and associated high bacterial counts in water

• Current consensus is to use a combination of methods with different targets

• Results must be interpreted carefully, combined with local observations and based on multiple samples collected over a period of time

ACKNOWLEDGMENT OF TEAM MEMBERS

• Food and Agricultural Policy Research Institute• U.S. Environmental Protection Agency • U.S. Geological Survey• College of Agriculture and Natural Resources• Department of Agriculture Engineering• Department of Agriculture Economics• College of Veterinary Medicine• U.S. Department of Agriculture• Missouri Department of Natural Resources• University of Missouri Extension Services