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Bacterial Source Tracking: Methods Comparison and Field Application. Ken Hyer, U.S. Geological Survey Richmond, VA. Cooperators. Cooperators. VA Department of Environmental Quality. VA Department of Environmental Quality. Berkeley County, WV. Berkeley County, WV. - PowerPoint PPT Presentation
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Ken Hyer, U.S. Geological SurveyRichmond, VA
Bacterial Source Tracking:Methods Comparison and
Field Application
WV Department of Environmental ProtectionWV Department of Environmental Protection
VA Dept of Conservation and RecreationVA Dept of Conservation and Recreation
Berkeley County, WVBerkeley County, WV
WV Department of AgricultureWV Department of Agriculture
CooperatorsCooperators
VA Department of Environmental QualityVA Department of Environmental Quality
Fairfax County, VAFairfax County, VA
Objectives for Talk
• Describe a methods comparison study that evaluated 7 source tracking methods.
• Describe a field application of BST and the associated quality control activities.
Bacteria Source TrackingBacteria Source Tracking
WaterSamples
SourceSamples
IsolatedE. coli
IsolatedE. coli
rRNAC
rRNAB
rRNAA
rRNADog
rRNAHuman
rRNAGoose
UNKNOWN KNOWN
BST Methods Comparison Study• Sampling in Berkeley County, West Virginia.• Involves researchers from across the nation and 3 different
USGS district offices (Melvin Mathes of WV and Don Stoeckel of Ohio).
• Five Genotypic Methods (and investigators)– Ribotyping using two different enzyme sets
(George Lukasik, Mansour Samadpour)– Pulsed-field Gel Electrophoresis
(West Virginia Department of Agriculture)– rep-PCR using two different primer sets
(Howard Kator, Don Stoeckel)• Two Phenotypic Methods (and investigators)
– Antibiotic Resistance Analysis (Bruce Wiggins)– Carbon Substrate Utilization (Chuck Hagedorn)
BST Methods Comparison Study
• Prominent sources of fecal pollution being considered (based on NRCS input for Berkeley County):
– Humans
– Cattle (beef and dairy)
– Chickens
– Swine
– Horses
– Dogs
– Canada Geese
– Deer
Methods Comparison - Study Design
• Collect feces from at least 20 individuals per source.
• Isolate and confirm a library of known E. coli from the fecal samples:– Total of 70-100 confirmed E. coli per source– Total known library size of 900 isolates
• Prepare a blind sample set comprised of 200 isolates that included three subsets:– 26 Replicates from the known library– 150 Fresh isolates from the 9 prominent sources– 24 Fresh isolates from sources that were not in the
original known library (mice, cats, raccoons, etc.)
Methods Comparison - Study Design
• Each researcher identified the source of each blind isolate.
• Results were scored and the following were considered for each method:– Accuracy of isolate identification – Precision (reproducibility of replicate isolate
analyses)– Robustness (isolates from sources not in the
library are identified as unknown)
Methods Comparison - Results
• In a general sense, we found that:-In this study, under these conditions…
-Most methods did not perform as well as we expected, based on published literature.
-Detailed study manuscript is in press at Environmental Science and Technology.
Results - Replicates
• The first 3 methods used discriminant analysis (DA), the other 4 used direct matching techniques.
• In scoring the replicates, the response “unknown” was considered incorrect (all isolates were in the known library).
• For each method, considering an 8-way source classification:-ARA: 6 of 26 (23% correct)-CUP: 6 of 25 (24% correct)-RT-HindIII: 3 of 23 (13% correct)-RT-EcoR1: 14 of 26 (54% correct)-PFGE: 24 of 24 (100% correct)-BOX-PCR: 17 of 26 (65% correct)-REP-PCR: 10 of 23 (43% correct)
Results - Accuracy
• For the accuracy subset, the response “unknown” was considered neutral (neither correct nor incorrect) – thus the number of isolates attempted is very important.
• For each method, considering an 8-way source classification:-ARA: 36 of 150 (24% correct)-CUP: 20 of 143 (14% correct)-RT-HindIII: 19 of 147 (13% correct)-RT-EcoR1: 7 of 8 (88% correct)-PFGE: 15 of 40 (38% correct)-BOX-PCR: 32 of 149 (21% correct)-REP-PCR: 23 of 93 (25% correct)
Results - Ringers• In scoring the ringers, the response “unknown” was considered
the only correct response because none of these isolates were in the library.
• None of the DA methods attempted to identify and reject ringers.
• For each method, considering an 8-way source classification:-ARA: 0 of 24 (0% correct)-CUP: 0 of 24 (0% correct)-RT-HindIII: 0 of 24 (0% correct)-RT-EcoR1: 24 of 24 (100% correct)-PFGE: 16 of 24 (67% correct)-BOX-PCR: 0 of 24 (0% correct)-REP-PCR: 8 of 24 (33% correct)
Reasons For Method Underperformance
• Inadequate library size or structure
• Temporal component in the source-library collection
• Presence of many repetitive subtypes (transient strains)
• Different statistical analyses may be needed
• Regardless of these possible reasons, the study clearly demonstrates the need for QA/QC and proofing of methods.
Methods Comparison - Conclusions
• This is only one of several ongoing comparison studies. It demonstrates that under these study conditions, none of these methods are performing at the levels we anticipated.
• We can offer these recommendations:-Perform considerable QA/QC in your BST work! This may
include (1) analyzing blind collections of known isolates, (2) use of multiple BST methods, and (3) the use of other tracers to support the BST work.
-Perform your QA/QC in such a way that you can detect if your method is working or failing.
Example of an Applied StudyExample of an Applied Study
1. Accotink Creek – Urban 1. Accotink Creek – Urban
11
2. Blacks Run – Mixed Urban/Agricultural2. Blacks Run – Mixed Urban/Agricultural
22
3. Christians Creek - Agricultural3. Christians Creek - Agricultural
33
Study Design
• Field Data Collection
– Water-sample collection
• Baseflow
• Stormflow
• Continuum
– Source sample collection
• Bacteria Source Tracking Analysis (Ribotyping)
Results of MST:Results of MST:By Individual ContributorBy Individual Contributor
Blacks RunBlacks Run
Christians CreekChristians Creek
Accotink CreekAccotink Creek
00
55
1010
1515
2020
2525
3030
3535G
oo
seG
oo
se
Hu
ma
nH
um
an
Do
gD
og
Du
ckD
uck
Ca
tC
at
Se
a G
ull
Se
a G
ull
Ra
cco
on
Ra
cco
on
Ca
ttle
Ca
ttle
De
er
De
er
Ho
rse
Ho
rse
Po
ultr
yP
oul
try
Pe
rce
nt o
f Kn
ow
nP
erc
en
t of K
no
wn
Seasonal Patterns in MST Data:Seasonal Patterns in MST Data:Comparison of Warm and Cool SeasonsComparison of Warm and Cool Seasons
00
1010
2020
3030
4040C
attl
eC
attl
e
Hu
ma
nH
um
an
Do
gD
og
Ho
rse
Ho
rse
De
er
De
er
Wa
terf
ow
lW
ate
rfo
wl
Po
ultr
yP
oul
try
Ca
tC
at
Pe
rce
nt o
f Con
trib
utio
nP
erc
en
t of C
ontr
ibu
tion
Warm (May-Sept)Warm (May-Sept)
Cool (Oct-March)Cool (Oct-March)
Validation of MST:Validation of MST:Human SignatureHuman Signature
Accotink CreekAccotink Creek Christians CreekChristians Creek Blacks RunBlacks Run
CaffeineCaffeine
00
0.050.05
0.10.1
0.150.15
0.20.2
0.250.25
Ca
ffein
e (
µg
/L)
Ca
ffein
e (
µg
/L)
CotinineCotinine
00
0.010.01
0.020.02
0.030.03
0.040.04
Co
tinin
e (
µg
/L)
Co
tinin
e (
µg
/L)
Validation of MST:Validation of MST:Poultry SignaturePoultry Signature
00
100100
200200
300300
400400
500500
1010 2020 3030 4040 5050 6060
Time (Hours)Time (Hours)
Dis
char
ge (
cfs)
Dis
char
ge (
cfs)
00
0.10.1
0.20.2
0.30.3
0.40.4
To
tal A
rse
nic
(µg/
L)
To
tal A
rse
nic
(µg/
L)
DischargeDischarge
ArsenicArsenic
OtherOther21.1%21.1%
OtherOther12.0%12.0%
Accotink Creek, BST ResultsAccotink Creek, BST Results
(N=279)(N=279)
DogDog13.3%13.3%
DogDog9.0%9.0%
DeerDeer1.4%1.4%
DeerDeer10.0%10.0%
Four Mile Run, BST ResultsFour Mile Run, BST Results
(N=278)(N=278)
WaterfowlWaterfowl38.7%38.7%
WaterfowlWaterfowl37.0%37.0%
RaccoonRaccoon5.4%5.4%
RaccoonRaccoon15.0%15.0%
Comparison of MST Results:Comparison of MST Results:Comparison of Accotink Creek and Four Mile RunComparison of Accotink Creek and Four Mile Run
HumanHuman20.1%20.1%
HumanHuman17.0%17.0%
Take Home Messages
1. Perform considerable QA/QC to ensure that you have confidence in your results.
2. Many different tools that can be applied to quality assure your BST data.
3. Under appropriate conditions, it appears BST can be used to successfully identify bacterial sources.
USGS Contact InformationUSGS Contact Information
Ken HyerKen Hyer1730 E. Parham Rd1730 E. Parham RdRichmond, VA 23228Richmond, VA 23228Email: Email: [email protected]: 804-261-2636Phone: 804-261-2636On the web: http://va.water.usgs.gov/On the web: http://va.water.usgs.gov/