abundance & fate of fecal indicators, pathogens & antibiotic resistant bacteria in a...
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Abundance and Fate of Fecal Indicators, Pathogens and Antibiotic Resistant Bacteria in a Vegetative Treatment
System Receiving Cattle Feedlot runoff
Lisa Durso1, Dan Miller1, Chris Henry2, Dan Snow3
1USDA-ARS Agroecosystem Management Research Unit2University of Arkansas
3University of Nebraska
Traditional Beef Feedlot Waste Control
Traditional
Vegetative Treatment System (VTS)
X
X
X
X
X
X
Vegetative Treatment System (VTS)
Basin
Treatment Cells (1-8)
Cattle Pens1
2
3
4
5
Treatment CellsTreatment Cells
Pen
Pen
Pen
Pen Pen
Treatment Cells
1 2 3 4 5 6 7 8
Pump Station
Sampling Scheme - runoff
• Depend upon natural rain events
– Three events per year (spring, summer, fall)
• Three types of water samples
– Rainfall (clean) at the bottom of VTA prior to wastewater application
– Feedlot runoff (wastewater) as it is applied to VTA
– Excess wastewater at the bottom of VTA (1.5 hour after application)
2010
Event 1N=4
5/25/2010
Event 2N=35
6/9/2010
Event 3N=50
7/14/2010
Event 4Well Water9/7/2010
Event 6N=46
4/18/11
Event 7On/off6/2/11
Event 8N=18
6/6/11
Event 10N=34
4/17/12
Event 11N=24
5/29/12
Event 8Soil Survival6/6 – 6/20
2011
Event 5Soil Cores11/2/2010
Event 9Soil Cores9/26/2011
Event 12Soil Cores11/5/2012
2011 2012
2010
Event 1N=4
5/25/2010
Event 2N=35
6/9/2010
Event 3N=50
7/14/2010
Event 4Well Water9/7/2010
Event 6N=46
4/18/11
Event 7On/off6/2/11
Event 8N=18
6/6/11
Event 10N=34
4/17/12
Event 11N=24
5/29/12
Event 8Soil Survival6/6 – 6/20
2011
Event 5Soil Cores11/2/2010
Event 9Soil Cores9/26/2011
Event 12Soil Cores11/5/2012
2011 2012
Event 6N=46
4/18/11
Collect “Clean” rainwater that is
collected at bottom of VTA
cells
Open valves to drain
rainwater into pump, and runoff into pump
Apply runoff to first VTA
cell
After 10 minutes of application,
collect runoff samples
When runoff reaches
bottom of VTA cell, collect
“excess” samples
Apply runoff to
second VTA cell
Sampling Scheme - soil
• Collect soil cores from four VTA cells at five locations along transect
• Subsample with depth
Analyses (n=775 samples)
• Quantification of total coliforms, E. coli, Enterococcus• Enrichment for Pathogens (STEC, Salmonella)• Antibiotic resistance (phenotypic quantification)
Analyses (n=950 confirmed E. coli isolates)
• Disk diffusion assays for 12 drugs• PCR characterization for 14 tetracycline resistance
genes
Part I: Pathogens and IndicatorsA. In runoffB. In soil
Human Pathogens in Runoff
STEC ON
Salmonella ON
SPRING 2010
Percent of samples positive for pathogens from runoff water applied to, and collected from
vegetative areas
SUMMER 2010
0
20
40
60
80
100
0
20
40
60
80
100
STEC OFF
Salmonella OFFPe
rce
nt o
f sa
mp
les p
ositiv
e
Pe
rce
nt o
f sa
mp
les p
ositiv
e
0% of the “clean” Rainfall samples were STEC O157 positive
SOLID = ONSTRIPE = OFF
2010
ND = Not Detected
SoilFeedlot
Runoff
0
20
40
60
80
10096%
88%
5%ND ND ND ND ND ND ND ND ND
0-5 cm
Soil Soil Soil Soil Soil
5-10 cm 10-20 cm 20-30 cm 30-40 cm 40-50 cm
5% 5%
STEC O157
Salmonella
Perc
en
t o
f sam
ple
s p
osit
ive
2011 & 2012: One STEC O157 and zero Salmonella detections in soil
Human Pathogens in SoilPercent of soil samples positive for pathogens
ND = Not Detected
0
1
2
3
4
0-5 cm 5-10 cm 10-20 cm 20-30 cm 30-40 cm 40-50 cm
Log
MP
N/g
so
il
Berm soil
Treatment cell soil
E. coli in soil
0
1
2
3
4
0-5 cm 5-10 cm 10-20 cm 20-30 cm 30-40 cm 40-50 cm
Log
MP
N/g
so
il
Berm soil
Treatment cell soil
Enterococcus in soil
0
1
2
3
4
0-5 cm 5-10 cm 10-20 cm 20-30 cm 30-40 cm 40-50 cm
Log
MP
N/g
so
il
Berm soil
Treatment cell soil
Total Coliforms in soil
• Fecal indicator numbers not significantly different between applied and excess feedlot runoff
• No evidence that fecal indicator are enriched (or becoming enriched) in treatment soils
Summary: Pathogens and Indicators
Part II: Antibiotic ResistanceA. In runoffB. In soil
Wastewater Tetracycline-R Phenotype* Temporal Trends
0
1
2
3
4
5
6
Tetr
acyc
line
-R, l
og
CFU
/mL
Rainfall Runoff
Feedlot Runoff
Excess Feedlot Runoff
* Ability to grow on MAC plate with 16 µg/ml tetracycline
Wastewater Isolates:Multiple Resistances*
MAC Plates
0
20
40
60
80
100
120
140
160
180
0 2 4 6 8 10 12
Nu
mb
er
of
Iso
late
s
Rainfall Runoff
Feedlot Runoff
Excess FeedlotRunoff
TMAC Plates
0
20
40
60
80
100
120
140
160
180
0 2 4 6 8 10 12
* Isolates tested fro resistance to 12 drugs using CLSI standard disk diffusion methods
0-10 cm
10-20 cm
20-30 cm
30-40 cm
40-50 cm
N=6
N=3
N=1
N=45 coliform isolates total from 121 samples
Multidrug Resistance Patterns
Antibiotics # of Isolates Resistant
Ampicillin & Cefoxitin 1
Tetracycline & Streptomycin 2
Ampicillin, Cefoxitin & Chloramphenicol 1
Cefoxitin, Ceftriaxone & Streptomycin 1
Ampicillin, Cefoxitin & Amoxicillin w/Clavulanic Acid 1
Ampicillin, Tetracycline & Streptomycin 3
Vertical Transport of Antibiotic resistant bacteria in VTA
Tet gene prevalence in runoff E. coli isolates picked from MAC plates – by source
Total n=3,360 assays
Tet gene prevalence runoff E. coli isolates – by source
MAC TMAC
Antibiotic Resistant Bacteria and Genes
Tetracycline resistance in the wastewater and excess wastewater was 2 to 3 orders of magnitude higher compared to ‘clean’ VTA runoff.
Although antibiotic resistance was routinely detected in the wastewater, soil sampling indicated that these antibiotic resistant microorganisms in the wastewater that was applied to the VTA did not survive in the soil.
Summary: ARB/G
ACKNOWLEDGEMENTS
Christopher Henry, PhD, PE EngineerUniversity of [email protected]
Dan Miller, Ph.D.Microbiologist - NutrientsUSDA-ARS, Lincoln [email protected]
Lisa Durso, Ph.D.Microbiologist - PathogensUSDA-ARS, Lincoln [email protected]
Jennifer McGheeAmy MantzAlisha CraftJaime LaBrieEmily Hubel
Angel IversonRyan McGheeKatie HermanCrystal Powers
NE DEQ Grant : Evaluation of a Vegetative Treatment System to Remove Nutrients, Microorganisms, and Pharmaceutically Active Compounds in Runoff from a Concentrated Beef Cattle Feeding Operation in Central Nebraska (#56-0834)
USDA-ARS Project: Environmentally sound manure management for reduction of gas emissions, nutrients, and pathogens (#5440-12630-001-00D)
Dan Snow, Ph.D.Director Water Services CenterUniversity of [email protected]
Thank You