transport of antibiotic resistant bacteria into tile - · pdf file ·...
TRANSCRIPT
Transport of antibiotic resistant bacteria into tile drainage systems
Michelle Soupir
Agricultural & Biosystems Engineering, Iowa State University
Collaborators:
Trang Hoang, Alok Bhandari and Tom Moorman
11th Annual Drainage Research Forum
Owatonna, Minnesota
November 23rd, 2010
Pathogens are a leading cause of water quality impairments in streams and rivers
• Agricultural practices – Leading source of all
impairments
• Causes of waterborne
disease outbreaks resulting in gastroenteritis
• Enterococci are a potential indicator of pathogens
• Developing resistance to many antibiotics
Microbial
Indicator
Federal Standard
for primary
contact
Fecal
coliform 200 CFU/100 mL
E. coli 126 CFU/100 mL
Enterococci 33 CFU/100 mL
http://greenerloudoun.wordpress.com/2009
/02/16/thanks-for-the-free-cow-urine-in-my-
drinking-water/
Factors we considered in this study:
• 1/3 of land area in Iowa is drained
• Bacteria move into tiles via macropores – Consider only no-till
• Tylosin commonly administered at sub-therapeutic levels – Up to 90% excreted
• Relationships between sources and resistant organisms typically inferred
Image by Alok Bhandari
swine farm
surface transport
1. 2. Representative scenario What is lacking?
Objectives of the study:
1. To detect and quantify the occurrence of tylosin-resistant enterococci in:
• manure from swine facilities feeding tylosin at sub-therapeutic doses;
• soils amended with swine waste and
• tile drain flow from swine waste amended agricultural fields;
2. To assess the effects of hydrology and solids transport on the enterococci in tile water;
3. To test enterococci isolates for known macrolide resistant genes
www.scq.ubc.ca
Approach
• ISU Northeast Research Farm
• 36 plots (0.4 ha each)
– Soil type: moderate to poorly drained
– Tile drainage: 1979
• Corn-soybean rotation
7
25
20
Field Study: Nashua, Iowa Manure & Soil Sampling
Approach
• ISU Northeast Research Farm
• 36 plots (0.4 ha each)
– Soil type: moderate to poorly drained
– Tile drainage: 1979
• Corn-soybean rotation
8
Rainfall Simulation Water Sampling
• Spring: sampling started after flow started
• Fall: sampling started after rainfall simulation started
Approach
• Membrane Filtration Technique
(APHA, 1998)
• mEnterococcus agar with tylosin at
35 mg/L (resistant) and without
tylosin (control)
• Total Suspended Solids
Analysis for Enterococci Confirmation
• EPA Method 1600
Incubate
at 35oC
for 48 hrs
Transfer Incubate
Incubate
Transfer
BHI Broth
BEA
BHI Broth 6.5% NaCl
BHI Broth
Gram stain
Transfer Incubate
Transfer Incubate
Incubate
Transfer
Approach
• Select isolates
• DNA extraction
• PCR Amplification
(Denaturation, annealing, and
extension)
• Positive controls: Dr. Roberts,
University of Washington
• Negative controls: ATCC
Resistant Gene Detection by PCR
Results
Enterococci in manure and soil samples
11
1.2x106
6.7x104
3.3x101
5.7x105
1.3x105
0
1.4x106
5.0x104
0
3.8x105
1.0x105
0 1.E+00
1.E+01
1.E+02
1.E+03
1.E+04
1.E+05
1.E+06
1.E+07
Swine manure Soil aftermanure
application
Soil beforemanure
application
Swine manure Soil aftermanure
application
Soil beforemanure
application
Spring Fall
Total
Resistant
Enterococci and tile flow
12
Total : 33 to 4967 CFU/100 mL
Resistant: 67 to 1167 CFU/100 mL
0
50
100
150
200
250
300
350
0
1000
2000
3000
4000
5000
6000
0 100 200 300 400
Flo
w(L
/h)
Ente
rococci concentr
ation (
cfu
/100m
L)
Elapsed time since flow started (min)
Total
Resistant
Flow
80 min
26 min
Spring 2009
Enterococci concentration and tile drained flow
13
Total : up to 2567 cfu/100 ml
Resistant: up to 1433 cfu/100 ml
0
50
100
150
200
250
300
350
0
1000
2000
3000
4000
5000
6000
0 200 400 600 800 1000
Flo
w (
L/h
)
Ente
roco
cci c
on
cen
trat
ion
(cf
u/1
00
mL)
Elapsed time since rainfall simulation started (min)
TotalResistantFlow135 min
45 min
Fall 2009
Results: Enterococci loading
cfu/ha
14
Fall 2009
1.E+07
1.E+08
1.E+09
1.E+10
1.E+11
1.E+12
0 200 400 600 800 1000
Ente
rococci concentr
atio
n
(log10 c
fu/h
a)
Elapsed time since rain fall simulation started (min)
Total Resistant
Resistant/Total = 73%
p-value << 0.05
1.12x1011
8.25x1010
1.E+07
1.E+08
1.E+09
1.E+10
1.E+11
1.E+12
0 100 200 300 400Ente
rococci concentr
atio
n
(log10 c
fu/h
a)
Elapsed time since flow started (min)
Total Resistant
Resistant/Total = 23%
p-value << 0.05
1.1x1010
4.6x1010
Spring 2009
Enterococci concentration and TSS: Spring Simulation
• Poor regression because sediment was deposited in the tile line
15
0100200300400500600700
0 2000 4000
TSS
(mg
/L)
Total enterococi concentration (cfu/100 mL)
0
100
200
300
400
500
600
700
0
1000
2000
3000
4000
5000
6000
0 100 200 300 400
TS
S (
mg
/L)
En
tero
co
cci co
nce
ntr
ation
(c
fu/1
00
ml)
Elapsed time since drain-flow started (minutes)
Total
Resistant
TSS
Enterococci concentration and TSS: Fall Simulation
• Good correlation because of base -flow
R² = 0.6464
0
100
200
300
400
0 1000 2000 3000
TSS
(mg
/L)
Total enterococci concentration (cfu/100 mL)
0
100
200
300
400
500
600
700
0
500
1000
1500
2000
2500
3000
0 500 1000
TS
S (
mg
/L)
Ente
rococci c
oncentr
ation
(cfu
/10
0 m
L)
Elapsed time since rainfall simulation started (mins)
Total
Resistant
TSS
Resistant gene detection
0
10
20
30
40
50
60
70
80
90
100
msrA ErmF ErmB ErmT ermC msrA ErmF ErmB ErmT ermC
Spring Fall
Gen
e d
etec
tio
n f
req
uen
cy (
%)
Manure Soil Tile water
Conclusions
1. This study has provided the first data on tracking the transport pathway of tylosin-resistant enterococci from a specified source through a subsurface drainage system
• Transport of resistant enterococci from swine manure through soil and into tile drainage was confirmed
• Enterococci control and tylosin –resistant concentrations were significantly different
2. In the presence of base-flow, good correlation between TSS and enterococci was observed.
3. Tylosin resistant genes were detected in enterococci isolates from manure, soil and tile water: msrA, ermB, ermF, ermT, ermA, ermX.
• Both resistance mechanisms to tylosin occurred in enterococci isolates.
18
Acknowledgements
• Multi-university USDA-NRI grant on Role of Directly Connected Macropores in Pathogen Transport to Subsurface Drainage
• VOSP scholarship from Vietnamese government
• Graduate & undergraduate students: Chi Hoang, Pramod Pandey, Martha Zwonitzer, Ping Liu, Rachel McDaniel, Christina Goeddel and Bridgette Huss
19