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

Image by Christina Goeddel

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?

Factors we considered in this study:

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3. What is causing the resistance?

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

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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

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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.

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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

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