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Tracking Antibiotics and Tetracycline Resistance Genes

through the Poultry Waste Composting Process – Lessons

Learned

Patricia Keen, PhD

Institute for Resources, Environment & Sustainability

Tracking Antibiotics through Poultry Compost Process

Outline

1. Project Introduction

2. Experiment design/methods

3. Results

4. Lessons learned

Tracking Antibiotics through Poultry Compost Process

Why are Antibiotics in the Environment a concern?

The surviving bacteria can resist the action of the antibiotic

The ‘fittest” bacteria survive

Bioactive compounds act on the organism

Prescription drugs Involuntary exposure

Antimicrobial Resistance

Natural Selection of Bacteria

Changes in Cellular Biochemistry

Exposure of Bacteria to Antibiotics

Antibiotics in an Agricultural Watershed

AMR in the Environment

Exchange genetic

determinants of

resistance

Transport in

Environment

Development of

Resistance

Tracking Antibiotics through Poultry Compost Process

Objectives

To refine LC ESI-MS/MS methodology for analyses of low level antibiotic concentrations in environmental samples.

To determine the concentration of antibiotics and tetracycline resistance genes in samples of waste from poultry production to track through the composting process.

To investigate whether monitoring antibiotic residues or antibiotic resistance genes could indicate potential for spread of antimicrobial resistance through environmental pathways.

To follow the persistence of four selected tetracycline resistance genes on a fertilized field

Tracking Antibiotics through Poultry Compost Process

Sumas Watershed

Headwaters in Whatcom County, USA

Flows across the international border

Drains into the Fraser River at the mouth of the Vedder Canal

Lower Fraser Valley, British Columbia

The Sumas River

Tracking Antibiotics through Poultry Compost Process

Study Design

Measure antibiotics/tetracycline

resistance genes compost samples

Monitor the analytes through

the composting process

Follow the tetracycline resistance

genes after application on field

To examine how

composting affects the

pathways by which

bacteria could develop

AMR via environmental

exposure

Tracking Antibiotics through Poultry Compost Process

Antibiotics Monitored 2004-2005 in the Sumas Watershed

Oxytetracycline Sulphamethazine Monensin

Chloroxytetracycline Sulphathiazole Trimethoprim

Democlocycline Sulphadimethoxine Ormetroprim

Doxycyline Sulphapyridine Narasin

Tetracycline Sulphaguanidine Salinmycin sodium

Tylosin Sulphamethizole Lasolicid sodium

Tilmicosin Sulphachloropyridazine Gentimycin

Virginiamycin Sulphamethoxazole Spectinomycin

Lincomycin Sulphamerizine Erythromycin

Tracking Antibiotics through Poultry Compost Process

Antibiotics Measured in the Sumas Watershed between 2-100 ppb

Oxytetracycline

Chloroxytetracycline

Democlocycline

Doxycyline

Tetracycline

Tylosin

Tilmicosin

Virginiamycin

Lincomycin

Sulphamethazine

Sulphathiazole

Sulphadimethoxine

Sulphapyridine

Sulphaguanidine

Sulphamethizole

Sulphachloropyridazine

Sulphamethoxazole

Sulphamerizine

Monensin

Trimethoprim

Ormetroprim

Narasin

Salinmycin sodium

Lasolicid sodium

Gentimycin

Spectinomycin

Erythromycin

Tracking Antibiotics through Poultry Compost Process

Antibiotic Residues Detected in Receiving Water and Soil – Sumas Watershed

doxycycline 3.7 – 11.1

democlocycline 3.8 – 27.0

oxytetracycline 3.1 – 57.9

chloroxytetracycline 3.9 – 37.2

tetracycline 3.2 – 9.1

Receiving Water (ppb)

(July 04 – April 05)

doxycycline n/d

democlocycline n/d

oxytetracycline 20 – 40

chloroxytetracycline n/d

tetracycline n/d

sulphamethizole 20 – 25

sulphathiazole 20 – 25

virginiamycin 20 – 40

lincomycin 20 – 25

erythromycin 20 – 25

Soil (ppb)

(April 05)

Total Tetracycline Resistance Genes in Compost & Soil

Tracking Antibiotics through Poultry Compost Process

Total tet Resistance Genes - Poultry Compost & Soils

1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

1.00E+09

Jan

25/0

4 ch

icke

n litte

r bef

ore

bird

s

June

2/0

4 co

mpo

st

July 2

3/04

com

post

Aug

20/

04 com

post

Sep

t 6/0

4 co

mpo

st

Sep

t PRE s

pray

soi

l

Sep

t 8/0

4 so

il

Sep

t 15/

04 s

oil

Sep

t 24/

04 s

oil

Oct 8

/04

soil

Oct

14/

04 s

oil

Oct 2

0/04

soi

l

Oct

29/

04 s

oil

Nov

4/0

4 so

ilDec

3/0

4 so

il

Dec

10/

04 soi

l

Jan

25/0

5 so

il

Sample date

Ab

un

da

nc

e C

op

ies

/mg

tet M

tet Q

tet O

tet W

Total Tcr and 16S rRNA genes

Tracking Antibiotics through Poultry Compost Process

1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

1.00E+09

1.00E+10

Jan

25/0

4 ch

icke

n litte

r bef

ore

bird

s

June

2/0

4 co

mpo

st

July 2

3/04

com

post

Aug

20/

04 com

post

Sep

t 6/0

4 co

mpo

st

Sep

t PRE s

pray

soi

l

Sep

t 8/0

4 so

il

Sep

t 15/

04 s

oil

Sep

t 24/

04 s

oil

Oct 8

/04

soil

Oct

14/

04 s

oil

Oct 2

0/04

soi

l

Oct

29/

04 s

oil

Nov

4/0

4 so

il

Dec

3/0

4 so

il

Dec

10/

04 soi

l

Jan

25/0

5 so

il

Sample Date

Co

pie

s/m

g

Total tet resistance genes

16S rRNA

Tracking Antibiotics through Poultry Compost Process

Prevalence of multi-drug resistance Salmonelle spp. isolates recovered from broiler chickens to 16 antimicrobials on the

NARMS panel (n=16)

Tracking Antibiotics through Poultry Compost Process

Prevalence of multi-drug resistance E.coli isolates recovered from broiler chickens to 16 antimicrobials on the NARMS

panel (n=16)

Tracking Antibiotics through Poultry Compost Process

Project Results - Summary

Some antibiotics require further method development for accurate and reliable results in compost/soil matrices (eg. Aminoglycocides)

Some antibiotic residues have been observed in ‘organic’ compost

Antibiotics present in the Sumas watershed coincide with local land use practices

There appears to be no correlation between tetracyclinesfound in the poultry compost samples and tetracycline resistance genes

Results appeared to be in line with observations of other global researchers

Lessons Learned

Measuring the antibiotic residues did not yield sufficient evidence to reliably monitor their presence or persistence

Analytical methodology required further refinement

Measuring selected tetracycline resistance genes allowed some observation of effects

Selecting an agricultural control site is extremely difficult –implications for experimental design

Some management practises can be recommended

Antibiotics in an Agricultural Watershed

Tracking Antibiotics through Poultry Compost Process

Project Collaborators

Health Santé

Canada Canada

Dr. Patricia Keen, University of British ColumbiaDr. Ken Hall, University of British ColumbiaDr. Bob Hancock, OC, University of British ColumbiaDr. Pierre Berubé, University of British ColumbiaDr. Julian Davies, OC, University of British Columbia

Dr. David Graham, University of KansasDr. Charles Knapp, University of Kansas

Dr. Merv Wetzstein, BC MALDr. Nancy deWith, BC MALDr. Heather Hannah, BC MALDr. Jane Pritchard, BC MAL

Dr. Dayue Shang, Health Canada Derek Smith, UBC Department of Chemistry

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Antibiotics in an Agricultural Watershed