The Impact on Public Health of Environmental Contamination with

Antimicrobial Residues

Randall Singer, DVM, MPVM, PhD

Will not focus on toxicology

How much antimicrobial residue is in the environment?

Is there biological activity of these residues?

How can we predict how much residue will be loaded into the environment?

An example One Health approach

Overview

The Ecological Model (Confusogram)

Residues in the Environment

Residues in the Environment

Residues in the Environment

Residues in the Environment

Residues in the Environment

Residues in the Environment

Ecology of Resistance

Cluster Count AMP AXO FOX TIO AMI GEN KAN STR NAL CIP FLO CHL TET AMC COT FIS Rivers Farms stx 1 stx 2 eae

A-F 1,054 ≥13 ≥13 ≥14 ≥17 ≥14 ≥12 ≥13 ≥11 ≥13 ≥15 ≥14 ≥12 ≥14 ≥13 ≥10 ≥12 318 736 1 11 23G 47 17.0 24.1 22.0 19.3 17.0 16.0 15.1 12.5 19.4 22.0 19.4 19.0 17.8 17.9 20.3 6.3 1 46 0 2 0H 45 19.9 27.0 24.4 22.0 19.0 17.8 18.2 13.7 21.5 29.4 22.7 22.2 7.3 21.0 24.8 20.9 7 38 0 0 1I 9 19.8 26.3 24.2 21.8 19.3 18.0 19.0 14.6 8.0 23.1 25.9 23.1 19.1 22.0 26.6 25.4 0 9 0 0 0J 12 6.0 24.9 24.4 20.3 18.0 16.8 17.1 13.3 21.2 23.9 21.8 21.8 14.6 17.1 25.3 19.4 11 1 0 0 1K 28 18.3 26.6 22.6 21.5 18.9 17.5 18.4 9.7 16.5 26.0 21.9 22.1 6.0 19.6 19.4 6.1 0 28 0 0 0L 16 7.8 25.5 24.5 21.2 18.3 17.2 16.2 8.5 21.9 29.7 22.3 22.1 16.0 18.6 11.2 6.0 11 5 0 0 1M 8 16.6 25.0 23.3 20.0 18.4 17.8 17.8 6.0 20.3 27.4 6.0 6.0 6.0 18.6 17.6 6.0 0 8 0 0 0N 7 6.0 26.0 22.6 20.4 18.4 13.0 10.3 9.4 6.0 10.1 22.9 15.4 6.0 15.1 16.9 13.7 7 0 0 0 0O 3 6.0 10.0 6.0 6.0 13.3 7.7 9.0 9.0 9.7 12.0 6.0 6.0 12.7 9.7 9.7 9.0 0 3 0 0 0

Total 1229 355 874 1 13 26

≤13 ≤13 ≤14 ≤17 ≤14 ≤12 ≤13 ≤11 ≤13 ≤15 ≤14 ≤12 ≤14 ≤13 ≤10 ≤12

Antibiotics Tested Virulence Genes

Breakpoints

Ecology of Resistance

Los Lagos

Valdivia

Mariquina

Máfil

Corral

Lanco

Paillaco

Panguipulli

FutronoLa Unión

PL

PK

PJ

PI

PH

PG

PFPE

PD

PC

PB

PA

0 10 20 30 405Kilometers

.

Clusters

O

N

M

L

K

J

I

H

G

A-F

Ecology of Resistance

WC19

WC18

WC17

WC16

WC15 WC14

WC13

WC12

WC11

WC10

WC09

WC08

WC07 WC06

WC05

WC04

WC03

WC02 WC01

WS10

WS09

WS08

WS07WS06

WS05

WS04

WS03 WS02

WS01

Los Lagos

Valdivia

Mariquina

Máfil

Corral

Lanco

Paillaco

Panguipulli

FutronoLa Unión 0 10 20 30 405

Kilometers

.

Clusters

O

N

M

L

K

J

I

H

G

A-F

Do low concentrations of chlortetracycline or tylosin have a biological effect on the bacterial community in river water?

Samples taken from the Mississippi River

Activity of Antibiotics in the Water

Chemostats inoculated with 8 µg/L, 800 µg/L and 32 µg/ml of chlortetracycline (CTC) or 10 µg/L and 50,000 µg/L of tylosin twice daily for 10 days. One chemostat was used as a control.

Activity of Antibiotics in the Water

For chlortetracycline, total bacteria counts did not differ among chemostats (P = 0.51)

High-CTC chemostat selected for CTC resistance (P = 0.03)

Activity of Antibiotics in the Water

0

2

4

6

8

10

0 2 4 6 8 10

Time (Days)

Cou

nt (l

og c

fu /

ml)

Control

8 ug / L

800 ug / L

32000 ug / L

0

2

4

6

8

10

0 2 4 6 8 10

Time (Days)

Cou

nt (l

og c

fu /

ml)

Control

8 ug / L

800 ug / L

32000 ug / L

For tylosin, total bacterial load decreased with increasing concentration (P < 0.01)

No significant difference among chemostats for total growth on plates with tylosin (P < 0.17)

Activity of Antibiotics in the Water

0123456789

10

0 2 4 6 8 10

Time (days)

Cou

nt

(log

cfu

/ m

l)

Control

10 μg / L

50000 μg / L

0

1

2

3

4

5

6

7

8

9

0 2 4 6 8 10

Time (days)

Cou

nt

(log

cfu

/ m

l)Control

10 μg / L

50000 μg / L

High tylosin chemostats had significantly higher ratios of counts on CNA-TYL / CNA (P = 0.025)

High tylosin chemostats had higher ratios on LB-CTC / LB (P = 0.08)

Activity of Antibiotics in the Water

00.10.20.30.40.50.60.70.80.9

1

0 2 4 6 8 10

Rat

io o

f co

un

ts

Time (days)

Control

10 μg / L

50000 μg / L

0

0.02

0.04

0.06

0.08

0.1

0.12

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Rat

io o

f cou

nts

Time (days)

Control

10 μg / L

50000 μg / L

Activity of Excreted Residues

Activity of Excreted Residues

Medicated pelleted feed used in aquaculture

Florfenicol is a commonly used antibiotic in aquaculture in many countries

Multidrug Resistance Plasmids

Call et al., Antimicrob. Agents Chemother., 2010

Resistance in Aquaculture

Fernández-Alarcón et al., Zoonoses Pub Health., 2010

Activity of Excreted Residues

How do we develop accurate estimates of environmental loading of antibiotics excreted in animal agriculture?

Track concentrations in each compartment to develop excretion profile

Cmax and half-life within each compartment

Volume excreted from each compartment

Estimate percentage of total dose in each compartment to develop excretion profile

Half-life of compound in each compartment

Back-calculate to get estimate of concentration

Modeling Excretion of Residues

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Feca

l Con

cent

ratio

n (µ

g/g)

Feca

l Exc

retio

n (m

g)

Time (days)

Cow Fecal Excretion

Fecal Concentration

Modeling Excretion of Residues

Modeling Excretion of Residues

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Milk

Con

cent

ratio

n (µ

g/L)

Milk

Exc

retio

n (m

g)

Time (days)

Cow Milk Excretion Milk Concentration

Modeling Excretion of Residues

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Feca

l Con

cent

ratio

n (µ

g/g)

Feca

l Exc

retio

n (m

g)

Time (days)

Calf Fecal Excretion

Fecal Concentration

Few data available concerning concentration of compound in each compartment

Older compounds have very sparse data

If environmental loading is of interest, additional trials might need to be conducted

Dairy example highlights dilemma of discarding waste milk or feeding it to the calves

Excretion Data Gaps and Concerns

Background: 20%

Transmission: 0 – 12%

Selection: 20 – 60%

0

0.5

1

1.5

2

2.5

1 1.5 2

Selection Pressure

Ris

k R

atio

Baseline

Scenario 1

Scenario 2

Scenario 3

A One Health Model

A One Health Model

A One Health Model

Ministry of Health

Ministry of Agriculture

Ministry of the Environment

Ministry of Tourism

Ministry of Indigenous Peoples

National Water Authority

NGOs

Private Foundations

Private Business

Salmon Coalition

Academia

A One Health Model

First meeting in January 2013, Panguipulli, Chile

Environmental concerns and waste management will continue to be key issues

Residues will decrease with time in the environment, but the rate of decay will vary with many factors

Biological activity in environment remains highly uncertain

Excretion data from animals is lacking

Take Home Messages

Compounds can bind in the environment, analogous to binding in gut or in serum/plasma

Not a blame game; to minimize impacts of residues in the environment, we must develop methods for attribution

Take Home Messages

United StatesDoug CallTim JohnsonAbigail SalyersTom Shryock

ChileClaudia CampilloSantiago ErnstGerardo GonzalezKeith HoodGuillermo Ramirez

United KingdomPeter Silley

Singer labJanet AndersonClaudia FernándezKevin LangJeannette MuñozSheila Patterson