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Impacts on antimicrobial susceptibilities in stream

periphyton bacteria exposed to triclosan

Kirsten TrowbridgeAnne Hershey

University of North Carolina at Greensboro

WRRI Annual Conference

March 17, 2016

Triclosan does more than just wash hands!

Dhillon et al, 2015 (figure)

Added to thousands of products

PPCPs, medical, plastic, textile,

household, industrial…

Trowbridge (photos)

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Human Exposure

Triclosan can be absorbed through skin or mouth

Detected in human samples

Urine

Up to 100% of people tested

Health care workers

Pregnant women

Breast milk

Cord blood plasma

Calafat et al. 2008, MacIsaac, Adolfsson‐Erici et al.

2002, Allmyr et al. 2006

Pycke et al.

2014

Cherednichenko et al. 2012, Gee et al. 2007, Ishibashi et al. 2004, James et al. 2010, Kumar et al.

2009, Stoker et al. 2010, Veldhoen et al. 2006, Zorrilla et al. 2009, Dinwiddie et al. 2014, Bertelsen et al.

2012, Savage et al. 2012, CDC, 2013, Yazdankhah et al. 2006, Pycke et al. 2010, Ciusa et al. 2012,

Drury et al. 2013, Nietch et al 2013

Health effects in mammals

Endocrine, cancer, muscular, allergy, skin irritation

Unwanted By-products

Resistant bacteria

Dioxins

Chloroform and chlorinated organics

Triclosan does more than just wash hands!

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Triclosan in Environmental Samples

Water

Wastewater influents and effluents

Streams/rivers

Estuarine waters

Sea water

Pore water

Solids

Sediments

Biosolids

Activated Sludge

Dann and Hontela, 2011, Heidler and Halden 2006, Fang et al. 2010, Nietch et al. 2013, Kookana

et al. 2011, Lawrence et al. 2005, Capdevielle et al. 2009, Jones et al. 2005, Kolpin et al. 2002 Need to update

references on this slide

Triclosan in periphyton and water

Modified from Coogan et al. 2007 Trowbridge, unpublished data

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Triclosan goes through WWTP,

where some is passed to effluents…

Does periphyton mitigate

Triclosan levels in streams?

Do stream bacteria exhibit

changes in susceptibility?

Photo

by Lisa

Bracken

Periphyton communities mitigate

triclosan in streams

Environmental exposure results in

decreased susceptibility to

triclosan

Hypotheses

Two study streams in NC Piedmont

Reference

WWTP

Reference

NC map

www.thomaslegion.net

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Hypotheses

Reference periphyton bacteria exhibit greater susceptibility to

triclosan than wastewater-associated periphyton bacteria

Resistance to triclosan is associated with resistance to other

antibiotics

Triclosan exposure results in decreased susceptibility to

triclosan

Expect more pronounced effect in reference stream bacteria

compared to WW-associated

Triclosan Microcosm Studies

Approach

Expose periphyton to

environmentally-relevant level of

triclosan in recirculating stream

microcosms

Randomly select isolates and

measure susceptibilities

Stone tiles used as periphyton

colonization substrate

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Triclosan-exposure Microcosms

Stream microcosms with periphyton-colonized tiles

Isolate unexposed periphyton bacteria from both

streams

Isolate periphyton bacteria after exposures of: 2, 11,

22, and 33 hours; also after 1, 2, 3 and 4 weeks

WW-associated

periphyton

Spread plate

Periphyton

And randomly select

isolates for

susceptibility testing

Reference

periphyton

All microcosms contain reference stream water +10 ppb triclosan

Susceptibility Studies

Susceptibility testing

Isolate individual colonies (random)

Collect stream bacteria

from periphyton exposed in lab (WW-associated or forested reference)

pure cultures (random isolates; collected after

varying times of Triclosan exposure*)

(No Triclosan)

*Repeated after 2,

11 and 33 hours,

then 1,2,3 and 4

weeks of exposure

to triclosan in

microcosms

% inhibition, broth

microdilution assays

Susceptibility screen,

agar dilution assays

Triclosan concentration in agar

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Triclosan susceptibility screen

Agar dilution assay

0

µg/mL

Triclosan concentration in agar

0

5

10

15

20

25

30

35

WW Ref

[Tri

clo

sa

n] (p

pm

)

to in

hib

it g

row

th

0 2-33 ≥168

Triclosan Exposure (hours)

10

µg/mL

50

µg/mL5

µg/mL

1

µg/mL0

µg/mL

0.1

µg/mL

As we move forward…

Agents – Triclosan, Carbenicillin, Chloramphenicol,

Trimethoprim, Erythromycin, Ciprofloxacin

For each agent

2 doses tested (most data shown today from low dose tests)

Isolates – Randomly selected from Reference or WW-

associated stream periphyton

Unexposed

Exposed

Varying times of exposure in microcosms

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Microdilution Assay

Two doses of each agent (antibiotic/antimicrobial)

Dosed wells contain:Broth + agent (dissolved in Vehicle) + isolate culture

Δ Agent = ODAgent + bacteria – ODAgent + broth only

Δ Agent = cloudiness due to bacterial growth with agent

Vehicle control wells containBroth + Vehicle + isolate culture

Δ Vehicle = ODVeh + bacteria – ODVeh + broth only

Δ Vehicle is cloudiness due to bacteria

Blank wells containBroth + agent in 2% DMSO (Vehicle)

% inhibition =100 * Δ Vehicle - Δ Agent Percentage of growth prevented

Δ Vehicle by tested dose of agent

Triclosan

Carbenicillin

Chloramphen…

Trimethoprim

Erythromycin

Ciprofloxacin

0.0

20.0

40.0

60.0

80.0

100.0

% Inhibition Profiles

0% inhibition

“bar” on graph =

The isolate is resistant to the

level of agent tested in this

assay

100% inhibition

The agent effectively

prevented growth of the isolate

Each horizontal row of bars

represents a different agent

Each vertical column of bars

is a different isolate

Low doses tested (µg/mL):

Triclosan 0.125

Carbenicillin 0.25

Chloramphenicol 0.5

Trimethoprim 1

Erythromycin 0.625

Ciprofloxacin 0.125

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Unexposed Exposed 2-33 hrs

% in

hib

itio

n

S

usce

ptib

ility

Forested

Reference

WW-

associated

5% or less

53

144

37%

5% or less

47

162

29%

5% or less

33

138

24%

5% or less

29

150

19%

5% or less

53

144

37%

5% or less

29

150

19%

Unexposed Exposed 2-33 hrs

Forested

Reference

WW-

associated

5% or less

53

144

37%

5% or less

29

150

19%

5% or less

18

54

33%

5% or less

19

42

45%

2 hour

% in

hib

itio

n

S

usce

ptib

ility

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Unexposed Exposed 1-4 weeks

Forested

Reference

WW-

associated

5% or less

53

144

37%

5% or less

29

150

19%

5% or less

18

114

16%

5% or less

21

156

13%

% in

hib

itio

n

S

usce

ptib

ility

Proportion showing 5% inhibition or less

Inhibition by “low” doses

Forested Reference WW-associated

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m

TriclosanCarbenicillin

ChloramphenicolTrimethoprim

ErythromycinCiprofloxacin

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

WW

-0

WW

-0

WW

-0

WW

-2

WW

-0

WW

-33

WW

-2

WW

-33

WW

-2

WW

-0

WW

-2

WW

-2

WW

-0

WW

-0

WW

-2

WW

-0

WW

-0

WW

-0

R-5

29 R-0

R-0

R-0

R-0

R-1

60 R-0

R-2

% Inhibition profiles to low doses in isolates resistant to low dose Triclosan

Triclosan Carbenicillin Chloramphenicol Trimethoprim Erythromycin Ciprofloxacin

How many triclosan survivors also survive

other antibiotics?

Of isolates uninhibited by 0.125 µg/mL Triclosan

Proportion of isolates inhibited 5% or less

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Triclosan

Carbenicillin

Chloramphenicol

Trimethoprim

Erythromycin

Ciprofloxacin

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

% Inhibition profiles to low doses in isolates resistant to Triclosan

Triclosan Carbenicillin Chloramphenicol Trimethoprim Erythromycin Ciprofloxacin

Unexposed triclosan-resistant isolates

0 hr

WW

1 wk

WW

exposed

Reference

% in

hib

itio

n

Incre

asin

g a

ge

nt e

ffe

ctive

ne

ss

D

ecre

asin

g s

usce

ptib

ility

Forested

Reference

WW-

associated

5% or less

53

144

37%

5% or less

29

150

19%

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Review

Background

Triclosan has not been regulated well thus far and is in thousands of

products

Triclosan is nearly ubiquitous in the environment

Triclosan poses diverse risks to human and environmental health

Hypotheses

Reference periphyton bacteria exhibit greater susceptibility to triclosan

than wastewater-associated periphyton bacteria

Resistance to triclosan is associated with resistance to other

antibiotics

Triclosan exposure results in decreased susceptibility to triclosan

Expect more pronounced effect in reference stream bacteria compared to WW-

associated

Overall Summary

Observations in the random isolates tested

Unexposed: overall less susceptibility in WW-associated

compared to reference isolates (p=0.0484)

Upon environmentally-relevant exposures to triclosan

Unexposed and exposed WW-associated isolates show similar

overall susceptibility (p=0.6427)

Exposed reference stream isolates show less susceptibility

compared to unexposed reference isolates (p=0.0099)

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Continuing endeavors…

Identify isolates

16S rRNA gene sequencing

Were there community shifts resulting from microcosm

exposures?

qPCR targeting several select taxa

Acknowledgements

UNCG Biology

Laura Fondario

Megan Corum

Tatsiana Shymanovich

Hershey Lab

Allison Bullard

Matt Monteverde

Peter Blum

Josh Brigham

Kimber Corson

Undergraduate assistants

Chemistry & Biochemisty

Emily Britton

Daniel Todd

Tyler Graf

Amninder Kaur

Vincent Sica

Other support

Mary Hall-Brown

Mike Shelton

Ray Carney

UNCG Biology

Department

UNCG Graduate

School and GSA

SETAC

Committee

Anne Hershey

Nadja Cech

Parke Rublee

Zhenquan Jia

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And…

Thank You