Célia M. ManaiaCatholic University of Portugalcmanaia@porto.ucp.pt

Antibiotic resistant bacteria as

contaminants of emerging concern

Clatworthy et al. (2007) Nature Chemical Biology 3, 541 - 548

~75 YEARS OF ANTIBIOTHERAPY …

Lewis (2013) Nature Reviews Drug Discovery 12, 371–387

ANTIBIOTICS TARGET SPECIFIC STRUCTURES IN THE BACTERIAL CELL

Cell wall

DNA

Ribosomes

THREE MAJOR TARGETS

FIVE GENERAL RESISTANCE MECHANISMS

HUNDREDS OF GENES…Adaptado de: http://www.reactgroup.org/toolbox/category/understand/the-rise-and-spread-of-antibiotic-resistance/resistance-mechanisms-in-bacteria// (Julho 2016)

Andersson & Hughes (2010) Nature Reviews Microbiology, 8, 260-271

HORIZONTAL GENE TRANSFER /MUTATION

HORIZONTAL GENE TRANSFERMUTATION

Adapted: http://www.reactgroup.org/toolbox/category/understand/the-rise-and-spread-of-antibiotic-resistance/mutation-and-selection/ (Julho 2016)

Resistantbacteria

Susceptiblebacteria

RESISTANCE BECOMES

PREDOMINANT

SUSCEPTIBLE BACTERIA: DIE+

RESISTANT BACTERIA: MULTIPLY

SELECTION

SELECTIVE PRESSURE

Clatworthy et al. (2007) Nature Chemical Biology 3, 541 - 548

~75 YEARS OF ANTIBIOTHERAPY …

= 75 YEARS OF ANTIBIOTIC RESISTANCE

ANTIBIOTIC RESISTANCE…

BE

NL

DECZ

AU

PL

IT

FR

ESPT

UKIE DK

NOSE

FI

LT

SK

RO

BU

SI

HR

HU

GR

CH

CY

ANTIBIOTIC CONSUMPTION…

Vaz-Moreira et al., 2014. FEMS Microbiol Rev. 38:761-78

ANTIBIOTIC RESISTANCE IS NATURAL…AND ALSO… CONTAMINANT

NATURAL

Top superbugs

CONTAMINANT

Vaz-Moreira et al., 2014. FEMS Microbiol Rev. 38:761-78

CONTAMINANTS OF (SPECIAL) EMERGING CONCERN

PROPAGATIONSelection

HGT

WATER AS A MAJOR ROUTE OF PROPAGATION

Davies and Davies Microbiol. Mol. Biol. Rev. 2010;74:417-433

WATER

HABITAT

TRANSPORT

WASTEWATER TREATMENT IS ESSENTIAL TO REMOVE

ANTIBIOTIC RESISTANCE106 CFU/100 mL

104 CFU/100 mL

20% Resistance prevalence

Ferreira da Silva, et al. 2007. FEMS Microbiology Ecology, 60:166-176

p < 0.0001

Escherichia coliQuinolone-resistant

Raw and treated wastewater

0

5

10

15

20

25

30

35

40

45

50

AML CIP SXT TET CP GEN

% o

f re

sis

tan

ce

Raw inflow Final effluent

100% due to mutation in chromosomal genes (gyrA and parC)

Figueira et al. 2011 Science of the Total Environment, 409:1017–1023

1019

1018

1017

1016

1015

1014

1013

1012

1011

1010

109

108

107

106

105

104

103

102

101

100

Ge

ne

co

py

nu

mb

er

pe

r d

ay

ES FI EE EE FI EE EE ES FI EE EE ES ES PT ES FI EE EE FI EE EE CN US CN FI EE EE US US FI EE EE US US US ES US ES ES ES ES

blaOXAblaCTX-M

blaSHV

blaTEM

sul1 sul2

tetA

tetCtetG

tetM

tetQtetWtetX

qnrA,B,S

It is estimated that more than 1010 to 1014 copies of genes encoding

for tetracycline or beta-lactam resistance are released per minute to

the surrounding environment

Manaia et al., 2016. Applied Microbiology and Biotechnology. 100:1543–1557

WASTEWATER TREATMENT IS ESSENTIAL TO REMOVE

ANTIBIOTIC RESISTANCE

OPERATIONAL CONDITIONS (oxygen, organic matter, sludge age, etc)

SELECTIVE PRESSURES (antibiotic residues, metals, nanoparticles, etc)

INFLUENT QUALITY (hospital, animal production, etc)

Varela et al., 2014. Water Research, 54, 327-336; Narciso da Rocha et al., 2014. Journal of Global Antimicrobial Resistance, 2:309–315

Antimicrobial Residues

Axis

2 (

6.5

%)

Axis 1 (11.4%)-1.0 1.0

-1.0

1.0

B1

B2

B3

B4

B5

B6

B8

B9

B10

B11

B13B14

B1

5

B16

B17

B18

B19

B20

B21

B22

B23

B24

B26

TET

PEN SUL

CIP

ASOFX

B12

(AX1 -0.22, AX2 0.53)(AX1 0.20, AX2 0.49)

(AX2 0.31)

(AX1 0.68, AX2 0.11)

(AX1 0.43, AX2 -0.14)

(AX1 -0.15,

AX2 0.11)

AntibioticResistantBacteria

PCACIPGSPCIPmFCCIP

Bacterial Communities

(B7) Bacteroidia

Gammaproteobacteria

(B25)ClostridiaBacteroidia

Clostridia

Clostridia

Gammaproteobacteria

AntibioticResistance Genes

intI1

blaTEM

vanA

Total bacteria vs. Antimicrobial residues vs. ARG vs. ARBSELECTIVE PRESSURES

HOSPITAL EFFLUENT VS. THE MUNICIPAL WWTP

intI1/marA/blaTEM/vanA - Prevalence / 16S rRNA

Narciso da Rocha et al., 2014. Journal of Global Antimicrobial Resistance, 2:309–315

1 000 m3/day

54 000 m3/dia

(~2%)

INFLUENT QUALITY

Narciso da Rocha et al., 2014. Journal of Global Antimicrobial Resistance, 2:309–315

intI1/marA/blaTEM/vanA

HOSPITAL EFFLUENT VS. THE

MUNICIPAL WWTPINFLUENT QUALITY

WA20 TWWSVu32 Columbidae

WA23 streamWZ5 stream

A

SG20 Larus fuscusSG16 Larus fuscus (Ile80Ala84)

WA7 RWWSG15 Larus fuscus (Ile80Ala84)

SG7 Larus fuscusSG5 Larus fuscus

WA24 RWW (aac(6')-Ib-cr, qnrS)SG3 Larus fuscus

SP480 Larus michaelisWZ2 stream

SP281 Larus michaelisSP422 Larus michaelis (Leu83Asn87)

BR8 Hieraaetus pennatusBR6 Buteo buteo

BR7 Hieraaetus pennatusBR1 Buteo buteoWA12 TWW

SP367 Larus michaelisSP254 Larus michaelis (Leu83Asn87)

BR5 Buteo buteoBR4 Hieraaetus pennatusBR3 Buteo buteo

WA8 RWW (Leu83, Arg80)BR9 Milvus migrans

SG6 Larus fuscusSG12 Larus fuscus

B

SG9 Larus fuscus (aac(6')-Ib-cr)SG8 Larus fuscus (aac(6')-Ib-cr)

SG11 Larus fuscus (aac(6')-Ib-cr)SG10 Larus fuscus (aac(6')-Ib-cr)

SG4 Larus fuscus (aac(6')-Ib-cr)WA9 TWW

SP238 Larus michaelis (Leu83, qnrS)WA19 RWW

WA15 TWWWA5 TWW

SP444 Larus michaelis

C

WA18 RWWWZ10 TWW (Leu83)

WA2 stream (Leu83)SP418 Larus michaelis

WA16 RWWWZ4 stream

SG17 Larus fuscusWZ9 TWW (Ile80Gly84)

WA10 TWWUP2 stream

SG14a Larus fuscus (aac(6')-Ib-cr)WZ11 TWW

WA6 TWW (Ile80Gly84)SV32 Larus argentatus (aac(6')-Ib-cr, Ile80Gly84)SV83 Larus argentatus (Ile80Gly84)SV230 Larus argentatus (Ile80Gly84)

D

WZ6 RWWSVu22 LaridaeUR2 stream

WZ3 streamSG1 Larus fuscus (aac(6')-Ib)

SG19 Larus fuscus (aac(6')-Ib-cr)

E

WZ7 RWWWA21 TWWSG2 Larus fuscus (Leu83Tyr87, Arg80)

SG18 Larus fuscus (aac(6')-Ib-cr)BR19 Bubo bubo (Leu83Gly87)

BR12 Aegypius monachus (Leu83Gly87)BR15 Accipiter nisus (Leu83Gly87)

BR17 Hieraaetus fasciatus (Leu83Gly87)BR16 Buteo buteo (Leu83Gly87)BR18 Bubo bubo (Leu83Gly87)

BR10 Aegypius monachus (Leu83Gly87)BR11 Aegypius monachus (Leu83Gly87)

BR13 Strix aluco (Leu83Tyr87)

F

100

100100

100

100

91

89

86100

83100

79

78

70

7074

98

56

55

5062

100

69

10082

74

90

100

98

67

64

100

99

0.002

Quinolone resistant Escherichia colibirds of prey, water environments and gulls

Portugal, Spain and Sweden

Vredenburg et al., 2014. Environ Microbiology, 16(4):995-1004;

Gulls

Wastewater

Surface water

Birds of prey

Natural reserve

Varela et al., J Global Antimicrobial Resistance, 3:286-289 Varela et al. 2015. FEMS Microbiol Ecol. 91(5).;

Hospital Effluent

Hospital Effluent

Hospital Effluent

Hospital EffluentST131

vanAblaCTXM-15…

Mitigation measures

METHODS HARMONIZATION

LOWER THE QUANTIFICATION LIMITS

EXPAND ARG DATABASES

ENVIRONMENT+ ANIMAL+HUMAN

Berendonk et al. 2015 Nature Reviews Microbiology. 13(5):310-7.

Davies and Davies Microbiol. Mol. Biol. Rev. 2010;74:417-433

I. ASSESS THE RISKS OF TRANSMISSION FROM ENVIRONMENT

TO HUMANS

II. IDENTIFICATION OF CRITICAL CONTROL POINTS

Berendonk et al. 2015 Nature Reviews Microbiology. 13(5):310-7.

Davies and Davies Microbiol. Mol. Biol. Rev. 2010;74:417-433

INCOME OF ANTIBIOTIC RESISTANT BACTERIA

BACTERIAL GROWTH

BIOFILM/CELL AGGREGATES

SELECTIVE PRESSURES

ADAPTATIVE MUTATIONS

HORIZONTAL GENE TRANSFER

0

1

2

3

4

5

6

7

8

9

10

16S rRNA intI1 blaTEM qnrS sul1

Lo

g (g

en

e c

op

y n

um

be

r/1

00

mL

)

0

1

2

3

4

5

6

7

8

9

10

16S rRNA intI1 blaTEM qnrS sul1L

og

(g

en

e c

op

y n

um

be

r/1

00

mL

)

LO

Q

LO

Q

LO

Q

LO

Q

LO

Q

LO

Q

Ozonation UV

Untreated control Treated Three days storage

Antibiotic resistance genes

J Hazard Mater. 2017 5;323(Pt A):434-441.

WASTEWATER DISINFECTION

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

C0 C3 T3 C0 C3 T3 C0 C3 T3 C0 C3 T3

WW1UV WW1O3 WW2PO SWPO

% o

f re

ads

TM7-1

Gammaproteobacteria

Deltaproteobacteria

Betaproteobacteria

Alphaproteobacteria

Clostridia

Sphingobacteriia

Flavobacteriia

Bacteroidia

[Saprospirae]

Actinobacteria

Acidimicrobiia

Other classes with abundance < 5 % reads

Unclassified at class level

TM7

Proteobacteria

Firmicutes

Actinobacteria

Bacteroidetes

Classes Phyla

A

WW1-UV254 nm WW1-O3 WW2-PO SW-PO

REGROWTH AFTER DISINFECTION

Sci Total Environ. 2016 ;573:313-323.

Besides treatment, also storage and transportare key issues in wastewater reuse

WASTEWATER TREATMENT IS ESSENTIAL TO REMOVE

ANTIBIOTIC RESISTANCE

Improved wastewater treatment processes

Bacterial removal (disinfection)

Environmental friendly (low impact on the

environment in particular on the microbiota)

Cost effective

Wastewater plumbing/storage

StAREStopping Antibiotic Resistance

Evolution

2013 Water JPI Pilot Call focused on “Emerging water contaminants-anthropogenic pollutants and pathogens”

December 2014 – December 2017National Funding AgenciesPortugalSpainCyprusGermanyIrelandNorwayFinland

Partners7 countries, 10 institutions

Universidade Católica Portuguesa (UCP)

University of Aveiro (UA)

University of Helsinki (UHel)

Nireas International Water Research Center, Univesity of Cyprus (Nireas-IWRC)

Fundació Institut Català de Recerca de l’ Aigua (ICRA

Aquantec GmbH

National University of Ireland, Maynooth (NUIM)

Agencia Estatal Consejo Superior de Investigaciones Científicas (CSIC)

Norwegian School of Veterinary Science (NSVS)

Technische Universität Dresden (TUD)

On-going projects on antibiotic

resistance in wastewater (and its reuse)

HEARD

WG5

NORMAN

NEREUS

ANSWERS

StARE

OVERSEAS

EU

EU

EU

USEU

Asia

EUUS

Australia

Asia

Partners (EU)

COST-European Cooperation in Science and Technology, to the

COST Action ES1403: New and emerging challenges and opportunities in wastewater reuse (NEREUS) - http://www.nereus-

cost.eu/

Olga Nunes (Portugal)

Paulo Costa (Portugal)

Manuela Caniça (Portugal)

Adrian Silva (Portugal)

Sérgio Silva (Portugal)

Isabel Henriques (Portugal)

Despo Fatta-Kassinos (Cyprus)

Thomas Berendonk (Germany)

Thomas Schwartz (Germany)

José Luis Martinez (Spain)

Sara Rodriguez (Spain)

Marko Virta (Finland)

Fiona Walsh (Ireland)

Henning Sörum (Norway)

Eddie Cytryn (Israel)

Christophe Merlin (France)

Luigi Rizzo (Italy)

Norbert Kreuzinger (Austria)

Network of reference laboratories, research centres and related

organisations for monitoring of emerging environmental substances -http://www.norman-network.net/?q=node/19

Stopping Antibiotic Resistance Evolutionhttps://stareeurope.wordpress.com/

UID/Multi/50016/2013-CBQF

OVERSEAS (FLAD 298/2015)

ANtibioticS and mobile resistance elements in WastEwater Reuse

applications: risks and innovative solutions – ANSWERS. European Commission Horizon 2020 - MSCA-ITN-2015-ETN: Marie Sklodowska-

Curie Innovative Training Networks (ITN-ETN)

Bacterial diversity groupESB-UCP, Porto, Portugal