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Célia M. ManaiaCatholic University of [email protected]
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