AMR and Water Reuse Workshop Program

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AMR and Water Reuse Workshop in Dijon, 2024.10.22

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Workshop Program Tuesday, October 22 2024

Session I – The science of AMR:I

Beatriz Guerra (EFSA) / Celia Manaia (Universidade Católica Portuguesa) / Will Gaze (University of Exeter) / Andrea Di Cesare (National Research Council Italy) / Pablo Gago Ferrero (CSIC, Spain) Session II – Regulatory framework for water reuse

Pietro Drei on behalf of Roberta Maffettone (JRC, European Commission) / Danny Greenwald (Israel Water Authority) / Jeff LeJeune (FAO) / Charlotte Arnal (Veolia, France) / [Group Discussion]

Wednesday, October 23 2024

Plenary reporting from discussion groups A and B Session III – AMR Science II

Didier Hocquet (University Hospital Besancon) / Thomas Berendonk (University of Dresden) / Ryo Honda (Kanazawa University) / Eddie Cytryn (Volcani Institute) / Caroline Le Marechal (ANSES) / Amy Pruden (Virginia Tech) / Nathalie Wery (INRAE) / Dominic Frigon (McGill University)

Session IV – Risk Science

Carolee Carson (Public Health Agency of Canada) / Karina Yew- Hoong Gin (National University of Singapore) / Steven Djordjevic (University of Technology Sydney) / Erica Donner (University of South Australia) / [Group Discussion]

Thursday, October 24 2024

Plenary reporting from discussion / [Group Discussion]

2023 Report by Quadripartite of FAO/UNEP/WHO/WOAH

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Transition of Antimicrobial Resistome during Wastewater Treatment

AMR and Water Reuse Workshop in Dijon, 2024.10.22

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Professor, Kanazawa University

Collaborative Researcher, Osaka University

Ryo Honda <[email protected]>

+3.7 log

Annual discharge of AMR E. coli

Honda et al. (2020) npj Clean Water 3, 15.

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Wastewater treatment plants as barriers and reservoirs of AMR

Treated effluent Combined sewer

overflow (CSO)

WWTP

Activated sludge is a reservoir of

microbes and AMR.

Retention time of bacteria:

WWTP – 3~14 days Human gut – 1~3 days Oxygen & temperature WWTP – aerobic, 15-25 ° C

Human gut – anaerobic, 35-37 ° C

Wastewater is a major source of AMR from human to environ.

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Transition of AMR in WWTPs

1. Two major players

2. Sludge is always diluted.

Which AMR is reserved in WWTPs and how?

To address this question…

Transition of AMR in WWTPs

Which AMR is reserved in WWTPs and how?

To address this question…

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Created by Honda from Novo & Manaia 2010.

Appl. Microbiol. Biotechnol. 87,

1. Two major player

ü No difference in removal rates

between ARB and non-ARB

Fate of ARB in WWTP by culture-based method

Influent

wastewater Cl

PST

Aeration Tank

FST

Efflent

E. coli population

Ratio of AMR E.coli

Honda et al. (2020) J. Water Health 18 (6): 879–889.DOI: 10.2166/wh.2020.013

in Japan in Portugal

LR V= 1.3 2

AMX resistant TC resistant CIP resistant Total population Heterotrophs, Enterobacteria, Enterococci

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1. Two major players

Fate of ARB in WWTP by molecular-based method

Shotgun Metagenomic Sequencing

Sample collection

Wastewater, Sludge, Effluent at 5 WWTPs,

in Summer & Winter, 2018-2019

MiSeq

次世代シークエンサー

Illumina HiSeq 2x150 bp, 5Gb

ARG profiles

Entire ARGs

(unit: RPK)

efflux

inactivation target alteration

target protection

target replacement

グラフタイトル

efflux efflux;reduced permeability to antibiotic efflux;target alteration

inactivation reduced permeability to antibiotic target alteration

target alteration;target replacement target protection target replacement

ARG composition (unit: proportion of RPK)

5 WWTPs in Japan

with 5 different treatment systems

Honda et al. (2023) npj Clean Water 6, 46

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Fate of ARB in WWTP by molecular-based method

5 WWTPs in Japan

ü Molecular-based AMR abundance decreased in sludge in all locations and treatment system configurations.

Shotgun Metagenome Sequencing

1. Two major players Japan

MiSeq

次世代シークエンサー

HiSeq 2x150 bp 5Gb Sample collection Wastewater & Sludge at 5 WWTPs,

in Summer & Winter, 2018-2019

USA, China, Europe

Search Keyword:

“metagenomic & (wastewater or sludge)”

Selection criteria:

ü Commercial-scale WWTP ü Sampling date: 2015~2018 ü location information is provided ü HiSeq compatible, 2x150-200 bp ü Data size: 3.0~20GB

• Japan: 10

• USA: 7

• China: 23

• Europe: 85

• Japan: 12

• USA: 10

• China: 8

• Europe: 33

Sludge Wastewater

Global comparison of ARG profiles in WWTPs

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Molecular-based AMR abundance in WWTPs worldwide

ü Molecular-based AMR abundances are lower in sludge than in wastewater regardless of country and system configurations.

ü The same trends were reported in literature using qPCR.

Sabar, Kani, Morinaga, Honda, in preparation

Ave.

Japa

n Chin

a

India Sri Lank a

US Franc

e German

y Belgiu

m Switzerla

nd

Spain Denmar

k Swede

n Japa

n Chin

a

India US Denmar k

Asia Europe + USA Asia Europe+USA

Switzerlan d Th

ai lan d Thai

land

Influent Wastewater Activated sludge

To ta l A RG a bu nd an ce

[RPK/RPK-16S]

Some common mechanisms in WWT processes make this happen.

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ü Primary clarifier üFinal clarifier ü Return sludge

Decline of AMR ratio as a whole

Reduction of total bacterial population

If this hypothesis is true...

Sludge bacteria have lower AMR ratio than fecal bacteria

Po pu la tio n

(Linear-scale)

Influent Activated

sludge Treated

effluent

To ta l p op ul at io n AR B

Fecal bacteria (many culturable, prefer anaerobic & 35-37 ° C)

Sludge bacteria (many viable but non-culturable, prefer aerobic & 15-25 ° C) u Two key players

Influent

wastewater Cl

PST

Aeration Tank

FST

Efflent

Proportion of cells carrying ARGs by single-cell sequencing

0%

10%

20%

30%

40%

50%

60%

Influent

(681) Sludge

(341) Effluent (225)

Proportion of cells harboring ARGs

by single-cell genome sequencing

(unpublished)

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1. Two major players

ü Sludge bacteria carry different ARGs from fecal bacteria.

Correlations of proportion between ARG and phylogenetic classes

Anerobic fecal bacteria

Bacteroidetes, Firmicutes, Fusobacteria

Aerobic sludge bacteria

Acidobacteria,

Actinobacteria, Nitrospirae,

! , " -Proteobacteria

Wastewater Sludge

Effluent

Difference of ARG profiles by principal component analysis (PCA)

Sludge resistome is less diverse than wastewater resistome

Balance of

fecal bacteria and sludge bacteria shapes AMR-ome in WWTPs

Transition of AMR in WWTPs

Which AMR is reserved in WWTPs

and how?

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2. Sludge is always diluted

ü Only particular ARGs are dominant in wastewater and sludge.

ü Some ARGs are commonly abundant in both, but some are not.

Proportion of ARG in WWTP by drug class

Jap

an Chi

na Indi

a Sri Lan ka

US Fran

ce German

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Swiss Denmar Spain

k Swede

n Jap

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US Denmar

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Asia Europe + USA Asia Europe+USA

Swiss Th ai la nd Thai

land

β-lactam

Jap

an Chi

na Indi

US Fran

ce German

y Belgiu

m Swiss Denmar Spain

k Swede

n Jap

an Chi

na Indi

aUS Denmar

k

Asia Europe +

USA

Asia Europe+USA

land

aminoglycoside

Jap

an Chi

na Indi

US Fran

ce German

y Belgiu

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k Swede

n Jap

an Chi

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US Denmar

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land

Macrolide

Jap

an Chi

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US Denmar

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Tetracycline OXA

aadA6

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ü Only particular ARGs are dominant in wastewater and sludge.

ü Some ARGs are commonly abundant in both, but some are not.

Proportion of ARG in WWTP by drug class

Jap

an Chi

na Indi

US Fran

ce German

y Belgiu

m

k Swede

n Jap

an Chi

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US Denmar

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land

Multidrug efflux

Japan China IndiaSri LankaUS DenmarkSwitzerlandFrance GermanyBelgium SpainSweden Japan China India USDenmark

Switzerland Thai

land Thai

land

Fluoroquinolone

ARGs commonly found at higher abundances in sludge (in most WWTPs) Aminoglycoside: AAC(6’)-Ib7

Macrolide: carA, srmB, tlrC, oleB, oleC Quinolone: qnrS2

Multidrug efflux: MexF/W/Q, MuxC/D, AxyY Antiseptic: qacEdelta1

Why are these ARGs more abundant in activated sludge?

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% = &^"#

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(sludge) SRT = 3~14 days

è Dilution rate = 0.07 ~ 0.33 /day Waste-

water

Aeration Tank

RT distribution in CSTR

(as doubling time = 2 ~ 10 days)

(i) Increase rate > Dilution rate:

Abundance in sludge > Abundance in wastewater (ii) Increase rate < Dilution rate:

Abundance in sludge = Abundance in wastewater Abundance of a component in activated sludge

Requirements to retain ARG/microbes in activated sludge

Higher abundance in sludge à They increase in sludge.

(2) Cell growth (1) Horizontal gene transfer

Q

_in

Q

_r

Q

r

/ Q

in

=0.25~0.4

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2. Sludge is always diluted – selection mechanisms of ARGs (1) Horizontal gene transfer

HGT rates of in vitro study

are mostly slower than the dilution rate.

Reference Donor Recipient Temp Contact

time [hr] Frequency [log] Quantification Max HGT rate, estimated [1/d]

Li et al. 2019 E. coli E. coli 26 4 -1.5 Molecular 0.18

Kenzaka et al. 2007 Phage E. coli 37 0.33 -3 to -4 Molecular 0.07

Phage E. coli 37 0.33 -6 to -7 Culture 7.2.E-05

Johnsen & Kroer 2007 E. coli P. putida 29 18 -3 to -4 Culture 1.3.E-03

Hutinel et al. 2021 WWTP Effluent E. coli 30 3 -4 to -6 Culture 8.0.E-04

Jutkina et al. 2016 WWTP Effluent E. coli 30 3 -5 to -6 Culture 8.0.E-05

WWTP Effluent E. coli 30 16 -5 to -6 Culture 1.5.E-05

Mishra et al. 2021 E. coli E. coli 37 2 -12 to -13 Culture 1.2.E-11

E. coli E. coli 30 2 -12 to -13 Culture 1.2.E-11

E. coli E. coli 20 6 -13 to -14 Culture 4.0.E-13

Dilution rate of sludge = 0.07 ~ 0.33 /day

1 Transfer rate to recipient in log 10

[1/ d]

0 -2 -4 -6 -8 -10 -12 -14

Culture Molecular

1 Transfer rate to recipient in log 10

[1/ hr ]

0 -2 -4 -6 -8 -10 -12 -14 -16

Culture Molecular

Dilution rate in WWTP

HGT alone probably cannot retain particular ARGs in WWTP systems (unless operated with long SRT).

HGT rates in real aeration tank could be smaller because of diversity of species

Tr an sf er ra te fr om d on or s to re ci pi en ts in lo g1 0 [1 /d ]

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1 Estmated specific growth rate [1/d]

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Estimated specific growth rate of activated sludge

Domestic sewage (n=17) Synthetic (n=3)

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2. Sludge is always diluted – selection mechanisms of ARGs

• Specific growth rate of

activated sludge = 0.25~2.6 /day* (average 1.1 /day)

Ø Intrinsically Ø HGT

(2) Cell growth

Cell growth is the primary driver to retain specific ARGs in activated sludge.

• HGT rate = 0.18 /day max. (in vitro, E. coli to E. coli)

• Dilution rate of sludge = 0.07 ~ 0.33 /day

* Estimated assuming BOD=5 mg/L from kinetic parameters

by Sozen et al. (1998) Water Res 32, 476–488.

1 Estmated specific growth rate [1/d]

0.0 1.0 2.0 3.0 4.0 5.0 6.0

Estimated specific growth rate of activated sludge

Domestic sewage (n=17) Synthetic (n=3)

How did the sludge bacteria initially acquire the ARGs…?

Aminoglycoside: AAC(6’)-Ib7

Macrolide: carA, srmB, tlrC, oleB, oleC Quinolone: qnrS2

Multidrug efflux: MexF/W/Q, MuxC/D, AxyY Antiseptic: qacEdelta1

u Two key players – Their balance shapes AMR-ome in WWTPs.

Ø fecal bacteria: high AMR ratio on clinically important drugs

Ø sludge bacteria: low AMR ratio some specific ARGs (AAC, ole, Mex/Mux, qacEdelta, etc.)

u Some particular ARGs are retained selectively in sludge.

– The main driver is cell growth of sludge bacteria carrying these ARGs.

Ø Sludges has less diverse AMR-ome than influent (in many countries).

Ø Mechanism of retention: Cell growth > HGT

Takeaways

Transition of AMR in WWTPs

20 Ryo Honda <[email protected]>

We need to know…

p Host species carrying ARGs of concern/interest

p Competitive selection & exclusion among species

To elucidate dynamics of AMR in environment

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Development of National Monitoring Framework of AMR in Japan

Research Project for “Development of Environmental Monitoring Framework of Antimicrobial Resistance and Substances”

funded by Ministry of Environment, Apr 2025- Mar 2028

PI: Ryo Honda (Kanazawa Univ), co-PI: Eiji Haramoto (Yamanashi Univ), Hiroshi Yamamoto (NIES) ü Rivers / lakes

ü WWTPs / hospitals ü Livestock industry ü Cropland

Antimicrobials

E. coli Genome Library from Environment

National guidelines for environ AMR monitoring

Selection of markers to identify AMR sources

Ecological toxicity, pollution load modelling

AMR One-Health Platform Nation-wide pilot survey AMR indicators

MST markers comparison & evaluation - ESKAPE, intI1, NGS, etc.

Knowledge base for One Health studies

ü To develop monitoring methodology of AMR in aquatic environment

ü To develop AMR source tracking methodology

ü To identify antimicrobials with high ecological toxicity

Objectives