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Predicting selective windows in wastewater
treatment plants: effect of plasmid transfer versus
antibiotic concentration
Jan-Ulrich Kreft
University of Birmingham, UK
Roberto
de la Cruz
Questions
• Does antimicrobial resistance (AMR) increase in WWTPs
due to selection?
• Does it increase due to plasmid transfer?
Selective window
Gullberg E, Cao S, Berg OG, Ilbäck C, Sandegren L, Hughes D, and Andersson DI (2011).
Selection of Resistant Bacteria at Very Low Antibiotic Concentrations. PLOS Pathogens 7: e1002158
resistantsensitive
Standard dose response curve vs MIC
Regoes et al. 2004
Conversion between EC50 and zMIC: 𝑧𝑀𝐼𝐶ℎ = 𝐸𝐶50ℎΨ𝑚𝑎𝑥
−Ψ𝑚𝑖𝑛
Example dose response curves
Regoes et al. 2004
Change of ARGs in 62 Dutch WWTPs
Pallares-Vega, …, Heike Schmitt (2019)
Absolute abundance Relative abundance
How to answer the questions?
• Mathematical models can piece together the puzzle of
factors that may or may not be relevant under real world
conditions in WWTPs
– Dose response curves measured in the lab
– Growth kinetics measured in the lab
– Plasmid transfer kinetics measured in the lab
– That is, elements studied in isolation under controlled
conditions (should reflect WWTP though)
• Find out what happens when these elements are combined
– What factors are important apart from MIC and fitness cost?
– Are predictions approximately correct?
Models as logical machines
Gamze Gülez
MICRO-C-OMICS
Simple Plasmid Model
within Activated Sludge
Model 1Processes:• Growth
• Substrate consumption
• Decay
• Plasmid transfer/loss
• Selection by antimicrobial (Emax model)
• Degradation of antimicrobial
• Inflow/Outflow
• SRT > HRT
Bacterial Populations
Non-recipient
Recipient
Transconjugant
Donor
Ente
rics
Wa
ste
Wa
ter
Typ
es
Inflow
Outflow
Enterics (E)
Antimicrobial
Substrate
(for
Wastewater
bacteria and
Enterics)
𝑪𝒖𝟐+
𝑪𝒖𝟐+𝑪𝒖𝟐+
Conditions:
• Temperature
• Solid Retention Time
• (SRT)
• Hydraulic Retention Time
• (HRT)
ASM1+++
Specific growth rate of Enterics (E) [aerobic] + [anaerobic growth] including substrate and temperature dependence, fitness cost of plasmid (if present) and effect of antimicrobial:
𝜇𝐸(∙)=
𝑆𝑂𝐾𝑂𝐸 + 𝑆𝑂
Ƹ𝜇𝐸𝑆𝑠𝐸
𝐾𝑠𝐸 + 𝑆𝑠𝐸𝜃𝐸𝛤 + 𝜅𝐸 1 − 𝐶𝜎,𝜌 +
𝐾𝑂𝐸𝐾𝑂𝐸 + 𝑆𝑂
Ƹ𝜇𝐸 𝜂𝑔𝑆𝑠𝐸
𝐾𝑠𝐸 + 𝑆𝑠𝐸
𝑆𝑁𝑂𝐾𝑁𝑂𝐸 + 𝑆𝑁𝑂
𝜃𝐸𝛤 + 𝜅𝐸 1 − 𝐶𝜎,𝜌
𝑑𝐸
𝑑𝑡= 𝜇𝐸
(∙)− 𝑏 −
𝐸𝑚𝑎𝑥𝜎,𝜌 (𝑆𝐴)𝐻
(𝐸𝐶50𝜎,𝜌)𝐻 + (𝑆𝐴)
𝐻 𝐸
Default scenario
Default scenario
• Selective window may stop below MIC of resistant
• Same EC50 for E and W gives different MICs
Default scenario versus no plasmid transfer
• Plasmid transfer extends and shifts ‘selective’ window towards
low concentrations of antibiotics
Plasmid transfer from E to E, E to W,
W to W, W to ENo plasmid transfer
Default scenario versus higher level resistance
• Larger difference in EC50 between resistant and sensitive
extends and shifts selective window towards higher
concentrations
EC50 of resistant = 10 x EC50 sensitive EC50 of resistant = 100 x EC50 sensitive
Default scenario versus 10-fold diluted sewage
• Diluted sewage (lower nutrients) shrinks ‘selective window’
Default scenario versus 18-fold concentrated sewage
• More concentrated sewage (higher nutrients) expands
‘selective’ window, mostly due to plasmid transfer being more
frequent at higher bacterial abundance
Effect of retention times
Default:
SRT/HRT = 10
HRT = 16.7 h
• Longer solid residence times broaden selective window
Default but 100 fold higher plasmid transfer rate
• Higher plasmid transfer rate can maintain resistance plasmids
in the absence of antibiotics
Default but 100 fold boost of plasmid transfer ratein transconjugants only
• Transient boost of plasmid transfer in transconjugants is
enough to maintain plasmids in the absence of antibiotics
Selective window depends on decay rate
• High mortality/predation/decay of biomass shrinks selective
window
Default biomass
decay rate 0.026 h-1
Answers from ‘simple’ plasmid model
• Selective window concept based on simple lab experiments
insufficient, need to consider
– Biomass decay/mortality
– Solid retention time
– Plasmid transfer and fitness cost shape selective window
• Resistant strains can be eliminated well below their EC50 and MIC
• Selection unlikely to happen at the low concentrations of
antibiotics typically found in WWTPs
• In concentrated sewage, plasmids maintained by transfer, no
need for selection by antibiotics
• In ‘normal’ sewage, plasmids can be maintained by transfer if
transfer rate of transconjugants boosted
• Mathematical models can predict ‘selective window’ in the real
world from simple lab experiments – are predictions true?
Thanks to
• Barth Smets, DTU
• David Graham, Newcastle University