Jan-Ulrich Kreft University of Birmingham, UK · 2020. 5. 28. · Jan-Ulrich Kreft University of...
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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
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Questions
• Does antimicrobial resistance (AMR) increase in WWTPs
due to selection?
• Does it increase due to plasmid transfer?
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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
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Standard dose response curve vs MIC
Regoes et al. 2004
Conversion between EC50 and zMIC: 𝑧𝑀𝐼𝐶ℎ = 𝐸𝐶50ℎΨ𝑚𝑎𝑥
−Ψ𝑚𝑖𝑛
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Example dose response curves
Regoes et al. 2004
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Change of ARGs in 62 Dutch WWTPs
Pallares-Vega, …, Heike Schmitt (2019)
Absolute abundance Relative abundance
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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?
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Models as logical machines
Gamze Gülez
MICRO-C-OMICS
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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)
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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
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Default scenario
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Default scenario
• Selective window may stop below MIC of resistant
• Same EC50 for E and W gives different MICs
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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
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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
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Default scenario versus 10-fold diluted sewage
• Diluted sewage (lower nutrients) shrinks ‘selective window’
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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
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Effect of retention times
Default:
SRT/HRT = 10
HRT = 16.7 h
• Longer solid residence times broaden selective window
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Default but 100 fold higher plasmid transfer rate
• Higher plasmid transfer rate can maintain resistance plasmids
in the absence of antibiotics
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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
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Selective window depends on decay rate
• High mortality/predation/decay of biomass shrinks selective
window
Default biomass
decay rate 0.026 h-1
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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?
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Thanks to
• Barth Smets, DTU
• David Graham, Newcastle University