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