The Antibiotic Dosage of Fastest Resistance Evolution: Gene Amplifications Underpinning the Inverted-U.

Carlos Reding,Pablo Catalán,Emily Wood,Ivana Gudelj,Hinrich Schulenburg,Gunther Jansen,Robert Beardmore,Phillip Rosenstiel,Tobias Bergmiller

doi:10.1093/molbev/msab025

Abstract

To determine the dosage at which antibiotic resistance evolution is most rapid, we treated Escherichia coli in vitro, deploying the antibiotic erythromycin at dosages ranging from zero to high. Adaptation was fastest just below erythromycin’s minimal inhibitory concentration (MIC) and genotype-phenotype correlations determined from whole genome sequencing revealed the molecular basis: simultaneous selection for copy number variation in three resistance mechanisms which exhibited an “inverted-U” pattern of dose-dependence, as did several insertion sequences and an integron. Many genes did not conform to this pattern, however, reflecting changes in selection as dose increased: putative media adaptation polymorphisms at zero antibiotic dosage gave way to drug target (ribosomal RNA operon) amplification at mid dosages whereas prophage-mediated drug efflux amplifications dominated at the highest dosages. All treatments exhibited E. coli increases in the copy number of efflux operons acrAB and emrE at rates that correlated with increases in population density. For strains where the inverted-U was no longer observed following the genetic manipulation of acrAB, it could be recovered by prolonging the antibiotic treatment at subMIC dosages.

Highlights

We treat the bacterium Escherichia coli at different antibiotic dosages in vitro to ascertain which one supports the most rapid resistance adaptation for a variety of genotypic and phenotypic statistics
Prediction: Greater Antibiotic Sensitivity Supports Fastest Adaptation at Lower Dosages Before presenting any data, we first turn to two simple theoretical models to show that whereas inverted-Us likely depend on many parameters, they should be independent of a bacterium’s minimal inhibitory concentration (MIC) (Day and Read 2016)
In order to quantify phenotypic increases in resistance, one could sample populations each day and determine their respective changes in erythromycin MIC. As this requires a dose-response assay for each replicate of each lineage each day, we reduced the overhead by introducing a measure, the effective antibiotic dose (EAD), which can be determined directly from population density data as it is collected

Summary

Introduction

We treat the bacterium Escherichia coli at different antibiotic dosages in vitro to ascertain which one supports the most rapid resistance adaptation for a variety of genotypic and phenotypic statistics. No study addresses this question directly, to the best of our knowledge, the literature makes relevant predictions. The inverted-U is said to be (Day and Read 2016) “arguably the single-most robust finding in all of the empirical literature.” Our data address both this techpopulations at sub and super-MIC dosages and seek the nical disagreement and the robustness of the inverted-U

Methods

Results

Conclusion