Antibiotic Resistance Protocols (Methods in Molecular Biology)

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Stephen H. Gillespie (ed.), Antibiotic Resistance Protocols, Methods in Molecular Biology, vol. 1736,
https://doi.org/10.1007/978-1-4939-7638-6_9, © Springer Science+Business Media, LLC 2018

Chapter 9

Methods to Determine Mutational Trajectories After

Experimental Evolution of Antibiotic Resistance

Douglas L. Huseby and Diarmaid Hughes

Abstract

The evolution of bacterial resistance to antibiotics by mutation within the genome (as distinct from hori-
zontal gene transfer of new material into a genome) could occur in a single step but is usually a multistep
process. Resistance evolution can be studied in laboratory environments by serial passage of bacteria in
liquid culture or on agar, with selection at constant, or varying, concentrations of drug. Whole genome
sequencing can be used to make an initial analysis of the evolved mutants. The trajectory of evolution can
be determined by sequence analysis of strains from intermediate steps in the evolution, complemented by
phenotypic analysis of genetically reconstructed isogenic strains that recapitulate the intermediate steps in
the evolution.

Key words Serial passage, Whole genome sequencing, Minimal inhibitory concentration, Relative

fitness, Isogenic strains

1 Introduction

Antibiotics inhibit cell growth by targeting essential bacterial func-

tions including the enzymatic activities of DNA gyrase, RNA poly-

merase, and the ribosome. Mutations in these enzymes that reduce

the affinity of the drug for its target are one cause of resistance to

antibiotics. Because these drug targets are highly conserved, and

under strong selection for maximal functionality, most mutations

that reduce drug-target interactions also reduce enzymatic func-

tionality [ 1 ]. The reduced functionality associated with mutations

causing antibiotic resistance sets up a dynamic situation where the

mutant bacteria must continue to adapt by the acquisition of addi-

tional mutations. These additional mutations serve at least two dif-

ferent adaptive purposes: increasing resistance to the drug, and/or

increasing relative fitness in the presence or absence of the selective

drug. Examples of this type of multistep process are the evolution

of resistance to fluoroquinolones in E. coli that requires the

accumulation of several mutations to generate clinical resistance