Elucidating the Molecular Factors Implicated in the Persistence and Evolution of Transferable Antibiotic Resistance

Abstract

Being the most diverse and abundant domain of life, bacteria exemplify the remarkable ability of evolution to fit organisms into almost any imaginable niche on the planet. Although the capacity of bacteria to diversify and adapt is fundamental to natural ecosystems and modern biotechnology, the same adaptive mechanisms constantly threaten human health. Less than a century ago, infectious disease was among the most common causes of mortality, but luckily this situation was drastically improved with the introduction of vaccination and effective antimicrobial drugs. Unfortunately, this situation is changing with the rapid emergence of multidrug resistant bacteria that do not respond to our current treatments. This process is to a large extent driven by gene exchange that allows bacteria to rapidly acquire ready-made adaptive features. The aim of this thesis has been to understand the adaptive mechanisms governing the dynamics of bacterial gene-sharing. Specifically, the focus has been on antibiotic resistance genes and their genetic vectors due to the profound implications of these genetic elements in human health. To observe the extend and impact of gene transfer events in a highly relevant natural environment, we looked into the genomes of Escherichia coli longitudinally sampled from the infant gut over the first year of life. Sequence analysis revealed a high degree of genomic plasticity, with frequent gene acquisition and loss events. While the acquisition of new genetic material is often deleterious, we show that plasmids encoding resistance and virulence factors may indeed be stably maintained in the gut despite imposing a measurable fitness cost to their bacterial hosts in vitro. In two studies investigating the stability of genetic elements, we zoom in on the molecular mechanisms enabling conflict resolution between incoming genetic elements and naïve recipient genomes. In both studies, the burden of initially costly genetic elements is ameliorated via adaptive evolution over time. In the case of a large multi-drug resistance plasmid, adaptation happens through IS26 mediated deletions of costly genes that (collaterally) sacrifice the transfer proficiency of the plasmid. For the industrially relevant mevalonate production pathway, we observe similar population-level loss dynamics. Using ultra-deep sequencing we show that the cost-attenuated pathway variants are interrupted by different IS-element insertions that enrich over time due to the fitness benefit of production loss. For both studies, the compensatory activity depends on the host background, and we suggest measures that can harness evolution to increase genetic stability of the costly production pathway. The final study of this thesis investigates the phenotypic effects of expressing 200 antibiotic resistance genes in E. coli. As the currency of evolution, genes are subject to selection at different levels that may promote or limit their success when transferred to a new host. Through sequence analysis and experimental interrogations, we suggest that functional constrains, rather than sequence composition, is the main challenge that acquired genes encounter when transferred across phylogeny. The work conducted in this thesis provides novel insight into the persistence and evolution of highly relevant genetic elements in vitro, In vivo and in situ. The conclusions shed light on fundamental evolutionary questions of genome dynamics and bacterial adaptation, which may ultimately improve our ability to predict and prevent the spread of antibiotic resistance and guide the engineering of robust biological systems.