Evolutionary Trajectories to Daptomycin Resistance in Enterococcus faecalis
Olson, John S.
Doctor of Philosophy
With increasing amounts of hospital-acquired antibiotic resistant infections each year and staggering healthcare costs, there is a clear need for new antimicrobial agents, as well as novel strategies to extend their clinical efficacy. While genomic studies have provided a wealth of information about the alleles associated with adaptation to antibiotics, they do not provide essential information about relative importance of genomic changes, their order of appearance, or potential epistatic relationships between adaptive changes. In this thesis, I have combined experimental evolution, comparative whole genome sequencing, and allelic frequency measurements to study daptomycin (DAP) resistance in the vancomycin resistant clinical pathogen Enterococcus faecalis strain S613. Maintaining cells inside a turbidostat, a single polymorphic culture was grown sustaining both planktonic and non-planktonic (e.g. biofilm) populations in co-culture as the concentration of antibiotic was raised, facilitating the development of more ecological complexity than is typically observed in laboratory evolution. This approach revealed a clear order and hierarchy of genetic changes leading to resistance, the signaling and metabolic pathways responsible, and the relative importance of these mutations to the evolution of DAP resistance. Genetic and phenotypic comparisons between resistant isolates also identified convergent evolutionary trajectories, suggesting a common biochemical mechanism of resistance. Despite the relative ecological simplicity of this approach compared to the complexity of the human body, I show that experimental evolution can be used to rapidly identify clinically relevant adaptive molecular pathways and new targets for drug design in pathogens.