Abstract
Antibiotic resistance is a rapidly rising global health threat for which new strategies required to combat multi-drug resistant infections. Enterococci are opportunistic pathogens that are a major cause of hospital acquired infections. Understanding the development of resistance against antibiotics is essential in maintaining viable treatment options for severe enterococcal infections. Ampicillin and daptomycin are cell-wall targeting antibiotics used in the treatment against multi-drug resistant enterococci. In this study, a combination of transposon and spontaneous mutagenesis was undertaken to isolate mutants with altered ampicillin and daptomycin susceptibility. A transposon screen was used to disrupt whole genes while serial passaging was undertaken to evolve point mutations within E. faecalis with modification of proteins resulting in changes in susceptibility. Whole-genome sequencing and antibiotic susceptibility assays were used to isolate and identify various mutations which play a role in the evolution of ampicillin and daptomycin resistance. Establishing resistance to ampicillin involved key mutations in genes encoding for the signal transduction regulatory systems of a serine/threonine phosphatase kinase pair IreP/K and the two-component system CroRS. Changes were also observed in cyclic-di-AMP phosphodiesterases which regulate the associated second messenger molecule. In daptomycin resistant isolates, the wildtype strains developed mutations in liaF, a negative regulator of the cell wall stress response system LiaRS, which coevolved with mutations associated with membrane phospholipid composition including mprF and cardiolipin synthases. Importantly, mutations in key genes were observed across both E. faecalis strains tested, suggesting a homologous mode of resistance acquisition. In summary, the results presented here highlight the evolution of mutations in cell wall homeostasis and cell wall composition pathways and how they significantly contribute to the development of ampicillin and daptomycin resistance in E. faecalis. In addition, I show that the accumulation of multiple mutations is necessary for high-levels of resistance.