From susceptible to resistant: evolutionary routes via heteroresistance

Antimicrobial resistance is a major challenge in modern medicine. The origin of stable resistance (R) has mostly been associated with the emergence of point mutations or the horizontal acquisition of genes. Heteroresistance (HR), the presence of subpopulations with increased resistance within otherwise susceptible (S) bacterial populations, has been recognized as an overlooked phenomenon regarding treatment failures and the emergence of stable resistance. In practical terms, HR is defined when the frequency of cells able to grow at an 8-fold higher MIC than the original population is at least 10-7. HR is a result of genomic instability, and in Gram-negative bacteria, it is mainly associated with gene copy-number variation (CNV), which can be plasmid-associated or chromosome-associated. Apart from medical importance, HR is an interesting phenomenon in the broader context of the evolution of switchable phenotypes (“bet-hedging”): in the absence of antibiotics, a heteroresistant strain behaves like a susceptible strain, minimizing the resistance-associated fitness costs, while in the presence of antibiotics, the higher-resistance subpopulation can rapidly be enriched and keep the population size high. Stable resistance can emerge within HR populations, indicating that HR can serve as an intermediate state and open novel evolutionary trajectories towards R, which are unlikely to be explored in the traditional one-step S tot R process. The main question in this research project is how these switchable phenotypes arise as a response to antibiotic exposure in initially fully susceptible strains, and how this affects the further evolution of resistance. The project aims to identify key genomic requirements for the emergence of HR, in particular HR associated with gene copy number variation in Gram-negative bacteria, develop methods for easier detection of unstable resistance, understand the association between gene copy number and resistance level, and explore the link between HR and horizontal gene transfer. The core part of the research will be utilizing experimental evolution on Escherichia coli.