Evolution of Heteroresistance

Antibiotic resistance is a major threat to global health, associated with ≈ 5 million deaths annually. While the field has largely focused on the emergence of stable resistance through mutation or horizontal gene transfer, a critical evolutionary unstable intermediate remains underexplored: heteroresistance (HR). In HR, a rare subpopulation within a main susceptible bacterial population exhibits transient high-level resistance - a phenotype that enables survival and growth under antibiotic pressure and paves the way to stable resistance. HR is often caused by increased copy number of resistance genes with low expression or activity. While cells carrying a single gene copy are susceptible, those with elevated copy numbers become resistant, and can replicate under antibiotic exposure, potentially leading to treatment failure. This project proposes a paradigm shift in our understanding of resistance evolution. Recent discoveries from our lab have revealed, across diverse bacterial pathogens, three novel and prevalent mechanisms by which HR emerges through increased gene copy number. These mechanisms generate subpopulations that evade antibiotic killing, yet revert rapidly in the absence of selection allowing the main population to remain fit. This duality makes HR an exceptionally flexible, but clinically dangerous, adaptive strategy. Despite its significance, the evolutionary origin(s) and consequences of HR remain unknown. This proposal seeks to fill that critical gap. We will (i) define the genetic and selective conditions that allow susceptible populations to evolve HR; (ii) determine how HR acts as a stepping stone toward stable resistance; and (iii) explore the potential of HR to promote horizontal gene transfer of resistance genes. By resolving these fundamental unknowns, this project will establish HR as a central driver in the evolution of antimicrobial resistance and inform future diagnostics and treatment strategies.