Population size modulates the transition from gene amplification-mediated heteroresistance to stable resistance

Heteroresistance is characterized by antibiotic-resistant subpopulations which can be underpinned by reversible gene duplication-amplification (GDA) of antibiotic resistance genes. It is often considered a precursor of stable resistance mutations. We investigated the evolutionary dynamics between GDA-mediated heteroresistance and stable resistance mutations in a clinical Enterobacter cloacae complex isolate. Exposure to ceftazidime resulted in enrichment of ampC β-lactamase GDAs in a concentration-dependent manner. Long-term exposure and high concentrations of ceftazidime selected for stable resistance mutations in dacB and ampD, a decrease of GDAs and a loss of population heterogeneity. Evolution experiments and mathematical modelling suggest that, in large populations, competition with GDA-carrying subpopulations can prolong fixation of stable resistance mutations in the population. By contrast, heteroresistance rescues small populations upon sudden antibiotic challenge, thereby acting as a precursor of stable resistance. This study highlights a previously underappreciated dual, population-size dependent role for GDAs in the transition from heteroresistance to stable resistance.