Copy number flexibility facilitates heteroresistance to increasing antibiotic pressure and threatens the beta-lactam pipeline

It is unclear how bacteria rapidly adapt to resist novel drugs upon their introduction. Heteroresistance (HR) is a form of antibiotic resistance where a phenotypically unstable minority resistant subpopulation co-exists with a susceptible population. We observed HR to cefiderocol, a novel beta-lactam developed to resist beta-lactamases including extended-spectrum-beta-lactamases (ESBLs), among clinical isolates collected before its use. The resistant subpopulation in Enterobacter was a continuum; increasing gene copy number of an otherwise ineffective ESBL mediated increased resistance in decreasing numbers of cells. The factors that control the magnitude of amplification are unclear. We observed that ESBL activity controlled the level of amplification, thus increased copy number can compensate for poor enzymatic activity. A Klebsiella isolate from a clinical treatment failure also demonstrated amplification, highlighting the relevance of this phenomenon. These data provide insights into factors controlling dynamics of HR and how bacteria use phenotypic resistance to flexibly confront new antibiotic threats.