Heteroresistance via beta-lactamase gene amplification threatens the beta-lactam pipeline

Novel antibiotics are threatened by rapid emergence of resistance. Heteroresistance (HR) is a form of resistance where a phenotypically unstable and often undetected minority resistant subpopulation co-exists with a susceptible population. We observed HR to cefiderocol, a novel beta-lactam developed to resist beta-lactamases including ESBLs. The resistant subpopulation was a continuum; increasing gene amplification of otherwise ineffective ESBLs mediated increased resistance in decreasing numbers of cells. Surprisingly, ESBL amplification further increased when mutations or beta-lactamase inhibitors (BLI) were employed to inhibit activity, demonstrating that amplification overcomes inefficient activity. After one exposure to sub-breakpoint concentrations of cefiderocol/BLI, this enhanced amplification mediated resistance in an isolate collected before clinical introduction of the drugs. Therefore, amplification allows HR isolates to employ pre-existing, otherwise ineffective beta-lactamases to overcome novel beta-lactam/BLIs without new, stable evolution. This phenotypic flexibility is undetected during drug development and reveals an unappreciated threat to beta-lactam/BLIs that dominate the antibiotic pipeline. ### Competing Interest Statement DSW is listed as an author on a pending patent broadly related to this work.