Evolution of cefiderocol resistance in using serial passage techniques.

Objectives: Cefiderocol is a siderophore cephalosporin active against MDR Gram-negatives including . Cefiderocol resistance remains uncommon and incompletely understood. We selected for cefiderocol-resistant and characterized the genetic mechanisms and potential for cross-resistance to other antimicrobials.

Methods: We selected cefiderocol resistance in three clinical strains of by serial passage in escalating concentrations of cefiderocol. Emergent cefiderocol-resistant isolates were subjected to repeat susceptibility testing against a panel of relevant antimicrobials. Isolates with confirmed MIC changes were whole genome sequenced.

Results: Each parent strain was initially susceptible to cefiderocol (MICs of 0.03125, 0.03125 and 0.125 mg/L), and one initially tested susceptible to ceftazidime/avibactam (MIC 4 mg/L). We recovered evolved isolates achieving cefiderocol resistance at MICs of 8-32 mg/L from each parental strain. Some cefiderocol resistant isolates reverted following one to four drug-free passages. Ceftazidime/avibactam MICs of passaged isolates repeatedly increased to ≥256 mg/L, and while other MICs were largely unchanged, trimethoprim/sulfamethoxazole MICs declined 4-fold in two strains. WGS revealed one evolved isolate carrying six coding mutations, while four were isogenic mutants of , , and the promoter Mutation of the promoter downregulated the efflux pump and reduced susceptibility to penicillins but increased susceptibility to several other classes including sulphonamides. Other mutations occurred in genes putatively involved in iron metabolism including and .

Conclusions: strains evolved cefiderocol resistance through different genetic pathways, but often involved iron transport. Future work is required to fully understand the role(s) of other genes in cefiderocol resistance.