To analyze the dynamics and mechanisms of stepwise resistance development to the novel siderophore-cephalosporin cefiderocol in wild-type, mutator and XDR high-risk clones of P. aeruginosa

Objective: To analyze the dynamics and mechanisms of stepwise resistance development to the novel siderophore-cephalosporin cefiderocol in wild-type, mutator and XDR high-risk clones of P. aeruginosa. Methods: Cefiderocol resistance evolution trajectories were analyzed in wild-type PAO1, PAOMS (mutS mutator derivate) and 3 XDR clinical isolates belonging to ST111, ST175, and ST235 clones. Each strain was incubated in triplicate experiments for 24h in iron-depleted cation-adjusted Müeller-Hinton broth with 0.06-128 mg/L of cefiderocol. Tubes from the highest antibiotic concentration showing growth were reinoculated into fresh medium containing concentrations up to 128 mg/L for seven consecutive days. Two colonies per strain and experiment were characterized by determining the susceptibility profiles and whole genome sequencing (WGS). Results: Evolution of resistance was significantly enhanced in PAOMS compared to PAO1. Resistance development was variable for the XDR clinical strains, including levels similar to PAOMS (ST235), similar to PAO1 (ST175) or even well below PAO1 (ST111). WGS revealed 2-5 mutation for PAO1 lineages and 35-58 for PAOMS. The number of mutations in the XDR clinical strains ranged 2-4 except for one of the ST235 experiments in which a mutL lineage was selected, thus increasing the number of mutations (10-15). The most frequently mutated genes were piuC, followed by fptA and pirR, all related with iron-uptake. Additionally, a L320P AmpC mutation was selected in multiple lineages and cloning confirmed its major impact in cefiderocol (but not ceftolozane/tazobactam or ceftazidime/avibactam) resistance. Mutations in the CpxS two-component sensor were also frequently documented. Conclusions: This work deciphers the potential resistance mechanisms that may emerge upon the introduction of the novel siderophore-cephalosporin cefiderocol in the clinical practice, and highlights that the risk of resistance development might be strain-specific even for XDR high-risk clones.