The mechanisms of action and resistance of cefiderocol (FDC) in Acinetobacter baumannii are still not fully elucidated, but iron transport systems have been evoked in its entry into the cell to reach the penicillin-binding proteins (PBPs). To capture the dynamics of gene expression related to FDC action in various conditions, we report on the genomic and transcriptomic features of seven A. baumannii strains with different FDC susceptibility, focusing on the variants in genes associated with β-lactam resistance and the expression of the siderophore biosynthesis and transport systems acinetobactin and baumannoferrin. We also investigated the expression of the TonB energy transduction system (ETS) and siderophore receptors piuA and pirA. The four clinical samples belonged to the same clonal complex (CC2), and the two strains with the highest FDC MICs showed peculiar variants in PBP2 and ampC. Similarly, the two clinical strains with the lowest MICs shared variants in an outer membrane protein as well as ampC. Gene expression analyses highlighted the up-regulation of the acinetobactin and baumannoferrin genes in response to iron depletion and a down-regulation in the presence of high iron concentrations. In response to FDC, gene expression seemed strain-dependent, probably due to the different metabolic features of each strain. Overall, FDC activates the ETS, confirming the active import of the drug; baumannoferrin, more than acinetobactin, appeared stimulated by FDC in an iron-depleted medium. In conclusion, iron transport systems play a clear role in the FDC uptake, and their expression likely contributes to MIC variation together with β-lactam resistance determinants.IMPORTANCEAcinetobacter baumannii poses a threat to healthcare due to its ability to give difficult-to-treat infections as a consequence of our shortage of antibiotic molecules active on this multidrug-resistant bacterium. Cefiderocol (FDC) represents one of the few drugs active on A. baumannii, and to preserve its activity, this study explored the transcriptomic and genomic features of seven strains with varying susceptibility to FDC. Transcriptomic analyses revealed the different effects of FDC on iron transport systems, promoting mainly baumannoferrin expression-thus more likely related to FDC entry-and the energy transduction systems. These findings suggest that not all iron transport systems are equally involved in FDC entry into A. baumannii cells. Finally, mutations in PBPs and β-lactamases may contribute to the resistance onset. Overall, the study sheds light on the importance of iron availability and metabolic differences in FDC resistance, offering insights into understanding the evolution of resistance in A. baumannii strains.

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