Understanding mechanisms of reduced cefiderocol susceptibility in pvdS and fecI mutants of Pseudomonas aeruginosa

OBJECTIVES: Understand how mutations upstream of pvdS and fecI in Pseudomonas aeruginosa PAO1 lead to reduced susceptibility to cefiderocol, a siderophore cephalosporin. MATERIALS AND METHODS: Whole-genome sequencing, gene complementation and inactivation, mRNA expression, siderophore quantification and supplementation were carried out with mutants that showed reduced susceptibility to cefiderocol. RESULTS: Whole-genome sequencing, combined with gene complementation studies, with isolated mutants that showed 4-fold reduced susceptibility to cefiderocol identified mutations within the Fur-box of promoter regions upstream of pvdS and fecI. The pvdS promoter mutation led to increased pvdS transcription, resulting in increased pyoverdine production, as well as reduced transcription of other iron transport-related genes, including piuA an iron transporter responsible for cefiderocol uptake. The down-regulation of piuA, rather than competition for iron between pyoverdine and cefiderocol, was responsible for the decreased cefiderocol susceptibility. The fecI mutant demonstrated increased transcription of fecI and fecA, and the expression level of piuA in the fecI mutant was not related to reduced cefiderocol susceptibility, indicating a different mechanism for the reduced cefiderocol susceptibility in this mutant. Sequence analyses of over 35 000 clinical P. aeruginosa isolates did not identify isolates with the pvdS mutation, and only one isolate with the fecI mutation. CONCLUSIONS: These data highlight different mechanisms by which P. aeruginosa attempts to evade the action of cefiderocol by exploiting the complexities of iron haemostasis in bacteria. The absence of such mutants in the clinic suggests that such mutations may be detrimental for P. aeruginosa to survive in the clinical environment.