Susceptibility, methodological discrepancies, heteroresistance, and iron acquisition-mediated cefiderocol resistance in Italian Pseudomonas aeruginosa: results from a multicenter prospective study

Cefiderocol is a siderophore cephalosporin that leverages bacterial iron uptake systems to overcome resistance in Gram-negative pathogens. Despite strong in vitro activity against Pseudomonas aeruginosa, uncertainty remains regarding susceptibility testing accuracy and interpretation of borderline phenotypes. We conducted a prospective multicenter study of 352 non-duplicate clinical P. aeruginosa isolates from eight tertiary-care hospitals in Italy. The study assessed carbapenem resistance epidemiology, cefiderocol activity using reference and commercial methods, and methodological variability, including EUCAST-defined areas of technical uncertainty (ATU). Selected isolates underwent population analysis profiling, including strains showing growth within inhibition zones, and whole-genome sequencing of susceptible, heteroresistant, and resistant isolates. Carbapenem resistance was detected in 17.5% of isolates, with significant inter-hospital variation in carbapenemase distribution. Cefiderocol resistance was rare (0.3%) by reference testing, confirming sustained in vitro activity. Commercial susceptibility methods showed some variability in MIC values, particularly near clinical breakpoints. Disk diffusion occasionally identified isolates within the ATU, sometimes associated with discordant MIC results, although these discrepancies did not affect susceptibility categorization. Heteroresistance was uncommon (0.3%) and observed in a single isolate. Genomic analysis revealed alterations in iron acquisition pathways, including loss of pyoverdine biosynthesis genes in the resistant isolate, but no single genetic determinant fully explained reduced susceptibility, supporting a multifactorial resistance mechanism. Overall, cefiderocol retains strong in vitro activity against Italian P. aeruginosa isolates. However, variability in testing methods and the presence of intermediate phenotypes complicate interpretation. These findings underscore the need to refine susceptibility testing and better link MIC values, resistance mechanisms, and clinical outcomes.