Abstract
Accurate antimicrobial susceptibility testing (AST) of cefiderocol remains a diagnostic challenge, especially in infections caused by metallo-β-lactamase (MBL)-producing Klebsiella pneumoniae. While disk diffusion offers a cost-effective alternative to broth microdilution, it is highly sensitive to factors such as media composition and the presence of atypical colony morphology. The objective of this study was to evaluate how different agar media and interpretations of isolated colonies affect the performance and reliability of cefiderocol AST by disk diffusion. A total of 50 clinical K. pneumoniae MBL isolates were tested using disk diffusion on Columbia with blood, MacConkey, and chromogenic agars from three manufacturers. Inhibition zones were compared with MICs from broth microdilution. Statistical analyses included paired t-tests and Spearman correlation to assess media effects and zone morphology impact. Variability in inhibition zone diameters was observed between media, notably with chromogenic agar. The most consistent results were obtained using Graso Biotech and Thermo Fisher Columbia with blood agar. Isolated colonies were observed in over half the samples and, depending on how they were interpreted, led to major changes in classification accuracy. Up to 64% of results fell into the EUCAST area of technical uncertainty (ATU), and categorical agreement varied across media and interpretive criteria. Disk diffusion for cefiderocol may be used in resource-limited settings but only if rigorously standardized using validated media, consistent zone reading, and ATU-aware interpretive strategies. In borderline cases or when morphological anomalies are present, broth microdilution should be considered the sole reliable method. Clinical microbiologists are advised to exercise caution with ambiguous results and seek expert or confirmatory testing when needed.