Cefiderocol

Disk diffusion was performed with the standard Kirby-Bauer disk diffusion method according to the EUCAST guidelines. Briefly, 0.5 McFarland standard of a bacterial suspension in 0.9% NaCl was streaked in three different directions on Mueller-Hinton agar (bioMérieux GmbH, Germany), and 30 μg of Cefiderocol disk (Liofilchem, Italy) was placed on the agar and incubated at 35 ± 1°C for 18 h. Cefiderocol MIC was determined by broth microdilution (range was 0.25 to 128 mg/liter) using iron-depleted CA-MHB, which was prepared according to the protocol by Hackel et al. (28 (link)), following CLSI and EUCAST recommendations for Cefiderocol susceptibility testing. To determine the Cefiderocol MIC without the influence of metallo-β-lactamases, a broth microdilution was performed using iron-depleted CA-MHB supplemented with 100 μg/mL pyridine-2,6-dicarboxylic acid (DPA) as a metallo-β-lactamases inhibitor. The MICs of the cirA mutants generated by serial passaging were determined using a Cefiderocol concentration range of 0.03 to 32 mg/liter with and without DPA. The MIC was interpreted manually according to EUCAST broth microdilution interpretation guidelines and photometrically at OD₆₀₀. MIC and disk diffusion were interpreted according to the EUCAST clinical breakpoints v.11.0.

The isolates were initially processed using disk-diffusion antimicrobial susceptibility testing for amikacin, aztreonam, cefepime, ceftazidime, ciprofloxacin, doripenem, gentamicin, imipenem, levofloxacin, meropenem, netilmicin, piperacillin-tazobactam, ticarcillin-clavulanate, and tobramycin with Oxoid (Basingstoke, United Kingdom) disks. Complementarily, an in-house broth microdilution method using cation-adjusted Mueller–Hinton Broth (Sigma-Aldrich, St. Louis, MO, USA) was performed to determine the minimum inhibitory concentration (MIC) for amikacin gentamicin, imipenem, meropenem, colistin, polymyxin B, tigecycline, and ceftazidime-avibactam, and all salts were purchased from Sigma-Aldrich (St. Louis, MO, USA), except for avibactam, which was donated by Pfizer Inc. To complete the antimicrobial susceptibility panel, novel antimicrobials/combinations were evaluated with Liofilchem (Roseto degli Abruzzi, Italy) MIC test strips for ceftolozane–tazobactam, meropenem–vaborbactam, imipenem–relebactam, cefoperazone–sulbactam, cefiderocol, plazomicin, eravacycline, and fosfomycin.

Bacteria were isolated from samples processed for routine diagnostics; the species was identified by the matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) system (Bruker Daltonik GmbH, Bremen, Germany). Antimicrobial susceptibility was tested using the MicroScan WalkAway system (Beckman Coulter Inc., Brea, CA, USA), and the CZA and meropenem-vaborbactam (MVA) MICs were confirmed using Etest strips (Liofilchem, Roseto degli Abruzzi, Italy). The cefiderocol MICs were tested using the ComASP compact antimicrobial susceptibility panel for 0.008 to 128 mg/L cefiderocol (Liofilchem). All CZA-resistant strains were tested for bla_KPC genes (GeneXpert, Cepheid, CA, USA).