title: cefiderocol-naive P. aeruginosa blood culture isolate
design: Diagnostic routine microbiology was performed as described previously (11). Isolates were identified to the species level by MALDI-TOF spectroscopy using a Microflex instrument (Bruker Daltonics, Bremen, Germany). Susceptibility testing was performed according to EUCAST standard protocol for agar diffusion testing EUCAST disk diffusion protocol v 10.0) using disk from Oxoid (Thermo Fischer Scientific, Waltham, USA). Zone diameters were interpreted according to EUCAST clinical breakpoints v 10.0. MICs were determined using gradient strip assays (Liofilchem, Roseto degli Abruzzi, Italy) according to the manufacturers protocol. For quantification of pyoverdine a standard reference protocol was followed (12). In brief, strains were inoculated into 2mL of MHB from blood agar plates and incubated at 37C for 20h. Cultures were then diluted 1:100 in MHB or iron-depleted cation-adjusted MHB. 200l of the suspension were then transferred to a 96-well cell culture plate (Nunclon Delta Surface, Thermo Fisher Scientific, Waltham, MD, USA) and incubated for 20h at 37C under static growth conditions. OD600was recorded and the plates were centrifuged at 3220 g for 10 min. 100l of cell-free supernatant was transferred to black Greiner transparent bottom plates and the concentration of pyoverdine was quantified using a fluorescence spectrophotometer at an excitation wavelength of 395 nm and an emission wavelength of 470 nm. For whole genome sequencing, the DNA of P. aeruginosa Pae-A and Pae-B was isolated and sequenced on the Illumina MiSeq platform using paired-end chemistry (2x150bp), as described previously (13). Adapter sequences of the reads were trimmed, and any reads shorter than 35 nt removed using Trimmomatic v0.36 (PMID: 24695404). The trimmed reads were mapped to the reference genome strain P. aeruginosa PAO1 (NC_002516.2) with the aligner BWA (PMID: 19451168). The variants were called with the tool "GATK4 HaplotypeCaller" (PMID: 20644199) from the alignments and annotated using SnpEff (PMID: 22728672). The antimicrobial resistance genes were identified by the tool RGI 6.0.0, which utilizes the database CARD 3.2.5 (17)