Microflex lt sh mass spectrometer

Overnight cultured C. auris isolates grown on SDA were used for spectra acquisition. Full extraction was performed according to the MALDI Biotyper standard protocol as described elsewhere [54 (link),55 (link),56 (link)]. Briefly, from each isolate, biomass was taken with a 10 µL inoculation loop and suspended in 500 µL distilled water and centrifuged 3 min at 14,000× g, the supernatant was discarded, and 1 mL of 70% ethanol was added. The suspension was homogenized and then centrifuged at 3 min at 14,000× g. Following this, based on the pellet size, equal amounts of 70% formic acid and 100% acetonitrile were added. After centrifugation for 5 min at 14,000× g, 1 μL of supernatant was spotted eight times onto a polished steel target plate (Bruker Daltonik) and overlaid with 1 µL MALDI matrix (10 mg/mL of α-cyano-4-hydroxy-cinnamic acid (α-HCCA) in 50% acetonitrile–2.5% trifluoroacetic acid; Bruker Daltonik) [57 (link)]. MALDI-TOF MS spectra were acquired with a Microflex LT/SH mass spectrometer (Bruker Daltonik) calibrated with the Bruker Bacterial Test Standard in the mass range between 2 and 20 kDa [25 (link)]. Up to 24 raw spectra were analyzed by the MALDI Biotyper Compass Explorer 4.1 software (Bruker Daltonik) to generate the reference spectra (MSP—Main Spectra Projection) and a UPGMA dendrogram was created with BioloMICS v12 (BioAware).

A. baumannii isolates (n = 78) from horses were randomly collected in our routine veterinary microbiological diagnostic laboratory from 2008 to 2020. The isolates were obtained from the genital and urinary tract (n = 21), wounds and abscesses (n = 19), the respiratory tract (n = 14), and various other clinical sites, including eyes, feces, gastrointestinal tract, organs, skin, hair, and hoof (n = 24). Species identification was performed with matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) using the Microflex LT/SH mass spectrometer and the biotyper database (MALDI Biotyper V3.3.1.0) (both Bruker Daltonics, Bremen, Germany). Furthermore, the identification was confirmed by rMLST using the online tool “Identify species” provided by PubMLST (https://pubmlst.org/species-id (accessed on 30 December 2022)). All isolates were stored at −70 °C in Brain Heart Infusion Broth (Oxoid, Wesel, Germany) containing 30% glycerol.