Bacterial Species Identification by MALDI-TOF MS

The identification of bacterial species was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) according to the procedure described previously [29 (link)]. Briefly, a water suspension of cultured bacteria (300 μl) was precipitated with 900 µL of ethanol (96% vol/vol; Carl Roth GmbH, Karlsruhe, Germany) and centrifuged for 5 min at 10,000 × g. The pellet was resuspended in 50 μl of 70% (vol/vol) formic acid (Sigma-Aldrich Chemie GmbH, Steinheim, Germany), followed by adding 50 μl of acetonitrile (Carl Roth GmbH), mixing, and centrifugation for 5 min at 10,000 × g. Then, 1.5 μl of the supernatant were spotted onto a MTP 384 Target Plate Polished Steel TF (Bruker Daltonik GmbH, Bremen, Germany) and air-dried. The dried material was overlaid with 2 μl of a saturated solution of α-cyano-4-hydroxycinnamic acid (in a solution of 50% acetonitrile, 2.5% trifluoroacetic acid) (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). Air-drying at room temperature followed. Spectra recording and data analysis were done with an Ultraflex instrument and Biotyper 3.1 software (Bruker Daltonik GmbH) [30 (link)].