MALDI-TOF-MS Identification of Coagulase-Negative Staphylococci

Milk samples growing coagulase-negative staphylococci at the routine milk analysis were sent frozen to the milk quality laboratory at the University of Milan for microbial identification by MALDI-TOF-MS. There, milk samples were thawed at room temperature, and 100 µL of milk was spread on blood agar half-plates to obtain bacterial colonies for MALDI-TOF-MS identification. After incubation for 24 h at 37 °C, the plates were examined for microbial growth. When no more than two different colony types were present, these were counted and assessed by MALDI-TOF-MS for microbial identification as described previously^{46 (link)}, with minor modifications. One colony was deposited in duplicate on the target plate using a toothpick, overlaid with 1 µL of α-cyano-4-hydroxycinnamic acid (Bruker Daltonik GmbH, Bremen, Germany) and left to dry. The spectra were acquired with a microFlex™ mass spectrometer (Bruker Daltonik GmbH) in the positive mode, including two spots of Bacterial Test Standard (Bruker Daltonik GmbH) for calibration in each target plate. The obtained spectra were interpreted against the MBT Compass^® 4.1 database. Log scores of ≥ 1.7 and ≥ 2.0 were the thresholds for the genus and species level identification, respectively.