Bacterial test standard

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.

For database construction and sample identification, measurements were performed in AutoExecute mode using an UltraFlex II MALDI TOF/TOF mass spectrometer (Bruker Daltonics GmbH, Leipzig, Germany) with ion source 1: 25 kV; ion source 2: 23.50 kV; and a 50.0 Hz nitrogen laser. Spectra were recorded in the positive linear mode for the relative molecular mass to charge ratio (m/z) range of 2000–20,000. Each spectrum was obtained by averaging 600 laser shots. The spectra were externally calibrated by using a Bacterial Test Standard that covers a mass range of 3600–17,000 Da (Bruker Daltonics).

Following the protocol of Djalali Farahani-Kofoet et al. [31 (link)] for the MALDI-TOF analysis, 1 µL of the protein extract was spotted on a polished steel target (Bruker Daltonik, Bremen, Germany) and allowed to dry. Then, 1 µL of the saturated HCCA (α-cyano-4-hydroxycinnamic acid solution) was added as matrix and allowed to dry. The MALDI method was calibrated using a bacterial test standard (Bruker Daltonik). The MALDI target plate containing the protein extracts was read with an ultrafleXtreme MALDI-TOF mass spectrometer (Bruker Daltonik) working in linear positive mode and acquiring mass spectra in the range of m/z 200–20,000. Measurements were performed by flexControl v3.4 software (Bruker Daltonik). The protein spectra served as the fingerprint of the test fungi and were compared with the reference database (1832 entries) of the Bruker Filamentous Fungi Library, including 13 entries for Trichoderma sp. The MALDI Biotyper v3.1 software (Bruker Daltonik) was used to process the raw spectra, generate peak lists and perform a database search for isolate identification. A dendrogram based on PCA clustering of m/z values was constructed using the hierarchical method, correlation distance measure, and average linkage algorithm.