Bacteria Identification by MALDI-TOF-MS

Single bacterial colonies were picked up three times with pipette tips and separately smeared as a thin layer onto a ground steel matrix-assisted-laser-desorption/ionization (MALDI) target according to the manufacturer’s instructions (Bruker Daltonics, Bremen, Germany). The on-target deposits were overlaid with 1 μL of 70% formic acid solution, dried at room temperature, overlaid again with 1 μL of matrix solution [10 mg/ml of HCCA dissolved in ACN/water/TFA (50/47.5/2.5; v/v/v)] and dried again (Nacef et al., 2016).

Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry analyses were performed on an Autoflex Speed^TM (Bruker Daltonics) running Flexcontrol 3.4 software. The MALDI-TOF mass spectrometer calibration was performed using the BTS as per Bruker’s recommendations. This calibration kit contains a typical protein extract of E. coli DH5alpha spiked with two additional pure proteins (RNAse A and myoglobin) to cover an overall mass range from 4 to 17 kDa. MALDI-MS profiles were acquired in positive linear mode across the m/z range of 2,000–20,000 Da using the manufacturer’s automatic method MBT_FC.par. Each MALDI-MS profile was the sum of the ions obtained from 480 laser shots performed randomly on different regions of the same spot.

Mass spectra were processed using Biotyper software (version 3.0; Bruker Daltonics) running with the Biotyper database version DB-5989, containing 5989 reference MALDI-MS profiles (5298 of bacteria, 626 of yeasts and 65 of filamentous fungi) called MSP. The experimental MALDI-MS profiles obtained from bacteria isolates were matched on the reference MALDI-MS profiles and the matches were restituted by Biotyper along with a Log(score) and an associated color code (green, yellow, and red). Briefly, a Biotyper Log(score) exceeding 2.3 (green color) indicates a highly probable identification at the species level. A Log(score) between 2.0 and 2.3 corresponds to highly probable identification at the genus level (green color) and probable identification at the species level. A Log(score) between 1.7 and 2.0 (yellow color) implies only probable genus identification; while score value lower than 1.7 (red color) means no significant similarity between the unknown profile and any of those of the database.