MALDI-TOF Mass Spectrometry
Inactivation and preparation of the isolates for MALDI‐TOF MS analysis was performed using the Mycobacteria Extraction Method (MycoEX) in accordance with the manufacturer. In order to enable optical evaluation of the tested colonies and because the quality of the spectra obtained from isolates grown on solid media is better than those obtained from liquid media, 7H10 agar-plates were chosen as culture medium for MALDI‐TOF MS analysis (Kodana et al., 2016, Lotz et al., 2010). A loopful of culture from solid medium was transferred into a 1.5 ml Eppendorf tube with 300 μL of HPLC-water and inactivated for 30 min at 99°C under biosafety level 3 conditions. After a centrifugation step of 2 min at 13000 g , the supernatant was discharged the pellet was re-suspended in 300 μL of HPLC-water and 900 μL of ethanol. Thereafter, centrifugation was repeated and the supernatant was discharged. The tubes were left open enabling the pellets to dry at room temperature. A spatula-tip full of bead suspension (Zirconia/Silica; BioSpec, Bartlesville, USA) and 10-50µL μl of acetonitrile were added to the pellets, depending on the volume of the pellets. Mycobacterial cells were disrupted by vortexing at maximal speed for 1 min and 25–50 μl of 70% formic acid were added, depending on the volume of the pellets. In conclusion, the tubes were centrifuged at the same conditions as above and 1 μl of the supernatant was spotted on the MALDI‐TOF target plates (MSP 96 target ground steel; Bruker Daltonics, Billerica, MA, USA) in duplicates. At this point, the target plates were allowed to dry at room temperature and then taken to biosafety level 2 conditions. Thereafter 1 μl of matrix was added to each spot (HCCA, α‐cyano‐4‐hydroxycinnamic acid).
Peptide mass spectra were acquired in a linear positive ion mode at a maximum laser frequency of 60 Hz across a mass to charge ratio (m/z) of 2,000 to 20,000 Da using the Microflex LT benchtop operating system (Bruker Daltonik GmbH, Fällanden, Switzerland). Each spot was measured twice using the MBT_FC.par FlexControl method and analysed by the FlexAnalysis 3.3 software (Bruker Daltonik GmbH). The highest log score value was compared with the MBT Mycobacteria Library 4.0, containing 880 main spectrum profiles (MSP), representing 159 mycobacterial species. AnEscherichia coli reference strain provided by the manufacturer was used in each run as a calibrator and for quality control. Log scores values (LSV) between 2.0 and 3.0 were considered as acceptable, between 1.8 and 2.0 were treated with caution and considered consistent when the same species was the only one suggested by the software with a LSV above 1.8. Lower LSV (< 1.8) were interpreted as incorrect and recorded as “no identification possible” (Saleeb et al., 2011). For each species isolated, the measured spectra of one isolate were exported in specific main spectrum profiles (MSP) using the MALDI Biotyper Compass Explorer 4.1 software. Each MSP was matched against the MBT Mycobacteria Library 4.0.