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.