Formic acid

Directly from Nocardia 48–72 h culture on COS, colonies were picked and deposited using the VITEK^®-PICKME^TM pen on 2 wells of a single-use target slide (=1 run). Next, the deposits were overlaid with 0.5 μL of formic acid (bioMérieux) and were allowed to dry; they were then overlaid with 1 μL of CHCA matrix solution (bioMérieux) and were allowed to dry again. The Escherichia coli reference strain ATCC 8739 was used on each plate for instrument calibration according to the manufacturer’s instructions. Finally, the slide was loaded into the VITEK^®-MS instrument. The mass spectra obtained were compared with the VITEK^®-MS IVD V3.2 database according to the manufacturer’s recommended settings. The software produced an identification associated with a confidence level. An identification was considered as interpretable when the confidence level was ≥99.9% on 1 or 2/2 wells. In case of failure, another run of 2 wells was deposited using the same interpretation criteria. After 3 failed runs, the strain was considered as unidentifiable (no identification) by VITEK^®-MS IVD.

Nocardia strains were incubated for 24-72 hours on Columbia blood agar (Becton Dickinson, Heidelberg, Germany) until colonies were visible. For MALDI-TOF-based identification, we used two different protocols. (I) A bacterial colony was spotted onto a polished steel MALDI-TOF target plate. Samples were overlaid with 0.5 µl of formic acid (BioMérieux, Nuertingen, Germany) and, once dried, 1 µl of matrix (CHCA, alpha-cyano-4-hydroxy-cinnamic acid) was added. (II) A bacterial colony was suspended in 10 μl of 70 % formic acid and subsequently in 10 μl of pure acetonitrile. After a centrifugation step of 2 minutes at 14,000 x g, 1 µl of the supernatant was spotted onto the MALDI-TOF target plate. Once dried, 1 µl of matrix (CHCA, alpha-cyano-4-hydroxy-cinnamic acid) was added. All isolates were analyzed in duplicates.