Vitek ms mycobacterium nocardia kit

All procedures were performed with the VITEK® MS Mycobacterium/Nocardia Kit (bioMérieux SA, Marcy L’Étoile, France). In brief, a 1-µL loopful of organisms was picked and transferred into a tube with glass beads and 70% ethanol, followed by vortexing for 15 min and incubation at room temperature for another 10 min. After the subsequent transfer and centrifugation, the dried pellets were uniformly dispersed with 70% formic acid and acetonitrile in turn. After centrifugation, 1 µL of the supernatant was transferred to the target slide. Once the sample was dry, 1 µL of CHCA matrix (bioMérieux SA, Marcy L’Eoile, France) was applied and allowed to dry prior to analysis. All slides were run within 3 h after preparation, and the identifications were run in automatic mode. For each isolate, a single extraction was performed and spotted in duplicate. For any isolates where discrepancies were observed, the isolate was confirmed by gene sequencing (16 S rRNA, hsp65 PRA or erm). For each run, Escherichia coli (ATCC 8739; American Type Culture Collection, Manassas, VA, USA) was used for calibration and quality control according to the manufacturer’s protocols. Mycobacterium abscessus ATCC 19977 was used as a positive control, while negative controls were simultaneously analyzed, which was performed with spotted matrix only⁴⁹ .

Species identification was performed for all samples, by using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Vitek MS Mycobacterium/Nocardia Kit, bioMérieux SA, Marcy L’Étoile, France). Briefly, 1 μL of NTM colonies grown on LJ medium was transferred to a tube containing glass beads and 70% ethanol, vortexed for 15 min, and incubated for additional 10 min at room temperature. The dried pellets were then uniformly dispersed in 70% formic acid and acetonitrile. After centrifugation, 1 μL of the supernatant was transferred to a slide. After drying, 1 μL of CHCA matrix (bioMérieux SA, Marcy L’Eoile, France) was applied and allowed to dry before the analysis. Identification was performed automatically. For calibration and quality control, Escherichia coli (ATCC 8739; American Type Culture Collection, Manassas, VA, USA) was used for each run according to the manufacturer’s protocol [19 (link)].