Chca matrix

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)].

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 .

The NTM strains were isolated from clinical specimens of patients who visited or admitted to the University of the Ryukyus Hospital between May 2015 and August 2016, and stored in skim milk at −80 °C. These clinical isolates were inoculated on 2% Ogawa medium, and the cultures were incubated at 35 °C. A 1-µL loopful of colonies was picked and transferred into a tube with 0.6 mm zirconia/silica beads and 70% ethanol, followed by vortexing for 15 min and incubation at room temperature (approximately 25°C) for another 10 min. After centrifugation, the pellets were uniformly dispersed by successive washing with 70% formic acid and acetonitrile. After centrifugation, 1 µL of each 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 to each slide and allowed to dry prior to analysis. The identifications were run in automatic mode of Vitek MS v3.0 system (bioMérieux SA). For each isolate, a single extraction was performed and spotted in quadruplicate. For each run, Escherichia coli (ATCC 8739; American Type Culture Collection, Manassas, VA, USA) was used for calibration and quality-control testing, according to the manufacturer’s protocols.