M7h10 agar

A total of 194 clinical M. abscessus isolates were acquired retrospectively from sputum and bronchoalveolar lavage fluid specimens that were obtained between January 2014 and December 2017 and stored at Shanghai Pulmonary Hospital. Shanghai Pulmonary Hospital is one of the designated treatment centers for Chinese tuberculosis and NTM infections, attracting NTM cases from across the country. Both MGIT960 medium culture and p-nitrobenzoic acid testing were used as preliminary screening methods for NTM. The positive isolates were divided into subsp. abscessus and subsp. massiliense based upon multilocus sequencing of the rpoB and erm(41) genes. Staphylococcus aureus ATCC 29213, Mycobacterium peregrinum (ATCC 700686), and M. abscessus ATCC 19977 reference strains were purchased from the American Type Culture Collection (ATCC; Manassas, VA). M. abscessus was grown at 37°C on Middlebrook 7H10 (M7H10) agar plates supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC) and 0.2% glycerol or with continuous shaking in Middlebrook 7H9 (M7H9) broth supplemented with 10% OADC and 0.05% Tween 80 (7H9sB). Suspensions of all isolates were stored at –80°C and cultured fresh before each assay. Middlebrook 7H9 broth, M7H10 agar, cation-adjusted Mueller-Hinton II broth (CAMHB), and OADC supplement were purchased from Becton, Dickinson, and Company (BD; Franklin Lakes, NJ).

A total of 10 NTM reference strains and 194 clinical, M. abscessus isolates were evaluated. The following reference strains: M. abscessus subsp. abscessus (ATCC 19977), M. avium (ATCC 25291), M. intracellulare (ATCC 13950), M. kansasii (ATCC 12478), M. fortuitum (ATCC 6841), M. gordonae (ATCC 14470), M. scrofulaceum (ATCC19981), M. peregrinum (ATCC700686), and M. xenopi (ATCC19250) were purchased from the American Type Culture Collection (ATCC; VA, United States). M. abscessus subsp. massiliense (CIP108297) was purchased from the Biological Resource Center of Institut Pasteur (CIP; Paris, France). Detailed information regarding the clinical isolates was provided in a previous study (Guo et al., 2020 (link)). All strains were grown at 37°C on Middlebrook 7H10 agar plates supplemented with 10% OADC and 0.2% glycerol, or with continuous shaking in Middlebrook 7H9 broth supplemented with 10% OADC and 0.05% Tween 80. Middlebrook 7H9 broth, M7H10 agar, cation-adjusted Mueller-Hinton II broth and OADC were purchased from Becton Dickinson and Company (NJ, United States).

All Mycobacterium spp. strains were grown to mid-exponential phase from a single isolated colony in Middlebrook 7H9 broth (BD, Sparks, MD, USA) containing 0.5% (v/v) glycerol and 10% (vol/vol) oleic acid albumin with aeration (60 rpm). Growth rates were measured as turbidity (absorbance at 540 nm) in 50 mL of M7H9 broth in 500 mL Nephalometry flasks (Bellco Glass, Inc., Vineland, NJ, USA) inoculated with 1 mL of a 7-day, 2 mL M7H9 broth culture grown at 37 °C with aeration (120 rpm), and cells were harvested when the culture reached mid-log phase. A loopful of each culture was streaked on M7H10 agar (Becton Dickinson, Sparks, MD, USA) containing 0.5% (vol/vol) glycerol and 10% (vol/vol) oleic acid albumin and incubated at 37 °C for 10–14 days to confirm purity and colonial type.