Oadc supplement

PerM::tn, the Mtb H37Rv transposon mutant of gene perM (rv0955) containing a ΦMycoMarT7 transposon insertion at nucleotide 701, was isolated in a screen for acid-sensitive mutants described previously [13 (link)]. Mtb strains were grown in a humidified incubator at 37°C with 5% CO₂ in Sauton’s media with 0.05% Tween 80 or 0.05% tyloxapol; Middlebrook 7H9 medium (Difco) containing 0.2% glycerol, 0.5% bovine serum albumin, 0.2% dextrose, 0.085% NaCl, and 0.05% Tween 80; or Middlebrook 7H11 agar (Difco) containing 10% OADC supplement (Becton Dickinson) and 0.5% glycerol. Nominally magnesium-free Sauton’s media was prepared with 0.8 mM citric acid, 9 mM sodium citrate, 3 mM potassium phosphate, 30 mM L-asparagine, and 6% glycerol, chelated overnight with 20 g/L Chelex 100 resin (Bio-Rad), filtered to remove Chelex, supplemented with 0.2 mM ferric ammonium citrate and 5 μM zinc sulfate, and adjusted to pH 7.4. Before use, 0.05% Tween 80 and 2 mM MgCl₂ were added unless otherwise indicated. We call this medium “nominally magnesium-free” as trace residual magnesium is likely present. Hygromycin B (50 μg/ml), kanamycin (15 μg/ml) and streptomycin (20 μg/ml) were included when required for selection.

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).

M. tuberculosis H37Rv (ATCC25618) was grown in Middlebrook 7H9 medium plus 10% v/v OADC supplement (Becton Dickinson) and 0.05% w/v Tween 80 or on Middlebrook 7H10 agar plus 10% v/v OADC. Hygromycin was added at 100 μg/ml, kanamycin at 20 μg/ml, gentamicin at 10 μg/ml, X-gal at 50 μg/ml.