Middlebrook 7h9

Moxifloxacin, isoniazid, ethambutol, linezolid, clarithromycin, rifampicin, tyloxapol, and Tween80 were purchased from Sigma–Aldrich. Middlebrook 7H9 and Middlebrook 7H10 were purchased from Becton Dickinson. PBS was purchased from Invitrogen, Life Technologies (14080055).

The solvents were purchased from Merck, India. Middlebrook 7H9 (Becton-Dickinson) supplemented with 0.1% casitone, 0.2% glycerol and 10% OADC (oleic acid, albumin, dextrose and catalase), Resazurin dye [Resazurin sodium salt (Sigma®, USA)], MTT [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide] Sigma-Aldrich, 96-well plate with lid (Becton Dickinson), Acrodisc Syringe filter (Pall Life Sciences) with 0.2 μm filters, >95% pure rifampicin (RIF), amphotericin B and miltefosine standard (≥98%) (Sigma-Aldrich) were used to conduct the following biological experiments. Pre-coated silica gel 60 F₂₅₄ plates were used for analytical TLC.

M. smegmatis mc²155 was routinely cultured at 37 °C on Middlebrook 7H9 (Becton, Dickinson and Company) liquid media supplemented with 10% (v/v) Albumin-Dextrose Complex (0.5% bovine serum albumin, 0.2% dextrose, 0.085% NaCl), 0.2% (v/v) glycerol and Tween 80, 0.05% (w/v). Antibiotics were used as follows: the conditional mutant pstP-CM was routinely cultured in the presence of hygromycin (50 μg/ml) and pristinamycin (2 μg/ml), and pstP-CM transformed with pMV306 or derivatives was routinely cultured in the presence of hygromycin, pristinamycin and kanamycin (30 μg/ml).

Mycobacterium isolates were cultured in Middlebrook 7H9 (Beckton Dickinson, Difco) complete broth medium supplemented with 0.05% Tween 80, 0.2% glycerol, and 10% albumin-dextrose-catalase (ADC, Beckton Dickinson, Difco) at 37 °C. Bacterial cultures were centrifuged at 3000xg for 10 min and re-suspended into 1 mL lysis buffer (100 mM Tris–HCl (pH8.0), 250 mM NaCl, 1 mM CaCl₂ and 3% SDS) and heat-inactivated at 95 °C for 30 min. Heat-inactivated samples were transferred to BSL2 lab, added 20 µl of Proteinase K (10 mg/mL) and incubated for 30 min at 56 °C. Samples were placed into the 2 mL tube with lysing matrix B (MP biomedicals) and bead-beat (2 cycles of 6 m/sec, 45 s) with FastPrep-24 5G (MP biologicals). Bacterial cell lysates were transferred into a new microcentrifuge tubes and genomic DNA was purified with standard phenol–chloroform DNA extraction procedure. Purified DNA was analyzed with NanoDrop 2000 spectrophotometer (Thermo Scientific) and agarose gel electrophoresis.
The genomic DNA of the samples were first fragmented using Covaris prior to library preparation using a commercially available kit, NEBNext® Ultra™ DNA Library Prep Kit for Illumina® following the manufacturer’s protocol. The samples were then pooled to be sequenced in 1 lane of a standard Illumina HiSeq4000 2 × 151 bp (multiplexed) run at the Genome Institute of Singapore.

Samples from Swiss TPH were incubated in liquid media Middlebrook 7H9 (Becton Dickinson) at 37 °C during two weeks, while as those from our laboratory were grown in commercial Middlebrook 7H10 agar (Becton Dickinson) with OADC supplement. In all cases, DNA extraction method was performed following the CTAB method³¹ . Purified DNA was used to perform our molecular approaches.

MIC determination was performed using the microplate alamar blue method, as described in the literature (21 (link)). The H37Rv strain (ATCC 27294) was used as a sensitive strain control. Firstly, the efflux pump inhibitor VP (MCE, China) was serially diluted twofold to a concentration ranging from 8 to 256 µg/mL, and its MIC was determined for all clinical isolates. Then the INH or RIF with Middlebrook 7H9 (Becton Dickinson Biosciences) medium containing 0.2% glycerol and 10% OADC (Hopebio, China) with or without one-half MIC of VP was serially diluted twofold to a concentration ranging from 0.001 to 128 µg/mL for INH) or from 0.001 to 256 µg/mL (for RIF), respectively, to determine the MICs of INH or RIF for all clinical isolates with or without the supplement of one-half MIC of VP. All tests were conducted twice. The MIC was defined as the lowest drug concentration which prevented a color change.