Isoniazid

Mycobacterium tuberculosis H37Ra was grown at 37°C in Middlebrook 7H9 liquid medium or on 7H11 agar plates supplemented with 10% (v/v) albumin–dextrose–catalase (ADC, Becton Dickinson, Sparks, MD, United States) plus 0.5% (v/v) glycerol, 0.25% (v/v) Tween 80. Plasmids p2NIL and pGOAL19 used in this study were obtained from Addgene (Cambridge, MA, United States). isoniazid (INH), rifampicin (RIF), streptomycin (SM), ethambutol, pyrazinamide (PZA), levofloxacin, amikacin, cycloserine, p-aminosalicylic acid (PAS), clofazimine (CFZ), tetracycline, linezolid, clarithromycin, and piperine were purchased from Sigma-Aldrich (St Louis, MO, United States). The drugs were dissolved in DMSO and further diluted to obtain the desired concentrations in culture media for drug susceptibility testing (see below).

Isoniazid (INH), Rifampicin (RIF), Ethionamide (ETH), and Pretomanid (PMD) were purchased from Sigma; Delamanid (DMD) was procured from MedKoo Biosciences. All antibiotics were dissolved in DMSO with exception of INH, which when used for treatment of mice, was dissolved in H₂O.

50 μg/ml Hygromycin (Invitrogen), 20 μg/ml Kanamycin (GoldBio), 12.5 μg/ml zeocin (Invitrogen), 0.2% acetamide (Sigma), and 50 ng/ml of anhydrous tetracycline (ATc) (Sigma) were used unless otherwise indicated. H₂O₂, MMS, streptomycin, isoniazid, rifampicin, ciprofloxacin (all from Sigma) were used at the indicated concentrations.

Certified reference materials (>97% purity) of the drug analytes isoniazid, rifampicin, ethambutol, pyrazinamide, and bedaquiline were obtained from Sigma Aldrich. All solvents (methanol (MeOH), ethanol (EtOH), water (H₂O), isopropanol (IPA), acetonitrile (ACN) and formic acid (FA)) were Optima™ LC-MS grade, obtained from Fischer Scientific.
The culture media for the cell culture procedure was prepared as previously described in Wishart et al.⁴³ Dulbecco's modified Eagle's medium (DMEM) with high glucose (Sigma-Aldrich, Merck, UK) was supplemented with 10% fetal bovine serum (Fisher Scientific, Loughborough, UK), 1% penicillin/streptomycin (Fisher Scientific, UK), and 2 mM l-glutamine (Sigma-Aldrich, Merck UK).

Pentylenetetrazole, isoniazid, gamma aminobutyric acid, glutamate, diazepam (D-907), phenytoin (PHR1139), and vigabatrin were purchased from Sigma Ltd., USA. ELISA kit (MBS939900; MyBioSource, Inc., San Diego, CA, USA) was used for the estimation of GABA-T activity.

Middlebrook 7H11 plates were prepared to contain rifampin (Sigma; 0.1 μg/ml) or 0.2 μg/ml isoniazid (Sigma; 0.2 μg/ml). Phage lysate diluted to 10⁵ PFU in 0.1 ml was spread onto 7H11 plates with or without antibiotics and allowed to dry in a laminar flow biosafety cabinet; 0.1 ml of an M. tuberculosis H37Rv culture was then spread into plates and incubated for 6 weeks at 37°C.

Rifampicin (RMP), isoniazid (INH), ethambutol (EMB), streptomycin (SM), ofloxacin (OFX) and kanamycin (KAN) were purchased from Sigma. Racemic R,S-1′-acetoxychavicol acetate (rac-ACA) was purchased from LKT laboratories (St. Paul, MN, USA; Product Number A0817). The MICs were determined as described previously [16 (link)]. Briefly, all mycobacterial strains were prepared from scoops of solid culture and suspended in PBS plus Tween-80, to reach a concentration equivalent to McFarland Standard No. 1 (approximately 3 × 10⁷ Colony Forming Units (CFU)/mL) and then diluted by Middlebrook 7H9 medium supplemented with 10% OADC to about 10⁵ CFU/mL. One hundred microliters of the bacterial suspensions were added to microtiter plates containing, in each well, two-fold diluted drugs in 100 µL of Middlebrook 7H9 medium with 10% OADC. The volume of drug-free and bacteria-free wells was adjusted to 200 µL and used as the control. The plates were incubated at 37 °C for 5 days. Twenty microliters of Alamar Blue dye (Accumed International, Westlake, OH, USA) and 12.5 µL of 20% sterilized Tween 80 reagent were added. The plates were re-incubated at 37 °C for 24 h, and the colors of all wells were recorded. The MIC was defined as the lowest drug concentration that prevented a color change from blue to pink.