Pgoal19

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).

The putative resistance-associated rpoB mutations were amplified by PCR from the genomic DNA of the resistant strains as a template and then cloned into the ScaI site of the suicide plasmid pGOAL19 (Addgene; MA, United States) (Parish and Stoker, 2000 (link)). The primers used are shown in Supplementary Table 1. The resulting plasmids were transformed into M. tuberculosis H₃₇Rv, and point mutants were selected after two rounds of homologous recombination, as previously described (Parish and Stoker, 2000 (link); Larsen et al., 2007 ).