Middlebrook 7h9 medium

The in vitro antimycobacterial activity of the tested compounds was assessed according to the EUCAST broth microdilution reference method for MIC determination [78 (link)]. The M. tuberculosis H37Rv strain (ATCC 27294) was cultured at 37 °C in Loewenstein-Jensen medium until log phase growth; then, a cell suspension was prepared at a concentration of approximately 2 × 10⁶ cells/mL and further diluted 1:20 in Middlebrook 7H9 medium with 10% OADC (oleic acid-albumin-dextrose-catalase) (Becton Dickinson and Co., Sparks, MD, USA). Ninety-six-well microplates were used. The Middlebrook 7H9 medium was added dropwise at the appropriate concentration of the test compound (range of 0.25 to 32 mg/L) and M. tuberculosis suspension. Ethambutol and isoniazid were used as controls. Reading was performed after 7, 14, and 21 days of incubation at 37 °C using an inverted mirror. The MIC was the lowest concentration without visual growth.

From the logarithmic phase culture of M. tuberculosis H37Rv, 2 mL was withdrawn and adjusted to a concentration equivalent to the 0.5 McFarland Standard (approximately 1.5 × 10⁸ CFU/ml) in Middlebrook 7H9 medium (Becton Dickinson) using a nephelometer (Biomerieux Vitek, Hach Co, Loveland, United States). Serial dilutions were made until a concentration of 1.5 × 10⁴ CFU/mL was obtained. In a 24-well plate, 1 mL of 1.5 × 10⁴ CFU/mL M. tuberculosis H37Rv was added in triplicate. Some of the samples were cultured in the presence of simvastatin or simvastatin acid, while others were cultured without treatment or with the vehicle (0.024% DMSO and 0.056% EtOH in PBS). Simvastatin and simvastatin acid were added at the following concentrations: 0.1, 0.5, 1, 2, 5, 10, 15, and 20 μM. The plates were incubated at 37°C in a 7.5% CO₂ atmosphere for 96 h (Schaefer, 1957 (link)). To quantify the mycobacterial growth, serial dilutions were made. Ten microliter aliquots were obtained and inoculated onto Middlebrook 7H10 agar plates (Becton Dickinson) followed by incubation at 37°C in a 7.5% CO₂ atmosphere. The plates were examined, and the CFU/mL counts were collected on days 7, 14, and 21.

M. ulcerans 98–912 (ITM collection, Antwerp, Belgium) is a mycolactone D producing strain isolated in China from an ulcerative lesion [39 (link)]. M. ulcerans 1615 is a mycolactones A/B producing strain isolated in Malaysia from an ulcerative case. The isolates were grown on Middlebrook 7H9 medium (Becton, Dickinson and Company) with agar at 32°C for approximately 6–8 weeks. For the preparation of the inoculum, M. ulcerans was recovered, diluted in phosphate-buffered saline (PBS) and vortexed using glass beads.

M. smegmatis cultures were grown in Middlebrook 7H9 medium (Becton Dickinson, Sparks, MD) supplemented with ADS enrichment (Albumin-Dextrose Saline containing 5% (w/v) Bovine serum albumin fraction V, 2% (w/v) D-Dextrose and 8.1% (w/v) NaCl) (Jacobs et al., 1991 (link)), 0.05% (v/v) Tween 80, and 0.5% (v/v) glycerol (Beijing Modern Eastern Finechemical Co. Ltd, Beijing). Middlebrook 7H10 medium supplemented with ADS enrichment and 0.5% (v/v) glycerol was used as the solid medium for examination of growth status. Growth was also examined in minimal Sauton's medium (4 g asparagine, 2 g sodium citrate, 0.5 g K₂HPO₄·3H₂O, 0.5 g MgSO₄·7H₂O, 0.05 g ferric ammonium citrate, 60 g glycerol in 1 L of H₂O supplemented with 0.05% (v/v) Tween 80) supplemented with antibiotics as indicated. Hygromycin (75 mg/L for M. smegmatis, 150 mg/L for Escherichia coli; Roche) and kanamycin (25 mg/L for M. smegmatis, 50 mg/L for Escherichia coli; Amresco) were added to the medium as needed. All bacterial strains used in this study are listed in Table S1.

M. tuberculosis H37Rv (London Pride, ATCC 25,618) expressing DsRed was used for the primary screen^{17 (link)}. M. tuberculosis isolates containing SNPs within genes of interest were originally isolated from wild-type ATCC 25,618^{9 (link)–11 (link)}. M. tuberculosis was grown under aerobic conditions in Middlebrook 7H9 medium (Becton Dickinson) supplemented with 10% v/v OADC (oleic acid, albumin, dextrose, catalase), 0.05% w/v Tween 80 (7H9-Tw-OADC) or Middlebrook 7H10 agar supplemented with 10% v/v OADC.

Mtb H37Rv strain was grown at 37°C in Middlebrook 7H9 medium supplemented with 10% albumin-dextrose-catalase (both from Becton Dickinson, NJ, United States) and 0.05% Tween-80 (Sigma-Aldrich). The Mtb γ-irradiated H37Rv strain (NR-49098) and its total lipids´ preparation (NR-14837) were obtained from BEI Resource, Manassas, VA, USA. The RFP-expressing Mtb strain was gently provided by Dr. Fabiana Bigi (INTA, Castelar, Argentina).