Potassium clavulanate cla

Eight Mtb clinical strains and the reference strain H37Rv were used in this study (Table S1). Antimycobacterial drug susceptibility testing under standardized guidelines, in silico lineage determination, and spoligotyping were previously performed for the clinical strains at the Portuguese National Institute of Health (10 (link)). Screening for bedaquiline and linezolid susceptibility was not part of standard routine during the timeframe the strains were tested. Regarding antimycobacterial drug resistance profile, clinical strains were classified according to the most recent definitions by WHO as susceptible, MDR (resistance to both rifampicin and INH), or pre-extensively drug-resistant (pre-XDR; resistance to rifampicin and any fluoroquinolone) (22 ). Bacteria were grown in Middlebrook 7H9 medium (BD Biosciences) or in Middlebrook 7H10 medium (BD Biosciences), supplemented with 0.2% or 0.5% of glycerol, respectively. Both media were supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC) (BD Difco), and tyloxapol (Sigma-Aldrich) was added to liquid medium to a final concentration of 0.05%. Stocks of amoxicillin (AMX), EMB, meropenem (MEM), and vancomycin (VAN) (Sigma-Aldrich) were prepared in purified water. Potassium clavulanate (CLA) (Sigma-Aldrich) was prepared in phosphate buffer pH 6.0, 0.1 M, and INH was prepared in dimethyl sulfoxide (DMSO; PanReac AppliChem).

Mtb H37Rv, Msm mc²-155 and Msm PM965 (ept-1 rpsL4 ΔblaS1), a mutant strain deficient for the major beta-lactamase BlaS (Raymond et al., 2005 (link)), were used in this study. Bacteria were grown in Middlebrook 7H9 medium (BD Biosciences) or in Middlebrook 7H10 medium (BD Biosciences), supplemented with 0.2% or 0.5% of glycerol, respectively. Both media were supplemented with 10% oleic acid-albumin-dextrose-catalase (BD Difco) for Mtb and 0.5% glucose for Msm. Tyloxapol (Sigma-Aldrich) was added to liquid medium to a final concentration of 0.05% to prevent clump formation. Stocks of amoxicillin (AMX), biapenem (BIA), cefotaxime (CTX), doripenem (DOR), ertapenem (ETP), ethambutol (EMB), meropenem (MEM) and vancomycin (VAN) (Sigma-Aldrich) were prepared in purified water. Potassium clavulanate (CLA) (Sigma-Aldrich) was prepared in phosphate buffer pH 6.0, 0.1 M. Isoniazid (INH) and rifampicin (RIF) (Sigma-Aldrich) were prepared in dimethyl sulfoxide or methanol, respectively.

The bacterial strains, plasmids and cell lines used in this study are listed in Table 1. To amplify the CRISPRi backbone PLJR962 (Addgene #115162) and for cloning, E. coli was cultured in Luria-Bertani medium (Merck), supplemented with 25 μg/mL of kanamycin (NZYTech) when appropriate. M. smegmatis strains were cultured in Middlebrook 7H9 broth (BD™ Biosciences) supplemented with 0.2% glycerol (ThermoScientific), 0.5% glucose (Sigma-Aldrich) and 0.05% tyloxapol (Sigma-Aldrich) or 7H10 agar (BD™ Biosciences) supplemented with 0.5% glycerol. When necessary, the medium was supplemented with 25 μg/mL of kanamycin. Bacteria were grown at 37°C with (broth) or without (agar) shaking at 180 rpm. To induce sgRNA and dCas9_Sth1 expression, 100 ng/mL of ATc were added to mycobacterial cultures every 24 h. The J774.A1 cells were cultured in DMEM media (Gibco) supplemented with 10% fetal bovine serum, 1% sodium pyruvate, 1% L-Glutamine, and 1% HEPES buffer. The growth of J774.A1 cells occurred at 37°C, with 5% CO₂. Stocks of amoxicillin (AMX), cefotaxime (CTX), ethambutol (EMB), meropenem (MEM) and isoniazid (INH) (Sigma-Aldrich) were prepared with purified MilliQ water. ATc (Sigma-Aldrich) was prepared at a stock concentration of 10 mg/mL in dimethyl sulfoxide (DMSO) cell culture grade (AppliChem). Potassium clavulanate (CLA) (Sigma-Aldrich) was prepared in phosphate buffer pH 6.0, 0.1 M.