Clarithromycin

A total of 2252 drugs/compounds comprising 1576 FDA-approved drugs and 676 compounds in clinical development were purchased from MedChemExpress. The compounds were dissolved in DMSO to a stock concentration of 10 mM. The clarithromycin resistance in M. abscessus was induced for 3 days under 0.5 mg/L of clarithromycin (Merck) before being subjected to compound screening. M. abscessus strain M422 was selected for the primary screening as it exhibited robust inducible clarithromycin resistance. After 3 days of incubation in an airtight container (Lock and Lock container) with moist paper towels at 37°C, the MIC of clarithromycin increased from 4 mg/L to >64 mg/L. MIC values were determined from dose–response curves using GraphPad Prism version 9 (Dotmatics).
Primary screening was carried out in 96-well flat-bottom cell culture plates (Corning Costar) at 50 µM compound along with 4 mg/L clarithromycin in a starting inoculum of preinduced M. abscessus cells at OD₆₀₀ of 0.005 in a final volume of 100 µL. The plates were put in an airtight container with moist paper towels and incubated for 4 days at 30°C. The cultures in the wells were resuspended before OD₆₀₀ was read in a BioTek Synergy 4 plate reader. Compounds were defined as hits if they showed growth inhibition >90% compared with the untreated control.

ECs were exposed in vitro to septic stimulation for 24 h, by an association of proinflammatory cytokines increased during sepsis: Interferon γ (IFN‐γ) 15 ng/ml (R&D Systems), TNF‐α 5 ng/ml (Peprotech) and lipopolysaccharide (LPS) 100 ng/ml extracted from Escherichia coli. After 24 h of septic stimulation, ECs were incubated with macrolides: Spiramycin, Erythromycin, or Clarithromycin (Merck) at the indicated concentrations for another 24 h. Parallel cultures were carried out with the relevant diluent before phenotypic, quantitative polymerase chain reaction (qPCR), and functional assays.

The target pharmaceutical compounds in this study were carbamazepine, ibuprofen, ciprofloxacin, sulfamethoxazole, erythromycin and clarithromycin, and were purchased from Merck Millipore (Gauteng, South Africa). The target compounds were selected based on global use. The standard solution (1000 µg/mL) of the target compounds were prepared by dissolving the weighed standard powder into methanol (High Performance Liquid Chromatography gradient-grade) purchased from Merck Millipore (Gauteng, South Africa). The structure and molecular weight of the target pharmaceuticals are shown in Table 2.

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).

M. avium subsp. hominissuis 109 (14 (link)) was provided by Petros Karakousis (Johns Hopkins University). M. abscessus subsp. abscessus ATCC 19977 (15 (link)) was purchased from the American Type Culture Collection.
Strains were grown in complete Middlebrook 7H9 broth (BD Difco) supplemented with 0.05% Tween 80, 0.2% glycerol, and 10% albumin-dextrose-catalase with orbital shaking at 90 rpm (INFORS HT Multitron). For determination of CFU, bacterial cultures were spread onto Middlebrook 7H10 agar (BD Difco) supplemented with 10% (vol/vol) Middlebrook oleic acid-albumin-dextrose-catalase and 0.5% glycerol and grown at 37°C for 3 weeks (M. avium) or 1 week (M. abscessus). When appropriate, agar was supplemented with SPR719 or Clarithromycin for isolation of resistant mutants. SPR719 was obtained from Spero Therapeutics. Clarithromycin and Moxifloxacin were purchased from Sigma-Aldrich, USA.