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BCG was grown in Middlebrook 7H9 broth (BD Biosciences, UK) with 50 μg/ml hygromycin (Merck Life Science UK Limited), 10% OADC enrichment (BD Biosciences, UK), 0.2% glycerol, and 0.05% tyloxapol (Sigma-Aldrich, UK), at 37°C with 200 rpm shaking