7h11 agar

The Mtb H37Rv strain (obtained from C. Sassetti) was grown at 37 °C in a minimal medium (Difco Middlebrook 7H9 broth (BD, 271310) supplemented with 0.5% (v/v) glycerol, 0.05% (v/v) tyloxapol (Sigma, T8761), 0.2 g l⁻¹ casamino acids (BD, 223050), and 10% (v/v) OADC (oleic acid, albumin, dextrose and catalase; BD, 212351)). The double-auxotrophic Mtb mc²6206 strain (H37Rv ΔpanCDΔleuCD)^{42 (link)} (obtained from W. Jacobs Jr) was grown in the minimal medium with an additional 50 mg l⁻¹l-leucine (Sigma, L8000) and 24 mg l⁻¹ pantothenic acid (Sigma, P5155). The Msm mc²155 strain (obtained from S. Fortune) was grown in the Middlebrook 7H9 medium supplemented with 0.2% (v/v) glycerol, 0.05% (v/v) Tween-80 (VWR, M126), and 10% (v/v) albumin–dextrose–catalase. Liquid Mtb and Msm cultures were grown at 37 °C in Nalgene sterile square PETG medium bottles with constant agitation. The solid Mtb culture was grown on 7H11 agar (Sigma, M0428) supplemented as described above except for tyloxapol.

The M. avium ATCC 25291 strain was cultured in 7H9 medium (Hi Media, Santa Maria, CA, USA) supplemented with albumin dextrose complex (ADC) (GeminiBio, West Sacramento, CA, USA) and incubated at 37 °C and grown until reaching the logarithmic growth phase (indicated by an optical density between 0.5 and 0.8 at A600). M. avium cultures were centrifuged at 4000 rpm to form a pellet. The supernatant was discarded, and the bacterial pellet was washed with PBS (Sigma, St Louis, MO, USA) and subsequently disaggregated by vortexing five times with 3 mm sterile glass beads at 3 min intervals. M. avium suspension was then filtered using a 5 μm syringe filter to remove any remaining bacterial aggregations. The single-cell suspension of processed M. avium was serially diluted and plated on 7H11 agar (Sigma, St Louis, MO, USA) to determine the bacterial numbers present in the processed stock. Aliquots of processed bacterial stocks were frozen and stored in a cryogenic freezer at −80 °C. The frozen processed stocks of M. avium were thawed and used at the time of the experimental study.