Middlebrook 7h11 agar

Mycobacteria from positive MGIT cultures were first identified as NTM by Genotype CM (Bruker, Germany) and then identified as M. chimaera by Genotype NTM-DR (Bruker, Germany). Positive MGIT were subcultured in Middlebrook 7H11 Agar (7H11 plate, Becton Dickinson, USA) for approximately 2 weeks, in order to obtain cultures of “pure” M. chimaera. All the isolated M. chimaera strains were stored at −20°C using Microbank cryovial (Pro-Lab Diagnostics, Canada), after further identification by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry using MALDI Biotyper system (Bruker, Germany).
As requested by the Italian Ministry of Health as part of the national surveillance program, unfrozen M. chimaera strains were subcultured in a new MGIT and sent to the National Institute from Infectious Diseases “L. Spallanzani” (INMI) to carry out molecular epidemiological investigation. Subsequent DNA extraction, WGS, and bioinformatic analysis were then performed at INMI.

M. tuberculosis H37Rv (American Type Culture Collection, #27294) or its derivative expressing pGFPHYG2 replicative plasmid (Addgene #30173; deposited by Lalita Ramakrishnan; Cosma et al., 2006 (link)) was grown in Middlebrook 7H9 broth (Becton Dickinson) supplemented with albumin-dextrose-catalase enrichment (Becton Dickinson), 0.2% glycerol, 0.05% Tween 80, or on Middlebrook 7H11 agar (Becton Dickinson) containing 10% v/v oleic acid-albumin-dextrose-catalase enrichment (Becton Dickinson) and 0.2% glycerol. Ten milligrams of FX11 (Merck Millipore) were dissolved in 1 ml of DMSO.

Lungs and spleen were removed aseptically and homogenised in phosphate buffer saline (PBS). Aliquots (0.1 mL) of homogenates (undiluted and serial tenfold dilutions) were plated on Middlebrook 7H11 agar (Becton–Dickinson, USA) enriched with 10% oleic acid-albumin-dextrose-catalase (OADC) (Difco-Becton Dickinson, USA). 4 mg/ml of 2-thiophenecarboxylic acid hydrazide (TCH) (Sigma Aldrich) was added in 7H11 agar media to select for M. tuberculosis^{45 (link)}. Plates were incubated at 37 °C in a 5% CO₂ environment and colony forming unit (CFU) counts were determined after 28 days of incubation.

Mycobacterium bovis AF2122/97, a fully virulent strain isolated from lung caseous lesions of a tuberculin test reactor cow in Great Britain in 1997, has been entirely sequenced and its genome annotation recently updated and is the reference model for M. bovis for bovine TB investigation [26 (link), 27 (link)]. Thus, the benchmark AF2122/97 strain was used for our intranasal infections of mice as previously described [28 (link)]. The bacteria were originally grown in 7H9 medium supplemented with 4.16 g/L pyruvic acid (Sigma), 10% v/v ADC (Becton–Dickinson) and 0.05% v/v Tween 80 (Sigma). Bacteria were harvested at mid-exponential growth phase and frozen at −80 °C until use. CFU were counted after plating dilutions on Middlebrook 7H11 agar (Becton–Dickinson) supplemented with 4.16 g/L pyruvic acid (Sigma) and with OADC (ADC supplemented with 0.05% oleic acid, Becton–Dickinson).

M. smegmatis strains were routinely cultured at 37 °C in Middlebrook 7H9 broth (Becton–Dickinson, Franklin Lakes, NJ, USA) supplemented with albumin-dextrose-catalase, 0.2% glycerol, and 0.05% Tween 80 with shaking at 130 rpm, or on Middlebrook 7H11 agar (Becton–Dickinson) plates with oleic acid-albumin-dextrose-catalase enrichment and 0.5% glycerol. Unless otherwise stated, 7H9 and 7H11 media contained the abovementioned supplements in any other experiments. Escherichia coli DH5α, which was used for plasmid manipulation, was cultured in Luria–Bertani broth or agar plates.

M. tuberculosis H37Rv (American Type Culture Collection, #27294) was grown in Middlebrook 7H9 broth (Becton Dickinson) supplemented with albumin-dextrose-catalase enrichment (Becton Dickinson), 0.2% glycerol, 0.05% Tween 80 or on Middlebrook 7H11 agar (Becton Dickinson) containing 10% v/v oleic acid-albumin-dextrose-catalase enrichment (Becton Dickinson) and 0.2% glycerol. Infection stocks were prepared from mid-log phase cultures. For c.f.u. determinations, serial dilutions were performed in PBS/0.05% Tween 80 and plated onto Middlebrook 7H11 agar. Plates were incubated at 37 °C for 3–4 weeks prior to c.f.u. counting.

The MF GZ001 clinical strain was collected from the sputum of a 45-year-old male patient with a pulmonary infection. The patient with the symptoms of non-paroxysmal irritant cough with yellow and white sputum, chest pain, shortness of breath, headache, and low fever was admitted to the Guangzhou Chest Hospital, Guangzhou, China. The isolate was grown on Middlebrook 7H11 agar (Becton, Dickinson, and Company) supplemented with 0.2% glycerol (Shanghai Macklin Biochemical, Shanghai, China) and 10% oleic acid-albumin-dextrose-catalase (OADC) and in Middlebrook 7H9 (Difco, Becton, Dickinson and Company, New Jersey, USA) broth medium supplemented with 10% OADC, (Difco) and 0.05% Tween-80 (Amresco, USA).