Mycobacterial Culture and Antibiotic Resistance

Mtb H37Rv (ATCC 27294) and M. bovis BCG (ATCC 35734) strains were maintained in complete Middlebrook 7H9 medium (BD Difco) supplemented with 0.05% (vol/vol) Tween 80, 0.5% (vol/vol) glycerol, 0.5% albumin, 0.2% glucose, 0.085% sodium chloride, and 0.0003% catalase at 310 K with agitation at 80 rpm. POA-resistant strain Mtb H37Rv POA^R18 (clpC1: A625G/Lys209Glu) was isolated and described previously⁷. The Mtb H37Rv ΔprcAB strain (hygromycin-resistant) and its complemented mutant (hygromycin-resistant and Kanamycin-resistant) were previously generated and described^{18 (link)}. Hygromycin B (Roche) and Kanamycin (Sigma-Aldrich) were used for selection when required at 50 and 25 µg/mL, respectively. PZA, POA, and Isoniazid were purchased from Sigma-Aldrich. Bortezomib was purchased from Chembridge. Antibiotics were dissolved in 90% DMSO and sterilized using 0.2 μm PTFE membrane filters (Acrodisc PALL).

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Gopal P., Sarathy J.P., Yee M., Ragunathan P., Shin J., Bhushan S., Zhu J., Akopian T., Kandror O., Lim T.K., Gengenbacher M., Lin Q., Rubin E.J., Grüber G, & Dick T. (2020). Pyrazinamide triggers degradation of its target aspartate decarboxylase. Nature Communications, 11, 1661.

Publication 2020

Hygromycin B Kanamycin Isoniazid

Albumin Antibiotics Bortezomib Catalase Dmso Glucose Glycerol Hygromycin Hygromycin b Isoniazid Kanamycin Membrane Ptfe Sodium chloride Tween 80

Corresponding Organization :

Other organizations : National University of Singapore, Nanyang Technological University, Harvard University, Center for Discovery, Hackensack Meridian Health

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Variable analysis

independent variables

Mtb H37Rv (ATCC 27294) strain
M. bovis BCG (ATCC 35734) strain
POA-resistant strain Mtb H37Rv POA^R18 (clpC1: A625G/Lys209Glu)
Mtb H37Rv ΔprcAB strain (hygromycin-resistant)
Mtb H37Rv ΔprcAB complemented mutant (hygromycin-resistant and Kanamycin-resistant)

dependent variables

Growth and maintenance of bacterial strains

control variables

Complete Middlebrook 7H9 medium (BD Difco) supplemented with 0.05% (vol/vol) Tween 80, 0.5% (vol/vol) glycerol, 0.5% albumin, 0.2% glucose, 0.085% sodium chloride, and 0.0003% catalase
Incubation conditions: 310 K with agitation at 80 rpm
Hygromycin B used for selection at 50 μg/mL
Kanamycin used for selection at 25 μg/mL
PZA, POA, and Isoniazid dissolved in 90% DMSO and sterilized using 0.2 μm PTFE membrane filters
Bortezomib dissolved in 90% DMSO and sterilized using 0.2 μm PTFE membrane filters

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