Bead beating device

For isolation of bacterial genomic DNA, inactivated culture material (from Lowenstein-Jensen slants) was resuspended in 1 ml TET buffer (Tris, ETDA, Tween 20) and filtered through a 5-μm-pore-size filter (Omnifix Braun, Melsungen, Germany) to obtain single cells. The bacterial solution was washed twice with TET buffer, centrifuged at 5,000 × g for 5 min, and, finally, adjusted to an optical density of 0.01 in TET buffer. Subsequently, 400 μl of the bacterial solution was disrupted using a bead beating device (VWR, Erlangen, Germany) and 300 mg of glass beads (<106 μm; Sigma-Aldrich, St. Louis, MO, USA), which were centrifuged at 5,000 × g for 80 s. The cell lysate obtained was centrifuged at 5,000 × g for 10 s, and the supernatant was filtered through a 10-μm-pore-size filter (Mobitec, Eupen, Belgium) and centrifuged at 5,000 × g for 1 min. The genomic DNA was extracted from 100 μl of this cell lysate using a QIAamp blood minikit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. DNA quantification was performed using a Quant-iT PicoGreen double-stranded DNA kit (Invitrogen, Carlsbad, CA, USA). Finally, the DNA sequences of the MBTC strains were determined by GATC Biotech AG (Constance, Germany) and analyzed using GENtle software (http://gentle.magnusmanske.de).

To isolate the bacterial genomic DNA of the inactivated culture material the cell suspension was filtered through 5 μm filter pore size (Omnifix Braun, Melsungen, Germany) to obtain single cells. Then the bacteria solution was washed two times with TET buffer (10 mM Tris-HCl, 1 mM EDTA·Na₂, 0.05% Tween 80), centrifuged at 5,000 × g for 5 min and finally adjusted to an optical density of 0.01 in TET buffer. 400 μL of the bacteria cells were disrupted applying a bead beating device (VWR, Erlangen, Germany) and 300 mg of <106 μm glass beads (Sigma Aldrich, St. Louis, USA) at 5,000 × g for 80 s. The obtained cell lysate was centrifuged at 5,000 × g for 10 s. The supernatant was filtered through a 10 μm pore size filter (Mobitec, Eupen, Belgium) and centrifuged at 5,000 × g for 1 min. Subsequently the genomic DNA was extracted from 100 μL cell lysate using the QIAamp Blood Mini Kit (Qiagen, Hilden, Germany) according to manufactures instructions. The isolated DNA was quantified using the Quant-iT PicoGreen dsDNA Kit (Invitrogen, Carlsbad, USA). M. tuberculosis target-specific amplicon were sequenced by GATC Biotech AG (Konstanz, Germany) and analyzed using the GENtle software (http://gentle.magnusmanske.de).