106 μm glass beads

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).

Samples were transferred to a 2 mL tube containing 200 mg of ≤106 μm glass beads (Sigma, St. Louis, MO) and 0.3 mL of Qiagen ATL buffer (Valencia, CA), supplemented with 20 mg/mL lysozyme (Thermo Fisher Scientific, Grand Island, NY). The suspension was incubated at 37°C for 1 h with occasional agitation. Subsequently the suspension was supplemented with 600IU of Qiagen proteinase K and incubated at 60°C for 1 h. Finally, 0.3 mL of Qiagen AL buffer was added and a final incubation at 70°C for 10 minutes was carried out. Bead beating was then employed for 3 minutes in a Qiagen TissueLyser II at 30Hz. After a brief centrifugation, supernatants were aspirated and transferred to a new tube containing 0.3 mL of ethanol. DNA was purified using a standard on-column purification method with Qiagen buffers AW1 and AW2 as washing agents and eluted in 10mM Tris (pH 8.0).

We performed whole genome shotgun (WGS) sequencing of bacterial DNA extracted from vaginal swabs to quantify relative abundance and diversity of bacterial taxa. Vaginal swab samples were transferred to a 2 mL tube containing 200 mg of ≤ 106 μm glass beads (Sigma, St. Louis, MO) and 0.3 mL of Qiagen ATL buffer (Valencia, CA), supplemented with 20 mg/mL lysozyme (Thermo Fisher Scientific, Grand Island, NY). The suspension was incubated at 37°C for 1 hour with occasional agitation. Subsequently, the suspension was supplemented with 600 IU of Qiagen proteinase K and incubated at 60°C for 1 h. Finally, 0.3 mL of Qiagen AL buffer was added and a final incubation at 70°C for 10 minutes was carried out. Bead beating was then employed for 3 minutes in a Qiagen TissueLyser II at 30 Hz. After a brief centrifugation, supernatants were aspirated and transferred to a new tube containing 0.3 mL of ethanol. DNA was purified using a standard on-column purification method with Qiagen buffers AW1 and AW2 as washing agents, and eluted in 10mM Tris (pH 8.0).