Septifast lys kit

The extraction of bacterial DNA was performed with the QIAamp Fast Stool DNA Mini Kit (Qiagen) after the mechanical disruption (speed 7,000 for 70 s) with the SeptiFast Lys Kit (Roche) on MagNA Lyser (Roche). The DNA concentration was verified using Quant-iT PicoGreen dsDNA Kit (Thermo Fisher Scientific). Bacterial community composition was determined by paired-end sequencing on an Illumina MiSeq platform, targeting the V3–V4 hypervariable region of the 16S rRNA gene. Library preparation was carried out using the 341F (5′-CCTACGGGNGGCWGCAG-3′) – 805R (5′-GACTACHVGGGTATCTAATCC-3′) set of primers. On the same run a template-free sample was included as a negative sequencing control.
Sequencing data was deposited at metagenomic analysis server MG-RAST (Meyer et al., 2008 (link)) and is accessible at https://www.mg-rast.org/linkin.cgi?project=mgp85501.

For the mechanical workup of the samples, two kits were used in the first step of the extraction process. For tissues, MagNA Lyser Green Beads (Roche, Basel, Switzerland) were used, while BALs and other fluids were processed with the SeptiFast Lys Kit (Roche). Subsequent DNA extraction from all clinical samples was performed with the High Pure PCR Template Preparation Kit (Roche). DNA was eluted in 100 µl elution buffer. Elution buffer was used as a reagent control.

After the individual colonies with lysis zones were picked, the entire plate was swabbed and resuspended in 1 mL of Inhibitex buffer (QIAGEN). For each individual sample and each cultivation approach (i.e., enriched/nonenriched, aerobic/anaerobic), swabs from all plated dilutions were pooled and stored at −80 °C for 16S rRNA amplicon sequencing.
From fecal samples and pellets of cultivated samples (saliva and feces), total DNA for 16S RNA sequencing was isolated using the QIAamp Fast DNA Stool Mini Kit (QIAGEN) according to the manufacturer’s instructions. Cells were lysed with SeptiFast Lys kit and MagNA Lyser (Roche Diagnostics, Mannheim, Germany). Sequencing of the bacterial 16S RNA metagenome (V3V4 variable region) was carried out as previously described [19 (link)]. Briefly, mothur (v.1.44.0) was used for quality filtering of sequence reads, as well as downstream analysis. Taxonomy was inferred using the RDP 16S rRNA reference base (Ribosomal Database Project, version 18). In total, we obtained 3,969,337 quality filtered reads, on average 30,533.4 per sample (SD = 11,475.3). We removed reads with an abundance of less than 0.001%, and each sample was subsampled to 10,000 reads.
Downstream statistics included analysis of alpha diversity (community richness and diversity) and beta diversity (AMOVA), all performed in mothur (v.1.44.0).