2.2 16S rRNA and ITS2 amplification and sequencing
Total DNA was extracted from 200 μL of lysate using Cador Pathogen 96 QIAcube HT Kit (Qiagen, Hilden, North Rhine-Westphalia, Germany) according to the instructions provided by the manufacturer. The V3-V4 region of bacterial 16S rRNA was amplified using the primers Pro341F (5′-CCTACGGGNBGCASCAG -3′) and Pro805R (Rev 5′-GACTACNVGGGTATCTAATCC -3′)(26). ITS2 region of the fungal ribosomal small subunit RNA was amplified using the primers ITS3f (5′- GCATCGATGAAGAACGCAGC -3′) and ITS4r (5′- TCCTCCGCTTATTGATATGC -3′). PCR was performed with the following conditions: 94°C for 1 minute, 35 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 68°C for 45 seconds, followed by final extension at 68°C for 7 minutes. PCR products were analyzed by electrophoresis on 1.5% agarose gel. Negative controls (PCR reagents without template DNA and template from blank swabs) were included to verify the possible presence of contaminating DNA. The PCR-amplified amplicons were purified using Agencourt AMPure XP kit (Beckman Coulter, Inc., Brea, California, USA), quantified with a Qubit 2.0 instrument (Thermofisher, Waltham, Massachusetts, US) and sequenced with the Illumina MiSeq platform (MiSeq ver. 3, 600 cycles, Illumina, Inc., San Diego, California, USA) to generate paired-end reads with a nominal length of 300 bp in each direction.