DNA extraction and 16S rRNA gene sequencing.
DNA extraction from sputum and oral wash samples, paralleled with experimental control samples, was performed using the Maxwell RSC whole blood DNA kit (Promega, USA). A sample of 750 μL was transferred to the Eppendorf tube and centrifuged for 5 min at 14,000 × The V3-V4 hypervariable region of the 16S rRNA gene of all samples was amplified by PCR according to the 16S Metagenomic Sequencing Library Preparation guide provided by Illumina (