Characterization of lung microbiome in autopsied infants and its interaction with Pneumocystis colonization.
A total of 54 infants were selected on the basis of unexpected death were included in this study. We compared the bacterial and fungal composition of lung tissue from infants highly, moderately and not-colonized by Pneumocystis, according to their Ct values.For each autopsied subject, the right upper lobe was removed, small samples were obtained from deep lung tissue, cut into small pieces, divided in five aliquots (0.4 g) and frozen at -80ºC for DNA extraction. A DNA extraction protocol was applied on each piece o sample: constisting of a pre-treatment step followed by the bead-beating and phenol-chloroform steps.Pneumocystis-colonized samples were identified using a nested-PCR and qPCR procedures.For all samples, the 16S rRNA gene analysis of the bacterial community, and the internal transcribed spacer (ITS) region for fungal community were carried out, respectively. PCR amplification of 469  bp of the V3-V4 regions in the case of bacteria, and  spanning about 289 bp of the small subunit and the 5.8S region of the rRNA operon in the case of fungi were performed and sequencing of the amplicons using the Illumina MiSeq with 300 bp x2 chemistry.