Laboratory Assays

DNA extractions were performed using the Qiagen EZ1 Advanced automated extraction platform (Qiagen Inc., Valencia, CA) with the Tissue Kit and Bacterial card per manufacturer's instructions. Total bacterial load (TBL) (gene copies/reaction) was estimated for each sample and negative control blanks using a TaqMAN assay [21,22]. Two PCR negative controls were included on the same TBL plate used for these samples, the blanks returned values of 0 and 36.6 copies/reaction. The higher value was used for comparison to the TBL values from the samples and corresponds to 6.3 log₁₀ copies/mL after multiplying by a 50,000 dilution factor. 16S Amplicon Library Construction: Bacterial profiles were determined by broad-range amplification and sequence analysis of 16S ribosomal RNA (rRNA) genes following our previously described methods [23,24]. Amplicons were generated using MiSeq compatible primers that target approximately 300 base pairs of the V1V2 variable region (27F/338R) of the 16S rRNA gene. A negative control reaction was run for each sample (unique paired barcode combination) to ensure unintended DNA input was not responsible for any amplification products observed. None of the negative control reactions resulted in a visible band on an agarose gel. Polymerase chain reaction (PCR) products were normalized using agarose gel densitometry, pooled, purified and concentrated using a DNA Clean and Concentrator Kit (Zymo, Irvine, CA). Pooled amplicons were quantified using Qubit Fluorometer 2.0 (Invitrogen, Carlsbad, CA). The pool was diluted to 4nM and denatured with 0.2 N NaOH at room temperature. The denatured DNA was diluted to 20pM and spiked with 10% of the Illumina PhiX control DNA prior to loading the sequencer. Illumina paired-end sequencing was performed on the Miseq platform using a 500 cycle version 2 reagent kit. Analysis of Illumina Paired-end Reads: Illumina Miseq paired-end reads were aligned to human reference genome Hg19 with bowtie2 and matching sequences discarded [25]. As previously described, the remaining non-human paired-end sequences were sorted by sample via barcodes in the paired reads with a python script [24]. Sorted paired end sequence data were deposited in the NCBI Short Read Archive under accession number SRP066906. The sorted paired reads were assembled using phrap [26,27]. Pairs that did not assemble were discarded. Assembled sequence ends were trimmed over a moving window of 5 nucleotides until average quality met or exceeded 20. Trimmed sequences with more than 1 ambiguity or shorter than 200 nt were discarded. Potential chimeras identified with Uchime (usearch6.0.203_i86linux32) [28] using the Schloss [29] Silva reference sequences were removed from subsequent analyses. Assembled sequences were aligned and classified with SINA (1.2.11) [30] using the 418,497 bacterial sequences in Silva 115NR99 [31] as reference configured to yield the Silva taxonomy. Operational taxonomic units (OTUs) were produced by clustering sequences with identical taxonomic assignments. This process generated 390,248 sequences for 11 samples (average sequence length: 316 nt; average sample size: 35,477 with a range 18,043 to 60,288 sequences). 225 taxa were identified across the 11 samples. The median Goods coverage score was ≥ 99.85% at the rarefaction point of 18,043. The software package Explicet (v2.10.5, www.explicet.org) was used for display and calculation of common sequencing measures (rarefied values for median Good’s coverage and Shannon alpha diversity) [32].

BALF neutrophil elastase was quantified by a spectrophotometric assay based on the hydrolysis of the specific substrate MeO-suc-Ala-Ala-Pro-Ala-p-nitroanilide (Sigma Chemical Co.; St. Louis, MO) and interleukin-8 concentration was measured as part of an eight-plex panel (EMD Millipore Corporation; Millerica, MA).