Air samples collected (n = 63).

<p>Air samples collected (n = 63).</p

NGS summary of air samples (n = 61).

<p>NGS summary of air samples (n = 61).</p

Longitudinal Metagenomic Analysis of Hospital Air Identifies Clinically Relevant Microbes

<div><p>We describe the sampling of sixty-three uncultured hospital air samples collected over a six-month period and analysis using shotgun metagenomic sequencing. Our primary goals were to determine the longitudinal metagenomic variability of this environment, identify and characterize genomes of potential pathogens and determine whether they are atypical to the hospital airborne metagenome. Air samples were collected from eight locations which included patient wards, the main lobby and outside. The resulting DNA libraries produced 972 million sequences representing 51 gigabases. Hierarchical clustering of samples by the most abundant 50 microbial orders generated three major nodes which primarily clustered by type of location. Because the indoor locations were longitudinally consistent, episodic relative increases in microbial genomic signatures related to the opportunistic pathogens <i>Aspergillus</i>, <i>Penicillium</i> and <i>Stenotrophomonas</i> were identified as outliers at specific locations. Further analysis of microbial reads specific for <i>Stenotrophomonas maltophilia</i> indicated homology to a sequenced multi-drug resistant clinical strain and we observed broad sequence coverage of resistance genes. We demonstrate that a shotgun metagenomic sequencing approach can be used to characterize the resistance determinants of pathogen genomes that are uncharacteristic for an otherwise consistent hospital air microbial metagenomic profile.</p></div

<i>S</i>. <i>maltophilia</i> K279a efflux pump operon read coverage depth.

<p>(A) smeSF-smeABC. (B) smeT-smeDEF. The x-axis labels indicate the position on the K279a genome. The white lines represent intergenic gaps and the black lines represent gene distinction.</p

Correction: Longitudinal Metagenomic Analysis of Hospital Air Identifies Clinically Relevant Microbes

<p>Correction: Longitudinal Metagenomic Analysis of Hospital Air Identifies Clinically Relevant Microbes</p

Enrichment workflow using McrB-N.

<p>(A) Biotinylated McrB-N enzyme is added to a DNA mixture. Following the addition of strepatavidin coated magnetic beads, the mixture is segregated into fractions that are bound (containing majority of human) or unbound (containing majority of microbes). (B) Sequence analysis demonstrates that McrB-N segregates human and rice DNA away from microbial genomes in the unbound fraction. The fold enrichment for each taxa is plotted for the unbound (blue) fraction.</p

Sequence analysis of HpaII mediated enrichment of a genomic mixture.

<p>The fold enrichment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146064#pone.0146064.e001" target="_blank">Eq 1</a>) for each Eukaryote, Prokaryote and virus genome is listed. The GC content of microbial genomes are plotted above.</p

HpaII mediated enrichment of DNA from a pooled sputum sample improves microbe sequencing detection and coverage.

<p>(A) The percent of microbial ID reads (blue) increases and human ID reads (red) decrease with enrichment. (B) Normalized microbial Order sequence Identification reads are plotted for bound and input samples. Greater than 95% of identified microbes have increased sequenced reads. Many microbes (red points) are only detectable after enrichment. (C) Comparison of ratio of microbial sequence ID reads in sputum input and sputum bound samples. (D) Genomic sequencing coverage of bacteria such as <i>P</i>. <i>aeruginosa</i> improves with enrichment. The input DNA sample coverage (red line) and HpaII bound coverage (blue line) are plotted across the genome position of <i>P</i>. <i>aeruginosa</i>.</p

<i>Y</i>. <i>pestis</i> genomic DNA is efficiently segregated from human DNA.

<p>(A) Recovery of decreasing levels of <i>Y</i>. <i>pestis</i> DNA (blue bars) from a fixed 1000 ng human DNA (green bars). (B) DNA recovery using 100 ng and 1000 ng <i>Y</i>. <i>pestis</i> DNA in a background of 1000 ng human DNA. (C) Recovery of a fixed 1 ng of <i>Y</i>. <i>pestis</i> DNA (blue bars) from increasing levels of human DNA (green bars).</p

Longitudinal sampling identifies changes in location profiles.

<p>Microbial metagenomic profiles representing the percent distribution of normalized (to 1 M) read counts for 26 microbial orders with >1% abundance aid in the identification of samples with unusual profiles (A) in lobby L2 between April 30 –May 5 and (B) in duct D2 between September 5–26. Sample numbers and collection dates are indicated. Color schemes represent orders grouped by class or phylum. “Other” is represented by the percent sum of the remaining 24 orders.</p