Schematic of experimental design.

<p>Schematic of experimental design.</p

Abundances of dominant phyla in samples.

<p>Abundances (% of total 16S rRNA sequences) of the predominant bacterial phyla in healthy control and IBS patient fecal sample aliquots exposed to room temperature and −80°C for different lengths of time.</p

OTUs most significantly altered between room temperature and −80°C storage for all samples and each individual.

<p>OTUs most significantly altered between room temperature and −80°C storage for all samples and each individual.</p

β-diversity analysis of samples.

<p>Principal coordinates analysis (PCoA) of weighted and unweighted UniFrac distances of IBS patient (green and orange triangles) and healthy control (blue squares and red circles) fecal sample aliquots exposed to room temperature and −80°C for different lengths of time (room temperature - 1, 4, 6, 8 and 24 hours; −80°C −1 week and 1, 2, 3, 4, 5 and 6 months). PCoA plots illustrate the subject each sample aliquot originated from (<b>A&E</b>) and the temperature they were stored at (<b>B&F</b>). Average weighted UniFrac distances for all sample aliquots based on storage at room temperature (<b>C&G</b>) or −80°C (<b>D&H</b>) indicate that sample aliquot microbiotas show significantly similarity (*<i>p</i><0.05).</p

Operational Taxonomic Unit (OTU) network analysis of bacterial communities from samples with high and low fecal protease (FP) activity.

<p>Nodes represent high FP activity samples (<i>n</i>=13, blue circles), low FP activity samples (<i>n</i>=13, yellow circles) samples, and OTUs (white circles). Edges (lines) connecting samples with high FP activity nodes (blue edges) or low FP activity nodes (yellow edges) to OTUs indicate whether a given OTU was found in that sample. The clustering of blue and yellow nodes and edges indicates that samples with high FP activity share numerous OTUs in common, and segregate from the shared OTUs between low FP activity samples. </p

Levels of protease activity in fecal supernatants from all subjects (healthy controls and IBS patients) that participated in this study.

<p>Top and bottom quartiles representing samples high and low FP activity, respectively, are indicated. </p

Bacterial community composition analysis between samples with high (<i>n</i>=13) and low (<i>n</i>=13) FP activity.

<p>Principal coordinates analysis (PCoA) plots of un-weighted and weighted UniFrac distances for samples with high (blue circles) and low (yellow squares) FP activity are shown. Analysis of similarity (ANOSIM) demonstrated a significant separation in the composition of fecal microbiotas between high and low FP activity samples using both un-weighted (<i>p</i>=0.001) and weighted (<i>p</i>=0.003) UniFrac distances. The R statistic (where R=1 and R=0 signifies differences and no differences between groups, respectively) is higher in the un-weighted analysis suggesting the separation between microbiotas is a result of both high and low abundances bacterial species.</p

Abundances of Family level taxa in samples with high (<i>n</i>=13) and low (<i>n</i>=13) FP activity.

<p>The composition and abundances of bacterial families differ between the microbiotas of fecal samples exhibiting high and low FP activity. </p

Microbial richness of samples with high (<i>n</i>=13, blue) and low (<i>n</i>=13, yellow) FP activity.

<p>Both the number of observed bacterial species (based on species-level OTUs) and Shannon index of diversity are significantly lower in fecal samples with high compared to low protease activity (<i>p</i>=0.002). Error bars represent the standard error. </p

Correlation of <i>Faecalibacterium prausnitzii</i> with FP activity.

<p><i>F. prausnitzii</i> exhibits a significant (<i>p</i>=0.01) negative correlation with FP activity. Blue and yellow circles indicate high and low FP activity samples used in previous analyses, respectively. </p