Volcano plots showing differentially expressed genes following incubation of primary mesothelial cells with <i>S</i>. <i>epidermidis</i> isolates for 1 hour.

<p>Volcano plots showing differentially regulated genes (adj. <i>p</i>-value < 0.05) following exposure of primary mesothelial cells to <i>S</i>. <i>epidermidis</i> ATCC^® 14990 (A) or <i>S</i>. <i>epidermidis</i> ATCC^® 12228 (B). A positive Log Fold Change indicates up-regulation; a negative Log Fold Change indicates down-regulation. The Log Odds (B value) is the log of the probability that a gene is differentially expressed. A Log Odds value of 0 corresponds to a 50–50 chance that the gene is differentially expressed.</p

Analysis of early mesothelial cell responses to <i>Staphylococcus epidermidis</i> isolated from patients with peritoneal dialysis-associated peritonitis

<div><p>The major complication of peritoneal dialysis (PD) is the development of peritonitis, an infection within the abdominal cavity, primarily caused by bacteria. PD peritonitis is associated with significant morbidity, mortality and health care costs. <i>Staphylococcus epidermidis</i> is the most frequently isolated cause of PD-associated peritonitis. Mesothelial cells are integral to the host response to peritonitis, and subsequent clinical outcomes, yet the effects of infection on mesothelial cells are not well characterised. We systematically investigated the early mesothelial cell response to clinical and reference isolates of <i>S</i>. <i>epidermidis</i> using primary mesothelial cells and the mesothelial cell line Met-5A. Using an unbiased whole genome microarray, followed by a targeted panel of genes known to be involved in the human antibacterial response, we identified 38 differentially regulated genes (adj. <i>p</i>-value < 0.05) representing 35 canonical pathways after 1 hour exposure to <i>S</i>. <i>epidermidis</i>. The top 3 canonical pathways were TNFR2 signaling, IL-17A signaling, and TNFR1 signaling (adj. <i>p</i>-values of 0.0012, 0.0012 and 0.0019, respectively). Subsequent qPCR validation confirmed significant differences in gene expression in a number of genes not previously described in mesothelial cell responses to infection, with heterogeneity observed between clinical isolates of <i>S</i>. <i>epidermidis</i>, and between Met-5A and primary mesothelial cells. Heterogeneity between different <i>S</i>. <i>epidermidis</i> isolates suggests that specific virulence factors may play critical roles in influencing outcomes from peritonitis. This study provides new insights into early mesothelial cell responses to infection with <i>S</i>. <i>epidermidis</i>, and confirms the importance of validating findings in primary mesothelial cells.</p></div

TNF is an upstream regulator of ten of the differentially regulated genes.

<p>The connections between nodes represent direct (solid lines) and indirect (dashed lines) relationships between genes, as supported by information in the IPA database. Up-regulated genes are shaded red, and down-regulated genes are shaded blue, with the intensity of the colour indicative of the magnitude of regulation. Feedback loops indicate auto regulation.</p

Changes in mesothelial cell gene expression in response to <i>S</i>. <i>epidermidis</i>.

<p>Confluent Met-5A or primary mesothelial cells were exposed to 10⁷ cfu/mL isolates of <i>S</i>. <i>epidermidis</i> or 10 μg/mL lipoteichoic acid (LTA) for 1 hour. Changes in gene expression were analysed using the RT² human antibacterial response PCR array. 36 of the 84 genes on the RT² panel were differentially regulated (>2-fold) in ≥2 <i>S</i>. <i>epidermidis</i> isolates and are shown grouped by category.</p

Viability of Met-5A and primary mesothelial cells exposed to <i>S</i>. <i>epidermidis</i> and lipoteichoic acid.

<p>Confluent Met-5A mesothelial cells were exposed to 10⁷ cfu/mL of two <i>S</i>. <i>epidermidis</i> reference isolates (ATCC^® 14990 and ATCC^® 12228), five <i>S</i>. <i>epidermidis</i> clinical isolates from PD peritonitis patients (C015, C016, C017, C018, C019) and 10 μg/mL lipoteichoic acid (LTA) for 1 hour at 37°C. Confluent primary mesothelial cells were exposed to 10⁷ cfu/mL of the clinical <i>S</i>. <i>epidermidis</i> isolate C016. Viability was determined using flow cytometry and a LIVE/DEAD^® Fixable Near-IR Dead Cell Stain, and data reported as the mean percentage of cell death across a minimum of biological triplicates (error bars are standard deviation). There was no statistically significant (<i>p</i> < 0.05) difference in the percent of cell death between any of the samples and the media control.</p

Primary mesothelial cell genes significantly up-regulated by <i>S</i>. <i>epidermidis</i> at 1 hour.

<p>Primary mesothelial cell genes significantly up-regulated by <i>S</i>. <i>epidermidis</i> at 1 hour.</p

Canonical pathways represented by the differentially expressed genes following incubation of primary mesothelial cells with <i>S</i>. <i>epidermidis</i> for 1 hour.

<p>28 differentially expressed genes identified by microarray following incubation of <i>S</i>. <i>epidermidis</i> with primary mesothelial cells were analysed using Ingenuity Pathway Analysis (IPA) and 35 canonical pathways were represented in our dataset. The top 15 canonical pathways are shown above, with the full list of canonical pathways shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178151#pone.0178151.s001" target="_blank">S1 Table</a>. A -log(B-H <i>p</i>-value) (shown in gold) of >2 represents data with an adjusted <i>p</i>-value < 0.01 (threshold for significance shown as a vertical line at 2.00). The ratio (shown in purple) indicates the proportion of differentially expressed genes relative to the total number of genes in each pathway.</p

Flow chart demonstrating the experimental approach.

<p>Experimental steps are shown in dark grey and analysis and experimental questions are shown in light grey. IPA = Ingenuity Pathway Analysis.</p

Primary mesothelial cell genes significantly down-regulated by <i>S</i>. <i>epidermidis</i> at 1 hour.

<p>Primary mesothelial cell genes significantly down-regulated by <i>S</i>. <i>epidermidis</i> at 1 hour.</p

tPA and DNase do not influence antimicrobial activity.

<p>The effect of tPA and DNase on antimicrobial activity was assessed by measuring the survival of bacteria (with known sensitivity) to the antimicrobial agents over time in the presence or absence of tPA and DNase. All assessments were performed in triplicate and bacterial counts were calculated through spot counting of serial dilutions of bacteria. Data show is the mean number of cfu/mL at 6 and 24 hours as a % of baseline (error bars are the standard deviation). A = Amoxicillin, C = Cefazolin, V&G = Vancomycin and Gentamicin.</p