Time-kill kinetics of SPI031 against <i>S</i>. <i>aureus</i> and <i>P</i>. <i>aeruginosa</i>.

<p>(A) Concentration-dependent killing of <i>S</i>. <i>aureus</i> by SPI031 and vancomycin (VAN). (B) Concentration-dependent killing of <i>P</i>. <i>aeruginosa</i> by SPI031 and polymyxin B (PMB). All data represent means ± standard error of the mean (SEM) from 3 independent experiments (*p < 0.05; **p < 0.01; ***p < 0.001). The black dotted lines indicate the lower limit of detection.</p

Integrity of <i>S</i>. <i>aureus</i> liposomes after treatment with SPI031 at 1x MIC and 4x MIC.

<p>SDS (5%) and tetracycline (TET) (4x MIC) were used as positive and negative controls, respectively. Means and standard deviation (SD) of 3 independent experiments are shown (*p < 0.05; **p < 0.01; ***p < 0.001 compared to treatment with tetracycline).</p

Structure of compound SPI031.

<p>Structure of compound SPI031.</p

Microscopic analysis of the cell membrane after treatment with SPI031.

<p>Fluorescent images of <i>S</i>. <i>aureus</i> cells (upper row) and <i>P</i>. <i>aeruginosa</i> cells (lower row) stained with FM 4–64 in the absence or presence of 1x MIC of SPI031. Scale bar corresponds to 2 μm. Images were processed with unsharp mask of Zen 2.0.</p

Effect of SPI031 on membrane permeability.

<p>(A) Effect of increasing concentrations of SPI031 on the membrane permeability of <i>S</i>. <i>aureus</i>, monitored by the uptake of SYTOX green. Cells treated with melittin (MEL) (1x MIC) served as a positive control. (B) Inner membrane permeabilization of <i>P</i>. <i>aeruginosa</i> after treatment with different concentrations of SPI031, determined by measuring SYTOX green uptake. Melittin (MEL) (1x MIC) was used as a positive control. (C) Outer membrane permeabilization of <i>P</i>. <i>aeruginosa</i> after treatment with different concentrations of SPI031, assessed by quantifying NPN uptake. Cells treated with polymyxin B (PMB) (1x MIC) were used as a positive control. Data represent the means of three independent replicates ± SEM (*p < 0.05; **p < 0.01; ***p < 0.001 compared to untreated control).</p

Network analysis of differential expression data.

<p>Resulting subnetwork from network analysis of RNAseq data using PheNetic [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155139#pone.0155139.ref027" target="_blank">27</a>]. This subnetwork shows molecular mechanisms which are differentially active when comparing <i>P</i>. <i>aeruginosa</i> cells treated with 0.2x MIC of SPI031 to <i>P</i>. <i>aeruginosa</i> cells treated with DMSO. Nodes and connecting lines represent genes and interactions between these genes, respectively. Red dots represent genes overexpressed in the SPI031-treated organisms with respect to the DMSO-treated organism and vice versa for green dots. The color of the connecting lines represents the type of interaction in between genes. Yellow lines represent metabolic interactions, red lines represent regulatory interactions and green lines represent protein-protein interactions.</p