Biophotonic imaging of burn wounds.

<p>(<b>A</b>) Timeline of scald injury experiments. (<b>B</b>) Dorsal images of representative BALB/c mice challenged with approx. 1 × 10⁷ CFU of bioluminescent <i>S</i>. <i>aureus</i> ATCC 12600 (Xen29). Mice were subjected to bioluminescent imaging on IVIS Lumina XRMS Series III system at the indicated times. (<b>C</b>), Quantification of photon intensities of bacterial burden showing significant reduction in photon intensities in mupirocin-treated mice. Total flux (means±SEM photons/s; n = 6 mice). *<i>p</i><0.05; **<i>p</i><0.01; multiple <i>t</i>-tests). (<b>D</b>), Total bacterial counts from tissues of control, infected but not treated, and infected + mupirocin-treated mice at the conclusion of the experiment, showing strong correlation with total photon intensities obtained from each treatment group.----denotes limit of detection; ^_____ denotes geometric mean counts.</p

Luminescence signal comparison between groups of CD1 mice challenged IP with <i>S</i>. <i>aureus</i> ATCC 12600 (Xen29).

<p>(<b>A</b>), Quantification of photon intensities on a Xenogen IVIS Lumina XRMS Series III live animal biophotonic imaging system, showing significant reduction in photon intensities in daptomycin-treated mice. Total flux (means±SEM photons/s; n = 6 mice). **<i>p</i><0.01; multiple <i>t</i>-tests). (<b>B</b>), Survival times for CD1 male mice (n = 6) challenged IP with approx. 2.5 × 10⁷ CFU of bioluminescent <i>S</i>. <i>aureus</i> Xen29 and administered the drug vehicle only or daptomycin (6 mg/kg) IP at 2 and 6 h post-infection. Differences in median survival times (time to moribund) for mice between groups were analyzed by the log-rank (Mantel-Cox) tests. ***, <i>P</i><0.001. (<b>C</b>), Total bacterial counts from blood of control and infected + daptomycin-treated mice at 2, 4 and 6 h post-infection.----denotes limit of detection; ^_____ denotes geometric mean counts.</p

Biophotonic ventral and dorsal images of 2 representative CD1 male mice challenged IP with approx.

<p><b>2.5 × 10⁷ CFU of bioluminescent <i>S</i>. <i>aureus</i> ATCC 12600 (Xen29) and then administered the drug vehicle only or daptomycin IP at 2 and 6 h post-infection</b>. Mice were subjected to bioluminescent imaging on IVIS Lumina XRMS Series III system at the indicated times.</p

Analysis of wound healing in non-infected, infected but not treated, and infected + mupirocin-treated scald burn wounds.

<p>(<b>A</b>), Representative images and graphical analysis of scald wounds over a time-course of 10 days illustrating macroscopic differences in rate of healing between non-infected, infected but not treated, and infected + mupirocin-treated mouse scald wounds. (<b>B</b>), Representative haematoxylin and eosin-stained sections of partial-thickness scald wounds in mice with non-infected, infected but not treated, and infected + mupirocin-treated wounds. Microscopic analysis of scald wounds at day 10 post-wounding suggests that mupirocin treatment of scald wounds is effective in treating <i>S</i>. <i>aureus</i> infected wounds leading to significantly decreased wound length, dermal gape and significantly increased wound re-epithelisation compared to infected wounds. Arrows indicate dermal wound gape distance. Magnification ×4 stitched image. Scale bar = 100 μm. Results represent means and SEM, n = 6 wounds per mice group with a single time-point.</p

NCL812 compounds exert their antibacterial action on the cell membrane of <i>S</i>. <i>pneumoniae</i> and <i>S</i>. <i>aureus</i>.

<p>(<b>A and B</b>), <i>S</i>. <i>pneumoniae</i> strain D39 exposed to 16 μg/ml NCL812 for 6 h exhibited significantly thicker cell membranes compared to untreated samples (<b>A</b>) (<i>p</i> < 0.0001; two-tailed unpaired <i>t</i>-test) and displayed significantly wider periplasmic space compared to untreated samples (<b>B</b>) (<i>p</i> < 0.001; two-tailed unpaired <i>t</i>-test. Data presented are an example from 12 different bacterial cells, each with at least 10 measurements per bacteria for both treated and untreated samples. (<b>C and D</b>) NCL812 affects macromolecular synthesis (<b>c</b>) and ATP release (<b>D</b>) in <i>S</i>. <i>aureus</i>. (<b>E and F</b>), NCL Compounds dissipate the membrane potential of <i>S</i>. <i>pneumoniae</i> and <i>S</i>. <i>aureus</i>. Membrane potential measurements of <i>S</i>. <i>pneumoniae</i> D39 (<b>E</b>) and <i>S</i>. <i>aureus</i> ATCC49775 (<b>F</b>). Bacterial suspensions were exposed to 16 μg/ml NCL812, NCL195, NCL219, or ampicillin (control) for 5 min after which DiOC₂(3) was added and the fluorescence monitored until it plateaued. Cells were then re-energized with 0.5% glucose and the establishment of a membrane potential was measured as an increase in fluorescence until it plateaued. The membrane potential was then disrupted by the addition of the proton ionophore (CCCP). Data presented is representative of two experiments. For full description, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183457#sec002" target="_blank">Materials and Methods</a>.</p

Antimicrobial activity of NCL195 against Gram-negative bacteria alone and in combination with EDTA or polymyxin B.

<p>Antimicrobial activity of NCL195 against Gram-negative bacteria alone and in combination with EDTA or polymyxin B.</p

Physicochemical and metabolism parameters for NCL812, NCL195 and NCL219 in human and mouse liver microsomes.

<p>Physicochemical and metabolism parameters for NCL812, NCL195 and NCL219 in human and mouse liver microsomes.</p

Structure activity relationship between NCL812, NCL195 and NCL219.

<p>Installation of a 4-<i>tert</i>-butyl and a C-methyl imine moiety provided NCL219 with considerably enhanced hydrolytic stability while retaining the excellent antimicrobial activity of NCL812, while guanidine to 2,4,6-triaminopyrimindine bioisosteric modification yielded NCL195, which allowed potency and drug-like character enhancement.</p

MIC range values for NCL812, NCL195 and NCL219 compared to MIC₉₀ values for daptomycin and ampicillin against <i>S</i>. <i>pneumoniae</i>, <i>S</i>. <i>aureus</i> and vancomycin resistant enterococci (VRE).

<p>MIC range values for NCL812, NCL195 and NCL219 compared to MIC₉₀ values for daptomycin and ampicillin against <i>S</i>. <i>pneumoniae</i>, <i>S</i>. <i>aureus</i> and vancomycin resistant enterococci (VRE).</p

NCL195 demonstrates limited cytotoxicity to mammalian cell lines.

<p>Real-time cell viability measurements for Hep G2 (<b>A, B, C</b>) and MDBK (<b>D, E, F</b>) cells after treatment with 2 or 8 μg/ml NCL812, NCL195 or NCL219. Cell viability was measured every 60 min for 20 h at 37°C and 5% CO₂ on a Cytation 5 Cell Imaging Multi-Mode Reader (BioTek) using the RealTime-Glo MT Cell Viability Assay reagent (Promega). Data are means (± s.e.m.) relative light units (RLU) for each treatment per time point (in duplicate).</p