The demographic, clinical, and relevant microbiological data of the 353 patients with nosocomial <i>S</i>. <i>aureus</i> bacteremia by strains.

<p>The demographic, clinical, and relevant microbiological data of the 353 patients with nosocomial <i>S</i>. <i>aureus</i> bacteremia by strains.</p

Drug susceptibilities (%) and carriage of SCC<i>mec</i> elements (%) of the 261 <i>S</i>. <i>aureus</i> blood isolates.

<p>Drug susceptibilities (%) and carriage of SCC<i>mec</i> elements (%) of the 261 <i>S</i>. <i>aureus</i> blood isolates.</p

The demographic, clinical, and relevant microbiological data of the 353 patients with nosocomial <i>S</i>. <i>aureus</i> bacteremia stratified by in-hospital mortality and univariate analysis for all-cause in-hospital mortality.

<p>The demographic, clinical, and relevant microbiological data of the 353 patients with nosocomial <i>S</i>. <i>aureus</i> bacteremia stratified by in-hospital mortality and univariate analysis for all-cause in-hospital mortality.</p

Multivariate analysis for risk factors associated with all-cause in-hospital mortality.

<p>Multivariate analysis for risk factors associated with all-cause in-hospital mortality.</p

Dose response effects of LPS and LPS antibody (triangle) on the bactericidal activity of HBc147-183.

<p>LPS from <i>P. aeruginosa</i> (square) and <i>E. coli</i> (diamond), and LPS antibody (triangle) were mixed with <i>P. aeruginosa</i> and HBc147-183 (1×MBC) for 3 hours. The bacteria were then plated on MH agar for the measurement of viability. Samples were measured in triplicates.</p

Antimicrobial activity of ARD peptide HBc147-183 against colistin-resistant and sensitive <i>P. aeruginosa</i> and <i>A. baumannii</i>.

<p>Antimicrobial activity of ARD peptide HBc147-183 against colistin-resistant and sensitive <i>P. aeruginosa</i> and <i>A. baumannii</i>.</p

The killing kinetics of HBc147-183 against <i>P. aeruginosa</i> (circle), <i>K. pneumoniae</i> (diamond), <i>E. coli</i> (square) and <i>S. aureus</i> (triangle).

<p>Bacteria were treated with HBc147-183 (1×MBC). The viability of bacteria was measured at indicated time point. Samples were measured in triplicates.</p

<i>In vivo</i> studies of the protection activity of ARD peptide HBc147-183 against <i>S. aureus</i>.

<p>(A) Three-week old male ICR mice were challenged with a lethal dose of <i>S. aureus</i> ATCC 19636 and then divided into five separate groups for five different time points. At each indicated time point (n = 5), blood samples were collected, diluted and plated on BHI agar. The number of bacteria was counted the following day. A maximal bacterial load in the blood was observed at 2 h post-inoculation. The data were shown in mean ± SD. (B) ICR mice inoculated with a lethal dose of <i>S. aureus</i> as described above were treated by intraperitoneal injection with ARD peptide (10 mg/kg) at 1, 1.5 or 2 h post-inoculation, respectively. Each group contained 10 mice. All mice (100%) treated with the PBS control died at day 1, while treatment of ARD peptide at 1, 1.5 or 2 h post-inoculation protected the mice with survival rates of 100%, 70% and 40% after 7 days, respectively. (C) As described above, ICR mice were i.p. inoculated with <i>S. aureus</i>, followed by i.p. injection with PBS (n = 5) or 10 mg/kg ARD peptide (n = 5) at 1 h post-inoculation. At 4 h post-inoculation, blood, liver and spleen were collected. Liver and spleen samples were homogenized, diluted and, together with blood samples, plated on BHI agar. The number of bacteria was counted the following day. In comparison to mice treated with PBS, treatment of ARD peptide effectively reduced the bacterial load in blood, liver and spleen. (D) Quantitative comparison of bacterial loads in blood, liver and spleen samples of mice treated with PBS (open circle, diamond and square) versus ARD peptide HBc147-183 (solid circle, diamond and square). The line indicated the mean of bacterial load. **<i>P</i><0.01 (Mann-Whitney U test) for PBS and ARD peptide HBc147-183.</p

Amino acid sequences of various HBc ARD peptides tested for bactericidal activity in this study.

<p>The lower panel presents various phosphorylated peptides and an R-to-A mutant peptide with a total of four arg-to-ala substitutions in ARD-III and ARD-IV of HBc147-183.</p

Cytotoxicity assays of ARD peptide HBc147-183.

<p>% human red blood cells (RBC). Compared to melittin, HBc147-183 showed no hemolytic activity. (B) Huh7, HepG2, Vero and HEK293 cells were incubated with varying concentrations (0 to 100 µM) of HBc147-183 (black) and melittin (white) for 1 hour at 37°C. The effects on cell viability were determined by MTT assay. Melittin was used as a positive control. HBc147-183 showed no detectable effect on cell viability, while melittin exhibited strong toxicity. (C) Kidney cells Vero and HEK293 were stained with CFSE and seeded at day 0 (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003425#s2" target="_blank">Materials and Methods</a>). At day 1, cells were incubated with varying concentrations (0 to 100 µM) of HBc147-183 for 1 hour. Cell proliferation at day 1 and day 3 were determined by flow cytometry. Similar to the mock control experiment, no significant effect on Vero and HEK293 cells was detected. Samples assayed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003425#ppat-1003425-g007" target="_blank">Figure 7A–C</a> were always measured in triplicates. (D) <i>In vivo</i> toxicity of ARD peptide HBc147-183 was determined using three-week old male ICR mice. The mice were injected intraperitoneally with peptide (10 and 20 mg/kg of body weight). All mice were alive after 7 days.</p