<i>In vivo</i> tracking of bioluminescently labeled (live) bacterial infections.

<p>CD-1 female mice were injected with individual bacterial strains carrying plasmids constitutively expressing <i>lux</i> reporter genes. Bacterial strains were injected subcutaneously at a dose of 1 × 10⁹ CFU <i>E</i>. <i>faecium</i>, 5 × 10⁷ CFU <i>S</i>. <i>aureus</i>, 1 × 10⁹ CFU <i>K</i>. <i>pneumoniae</i>, 1 × 10⁹ CFU <i>A</i>. <i>baumannii</i>, 5 × 10⁷ CFU <i>P</i>. <i>aeruginosa</i>, 2.5 × 10⁸ CFU <i>E</i>. <i>cloacae</i>, and 1 × 10⁸ CFU <i>E</i>. <i>coli</i>. The infection was monitored 1 h post infection and then every 24 h until day 3. Representative images for day 0 and day 3 are shown. Mice were imaged using a Perkin Elmer <i>in vivo</i> imaging system (IVIS) and the experiment was repeated twice with three mice/group. The scale at the bottom indicates radiance x 10⁶.</p

Synergy between conventional antibiotics and anti-biofilm peptides in a murine, sub-cutaneous abscess model caused by recalcitrant ESKAPE pathogens

<div><p>With the antibiotic development pipeline running dry, many fear that we might soon run out of treatment options. High-density infections are particularly difficult to treat due to their adaptive multidrug-resistance and currently there are no therapies that adequately address this important issue. Here, a large-scale <i>in vivo</i> study was performed to enhance the activity of antibiotics to treat high-density infections caused by multidrug-resistant Gram-positive and Gram-negative bacteria. It was shown that synthetic peptides can be used in conjunction with the antibiotics ciprofloxacin, meropenem, erythromycin, gentamicin, and vancomycin to improve the treatment outcome of murine cutaneous abscesses caused by clinical hard-to-treat pathogens including all ESKAPE (<u><i>E</i></u><i>nterococcus faecium</i>, <u><i>S</i></u><i>taphylococcus aureus</i>, <u><i>K</i></u><i>lebsiella pneumoniae</i>, <u><i>A</i></u><i>cinetobacter baumannii</i>, <u><i>P</i></u><i>seudomonas aeruginosa</i>, <u><i>E</i></u><i>nterobacter cloacae</i>) pathogens and <i>Escherichia coli</i>. Promisingly, combination treatment often showed synergistic effects that significantly reduced abscess sizes and/or improved clearance of bacterial isolates from the infection site, regardless of the antibiotic mode of action. <i>In vitro</i> data suggest that the mechanisms of peptide action <i>in vivo</i> include enhancement of antibiotic penetration and potential disruption of the stringent stress response.</p></div

Antibiotic and synthetic peptide mono- and combinatorial therapy in a murine cutaneous abscess model using female CD-1 mice and clinical drug-resistant bacterial isolates.

<p>Bacterial strains were injected subcutaneously and treated one hour post infection with either saline (control), synthetic peptides, antibiotics, or antibiotic-peptide combinations. Synthetic peptide concentrations for all conditions were as follows: 1002, 10 mg/kg (3 mg/kg for <i>E</i>. <i>faecium</i>); 1018, 10 mg/kg; HHC-10 10 mg/kg, and DJK-5, 3 mg/kg (0.25 mg/kg for <i>S</i>. <i>aureus</i>). Infected and inflamed tissue was measured three days post infection and pus-containing abscess lumps excised to determine CFU. Abscess sizes are in box and whiskers plots (left panel) and counted CFU/abscess data expressed with geometric mean (right panel). <b>A</b>. <i>P</i>. <i>aeruginosa</i> LESB58, ciprofloxacin 0.4 mg/kg. <b>B</b>. <i>A</i>. <i>baumannii</i> Ab5075, erythromycin 6 mg/kg, meropenem 6 mg/kg. <b>C</b>. <i>K</i>. <i>pneumoniae</i> KPLN649, meropenem 10 mg/kg, ciprofloxacin 30 mg/kg. <b>D</b>. <i>E</i>. <i>cloacae</i> 218 R1, ciprofloxacin 0.006 mg/kg. <b>E</b>. <i>E</i>. <i>coli</i> E38, ciprofloxacin 4 mg/kg, F. <i>E</i>. <i>faecium</i> #1–1, gentamicin 16 mg/kg. G. <i>S</i>. <i>aureus</i> LAC, clindamycin 0.01 mg/kg; vancomycin 0.15 mg/kg. <b>(A-G)</b> <i>n</i> = 368 biologically independent animals. All experiments were done at least three times with 2–4 mice/group. Statistical analysis was performed using One-way ANOVA, Kruskal-Wallis test with Dunn’s correction (two-sided). The asterisk indicates significant differences to the wild-type (*, <i>p</i> < 0.05; **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001). The hash indicates significant differences of the combination therapy over the sum of the effects of each agent alone (#, <i>p</i> < 0.05; ##, <i>p</i> < 0.01; ###, <i>p</i> < 0.001).</p

Outer membrane permeabilization by peptides cf. antibiotics at their corresponding MICs.

<p>The uptake of the fluorophore NPN in the presence of different antibiotics and synthetic peptides was determined by assessing increased fluorescence at an excitation wavelength of 350 nm and an emission wavelength of 420 nm due to partition of the normally impermeable hydrophobic NPN into bacterial membranes. Relative fluorescence values of at least three biological replicates were determined by subtracting the fluorescence value without test substance.</p

<i>In vitro</i> MICs of antibiotics and peptides against clinical isolates.

<p><i>In vitro</i> MICs of antibiotics and peptides against clinical isolates.</p

Bacterial strains used in this study.

<p>Bacterial strains used in this study.</p

Schematic presentation of the genomic region of the <i>P. aeruginosa</i> PA14 dipeptide transport machinery.

<p>(A) Region surrounding the dipeptide transporter operon <i>dppBCDF</i> and its substrate-binding proteins <i>dppA1–A4</i>. The 3,952-bp deletion of the ABC transporter <i>dppBCDF</i> operon is indicated. (B) Genomic region surrounding <i>dppA5</i>. The subcellular location of the proteins is indicated by colors. Black arrows indicate operon structures <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111311#pone.0111311-Winsor1" target="_blank">[77]</a>.</p

Global utilization pattern of dipeptides by PA14, the dipeptide transporter DppBCDF and the SBPs DppA1–A5.

<p>The rings show the total number of dipeptides utilized by the wild-type strain PA14 (outer purple ring), transported by the dipeptide transporter system DppBCDF (middle blue ring), and recognized by the substrate-binding proteins (inner dark green ring). The oval rings (green) present different pools of dipeptides (nonpolar, acidic, uncharged) containing a specific amino acid side-chain either at the N- or C-terminal end of the dipeptide (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111311#pone-0111311-t002" target="_blank">Table 2</a>). Dipeptides containing positively charged amino acid residues were excluded from the analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111311#s4" target="_blank">Materials and Methods</a>).</p

Differences in dipeptide utilization efficiency of the SBPs DppA1–DppA4 determined by differences in total growth^a.

<p>Differences in dipeptide utilization efficiency of the SBPs DppA1–DppA4 determined by differences in total growth^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111311#nt104" target="_blank">a</a>}.</p

Total numbers of dipeptides utilized by PA14 (WT), the <i>dppBCDF</i> mutant (Dpp) and the SBP penta mutant (SBP) (see also Figure 3).

<p>Total numbers of dipeptides utilized by PA14 (WT), the <i>dppBCDF</i> mutant (Dpp) and the SBP penta mutant (SBP) (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111311#pone-0111311-g003" target="_blank">Figure 3</a>).</p