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. 2019 Jul 24:10:1688.
doi: 10.3389/fmicb.2019.01688. eCollection 2019.

Evaluation of the Antimicrobial Peptide, RP557, for the Broad-Spectrum Treatment of Wound Pathogens and Biofilm

Kathryn Wynne Woodburn  1 , Jesse M Jaynes  2 , L Edward Clemens  1
Affiliations

Evaluation of the Antimicrobial Peptide, RP557, for the Broad-Spectrum Treatment of Wound Pathogens and Biofilm

Kathryn Wynne Woodburn et al. Front Microbiol. .

Abstract

The relentless growth of multidrug resistance and generation of recalcitrant biofilm are major obstacles in treating wounds, particularly in austere military environments where broad-spectrum pathogen coverage is needed. Designed antimicrobial peptides (dAMPs) are constructed analogs of naturally occurring AMPs that provide the first line of defense in many organisms. RP557 is a dAMP resulting from iterative rational chemical structural analoging with endogenous AMPs, human cathelicidin LL-37 and Tachyplesin 1 and the synthetic D2A21 used as structural benchmarks. RP557 possesses broad spectrum activity against Gram-positive and Gram-negative bacteria and fungi, including recalcitrant biofilm with substantial selective killing over bacterial cells compared to mammalian cells. RP557 did not induce resistance following chronic passages of Pseudomonas aeruginosa and Staphylococcus aureus at subinhibitory concentrations, whereas concurrently run conventional antibiotics, gentamycin, and clindamycin, did. Furthermore, RP557 was able to subsequently eliminate the generated gentamycin resistant P. aeruginosa and clindamycin resistant S. aureus strains without requiring an increase in minimum inhibitory concentration (MIC) concentrations. RP557 was evaluated further in a MRSA murine wound abrasion infection model with a topical application of 0.2% RP557, completely eliminating infection. If these preclinical results are translated into the clinical setting, RP557 may become crucial for the empirical broad-spectrum treatment of wound pathogens, so that infections can be reduced to a preventable complication of combat-related injuries.

Keywords: antibacterial; antimicrobial peptides; bacterial resistance; biofilm; multidrug resistance; wound infection.

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Figures

Figure 1
Figure 1
Schematic representation of the endogenous AMPs, LL-37 and Tachyplesin 1 and the dAMPs, D2A21 and RP557. Hydrophobic residues are in blue, while those residues with a net positive charge are in red and those with a negative charge are in green.
Figure 2
Figure 2
RP557 is stable in serum. RP557, at 200 μg/mL, was incubated with human serum for 72 h at 37°C and peptide monitored via LC-MS/MS. Data represent mean ±SE. Assays were performed in triplicate.
Figure 3
Figure 3
RP557 immediately destroys P. aeruginosa and S. aureus in an increasing dose-dependent manner. The bactericidal effectiveness of RP557, tobramycin and vancomycin were evaluated against bioluminescent P. aeruginosa 19660 (A) and S. aureus 49525 (B) through 60 min of exposure. The concentration dependence of RP557, tobramycin and vancomycin following 30 min of exposure with P. aeruginosa and S. aureus is shown in (C,D), respectively. The bioluminescence of viable cells was quantitated non-invasively with an IVIS Lumina bioimaging system. Bioluminescence reflects bacterial viability as is directly correlated with number of colony forming units. Data represent the mean ± SE of triplicate replicates from two independent experiments; statistically significant (*P < 0.05; **P < 0.01, ***P < 0.001, ****P < 0.0001), using one-way ANOVA followed by Tukey analysis. For some points, the error bars are shorter than the height of the symbols.
Figure 4
Figure 4
RP557 exhibits minimal cytotoxicity. Keratinocyte and fibroblast cytotoxicity was non-invasively assayed using bioluminescent strains of human HaCaT keratinocytes (A) and murine L929 fibroblast cells (B) and viability assayed using an IVIS Lumina imaging system. Hemolysis toward human RBCs is shown in (C). Data represents the mean ±SE. Bioluminescent experiments were performed in triplicate from two independent experiments. The red blood cell viability assay involved two replicates; statistically significant in comparison to RP557 (*P < 0.05; **P < 0.01, ***P < 0.001), using one-way ANOVA followed by Tukey analysis.
Figure 5
Figure 5
RP557 possesses potent selectivity for bacterial pathogens. The Selectivity Index is defined by the dose required to hemolyze 10% human red blood cells compared to the S. aureus and P. aeruginosa minimal inhibitory concentration (MIC).
Figure 6
Figure 6
P. aeruginosa and S. aureus did not develop resistance to RP557. Sub-inhibitory concentrations of RP557, gentamicin and clindamycin were cultured with P. aeruginosa ATCC 27853 (A) and S. aureus ATCC 29213 (B) for 24 h. Bacteria showing growth in the highest concentration were re-passaged in fresh dilutions containing sub-MIC levels of each component for 30 consecutive passages; means are shown.
Figure 7
Figure 7
RP557 disrupts P. aeruginosa, multidrug resistant S. epidermidis and MRSA biofilms, including mature biofilm. Dose-dependent concentration response of RP557 against 6 hr MRSA ATCC 1556 (A), mature (120 h) MRSA ATCC 1556 (B), P. aeruginosa P14 (C), and MDR S. epidermidis ATCC 700578 (D) biofilms. Biofilms were established in plastic 96-well plates and then treated overnight with increasing concentrations of RP557 or daptomycin and then CFUs evaluated. Three technical replicates were performed for each condition tested. The dashed lines represent the lower limit of detection (LLOD) which is 2.6 log CFU/mL.
Figure 8
Figure 8
A single topical treatment of 0.2% RP557 reduced MRSA infection in a murine skin abrasion wound infection model. Infected scratch wounds were created on the backs of immunosuppressed BALB/c mice with bioluminescent MRSA (Xen31, MRSA ATCC 33591). After 4 h, 0.2% RP557 in 2% hydroxypropyl methylcellulose was applied to one group, with the other group serving as a non-treatment control. The bioluminescence of viable cells was quantitated non-invasively using bioluminescence imaging (A) and RP557 bactericidal effectiveness measured by the magnitude of the bioluminescent signal (B). Data is expressed as mean ± SE of 5 mice, except for the untreated control group where an animal was found dead on Day 4. Statistical significance, compared to untreated control, determined by two-tailed unpaired-t test (*p < 0.05, **p < 0.01).
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