Enhanced antimicrobial activity of peptide-cocktails against common bacterial contaminants of ex vivo stored platelets

Abstract

Bacterial contamination of blood components such as ex vivo-stored platelets is a major safety risk in transfusion medicine. We have recently shown that synthetic antimicrobial peptides named PD1-PD4 derived from the thrombin-induced human platelet-derived antimicrobial proteins, and repeats of Arg-Trp (RW1-RW5) demonstrate microbicidal activity against selected bacteria and viruses. In the present study, we selected PD3, PD4, RW2, RW3 and RW4 and evaluated each individual peptide and their various combinations to see whether the cocktail regimen enhances the antimicrobial activity above and over the individual peptides. Stored platelet or plasma samples spiked with known titres of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Bacillus cereus were treated with either individual peptides or with peptides in various combinations. Analyses revealed that individual peptides show moderate microbicidal activity (10- to 100-fold reduction) against the tested bacteria relative to their combined regimen. The peptide combinations (RW2 + RW4, RW2 + RW3 + RW4 and PD4 + RW3 + RW4) on the other hand enhanced the microbicidal activity (c.10 000-fold reduction) and revealed a minimal inhibitory concentration of 5 μM. Time-kill kinetics indicated that these three peptide combinations exhibited enhanced antimicrobial activity bringing about a 100-fold reduction of bacterial titres within 20 min of incubation. The present study therefore demonstrates the synergistic effect of antimicrobial peptides when used in combinations and provides a proof-of-concept of its potential application as a molecular tool towards pathogen reduction and further extends the possibility of using peptide combinatorial therapeutics as broad-spectrum antibiotics or as alternatives to combat drug-resistant bacteria.

Antimicrobial peptides: an effective approach to prevent bacterial biofilm formation in platelet concentrates

“…Our time‐killing assays demonstrated that a combination of the three AMPs PD4‐RW3‐RW4 were effective in reducing viable counts of three S. epidermidis isolates by approximately 4 log after 120 minutes of AMP treatment. Similar results were shown by Mohan and coworkers who reported that synthetic AMPs had a bactericidal effect in spiked PCs with bacterial reductions greater than 3 log after 90 minutes of AMP treatment. Importantly, no differences in the action of the AMPs were observed between these isolates despite their origin and differences in biofilm‐associated factors.…”

“…Previously Mohan and colleagues have demonstrated that the combination of synthetic AMPs used in this study (PD4‐RW3‐RW4) was effective in reducing the viability of common bacterial contaminants in PCs. Furthermore, Bosch‐Marcé and coworkers have confirmed that when used individually, these AMPs do not elicit immune response in rabbits and do not significantly affect PCs recovery in a SCID mouse model.…”

“…A stock solution of the combination of AMPs was made to 10 mmol/L in PBS and stored at −20°C until required. On the day of the assay, a 10 µmol/L final concentration of AMPs combination was prepared as it has been shown that this concentration is optimal for bactericidal assays while maintaining PC quality …”

“…During PC storage, PLTs release proteins with microbicidal activity from alpha granules when they are activated as a defense mechanism against infection . Yeaman and colleagues have demonstrated that a synthetic PLT‐derived peptide (PD4) from the PLT microbicidal protein exhibits bactericidal effect against Staphylococcus aureus and Salmonella typhimurium . In addition, short synthetic peptides containing arginine‐tryptophan (RW) repeats have been shown to exhibit significant bactericidal activity in contaminated PCs .…”

“…17 In addition, short synthetic peptides containing argininetryptophan (RW) repeats have been shown to exhibit significant bactericidal activity in contaminated PCs. [17][18][19] Importantly, PCs treated with the RW3 and RW4 peptides maintained their in vitro quality properties during storage for up to 7 days. 20 Combination antimicrobial therapy, which uses a mixture of two AMPs or more, can synergistically enhance their bactericidal activity.…”