PLP-3: a novel bicyclic peptide active against multidrug-resistant bacteria

Introduction

Antimicrobial resistance (AMR) is a global health crisis and one of the top ten public health threats according to the WHO. In recent decades, few antibiotics have reached the market to tackle infections caused by multidrug-resistant (MDR) bacteria. Thus, new antimicrobials are needed.

This project evaluates the activity of PLP-3, a synthetic antimicrobial peptide designed in silico as a Protegrin-1 analogue, against a panel of MDR pathogens.

Methods

The antibacterial activity of PLP-3 was evaluated by determining the Minimum Inhibitory Concentration (MIC) against a clinical collection of MDR strains, including Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecalis and Enterococcus faecium. Time–kill curves (TKCs) were performed to assess bactericidal activity, and checkerboard assays were used to evaluate synergies with conventional antibiotics. The Minimum Biofilm Inhibitory Concentration (MBIC) and Minimum Biofilm Eradication Concentration (MBEC) were also determined. Finally, PLP-3-resistant mutants were generated and sequenced to elucidate the mechanism of action.

Results

PLP-3 MIC₅₀ and MIC₉₀ ranged between 1-4mg/L and 1-16mg/L across all Gram-negative and Gram-positive strains tested, respectively. TKC confirmed that PLP-3 is bactericidal at concentrations ≥ 2xMIC after 24h, with viable counts falling below the detection limit. Checkerboard assays showed that most antibiotic combinations were additive, achieving synergy with meropenem against a clinical strain of A. baumannii. As for PLP-3 antibiofilm activity, both MBIC₅₀ and MBEC₅₀ ranged between 4-32mg/L and 64->64mg/L for Gram-negative strains, respectively, and 1-16mg/L and 16->64mg/L for Gram-positive strains. Regarding resistant mutants, variant calling analysis indicated that the prevalent mechanism appears to involve a reduction in surface negative charge to minimize electrostatic interactions with PLP-3.

Conclusion

PLP-3 shows broad-spectrum bactericidal activity against MDR bacteria for planktonic and biofilm-associated cells, which is a promising candidate for the development of new antimicrobials to combat AMR.