Antimicrobial Peptides SET-M33L and SET-M33L-PEG Are Promising Agents Against Strong Biofilm-Forming Clinical Strains of P. aeruginosa, Including Multidrug-Resistant Isolates

Alessio Fontanot; Clelia Cortese; Adrianus de Bruijn; Peter Croughs; Chiara Falciani; Alessandro Pini; Rupert Ecker; Felicitas Mungenast; Wendy Unger; Isabella Ellinger; John Hays

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Antimicrobial Peptides SET-M33L and SET-M33L-PEG Are Promising Agents Against Strong Biofilm-Forming Clinical Strains of P. aeruginosa, Including Multidrug-Resistant Isolates

Alessio Fontanot

^{1, 2, 3, 4},

Clelia Cortese

⁵,

Adrianus C. J. M. de Bruijn

³,

Peter D. Croughs

⁴,

^{5, 6},

^{5, 6, 7},

^{1, 8},

¹,

³,

^{*

2},

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4}

¹ TissueGnostics GmbH, Taborstraße 10, 1020 Vienna, Austria
² Institute of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Währingergürtel 18-20, 1090 Vienna, Austria
³ Laboratory of Pediatrics, Department of Pediatrics, Sophia Children’s Hospital, Erasmus University Medical Center (Erasmus MC), 3015 GD Rotterdam, The Netherlands
⁴ Department of Medical Microbiology & Infectious Diseases, Erasmus University Medical Center (Erasmus MC), 3015 GD Rotterdam, The Netherlands
⁵ Department of Medical Biotechnology, University of Siena, via A. Moro 2, 53100 Siena, Italy
⁶ SetLance srl, via Fiorentina 1, 53100 Siena, Italy
⁷ Clinical Pathology Unit, Santa Maria alle Scotte Hospital, Viale M. Bracci, 53100 Siena, Italy
⁸ School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, 4059, Australia

Academic Editor: Marc Maresca

Published: 04 May 2026 by MDPI in Antibiotics 2026—Advances in Antimicrobial Action and Resistance session Antimicrobials, Antimicrobial Resistance, and One Health

Abstract:

Background: Biofilm formation by multidrug resistant (MDR) Pseudomonas aeruginosa contributes to increased morbidity and mortality in patients with pulmonary diseases. SET-M33L is a synthetic tetra-branched peptide that previously showed antimicrobial activity on standard strains of MDR gram-negative bacteria and improved stability in biological fluids than linear versions. PEGylated version of the peptide, SET-M33L-PEG, further displayed enhanced resistance to P. aeruginosa elastase. Therefore, we investigated these two antimicrobial peptides (AMPs) for their antimicrobial and antibiofilm activity against 10 selected P. aeruginosa clinical isolates, including MDR strains.

Methods: The effect of AMPs on outer membrane (OM) integrity was investigated by measuring N-Phenyl-1-naphthylamine (NPN) uptake. Their minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs), minimum biofilm inhibitory concentrations (MBICs) were evaluated with gold standard methodologies. Effect on pre-formed biofilm was evaluated measuring bacterial viability assessed by resazurin dye assay. Conventional antimicrobial compounds tobramycin, ceftazidime, and polymyxin B were used as comparator controls.

Results: Dose-dependent increase of NPN fluorescence intensity after treatment with AMPs indicated a disruption of OM integrity in all tested strains. MICs and MBCs values of the AMPs were 7 to 100 times lower than for tobramycin, and 10 to 300 times lower than for ceftazidime, for the respective MDR strains. AMPs MBICs values ranged from 0.3 μM to 21.8 μM, while tobramycin presented the highest MBIC values among the compounds, ranging between 2.1 μM and 273.8 μM. With increasing concentrations of AMPs, a reduction in cell viability of 50% to 100% was observed in pre-formed biofilms. Fractional inhibitory concentration (FIC) indices showed an additive effect (0.5 < FBC < 1), while fractional bactericidal concentration (FBC) indices showed synergistic effects (FBC < 0.5) for most isolates when the AMPs were combined with tobramycin or ceftazidime.

Conclusion: SET-M33L and SET-M33L-PEG are promising antimicrobial agents against strong biofilm-forming P. aeruginosa, including MDR isolates.

Keywords: Pseudomonas aeruginosa; antimicrobial peptides; multidrug resistance; biofilm; SET-M33L; SET-M33L-PEG