Biofilms are the predominant lifeforms of microorganisms, contributing to over 80% of infections, including those caused by Candida species like C. albicans, C. parapsilosis and Candidozyma auris. These species form biofilms on medical devices, making infections challenging to treat, especially with the rise in drug-resistant strains. Candida infections, particularly hospital-acquired ones, are a significant health threat due to their resistance to antifungals and the risk of developing systemic infections (i.e., sepsis). We have previously shown that C14R reduces the viability of C. albicans and C. auris, but not of C. parapsilosis. Here, we show that C14R not only inhibits viability by pore formation, shown in a resazurin reduction assay, and in a C. parapsilosis and fluorescence-based permeabilization assay, but it also halts biofilm maturation and significantly reduces the biomass of preformed biofilms by over 70%. These findings suggest C14R could be an effective option for treating severe fungal infections, offering a potential new treatment approach for biofilm-related diseases. Further research is needed to fully understand its biofilm dispersal potential and to optimize its use for future applications as an antifungal in clinical settings.

The World Health Organization (WHO) prioritized 19 fungal species based on the significant impact of these pathogens on human health, including the emergence of antifungal resistance, which highlights the necessity of finding new antifungal therapies. Among these novel therapeutic approaches, the antimicrobial pore-forming peptide C14R has shown to be promising against Candida albicans and Candida auris. In this study, the antifungal in vitro efficacy of C14R was assessed against six additional species from the WHO priority list, Cryptococcus neoformans, Cryptococcus gattii, Candida glabrata, Candida tropicalis, Candida parapsilosis and Candida krusei, as well as against Candida dubliniensis. This study shows that C14R has good antifungal activity against several clinical isolates of the studied species, with MIC values between 0.8476 and 10.88 µg/mL. Most notably, some of the studied isolates are resistant to commonly used antifungal drugs but are susceptible to the peptide. C14R showed, moreover, its capacity to disrupt Cryptococcus capsules, beyond its already proven capacity to disrupt plasma membranes, and its antifungal activity was not affected depending on the serotype or species assessed. The inclusion of basidiomycete and ascomycete yeasts allowed us to display the broad-spectrum potential of C14R, highlighting it as a promising candidate as an antifungal agent.

Background/Objectives: Infections caused by Candida albicans, Candidozyma auris, and Candida parapsilosis increasingly challenge current treatment options as resistance to currently used antifungals is continuously developing. Neutralizing antimicrobial peptides (nAMPs), which modulate pathogenic behavior rather than inducing cell death, represent a promising approach to fighting against fungal infections. Methods: This study established a whole-cell phage display workflow to identify novel nAMPs, and therefore three independent biopanning processes with the Ph.D.-12 phage display library against C. albicans, C. auris, and C. parapsilosis cells were conducted. Results: Phage display produced species-selective, high-affinity peptides that were non-cytotoxic to human cells and did not affect planktonic Candida viability. These peptides inhibited early biofilm formation, and several also slowed early biofilm maturation down. Conclusions: These findings demonstrate that whole-cell phage display as a powerful and adaptable discovery tool is suitable for identifying nAMPs that neutralize biofilm development without toxicity towards human cells. Beyond the peptides described here, this approach expands the methodological toolbox for antifungal research and provides a sustainable approach for generating targeted peptides.

The global rise in antimicrobial resistance among the ESKAPE pathogens represents a major challenge to public health. Here, we report the broad-spectrum antibacterial activity of the synthetic antimicrobial and pore-forming peptide C14R against all six ESKAPE species. Using a radial diffusion assay and resazurin-based viability testing, C14R exhibited a potent bactericidal effect with minimum inhibitory concentrations (MICs), defined as the lowest concentration of an antimicrobial agent that completely inhibits visible growth of planktonic microorganisms, ranging from 3.4 µg/mL (Enterococcus faecium, vancomycin-resistant) to 45.2 µg/mL (Klebsiella quasipneumoniae, ESBL). C14R also inhibited biofilm formation by Gram-positive pathogens, with minimum biofilm inhibitory concentrations (MBICs), referring to the minimal concentration required to prevent the development of biofilms, of 15.0 µg/mL (Staphylococcus aureus, MRSA) and 22.0 µg/mL (E. faecium, VRE), whereas Gram-negative biofilms showed higher tolerance. Together, these findings demonstrate that C14R retains high activity against multidrug-resistant ESKAPE strains, highlighting its potential as a lead compound for the development of next-generation antimicrobial drugs to expand the portfolio of available antibiotics and brace health systems against emerging severe infections.

Biofilms are the predominant lifestyle for microorganisms, contributing to over 80% of infections, including those caused by Candida species like C. albicans, C. auris, and C. parapsilosis. These species form biofilms on medical devices, making infections challenging to treat, especially with the rise of drug-resistant strains. Candida infections, particularly hospital-acquired ones, are a significant health threat due to their resistance to antifungals and the risk of developing systemic infections (i.e. sepsis). C14R, a classical pore-forming antimicrobial peptide (AMP), shows promising activities against both planktonic and biofilm forms of Candida. The 16-amino acid peptide disrupts cell membranes by integrating into lipid bilayers and forming pore-like structures. Studies demonstrate that C14R reduces the viability of C. albicans, C. auris, and C. parapsilosis, including clinical isolates. Here, we show that C14R not only inhibits biofilm growth, but also halts biofilm maturation and significantly reduces the biomass of preformed biofilms by over 70%. These findings suggest C14R could be an effective option for treating severe fungal infections, offering a potential new treatment approach for biofilm-related diseases. Further research is needed to fully understand its biofilm dispersal potential and to optimize its use for future applications as an antifungal in clinical settings.