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.

Candida species are major contributors to nosocomial infections, with biofilm formation being a critical virulence factor that enables persistence in clinical settings and resistance to antifungal therapies. Central to biofilm development is the adhesion of fungal cells, a process mediated by surface proteins such as Als5p in Candida albicans. The amyloid-forming peptide sequence within Als5p (³²²SNGIVIVATTRTV³³⁴) has been implicated in mediating adhesion and biofilm formation; however, its role in shaping the biofilm architecture has not been fully elucidated. In this study, we demonstrated that the addition of Als5pFP promoted biomass accumulation in C. albicans biofilms under laboratory conditions, including complex media and at temperatures compatible with clinical biofilm assays. Using advanced image analysis of microscopy images, we show that the Als5p peptide induces a distinct morphological effect on biofilms: a shape-edging of microcolony structures, characterized by the concentration of fungal cells into denser aggregates and the reduction of cells in intermediate spaces. These observations suggest a potential role of amyloid-like fibrils formed by the Als5p peptide in influencing the spatial organization of C. albicans biofilms. This discovery presents a novel aspect on how these fibrils affect the biofilm architecture extending beyond previous studies, which primarily focused on biomass accumulation. Our findings contribute to the understanding of the architectural development of C. albicans biofilms and provide a foundation for future research aimed at targeting the amyloid structures within fungal biofilms. Furthermore, the results may support the design of biofilm-targeting antifungal agents and development of biosensors for monitoring amyloid formation during infection.

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.