Valutazione dell'effetto sinergico di hLF 1-11 e caspofungina su cellule planctoniche e biofilm di Candida spp.

Candida species are the main fungal opportunistic pathogens causing mucosal and systemic infections often associated with drug resistance and biofilm production on medical device. Biofilm associated infection are difficult to treat and could evolve in Candidaemia, often associated with an increase of morbidity and mortality. The difficulty to find new antifungal drugs has led to an increased interest in the use of antimicrobial peptides, alone or in combination with conventional drugs. The present study was aimed at investigating the possible synergistic activity of a synthetic peptide hLF1–11 and caspofungin against different strains of C. albicans , C. parapsilosis and C. glabrata planctonic cells and biofilm in vitro. The interaction between caspofungin and hLF1–11 and was first evaluated by the checkerboard assay using 96-well bottom polystyrene microtiter plates and a killing assay on planctonic cells. Then, synergistic activity was evaluated in biofilm producer strains by XTT reduction metabolic assay in the checkerboard plate and then, reduction of cell viability was evaluated in selected concentrations with low metabolic activity. Live/dead staining was performed by combining the green-fluorescent nucleic acid stain SYTO 9 and the red-fluorescent nucleic acid stain propidium iodide in PBS, then epifluorescence images were acquired using the Operetta CLS High-Content Imager and the Harmony software. A strong synergistic effect in biofilm inhibition was observed against all the tested C. albicans, C. parapsilosis and C. glabrata strains. A fractional inhibitory concentration (FIC) index ≤0.5 was interpreted as synergy as a decrease in CFU/ml of ≥2 Log by the combination of hLF1–11 and caspofungin in comparison with the most active constituent. Checkerboard assay shows a strong synergistic effect against all the tested C. albicans, C. parapsilosis and C. glabrata strains, in the planktonic cells, biofilm inhibition and biofilm eradication. On C. albicans caspofungin-resistant strains (CACR) biofilm inhibitory concentration (BIC) of drugs in combination was reduced up to 5 serial dilution, and comparable results were obtain for other strains. In viability assay performed on planktonic cells and sessile cells, synergy was found with various combinations of concentrations. Fluorescence imaging analysis confirmed the synergistic effect observed between caspofungin and hLF1-11 against biofilm, interesting the combinations of two drugs show the ability to inhibit hyphae and pseudohyphae formation in all strains tested. In conclusion, synergistic effect observed between caspofungin and hLF1-11 against Candida biofilm representing a possible interesting novel approach to target drug resistant fungal biofilm infections