Abstract
Candida tropicalis is a WHO-prioritised opportunistic fungal pathogen capable of causing superficial-to-systemic fungal infections and forming biofilms on both biotic and abiotic surfaces, such as tissues and medical implants. The rising incidence of fungal infections and the emergence of drug resistance underscore the urgent need for alternative, natural therapeutic agents such as antifungal macromolecules. AF(4) and AF(5), natural antifungal lipopeptide homologs derived from the Bacillus subtilis RLID strain, were purified to homogeneity from the cell free supernatant using semi-preparative scale HPLC with a C18 column at the final step. Using these lipopeptide fractions AF(4) and AF(5), the anti-biofilm efficacies were evaluated against C. tropicalis ATCC 750 biofilms and their synergistic potential with the standard antifungal fluconazole. Two compounds with amphiphilic nature were highly effective in preventing adhesion and inhibiting the formation of biofilm within 6 h. Additionally, they eradicated 24 h mature biofilms, as were demonstrated through various biofilm quantification techniques, including the crystal violet assay, XTT reduction assay, quantification of EPS, and ATR-FTIR analysis of biochemical composition of treated biofilms. In vitro studies revealed that AF(4) and AF(5) significantly reduced biofilm biomass and metabolic activity of cells embedded within the biofilm. Furthermore, morphological analysis of biofilms stained with Con A and FUN-1 using CLSM and COMSTAT analyses demonstrated a strong antibiofilm effect, including damaged cell surfaces and reduced hyphae structures, indicating effective biofilm disruption. Combinations of fluconazole with AF(5) exhibited improved synergistic anti-biofilm effects. However, AF(4) alone demonstrated strong potential to combat all phases of C. tropicalis biofilms.