Therapeutic potential and synergistic effects of antifungal and off target drugs against clinical isolates of Cryptococcus laurentii

Cryptococcus laurentii is an emerging opportunistic yeast pathogen associated with unpredictable antifungal susceptibility and biofilm-mediated resistance. This study investigated the therapeutic potential and synergistic interactions of standard antifungals (fluconazole, amphotericin B) and off-target drugs against clinical isolates of C. laurentii. Three clinical isolates recovered from blood (n = 2) and cerebrospinal fluid (n = 1) were confirmed by phenotypic characteristics and PCR amplification of the 18 S rRNA gene (~ 480 bp). Biofilm formation was screened using Congo red agar and quantified via crystal violet assay. Susceptibility testing employed CLSI-guided disc/well diffusion and broth microdilution methods for antifungals and 14 off-target drugs. Minimum biofilm inhibitory concentrations (MBICs) were determined, and synergistic effects were evaluated using checkerboard assays (fractional inhibitory concentration index, FICI) in planktonic cells and biofilms, supplemented by coverslip biofilm reduction assay. Isolates showed moderate biofilm production (mean OD₅₉₅ = 0.32 ± 0.008), fluconazole susceptibility (mean MIC = 0.77 µg/mL), and amphotericin B resistance (mean MIC = 4.48 µg/mL). Diclofenac sodium exhibited the strongest off-target activity (mean MIC = 1.32 µg/mL). Checkerboard assays revealed synergy between fluconazole + amphotericin B (FICI = 0.5) and fluconazole + diclofenac sodium (FICI = 0.32) in planktonic cells, with additive effects against biofilms (FICI = 0.9). The fluconazole-diclofenac combination significantly disrupted biofilm matrix. Diclofenac sodium enhances fluconazole efficacy and disrupts biofilms, supporting drug repurposing as an adjuvant strategy for managing biofilm-associated C. laurentii infections.