Fluconazole and biogenic silver nanoparticles-based nano-fungicidal system for highly efficient elimination of multi-drug resistant Candida biofilms

Abstract

HypothesisSecretion of extracellular hydrolytic enzymes such as secreted aspartyl proteinases (SAP) and biofilm formation are considered as important virulence determinants in resistant strains of Candida sp. which facilitate the pathogenicity towards host cells. The resistance in fungal pathogens results in use of high dosage of antifungal drug, therefore in order to study the effect of reduced amount of antifungal, combination therapy was utilized. Currently, no data related to combined effect of mycogenic silver nanoparticles and fluconazole, in SAP inhibition by resistant Candida spp. has been described. In this study, synergistic combination along with anti-virulence was used as a way for discovering new antifungal potential of mycogenic silver nanoparticles (Ag-NPmyc). ExperimentsThis study utilized Ag-NPmyc, synthesized using Aspergillus oryzae SZ1 and their combined effect with fluconazole on planktonic and 24 h (h) biofilm forms of clinical isolates of six Candida species (C. albicans, C. galabrata, C. parapsilosis, C. krusie, C. tropicalis, C. albicans ATCC 24433). The cytotoxic effects of Ag-NPmyc mycogenic using brine shrimp lethality assay was also determined. Ag-NPmyc and fluconazole (FLC) suspensions were used to treat Candida sp. biofilms grown on polystyrene surfaces for 24 h (h) and their efficacy was determined by MIC, time-kill growth curve analysis, cell viability analysis (XTT reduction assay), inhibition of virulence factors exopolymeric substances (EPS) and SAP activity. Nanoparticles were characterized using Ultraviolet–Visible (UV–Vis) spectroscopy, X-Ray Diffraction (XRD) and transmission electron microscopy (TEM). FindingsThe surface plasmon resonance band of Ag-NPmyc was observed at 430 nm by Ultraviolet–Visible spectroscopy, X-Ray Diffraction and TEM confirmed with uniform, spherical shaped and monodispersed with size range of 1–50 nm. Maximum cytotoxicity was achieved after 0.3 mg/ml. The results showed that combination use of Ag-NPmyc with FLC exhibit strong in vitro antifungal synergy (FICI values ranged from 0.2812 to 0.375) against the majority of the Candida strains tested with gradual decrease in CFU till 12 h, and after 15 h fungistatic growth was observed. Comparable effects on biofilms of Candida sp. were obtained. Overall, >60% reduction of metabolic activity was observed in different Candida sp. at 16 μg/ml FLC in combination with Ag-NPmyc. The EPS inhibition (%) in planktonic cells ranged from 2 to 73, 3–82 and 1–19 by 15 ppm Ag-NPmyc/FLC, 25 ppm Ag-NPmyc/FLC and FLC alone, respectively. Whereas in case of biofilm forms, EPS inhibition (%) ranged from 1 to 84, 1–93 and 1–32 by 15 ppm Ag-NPmyc/FLC, 25 ppm Ag-NPmyc/FLC and FLC alone, respectively. Highest EPS inhibition in planktonic cells was observed with Ag-NPmyc/FLC, 25 ppm which ranged 3–82%. Whereas in case of biofilm forms, EPS inhibition ranged from 1 to 93% with the same combination. Less inhibition seen in case of FLC treated Candida spp. planktonic (1–15%) and biofilm (1–14%) cells. The results obtained, showed that combinations of 15 ppm and 25 ppm Ag-NPmyc with FLC effectively inhibited the SAP in Candida spp. with an inhibition range of 1–82% and 1–79%, respectively in case of planktonic cells. Whereas 1–78% and 2–92% in case of biofilm cells as compared to less inhibition (<20%) seen in case of FLC treated Candida spp. planktonic and biofilm cells. ConclusionAg-NPmyc in combination with fluconazole promoted significant reduction in virulence factors of resistant forms of Candida sp. Ag-NPmyc at different concentrations were not found cytotoxic to brine shrimps. The results highlight the improved antifungal effect of Ag-NPmyc in combination with FLC against planktonic and biofilm forms of different species of Candida while inhibiting (>70%) in SAP production. The effect of azole-NP combination on EPS inhibition was also significant. Synergistic effect showed inhibition in SAP production both in planktonic and biofilm forms of Candida spp.