Combating multidrug-resistant Candida species and their virulence attributes using Saussurea costus leaf extract: Chemical profiling and bioactivity assessment

Abstract

Multidrug-resistant Candida infections represent a critical therapeutic challenge, particularly among immunocompromised patients and in healthcare-associated settings. In this study, the in vitro antifungal and anti-virulence activities of the ethyl acetate extract of Saussurea costus leaves were systematically investigated against clinical Candida isolates. Among 80 clinical specimens examined, 57 Candida isolates were recovered and identified through conventional phenotypic methods, CHROMagar™, and the VITEK® 2 automated identification system. Candida albicans was the predominant species (50.9%), followed by C. tropicalis and C. krusei (24.6% each). Antifungal susceptibility analysis revealed significant variability among tested agents (p < 0.001), with clotrimazole, nystatin, ketoconazole, and amphotericin B showing the highest inhibitory activity, whereas itraconazole and fluconazole exhibited comparatively lower activity and higher resistance rates. Hemolytic profiling demonstrated that the majority of isolates were non-hemolytic (75.42%), while 24.56% exhibited β-hemolytic activity, with significant differences among isolates (p < 0.05). The ethyl acetate extract of S. costus leaves (extraction yield: 1.4% w/w) demonstrated reproducible antifungal activity against multidrug-resistant Candida isolates, with inhibition zones ranging from 14.0 ± 2.1 to 23.0 ± 2.5 mm. The majority of isolates exhibited MIC values of 31.25 mg/mL, whereas isolate 23 showed reduced susceptibility with a higher MIC of 62.5 mg/mL (p < 0.001). The extract significantly inhibited biofilm formation, with MBIC values ranging from 0.488 to 7.813 mg/mL (p < 0.05). In the hyphae-competent isolate 32, extract treatment markedly suppressed filamentation, reducing hyphal formation from 78 ± 4% in untreated controls to 8 ± 2%, accompanied by significant decreases in hyphal length (31.5 ± 2.1 to 4.7 ± 1.3 μm) and hyphal density (120 ± 10 to 15 ± 3 cells/field) (p < 0.001). Scanning electron microscopy further confirmed pronounced disruption of biofilm architecture, characterized by reduced surface coverage (approximately 85% to 48–50%), increased porosity (approximately 10–12% to 32–35%), and significant reductions in biofilm cluster size and density (p < 0.001). GC–MS profiling revealed a chemically diverse composition, with multiple constituents putatively identified by spectral library matching, including eugenol, caryophyllene oxide, myristicin, fatty acid esters, and a major compound tentatively assigned as dehydrocostus lactone. Collectively, these findings demonstrate that the ethyl acetate extract of S. costus exhibits statistically supported in vitro antifungal and anti-virulence activities against multidrug-resistant Candida isolates. However, these findings remain preliminary and restricted to in vitro evaluation; further studies involving bioassay-guided fractionation, compound-level validation, safety assessment, mechanistic investigations, and in vivo evaluation are required to determine its biological significance and translational potential.