Synergistic inhibition of pathogenic fungi by oleanolic acid combined with azoles

ABSTRACT Invasive fungal infections and emerging antifungal resistance mediated by pathogenic fungi, notably Candida spp., Aspergillus spp., Cryptococcus neoformans , and Exophiala dermatitidis, pose a critical challenge to global public health systems. This study evaluated the synergistic antifungal activity of oleanolic acid (OA) combined with five azole agents (itraconazole [ITR], voriconazole [VOR], posaconazole [POS], isavuconazole [ISA], and fluconazole) against Candida spp., Aspergillus spp., E. dermatitidis , and C. neoformans . Using CLSI-compliant broth microdilution assays (M27-A3/M38-A2), we determined the minimum inhibitory concentrations (MICs) of oleanolic acid against four fungal pathogens and evaluated in vitro synergistic interactions with five azoles to assess their therapeutic potential. OA exhibited no intrinsic fungicidal activity when administered alone. However, synergistic inhibition was observed in specific azole combinations across tested pathogens: Candida spp. ( n = 18) demonstrated 33% (6/18) and 56% (10/18) synergy rates with ITR and POS, respectively. Aspergillus spp. ( n = 30) showed enhanced synergism at 80% (24/30) for ITR and 97% (29/30) for POS. All OA-azole combinations were synergistic against C. neoformans ( n = 8), with rates of 75% (6/8) for ITR, 75% (6/8) for VOR, 87.5% (7/8) for ISA, and 100% (8/8) for POS. In contrast, E. dermatitidis ( n = 21) responded only to OA/POS (52%, 11/21). Overall synergistic frequencies were POS (75%, 58/77) > ITR (47%, 36/77) > ISA (9%, 7/77) > VOR (8%, 6/77). The OA-azole combinations demonstrated significant reductions in azole MIC values and exhibited synergistic antifungal activity, particularly against Aspergillus spp. and C. neoformans . While the mechanistic basis of OA’s chemosensitization remains uncharacterized, its multi-target pharmacological profile underscores potential utility in combating antifungal resistance. IMPORTANCE Invasive fungal infections and antifungal resistance are significant global health challenges. This study highlights the potential of oleanolic acid (OA) combined with azoles to combat these issues. OA alone had no antifungal activity, but its combinations with azoles exhibited notable synergy against major pathogens, particularly Aspergillus spp. and Cryptococcus neoformans . Notably, OA combined with posaconazole showed synergy against Exophiala dermatitidis for the first time. These findings suggest that OA could enhance the efficacy of existing antifungal agents, reduce their required doses, and potentially overcome resistance. This research underscores the importance of exploring OA as a novel adjuvant to improve antifungal therapy.