The work addresses one of the most pressing problems in infectious disease medicine. Invasive fungal infections cause substantial illness and death among immunocompromised patients, including people with neutropenia, transplant recipients, and those in intensive care. The arsenal of antifungal drugs is narrow, and the azole class, which includes itraconazole, voriconazole, posaconazole, isavuconazole, and fluconazole, has become a cornerstone of both prevention and treatment. Azoles work by inhibiting lanosterol 14-alpha-demethylase, the CYP51 enzyme essential for building ergosterol, the structural backbone of fungal cell membranes. Decades of widespread and sometimes excessive use have driven the emergence of resistant strains through CYP51 mutations, overexpression of drug efflux pumps, and biofilm formation, and resistance frequently translates into clinical treatment failure.

Rather than waiting for entirely new drugs, which is a prolonged and costly endeavor, the research team from Yangtze University and Jingzhou Hospital in Hubei, China, pursued the adjuvant strategy: combining existing antifungals with a second compound that enhances their effect. Adjuvants can allow lower doses of the primary drug, reducing toxicity, slowing the evolution of resistance, and extending the clinical lifespan of established therapies. Usnic acid was a natural candidate. Long used in traditional medicine for its antimicrobial and anti-inflammatory properties, it has documented activity against Gram-positive bacteria and mycobacteria, and prior studies suggested it can inhibit bacterial efflux pumps and disrupt microbial membranes. Its potential as a broad-spectrum azole adjuvant, however, had never been systematically evaluated in fungi.

The team tested usnic acid against five azoles using CLSI-standardized broth microdilution and checkerboard assays on clinical isolates of Aspergillus species, Candida species, the black yeast Exophiala dermatitidis, and Cryptococcus neoformans. Interactions were classified with the fractional inhibitory concentration index, with synergy defined as an FICI of 0.5 or less, indifference between 0.5 and 4.0, and antagonism above 4.0. Isavuconazole was tested against molds and fluconazole against Candida and Cryptococcus, reflecting their clinical uses. Rates were reported with 95 percent Wilson confidence intervals, and comparisons employed Fisher-Freeman-Halton and pairwise Fisher exact tests with Benjamini-Hochberg correction, a statistically rigorous framework unusual for its thoroughness in this field.

The results were strikingly lopsided. Usnic acid alone showed no appreciable antifungal activity, confirming its role purely as a helper compound. Combined with posaconazole, however, it achieved synergy in 74 of 110 isolates. The corresponding rates were 11.82 percent for itraconazole, 9.09 percent for fluconazole, 5.45 percent for voriconazole, and zero percent for isavuconazole. Within individual pathogens, the posaconazole pairing reached 70.31 percent synergy against Aspergillus species, 66.67 percent against Candida species including 80 percent against the multidrug-resistant yeast Candida auris, and 92.86 percent against Exophiala dermatitidis. Where synergy occurred, minimum inhibitory concentrations of posaconazole fell to as low as 0.03 to 0.13 micrograms per milliliter, meaning dramatically less drug would be needed to inhibit fungal growth.

Just as important was where the combination failed. Against Cryptococcus neoformans, usnic acid paired with voriconazole showed antagonism in all eight isolates, and paired with fluconazole it was antagonistic in seven of eight, a finding with direct clinical safety implications for patients with cryptococcosis. The posaconazole combination, by contrast, caused antagonism in only one of 110 isolates overall, and the itraconazole combination caused none. When the team stratified isolates by posaconazole susceptibility, the pattern grew more nuanced: synergy in Aspergillus fumigatus was largely confined to susceptible strains, dropping from 100 percent in sensitive isolates to 16.67 percent in resistant ones, whereas Aspergillus terreus and Exophiala dermatitidis showed broad synergy regardless of resistance status. Species such as A. niger, C. tropicalis, and C. auris showed minimal or no synergy in their sensitive isolates. The lesson, the authors emphasize, is that a one-size-fits-all approach will not work; the adjuvant must be matched to the species and resistance phenotype.

To understand the mechanism, the researchers probed two cellular processes in nine selected isolates, seven of them synergistic. First, they measured reactive oxygen species using a fluorescent probe and flow cytometry. Posaconazole alone elevated intracellular ROS in all tested strains, but adding usnic acid pushed ROS levels significantly higher in five of the seven synergistic strains. This aligns with prior work showing that usnic acid targets fungal mitochondria, inhibiting respiratory chain complexes I and III and uncoupling oxidative phosphorylation. The combined assault may generate an oxidative burst that overwhelms the fungus’s antioxidant defenses, causing irreversible damage and cell death.

Second, the team ran rhodamine 6G efflux assays to measure the activity of the multidrug transporters fungi use to pump azoles out of their cells. In four of the seven synergistic strains, the combination significantly suppressed efflux pump function compared with posaconazole alone. Two strains, 382 and PY1b, displayed both heightened ROS accumulation and efflux inhibition simultaneously, suggesting the synergy operates through mechanistically diverse and potentially interconnected pathways rather than a single unified mechanism. Notably, three synergistic isolates showed no efflux change at all, their synergy apparently driven purely by oxidative stress, mirroring earlier observations that even isolates carrying identical CYP51 mutations can differ in their gene expression profiles.

Why posaconazole benefits most remains partly a matter of pharmacology. Posaconazole is the most lipophilic azole tested and a well-documented substrate of fungal multidrug efflux transporters, so usnic acid’s membrane-perturbing activity likely enhances its uptake into fungal cells while its efflux inhibition prevents the drug from being pumped back out. Voriconazole, by contrast, is the most hydrophilic azole and largely enters cells by passive diffusion; membrane perturbation may actually reduce its accumulation, and in Cryptococcus, whose large transporter repertoire differs substantially from that of ascomycete pathogens, the combination appears to trigger adaptive responses that counteract azole action. The sharp divergence between itraconazole and posaconazole, which share the same CYP51 target, underscores that drug-specific membrane partitioning and transporter specificity, not the molecular target, decide whether an adjuvant succeeds.

The authors acknowledge the study’s limits: everything was conducted in vitro, and definitive proof of the mechanistic hypotheses, including direct measurement of intracellular drug accumulation, awaits further experiments, as do animal studies of efficacy and safety. Still, the translational implications are considerable. Boosting posaconazole, a first-line therapy for invasive mold infections, could enable lower, safer dosing while tackling resistance in formidable pathogens such as multidrug-resistant Exophiala dermatitidis and Candida auris. There may even be agricultural dividends, since the combination showed 100 percent synergy against sensitive Aspergillus flavus isolates, a major crop pathogen behind aflatoxin contamination of maize and peanuts. If in vivo work confirms what the Petri dishes suggest, a compound that lichens have quietly produced for millennia could become an unlikely ally in the escalating war against drug-resistant fungi.

Subject of Research: Synergistic enhancement of azole antifungal drugs by the lichen-derived compound usnic acid against clinical fungal isolates

Article Title: Enhanced antifungal efficacy of azole drugs through combination with usnic acid

Article References: Tang, M., Yang, X., Ge, L., Xie, S., Zeng, Y., Chen, T., Zhang, H., & Sun, Y. (2026). Enhanced antifungal efficacy of azole drugs through combination with usnic acid. Frontiers in Microbiology, 17, Article 1864975. https://doi.org/10.3389/fmicb.2026.1864975

Image Credits: AI Generated

DOI: 10.3389/fmicb.2026.1864975

Keywords: usnic acid, posaconazole, azole resistance, antifungal adjuvant, Aspergillus, Candida auris, Cryptococcus neoformans, efflux pump inhibition, reactive oxygen species, drug synergy, invasive fungal infections, lichen natural product