Silybin boosts posaconazole against Candida auris in vitro

A laboratory study of 10 clinical Candida auris isolates found that combining the natural compound silybin with the antifungal drug posaconazole produced synergistic activity in seven isolates. Using a broth-microdilution checkerboard assay based on the CLSI M27-A4 standard, the researchers classified an interaction as synergistic when the fractional inhibitory concentration index was no greater than 0.5. The posaconazole-silybin combination showed the highest synergy rate among the four azole combinations tested, although silybin alone did not inhibit fungal growth at the concentrations used.

The result was specific rather than universal. Silybin combined with voriconazole was synergistic against five of the 10 isolates, while the combination with itraconazole was synergistic against three. No synergy was detected with fluconazole, and one isolate showed antagonism with silybin plus itraconazole. Compared with the silybin-fluconazole reference, the difference in synergy rate was statistically significant only for the posaconazole combination after Holm adjustment. The exact 95% confidence interval for the 70% posaconazole-silybin synergy rate was 34.8% to 93.3%, emphasizing the preliminary scale of the evidence.

Mechanistic experiments focused primarily on the seven isolates that responded synergistically to posaconazole plus silybin. The combination lowered cellular ergosterol more than posaconazole alone in all seven, with significant differences ranging from p = 0.0291 to p less than 0.0001. An eighth isolate classified as showing no interaction did not exhibit a significant change. Ergosterol is a major fungal membrane sterol, and the azole target CYP51 participates in its synthesis. The findings are consistent with interference in membrane sterol production, but the researchers did not establish that silybin directly inhibits CYP51 or that this mechanism causes the observed synergy.

Reactive oxygen species measurements provided another correlate of antifungal action. In six of eight tested isolates, posaconazole plus silybin produced higher fluorescence than posaconazole alone, indicating greater intracellular reactive oxygen species accumulation; the differences were significant at p = 0.0217. Two isolates did not show significant increases. The authors were careful to distinguish association from mechanism: because the experiments did not test whether neutralizing reactive oxygen species reversed the combination effect, the elevated signal cannot yet be described as the definitive mediator of fungal killing or reduced biofilm survival.

Biofilm assays produced convergent results across developmental stages. In the crystal-violet assay, posaconazole alone inhibited biofilm formation by 13.7% to 61.5%, depending on the isolate. Adding silybin raised inhibition to 24.2% to 79.7%. The combination was significantly more effective than posaconazole alone in six of eight strains, improving inhibition by 16.5 to 46.5 percentage points, although no significant difference was detected in strains 382 and 390. An XTT assay, which measures fungal metabolic activity, likewise found stronger suppression by the combination in five of eight isolates, all with p less than 0.0001; three strains showed no significant difference between treatments.

The combination also affected established microbial communities. In SYTO9 and propidium iodide staining assessed by confocal laser scanning microscopy, posaconazole alone had limited activity against preformed mature biofilms. Posaconazole plus silybin decreased green fluorescence from viable cells and increased red fluorescence associated with membrane-compromised or dead cells. These observations indicate lower biofilm-cell viability after combination treatment. Together with the formation and metabolic assays, the results show activity against both developing and mature biofilms, but the microscopy was performed in laboratory culture rather than in an infected host or medical device.

Real-time quantitative PCR suggested that the combination altered several pathways relevant to adhesion, biofilm regulation, cell-wall integrity, and sterol synthesis. Compared with posaconazole monotherapy, the combination significantly reduced expression of bcr1, mkc1, erg2, and als5 in all seven synergistic isolates. Expression of efg1 fell in six, erg11 in six, and scf1 in five. The non-synergistic isolate showed significant reductions only in mkc1 and als5. These measurements were made in planktonic cells, so they may not represent gene activity within a biofilm. The authors also noted that altered messenger RNA levels do not by themselves prove loss of adhesion, biofilm formation, or other biological functions.

Silybin is a flavonolignan obtained from milk thistle and is used as a medicinal agent and dietary supplement. Posaconazole is an established antifungal, and each has an individual safety profile. That does not establish the safety or effectiveness of their combination. No direct cytotoxicity testing was performed in mammalian cells, no animal efficacy or safety study was conducted, and no clinical outcomes were evaluated. The experimental concentration is also a major translational obstacle: the lowest silybin concentration producing synergy was 4 micrograms per milliliter, compared with an effective in-vivo concentration of approximately 0.0965 micrograms per milliliter and typical oral exposure constrained by silybin’s reported absolute bioavailability of about 0.45%.

Taken together, the study provides proof-of-concept evidence for a combination that was most consistently synergistic with posaconazole in a small collection of multidrug-resistant C. auris isolates. Its effects included reduced ergosterol, increased reactive oxygen species, stronger inhibition of biofilm formation and metabolism, reduced viability in mature biofilms, and lower expression of selected biofilm- and sterol-associated genes. The findings support further investigation rather than immediate clinical use. Larger and more diverse strain panels, direct mechanistic tests, mammalian-cell toxicity studies, animal pharmacodynamic experiments, and improved silybin formulations would be needed to determine whether the laboratory synergy can be reproduced in vivo and translated into a treatment that balances efficacy, exposure, and safety.

Subject of Research: In-vitro synergistic antifungal and antibiofilm effects of silybin combined with posaconazole against Candida auris.

Article Title: In vitro synergistic antifungal activity of silybin-posaconazole combination against Candida auris

Article References: Peng, M., Zhu, X., Hu, Q., Fan, Y., Mao, Y., Chen, X., Qin, H., Zhang, H., & Sun, Y. (2026). In vitro synergistic antifungal activity of silybin-posaconazole combination against Candida auris. Frontiers in Microbiology, 17, Article 1881511. https://doi.org/10.3389/fmicb.2026.1881511

Image Credits: AI Generated

DOI: 10.3389/fmicb.2026.1881511

Keywords: Candida auris, silybin, posaconazole, antifungal synergy, drug resistance, biofilms, ergosterol, oxidative stress, azole resistance, in-vitro research, vitro, synergistic