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

The research team, led by Ahmed Rezk, Heba S. Said, Sherin M. Elfeky, Mohammed El-Mowafy, and Ramadan Hassan of Mansoura University, began by surveying the local resistance landscape. They collected 64 clinical isolates of C. albicans from hospitals in Dakhlya governorate, drawn from urine samples, vaginal swabs, blood cultures, and other clinical sources between May and July 2022. Using standard disk diffusion and broth microdilution methods aligned with Clinical and Laboratory Standards Institute guidelines, they found that 16 isolates, a full 25 percent of the collection, were resistant to fluconazole. Minimum inhibitory concentrations among these resistant strains ranged from 64 to 2048 micrograms per milliliter, levels far above what fluconazole can achieve safely in patient tissue.

That resistance is not accidental. Fluconazole works by inhibiting the fungal enzyme lanosterol demethylase, encoded by the ERG11 gene, which is essential for building ergosterol, the structural backbone of the fungal cell membrane. Resistant fungi fight back in several ways: they mutate ERG11 so the drug binds poorly, they overproduce the target enzyme, and, most importantly for this study, they crank up expression of efflux pumps that simply eject the drug from the cell before it can do damage. The dominant player among these pumps is Cdr1, an ATP-binding cassette transporter embedded in the plasma membrane. Cdr1 operates like a molecular bilge pump: azole drugs drift into the cytoplasm, slip into a hydrophobic cavity in the transporter’s transmembrane domains, and are then expelled outward when ATP binding and hydrolysis at the nucleotide-binding domains drive a conformational flip from an inward-facing to an outward-facing state.

Disulfiram, chemically known as tetraethylthiuram disulfide, has a long and curious pharmacological history. Approved for aversion therapy in alcohol use disorder, it inhibits aldehyde dehydrogenase in the liver, causing acetaldehyde to accumulate and producing the flushing, nausea, and headaches that discourage drinking. But over the past two decades it has attracted attention as a repurposing candidate in oncology and infectious disease. Previous work had already hinted at antifungal potential: an earlier study showed disulfiram directly modulates Cdr1p, and a non-toxic concentration of the drug enhanced the sensitivity of yeast cells expressing the pump to fluconazole and miconazole. It has also shown activity against methicillin-resistant Staphylococcus aureus, Helicobacter pylori, and metallo-beta-lactamase enzymes.

To test whether that promise held up against real clinical resistance, the Mansoura team measured disulfiram’s own minimum inhibitory concentration against six representative fluconazole-resistant isolates, obtaining values between 10 and 20 micrograms per milliliter. They then repeated fluconazole susceptibility testing in the presence of a subinhibitory dose of disulfiram, one too low to inhibit fungal growth on its own. The results were striking: fluconazole’s MIC dropped by factors ranging from 2-fold to as much as 128-fold across the resistant isolates. In other words, a dose of disulfiram that by itself could not harm the fungus dramatically re-sensitized it to a drug it had previously shrugged off.

Checkerboard assays, in which both drugs are titrated in two dimensions across a microtiter plate, confirmed the synergy quantitatively. Using the fractional inhibitory concentration index, the team classified the combination as fully synergistic in three isolates, partially synergistic in two, and additive in one. No antagonism was observed. Under the conventional interpretation scheme, a FICI of 0.5 or below indicates synergy, meaning the drugs together accomplish more than the sum of their individual effects, a hallmark of a genuine resistance-reversing interaction rather than simple additive killing.

The molecular mechanism behind this synergy emerged from two complementary lines of evidence. First, quantitative reverse-transcription PCR on two resistant isolates, Ca15 and Ca39, showed that Cdr1 was overexpressed when the fungi were exposed to fluconazole alone, consistent with the pump being the engine of resistance. When disulfiram was added at subinhibitory concentrations, either alone or in combination with fluconazole, Cdr1 expression was downregulated relative to untreated controls. Second, molecular docking simulations using the recently solved cryo-EM structure of Cdr1 bound to fluconazole revealed that disulfiram can occupy two strategic positions on the transporter. At the hydrophobic substrate cavity in the transmembrane domain, disulfiram binds in a mode nearly identical to fluconazole, forming a hydrogen bond with Asn1240 and hydrophobic contacts with residues including Phe552, Leu555, Phe559, and Ile1237, with a calculated binding affinity of minus 6.46 kilocalories per mole, slightly stronger than fluconazole’s minus 6.07. At the ATP-binding site in the nucleotide-binding domain, disulfiram mimics ATP itself, hydrogen bonding with Thr195, Cys193, Gly190, Ser194, and Gly192, residues essential for ATP binding and hydrolysis. By competing at both sites simultaneously, disulfiram appears to jam the pump’s fuel supply and its cargo door at once, allowing fluconazole to accumulate in the cytoplasm and attack its target enzyme.

Beyond reversing resistance, disulfiram delivered a second blow to the fungus: it interfered with biofilm formation. Biofilms, the organized communities of cells encased in a protective matrix that adhere to catheters, mucosal surfaces, and medical devices, are notoriously tolerant of antifungals and a major driver of persistent infection. Using a tissue culture plate assay with crystal violet staining, the researchers showed that disulfiram reduced biofilm formation in a dose-dependent manner at sub-MIC concentrations of one-half, one-quarter, one-eighth, and one-sixteenth of its MIC. Remarkably, even at the weakest dose tested, one-sixteenth of the MIC, the drug still achieved roughly 14 to 37 percent inhibition of biofilm formation and significant eradication of mature, 24-hour-old biofilms. This dual activity, chemosensitization plus anti-biofilm action, suggests disulfiram could undermine two of the most stubborn survival strategies C. albicans deploys in the human host.

The study’s authors are careful to frame these results as a foundation rather than a finished therapy. All experiments were conducted in vitro, and the researchers explicitly call for follow-up work including site-directed mutagenesis, functional binding assays, and investigation of other resistance mechanisms such as ERG11 point mutations and the efflux genes Cdr2 and Mdr1. In vivo efficacy and pharmacodynamic studies will be essential before any clinical conclusions can be drawn. Still, the appeal of the strategy is considerable. Drug repurposing bypasses the years of safety profiling required for entirely new molecules, and combination therapy can lower effective doses of both agents, potentially reducing toxicity and slowing the further evolution of resistance. With fluconazole resistance rates in Egyptian bloodstream isolates recently reported as high as 58.3 percent, and with the researchers highlighting the infrequent use of routine antifungal susceptibility testing in the region, the need for such adjuvant approaches is urgent. If subsequent animal and clinical studies validate what the Mansoura team observed in the laboratory, a fifty-year-old sobriety drug could become an unexpected ally in preserving the usefulness of modern antifungal medicine.

Subject of Research: Reversal of fluconazole resistance in Candida albicans by disulfiram through Cdr1 efflux pump inhibition and biofilm disruption

Article Title: Disulfiram reverses fluconazole-resistance mediated by Cdr1 in Candida albicans and interferes with biofilm formation

Article References: Rezk, A., Said, H. S., Elfeky, S. M., El-Mowafy, M., & Hassan, R. (2026). Disulfiram reverses fluconazole-resistance mediated by Cdr1 in Candida albicans and interferes with biofilm formation. Applied Microbiology and Biotechnology, 110(1), Article 269. https://doi.org/10.1007/s00253-026-13977-w

Image Credits: AI Generated

DOI: 10.1007/s00253-026-13977-w

Keywords: Candida albicans, fluconazole resistance, disulfiram, Cdr1 efflux pump, drug repurposing, antifungal synergy, biofilm inhibition, ATP-binding cassette transporter, molecular docking, candidiasis, antimicrobial resistance, combination therapy