The clinical context behind this bioprospecting effort is sobering. While Candida albicans remains the leading cause of candidiasis, infections caused by non-albicans Candida (NAC) species are continuously rising, now affecting an estimated 1.6 billion individuals worldwide and posing particular danger to immunocompromised patients in healthcare settings. NAC species often display reduced or intrinsic susceptibility to azole antifungals, contributing to higher rates of therapeutic failure. Candida krusei, a dimorphic yeast capable of causing skin, mucous membrane and systemic infections, is intrinsically resistant to fluconazole and rapidly develops acquired resistance to other antifungal drugs. The isolate used in this study, obtained from a clinical laboratory in Florianópolis, was confirmed resistant to fluconazole and intermediate to ketoconazole and itraconazole.

Biofilms compound the problem dramatically. When Candida species adhere to surfaces such as catheters and other medical devices, they encase themselves in a protective extracellular matrix that shields them from both host defenses and antifungal drugs. The mortality rate associated with biofilm-forming Candida infections is nearly two-fold higher than for planktonic infections, with deaths related to biofilm formation reaching up to 70 percent in candidemia cases. For C. krusei specifically, mortality in biofilm-related infections ranges from 40 to 59 percent. Against this backdrop, the Brazilian team, led by Leticia Bachmann of the Universidade Regional de Blumenau, set out to evaluate whether the complex mixture of phenolic compounds found in M. neoobscura leaves could attack the pathogen through multiple routes simultaneously.

The researchers collected leaves of M. neoobscura in May 2022 from a municipal natural park in Blumenau, dried and ground them, and extracted the material by maceration in a 70 percent hydroalcoholic solution for seven days. The resulting crude hydroalcoholic extract was then partitioned into fractions of increasing polarity: hexane, dichloromethane, ethyl acetate and an aqueous fraction, along with an insoluble fraction separated from the resuspended extract. Previous phytochemical characterization by the same research group had revealed that the extract is rich in phenolic compounds, with gallic acid as the major constituent, followed by chlorogenic acid and p-coumaric acid, alongside catechin, isoorientin, taxifolin, resveratrol, naringenin and eriodictyol.

The antifungal evaluation followed the Clinical and Laboratory Standards Institute protocol M27-A4, using broth microdilution in 96-well plates with RPMI 1640 medium buffered with MOPS, and amphotericin B as the positive control. Four clinical Candida isolates were tested: C. albicans, C. glabrata, C. tropicalis and C. krusei. The results were strikingly selective. Against C. krusei, the aqueous fraction stood out with a minimum inhibitory concentration (MIC) of 7.81 micrograms per milliliter, followed by the crude extract, ethyl acetate and insoluble fractions at 15.62 micrograms per milliliter, and the hexane and dichloromethane fractions at 62.50 micrograms per milliliter. According to the classification adopted in the study, values of 100 micrograms per milliliter or less are considered strong inhibitory activity. No activity was found against the other three isolates at concentrations up to 1000 micrograms per milliliter.

To probe the mechanism of action, the team employed two complementary assays. In the ergosterol binding assay, exogenous ergosterol was added to the test medium; if a compound complexes with membrane ergosterol, the added sterol sequesters it and MIC values rise. In the sorbitol protection assay, the osmoprotectant sorbitol shields fungal cells whose cell walls have been compromised, so increased MIC values in its presence indicate cell wall targeting. For all M. neoobscura samples, MIC values increased up to 128-fold, from 7.81 to 1000 micrograms per milliliter, in the presence of either ergosterol or sorbitol, suggesting that the extract and its fractions act concomitantly on both the fungal membrane and the cell wall. This dual mechanism is particularly significant because the cell wall exists only in fungal cells and not in human cells, meaning antifungal agents that inhibit cell wall synthesis are generally less harmful to patients.

The biofilm experiments added another dimension to the findings. Using crystal violet staining in polystyrene microtiter plates, the researchers determined that the C. krusei isolate is a moderate biofilm producer, with an average optical density of 0.636. When mature 48-hour biofilms were challenged with the extract and fractions at MIC and twice-MIC concentrations, all samples dissolved the biofilm by 45.6 to 60.2 percent relative to the negative control, with the dichloromethane fraction achieving the maximum inhibition of 60.2 percent. Amphotericin B, the positive control, inhibited biofilm by more than 80 percent. Notably, the aqueous fraction achieved antibiofilm activity similar to that of the dichloromethane and hexane fractions but at concentrations roughly threefold lower, reinforcing the potential contribution of its higher phenolic and flavanol content, which reached 297.0 milligrams of gallic acid equivalents per gram and 302.9 milligrams of catechin equivalents per gram.

The statistical framework underpinning these comparisons was distinctly modern. Rather than relying solely on classical frequentist tests, the team fitted a Bayesian log-normal model using the brms package in R, sampling via Markov Chain Monte Carlo. Convergence was verified with the Gelman-Rubin diagnostic, and posterior predictive checks confirmed that the model accurately replicated the observed absorbance data. Pairwise contrasts, adjusted by Bonferroni and evaluated through 95 percent credibility intervals, confirmed that all extract and fraction treatments significantly inhibited biofilm growth relative to the negative control. The hexane fraction at twice-MIC showed substantial inhibition with an estimate of −0.692 and a credibility interval strictly excluding zero, while the dichloromethane fraction at MIC followed closely at −0.670. Comparisons among treatments revealed distinct clusters of performance, with the hexane, dichloromethane and aqueous fractions forming the most potent group.

The pronounced activity of the aqueous and ethyl acetate fractions aligns with their phytochemical profiles. The aqueous fraction contained the highest levels of total phenolics and flavanols, while the ethyl acetate fraction concentrated the second-highest phenolic content and the highest flavonoid level. Phenolic compounds are well documented as antifungal agents, and the individual constituents of M. neoobscura have each shown activity against Candida species in prior studies. Gallic acid, with its multiple hydroxyl groups, can disrupt microbial cell membranes and has been reported to inhibit sterol 14α-demethylase and squalene epoxidase, reduce CYP51 enzyme activity essential for sterol biosynthesis, and induce apoptotic cell death in C. albicans. Chlorogenic acid can inhibit and destroy C. albicans hyphae, injure cellular membranes, and trigger apoptosis through mitochondrial membrane depolarization, reactive oxygen species generation and DNA fragmentation. Resveratrol also acts through apoptosis, while p-coumaric acid generates oxidative stress and interferes with ergosterol biosynthesis.

Several limitations temper the enthusiasm. The study evaluated only one clinical isolate per Candida species, and the substantial genotypic and phenotypic diversity among clinical isolates means the findings should be interpreted cautiously regarding broader clinical applicability. A reference C. krusei strain was unavailable, biofilm disruption was tested only at MIC and twice-MIC concentrations, and future work must determine whether the activity is fungistatic or fungicidal. Comprehensive safety evaluation, including hemolysis and in vivo toxicity studies, remains necessary before clinical translation, although previous MTT assays in L929 fibroblasts by the same group detected no cytotoxic effects. Nevertheless, the dual action on cell wall and membrane, the significant dissolution of mature biofilms, and the previously reported absence of cytotoxicity together position M. neoobscura as a promising bioprospecting lead from the Atlantic Rainforest, underscoring the importance of sustainable use of native Brazilian flora in the search for new antifungal therapies.

Subject of Research: Biology

Article Title: Antifungal and antibiofilm activity of crude hydroalcoholic extract and fractions of Myrcia neoobscura leaves against a clinical isolate of Candida krusei

Article References: Bachmann, L., Botelho, E. R., Arrabal, P. H., Munzlinger, P. E., da Silva Dantas, P. L., Krepsky, L. M., Mariotto, L. F., Alberton, M. D., & Rensi Botelho, T. K. (2026). Antifungal and antibiofilm activity of crude hydroalcoholic extract and fractions of Myrcia neoobscura leaves against a clinical isolate of Candida krusei. Frontiers in Natural Products, 5, Article 1914843. https://doi.org/10.3389/fntpr.2026.1914843

Image Credits: AI Generated

DOI: 10.3389/fntpr.2026.1914843

Keywords: Antifungal, antibiofilm, activity, crude, hydroalcoholic, extract, fractions, Myrcia, neoobscura, leaves, against, clinical