Exploring essential oils and propolis as adjuncts or alternatives to azole antifungals for Candida infections: Laboratory experimental study

Background. Fungal infections caused by Candida species represent a significant and growing threat to human health, particularly among immunocompromised individuals, such as those undergoing chemotherapy, organ transplant recipients, and patients in intensive care units. These opportunistic pathogens are responsible for a wide spectrum of clinical manifestations, ranging from superficial mucosal infections to life-threatening invasive candidiasis. The increasing resistance of Candida spp. to conventional antifungal agents, especially the azoles, has prompted the scientific community to explore alternative therapeutic options, including natural products. Essential oils derived from plants of the Lamiaceae family and propolis, a resinous substance produced by honeybees, have emerged as promising candidates due to their diverse bioactive compounds and broad-spectrum antimicrobial properties. Objective. the primary objective of this study was to evaluate the in vitro susceptibility of clinical Candida isolates to both conventional azole antifungals and plant-derived agents, and to assess their ability to inhibit biofilm formation. Methods. A total of fifty clinical Candida isolates were obtained from urine samples and identified using PCR with standard species-specific primers and MALDI-TOF mass spectrometry, following the manufacturer’s protocols. The study employed two main experimental approaches: the disk diffusion method to assess antifungal activity against planktonic cells, and a static microtiter biofilm assay to evaluate antibiofilm effects. The agents tested included marjoram oil, sage oil, thyme oil, and propolis, while the conventional antifungals used for comparison were clotrimazole (10 µg), itraconazole (10 µg), and fluconazole (25 µg). Biofilm inhibition was calculated based on optical density measurements at 570 nm (OD₅₇₀). Results. Propolis exhibited the highest antifungal activity among the natural products tested. Its inhibition zones measured 20.46 ± 2.87 mm against C. glabrata and 21.70 ± 1.16 mm against C . parapsilosis . Marjoram oil followed, with zones of 16.11 ± 1.48 mm and 15.50 ± 0.85 mm, respectively, while sage oil showed moderate activity (11.56 ± 1.56 mm and 10.90 ± 1.20 mm). Thyme oil produced no inhibition zones against either species, indicating a lack of activity in the disk diffusion assay. Among the conventional antifungals, clotrimazole was the most effective against C. glabrata (23.96 ± 6.41 mm), whereas against C. parapsilosis , propolis surpassed clotrimazole, with an inhibition zone of 21.70 mm compared to 13.80 mm for clotrimazole. In terms of biofilm inhibition, propolis again demonstrated the strongest effect, reducing biofilm formation by 44.9% in C. glabrata and 41.2% in C. parapsilosis . Marjoram oil inhibited biofilm formation by 33.9% and 28.6%, sage oil by 24.6% and 19.3%, and thyme oil by 19.5% and 39.5%, respectively. Interestingly, thyme oil, despite being inactive in the disk diffusion test, showed a notable species-dependent antibiofilm effect, particularly against C. parapsilosis . Conclusion. Propolis and marjoram oil demonstrate promising antifungal and antibiofilm activity against C. glabrata and C. parapsilosis . Thyme oil is inactive in disk diffusion but exhibits species-dependent antibiofilm effects. Clotrimazole remains highly active against C. glabrata , but propolis may be superior against C. parapsilosis . These findings suggest that natural agents warrant further investigation as adjuncts or alternatives to conventional azole antifungals, particularly in cases of azole-resistant Candida infections.