Candida albicans, a commensal yeast of the human microbiota, can become an opportunistic pathogen, causing a range of infections from superficial thrush to life-threatening systemic candidiasis. A significant challenge in treating C. albicans infections is its ability to form biofilms. Biofilms are complex communities of microorganisms encased in a self-produced extracellular matrix (ECM), adhering to biotic and abiotic surfaces. This biofilm lifestyle confers increased resistance to conventional antifungal agents such as fluconazole and echinocandins (e.g., caspofungin), making treatment significantly more difficult. The high prevalence of multi-drug resistant (MDR) C. albicans strains further exacerbates this problem. The current study focuses on exploring alternative antifungal strategies by investigating the potential of aqueous extracts from commonly used spices—garlic, clove, and Indian gooseberry—to inhibit biofilm formation and control established biofilms of MDR C. albicans isolates. These spices have a long history of use in traditional medicine and possess known antimicrobial properties. The investigation aims to determine the efficacy of these extracts in combating MDR C. albicans biofilms, identifying the active compounds responsible, and characterizing the morphological changes induced in the biofilms by these extracts. The ultimate goal is to explore the possibility of developing novel, natural, safe, and cost-effective therapeutics to combat this growing clinical challenge.

Extensive research highlights the significant role of biofilms in the pathogenesis of C. albicans infections. Biofilms provide a protective environment, shielding the yeast cells from antifungal drugs and the host's immune response. Several studies have demonstrated the enhanced resistance of biofilm-associated C. albicans to various antifungal agents, including azoles and echinocandins. The mechanisms underlying this resistance include reduced drug penetration, altered drug targets, and the presence of persister cells within the biofilm. The rising incidence of MDR C. albicans strains adds to the urgency for the development of new therapeutic strategies. Traditional medicine offers a rich source of potential antimicrobial agents, with several plant-derived compounds exhibiting antifungal activity against C. albicans. Previous studies have explored the antifungal properties of individual spices, including garlic, clove, and Indian gooseberry, demonstrating their inhibitory effects on C. albicans growth in planktonic cultures. However, their effectiveness against established biofilms of MDR strains remains largely uninvestigated. Therefore, this study fills an important gap in understanding the efficacy of these readily available, cost-effective, and safe extracts against drug-resistant C. albicans biofilms.

The study employed a multi-faceted approach involving various microbiological and analytical techniques. Eleven C. albicans isolates, including ten clinical isolates from patients with invasive candidiasis and a standard strain (MTCC-3017), were used. Biofilm formation ability was assessed using a biofilm tube test and point inoculation on Trypticase Soy Agar (TSA). Two high biofilm-forming isolates (M-207 and S-470) exhibiting resistance to fluconazole and caspofungin were selected for further investigation. Aqueous and organic extracts of various spices were prepared using a standardized protocol. Agar well and disc diffusion assays were performed to determine the zone of inhibition (ZOI). The minimum inhibitory concentration (MIC) was determined using a broth microdilution assay, and cell viability was assessed with an MTT assay. The effect of the aqueous extracts on pre-formed biofilms was also investigated. High-performance thin-layer chromatography (HPTLC) and liquid chromatography-mass spectrometry (LC-MS) were used to identify the major bioactive compounds in the aqueous extracts of garlic, clove, and Indian gooseberry. Microscopic techniques, including bright-field microscopy, phase-contrast microscopy, and fluorescence microscopy (using calcofluor white staining), were employed to visualize the morphology and structure of the biofilms at different growth stages and following treatment with the extracts. Statistical analysis, including one-way ANOVA followed by Tukey's multiple comparison test, was used to evaluate statistical significance.

The study revealed that the selected C. albicans isolates (M-207 and S-470) exhibited significant resistance to fluconazole (25 mcg) and caspofungin (8 mcg). However, the aqueous extracts of garlic, clove, and Indian gooseberry demonstrated notable antimycotic activity against these MDR isolates. The aqueous garlic extract showed the strongest inhibitory effect against both M-207 and S-470 biofilms, achieving a 50% reduction in growth (MIC50) within 12 hours of incubation at concentrations of 1 mg/mL for M-207 and 1.25 mg/mL for S-470. Clove and Indian gooseberry extracts were also effective against S-470 biofilms, with MIC50 values of 0.215 mg/mL and 0.537 mg/mL, respectively, at 12 h. HPTLC and LC-MS analysis confirmed the presence of allicin in garlic, ellagic acid in clove, and gallic acid in Indian gooseberry extracts. Microscopic observations revealed significant morphological alterations in the biofilms following treatment with the aqueous extracts. Treated biofilms exhibited reduced biomass, altered cell arrangements, and decreased extracellular matrix compared to untreated controls. The MTT assay further confirmed the reduction in cell viability following treatment with the aqueous spice extracts, with the greatest effect observed at 12 hours of incubation. Notably, even visual inspection of treated biofilm cultures in Petri dishes revealed less turbidity and reduced biofilm formation on glass slides, indicating the efficacy of these extracts.

The findings of this study strongly support the potential of using aqueous extracts of garlic, clove, and Indian gooseberry as alternative antimycotics to combat MDR C. albicans biofilms. The observed inhibitory effects, coupled with the identification of major bioactive compounds, provide a strong foundation for further research. The efficacy of these extracts against MDR isolates is particularly significant, given the increasing clinical challenge posed by drug resistance. The ease of preparation, low cost, and generally recognized safety of these spice extracts make them attractive candidates for the development of novel antifungal therapies. The observed morphological changes in biofilms further suggest a potential mechanism of action involving disruption of biofilm architecture and cell viability. Future research should focus on the purification and characterization of the active compounds within these extracts to optimize their antifungal activity and explore potential synergistic effects with existing antifungal agents. In vivo studies are crucial to validate the effectiveness of these extracts and to evaluate their safety and efficacy in treating C. albicans infections in animal models.

This study provides compelling evidence for the potential of using aqueous extracts from garlic, clove, and Indian gooseberry to control drug-resistant C. albicans biofilms. The extracts demonstrated significant antimycotic activity, identified bioactive compounds, and showed visible biofilm disruption. This research paves the way for further investigation towards developing safe and cost-effective alternative therapies for Candida infections, potentially leading to novel treatments and formulations (tablets, injections, powders, sprays, douches, and coated catheters).

The study primarily focused on in vitro investigations, and further in vivo studies are needed to confirm the findings. The use of whole aqueous extracts limits the precise identification and quantification of all bioactive compounds responsible for the observed effects. Additional research is necessary to optimize extraction methods and explore the potential synergistic effects with existing antifungal treatments. The study used a limited number of clinical isolates, and a more comprehensive analysis involving diverse strains would strengthen the conclusions.