Aqueous spice extracts as alternative antimycotics to control highly drug-resistant extensive biofilm-forming clinical isolates of Candida albicans

Candida albicans biofilms confer enhanced resistance to conventional antifungals, complicating treatment of invasive candidiasis. The study investigates whether whole aqueous extracts of commonly used spices—garlic (Allium sativum), clove (Syzygium aromaticum), and Indian gooseberry (Phyllanthus emblica)—can inhibit biofilm formation by multidrug-resistant, high biofilm-forming clinical isolates of C. albicans. The rationale is based on longstanding traditional use and reported antimicrobial properties of these spices, coupled with the need for safe, cost-effective alternatives to current antimycotics. The study aims include screening clinical isolates for biofilm capacity, assessing the antimycotic activity and MIC of spice extracts, profiling major bioactive constituents, and visualizing structural effects on biofilms.

Prior work highlights that C. albicans biofilms are associated with increased drug resistance and virulence and that media composition and serum can influence biofilm formation and adhesion. Various plant-derived products, including garlic and clove, have shown antifungal activity against Candida species; Indian gooseberry has documented antimicrobial and health-promoting properties. Studies report that garlic constituents (e.g., allicin) can impact virulence factors and enhance antimicrobial activity; clove contains phenolics like ellagic acid and eugenol with antimicrobial and anti-inflammatory activities; Indian gooseberry is rich in gallic acid and exhibits antioxidant and cytotoxic effects at higher concentrations. Literature also shows mixed effects of serum (FBS) on biofilm formation across organisms and conditions, and that clinical isolates may behave differently from reference strains. These findings support exploring aqueous spice extracts as antimycotics against biofilm-forming, drug-resistant C. albicans.

- Strains: Standard C. albicans MTCC-3017 (ATCC-90028) and 10 clinical isolates (M-207, M-529, S-470, U-2647, U-3713, U-3800, U-3893, U-427, U-499, D-4) from various clinical samples of patients with invasive candidiasis. - Identification: Lactophenol cotton blue staining, germ tube test, and CHROMagar Candida colony color to confirm C. albicans. - Culture maintenance: Subculture on Malt Yeast Agar (MYA), 37°C, 24 h; glycerol stocks (15% v/v) stored at −86°C. - High biofilm former screening: Point inoculation on TSA, YEPD, SDA; incubation 37°C for 16 h to observe lawn formation. Biofilm tube test with crystal violet to categorize biofilm formers. - In vitro biofilm induction: 96-well polystyrene microtiter plates; adhesion for 90 min at 37°C with 10^6 cells/mL inoculum; wash with PBS; growth in various media (MYB, YEPD, PDB, SDB, YMB, TSB, RPMI-1640). Some plates pre-coated with FBS (100 μL/well, 37°C, 24 h) to assess effect on adhesion; OD600 measured over time. - Preparation of extracts: Fresh whole aqueous extracts—garlic and Indian gooseberry prepared by crushing 10 g in 5 mL sterile water; clove by mixing 5 g powder with 10 mL water. Centrifugation (10,000 rpm, 10 min, 4°C), filter, use supernatant. Also prepared organic solvent extracts (petroleum ether, ethyl acetate, chloroform, methanol, ethanol, butanol) similarly for initial screening. Estimated total solids: garlic 200 mg, clove 43 mg, gooseberry 86 mg (dry weight in starting aliquots). - Antimicrobial screening: Agar well and disc diffusion on Mueller-Hinton Agar (MHA) with 10^6 cells/mL lawn; apply 100 μL extract to wells or supersaturated 5 mm discs. Controls: water (aqueous) and respective organic solvents; standard antifungals: fluconazole 25 μg and caspofungin 8 μg discs. Measure zones of inhibition (ZOI) after 16 h at 37°C. - MIC determination (broth microdilution): After 90 min adhesion and PBS wash, add fresh medium plus aqueous extracts in microtiter wells; concentration ranges: garlic 0.25–10 mg (and tested up to 200 mg in diffusion), clove 0.05–0.537 mg, gooseberry 0.107–4.3 mg (dry weight equivalents). Incubate 0–24 h; measure OD600 to determine MIC50 (lowest concentration yielding ≥50% growth reduction at a given time point). Sterile water control. Only aqueous extracts advanced beyond ZOI screening. - Viability (MTT assay): After incubation, wash with PBS, add 50 μL of 5 mg/mL MTT, incubate 3 h at 37°C; dissolve formazan with 150 μL acidified isopropanol; read A540. Performed at MIC50 and additional concentrations; also on preformed biofilms (24 h and 48 h) treated with garlic. - Biofilm on glass slides: Grow biofilm in TSB in Petri plates with immersed glass slides; treat at 24 h with aqueous extracts at MIC50 equivalents (M-207: garlic 1 mg; S-470: garlic 1.25 mg, clove 0.215 mg, gooseberry 0.537 mg). Record visual turbidity and microscopy. - Chemical profiling: HPTLC using silica gel 60 F254 plates; mobile phases optimized per spice. Visualize at 254/366 nm and derivatize (ninhydrin, ferric chloride). Standards: alliin (garlic), ellagic acid (clove), gallic acid (gooseberry). LC-MS: Shimadzu LC with Luna C18 column, gradient programs per extract; detection at 210 nm (garlic), 254/215 nm (clove), 275 nm (gooseberry). Identify allicin, ellagic acid, gallic acid by retention time and mass (protonated/deprotonated ions). - Microscopy: Bright-field with crystal violet and lactophenol cotton blue staining at multiple time points (1–120 h, and up to 12 days for growth observation). Phase-contrast imaging directly in 96-well plates at 1–120 h (20×). Fluorescence microscopy with calcofluor white (1 mg/mL) and 10% KOH, excitation 380 nm/emission 475 nm at 3, 6, 12, 24 h; image quantification by ImageJ. - Statistics: One-way ANOVA with Tukey’s multiple comparisons to assess FBS vs non-FBS effects on biofilm formation.

- Two clinical isolates, C. albicans M-207 and S-470, were identified as high biofilm formers via point inoculation on TSA (complete lawn at 16 h) and biofilm tube test. - Choice of growth medium influenced biofilm growth: TSB supported highest growth to 48 h; YEPD showed reduced growth beyond 24 h. FBS pre-coating did not significantly affect adhesion or biofilm formation for M-207 and S-470. - Drug resistance: Both isolates showed resistance to fluconazole (25 μg) and caspofungin (8 μg) by disc diffusion (no ZOI). - Antimicrobial screening: Whole aqueous extracts exhibited stronger antimycotic activity than most organic solvent extracts. Garlic aqueous extract inhibited biofilms of both M-207 and S-470; clove and Indian gooseberry aqueous extracts inhibited S-470 but not M-207. - MIC50 at 12 h (by OD600) for developing biofilms: • M-207: garlic 1.0 mg. • S-470: garlic 1.25 mg; clove 0.215 mg; Indian gooseberry 0.537 mg (dry weight equivalents per well). - Cell viability at MIC50 (MTT): • M-207 + garlic: 24.4% viability. • S-470 + garlic: 26.9% viability; + clove: 29.9%; + gooseberry: 36.1%. - Preformed biofilms (garlic only): At MIC50, viability was higher than in developing biofilms—24 h preformed: 49.27% (M-207), 53.1% (S-470); 48 h preformed: 50.49% (M-207), 56.57% (S-470). - Visual and microscopic assessments showed reduced turbidity and biomass in treated samples, with maximal inhibition around 12 h; at 24 h, increased extracellular matrix in treated sets suggested decreased efficacy and potential persistence of cells. - Chemical profiling confirmed key phytochemicals: allicin in garlic (LC-MS RT ~19.79 min; m/z 163.1 [M+H]+), ellagic acid in clove (RT ~23.54 min; m/z 300.9 [M−H]−), and gallic acid in Indian gooseberry (RT ~18.73 min; m/z ~168.6 [M−H]−). HPTLC Rf bands adjacent to standards supported their presence. - Unique morphology with low garlic concentrations showed creased biofilm near wells; higher concentrations produced clear ZOI and smoother distant biofilm morphology.

The study demonstrates that whole aqueous extracts of garlic, clove, and Indian gooseberry can inhibit biofilm formation by multidrug-resistant C. albicans isolates, directly addressing the challenge of antifungal resistance inherent to biofilms. Aqueous garlic extract was broadly effective (M-207 and S-470), while clove and Indian gooseberry were effective against S-470, indicating strain-dependent susceptibility. The lack of impact from FBS on biofilm adhesion for the high biofilm-forming isolates simplifies in vitro modeling and suggests strong inherent adhesiveness. Media selection critically influenced growth and biofilm development, with TSB optimal for subsequent assays. The superior activity of aqueous extracts over organic solvent extracts supports eco-friendly, potentially safer extraction approaches and aligns with traditional usage. The MIC50 and MTT data showed substantial reductions in biomass and metabolic activity within 12 h, though treatment of preformed biofilms was less effective than prevention/inhibition during development, reflecting known biofilm tolerance. Microscopy corroborated quantitative assays, showing reduced cell density and biofilm matrix upon treatment; however, increased ECM by 24 h in treated sets suggests persistence and possible adaptive responses diminishing efficacy over time. Chemical analyses verified the presence of major bioactives—allicin, ellagic acid, and gallic acid—likely contributing to the observed antimycotic effects. Collectively, the findings support the potential of these readily available spices, particularly garlic, as adjunct or alternative antimycotics targeting biofilm-associated C. albicans, with implications for developing affordable, safe therapeutics.

This work reports a simple, cost-effective approach using whole aqueous extracts of garlic, clove, and Indian gooseberry to control biofilm formation by multidrug-resistant, high biofilm-forming C. albicans clinical isolates. FBS did not significantly influence adhesion in the studied isolates, while culture media selection did. Effective inhibitory concentrations at 12 h were 1.0 mg garlic (M-207) and 1.25 mg garlic, 0.215 mg clove, 0.537 mg Indian gooseberry (S-470). Phytochemical profiling confirmed allicin, ellagic acid, and gallic acid as dominant components in the respective extracts. Structural and biochemical analyses showed reduced cell numbers and transitions away from robust biofilm phenotypes upon treatment. These results suggest that whole aqueous spice extracts are effective and could be further optimized; purified fractions may offer even greater potency. Future directions include purification and characterization of active constituents, mechanistic studies, evaluation against broader clinical panels and mixed-species biofilms, biocompatibility and toxicity assessments, in vivo efficacy studies, and formulation development (e.g., tablets, sprays, douches, coatings for medical devices such as catheters).

The study was conducted in vitro on a limited number of isolates, with detailed efficacy characterized primarily for two high biofilm-forming strains (M-207 and S-470). While aqueous extracts showed strong early inhibition (notably at 12 h), reduced efficacy and increased extracellular matrix were observed by 24 h, suggesting persistence phenomena. No in vivo evaluations or comprehensive toxicity/biocompatibility assessments were performed, and mechanisms of action were not dissected. The amounts/concentrations reported are based on crude dry-weight equivalents and may not directly translate to clinical dosing. Strain-dependent differences (e.g., M-207 not inhibited by clove or gooseberry) indicate variability that requires broader validation.