Table 2.
Inhibition of planktonic growth (A) of Candida albicans isolates and strains (n = 16), biofilm progression (development and maturation) of C. albicans (ATCC 10231) (B) by menthol alone and in combination with FLC (C).
| Assay | Planktonic growth | |||||
|---|---|---|---|---|---|---|
| Number of isolates | ||||||
| A. Broth micro dilution assay | MIC∗ of menthol (mM) | Susceptible (N = 8) | S-DD (N = 2) | Resistant (N = 6) | ||
|
| ||||||
| 1.79 | 3 | — | 2 | |||
|
| ||||||
| 3.58 | 5 | 2 | 4 | |||
|
| ||||||
| B. Biofilm assay | Biofilm formation (%) | |||||
|
| ||||||
| Menthol (mM) | 0 | 3.58 | 7.16 | 14.32 | 28.65 | 57.3 |
|
| ||||||
| Developing biofilm | 100 ± 0 | 21 ± 9.24 | 14 ± 0.97 | 13 ± 0.65 | 12 ± 1.14 | 12 ± 0.32 |
|
| ||||||
| Mature biofilm | 100 ± 0 | 45 ± 11 | 30 ± 5.17 | 25 ± 2.08 | 25 ± 3.78 | 25 ± 7.02 |
|
| ||||||
| C. Synergistic assay | ||||||
|
| ||||||
| C1. Planktonic growth (MIC) | ||||||
|
| ||||||
| Combinations | MIC FLZ (mg/L) | Menthol (mM) | FICs FLZ | Menthol | FICI | Interaction |
|
| ||||||
| FLZ alone | 128 | 0.0 | 1.0 | 0.0 | 1.0 | NI |
|
| ||||||
| Menthol + FLZ | 8.0 | 0.44 | 0.062 | 0.12 | 0.182 | S |
|
| ||||||
| Menthol alone | 0.0 | 3.58 | 0.0 | 1.0 | 1.0 | NI |
|
| ||||||
| C2. Developing biofilm (MIC90) | ||||||
|
| ||||||
| FLZ alone | ≥256 | 0.0 | 1.0 | 0.0 | 1.0 | NI |
|
| ||||||
| Menthol + FLZ | 8.0 | 1.79 | 0.031 | 0.062 | 0.093 | S |
|
| ||||||
| Menthol alone | 0.0 | 28.65 | 0.0 | 1.0 | 1.0 | NI |
|
| ||||||
| C3. Matured biofilm (MIC70) | ||||||
|
| ||||||
| Menthol + FLZ | 8.0 | 1.79 | 0.031 | 0.031 | 0.093 | S |
|
| ||||||
| Menthol alone | 0.0 | 28.65 | 0.0 | 1.0 | 1.0 | NI |
∗ MIC of menthol is fungicidal; thus, MIC and minimum fungicidal concentration (MFC) is same. S-DD: susceptible dose-dependent to fluconazole (According to CLSI M27 A3). ∗Stage of development: Developing biofilm = 24 h. Mature biofilm = 48 h. FIC = fractional inhibitory concentrations. FICI = ∑FICs. FLZ = fluconazole. (FICI value ≤ 0.5 = Synergy. FICI ≥0.5- ≤4.0 = No interaction and FICI ≥4 = antagonistic [22].