1. Background
The growing emergence of candidiasis and its epidemiological changes and drug resistance clearly justify the need for conducting studies on fungal infections. Among all the Candida species, C. albicans is considered the most frequent species that forms part of the natural flora of mucus and the skin. These yeasts are a major cause of mortality especially in immunocompromised patients (1).
An antifungal agent for treating candidiasis belongs to the group of azoles. Despite having a low toxicity, successful treatments with azoles such as fluconazole are rare due to the extensive resistance of different species of Candida (2). In recent years, the clinical isolates of fluconazole-resistant Candida have emerged in Iran. Using different antimicrobial compounds, including medicinal herbs, is therefore recommended for preventing or controlling this resistance. A long history of applying herbal medicine for treating diseases can be traced in Iran. Silybum marianum, commonly known as milk thistle, is an ancient medicinal plant that widely grows in Iran (3, 4) and has long been used to treat different diseases, including liver and gallbladder disorders. The active constituents of Silybum marianum are obtained from its dried seeds containing approximately 70% - 80% silymarin (5, 6), which is a mixture of flavonolignan isomers, including silybin, isosilybin, dehydrosilybin, silychristin, silydianin and a few flavonoids mainly taxifolin (7). Silymarin presents a wide range of biological and pharmacological activities, including anti-oxidative and anti-inflammatory metastatic effects (8).
2. Objectives
The present study was conducted to determine the effect of Silybum marianum, individually and in combination with fluconazole, on clinical Candida species isolated from patients in- vitro using a community approach to traditional medicine and treatment with natural substances.
3. Methods
3.1. Phenotypic Identification of Candida Species
The present cross-sectional study was conducted on 85 clinical samples randomly collected from the skin and mucosa of patients, aged 15 - 60 years, suspected of candidiasis and presenting to four hospitals in Iran’s northern city of Gorgan. A total of 55% of the study population were female.
To identify the isolated microorganisms, swab samples were cultured on Sabouraud dextrose agar (SDA, Merck, Germany) and incubated for 48 hours at 35°C. After performing direct microscopic checks using 10% KOH and sub-culturing on SDA, other tests were conducted, including germ tube test, culturing on Chromogenic CHROMagar (Hi Media, India) and the carbohydrate assimilation test using API 20CAUX kits (BioMe’rieux, France).
3.2. Antifungal Susceptibility Test
The drug susceptibility test was performed using the broth microdilution test according to the CLSI-M27-A3 guideline (9). After preparing the fluconazole solution (Gibco, Germany) in water and diluting it at a range of 1024 - 2 µg/mL, dilutions were poured into the 96-well ELISA microplates containing RPMI-1640 (with glutamines, without bicarbonate and with a PH indicator) and MOPS buffer (Sigma, USA). Yeast suspensions (1 × 103 cfu/mL) were then inoculated in microplate wells and incubated for 48 hours at 35°C. The ELISA Reader was used to confirm the results when reporting MIC. To determine the minimum fungicidal concentration (MFC), 100 µL of the wells’ content with a concentration higher than the MIC together with 100 µL of the positive control were separately inoculated on SDA and incubated for 48 hours at 35°C.
According to the instructions of this committee, Candida strains with MIC ≤ 8 µg/mL were considered susceptible, ones with MIC = 16 - 32 µg/mL were considered susceptible-dose dependent, and ones with MIC ≥ 64 µg/mL were considered resistant to fluconazole. The present study used Candida albicans ATCC90028 as the control strain.
3.3. Preparing Silybum marianum Extract
To obtain the plant extract, Silybum marianum was collected from Gorgan’s plains. After extracting the seeds from the Silybum marianum flower, they were was mixed and soaked in 70% ethanol at a ratio of 1:10 (each 100 cc of the extract was soaked in 1000 cc of ethanol), and placed in a shaker. After 24 hours, the mixture was filtered and placed in a rotary container to evaporate the solvent (ethanol). The aqueous extract was obtained through maceration, and the filtrate solution was therefore heated for 15 minutes up to boiling. The mixture was then stored in a container at room temperature to be cooled. Afterwards, the mixture was passed through Whatman grade 1 filter paper. The solution was placed in a closed glass at 4°C during usage.
3.4. Evaluating the Inhibitory Ability of Silybum marianum Extract
Broth microdilution was performed with concentrations of 4096 - 8 µL/mL to investigate the anti-candidiasis effects of Silybum marianum extract. A microbial suspension with a concentration of 103 cfu/mL was therefore prepared first and the plant stock was made by dissolving Silybum marianum extract in dimethyl sulfoxide (DMSO). Afterwards, 100 µL of the stock was inoculated in the microplate wells containing 100 µL of RPMI medium, and was serially diluted. One hundred µL of the yeast suspension was also added to each well. The minimum concentration inhibiting fungal growth up to 90% compared to positive controls is considered MIC90. The negative control well contained the stock with RPMI, and the positive control well contained RPMI with yeast suspensions. MFC was ultimately determined.
3.5. Susceptibility Test of Fluconazole in Combination with Silybum marianum Extract
To make the stock solution and reach a concentration of 1024 µg/mL, 1 mg of fluconazole powder was dissolved in water and 1 mL of Silybum marianum extract in DMSo. To prepare serial dilutions, 25 μL of the drug and 25 μL of the extract were added to the first well of the 96-well microplate containing 50 μL of RPMI medium. After adding 50 μL of yeast suspensions at a concentration of 103 cfu/mL and performing a 48-h incubation at 35°C, MIC and MFC were evaluated and interpreted.
4. Results
Direct microscopic tests revealed 73 cases of yeast cells in 85 study patients, 90.4% of which were identified as Candida species through phenotypic tests. Most of the isolated species of Candida strains were C. albicans (72.7%) (Table 1). The present study found the highest resistance to fluconazole to be associated with C. glabrata (81.8%) and C. albicans (72.9%) (Table 2).
| Candida spp. | Absolute Frequency | Relative Frequency |
|---|---|---|
| C. albicans | 48 | 72.7 |
| C. glabrata | 11 | 16.7 |
| C. tropicalis | 3 | 4.5 |
| C. parapsilosis | 4 | 6.1 |
| Total | 66 | 100 |
Frequency of the Candida Species Isolated from Hospitalized Patients
| Species | Resistant | S-DD | Suseptibility |
|---|---|---|---|
| C. albicans | 35 (72.9) | 9 (18.8) | 4 (8.3) |
| C. glabrata | 9 (81.8) | 0 (0) | 2 (18.2) |
| C. tropicalis | 1 (33.3) | 0 (0) | 2 (66.7) |
| C. parapsilosis | 0 (0) | 0 (0) | 4 (100) |
Frequency Distribution of Drug- Resistance Among Candida Isolatesa
The mean MIC of fluconazole against C. albicans and C. glabrata was also determined at 210 µg/ mL, and most growth fluctuations were observed at concentrations of 128 and 64.
The changes in the MIC of Silybum marianum against C. albicans and C. glabrata indicated significant differences between the growth and non-growth of fluconazole-resistant Candida (Table 3). The MFC value of Silybum marianum extract determined at a concentration of 4096 μL/mL for 86% of fluconazole-resistant Candida isolates suggested fungicidal properties in this extract at high concentrations.
| Extract/Density, µL/mL | Strains 1 × 103 cfu/mL | No Growth | Growth | Comparison |
|---|---|---|---|---|
| 2048 | C. albicans | 31 (88.6) | 4 (11.4) | x2 = 0, Significant |
| 2048 | C. glabrata | 7 (77.8) | 2 (22.2) | x2 = 0, Significant |
Comparison of Growth and No-Growth of Fluconazole-Resistant Candida albicans and Candida glabrata in Concentration of 2048 μL/mL of Silybum marianum Extracta
The mean MIC of Silybum marianum extract in combination with fluconazole against C. albicans and C. glabrata was reported at 60 µg/mL, and the most growth fluctuations were observed at concentrations of 32 and 64. Significant differences were also observed between growth and non-growth of the drug-resistant isolates (Table 4). The concentration of Silybum marianum extract in combination with fluconazole that inhibited 90% of C. glabrata isolates (MIC90) was found to be 128 μg/mL, four times lower than that of fluconazole (MIC90 = 512 μg/mL) (P < 0.01). This rate was also fourfold for C. albicans isolates. The concentration of Silybum marianum extract in combination with fluconazole that inhibited 90% of Candida isolates was 16 times lower than that of the extract individually. Comparing the in-vitro antifungal susceptibility of C. albicans and C. glabrata revealed differences in MIC50 and MIC90 between the isolates.
| Extract + Fluconazole, µL/mL | Strains 1 × 103 cfu/mL | No Growth | Growth | Comparison |
|---|---|---|---|---|
| 128 | C. albicans | 33 (94.3) | 2 (5.7) | x2 = 0, Significant |
| 128 | C. glabrata | 8 (89) | 1 (11) | x2 = 0, Significant |
Comparison of Growth or No-growth of Candida albicans and Candida glabrata Strains in the Presence of the Combination of Silybum marianum Extract and Fluconazolea
The MFC of Silybum marianum extract in combination with fluconazole against fluconazole-resistant C. glabrata was found to be four times lower and against C. albicans isolates to be 16 times lower than the MFC of the single extract.
5. Discussion
Research suggests a significant increase in the prevalence of opportunistic infections caused by Candida species. Although C. albicans is the predominant yeast, Candidiasis has been reported to be caused by other Candida species, including C. glabrata, C. krusei, C. tropicalis, C. paraposilosis, C. guilliermondii, C. famata and C. lusitaniae (10, 11). Prolonged or frequent exposure to antifungal drugs can be associated with the risk of antifungal resistance among Candida strains (12).
The present study found 73% of C. albicans isolates to be resistant to fluconazole. A study conducted by Diba et al. using the disk-diffusion method found C. albicans species to present low sensitivity to fluconazole (13). Matar et al. showed that 4% of the isolates were resistant to fluconazole. A study conducted in South Africa reported 100% susceptibility to fluconazole among C. albicans isolates (14). Badiee et al. assessed the susceptibility of 178 C. albicans isolates to fluconazole using broth microdilution and the frequency of isolated fluconazole-resistant C. albicans was estimated at 4.6% (12). Kabli found 26.1% of 107 C. albicans isolates to be resistant to fluconazole using the disk-diffusion method (15). Bagg et al. reported a resistance to fluconazole of about 3% in 93 C. albicans isolated from patients with advanced cancer (16).
These figures are less than those found in the present study. Different levels of antibiotic resistance reported in different regions of Iran and across the word can be associated to genetic changes in the causative strains, differences in the amount of antibiotics used and changes in the availability of new antibiotics in different regions.
The present study found high resistance to fluconazole in C. albicans. Future research is therefore strongly recommended to focus on drug resistance in Candida species, especially to azole agents.
A review of literature shows that the MIC of fluconazole on Candida species such as C. albicans, C. glabrata, C. tropicalis and C. parapsylosis are 0.06 - 128, 0.03 - 8 and 0.06 - 16 μg/mL (17), which are comparable with the present findings. The present study found C. parapsylosis to show the lowest resistance (0%) to fluconazole at a concentration of 16 μg/mL, as 99% of them were eliminated. Fluconazole has been shown to exert its maximum antifungal effect on C. parapsylosis, and its MIC to be about 0.5 - 16 μg/mL. Moreover, a study conducted in China reported reductions in azole susceptibility among C. tropicalis isolates (18).
Scientists therefore seek modern therapeutic approaches and alternative compounds with antimicrobial properties. Given the diversity of plant species in Iran and the stress placed by the country’s top-level authorities on food and medicine, including the vice president, herbal compounds have been particularly emphasized in Iran in recent years.
Research confirms the inhibitory effects of marjoram essential oil on Aspergillus, Honsonella and Dermatophyte fungi, including Trichophyton rubrum and C. albicans (19). The present research also confirmed the inhibitory effect of Silybum marianum extract on Candida species at high concentrations.
Anti-Candida activities caused by high levels of thymol and carvacrol have been associated to marjoram essential oil (20). Karaman et al. observed the inhibitory effect of the essential oil obtained from the aerial parts of Thyme on the growth of C. albicans, C. tropicalis and Saccharomyces cerevisiae (21). The antifungal effect of Silybum marianum on Dermatophytes has been reported to be higher compared to other plants such as Allium, Juglans regia, Sativum, Piper betle, Azadirachta (22, 23). The present study therefore sought to inspect the antifungal effect of this plant on fluconazole-resistant Candida species. In certain types of fungi, the combination of two drugs may weaken the effects of individual drugs (24). Researchers have therefore focused on the effects of antibiotics combined with other antimicrobial compounds.
The antifungal activity of fluconazole combined with lovastatin and its effect on gene expression in the pathway of ergosterol biosynthesis in C. albicans have been evaluated. Studies reporting an MIC of growth suggest that lovastatin acts synergistically with fluconazole in-vitro (25). The present findings suggest that Silybum marianum extract combined with fluconazole significantly affects fluconazole-resistant Candida. After combining fluconazole with the extract, the levels of MIC50, MIC90 and MFC decreased in yeasts.
The present study found silybin or silidianin to be a potential inhibitor of Candida species. The observed antifungal effects can therefore be associated with one of the compounds cited.
5.1. Conclusions
The results of the present study showed that a combination of fluconazole and aqueous extracts of Silybum marianum exerts greater antifungal effects on the resistant strains of C. albicans and C. glabrata compared to fluconazole or the plant extract individually.
