1. Background
In recent years, morbidity and mortality are increased significantly by severe fungal infections (1). Candida species have been one of the most common pathogens responsible for fungal infections, which cause hospital-acquired sepsis with annually mortality rate of up to 40% (2). Opportunistic fungi cause fungal infections, especially in vulnerable people with special conditions such as pregnancy or HIV-positive and immune-compromised patients who need intensive treatment with broad-spectrum antibiotics (3-5). Nowadays most of the available effective antifungal agents are based on polyenes (amphotericin B), echinocandins (caspofungin, micafungin, and anidulafungin) and triazoles (fluconazole, itraconazole, voriconazole, and posaconazole) (6, 7). However, scientists are looking for new natural and inorganic antimicrobial agents (8, 9). The recent research on metal nanoparticles showed that silver nanoparticles (nanosilvers) have received special attention as a possible antimicrobial agent (10-16). Since ancient times, silver has been used widely to treat infections and has strong inhibitory effects as well as a broad spectrum of antimicrobial activities against microorganisms, which has been thoroughly investigated (1, 9, 15, 17). This toxicity effect on bacteria has been investigated for more than 60 years (16) and in comparison to other metals, silver exhibits lower toxicity to mammalian cells and higher toxicity to microorganisms (18). Nanosilver exerts antimicrobial effects through interacting with main components of microorganisms including DNA (19), microbial proteins (20), and cell wall (20, 21); moreover, nanosilver produces reactive oxygen species (ROS) (21). The accumulation of intracellular ROS is as an important regulator for starting early apoptosis phase (22). Subsequently, increasing level of intracellular ROS lead to initiation of mitochondrial fragmentation (23).
2. Objectives
Regarding comparison of difference between antimicrobial effect of nanosilver and some antibiotic agents on Candida albicans, we compared the effect of nanosilver with fluconazole and their combination on collected fluconazole-resistant and fluconazole-sensitive C.albicans.
3. Materials and Methods
In this experimental study, 18 fluconazole-resistant C. albicans isolated from the patient’s blood at the Shiraz University of Medical Sciences, Shiraz, Iran, two fluconazole-resistant C. albicans isolated from the patient’s blood at the Mycology Laboratory of Tehran University, Tehran, Iran, and one standard sample (ATCC10261) were used. We studied effect of fluconazole, nanosilver, and their combination on these C. albicans samples. The yeasts were identified using conventional mycological procedures.
In first stage, to identifying the fluconazole minimal inhibitory concentration (MIC) we used Clinical and Laboratory Standards Institute (CLSI) (M27-A3) protocol “Broth micro dilution”, which included:
- Preparation of fluconazole stock solution (2048 µg/mL; potency, 999 µg/mL) (Pars Daru, 99.95% potency): Briefly, 21.5 mg of fluconazole was dissolved in 500 µL of absolute ethanol, and 10 mL of distilled water was added. The stocks were stored in -20°C.
- Preparation of RPMI 1640 Medium: Briefly, 1.73 g MOPS [3- (N-morpholino propane sulfonic acid)] was dissolved in 50 mL of RPMI1640 Medium after filtration and was stored in 4°C.
- Preparation some yeast suspension: After 24 hours incubation at 35°C, five C. albicans colonies in PDA (Potato Dextrose Agar) medium were mixed in 5 mL of physiology serum. After 15 second of vortex, they were diluted by RPMI-1640 medium to 1/100 and after that 1/20; therefore, this suspension had 0.5 × 103 to 2.5 × 103 cells.
- Preparation of serial dilution of fluconazole: Chosen plate had 12 wells; we added 1 mL of RPMI to each one and then added 1 mL of fluconazole stock (2048 µg/mL) to first well. After turning up and down, we infused 1 mL of this well to second well and went on to tenth well. Therefore, tenth well had 0.5 µg/mL of fluconazole stock. Eleventh well had just 1 mL of RPMI (Negative control) and twelfth well as a positive control had 1 mL of RPMI and 100 µL of fungi suspension.
- Identification of drug sensitivity tests: We added 100 µL of yeast suspension to each well except negative control, and incubated them at 35°C for 48 hours.
In second stage, we used this protocol to identify the nanosilver’s MIC:
- Preparation of Nanosilver stock solution (100 µg/mL): Klebsiella pneumoniae was injected in Muller Hinton, incubated in 37°C for 24 hours, and then centrifuged at 12000 rpm for five minutes. Then 1 mL of floating liquid was added to 100 mL of silver nitrate (AgNO3, 1 mM) and after five minutes, existence of nanosilver was shown by color changing to light brown. Spectrum analysis was performed by spectrophotometer UV-Vis (Model Sessile 9200; resolution, 1 nm; Japan). Specification of nanosilver was illustrated by transmission electron microscope (model EM Philips, Eindhoven, the Netherlands) and energy-dispersive spectroscopy.
- Preparation of RPMI 1640 Medium: Briefly, 1.73 g of MOPS [3- (N-morpholino propane sulfonic acid)] was dissolved in 50 mL of RPMI1640 medium after filtration and was stored at 4°C.
- Preparation of some yeast suspension: After 24 hours of incubation in 35°C, five C. albicans colonies in PDA medium (diameter, 1 mm) were mixed in 5 mL of physiology serum. After 15 second of vortex, they were diluted by RPMI medium to 1/100 and after that 1/20. Therefore, this suspension had 0.5 × 103 to 2.5 × 103 cells.
- Preparation serial dilution of Nanosilver: Selected plate had nine wells. We added 1 mL of RPMI to each on and then added 1 mL of nanosilver stock solution (64 µg/mL) to first well. After turning it up and down, we infused 1 mL of this well to second well and went on to seventh well. Therefore, seventh well had 0.5 µg/mL of Nanosilver stock solution. Eighth well had just 1 mL of RPMI (Negative control) and ninth well, as a positive control, had 1 mL of RPMI and 100 µL of fungi suspension.
- Identification of drug sensitivity tests: We added 100 µL of yeast suspension to each well except negative control, and incubated them at 35°C for 48 hours.
In third stage, to identifying the MIC of nanosilver plus fluconazole was performed as follows:
- Preparation of Fluconazole plus Nanosilver stock solution Fluconazole (2048 µg/mL) diluted by RPMI medium solution to 16 µg/mL, and Nanosilver stock solution (100 µg/mL) diluted by distilled water to 4 µg/Ml.
- Preparation of RPMI 1640 Medium: Briefly, 1.73 g of MOPS [3- (N-morpholino propane sulfonic acid)] was dissolved in 50 mL of RPMI1640 medium and after filtration, was stored at 4°C.
- Preparation of yeast suspension: After 24 hours incubation at 35°C, five C. albicans colonies in PDA medium (diameter, 1mm) were mixed in 5 mL of physiology serum. After 15 seconds of vortex, they were diluted by RPMI medium to 1/100 and after that 1/20. Therefore, this suspension had 0.5 × 103 to 2.5 × 103 cells.
- Preparation of serial dilution of fluconazole plus nanosilver: chosen plate had 12 wells. We added 1 mL of RPMI to each one and then added 1 mL of nanosilver stock solution (4 µg/mL) to first well; After turning up and down, we infused 1 mL of this well to second well and went on to sixth well. Therefore, sixth well had 0.0625 µg/mL of Nanosilver stock. Seventh well had just 1 mL of RPMI (Negative control) and eighth well, as a positive control, had 1 mL of RPMI and 100 µL of fungi suspension. Then we added 1 mL of fluconazole (16 µg/mL) to each one except seventh and eighth wells.
- Drug sensitivity tests: we added 100 µL of yeast suspension to each well except negative control, and incubated them at 35°C for 48 hours.
4. Results
Result of fungi static and fungicidal activities of fluconazole against C. albicans showed: 1) The MIC of fluconazole concentration for standard sample was 16 µg/mL (Table 1). 2) The growth of 20 fluconazole-resistant C. albicans was inhibited at MICs > 512 µg/mL (Table 1).
Result of fungi static and fungicidal activities of nanosilver against C. albicans showed: 1) The MIC of nanosilver for standard sample was 4 µg/mL. (Table 2). 2) The MICs of nanosilver for 20 resistant C. albicans were 2 µg/mL (58%) and 4 µg/mL (42%).
Result of fungi static and fungicidal activities of nanosilver plus fluconazole (8 µg/mL) on C. albicans showed: 1) the combination had better inhibitory effect on the growth of standard C. albicans when MIC of fluconazole (8 µg/mL) was combined with MIC of Nanosilver (0.0625 µg/mL) (Table 3). 2) Results on 20 resistant C. albicans showed there are several MIC of nanosilver: 40% of resistant C. albicans samples grew on 0.25 µg/mL, 11% on 0.0625 µg/mL, 22% on 0.03125 µg/mL, and 27% had no growth on 0.03125 (Table 3).
Table 1. The Growths of Standard and Fluconazole-Resistant Candida albicans on Difference Fluconazole Concentrations
| Variables | Fluconazole Concentrations, µg/mL | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| 0.5 | 1 | 2 | 4 | 8 | 16 | 32 | 64 | 128 | 256 | 512 | |
| ResistantCandida albicans samples | + | + | + | + | + | + | + | + | + | + | + |
| Standard sample | + | + | + | + | + | - | - | - | - | - | - |
Table 2. The Growths of Standard Sample and Fluconazole-Resistant Candida albicans on Difference Nanosilver Concentrations
| Variables | Nanosilver Concentrations, µg/mL | |||||||
|---|---|---|---|---|---|---|---|---|
| 0.5 | 1 | 2 | 4 | 8 | 16 | 32 | 64 | |
| 58% of resistantCandida albicans samples | + | - | - | - | - | - | - | - |
| 42% of resistant Candida albicans samples | + | + | - | - | - | - | - | - |
| Standard sample | + | + | + | - | - | - | - | - |
Table 3. The Growth of Standard and Fluconazole-resistant Candida albicans on Difference Nanosilver Concentrations Combined with 8 µg/mL Fluconazole
| Variables | Nanosilver Concentrations (0.5-0.0625 µg/mL) Plus 8 µg/mL of Fluconazole | ||||
|---|---|---|---|---|---|
| 0.015625 | 0.03125 | 0.0625 | 0.125 | 0.25 | |
| 22% of resistant Candida albicans samples | + | + | - | - | - |
| 11% of resistant Candidaalbicans samples | + | + | + | - | - |
| 40% of resistant Candida albicans samples | + | + | + | + | + |
| 27% of resistant Candida albicans samples | + | - | - | - | - |
| Standard sample | + | - | - | - | - |
5. Discussion
Between azoles, Fluconazole has excellent in vitro activity against C. albicans at a wide range of body sites and tissues (24, 25). In addition, fluconazole is effective against some other Candida species, including Candida tropicalis, Candida parapsilosis, and Candida glabrata (25, 26). While the most important problem in treatment by the chemical antimicrobial agents is multidrug resistance, silver, as an antimicrobial agent in various fields, has brought some hope (17). It is well known that inorganic drug such as silver ions and its compounds have strong antimicrobial effects (27). In our study, the MICs of fluconazole and nanosilver against Standard C. albicans were 8 µg/mL and 2 µg/mL, respectively. Investigation of our finding shows the followings:
- The study of results in first stage showed that MIC for standard and drug resistant C. albicans were 256 to 512 µg/mL and ≥ 64 µg/mL, respectively. Moreover, these results were similar to another researches such as a study by Pfaller et al. that reported MIC ≥ 64 μg/mL (28), or study by Enwuru et al. on HIV-positive patients that showed fluconazole’s MIC of 64 μg/mL against C. albicans (29).
- In second stage, comparison between MIC of nanosilver and fluconazole showed that nanosilver inhibited C. albicans growth seven-fold to nine-fold more than fluconazole did.
- In third stage, the MIC analysis showed that nanosilver combined with fluconazole had the most effective activity against C. albicans. Another study by Kim et al. in 2008 showed that amphotericin plus nanosilver and fluconazole plus nanosilver were the most effective combinations against trichophyton/mentagrophytes and C. albicans, respectively (30).
According to our study, fluconazole with insignificant amount of nanosilver exhibit higher antifungal activity against pathogenic C. albicans compare with fluconazole and nanosilver alone. The nanosilver inhibits growth of these fungi at very low concentrations that are comparable to those of current antifungal drugs. Although the nanosilver exhibit no significant cytotoxic effects on human fibroblasts in these concentrations (31), clinically prescription of these particles needs more clinical trial studies.
