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. 2014 Oct 7;9(10):e108876.
doi: 10.1371/journal.pone.0108876. eCollection 2014.

Ultrastructural Analysis of Candida Albicans When Exposed to Silver Nanoparticles

Free PMC article

Ultrastructural Analysis of Candida Albicans When Exposed to Silver Nanoparticles

Roberto Vazquez-Muñoz et al. PLoS One. .
Free PMC article


Candida albicans is the most common fungal pathogen in humans, and recently some studies have reported the antifungal activity of silver nanoparticles (AgNPs) against some Candida species. However, ultrastructural analyses on the interaction of AgNPs with these microorganisms have not been reported. In this work we evaluated the effect of AgNPs on C. albicans, and the minimum inhibitory concentration (MIC) was found to have a fungicidal effect. The IC50 was also determined, and the use of AgNPs with fluconazole (FLC), a fungistatic drug, reduced cell proliferation. In order to understand how AgNPs interact with living cells, the ultrastructural distribution of AgNPs in this fungus was determined. Transmission electron microscopy (TEM) analysis revealed a high accumulation of AgNPs outside the cells but also smaller nanoparticles (NPs) localized throughout the cytoplasm. Energy dispersive spectroscopy (EDS) analysis confirmed the presence of intracellular silver. From our results it is assumed that AgNPs used in this study do not penetrate the cell, but instead release silver ions that infiltrate into the cell leading to the formation of NPs through reduction by organic compounds present in the cell wall and cytoplasm.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.


Figure 1
Figure 1. Characterization of silver nanoparticles (AgNPs) used in this study.
A) Transmission electron micrograph of AgNPs functionalized with Polyvinylpyrrolidone (Vector-Vita Ltd., Russia), B) histogram of measured nanoparticles, C) High-angle annular dark-field imaging (HAADF) of AgNPs, D) Typical EDS analysis of particles along the trace indicated by a white line in (C).
Figure 2
Figure 2. The combined effect of silver nanoparticles (AgNPs) and fluconazole (FLC) in C. albicans reduces cell proliferation.
Liquid cultures were exposed to the IC50 of FLC and several combinations of AgNPs-FLC.
Figure 3
Figure 3. Subcultures of C. albicans in YPD agar plates after 24 h of incubation.
A) Representative image of a culture to determine the IC50 of AgNPs and FLC; B) subcultures of the combined effect of the IC50 of AgNPs (18 µg/mL) and two concentrations of FLC; C) subcultures of the combined effect of the MIC of AgNPs (42 µg/mL) and two concentrations of FLC. In B–C the IC50 of FLC (31 µg/mL) was used in the inoculations on the left side of the plate and 300 µg/mL of FLC on the right side.
Figure 4
Figure 4. Microscopic analysis of C. albicans from liquid cultures.
A, B) Cells from control cultures observed under optical bright field microscopy and TEM, respectively; C, D) Cells exposed to silver nanoparticles were agglomerated and surrounded by AgNPs as seen by optical bright-field microscopy (C) and confirmed by TEM (D). Black arrows indicate Candida cells and white arrows indicate AgNPs aggregation.
Figure 5
Figure 5. Chemical characterization of C. albicans ultrathin sections.
A, D) High-angle annular dark-field imaging (HAADF) of analyzed cells; (B, E) EDS analysis showing the absence of silver in (A) and the presence of silver in (D); (C, F) Lineal EDS spectrum of (A) and (D), respectively. Red line in A and D indicates the transect where chemical analysis was performed.
Figure 6
Figure 6. Chemical characterization of intracellular nanoparticles.
A) HAADF image in which analysis of internal AgNPs was carried out, B) Closer view of internal-external particles, C) Amplified image of analyzed internal particle, (D–F) Images of analyzed internal particle, G) EDS analysis showing the presence of silver, H) Variation of Ag and Os along a trace line at neighbor points near the particle indicated by a yellow arrow in Figs A to E; sampled points can be seen as black dots in D and E. Yellow arrows point out analyzed particle, red arrows point out extracellular AgNPs. Enclosed area in (F) indicates the zone where chemical analysis was conducted, and the image in the upper corner is the diffraction pattern of analyzed particle, confirming the presence of crystalline silver.
Figure 7
Figure 7. Crystallographic analysis of intracellular nanoparticles in C. albicans.
Silver nanoparticle shows (111) planes with 0.24 nm spacing.
Figure 8
Figure 8. TEM images showing interaction of silver nanoparticles with C. albicans.
A, B) Sections of cells in which extracellular agglomeration of AgNPs coincided with the accumulation of smaller AgNPs in the cell wall and cytoplasm, C) Size distribution of intracellular AgNPs.

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This work was partially supported by a SEP-CONACyT grant (CB2011/169154). The authors also thank CONACyT for a grant to Roberto Vazquez-Muñoz. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.