Activities of Nanoparticles Against Fluconazole-Resistant Candida parapsilosis in Clinical Isolates

Abstract

Candida parapsilosis is a non-albicans Candida spp. associated with bloodstream infections in critically ill patients. Failure to treat it effectively due to delay in diagnosis often leads to serious illnessess. The present research aimed to investigate the antifungal activities of nanoparticles (NPs) against fluconazole-resistant C. parapsilosis strains. Ten strains were used from archived clinical isolates. Antifungal activities of NPs were examined based on the Clinical and Laboratory Standards Institute (M27–A3/S4) guideline. The morphological changes of strains exposed to each NP were observed by scanning electron microscope (SEM). The effect of NP on the membrane permeability of C. parapsilosis and the viability of the cells was assessed using the confocal laser scanning microscopy and 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, respectively. The cytotoxicity was evaluated against three mammalian cell lines. Minimum Inhibitory Concentration of NPs of 10 strains was in the concentration range of 0.5–4 μg/mL; these results were confirmed with the viability test. The antifungal activity of synthesized silver NPs (AgNPs) against resistant C. parapsilosis was greater in comparison with the gold NPs (AuNPs). The SEM images indicated a difference in the fungal morphology of the fungi. The propidium iodide uptake by C. parapsilosis cells showed concentration-dependent mortality in NPs treatment with a confocal laser scanning microscope. There was a notable difference (p < 0.01) in the cell viability in the concentration range of 0.5–4 μg/mL between NPs based on the MTT assay. In addition, these NPs exhibited very low toxicity for three mammalian cell lines, specially at 0.5 μg/mL. AgNPs and AuNPs had fungicidal activities against fluconazole-resistant C. parapsilosis strains. It is crucial to have knowledge based on fundamental research to find new ways to overcome resistant microorganisms.

Introduction

Candida parapsilosis is a non-albicans Candida spp. associated with bloodstream infections due to prosthetic devices and indwelling catheters in the intensive care and neonatal units.¹ Despite the fact that C. parapsilosis is not known as immune to antifungal resistance,² a recent increase in the resistance of C. parapsilosis to azoles has become a cause for concern.³ The advent of C. parapsilosis isolates with decreased sensitivity to fluconazole (FLC) may be linked to the broad consumption of FLC in patients with severe conditions.⁴

With the emergence of antifungal-resistant species, which cause life-threatening infections, many researchers started to investigate the antifungal efficacy of nanoparticles (NPs).⁵ The nanomedicine technology could be an effective solution for combating drug resistance.⁶

The present research aimed to assess the susceptibility of 10 FLC-resistant C. parapsilosis clinical isolates to silver NPs (AgNPs) and gold NPs (AuNPs). It was hypothesized that the NPs may make pores in the cell wall and membrane of strains and could possibly decrease the resistance to FLC. Herein, scanning electron and confocal microscopes were used, and the survival rate and cytotoxicity of the species were evaluated with 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT).

Materials and Methods

Candida Strain

Ten archived resistant C. parapsilosis strains were isolated⁷ and C. parapsilosis (ATCC 22019) was as a control strain. The antifungal activities of two NPs were examined based on the Clinical And Laboratory Standards Institute (CLSI M27–A3/S4).⁸ This study was performed at the Medical Mycology Department of Shahid Beheshti University of Medical Sciences, Tehran, Iran.

Yeast Inoculation Preparation

Resistant C. parapsilosis isolates were cultured on Sabouraud Dextrose Agar medium (Sigma, USA) for 24 h at 35°C, and colonies were suspended in 1 mL of normal saline solution and transferred to Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma). The inoculum concentration of about 0.5 × 10³–2.5 × 10³ colony-forming unit (CFU)/mL was achieved as a concentration adjustment.

Synthesis and Characterization of NPs

Klebsiella pneumoniae was briefly inoculated in Muller Hinton Broth medium (Sigma, USA) to prepare the AgNPs stock solution (100 μg/mL). Then 100 mL of silver nitrate (AgNO₃, 1 mM) was supplemented by 1 mL of supernatant. It should be noted that in the case of the presence of AgNPs, the color changes to light brown.⁹

To prepare the AuNPs, AuCl4 (20 mg) (Sigma-Aldrich, St. Louis, MO) was dissolved in 100 double-distilled water using the magnetic stirrer. Then, 150 mg of trisodium citrate (Sigma-Aldrich) was added to the mixture, and the solution temperature was adjusted to 80°C. The solution mixture was stirred until its color turned to deep red. The solution was cooled down to room temperature. Subsequently, 5 mL of concentrated glycerol was added while being vigorously stirred.¹⁰ The zeta potential and Z-average size were determined by the Malvern Zetasizer Nano ZS-90 instrument (Malvern, England).

Minimum Inhibitory Concentration of NPs

Minimum inhibitory concentration (MIC₉₀) of NPs for FLC-resistant isolates was performed according to CLSI (M27-A3/S4) guideline. In brief, twofold serial dilutions of NPs were prepared in concentrations within the range of 0.125–4 μg/mL. Since both NPs were soluble and hydrophilic, they were shaken completely before adding to the 96-well microplate to be completely homogenized. One hundred microliters diluted NPs were added to 96-well microtiter plates to prepare inocula. Afterward, the C. parapsilosis cells were added to RPMI 1640 medium and adjusted to final inoculum concentration 0.5 × 10³ to 2.5 × 10³ CFU/mL. Moreover, the wells containing the diluted NPs were supplemented by 100 μL of each suspension. All plates were incubated at 37°C for 48 h. The experiments were performed for each NP two times.

Scanning Electron Microscopy

The C. parapsilosis strains were exposed to the NPs and changed morphologically. These changes were observed with the scanning electron microscope (SEM) (AIS2100; Seron Technology, South Korea). To make the strains ready, the agar was cut, fixed for a minimum of 3 h in 2.5% (v/v) glutaraldehyde (100 mM phosphate buffer solution, pH 7.2) followed by 1% (w/v) osmium tetroxide for 1 h. The dehydration of the agar blocks was performed via a graded series of ethanol (30%, 50%, 60%, 70%, 80%, 90%, 95%, and 100%; each level was applied twice for 15 min each time) as well as ethanol:isoamyl acetate (3:1, 1:1, 1:3, and 100% isoamyl acetate twice for 30 min). A critical-point dryer (Sc7620; Emitech, England) with liquid CO₂ was used to dry the agar blocks, and a gold-coater was used to coat the agar blocks for 5 min. The coated samples were visualized with an accelerating voltage of 9 kV.¹¹

Effect of NPs on the Membrane Permeability of C. parapsilosis

The changes in the membrane permeability of C. parapsilosis strains exposed to AgNPs and AuNPs were confirmed by uptake of propidium iodide (PI). Both the treated (MIC and 2 × MIC) and untreated cells were incubated with PI (5 μg/mL) for 15 min. Afterward, phosphate-buffered saline was used to wash the samples to remove the overstraining. Eventually, the fluorescent images of C. parapsilosis cells were obtained via the confocal laser scanning microscope (CLSM) (Carl Zeiss, Jena, Germany). Experiments were performed for all strains.

Effects of NPs on the Yeast Cell Viability Based on MTT Assay

An aliquot of 200 μL of C. parapsilosis (5 × 10³–2.5 × 10³ CFU/mL) with different concentrations of NPs (0.125–4 μg/mL) were incubated at 30°C for 24 h shaking at 160 rpm. Afterward, 25 μL of RPMI medium, which included 5 mg MTT (Sigma Aldrich), was added to the wells, and the plates were incubated for 3 h at 37°C. Then the plates were centrifuged at 1,000 g for 10 min, and supernatant solutions were aspirated.

Of the acid isopropanol (95 mL of isopropanol, 5 mL of 1NHCl), 0.1 mL was added, the plates were put on a shaker for 5 min to dissolve formazan crystals. Then, the amount of formazan was measured using optical density at 550 nm via a microplate reader (Bio-Rad, USA).The wells containing fungi and acid propanol without menadione-formazan were provided as background controls.¹²

In vitro Cytotoxicity Assays

According to the eukaryotic nature of fungi, it is essential to check the selectivity of new antifungal structures against fungi cells and the proper safety profile to prevent toxicity to mammalian cells.¹³ Hence, the AgNPs and AuNPs were tested against three different cell lines, including human pancreatic cancer (PANC-1), epidermoid carcinoma (A-431), and human dermal fibroblast (HDF) normal cells with fluconazole (FLC) as the positive control.

Three mammalian cancer cell lines were used, which were provided by the National Cell Bank of Iran (Pastor Institute, Tehran, Iran). All the cells were cultured in RPMI-1640 medium and Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (Gibco, Milano, Italy). Then a cell suspension of 8,000 cells was poured into 96-well plate and incubated in a humidified incubator containing 5% CO₂ at 37°C for 24 h. Subsequently, different concentrations (0.5, 1, 2, and 4 μg/mL) of the NPs and FLC were added to these plates and incubated for 48 h under the above conditions, and 0.1% dimethyl sulfoxide and cisplatin served as negative and positive controls, respectively. After incubation, the solution containing 5% MTT was added to all the wells and then incubated for another 4 h. Color intensity was recorded at 570 nm by Bio-Rad microplate reader (Model 680) and the percentage of live cells was obtained by following equation: % cell survival = [optical density (OD) of sample/OD of positive control] × 100. The experiments were performed in triplicate.¹⁴ Each test was performed in triplicate.

Statistical Analysis

The data are expressed as the mean ± SD values via the GraphPad Prism software (ver.6, Inc.). For comparison between two groups, the Student t-test was used with a p < 0.01 as statistically significant.

Results

Characteristics of NPs

The zeta potentials of AgNPs and AuNPs were obtained at −21.2 and −20.7 Mv, respectively. The zeta-average sizes of AgNPs and AuNPs were 30 and 13 nm, respectively.

MIC₉₀ of the NPs

The MICs₉₀ of 10 FLC-resistant C. parapsilosis strains were in the concentration range 0.5 to 4 μg/mL (Table 1). The growth was completely inhibited at 0.5 μg/mL or more compared to the control and no inhibition was observed at concentrations less than 0.5. The AgNPs had greater antifungal activity compared to AuNPs.

Table 1.

Minimum Inhibitory Concentration of 10 Fluconazole-Resistant Candida parapsilosis Strains Exposed to Silver Nanoparticles and Gold Nanoparticles

Isolates	Antifungal	Nanoparticles
	FLC	AgNPs	AuNPs
	MIC₉₀ range (μg/mL)
	R: ≥8, I: 4, S: ≤2	—	—
Candida parapsilosis R1	8	0.5	2
C. parapsilosis R2	16	2	2
C. parapsilosis R3	8	2	4
C. parapsilosis R4	8	0.5	4
C. parapsilosis R5	8	1	4
C. parapsilosis R6	8	1	4
C. parapsilosis R7	16	0.5	2
C. parapsilosis R8	16	0.5	2
C. parapsilosis R9	8	2	4
C. parapsilosis R10	8	2	4
GM ± SD	9.84 ± 3.8	1 ± 0.71	3.03 ± 1.03
C. parapsilosis ATCC (22019)	2	0.5	1

No. of resistant Candida parapsilosis strains (R1–R10). MIC required to inhibit the growth of 90% of yeasts.

AgNPs, silver NPs; AuNPs, gold NPs; FLC, fluconazole; GM, geometric range; I, intermediate; MIC₉₀, minimum inhibitory concentration; R, resistant; S, susceptible; SD, standard deviation.

Scanning Electron Microscopy Image

Figure 1 shows the SEM micrograph of C. parapsilosis before and after treatment with AgNPs for 48 h. Based on the SEM images, there was a clear variation between the fungal morphology of the untreated (control) and treated fungi. In untreated C. parapsilosis (Fig. 1A), the only spherical shape was blastoconidia. After the treatment of C. parapsilosis with AgNPs (Fig. 1B), abnormal morphology, more pores, and distorted membranes emerged on the yeasts (red circle on the image). According to Figure 1C, AuNPs were attached to the surface of yeasts and produced a little pore (red circle on the image).

Fig. 1.

Scanning electron microscopy images of Candida parapsilosis (ATCC 22019) treated with AgNPs and AuNPs; 8,000 × magnification. (A) Untreated C. parapsilosis; the red arrow indicates the smooth cell membrane of normal yeast. (B) C. parapsilosis exposed to 2 μg/mL AgNPs. (C) C. parapsilosis exposed to 2 μg/mL AuNPs. The red circle indicates the damaged membranes and pores in (B, C). AgNPs, silver NPs; AuNPs, gold NPs. Color images are available online.

After the fungi were treated with NPs at the MIC dose, its cell membrane lost its smoothness, which caused the appearance of uncommon surface bulges, such as impairment of cell membrane integrity. At a 2 × MIC dose concentration of AgNPs (Fig. 1B), the fungal morphology significantly altered and turned into a contorted cell structure with the breakdown of the cell membranes. Nevertheless, at the 2 × MIC level concentration of AuNPs (Fig. 1C), the fungal morphology did not alter significantly.

Effect of NPs on the Membrane Permeability by PI Staining

Figure 2 shows the CLSM images of stained cells of C. parapsilosis prior and posterior to their exposure to NPs. The PI uptake by C. parapsilosis cells showed concentration-dependent mortality in treatment with NPs. No or very few PI-stained cells were found in the control group (Fig. 2A), which signifies the intact cell wall structure of C. parapsilosis. Nevertheless, a significant number of PI-stained cells showed red fluorescence in the groups treated with NPs (Fig. 2B and C ). In addition, at comparable MIC levels (i.e., 2 × MIC), the cells that were exposed to AgNPs (Fig. 2B) showed an increase in the level of red fluorescence in comparison with the AuNPs (Fig. 2C).

Fig. 2.

Confocal laser scanning microscope images of propidium iodide-stained Candida parapsilosis (ATCC 22019) cells. (A) Control (untreated); (B) C. parapsilosis treated with 2 μg/mL AgNPs; (C) C. parapsilosis treated with 2 μg/mL AuNPs. Color images are available online.

Effects of Silver and AuNPs on the Cell Viability Using the MTT Assay

The effects of NPs on the viability were investigated using the MTT assay.

The exposure to AgNPs had a strong influence on the cell viability of C. parapsilosis. As can be seen in Figure 3, the viability of C. parapsilosis cells reduced with the increase in the concentration of AgNPs. A significant difference (p < 0.01) was found in the cell viability of C. parapsilosis treated with AgNPs and AuNPs in the concentration range of 0.5–4 μg/mL. The cell viability of C. parapsilosis exposed to AuNPs at the MIC ranges of 0.5 μg/mL was 99–100%. Moreover, cell viabilities of C. parapsilosis treated with AgNPs at the MIC levels of 1 and 4 μg/mL were 27% and 15%, respectively. This percentage reduction in the cell viability rate of AgNPs revealed a better effect on the resistant cells, compared to AuNPs.

Fig. 3.

Viability of Candida parapsilosis (ATCC 22019) after treatment with silver and AuNPs based on MTT assay. Data indicate the mean of triplicate experiment samples. A p-value of <0.01 was considered significant. MTT, 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide.

In vitro Cytotoxicity Assays

Against PANC-1, the AgNPs and AuNPs exhibited no toxicity at 0.5, 1, 2, and 4 μg/mL. However, the AgNPs showed a better activity at 1 μg/mL (85% cell survival). As illustrated in Figure 4, the viability of A-431 cancer cells for the AgNPs and AuNPs was affected by more than 95% and 90% at the concentration of 0.5 μg/mL, respectively. But the AuNPs demonstrated a few toxicity levels at 4 μg/mL (≤60% cell survival). Against the HDF normal cell line, the AgNPs exhibited 80% viability at 0.5 μg/mL. However, the AuNPs showed some toxicity levels (≤50% cell survival) at 2 and 4 μg/mL. These data are presented in Figure 4.

Fig. 4.

Representative cytotoxicity assays of AgNP and AuNP against three mammalian cell lines: PANC-1, A-431, and HDF. Cells were treated with various concentrations of the compounds and FLC. The positive control consisted of cells treated with cisplatin (<10% cell survival, not shown). The negative control consisted of cells treated with DMSO (no drug). A p-value of less than 0.01 was considered significant. **p < 0.01 was observed for all groups compared to the positive control group. A-431, epidermoid carcinoma; DMSO, dimethyl sulfoxide; FLC, Fluconazole; HDF, human dermal fibroblast; PANC1, pancreatic cancer.

Discussion

C. parapsilosis is one of the most common fungal agents related to increased morbidity and mortality of invasive infections.¹⁵ Resistance to available antifungals is an important point in the management of severe candidiasis.^16,17 Therefore, there is an increasing interest in discovering alternative antifungals to overcome emerging resistant fungal isolates.^{9,11,17
–19} In recent studies, metal NPs have drawn attention due to their fungicidal activities.^{9,11,17,20,21}

Our results suggest that AgNPs and AuNPs had 0.5 ≤ MIC ≤2 μg/mL and 2 ≤ MIC ≤4 μg/mL against resistant C. parapsilosis strains, respectively. The AgNPs were shown to have more potent activity against strains than AuNPs. This is consistent with the findings of a study conducted by Alimehr et al., which reported that MICs of AgNPs for 20 FLC-resistant Candida albicans were 2 and 4 μg/mL.⁹ In another study, AgNPs had potent efficacy on C. albicans (MIC = 0.5 μg/mL).¹¹ Wady et al. also proved that AgNP solution has significant activity against Candida spp.²² In our previous study, AgNPs were activated against FLC-resistant C. parapsilosis with MIC = 2 μg/mL.¹⁷ Rónavári et al. highlighted that AgNPs could inhibit the growth of C. parapsilosis (CBS 604), but it is unable to inhibit Candida tropicalis (CBS 94). Inconsistent with the results of the present study, they reported that AuNPs could not affect C. parapsilosis (CBS 604).²³

Further tests with SEM highlighted that the cell membrane underwent significant changes after 48 h of exposure to NPs. Nasrollahi et al. expressed that after treatment with AgNPs (MIC₅₀ = 0.5 mg/mL), several pores were formed in C. albicans cell membranes resulting in the destruction of the cells. Findings of their study revealed that a reciprocal association between AgNPs and cell membrane destroys fungal cells.¹¹ El Rabey et al. studied the morphological variations of C. parapsilosis cells following their treatment by FLC-chitosan NPs conjugate.²⁴ In another study, the SEM analysis on C. glabrata showed that after treatment with AgNPs, AuNPs, and SeNPs, fungal cells exhibited significant morphological changes leading to cell death.²⁰

The CLSM images of stained cells of C. parapsilosis underlined that in the NPs-treated group, a noticeable quantity of PI-stained cells exhibited red fluorescence. Pereira et al. studied the antifungal activity of the n-butanol fraction on the viability of C. albicans biofilms according to the CLSM.²⁵ CLSM examinations were used to determine the photodynamic inactivation activity of hypocrellin B on C. albicans isolates.²⁶ CLSM revealed the detailed morphology and architecture of C. albicans biofilms after 5-Aminolevulinic acid photodynamic therapy.²⁷

According to the MTT assay, there was a reduction in the viability of C. parapsilosis cells, while the concentration of both NPs increased. However, the decreased cell viability rate of AgNPs had a better effect on the FLC-resistant fungal cells, compared to AuNPs. Silva Viana et al. used the MTT assay to evaluate the cytotoxicity of xylan and nanoxylan.²⁸ Bansal et al. concluded that conjugation of biosurfactant with graphene quantum dots significantly reduced the viability of cells with the increase of time and dose.²⁹

Interestingly, none of the NPs displayed toxicity against three mammalian cell lines up to 0.5 μg/mL. When considering these NPs' extremely low MIC values against fluconazole-resistant C. parapsilosis in clinical isolates, these cytotoxicity data are encouraging and provide a rational therapeutic window.

Conclusions

Scientists have continuously tried to discover new antifungals or develop existing ones to combat resistant agents. This report can be used in encapsulating conventional drugs with these NPs to reduce their adverse effects and enhance their activity. However, the confirmation of these findings needs more investigation.

Footnotes

Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for this article.

Abbreviations Used

References

Ghannoum

, Chen

, Buhari

, et al.: Differential in vitro activity of anidulafungin, caspofungin and micafungin against Candida parapsilosis isolates recovered from a burn unit. Clin Microbiol Infect, 2009; 15:274–279.

Van Asbeck

, Clemons

, Martinez

, Tong

, Stevens

: Significant differences in drug susceptibility among species in the Candida parapsilosis group. Diagn Microbiol Infect Dis, 2008; 62:106–109.

Pfaller

, Diekema

, Gibbs

, et al.: Geographic and temporal trends in isolation and antifungal susceptibility of Candida parapsilosis: a global assessment from the artemis disk antifungal surveillance program, 2001 to 2005. J Clin Microbiol, 2008; 46:842–849.

Silva

, Miranda

, Lisboa

, Pina-Vaz

, Rodrigues

: Prevalence, distribution, and antifungal susceptibility profiles of Candida parapsilosis, c. Orthopsilosis, and c. Metapsilosis in a tertiary care hospital. J Clin Microbiol, 2009; 47:2392–2397.

Talebian

, Sadeghi Haddad Zavvare

: Enhanced bactericidal action of sno2 nanostructures having different morphologies under visible light: influence of surfactant. J Photochem Photobiol B, 2014; 130:132–139.

Thorley

, Tetley

: New perspectives in nanomedicine. Pharmacol Ther, 2013; 140:176–185.

Lotfali

, Ghajari

, Kordbacheh

, et al.: Regulation of ERG3, ERG6, and ERG11 genes in antifungal-resistant isolates of Candida parapsilosis . Iran Biomed J, 2017; 21:275–281.

Clinical and Laboratory Standards Institute. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. Clinical and Laboratory Standards Institute, Wayne, PA, 2008.

Alimehr

, Shekari Ebrahim Abad

, Shahverdi

, et al.: Comparison of difference between fluconazole and silver nanoparticles in antimicrobial effect on fluconazole-resistant Candida albicans strains. Arch Pediatr Infect Dis, 2015; 3.

10.

Tabrizi

, Ayhan

: Gold nanoparticle synthesis and characterisation. Hacettepe J Biol Chem, 2009; 37:217–226.

11.

Nasrollahi

, Pourshamsian

, Mansourkiaee

: Antifungal activity of silver nanoparticles on some of fungi. IJND, 2011; 1:233–239.

12.

Meletiadis

, Meis

, Mouton

, Donnelly

, Verweij

: Comparison of NCCLS and 3-(4, 5-dimethyl-2-thiazyl)-2, 5-diphenyl-2h-tetrazolium bromide (MTT) methods of in vitro susceptibility testing of filamentous fungi and development of a new simplified method. J Clin Microbiol, 2000; 38:2949–2954.

13.

Fuentefria

, Pippi

, Dalla Lana

, Donato

, De Andrade

. Antifungals discovery: an insight into new strategies to combat antifungal resistance. Lett Appl Microbiol, 2018; 66:2–13.

14.

Thamban Chandrika

, Shrestha

, Ngo

, et al.: Alkylated piperazines and piperazine-azole hybrids as antifungal agents. J Med Chem, 2018; 61:158–173.

15.

Lotfali

, Kordbacheh

, Mirhendi

, et al.: Antifungal susceptibility analysis of clinical isolates of Candida parapsilosis in Iran. Iran J Public Health, 2016; 45:322–328.

16.

Pemán

, Canton

, Espinel-Ingroff

: Antifungal drug resistance mechanisms. Expert Rev Anti Infect Ther, 2009; 7:453–460.

17.

Lotfali

, Shahverdi

, Mohammadi

, et al.: In vitro activity of two nanoparticles on clinical isolates of Candida parapsilosis, showing resistance against antifungal agents in children. Archiv Clin Infect Dis, 2017; 12.

18.

Dehkordi

, Hosseinpour

, Kahrizangi

: An in vitro evaluation of antibacterial effect of silver nanoparticles on Staphylococcus aureus isolated from bovine subclinical mastitis. Afr J Biotechnol, 2011; 10:10795–10797.

19.

Kim

K-J

, Sung

W-S

, Moon

S-K

, Choi

J-S

, Kim

J-G

, Lee

D-G

: Antifungal effect of silver nanoparticles on dermatophytes. J Microbiol Biotechnol, 2008; 18:1482–1484.

20.

Lotfali

, Toreyhi

, Makhdoomi Sharabiani

, et al.: Comparison of antifungal properties of gold, silver, and selenium nanoparticles against amphotericin b-resistant Candida glabrata clinical isolates. Avicenna J Med Biotechnol, 2021; 13:47–50.

21.

Moazeni

, Kelidari

, Saeedi

, et al.: Time to overcome fluconazole resistant Candida isolates: solid lipid nanoparticles as a novel antifungal drug delivery system. Colloids Surf B Biointerfaces, 2016; 142:400–407.

22.

Wady

, Machado

, Foggi

, et al.: Effect of a silver nanoparticles solution on staphylococcus aureus and Candida spp. J Nanomater, 2014; 2014.

23.

Rónavári

, Igaz

, Gopisetty

, et al.: Biosynthesized silver and gold nanoparticles are potent antimycotics against opportunistic pathogenic yeasts and dermatophytes. Int J Nanomedicine, 2018; 13:695.

24.

El Rabey

, Almutairi

, Alalawy

, et al.: Augmented control of drug-resistant Candida spp. Via fluconazole loading into fungal chitosan nanoparticles. Int J Biol Macromol, 2019; 141:511–516.

25.

Pereira

, Freires

, Castilho

, Da Cunha

, Alves

HDS

, Rosalen

: Antifungal potential of sideroxylon obtusifolium and syzygium cumini and their mode of action against Candida albicans . Pharm Biol, 2016; 54:2312–2319.

26.

Jan

, Liu

, Deng

, et al.: In vitro photodynamic inactivation effects of hypocrellin b on azole-sensitive and resistant Candida albicans . Photodiagnosis Photodyn Ther, 2019; 27:419–427.

27.

Shi

, Li

, Zhang

, Sun

: Effect of 5-aminolevulinic acid photodynamic therapy on Candida albicans biofilms: an in vitro study. Photodiagnosis Photodyn Ther, 2016; 15:40–45.

28.

Silva Viana

, Pereira Fidelis

, Jane Campos Medeiros

, et al.: Green synthesis of antileishmanial and antifungal silver nanoparticles using corn cob xylan as a reducing and stabilizing agent. Biomolecules, 2020; 10:1235.

29.

Bansal

, Singh

, Kumari

, et al.: Development of biosurfactant-based graphene quantum dot conjugate as a novel and fluorescent theranostic tool for cancer. Int J Nanomedicine, 2019; 14:809.

Activities of Nanoparticles Against Fluconazole-Resistant Candida parapsilosis in Clinical Isolates

Abstract

Introduction

Materials and Methods

Candida Strain

Yeast Inoculation Preparation

Synthesis and Characterization of NPs

Minimum Inhibitory Concentration of NPs

Scanning Electron Microscopy

Effect of NPs on the Membrane Permeability of C. parapsilosis

Effects of NPs on the Yeast Cell Viability Based on MTT Assay

In vitro Cytotoxicity Assays

Statistical Analysis

Results

Characteristics of NPs

MIC90 of the NPs

Scanning Electron Microscopy Image

Effect of NPs on the Membrane Permeability by PI Staining

Effects of Silver and AuNPs on the Cell Viability Using the MTT Assay

In vitro Cytotoxicity Assays

Discussion

Conclusions

Footnotes

Disclosure Statement

Funding Information

Abbreviations Used

References

MIC₉₀ of the NPs