In Vitro Antifungal Activity of Silver Nanoparticles Against Ocular Pathogenic Filamentous Fungi

Abstract

Purpose:

Fungal keratitis is emerging as a major cause of vision loss in a developing country such as China because of higher incidence and the unavailability of effective antifungals. It is urgent to explore broad-spectrum antifungals to effectively suppress ocular fungal pathogens, and to develop new antifungal eye drops to combat this vision-threatening infection. The aim of this study is to investigate the antifungal activity of silver nanoparticles (nano-Ag) in comparison with that of natamycin against ocular pathogenic filamentous fungi in vitro.

Methods:

Susceptibility tests were performed against 216 strains of fungi isolated from patients with fungal keratitis from the Henan Eye Institute in China by broth dilution antifungal susceptibility test of filamentous fungi approved by the Clinical and Laboratory Standards Institute M38-A document. The isolates included 112 Fusarium isolates (82 Fusarium solani species complex, 20 Fusarium verticillioides species complex, and 10 Fusarium oxysporum species complex), 94 Aspergillus isolates (61 Aspergillus flavus species complex, 11 Aspergillus fumigatus species complex, 12 Aspergillus versicolor species complex, and 10 Aspergillus niger species complex), and 10 Alternaria alternata isolates. The minimum inhibitory concentration (MIC) range and mode, the MIC for 50% of the strains tested (MIC₅₀ value), and the MIC₉₀ value were provided for the isolates with the SPSS statistical package.

Results:

MIC₅₀ value of nano-Ag were 1, 0.5, and 0.5 μg/mL for Fusarium spp., Aspergillus spp., and Al. alternata, respectively. MIC₉₀ values of nano-Ag were 1, 1, and 1 μg/mL for Fusarium spp., Aspergillus spp., and Al. alternata, respectively. MIC₅₀ values of natamycin were 4, 32, and 4 μg/mL for Fusarium spp., Aspergillus spp., and Al. alternata, respectively. MIC₉₀ values of natamycin were 8, 32, and 4 μg/mL for Fusarium spp., Aspergillus spp., and Al. alternata, respectively.

Conclusions:

Nano-Ag, relative to natamycin, exhibits potent in vitro activity against ocular pathogenic filamentous fungi.

Introduction

Fungal keratitis is emerging as a major cause of vision loss in a developing county such as China because of higher incidence and the unavailability of effective antifungal agents.^1–3 Fungal keratitis may constitute 46.7%–61.9% of all cases of suppurative keratitis inpatients, and 35.1% of all cases of infectious keratitis underwent penetrating keratoplasty in some regions in China.^2,3 To date, only fluconazole and natamycin are commercially available for fungal keratitis in China. Fluconazole has high bioavailability against Candida spp. but it is inactive against Fusarium and Aspergillus species.^4–6 Filamentous fungi, mainly Fusarium spp. (65%–77.6%) and Aspergillus spp. (10.8%–20.5%), are most commonly associated with fungal keratitis, while Candida spp. (1%) is rarely implicated as an etiological agent for fungal keratitis in China.^1–3,7 Natamycin is the only topical ophthalmic antifungal compound approved in the United States.^8,9 Natamycin is insoluble in water and is stable in 5% suspension. After topical application, natamycin penetrates the cornea and conjunctiva poorly; effective drug levels are not achieved in either the cornea or aqueous,¹⁰ and it is, therefore, useful only in the treatment of superficial infections that are not severe. Natamycin is the drug of choice for therapy of fungal keratitis in many countries, particularly for keratitis due to filamentous fungi.^8,9 Some studies, however, have shown that Aspergillus spp. are resistant to natamycin and there are poor outcomes with natamycin for patients with fungal keratitis.^4,11–14 Since the clinical management of fungal keratitis has been limited due to the relative unavailability of effective antifungals, corneal lesions fail to resolve in many of the patients who receive antifungal treatment and some patients get marked loss of vision and eventually perforation of the cornea, ultimately require penetrating keratoplasty, or even enucleation or evisceration.^2,8,9 Therefore, it is inevitable and urgent to explore broad-spectrum antifungals to effectively suppress ocular fungal pathogens, and to develop new antifungal eye drops to combat this sight-threatening infection.

The use of metallic silver as an antimicrobial agent has long been recognized.^15,16 Silver nitrate has been used since the nineteenth century to prevent ophthalmia neonatorum.¹⁷ Silver compounds possess the advantage of having broad antimicrobial activities against bacteria, viruses, and fungi, with minimal development of microbial resistance.¹⁸ In particular, due to the recent advances in research on metal nanoparticles, silver nanoparticles (nano-Ag) have received special attention as a possible antimicrobial agent.^19–21 The bactericidal effect of nano-Ag has been intensively studied recently, mostly due to the growing bacterial resistance to common antibiotics.^19,20 The already published studies on bactericidal activity have proved that nano-Ag kill bacteria at such low concentrations (units of μg/mL), which do not reveal acute toxic effects on human cells.^19–22 Nano-Ag, exhibiting very strong bactericidal activity against both gram-positive and gram-negative bacteria including multiresistant strains, can be considered a potential antifungal agent.²³ However, the antifungal effect of nano-Ag has received only marginal attention, and just a few studies on antifungal activity of nano-Ag against Candida spp. and dermatophytes have been published.^23–25 The efficacy of nano-Ag against ocular pathogenic fungi has not been evaluated so far. The present study was performed to determine the antifungal activity of nano-Ag against ocular pathogenic fungi in vitro.

Methods

Antifungal agents

Nano-Ag (NTX-330WT; Nanux Inc.) of sizes ranging from 20 to 30 nm and stabilized in polymers was used. The Nano-Ag was a colloidal solution with nankeen color, pH 4.9±0.5, and viscosity <5. The concentration of colloidal silver was 2,000 ppm. For the comparisons of the antifungal activity, natamycin (Yinxiang Biotechnology Co. LTD; minimum 95%) was used. They were dissolved in 100% dimethyl sulfoxide, respectively. The stock solutions were prepared at a concentration of 800 μg/mL for nano-Ag, 1,600 μg/mL for natamycin. Drug dilutions were made in RPMI 1640 medium (with l-glutamine, without sodium bicarbonate; Gibco BRL–Life Technologies) buffered to pH 7.0 with 0.165 M morpholinepropanesulphonic acid (Serva, Feinbochemica GmbH & Co.). Final concentrations ranged from 0.0156 to 8 μg/mL for nano-Ag, from 0.0313 to 16 μg/mL for natamycin.

Fungi isolates

Antimycotic activity was tested using 216 strains of fungi isolated from patients with fungal keratitis from the Henan Eye Institute in Zhengzhou, China. The clinical samples of fungi were obtained between January 2007 and December 2007. We chose the isolates for the antimycotic activity according to two principles. First, we mainly chose the isolates of filamentous fungi, particularly Fusarium spp. and Aspergillus spp., as filamentous fungi; mainly Fusarium spp. and Aspergillus spp. are the most commonly associated with fungal keratitis in China.^1–3,7 Second, we only chose the isolates that were obtained 10 strains or more than 10 strains for every “species complex,” as 90% minimum inhibitory concentration (MIC₉₀) data are not valid for <10 strains. All isolates were identified to the species level based on morphology by standard methods.^26,27 They included 112 Fusarium isolates (82 Fusarium solani species complex, 20 Fusarium verticillioides species complex, and 10 Fusarium oxysporum species complex), 94 Aspergillus isolates (61 Aspergillus flavus species complex, 11 Aspergillus fumigatus species complex, 12 Aspergillus versicolor species complex, and 10 Aspergillus niger species complex), and 10 Alternaria alternata isolates.

Antifungal susceptibility tests

Inocula were prepared according to Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS) document M38-A.²⁸ Simply, the isolates were passaged twice at an interval of 7 days on potato dextrose agar slants at 35°C for 7 days (Aspergillus spp.) or at 35°C for 72 h and then at 26°C until day 7 (Fusarium spp. and Al. alternata). A seven-day-old colony was covered with 1 mL of sterile 0.85% saline, and then a suspension was made. The resulting mixture of conidia and hyphal fragments was withdrawn. After heavy particles had been allowed to settle for 3–5 min, the upper homogeneous suspension was collected and mixed with a vortex mixer for 15 s. The turbidity of the supernatants was measured spectrophotometrically at a wavelength of 530 nm, and transmission was adjusted to 68%–70% (Fusarium spp. and Al. alternata) or 80%–82% (Aspergillus spp.). These suspensions were diluted 1:50 in RPMI 1640. The 1:50 inoculum dilutions corresponded to twice the density needed of ∼0.4×10⁴ to 5×10⁴ colony forming unit (CFU)/mL.

A broth microdilution method was performed by following CLSI M38-A document.²⁸ The tests were performed in duplicate in 96-well flat-bottom microtitration plates. Each well was inoculated with 0.1 mL of the 2×conidial inoculum suspension. The growth control wells contained 0.1 mL of the corresponding diluted inoculum suspension and 0.1 mL of RPMI 1640 broth without antifungal agents (with dimethyl sulfoxide 2%). Candida parapsilosis ATCC22019 was used as a quality control. The quality control was tested in the same manner and was included in each test. Plates were incubated at 35°C for 48 h.

The MIC was determined as the lowest concentrations of nano-Ag or natamycin that prevented any discernible growth. The MIC range and mode, the MIC for 50% of the strains tested (MIC₅₀ value), and the MIC₉₀ value were provided for the isolates with the SPSS statistical package (version 17.0). For calculation, any high off-scale MIC was converted to the next higher concentration.

Results

All the isolates produced detectable growth after 48 h of incubation at 35°C. The MIC of the quality control strain was within the reference ranges for each test. The in vitro activities of nano-Ag and natamycin against the ocular pathogens were summarized in Tables 1 and 2, respectively.

Table 1.

In Vitro Susceptibilities of Ocular Fusarium Isolates to Nano-Ag and Natamycin

	MICs (μg/mL)
Organism (No.) and antifungal agents	Range	Mode	MIC₅₀ value	MIC₉₀ value
Fusarium solani species complex (82)
Nano-Ag	0.25–2	1	1	1
Natamycin	4–32	4	4	8
Fusarium verticillioides species complex (20)
Nano-Ag	0.5–2	1	1	2
Natamycin	4–8	4	4	8
Fusarium oxysporum species complex (10)
Nano-Ag	0.5–2	0.5	0.5	1
Natamycin	4–8	4	4	8
Fusarium spp. (112)
Nano-Ag	0.25–2	1	1	1
Natamycin	4–32	4	4	8

MIC, minimum inhibitory concentration.

Table 2.

In Vitro Susceptibilities of Ocular Aspergillus and Alternaria alternata Isolates to Nano-Ag and Natamycin

	MICs (μg/mL)
Organism (No.) and antifungal agents	Range	Mode	MIC₅₀ value	MIC₉₀ value
Aspergillus flavus species complex (61)
Nano-Ag	0.5–1	0.5	0.5	1
Natamycin	4–32	32	32	32
Aspergillus fumigatus species complex (11)
Nano-Ag	0.25–1	0.5	0.5	0.5
Natamycin	4–32	4	4	4
Aspergillus versicolor species complex (12)
Nano-Ag	0.125–0.5	0.5	0.25	0.5
Natamycin	4–32	8	8	32
Aspergillus niger species complex (10)
Nano-Ag	0.0625–0.5	0.25	0.25	0.5
Natamycin	0.25–16	4	4	8
Aspergillus spp. (94)
Nano-Ag	0.0625–1	0.5	0.5	1
Natamycin	0.25–32	32	32	32
Alternaria alternata (10)
Nano-Ag	0.25–1	0.5	0.5	1
Natamycin	2–8	4	4	4

MIC₅₀ values of nano-Ag were 1, 0.5, and 0.5 μg/mL for Fusarium spp., Aspergillus spp., and Al. alternata, respectively. MIC₉₀ values of nano-Ag were 1, 1, and 1 μg/mL for Fusarium spp., Aspergillus spp., and Al. alternata, respectively. MIC₅₀ values of natamycin were 4, 32, and 4 μg/mL for Fusarium spp., Aspergillus spp., and Al. alternata, respectively. MIC₉₀ values of natamycin were 8, 32, and 4 μg/mL for Fusarium spp., Aspergillus spp., and Al. alternata, respectively.

Discussion

In this study, nano-Ag, relative to natamycin, exhibited very high activity against ocular pathogenic filamentous fungi. When comparing the MIC₉₀ values of nano-Ag with those of natamycin, the activity of nano-Ag against Fusarium spp., Aspergillus spp., and Al. alternata was 8 times, 32 times, and 4 times, respectively, greater than that of natamycin. Therefore, the antifungal activity of nano-Ag was significantly superior to that of natamycin against ocular pathogenic filamentous fungi in vitro. The result suggests the possibility of using nano-Ag to eradicate ocular pathogenic filamentous fungi. This is an important result, particularly when the clinical management of fungal keratitis has been limited due to the relative unavailability of effective antifungals. It should be emphasized that all the multiplicity fungi used in this article were derived from clinical patients with fungal keratitis. To our knowledge, this is the first study that applies nano-Ag successfully to ocular pathogenic filamentous fungi in vitro. Future work based on animal model tests should address the suitability of nano-Ag for treatment of fungal keratitis.

Susceptibility breakpoints for natamycin have not been described so far in CLSI guidelines; however, an MIC value of 16 μg/mL or less is considered to indicate susceptibility of a fungal isolate.^29,30 In our study, Fusarium spp. and Al. alternata isolates were susceptible to natamycin. However, natamycin shows various activities against different Aspergillus spp. Most Aspergillus spp. were not susceptible, but As. fumigatus complex and As. niger complex were susceptible to natamycin.

The antifungal activity of nano-Ag against fungal pathogens of the eye has not been studied and probably only the work by us, in which we determined that the MIC₉₀ values of silver nitrate were 2 μg/mL for Fusarium spp. and Al. alternata, respectively, and 1 μg/mL for Aspergillus spp., can be used for the qualitative comparison.^5,12 Comparing the antifungal activity of nano-Ag with the activity of silver nitrate, nano-Ag shows higher antifungal activities against Fusarium spp. and Al. alternata, and the same antifungal activities against Aspergillus spp. in vitro. We utilized the same protocol as described by CLSI's publication M38-A in our previous publication on silver nitrate; therefore, the results published by us can be used for the qualitative comparison, and the comparison of the MICs between silver nitrate and nano-Ag has validity.

The nano-Ag shows efficient antimicrobial characteristics compared with other salts due to their extremely large surface area, which provides better contact with microorganisms.³¹ Nano-Ag are usually within a size range of <100 nm. As is the case with all nanomaterials, the principle characteristic of nano-Ag is their ultra-small size. Ultra-small particle size leads to ultra-large surface area per mass where a large proportion of atoms are in immediate contact with ambiance and readily available for reaction. Nano-Ag in the sub-50 nm range exhibit increased efficacy in inhibiting a wide range of bacteria and fungi.^22,31–35

Panacek et al.²³ have recently investigated the toxicity of ionic silver and nano-Ag against the tested human fibroblast. The results indicate that ionic silver is cytotoxic against the tested eukaryotic cell lines at LC100 values (the concentrations of silver lethal to 100% of the cells)=1 μg/mL, whereas the nano-Ag shows a cytotoxic effect only at concentrations higher than 30 μg/mL. The fungistatic activity of the nano-Ag is determined at the concentrations 100 times lower than their cytotoxic level, and, therefore, the use of nano-Ag as the antifungal agents is less complicated in terms of their cytotoxicity to human eukaryotic cells when compared with ionic silver. Similar relations between cytotoxicity and antibacterial activity of ionic silver and nano-Ag have been already published for the study of nano-Ag as a source of antimicrobial silver for possible controlled-release contact lens controlled delivery formulations.³⁶ The study proves that silver nitrate convincingly kills human corneal epithelial cells and a murine macrophage cell line at concentrations of 8 and 10 μM in serum-free media, but nano-Ag shows no significant impact on cell toxicity for either cell line, at any dosing, versus untreated controls.³⁶

In conclusion, in this study, nano-Ag, relative to natamycin, exhibits potent in vitro activity against ocular pathogenic filamentous fungi Fusarium spp., Aspergillus spp., and Al. alternata. These results indicate that nano-Ag may have potential as an antifungal agent in treating fungal keratitis and should be further tested in vivo for evaluation of efficacy and safety to ascertain the real clinical value.

Footnotes

Acknowledgments

This research was supported by the National Natural Science Fund of China (81241033), Fund of Bureau of Health of Henan Province (201002012), and Fund of Bureau of Science and Technology of Henan Province (0641130305).

Author Disclosure Statement

No competing financial interests exist.

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